Identification of novel genes expressed during spermatogenesis in stage-synchronized rat testes by differential display.

The molecular mechanism regulating spermatogenesis at different developmental stages remains largely unknown. In a vitamin A-deficiency (VAD) rat model, five distinct histologically defined, stage-synchronized testes: (i) resting spermatogonia and preleptotene spermatocytes at Day 0 of post-vitamin A treatment (PVA); (ii) early pachytene spermatocytes at Day 7 PVA; (iii) late pachytene at Day 15 PVA; (iv) round spermatids at Day 25 PVA; and (v) elongated spermatids at Day 35 PVA were used to study gene expression profiles by mRNA differential display. Twenty-four differentially expressed cDNA fragments were identified and cloned; oligonucleotide sequence analyses indicated that there are 12 novel gene sequences, half of which share no apparent match in current GenBank/EMBL databases. Other 12 VAD clones share sequence homology to membrane channel and transport, transcription and translation, cell cycle and morphogenesis, inducer and transducer, surface or secreted glycoproteins or enzymes, and other miscellaneous molecules. Semi-quantitative RT-PCR analyses against different stages of VAD testes demonstrated: (i) restricted expression of VAD1.2 and 1.3 (novel) on Day 25 PVA when round spermatids form; (ii) escalating pattern of VAD12 (Cx43) in Sertoli cells; and (iii) relative constant levels of VAD4 (A5D3), VAD26.1 (ribonuclease), and VAD27 (GRP8) in spermatogenesis.

Differential expression of gap-junction gene connexin 31 in seminiferous epithelium of rat testes.

Spermatogenesis, a tightly regulated developmental process of male germ cells in testis, is associated with temporal and spatial expression of certain gap-junction connexins. Our findings by RT-PCR indicate that the Cx31 gene is expressed in testis tissue of adult and postnatal rats. During the postnatal spermatogenic process, the Cx31-specific signal became detectable at 15 dpp and onward by in situ hybridization, and apparently localized in the basal compartment of seminiferous epithelium where active spermatogonia and early primary spermatocytes reside. No signal was found in the luminal region. In adult testes, spermatids of elongation phase were also Cx31 positive. Immunohistochemical analysis with mouse anti-Cx31 antibody gave a similar staining pattern, providing further evidence that the gap-junction protein is abundant in the basal seminiferous epithelium, in accordance with the cellular distribution of Cx31 mRNA. These results represent the first demonstration of Cx31 expression at both transcriptional and protein levels in the seminiferous epithelium of rat testes. Thus, Cx31 may play a role in cell-cell communication during spermatogenesis.