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Context The establishment of pregnancy in cows requires uterine activity regulation of the main Hippo signalling effector yes-associated protein 1 (YAP). It remains unknown (1) how YAP activity at the corpus luteum (CL) correlates with early pregnancy-related events in ruminants; and (2) if YAP activity in the uterus and CL can be affected by metabolic disorders that may lead to pregnancy failure in ruminants. Aims and methods To determine the effect of early pregnancy on total and phospho-YAP expression and its transcriptional activity in the CL, we compared non-pregnant vs pregnant ewes. To understand the YAP activity dysregulation with disorders that may result in pregnancy loss, we induced negative energy balance in pregnant ewes. Key results and conclusions Our main results indicate that early pregnancy alters the expression and activity patterns of YAP in the ovine CL but not in the endometrium. In addition, while our NEB-induced model fails to alter YAP activity at the endometrium level, we found that fasting during the first but not second week of pregnancy affects YAP activity in the CL of pregnant ewes. Implications The data presented herein provide considerable insight into the activity of a signalling pathway that may be a key player in pregnancy recognition and establishment in ewes.

Made available in DSpace on 2018-11-28T03:24:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) ACSC There is evidence that regulation of follicle selection in cattle involves locally produced growth factors. In the present study, we investigated the expression of members of the fibroblast growth factor (FGF) 7 family during follicle deviation. The largest and second largest follicles were recovered during the second day of a synchronised follicle wave and the future dominant and future subordinate follicles were identified based on diameter and cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1) mRNA levels in granulosa cells. Theca cells of the future dominant follicle contained less mRNA encoding FGF7 and FGF10 compared with those from the future subordinate follicle 2.5 days after ovulation, before a significant difference between the diameters of the future dominant and future subordinate follicles could be observed, but FGF22 mRNA levels did not change. Levels of mRNA encoding FGF receptors FGFR1B and FGFR2B in theca and granulosa cells, respectively, were lower in the future dominant follicle compared with the future subordinate follicle. Addition of FGF10 to granulosa cells in vitro significantly decreased oestradiol secretion, as well as CYP19A1, FSH receptor (FSHR) and insulin-like growth factor 1 receptor (IGF1R) mRNA abundance, whereas FGF22 had no effect. We conclude that FGF10 and FGFR2B expression is increased in the future subordinate follicle before morphological deviation, which may contribute to follicle selection. Univ Estadual Paulista, Dept Farmacol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil Univ Montreal, Fac Med Vet, Ctr Rech Reprod Anim, 3200 Sicotte, St Hyacinthe, PQ J2S 7C6, Canada Univ Fed Santa Maria, Lab Biotecnol & Reprod Anim, Ave Roraima 1000, BR-97105900 Santa Maria, RS, Brazil Univ Estadual Paulista, Dept Fisiol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil Univ Estadual Paulista, Dept Farmacol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil Univ Estadual Paulista, Dept Fisiol, Inst Biociencias, BR-18618970 Sao Paulo, Brazil FAPESP: 2008/57365-2

The present study evaluated whether the gonadotrophin surge modulates components of the renin–angiotensin system and whether angiotensin II (Ang II) plays a role in the production of hormones by follicular cells during the ovulatory process. In Experiment 1, cows were ovariectomised at various times (0, 3, 6, 12 and 24h) after GnRH injection to obtain preovulatory follicles. The concentration of Ang II in follicular fluid increased after GnRH and reached a peak at 24h, concomitant with the peak of angiotensinogen (AGT) mRNA expression in granulosa cells. AGT mRNA was not expressed in theca cells. Ang II receptor type 2 and angiotensin-converting enzyme mRNA levels were transiently upregulated in theca cells. In Experiment 2, an in vitro culture was used to determine whether Ang II could modulate hormone production by healthy dominant follicles. In the absence of LH, Ang II did not alter hormonal production by either theca or granulosa cells. Ang II plus LH increased progesterone and prostaglandin secretion by granulosa cells. In summary, the renin–angiotensin system is actively controlled during the preovulatory period and Ang II amplifies the stimulatory effects of LH on the secretion of progesterone and prostaglandins by granulosa cells.

Nuclear maturation of the oocytes, characterised by a resumption of meiosis, occurs at the end of the growing phase in preovulatory follicles, after the peak of gonadotropins. The mechanisms and substances involved, by which gonadotropins lead to the final nuclear maturation, are still unknown. The withdrawal of immature oocytes from the follicular environment is sufficient to trigger the spontaneous resumption of meiosis, hindering the study of these events in the in vitro model. However, the co-culture of immature oocytes with hemi-sections (HS) of follicular walls holds the meiosis arrest and is therefore an alternative system for evaluating the role of certain substances in the process of oocyte maturation. Nitric oxide is an important signalling molecule. There is evidence that NO is involved in the mechanisms that hold the meiotic arrest, and also in the events that lead to the resumption of nuclear maturation. Through the action of the NO synthase enzyme, l-arginine releases NO as a by-product. Thus, l-arginine may be considered a natural NO donor. This study tested the possible involvement of NO in the oocyte maturation process in in vitro conditions mimicking the in vivo environment. Different concentrations of l-arginine (2.5, 4, 5, 6, 10, and 50 mM) were added in the culture medium (200 µL of TCM-199/albumin). Groups of 20 cumulus–oocyte complexes were matured for 22 h at 38.5°C with 8 HS from the follicular wall. In addition, two controls (with and without HS) were processed. Membrane integrity of cumulus cells (CC) was evaluated, as well as the nuclear maturation of the oocytes at 22 h of culture, by double staining with Hoechst 33342 (10 µg mL–1) and propidium iodide (10 µg mL–1) and staining with 1% orcein, respectively. The results were assessed through ANOVA, and means were compared by t-test. There was an increase in the proportion of CC with intact membranes (P

