Comparison of the steroidogenic response of luteinized granulosa cells from rhesus monkeys to luteinizing hormone and chorionic gonadotropin.

The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum

Two experiments were conducted to examine the effect of treatment with human chorionic gonadotropin (hCG) or ovine luteinizing hormone (LH) on the number and size distribution of steroidogenic luteal cells. In Experiment I, 27 ewes were assigned to one of three groups: 1) hCG (300 IU, i.v.) administered on Days 5 and 7.5 of the estrous cycle (Day 0 = Estrus); 2) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the cycle; 3) saline (i.v.) administered as in the LH treatment group. Blood samples were drawn daily from the jugular vein for quantification of progesterone. On Day 10, corpora lutea were collected, decapsulated, weighed, and dissociated into single cell suspensions. Cells were fixed, stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity, and the size distribution of 3 beta HSD-positive cells was determined. Treatment with hCG, but not LH, increased (p less than 0.05) concentrations of progesterone in serum and the weight of corpora lutea. Treatment with either hCG of LH increased the proportion of cells greater than 22 micron in diameter and decreased the proportion of cells less than or equal to 22 micron (p less than 0.01). The ratio of small to large luteal cells decreased after treatment with either hCG or LH (p less than 0.05). In Experiment II, 9 ewes were assigned to one of two groups: 1) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the estrous cycle, and 2) saline (i.v.) administered as in the LH treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of human chorionic gonadotropin administration on pituitary and luteal response to gonadotropin-releasing hormone

The effect of human chorionic gonadotropin (hCG) administration on the pituitary and luteal responses to acute gonadotropin-releasing hormone (GnRH) administration at the mid luteal phase (LP) were studied in 20 infertile women. Patients were divided into 2 groups. In 1 group (n = 8), hCG (5,000 IU i.m.) was injected in a single shot on day 5 of LP. Sixty hours later (day 8 of LP) blood samples were taken every 15 min for 180 min; then 25 micrograms GnRH were acutely administered intravenously and blood samples taken at 185, 195, 210, 225, 240, 255, 270, 285 and 300 min. In the other 12 patients the same experimental design with GnRH was performed on day 8 of an untreated LP. Plasma LH, FSH, beta-hCG, progesterone and estradiol (E2) were assayed. The responsiveness of different hormones to GnRH was evaluated as integrated secretory area for 120 min after injection (sISA) and as the absolute increase with respect to the area under basal conditions before a GnRH administration (bISA). hCG-treated patients showed higher basal and bISA plasma values of LH/hCG than controls (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene

LH/hCG receptors were disrupted by gene targeting in embryonic stem cells. The disruption resulted in infertility in both sexes. The gonads contained no receptor mRNA or receptor protein. Serum LH levels were greatly elevated, and FSH levels were moderately elevated in both sexes; estradiol and progesterone levels decreased but were not totally suppressed in females; testosterone levels were dramatically decreased and estradiol levels moderately elevated in males. The external and internal genitalia were grossly underdeveloped in both sexes. Abnormalities included ambiguous vaginal opening, abdominal testes, micropenis, dramatically decreased weights of the gonads and reproductive tract, arrested follicular growth beyond antral stage, disarray of seminiferous tubules, diminished number and hypotrophy of Leydig cells, and spermatogenic arrest beyond the round spermatid stage. LH/hCG receptor gene disruption had no effect on FSH receptor mRNA levels in ovaries and testes, progesterone receptor (PR) levels in ovaries and androgen receptor (AR) levels in testes. However, it caused a dramatic decrease in StAR and estrogen receptor-alpha (ERalpha) mRNA levels and an increase in ERbeta mRNA levels in both ovaries and testes. Estradiol and progesterone replacement therapy in females and testosterone replacement in males, to determine whether phenotype and biochemical changes were a consequence of decreased gonadal steroid levels or due to a loss of LH signaling, revealed complete restoration of some and partial restoration of others. Nevertheless, the animals remained infertile. It is anticipated that the LH receptor knockout animals will increase our current understanding of gonadal and nongonadal actions of LH and hCG.     

