Accelerated follicle growth during the culture of isolated caprine preantral follicles is detrimental to follicular survival and oocyte meiotic resumption

Accepted Manuscript Accelerated follicle growth during the culture of isolated caprine preantral follicles is detrimental to follicular survival and oocyte meiotic resumption Livia Brunetti Apolloni, Jamily Bezerra Bruno, Benner Geraldo Alves, Anna Clara Accioly Ferreira, Victor Macêdo Paes, Jesus de los Reyes Cadenas Moreno, Francisco Léo Nascimento de Aguiar, Felipe Zandonadi Brandão, Johan Smitz, Gary Apgar, José Ricardo de Figueiredo PII:

S0093-691X(16)30212-6

DOI:

10.1016/j.theriogenology.2016.05.012

Reference:

THE 13681

To appear in:

Theriogenology

Received Date: 16 November 2015 Revised Date:

14 April 2016

Accepted Date: 13 May 2016

Please cite this article as: Apolloni LB, Bruno JB, Alves BG, Ferreira ACA, Paes VM, Moreno JdlRC, de Aguiar FLN, Brandão FZ, Smitz J, Apgar G, de Figueiredo JR, Accelerated follicle growth during the culture of isolated caprine preantral follicles is detrimental to follicular survival and oocyte meiotic resumption, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.05.012. 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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Accelerated follicle growth during the culture of isolated caprine preantral follicles

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is detrimental to follicular survival and oocyte meiotic resumption

3 Livia Brunetti Apollonia, Jamily Bezerra Brunoa, Benner Geraldo Alvesa, Anna Clara

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Accioly Ferreiraa, Victor Macêdo Paesa, Jesus de los Reyes Cadenas Morenoa,

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Francisco Léo Nascimento de Aguiara, Felipe Zandonadi Brandãob, Johan Smitzc, Gary

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Apgard, José Ricardo de Figueiredoa

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Laboratory of Manipulation of Oocyte Enclosed in Preantral Follicles – LAMOFOPA,

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Faculty of Veterinary, University of Ceará, Fortaleza, Brazil

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Fluminense, Niterói, Rio de Janeiro, Brazil

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Laarbeeklaan 101, B-1090 Brussels, Belgium

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Carbondale, USA.

Department of Animal Reproduction, Faculty of Veterinary, Federal University

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Follicles Biology Laboratory, Center for Reproductive Medicine, UZ Brussel,

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Department of Animal Science, Food and Nutrition, Southern Illinois University,

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E-mail address: [email protected] (José Ricardo de Figueiredo).

Corresponding author. Tel: +55 85 3101 9852; Fax: + 55 85 3101-9840.

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ABSTRACT

26 This study investigated the effect of androstenedione (A4) alone or in association with

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different concentrations of bovine recombinant FSH on the in vitro culture of isolated

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goat preantral follicles. Follicles were mechanically isolated from ovarian tissue and

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cultured for 18 days in -MEM supplemented or not with A4 (10 ng/ml) alone or in

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association with fixed (A4+FixFSH: 100 ng/ml) or sequential (A4+SeqFSH: Day 0: 100

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ng/ml; Day 6: 500 ng/ml; Day 12: 1000 ng/ml) concentrations of FSH. After 18 days,

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the oocytes were recovered for in vitro maturation and fluorescence analysis. At Day

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18 of culture, only A4+SeqFSH treatment showed a lower (P < 0.05) rate of intact

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follicles, survival probability and meiotic resumption, as well as higher (P < 0.05)

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percentage of degeneration and/or extrusion after antrum formation. Taken together,

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these results showed a positive correlation between fast-growing follicles and follicles

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that degenerated and/or extruded after antrum formation. When compared to control, the

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addition of A4 alone or in association of FSH did not increase (P > 0.05) the estradiol

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production or androstenedione levels on Day 6. However on Day 18, the

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androstenedione levels were significantly lower in A4+SeqFSH treatment when

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compared to A4 alone or to A4+FixFSH treatments, while the estradiol production did

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not differ (P > 0.05). In summary, this study demonstrated that accelerated follicle

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growth negatively impacted the morphology of caprine preantral follicle cultured in

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vitro. In addition, the association of androstenedione with increasing concentration of

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FSH was detrimental to follicular survival and oocyte meiotic resumption.

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Keywords: secondary follicles, caprine, FSH, androgens, growth rates.

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1. Introduction

50 Ovarian function depends on multiple integrated processes that culminate in the

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production of competent oocytes during folliculogenesis. FSH plays an important role in

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folliculogenesis [1]. Regardless the FSH addition (i.e., fixed or increased concentrations)

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in in vitro culture media [2,3] it provides for the maintenance of follicular growth and

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survival as well as stimulates steroidogenesis [4]. Among the steroidal hormones, addition

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of

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testosterone) promotes follicular growth in different species [5-7] through the stimulation

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of their receptors. Androgen receptors are located in the cytoplasm of granulosa cells

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(GC), theca cells (TC) and oocytes [8] of preantral follicles [9-11]; and antral [9, 10, 12],

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being the GC more responsive to androgens [13].

(androstenedione,

dihidroandrostenediona,

dihydrotestosterone

and

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androgens

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An important tool to investigate in vitro folliculogenesis, including the effect of

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different substances, is the in vitro culture of preantral follicles [14]. Studies evaluating the

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role of androstenedione on in vitro follicle culture have been controversial with findings

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being both concentration-dependent and species specific. For example, addition of

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androstenedione in a dose dependent manner reduced the survival rate of isolated mice

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preantral follicles [15]. On the other hand, culture of isolated swine preantral follicles in

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medium containing androstenedione improved antrum formation rates [16]. In another

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study, after in vitro culture of caprine preantral follicles (primordial and primary) enclosed

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in ovarian tissue, androstenedione associated with fixed FSH maintained the percentage of

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normal follicles during the culture [17]. However, the effect of androstenedione and FSH

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on in vitro culture of isolated secondary preantral follicles has not been fully investigated

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in caprines. It is well known that goats are spread worldwide, having social and economic

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importance in many countries. In addition, the goat has been proposed as an important

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animal model for women [18]. Therefore, the objective of this study was to investigate the effect of

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androstenedione addition, associated or not with FSH (fixed or sequential concentration)

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into the culture medium, by evaluating the following endpoints: (i) follicular survival and

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growth, (ii) antrum formation, (iii) hormone production (androstenedione and estradiol) of

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caprine isolated secondary follicles, and (iv) oocyte meiotic resumption.

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80 2. Material and Methods

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2.1 Chemicals

Unless stated otherwise, all the chemicals and culture media used for this study were purchased from Sigma Chemical Company (St. Louis, MO, USA).

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2.2 Source of ovaries

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Ovaries (n= 60) from 30 adults (between 1 and 3 years old), non-pregnant,

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mixed-breed goats (Capra hircus) were collected at a slaughterhouse. After slaughter,

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the pairs of ovaries were washed in 70% alcohol for 10 seconds and then twice in

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minimum essential medium plus HEPES (MEM-HEPES) supplemented with 100

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µg/mL penicillin and 100 µg/mL streptomycin. After which, the ovaries were

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transported within 4 h to the laboratory in MEM-HEPES at 4°C [19].

