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Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue
Accepted Manuscript Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue G.D.A. Gastal, F.L.N. Aguiar, G.M. Ishak, C.A. Cavinder, S.T. Willard, P.L. Ryan, J.M. Feugang, E.L. Gastal PII:
S0093-691X(18)30225-5
DOI:
https://doi.org/10.1016/j.theriogenology.2018.11.034
Reference:
THE 14792
To appear in:
Theriogenology
Received Date: 19 May 2018 Revised Date:
30 November 2018
Accepted Date: 30 November 2018
Please cite this article as: Gastal GDA, Aguiar FLN, Ishak GM, Cavinder CA, Willard ST, Ryan PL, Feugang JM, Gastal EL, Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue, Theriogenology (2019), doi: https://doi.org/10.1016/j.theriogenology.2018.11.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of cryopreservation techniques on proliferation and
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apoptosis of cultured equine ovarian tissue
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G.D.A. Gastal1, F.L.N. Aguiar1, G.M. Ishak1,2, C.A. Cavinder3, S.T. Willard3, P.L. Ryan3, J.M.
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Feugang3, E.L. Gastal1*
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lllinois, USA
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Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale,
Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Baghdad,
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Baghdad, Iraq
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USA
Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS,
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Short title: Cryopreservation and in vitro culture of equine ovarian tissue
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Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA. Tel.: + 618 453
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1774; fax: + 618 453 5231. E-mail: [email protected]
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Correspondence: Eduardo Gastal, Department of Animal Science, Food and Nutrition, Southern
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Abstract
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Preservation of cellular integrity and its mechanisms after ovarian tissue cryopreservation (OTC)
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and in vitro culture (IVC) procedures are crucial aspects for the success of preservation and
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recovery of female fertility. This study aimed to evaluate the effects of two cryopreservation
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methods (slow-freezing, SF, and vitrification, VIT) on the equine ovarian tissue after 1, 3, and 7
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days of IVC by assessing: (i) preantral follicle morphology and distribution of follicle classes;
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(ii) protein expression of markers of cell proliferation for EGFR and Ki-67; (iii) markers of
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apoptosis for Bax and Bcl-2; and (iv) DNA fragmentation. Percentages of normal primordial
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follicles were similar (P > 0.05) among SF-control, VIT-control, and fresh control groups. After
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7 days of culture, VIT-IVC7 had a greater (P < 0.05) total percentage of normal preantral
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follicles when compared with SF-IVC7, but both had a lower (P < 0.05) percentage than fresh
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IVC7 group. Prior to and after 7 days of culture, expression of EGFR and Ki-67 were similar (P
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> 0.05) among fresh, SF, and VIT groups. After 7 days of culture, VIT had higher (P < 0.05) Bax
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expression than the fresh and SF tissues, but Bcl-2 was similar (P > 0.05) among groups. Prior to
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IVC, TUNEL signals were similar (P > 0.05) among groups; however, VIT-IVC7 had greater
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(P<0.05) TUNEL signals when compared with the fresh IVC7 group. In conclusion, findings
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demonstrated: (i) similar efficiency between SF and VIT compared with fresh control to preserve
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morphologically normal follicles; and (ii) similar tissue functionality and cell proliferation
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capability after equine OTC by either SF and VIT methods following IVC for 7 days. The results
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herein presented shed light on equine fertility preservation programs using OTC techniques.
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Keywords: equine; preantral follicles; cryopreservation; in vitro culture; proliferation
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1. Introduction Ovarian tissue cryopreservation (OTC) has been a fundamental technique to preserve fertility
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for cancer patients, domestic animals, and endangered species [1, 2]. Animal models have played
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a major role in the improvement of OTC protocols [1, 3-5], allowing the success and expansion
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of OTC and ovarian tissue transplantation techniques; these fertility restoration procedures have
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been able to generate more than 80 successful live births in humans [6]. Moreover, OTC has
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several advantages over cryopreservation of oocytes and embryos, which include but are not
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limited to: a larger number of primordial germ cells is available in the ovarian cortex [7], the
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method of ovarian tissue biopsy can be performed independently of age and stage of the
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reproductive cycle [8-10], part of the ovarian tissue can be thawed and submitted to in vitro
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culture (IVC) [11-16], the ovarian tissue tolerates transport at low temperatures [17, 18], and
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fertilization is not immediately required [19]. Therefore, new protocols of OTC using either
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slow-freezing (SF) or vitrification (VIT) methods have been constantly tested, improved, and
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simplified to determine their success in preserving ovarian tissue quality. However, the impact of
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OTC techniques on cellular/molecular mechanisms is still not fully understood; therefore, efforts
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are necessary to understand those effects in order to improve the methodologies to better
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preserve and recover female fertility.
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Currently, to determine the quality of ovarian tissue after the cryopreservation procedure, the
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tissue is evaluated immediately after cryopreservation/thawing [4, 20], and can be submitted to
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IVC for determining the capability of preantral follicles to develop and grow [4, 11, 21, 22].
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Therefore, efficient IVC methods have been developed in different animal models to obtain
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valuable information regarding the quality of the fresh ovarian tissue, such as the preantral
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follicle morphology, activation and development, and the expression of cell proliferation,
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apoptosis, and angiogenic factors [14, 23-26]. Recently, suitable IVC media for equine preantral
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follicles enclosed in fresh ovarian tissue has been developed [14] and improved [4] to ensure the
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maintenance of stromal cell survival and proliferation. However, information about the potential
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of cryopreserved-thawed equine ovarian tissue to survive and proliferate when submitted to IVC
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is still scarce [4].
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The culture of preantral follicles enclosed in the ovarian tissue allows a better cell-cell
interaction among the surfaces of adjacent cells [25]. This plays a crucial role in the development
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and function of multicellular organs [27] such as the ovary which contains stromal, theca and
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granulosa cells, and oocytes. Indeed, the in situ culture allows the evaluation of factors related to
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cell proliferation and apoptosis during neoangiogenesis [28]. In this regard, the stromal cells for
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example are responsible for expressing proteins related to cell proliferation, such as epidermal
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growth factor receptor (EGFR) [29, 30] and Ki-67 [5], and for interacting with the follicles to
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produce competent oocytes [31]. Only a few studies have investigated the influence of
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cryopreservation on ovarian stromal cells during IVC [32, 33]. EGFRs have been found in
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stromal cells located near primordial follicles; this has been believed to contribute to follicle
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activation and development, and it is thought that disrupting the EGFR protein may lead to
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apoptosis and early cell death [32]. In respect to Ki-67 antibody, it recognizes a nuclear antigen
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that is expressed in all stages of the cell cycle except G0 [34]; the expression of Ki-67 has been
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demonstrated in frozen-thawed ovarian stromal cells and is highly correlated with EGFR
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expression [5]. Therefore, it is important to understand the impact of cryopreservation on EGFR
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and Ki-67 expression in the ovarian tissue after thawing and during the IVC.
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The aim of this study was to evaluate the effects of two cryopreservation methods – SF and VIT – on equine ovarian tissue after 1, 3, and 7 days of in vitro culture by assessing: (i) preantral
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follicle morphology and distribution of follicle classes, (ii) markers of cell proliferation (EGFR
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and Ki-67), (iii) markers of apoptosis (Bax and Bcl-2), and (iv) DNA fragmentation.
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2. Materials and methods
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2.1. Chemicals
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise
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2.2. Ovarian tissue collection and processing
All experimental procedures were performed according to the United States Government
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Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and
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Training (grants.nih.gov/grants/olaw/references/phspol.htm). The use of animals and procedures
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were approved by Mississippi State University Institutional Animal Care and Use Committee.
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Ovaries were harvested, during the anestrous season (February), from 5 Quarter horse type mares
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(7-19 years old) slaughtered at Mississippi State University. Briefly, ovaries were rinsed in
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alcohol 70%, followed by three washes in saline solution (0.9% NaCl) supplemented with
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antibiotics (100 IU/ml penicillin and 100 µg streptomycin/ml). Subsequently, ovaries were
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placed in a petri dish with α-MEM containing 0.4% BSA, 93.3 U/ml penicillin G, 36.8 U/ml
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streptomycin sulphate, 0.047 mmol/l pyruvic acid (sodium salt), and 2.5 mM Hepes [10]. Then,
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ovaries were divided into three longitudinal portions (two lateral and one middle); only the
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middle portion of the ovary was used to collect fragments [18] for this study. Fragments were
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sliced into small pieces (3 x 3 x 0.5 mm) using scalpels, tweezers, and the Thomas Stadie-Riggs
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Tissue Slicer (Thomas Scientific®, Swedesboro, NJ, USA) to obtain a standard thickness (0.5
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mm). Only one ovary of each mare was used and considered as a replicate. Therefore, five
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replicates were performed.
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2.3. Experimental design
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Two cryopreservation methods (SF vs. VIT) were compared to evaluate the viability of
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cryopreserved-thawed equine ovarian tissue submitted to in vitro culture for 0 (noncultured
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control), 1, 3, or 7 days. The study involved twelve groups: fresh noncultured control, fresh IVC-
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1 day, fresh IVC-3 days, fresh IVC-7 days, SF noncultured, SF IVC-1 day, SF IVC-3 days, SF
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IVC-7 days, VIT noncultured, VIT IVC-1 day, VIT IVC-3 days, and VIT IVC-7 days. Fresh
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tissue (standard control) was evaluated at the same time points of the cryopreservation
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treatments. A total of 96 small fragments from each ovary were randomly distributed among
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twelve groups (n = 8 fragments/group/replicate; total = 480 fragments) to evaluate the following
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endpoints: preantral follicle morphology and distribution of follicle classes; protein expression of
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proliferative (Ki-67 and EGFR) and pro- and anti-apoptotic (Bax and Bcl-2, respectively)
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markers, and DNA fragmentation.
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2.4. Slow-freezing of ovarian tissue
Cryopreservation of ovarian tissue fragments by the slow-freezing method was performed
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according to protocol described previously [4]. Briefly, ovarian fragments were placed in 1.5 ml
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cryovials containing the following cryoprotectant solution: α-MEM supplemented with 2.5 mM
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Hepes, 10% fetal equine serum (FES), 0.25 M sucrose, and 1.5 M DMSO. After equilibration
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time in the cryoprotectant at room temperature (RT), the cryovials were placed in a
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programmable freezing machine (Bio-Cool IV40 - Controlled Rate Freezer, SP Scientific
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Company, Warminster, PA, USA). The cooling curve was programmed for a rate of 2°C/min to
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‒7°C and the seeding was done manually by touching the vials with a forceps dipped into liquid
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nitrogen (LN2). Then, the freezing curve was set for a rate of 0.3°C/min to ‒40 ºC, and vials were
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plunged in LN2, and stored for 1 week. For the thawing process, the cryovials were exposed to
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RT for 30 sec and then immersed in water at 37ºC for 1 min. Frozen-thawed fragments were
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washed in three step solutions (5 min each) in the following order: α-MEM + 2.5 mM Hepes +
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10% FES + 0.5 M sucrose; α-MEM + 2.5 mM Hepes + 10% FES + 0.25 M sucrose; and α-MEM
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+ 2.5 mM Hepes + 10% FES.
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2.5. Vitrification of ovarian tissue
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Cryopreservation of equine ovarian tissue fragments by the vitrification method was
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performed according to a protocol previously described [4]. Briefly, fragments submitted to
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vitrification were placed in petri dishes containing the following solutions and periods of
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equilibration: 1st step: 0.3 M ethylene glycol (EG) and 0.5 M trehalose in base medium (α-MEM
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+ 2.5 mM Hepes + 6% FES) for 3 min at RT; 2nd step: 1.5 M EG in base medium for 1 min at
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RT; and 3rd step: 3 M EG in base medium for 1 min at RT. Fragments were lightly dried, placed
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in cryovials (1 ml), plunged in LN2, and stored for 1 week. For the thawing process, the cryovials
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were exposed to RT for 30 sec and then immersed in water at 37ºC for 1 min. Then, to remove
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the cryoprotectant solution, fragments were submitted to decreasing concentrations of sucrose
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(0.5, 0.25 and 0 M in the base medium) for 5 min within each solution.
