Cell interactions in testis development: Overexpression of c‐mos in spermatocytes leads to increased germ cell proliferation

Abstract

Possible functions of the c-mos proto-oncogene during spermatogenesis were investigated through perturbations of its expression in transgenic mice. Two promoters, one from the pre-meiotic male germ cell-specific mouse phosphoglycerate kinase 2 gene, and the other from the post-meiotic male germ cell-specific rat RT7 gene were used to direct expression of c-mos. Northern blot analysis of testis RNA from transgenic PGK-c-mos mice indicated elevated levels of c-mos RNA in spermatocytes and spermatids compared to controls. No transgene expression was detected in any other tissue examined, suggesting that the mouse PGK2 promoter, like the previously used human PGK2 promoter, confers correct cell-specific expression onto c-mos. The promoter from a newly characterized rat gene, RT7, was shown to direct expression specific to post-meiotic spermatids. Transgenic mice carrying an RT7-lacZ construct displayed immunoreactive bacterial beta-galactosidase as well as enzyme activity in round spermatids. The cellular specificity for beta-galactosidase expression observed in RT7-lacZ transgenic animals was in agreement with endogenous RT7 transcript expression. Northern blot analysis of testis RNA of RT7-c-mos transgenic mice showed elevated levels of c-mos in spermatids, but not in other cells or tissues examined. Western blot analysis demonstrated elevated levels of p43c-mos in spermatids of both PGK-c-mos and RT7-c-mos transgenic animals, but only PGK-c-mos transgenics had increased p43c-mos levels in spermatocytes. Both RT7-c-mos and PGK-c-mos transgenic mice are fertile and show no tendency toward transformation. RT7-c-mos mice have no discernible phenotype associated with the c-mos overexpression in spermatids. However, PGK-c-mos transgenic males exhibited a significant increase in germ cell number, as determined by cell counts using total germ cells and germ cells fractionated by centrifugal elutriation. Because mitotic divisions of germ cells occur prior to PGK-c-mos transgene expression, our observations suggest that c-mos overexpression in spermatocytes causes an alteration in cell-cell interactions.

Spermatocyte/Spermatid-specific Thioredoxin-3, a Novel Golgi Apparatus-associated Thioredoxin, Is a Specific Marker of Aberrant Spermatogenesis

“…Western Blot Analysis-Elutriated rat germ cells were prepared according to Higgy et al (37). Elutriated fractions, of ϳ90% purity, were obtained for elongated spermatids, round spermatids, and spermatocytes.…”

Interactional Cloning of the 84-kDa Major Outer Dense Fiber Protein Odf84

“…Spermatocytes and spermatids were obtained by centrifugal elutriation of SD rat spermatogenic cells as described previously (30,41), and RNA was isolated as described above. RNA samples were analyzed by Northern blotting/hybridization using Duralon-UV membranes (Stratagene) and indicated random-primed probes as described (39).…”

“…The odf27 gene is abundantly transcribed in spermatids and translated in elongating spermatids. We demonstrated that the odf27 promoter confers spermatidspecific expression (29,30) and that it is activated by the spermatid-specific Crem transcription factor (31). Analysis of the predicted Odf27 amino acid sequence revealed two putative domains.…”

Outer Dense Fibers Serve as a Functional Target for Cdk5·p35 in the Developing Sperm Tail

“…A single cell suspension was obtained by repeated pipetting of tubules and filtration through a 120-m nylon mesh. Pachytene spermatocytes, round spermatids, and elongating spermatids were then isolated by centrifugal elutriation (33,34) of released germ cells.…”