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2.3 Isolation and selection of preantral follicles In the laboratory, the surrounding fatty tissue and ligaments were stripped from

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the ovaries. Ovarian cortical slices (1–2 mm thick) were excised from the ovarian

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surface using a surgical blade under sterile conditions and subsequently placed in

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holding medium consisting of MEM-HEPES with antibiotics. Preantral follicles,

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approximately 150-200 µm in diameter without antral cavities (secondary follicles),

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were visualized under a stereomicroscope (model SMZ 645; Nikon, Tokyo, Japan) and

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mechanically isolated by using 26-gauge (26 G) needles. Approximately 10 secondary

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follicles were isolated from each ovarian pair. These follicles were then transferred to

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100 µL droplets containing basic culture medium for the evaluation of quality. Only

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secondary follicles that displayed the following characteristics were selected for culture:

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an intact basement membrane, two or more layers of granulosa cells and a visible

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oocyte that was round and centrally located within the follicle, without any dark

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cytoplasm. Then, isolated follicles were pooled and randomly allocated to the

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treatments [20].

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2.4 In vitro culture of preantral follicles

After selection, the follicles were individually cultured (one follicle per droplet)

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in 100 µL droplets of culture medium under mineral oil in Petri dishes (60x15 mm;

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Corning, Sarstedt, Newton, NC, USA). The medium used was α-MEM (pH 7.2– 7.4;

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Gibco, Invitrogen, Karlsruhe, Germany) supplemented with 3.0 mg/mL bovine serum

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albumin (BSA), 10 ng/mL insulin, 5.5 g/mL transferrin, 5 ng/mL selenium, 2 mM

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glutamine, 2 mM hypoxanthine and 50 g/mL ascorbic acid, here in after referred as α-

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MEM+ [20]. Incubation was conducted at 39°C and 5% CO2 in air for 18 days. Fresh

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media were prepared and incubated for 1 h prior to use, with 60 µL medium being

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changed in each drop every 2 days. The experimental conditions were replicated four

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times and at least 40 follicles were used per treatment.

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2.5 Experimental design Follicles were randomly assigned to four different treatments as follows: 1) α-

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MEM+ alone (control group); 2) α-MEM+ supplemented with androstenedione (A4: 10

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ng/ml); 3) α-MEM+ plus A4 (10 ng/ml) associated with recombinant bovine FSH -

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rbFSH (Nanocore, São Paulo, Brazil) in increasing concentrations (Sequential FSH:

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from Day 0 to Day 6 = 100 ng/mL; from Day 6 to Day 12 = 500 ng/mL; from Day 12

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to Day 18 = 1000 ng/mL; A4+SeqFSH); 4) α-MEM+ plus A4 (10 ng/ml) associated with

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a fixed concentration of rbFSH (FixFSH: 100 ng/ml; A4+FixFSH). Concentrations of

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A4 and rbFSH were chosen based on previous work performed by our group (A4: [17];

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SeqFSH: [3]; FixFSH: [2]).

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2.6 Morphological evaluation of follicle development Follicles were evaluated during culture (Days 0, 6, 12, and 18) to the following

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end-points: integrity of the basement membrane, morphology of the oocyte and

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surrounding granulosa cells, and morphological signs of degeneration such as darkness.

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The percentage of morphologically intact follicles was calculated by excluding the

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follicles which experienced rupture of the basement membrane and had degenerated.

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The percentage of degenerated follicles was calculated using the number of follicles that

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exhibited dark and/or misshapen cytoplasm of the oocyte and surrounding cumulus

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cells. The percentage of extruded follicles was then calculated using the number of

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follicles that showed rupture of the basement membrane. The follicular diameter was

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measured only in morphologically intact follicles every 6 days with the aid of an ocular

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micrometer attached to a stereomicroscope (SMZ 645 Nikon, Tokyo, Japan;

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magnification: X 100). Two perpendicular diameters were recorded for each follicle and

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the average of these two values was reported as the follicular diameter. To evaluate

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daily follicular growth, the mean increase in follicular diameter was calculated as

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follows: the diameter of morphologically intact follicles at Day 18 minus their diameter

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at Day 0, divided by 18 days. In vitro cultured follicles were divided into three growing

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categories: (i) non-growing: follicles that did not grow during the culture; (ii) slow-

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growing: follicles with a daily growth between 0.1 - 7.0 µm/day; and (iii) fast-growing:

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follicles with a daily growth between 7.1 - 34 µm/day. Also, antral cavity formation was

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defined as a visible translucent cavity within the granulosa cell layers.

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2.7 In vitro maturation

At the end of the culture period, all the morphologically intact follicles were

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carefully and mechanically opened with 26 G needles under a stereomicroscope for

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oocyte recovery. Only oocytes (≥ 110 µm) with homogeneous cytoplasm and

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surrounded by at least one compact layer of cumulus cells were selected for in vitro

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maturation (IVM). The oocyte recovery rate was calculated by the ratio between the

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number of oocytes ≥ 110 µm (including zona pellucida) and the total number of normal

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and degenerated oocytes (smaller and larger than 110 µm). The cumulus-oocyte

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complexes were selected and washed three times in maturation medium consisting of

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TCM-199 supplemented with 1% BSA, 5 µg/ml of luteinizing hormone (LH), 0.5

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µg/mL of rbFSH, 10 ng/mL of epidermal growth factor (EGF), 50 ng/mL of

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recombinant insuline-like growth factor (IGF), 100 µM of cysteamine, 1 mM of

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pyruvate, and 1 µg/mL of estradiol [21]. After washing, oocytes were transferred to 100

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µL drops of maturation medium under mineral oil and then incubated for 32 hours at

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39oC with 5% CO2 in air.

171 2.8 Assessment of oocyte viability and chromatin configuration

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Fluorescence microscopy was used to analyze the viability of the oocytes from

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the caprine follicles classified as morphologically normal under the stereomicroscope,

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after culture. Briefly, the oocytes were incubated in 100 µL TCM 199 HEPES,

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supplemented with 5.5 µL calcein-AM, 88.5 µl ethidium homodimer-1 (Molecular

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Probes, Invitrogen, Karlsruhe, Germany), 5.0 µL Hoechst 33342 and 1.0 µL

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glutaraldehyde (Sigma, Deisenhofen, Germany) at 37◦C, for 30 min.

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After the incubation, oocytes were washed three times in TCM 199 HEPES and

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placed in 3 µL droplets of mounting medium (6.25 mL of PBS, 6.25 mL of glycerol and

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6.25 µL Hoechst), covered with a cover slip on the slide and evaluated under a

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fluorescence microscope (Nikon, Eclipse 80i, Tokyo, Japan). Oocytes with cytoplasm

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stained by calcein-AM (green) were classified as viable and those that had chromatin

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marked with ethidium homodimer-1 (red) were considered to be degenerated. Hoechst

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was used to analyze the oocyte chromatin configuration for intact germinal vesicles

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(GV), meiotic resumption [germinal-vesicle breakdown (GVBD), metaphase I (MI),

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and metaphase II (MII)].

