Relaxin positively regulates matrix metalloproteinase expression in human lower uterine segment fibroblasts using a tyrosine kinase signaling pathway.

Despite the importance of relaxin to normal parturition in various species and its potential as an etiological agent in preterm delivery in women, knowledge regarding the mechanisms by which relaxin alters cervical connective tissue is extremely limited. An established in vitro model for human pregnancy cervix, human lower uterine segment fibroblasts, was used to determine the effects of relaxin as well as those of progesterone on the expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1. The results demonstrate that relaxin is a positive regulator of matrix metalloproteinase expression, as it stimulates the expression of procollagenase protein and mRNA levels, stimulates prostromelysin-1 protein and mRNA levels, and inhibits tissue inhibitor of metalloproteinase-1 protein expression. Stimulation of procollagenase and prostromelysin-1 expression by relaxin does not involve phorbol-12-myristate-13-acetate- sensitive PKCs. Relaxin-stimulated tyrosine phosphorylation of the putative receptor and inhibition by a receptor tyrosine kinase inhibitor suggest that the relaxin receptor is probably a tyrosine kinase receptor. Inhibition of c-Raf protein expression using an antisense oligonucleotide inhibits relaxin regulation of matrix metalloproteinase and tissue inhibitor of metalloproteinase-1, suggesting that a signaling pathway involving c-Raf kinase mediates relaxin action.

The preterm prediction study: maternal serum relaxin, sonographic cervical length, and spontaneous preterm birth in twins.

OBJECTIVE: The risk of spontaneous preterm birth has been related to decreased cervical length and to increased serum relaxin. To explore a relationship between these findings, we used data collected from two prior studies to correlate relaxin levels with cervical length and risk of spontaneous preterm birth in women with twin pregnancies. METHODS: In a secondary analysis of data collected in two previous observational studies of risk factors for preterm birth, relaxin levels in maternal serum and cervical length were measured at 24 (n= 188) and 28 (n= 145) weeks in women with spontaneous twin pregnancies. Relaxin, as a continuous variable, was related by logistic regression analysis to risk of spontaneous preterm birth before 37, 35, and 32 weeks' gestation, and by Spearman correlation coefficients to cervical length at 24 and 28 weeks. Cervical length at 24 weeks was known to be correlated with spontaneous preterm birth before 37, 35, and 32 weeks (P =.03,.01, and.002, respectively) in this study population. RESULTS: Cervical length did not correlate with relaxin levels at 24 (P=.601) or 28 (P=.304) weeks. Relationships between relaxin and spontaneous preterm birth were observed at 24 weeks for births before 37 weeks (odds ratio [OR] 1.56, 95% confidence interval [CI] 1.00, 2.44; P=.05), and at 28 weeks for births before 35 weeks (OR 1.97, 95% CI 1.05, 3.70; P=.034) and 32 weeks (OR 2.43, 95% CI 1.01, 5.83; P=.048). CONCLUSION: The absence of an association between relaxin and cervical length suggests that increased relaxin does not explain the inverse correlation between cervical length and spontaneous preterm birth in women with spontaneous twin pregnancies.

Luteal progesterone relates to histological endometrial maturation in fertile women.

To examine the relationship between endometrial histological maturation and reproductive hormones, we studied 11 fertile women, aged 18-37 yr. All participants had had at least 1 previous pregnancy and cycled regularly, every 25-35 days. Women collected daily, first morning voided urine for measurement of estradiol and progesterone metabolite excretion, estrone conjugates (E1c), and pregnanediol glucuronide (Pdg), respectively, throughout the cycle of study. Hormones were normalized for creatinine. Between 7-9 days after home detection of a LH surge (Sure Step), participants underwent an endometrial biopsy using a small bore (Pipelle) catheter. Tissue was prepared for histological and biochemical analyses. The histological analysis is reported herein. Endometrium was dated by 3 authors (N.S., D.H., and S.P.), all of whom were blinded to the participant's identity or timing of biopsy within her cycle. Final dating was agreed upon based upon the method of Noyes et al. E1c and Pdg were integrated throughout the cycle using the trapezoidal rule, and correlations were sought between deviation from expected histology (based upon urinary hormones and LH surge) and integrated hormone values. E1c varied over a 2-fold range in these normal women, from 1196-2040 ng/cycle. Pdg excretion was much more variable, ranging from 22-119 microg/cycle. No relationship could be found between histological lagging of endometrial maturation and lower excretion of E1c. A moderate correlation was observed (Spearman's r = 0.6; P < 0.05) between degree of histological maturation and integrated Pdg. Of two women with evidence of a disparity between gland and stromal development (glands lagging behind stroma by >2 days), one excreted 24 microg Pdg/cycle, the next to lowest value. We conclude that normal fertile women experience a wide range of hormone concentrations in the face of normal endometrial maturation. Progesterone appears to exert a dose-related effect on endometrial maturation, and the techniques we used, although relatively crude clinical measures, appeared to be sufficient to detect this relationship.

