Female bovine blastocysts are more prone to apoptosis than male ones

Accepted Manuscript Female bovine blastocysts are more prone to apoptosis than male ones Emmanuelle Ghys, Matthew Dallemagne, Delphine De Troy, Caroline Sauvegarde, Abdelmounaim Errachid, Isabelle Donnay PII:

S0093-691X(15)00523-3

DOI:

10.1016/j.theriogenology.2015.09.050

Reference:

THE 13355

To appear in:

Theriogenology

Received Date: 21 May 2015 Revised Date:

13 July 2015

Accepted Date: 28 September 2015

Please cite this article as: Ghys E, Dallemagne M, De Troy D, Sauvegarde C, Errachid A, Donnay I, Female bovine blastocysts are more prone to apoptosis than male ones, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.09.050. 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.

ACCEPTED MANUSCRIPT Title: Female bovine blastocysts are more prone to apoptosis than male ones

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Authors: Emmanuelle Ghys1, Matthew Dallemagne1, Delphine De Troy1, Caroline Sauvegarde1, Abdelmounaim Errachid1, Isabelle Donnay1

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Affiliation : 1Université catholique de Louvain, Institut des Sciences de la Vie, Belgium

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Corresponding author : Isabelle Donnay, Université catholique de Louvain, Institut des Sciences de la Vie, Croix du Sud 4-5, box L7.07.10, B-1348 Louvain-la-Neuve (Belgium), email : [email protected]

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Keywords : bovine embryo, apoptosis, sex effect, sex-sorted semen, blastocyst

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ACCEPTED MANUSCRIPT Summary

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Female and male embryos show differences in gene expression and metabolism from the onset of their genome. Those differences are affected by environmental factors. The objective of the study was to compare the apoptotic rates of in vitro produced female and male bovine blastocysts cultured in different conditions. Day-7 blastocysts obtained after in vitro fertilization with sex-sorted semen and culture in two SOF-based media (containing foetal calf serum [FCS] or bovine albumin, insulin, transferrin and selenium [BSA-ITS]) were simultaneously evaluated for two markers of apoptosis after 3D reconstruction from confocal images: active caspase-3 by immunofluorescence, and DNA fragmentation by TUNEL. Higher levels of apoptotic cells were observed in female embryos whatever the culture condition, but with a more pronounced difference in FCS medium. This result was confirmed using the unsexed semen of two bulls. The sex effect on apoptosis was detected in both the inner cell mass and the trophectoderm but was dependent on the embryonic size. In conclusion, this study demonstrated that female bovine blastocysts are more prone to apoptosis than male ones but that culture in FCS exacerbates the differences in apoptosis between sexes, especially in small blastocysts.

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1. Introduction

Differences in gene expression between male and female preimplantation embryos can lead to sexual dimorphism in developmental kinetics, cell number, energy metabolism or sensitivity to environmental conditions (reviewed by [1]). This sexual dimorphism appearing before gonadal differentiation is related to the fact that X-chromosome inactivation, occurring in females and allowing for a functional equivalency between male and female cells, starts at the blastocyst stage in most species and will take several days to be completed. As a consequence, between the major activation of the embryonic genome (at D2 post-insemination in the bovine) and the completion of the random inactivation of one Xchromosome (probably around D14 in the bovine) [2], higher expression of several X-linked genes is observed in female embryos that contributes to differences in metabolism between female and male embryos and in their ability to cope with their environment [3]. Bermejo and colleagues also showed that roughly one third of the detected transcripts exhibited sexual dimorphism indicating that genes from sex chromosomes could impose an extensive transcriptional regulation upon autosomal genes [4]. Differences between sexes for the expression of X-linked genes seem to vary depending on the origin of the embryo (in vivo, in vitro or obtained after nuclear transfer), but also between culture conditions [5-7]. For example, G6PD, a X-linked gene, was found more expressed in female than male embryos in some studies [5, 7, 8] but less expressed in another study [6]. If some authors hypothesized that those apparent divergent results are related to variations in the pattern of Xinactivation process, it is also possible that they are due to metabolic adaptations to environmental factors.

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The aim of the present study is to compare the apoptotic rates between male and female bovine blastocysts grown in two different culture conditions known to differently impact embryo quality: a medium containing fetal calf serum (FCS) and a medium containing bovine serum albumin and a serum substitute, a mixture of Insulin, Transferrin and Selenium (BSAITS) [16]. Cohorts of male and female blastocysts were produced with sex-sorted semen and their apoptotic rates evaluated using simultaneously two complementary commonly used apoptotic markers: DNA fragmentation by the TUNEL technique and activation of the caspase-3 by immunohistochemical detection. Embryo evaluation was made after 3D reconstruction from confocal images. The results were then confirmed using unsorted semen of two different bulls.