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2.9 Androstenedione and Estradiol Assay

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To evaluate the relationship between follicle development and hormone

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production, 60 µL of culture medium from all the follicles was collected every 6 days

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(Days 6, 12 and 18) to quantify androstenedione (A4) and estradiol (E2) production.

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Androstenedione was measured by direct radioimmunoassay from Biosource

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(androstenedione-RIA-CT, BioSource Europe, Nivelles, Belgium) with a sensitivity of

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70 ng/l and a total imprecision profile (CV = 10%) for concentrations between 100 and

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7,000 ng/l. Hormone Assay for E2 (DSL4800) secretion was determined by double

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antibody radioimmunoassay (RIA) using a commercial kit Immunotech (catalog No:

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DSL4800, IMMUNOTECH s.r.o. - Radiová 1 - 102 27 Prague 10 - Czech Republic).

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The analytical sensitivity of the assay was 2.2 pg/ml (assay range, 2.2-750 pg/ml). Intra-

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assay: samples were assayed 12 times in the same run. The coefficients of variation

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were ≤ 8.9%. Inter-assay coefficients of variation were assayed in duplicate within 8

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different runs and were ≤ 12.2%. All samples were assayed in the same RIA to

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eliminate inter- assay variability.

204 2.10 Statistical analysis

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All statistical analyses were performed using Sigma Plot 11 (Systat Software

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Inc., USA). Data that were not normally distributed (Shapiro-Wilk test) were submitted

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to logarithmic transformation. Follicle and oocyte diameter, and follicular growth

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among treatments were compared by Kruskal-Wallis test, while the Wilcoxon signed

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test was used to analyze the effect of treatment within days of culture. The proportion of

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follicular variables (intact, degenerated, extruded, antrum formation, and meiotic

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resumption) among treatments and days of culture were analyzed by Fisher’s exact test.

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A linear regression analysis was performed to evaluate the association of oocyte

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diameter with chromatin configuration and the relationship of follicular growth and

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antrum formation, while the logistic regression analyzed the association between follicle

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diameter and antrum formation. The follicle survival probability throughout the culture

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was plotted using Kaplan-Meier method and the difference among survival curves

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(treatments) was evaluated using log rank test. Data are presented as mean (±SEM) and

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percentage and the results were considered different when P < 0.05. Probability values

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> 0.05 and ≤ 0.1 indicated that a difference approached significance.

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3. Results

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223 3.1 Follicular morphology

A total of 169 follicles were selected for in vitro culture with a mean of 42.2 ±

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0.6 follicles analyzed per treatment. The percentage of intact follicles decreased (P <

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0.05) from Day 0 to Day 18 in all treatments, except for the control. Moreover, this

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percentage tended to decrease (P < 0.08) only in the A4+SeqFSH treatment from Day

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12 to Day 18 of culture (Fig. 1). Except for the control, the percentage of intact follicle

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decreased (P < 0.05) from Day 0 to Day 18 and tended to decrease (P < 0.08) only in

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the A4+SeqFSH treatment from Day 12 to Day 18. In addition, this treatment was

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lower on Day 18 compared to the control (P < 0.05). The effect of treatments on follicle

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survival probability was evaluated by Kaplan Meier method (Fig. 2). The only

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treatment that showed a reduction (73.8%; P < 0.05) in follicle survival on Day 18 was

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the A4+SeqFSH when compared to the control group.

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The rate of degenerated follicles was similar (P > 0.05) among the treatments

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(Fig. 3). However, from Day 12 to Day 18 the A4+SeqFSH treatment showed an

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increase (P < 0.05) in the percentage (11.9%) of degenerated follicles. The percentage

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of follicular extrusion is shown (Fig. 4). No difference was observed among treatments

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(P > 0.05). However, treatments containing FSH increased (P < 0.05) the percentage of

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extruded follicles from Day 0 to Day 18.

242 3.2 Follicular diameter and growth rate

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The average values of follicular diameter during in vitro culture of isolated

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preantral follicles are shown (Table 1). A progressive increase (P < 0.05) in follicular

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diameter was observed in all treatments during the culture period. However, the follicle

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diameter was similar (P > 0.05) among treatments irrespective of the culture period.

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The daily growth rate is shown (Table 2), and the A4+SeqFSH treatment tended (P <

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0.09) to be higher than control group in the second third of culture period (D6-D12).

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Moreover, only A4+SeqFSH treatment the growth rate tended to be higher (P < 0.07) in

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the second third compared to the first third of culture. The frequency distributions of

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follicles classified according to growing rates is shown (Fig. 5A,B). We observed that

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the A4+SeqFSH treatment presented a reduction (P < 0.05) in the percentage of slow-

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growing follicles compared to control and A4 treatments. Additionally, FSH treatments

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showed a higher (P > 0.05) percentage of fast growing follicles compared to control

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(Fig. 5B).

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3.3 Antrum formation

Regardless of treatment, the percentage of antrum formation increased (P <

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0.05) from Day 0 until Day 12 of culture and remained constant until the end of the

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culture period (Table 3). A positive correlation between antrum formation and

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follicular diameter during in vitro culture was observed by logistic regression analysis

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(Fig. 6). Therefore, the mean growth rate of follicles that formed antrum followed by

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degeneration and extrusion was 1.8-fold superior than follicles that maintained the

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antrum (Fig. 7). When the treatments were compared to each other using these end

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points (Fig. 8), the A4+SeqFSH had a decrease (P < 0.05) in the percentage of follicles

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that maintained the antrum but an increase (P < 0.05) in the percentage of follicles that

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degenerated or extruded after antrum formation compared control group. These results

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could be confirmed by the positive association between antrum formation phenotypes

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and follicular growth rates (Fig. 9).

271 3.4 Oocyte parameters and chromatin configuration

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No difference in the oocyte recovery rate (≥ 110 µm), oocyte diameter (µm) and

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oocyte viability (%) were observed among the treatments (P > 0.05) after 18 days of

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culture (data not shown). The A4+SeqFSH treatment had a lower percentage of meiotic

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resumption (P < 0.05) than the control group and tended to differ (P < 0.06) from

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A4+FixFSH treatment. Metaphase II (MII) was observed only in the control and

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A4+FixFSH treatments (Fig. 10). Finally, as expected a positive relationship between

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chromatin configuration and oocyte diameter was observed using a regression analysis

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(Fig. 11).

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3.5 Hormonal production Regardless of treatment, androstenedione concentrations were similar (P > 0.05)

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between 6 and 18 days of culture. However, at Day 18 of culture there was a decrease

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in androstenedione levels of A4+SeqFSH compared with A4 (tended to differ; P < 0.07)

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and A4+FixFSH treatments (P < 0.05; Fig. 12). No difference (P > 0.05) was observed

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among treatments on the estradiol concentration during the in vitro culture (data not

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shown).