Endothelin stimulation of rat uterine segment contractility is estrogen-dependent.

To determine whether the effect of endothelin upon in vitro uterine contractility requires estrogen, immature female Long Evans rats were subcutaneously injected daily for 3 days with either estradiol benzoate or vehicle. The uterine contractile response to endothelin (5 or 100 nM) was measured. A response to endothelin in vehicle-treated animals exposed to physiologic (5.6 mM) or high (70.6 mM) potassium levels was also noted. Vehicle-treated uteri did not respond to endothelin; the contractions in estrogen-treated uteri increased by 288%. With high potassium, and without estrogen, no response to endothelin was seen. These data indicate that the contractile effect of endothelin on the uterus requires estrogen.

Elevated maternal serum relaxin concentrations throughout pregnancy in singleton gestations after superovulation.

OBJECTIVE: To test the hypothesis that superovulation results in elevated maternal circulating relaxin concentrations throughout the second and third trimesters of pregnancy, independent of the pattern of hCG secretion. METHODS: Two groups of women with singleton gestations were studied: a group of nine women who achieved pregnancy after stimulation with human menopausal gonadotropin and a group of six women who achieved pregnancy without prior stimulation. Peripheral blood samples were drawn approximately every 5 weeks throughout the second and third trimesters. Serum relaxin concentrations were measured using a human relaxin-specific enzyme-linked immunosorbent assay; hCG was measured by an immunofluorometric assay. RESULTS: The stimulated group had significantly higher relaxin levels throughout pregnancy (P=.007, multivariate analysis of variance) than did nonstimulated controls. The mean relaxin level in stimulated patients was 1.78 ng/mL (95% confidence interval [CI] 1.5, 2.17) and in nonstimulated subjects the level was 0.73 ng/mL (95% CI 0.59, 1.25). Spline fits demonstrated that stimulated patients had higher relaxin levels throughout the second and third trimesters. There was no significant difference in hCG concentrations between the two groups (P=.61). CONCLUSION: In singleton gestations after superovulation, maternal serum relaxin concentrations are significantly higher throughout the second and third trimesters of pregnancy. These differences are independent of the pattern of hCG secretion. It appears that luteal relaxin secretion is controlled by factors in addition to hCG.

Dogfish shark (Squalus acanthias) testes contain a relaxin.

Relaxin is a 6-kd polypeptide that exerts important hormonal effects in many female mammals. Relaxin is produced by the ovary, placenta, or uterus in many mammalian species. The functions of relaxin in the male mammal are not yet firmly established, but there is some evidence suggesting an exocrine effect on sperm motility and fertilizability. In the male mammals that have been studied, relaxin is produced by the prostate gland (human) or seminal vesicles (boar). However, in the bird, the testis is the likely source of relaxin. Among the elasmobranchs, ovaries obtained from dogfish sharks have been shown to contain a polypeptide hormone that is structurally, biologically, and immunologically similar to mammalian relaxins, but the male reproductive tract of this species has not previously been investigated as a potential source of relaxin. Extracts of testes obtained from mature dogfish sharks have now been tested by a specific relaxin bioassay and by a homologous porcine radioimmunoassay for the presence of relaxin. Both crude and partially purified testicular extracts contained unmistakable guinea pig pubic symphysis-"relaxing" activity and relaxin-like immunoactivity. Following immunoaffinity purification, the shark testis polypeptide had an apparent specific activity of 88 microg porcine relaxin equivalents per milligram in the radioimmunoassay, which is similar to the immunoactivity of pure shark ovarian hormones. These data, therefore, strongly support the view that in dogfish sharks, the male as well as the female gonad produces relaxin. Furthermore, as the dogfish shark has existed as a species for about 200 million years, the data suggest that testicular relaxin appeared early in vertebrate evolution.