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Apoptosis is described both in in vitro and in vivo preimplantation embryos, after the onset of the embryonic genome, and is involved in the regulation of cell populations as well as in the elimination of defective or damaged cells or cells undergoing an inappropriate differentiation [9]. Apoptosis is thus considered as a physiological and indispensable part of normal development that has the characteristics to be stage-specific regulated and to occur within a precise window of incidence. The occurrence of the apoptotic process can be perturbed by internal factors such as the intrinsic quality of the blastocyst but also by external factors such as suboptimal culture conditions (reviewed by [10]). Indeed, some culture media containing fetal calf serum (FCS - [11]), pro-oxidant agents [12], high concentrations of glucose [13], or lacking growth factors [14] have been shown to increase the level of apoptosis. Its level is therefore related to embryo quality but could also serve as an indicator of suboptimal embryo production systems (reviewed by [15]). Nonetheless, the consequences of apoptosis on embryo survival depend on its extent even if no threshold has been determined yet that would provoke an irremediable cell loss impairing embryo survival.

2. Material and methods

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2.1 In vitro production of embryos

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Bovine embryos were produced as previously described [17]. Briefly, bovine ovaries were obtained from a local slaughterhouse and transported to the lab in an insulated device. Oocytes were recovered by puncturing follicles 3-8 mm in diameter under a controlled pressure and collected in 50 ml conical tubes. The pellet containing COC’s was recovered and only COC’s with a homogenous dark cytoplasm and surrounded by at least three layers of compact cumulus cells were selected and washed four times in HEPES buffered TCM-199. Groups of 50-80 COC’s were transferred to 4-well dishes and matured in 500 µl of enriched serum-free maturation medium [18] for 24-26h at 39°C under a humidified atmosphere of 5% CO2 in air.

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Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich (Steinheim, Germany)

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Sex-sorted and unsorted semen from the same bull (bull 1 - Holstein) were kindly provided by CRV BV (The Netherlands). Unsorted semen from bull 2, (Belgian blue) routinely used for IVF in the lab was kindly provided by the AWE (Ciney, Belgium). In preliminary experiments, the purity of sex-sorted semen was verified through embryo sexing by PCR. Forty-two out of 44 (95.5%) embryos generated with Y-sorted semen were of the predicted sex whereas after fertilization with X-sorted semen 45 out of 46 embryos (97.8%) were females. The same in vitro fertilization procedure was followed for both sex-sorted and unsorted semen. Based on the recommendations kindly provided by P. Bermejo-Alvarez (personal communication), the fertilization procedure was adapted to the use of sex-sorted semen. Briefly, matured COC’s were washed in fertilization medium consisting of Tyrode Albumin Lactate Pyruvate containing 6 mg/ml BSA and supplemented with 10 µg/ml heparin, 15 µM hypotaurine, 1.5 µM epinephrine, 30 µM penicillamin (Fert-TALP) and transferred in 25 µl fertilization medium drops covered with mineral oil. Motile spermatozoa were selected by centrifugation of frozen-thawed semen on a Percoll discontinuous gradient (45-90%, Pharmacia, Uppsala, Sweden) and washed in Fert-TALP. Fifteen µl of diluted sperm suspension were added to the drops containing the COC’s in order to achieve a final concentration of 106 spz/ml. Coincubation was performed for 18h, in the same conditions as for the maturation step.

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2.2 Embryo staining procedure

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Day-7 blastocysts were washed four times in PBS containing 1 mg PVP/ml (PBS-PVP) before fixation in 2% paraformaldehyde for one hour at RT. They were then stored at 4°C in PBS and processed within two weeks. The day of staining, fixed embryos were permeabilized in PBS with 1% Triton-X-100 (Roche, Mannheim, Germany) and 0.1% tri-citrate sodium for one hour. Positive controls were treated with DNase (0.1 mg/ml) (Roche) for one hour at 37°C. Samples and positive controls were then submitted to TUNEL reaction (In Situ Cell Death Detection kit, Fluorescein, Roche), whereas negative controls were incubated in TUNEL mixture without transferase. Following three washes in PBS with 0.5 % Tween (PBS-Tween), immunohistochemical detection of the activated caspase-3 was performed after an initial step of blocking in PBS with 10% normal goat serum and 0.3% Triton-X-100. Embryos were incubated overnight at 4°C with the primary anti-caspase-3 antibody (Cleaved caspase-3 (Asp175) antibody, Cell Signaling Technology, Leiden, The Netherlands) diluted in PBS with

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After IVF, presumptive zygotes were mechanically denuded by vortexing to remove cumulus cells and washed in HEPES buffered TCM-199. Groups of about 30 zygotes were then cultured in 30 µl droplets of Synthetic Oviduct Fluid (SOF) modified according to [19] and containing 0.7 mM Na-pyruvate, 4.2 mM Na-lactate, 2.8 mM myo-inositol, 0.2 mM glutamine (Gibco, Paisley, Scotland), 0.3 mM citrate, 30 ml/l essential amino acids, 10 ml/l non-essential amino acids, 50 mg/ml gentamycin (Gibco) supplemented either with 5% FCS (FCS, ICN Biomedicals) or BSA (4mg/ml) and ITS (5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium) (BSA-ITS). Culture was performed under mineral oil (Fertipro, Beernem, Belgium) in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2.

