hCG Binding Sites Research Articles

Hormonal regulation of testicular LH/hCG receptors in rat.

A single injection of 60 IU of hCG to 25-day-old rats led to a loss of LH/hCG receptors in the testis. The content of [125I] hCG binding sites reached a nadir 2 days after hCG injection. At this time the effect of daily treatment of hormones and other agents on the formation of LH/hCG receptors was studied. Gonadotropin receptors were estimated 2, 4, 6, 8 and 10 days after injection of hCG. Daily treatment of rats with PMSG (30 IU/day) increased testicular content of hCG binding sites. The presence of the pituitary in these animals was necessary during the formation of receptors after hCG-induced loss of binding sites. LH/hCG receptor content remained low in rats treated with testosterone (250 microgram/day), hCG (100 IU/day) or inhibitors of the synthesis and the secretion of prolactininhibitors-bromocryptine (250 microgram/day), ergocryptine (200 microgram/day) and deprenon (50 and 600 micrograms/day). Administration of cortisol (1 mg/day) had no effect on restoration of LH/hCG receptors. These data suggest an important role for FSH and prolactin in the formation of LH/hCG receptors in rat testis.

Thermal destabilization of ovarian LH/hCG receptors by negatively charged lipids.

The stabilizing effect of BSA on the rat ovarian LH/hCG receptor was analyzed by thermal perturbation technique. Thermal destabilization of the receptor with arachidonic acid along with digestion of membrane with phospholipase A2 and reversal of these effects when BSA was used as fatty acids scavenger, may indicate that free fatty acids are responsible for instability of the LH/hCG receptor. This destabilizing effect may be caused by the presence of a net negative surface charge provided by fatty acids. This presumption was corroborated by the reconstitution of delipidated LH/hCG receptor into proteoliposomes. Delipidated receptor lost to a great extent its binding activity and thermal stability. The receptor was fully reactivated by the reconstitution into proteoliposomes with neutral phosphatidylcholine but not with negatively charged phosphatidylserine and phosphatidylglycerol. Thermal inactivation of the LH/hCG receptor by delipidation was entirely inverted by treatment with phosphatidylcholine but the presence of negatively charged phospholipids did not change the heat inactivation profile of hCG-binding sites.

Expression of a Cloned Full-Length cDNA Encoding Bovine Luteinizing Hormone Receptor in COS-7 Cells.

A cloned full-length cDNA encoding bovine luteinizing hormone (LH) receptor was expressed in COS-7 cells and the binding activity of the protein to human chorionic gonadotropin (hCG) was examined. Molecular cloning of the bovine LH receptor cDNA was carried out by reverse transcription-polymerase chain reaction using total RNA extracted from a bovine corpus luteum. Three overlapping partial fragments of the receptor cDNA were amplified, sequenced, and engineered to generate the entire cDNA coding sequence and it was subcloned into a mammalian expression vector. Sequence analysis showed a 2,103-nucleotide open reading frame encoding the bovine LH receptor, predicting a putative 26-amino acid signal peptide, and a 675-amino acid mature receptor protein that is 94, 88, 86 and 85% identical to the porcine, human, rat and mouse homologs, respectively. Scatchard analysis of the intact COS-7 cells transfected with the bovine LH receptor cDNA showed high affinity and low capacity of [125I]-hCG binding sites on the cells. These results suggest that the protein expressed from the cloned bovine LH receptor cDNA is located on the cell surface with its binding activity to LH/hCG.

Structure-stabilizing effect of albumin on rat ovarian LH/hCG receptors

The stabilizing effect of albumin on structure-functional alteration of LH/hCG receptors was analyzed by thermal perturbation technique. On exposing the membranes to bovine serum albumin (BSA) the heat inactivation profile of hCG-binding sites was shifted to a temperature higher by about 5°C ( T 50 values). The receptor destabilizing action of arachidonic and oleic acids incorporated into ovarian membranes and reversal of this effect when BSA was used as fatty acid scavenger, may indicate that free fatty acids are responsible for the thermal instability of hCG-binding sites. This presumption was corroborated by digestion of membranes with phospholipase A 2 (PLA 2). This enzyme exerted effects on the thermal stability of the receptor protein resembling those observed upon insertion of fatty acids. The membrane fluidization induced by arachidonic acid can be reversed by BSA. However, alterations of lipid fluidity in membranes were not found to be a necessary prerequisite for stabilization of the LH/hCG receptor structure. Fluorescence quenching studies indicated that incorporation of oleic acid or digestion of membrane phospholipids with PLA 2 elevated the accessibility of fluorophores for acrylamide. BSA scavenging of free fatty acids approached the quenching rate of control membranes. Analysis of fluorescence of membranes bound to monodansylcadaverine probe revealed that the negative surface charge derived from free fatty acids resulted in destabilization of the receptor protein. The effects of free fatty acids on membranes suggest that altered lipid-protein interactions may directly affect the stability of the LH/hCG receptor structure.

