Investigating the role of activin, inhibin and bone morphogenetic proteins during the first wave of mouse spermatogenesis

Abstract

Transforming Growth Factor β (TGFβ) superfamily proteins regulate many aspects of normal development, including spermatogenesis. However, their specific expression patterns during postnatal testis development and how they function to coordinate spermatogenesis is not well understood. This thesis addressed the hypothesis that differential synthesis of specific TGFβ superfamily members is central to normal male fertility. Developmentally regulated patterns of activin βA, βB and βC synthesis were documented in developing and adult mouse testes. Activin A levels decreased significantly during the first days and declined further within two weeks after birth. This was concordant with significant elevation of activin antagonists, Fst and inhibin/activin βC. This suggested that modulated synthesis and action of activins is required at the onset of spermatogenesis. Differential synthesis of other TGFβ superfamily members during testis development was identified using a cDNA array, leading to novel identification of BMP3 in the testis. Further assessment of BMPs 2, 3, 3b and 4 expression was performed. Similar to activin βA, BMP2 and BMP3b transcript levels were highest during the first postnatal week, when spermatogonia first differentiate. In contrast BMP4 mRNA levels peaked at the time of spermatocyte emergence, and BMP3 was detected in elongating spermatids. Recombinant BMP3 induced Smad3 nuclear localisation in primary cultures of Sertoli cells in the absence of BMP- or activin-responsive Smad phosphorylation. Thus modulated BMP synthesis is also a feature of normal testis development. The roles of inhibin and activin at the onset of spermatogenesis were investigated in vivo using two mouse models, one with reduced activin bioactivity (InhbaBK) and one deficient in inhibin production (Inha-/-). Testes from InhbaBK/BK mice were smaller from birth, while those from Inha-/- mice were larger from 4 dpp relative to wildtype littermates. Sertoli cell numbers were reduced in 0 dpp InhbaBK/BK testes, while gonocyte numbers were reduced in 0 dpp Inha-/- testes, demonstrating the converse affect of activin on somatic and germ cells. In newborns, gonocyte migration to the basement membrane appeared advanced in Inha-/- testes compared to wildtype littermates, and FSH levels were elevated in Inha-/- mice from 4 dpp. These data confirm that activin A enhances Sertoli cell proliferation, thereby contributing to fetal testis growth. In contrast, activin inhibition appears required for the growth and timely migration of gonocytes at the beginning of spermatogenesis. Collectively, this evidence indicates that the impact of inhibin changed from local control of gonocyte development at 0 dpp to that of endocrine regulation of FSH secretion after 4 dpp. The role of inhibin in the testis after this age was difficult to determine due to the elevated FSH levels in Inha-/- mice, which would independently affect the testicular phenotype. The novel information in this thesis demonstrates how several TGFβ superfamily ligands contribute to testis development and affect the onset of spermatogenesis. In addition, they provide a foundation for future analysis of their integration with other signalling systems that influence fertility.