Cloning of a novel inhibin alpha cDNA from rhesus monkey testis

Background  

Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA.     

Adrenal renin: a possible local regulator of aldosterone production

Extrarenal renin has been identified in a number of tissues, including the brain, the submaxillary gland, uterus, ovary, vascular endothelium, testes, pituitary gland, and the adrenal cortex. In some tissues, including the adrenal cortex, all of the components of the renin-angiotensin system have been identified; however, no specific physiologic role has been clearly demonstrated for these extrarenal renin-angiotensin systems. We have studied the role of the renin-angiotensin system in the adrenal cortex of the rat and have found that renin is localized and synthesized in the zona glomerulosa cells. Its production can be influenced by alterations in electrolyte balance, as well as the genetic background of the rat. In adrenal capsular explant cultures, a converting enzyme inhibitor can lower angiotensin II production and reduce the stimulation of aldosterone by potassium, suggesting that this system is involved in the aldosterone response to potassium. In addition to rat adrenals, renin has been identified in human adrenal tissue and human adrenal tumors, including aldosteronomas, and a patient with hypertension has been reported to have an adrenal tumor that appeared to be secreting renin into the circulation.     

Type-1 cannabinoid receptor expression in the frog, Rana esculenta, tissues: a possible involvement in the regulation of testicular activity

Endogenous cannabinoids and type-1 cannabinoid receptor (CB1) are widely produced and distributed in the central nervous system (CNS) and peripheral nerves in mammals. In addition, the detection of endocannabinoids and corresponding receptors in non nervous peripheral tissues indicates an involvement of the system in the control of a wide range of physiological activities, including reproduction. Recently, the existence of CB1 was also observed in lower vertebrates and in urochordate suggesting that the endocannabinoid system is phylogenetically conserved. Using RT-PCR, CB1 mRNA expression profiles were characterized in a wide range of tissues of the anuran amphibian, the frog, Rana esculenta. Besides a strong expression in the CNS, CB1 was also present in testis, kidney, liver, ovary, muscle, heart, spleen, and pituitary. The CB1 expression pattern has been characterized in both testis and CNS during the annual sexual cycle. In testis, CB1 is poorly expressed during the winter stasis of the spermatogenesis rising during the breeding season and resumption period. An expression profile mismatching to that observed in testis was detected in whole-brain preparations during the sexual cycle; in particular in the diencephalon, the encephalic area mainly involved in the control of reproductive functions. Furthermore, fluctuations inside isolated encephalic areas and spinal cord were observed all over the reproductive cycle. In conclusion, CB1 receptor is expressed in R. esculenta CNS and testis. As far as the gonad it concerns, our results suggest the involvement of the endocannabinoids in the control of reproductive function.     

The distribution of zinc in the subcellular fractions of the rhesus monkey testis

The zinc content in the testis of sexually immature, adult, and efferent duct-ligated adult rhesus monkeys was determined by atomic absorption spectrophotometry. Zinc content (μg/g wet wt) was found to be high in adult testis (165.9) when compared to immature (68.9) or efferent duct-ligated (104.2) animals. Analysis of subcellular fractions revealed that the concentration of zinc (expressed in relation to protein) was maximum in the microsomal fraction. The possible significance of this trace metal as a constituent of membrane proteins and enzymes, and as an activator of mitochondrial function in testis, is discussed.     

Ontogeny of androgen and estrogen receptor expression in porcine testis: effect of reducing testicular estrogen synthesis

Reducing endogenous estrogen leads to increased proliferation of porcine Sertoli cells during the first 2 months of life. The resulting increase in porcine Sertoli cell numbers is maintained through puberty. The reduced estrogen appears to be the direct hormonal mediator because essentially no changes are observed in other hormones. However, the mechanism for this effect on Sertoli cell proliferation is unknown. The objective of these studies was to evaluate estrogen receptors alpha and beta (ESR1 and ESR2) in conjunction with androgen receptor (AR) on Sertoli cells and other testicular cell types, as an initial step toward understanding how reduced estrogen leads to increased Sertoli cell numbers. Testis sections from treated animals (aromatase inhibition to decrease endogenous estrogen beginning at 1 week of age) and from littermate controls treated with vehicle were subjected to immunocytochemical labeling for ESR1, ESR2, and AR. Three observers scored Sertoli cells, interstitial cells, peritubular myoid cells, and germ cells for intensity of labeling (0: absent; 1+: weak; 2+: moderate; or 3+: strong labeling). AR in Sertoli cells was readily detected at 1 week of age, was very faint in 2-month vehicle controls, and labeling appeared to increase in 3-month vehicle controls. AR in Sertoli cells, interstitial cells, and apparently germ cells was increased in treated animals at 2 months of age compared with the vehicle controls. This increase was confirmed in western blots. ESR1 and ESR 2 were clearly present in Sertoli cells from 1-week-old animals; ESR in Sertoli cells generally decreased with age with the decrease more apparent for ESR2. ESR1 in Sertoli cells and peritubular myoid cells exhibited some treatment-related effects but reduction of endogenous estrogen did not appear to affect ESR2 in the boar testis. The observed alterations in AR and ESR1 may mediate the increases in Sertoli cell proliferation following inhibition of endogenous estrogen production or may reflect the altered function of the Sertoli cells and peritubular myoid cells.     