289 4. Discussion

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In general, we observed that androstenedione, associated with increasing

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concentrations of FSH (A4+SeqFSH) hampered the follicular survival as well as the

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ability of in vitro grown oocytes to resume meiosis.

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Previous studies in caprines have shown that the presence of increasing

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concentrations of FSH and the addition of others substances such as activin A [22]

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epidermal growth factor [23] and IGF-II [24] did not impair follicular development and

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improved meiosis resumption of the oocytes recovered from in vitro grown preantral

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follicles. Unlike the positive effects of those substances associated with increasing FSH

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concentrations, in our study the androstenedione caused a deleterious effect on the in

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vitro culture of preantral follicles, when associated with increasing concentrations of

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FSH and no effects when associated with fixed FSH concentration. Even though we

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expected positive results, these findings clearly show the timeliness of this study.

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In the current study, the percentage of morphologically intact follicles, as well as

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the follicular survival probability decreased, whereas the rate of degenerated follicles

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increased at the end of culture in the A4+SeqFSH treatment. Several authors have

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described that during the in vitro culture of preantral follicles, the addition of FSH either

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in increasing concentrations [3] or in decreasing concentrations associated to

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dihydrotestosterone [7] increased follicle survival. It is well known that follicular

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growth and atresia depends on a delicate balance between gonadotropin and paracrine

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factors [25]. Therefore, we suggest that increasing concentrations of FSH associated

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with androstenedione may cause a negative effect, compromising the follicular

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physiology through the inappropriate proliferation of GC [26]. In relation to extrusion rates, FSH treatments (fixed and sequential) presented an

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increase of the rate of extruded follicles between Day 0 and Day18. Androgens increase

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the expression of FSH receptor (FSHR) in the GC [27,28], therefore we propose that the

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association of androstenedione with FSH may have potentiated the action of this

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hormone in the intrafollicular environment, hyperstimulating the proliferation of GC

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[26] and consequently, causing rupture of the basal membrane and releasing the

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cumulus-oocyte complexes. These findings are supported by the fact that FSH

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treatments presented a higher proportion of follicles in the fast-growing category than

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control group. Additionally, using linear regression analysis it was possible to observe a

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correlation between fast-growing follicles and follicles that degenerated and/or extruded

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after antrum formation, implying that follicles require a slow growth rate to maintain

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follicular viability.

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In the present study, the A4+SeqFSH treatment was the only one that had lower

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percentage of follicles that formed antrum and remained intact until the end of the

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culture when compared to control. This can be explained by the high rates of

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degeneration and/or extrusion after antrum formation observed in this treatment. Other

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studies tested the effect of different concentrations of androstenedione (10 and 100

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ng/ml) in medium that was FSH-free on the in vitro culture of bovine early antral

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follicles and demonstrated that both concentrations were able to maintain the antrum

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intact until the end of culture [29].

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This is the first study that evaluated the production of androstenedione during

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the in vitro culture of goat preantral follicles. Despite the difference in androstenedione

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concentrations at the onset of culture (0 ng/ml-control vs 10 ng/ml-A4 treatments), the

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androstenedione levels on Day 6 were equivalent between control and A4 treatments. In

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other words, the concentration of androstenedione was reduced at least 66 times in A4

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treatments indicating a high rate of metabolization of androstenedione by cultured

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follicles in these treatments. In addition, follicles cultured in the medium containing low

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insulin were able to produce adequate levels of androstenedione, resulting in equivalent

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production of estradiol both on Day 6 and 18 of culture. This suggests that the excessive

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metabolization of androstenedione was not used expressly for estradiol production.

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During the steroidogenesis process, androstenedione can be converted either to: (i)

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testosterone by 17β-HSD (17β-hydroxysteroid dehydrogenase) which itself can then be

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aromatised into estradiol by P450arom (CYP19) in CG (A4
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E2), (ii) estrone by

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P450arom (CYP19) for subsequent conversion in estradiol by 17β-HSD in CG (A4


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E1
E2), or (iii) testosterone by 17β-HSD in TC which itself can then be converted

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into dihydrotestosterone by 5α-reductase in GC (A4
T
DHT) [8]. Therefore, in our

350

study, we suggest that the major pathway used by the follicles cultured in A4 treatments

351

was A4
DHT since the estradiol production did not differ among treatments

352

regardless the culture period and the testosterone production was below the detection

353

limit. This assumption can be support by Bogovish and Richards [30] which

354

demonstrated the ability of GC from rodents to generate dihydrotestosterone when

355

treated with androstenedione. Moreover, WU et al. [31] demonstrated that the addition

356

of testosterone in medium containing FSH or not, promoted a stimulatory effect of

357

CYP19 in mice GC, which correlated with estradiol production.

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The A4+SeqFSH treatment showed lower rates of meiosis resumption than

359

control after oocyte in vitro maturation. The control medium used in our experiment

360

was previously developed by our team [20] which defined the appropriated insulin

361

concentration as 10 ng/ml. Insulin is a major factor related to follicular survival, growth

362

and production of steroids hormones [20]. Like androgens [27], studies have reported

363

that insulin increases the expression of FSHR [32,33]. We believe that androstenedione

364

associated with insulin enhanced the action of FSH on the in vitro culture of preantral

365

follicles. The high FSH concentrations used in the A4+SeqFSH treatment caused a

366

deleterious effect on meiosis resumption. This result is in agreement with Sánchez et al.

367

[26] that reported that higher concentrations of FSH increased the levels of LH

368

receptors (LHR) in mice cumulus cells, compromising oocyte competence, since the

369

upregulation of LHR is related to low oocyte quality [34].

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In our study, surviving follicles were divided into three categories (non-, slow-

371

and fast-growing follicles), according to their growth rates. The growth rates are an

372

important parameter to evaluate the efficiency of follicular development in culture.

373

However, in our study, accelerated follicle growth was correlated with degeneration

374

and/or extrusion which may allow it to be used as a tool to predict follicular fate in

375

vitro. On the other hand, Xu et al. [35] demonstrated that in the culture of monkeys

376

preantral follicles, the production of anti-mullerian hormone (AMH) was correlated

377

with follicular growth, wherein early AMH production can predict further development

378

of cultured preantral follicles. Other studies using activin A in the culture medium of

379

isolated caprine preantral follicles showed that the treatment with the highest percentage

380

of fast-growing follicles also presented high meiotic resumption rates [23]. Differences

381

in the species, in the medium composition as well as in the classification on the follicle

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growth categories may explain this controversial finding. For instance, in the culture of

383

isolated caprine preantral follicles, Silva et al. [23] classified fast-growing follicles as

384

those which showed growth rate higher than 10 µm/day, while in our study the follicles

385

that had a daily growth between 7.1 - 34 µm/day were classified as fast-growing

386

follicles. Besides the presence of androstenedione in the majority of our treatments,

387

Silva et al. [23] used higher insulin concentration in the basic medium. This indicates

388

that in the presence of androstenedione and increasing concentrations of FSH the

389

acceleration of follicular development was detrimental for efficiency of caprine

390

preantral follicles cultured in vitro.