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2.6. In vitro culture
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Ovarian fragments were in vitro cultured according to the protocol reported previously [4]. Briefly, ovarian fragments were placed in culture plates (4 fragments per well) containing 1000
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µl of medium composed of α-MEM supplemented with insulin (10 ng/ml), transferrin (5 ng/ml),
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selenium (5 ng/ml), glutamine (2 mM), hypoxanthine (2 mM), BSA - bovine serum albumin
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(1.25 mg/ml), ascorbic acid (50 µg/ml), follicle stimulating hormone from porcine pituitary (50
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µg/ml), EGF - epidermal growth factor (50 ng/ml), penicillin (100 µg/ml), and streptomycin (100
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µg/ml). Fragments were cultured for 1, 3, or 7 days at 39°C in a humidified atmosphere with 5%
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CO2 in air. Culture medium was completely replaced on days 2, 4, and 6 of culture.
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2.7. Histological processing
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Fresh and cryopreserved-thawed fragments were fixed in 4% paraformaldehyde for 4 h and then kept in 70% alcohol at 4ºC until standard histological processing. The fragments were
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embedded in paraffin and cut into 7-µm serial sections [35]. Every section was mounted and
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stained with Periodic Acid-Schiff (PAS) and counterstained in hematoxylin. Histological
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sections were analyzed using a light microscope (Nikon E200, Tokyo, Japan) at 40× objective
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magnification and an image capture system (LEICA Imaging Software, Wetzlar, Germany).
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2.8. Morphological classification of follicles Preantral follicles with visualized oocyte nucleus were counted and morphologically
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classified as either normal (follicles containing an intact oocyte and oocyte nucleus surrounded
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by granulosa cells well organized in one or more layers) or abnormal (follicles with a retracted
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oocyte or disorganized granulosa cell layers detached from the basement membrane and oocyte
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with pyknotic nucleus). Preantral follicles were classified according to their developmental stage
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into primordial, transitional, primary, and secondary [36].
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2.9. Immunohistochemistry
Ovarian tissue sections mounted on slides were deparafinized, submitted to antigen retrieval
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in sodium citrate buffer for 30 min in a steamer, washed with PBS, and submitted to immediate
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standard in situ immunofluorescence detection of EGFR, Ki-67, Bax, and Bcl-2 proteins as
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described previously [4]. Briefly, slides were permeabilized in 1% Triton X-100 for 30 min at
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RT, non-specific binding sites blocked in 1% BSA solution for 60 min at RT, and incubated with
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100× diluted rabbit polyclonal anti-human EGFR (sc-03-G; Santa Cruz Biotechnology, Dallas,
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TX, USA), Ki-67 (PA5-19462; Thermo Fisher Scientific Inc., Waltham, MA, USA), Bax
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(orb312174; Biorbyt, Berkeley, CA, USA), or Bcl-2 (sc-492; Santa Cruz Biotechnology, Dallas,
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TX, USA) antibodies for 60 min at RT. Then, slides were incubated with FITC labeled goat anti-
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rabbit secondary antibody (200x dilution) for 60 min at RT. Three washes with PBS solution
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were performed between all steps. Slides were immediately covered with a DAPI-containing
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mounting medium to counterstain cell nuclei for a fluorescence evaluation using a fluorescent
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microscope (EVOS FL Cell Imaging System, Thermo Fisher Scientific Inc., Waltham, MA
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USA). Five images from randomly selected fields of one tissue section/treatment/replicate were
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obtained at 20x objective magnification and analyzed using ImageJ software (version 1.50f) to
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calculate the fluorescence intensity of all labeled cells in the ovarian stroma.
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2.10. TUNEL assay TUNEL staining was carried out using a commercially available kit (DeadEnd™ Fluorometric TUNEL System, Promega©, Madison, WI, USA) following the manufacturer’s
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instructions. Tissue sections were examined, and five images were obtained at 20x objective
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magnification to calculate the fluorescence intensity of TUNEL positive cells using the ImageJ
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software. TUNEL positive and negative controls were included in all evaluations, according to
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the manufacturer’s recommendations.
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2.11. Statistical analyses
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All statistical analyses were performed using R statistical software version 3.0.2 (R Foundation for Statistical Computing, Vienna, Austria). Data for the end points evaluated did not
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follow a normal distribution. Therefore, statistical difference among treatments was analyzed by
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Kruskal–Wallis test, and Mann–Whitney U test was used to compare mean values among
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groups. A probability of P < 0.05 indicated that a difference was significant. Data are presented
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as mean ± SEM and percentages.
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3. Results
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3.1. Preantral follicle morphology and class distribution
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A total of 620 preantral follicles were recorded with a mean of 51.7 ± 7.3 follicles per
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treatment (Table 1). Percentage of normal primordial follicles was similar (P > 0.05) among
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fresh noncultured control, SF-control, and VIT-control groups. However, follicle morphology
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was impaired (P < 0.05) in cryopreserved fragments (SF and VIT treatments) submitted to IVC
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for 1, 3, and 7 days when compared with fresh tissue. Therefore, data from morphologically
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normal preantral follicles at days 1, 3, and 7 of culture were grouped to evaluate the overall
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effect of IVC on follicle morphology after cryopreserved-thawed by SF and VIT methods in the
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total percentage of normal preantral follicles and according to follicle classes (Table 2). Thus,
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VIT treatment had a greater (P < 0.05) total percentage of normal preantral follicles when
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compared with SF treatment after IVC.
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3.2. Markers of proliferation
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Prior to and after 7 days of culture, protein expression of EGFR (Fig. 1) and Ki-67 (Fig. 2) in SF and VIT treatment groups was similar (P > 0.05) to that of the fresh noncultured control
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group. At day 1 of culture, SF-IVC1 and VIT-IVC1 groups had lower (P < 0.05) EGFR
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expression and similar (P > 0.05) Ki-67 expression compared to the fresh IVC1 group. At day 3
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of culture, SF-IVC3 and VIT-IVC3 groups had similar (P > 0.05) EGFR expression compared to
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fresh IVC3; however, the VIT-IVC3 group had higher (P < 0.05) EGFR expression compared to
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the SF-IVC3 group. Moreover, Ki-67 expression was similar (P > 0.05) between fresh IVC3 and
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VIT-IVC3 groups and lower (P < 0.05) in the SF-IVC3 group.
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3.3. Markers of apoptosis
All treatment groups had lower (P < 0.05) Bax (Fig. 3) and Bcl-2 (Fig. 4) expression when
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compared with the fresh noncultured control group. At day 1 of culture, the VIT-IVC1 group had
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lower (P < 0.05) Bax expression compared to the fresh IVC1 and SF-IVC1 groups; Bcl-2
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expression in the SF-IVC1 and VIT-IVC1 groups was similar (P > 0.05) compared to the fresh
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IVC1 group. At day 3 of culture, the fresh IVC3 group had similar (P > 0.05) Bax expression
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compared to the VIT-IVC3 group, but higher (P < 0.05) than the SF-IVC3 group; however, the
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VIT-IVC3 group had higher (P < 0.05) Bcl-2 expression compared with the fresh IVC3 and SF-
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IVC3 groups. After 7 days of culture, Bax expression was greater (P < 0.05) in the VIT-IVC7
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group compared with the fresh IVC7 and SF-IVC7 groups; however, Bcl-2 expression did not
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differ (P > 0.05) among groups.
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3.4. DNA fragmentation
Prior to in vitro culture, DNA fragmentation was similar (P > 0.05) among treatment groups
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(Fig. 5). At day 1 of culture, the SF-IVC1 and VIT-IVC1 groups had lower (P < 0.05) DNA
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fragmentation compared to the fresh IVC1 group. At day 3 of culture, SF-IVC3 had lower (P <
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0.05) DNA fragmentation compared to the fresh IVC3 and VIT-IVC3 groups. At day 7 of
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culture, the VIT-IVC7 group had greater (P < 0.05) DNA fragmentation compared to fresh
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IVC7, but similar (P > 0.05) to the SF-IVC7 group.
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4. Discussion
Ovarian tissue cryopreservation techniques may disturb cellular mechanisms involved with cell proliferation and apoptosis signaling after thawing [4]. To the best of our knowledge, the
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present study has shown for the first time the expression pattern of EGFR, Ki-67, Bax, and Bcl-2
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in equine ovarian stromal cells cryopreserved by either slow-freezing or vitrification methods
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during IVC for up to 7 days. Moreover, the study has shown the preantral follicle morphology
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and DNA degradation throughout the in vitro culture period for fresh and cryopreserved-thawed
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equine ovarian tissue.
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In the present study, EGFR expression in cryopreserved-thawed tissue by slow-freezing and vitrification methods after 7 days of culture was similar to that of fresh tissue, demonstrating
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tissue functionality and growth capability. Similarly, protein expression of Ki-67 after 7 days of
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culture corroborated the survivability and proliferative potential of cryopreserved-thawed equine
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ovarian tissue by slow-freezing and vitrification methods. Protein expression of Ki-67 has been
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well-described as an acceptable index of cell proliferation for cryopreserved ovarian tissue after
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thawing [37] and submission to in vitro culture [4]. A recent study [14] has shown that addition
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of EGF to the culture medium contributed to the maintenance of follicular survival and activation
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of preantral follicles during in vitro culture. EGFRs are involved in different cell signaling
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pathways, such as MAP-kinase (ERK), protein kinase C, phosphatidyl-inositol 3-kinase (PI3-
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kinase), and G protein coupled receptors [38]. Although low levels of EGFR are present in
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granulosa cells of small preantral follicles, primary and secondary follicles of the hamster
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responded to an EGF-stimulating effect on cellular growth [39]. Moreover, EGFR has been
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shown to be present in high concentrations in the theca interna and granulosa cells of equine
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antral follicles [40]. Notwithstanding, protein-receptor coupling is required for activating the
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aforementioned pathways contributing to tissue proliferation. Therefore, equine ovarian tissue
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can be cryopreserved and in vitro cultured following the methods applied in the present study
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without jeopardizing the EGF protein receptors.
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Equine ovarian tissue cryopreserved-thawed by slow-freezing and vitrification had lower
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expression of Bax and Bcl-2 compared with fresh noncultured tissue. The lower expression of
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Bax and Bcl-2 after thawing may have been caused by ice recrystallization during
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cryopreservation [41], which altered the abundance and expression of several proteins [42].
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However, slow-freezing and vitrification had higher Bax expression after 7 days of culture
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compared to fresh tissue, while the expression of Bcl-2 was similar among the treatment groups.
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The same pattern of DNA degradation was observed in cryopreserved-thawed and in vitro
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cultured ovarian tissues evaluated by TUNEL assay. In this regard, our recent study [4] has
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shown a similar pattern of apoptosis after the thawing process for equine ovarian tissue. Similar
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results have been reported for human [34, 37] and sheep [43] ovaries after OTC and IVC.
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However, no information was available up to now regarding apoptosis of cryopreserved-thawed
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equine ovarian tissue submitted to IVC. Bax and Bcl-2 proteins play a major role in the intrinsic
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apoptotic pathway triggered by mitochondrial dysfunction [44]. Therefore, an adequate balance
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between pro- (Bax) and anti-apoptotic (Bcl-2) markers seems to be vital to determine the fate of
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the cells [45].