Combined analysis of microRNome and 3'-UTRome reveals a species-specific regulation of progesterone receptor expression in the endometrium of rhesus monkey

The establishment of endometrial receptivity is a prerequisite for successful pregnancy, which is controlled by a complex mechanism. MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as important regulators of gene expression. However, the contribution of miRNAs in endometrial receptivity is still unknown. Here we used rhesus monkey as an animal model and compared the endometrial miRNA expression profiles during early-secretory (pre-receptive) phase and mid-secretory (receptive) phase by deep sequencing. A set of differentially expressed miRNAs were identified, 8 of which were selected and validated using quantitative RT-PCR. To facilitate the prediction of their target genes, the 3'-UTRome was also determined using tag sequencing of mRNA 3'-termini. Surprisingly, about 50% of the 10,677 genes expressed in the rhesus monkey endometrium exhibited alternative 3'-UTRs. Of special interest, the progesterone receptor (PGR) gene, which is necessary for endometrial receptivity, processes an ultra long 3'-UTR (~10 kb) along with a short variant (~2.5 kb). Evolutionary analysis showed that the 3'-UTR sequences of PGR are poorly conserved between primates and rodents, suggesting a species-biased miRNA binding pattern. We further demonstrated that PGR is a valid target of miR-96 in rhesus monkey and human but not in rodents, whereas the regulation of PGR by miR-375 is rhesus monkey-specific. Additionally, we found that miR-219-5p regulates PGR expression through a primate-specific long non-coding RNA immediately downstream of the PGR locus. Our study provides new insights into the molecular mechanisms underlying endometrial receptivity and presents intriguing species-specific regulatory roles of miRNAs.     

Circadian and circannual changes in the testicular function of adult rhesus monkeys

The endocrine and gametogenic status of the testes were studied in 9 healthy adult rhesus monkeys of proven fertility throughout a one-year period. Testosterone levels were estimated by radioimmunoassay in blood samples collected at 4 h intervals over a 24 h period once a month. Semen samples and testicular biopsies were also examined once a month. A well-defined circandian rhythm was evident in serum levels of testosterone. The rhythmicity was less pronounced in February and September. The 24 h mean levels of serum testosterone were high between the months of August to March and low in the months of May to July. All animals did not uniformly respond to electro-ejaculation in April and May. Semen volume and total number of spermatozoa were maximal between September and March and least from April to August. Testicular biopsies indicated that all stages of spermatogenesis were evident between September and March and the spermatogenic activity was less evident between April and August. The contents of Sertoli cells showed a seasonal cyclicity; they were laden with lipid droplets during April to August when spermatogenesis was quiescent and vacuolated during September to March when spermatogenesis was active. These studies indicate that the testing of contraceptive drugs needs to be restricted to months of September to March in male rhesus monkeys otherwise, it is possible that the naturally occurring reproductive quiscence may be attributed to the effect of the drug being tested. The data accrued from the present studies also provide quantitative information on circulating levels of testosterone which could be used as a reference background while evaluating the contraceptive drug-effects in male rhesus monkeys.     

Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu

Sex hormones play an important role in adipose tissue metabolism by activating specific receptors that alter several steps of the lipolytic and lipogenic signal cascade in depot- and sex-dependent manners. However, studies focusing on steroid receptor status in adipose tissue are scarce. In the present study, we analyzed steroid content [testosterone (T), 17beta-estradiol (17beta-E2), and progesterone (P4)] and steroid receptor mRNA levels in different rat adipose tissue depots. As expected, T levels were higher in males than in females (P = 0.031), whereas the reverse trend was observed for P4 (P < 0.001). It is noteworthy that 17beta-E2 adipose tissue levels were higher in inguinal than in the rest of adipose tissues for both sexes, where no sex differences in 17beta-E2 tissue levels were noted (P = 0.010 for retroperitoneal, P = 0.005 for gonadal, P = 0.018 for mesenteric). Regarding steroid receptor levels, androgen (AR) and estrogen receptor (ER)alpha and ERbeta densities were more clearly dependent on adipose depot location than on sex, with visceral depots showing overall higher mRNA densities than their subcutaneous counterparts. Besides, expression of ERalpha predominated over ERbeta expression, and progesterone receptor (PR-B form and PR-A+B form) mRNAs were identically expressed regardless of anatomic depot and sex. In vitro studies in 3T3-L1 cells showed that 17beta-E2 increased ERalpha (P = 0.001) and AR expression (P = 0.001), indicating that estrogen can alter estrogenic and androgenic signaling in adipose tissue. The results highlighted in this study demonstrate important depot-dependent differences in the sensitivity of adipose tissues to sex hormones between visceral and subcutaneous depots that could be related to metabolic situations observed in response to sex hormones.     

