Genetic Removal of Cyclin D2 Leads to Severe Hypogonadism and Infertility in FSHβ Null Male Mice.

Abstract

Follicle-stimulating hormone (FSH) is a pituitary-derived heterodimeric glycoprotein. It consists of a hormone-specific β subunit and an α subunit that is common to luteinizing hormone and thyroid-stimulating hormone. Our previous genetic studies indicated that male FSHβ null male mice demonstrate hypogonadism, reduced tubule size, and sperm number, but are fertile. Similarly, null males lacking cyclin D2, a key G1-S phase cell cycle protein are also fertile, display hypogonadism and reduced testicular tubule size and sperm number. Null males lacking other D-type cyclins such as cyclin D1 or D3 have also been generated and these are eugonadal and fertile. Our initial immunochemistry experiments confirmed that cyclins D1, D2 and D3 were all expressed in both Sertoli and spermatogonia at 7d postnatally, but their expression was spatially restricted in the adult testis. At postnatal day 42, cyclin D2 expression was undetectable in both Sertoli cells and spermatogonia, but it was barely detectable in early spermatocytes. Expression of cyclin D1 was restricted to mostly spermatogonia. Contrastingly, cyclin D3 showed a stage-dependent expression mostly restricted to later stages of spermatogenesis.To determine whether cyclin D1 or D3 compensate for cyclin D2 in cyclin D2 null mice, and whether D-type cyclins are affected in the absence of FSH, we performed western blot analysis using testes samples from adult control mice and mice lacking either FSHβ or cyclin D2.These data indicated that both cyclins D1 and D3 were upregulated in the absence of cyclin D2; whereas only cyclin D2 was slightly upregulated in the absence of FSH. To further characterize in vivo interactions between FSH signaling and cyclin D2, we generated double mutant mice lacking both FSHβ and cyclin D2 by a genetic intercross. Interestingly, male mice lacking both FSHβ and cyclin D2 display more severe hypogonadism and complete infertility. These double mutant phenotypes are distinct when compared to those of null mice lacking either FSHβ or cyclin D2 alone. Histological analysis of testes from adult double mutant mice indicated several classes of abnormal tubules including those with vacuolated Sertoli cells, or loss of germ cells, or hypospermatogenesis. In addition, ultrastructure analysis indicated abnormal sperm morphology in several tubules that show otherwise apparently normal stages of spermatogenesis. Further functional studies indicated defective Sertoli cell proliferation and differentiation/maturation as judged by aberrant expression of Sertoli cell markers including androgen receptor, clusterin and espin in testes of double mutants. Together, these results support the hypothesis that abnormal Sertoli cell proliferation/maturation contributed to severe hypogonadism, germ loss and infertility in FSHβ/cyclin D2 double mutant mice. Thus, we conclude that cyclin D2 is a critical determinant for FSH signaling in the testis and is presumably responsible for maintaining normal fertility of FSHβ null male mice. (Supported by NIH grants HD-043945, P20-RR024214 and The Hall Family Foundation, Kansas City, MO)