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The present study has shown that fresh ovarian fragments had approximately 60% morphologically normal preantral follicles after 7 days of in vitro culture. Moreover, frozen and
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vitrified tissues had more than 70% morphologically normal preantral follicles after the thawing
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process. However, a low percentage (10%) of morphologically normal preantral follicles
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enclosed in cryopreserved-thawed (slow-freezing and vitrificaion) ovarian tissues were found
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after 7 days of culture. Our present results are in agreement with our recent publication [4], in
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which 70% of preantral follicles enclosed in fresh ovarian tissue and 10% of preantral follicles
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enclosed in cryopreserved-thawed ovarian tissues maintained a normal morphology after 7 days
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in culture. Altogether, VIT-IVC7 had a greater percentage of morphologically normal preantral
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follicles compared to SF-IVC7, but a higher expression of Bax and DNA fragmentation was
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observed after 7 days of culture. Therefore, two hypotheses have been raised: (i) a greater
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percentage of normal follicles in VIT-IVC7 demands higher tissue metabolism and, therefore,
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causes higher Bax expression and DNA fragmentation; or (ii) although there is a greater
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percentage of normal follicles at day 7 of culture, the tissue metabolism is burdened and
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apoptosis pathways are activated, causing tissue death. In agreement with our findings, a recent
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study [46] has shown a significant decrease in preantral follicle density and morphology after 7
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days of in vitro culture of vitrified caprine ovarian tissue compared with fresh tissue; the authors
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suggested the need for different medium composition to culture vitrified tissues. In this regard,
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we believe that the tissue suffers two types of stress when submitted to cryopreservation and in
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vitro culture; firstly, the tissue must recover from the cryopreservation and thawing processes,
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where several modifications occur at the cell membrane and organelle levels; secondly, the tissue
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must adapt to the new in vitro culture environment, requiring readjustment of physiological
325
pathways to survive and continue to grow. Based on the prior assumptions, a better preservation
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of proliferative markers in the ovarian tissue may help to minimize the stress caused by the
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cryopreservation techniques. Therefore, adaptations in the cryopreservation and thawing
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procedures, medium compositions, and culture environment warrant further investigation to
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maximize the potential of fertility preservation using OTC.
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In conclusion, our study has shown that cryopreservation of equine ovarian tissue by slow-
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freezing or vitrification techniques had similar efficiency in preserving morphologically normal
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follicles. Moreover, tissue functionality and cell proliferation capability were preserved after
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OTC by either slow-freezing or vitrification methods following IVC for 7 days. The results
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herein presented shed light upon the equine fertility preservation programs using OTC
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techniques.
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Acknowledgements
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The authors thank Saffron Scientific Histology Services for technical assistance with histological
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preparation. Research was supported by Southern Illinois University (SIU) and USDA-ARS
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Grant # 58-6402-3- 018 (to J M Feugang). Gastal G.D.A. was the recipient of a PhD scholarship
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from The National Council for Scientific and Technological Development (CNPq; grant
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#246741/ 2012-0), Brazil. Ishak G.M. was the recipient of a PhD scholarship from the Ministry
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of Higher Education & Scientific Research, Baghdad, Iraq. Aguiar F. L. N. was the recipient of a
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doctoral scholarship from Fundação Cearense de Apoio ao Desenvolvimento Científico e
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Tecnológico (FUNCAP), Brazil.
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References
349
[1] Comizzoli P, Wildt DE. Mammalian fertility preservation through cryobiology: value of
350
classical comparative studies and the need for new preservation options. Reprod Fertil Dev
351
2014;26:91-8.
352
[2] Dolmans MM, Binda MM, Jacobs S, Dehoux JP, Squifflet JL, Ambroise J, et al. Impact of
353
the cryopreservation technique and vascular bed on ovarian tissue transplantation in cynomolgus
354
monkeys. J Assist Reprod Genet 2015;32:1251-62.
355
[3] Santos RR, Amorim C, Cecconi S, Fassbender M, Imhof M, Lornage J, et al.
356
Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of
357
endangered species and breeds. Anim Reprod Sci 2010;122:151-63.
358
[4] Gastal GD, Aguiar FL, Alves BG, Alves KA, de Tarso SG, Ishak GM, et al. Equine ovarian
359
tissue viability after cryopreservation and in vitro culture. Theriogenology 2017;97:139-47.
360
[5] Gastal GD, Aguiar FL, Rodrigues AP, Scimeca J, Apgar G, Banz W, et al. Cryopreservation
361
and in vitro culture of white-tailed deer ovarian tissue. Theriogenology 2018;113:253-60.
362
[6] Jensen AK, Macklon KT, Fedder J, Ernst E, Humaidan P, Andersen CY. 86 successful births
363
and 9 ongoing pregnancies worldwide in women transplanted with frozen-thawed ovarian tissue:
364
focus on birth and perinatal outcome in 40 of these children. J Assist Reprod Genet 2017;34:325-
365
36.
366
[7] Faddy M, Gosden R, Gougeon A, Richardson SJ, Nelson J. Accelerated disappearance of
367
ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 1992;7:1342-
368
6.
369
[8] Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Orthotopic and heterotopic
370
ovarian tissue transplantation. Hum Reprod Update 2009;15:649-65.
AC C
EP
TE D
M AN U
SC
RI PT
348
ACCEPTED MANUSCRIPT
"Clean copy"
18
[9] Alves KA, Alves BG, Gastal GD, de Tarso SG, Gastal MO, Figueiredo JR, et al. The mare
372
model to study the effects of ovarian dynamics on preantral follicle features. PloS One
373
2016;11:e0149693.
374
[10] Haag KT, Magalhaes-Padilha DM, Fonseca GR, Wischral A, Gastal MO, King SS, et al. In
375
vitro culture of equine preantral follicles obtained via the Biopsy Pick-Up method.
376
Theriogenology 2013;79:911-7.
377
[11] Isachenko V, Montag M, Isachenko E, van der Ven K, Dorn C, Roesing B, et al. Effective
378
method for in vitro culture of cryopreserved human ovarian tissue. Reprod Biomed Online
379
2006;13:228-34.
380
[12] Aguiar FL, Lunardi FO, Lima LF, Rocha RM, Bruno JB, Magalhaes-Padilha DM, et al.
381
FSH supplementation to culture medium is beneficial for activation and survival of preantral
382
follicles enclosed in equine ovarian tissue. Theriogenology 2016;85:1106-12.
383
[13] Aguiar FL, Lunardi FO, Lima LF, Rocha RM, Bruno JB, Magalhaes-Padilha DM, et al.
384
Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and
385
reduces reactive oxygen species production after culture. Theriogenology 2016;85:1063-9.
386
[14] Aguiar FL, Gastal GD, Ishak GM, Gastal MO, Teixeira DI, Feugang JM, et al. Effects of
387
FSH addition to an enriched medium containing insulin and EGF after long-term culture on
388
functionality of equine ovarian biopsy tissue. Theriogenology 2017;99:124-33.
389
[15] Sales AD, Duarte AB, Santos RR, Alves KA, Lima LF, Rodrigues GQ, et al. Modulation of
390
aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro
391
culture. Cell Tissue Res 2016;365:415-24.
AC C
EP
TE D
M AN U
SC
RI PT
371
ACCEPTED MANUSCRIPT
"Clean copy"
19
[16] Filatov M, Khramova Y, Kiseleva M, Malinova I, Komarova E, Semenova M. Female
393
fertility preservation strategies: cryopreservation and ovarian tissue in vitro culture, current state
394
of the art and future perspectives. Zygote 2016;24:635-53.
395
[17] Schmidt K, Ernst E, Byskov A, Andersen AN, Andersen CY. Survival of primordial
396
follicles following prolonged transportation of ovarian tissue prior to cryopreservation. Hum
397
Reprod 2003;18:2654-9.
398
[18] Gastal GD, Alves BG, Alves KA, Souza ME, Vieira AD, Varela AS, et al. Ovarian
399
fragment sizes affect viability and morphology of preantral follicles during storage at 4 °C.
400
Reproduction 2017;153:577-87.
401
[19] Shaw J, Cox S, Trounson A, Jenkin G. Evaluation of the long-term function of
402
cryopreserved ovarian grafts in the mouse, implications for human applications. Mol Cell
403
Endocrinol 2000;161:103-10.
404
[20] Poirot C, Vacher-Lavenu MC, Helardot P, Guibert J, Brugieres L, Jouannet P. Human
405
ovarian tissue cryopreservation: indications and feasibility. Hum Reprod 2002;17:1447-52.
406
[21] Hreinsson J, Zhang P, Swahn ML, Hultenby K, Hovatta O. Cryopreservation of follicles in
407
human ovarian cortical tissue. Comparison of serum and human serum albumin in the
408
cryoprotectant solutions. Hum Reprod 2003;18:2420-8.
409
[22] Hovatta O. Cryopreservation and culture of human ovarian cortical tissue containing early
410
follicles. Eur J Obstet Gynecol Reprod Biol 2004;113:50-4.
411
[23] Liu J, Van der Elst J, Van den Broecke R, Dhont M. Live offspring by in vitro fertilization
412
of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation
413
and in vitro maturation. Biol Reprod 2001;64:171-8.
AC C
EP
TE D
M AN U
SC
RI PT
392
ACCEPTED MANUSCRIPT
"Clean copy"
20
[24] Gandolfi F, Paffoni A, Papasso Brambilla E, Bonetti S, Brevini TA, Ragni G. Efficiency of
415
equilibrium cooling and vitrification procedures for the cryopreservation of ovarian tissue:
416
comparative analysis between human and animal models. Fertil Steril 2006;85:1150-6.
417
[25] Araujo VR, Gastal MO, Figueiredo JR, Gastal EL. In vitro culture of bovine preantral
418
follicles: a review. Reprod Biol Endocrinol 2014;12:78.
419
[26] Bandeira FT, Carvalho AA, Castro SV, Lima LF, Viana DA, Evangelista JS, et al. Two
420
methods of vitrification followed by in vitro culture of the ovine ovary: evaluation of the
421
follicular development and ovarian extracellular matrix. Reprod Domest Anim 2015;50:177-85.
422
[27] Peluso JJ, Hirschel MD. Factors controlling the growth of bovine primary and preantral
423
follicles in perifusion culture. Theriogenology 1988;30:537-46.
424
[28] Soares M, Dolmans MM, Donnez, J. Heterotopic ovarian tissue transplantation. In: Suzuki
425
N, Donnez J, editors. Gonadal tissue cryopreservation in fertility preservation. 1st ed. Japan:
426
Springer; 2016. p. 105-23.
427
[29] Kohler M, Janz I, Wintzer HO, Wagner E, Bauknecht T. The expression of EGF receptors,
428
EGF-like factors and c-myc in ovarian and cervical carcinomas and their potential clinical
429
significance. Anticancer Res 1989;9:1537-47.
430
[30] Doraiswamy V, Parrott JA, Skinner MK. Expression and action of transforming growth
431
factor alpha in normal ovarian surface epithelium and ovarian cancer. Biol Reprod 2000;63:789-
432
96.
433
[31] Huang W, Nagano M, Kang S-S, Yanagawa Y, Takahashi Y. Effects of in vitro growth
434
culture duration and prematuration culture on maturational and developmental competences of
435
bovine oocytes derived from early antral follicles. Theriogenology 2013;80:793-9.
AC C
EP
TE D
M AN U
SC
RI PT
414
ACCEPTED MANUSCRIPT
"Clean copy"
21
[32] Fujihara M, Comizzoli P, Keefer CL, Wildt DE, Songsasen N. Epidermal growth factor
437
(EGF) sustains in vitro primordial follicle viability by enhancing stromal cell proliferation via
438
MAPK and PI3K pathways in the prepubertal, but not adult, cat ovary. Biol Reprod 2014;90:86.
439
[33] Castro SV, Carvalho AA, Silva CM, Santos FW, Campello CC, de Figueiredo JR, et al.
440
Frozen and fresh ovarian tissue require different culture media to promote in vitro development
441
of bovine preantral follicles. Biopreserv Biobank 2014;12:317-24.
