Role of the Calcium-Sensing Receptor (CaSR) in bovine gametes and during in vitro fertilization

Accepted Manuscript Role of the Calcium-Sensing Receptor (CaSR) in bovine gametes and during in vitro fertilization Beatriz Macías-García, Graça Lopes, Antonio Rocha, Lauro González-Fernández PII:

S0093-691X(17)30107-3

DOI:

10.1016/j.theriogenology.2017.03.002

Reference:

THE 14025

To appear in:

Theriogenology

Received Date: 2 December 2016 Revised Date:

15 February 2017

Accepted Date: 6 March 2017

Please cite this article as: Macías-García B, Lopes G, Rocha A, González-Fernández L, Role of the Calcium-Sensing Receptor (CaSR) in bovine gametes and during in vitro fertilization, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.03.002. 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.

Revised highlighted

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Role of the Calcium-Sensing Receptor (CaSR) in bovine gametes and

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during in vitro fertilization

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Beatriz Macías-Garcíaa,b, Graça Lopesa, Antonio Rochaa, Lauro González-Fernándeza,*

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University of Porto, Portugal

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CECA/ICETA – Animal Sciences Centre; ICBAS – Abel Salazar Biomedical Institute,

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CCMIJU – Minimally Invasive Surgery Centre Jesús Usón, Cáceres, Spain

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∗ Corresponding author at: Centro de Estudos de Ciência Animal (CECA),

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Universidade do Porto, Rua Padre Armando Quintas 7, 4485-661,

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Vairão, Vila do Conde. Portugal. E-mail address: [email protected]

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ABSTRACT

27 Calcium Sensing Receptor (CaSR) is a G-protein coupled receptor which senses

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extracellular calcium and activates diverse intracellular pathways. The objective of our

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work was to demonstrate the presence of CaSR in bovine gametes and its possible role

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in fertilization and embryo development. The location of CaSR was demonstrated by

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immunofluorescence in bovine gametes; additionally bovine sperm were incubated with

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5, 10 and 15 µM of the specific CaSR inhibitor NPS2143 in a Tyrode's Albumin Lactate

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Pyruvate medium (4 h). Sperm viability was maintained for all concentrations tested

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while total motility decreased significantly at 10 and 15 µM. Addition of 15 µM of

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NPS2143 during oocyte in vitro maturation did not alter the maturation rate. When

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NPS2143 (15 µM) was added to the fertilization medium during sperm-oocyte co-

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incubation the cleavage, morula and blastocyst rates remained unchanged. To confirm if

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15 µM of NPS2143 exerted any effect on embryo development, NPS2143 was added to

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the embryo culture medium. Cleavage rates remained unchanged when 15 µM of

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NPS2143 was added to the culture medium (79.1 ± 6.8 vs. 73.7 ± 5.3; mean % ± SEM;

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p > 0.05, control vs. inhibitor). By contrast, development to the morula (46.6 ± 7.3 vs.

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24.3 ± 4.3; mean % ± SEM; p < 0.05) and blastocyst stages (29.9 ± 9.0 vs. 9.9 ± 3.6;

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mean % ± SEM; p < 0.05) decreased (control vs. inhibitor). Our results demonstrate a

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key role of CaSR on sperm motility and embryo development but not on oocyte

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maturation or fertilization in the bovine species.

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Keywords: IVF, sperm, oocyte, bovine, CaSR, development

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1. Introduction

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ACCEPTED MANUSCRIPT 51 The fertilization process is constituted by an intricate series of well-orchestrated events

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that lead to the formation of a blastocyst, eventually resulting in viable offspring.

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Although in vitro fertilization (IVF) can be achieved in the laboratory and is routinely

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used as a source of commercially transferable embryos in many livestock species

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including bovine [1-3], the extracellular signals involved in the process are still under

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investigation. Bovine oocytes used for IVF can be retrieved from live animals using

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ovum pick up or harvested directly from the ovaries post-mortem. When oocytes are

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retrieved from post-mortem ovaries, they are arrested in profase I of meiosis, and have

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to be subjected to in vitro maturation (IVM) to reach the metaphase II stage (MII) prior

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to fertilization [4]. On the other hand, ejaculated sperm have to acquire their fertilizing

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capacity in a process known as capacitation [5,6]. Capacitated sperm and MII oocytes

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are co-incubated and several events occur: the sperm crosses the zona pellucida (ZP)

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triggering the blockage of the ZP [7], the zygote begins to form and, after many cell

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divisions, embryo formation takes place [8]. Although the intracellular signaling

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involved in the embryo formation are regulated by many extracellular molecules and

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ions, calcium plays a major role regulating oocyte maturation and activation [9,10],

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sperm capacitation [11], and embryo cleavage [12]. However, it is not just the presence

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or absence of calcium but also its extracellular and intracellular concentrations that play

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a crucial role during these events [13-16]. CaSR is a G-protein coupled receptor, that

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has been described in many different somatic cell types including bovine parathyroid

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glands, and is capable to detect and transduce subtle changes in extracellular calcium

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[17,18]. CaSR has been shown to play a key role in numerous somatic intracellular

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pathways [19]. In addition, CaSR has also been studied in germ cells [20] showing a

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key role in porcine and equine oocyte maturation [16,21] and in porcine and rat sperm

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ACCEPTED MANUSCRIPT motility [22]. Recently, CaSR has been described to play a pivotal role regulating

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capacitation and motility in stallion sperm [23]. Despite its central role in many

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fertilization-related events, no reports have been published in bovine gametes, and the

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role that CaSR may play in sperm motility, oocyte maturation and subsequent

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fertilization remains unknown. Thus, the present study was designed to investigate the

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role of CaSR during oocyte maturation, fertilization and embryo development in cattle,

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which included key fertilization-related events such as meiosis resumption and

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regulation of sperm motility.

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2. Materials and methods

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2.1. Materials

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All reagents were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA) unless

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otherwise stated.

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2.2. Semen collection and processing

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Semen from four Holstein bulls was purchased from a commercial bull station (Centro

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de Recolhas de Vendas Novas, Portugal). Frozen straws (0.25 mL) were thawed at 37ºC

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for 1 min and semen from each bull was evaluated individually; for all bulls post-thaw

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total sperm motility ranged between 35-50%. Thawed sperm was resuspended in 5 mL

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of Tyrode's albumin lactate pyruvate (TALP) medium (fertilization medium), consisting

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of 114 mM NaCl, 3.2 mM KCl, 0.5 mM MgCl2·6H2O, 10 mM sodium lactate, 25 mM

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NaHCO3, 0.34 mM NaH2PO4·H2O, 1.0 mM pyruvic acid, 2 mM CaCl2·2H2O, 6 mg/mL

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ACCEPTED MANUSCRIPT of BSA, 1 µL/ mL phenol red, penicillin–streptomycin (10 U/mL of penicillin and 10

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µg/mL of streptomycin) and 10 µg/mL heparin. Diluted semen was centrifuged at 200 ×

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g for 5 min and the resulting pellet was resuspended in TALP medium at 10 × 106

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sperm/mL for IVF or at 50 × 106 sperm/mL for motility and viability experiments.

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Medium was incubated at least 3 hours at 38.5ºC in a 5% CO2/95% air incubator before

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the beginning of the experiment. Medium was set to an osmolality of 290-300

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mOsm/Kg and adjusted at pH 7.4.

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Immunofluorescence for detection of CaSR was performed as previously reported [23].