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382

In summary, the addition of androstenedione alone or associated with fixed

392

concentrations of FSH did not improve either follicular survival, development or steroidal

393

levels after in vitro culture. Moreover, increasing FSH concentrations associated with

394

androstenedione had a detrimental effect on follicle survival and oocyte meiotic

395

resumption. Finally, this study demonstrated that accelerated follicle growth negatively

396

affected the morphology of caprine preantral follicle cultured in vitro.

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399 400

Acknowledgments

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This work was carried out at the Laboratory of Manipulation of Oocytes and

401

Ovarian Preantral Follicles (LAMOFOPA), Faculty of Veterinary Medicine of Ceará

402

State University, Fortaleza, CE, Brazil, and supported by CNPq (407594/2013-2). Livia

403

Brunetti Apolloni is recipientes of a grant from CAPES Brazil and José Ricardo de

404

Figueiredo are recipientes of a grant from CNPq Brazil.

405

18

ACCEPTED MANUSCRIPT 406

Disclosures

407 408

Conflict of interest: None of the authors declared having any conflict of interest.

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409 410 411

Figure legends

SC

412

Figure 1. Percentage of intact follicles during in vitro culture in medium containing

414

androstenedione associated or not with fixed and sequential concentrations of FSH.

415

Control: α-MEM+; A4: α-MEM+ plus androstenedione; A4+SeqFSH: α-MEM+ plus

416

androstenedione and sequential concentrations of rbFSH; A4+FixFSH: α-MEM+ plus

417

androstenedione and fixed concentrations of rbFSH.

418

uppercase letters indicate difference (P < 0.05).

419

lowercase letters indicate difference (P < 0.05). ǂ Tended to differ from Day 18 (P <

420

0.08).

Within day, uncommon

Within treatment, uncommon

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a,b

A,B

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421

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413

Figure 2. Follicle survival probability (Kaplan-Meier plot) during in vitro culture in

423

medium containing androstenedione associated or not with fixed and sequential

424

concentrations of FSH. Control: α-MEM+; A4: α-MEM+ plus androstenedione;

425

A4+SeqFSH: α-MEM+ plus androstenedione and sequential concentrations of rbFSH;

426

A4+FixFSH: α-MEM+ plus androstenedione and fixed concentrations of rbFSH.

427

Differed from control ( P < 0.05).

428

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422

*

19

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Figure 3. Percentage of degenerated follicles during in vitro culture in medium

430

containing androstenedione associated or not with fixed and sequential concentrations

431

of FSH. Control: α-MEM+; A4: α-MEM+ plus androstenedione; A4+SeqFSH: α-MEM+

432

plus androstenedione and sequential concentrations of rbFSH; A4+FixFSH: α-MEM+

433

plus androstenedione and fixed concentrations of rbFSH.a,b Within treatment,

434

uncommon lowercase letters indicate difference (P < 0.05). Among treatments within

435

the same day (P > 0.05).

SC

RI PT

429

436

Figure 4. Percentage of extruded follicles during in vitro culture in medium containing

438

androstenedione associated or not with fixed and sequential concentrations of FSH.

439

Control: α-MEM+; A4: α-MEM+ plus androstenedione; A4+SeqFSH: α-MEM+ plus

440

androstenedione and sequential concentrations of rbFSH; A4+FixFSH: α-MEM+ plus

441

androstenedione and fixed concentrations of rbFSH.

442

lowercase letters indicate difference (P < 0.05). Among treatments within the same day

443

(P > 0.05).

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Within treatment, uncommon

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444

a,b

Figure 5. (A) Representation of follicles (n = 169) classified according to growth rate in

446

non-, slow-, and fast-growing.

447

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445

448

Figure 5. (B) Frequency distributions of follicles in growing categories during in vitro

449

culture in medium containing androstenedione associated or not with fixed and

450

sequential

451

androstenedione; A4+SeqFSH: α-MEM+ plus androstenedione and sequential

452

concentrations of rbFSH; A4+FixFSH: α-MEM+ plus androstenedione and fixed

concentrations

of

FSH.

Control:

α-MEM+;

A4:

α-MEM+

plus

20

ACCEPTED MANUSCRIPT a,b

453

concentrations of rbFSH.

454

letters indicate difference (P < 0.05).

455

Tended to differ from control (P < 0.07).

Within the same growing categories, uncommon lowercase #

Tended to differ from control (P < 0.06).

†

RI PT

456 Figure 6. Relationship between antrum formation and follicle diameter. Each point of

458

the graph is a follicle recorded during in vitro culture (n = 169). The follicles evaluated

459

were defined by binary values (without antrum formation = 0; presence of antrum

460

formation = 1) to dependent variable. A logistic regression (antrum predicted

461

probability) is represented by the equation and the black line [Logit P = -1.916 +

462

(0.0131 × follicle diameter); P < 0.05].

M AN U

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463 464

Figure 7. Mean (± SEM) daily growth rate of follicles according to their ability to form

465

antrum and maintain normal morphology.

466

difference (P < 0.05).

Uncommon lowercase letters indicate

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467

a,b,c

Figure 8. Effect of the addition of androstenedione associated or not with fixed and

469

sequential concentrations of FSH on the antrum formation and maintenance of normal

470

morphology. Control: α-MEM+; A4: α-MEM+ plus androstenedione; A4+SeqFSH: α-

471

MEM+ plus androstenedione and sequential concentrations of rbFSH; A4+FixFSH: α-

472

MEM+ plus androstenedione and fixed concentrations of rbFSH.

473

antrum conditions, uncommon lowercase letters indicate difference (P < 0.05).

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a,b

Within the same

474 475

Figure 9. Relationship between antrum phenotypes conditions and follicular growth.

476

Each point of graph is a follicle recorded during in vitro culture (n = 169). The follicles

21

ACCEPTED MANUSCRIPT

evaluated were defined by binary values (absence of antrum formation = 1; antrum

478

maintenance = 2; follicles degenerated and/or extruded after antrum formation = 3) to

479

dependent variable. A linear regression is represented by the equation and the black line

480

[Antrum formation conditions = 1.955 + (0.0162 × follicular growth); R2 = 0.08; r =

481

0.28; P < 0.001].

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477

482

Figure 10. Percentage of meiotic resumption, germinal vesicle (GV), germinal vesicle

484

breakdow (GVBD), metaphase I (MI) and metaphase II (MII) of oocytes from preantral

485

follicles cultured in vitro in medium containing androstenedione associated or not with

486

fixed and sequential concentrations of FSH. Control: α-MEM+; A4: α-MEM+ plus

487

androstenedione; A4+SeqFSH: α-MEM+ plus androstenedione and sequential

488

concentrations of rbFSH; A4+FixFSH: α-MEM+ plus androstenedione and fixed

489

concentrations of rbFSH.

490

differ (P < 0.05). # Tended to differ from A4+SeqFSH (P < 0.06).