442
[34] Fabbri R, Macciocca M, Vicenti R, Pasquinelli G, Caprara G, Valente S, et al. Long-term
443
storage does not impact the quality of cryopreserved human ovarian tissue. J Ovarian Res
444
2016;9:50.
445
[35] Alves KA, Alves BG, Rocha CD, Visonna M, Mohallem RF, Gastal MO, et al. Number and
446
density of equine preantral follicles in different ovarian histological section thicknesses.
447
Theriogenology 2015;83:1048-55.
448
[36] Haag KT, Magalhaes-Padilha DM, Fonseca GR, Wischral A, Gastal MO, King SS, et al.
449
Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy
450
Pick-Up method. Theriogenology 2013;79:599-609.
451
[37] Fabbri R, Vicenti R, Macciocca M, Pasquinelli G, Paradisi R, Battaglia C, et al. Good
452
preservation of stromal cells and no apoptosis in human ovarian tissue after vitrification. BioMed
453
Res Int 2014;2014:673537.
454
[38] Garnett K, Wang J, Roy SK. Spatiotemporal expression of epidermal growth factor receptor
455
messenger RNA and protein in the hamster ovary: follicle stage-specific differential modulation
456
by follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone. Biol Reprod
457
2002;67:1593-604.
AC C
EP
TE D
M AN U
SC
RI PT
436
ACCEPTED MANUSCRIPT
"Clean copy"
22
[39] Greenwald G, Roy S. Follicular development and its control. In: Knobil E, Neill JD, editors.
459
The physiology of Reproduction. Raven Press: New York; 1994. p.629–724.
460
[40] Ishak GM, Bashir ST, Dutra GA, Gastal GA, Gastal MO, Cavinder CA, et al. In vivo antral
461
follicle wall biopsy: a new research technique to study ovarian function at the cellular and
462
molecular levels. Reprod Biol Endocrinol 2018;16:71.
463
[41] Lee J, Kim SK, Youm HW, Kim HJ, Lee JR, Suh CS, et al. Effects of three different types
464
of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation. PLoS One
465
2015;10:e0126252.
466
[42] Raynel S, Padula MP, Marks DC, Johnson L. Cryopreservation alters the membrane and
467
cytoskeletal protein profile of platelet microparticles. Transfusion 2015;55:2422-32.
468
[43] Maffei S, Hanenberg M, Pennarossa G, Silva JR, Brevini TA, Arav A, et al. Direct
469
comparative analysis of conventional and directional freezing for the cryopreservation of whole
470
ovaries. Fertil Steril 2013;100:1122-31.
471
[44] Aitken RJ, Findlay JK, Hutt KJ, Kerr JB. Apoptosis in the germ line. Reproduction
472
2011;141:139-50.
473
[45] Albamonte MI, Albamonte MS, Stella I, Zuccardi L, Vitullo AD. The infant and pubertal
474
human ovary: Balbiani's body-associated VASA expression, immunohistochemical detection of
475
apoptosis-related BCL2 and BAX proteins, and DNA fragmentation. Hum Reprod 2013;28:698-
476
706.
477
[46] Donfack NJ, Alves KA, Alves BG, Rocha RMP, Bruno JB, Lima LF, et al. In vivo and in
478
vitro strategies to support caprine preantral follicle development after ovarian tissue vitrification.
479
Reprod Fertil Dev 2018. doi.org/10.1071/RD17315
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Table legends
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Table 1. Mean (± S.E.M.) percentage of morphologically normal equine preantral follicles in
483
fresh or cryopreserved-thawed (slow-freezing or vitrification) ovarian tissue before (control, day
484
0) and after 1, 3, and 7 days of in vitro culture.
485
Table 2. Mean (± S.E.M.) percentage of morphologically normal equine preantral follicles in
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fresh or cryopreserved-thawed (slow-freezing or vitrification) ovarian tissue after 1-7 days of in
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vitro culture‡.
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Figure legends
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Fig. 1. Mean (± S.E.M.) immunofluorescence of EGFR in fresh or cryopreserved-thawed (slow-
491
freezing or vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3,
492
and 7 days. Differ from fresh control (day 0) group. A,B Different superscripts within same day
493
of culture differ among treatments. a,b,c Different superscripts within same treatment differ among
494
days of culture.
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Fig. 2. Mean (± S.E.M.) immunofluorescence of Ki-67 in fresh or cryopreserved-thawed (slow-
496
freezing or vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3,
497
and 7 days. Differ from fresh control (day 0) group. A,B Different superscripts within same day
498
of culture differ among treatments. a,b,c Different superscripts within same treatment differ among
499
days of culture.
500
Fig. 3. Mean (± S.E.M.) immunofluorescence of BAX in fresh or cryopreserved-thawed (slow-
501
freezing or vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3,
502
and 7 days. Differ from fresh control (day 0) group. A,B Different superscripts within same day
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of culture differ among treatments. a,b,c Different superscripts within same treatment differ among
504
days of culture.
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Fig. 4. Mean (± S.E.M.) immunofluorescence of Bcl-2 in fresh or cryopreserved-thawed (slow-
506
freezing or vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3,
507
and 7 days. Differ from fresh control (day 0) group. A,B Different superscripts within same day
508
of culture differ among treatments. a,b,c Different superscripts within same treatment differ among
509
days of culture.
510
Fig. 5. Mean (± S.E.M.) DNA fragmentation in fresh or cryopreserved-thawed (slow-freezing or
511
vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3, and 7 days.
512
Differ from fresh control (day 0) group. A,B Different superscripts within same day of culture
513
differ among treatments. a,b,c Different superscripts within same treatment differ among days of
514
culture.
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Table 1 Mean (± S.E.M.) percentages of morphologically normal equine preantral follicles in fresh or
after 1, 3, and 7 days of in vitro culture.
Fresh
Group
Primordial
Developing†
Total
Control
81.2 ± 6.7 Aa
0.0 ± 0.0‡
81.2 ± 6.7 Aa
(n = 35)
(n = 0)
(n = 35)
69.7 ± 9.2 Aa
9.1 ± 6.3‡
78.8 ± 8.0 Aab
IVC-1
(n = 32)
(n = 2)
25.6 ± 10.5
Ab
(n = 17)
IVC-7 Slow-freezing
Control
48.4 ± 6.1
Ab
9.8 ± 4.0
(n = 33)
58.2 ± 6.1 Ac
(n = 8)
(n = 89)
77.7 ± 5.8 Aa
0.0 ± 0.0‡
77.7 ± 5.8 Aa
(n = 56)
(n = 0)
(n = 56)
0.0 ± 0.0 *Bb
5.0 ± 5.0 Aa
5.0 ± 5.0*Bb
(n = 14)
(n = 25)
3.9 ± 2.9
Ca
10.5 ± 4.7*Bb (n = 56)
IVC-7
0.0 ± 0.0 *Bb
2.4 ± 2.4 Aa
2.4 ± 2.4*Bb
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6.6 ± 3.9
*Bb
(n = 15)
(n = 16)
(n = 14)
(n = 30)
72.3 ± 6.4 Aa
3.9 ± 2.1‡
76.2 ± 6.1 Aa
(n = 101)
(n = 5)
(n = 106)
Control IVC-1 IVC-3 IVC-7
†
55.2 ± 18.4 Abc
(n = 41)
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Vitrification
(n = 34)
(n = 16)
Ab
(n = 11)
IVC-3
Aa
(n = 81)
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IVC-1
29.6 ± 10.5
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Treatment
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cryopreserved-thawed (slow-freezing or vitrification) ovarian tissue before (control, day 0) and
10.9 ± 6.3
*Bb
5.1 ± 3.5
Aab
15.9 ± 6.8*Bb
(n = 25)
(n = 15)
(n = 40)
1.2 ± 1.2 *Bb
14.7 ± 6.4 *Ba
15.9 ± 7.0*Bb
(n = 8)
(n = 37)
(n = 45)
6.7 ± 3.7 *Bb
1.1 ± 1.1‡
7.8 ± 3.8*Bb
(n = 68)
(n = 3)
(n = 71) *
Developing follicles are comprised of transition, primary, and secondary follicles. Differ from fresh control group. A,B Different superscripts within same group differ among treatments. a,b,c
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Different superscripts within same treatment differ among groups. ‡ No statistical analysis was performed due the low number of preantral follicles encountered. n, number of follicles found.
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Table 2 Mean (± S.E.M.) percentage of morphologically normal equine preantral follicles in fresh or
culture.
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cryopreserved-thawed (slow-freezing or vitrification) ovarian tissue after 1-7 days of in vitro
Primordial
Developing†
Fresh
51.8 ± 4.9a
11.9 ± 3.3a
(n = 130)
(n = 26)
3.2 ± 1.9b
3.8 ± 2.0b
7.0 ± 2.7c
(n = 68)
(n = 43)
(n = 111)
6.4 ± 2.5b
6.1 ± 2.2b
12.6 ± 3.3b
(n = 55)
(n = 156)
Vitrification
(n = 101) †
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Slow-freezing
SC
Treatment
Total
63.7 ± 4.5a (n = 156)
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Developing follicles are comprised of transition, primary, and secondary follicles. Data obtained on days 1, 3, and 7 of culture for each treatment were combined for statistical analyses. a,b,c Different superscripts within the same column differ among treatments. n, number of follicles found.
5x10
6
Aa
ABa
Aa Aa
Ba
ABb
Ab
4x106
*Ab *Bb *Bc
3x106
*Bb
2x106 1x106 0
0
1
3
Days in culture
TE D AC C
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Figure 1
Aa
SC
6x106
Vitrification
Slow-freezing
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Fresh
7x106
7
M AN U
EGFR fluorescence intensity
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6x106 5x10
Aa
6
Vitrification
Slow-freezing
Aa Aa Ab
4x106
Aa
Aab
*Ab
0
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1x106
1
EP
3
Days in culture
Figure 2
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Aa
SC
*Bb
2x106
0
Aa
*Ab
*Ab
3x106
TE D
Ki-67 fluorescence intensity
7x106
RI PT
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8x10 7x10 6x10 5x10 4x10 3x10 2x10 1x10
Fresh
6 6
Vitrification
Slow-freezing
Aa
RI PT
9x10
6 6 6
*Aa
Ba Bb
Ab
Ab *Ab
6
*Bb *ABc
*Ba
*Bd
6 6 6
0
1
3
Days in culture
7
TE D
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0
Cb
SC
Bax fluorescence intensity
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Figure 3
7x10 6x10 5x10 4x10 3x10 2x10 1x10
6 6
Aa
6 6
Bab
Ba
ABb *Aa *Bb
6 6 6 6
0
1
EP
Figure 4
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Bb *Bb
3
Days in culture
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0
Vitrification
Slow-freezing
*Aa
Ab
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8x10
Fresh
6
*Aa *Aa
SC
9x10
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Bcl-2 fluorescence intensity
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7x106 5x106
Aa
*Aa
Aa
ABa
Aa
Ba
Aab Ab
Bb
Bb
4x106
Ab Bb
3x106 2x106 1x106 0
1
Days in culture
EP
Figure 5
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3
7
M AN U
0
SC
6x106
Vitrification
Slow-freezing
RI PT
Fresh
8x106
TE D
TUNEL fluorescence intensity
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Highlights:
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This study demonstrated the feasibility of culturing in vitro cryopreserved equine ovarian tissue. Slow-freezing and vitrification had similar efficiency in preserving the morphology of preantral follicles. Expression of EGFR and Ki-67 proteins were preserved after cryopreservation and in vitro culture for 7 days. Expression of Bax and Bcl-2 protein in cryopreserved tissue was similar to that of fresh tissue after culture. DNA degradation in cryopreserved-thawed ovarian tissue was similar to that in fresh tissue.