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In brief, bovine thawed sperm (n = 3, each from a different bull) were fixed with

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methanol at -20ºC for 5 min at room temperature. On the other hand, the zona pellucida

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(ZP) was removed using 0.1 % pronase (wt/vol) and oocytes were fixed with 4%

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formaldehyde (v:v) in PBS added with 0.2% of polyvinylalcohol (PBS+PVA; wt/vol)

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overnigth at 4ºC. The next morning, the oocytes (n = 24, 7 replicates) were

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permeabilized with 0.2% Triton X-100 (v:v) in PBS for 30 min at room temperature.

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Gametes were blocked for 1 hour with 10% fetal bovine serum (FBS; Thermo Scientific

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HyClone, Logan, UT, USA) in PBS, and incubated overnight at 4ºC with anti-goat-

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CaSR antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted 1:20 in

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PBS supplemented with 1.5% FBS (PBS+FBS; v:v). The next morning, gametes were

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incubated with an anti-goat IgG (FITC)-conjugated secondary antibody (Santa Cruz

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Biotechnology Inc., Santa Cruz, CA, USA) diluted 1:50 in PBS+FBS. After washing in

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PBS+PVA the samples were mounted on a slide using glycerol and evaluated using an

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Olympus BX41 fluorescence microscope equipped with × 100 objective (New Hyde

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Park, NY).

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2.4. Sperm viability

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To determine the percentage of live cells the supravital eosin-nigrosin stain was used.

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Ten microliters of bovine sperm were mixed with an equivalent volume of the stain, and

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the mix was smeared on a pre-heated slide at 37ºC. The samples were air dried and

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examined using a light microscope (magnification × 100). One hundred sperm were

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counted per sample. Sperm excluding the nigrosin-eosin stain were considered alive,

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while sperm showing a “pinkish” colour were counted as dead. One sample per bull was

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evaluated (n = 4).

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2.5. Motility analysis

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Sperm motility was analyzed by a computer-assisted sperm analysis (CASA) system

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(ISAS 1.0.6; Proiser S.L., Valencia, Spain). An aliquot (6 µL) of each sample was

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placed in a pre-heated (37°C) motility chamber with a fixed height of 20 µm (Proiser

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S.L., Valencia, Spain). A minimum of three microscopic fields and 300 total sperm

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were evaluated at 25 frames per second. The parameter assessed was percent of total

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motility (TM); sperm with an average path velocity (VAP) less than 10 µm/s were

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considered immotile. One sample per bull was evaluated (n = 4).

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2.6. In vitro fertilization

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ACCEPTED MANUSCRIPT Bovine ovaries were collected at a nearby abattoir and were maintained in saline

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solution (0.9% NaCl) during transport (1 h total). Upon arrival at the laboratory, the

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ovaries were thoroughly rinsed with PBS at 37ºC. Cumulus oocyte complexes (COCs)

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were aspirated from 2 to 8 mm-diameter follicles using a 10 mL plastic syringe attached

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to a 20-ga hypodermic needle. Oocytes with three or more layers of compact cumulus

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cells were washed in tissue culture medium 199 (TCM-199) and transferred to a 4-well

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Nunc® plate added with 500 µL of maturation medium under mineral oil and incubated

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at 38.5ºC for 24 h in a 5% CO2/95% air incubator. The maturation medium consisted of

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TCM-199 (M2520) supplemented with 25 mM bicarbonate, 10% fetal bovine serum, 10

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mU/mL of follicle-stimulating hormone (FSH; Life Technologies Corporation), 10

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mU/mL of luteinizing hormone (LH; Life Technologies Corporation), and penicillin–

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streptomycin (10 U/mL of penicillin and 10 µg/mL of streptomycin). After oocyte

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maturation, COCs were washed in fertilization medium and transferred to a 90 µL

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droplet of fertilization medium under mineral oil. Ten microliters of thawed sperm were

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added to the fertilization droplet to reach a final sperm concentration of 1 x 106

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sperm/mL. Gametes were co-incubated for 18 h at 38.5°C in a 5% CO2/95% air

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atmosphere. Presumptive zygotes from each group were washed in TCM-199

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supplemented with 25 mM bicarbonate, penicillin–streptomycin (10 U/mL of penicillin

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and 10 µg/mL of streptomycin) and 10% FBS (culture medium) and transferred to 500

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µL of the same medium in a 4-well Nunc® plate and covered with mineral oil.

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Incubation of presumptive zygotes was performed in a humidified atmosphere at 38.5°C

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in a 5% CO2/95% air incubator. Embryos from IVF experiments were evaluated by

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stereomicroscope on day 2, 6 and 7 post-insemination for cleavage and development to

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the morula and blastocyst stages.

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2.7. DNA evaluation

176 After oocyte maturation, DNA evaluation for determination of maturational stage was

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performed as previously reported [24]. Briefly, oocytes were denuded, and fixed in 4%

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formaldehyde in PBS supplemented with 0.2% PVA. Then, the oocytes (n = 74, 6

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replicates for control and n = 78, 6 replicate for NPS2143 15µM) were washed in

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PBS+PVA and stained with Hoechst 33342 at 2.5 µg/mL at 37°C for 10 min in the dark.

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Oocytes were mounted on slides using glycerol and the DNA integrity and maturational

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stage were assessed using an Olympus BX41 fluorescence microscope (New Hyde

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Park, NY, USA) equipped with × 40 and × 100 objectives. Oocytes were considered in

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germinal vesicle stage (GV) when highly condensed chromatin or fibrillar chromatin

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was observed; oocytes with chromatids in a circular array conforming a metaphase plate

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were considered in metaphase I (MI), oocytes with a MI plate and a first polar body

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were considered in metaphase II (MII). The oocytes were considered as degenerated

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when no DNA or abnormal DNA conformations were found.

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2.8. Statistical Analysis

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The proportions of oocytes showing different chromatin configurations were compared

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among groups by Chi-square test with the Yates correction for continuity. The Fisher’s

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Exact Test was used when a value of less than 5 was expected in any treatment. The

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Kruskal-Wallis one-way analysis of variance by ranks following by Dunnett's pos-hoc

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test was used to compare sperm motility parameters and viability. When two treatments

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were compared, a t-test was used for normal data or a Mann-Whitney U-Test when

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normality test failed. Statistical significance was set at p < 0.05. Analyses were

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performed using SigmaPlot ver. 12.0 for windows (Systat Sofrware, Chicago, IL).

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3.1. Identification and distribution of CaSR in bull sperm

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The immunofluorescence assays demonstrated that CaSR is located in the acrosomal

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region and in the connecting piece of the sperm (Figure 1). In the female gametes,

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fluorescence was distributed throughout the oocyte’s cytoplasm in a diffuse pattern

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(Figure 2).

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3.2. Effect of NPS2143 inhibitor on motility and viability in bull sperm

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To explore the role of CaSR on sperm motility we used the specific inhibitor NPS2143

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at different concentrations (5, 10 and 15 µM), as it has been used in equine sperm [23].

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This inhibitor decreased the percentage of total motile sperm in a dose-dependent

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manner (Figure 3). However, only the 10 µM (5.2 ± 0.8; mean % ± SEM) and 15 µM

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(1.5 ± 0.7; mean % ± SEM) concentrations of NPS2143 demonstrated to significantly

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decrease sperm motility compared to control (12.9 ± 1.5; mean % ± SEM; p < 0.05).

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None of the dosages of NPS2143 exerted a toxic effect, as sperm viability remained

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unchanged between treatments (Figure 4).

