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The combination of estrus synchronization and superovulation treatments introduces molecular modifications whose effects are yet to be disclosed. Here, reproductive parameters and gene expression changes in ovaries and endometrium were explored on day 6 after artificial insemination (AI), when synthetic progestin altrenogest (ALT) was combined with gonadotropins. Sows were administered ALT for 7 d beginning on the day of weaning and superovulated with equine chorionic gonadotropin (eCG) 24 h later and human chorionic gonadotropins (hCG) at the onset of estrus (SS-7 group; n= 6). The controls were either superovulated sows with eCG 24 h postweaning and hCG at the onset of estrus (SCgroup; n= 6) or sows with postweaning spontaneous estrus (NCgroup; n= 6). Ovary examination and embryo and tissue collection were performed in all sows via laparotomy on day 6 post-AI. RNA-Seq was conducted to analyze differentially expressed genes (DEGs) between groups. Statistical analysis of the reproductive parameters was conducted with ANOVA and Tukey post hoc tests. DEGs were analyzed with an ANOVA (fold changes ≥2 or ≤2, Pvalue <0.05). Hormonal treatments almost doubled (P< 0.03) the number of corpora lutea (39.8 ± 10.2 and 38.3 ± 11.1 in SS-7 and SC sows, respectively) compared with that in the NC group (23.1 ± 3.8). In contrast, embryo viability significantly decreased (P< 0.003) in response to SS-7 treatment (75.1% ± 15.2%) compared to SC and NC groups (93.8 ± 7.6% and 91.8 ± 6.9%, respectively). RNA-Seq analyses revealed 675 and 1,583 DEGs in the SS-7 group compared to both SC and NC groups in endometrial and ovarian samples, respectively. Interestingly, many genes with key roles in the Wnt/β-catenin and Notch signaling pathways were differentially expressed in SS-7 sows relative to SC and NC groups (e.g., Ctnnb1, Myc, Gli3, Scyl2, Ccny, Daam1, Ppm1n, Rbpj,and Usp8). A key finding in this study was the downregulation of β-catenin (Ctnnb1) gene expression in the SS-7 endometrium, suggesting that this treatment influences embryo–uterine dialogue by triggering a cascade of events leading to embryo maldevelopment. These data explain the proliferative defects in SS-7 embryos and suggest a novel mechanism of a porcine embryo–maternal crosstalk.Genes with key roles in the Wnt/β-catenin and Notch signaling pathways were differentially expressed in the endometrium of sows subjected to synchronization and superovulation treatments, suggesting that these treatments influence embryo–uterine dialogue by triggering a cascade of events leading to embryo maldevelopment.Methods for porcine superovulation (increasing the number of ovulated oocytes per cycle) and estrus synchronization (grouping estrus sows on the same day) are available for assisted reproductive technologies, using hormonal treatments. The main goal of the present study was to understand how hormones used for these purposes influence gene expression patterns in the female reproductive tract (ovaries and endometrium). We observed that hormonal treatments (synchronization combined with superovulation) have the potential to alter ovarian and endometrial gene expression patterns, triggering improper follicle development and oocyte growth, and leading to abnormal embryonic development before implantation. Genes involved in two key metabolic pathways for embryo development (Wnt/β-catenin and Notch signaling pathways) were dysregulated in reproductive tissues.

The objective of the present study was to evaluate the effect of centrifugation and cytoskeletal stabilization with cytochalasin-B before vitrification on the survival and cytoskeleton integrity of vitrified porcine blastocysts. Unhatched blastocysts were collected by laparotomy from weaned crossbred sows (n=10) on day 6 of the estrous cycle. A subset of blastocysts (n=22) was not vitrified and cultured to evaluate the in vitro development of fresh embryos. The remaining blastocysts were randomly allocated to different experimental groups: a) blastocysts vitrified without pre-treatments (n=25); b) blastocysts centrifuged at 13.000 x g for 12 min (n=28); c) blastocysts equilibrated in 7.5 µg/ml cytochalasin-B for 20 min (n=30) and d) blastocysts equilibrated in cytochalasin-B for 20 min and then centrifuged at 13.000 × g for 12 min. Pretreated and non-pretreated blastocysts were vitrified (n=28). The basic medium for vitrification and warming (TCM-NBCS) was TCM199 Hepes supplemented with 20% (v/v) of newborn calf serum (NBCS) and 0.5 mM of L-Glutamine. Groups of 4 to 6 embryos were equilibrated in TCM-NBCS with 7.5% (v/v) dimethyl-sulfoxide (DMSO) and 7.5% (v/v) ethylene glycol (EG) for 3 min and in TCM-NBCS with 17% (v/v) DMSO, 17% (v/v) EG and 0.4 M sucrose for 1 min. During the last step, embryos were loaded into the narrow end of a superfine open pulled straw (SOPS) by capillary action and plunged into the liquid nitrogen. Straws were warmed using a one step dilution procedure. The narrow of the straw was immersed vertically in a well containing 800 µL TCM-NBCS with 0.13 M sucrose for 5 min. Fresh and vitrified-warmed blastocysts were cultured for 24 h in TCM199 supplemented with 10% fetal calf serum at 39°C, in 5% CO2in humidified air. After the culture, embryos were assessed by stereomicroscopy and classified as viable or degenerated. Blastocysts that reformed their blastocoelic cavities after warming, displaying a normal zona pellucida and excellent appearance, were considered viable. The in vitro survival rate was defined as the ratio of viable embryos divided by the total number of embryos cultured. The hatching rate was determined as the ratio of the number of embryos hatched to the total number embryos cultured. Some vitrified and fresh embryos were labeled after culture, with Alexa Fluor 488 phalloidin and wheat germ agglutinin-Alexa Fluor 594 conjugate, to assess cytoskeleton quality. Data were analyzed by Chi-square test and expressed as mean values ± SEM. The embryo survival (81.8 ± 1.2%) and the hatching rate (36.4 ± 8.6) for fresh embryos were not significantly different to those obtained with vitrified embryos (ranging from 75.0±8.3% to 80.0±8.2% and from 20.0 ± 8.2% to 39.3 ± 9.4%, respectively). No differences were found for in vitro suvirval, hatching rates and cytoskeleton quality among vitrification groups Embryos classified as degenerated by stereomicroscopy displayed wide cytoskeletal disruption. Vitrified embryos classified as viable by stereomicroscopy displayed similar cytoskeleton quality than fresh blastocysts, showing only few cells with microfilament disruption. In conclusion, the pretreatment with cytochalasin-B and/or the centrifugation prior vitrification did not improve the blastocysts developmental competence and the cytoskeletal quality after SOPS-vitrification. Supported by CICYT-FEDER (AGL2004-07546), CARM (2I05SU0012), MEC (BES-2005-8894) and Seneca Foundation (04543/GERM/07).