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S0093-691X(18)30225-5
DOI:
https://doi.org/10.1016/j.theriogenology.2018.11.034
Reference:
THE 14792
To appear in:
Theriogenology
Received Date: 19 May 2018 Revised Date:
30 November 2018
Accepted Date: 30 November 2018
Please cite this article as: Gastal GDA, Aguiar FLN, Ishak GM, Cavinder CA, Willard ST, Ryan PL, Feugang JM, Gastal EL, Effect of cryopreservation techniques on proliferation and apoptosis of cultured equine ovarian tissue, Theriogenology (2019), doi: https://doi.org/10.1016/j.theriogenology.2018.11.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of cryopreservation techniques on proliferation and
2
apoptosis of cultured equine ovarian tissue
3
RI PT
1
4
G.D.A. Gastal1, F.L.N. Aguiar1, G.M. Ishak1,2, C.A. Cavinder3, S.T. Willard3, P.L. Ryan3, J.M.
5
Feugang3, E.L. Gastal1*
7
1
8
lllinois, USA
9
2
M AN U
Department of Animal Science, Food and Nutrition, Southern Illinois University, Carbondale,
Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Baghdad,
10
Baghdad, Iraq
11
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Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS,
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Short title: Cryopreservation and in vitro culture of equine ovarian tissue
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Illinois University, 1205 Lincoln Drive, MC 4417, Carbondale, IL, 62901, USA. Tel.: + 618 453
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1774; fax: + 618 453 5231. E-mail: [email protected]
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Correspondence: Eduardo Gastal, Department of Animal Science, Food and Nutrition, Southern
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Abstract
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Preservation of cellular integrity and its mechanisms after ovarian tissue cryopreservation (OTC)
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and in vitro culture (IVC) procedures are crucial aspects for the success of preservation and
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recovery of female fertility. This study aimed to evaluate the effects of two cryopreservation
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methods (slow-freezing, SF, and vitrification, VIT) on the equine ovarian tissue after 1, 3, and 7
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days of IVC by assessing: (i) preantral follicle morphology and distribution of follicle classes;
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(ii) protein expression of markers of cell proliferation for EGFR and Ki-67; (iii) markers of
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apoptosis for Bax and Bcl-2; and (iv) DNA fragmentation. Percentages of normal primordial
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follicles were similar (P > 0.05) among SF-control, VIT-control, and fresh control groups. After
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7 days of culture, VIT-IVC7 had a greater (P < 0.05) total percentage of normal preantral
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follicles when compared with SF-IVC7, but both had a lower (P < 0.05) percentage than fresh
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IVC7 group. Prior to and after 7 days of culture, expression of EGFR and Ki-67 were similar (P
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> 0.05) among fresh, SF, and VIT groups. After 7 days of culture, VIT had higher (P < 0.05) Bax
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expression than the fresh and SF tissues, but Bcl-2 was similar (P > 0.05) among groups. Prior to
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IVC, TUNEL signals were similar (P > 0.05) among groups; however, VIT-IVC7 had greater
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(P<0.05) TUNEL signals when compared with the fresh IVC7 group. In conclusion, findings
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demonstrated: (i) similar efficiency between SF and VIT compared with fresh control to preserve
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morphologically normal follicles; and (ii) similar tissue functionality and cell proliferation
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capability after equine OTC by either SF and VIT methods following IVC for 7 days. The results
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herein presented shed light on equine fertility preservation programs using OTC techniques.
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Keywords: equine; preantral follicles; cryopreservation; in vitro culture; proliferation
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1. Introduction Ovarian tissue cryopreservation (OTC) has been a fundamental technique to preserve fertility
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for cancer patients, domestic animals, and endangered species [1, 2]. Animal models have played
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a major role in the improvement of OTC protocols [1, 3-5], allowing the success and expansion
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of OTC and ovarian tissue transplantation techniques; these fertility restoration procedures have
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been able to generate more than 80 successful live births in humans [6]. Moreover, OTC has
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several advantages over cryopreservation of oocytes and embryos, which include but are not
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limited to: a larger number of primordial germ cells is available in the ovarian cortex [7], the
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method of ovarian tissue biopsy can be performed independently of age and stage of the
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reproductive cycle [8-10], part of the ovarian tissue can be thawed and submitted to in vitro
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culture (IVC) [11-16], the ovarian tissue tolerates transport at low temperatures [17, 18], and
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fertilization is not immediately required [19]. Therefore, new protocols of OTC using either
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slow-freezing (SF) or vitrification (VIT) methods have been constantly tested, improved, and
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simplified to determine their success in preserving ovarian tissue quality. However, the impact of
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OTC techniques on cellular/molecular mechanisms is still not fully understood; therefore, efforts
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are necessary to understand those effects in order to improve the methodologies to better
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preserve and recover female fertility.
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Currently, to determine the quality of ovarian tissue after the cryopreservation procedure, the
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tissue is evaluated immediately after cryopreservation/thawing [4, 20], and can be submitted to
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IVC for determining the capability of preantral follicles to develop and grow [4, 11, 21, 22].
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Therefore, efficient IVC methods have been developed in different animal models to obtain
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valuable information regarding the quality of the fresh ovarian tissue, such as the preantral
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follicle morphology, activation and development, and the expression of cell proliferation,
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apoptosis, and angiogenic factors [14, 23-26]. Recently, suitable IVC media for equine preantral
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follicles enclosed in fresh ovarian tissue has been developed [14] and improved [4] to ensure the
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maintenance of stromal cell survival and proliferation. However, information about the potential
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of cryopreserved-thawed equine ovarian tissue to survive and proliferate when submitted to IVC
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is still scarce [4].
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The culture of preantral follicles enclosed in the ovarian tissue allows a better cell-cell
interaction among the surfaces of adjacent cells [25]. This plays a crucial role in the development
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and function of multicellular organs [27] such as the ovary which contains stromal, theca and
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granulosa cells, and oocytes. Indeed, the in situ culture allows the evaluation of factors related to
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cell proliferation and apoptosis during neoangiogenesis [28]. In this regard, the stromal cells for
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example are responsible for expressing proteins related to cell proliferation, such as epidermal
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growth factor receptor (EGFR) [29, 30] and Ki-67 [5], and for interacting with the follicles to
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produce competent oocytes [31]. Only a few studies have investigated the influence of
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cryopreservation on ovarian stromal cells during IVC [32, 33]. EGFRs have been found in
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stromal cells located near primordial follicles; this has been believed to contribute to follicle
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activation and development, and it is thought that disrupting the EGFR protein may lead to
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apoptosis and early cell death [32]. In respect to Ki-67 antibody, it recognizes a nuclear antigen
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that is expressed in all stages of the cell cycle except G0 [34]; the expression of Ki-67 has been
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demonstrated in frozen-thawed ovarian stromal cells and is highly correlated with EGFR
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expression [5]. Therefore, it is important to understand the impact of cryopreservation on EGFR
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and Ki-67 expression in the ovarian tissue after thawing and during the IVC.
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The aim of this study was to evaluate the effects of two cryopreservation methods – SF and VIT – on equine ovarian tissue after 1, 3, and 7 days of in vitro culture by assessing: (i) preantral
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follicle morphology and distribution of follicle classes, (ii) markers of cell proliferation (EGFR
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and Ki-67), (iii) markers of apoptosis (Bax and Bcl-2), and (iv) DNA fragmentation.
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2. Materials and methods
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2.1. Chemicals
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise
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2.2. Ovarian tissue collection and processing
All experimental procedures were performed according to the United States Government
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Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and
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Training (grants.nih.gov/grants/olaw/references/phspol.htm). The use of animals and procedures
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were approved by Mississippi State University Institutional Animal Care and Use Committee.
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Ovaries were harvested, during the anestrous season (February), from 5 Quarter horse type mares
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(7-19 years old) slaughtered at Mississippi State University. Briefly, ovaries were rinsed in
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alcohol 70%, followed by three washes in saline solution (0.9% NaCl) supplemented with
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antibiotics (100 IU/ml penicillin and 100 µg streptomycin/ml). Subsequently, ovaries were
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placed in a petri dish with α-MEM containing 0.4% BSA, 93.3 U/ml penicillin G, 36.8 U/ml
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streptomycin sulphate, 0.047 mmol/l pyruvic acid (sodium salt), and 2.5 mM Hepes [10]. Then,
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ovaries were divided into three longitudinal portions (two lateral and one middle); only the
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middle portion of the ovary was used to collect fragments [18] for this study. Fragments were
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sliced into small pieces (3 x 3 x 0.5 mm) using scalpels, tweezers, and the Thomas Stadie-Riggs
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Tissue Slicer (Thomas Scientific®, Swedesboro, NJ, USA) to obtain a standard thickness (0.5
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mm). Only one ovary of each mare was used and considered as a replicate. Therefore, five
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replicates were performed.
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2.3. Experimental design
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Two cryopreservation methods (SF vs. VIT) were compared to evaluate the viability of
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cryopreserved-thawed equine ovarian tissue submitted to in vitro culture for 0 (noncultured
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control), 1, 3, or 7 days. The study involved twelve groups: fresh noncultured control, fresh IVC-
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1 day, fresh IVC-3 days, fresh IVC-7 days, SF noncultured, SF IVC-1 day, SF IVC-3 days, SF
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IVC-7 days, VIT noncultured, VIT IVC-1 day, VIT IVC-3 days, and VIT IVC-7 days. Fresh
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tissue (standard control) was evaluated at the same time points of the cryopreservation
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treatments. A total of 96 small fragments from each ovary were randomly distributed among
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twelve groups (n = 8 fragments/group/replicate; total = 480 fragments) to evaluate the following
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endpoints: preantral follicle morphology and distribution of follicle classes; protein expression of
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proliferative (Ki-67 and EGFR) and pro- and anti-apoptotic (Bax and Bcl-2, respectively)
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markers, and DNA fragmentation.
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2.4. Slow-freezing of ovarian tissue
Cryopreservation of ovarian tissue fragments by the slow-freezing method was performed
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according to protocol described previously [4]. Briefly, ovarian fragments were placed in 1.5 ml
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cryovials containing the following cryoprotectant solution: α-MEM supplemented with 2.5 mM
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Hepes, 10% fetal equine serum (FES), 0.25 M sucrose, and 1.5 M DMSO. After equilibration
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time in the cryoprotectant at room temperature (RT), the cryovials were placed in a
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programmable freezing machine (Bio-Cool IV40 - Controlled Rate Freezer, SP Scientific
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Company, Warminster, PA, USA). The cooling curve was programmed for a rate of 2°C/min to
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‒7°C and the seeding was done manually by touching the vials with a forceps dipped into liquid
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nitrogen (LN2). Then, the freezing curve was set for a rate of 0.3°C/min to ‒40 ºC, and vials were
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plunged in LN2, and stored for 1 week. For the thawing process, the cryovials were exposed to
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RT for 30 sec and then immersed in water at 37ºC for 1 min. Frozen-thawed fragments were
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washed in three step solutions (5 min each) in the following order: α-MEM + 2.5 mM Hepes +
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10% FES + 0.5 M sucrose; α-MEM + 2.5 mM Hepes + 10% FES + 0.25 M sucrose; and α-MEM
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+ 2.5 mM Hepes + 10% FES.
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2.5. Vitrification of ovarian tissue
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Cryopreservation of equine ovarian tissue fragments by the vitrification method was
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performed according to a protocol previously described [4]. Briefly, fragments submitted to
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vitrification were placed in petri dishes containing the following solutions and periods of
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equilibration: 1st step: 0.3 M ethylene glycol (EG) and 0.5 M trehalose in base medium (α-MEM
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+ 2.5 mM Hepes + 6% FES) for 3 min at RT; 2nd step: 1.5 M EG in base medium for 1 min at
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RT; and 3rd step: 3 M EG in base medium for 1 min at RT. Fragments were lightly dried, placed
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in cryovials (1 ml), plunged in LN2, and stored for 1 week. For the thawing process, the cryovials
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were exposed to RT for 30 sec and then immersed in water at 37ºC for 1 min. Then, to remove
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the cryoprotectant solution, fragments were submitted to decreasing concentrations of sucrose
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(0.5, 0.25 and 0 M in the base medium) for 5 min within each solution.