Biological actions of monoclonal luteinizing hormone/human chorionic gonadotropin receptor antibodies.

Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.     

Effects of luteinizing hormone and human chorionic gonadotropin on corpus luteum cells in a spheroid cell culture system

The human corpus luteum (CL) is a highly vascularized, temporarily active endocrine gland and consists mainly of granulosa cells (GCs), theca cells (TCs), and endothelial cells (ECs). Its cyclic growth and development takes place under the influence of gonadotropic hormones. If pregnancy does occur, human chorionic gonadotropin (hCG) takes over the function of luteinizing hormone (LH) and, in contrast to LH, extends the functional life span of the CL. In this study, we investigated the effects of hCG and LH in a spheroidal cell culture model of CL development. Our data indicate that GCs secrete factors under the control of hCG that increase sprout formation of EC-spheroids. We demonstrate that the most prominent of these factors is VEGF-A. Furthermore, we found that both LH and hCG decrease sprout formation of GC-spheroids. After forming EC-GC coculture spheroids and consequently bringing GCs and ECs in close contact, sprouting increased under the influence of hCG, however not under LH. These experiments provide evidence for an hCG dependent functional switch in the GCs after coming in contact with ECs. Moreover, it demonstrates the considerably different effects of hCG and LH on GCs although their signaling is transmitted via the same receptor.     

Selective radioimmunoassays for human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG).

Highly specific radioimmunoassay systems were developed for measurement of hLH and hCG using antisera purified by affinity chromarography or simple adsorption to select the antibodies reacting specifically with either gonadotropin. Such systems permit specific measurement of lLH and hCG in samples containing both. These assays are suitable for various clinical and physiological studies, particularly, study of pituitary functions in the presence of chorionic or trophoblastic secretion.     

Inhibition of testicular luteinizing hormone receptor level by treatment with a potent luteinizing hormone-releasing hormone agonist of human chorionic gonadotropin.

Two proteins larger than proinsulin (estimated molecular weight 11,000 and 10,000 daltons), were observed when labeled rat islet proteins were electrophoresed on sodium dodecyl sulfate gels. The proteins are synthesized before proinsulin, turn over more rapidly than proinsulin, their synthesis is stimulated by glucose, and they are specifically bound by anti-insulin antibodies.     

Labeling of bovine corpus luteal plasma membrane human chorionic gonadotropin or luteinizing hormone (hCG/LH) receptor and its purification and properties.

A method has been developed for labeling receptors for human chorionic gonadotropin or luteinizing hormone ($\text{hCG}\text{LH}$) present on bovine corpus luteal plasma membranes. It consists of four steps: (a) protection of the receptor by treating the plasma membranes with hCG; (b) iodination of the membranes with KI using glucose, glucose oxidase, and lactoperoxidase; (c) unmasking the receptor with either 2 m NaCl, 1 m guanidine hydrochloride, or rabbit anti-hCG; and (d) reiodination of the membranes using Na¹³¹I. After solubilization by successive treatments with Sepharose-concanavalin A and Sepharose-hCG and finally by preparative disc electrophoresis, the resulting purified receptor after electrophoresis in polyacrylamide gel showed a single radioactive band containing receptor activity. This highly purified receptor is fairly stable and retains its hormonal specificity, binding affinity, and pH optimum. It was observed that the receptor alone or as a complex with the hormone tends to aggregate. The receptorhormone complex does not dissociate during polyacrylamide-gel electrophoresis.     

