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We and others have shown that the inhibin/activin beta B-subunit gene is expressed differently in the gonads. Two species of 4.8- and 3.7-kilobase (kb) beta B-subunit messenger RNA (mRNA) with equal concentrations were identified in the testis, whereas 1 predominant 4.8-kb and a minor 3.7-kb mRNA were observed in the ovary. In this study, we analyzed the structures of these 2 mRNAs in rat testis and showed that both 4.8- and 3.7-kb beta B-subunit mRNAs were terminated at the region proximal to 2.2 kb down-stream from the translation stop codon. However, only 4.8-kb mRNA could be detected when RNA probes prepared from the 5'-region 1 kb up-stream from the translation start site were used for Northern blot analysis. Our observations suggested that the 2 heterogeneously sized beta B-subunit mRNAs are transcribed from different initiation sites. Transcription of the 4.8-kb mRNA was initiated at 3 adjacent nucleotides, GGA, 1.1 kb up-stream from the translation start codon ATG, whereas multiple transcription initiation sites spreading over 150 nucleotides upstream from the ATG codon were previously identified for 3.7-kb mRNA. Neither of the 2 transcripts contained TATA and CAAT boxes in their promoters. The 5'-flanking DNAs required for transcription of the 4.8- and 3.7-kb mRNA were examined by their ability to induce transient expression of the chloramphenicol acetyltransferase (CAT) gene in MA-10 Leydig tumor cells. A marked increase in CAT activity was detected when the 5'-flanking DNA for the 4.8- or 3.7-kb transcript was progressively shortened from its 5'-end. Maximal CAT activity was observed when -409 and -139 basepair beta B-subunit DNA up-stream from the 4.8- and 3.7-kb transcription initiation site, respectively, were fused to the CAT gene, suggesting the presence of a negative regulatory element(s) at the up-stream regions of these promoters. Although putative AP-2 sites were identified, treatment of the transfected cells with cAMP and/or phorbol 12-myristate 13-acetate did not apparently change CAT activity driven by either the 4.8- or 3.7-kb promoter. Our results concluded that 1) the two inhibin/activin beta B-subunit mRNAs were transcribed from different initiation sites; 2) both promoters may be controlled by up-stream negative regulatory elements; and 3) neither of these promoters is responsive to cAMP and/or phorbol esters under the conditions employed.

In addition to the feedback regulation of pituitary FSH secretion, gonadal activins have been shown to modulate physiological functions in the reproductive tissues. These observations suggested that activins and the receptors for these peptides may be coexpressed in gonadal tissues. In this study, we have cloned cDNAs encoding two species of type II activin receptors (ActRII and ActRIIB) from rat testicular mRNA and have shown that the two rat activin receptors share 97% similarity in the nucleotide sequence with those reported in the mouse. Two species [6 and 3 kilobases (kb)] ActRII mRNA were identified in all reproductive tissues of adult male and female rats. The 6-kb ActRII mRNA was the abundant form in most of the reproductive tissues. In placenta, the 6- and 3-kb mRNA were present in equal intensity. Interestingly, the ratio of the expression of two species of ActRII mRNA in rat testis changed with age. The 6-kb mRNA was the predominant form in immature 15- and 20-day-old testis, while the 3-kb mRNA increased with age and became the major form in mature testis. In female rats, however, the 6-kb ActRII mRNA was the abundant species in all of the ovaries examined, including immature, normal cycling, and pregnant rats. One major 2.25-kb species of ActRIIB mRNA was identified in all of the reproductive organs examined. Nucleotide sequence analysis of the isolated ActRIIB cDNA clones revealed that ActRIIB2 was the major isoform found in rat testis. The levels of expression of ActRIIB gene in rat testis or ovary were not changed during development. We conclude that 1) both type II and IIB activin receptor genes are widely expressed in the male and female reproductive tissues; and 2) the expression of 6- and 3-kb ActRII mRNA is tissue dependent as well as age dependent in rat testis.

The efficacy and specificity of human recombinant inhibin alpha-subunit as an immunogen were determined by studying its effect on the ovulation rate and serum gonadotropin levels in ewes. Beginning on July 12 (day 1 of the experiment), 2-yr-old Rambouillet ewes were immunized four times at intervals of 20, 30, and 30 days, respectively, by im injections of the alpha-subunit each time with 100 micrograms. Blood was collected from the jugular vein as required before and after ovulation. Ovulation rates were determined, beginning on day 87, by laparotomy before estrous synchronization by means of vaginal progestagen sponges. After synchronization, ewes were examined again. The ovulation rate before estrous synchronization was over 4 times higher in immunized than control ewes [8.7 +/- (+/- SE) 1.9 vs. 2.0 +/- 0.0]. After estrous synchronization, ovulation rates in the immunized and control ewes were 6.3 +/- 2.4 and 1.3 +/- 0.3, respectively; this increase was accompanied by higher pre- and postovulatory FSH levels, but there was no change in the pattern of circulating LH. Furthermore, the postovulatory FSH surge persisted for longer periods of time in the immunized than control animals. These results demonstrate the potency of the human recombinant alpha-subunit as an immunogen and illustrate its specificity in suppressing the effects of endogenous inhibin. The results also point to inhibin as an important regulatory factor in controlling the ovulation rate by modulating both pre- and postovulatory FSH levels.

The clusterin protein and its messenger RNA were identified in many tissues including testis. In this report, we demonstrate the expression of clusterin gene in four Leydig tumor cell lines, including mouse MA-10 and I-10 and rat R2C and LC-540. When the cells were incubated with 0.1 mM 8-bromo-cAMP or (Bu)2cAMP for 17 h, an unexpected, profound suppression of clusterin mRNA accumulation was observed. A 60-70% decrease in clusterin mRNA was observed in MA-10 and R2C cells, 10% in I-10 cells, and no apparent change in LC-540 cells. The inhibitory effect of cAMP was specific to the clusterin gene, since in the same cells cholesterol side-chain cleavage enzyme mRNA was drastically elevated in MA-10 and I-10 cells while alpha-tubulin mRNA levels were not changed in all four cell lines. The reduction could be detected as early as 4 h, and was evident at 17 h after cAMP administration. Removal of cAMP from culture media at 17 h prevented the decline of clusterin mRNA. The suppression of clusterin gene expression can also be demonstrated by treatment with human CG or forskolin, which were known to elevate intracellular cAMP levels. Our observations suggest: 1) cAMP negatively regulates clusterin gene expression in two Leydig tumor cell lines, MA-10 and R2C; 2) The inhibitory effect of cAMP on clusterin gene expression is probably acting through the protein kinase A pathway; and 3) The four Leydig tumor cell lines respond differently to cAMP in the expression of clusterin and side-chain cleavage genes.