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2.6. In vitro culture
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Ovarian fragments were in vitro cultured according to the protocol reported previously [4]. Briefly, ovarian fragments were placed in culture plates (4 fragments per well) containing 1000
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µl of medium composed of α-MEM supplemented with insulin (10 ng/ml), transferrin (5 ng/ml),
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selenium (5 ng/ml), glutamine (2 mM), hypoxanthine (2 mM), BSA - bovine serum albumin
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(1.25 mg/ml), ascorbic acid (50 µg/ml), follicle stimulating hormone from porcine pituitary (50
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µg/ml), EGF - epidermal growth factor (50 ng/ml), penicillin (100 µg/ml), and streptomycin (100
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µg/ml). Fragments were cultured for 1, 3, or 7 days at 39°C in a humidified atmosphere with 5%
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CO2 in air. Culture medium was completely replaced on days 2, 4, and 6 of culture.
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2.7. Histological processing
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Fresh and cryopreserved-thawed fragments were fixed in 4% paraformaldehyde for 4 h and then kept in 70% alcohol at 4ºC until standard histological processing. The fragments were
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embedded in paraffin and cut into 7-µm serial sections [35]. Every section was mounted and
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stained with Periodic Acid-Schiff (PAS) and counterstained in hematoxylin. Histological
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sections were analyzed using a light microscope (Nikon E200, Tokyo, Japan) at 40× objective
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magnification and an image capture system (LEICA Imaging Software, Wetzlar, Germany).
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2.8. Morphological classification of follicles Preantral follicles with visualized oocyte nucleus were counted and morphologically
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classified as either normal (follicles containing an intact oocyte and oocyte nucleus surrounded
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by granulosa cells well organized in one or more layers) or abnormal (follicles with a retracted
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oocyte or disorganized granulosa cell layers detached from the basement membrane and oocyte
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with pyknotic nucleus). Preantral follicles were classified according to their developmental stage
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into primordial, transitional, primary, and secondary [36].
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2.9. Immunohistochemistry
Ovarian tissue sections mounted on slides were deparafinized, submitted to antigen retrieval
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in sodium citrate buffer for 30 min in a steamer, washed with PBS, and submitted to immediate
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standard in situ immunofluorescence detection of EGFR, Ki-67, Bax, and Bcl-2 proteins as
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described previously [4]. Briefly, slides were permeabilized in 1% Triton X-100 for 30 min at
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RT, non-specific binding sites blocked in 1% BSA solution for 60 min at RT, and incubated with
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100× diluted rabbit polyclonal anti-human EGFR (sc-03-G; Santa Cruz Biotechnology, Dallas,
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TX, USA), Ki-67 (PA5-19462; Thermo Fisher Scientific Inc., Waltham, MA, USA), Bax
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(orb312174; Biorbyt, Berkeley, CA, USA), or Bcl-2 (sc-492; Santa Cruz Biotechnology, Dallas,
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TX, USA) antibodies for 60 min at RT. Then, slides were incubated with FITC labeled goat anti-
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rabbit secondary antibody (200x dilution) for 60 min at RT. Three washes with PBS solution
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were performed between all steps. Slides were immediately covered with a DAPI-containing
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mounting medium to counterstain cell nuclei for a fluorescence evaluation using a fluorescent
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microscope (EVOS FL Cell Imaging System, Thermo Fisher Scientific Inc., Waltham, MA
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USA). Five images from randomly selected fields of one tissue section/treatment/replicate were
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obtained at 20x objective magnification and analyzed using ImageJ software (version 1.50f) to
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calculate the fluorescence intensity of all labeled cells in the ovarian stroma.
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2.10. TUNEL assay TUNEL staining was carried out using a commercially available kit (DeadEnd™ Fluorometric TUNEL System, Promega©, Madison, WI, USA) following the manufacturer’s
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instructions. Tissue sections were examined, and five images were obtained at 20x objective
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magnification to calculate the fluorescence intensity of TUNEL positive cells using the ImageJ
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software. TUNEL positive and negative controls were included in all evaluations, according to
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the manufacturer’s recommendations.
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2.11. Statistical analyses
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All statistical analyses were performed using R statistical software version 3.0.2 (R Foundation for Statistical Computing, Vienna, Austria). Data for the end points evaluated did not
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follow a normal distribution. Therefore, statistical difference among treatments was analyzed by
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Kruskal–Wallis test, and Mann–Whitney U test was used to compare mean values among
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groups. A probability of P < 0.05 indicated that a difference was significant. Data are presented
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as mean ± SEM and percentages.
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3. Results
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3.1. Preantral follicle morphology and class distribution
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A total of 620 preantral follicles were recorded with a mean of 51.7 ± 7.3 follicles per
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treatment (Table 1). Percentage of normal primordial follicles was similar (P > 0.05) among
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fresh noncultured control, SF-control, and VIT-control groups. However, follicle morphology
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was impaired (P < 0.05) in cryopreserved fragments (SF and VIT treatments) submitted to IVC
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for 1, 3, and 7 days when compared with fresh tissue. Therefore, data from morphologically
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normal preantral follicles at days 1, 3, and 7 of culture were grouped to evaluate the overall
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effect of IVC on follicle morphology after cryopreserved-thawed by SF and VIT methods in the
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total percentage of normal preantral follicles and according to follicle classes (Table 2). Thus,
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VIT treatment had a greater (P < 0.05) total percentage of normal preantral follicles when
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compared with SF treatment after IVC.
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3.2. Markers of proliferation
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Prior to and after 7 days of culture, protein expression of EGFR (Fig. 1) and Ki-67 (Fig. 2) in SF and VIT treatment groups was similar (P > 0.05) to that of the fresh noncultured control
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group. At day 1 of culture, SF-IVC1 and VIT-IVC1 groups had lower (P < 0.05) EGFR
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expression and similar (P > 0.05) Ki-67 expression compared to the fresh IVC1 group. At day 3
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of culture, SF-IVC3 and VIT-IVC3 groups had similar (P > 0.05) EGFR expression compared to
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fresh IVC3; however, the VIT-IVC3 group had higher (P < 0.05) EGFR expression compared to
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the SF-IVC3 group. Moreover, Ki-67 expression was similar (P > 0.05) between fresh IVC3 and
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VIT-IVC3 groups and lower (P < 0.05) in the SF-IVC3 group.
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3.3. Markers of apoptosis
All treatment groups had lower (P < 0.05) Bax (Fig. 3) and Bcl-2 (Fig. 4) expression when
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compared with the fresh noncultured control group. At day 1 of culture, the VIT-IVC1 group had
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lower (P < 0.05) Bax expression compared to the fresh IVC1 and SF-IVC1 groups; Bcl-2
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expression in the SF-IVC1 and VIT-IVC1 groups was similar (P > 0.05) compared to the fresh
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IVC1 group. At day 3 of culture, the fresh IVC3 group had similar (P > 0.05) Bax expression
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compared to the VIT-IVC3 group, but higher (P < 0.05) than the SF-IVC3 group; however, the
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VIT-IVC3 group had higher (P < 0.05) Bcl-2 expression compared with the fresh IVC3 and SF-
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IVC3 groups. After 7 days of culture, Bax expression was greater (P < 0.05) in the VIT-IVC7
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group compared with the fresh IVC7 and SF-IVC7 groups; however, Bcl-2 expression did not
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differ (P > 0.05) among groups.
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3.4. DNA fragmentation
Prior to in vitro culture, DNA fragmentation was similar (P > 0.05) among treatment groups
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(Fig. 5). At day 1 of culture, the SF-IVC1 and VIT-IVC1 groups had lower (P < 0.05) DNA
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fragmentation compared to the fresh IVC1 group. At day 3 of culture, SF-IVC3 had lower (P <
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0.05) DNA fragmentation compared to the fresh IVC3 and VIT-IVC3 groups. At day 7 of
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culture, the VIT-IVC7 group had greater (P < 0.05) DNA fragmentation compared to fresh
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IVC7, but similar (P > 0.05) to the SF-IVC7 group.
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4. Discussion
Ovarian tissue cryopreservation techniques may disturb cellular mechanisms involved with cell proliferation and apoptosis signaling after thawing [4]. To the best of our knowledge, the
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present study has shown for the first time the expression pattern of EGFR, Ki-67, Bax, and Bcl-2
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in equine ovarian stromal cells cryopreserved by either slow-freezing or vitrification methods
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during IVC for up to 7 days. Moreover, the study has shown the preantral follicle morphology
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and DNA degradation throughout the in vitro culture period for fresh and cryopreserved-thawed
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equine ovarian tissue.
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In the present study, EGFR expression in cryopreserved-thawed tissue by slow-freezing and vitrification methods after 7 days of culture was similar to that of fresh tissue, demonstrating
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tissue functionality and growth capability. Similarly, protein expression of Ki-67 after 7 days of
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culture corroborated the survivability and proliferative potential of cryopreserved-thawed equine
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ovarian tissue by slow-freezing and vitrification methods. Protein expression of Ki-67 has been
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well-described as an acceptable index of cell proliferation for cryopreserved ovarian tissue after
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thawing [37] and submission to in vitro culture [4]. A recent study [14] has shown that addition
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of EGF to the culture medium contributed to the maintenance of follicular survival and activation
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of preantral follicles during in vitro culture. EGFRs are involved in different cell signaling
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pathways, such as MAP-kinase (ERK), protein kinase C, phosphatidyl-inositol 3-kinase (PI3-
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kinase), and G protein coupled receptors [38]. Although low levels of EGFR are present in
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granulosa cells of small preantral follicles, primary and secondary follicles of the hamster
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responded to an EGF-stimulating effect on cellular growth [39]. Moreover, EGFR has been
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shown to be present in high concentrations in the theca interna and granulosa cells of equine
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antral follicles [40]. Notwithstanding, protein-receptor coupling is required for activating the
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aforementioned pathways contributing to tissue proliferation. Therefore, equine ovarian tissue
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can be cryopreserved and in vitro cultured following the methods applied in the present study
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without jeopardizing the EGF protein receptors.
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Equine ovarian tissue cryopreserved-thawed by slow-freezing and vitrification had lower
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expression of Bax and Bcl-2 compared with fresh noncultured tissue. The lower expression of
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Bax and Bcl-2 after thawing may have been caused by ice recrystallization during
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cryopreservation [41], which altered the abundance and expression of several proteins [42].
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However, slow-freezing and vitrification had higher Bax expression after 7 days of culture
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compared to fresh tissue, while the expression of Bcl-2 was similar among the treatment groups.
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The same pattern of DNA degradation was observed in cryopreserved-thawed and in vitro
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cultured ovarian tissues evaluated by TUNEL assay. In this regard, our recent study [4] has
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shown a similar pattern of apoptosis after the thawing process for equine ovarian tissue. Similar
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results have been reported for human [34, 37] and sheep [43] ovaries after OTC and IVC.
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However, no information was available up to now regarding apoptosis of cryopreserved-thawed
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equine ovarian tissue submitted to IVC. Bax and Bcl-2 proteins play a major role in the intrinsic
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apoptotic pathway triggered by mitochondrial dysfunction [44]. Therefore, an adequate balance
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between pro- (Bax) and anti-apoptotic (Bcl-2) markers seems to be vital to determine the fate of
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the cells [45].