Demonstration of a relaxin receptor and relaxin-stimulated tyrosine phosphorylation in human lower uterine segment fibroblasts.

To elucidate the mechanism of relaxin action, we studied the binding characteristics of human relaxin and its effects on intracellular concentrations of cAMP and tyrosine phosphorylation of cellular proteins in a model system of human cervix, human lower uterine segment fibroblasts. Human relaxin labeled with 125I bound specifically to a single class of high-affinity relaxin binding sites, distinct from insulin receptors, with a mean (+/-SEM) dissociation constant (Kd) of 4.36 +/- 1.7 x 10(-9) M and a mean of 3220 +/- 557 binding sites per cell in human lower uterine segment fibroblasts. Relaxin, in quantities that were shown previously to stimulate intracellular levels of cAMP in other cell types, had no effect on intracellular levels of cAMP in human lower uterine segment fibroblasts even in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). Incubation of the cells with relaxin caused a significant increase in tyrosine phosphorylation of a protein with an apparent Mr of approximately 220 kDa in these cells. In concert with results of recent studies that demonstrated that the Mr of the relaxin receptor is approximately 220 kDa, our data suggest that the phosphorylated protein is likely to be the relaxin receptor.

Gonadotropin-releasing hormone agonist and human menopausal gonadotropin (hMG) short protocol, but not hMG alone, rescues the corpus luteum from the preceding cycle.

OBJECTIVE: To determine whether ovulation induction regimens, particularly the short protocol, has an effect on the corpus luteum (CL) from the previous cycle. DESIGN: Infertility patients were followed in an academic research environment. Patients were treated with either the short protocol (GnRH agonist [GnRH-a] and hMG) or hMG alone in a controlled ovarian hyperstimulation cycle. SETTING: Infertility center in academic setting. PATIENTS: Patients requiring ovulation induction. INTERVENTIONS: The blood samples were drawn on day 2 or 3 as a baseline before initiation of any treatments, generally on day 5 and usually every other day thereafter until ovulation. Serum P, E2, and relaxin were determined. MAIN OUTCOME MEASURES: Serum P, E2 and relaxin. RESULTS: Patients receiving hMG alone showed no change in serum P levels in the first few days of treatment whereas most short-protocol patients (18/30; 60%) showed an increase of P within 3 days of the initiation of treatment. The increase in P almost always was associated with an increase in relaxin as a marker of the luteal production of this P. CONCLUSION: The short protocol with its flare of gonadotropins is able to stimulate the CL from the previous cycle, resulting in an early increase in P that comes from the CL as indicated by its association with an increased relaxin in the same subjects.

Use of synthetic canine relaxin to develop a rapid homologous radioimmunoassay.

Canine relaxin (cRlx) was synthesized by a combination of solid-phase methods and sequential site-directed disulfide bond formation. Proof that the intended molecule had been synthesized was obtained by analytical HPLC of the intact and reduced molecule, by amino acid and sequence analysis, and by receptor binding and in vivo mouse interpubic ligament bioassays. Antisera to synthetic cRlx were raised in six male rabbits; these cross-reacted with relaxins of other species, but not with insulin, LH, FSH, hCG, or prolactin (PRL). Three of the antisera neutralized relaxin-induced interpubic ligament formation in estrogen-primed mice in vivo. A new homologous cRlx RIA was developed through the use of rabbit antiserum 79888, synthetic cRlx for standards and 125l-labeled trace, and a goat anti-rabbit lgG-polyethylene glycol precipitant. The new RIA can be completed in 26 h and has a sensitivity of 0.195-0.39 ng cRlx/tube. Intra- and interassay coefficients of variation were 3% and 12.5%. During pregnancy in bitches, serum cRlx rose to about 10 micrograms/ml. Immunoactive cRlx was also detected in serum, colostrum, and milk of lactating bitches, but not in large volumes (100-300 microliters) of serum of pseudopregnant or estrous bitches. Immunoreactive cRlx was also found in seminal plasma, but not in serum, of male dogs. The new homologous cRlx RIA is simple, rapid, sensitive, and specific, and will be used in future studies of canine relaxin physiology.