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3.3. Effect of NPS2143 inhibitor on bovine oocyte in vitro maturation (IVM)

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oocytes from profase I arrest. Bovine oocytes were matured in vitro in the presence or

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absence of 15 µM NPS2143. After 24 h, the oocytes were denuded, fixed and stained

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with Hoechst 33342 for chromatin configuration assessment. The addition of NPS2143

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did not affect the percentage of oocytes reaching MII after in vitro maturation (72.9%

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vs. 78.2%; p > 0.05) (control vs. inhibitor respectively; Table 1).

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3.4. Effect of NPS2143 inhibitor on in vitro fertilization and embryo development

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Finally, we explored the role of CaSR in the fertilization process, cleavage and

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subsequent embryo development. For that purpose two experiments were run: 1) the

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sperm-oocyte co-incubation was performed in the presence or absence of 15 µM of

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NPS2143 in the fertilization medium for 18 hours and the presumptive zygotes were

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transferred to an embryo culture medium devoid of NPS2143 and allowed to develop

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for 7 days; n = 87 COCs (Control) and n = 90 COCs (Inhibitor) from 11 replicates; 2)

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the sperm-oocyte co-incubation was performed in absence of NPS2143 and presumable

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zygotes after fertilization were incubated to the blastocyst stage in presence or absence

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of 15 µM of NPS2143; n = 53 COCs (Control) and n = 56 COCs (Inhibitor) from six

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replicates.

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When the sperm-oocyte co-incubation was performed in presence of 15 µM of

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NPS2143 the cleavage (83.8 ± 4.0 vs. 83.6 ± 1.8; mean % ± SEM), morula (44.3 ± 4.3

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vs. 35.8 ± 2.3; mean % ± SEM) and blastocyst rates (22.9 ± 4.5 vs. 27.0 ± 2.3; mean %

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± SEM; control vs. inhibitor respectively) remained unchanged (Figure 5). However, in

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experiment 2, when the NPS2143 was added to the embryo culture medium after

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fertilization, the percentages of zygotes reaching the morula (46.6 ± 7.3 vs. 24.3 ± 4.3;

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mean % ± SEM; p < 0.05) and blastocyst stages (29.9 ± 9.0 vs. 9.9 ± 3.6; mean % ±

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SEM; p < 0.05; control vs. inhibitor respectively) significantly decreased with respect to

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control group (Figure 6).

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The present work demonstrates the presence of CaSR in bovine gametes as well as its

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central role on bovine sperm motility and embryo development.

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In the first part of this study, the presence of the Calcium Sensing Receptor in bovine

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gametes was analyzed by immunofluorescence. Distribution of CaSR was observed in

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the sperm acrosomal region and in the connecting piece (Figure 1), coinciding with

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previous results in which CaSR has been demonstrated to be located in the acrosomal

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region in stallion sperm [23]. In the oocyte, the immunolocalization yielded a diffuse

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distribution pattern throughout the oocyte (Figure 2), as previously reported in human

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[25] and equine oocytes [16].

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Once the presence of the CaSR was demonstrated, its function was subsequently

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explored in male and female gametes. In bovine sperm the inhibition of CaSR by 10 and

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15 µM NPS2143 in a capacitating medium (TALP) decreased the percentages of total

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motility, likely by the inability of bull sperm to uptake extracellular calcium, without

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affecting sperm viability (Figure 3 and 4). This result is in agreement with those of

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Macias-Garcia et al. (2016) who demonstrated a decline in total motility in stallion

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sperm incubated in presence of NPS2143 at the same concentrations. However, in the

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bovine species sperm viability remains unaffected, while in equine sperm, the

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percentage of viable sperm was significantly reduced [23]. These results seem to be

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ACCEPTED MANUSCRIPT species-specific as calcium has a inhibitory effect on equine sperm capacitation [26] but

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not in bovine sperm [27].

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To study the possible implication of CaSR in bovine oocytes meiosis resumption, 15

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µM of NPS2143 was added to the IVM medium. Our results demonstrated that in the

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bovine species inhibition of CaSR by NPS2143 did not affect the meiotic competence of

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the oocytes (Table 1). In contrast, in porcine oocytes CaSR inhibition by NPS2390

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significantly decreased the percentage of oocytes that reached MII after IVM [21,21].

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Thus, in view of our data and the previously mentioned reports, the implication of CaSR

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in oocytes’ meiosis progression and in sperm viability seems to be species-specific.

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Next, we investigated if CaSR inhibition interferes with the fertilization process. For

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this purpose, 15 µM of NPS2143 was added to the TALP fertilization medium during

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sperm-oocyte co-incubation. Our results did not show a significant effect of CaSR

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inhibition on the cleavage rate or the percentages of zygotes reaching the morula and

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blastocyst stages (Figure 5). These results demonstrate that despite CaSR inhibition,

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core fertilization related processes namely capacitation, acrosome reaction,

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hyperactivation or zona pellucida penetration are not significantly affected. To the

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authors’ best knowledge, this is the first report studying the role of CaSR in the in vitro

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fertilization process of any species. Previously, it has been reported that inhibition of

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CaSR by NPS2143 in stallion sperm recovered protein tyrosine phosphorylation

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inhibited by calcium [23]. However, our results may suggest that, unlike what happens

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in the equine species, inhibition of CaSR does not seem to be interfering with the

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capacitation process in the bovine species as sperm are capable to fertilize in presence

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of NPS2143. However, specific experiments in bull sperm capacitation should be

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performed to elucidate the exact role of CaSR on this event.

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ACCEPTED MANUSCRIPT In view of our results, despite the vivid effect that NPS2143 exerts on total sperm

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motility (1.5 ± 0.7; % mean ± SEM) after 4 hours, the fertilization capacity of bovine

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sperm and the developmental competence of bovine oocytes is maintained (Figure 5).

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As oocyte fertilization is achieved within 3-6 hours of gamete co-incubation, the effect

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of NPS2143 may have not reached its maximum by the time at which oocyte

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fertilization is accomplished [28,29].

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As the inhibition of CaSR did not interfere with the fertilization process the effect that

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CaSR inhibition could induce in the development of presumable zygotes was studied.

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Our results demonstrated that CaSR inhibition did not interfere with the first division

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post-fertilization (cleavage), although the morula and blastocyst rates decreased

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significantly (Figure 6); these results suggest that CaSR is playing a central role in pre-

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implantation embryo development. In this regard, it has been described an increase in

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the CaSR expression during the implantation process in rat uterus [30], and thus, in

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view of our results, this receptor needs to be more profoundly studied during the pre-

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implantation window.

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It is well established that intracellular calcium oscillations are crucial to trigger egg

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activation, fertilization and embryo development [31] and also that CaSR activation

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induces calcium oscillations in many cell types such as human embryonic kidney cells

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(HEK) or the pancreatic duct [32]. Calcium oscillations are well known inducers of the

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Mitogen Activated Protein Kinase (MAPK) pathways, which are also essential for

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embryo development [33,34]. This pathway has been reported to be regulated by

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calcium sensing receptor in human [25] equine [16], and porcine oocytes [21]; thus

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CaSR could be modulating intracellular calcium concentrations through the MAPK

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pathway in bovine embryos as well.

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In conclusion, our results demonstrate a key role of CaSR on sperm motility and

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embryo development in bovine. Further studies are required to increase our

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understanding regarding the intracellular pathways downstream CaSR and their

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implication in bovine gamete biology and embryo development.

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325 Acknowledgements

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LG-F hold a postdoctoral grant of the Fundação para a Ciência e a Tecnologia

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(Portuguese Ministry for Science, Technology and Higher Education) co-funded by

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Programa Operacional Potencial Humano (POPH) financed by European Social Fund

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(ESF) and Portuguese national funds from Ministry for Science, Technology and Higher

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Education (Grant reference: SFRH/BPD/85532/2012). BM-G holds a postdoctoral grant

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Juan de la Cierva Incorporación (IJC-2014-19428) from the Spanish Ministry of

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Economy, Industry and Competitiveness and was also partially funded by the Fundação

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para a Ciência e a Tecnologia (Grant reference: SFRH/BPD/84354/2012).

