Immunostainable inhibin alpha-subunit has been demonstrated in rat testes in a pattern consistent with localization in Sertoli cells. In the present study the distribution of alpha-subunit immunostaining was compared to those of beta-A- and beta-B-subunits. Immunostaining of alpha-subunit was present in the seminiferous epithelium of fetal, neonatal, pubertal, and adult rats as well as in Sertoli cells in culture. The distribution of inhibin beta-B-subunit immunostaining in this epithelium was consistent with Sertoli cell localization similar to that of the alpha-subunit. The predominant staining with antibodies against the beta-A-subunit was in nuclei of immature germ cells around the periphery of each seminiferous tubule. The most probable localization of this staining was in the nuclei of pachytene and zygotene spermatocytes. Specific immunostaining with beta-A-subunit antiserum was also evident in the seminiferous epithelium adjacent to the tubular lumen. Immunoreactive alpha- and beta-A-subunit staining was present in a Leydig cell line, and beta-A immunoreactivity was present in interstitial cells of neonatal rat testes. After hypophysectomy, inhibin alpha-subunit immunostaining decreased, beta-A-subunit staining did not change, and beta-B-subunit staining increased. We conclude that immunoreactive inhibin subunits are present in multiple cells in the testis and that the amounts of immunostainable subunits in the seminiferous epithelium are differentially regulated.
The genes encoding rat inhibin alpha- and beta-B-subunits were isolated and characterized. Both genes contain one intron that interrupts the region coding for the precursor portion of the alpha- and beta-B-subunits. The transcription start sites of alpha- and beta-B-subunit genes were determined by primer extension and nuclease mapping assay using mRNA from rat ovary and testis. Transcription of the alpha-subunit gene initiates predominantly at three adjacent sites with similar intensity. Several potential transcription start sites of beta-B-subunit gene are spread over 150 nucleotides upstream from translation initiation site. Neither of these two genes contains obvious TATA or CCAAT boxes. The alpha-subunit gene contains many GA clusters in the promoter region, while beta-B-subunit gene is highly GC rich. Several GGGCGG repeats and their inverted sequences, which are the potential binding sites for transcription factor Spl, were observed at the 5'-end as well as at the coding region of the beta-B-subunit gene. The potential cAMP-responsive element CTGCGTCAG was identified in alpha-but not beta-B-subunit gene. This sequence is identical to the cAMP- and phorbol ester-inducible DNA fragment found in human preproenkephalin gene. The different structure of the promoter region of rat alpha- and beta-B-subunit genes and the presence of a potential cAMP-inducible DNA sequence in alpha- but not beta-B-subunit gene is consistent with the hypothesis that transcription of alpha- and beta-B-subunit genes in rat is regulated by different mechanisms.
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