Adrenocorticotropin (ACTH), j3-endorphin, and the melanocyte-stimulating hormones (MSHs), which are products of a common precursor, pro-opiomelanocortin (POMC), are present in a variety of tissues other than pituitary. The recent detection of immunoreactive POMC-derived peptides in the male reproductive tract raised the possibility that these hormones might regulate reproductive function. To Pro-opiomelanocortin (POMC) is a precursor protein that contains the sequence for several bioactive peptides including adrenocorticotropin (ACTH), 8-endorphin, and melanocyte-stimulating hormones (MSHs) (1, 2). These POMC-derived peptides have been detected in a variety of tissues. The de novo synthesis of POMC-derived peptides has been demonstrated in the pituitary (for a review, see ref.2), hypothalamus (3-5), and human placenta (6, 7). Immunocytochemical studies have revealed the presence of POMC-derived peptides in the gastrointestinal tract (8, 9), pancreas (10), adrenal (11, 12), lymphocytes (13,14), and the unicellular organism, tetrahymena pyriformis (15). Recently, immunoreactive POMC-derived peptides also have been described in both male and female reproductive tissues (11,(16)(17)(18)(19)(20)(21). Materials similar to 3-endorphin and a-MSH were characterized in human semen (16) and rat testicular extract (17-19) by a variety of physicochemical techniques. Immunostainable POMC-derived peptides were detected in the Leydig cells of the testis and epithelium of epididymis and other male reproductive tissues (11,20). The concentrations of POMC-derived peptides in these tissues are very low, suggesting that they could only have local paracrine and/or autocrine functions (18,19 Ham's F-12/Dulbecco-modified Eagle's medium, 1:1 (vol/ vol) (Irvine Scientific) supplemented with 15 mM Hepes buffer, 20 mg of gentamycin per liter, 5% horse serum, and 2.5% newborn bovine serum. Sera, Hepes, and gentamycin were purchased from GIBCO. Cells were subcultured every 4-6 days when subconfluent. To prepare mRNA, cells were grown to confluency in 100-mm tissue culture dishes (Corning), washed twice with phosphate-buffered saline, and scraped. The cells were pelleted by centrifugation, and the pellets were stored at -70°C until RNA extraction. The derivations of the Leydig cell line, TM3, and the Sertoli cell line, TM4, have been described (22). I1OA cells, established from a mouse Leydig cell tumor by Shin (23), were obtained from the American Type Culture Collection.Blot Hybridization Analysis. Total RNA from various tissues was isolated by the urea/lithium chloride precipitation method (24), and poly(A)-containing RNAs were obtained by oligo(dT)-cellulose column chromatography (25). The poly(A)+ RNAs were denatured with 1 M glyoxal and 50% dimethylsulfoxide and were fractionated in 1.5% agarose gel as described by Thomas (26). The poly(A)+ RNAs were then transferred to nitrocellulose paper, and POMC-like mRNAs were identified by hybridization with a radioactive POMC complementary DNA (cDNA) probe and exposed to x-ray film. D...

The aim of this study was to determine the bidirectional release of immunoreactive inhibin-alpha (irINH-alpha) by different testicular compartments in the adult ram and to assess the effects of FSH on the distribution of inhibin in the testis. Immunoreactive INH-alpha was measured by RIA in fluid samples collected concurrently from the three testicular compartments--the seminiferous tubules, the interstitium, and the vascular system--through catheters inserted surgically into the rete testis, testicular lymphatic duct system, and spermatic veins, respectively. Overall, the concentration of irINH-alpha in rete testis fluid was 25 times the level in testicular lymph and over 500 times the concentration in peripheral blood. The pattern of irINH-alpha concentration in rete testis fluid was inversely related to that in testicular lymph, but i.v. administration of FSH had a decoupling effect on this relationship by depressing inhibin concentration in testicular lymph without affecting inhibin levels in rete testis fluid. Nevertheless, increased flow of testicular lymph more than compensated for the transient fall in irINH-alpha concentration so that, overall, the total output of inhibin via the testicular lymphatic duct system (and the vascular system) increased significantly. No persistent or significant changes were observed in the flow rate of rete testis fluid or concentration of irINH-alpha in the fluid after administration of FSH. The time frame for the response of the testis to FSH is indicative of the involvement of a mediator. Electrophoretic analysis of serially collected testicular lymph samples consistently revealed an FSH-induced release of a series of proteins in the M(r) range of 30,000-32,000.(ABSTRACT TRUNCATED AT 250 WORDS)