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The authors wish to thank the Laboratory of Applied Physiology (Department of

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Aquatic Production) of the ICBAS and especially to Mariana Hinzmann for allowing us

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to use their fluorescence microscope.

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Disclosures

The authors declare no conflict of interest.

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References

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[10] Parrish JJ. Bovine in vitro fertilization: in vitro oocyte maturation and sperm capacitation with heparin. Theriogenology 2014;81:67-73. [11] Rodriguez PC, Satorre MM, Beconi MT. Effect of two intracellular calcium modulators on sperm motility and heparin-induced capacitation in cryopreserved

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bovine spermatozoa. Anim Reprod Sci 2012;131:135-142.

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2015;59:261-270.

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[12] Gallo A, Tosti E. Ion currents involved in gamete physiology. Int J Dev Biol

[13] Navarrete FA, Garcia-Vazquez FA, Alvau A, Escoffier J, Krapf D, Sanchez-

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Cardenas C, et al. Biphasic Role of Calcium in Mouse Sperm Capacitation

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Signaling Pathways. J Cell Physiol 2015;230:1758-1769.

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[14] Bhoumik A, Saha S, Majumder GC, Dungdung SR. Optimum calcium concentration: a crucial factor in regulating sperm motility in vitro. Cell Biochem

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Biophys 2014;70:1177-1183.

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[15] Chun JT, Puppo A, Vasilev F, Gragnaniello G, Garante E, Santella L. The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin

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polymerization and Ca2+ signaling. PLoS One 2010;5:e14100.

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[16] De Santis T, Casavola V, Reshkin SJ, Guerra L, Ambruosi B, Fiandanese N, et al. The extracellular calcium-sensing receptor is expressed in the cumulus-oocyte complex in mammals and modulates oocyte meiotic maturation. Reproduction 2009;138:439-452. [17] Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 2001;81:239-297.

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[18] Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, et al. Cloning

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and characterization of an extracellular Ca(2+)-sensing receptor from bovine

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parathyroid. Nature 1993;366:575-580.

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Calcium 2004;35:183-196.

[20] Ellinger I. The Calcium-Sensing Receptor and the Reproductive System. Front Physiol 2016;7:371.

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[19] Chang W, Shoback D. Extracellular Ca2+-sensing receptors--an overview. Cell

[21] Liu C, Wu GQ, Fu XW, Mo XH, Zhao LH, Hu HM, et al. The Extracellular

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Calcium-Sensing Receptor (CASR) Regulates Gonadotropins-Induced Meiotic

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Maturation of Porcine Oocytes. Biol Reprod 2015;93:131. [22] Mendoza FJ, Perez-Marin CC, Garcia-Marin L, Madueno JA, Henley C, Aguilera-

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Tejero E, et al. Localization, distribution, and function of the calcium-sensing

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receptor in sperm. J Androl 2012;33:96-104.

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[23] Macias-Garcia B, Rocha A, Gonzalez-Fernandez L. Extracellular calcium regulates protein tyrosine phosphorylation through calcium-sensing receptor

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(CaSR) in stallion sperm. Mol Reprod Dev 2016;83:236-245.

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[24] Gonzalez-Fernandez L, Macedo S, Lopes JS, Rocha A, Macias-Garcia B. Effect of Different Media and Protein Source on Equine Gametes: Potential Impact During In Vitro Fertilization. Reprod Domest Anim 2015;50:1039-1046.

[25] Dell'Aquila ME, De ST, Cho YS, Reshkin SJ, Caroli AM, Maritato F, et al.

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Localization and quantitative expression of the calcium-sensing receptor protein in

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human oocytes. Fertil Steril 2006;85 Suppl 1:1240-1247.

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[26] Gonzalez-Fernandez L, Macias-Garcia B, Velez IC, Varner DD, Hinrichs K.

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Calcium-calmodulin and pH regulate protein tyrosine phosphorylation in stallion

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sperm. Reproduction 2012;144:411-422. [27] Galantino-Homer HL, Florman HM, Storey BT, Dobrinski I, Kopf GS. Bovine

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sperm capacitation: assessment of phosphodiesterase activity and intracellular

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alkalinization on capacitation-associated protein tyrosine phosphorylation. Mol

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Reprod Dev 2004;67:487-500.

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[28] Ward F, Enright B, Rizos D, Boland M, Lonergan P. Optimization of in vitro bovine embryo production: effect of duration of maturation, length of gamete co-

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incubation, sperm concentration and sire. Theriogenology 2002;57:2105-2117.

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[29] Dode MA, Rodovalho NC, Ueno VG, Fernandes CE. The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

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Anim Reprod Sci 2002;69:15-23.

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[30] Xiao LJ, Yuan JX, Li YC, Wang R, Hu ZY, Liu YX. Extracellular Ca2+-sensing receptor expression and hormonal regulation in rat uterus during the peri-

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implantation period. Reproduction 2005;129:779-788.

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[31] Ducibella T, Fissore R. The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development. Dev Biol 2008;315:257-279.

[32] Ward DT. Calcium receptor-mediated intracellular signalling. Cell Calcium 2004;35:217-228.

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[33] Asaoka Y, Nishina H. Diverse physiological functions of MKK4 and MKK7 during early embryogenesis. J Biochem 2010;148:393-401. [34] Colella M, Gerbino A, Hofer AM, Curci S. Recent advances in understanding the extracellular calcium-sensing receptor. F1000Res 2016;5.

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Figure Legends

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Figure 1. Distribution of CaSR in bovine sperm. Thawed sperm (n = 3, each from a

441

different bull) were fixed and incubated with CaSR antibody. Fluorescence image (A)

442

bright image (B); and overlay of the two images (C) are shown. Negative control was

443

performed omitting primary antibody; fluorescence image (D) and bright image (E).

444

Micrographs were captured at × 100; bar = 20 µm.

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Figure 2. Distribution of CaSR in bovine oocytes (n = 24, 7 replicates). Representative

447

images of bovine female gametes after CaSR labeling are shown. DNA was stained in

448

blue with Hoechst 33342 (A); CaSR was visualized in green (B) and the overlay of both

449

images is shown (C). Negative controls were made omitting primary antibody; Hoescht

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33342 (A’); fluorescence image (B’) and overlay of the two images (C’). Micrographs

451

were captured at × 40; bar = 100 µm.

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Figure 3. Effect of different concentrations of NPS2143 on sperm motility. After 4 h at

454

37ºC in TALP medium in a 5% CO2/95% air atmosphere, sperm motility was analyzed

455

using a CASA system. Motility values are expressed as mean ± SEM. Four different

456

bulls were used (one ejaculate per bull; n = 4). * p < 0.05 compared to the control.

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ACCEPTED MANUSCRIPT 458

Figure 4. Effect of different concentrations of NPS2143 on bovine sperm viability.

459

After 4 h at 37ºC in TALP medium sperm motility was analyzed by eosin-negrosin

460

staining. Viability values are expressed as mean ± SEM of four bulls, one ejaculate per

461

bull (n = 4; p > 0.05).

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Figure 5. Effect of NPS2143 on oocyte fertilization. The sperm-oocyte co-incubation

464

was run in presence or absence of 15 µM of NPS2143 for 18 h and the presumptive

465

zygotes were cultured. Embryos were evaluated on day 2 (cleavage), 6 (morula) and 7

466

(blastocyst) post-insemination. Data are presented as the mean percentage ± SEM

467

(results of eleven replicates); n = 87 COCs (Control) and n = 90 COCs (Inhibitor). p >

468

0.05.