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The present study has shown that fresh ovarian fragments had approximately 60% morphologically normal preantral follicles after 7 days of in vitro culture. Moreover, frozen and
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vitrified tissues had more than 70% morphologically normal preantral follicles after the thawing
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process. However, a low percentage (10%) of morphologically normal preantral follicles
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enclosed in cryopreserved-thawed (slow-freezing and vitrificaion) ovarian tissues were found
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after 7 days of culture. Our present results are in agreement with our recent publication [4], in
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which 70% of preantral follicles enclosed in fresh ovarian tissue and 10% of preantral follicles
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enclosed in cryopreserved-thawed ovarian tissues maintained a normal morphology after 7 days
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in culture. Altogether, VIT-IVC7 had a greater percentage of morphologically normal preantral
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follicles compared to SF-IVC7, but a higher expression of Bax and DNA fragmentation was
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observed after 7 days of culture. Therefore, two hypotheses have been raised: (i) a greater
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percentage of normal follicles in VIT-IVC7 demands higher tissue metabolism and, therefore,
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causes higher Bax expression and DNA fragmentation; or (ii) although there is a greater
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percentage of normal follicles at day 7 of culture, the tissue metabolism is burdened and
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apoptosis pathways are activated, causing tissue death. In agreement with our findings, a recent
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study [46] has shown a significant decrease in preantral follicle density and morphology after 7
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days of in vitro culture of vitrified caprine ovarian tissue compared with fresh tissue; the authors
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suggested the need for different medium composition to culture vitrified tissues. In this regard,
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we believe that the tissue suffers two types of stress when submitted to cryopreservation and in
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vitro culture; firstly, the tissue must recover from the cryopreservation and thawing processes,
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where several modifications occur at the cell membrane and organelle levels; secondly, the tissue
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must adapt to the new in vitro culture environment, requiring readjustment of physiological
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pathways to survive and continue to grow. Based on the prior assumptions, a better preservation
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of proliferative markers in the ovarian tissue may help to minimize the stress caused by the
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cryopreservation techniques. Therefore, adaptations in the cryopreservation and thawing
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procedures, medium compositions, and culture environment warrant further investigation to
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maximize the potential of fertility preservation using OTC.
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freezing or vitrification techniques had similar efficiency in preserving morphologically normal
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follicles. Moreover, tissue functionality and cell proliferation capability were preserved after
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OTC by either slow-freezing or vitrification methods following IVC for 7 days. The results
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herein presented shed light upon the equine fertility preservation programs using OTC
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techniques.
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Acknowledgements
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The authors thank Saffron Scientific Histology Services for technical assistance with histological
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preparation. Research was supported by Southern Illinois University (SIU) and USDA-ARS
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Grant # 58-6402-3- 018 (to J M Feugang). Gastal G.D.A. was the recipient of a PhD scholarship
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from The National Council for Scientific and Technological Development (CNPq; grant
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#246741/ 2012-0), Brazil. Ishak G.M. was the recipient of a PhD scholarship from the Ministry
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of Higher Education & Scientific Research, Baghdad, Iraq. Aguiar F. L. N. was the recipient of a
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doctoral scholarship from Fundação Cearense de Apoio ao Desenvolvimento Científico e
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Tecnológico (FUNCAP), Brazil.
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References
349
[1] Comizzoli P, Wildt DE. Mammalian fertility preservation through cryobiology: value of
350
classical comparative studies and the need for new preservation options. Reprod Fertil Dev
351
2014;26:91-8.
352
[2] Dolmans MM, Binda MM, Jacobs S, Dehoux JP, Squifflet JL, Ambroise J, et al. Impact of
353
the cryopreservation technique and vascular bed on ovarian tissue transplantation in cynomolgus
354
monkeys. J Assist Reprod Genet 2015;32:1251-62.
355
[3] Santos RR, Amorim C, Cecconi S, Fassbender M, Imhof M, Lornage J, et al.
356
Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of
357
endangered species and breeds. Anim Reprod Sci 2010;122:151-63.
358
[4] Gastal GD, Aguiar FL, Alves BG, Alves KA, de Tarso SG, Ishak GM, et al. Equine ovarian
359
tissue viability after cryopreservation and in vitro culture. Theriogenology 2017;97:139-47.
360
[5] Gastal GD, Aguiar FL, Rodrigues AP, Scimeca J, Apgar G, Banz W, et al. Cryopreservation
361
and in vitro culture of white-tailed deer ovarian tissue. Theriogenology 2018;113:253-60.
362
[6] Jensen AK, Macklon KT, Fedder J, Ernst E, Humaidan P, Andersen CY. 86 successful births
363
and 9 ongoing pregnancies worldwide in women transplanted with frozen-thawed ovarian tissue:
364
focus on birth and perinatal outcome in 40 of these children. J Assist Reprod Genet 2017;34:325-
365
36.
366
[7] Faddy M, Gosden R, Gougeon A, Richardson SJ, Nelson J. Accelerated disappearance of
367
ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 1992;7:1342-
368
6.
369
[8] Demeestere I, Simon P, Emiliani S, Delbaere A, Englert Y. Orthotopic and heterotopic
370
ovarian tissue transplantation. Hum Reprod Update 2009;15:649-65.
AC C
EP
TE D
M AN U
SC
RI PT
348
ACCEPTED MANUSCRIPT
"Clean copy"
18
[9] Alves KA, Alves BG, Gastal GD, de Tarso SG, Gastal MO, Figueiredo JR, et al. The mare
372
model to study the effects of ovarian dynamics on preantral follicle features. PloS One
373
2016;11:e0149693.
374
[10] Haag KT, Magalhaes-Padilha DM, Fonseca GR, Wischral A, Gastal MO, King SS, et al. In
375
vitro culture of equine preantral follicles obtained via the Biopsy Pick-Up method.
376
Theriogenology 2013;79:911-7.
377
[11] Isachenko V, Montag M, Isachenko E, van der Ven K, Dorn C, Roesing B, et al. Effective
378
method for in vitro culture of cryopreserved human ovarian tissue. Reprod Biomed Online
379
2006;13:228-34.
380
[12] Aguiar FL, Lunardi FO, Lima LF, Rocha RM, Bruno JB, Magalhaes-Padilha DM, et al.
381
FSH supplementation to culture medium is beneficial for activation and survival of preantral
382
follicles enclosed in equine ovarian tissue. Theriogenology 2016;85:1106-12.
383
[13] Aguiar FL, Lunardi FO, Lima LF, Rocha RM, Bruno JB, Magalhaes-Padilha DM, et al.
384
Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and
385
reduces reactive oxygen species production after culture. Theriogenology 2016;85:1063-9.
386
[14] Aguiar FL, Gastal GD, Ishak GM, Gastal MO, Teixeira DI, Feugang JM, et al. Effects of
387
FSH addition to an enriched medium containing insulin and EGF after long-term culture on
388
functionality of equine ovarian biopsy tissue. Theriogenology 2017;99:124-33.
389
[15] Sales AD, Duarte AB, Santos RR, Alves KA, Lima LF, Rodrigues GQ, et al. Modulation of
390
aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro
391
culture. Cell Tissue Res 2016;365:415-24.
AC C
EP
TE D
M AN U
SC
RI PT
371
ACCEPTED MANUSCRIPT
"Clean copy"
19
[16] Filatov M, Khramova Y, Kiseleva M, Malinova I, Komarova E, Semenova M. Female
393
fertility preservation strategies: cryopreservation and ovarian tissue in vitro culture, current state
394
of the art and future perspectives. Zygote 2016;24:635-53.
395
[17] Schmidt K, Ernst E, Byskov A, Andersen AN, Andersen CY. Survival of primordial
396
follicles following prolonged transportation of ovarian tissue prior to cryopreservation. Hum
397
Reprod 2003;18:2654-9.
398
[18] Gastal GD, Alves BG, Alves KA, Souza ME, Vieira AD, Varela AS, et al. Ovarian
399
fragment sizes affect viability and morphology of preantral follicles during storage at 4 °C.
400
Reproduction 2017;153:577-87.
401
[19] Shaw J, Cox S, Trounson A, Jenkin G. Evaluation of the long-term function of
402
cryopreserved ovarian grafts in the mouse, implications for human applications. Mol Cell
403
Endocrinol 2000;161:103-10.
404
[20] Poirot C, Vacher-Lavenu MC, Helardot P, Guibert J, Brugieres L, Jouannet P. Human
405
ovarian tissue cryopreservation: indications and feasibility. Hum Reprod 2002;17:1447-52.
406
[21] Hreinsson J, Zhang P, Swahn ML, Hultenby K, Hovatta O. Cryopreservation of follicles in
407
human ovarian cortical tissue. Comparison of serum and human serum albumin in the
408
cryoprotectant solutions. Hum Reprod 2003;18:2420-8.
409
[22] Hovatta O. Cryopreservation and culture of human ovarian cortical tissue containing early
410
follicles. Eur J Obstet Gynecol Reprod Biol 2004;113:50-4.
411
[23] Liu J, Van der Elst J, Van den Broecke R, Dhont M. Live offspring by in vitro fertilization
412
of oocytes from cryopreserved primordial mouse follicles after sequential in vivo transplantation
413
and in vitro maturation. Biol Reprod 2001;64:171-8.
AC C
EP
TE D
M AN U
SC
RI PT
392
ACCEPTED MANUSCRIPT
"Clean copy"
20
[24] Gandolfi F, Paffoni A, Papasso Brambilla E, Bonetti S, Brevini TA, Ragni G. Efficiency of
415
equilibrium cooling and vitrification procedures for the cryopreservation of ovarian tissue:
416
comparative analysis between human and animal models. Fertil Steril 2006;85:1150-6.
417
[25] Araujo VR, Gastal MO, Figueiredo JR, Gastal EL. In vitro culture of bovine preantral
418
follicles: a review. Reprod Biol Endocrinol 2014;12:78.
419
[26] Bandeira FT, Carvalho AA, Castro SV, Lima LF, Viana DA, Evangelista JS, et al. Two
420
methods of vitrification followed by in vitro culture of the ovine ovary: evaluation of the
421
follicular development and ovarian extracellular matrix. Reprod Domest Anim 2015;50:177-85.
422
[27] Peluso JJ, Hirschel MD. Factors controlling the growth of bovine primary and preantral
423
follicles in perifusion culture. Theriogenology 1988;30:537-46.
424
[28] Soares M, Dolmans MM, Donnez, J. Heterotopic ovarian tissue transplantation. In: Suzuki
425
N, Donnez J, editors. Gonadal tissue cryopreservation in fertility preservation. 1st ed. Japan:
426
Springer; 2016. p. 105-23.
427
[29] Kohler M, Janz I, Wintzer HO, Wagner E, Bauknecht T. The expression of EGF receptors,
428
EGF-like factors and c-myc in ovarian and cervical carcinomas and their potential clinical
429
significance. Anticancer Res 1989;9:1537-47.
430
[30] Doraiswamy V, Parrott JA, Skinner MK. Expression and action of transforming growth
431
factor alpha in normal ovarian surface epithelium and ovarian cancer. Biol Reprod 2000;63:789-
432
96.
433
[31] Huang W, Nagano M, Kang S-S, Yanagawa Y, Takahashi Y. Effects of in vitro growth
434
culture duration and prematuration culture on maturational and developmental competences of
435
bovine oocytes derived from early antral follicles. Theriogenology 2013;80:793-9.
AC C
EP
TE D
M AN U
SC
RI PT
414
ACCEPTED MANUSCRIPT
"Clean copy"
21
[32] Fujihara M, Comizzoli P, Keefer CL, Wildt DE, Songsasen N. Epidermal growth factor
437
(EGF) sustains in vitro primordial follicle viability by enhancing stromal cell proliferation via
438
MAPK and PI3K pathways in the prepubertal, but not adult, cat ovary. Biol Reprod 2014;90:86.
439
[33] Castro SV, Carvalho AA, Silva CM, Santos FW, Campello CC, de Figueiredo JR, et al.
