Involvement of bone morphogenetic proteins in final follicular growth, ovulation and luteal function

Factors produced in the ovary, such as transforming growth factors beta members (TGFβ) and their receptors, play a key role during follicular deviation and ovulation. The interactions between ligands and receptors are involved in different ovarian functions under physiological or pathological conditions. In particular, factors produced by oocytes such as bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are involved in the regulation of ovarian functions such as follicular development and differentiation. However, most of the knowledge about these factors has been obtained using in vitro models or through spontaneous mutations in ewes and, despite the great relevance, little is known about the function of the oocyte factors in the ovarian physiology. In addition, the regulation and function of the TGFβ system in the regression process of the corpus luteum has not been evaluated in domestic species. In this regard, this thesis presents a series of studies about the involvement of TGFβ family members, specially BMPs, in the regulation of ovarian function including final follicular growth, ovulation, corpus luteum formation and luteolysis. The first study aimed to review the information available in the literature on the regulation and function of TGFβ family members in ovarian function. The objective of the second study was to investigate mRNA regulation of some TGFβ family ligands and their receptors during induced-luteolysis in vivo. Cows received an injection of prostaglandin F2α (PGF2α) on day 10 of the estrous cycle and luteal samples were obtained at 0, 2, 12, 24 or 48 h after treatment. The mRNA levels of BMP4, BMP6 and INHBA were upregulated at 2 h after administration of PGF2α (P<0.05) in comparison to 0 h. The relative mRNA expression of BMP2, BMP4, ACVR1B, INHBA and INHBB was upregulated at 12 h post PGF2α (P<0.05), whereas TGFBR3 was downregulated at 48 h. Collectively, the findings demonstrate that mRNA encoding several TGFβ family members are regulated after PGF2α administration in a time-specific manner, which suggests that this system is involved in both functional and morphological luteolysis in cattle. The objective of the third study was to evaluate the effect of BMP15 intrafollicular treatment on final follicular growth, ovulation and luteinization in cattle. Furthermore, it was evaluated the effect of gonadotrophins treatment in granulosa cells in vitro on the levels of transcripts encoding BMP15 receptors. Intrafollicular injections of rhBMP15 were performed at concentrations of 100 ng/mL, in dominant follicles, and 500 ng/mL, in preovulatory follicles. The intrafollicular injection of rhBMP15 tended (P=0.09) to inhibit follicular growth, whereas the treatment of preovulatory follicles support the hypothesis of an inhibitory effect on follicular growth and ovulation, without inhibiting luteinization. Follicles treated with BMP15 did not ovulate and became luteinized cysts, which was confirmed by serum progesterone levels. The mRNAexpression of BMPR2 and BMPR1B in granulosa cells in vitro was not altered by the treatment with LH or FSH. In conclusion, the treatment with BMP15 inhibits follicular development and ovulation, inducing luteinization and cysts formation in cattle. Future molecular studies are needed to elucidate the mechanism of action of BMP15. The fourth study was conducted to validate a technique for in vivo granulosa cells collection for gene expression studies. Six different protocols for granulosa cells isolation after follicular aspiration were evaluated aiming to identify the more appropriate procedure to extract RNA without the interference of other cells potentially present in the samples. The results suggest that the treatment of granulosa cells pellets with red blood cell lysis buffer, just after follicular aspiration, allows obtaining samples with higher RNA quality. With a well-validated protocol for cells isolation, it will be possible to continue the investigation of the function of follicular factors on granulosa cells in vivo. Taken together, the above-described studies besides generating basic knowledge to understand the role of local factors in ovarian physiology, may contribute to the understanding of pathological processes and to the development of technologies to promote contraception or to increase ovulation rate.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESFatores produzidos no ovário, como os membros da família dos fatores de crescimento transformantes beta (TGFβ) e seus receptores, exercem papel essencial durante a divergência folicular e ovulação. Interações entre ligantes e seus receptores estão envolvidas em distintas funções ovarianas sob condições fisiológicas ou patológicas. Em especial os fatores produzidos pelos oócitos como a proteína morfogenética óssea 15 (BMP15) e o fator de crescimento e diferenciação 9 (GDF9), desempenham papéis importantes na regulação das funções ovarianas como crescimento e diferenciação folicular. Entretanto, grande parte do conhecimento sobre esses fatores tem sido obtido em modelos in vitro ou através da observação de mutações espontâneas em ovelhas e, apesar da grande relevância, a função destes na fisiologia ovariana ainda é pouco conhecida. Além disso, o papel do sistema TGFβ na modulação do processo de regressão do corpo lúteo ainda não foi investigado nas espécies domésticas. Neste contexto, esta tese apresenta uma série de estudos acerca da participação de membros da família TGFβ, em especial das BMPs, na regulação de eventos ovarianos envolvendo o crescimento folicular final, ovulação, formação do corpo lúteo e luteólise. O primeiro estudo trata-se de uma revisão que teve por objetivo reunir a informação disponível na literatura sobre o tema. Posteriormente, um segundo estudo objetivou investigar o nível de transcritos de membros da família TGFβ e seus receptores durante a luteólise induzida in vivo. Foram utilizadas amostras de corpos lúteos bovinos coletados in vivo nos momentos 0, 2, 12, 24 e 48 h após a administração de uma dose de PGF2α no dia 10 do ciclo estral. Os níveis de RNAm de BMP4, BMP6 e INHBA foram regulados positivamente às 2 h após a administração de PGF2α (P<0,05) em comparação com 0 h. A expressão relativa de RNAm de BMP2, BMP4, ACVR1B, INHBA e INHBB foi regulada positivamente 12 h após a aplicação de PGF2α (P