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Figure 6. Effect of NPS2143 on embryo development. After fertilization, presumable

471

zygotes were incubated in presence or absence of 15 µM of NPS2143. Embryos were

472

evaluated on day 2 (cleavage), 6 (morula) and 7 (blastocyst) post-insemination. Data are

473

presented as the mean percentage ± SEM (results of six replicates); n = 53 COCs

474

(Control) and n = 56 COCs (Inhibitor). * p < 0.05.

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Role of the Calcium-Sensing Receptor (CaSR) in bovine gametes and

484

during in vitro fertilization

485 486

Beatriz Macías-Garcíaa,b, Graça Lopesa, Antonio Rochaa, Lauro González-Fernándeza,*

488

a

489

University of Porto, Portugal

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b

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CECA/ICETA – Animal Sciences Centre; ICBAS – Abel Salazar Biomedical Institute,

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CCMIJU – Minimally Invasive Surgery Centre Jesús Usón, Cáceres, Spain

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∗ Corresponding author at: Centro de Estudos de Ciência Animal (CECA),

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Universidade do Porto, Rua Padre Armando Quintas 7, 4485-661,

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Vairão, Vila do Conde. Portugal. E-mail address: [email protected]

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ABSTRACT

509 Calcium Sensing Receptor (CaSR) is a G-protein coupled receptor which senses

511

extracellular calcium and activates diverse intracellular pathways. The objective of our

512

work was to demonstrate the presence of CaSR in bovine gametes and its possible role

513

in fertilization and embryo development. The location of CaSR was demonstrated by

514

immunofluorescence in bovine gametes; additionally bovine sperm were incubated with

515

5, 10 and 15 µM of the specific CaSR inhibitor NPS2143 in a Tyrode's Albumin Lactate

516

Pyruvate medium (4 h). Sperm viability was maintained for all concentrations tested

517

while total motility decreased significantly at 10 and 15 µM. Addition of 15 µM of

518

NPS2143 during oocyte in vitro maturation did not alter the maturation rate. When

519

NPS2143 (15 µM) was added to the fertilization medium during sperm-oocyte co-

520

incubation the cleavage, morula and blastocyst rates remained unchanged. To confirm if

521

15 µM of NPS2143 exerted any effect on embryo development, NPS2143 was added to

522

the embryo culture medium. Cleavage rates remained unchanged when 15 µM of

523

NPS2143 was added to the culture medium (79.1 ± 6.8 vs. 73.7 ± 5.3; mean % ± SEM;

524

p > 0.05, control vs. inhibitor). By contrast, development to the morula (46.6 ± 7.3 vs.

525

24.3 ± 4.3; mean % ± SEM; p < 0.05) and blastocyst stages (29.9 ± 9.0 vs. 9.9 ± 3.6;

526

mean % ± SEM; p < 0.05) decreased (control vs. inhibitor). Our results demonstrate a

527

key role of CaSR on sperm motility and embryo development but not on oocyte

528

maturation or fertilization in the bovine species.

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Keywords: IVF, sperm, oocyte, bovine, CaSR, development

531 532

1. Introduction

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ACCEPTED MANUSCRIPT 533 The fertilization process is constituted by an intricate series of well-orchestrated events

535

that lead to the formation of a blastocyst, eventually resulting in viable offspring.

536

Although in vitro fertilization (IVF) can be achieved in the laboratory and is routinely

537

used as a source of commercially transferable embryos in many livestock species

538

including bovine [1-3], the extracellular signals involved in the process are still under

539

investigation. Bovine oocytes used for IVF can be retrieved from live animals using

540

ovum pick up or harvested directly from the ovaries post-mortem. When oocytes are

541

retrieved from post-mortem ovaries, they are arrested in profase I of meiosis, and have

542

to be subjected to in vitro maturation (IVM) to reach the metaphase II stage (MII) prior

543

to fertilization [4]. On the other hand, ejaculated sperm have to acquire their fertilizing

544

capacity in a process known as capacitation [5,6]. Capacitated sperm and MII oocytes

545

are co-incubated and several events occur: the sperm crosses the zona pellucida (ZP)

546

triggering the blockage of the ZP [7], the zygote begins to form and, after many cell

547

divisions, embryo formation takes place [8]. Although the intracellular signaling

548

involved in the embryo formation are regulated by many extracellular molecules and

549

ions, calcium plays a major role regulating oocyte maturation and activation [9,10],

550

sperm capacitation [11], and embryo cleavage [12]. However, it is not just the presence

551

or absence of calcium but also its extracellular and intracellular concentrations that play

552

a crucial role during these events [13-16]. CaSR is a G-protein coupled receptor, that

553

has been described in many different somatic cell types including bovine parathyroid

554

glands, and is capable to detect and transduce subtle changes in extracellular calcium

555

[17,18]. CaSR has been shown to play a key role in numerous somatic intracellular

556

pathways [19]. In addition, CaSR has also been studied in germ cells [20] showing a

557

key role in porcine and equine oocyte maturation [16,21] and in porcine and rat sperm

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ACCEPTED MANUSCRIPT motility [22]. Recently, CaSR has been described to play a pivotal role regulating

559

capacitation and motility in stallion sperm [23]. Despite its central role in many

560

fertilization-related events, no reports have been published in bovine gametes, and the

561

role that CaSR may play in sperm motility, oocyte maturation and subsequent

562

fertilization remains unknown. Thus, the present study was designed to investigate the

563

role of CaSR during oocyte maturation, fertilization and embryo development in cattle,

564

which included key fertilization-related events such as meiosis resumption and

565

regulation of sperm motility.

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2. Materials and methods

568 569

2.1. Materials

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All reagents were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA) unless

572

otherwise stated.

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2.2. Semen collection and processing

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Semen from four Holstein bulls was purchased from a commercial bull station (Centro

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de Recolhas de Vendas Novas, Portugal). Frozen straws (0.25 mL) were thawed at 37ºC

578

for 1 min and semen from each bull was evaluated individually; for all bulls post-thaw

579

total sperm motility ranged between 35-50%. Thawed sperm was resuspended in 5 mL

580

of Tyrode's albumin lactate pyruvate (TALP) medium (fertilization medium), consisting

581

of 114 mM NaCl, 3.2 mM KCl, 0.5 mM MgCl2·6H2O, 10 mM sodium lactate, 25 mM

582

NaHCO3, 0.34 mM NaH2PO4·H2O, 1.0 mM pyruvic acid, 2 mM CaCl2·2H2O, 6 mg/mL

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ACCEPTED MANUSCRIPT of BSA, 1 µL/ mL phenol red, penicillin–streptomycin (10 U/mL of penicillin and 10

584

µg/mL of streptomycin) and 10 µg/mL heparin. Diluted semen was centrifuged at 200 ×

585

g for 5 min and the resulting pellet was resuspended in TALP medium at 10 × 106

586

sperm/mL for IVF or at 50 × 106 sperm/mL for motility and viability experiments.

587

Medium was incubated at least 3 hours at 38.5ºC in a 5% CO2/95% air incubator before

588

the beginning of the experiment. Medium was set to an osmolality of 290-300

589

mOsm/Kg and adjusted at pH 7.4.

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590 2.3. Immunofluorescence

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Immunofluorescence for detection of CaSR was performed as previously reported [23].