440
Frozen and fresh ovarian tissue require different culture media to promote in vitro development
441
of bovine preantral follicles. Biopreserv Biobank 2014;12:317-24.
442
[34] Fabbri R, Macciocca M, Vicenti R, Pasquinelli G, Caprara G, Valente S, et al. Long-term
443
storage does not impact the quality of cryopreserved human ovarian tissue. J Ovarian Res
444
2016;9:50.
445
[35] Alves KA, Alves BG, Rocha CD, Visonna M, Mohallem RF, Gastal MO, et al. Number and
446
density of equine preantral follicles in different ovarian histological section thicknesses.
447
Theriogenology 2015;83:1048-55.
448
[36] Haag KT, Magalhaes-Padilha DM, Fonseca GR, Wischral A, Gastal MO, King SS, et al.
449
Quantification, morphology, and viability of equine preantral follicles obtained via the Biopsy
450
Pick-Up method. Theriogenology 2013;79:599-609.
451
[37] Fabbri R, Vicenti R, Macciocca M, Pasquinelli G, Paradisi R, Battaglia C, et al. Good
452
preservation of stromal cells and no apoptosis in human ovarian tissue after vitrification. BioMed
453
Res Int 2014;2014:673537.
454
[38] Garnett K, Wang J, Roy SK. Spatiotemporal expression of epidermal growth factor receptor
455
messenger RNA and protein in the hamster ovary: follicle stage-specific differential modulation
456
by follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone. Biol Reprod
457
2002;67:1593-604.
AC C
EP
TE D
M AN U
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[39] Greenwald G, Roy S. Follicular development and its control. In: Knobil E, Neill JD, editors.
459
The physiology of Reproduction. Raven Press: New York; 1994. p.629–724.
460
[40] Ishak GM, Bashir ST, Dutra GA, Gastal GA, Gastal MO, Cavinder CA, et al. In vivo antral
461
follicle wall biopsy: a new research technique to study ovarian function at the cellular and
462
molecular levels. Reprod Biol Endocrinol 2018;16:71.
463
[41] Lee J, Kim SK, Youm HW, Kim HJ, Lee JR, Suh CS, et al. Effects of three different types
464
of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation. PLoS One
465
2015;10:e0126252.
466
[42] Raynel S, Padula MP, Marks DC, Johnson L. Cryopreservation alters the membrane and
467
cytoskeletal protein profile of platelet microparticles. Transfusion 2015;55:2422-32.
468
[43] Maffei S, Hanenberg M, Pennarossa G, Silva JR, Brevini TA, Arav A, et al. Direct
469
comparative analysis of conventional and directional freezing for the cryopreservation of whole
470
ovaries. Fertil Steril 2013;100:1122-31.
471
[44] Aitken RJ, Findlay JK, Hutt KJ, Kerr JB. Apoptosis in the germ line. Reproduction
472
2011;141:139-50.
473
[45] Albamonte MI, Albamonte MS, Stella I, Zuccardi L, Vitullo AD. The infant and pubertal
474
human ovary: Balbiani's body-associated VASA expression, immunohistochemical detection of
475
apoptosis-related BCL2 and BAX proteins, and DNA fragmentation. Hum Reprod 2013;28:698-
476
706.
477
[46] Donfack NJ, Alves KA, Alves BG, Rocha RMP, Bruno JB, Lima LF, et al. In vivo and in
478
vitro strategies to support caprine preantral follicle development after ovarian tissue vitrification.
479
Reprod Fertil Dev 2018. doi.org/10.1071/RD17315
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Table legends
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Table 1. Mean (± S.E.M.) percentage of morphologically normal equine preantral follicles in
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fresh or cryopreserved-thawed (slow-freezing or vitrification) ovarian tissue before (control, day
484
0) and after 1, 3, and 7 days of in vitro culture.
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Table 2. Mean (± S.E.M.) percentage of morphologically normal equine preantral follicles in
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fresh or cryopreserved-thawed (slow-freezing or vitrification) ovarian tissue after 1-7 days of in
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vitro culture‡.
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Figure legends
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Fig. 1. Mean (± S.E.M.) immunofluorescence of EGFR in fresh or cryopreserved-thawed (slow-
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freezing or vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3,
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and 7 days. Differ from fresh control (day 0) group. A,B Different superscripts within same day
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of culture differ among treatments. a,b,c Different superscripts within same treatment differ among
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days of culture.
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Fig. 2. Mean (± S.E.M.) immunofluorescence of Ki-67 in fresh or cryopreserved-thawed (slow-
496
freezing or vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3,
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and 7 days. Differ from fresh control (day 0) group. A,B Different superscripts within same day
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of culture differ among treatments. a,b,c Different superscripts within same treatment differ among
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days of culture.
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Fig. 3. Mean (± S.E.M.) immunofluorescence of BAX in fresh or cryopreserved-thawed (slow-
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freezing or vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3,
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and 7 days. Differ from fresh control (day 0) group. A,B Different superscripts within same day
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of culture differ among treatments. a,b,c Different superscripts within same treatment differ among
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days of culture.
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Fig. 4. Mean (± S.E.M.) immunofluorescence of Bcl-2 in fresh or cryopreserved-thawed (slow-
506
freezing or vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3,
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and 7 days. Differ from fresh control (day 0) group. A,B Different superscripts within same day
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of culture differ among treatments. a,b,c Different superscripts within same treatment differ among
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days of culture.
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Fig. 5. Mean (± S.E.M.) DNA fragmentation in fresh or cryopreserved-thawed (slow-freezing or
511
vitrification) equine ovarian tissue before (day 0) and after in vitro culture for 1, 3, and 7 days.
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Differ from fresh control (day 0) group. A,B Different superscripts within same day of culture
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differ among treatments. a,b,c Different superscripts within same treatment differ among days of
514
culture.
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Table 1 Mean (± S.E.M.) percentages of morphologically normal equine preantral follicles in fresh or
after 1, 3, and 7 days of in vitro culture.
Fresh
Group
Primordial
Developing†
Total
Control
81.2 ± 6.7 Aa
0.0 ± 0.0‡
81.2 ± 6.7 Aa
(n = 35)
(n = 0)
(n = 35)
69.7 ± 9.2 Aa
9.1 ± 6.3‡
78.8 ± 8.0 Aab
IVC-1
(n = 32)
(n = 2)
25.6 ± 10.5
Ab
(n = 17)
IVC-7 Slow-freezing
Control
48.4 ± 6.1
Ab
9.8 ± 4.0
(n = 33)
58.2 ± 6.1 Ac
(n = 8)
(n = 89)
77.7 ± 5.8 Aa
0.0 ± 0.0‡
77.7 ± 5.8 Aa
(n = 56)
(n = 0)
(n = 56)
0.0 ± 0.0 *Bb
5.0 ± 5.0 Aa
5.0 ± 5.0*Bb
(n = 14)
(n = 25)
3.9 ± 2.9
Ca
10.5 ± 4.7*Bb (n = 56)
IVC-7
0.0 ± 0.0 *Bb
2.4 ± 2.4 Aa
2.4 ± 2.4*Bb
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6.6 ± 3.9
*Bb
(n = 15)
(n = 16)
(n = 14)
(n = 30)
72.3 ± 6.4 Aa
3.9 ± 2.1‡
76.2 ± 6.1 Aa
(n = 101)
(n = 5)
(n = 106)
Control IVC-1 IVC-3 IVC-7
†
55.2 ± 18.4 Abc
(n = 41)
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Vitrification
(n = 34)
(n = 16)
Ab
(n = 11)
IVC-3
Aa
(n = 81)
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IVC-1
29.6 ± 10.5
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Treatment
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cryopreserved-thawed (slow-freezing or vitrification) ovarian tissue before (control, day 0) and
10.9 ± 6.3
*Bb
5.1 ± 3.5
Aab
15.9 ± 6.8*Bb
(n = 25)
(n = 15)
(n = 40)
1.2 ± 1.2 *Bb
14.7 ± 6.4 *Ba
15.9 ± 7.0*Bb
(n = 8)
(n = 37)
(n = 45)
6.7 ± 3.7 *Bb
1.1 ± 1.1‡
7.8 ± 3.8*Bb
(n = 68)
(n = 3)
(n = 71) *
Developing follicles are comprised of transition, primary, and secondary follicles. Differ from fresh control group. A,B Different superscripts within same group differ among treatments. a,b,c
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Different superscripts within same treatment differ among groups. ‡ No statistical analysis was performed due the low number of preantral follicles encountered. n, number of follicles found.
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Table 2 Mean (± S.E.M.) percentage of morphologically normal equine preantral follicles in fresh or
culture.
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cryopreserved-thawed (slow-freezing or vitrification) ovarian tissue after 1-7 days of in vitro
Primordial
Developing†
Fresh
51.8 ± 4.9a
11.9 ± 3.3a
(n = 130)
(n = 26)
3.2 ± 1.9b
3.8 ± 2.0b
7.0 ± 2.7c
(n = 68)
(n = 43)
(n = 111)
6.4 ± 2.5b
6.1 ± 2.2b
12.6 ± 3.3b
(n = 55)
(n = 156)
Vitrification
(n = 101) †
M AN U
Slow-freezing
SC
Treatment
Total
63.7 ± 4.5a (n = 156)
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Developing follicles are comprised of transition, primary, and secondary follicles. Data obtained on days 1, 3, and 7 of culture for each treatment were combined for statistical analyses. a,b,c Different superscripts within the same column differ among treatments. n, number of follicles found.
5x10
6
Aa
ABa
Aa Aa
Ba
ABb
Ab
4x106
*Ab *Bb *Bc
3x106
*Bb
2x106 1x106 0
0
1
3
Days in culture
TE D AC C
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Figure 1
Aa
SC
6x106
Vitrification
Slow-freezing
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Fresh
7x106
7
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EGFR fluorescence intensity
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6x106 5x10
Aa
6
Vitrification
Slow-freezing
Aa Aa Ab
4x106
Aa
Aab
*Ab
0
M AN U
1x106
1
EP
3
Days in culture
Figure 2
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Aa
SC
*Bb
2x106
0
Aa
*Ab
*Ab
3x106
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Ki-67 fluorescence intensity
7x106
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8x10 7x10 6x10 5x10 4x10 3x10 2x10 1x10
Fresh
6 6
Vitrification
Slow-freezing
Aa
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6 6 6
*Aa
Ba Bb
Ab
Ab *Ab
6
*Bb *ABc
*Ba
*Bd
6 6 6
0
1
3
Days in culture
7
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Cb
SC
Bax fluorescence intensity
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7x10 6x10 5x10 4x10 3x10 2x10 1x10
6 6
Aa
6 6
Bab
Ba
ABb *Aa *Bb
6 6 6 6
0
1
EP
Figure 4
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3
Days in culture
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Vitrification
Slow-freezing
*Aa
Ab
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8x10
Fresh
6
*Aa *Aa
SC
9x10
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Bcl-2 fluorescence intensity
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7x106 5x106
Aa
*Aa
Aa
ABa
Aa
Ba
Aab Ab
Bb
Bb
4x106
Ab Bb
3x106 2x106 1x106 0
1
Days in culture
EP
Figure 5
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3
7
M AN U
0
SC
6x106
Vitrification
Slow-freezing
RI PT
Fresh
8x106
TE D
TUNEL fluorescence intensity
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Highlights:
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This study demonstrated the feasibility of culturing in vitro cryopreserved equine ovarian tissue. Slow-freezing and vitrification had similar efficiency in preserving the morphology of preantral follicles. Expression of EGFR and Ki-67 proteins were preserved after cryopreservation and in vitro culture for 7 days. Expression of Bax and Bcl-2 protein in cryopreserved tissue was similar to that of fresh tissue after culture. DNA degradation in cryopreserved-thawed ovarian tissue was similar to that in fresh tissue.
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