The effect of ovulation induction on the concentration of maternal serum relaxin in twin pregnancies.

OBJECTIVE: Our purpose was to determine the effects of fetal number, various ovulation induction treatments, and placental hormones on the concentration of maternal serum relaxin. STUDY DESIGN: The concentrations of relaxin, human chorionic gonadotropin, estriol, and alpha-fetoprotein were determined in blood samples drawn at 16 to 18 weeks for prenatal diagnosis in 72 singleton and 115 twin pregnancies and analyzed by one-way analysis of variance, correlation analysis, and stepwise multiple linear regression of the log-transformed data. RESULTS: The maternal serum concentrations of each of the four measured hormones were significantly higher in the twin pregnancies than in the singleton pregnancies: 1.4-fold for relaxin, 1.9-fold for human chorionic gonadotropin, 1.9-fold for estriol, and 2.2-fold for alpha-fetoprotein (all p < 0.01). The concentrations of each of the four hormones were significantly correlated with each of the others and with the number of fetuses (p < 0.01), except that estriol was not significantly correlated with human chorionic gonadotropin. The serum relaxin concentration in twin pregnancies after treatment with follicle-stimulating hormone and luteinizing hormone (menotropins) (n = 10) was 3.3-fold that in twins resulting from spontaneous ovulation (n = 89, p < 0.01). In twins resulting from in vitro fertilization or gamete intrafallopian transfer (n = 9) the serum relaxin concentration was 2.6-fold higher than in twins resulting from spontaneous ovulation (p < 0.01). The effect of clomiphene citrate (1.2-fold, n = 7) failed to reach statistical significance. CONCLUSIONS: The second fetus causes a 1.4-fold increase in the concentration of maternal serum relaxin in twin pregnancies. Induction of ovulation with menotropins causes an additional 3.3-fold increase, whereas in vitro fertilization or gamete intrafallopian transfer treatment causes an additional 2.6-fold increase over that seen in twin pregnancies that followed spontaneous ovulation.

Relaxin secretion in in vitro fertilization pregnancies.

OBJECTIVE: This study was designed to determine whether the late luteal functional status of the corpora lutea in in vitro fertilization cycles alters the secretion of relaxin during pregnancy. STUDY DESIGN: Analysis of serum relaxin, human chorionic gonadotropin, and steroid concentrations in sera of women with pregnancies viable beyond the twelfth week as a result of in vitro fertilization treatment was performed. RESULTS: The serum estradiol and progesterone concentrations decreased 5.5- and 4-fold from days 5 to 6 after human chorionic gonadotropin to days 11 to 13 after human chorionic gonadotropin, respectively. The serum relaxin concentration increased 8-fold between the 11- to 15-day interval and the 16- to 50-day interval after human chorionic gonadotropin and another 6-fold to the 51- to 90-day interval after human chorionic gonadotropin (all p < 0.01). Multiple linear regression analysis showed that the serum estradiol level 11 to 13 days after human chorionic gonadotropin and the serum human chorionic gonadotropin level 11 to 15 days after human chorionic gonadotropin were the most powerful paired predictors of the concentration of serum relaxin measured in the 11- to 15-day interval after human chorionic gonadotropin interval (R2 = 0.39, n = 50), the 16- to 50-day interval (R2 = 0.61, n = 51), and the 51- to 90-day interval (R2 = 0.55, n = 39). CONCLUSION: Secretion of relaxin is determined by an interaction of the late luteal functional status of the corpora lutea and the human chorionic gonadotropin secreted by the implanting pregnancy. These data allow for the hypothesis that inducing functional luteolysis by substituting one or more injections of luteinizing hormone for the human chorionic gonadotropin injection may decrease secretion of steroids, relaxin, and other factors from the corpora lutea during pregnancy, decreasing the risk of premature delivery in multiple gestations and the ovarian hyperstimulation syndrome.