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0.4% BSA and 0.3% Triton-X-100. The next day, embryos were washed in PBS-Tween and incubated with AlexaFluor conjugated secondary antibody (Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor®555 conjugate), Cell Signaling Technology) for one hour at RT. Finally, embryos were washed several times in PBS-Tween and mounted in Vectashield with DAPI (Labconsult, Brussels, Belgium) either on Lab-tek© (Thermo Fisher Scientific, St Leon-Rot, Germany) or on a microscope slide under coverslip according to the experiment. Embryos containing less than 80 cells were discarded from analysis to keep only expanding/expanded blastocysts. This avoids including late morulae or very bad quality/degenerating blastocysts in the analysis. Moreover it was often difficult to distinguish inner cell mass from trophectoderm cells in those embryos.

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2.3 Embryo sexing procedure

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The procedure relies on detection of X/Y Amelogenin polymorphism and is based on the protocol described in [20]. Blastocysts were placed individually in 0.5 ml tube in a minimal volume of PBS-PVP and stored for a maximum of one month at -80°C prior to sexing. DNA was extracted in 20 µl lysis buffer containing 15 mM Tris-HCL pH 8.5, 50 mM KCl, 2.5 mM MgCl2, 0.1% Triton-X-100 and 150 µg/ml proteinase K. After 1 minute centrifugation at 13000 rpm, the tubes were incubated for one hour at 56°C followed by inactivation of the proteinase K during 10 min at 90°C. Five microliters of lysates were then transferred into PCR tube and 20 µl PCR mix were added consisting of 6.5 µl milli-Q water with 12.5 µl GoTaq®Green Master Mix, 2x (400 µM dATP, 400 µM dTTP, 400 µM dCTP, 400 µM dGTP and 3 mM MgCl2) and 0.5 µl of 25 µM of each primer (F : 5’ - CAG CCA AAC CTC CCT CTG C - 3’ , R : 5’ - CCC GCT TGG TCT TGT CTG TTG C - 3’). Positive controls (DNA from bovine ovarian and testicular tissues) were included in each assay as well as two negative controls (water, lysis buffer). The amplification was performed as follows: denaturation at 95°C for 15 min, 34 cycles consisting of denaturation at 94°C for 20 s annealing at 60°C for 40 s, and extension at 72°C for 20 s. A final extension was performed for 10 min at 72°C. Migration of PCR products was then run on a 2% agarose gel for 22 min at 150V followed by staining with ethidium bromide for 20 min. When three bands were visible, embryo was considered as a male whereas one band corresponded to a female embryo.

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2.4 Statistical analysis

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As most of generated data were proportions, a preliminary arcsin square root transformation was required in order to fulfill the required normality of distribution and variance homoscedasticity. Data were then submitted to several analyses using SAS EG 5.1

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In order to assess the specificity of the immunohistochemical staining several controls were designed. Briefly, these included (1) omission of the primary antibody (2) omission of the secondary antibody (3) pre-absorption of primary antibody with immunogenic peptide (Cleaved caspase-3 (Asp175) blocking peptide, Cell Signaling Technology) (4) Western blot on cumulus cells incubated with Doxorubicine.

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2.5 Experimental design

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2.5.1 Experiment 1: Apoptotic rates in male and female Day-7 blastocysts produced with sexsorted semen in two culture media

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Fifty-five male and 57 female blastocysts cultured in BSA-ITS and 69 male and 58 female blastocysts cultured in FCS were collected at Day 7 post-insemination (pi) and stained. Three replications in each medium were performed. In order to maintain their three-dimensional structure, blastocysts were mounted on Lab-tek© and observation was realized with a Zeiss LSM710 confocal laser scanning microscope (Oberkauchen, Germany). One image was taken every 1 µm throughout the embryo and a 3D embryo reconstruction was made using the Imaris© software (Zurich, Switzerland). The software allowed rotating the 3D reconstruction in order to accurately count single and double stained cells and to localize them precisely either in inner cell mass (ICM) or in trophectoderm (TE) cell line. Staining of nuclei with DAPI ensured counting all cells. Stained cells were classified into three categories. Cells exhibiting both staining were referred to as double stained cells. The caspase-3 or TUNEL stained cells include double stained cells as well as single caspase-3 or TUNEL labelled cells. Cell counts (total cells, ICM or TE cells per blastocyst) and percentages of stained cells (single or double staining) were analyzed through mixed model with sex and culture medium as fixed factors and replication as random factor.