594

In brief, bovine thawed sperm (n = 3, each from a different bull) were fixed with

595

methanol at -20ºC for 5 min at room temperature. On the other hand, the zona pellucida

596

(ZP) was removed using 0.1 % pronase (wt/vol) and oocytes were fixed with 4%

597

formaldehyde (v:v) in PBS added with 0.2% of polyvinylalcohol (PBS+PVA; wt/vol)

598

overnigth at 4ºC. The next morning, the oocytes (n = 24, 7 replicates) were

599

permeabilized with 0.2% Triton X-100 (v:v) in PBS for 30 min at room temperature.

600

Gametes were blocked for 1 hour with 10% fetal bovine serum (FBS; Thermo Scientific

601

HyClone, Logan, UT, USA) in PBS, and incubated overnight at 4ºC with anti-goat-

602

CaSR antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted 1:20 in

603

PBS supplemented with 1.5% FBS (PBS+FBS; v:v). The next morning, gametes were

604

incubated with an anti-goat IgG (FITC)-conjugated secondary antibody (Santa Cruz

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Biotechnology Inc., Santa Cruz, CA, USA) diluted 1:50 in PBS+FBS. After washing in

606

PBS+PVA the samples were mounted on a slide using glycerol and evaluated using an

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Olympus BX41 fluorescence microscope equipped with × 100 objective (New Hyde

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Park, NY).

609 610

2.4. Sperm viability

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To determine the percentage of live cells the supravital eosin-nigrosin stain was used.

613

Ten microliters of bovine sperm were mixed with an equivalent volume of the stain, and

614

the mix was smeared on a pre-heated slide at 37ºC. The samples were air dried and

615

examined using a light microscope (magnification × 100). One hundred sperm were

616

counted per sample. Sperm excluding the nigrosin-eosin stain were considered alive,

617

while sperm showing a “pinkish” colour were counted as dead. One sample per bull was

618

evaluated (n = 4).

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2.5. Motility analysis

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Sperm motility was analyzed by a computer-assisted sperm analysis (CASA) system

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(ISAS 1.0.6; Proiser S.L., Valencia, Spain). An aliquot (6 µL) of each sample was

624

placed in a pre-heated (37°C) motility chamber with a fixed height of 20 µm (Proiser

625

S.L., Valencia, Spain). A minimum of three microscopic fields and 300 total sperm

626

were evaluated at 25 frames per second. The parameter assessed was percent of total

627

motility (TM); sperm with an average path velocity (VAP) less than 10 µm/s were

628

considered immotile. One sample per bull was evaluated (n = 4).

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2.6. In vitro fertilization

631

26

ACCEPTED MANUSCRIPT Bovine ovaries were collected at a nearby abattoir and were maintained in saline

633

solution (0.9% NaCl) during transport (1 h total). Upon arrival at the laboratory, the

634

ovaries were thoroughly rinsed with PBS at 37ºC. Cumulus oocyte complexes (COCs)

635

were aspirated from 2 to 8 mm-diameter follicles using a 10 mL plastic syringe attached

636

to a 20-ga hypodermic needle. Oocytes with three or more layers of compact cumulus

637

cells were washed in tissue culture medium 199 (TCM-199) and transferred to a 4-well

638

Nunc® plate added with 500 µL of maturation medium under mineral oil and incubated

639

at 38.5ºC for 24 h in a 5% CO2/95% air incubator. The maturation medium consisted of

640

TCM-199 (M2520) supplemented with 25 mM bicarbonate, 10% fetal bovine serum, 10

641

mU/mL of follicle-stimulating hormone (FSH; Life Technologies Corporation), 10

642

mU/mL of luteinizing hormone (LH; Life Technologies Corporation), and penicillin–

643

streptomycin (10 U/mL of penicillin and 10 µg/mL of streptomycin). After oocyte

644

maturation, COCs were washed in fertilization medium and transferred to a 90 µL

645

droplet of fertilization medium under mineral oil. Ten microliters of thawed sperm were

646

added to the fertilization droplet to reach a final sperm concentration of 1 x 106

647

sperm/mL. Gametes were co-incubated for 18 h at 38.5°C in a 5% CO2/95% air

648

atmosphere. Presumptive zygotes from each group were washed in TCM-199

649

supplemented with 25 mM bicarbonate, penicillin–streptomycin (10 U/mL of penicillin

650

and 10 µg/mL of streptomycin) and 10% FBS (culture medium) and transferred to 500

651

µL of the same medium in a 4-well Nunc® plate and covered with mineral oil.

652

Incubation of presumptive zygotes was performed in a humidified atmosphere at 38.5°C

653

in a 5% CO2/95% air incubator. Embryos from IVF experiments were evaluated by

654

stereomicroscope on day 2, 6 and 7 post-insemination for cleavage and development to

655

the morula and blastocyst stages.

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ACCEPTED MANUSCRIPT 657

2.7. DNA evaluation

658 After oocyte maturation, DNA evaluation for determination of maturational stage was

660

performed as previously reported [24]. Briefly, oocytes were denuded, and fixed in 4%

661

formaldehyde in PBS supplemented with 0.2% PVA. Then, the oocytes (n = 74, 6

662

replicates for control and n = 78, 6 replicate for NPS2143 15µM) were washed in

663

PBS+PVA and stained with Hoechst 33342 at 2.5 µg/mL at 37°C for 10 min in the dark.

664

Oocytes were mounted on slides using glycerol and the DNA integrity and maturational

665

stage were assessed using an Olympus BX41 fluorescence microscope (New Hyde

666

Park, NY, USA) equipped with × 40 and × 100 objectives. Oocytes were considered in

667

germinal vesicle stage (GV) when highly condensed chromatin or fibrillar chromatin

668

was observed; oocytes with chromatids in a circular array conforming a metaphase plate

669

were considered in metaphase I (MI), oocytes with a MI plate and a first polar body

670

were considered in metaphase II (MII). The oocytes were considered as degenerated

671

when no DNA or abnormal DNA conformations were found.

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2.8. Statistical Analysis

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The proportions of oocytes showing different chromatin configurations were compared

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among groups by Chi-square test with the Yates correction for continuity. The Fisher’s

677

Exact Test was used when a value of less than 5 was expected in any treatment. The

678

Kruskal-Wallis one-way analysis of variance by ranks following by Dunnett's pos-hoc

679

test was used to compare sperm motility parameters and viability. When two treatments

680

were compared, a t-test was used for normal data or a Mann-Whitney U-Test when

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ACCEPTED MANUSCRIPT 681

normality test failed. Statistical significance was set at p < 0.05. Analyses were

682

performed using SigmaPlot ver. 12.0 for windows (Systat Sofrware, Chicago, IL).

683 3. Results

685 686

3.1. Identification and distribution of CaSR in bull sperm

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The immunofluorescence assays demonstrated that CaSR is located in the acrosomal

689

region and in the connecting piece of the sperm (Figure 1). In the female gametes,

690

fluorescence was distributed throughout the oocyte’s cytoplasm in a diffuse pattern

691

(Figure 2).

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3.2. Effect of NPS2143 inhibitor on motility and viability in bull sperm

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To explore the role of CaSR on sperm motility we used the specific inhibitor NPS2143

696

at different concentrations (5, 10 and 15 µM), as it has been used in equine sperm [23].

697

This inhibitor decreased the percentage of total motile sperm in a dose-dependent

698

manner (Figure 3). However, only the 10 µM (5.2 ± 0.8; mean % ± SEM) and 15 µM

699

(1.5 ± 0.7; mean % ± SEM) concentrations of NPS2143 demonstrated to significantly

700

decrease sperm motility compared to control (12.9 ± 1.5; mean % ± SEM; p < 0.05).

