In vitro maturation (IVM) and parthenogenesis of adult murine primary ovarian follicles is ameliorated in the absence of anti-mÜllerian hormone (AMH)

Abstract

OBJECTIVE: To improve the IVM outcome of adult primary ovarian follicles in a murine model with the emphasis on the role of AMH in folliculogenesis.DESIGN: Primary(1°) and secondary(2°) ovarian follicles were isolated from adult mice and cultured in different serum and AMH conditions. The competence of the oocytes grown was assessed through parthenogenesis followed by cleavage and blastocyst formation.MATERIALS AND METHODS: 1° and 2° follicles were isolated from CBA/BL6 F1 mice at 9 weeks. 1° follicles were grown in droplets of aMEM with 1% ITS, 10 mIU/mL of LH, 100 mIU/mL of recombinant human (rh) FSH, with the following conditions 1)FBS containing AMH from male fetuses, 2)female FBS, and 3)female FBS with 5ug/mL of rhAMH. 2° follicles were grown in aMEM with 1% ITS, 100 mIU/mL of rhFSH in the same serum and AMH conditions as 1° follicles. Maturation was performed with hCG and EGF. Parthenogenesis was performed in Ca2+-free KSOM with SrCl2 and CCB and cultured in CZB medium up to 5 days for blastocyst development.RESULTS: 1° follicle growth into 2° follicles was enhanced with female FBS compared to unsexed FBS(86.4% : 64.9% p<0.01) and blocked by AMH. MII yield, in the female FBS group was improved in comparison with those cultured in unsexed FBS for both 1° and 2° follicles(69.5% : 50.9% p<0.01; 80.9% : 63.5% p<0.01). For 2° follicles, AMH significantly compromised MII oocytes yield(80.9% : 33.3 p<0.01) but the complete blockage observed in 1° follicles did not occur. The yield of competent oocytes via parthenogenesis was improved in both the 1° and 2° follicles(26.6% : 15.1% p<0.05; 57.2% : 30.4% p<0.05) when cultured in female FBS and adversely affected by AMH. 2 blastocysts were derived from 85 2° follicles in female FBS and 1 blastocyst out of 89 2° follicles in unsexed FBS.CONCLUSION: AMH plays a vital role in suppressing adult 1° to 2° follicle development in vitro. The yield of primary follicle growth, maturation, and developmental competence of oocytes is significantly improved in conditions of absent AMH. OBJECTIVE: To improve the IVM outcome of adult primary ovarian follicles in a murine model with the emphasis on the role of AMH in folliculogenesis. DESIGN: Primary(1°) and secondary(2°) ovarian follicles were isolated from adult mice and cultured in different serum and AMH conditions. The competence of the oocytes grown was assessed through parthenogenesis followed by cleavage and blastocyst formation. MATERIALS AND METHODS: 1° and 2° follicles were isolated from CBA/BL6 F1 mice at 9 weeks. 1° follicles were grown in droplets of aMEM with 1% ITS, 10 mIU/mL of LH, 100 mIU/mL of recombinant human (rh) FSH, with the following conditions 1)FBS containing AMH from male fetuses, 2)female FBS, and 3)female FBS with 5ug/mL of rhAMH. 2° follicles were grown in aMEM with 1% ITS, 100 mIU/mL of rhFSH in the same serum and AMH conditions as 1° follicles. Maturation was performed with hCG and EGF. Parthenogenesis was performed in Ca2+-free KSOM with SrCl2 and CCB and cultured in CZB medium up to 5 days for blastocyst development. RESULTS: 1° follicle growth into 2° follicles was enhanced with female FBS compared to unsexed FBS(86.4% : 64.9% p<0.01) and blocked by AMH. MII yield, in the female FBS group was improved in comparison with those cultured in unsexed FBS for both 1° and 2° follicles(69.5% : 50.9% p<0.01; 80.9% : 63.5% p<0.01). For 2° follicles, AMH significantly compromised MII oocytes yield(80.9% : 33.3 p<0.01) but the complete blockage observed in 1° follicles did not occur. The yield of competent oocytes via parthenogenesis was improved in both the 1° and 2° follicles(26.6% : 15.1% p<0.05; 57.2% : 30.4% p<0.05) when cultured in female FBS and adversely affected by AMH. 2 blastocysts were derived from 85 2° follicles in female FBS and 1 blastocyst out of 89 2° follicles in unsexed FBS. CONCLUSION: AMH plays a vital role in suppressing adult 1° to 2° follicle development in vitro. The yield of primary follicle growth, maturation, and developmental competence of oocytes is significantly improved in conditions of absent AMH.