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To verify a potential link between cell number (blastocyst size) and proportion of stained cells, embryos from each medium were divided in two groups of equal sizes (BSA-ITS: embryos ˃ or ≤ 179 cells; FCS: embryos ˃ or ≤175 cells). A mixed model analysis of variance was applied with sex and size as fixed factors and replication as random factor.

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2.5.2 Experiment 2: Apoptotic rates of male and female Day-7 blastocysts produced with unsorted semen from two bulls in FCS.

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or JMP 10.0.2 (Cary, USA). In case of significant interaction between factors, a test of effect slices was realized using LSMEANS with SLICES option. The differences were considered as significant at p<0.05.

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In order to exclude potential bias introduced by the use of sex-sorted semen on results of experiment 1, apoptotic rates were evaluated in blastocysts produced with unsorted semen of bull 1. Another bull (bull 2) was also used to verify the absence of a bull effect on the results. Those verifications were carried out in FCS because differences between sexes were more important in experiment 1 for this culture condition. Five replications were performed for bull 1 with a total of 167 Day-7 blastocysts analyzed. The staining was performed as in experiment 2 but quantification of stained cells was assessed through a DMIRB fluorescence microscope (Leitz, Oberkauchen, Germany). For that purpose, embryos were mounted on a microscope slide under coverslip in a minimal volume of Vectashield with DAPI. This simplified procedure was used to reduce the time needed to analyze each embryo (roughly one hour per embryo using the confocal microscope) and also allowed to compare the 6

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2.5.3 Experiment 3: Developmental rates of male and female embryos produced with sexsorted semen in two culture media In order to study the sex effect on developmental rates as well as a potential interaction between sex and culture medium effects, culture of male and female bovine embryos were performed in parallel in FCS and BSA-ITS. A total of 1126 embryos from 4 replicates were produced. Cleavage rates and rates of embryos at the 5-8 cell stage were evaluated at Day 2 post-insemination (pi). Blastocyst rates were evaluated at Day 6, Day 7 and Day 8 pi and hatching rates at Day 8. Developmental rates were calculated on cleaved embryos to reduce the impact of a potential difference arising from the sex sorting procedure itself or following the preparation of semen just prior to fertilization. Comparison of developmental rates was performed using a linear mixed model with sex and culture medium as fixed factors and replication as random factor.

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3. Results

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The expected staining was observed in all control samples and was consistent with what has been previously described in other studies. 3.1 Experiment 1: Apoptotic rates in male and female Day-7 blastocysts produced with sexsorted semen in two culture media

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obtained data with those of the literature, as most previous studies on apoptosis used the fluorescence microscope to count the cells. As a consequence, it was not possible to distinguish ICM and TE cells or to determine double stained cells. Directly after evaluation, embryos were recovered for further sex determination. A total of 256 Day-7 blastocysts were produced in 4 replications with semen of bull 2 and processed as described for embryos produced with semen of bull 1. Comparison between male and female embryos regarding proportion of stained cells was achieved through mixed model (sex: fixed factor, replication: random factor).

Male embryos displayed a higher total and TE cell numbers than female ones, whatever the culture medium (total cell number: BSA-ITS: male: 195±8 vs female: 169±7; FCS: male: 186±6 vs female: 177±7 – p=0.01; TE: BSA-ITS: male: 133±6 vs female: 115±5; FCS: male: 132±5 vs female: 123±5 – p=0.01). Culture in BSA-ITS but not in FCS led to higher number of ICM cells in male embryos compared to female ones (BSA-ITS: male: 62±3, female: 54±3 - p=0.02; FCS: male: 54±2, female: 54±3 - p=0.92).

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The mean number of cells or the ratio between ICM and TE cells did not differ between culture conditions (data not shown). The proportion of caspase-3 stained cells (p≤0.0001) and TUNEL positive cells (p=0.02) was higher in FCS than in BSA-ITS whatever the sex but no difference between culture media was observed for the rate of double stained cells (p=0.38). Similar results were obtained in both cell lineages except for double stained TE cells which were more abundant in FCS. 7

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The proportion of cells displaying only the caspase-3 staining (single caspase-3 stained cells) were also higher in female than in male blastocysts (BSA-ITS: male: 3.3±0.3%; female: 4.5±0.3%; FCS: male: 4.5±0.3%; female: 7.2±0.5% - p≤0.0001) contrary to the proportion of cells displaying only the TUNEL labelling (single TUNEL labelled cells) for which no difference between sexes was detected (BSA-ITS: male: 5.8±0.5%; female: 5.5±0.5%; FCS: male: 8.4±0.5%; female: 9.5±0.5% - p= 0.63).

