Role of FSH, numbers of FSH receptors and testosterone in the regulation of inhibin secretion during the seasonal testicular cycle of adult rams.

The regulation of inhibin secretion has not been elucidated fully in male ruminants. The aim of this study was to determine the relative importance of FSH and testosterone concentrations, and FSH receptors, in the control of secretion of immunoactive inhibin in rams. In Expt 1, temporal changes in hormone concentrations and testicular FSH binding were determined for two groups of rams (n = 4) kept under opposite, alternating 4 month periods of long (16 h light:8 h dark) and short (8 h light:16 h dark) days.

Alternative splicing converts the G-protein coupled follitropin receptor gene into a growth factor type I receptor: implications for pleiotropic actions of the hormone.

Pituitary follitropin (FSH) has pleiotropic actions on gonads, but it is not certain if all these events are mediated by a single receptor. A single gene for the FSH receptor undergoes extensive alternate splicing generating multiple transcripts, and several of these have been cloned and characterized from the sheep testis. In this study we have investigated the expression in HEK (human embryonic kidney) 293 cells of a cloned cDNA encoding the first eight exons of the FSH receptor along with a carboxyterminal extension that contributed a hypothetical single transmembrane domain.

Molecular cloning, structure, and expression of a testicular follitropin receptor with selective alteration in the carboxy terminus that affects signaling function.

During the molecular cloning of the ovine testicular follicle-stimulating (FSH) receptor that couples to the Gs-type effector systems, we discovered novel cDNA clones that were highly homologous. Some of these clones contained an insert of 1,584 bp, which consisted of a divergent 3' region spliced with a 5' region that was identical to nucleotides 724-1,924, forming part of the 9th and 10th exons, of the coding region of the ovine FSH receptor gene. The prominence of alternately spliced clone, which suggested important functional implications, prompted this detailed investigation.

Recognition of follicle stimulating hormone (alpha-subunit) by a recombinant receptor protein domain coded by an alternately spliced mRNA and expressed in Escherichia coli.

To assess the functional significance of putative proteins encoded by alternately spliced mRNA of the sheep testicular FSH receptor, a short form cDNA comprising of the first four exons (117 residues mature protein) was engineered for expression in Escherichia coli. The expressed protein of molecular mass 15 kDa was purified to homogeneity and verified by reaction with an antibody against a synthetic peptide sequence unique to the amino (N)-terminal region FSH receptor. The purified FSH receptor domain protein bound 125I-labeled hFSH in a ligand blot on polyvinylidine difluoride membranes.

Follitropin signal transduction: alternative splicing of the FSH receptor gene produces a dominant negative form of receptor which inhibits hormone action.

We have studied the functional properties of an alternately spliced form of sheep testicular FSH receptor cDNA that codes for a protein similar to a previously described active receptor but differs in the carboxy terminus in sequence and is also shorter by 25 residues. The receptor expressed in HEK 293 cells fails to activate adenylate cyclase. Cotransfection of stably expressing cells bearing FSH receptor that activates (Gs) adenylate cyclase with the altered receptor cDNA abrogates hormone response.

Bacterial expression of human chorionic gonadotropin alpha subunit: studies on refolding, dimer assembly and interaction with two different beta subunits.

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunit. The common alpha subunit contains two asparagine (N)-linked oligosaccharides. To study the function of carbohydrates on in vitro refolding of alpha subunit and dimer assembly, we generated recombinant non-glycosylated hCG alpha subunit (rNGl-hCGalpha) from E.

Pubertal development of ram lambs: Physical and endocrinological traits in combination as indices of postpubertal reproductive function.

The feasibility of using combinations of prepubertal measurements of body weight (BW), testicular size, and blood hormone concentrations (LH, FSH, PRL and testosterone) as indices of postpubertal reproductive function of rams was investigated. Data obtained on a group of 14 Suffolk rams born in March was analyzed by step-wise regression. Between the ages of 30 and 190 d, BW and testicular diameter (TD) were measured every 10 d, and a series of blood samples were collected (20-min intervals for 6 h) from the jugular vein every 20 d.

Molecular cloning and expression of the ovine testicular follicle stimulating hormone receptor.

A sheep testicular cDNA library constructed in pcDNA1 vector was screened with a probe generated by polymerase chain reaction (PCR) and corresponding to a 1.6 kb fragment of the rat luteinizing hormone receptor cDNA. Several clones hybridizing to the rat probe at low stringency were sequenced to obtain 95% of the putative full-length ovine follicle-stimulating hormone receptor (oFSH-R) cDNA.

Cloning of alternately spliced mRNA transcripts coding for variants of ovine testicular follitropin receptor lacking the G protein coupling domains.

