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Anthony R. Bellvé, PhD.


E-mail: bellve@cuccfa.ccc.columbia.edu

Regulation of spermatogenesis during development

Our research is focused on growth factors regulating the initial onset of spermatogenesis, and on the differentiation of the germ cell nucleus during its morphological transformation and subsequent fertilization of the egg. At the onset of spermatogenesis in the male, which occurs just after birth in most species, spermatogonial stem cells are quiescent and arrested in G2 of the cell cycle. The mechanisms controlling the subsequent renewal of DNA synthesis and cell proliferation, remain unknown. In our search for relevant factors, we found activity present in the mammalian testes that dramatically stimulates DNA synthesis in previously quiescent fibroblasts in culture. Significantly, the partially purified activity also stimulated DNA synthesis in spermatogonial stem cells in organ culture, based on an increase in the incorporation of bromo-deoxyuridine. The factor(s) is active in a dose-dependent manner, and, when added at maximal concentration, its effect can be down-regulated completely by addition of Mullerian inhibiting substance, transforming growth factor b, or inhibin, but not by activin. The last four factors are all members of the TGFb superfamily. The mitogenic activity, when purified 300,000-fold from bovine testes, by ion exchange, affinity, and reverse-phase chromatography, proved primarily to be 15-16K proteins. Studies by SDS-PAGE, peptide mapping, and microsequencing, identified fibroblast growth factor-1 and -2. Native or recombinant FGF-2, stimulates DNA synthesis in spermatogonial stem cells in a dose-dependent manner, with an ED50 of 3.7 pM. Moreover, both FGF-1 and FGF-2 mRNAs, and the type II FGF receptor, are synthesized by developing mouse testes, based on Northern blots, PCR, and immunoblots analyses. In the future we will be defining the role of FGFs in this essential process, and searching for novel factors, by applying contemporary molecular strategies.

In studies of germ cell nuclear differentiation, we have identified and purified a novel protein (thecin) associated with the sperm nucleus during spermiogenesis, the last phase of spermatogenesis. During its assembly, thecin, an 80-75K protein covers the anterior pole of the condensing nucleus, based on immunocytochemistry and immunoelectron microscopy. As the sperm leave the testis and enter the epididymis, the protein acquires a bipolar distribution, on the anterior-dorsal and posterior-ventral margins of the nucleus. Concomitantly, the protein undergoes an endoproteolytic cleavage, to yield a 49K peptide. This dynamic transition occurs in a period when the genome is inactive, and therefore provides a new avenue to study the process of sperm maturation. Purified sperm thecin proved to be a rare and novel protein. Moreover, the encoding cDNA contains an unusual motif of cysteines, a domain of glutamine repeats, a region rich in serine and threonine, and two putative nuclear localization signals. Regions of the protein share homology to the transcription factors, TFIID, SNF5, and SP1. Further, thecin shares structural homology to mitosis-inducing factors. What function this novel protein actually serves during spermatogenesis, epididymal maturation, and/or fertilization, remains to be determined and characterized.

Selected references:
Bellvé, A.R., Chandrika, R. and Barth, A. (1990). Polar distribution and transition of an epitope domain in the perinuclear theca during mouse spermatogenesis. J. Cell Sci. 96: 745-756.

Bellvé, A.R., Chandrika, R., Martinova, Y.S. and Barth, A.H. (1992). The perinuclear matrix as a structural element of the mouse sperm nucleus. Biol. Reprod. 47: 451-465.

Moss, S.B., Burnham, B.L. and Bellvé, A.R.. (1993). The differential expression of lamin epitopes during mouse spermatogenesis. Molec. Reprod. Devel. 34: 164-174.

Liévano, A., Santi, C.M., Serrano, J., Treviño, C.L., Bellvé, A.R., Hernandez-Cruz, A. and Darszon, A. (1996). T-type Ca2+ channels and a1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction. FEBS Letters 388: 150-155.

Baker, J., Hardy, M.P., Zhiu, J., Bondy, C., Lupu, F., Bellvé, A.R. and Efstradiatis, A. (1996). Effects of an Igf1 gene null mutation on mouse reproduction.. Molec. Endocrin. 10: 903-918.

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