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The analysis for ICM and TE cells are presented in table 1 and 2. The higher rates of caspase3 and double stained cells observed in females were globally confirmed in both cell lineages. However, the proportion of caspase-3 stained cells in ICM tended to be significantly higher in female embryos only when culture was performed in FCS but not in BSA-ITS (table 1). No significant difference between sexes was observed for the TUNEL labelled cells in the ICM whatever the medium, but a tendency was observed for TE lineage (table 2).

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In FCS the sex effect is more pronounced in small than in large embryos as highlighted by a significant interaction between the effects of size and sex of embryos (table 3). Indeed female embryos display higher rates of stained cells among the small group while no difference between sexes was found in large embryos (figure 4). Similar observations were made for both lineages except for TUNEL staining in TE (data not shown). Such effect of the size on the differences between sexes was not observed in BSA-ITS (table 3).3.2 Experiment 2: Apoptotic rates of male and female Day-7 blastocysts produced with unsorted semen from two bulls in FCS.

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A higher proportion of cells showing caspase-3 staining was observed in female than in male embryos (BSA-ITS: male: 6.7±0.6%; female: 9.1±0.8%; FCS: male: 9.2±0.6%; female: 14.2±1.0% – figure 1). The same observation was made for the proportion of cells showing TUNEL labelling, with higher rates in female than in male embryos (BSA-ITS: male: 9.3±0.8%; female: 10.2±0.9%; FCS: male: 13.1±0.9%; female: 16.5±1.1% - figure 2). Concerning cells showing both staining, higher rates were also observed for females than males, whatever the medium (BSA-ITS: male: 3.4±0.4%; female: 4.7±0.6%; FCS: male: 4.8±0.5%; female: 7.0±0.7% - figure 3). However, differences between sexes for the proportion of stained cells are more pronounced in FCS than in BSA-ITS medium.

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Male embryos produced with semen of Bull 2 displayed more cells than females (male: 165±4; female: 149±4 - p=0.01). A similar tendency was observed for embryos produced with semen of Bull 1 (male: 143±4; female: 132±4 - p=0.07). With both bulls, proportion of caspase-3 stained cells was higher in female than in male Day-7 blastocysts (figure 5). The proportion of TUNEL labelled cells was also higher in female embryos produced with semen of Bull 2 but the difference was not significant for Bull 1 (figure 6). 3.3 Experiment 3: Developmental rates of male and female embryos produced with sexsorted semen in two culture media

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4. Discussion This study compared for the first time the differences in apoptotic rates between male and female bovine blastocysts cultured in two culture media known to differentially impact embryo metabolism and quality. Two common markers of apoptosis were used and 3D reconstruction of embryos from images obtained by confocal microscopy allowed simultaneously evaluating the two markers and localizing the stained cells in the trophectoderm or in the inner cell mass. This study was performed with sex-sorted semen but the main results were then confirmed using the unsorted semen of two bulls and the classical evaluation by epifluorescence microscopy.

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At Day 8 blastocyst rates (on cleaved embryos) were higher for male than for female embryos, whatever the medium (table 4). Hatching rates at Day 8 were higher in male than in female embryos in both media. Globally, developmental rates were significantly higher in FCS than in BSA-ITS medium.

Female blastocysts displayed significantly higher proportions of caspase-3 stained cells than male ones in both media. Similar results were obtained for the TUNEL labelled cells. But we also showed that the sex effect on apoptosis is more pronounced when embryos are cultured in the FCS than in the BSA-ITS medium (experiment 1, figures 1 and 2).

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As previously observed by Byrne and colleagues, a higher rate of TUNEL labelled cells is observed when the culture medium is supplemented with FCS than with BSA [11]. The level of cells showing caspase-3 staining is also higher in our study in the FCS than in the BSA-ITS medium. These higher apoptotic rates in FCS are in agreement with the higher level of Bax transcripts, a pro-apoptotic member of the Bcl-2 family, observed in embryos cultured with FCS compared to those cultured with BSA [21]. Although the addition of FCS has been reported to increase [22, 23] or decrease [11] the number of cells, as well as to disturb the ICM:TE ratio [23], in our study those parameters did not differ between culture media. Differences between studies could be due to the high variability between FCS batches.