701

None of the dosages of NPS2143 exerted a toxic effect, as sperm viability remained

702

unchanged between treatments (Figure 4).

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3.3. Effect of NPS2143 inhibitor on bovine oocyte in vitro maturation (IVM)

705

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ACCEPTED MANUSCRIPT Next, we studied the effect of CaSR inhibition by NPS2143 on alleviation of bovine

707

oocytes from profase I arrest. Bovine oocytes were matured in vitro in the presence or

708

absence of 15 µM NPS2143. After 24 h, the oocytes were denuded, fixed and stained

709

with Hoechst 33342 for chromatin configuration assessment. The addition of NPS2143

710

did not affect the percentage of oocytes reaching MII after in vitro maturation (72.9%

711

vs. 78.2%; p > 0.05) (control vs. inhibitor respectively; Table 1).

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3.4. Effect of NPS2143 inhibitor on in vitro fertilization and embryo development

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Finally, we explored the role of CaSR in the fertilization process, cleavage and

716

subsequent embryo development. For that purpose two experiments were run: 1) the

717

sperm-oocyte co-incubation was performed in the presence or absence of 15 µM of

718

NPS2143 in the fertilization medium for 18 hours and the presumptive zygotes were

719

transferred to an embryo culture medium devoid of NPS2143 and allowed to develop

720

for 7 days; n = 87 COCs (Control) and n = 90 COCs (Inhibitor) from 11 replicates; 2)

721

the sperm-oocyte co-incubation was performed in absence of NPS2143 and presumable

722

zygotes after fertilization were incubated to the blastocyst stage in presence or absence

723

of 15 µM of NPS2143; n = 53 COCs (Control) and n = 56 COCs (Inhibitor) from six

724

replicates.

725

When the sperm-oocyte co-incubation was performed in presence of 15 µM of

726

NPS2143 the cleavage (83.8 ± 4.0 vs. 83.6 ± 1.8; mean % ± SEM), morula (44.3 ± 4.3

727

vs. 35.8 ± 2.3; mean % ± SEM) and blastocyst rates (22.9 ± 4.5 vs. 27.0 ± 2.3; mean %

728

± SEM; control vs. inhibitor respectively) remained unchanged (Figure 5). However, in

729

experiment 2, when the NPS2143 was added to the embryo culture medium after

730

fertilization, the percentages of zygotes reaching the morula (46.6 ± 7.3 vs. 24.3 ± 4.3;

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ACCEPTED MANUSCRIPT 731

mean % ± SEM; p < 0.05) and blastocyst stages (29.9 ± 9.0 vs. 9.9 ± 3.6; mean % ±

732

SEM; p < 0.05; control vs. inhibitor respectively) significantly decreased with respect to

733

control group (Figure 6).

734 4. Discussion

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The present work demonstrates the presence of CaSR in bovine gametes as well as its

738

central role on bovine sperm motility and embryo development.

739

In the first part of this study, the presence of the Calcium Sensing Receptor in bovine

740

gametes was analyzed by immunofluorescence. Distribution of CaSR was observed in

741

the sperm acrosomal region and in the connecting piece (Figure 1), coinciding with

742

previous results in which CaSR has been demonstrated to be located in the acrosomal

743

region in stallion sperm [23]. In the oocyte, the immunolocalization yielded a diffuse

744

distribution pattern throughout the oocyte (Figure 2), as previously reported in human

745

[25] and equine oocytes [16].

746

Once the presence of the CaSR was demonstrated, its function was subsequently

747

explored in male and female gametes. In bovine sperm the inhibition of CaSR by 10 and

748

15 µM NPS2143 in a capacitating medium (TALP) decreased the percentages of total

749

motility, likely by the inability of bull sperm to uptake extracellular calcium, without

750

affecting sperm viability (Figure 3 and 4). This result is in agreement with those of

751

Macias-Garcia et al. (2016) who demonstrated a decline in total motility in stallion

752

sperm incubated in presence of NPS2143 at the same concentrations. However, in the

753

bovine species sperm viability remains unaffected, while in equine sperm, the

754

percentage of viable sperm was significantly reduced [23]. These results seem to be

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ACCEPTED MANUSCRIPT species-specific as calcium has a inhibitory effect on equine sperm capacitation [26] but

756

not in bovine sperm [27].

757

To study the possible implication of CaSR in bovine oocytes meiosis resumption, 15

758

µM of NPS2143 was added to the IVM medium. Our results demonstrated that in the

759

bovine species inhibition of CaSR by NPS2143 did not affect the meiotic competence of

760

the oocytes (Table 1). In contrast, in porcine oocytes CaSR inhibition by NPS2390

761

significantly decreased the percentage of oocytes that reached MII after IVM [21,21].

762

Thus, in view of our data and the previously mentioned reports, the implication of CaSR

763

in oocytes’ meiosis progression and in sperm viability seems to be species-specific.

764

Next, we investigated if CaSR inhibition interferes with the fertilization process. For

765

this purpose, 15 µM of NPS2143 was added to the TALP fertilization medium during

766

sperm-oocyte co-incubation. Our results did not show a significant effect of CaSR

767

inhibition on the cleavage rate or the percentages of zygotes reaching the morula and

768

blastocyst stages (Figure 5). These results demonstrate that despite CaSR inhibition,

769

core fertilization related processes namely capacitation, acrosome reaction,

770

hyperactivation or zona pellucida penetration are not significantly affected. To the

771

authors’ best knowledge, this is the first report studying the role of CaSR in the in vitro

772

fertilization process of any species. Previously, it has been reported that inhibition of

773

CaSR by NPS2143 in stallion sperm recovered protein tyrosine phosphorylation

774

inhibited by calcium [23]. However, our results may suggest that, unlike what happens

775

in the equine species, inhibition of CaSR does not seem to be interfering with the

776

capacitation process in the bovine species as sperm are capable to fertilize in presence

777

of NPS2143. However, specific experiments in bull sperm capacitation should be

778

performed to elucidate the exact role of CaSR on this event.

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ACCEPTED MANUSCRIPT In view of our results, despite the vivid effect that NPS2143 exerts on total sperm

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motility (1.5 ± 0.7; % mean ± SEM) after 4 hours, the fertilization capacity of bovine

781

sperm and the developmental competence of bovine oocytes is maintained (Figure 5).

782

As oocyte fertilization is achieved within 3-6 hours of gamete co-incubation, the effect

783

of NPS2143 may have not reached its maximum by the time at which oocyte

784

fertilization is accomplished [28,29].

785

As the inhibition of CaSR did not interfere with the fertilization process the effect that

786

CaSR inhibition could induce in the development of presumable zygotes was studied.

787

Our results demonstrated that CaSR inhibition did not interfere with the first division

788

post-fertilization (cleavage), although the morula and blastocyst rates decreased

789

significantly (Figure 6); these results suggest that CaSR is playing a central role in pre-

790

implantation embryo development. In this regard, it has been described an increase in

791

the CaSR expression during the implantation process in rat uterus [30], and thus, in

792

view of our results, this receptor needs to be more profoundly studied during the pre-

793

implantation window.

794

It is well established that intracellular calcium oscillations are crucial to trigger egg

795

activation, fertilization and embryo development [31] and also that CaSR activation

796

induces calcium oscillations in many cell types such as human embryonic kidney cells

797

(HEK) or the pancreatic duct [32]. Calcium oscillations are well known inducers of the

798

Mitogen Activated Protein Kinase (MAPK) pathways, which are also essential for

799

embryo development [33,34]. This pathway has been reported to be regulated by

800

calcium sensing receptor in human [25] equine [16], and porcine oocytes [21]; thus

801

CaSR could be modulating intracellular calcium concentrations through the MAPK

802

pathway in bovine embryos as well.