We report the cloning and characterization of two alternately spliced forms of ovine testicular follitropin receptor mRNA. A smaller receptor cDNA (151 A1) of 727 bp codes for a possible soluble receptor protein of 134 amino acids arising from exons 1-4 of the full length receptor. The 1.

Differences in properties of the sheep testicular LH and FSH receptors.

Differences in binding and structural properties of ovine testicular FSH and LH receptors were investigated. The ovine FSH receptor did not discriminate between FSH of different species, although equine FSH was more reactive. In the same tissue, however, the LH receptor showed marked preference for ovine and bovine LH, reacting very weakly with other preparations of pituitary LH.

Hormone glycosylation required for lutropin receptor recognition in sheep testis.

The high affinity binding sites for ovine pituitary lutropin (oLH) present in DLS-1 sheep testis recognized only the fully glycosylated ovine or bovine hormone (bLH) in receptor binding assays using 125I-labeled oLH. Chemically deglycosylated (DG-) oLH or bLH which were fully active with other lutropin receptors (rat/pig) were completely inert in the DLS-1 receptor assay. In the same membranes, the FSH (follitropin) receptor reacted well with both glycosylated FSH and DG-oFSH.

Cytosolic glucocorticoid receptors in the porcine lung during development and after hypophysectomy or thyroidectomy.

The ontogeny of fetal lung glucocorticoid receptors and their regulation by the fetal pituitary, adrenal and thyroid gland during lung maturation were investigated. Sites with a specificity typical of glucocorticoid receptors were detectable in lung cytosol, with the order of potency of steroids being dexamethasone greater than cortisol greater than corticosterone greater than 11-deoxycorticosterone greater than progesterone greater than 17 alpha-hydroxyprogesterone greater than oestradiol-17 beta congruent to testosterone congruent to androstenedione congruent to oestrone. The binding affinity for [3H]dexamethasone was high (Kd = 0.

Purification and characterization of lutropin receptor from membranes of pig follicular fluid.

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000.

Gonadotrophin-binding components in porcine follicular fluid.

The follicular fluid is an important milieu for the growing and maturing oocyte and granulosa cells. In this study we investigated: (1) the properties of gonadotrophin-binding sites in the supernatant fraction of porcine follicular fluid (pFF) and compared them with those of membrane-bound receptors, and (2) the relative changes that occur in pFF and granulosa cell receptor-binding activity following hormone priming of gilts. 125I-Labelled human chorionic gonadotrophin (hCG) and 125I-labelled ovine FSH (oFSH) binding to particulate and supernatant fractions of pFF were hormone-specific and saturable.

Pubertal changes in the secretion of gonadotropic hormones, testicular gonadotropic receptors and testicular function in the ram.

Developmental changes in pituitary content and secretory patterns of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL), testicular size and steroidogenic function, testicular LH- and FSH-binding activity, and growth of the accessory sex organs were examined for 24 Dorset X Leicester X Suffolk rams (born in March) every 30 days from 30 to 150 days of age, and again at 200 days. Pituitary LH and FSH contents increased between 30 and 60 days of age and remained constant until 150 days, when contents were somewhat greater than on either 120 or 200 days. LH-pulse amplitude and frequency, and mean FSH concentration, were highest at 60 and (or) 90 days of age.

Sheep testicular gonadotropin binding sites: characterization and changes with surgical shortening of the scrotum.

Gonadotropin receptors in previously frozen (-70 degrees C) sheep testicular tissue were characterized, and methods of assessment of receptor binding activity were established and applied to an investigation of testicular function in the short scrotum ram. Binding of 125I-labelled ovine luteinizing hormone (125I-oLH) and 125I-labelled ovine follicle-stimulating hormone (125I-oFSH) to testicular membranes was highly specific and saturable. Uptake of labelled gonadotropins was proportional to the amount of membrane protein, with 125I-oFSH showing greater specific binding.

Conformational restrictions of the sheep testicular receptor discriminates pituitary lutropin and placental gonadotropins.

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation.

The reproductive--endocrine response of adult rams to sexual encounters with estrual ewes is season dependent.

An experiment was conducted with four adult, sexually inexperienced Finnish Landrace rams during the ovine nonbreeding (July) and breeding (October) seasons to determine the influence of components of the rams' mating behavior on the secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and testosterone. On four occasions in both seasons, blood was collected by jugular venipuncture at 20-min intervals during an 8-hr period while rams were (1) separated from, (2) observing with minimal direct physical contact, (3) mounting without intromission, or (4) mating estrous-induced ewes. In comparison with separation periods, mating activity in July was associated with increased mean LH (P less than 0.