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In the first experiment, both stainings were simultaneously localized in the 3D reconstructed embryos. Surprisingly, only roughly half of the stained cells showed the double staining (double staining: 5.0%±0.3 vs single active caspase- 3 staining: 4.9%±0.2 and single TUNEL labelling: 7.4%±0.3). Such disparity between these two apoptotic markers has already been observed in the bovine embryo by Gjorret and colleagues [24]. Single active caspase-3 staining could be explained by the fact that caspase-3 activation is considered as an early event in the apoptotic cascade, known to occur before DNA fragmentation, which in turn is defined as a late event [25]. Concerning cells showing TUNEL labelling without concurrent immunostaining of the active caspase-3 (single TUNEL labelled cells), two hypotheses can be proposed. In a first hypothesis, the caspase-3 staining might have disappeared at the end of the apoptotic process, either after inactivation or degradation of the active caspase-3 by 9

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The higher proportion of stained cells observed in the ICM than in the TE lineage in both media (ICM caspase-3 stained cells: 14.6±0.6% vs TE caspase-3 stained cells: 8.0±0.4% p≤0.0001; ICM TUNEL labelled cells: 15.8±0.7% vs TE TUNEL labelled cells: 11.2±0.5% p≤0.0001) is consistent with previous reports in rodents [14, 32] and in the bovine [11, 31, 33], while no difference between cell lineages was observed in human and pig embryos [10, 34]. The sex effect on the apoptotic rate in the ICM (but not in the TE) might differ between media, as illustrated by the tendency for an interaction between sex and medium effects for the caspase-3 staining in the ICM (table 1): the sex effect tended to be more pronounced in the FCS than in the BSA-ITS medium. This could illustrate a differential sensitivity to cell death between ICM and TE cells in response to changing environmental conditions, as already documented by several authors in other species [10, 35, 36].

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Experiment 1 was performed using the sex-sorted semen of Bull 1. It is known that deleterious effects inherent to the sorting procedure itself (reviewed by [37]) could be in part responsible for a lower fertility in vivo [38] and in vitro [39, 40]. Morton and colleagues also showed that IVF using sex-sorted semen can lead to alteration in the expression of several genes in the resulting embryo [41], which was not observed by Bermejo-Alvarez and colleagues on other genes [42]. To verify if the use of sex-sorted semen could have affected the apoptotic rates in the blastocyst, 41 embryos were also produced with the unsorted semen of the same bull, stained and analyzed by confocal microscopy as in experiment 1 (data not shown). Globally, the rate of apoptosis was similar to what was observed with sexsorted semen (BSA-ITS culture medium - sex-sorted semen: 8.0±0.5% vs unsorted semen: 8.9±0.9% - p=0.55). We can thus conclude that the use of sex-sorted semen did not have an impact on the global rate of apoptosis.

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endogenous caspase inhibitors [26-28]. A second hypothesis could be that the DNA fragmentation detected by the TUNEL technique does not only result from a process involving activation of caspase-3 [29]. Indeed cells undergoing necrosis can be labelled by this technique [30] and several types of cell death could occur in the early embryo [31]. Those hypotheses were tested on PA-1 cells treated with Doxorubicin to induce apoptosis. In that model, while single active caspase-3 staining could be observed at the beginning of the treatment, we never observed TUNEL labelling without active caspase-3 staining, even in cells exhibiting a very late stage of apoptosis, arguing for a persistence of the active protein throughout the process (data not shown). The immunohistochemical staining of the caspase3 thus appeared to be a more reliable marker of apoptosis, which convinced us to restrict the appellation of “apoptotic cells” to single active caspase-3 and double stained ones, which correspond in our study to what we call the caspase-3 stained cells.

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Moreover, the differences in apoptotic rates between sexes were confirmed on embryos produced with unsorted semen of bull 1 and cultured in FCS medium. The embryos were stained and analyzed using an epifluorescent microscope before being sexed by PCR (experiment 2). This mode of evaluation led to a lower total number of counted cells, 10

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probably because of the difficulty to accurately count the ICM cells. However, it had no impact on the observed sex effect that was significant. To make sure that the sex effect was not related to the used bull, the experiment was repeated using the unsorted semen of a second bull from another breed. Once more, female blastocysts collected at Day 7 pi showed a significant higher level of apoptotic cells (figure 5).

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To what extent can we correlate the lower blastocyst rates observed for females with their higher apoptotic rates remains an open issue. A massive or inadequate incidence of cell death may result in early embryonic death. Nevertheless, up to now no threshold of apoptotic rate leading to embryonic degeneration has been established [48]. Several studies tried to link developmental arrest and blastocyst morphology with apoptosis but led to conflicting results. Indeed, while some studies reported correlation between embryo fragmentation and apoptosis [49-51], others did not [52, 53]. In our study, blastocyst rates at Day 7 and hatched blastocyst rates at Day 8 were higher in FCS than in BSA-ITS medium, whatever the sex, while the apoptotic rates were always higher in FCS. It is thus difficult to link the higher apoptotic rates of female embryos observed at Day 7 to their lower developmental rates at Day 8.