The interaction between GnRH and its receptor on gonadotropes within the anterior pituitary gland represents a key point for regulation of the reproduction. In addition, GnRH can act in multiple extrapituitary tissues via autocrine/paracrine mechanisms. Protein for GnRH and mRNA for both GnRH and its receptor have been detected in human uterine endometrium and oviduct as well as in embryos at the morula/blastocyst stage in the mouse and human. Therefore, we hypothesized that GnRH may have a critical role in the development of pre-implantation embryos. To address this question, we examined the effect of a GnRH agonist and antagonist on the development of mouse embryos in vitro. For these studies, 1-cell embryos were randomly allocated to culture in KSOM containing the appropriate treatment for 144 h at 37°C in a 5% CO2 in air environment. The medium was changed every 12 h and embryos were scored daily for development. The data were compared using a χ2 test. First, we wanted to determine if a GnRH agonist, histrelin, could enhance embryonic development. Embryos were cultured with (n = 35) or without (n = 36) 10 μM histrelin. The addition of histrelin did not increase morula or blastocyst formation v. control. Second, we cultured embryos in the presence of different concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μM) of the GnRH antagonist, SB-75 (cetrorelix; n = 22/treatment) in order to determine its effect on embryonic development. The 10 μM SB-75 treatment blocked embryo development beyond the compact morula stage (P < 0.001). To determine if this was a receptor mediated effect, we attempted to rescue development of SB-75 treated embryos with a histrelin challenge. Our treatments consisted of control (n = 30), 10 μM histrelin (n = 27), 10 μM SB-75 (n = 29), and 10 μM SB-75 in combination with either 1 μM (n = 27) or 10 μM (n = 25) histrelin. Both levels of histrelin partially rescued the inhibition of blastocyst formation by SB-75 (P < 0.01). Next, we were interested in examining the signaling cascade activated following binding of GnRH to its receptor in pre-implantation embryos. Toward this end, we treated embryos with inhibitors of either PKC or PKA. First, embryos were cultured in the presence of 0 (n = 33), 0.1 (n = 35), 1 (n = 35), or 10 (n = 35) μM GF109203X (GFX), a PKC inhibitor. Similar to the results obtained with SB-75, treatment with 10 μM GFX significantly reduced development to the compact morula stage and completely blocked blastocyst formation. Second, we treated embryos (n = 15 to 17/treatment) with different concentrations (0, 0.01, 0.1, 0.5, or 1 mM) of the PKA inhibitor, SQ22536. In contrast to treatment with GFX, rates of blastocyst formation were decreased only by 35% (P < 0.05) at the highest concentration of SQ22536. The percentage of embryos developing to the hatched blastocyst stage was decreased in a dose-dependent manner following SQ22536 treatment (P < 0.05); however, this effect was not consistent with SB-75 inhibition of blastocyst formation. We suggest that GnRH has an important autocrine effect on early embryonic development, potentially signaling via PKC. Funding for M Montagner was provided by CAPES, Brazil.

Development of improved protocols for cryopreservation of zona pellucida-intact porcine embryos could greatly impact the swine industry. Our aim was to investigate in vitro development following cryopreservation of embryos from Chinese Meishan (M) and occidental white cross (WC) breeds using a modified protocol described previously (Misumi K et al. 2003 Theriogenology 60, 253–260). First-parity M sows (n = 11) and WC gilts (n = 13) were observed for estrus every 12 h and inseminated at 12 and 24 h after estrous onset within breed using semen from 2 different boars. Females were sacrificed between Days 4.5 and 6 after estrus and embryos were collected using Beltsville embryo culture medium (BECM). Compact morula (CM) or blastocyst stage embryos from each female within breed were randomly allocated either directly into the culture system to serve as controls (68 M and 48 WC embryos) or to undergo cryopreservation. A total of 101 M and 78 WC embryos were cryopreserved using the following protocol: (1) 5 min in BECM + 10% ethylene glycol (EG); (2) 5 min in BECM + 10% EG + 0.27 M sucrose + 1% polyethylene glycol (PEG); and (3) 30 to 45 s in BECM + 40% EG + 0.36 M sucrose + 2% PEG. In the last solution, 5 to 10 embryos in a 5- to 10-μL microdrop attached to a fine glass pipette were exposed to the vapor phase of liquid nitrogen (LN2) for 15 s and then plunged into LN2. The pipette tip was broken and the tip and associated frozen microdrop were placed inside an LN2-submerged 2-mL cryotube containing a hole in the lid for 1 h. Next, embryos were thawed using a 4-step (5 min each) procedure: (1) BECM + 5% EG + 0.57 M sucrose; (2) BECM + 2.5% EG + 0.29 M sucrose; (3) BECM + 0.3 M sucrose; and (4) BECM alone. All procedures were performed with solutions maintained at 37°C. Cryopreserved and control embryos were cultured in 50 μL drops of modified Whitten's medium + 1.5% BSA under oil at 37°C in a 5% CO2 in air environment and scored daily for development. For embryos undergoing cryopreservation, retrieval rates from cryovials were 92% and 96% for M and WC, respectively. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than for WC (44%; P < 0.001; χ2-test). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than for frozen embryos from both breeds. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98% and 95%, respectively), but more M embryos developed to the hatched blastocyststage (22% for M v. 9% for WC; P < 0.05). Our results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos. Funding for M. Montagner was provided by CAPES, Brazil.