The effect of multifetal pregnancy reduction on serum relaxin.

OBJECTIVE: To determine the effect of multifetal pregnancy reduction on circulating relaxin levels. METHODS: Patients with multifetal pregnancies had relaxin levels determined on the day of multifetal pregnancy reduction, after the procedure, and late in pregnancy. RESULTS: Forty-eight women (26 presenting with three fetuses and 22 with four or more) were studied. All pregnancies followed some form of ovulation induction. All pregnancies (except for one, which was reduced to a singleton) were reduced to twins. Pre-procedure, post-procedure and late-pregnancy relaxin levels were significantly higher in the in vitro fertilization (IVF)-gamete intrafallopian transfer (GIFT) group compared with the human menopausal gonadotropin (hMG)-alone group (P < .05). The initial number of fetuses had no significant effect on relaxin levels. Although post-procedure relaxin levels were significantly lower than pre-procedure levels (P = .002), relaxin levels continued to decrease throughout pregnancy, as evidenced by even lower levels later on (P = .0001). CONCLUSIONS: Serum relaxin levels were significantly higher in the IVF-GIFT group than in the hMG-alone group, which probably reflects more aggressive ovulation induction in the former. Because relaxin levels continued to decrease throughout pregnancy, the difference observed between pre- and post-procedure levels are not considered to be due to the procedure itself.

Relaxin and its role in pregnancy.

Relaxin is a 6000-d polypeptide, structurally related to insulin and the insulin-like growth factors. Unlike insulin, the structure of which is remarkably well conserved among the vertebrates, relaxin sequences can vary by more than 50% between different species. Despite these large sequence variations, relaxins (with few exceptions) have very similar biologic activities in animal test systems. The reason for this has recently come to light: the receptor binding region of the B chain, in contrast to the rest of the molecule, is highly conserved between species. Relaxin is measured by bioassays employing interpubic ligament formation in mice and guinea pigs, and by inhibition of uterine motility. A more sensitive and efficient bioassay is urgently needed. In women, the target organs for relaxin are the uterine cervix, myometrium, endometrium, and decidua. Other presumptive but unproven targets are the pubic symphysis and sacroiliac joints, mammary glands, and pituitary gland. Circulating relaxin is secreted by the corpus luteum. The placenta, decidua, or both also produce relaxin, which does not enter the circulation but may act in an autocrine or paracrine fashion. hCG is a stimulus to luteal relaxin secretion. Other regulatory factors are poorly defined. Aluteal women are hyporelaxinemic, and yet are capable of normal vaginal delivery of their infants. Local effects of placental or decidual relaxin cannot be discounted in such subjects. Hyperrelaxinemia may occur in women with multiple gestations and ovarian stimulation, and may be associated with increased premature births. Serum relaxin also is elevated in pregnant diabetics, but its role in this condition has not been defined. Clearly, further investigations are needed to delineate the precise role of relaxin in human pregnancy.

Transmission of relaxin from lactating bitches to their offspring via suckling.

The 6-kDa polypeptide hormone relaxin (Rlx) has been identified in human and bovine milk, and we recently reported its presence in canine milk. We postulated that Rlx might be transferred via suckling to the newborn pups, where, by virtue of its known effects to increase the distensibility of the pelvic connective tissues, it could play a role in causing the excessive laxity of the capsule and ligaments of the coxofemoral joint that precedes the development of hip dysplasia in genetically predisposed animals. Rlx was found in the serum of dysplastic (HD+) bitches for up to 6 wk of lactation, whereas it was detected in the serum of nondysplastic (HD-) bitches for only 1-2 wk of lactation. Rlx concentrations in milk were up to 60-fold greater than in serum. Milk Rlx levels varied markedly, but were highest during the first week of lactation and decreased thereafter. There were no significant differences in milk Rlx concentrations between HD+ and HD- bitches. Although the source of Rlx in milk is unknown, it cannot be the ovary or uterus, since hystero-ovariectomy performed at the time of cesarean section did not eliminate Rlx from milk during subsequent lactation. In serum samples taken from newborn pups before suckling, there were significant quantities of Rlx, demonstrating that the hormone enters the fetus in utero. However, Rlx rapidly disappears from serum of pups prevented from suckling for five hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Relaxin secretion into human semen independent of gonadotropin stimulation.