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Higher blastocyst rates (on cleaved embryos) were observed at Day 8 when using Y-bearing instead of X-bearing semen for IVF (experiment 3). Moreover, hatching rates were also higher in male than female blastocysts. Such difference between sexes seems not related to a difference in quality between the batches of X- and Y-bearing semen as the cleavage rates were higher when using X-bearing semen (data not shown). Several studies have reported developmental differences between sexes leading in some cases to distortions of sex ratio at the blastocyst stage, particularly in the presence of FCS or glucose [43-45]. Such shift in the sex ratio, generally in favor of male embryos, could be explained either by a preferential degeneration of female embryos in some culture conditions or by a delay in their development [46, 47]. The developmental differences observed in experiment 3 are unlikely to be due to a delay in blastocyst formation since few female blastocysts appeared at Day 8. They are concordant with the results of Bermejo-Alvarez and colleagues who obtained higher blastocyst rates in the presence of FCS with Y- than with X-bearing semen from one of the three used bulls [42]. Nevertheless, those data were obtained with sex-sorted semen and have thus to be interpreted cautiously: it cannot be excluded that the sorting procedure of the semen differentially affects X- and Y-bearing semen in their ability to produce blastocysts.

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Male blastocysts possess more cells than females do in both media which is in accordance with the study of Xu and colleagues [44] but in contrast with the report of Morton and colleagues [41]. Similar results were observed for embryos produced with sex-sorted and unsorted semen. In addition, the sex effect on the number of TE cells is the same whatever the medium, contrary to the effect on the number of ICM cells where only males cultured in 11

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BSA-ITS display a higher cell number. This brought another piece of evidence of the divergent effect of the combination between sex and culture medium according to cell lineage.

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Could the higher apoptotic rate observed in the present study in female blastocysts explain their lower cell number? Indeed, if we take into account the embryonic size, it appears that the proportion of apoptotic cells is lower in large than in small embryos, which has already been observed in other studies [11, 33]. It should also be noted that the decrease of apoptotic rates with the increase of size seems to mainly come from a decline of apoptotic cells in the TE lineage. In the FCS medium, the difference in apoptotic rates between sexes was more pronounced in embryos of small size and was no more significant for the large size embryos. This significant impact of the size on the differences between sexes was not observed in the BSA-ITS medium. The small female blastocysts could thus be more prone to degeneration than male ones in FCS but not in BSA-ITS, which might be related to the shift in sex ratio in favor of male embryos regularly observed in FCS but rarely in BSA supplemented media (REF).

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In conclusion, this study demonstrated that female bovine Day-7 blastocysts exhibit a higher proportion of apoptotic cells than male ones in two different culture conditions. However culture in FCS exacerbated the differences in apoptosis between sexes, especially in the ICM and in small blastocysts.

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The contribution of the imaging platform IMABIOL to the confocal microscope analysis is warmly acknowledged. We are very grateful to the CRV BV and to the AWE for the gift of the bull semen. We thank the Plateforme de Support en Méthodologie et Calcul Statistique of the UCL, especially Catherine Rasse and Christian Ritter, for their contribution to the statistical analysis of the results. We also wish to thank all members of the AMCB team for their contribution to embryo production and to the critical discussion of the results. We thank Anne Van Langendonckt for her constructive help in improving the manuscript. This work was supported by a FSR grant from the Université catholique de Louvain.

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[53] Ikeda S, Prendes JM, Alonso-Montes C, Rodriguez A, Diez C, Kitagawa M, et al. Apoptosisindependent poor morphology of bovine embryos produced by multiple ovulation. Reproduction in domestic animals = Zuchthygiene. 2006;41:383-5.

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Figure 1 : Percentage of caspase-3 stained cells (mean percentage ± SEM) in BSA-ITS and FCS media for female and male Day-7 blastocysts produced with sex-sorted semen of Bull 1

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Figure 2 - Percentage of TUNEL positive cells (mean percentage ± SEM) in BSA-ITS and FCS media for female and male Day-7 blastocysts produced with sex-sorted semen of Bull 1

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Figure 3 - Percentage of double stained cells (mean percentage ± SEM) in BSA-ITS and FCS media for female and male Day-7 blastocysts produced with sex-sorted semen of Bull 1

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Figure 4 - Percentage of caspase-3 (A), TUNEL (B) and double stained cells (C) (mean percentage ± SEM) in female and male Day-7 blastocysts in relation with the blastocyst cell number.