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a,b

Within the same parameter, values with different letters

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491

SC

483

Figure 11. Regression analysis of the chromatin configuration (GV, germinal vesicle;

493

GVBD, germinal vesicle breakdow; MI, metaphase I; MII, metaphase II) and oocyte

494

diameter. Each point of graph is a oocyte evaluated after in vitro maturation (n = 68).

495

The oocytes evaluated were defined by values (GV = 2; GVBD = 3; MI = 4; and MII =

496

5) to chromatin configuration (dependent variable). A linear regression is represented

497

by the equation and the black line [chromatin configuration = 1.507 + (0.00791 ×

498

oocyte diameter); R2 = 0.06; r = 0.24; P < 0.05].

499

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Figure 12. Mean (± SEM) androstenedione levels from preantral follicles cultured in

501

vitro in medium containing androstenedione associated or not with fixed and sequential

502

concentrations of FSH. Control: α-MEM+; A4: α-MEM+ plus androstenedione;

503

A4+SeqFSH: α-MEM+ plus androstenedione and sequential concentrations of rbFSH;

504

A4+FixFSH: α-MEM+ plus androstenedione and fixed concentrations of rbFSH.

505

Within day, uncommon uppercase letters indicate difference (P < 0.05).

506

differ from A4 (P < 0.07). Within treatment between days (P > 0.05).

RI PT

500

Tended to

SC

507 5. References

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508

†

A,B,C

509

[1] Sirard MA, Desrosier S, Assidi M. In vivo and in vitro effects of FSH on oocyte

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maturation and developmental competence. Theriogenology 2007; 68:71–6.

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[2] Rodrigues GQ, Silva CMG, Faustino LR, Bruno JB, Pinto LC, Lopes CAP,

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Campello CC, Figueiredo JR. Efeito de diferentes concentrações de hormônio folículo-

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estimulante recombinante sobre o desenvolvimento in vitro de folículos pré-antrais

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[3] Saraiva MVA, Celestino JJH, Araújo VR, Chaves RN, Almeida AP, Lima- Verde

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IB, Duarte ABG, Silva GM, Martins FS, Bruno JB, Matos MHT, Campello CC, et al.

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preantral follicles. Zygote 2011; 19:205–214.

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[4] Taniguchi F, Couse JF, Rodriguez KF, Emmen JMA, Poirier D, Korach KS.

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[5] Sen A, Prizant H, Light A, Biswas A, Hayes E, Lee HJ, Barad D, Gleicher N,

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Hammes SR. Androgens regulate ovarian follicular development by increasing follicle

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stimulating hormone receptor and microRNA-125b expression. PNAS 2014; 111:3008-

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Allen C, Campbell BK. Effect of dehydroepiandrosterone on in vivo ovine follicular

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[7] Rodrigues JK, Navarro PA, Zelinski MB, Stouffer RL, Xu J. Direct actions of

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androgens on the survival, growth and secretion of steroids and anti-Mullerian hormone

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by individual macaque follicles during three-dimensional culture. Reproductive biology

535

2015; 0:1-11.

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[8] Gervásio CG, Bernuci MP, Silva-de-Sá MF, Rosa-e-Silva ACJS. The role of

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androgen hormones in early follicular development. ISRN Obstetrics and Gynecology

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2014; 2014:1-11.

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[9] Salvetti NR, Alfaro NS, Velázquez MM. Alteration in localization of steroid

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hormone receptors and coregulatory proteins in follicles from cows with induced

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ovarian follicular cysts. Reproduction 2012; 144:723-735.

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[10] Juengel JL, Heath DA, Quirke LD, McNatty KP. Oestrogen receptor ߙ and ߚ,

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androgen receptor and progesterone receptor mRNA and protein localisation within the

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developing ovary and in small growing follicles of sheep. Reproduction 2006; 131:81–

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[11] Mlodawska W, Okolski A. Immunolocaization of androgen receptors and

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assessment of steroidogenic activity of the ovaries in prepubertal and pubertal mare.

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Journal of Equine Veterinary Science 2014; 34:113-114.

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[12] Słomczyñska M, Tabarowski Z. Localization of androgen receptor and cytochrome

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P450 aromatase in the follicle and corpus luteum of the porcine ovary.

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Reproduction Science 2001; 65:127–134.

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[13] Walters KA. Role of androgens in normal and pathological ovarian function.

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Reproduction and Fertility 2015; 149:193-218.

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[14] Figueiredo JR, Rodrigues APR, Amorim CA, Silva JRV. Manipulação de oócitos

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inclusos em folículos ovarianos pré-antrais – MOIFOPA. In: Gonçalves PBD,

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Figueiredo JR, Freitas VJF. Biotécnicas aplicadas à reprodução animal. São Paulo:

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Roca, 2008. p. 303-327.

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[15] Tarumi W, Tsukamoto S, Okutsu Y, Takahashi N, Horiuchi T, Masanori TI,

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Ishizuka B. Androstenedione induces abnormalities in morphology and function of

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developing oocytes, which impairs oocyte meiotic competence. Fertility and Sterility

561

2012; 97:469–476.

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[16] Tasaki H, Iwata H, Sato D, Monjy Y, Kuwayama T. Estradiol has a major role in

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antrum formation of porcine preantral follicles cultured in vitro. Theriogenology 2013;

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79:809-814.

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[17] Lima-Verde LB, Rosseto R, Matos MHT, Celestino JJH, Bruno JB, Silva CMG,

566

Faustino LR, Mororó MBS, Araújo VR, Campello CC, Figueiredo JR. Androstenedione

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and follicle stimulating hormone involvement in the viability and development of goat

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preantral follicles in vitro. Animal Reproduction 2010; 7:80-89.

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[18] Figueiredo JR, Rodrigues APR, Silva JRV, Santos RR. Cryopreservation and in

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vitro culture of caprine preantral follicles. Reproduction, Fertility and Development

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2011; 23:40-47.

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[19] Chaves RN, Martins FS, Saraiva MVA, Celestino JJH, Lopes CAP, Correia JC,

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Lima-Verde IB, Matos MHT, Báo SN, Name KPO, Campello CC, Figueiredo JR.

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Chilling ovarian fragments during transportation improve viability and growth of goat

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preantral follicles cultured in vitro. Reproduction, Fertility and Development 2008;

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20:640–647.

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[20] Chaves RN, Duarte ABG, Rodrigues GQ, Celestino JJH, Silva GM, Lopes CAP,

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Almeida AP, Donato MAM, Peixoto CA, Moura AAA, Lobo CH, Locatelli Y,

579

Mermillod P, Campello CC, Figueiredo JR. The effects of insulin and follicle-

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stimulating hormone (FSH) during in vitro development of ovarian goat preantral

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follicles and relative mRNA expression for insulin and FSH receptors and cytochrome

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P450 aromatase in cultured follicles. Biology of reproduction 2012; 87:69-80.

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[21] Brito IR, Silva CMG, Duarte ABG, Lima IMT, Rodrigues GQ, Rossetto R, Sales

584

AD, Lobo CH, Bernuci MP, Rosa-E-Silva ACJS, Campello CC, Xu M, Figueiredo JR.

