Evidence for a Sertoli cell, FSH-suppressible inhibiting factor(s) of testicular steroidogenic activity

By using a model of immature porcine Leydig and Sertoli cells cultured in serum free defined medium, we evidenced a paracrine control of Leydig cell steroidogenic activity by Sertoli cells via a secreted inhibiting protein(s). This protein(s), partially purified using gel filtration (M.W. 20,000-30,000) suppresses the steroidogenic responsiveness to LH/hCG by decreasing the specific LH/hCG binding (52% decrease) and hormone steroid biosynthesis (73% decrease) at a level(s) located between cAMP production and pregnenolone formation. The suppression of this inhibitor(s) by FSH, in a dose dependent manner, is one mechanism by which FSH "sensitizes" Leydig cell response to LH/hCG stimulation.     

Nociceptin is upregulated by FSH signaling in Sertoli cells in murine testes

In postnatal testes, follicle-stimulating hormone (FSH) acts on somatic Sertoli cells to activate gene expression directly via an intracellular signaling pathway composed of cAMP, cAMP-dependent protein kinase (PKA), and cAMP-response element-binding protein (CREB), and promotes germ cell development indirectly. Yet, the paracrine factors mediating the FSH effects to germ cells remained elusive. Here we show that nociceptin, known as a neuropeptide, is upregulated by FSH through cAMP/PKA/CREB pathway in Sertoli cells in murine testes. Chromatin immunoprecipitation from Sertoli cells shows that CREB phosphorylated at Ser133 associates with prepronociceptin gene encoding nociceptin. Analyses with Sertoli cells and testes demonstrates that both prepronociceptin mRNA and the nociceptin peptide are induced after FSH signaling is activated. In addition, the nociceptin peptide is induced in testes after 9days post partum following FSH surge. Thus, our findings may identify nociceptin as a novel paracrine mediator of the FSH effects in the regulation of spermatogenesis.     

LH contamination may explain FSH effects on rat Leydig cells

Treatment of immature, hypophysectomized male rats with 50 micrograms ovine FSH (NIH-FSH-S12) twice a day for 5 days stimulated the maximum quantity of 17 beta-hydroxyandrogen produced by isolated Leydig cells in response to hCG. Pretreatment of the FSH preparation with an LH antiserum in one study markedly reduced and in another study completely abolished this stimulatory effect of FSH, but only slightly impaired the capacity of the hormone to stimulate the Sertoli cell in vivo (epididymal androgen-binding protein). Administration of another highly potent FSH preparation (LER-1881) had no discernible effects on the dose-response characteristics of the Leydig cells but was superior to the NIH-FSH-S12 in its capacity for stimulating the Sertoli cell. When all hormone preparations were tested for their ability to stimulate steroid secretion from normal Leydig cells in vitro, a close correlation was obtained between their Leydig cell-stimulating activity (a measure of LH contamination) and their capacity to alter Leydig cell responsiveness after in-vivo treatment. FSH treatment had no effects on specific LH binding per 10(6) Leydig cells. It is concluded that the stimulatory influence of FSH on rat Leydig cells may to some extent be a result of the LH contaminating the hormone preparation.     

Inhibitory effect of Sertoli cell-cultured media on LH binding to mouse Leydig cells in culture

The aim of this study is to examine the influence of Sertoli cells on LH binding to Leydig cells in culture in immature mice. Leydig cells and Sertoli cells were obtained from the testes of immature C57BL/6Ncrj mice and were cultured in serum-free medium for 7 days. The LH binding to Leydig cells and the FSH binding to Sertoli cells were dependent on incubation time, the number of cells, and the amount of labelled hormone added. The dissociation constant for LH binding to Leydig cells was 7.3 x 10(-10) M. Co-culture of Leydig cells with Sertoli cells for 7 days decreased LH binding to Leydig cells. The binding was 34.9% of that to Leydig cells cultured alone. After cultivation of Leydig cells with spent Sertoli cell-cultured medium (SM) for the last 4 days of the 7-day culture period, LH binding to Leydig cells decreased to as low as 17.4% of that of the controls. For the controls, LH binding was measured in Leydig cells cultured in spent Leydig cell-cultured medium (LM). There was no difference between SM- and LM-cultures in the final survival rate or the percentage of cells showing histochemically demonstrated 3 beta-hydroxysteroid dehydrogenase activity. These data suggest that some factor or factors are secreted from the cultured Sertoli cells and inhibit the binding of LH to Leydig cells in culture.     

恒河猴睾丸间质细胞对支持细胞分泌雌二醇的影响

采用无血清培养的方法,分析了促肾上腺激素皮质激素(adrenocorticotropic hormone,ACTH)、黄体生成素(luteinizing hormone,LH)、cAMP、内啡肽(endorphin)和纳络酮(naloxone)对原代共培养的恒河猴(Macaca mulatta)睾丸间质细胞与支持细胞雌二醇分泌水平的影响。结果显示:ACTH、LH、cAMP和纳络酮对原代共培养恒河猴睾丸间质细胞与支持细胞的雌二醇分泌水平具有促进作用,并且这种影响与共培养的间质细胞数量呈线性关系,即共培养的间质细胞数量增加,雌二醇分泌水平亦明显上升;而内啡肽对原代共培养恒河猴睾丸间质细胞与支持细胞的雌二醇分泌水平有明显的抑制作用。研究表明,恒河猴睾丸的间质细胞对支持细胞分泌雌二醇具有调节作用。     

Further characterization of the effects of hypophysectomy, FSH and estrogen on LH stimulation of testosterone production in Leydig cells isolated from immature rats.