HCG: its pancreatic and duodenal receptors and in vivo electrolyte secretion in female rats.

A state of fluid flux probably resulting from ion movement across the plasma membrane occurs during early pregnancy or trophoblastic disease, manifesting as emesis or hyperemesis gravidarum or hydatidiform moles. In emesis or hyperemesis gravidarum, excessive secretion induced by a humoral agent may trigger vomiting by distending and activating the gastrointestinal (GI) tract mechanoreceptors. This agent may be human chorionic gonadotropin (hCG). High-affinity hCG binding sites similar to those in the ovary were found in the duodenum and pancreas of female rats, with dissociation constant values of 0.11 +/- 0.02, 1.9 +/- 0.6, and 4.7 +/- 3.5 nM, respectively. The isoelectric point for duodenal and ovarian proteins was 5.5. With the use of two antisera directed against amino acid residues 24-33 and 239-249 of the lutropin receptor, positive immunohistochemical staining was seen in smooth muscle, Brunner's glands, parasympathetic ganglia, crypt cells, and blood vessels of the duodenum, in zymogen granules of acini, and in intralobular ducts and blood vessels of the pancreas. Under nonreducing conditions, 150- and 170-kDa proteins were seen, through Western blot analysis, in the pancreas, duodenum, and ovary. Administration of hCG to female rats in vivo caused a significant increase in HCO-3 and K+ secretion from the duodenum and pancreas. We hypothesize that during pregnancy hCG stimulates excessive secretion of electrolytes (and fluid) into the upper GI tract, which culminates in the vomiting during pregnancy.

Time-course effects of human recombinant luteinizing hormone on porcine Leydig cell specific differentiated functions

Since recombinant hormones are considered as safer and more reliable in their bioactivity than extractive hormones, the recently available human recombinant luteinizing hormone (r-hLH), will probably replace hCG in the near future, for clinical purposes. This prompted us to investigate whether or not, and by which mechanisms, r-hLH can induce a desensitization of signal transduction and/or an up-regulation of steroidogenic capacity in Leydig cells. The effects of a 30 min to 24 h exposure to r-hLH (10 −9 M) on the differentiated functions of cultured immature porcine Leydig cells were studied by measuring the following parameters: LH/hCG receptor number and mRNA, hCG-, cholera toxin- and forskolin-induced cAMP production, G protein α s subunit content of the membrane, hCG-, cholera toxin-, forskolin-, 8Br-cAMP-, 22R-OH-cholesterol-, progesterone-, 17OH-progesterone-, DHEA-, Δ4-androstenedione-induced testosterone secretion and StAR, 3 β-HSD, cytochrome P-450scc and P-450c17 mRNAs. hCG binding sites and LH/hCG receptor mRNA were slowly down regulated by r-hLH, reaching 47±1 and 18±7% of control at 24 h, respectively. Down-regulation of both hCG- and cholera toxin-induced cAMP production occurred earlier and was more marked, and at 24 h represented only 2.7±0.5 and 12.5±3.6% of control. Due to the synergistic effect of r-hLH and forskolin on cAMP production, the forskolin-induced cAMP was higher in r-hLH treated than in control cells, but this response also declines with time and was, at 24 h, only 32% of that observed at 30 min. This decreased cAMP production was associated with a less marked decline in the amount of membrane content of G α s protein. The testosterone production in response to hCG, cholera toxin, forskolin and 8Br-cAMP declined to reach a nadir at 6 h but increased thereafter and at 24 h was significantly higher than in control cells. In contrast, the conversion of several precursors into testosterone remained stable or increased slightly during the first hours of r-hLH treatment and significantly increased at 24 h and this was associated with an increase of StAR, 3 β-HSD, P-450scc and P-450c17 mRNAs. Taken together, the present results indicate that, despite the marked down-regulation of transmembrane signaling, r-hLH increased the steroidogenic capacity of Leydig cells by increasing the expression of several genes encoding the proteins involved in testosterone synthesis.