To investigate the control of relaxin (Rlx) secretion in men, we studied seminal plasma Rlx concentrations after physiologic and supraphysiologic gonadal stimulation. In the first experiment, 14 men with idiopathic hypogonadotropic hypogonadism provided semen samples at various time points before and during therapy with pulsatile GnRH. These data were compared to seminal plasma Rlx values in 5 normal men. In a second experiment, pharmacologic doses of hCG were administered in a fashion similar to that previously shown to have stimulated Rlx secretion from the CL of women. In men with idiopathic hypogonadotropic hypogonadism, no relationship was detected by linear regression analysis between seminal plasma Rlx and testosterone, testicular volume, ejaculate volume, or the appearance of sperm in the ejaculate. Rlx concentrations varied considerably between subjects (6-120 ng/ml) but remained fairly consistent within the same individual over time. Supraphysiologic gonadal stimulation with hCG similarly failed to alter seminal plasma Rlx (n = 5, mean +/- SEM; 48 +/- 9 ng/ml, 42 +/- 7 ng/ml, and 56 +/- 9 ng/ml on Days 1, 3, and 6, respectively; p < 0.05) in normal men despite dramatic increases in serum testosterone (763 +/- 25 ng/dl, 1702 +/- 136 ng/dl, and 1494 +/- 97 ng/dl on Days 1, 3, and 6, respectively; p < 0.05 vs. Day 1). Taken together, these data suggest that Rlx in men is secreted independently from direct gonadotropin control.

Elevated first-trimester serum relaxin concentrations in pregnant women following ovarian stimulation predict prematurity risk and preterm delivery.

OBJECTIVE: To determine whether ovarian stimulation would result in higher circulating relaxin concentrations and whether this hyperrelaxinemia would be associated with prematurity. METHODS: Two groups of women were studied: 1) women achieving pregnancy after ovarian stimulation (n = 114) and 2) women achieving pregnancy without treatment (n = 37). Serum was obtained at 6-12 weeks' gestational age; fetal number was determined by transvaginal ultrasound. Prematurity risk or preterm delivery was determined from the obstetric record. A specific human relaxin enzyme-linked immunosorbent assay was used to measure serum relaxin concentrations. Hyperrelaxinemia was defined as levels greater than 3 standard deviations above the weighted mean of levels in normal unstimulated singleton pregnancies at 6-12 weeks' gestation. RESULTS: An association was found between prematurity risk or premature delivery and peripheral relaxin concentrations during weeks 6-12 of pregnancy in women having ovarian stimulation and in women having multiple gestations. Circulating relaxin concentrations greater than 16 ng/mL in women having ovarian stimulation and levels greater than 7 ng/mL in women who had multiple gestations predicted prematurity risk or premature delivery in 50% of the women. CONCLUSIONS: These data demonstrate that after ovarian stimulation, some women have highly elevated circulating first-trimester relaxin concentrations. First-trimester hyperrelaxinemia identifies a group of women at risk for prematurity who can be monitored aggressively.

Human chorionic gonadotropin stimulation of relaxin secretion by luteinized human granulosa cells.

