Biosynthesis, glycosylation, and secretion of rat luteinizing hormone alpha- and beta-subunits: differential effects of orchiectomy and gonadotropin-releasing hormone.

We have studied the de novo biosynthesis and secretion of LH subunits in pituitary quarters from orchiectomized and intact control adult male rats and their regulation by GnRH. After labeling with [35S]cystine ([35S]Cys), [35S]methionine, or [3H]glucosamine ([3H]GlcN) in the presence or absence of 10(-8) M GnRH, tissue lysates and media were immunoprecipitated with antisera to LH beta, then LH alpha (after removal of TSH by immunoprecipitation with anti-TSH beta), and the products were analyzed by sodium dodecyl sulfate gradient gel electrophoresis. During a 12-min pulse labeling with [35S]methionine, three forms of immunoreactive alpha were labeled at 21,000, 18,000, and 12,000 mol wt. After a 30-min chase with excess unlabeled methionine, the 12,000 form decreased from 10% to 3% of total radioactivity, while the 21,000 form increased from 57% to 69%, implying a precursor-product relationship. Neither orchiectomy nor GnRH had any effect on [35S]Cys or [3H]GlcN incorporation into intracellular or secreted total proteins. After a 6-h continuous labeling, incorporation of [35S]Cys into intracellular combined LH alpha in castrates was 158% of the control value, combined LH beta was 304%, and free alpha was 466%. The [3H]GlcN to [35S]Cys ratio, reflecting relative glycosylation, was unchanged in castrates for total proteins or LH alpha and somewhat decreased for LH beta and free alpha. Orchiectomy increased [35S]Cys-labeled secreted LH beta and free alpha to 183% and 231% of control values, respectively. Relative glycosylation of secreted LH alpha, LH beta, and free alpha was unchanged in castrates. Incorporation of [35S]Cys into intracellular combined LH alpha, LH beta and free alpha-subunit was unaffected by GnRH in pituitaries from intact rats. In castrates, LH alpha was unchanged, but LH beta and free alpha were slightly increased. Incorporation of [3H]GlcN into intracellular combined LH alpha, LH beta, and free alpha was increased with GnRH in both intacts and castrates, such that the 3H to 35S ratio, reflecting relative glycosylation, was also increased with GnRH. In castrates, the ratios, as a percentage of the control, were, respectively 250%, 250%, and 223% for LH alpha, LH beta, and free alpha. In intact animals, the ratios were 221%, 281%, and 143%, respectively. Incorporation of both [35S]Cys and [3H]GlcN into secreted subunits was increased in most instances, such that the 3H to 35S ratio was increased only for LH beta.(ABSTRACT TRUNCATED AT 400 WORDS)

Sertoli cell maturation is impaired by neonatal passive immunization with antiserum to luteinizing hormone-releasing hormone.

Male rats treated with a single injection of antiserum to LHRH (LHRH-AS) at 5 days of age have small testes as adults. In the present investigation, the serial maturation of the hypothalamic-pituitary-gonadal axis was studied in young male rats passively immunized with LHRH-AS. Testicular and epididymal weights, serum androgen and gonadotropin levels, testicular receptors for human CG (hCG), and androgen binding protein (ABP) concentrations in serum, testis, and epididymis were compared in developing animals treated with a single ip injection of LHRH-AS or normal rabbit serum. Rats treated with LHRH-AS had lower serum concentrations of ABP at all ages; the highest levels were on days 22-24, which were several days later than controls. Testicular weight was about 60% that of the control at all ages from 10-90 days. A reduction in epididymal weight to 80% that of the control was seen only in adults at days 60 and 90. Testicular ABP content increased steadily with age, but its concentration peaked at day 17 for controls and day 22 for LHRH-AS treated animals. Both testicular and epididymal ABP content were commensurate with testicular weight in controls and treated rats through day 45. Similarly, hCG-receptor content and concentration increased steadily with age, but differences between control and treated groups paralleled testicular weight. These results suggest an effect of LHRH blockade at a critical period which impairs early testicular growth and causes a permanent reduction in growth. Sertoli cell function and hCG-receptor appearance are impaired in proportion to this reduction.

Orchiectomy in young rats results in differential regulation of follicle-stimulating hormone and luteinizing hormone content.

While it is generally accepted that GnRH stimulates release of pituitary gonadotropins, it is not clear what regulates synthesis. The orchiectomized immature rat, with sustained high plasma levels of LH and FSH, provides an opportunity to study how gonadotropin biosynthesis responds to loss of the gonad. We have measured plasma and pituitary LH and FSH in castrate and sham operated rats after orchiectomy at 15, 30, 45, and 60 days of age. Plasma FSH and LH concentrations by RIA were markedly elevated in castrates within one to three days after castration, and they remained elevated in all groups. By contrast, pituitary content measurements revealed differences between the two gonadotropins: while LH content in castrates consistently exceeded that in controls, FSH content in castrates was lower. Pituitary LH excess was evident by seven to ten days after castration. The pituitary FSH deficit in younger animals was similarly apparent by seven to ten days. In the older groups, however, FSH content decreased as early as three days, but returned toward normal by 21 days. In orchiectomized young rats, pituitary LH is elevated, but FSH content is depressed. This discrepancy is delayed, but more marked, in younger rats. In view of sustained high plasma levels after castration, our findings imply differential regulation of synthesis, processing, or storage of the two gonadotropins.

Kinetics of secretion of luteinizing hormone subunits and free alpha, and effects of gonadotropin releasing hormone.