Genetically determined hypercholesterolemia in a rhesus monkey family due to a deficiency of the LDL receptor

A family of rhesus monkeys comprising a sire, a dam, and four male offspring were fed a cholesterol-free Purina Chow diet for several months. The sire, 431-J, and two of the offspring, B-8204 and B-8806, had persistent plasma cholesterol levels in the range of 100-130 mg/dl, whereas the dam, 766-I, and the two other offspring, B-1000 and B-7643, exhibited a marked hypercholesterolemia in the 250-300 mg/dl range associated with an elevation of plasma LDL and apoB. When fed for 12 weeks a diet containing 12.5% lard and 0.25% cholesterol, sire, dam, B-1000 and B-7643 exhibited a marked hypercholesterolemia (500-800 mg/dl range), whereas B-8204 and B-8806 developed only a modest hypercholesterolemia (200-250 mg/dl). All animals were Lp[a]+. Skin fibroblasts from each animal and from control cells were grown in 10% fetal calf serum, transferred to 10% lipoprotein-deficient serum for 48 hr, and then incubated at 4 degrees C or 37 degrees C with 125I-labeled Lp[a]-free LDL. The fibroblasts from dam and offspring B-1000 and B-7643 bound and internalized 125I-labeled LDL less efficiently than control cells. Mathematical analyses of the 4 degrees C binding data indicated that there were no significant differences in LDL binding affinity between test and control cells suggesting that cells from the animals with a spontaneous hypercholesterolemia had a decreased number of LDL receptors. This conclusion was supported by the results of ligand and immunoblot analyses carried out on cell lysates separated by gradient gel electrophoresis. We conclude that a genetically determined LDL receptor deficiency was responsible, in part, for the spontaneous hypercholesterolemia observed in three out of the six family members and that this deficiency accounted for the hyperresponsiveness to a dietary fat and cholesterol challenge by the dam and the two offspring, B-1000 and B-7643. The hyperresponsiveness noted in the sire that had no evidence for LDL-receptor deficiency illustrates that factors other than the LDL receptor were responsible for the hypercholesterolemia attending the fat challenge.     

Pro-opiomelanocortin-derived peptides in the testis: evidence for a possible role in Leydig and Sertoli cell function

Pro-opiomelanocortin (POMC)-derived peptides such as beta-endorphin, ACTH, and MSHs were identified in the testis where they were exclusively localized in Leydig cells. Examination of testicular extracts by a variety of physicochemical and immunological techniques indicates that the processing of the POMC in the testis is very similar to that in the brain. By using a cDNA probe, the POMC-like mRNA present in total testis and cultured Leydig cells was 150-200 bases shorter than that in the hypothalamus and pituitary. In addition, POMC mRNA was localized to Leydig cells using in situ hybridization. The expression of the POMC-like gene and the accumulation of POMC-derived peptides in Leydig cell were shown to be under the control of gonadotropin. As the testis contains low concentrations of POMC-derived peptides, we suggested that they may be implicated in local regulatory events within this organ. This postulate was supported by results from in vivo and in vitro experiments suggesting that different portions of the POMC-molecule may have opposite effects on Sertoli cell functions. For example, MSHs increased cAMP accumulation and aromatase activity in these cells, while opioids inhibited Sertoli cell proliferation and androgen binding protein (ABP) secretion. Furthermore, following intratesticular administration of opiate antagonists, testosterone production was reduced, suggesting that Leydig cell function may be also modulated by beta-endorphin and/or other related peptides. Taken together, these studies support the hypothesis of a possible role of POMC-derived peptides in testicular function.     

Villus atrophy and malabsorption in a rhesus monkey

Villus atrophy involving the proximal small intestine was diagnosed in a rhesus monkey (Macaca mulatta) at necropsy. A juvenile, male, captive-born monkey exhibited weight loss, hypoalbuminemia, patchy alopecia, eosinophilia, and vomiting. D-xylose absorption was impaired. Contrast radiography revealed thickening of the gastric and intestinal walls, gaseous distension of intestines and a rapid transit rate. There was no clinicopathologic evidence of involvement of organs other than the gastrointestinal tract. The intestinal villus atrophy was similar in distribution and morphology to that of human celiac disease.