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In conclusion, our results demonstrate a key role of CaSR on sperm motility and

804

embryo development in bovine. Further studies are required to increase our

805

understanding regarding the intracellular pathways downstream CaSR and their

806

implication in bovine gamete biology and embryo development.

808

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807 Acknowledgements

809

LG-F hold a postdoctoral grant of the Fundação para a Ciência e a Tecnologia

811

(Portuguese Ministry for Science, Technology and Higher Education) co-funded by

812

Programa Operacional Potencial Humano (POPH) financed by European Social Fund

813

(ESF) and Portuguese national funds from Ministry for Science, Technology and Higher

814

Education (Grant reference: SFRH/BPD/85532/2012). BM-G holds a postdoctoral grant

815

Juan de la Cierva Incorporación (IJC-2014-19428) from the Spanish Ministry of

816

Economy, Industry and Competitiveness and was also partially funded by the Fundação

817

para a Ciência e a Tecnologia (Grant reference: SFRH/BPD/84354/2012).

818

The authors wish to thank the Laboratory of Applied Physiology (Department of

819

Aquatic Production) of the ICBAS and especially to Mariana Hinzmann for allowing us

820

to use their fluorescence microscope.

822 823 824

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Disclosures

The authors declare no conflict of interest.

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References

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[3] Paramio MT, Izquierdo D. Current status of in vitro embryo production in sheep

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[4] Downs SM. Nutrient pathways regulating the nuclear maturation of mammalian oocytes. Reprod Fertil Dev 2015;27:572-582.

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[18] Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, et al. Cloning

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and characterization of an extracellular Ca(2+)-sensing receptor from bovine

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parathyroid. Nature 1993;366:575-580.

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Calcium-Sensing Receptor (CASR) Regulates Gonadotropins-Induced Meiotic

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Maturation of Porcine Oocytes. Biol Reprod 2015;93:131. [22] Mendoza FJ, Perez-Marin CC, Garcia-Marin L, Madueno JA, Henley C, Aguilera-

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Tejero E, et al. Localization, distribution, and function of the calcium-sensing

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receptor in sperm. J Androl 2012;33:96-104.

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(CaSR) in stallion sperm. Mol Reprod Dev 2016;83:236-245.

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[25] Dell'Aquila ME, De ST, Cho YS, Reshkin SJ, Caroli AM, Maritato F, et al.

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Localization and quantitative expression of the calcium-sensing receptor protein in

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human oocytes. Fertil Steril 2006;85 Suppl 1:1240-1247.

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[26] Gonzalez-Fernandez L, Macias-Garcia B, Velez IC, Varner DD, Hinrichs K.

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Calcium-calmodulin and pH regulate protein tyrosine phosphorylation in stallion

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sperm. Reproduction 2012;144:411-422. [27] Galantino-Homer HL, Florman HM, Storey BT, Dobrinski I, Kopf GS. Bovine

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incubation, sperm concentration and sire. Theriogenology 2002;57:2105-2117.

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[29] Dode MA, Rodovalho NC, Ueno VG, Fernandes CE. The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

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Anim Reprod Sci 2002;69:15-23.

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[30] Xiao LJ, Yuan JX, Li YC, Wang R, Hu ZY, Liu YX. Extracellular Ca2+-sensing receptor expression and hormonal regulation in rat uterus during the peri-

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implantation period. Reproduction 2005;129:779-788.

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[31] Ducibella T, Fissore R. The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development. Dev Biol 2008;315:257-279.

[32] Ward DT. Calcium receptor-mediated intracellular signalling. Cell Calcium 2004;35:217-228.

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916 917 918

[33] Asaoka Y, Nishina H. Diverse physiological functions of MKK4 and MKK7 during early embryogenesis. J Biochem 2010;148:393-401. [34] Colella M, Gerbino A, Hofer AM, Curci S. Recent advances in understanding the extracellular calcium-sensing receptor. F1000Res 2016;5.

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Figure Legends

922

Figure 1. Distribution of CaSR in bovine sperm. Thawed sperm (n = 3, each from a

923

different bull) were fixed and incubated with CaSR antibody. Fluorescence image (A)

924

bright image (B); and overlay of the two images (C) are shown. Negative control was

925

performed omitting primary antibody; fluorescence image (D) and bright image (E).

926

Micrographs were captured at × 100; bar = 20 µm.

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927

Figure 2. Distribution of CaSR in bovine oocytes (n = 24, 7 replicates). Representative

929

images of bovine female gametes after CaSR labeling are shown. DNA was stained in

930

blue with Hoechst 33342 (A); CaSR was visualized in green (B) and the overlay of both

931

images is shown (C). Negative controls were made omitting primary antibody; Hoescht

932

33342 (A’); fluorescence image (B’) and overlay of the two images (C’). Micrographs

933

were captured at × 40; bar = 100 µm.

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Figure 3. Effect of different concentrations of NPS2143 on sperm motility. After 4 h at

936

37ºC in TALP medium in a 5% CO2/95% air atmosphere, sperm motility was analyzed

937

using a CASA system. Motility values are expressed as mean ± SEM. Four different

938

bulls were used (one ejaculate per bull; n = 4). * p < 0.05 compared to the control.

939

39

ACCEPTED MANUSCRIPT 940

Figure 4. Effect of different concentrations of NPS2143 on bovine sperm viability.

941

After 4 h at 37ºC in TALP medium sperm motility was analyzed by eosin-negrosin

942

staining. Viability values are expressed as mean ± SEM of four bulls, one ejaculate per

943

bull (n = 4; p > 0.05).

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Figure 5. Effect of NPS2143 on oocyte fertilization. The sperm-oocyte co-incubation

946

was run in presence or absence of 15 µM of NPS2143 for 18 h and the presumptive

947

zygotes were cultured. Embryos were evaluated on day 2 (cleavage), 6 (morula) and 7

948

(blastocyst) post-insemination. Data are presented as the mean percentage ± SEM

949

(results of eleven replicates); n = 87 COCs (Control) and n = 90 COCs (Inhibitor). p >

950

0.05.

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951

Figure 6. Effect of NPS2143 on embryo development. After fertilization, presumable

953

zygotes were incubated in presence or absence of 15 µM of NPS2143. Embryos were

954

evaluated on day 2 (cleavage), 6 (morula) and 7 (blastocyst) post-insemination. Data are

955

presented as the mean percentage ± SEM (results of six replicates); n = 53 COCs

956

(Control) and n = 56 COCs (Inhibitor). * p < 0.05.

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ACCEPTED MANUSCRIPT Table 1. Chromatin configuration after IVM GV (%)

MI (%)

MII (%)

DEG (%)

n

Control

1 (1.3)

18 (24.3)

54 (72.9)

1 (1.3)

74

NPS2143 (15µM)

1 (1.2)

14 (17.9)

61 (78.2)

2 (2.5)

78

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Treatment

Values are represented as total number (and percentage) of six replicates. GV: Germinal

vesicle; MI: Metaphase I; MII: Metaphase II; DEG: Degenerated or absent chromatin; n: total

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oocyte number (p > 0.05).

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ACCEPTED MANUSCRIPT Highlights

- Calcium sensing receptor (CaSR) is present in bovine gametes. - CaSR inhibition blunts bovine sperm motility.

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- Bovine embryo development decreases by CaSR blockage.

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- Bovine oocyte maturation and fertilization rates are not altered by CaSR inhibition.