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Alginate hydrogel matrix stiffness influences the in vitro development of caprine

586

preantral follicles. Molecular Reproduction & Development 2014; 81:636-645.

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[22] Silva CMG, Castro CV, Faustino LR, Rodrigues GQ, Brito IR, Rossetto R, Saraiva

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MVA, Campello CC, Lobo CH, Souza CEA, Moura AAA, Donato MAM, Peixoto AC,

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Figueiredo JR. Activin-A promotes the development of goat isolated secondary follicles

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in vitro. Zygote 2013; 23:41-52.

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[23] Silva CM, Castro SV, Faustino LR, Rodrigues GQ, Brito IR, Rossetto R, Saraiva

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MV, Campello CC, Lobo CH, Souza CE, Moura AA, Donato MA, Peixoto CA,

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Figueiredo JR. The effects of epidermal growth factor (EGF) on the in vitro

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development of isolated goat secondary follicles and the relative mRNA expression of

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EGF, EGF-R, FSH-R and P450 aromatase in cultured follicles. Research in Veterinary

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[24] Duarte ABG, Araújo VR, Chaves RN, Silva GM, Luz VB, Haag KT, Magalhães-

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Padilha DM, Almeida AP, Lobo CH, Campello CC, Figueiredo JR. Insulin-like growth

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factor II (IGF-II) and follicle stimulating hormone (FSH) combinations can improve the

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in vitro development of grown oocytes enclosed in caprine preantral follicles. Growth

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Hormone & IGF Research 2013; 23:37-44.

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[25] Adriens IA, Cortvrindt R, Smitz J. Differential FSH exposure in preantral follicle

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culture has marked effects on folliculogenesis and oocyte developmental competence.

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Human Reproduction 2004; 2:398-408.

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[26] Sánchez F, Adriaenssens T, Romero S, Smitz J. Different follicle-stimulating

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hormone exposure regimens during antral follicle growth alter gene expression in the

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cumulus-oocyte complex in mice. Biology of Reproduction 2010; 83:514-524.

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[27] Weil S, Vendola K, Zhou J, Bondy CA. Androgen and follicle-stimulating

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hormone interactions in primate ovarian follicle development. Journal of Clinical

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Endocrinology 1999; 84:2951-2956.

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[28] Garcia-Velasco JA, Rodrígues S, Agudo D, Pacheco A, Schneider J, Pellicer A.

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FSH receptor in vitro modulation by testosterone and hCG in human luteinized

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granulosa cells. European Journal of Obstetrics, Gynecology and Reproductive Biology

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2012; 165: 259-264.

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[29] Taketsuru H, Hirao Y, Takenouchi N, Kosuke I, Miyano T. Effect of

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androstenedione on the growth and meiotic competence of bovine oocytes from early

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antral follicles. Zygote 2011; 20:407-415.

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[30] Bogovich K, Richards JS. Androgen synthesis during follicular development:

619

evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal

620

regulation. Biology of Reproduction 1984; 31:122-131.

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[31] Wu YG, Bennett J, Talla D, Stocco C. Testosterone, not 5 -Dihydrotestosterone,

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stimulates LRH-1 leading to FSH-independent expression of Cyp19 and P450scc in

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granulosa cells. Molecular Endocrinology 2011; 25:656-668.

624

[32] Shimizu T, Murayama C, Sudo N, Kawashima C, Tetsuka M, Miyamoto A.

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Involvement of insulin and growth hormone (GH) during follicular development in the

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bovine ovary. Animal Reproduction Science 2008; 106:143-152.

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[33] Van Den Hurk R, Zhao J. Formation of mammalian oocytes and their growth,

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differentiation and maturation within ovarian follicles. Theriogenology 2005; 63:1717-

629

1751.

630

[34] Eppig JJ, O’Brien MJ. Development in vitro of mouse oocytes from primordial

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follicles. Biology of Reproduction 1996; 54:197-207.

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[35] Xu J, Lawson MS, Yeoman RR, Pau KY, Barret SL, Zelinski MB, Stouffer RL.

633

Secondary follicle growth and oocyte maturation during encapsulated three-dimensional

634

culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin. Human

635

Reproduction 2011; 26:1061-1072.

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1

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Table 1. Mean (± SEM) diameter of normal follicles cultured in vitro for 18 days in

2

medium containing androstenedione associated or not with fixed and sequential

3

concentrations of FSH. Follicular diameter (µm) Day 0

Day 6

Day 12

Day 18

Control

202.1 ± 7.4 a

238.7 ± 12.1 b

258.2 ± 13.9 c

289.3 ± 18.8 d

A4

213.4 ± 7.5 a

240.4 ± 11.1 b

271.6 ± 14.0 c

311.5 ± 21.3 d

A4+SeqFSH

202.1 ± 9.4 a

224.8 ± 12.8 b

278.9 ± 18.6 c

317.9 ± 29.9 d

A4+FixFSH

208.5 ± 7.9 a

240.9 ± 11.1 b

293.3 ± 18.4 c

325.9 ± 26.3 d

SC

RI PT

Treatments

Control: α-MEM+; A4: α-MEM+ plus androstenedione; A4+SeqFSH: α-MEM+ plus

5

androstenedione and sequential concentrations of rbFSH; A4+FixFSH: α-MEM+ plus

6

androstenedione and fixed concentrations of rbFSH.

7

uncommon lowercase letters indicate difference (P < 0.05). Among treatments within

8

the same day (P > 0.05).

AC C

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TE D

M AN U

4

a,b,c,d

Within treatment,

1

ACCEPTED MANUSCRIPT 1

Table 2. Mean (± SEM) daily growth of normal follicles cultured in vitro for 18 days

2

in medium containing androstenedione associated or not with fixed and sequential

3

concentrations of FSH. Follicular growth (µm/day) on different intervals of culture D0-D6

D6-D12

D12-D18

Overall (D0-D18)

Control

6.1 ± 1.4 Aa

3.2 ± 1.6 Aa

5.2 ± 1.5 Aa

A4

4.5 ± 1.4 Aa

5.2 ± 2.0 ABa

6.6 ± 1.6 Aa

A4+SeqFSH

3.8 ± 2.0 Aa

9.0 ± 2.2 B†b#

6.5 ± 2.7 Aab

6.4 ± 1.5 A

A4+FixFSH

5.4 ± 1.4 Aa

8.7 ± 2.1 ABa

5.4 ± 1.8 Aa

6.5 ± 1.2 A

RI PT

Treatments

4.8 ± 0.8 A

SC

5.4 ± 1.1 A

Control: α-MEM+; A4: α-MEM+ plus androstenedione; A4+SeqFSH: α-MEM+ plus

5

androstenedione and sequential concentrations of rbFSH; A4+FixFSH: α-MEM+ plus

6

androstenedione and fixed concentrations of rbFSH.

7

uncommon uppercase letters indicate difference (P < 0.05). † Tended to differ from

8

control group within the same interval of culture (P < 0.09).

