Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

C S Haas,M T Rovani,P B D Gonçalves,B G Gasperin,V Bordignon,A D Vieira,F C Oliveira,R Ferreira

doi:10.21451/1984-3143-ar789

Abstract

Mutations in growth and differentiation factor 9 (GDF9) gene are associated to sterility or, paradoxically, increased ovulation rate in ewes. Despite its importance, the exact function of GDF9 in ovarian physiology is still poorly understood. This study aimed to investigate GDF9 function during dominant follicle growth and its regulation in follicular fluid. The regulation of GDF9 receptors in GnRH/LH-stimulated granulosa cells was also investigated. In a first experiment, a new follicular wave was induced and the intrafollicular GDF9 treatment into the largest growing follicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml (n = 4) had no effect on follicular growth, estrus manifestation and ovulation compared to control (PBSinjected) follicles (n = 3). In a second experiment, follicles were obtained just after follicular deviation (day 4 after follicular emergence) and the abundance of GDF9 in follicular fluid did not differ between healthy dominant (n = 4) and atretic subordinate follicles (n = 4), as assessed by western blot analysis. Finally, mRNA expression of BMPR2 and TGFBR1 receptors was evaluated in granulosa cells obtained from preovulatory follicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 h after i.m. GnRH administration (n = 4-5 follicles/moment). Both receptors were significantly up regulated 12 h after GnRH treatment. Present results do not confirm the hypothesis that GDF9 inhibits dominant follicle growth and suggests a minor role in determining follicle fate. In the other hand, GDF9 receptors regulation in GnRH/LH-stimulated granulosa cells provides the first in vivo evidence of its involvement in the complex cascade of events that culminates in ovulation and luteinization in cattle.

Highlights

Folliculogenesis and follicle differentiation are processes that culminate in ovulation, being mainly coordinated by well-established endocrine mechanisms
This study aimed to investigate growth and differentiation factor 9 (GDF9) function during dominant follicle growth and its regulation in follicular fluid
No significant difference were observed in GDF9 protein abundance in follicular fluid from dominant compared to subordinate follicles collected after follicular deviation (P > 0.05; Fig. 2)

Summary

Introduction

Folliculogenesis and follicle differentiation are processes that culminate in ovulation, being mainly coordinated by well-established endocrine mechanisms. Since all the follicles are under the same endocrine environment, ovarian-produced factors are involved in dominant follicle selection, regulating important events such as proliferation of granulosa cells, differentiation and steroidogenesis In this context, the involvement of oocyte-secreted factors such as growth and differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and their receptors, has been demonstrated in sheep (Knight and Glister, 2006). Spontaneous heterozygous inactivating mutations in BMP15 and GDF9 genes induce superovulation, whereas homozygous mutation (absence of functional protein) causes sterility in ewes as a consequence of impaired preantral follicle development (Galloway et al, 2000; Hanrahan et al, 2004) These interesting findings were reproduced using passive and active immunization against these proteins. In both sheep and cattle, short-term immunization increased ovulation rate (Juengel et al, 2004b, 2009), with no negative effects on fertilization, embryo development and pregnancy maintenance in ewes (Juengel et al, 2004b)

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