OBJECTIVE: To determine the effect of human chorionic gonadotropin (hCG) on relaxin secretion by long-term cultures of luteinized human granulosa cells (GC). DESIGN: Luteinized human GC were collected from 10 women undergoing in vitro fertilization (IVF) cycles. Luteinized human GC from each woman were plated in replicate wells at 1 x 10(5) cells/well and exposed to medium 199 (GIBCO, Grand Island, NY), medium 199 with 1 IU/mL hCG, and/or medium 199 with 100 IU hCG/mL. Luteinized human GC were maintained for up to 40 days in culture. Spent media were changed every 2 days and assayed for relaxin and progesterone (P) at the conclusion of each experiment. SETTING: Tertiary care center. PATIENTS, PARTICIPANTS: Luteinized human GC were obtained from women undergoing controlled ovarian hyperstimulation for IVF with one of the following regimens: (1) clomiphene citrate with human menopausal gonadotropins (hMG); (2) hMG alone; or (3) hMG with leuprolide acetate. All women were less than 40 years of age, in good health, and were not taking medications other than those used in the ovulation-induction regimen. MAIN OUTCOME MEASURES: Levels of P and relaxin in spent media. RESULTS: Relaxin secretion by luteinized human GC was dependent on hCG stimulation and was detected only after a time lag in culture. After relaxin secretion was detected, it was maintained throughout the culture period (10 to 22 days). Luteinized human GC produced P immediately under both basal and stimulated conditions. Progesterone production continued throughout the culture period with hCG-stimulated cells producing significantly greater P after 4 to 8 days in culture. CONCLUSIONS: Luteinized human GC obtained at the time of oocyte retrieval secrete relaxin in response to hCG stimulation and secrete P under both basal and hCG-stimulated conditions, thereby serving as a model to explore luteal function and control.

Ovarian expression of cellular Ki-ras p21 varies with physiological status.

To elucidate the potential role of the ras protooncogene proteins in a specific tissue, the present study determined the levels of individual c-ras-encoded p21 proteins in the rat ovary during various stages of physiological function. p21 protein was extracted from ovaries taken from immature normal female rats, mature nonpregnant animals in the metestrus stage of the estrus cycle, rats at various stages of pregnancy, and actively lactating animals. Levels of individual p21s were evaluated by immunoblot analysis with specific antibodies to the p21 proteins encoded by the Kirsten, Harvey, and neuroblastoma c-ras protooncogenes, c-Ki-ras, c-Ha-ras, and N-ras. Results showed that c-Ki-ras p21 is at its lowest level in the immature ovary and increases with development of the corpora lutea to its highest levels at day 16 of pregnancy, after which levels decline and then rise again during lactation. This pattern, which mimics that of circulating progesterone levels, suggests that ovarian c-Ki-ras p21 levels are regulated and that c-Ki-ras p21 plays a role in the differentiated function of the rat ovary, likely the luteal compartment. In contrast, levels of c-N-ras p21 did not appear to vary with changes in the physiological function of the ovary but appeared to be constitutive. A preferential role for the c-Ki-ras p21 may be due to the documented unique differences in the structure of the carboxyl terminus of this particular c-ras p21.

Human seminal relaxin is a product of the same gene as human luteal relaxin.

Unlike that of other species, which have only one gene encoding relaxin, the human genome contains two nonallelic genes for relaxin, designated H1 and H2, which encode markedly different relaxin peptides. Whereas human relaxin gene H2 is selectively expressed in the ovary, no ovarian expression of gene H1 has been detected. Since relaxin is actively produced in the human male, it is possible to postulate divergent gene expression of relaxin in the male and female. We examined this question directly through the structural determination of human seminal relaxin and its comparison with the structure of human luteal relaxin. Partially purified relaxin, prepared from pooled human seminal plasma which had been delipidated by extraction with acid acetone and hexane, subjected to two cycles of HPLC and an additional purification step by ion-exchange chromatography, was further purified by immunoaffinity chromatography, using a monoclonal antibody to the H2 relaxin A chain which cross-reacts with synthetic H1 relaxin, followed by an additional HPLC step performed on a C4 reverse-phase column. The recovered, purified relaxin was then analyzed by N-terminal gas-phase sequencing and fast atom bombardment mass spectroscopy for determination of the amino acid sequence and molecular ions of the A and B chains, respectively. The results demonstrate that the structure of the predominant relaxin in human semen plasma is derived from the product of the H2 gene, consisting of a N-terminal pyroglutamic acid A-24 A chain and a mixture of B-26 and B-27 B chains. With the exception of degradation of the seminal relaxin B chain C-terminus, this structure is identical to the structure of human luteal relaxin. Therefore, both human seminal and luteal relaxin are products of the H2 gene.