Lipoprotein augmentation of human chorionic gonadotropin and prolactin stimulated progesterone synthesis by rat luteal cells

A collagenase dispersed cell suspension from PMSG-hCG primed immature rats responded to exogenously added hCG, cholera enteroxin, prolactin, and 8-Bromocyclic-AMP with increase in progesterone production in a dose dependent manner, and this stimulation was augmented by the plasma lipoprotein fractions hHDL and hLDL. The responsiveness to low doses of prolactin was not apparent when lipoprotein fractions were not included in the assay mixture. When the incubation mixture contained either LDL or HDL, the stimulatory effect of prolactin on progesterone production was evident at 5 and 10 micrograms prolactin/ml of the incubation mixture. Progesterone production, both basal and hormone stimulated, was maximum on day 7 of pseudopregnancy. Although the extent of hCG and prolactin stimulation of progesterone production and its potentiation by lipoprotein fractions was observed to be higher on days 3 and 5 than that seen on day 7, the net amount of progesterone produced was highest on day 7. The basal as well as hormone and lipoprotein stimulated progesterone production started to decline after day 7, reaching a nadir on day 14. These experiments show that prolactin is effective in stimulating progesterone production by rat luteal cells in vitro and that lipoprotein fractions, LDL and HDL further potentiate this response. This study further suggests that it is important to include LDL or HDL as a source of cholesterol for in vitro experiments in which the steroidogenic response of luteal cells to exogenous stimuli is tested.     

Effect of human chorionic gonadotropin and prostaglandin F2a on progesterone production by human luteal cells

To determine and compare the direct effects of prostaglandin F2a (PGF2a) and human chorionic gonadotropin (hCG) on luteal cell progesterone production in vitro, 9 human corpora lutea obtained at tubal ligation were minced and treated with collagenase to disaggregate luteal cells. Dispersed luteal cells (80% viable) were incubated in air at 37 degrees C in a shaking water bath for 3 h and total progesterone in the media and cells was determined by radioimmunoassay. Optimum progesterone production was obtained using 25,000 or more cells per incubate and an incubation time of 2-4 h. hCG-stimulated progesterone production increased significantly with 0.01 IU to as high as 100 IU. In the early luteal phase (days 1-5 post ovulation or days 15-20 of the luteal phase), PGF2a (10-1000 ng) significantly inhibited progesterone production but significantly stimulated progesterone production in the mid-luteal phase (days 21-25). PGF2a had no effect on luteal cell progesterone production in the late luteal phase (days 26-30). This age-dependent direct effect of PGF2a on human luteal cell progesterone production in vitro indicates a role for PGF2a in the total intragonadal regulation of progesterone output, possibly through a paracrine or autocrine manner directed towards synchronizing luteal progesterone secretion and endometrial preparation for nidation.     

Gonadal receptors I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone

The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (K_d) and number of hormone binding sites (B_max) are highly sensitive to changes in hormone and/ or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher K_d as well as B_max values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436–453).     

The effect of luteinizing hormone, human chorionic gonadotropin and adrenocorticotropic hormone on puberty in gilts reared in confinement

Three trials were conducted to determine the effect of human chorionic gonadotropin (HCG), luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) on the incidence of estrus in gilts which were reared in confinement, relocated and exposed to a boar. In trial 1, 33 gilts were given saline or 250 IU HCG at an average age of 191 days and then relocated and observed for estrus twice daily for 10 days. Treatment with HCG did not increase the proportion of gilts that exhibited estrus. In trial 2, 42 gilts were relocated at an average age of 200 days. The gilts were assigned to three treatment groups and injected with saline, 68 mug LH or 1 mg LH. After 10 days of estrous detection, a laparoscopic examination of the ovaries was conducted on all gilts failing to exhibit estrus. In groups 1 to 3, the proportions of gilts exhibiting estrus or ovulating during the 10 days after treatment were 13 of 21, 6 of 10, and 5 of 11, respectively. In trial 3, 12 gilts were relocated to pasture lots, given saline or 80 IU ACTH twice daily for 2 days and checked for estrus for 14 days. The proportions of gilts that exhibited estrus after the administration of saline or ACTH were 4 of 6 and 6 of 6, respectively. The results indicate that the incidence of estrus in gilts reared in confinement, relocated and exposed to a boar was not affected by pre-treatment with exogenous HCG, LH or ACTH.