9

uncommon lowercase letters indicate difference (P < 0.05).

TE D

EP

D0-D6 interval (P < 0.07).

AC C

10

M AN U

4

A,B

#

Within interval time,

a,b

Within treatment,

Tended to differ from

1

ACCEPTED MANUSCRIPT 1

Table 3. Percentage of antrum formation during in vitro culture in medium

2

containing androstenedione associated or not with fixed and sequential concentrations

3

of FSH. Antrum formation (%) Day 0

Day 6

Day 12

Day 18

Control

0.0 (0/42) a

66.6 (28/42) b

95.2 (40/42) c

95.2 (40/42) c

A4

0.0 (0/41) a

68.2 (28/41) b

90.2 (37/41) c

90.2 (37/41) c

A4+SeqFSH

0.0 (0/42) a

64.2 (27/42) b

85.7 (36/42) c

85.7 (36/42) c

A4+FixFSH

0.0 (0/44) a

65.9 (29/44) b

90.9 (40/44) c

90.9 (40/44) c

SC

RI PT

Treatments

Control: α-MEM+; A4: α-MEM+ plus androstenedione; A4+SeqFSH: α-MEM+ plus

5

androstenedione and sequential concentrations of rbFSH; A4+FixFSH: α-MEM+ plus

6

androstenedione and fixed concentrations of rbFSH. a,b,c Within treatment, uncommon

7

lowercase letters indicate difference (P < 0.05). Among treatments within the same

8

day (P > 0.05).

AC C

EP

TE D

M AN U

4

ACCEPTED MANUSCRIPT Control 100

Aa Aa Aa Aa

A4+SeqFSH

A4 Aa

Aab

Aa

Aab

A4+FixFSH

Aa Aab

Aa

‡

Aa Aab

80

RI PT

Bb

60

40

SC

Intact follicles (%)

ABb

ABb

0 Day 0

M AN U

20

Day 6

Day 12

AC C

EP

TE D

Days of culture

Day 18

ACCEPTED MANUSCRIPT

100

RI PT

Follicle survival probability (%)

95

90

SC

85

M AN U

80 Control

A4 A4+SeqFSH A4+FixFSH

75

0

2

4

6 8 10 12 14 16 18 Days of culture

AC C

EP

TE D

70

*

ACCEPTED MANUSCRIPT

20

20

15

15

10

10

5 0

30 25

a

a

D0

D6

a

a

0

D12 D18

A4+SeqFSH (n = 42)

20

15

b

10

0

30 25

20

5

5

15 10

a

a

a

5 0

D6

EP AC C

a

D0

a

a

a

D6

D12 D18

A4+FixFSH (n = 44)

a

D12 D18 D0 Days of culture

TE D

D0

A4 (n = 41)

RI PT

25

M AN U

Degenerated follicles (%)

25

30

Control (n = 42)

SC

30

a

D6

a

a

D12 D18

ACCEPTED MANUSCRIPT

20

20

15

15

10

10

5

a

a

a

a

0

30 25

a a a

a

D0

D6

0 D0

D6

D12 D18

A4+SeqFSH (n = 42)

20 ab

ab

10

30 25

b

15

5

5

A4 (n = 41)

20 15 10 5

a

0 D6

EP AC C

D12 D18

A4+FixFSH (n = 44)

a

0 D12 D18 D0 Days of culture

TE D

D0

RI PT

25

M AN U

Extruded follicles (%)

25

30

Control (n = 42)

SC

30

b

ab

ab

D6

D12 D18

ACCEPTED MANUSCRIPT A

35

Fast-grow

30

Slow-grow

20 15

RI PT

Follicular growth (µm/day)

25

No-grow

10 5

SC

0

-10

B

M AN U

-5

1 10 20 30 40 50 60 70 80 90 100110120130140150160169 Preantral follicles

100

TE D

80

a

70 60

40 30 20

b#

a

ab

ac b†c b

EP

50

AC C

Frequency distribution of follicles (%)

90

Control A4 A4+SeqFSH A4+FixFSH

b

ab a

a

10

0

Non growing

Slow growing

Fast growing

b

ACCEPTED MANUSCRIPT

Antrum formation data (yes and no) Antrum predicted probability

Yes

RI PT

Antrum formation

80

Antrum predicted probability (%)

100

60

SC

40

0

M AN U

No

50 100 150 200 250 300 350 400 450 500 550 600

AC C

EP

TE D

Follicle diameter (um)

20

0

ACCEPTED MANUSCRIPT

14

c

10 8

RI PT

b

6 4

0

Antrum Degeneration/extrusion maintenance after antrum formation

AC C

EP

TE D

No antrum formation

SC

a

2

M AN U

Follicular growth (µm/day)

12

ACCEPTED MANUSCRIPT

100

a ab b

70 60

SC

50 40 30

b

20 10

a

a

a

No antrum formation

a

EP AC C

ab

ab

Degeneration/extrusion Antrum maintenance after antrum formation

TE D

0

M AN U

Percentage of follicles (%)

80

ab

RI PT

90

Control A4 A4+SeqFSH A4+FixFSH

SC M AN U

Degeneration/extrusion after antrum formation

Antrum maintenance

TE D

Antrum formation phenotypes

RI PT

ACCEPTED MANUSCRIPT

EP

No antrum formation

AC C

-10

-5

0

5

10

15

20

Follicular growth (µm/day)

25

30

35

ACCEPTED MANUSCRIPT

Control

A4+SeqFSH

A4

100

A4+FixFSH

b

RI PT

90 80 ab

60

a# a#

50 40

a

ab

ab ab

30 20 10

b

b

Meiotic resumption

GV

AC C

EP

TE D

0

a

SC

a

M AN U

Percentage

70

GVBD

MI

a

a

MII

RI PT

ACCEPTED MANUSCRIPT

SC

MI

GVBD

GV

20

40

60

M AN U

Chromatin configuration

MII

80

100 120 140 160 180

AC C

EP

TE D

Oocyte diameter (µm)

ACCEPTED MANUSCRIPT

Day 0 Day 6 Day 18

9.00

0.24

A

0.08 0.00

A

B

0.16 A

A A

Control

A4

RI PT

8.00

†

C

A4+SeqFSH

AC C

EP

TE D

M AN U

Treatments

B

A4+FixFSH

SC

Androstenedione (ng/mL)

10.00

ACCEPTED MANUSCRIPT

Highlights: 1) Androstenedione associated with increasing concentrations of FSH showed a lower rate of intact follicles, survival probability and meiotic resumption.

RI PT

2) Androstenedione associated with increasing concentrations of FSH showed a higher percentage of degeneration and/or extrusion after antrum formation.

3) A positive correlation between fast-growing follicles and follicles that degenerated and/or extruded after antrum formation was demonstrated.

SC

4) A positive correlation between the oocyte diameter and the chromatin configuration was demonstrated.

M AN U

5) The addition of androstenedione alone or in association with different concentration of FSH did

AC C

EP

TE D

not increase the estradiol production during the culture in vitro.