While GnRH is known to stimulate release of pituitary gonadotropins, its acute effects on the kinetics of secretion of the various hormones and subunits are not well characterized. Pulse-chase experiments were therefore performed to compare the time course of secretion of newly synthesized LH subunits and free alpha from rat pituitary quarters, and to study the effects of GnRH. After a 1-h pulse labeling with [35S]cysteine in the presence or absence of 10(-8) M GnRH, cultures were chased with excess unlabeled cysteine for 1, 2, 4, 8, or 20 h. Tissue lysates and media were immunoprecipitated sequentially with antisera to PRL, GH, LH beta and LH alpha, and the products were analyzed by gel electrophoresis. Labeled LH alpha was completely secreted by 4 h of chase without GnRH, and by 2 h with GnRH, as shown by its appearance in media and depletion from pituitary. Newly synthesized LH beta was depleted only at 8 h with GnRH from pituitary, suggesting much slower secretion. Incorporation of 35S into LH beta was approximately half that into LH alpha. Newly synthesized free alpha subunit was secreted between 4 and 20 h without GnRH, and by 8 h with GnRH. Free alpha incorporated a similar amount of radioactivity as LH beta. GnRH had no effect on the timing of the secretion of labeled total protein, PRL, GH, or TSH subunits. The amount of label incorporated during the pulse was greatest for PRL, approximately 15% of total protein label. GH incorporated approximately 20% as much label as PRL, with the glycoprotein subunits somewhat lower. The total amount of 35S incorporated into each protein studied was not affected by GnRH. The data indicate that short term in vitro GnRH exposure during a 1-h pulse labeling and chase acts to accelerate early secretion of LH alpha, LH beta and free alpha but does not affect overall protein synthesis. The peptide hormones PRL and GH incorporated the greatest amount of label and were the most briskly secreted hormones, while LH alpha led the glycoprotein subunits in the amount of incorporation and rapidity of secretion.

NIH Funding and Career Development Opportunities for Women in Reproductive Immunology.

The National Institutes of Health (NIH) has a number of mechanisms available to support research, research training and research career development. The various institutes, centers and special offices within the NIH have, as part of their missions, a diverse array of basic and clinical research goals. Much of the scientific spectrum of reproductive immunology, however, falls within the mission of the National Institute of Child Health and Human Development (NICHD). There have been a number of recent innovations in the preparation, review and funding of NIH grant applications. Specific opportunities, such as fellowships, career awards, and new investigator status, are available for investigators at different career stages. A wealth of information is available on the NIH website at www.nih.gov. NIH staff are available to answer your questions and to provide administrative guidance in preparing grant applications.

Sperm morphology, motility, and concentration in fertile and infertile men.

BACKGROUND: Although semen analysis is routinely used to evaluate the male partner in infertile couples, sperm measurements that discriminate between fertile and infertile men are not well defined. METHODS: We evaluated two semen specimens from each of the male partners in 765 infertile couples and 696 fertile couples at nine sites. The female partners in the infertile couples had normal results on fertility evaluation. The sperm concentration and motility were determined at the sites; semen smears were stained at the sites and shipped to a central laboratory for an assessment of morphologic features of sperm with the use of strict criteria. We used classification-and-regression-tree analysis to estimate threshold values for subfertility and fertility with respect to the sperm concentration, motility, and morphology. We also used an analysis of receiver-operating-characteristic curves to assess the relative value of these sperm measurements in discriminating between fertile and infertile men. RESULTS: The subfertile ranges were a sperm concentration of less than 13.5 x 10(6) per milliliter, less than 32 percent of sperm with motility, and less than 9 percent with normal morphologic features. The fertile ranges were a concentration of more than 48.0 x 10(6) per milliliter, greater than 63 percent motility, and greater than 12 percent normal morphologic features. Values between these ranges indicated indeterminate fertility. There was extensive overlap between the fertile and the infertile men within both the subfertile and the fertile ranges for all three measurements. Although each of the sperm measurements helped to distinguish between fertile and infertile men, none was a powerful discriminator. The percentage of sperm with normal morphologic features had the greatest discriminatory power. CONCLUSIONS: Threshold values for sperm concentration, motility, and morphology can be used to classify men as subfertile, of indeterminate fertility, or fertile. None of the measures, however, are diagnostic of infertility.

Efficacy of superovulation and intrauterine insemination in the treatment of infertility. National Cooperative Reproductive Medicine Network.

BACKGROUND: Induction of superovulation with gonadotropins and intrauterine insemination are frequently used to treat infertility. We conducted a large, randomized, controlled clinical trial of these treatments. METHODS: We studied 932 couples in which the woman had no identifiable infertility factor and the man had motile sperm. The couples were randomly assigned to receive intracervical insemination, intrauterine insemination, superovulation and intracervical insemination, or superovulation and intrauterine insemination. Treatment continued for four cycles unless pregnancy was achieved. RESULTS: The 231 couples in the group treated with superovulation and intrauterine insemination had a higher rate of pregnancy (33 percent) than the 234 couples in the intrauterine-insemination group (18 percent), the 234 couples in the group treated with superovulation and intracervical insemination (19 percent), or the 233 couples in the intracervical-insemination group (10 percent). Stratified, discrete-time Cox proportional-hazards analysis showed that the couples in the group treated with superovulation and intrauterine insemination were 3.2 times as likely to become pregnant as those in the intracervical-insemination group (95 percent confidence interval, 2.0 to 5.3) and 1.7 times as likely as those in the intrauterine-insemination group (95 percent confidence interval, 1.2 to 2.6). The couples in the intrauterine-insemination group and in the group treated with superovulation and intracervical insemination were nearly twice as likely to conceive as those in the intracervical-insemination group. CONCLUSIONS: Among infertile couples, treatment with induction of superovulation and intrauterine insemination is three times as likely to result in pregnancy as is intracervical insemination and twice as likely to result in pregnancy as is treatment with either superovulation and intracervical insemination or intrauterine insemination alone.