Bovine Cyclic Endometrium Contains High-Affinity Luteinizing Hormone/Human Chorionic Gonadotropin Binding Sites1

High-affinity LH/hCG binding sites have been characterized in porcine, lepine, and murine uteri. In the present study, LH/hCG binding sites were characterized in bovine endometrium. Radioreceptor assays were performed with membrane homogenates of endometrial tissues and analyzed for binding site specificity and capacity. There was little competition for receptor occupancy between hCG and ovine FSH (5%) or ovine prolactin (< 0.1%), but there was a 20% cross-reaction with eCG. There was no affinity for LH/hCG in crude membrane preparations of kidney, skeletal muscle, or vascular tissues. Concentrations of endometrial LH/hCG binding sites were determined during the bovine estrous cycle. LH/hCG receptors were found in cell preparations from Days 2-4 and 15-17 of the cycle, but not in preparations from the other stages of the cycle tested (Days 8-12, pre- and post-estrus, and ovulation). The concentration of uterine LH/hCG receptor varied during the estrous cycle, with higher values at Days 15-17 (3.1 fmol/mg protein) and lower values at Days 2-4 (1.2 fmol/mg protein). However, the binding capacity of hCG by luteal cells (9.7 fmol/mg protein) was 3-fold higher (p < 0.01) than that by endometrial tissue on any day studied. No differences in affinity constant (Ka) were seen between endometrial LH/hCG receptors (either) from Days 2-4 or 15-17) and mid-cycle luteal cells (0.60 x 10(11) M-1). Using Western blot analysis, we determined the expression of cyclooxygenase (COX) during the estrous cycle of the cow. It was found that the signal for COX was strongest at 15-17 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of juvenile hormone on mammalian steroidogenesis

The effects of juvenile hormone-III (JH-III) and the JH analogue 2-(4-phenoxyphenoxy)-ethoxytetrahydropiran on testicular steroidogenesis were studied. By using cultured MA-10 Leydig tumor cells as a model, these compounds were found to be potent inhibitors of LH/hCG steroidogenic action in a dose-dependent manner. Scatchard plot analysis of the binding data indicated that the JH analogue did not significantly alter the affinity nor the number of hCG binding sites, as well as GTP binding to plasma membranes. JH analogue inhibited the stimulatory action of both cholera toxin and forskolin on cAMP production and the concomitant steroidogenic response. JH analogue inhibited (Bu) 2cAMP-stimulated progesterone synthesis, indicating that a process downstream to the adenylyl cyclase in the steroidogenic pathway is also affected.

Human chorionic gonadotropin binding sites in the human endometrium

The existence of high-affinity and low-capacity specific binding sites for luteinizing hormone (LH)/human chorionic gonadotropin (hCG) has been reported in porcine, rabbit and rat uteri. We have identified hCG binding sites in the human endometrium collected from 35-42-year-old ovulatory and anovulatory women. The binding characteristics of hCG to endometrial tissue preparations from ovulatory and anovulatory women showed saturability with high affinity and low capacity. Scatchard plot analysis showed the dissociation constant of specific binding sites in the ovulatory women to be 3.5 x 10(-10) mol/l and in anovulatory women to be 3.1 x 10(-10) mol/l. The maximum binding capacity varied considerably between ovulatory (3.85 nmol/kg protein) and anovulatory (6.12 nmol/kg protein) endometrium. Among the divalent metal ions tested (Zn2+, Mg2+, Mn2+, Ca(2+)--4 mol/l), Zn2+ effected a remarkable increase in [125I]hCG binding to the endometrium (p < 0.005) whereas Mn2+ showed a marginal increase and other metal ions did not have any effect. Data obtained with human endometrium indicate an influence of the functional state of the ovary on [125I]hCG binding to endometrium.

Leydig cells increase their numbers but decline in steroidogenic function in the adult rat after neonatal hypothyroidism.

Administration of the goitrogen, 6-propyl-2-thiouracil (PTU), to suckling rat pups from birth through day 24 postpartum as a 0.1% solution in the mother's drinking water increases adult testis size and sperm production by about 80% and 140%, respectively, without affecting peripheral testosterone levels. The objectives of this study were to determine whether adult Leydig cell numbers were altered in PTU-treated rats and whether the steroidogenic function of these cells was normal. The number of Leydig cells per testis at 180 days increased by 69% in PTU-treated compared to control rats, whereas the average Leydig cell volume declined by about 20%. Steroidogenic function assessed in isolated adult Leydig cells decreased after neonatal PTU treatment. LH-stimulated testosterone production was reduced by 55% in Leydig cells from treated rats, commensurate with a 50% decline in the number of hCG-binding sites in these cells. The difference in steroidogenic potential was even more striking after incubations with saturating concentrations of steroid substrate, 22(R)-hydroxycholesterol; Leydig cells from treated males produced 73% less testosterone than controls. Therefore, this decrease in testosterone production may be partially due to a reduction in the numbers of LH receptors, but also reflects the impaired steroidogenic potential of these cells. These results clearly show that the dramatic increase in adult Leydig cell number after neonatal PTU treatment is counterbalanced by a permanent decline in Leydig cell steroidogenic function, producing no net change in peripheral testosterone levels.