Hypophysectomy of immature rats results after 5 days in a loss of LH responsiveness of Leydig cells. LH responsiveness can be partly maintained by treatment with FSH for 5 days. When estradiol benzoate was administered together with FSH to hypophysectomized rats the maintenance of LH responsiveness was not observed. The loss in LH responsiveness after hypophysectomy in terms of testosterone production could not be explained by either a change in the amount of Leydig cells present in the Leydig cell preparation or to a higher conversion of testosterone. The LH-stimulated cAMP production in cells from hypophysectomized rats was very low compared to cells from intact rats. There was no difference between cAMP production of Leydig cells from untreated, FSH-treated or FSH plus estradiol benzoate treated hypophysectomized rats. During the first 2 days after hypophysectomy LH responsiveness in both untreated and FSH-treated rats showed a comparable decrease. From day 2 after hypophysectomy LH responsiveness remained at a constant level in cells from rats treated with FSH, but declined further in cells from untreated rats. A single injection of estradiol benzoate to hypophysectomized rats treated with FSH counteracted the effect of FSH on LH responsiveness, but only when estradiol was administered at that time after hypophysectomy, when the effect of FSH on LH responsiveness was clear.     

Novel alternatively spliced exon in the extracellular ligand-binding domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) selectively increases ligand affinity and alters signal transduction coupling during spermatogenesis

The expression of the paracrine signaling hormone pituitary adenylate cyclase-activating polypeptide (PACAP) is regulated in a cyclical fashion during the 12-day spermatogenic cycle of the adult rat testis. The precise functions of PACAP in the development of germ cells are uncertain, but cycle- and stage-specific expression may augment cAMP-regulated gene expression in germ cells and associated Sertoli cells. Here we report the existence of a heretofore unrecognized exon in the extracellular domain of the PACAP type 1 receptor (PAC1R) that is alternatively spliced during the spermatogenic cycle in the rat testis. This splice variant encodes a full-length receptor with the insertion of an additional 72 base pairs encoding 24 amino acids (exon 3a) between coding exons 3 and 4. The PAC1R(3a) mRNA is preferentially detected in seminiferous tubules and is expressed at the highest levels in round spermatids and Sertoli cells. Analyses of ligand binding and signaling functions in stably transfected HEK293 cells expressing the two receptor isoforms reveals a 6-fold increase in the affinity of the PAC1R(3a) to bind PACAP-38, and alterations in its coupling to both cAMP and inositol phosphate signaling pathways relative to the wild type PAC1R. These findings suggest that the extracellular region between coding exons 3 and 6 of PAC1R may play an important role in the regulation of the relative ligand affinities and the relative coupling to G(s) (cAMP) and G(q) (inositol phosphates) signal transduction pathways during spermatogenesis.     

Rat testis 17 beta-estradiol: identification by gas chromatography-mass spectrometry and age related cellular distribution

The aromatization of testosterone into 17 beta-estradiol (E2) was assessed in purified Leydig and Sertoli cells from rats aged 10-80 days. E2 was identified by gas chromatography-mass spectrometry (GC-MS) and measured both by radioimmunoassay (RIA) and GC-MS associated with stable isotope dilution. A potent competitive inhibitor of the aromatase activity, 4-hydroxyandrostenedione (4-OH-A) was used to test the enzymatic specificity. The basal aromatase activity was present in both cell types whatever the age of the animals. The basal E2 levels did not vary in Sertoli cells while a gradual increase was noted in Leydig cells until day 40, followed by a slight decrease in mature rats. In 10-day old animals, the aromatase activity was localized in Sertoli cells and highly stimulated by FSH; on day 20, both Sertoli and Leydig cells synthesized E2 although E2 from Sertoli cell origin was still predominant. Starting on day 20 until adulthood, the aromatase activity was under LH control in Leydig cells with a maximum around 40 days. The FSH and LH effects were mediated by cyclic AMP.     

Alternative transcriptional initiation and splicing of mouse Lamc2 message.

To investigate the mechanism of two forms of messages (5.1 and 2.4 kb) in mouse Lamc2, a gene encoding for the gamma2 chain of epithelial cell-specific laminin 5, we analyzed approximately 40 kb of genomic DNA containing the sequences from the first intron to the 23rd exon to the 3' untranscribed region. With the 5' rapid amplification of cDNA end (RACE) and primer extension technique using RNA from mouse kidney and thymic epithelial cells, we found that the smaller message starts at the 2895th base of the cDNA within the 19th exon. Upstream sequences of this alternative start site showed the promoter activity in the reporter assay with the secreted form of alkaline phosphatase (SEAP). We also identified a novel alternatively spliced exon (exon 19B) that includes two stop codons. These results revealed two possible additional open reading frame that are different from the previously described alternative human gamma2 peptide. Therefore, the mechanism for generating smaller message and the event of alternative splicing are quite distinct between mouse and human Lamc2, although genomic organization is highly conserved. The significance of the presence of alternative message is discussed.