Expression of luteinizing hormone receptor during development of bovine fetal ovary.

Steroids and gonadotrophins are essential for the regulation of late stages of preantral development and antral follicular development. Although the luteinizing hormone receptor (LHCGR) has been detected in the preantral follicles of rats, rabbits, and pigs, its expression, in bovine fetal ovary, has not been demonstrated. Based on this, we aimed to investigate the expression of the LHCGR and LHCGR mRNA binding protein (LRBP), as well as, to quantify bta-miR-222 (a regulatory microRNA of the LHCGR gene) during the development of bovine fetal ovary. In summary, LHCGR expression was observed in the preantral follicle in bovine fetal ovary, from oogonias to primordial, primary and secondary stages, and the mRNA abundance was lower on day 150 than day 60. However, the mRNA abundance of LRBP followed the opposite pattern. Similar to LRBP, the abundance of bta-miR-222 was higher on day 150 than day 60 or 90 of gestation. The LHCGR protein was detected in oogonia, primordial, primary, and secondary follicles. Moreover, both oocytes and granulosa cells showed positive immunostaining for LHCGR. In conclusion, we suggest the involvement of LHCGR/LRBP/bta-mir222 with mechanisms related to the development of preantral follicles in cattle.

Pre-fertilization approach using α-l-fucosidase modulates zona pellucida hardening during bovine in vitro embryo production.

The polyspermy occurrence is considerably lower under in vivo compared to in vitro embryo culture conditions, suggesting that the presence of some factors in the maternal environment is responsible for this. The α-L-fucosidase (FUCA) is a natural glycosidase present in the oviductal fluid, therefore, this study aimed at investigating the effect of adding FUCA to the hardening of the zona pellucida (ZP), polyspermy control, and embryonic yield and quality of bovine blastocysts produced in vitro. In the first experiment, the effect of FUCA (0.125 U/mL) was evaluated during the entire in vitro fertilization (IVF). However, it was demonstrated to be embryotoxic by completely inhibiting the blastocyst formation. In the second experiment, the FUCA (0.125 U/mL) was tested as short-term incubation before IVF (pre-fertilization step) for 30 min or 2 h, which demonstrated that FUCA treatment for 30 min resulted in ZP hardening. In the third experiment, a pre-fertilization FUCA treatment (1 h) at different concentrations (0, 0.0625, and 0.125 U/mL) showed that FUCA (0.0625 U/mL) improved pre-fertilization ZP hardening and tended to increase monospermic fertilization rates but did not improve embryo yield and quality. Together, it has been demonstrated that FUCA can induce oocyte pre-fertilization ZP hardening and might improve monospermic fertilization performance, and this effect is dependent on both variables (protein concentration and incubation time).

Molecular phenotypes of bovine blastocyst derived from in vitro-matured oocyte supplemented with PAPP-A.

Insulin-like growth factor-1 (IGF-1) regulates cellular lipid content, whereas pregnancy-associated plasma protein-A (PAPP-A) increases IGF-1 bioavailability. Using in vitro-matured cumulus-oocyte complexes, we aimed to evaluate the impact of PAPP-A on the blastocyst lipid content, embryo cryotolerance and embryonic transcriptional profile. We determined that PAPP-A did not affect the lipid content of oocytes, blastocysts, or blastocyst yield (P > 0.05). However, PAPP-A modulated the embryo transcriptional profiles by downregulating PPARGC1A and AKR1B1, which are related to lipid metabolism; CASP9, a pro-apoptotic gene; and IFN-τ, a marker of embryo quality (P < 0.05). Furthermore, the use of PAPP-A improved blastocyst re-expansion in the first 3 h of culture after vitrification (P < 0.05). Although PAPP-A did not affect the blastocyst lipid content or embryo production, we suggest that embryonic transcriptional modulation could contribute to maintain the balance in embryo lipid metabolism. Furthermore, PAPP-A's approach seems to control key intracellular pathways that improve post-cryopreservation development of blastocysts.