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Embryos cultured in FCS medium and obtained with sex-sorted semen of Bull 1. ** p<0.0001; * p<0.001 (mixed model, results from 127 embryos)

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Figure 5 - Percentage of caspase-3 stained cells (mean percentage ± SEM) in female and male Day-7 blastocysts cultured in FCS and produced A. with unsorted semen of Bull 1 (**sex effect: p≤0.0001 – results from 167 embryos) and B. with unsorted semen of Bull 2 (**sex effect: p≤0.0001 – results from 256 embryos) (mixed model)

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Figure 6 - Percentage of TUNEL positive cells (mean percentage ± SEM) in female and male Day-7 blastocysts cultured in FCS and produced A. with unsorted semen of Bull 1 (sex effect: p=0.13 – results from 167 embryos) and B. with unsorted semen of Bull 2 (**sex effect: p≤0.0001 – results from 256 embryos) (mixed model)

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Table 1 - Proportions (mean±SEM) of stained cells in the inner cell mass of Day-7 blastocysts depending on the sex of the embryo

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Females 57 12.8±1.1 11.5±1.2 5.0±0.7 Males 55 11.7±1.0 11.2±1.1 4.3±0.5 Females 58 19.7±1.4 21.7±1.4 8.0±0.8 Males 69 14.2±1.2 17.9±1.4 6.1±0.8 Sex effect p=0.005* p=0.28 p=0.01* Medium effect p=0.049* p=0.04* p=0.20 Interaction Sex x Medium p=0.05 p=0.19 p= 0.12 Embryos were cultured either in a SOF-based medium supplemented with a mixture of BSA and ITS or with FCS. n: number of analyzed blastocysts Caspase-3: caspase-3 stained cells; TUNEL: TUNEL labelled cells; T-C: double stained cells Results from 3 replicates (mixed model - * significant effect p<0.05)

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Females 57 7.6±0.9 9.8±1.0 4.7±0.7 Males 55 4.6±0.6 8.4±0.9 3.2±0.5 Females 58 12.1±1.1 14.5±1.2 6.6±0.8 Males 69 7.5±0.6 11.7±0.8 4.4±0.5 Sex effect p≤0.0001* p=0.05 p=0.01* Medium effect p≤0.0001* p=0.05 p=0.04* Interaction Sex x Medium p=0.65 p=0.52 p= 0.76 Embryos were cultured either in a SOF-based medium supplemented with a mixture of BSA and ITS or with FCS. n: number of analyzed blastocysts Caspase-3: caspase-3 stained cells; TUNEL: TUNEL labelled cells; T-C: double stained cells Results from 3 replicates (mixed model - * significant effect p<0.05) BSA-ITS BSA-ITS FCS FCS

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FCS BSA-ITS Caspase-3 TUNEL T-C Caspase-3 TUNEL T-C Sex effect ≤0.0001 0.01 0.005 0.15 0.50 0.79 Size effect 0.0003 0.0046 0.0005 ≤0.0001 ≤0.0001 0.0002 Interaction (Sex*Size) 0.0046 0.02 0.0038 0.94 0.66 0.80 Embryos were cultured either in a SOF-based medium supplemented with a mixture of BSA and ITS or with FCS. Caspase-3: caspase-3 stained cells; TUNEL: TUNEL labelled cells; T-C: double stained cells number of analyzed blastocysts: FCS: 127; BSA-ITS: 112 Results from 3 replicates (mixed model)

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5-8 cell Day-6 Day-7 Day-8 Hatched stage blastocysts blastocysts blastocysts blastocysts BSA-ITS Females 265 40.1 ± 2.6 3.4 ± 1.3 24.5 ± 5.7 28.6±3.7 5.0±1.8 BSA-ITS Males 317 42.5 ± 4.2 5.3 ± 1.4 27.4 ± 3.4 36.7±4.0 14.3±2.5 FCS Females 230 46.4 ± 4.0 11.0 ± 2.7 30.5 ± 3.7 31.7±4.5 14.3±3.6 FCS Males 288 42.0 ± 3.6 16.5 ± 2.8 37.6 ± 3.6 41.7±2.9 23.3±4.0 Sex effect p=0.62 p=0.10 p=0.21 p=0.02* p=0.007* Medium effect p=0.42 p=0.0003* p=0.04* p=0.29 p=0.01* Interaction (Sex*Medium) p=0.35 p=0.67 p=0.90 p=0.76 p=0.53 Embryos were cultured either in a SOF-based medium supplemented with a mixture of BSA and ITS or with FCS. Results from 4 replicates (mixed model)

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This study demonstrated for the first time that female bovine blastocysts display a higher apoptotic rate than male ones. An original approach (3D embryo reconstruction from confocal images) was used to simultaneously evaluate two markers of apoptosis and to localize the stained cells in the inner cell mass and in the trophectoderm. Results were obtained first with the use of sex-sorted semen to produce the embryos and then confirmed with unsorted semen of two bulls. This study also highlighted that the differences between sexes depend on the culture medium. Indeed, gender difference was more pronounced when embryos were cultured in the presence of foetal calf serum than in the same basal medium supplemented with bovine serum albumin and a mixture of insulin, transferrin and selenium. Differences between sexes also differ according to the size of the embryo or the cell lineage (inner cell mass or trophectoderm). Those results underline the importance to take into account the differential sensitivity of male and female embryos when designing culture media or when analyzing the impact of environmental factors on embryo development and quality.