Leydig cells increase their numbers but decline in steroidogenic function in the adult rat after neonatal hypothyroidism

Administration of the goitrogen, 6-propyl-2-thiouracil (PTU), to suckling rat pups from birth through day 24 postpartum as a 0.1% solution in the mother's drinking water increases adult testis size and sperm production by about 80% and 140%, respectively, without affecting peripheral testosterone levels. The objectives of this study were to determine whether adult Leydig cell numbers were altered in PTU-treated rats and whether the steroidogenic function of these cells was normal. The number of Leydig cells per testis at 180 days increased by 69% in PTU-treated compared to control rats, whereas the average Leydig cell volume declined by about 20%. Steroidogenic function assessed in isolated adult Leydig cells decreased after neonatal PTU treatment. LH-stimulated testosterone production was reduced by 55% in Leydig cells from treated rats, commensurate with a 50% decline in the number of hCG-binding sites in these cells. The difference in steroidogenic potential was even more striking after incubations with saturating concentrations of steroid substrate, 22(R)-hydroxycholesterol; Leydig cells from treated males produced 73% less testosterone than controls. Therefore, this decrease in testosterone production may be partially due to a reduction in the numbers of LH receptors, but also reflects the impaired steroidogenic potential of these cells. These results clearly show that the dramatic increase in adult Leydig cell number after neonatal PTU treatment is counterbalanced by a permanent decline in Leydig cell steroidogenic function, producing no net change in peripheral testosterone levels.

Osmolytes improve the reconstitution of luteinizing hormone/ human chorionic gonadotropin receptors into proteoliposomes

Rat ovarian membrane luteinizing hormone/ human chorionic gonadotropin (LH/hCG) receptor was reconstituted into proteoliposomes. The ability of sodium cholate to extract and reconstitute hCG binding activity was dependent on the protein/detergent ratio. Trypsinization of the LH/hCG receptor containing proteoliposomes indicated that approximately 57% of hCG binding sites were oriented extravesicularly. The presence of 20% glycerol or other osmolytes during reconstitution increased the accessibility of LH/hCG receptors but not the activity of adenylate cyclase in proteoliposomes. This beneficial effect was independent of any specific detergent or its presence during detergent solubilization of proteins. Dynamic properties of membranes were monitored by electron spin resonance of 16-, 12-, and 5-doxyl stearic acid. Reconstituted proteoliposomes contain less ordered membrane lipids than do native membranes. Addition of glycerol before reconstitution increased the order of lipid bilayer and shifted it to the physical state of the native membrane. These findings are consistent with the hypothesis that a rise in membrane ordering increases the accessibility of membrane-bound LH/hCG receptors.

Differences in LH receptor down-regulation between rat and mouse Leydig cells: Effects of 3',5'-cyclic AMP and phorbol esters

The role of cyclic AMP and phorbol esters in luteinizing hormone (LH) receptor down-regulation in Leydig cells has been studied. Dibutyryl cyclic AMP (db-cAMP) (0.01, 0.1 and 1 mM), forskolin (80 μM) and cholera toxin (1.19 nM) caused a 30–50% loss of [ 125I]hCG binding sites and an inhibition of receptor-[ 125I]hCG complex internalization in mouse tumour Leydig (MA10, MLTC-1) cells during 2 h. In contrast, db-cAMP had no effect on the level of binding sites or internalization of the hormone receptor complex in rat testis Leydig cells or a rat tumour (R2C) Leydig cell. Phorbol 12-myristate 13-acetate (PMA) at concentrations from 10 −9 to 10 −5 M had no effect on hormone binding or hormone-receptor complex internalization in any of the Leydig cells. In contrast a 2 h preincubation of MLTC-1 cells with 10 −7 M PMA caused a loss of subsequent LH-stimulated cyclic AMP and pregnenolone production. These results indicate that LH receptor down-regulation is mediated by cyclic AMP dependent kinase, but not protein kinase C, in mouse Leydig cells. No down-regulation of rat Leydig cell LH receptor occurs with either kinase.

Is deglycosylated human chorionic gonadotropin an antagonist to human chorionic gonadotropin? Characterization of deglycosylated human chorionic gonadotropin action in two testicular interstitial cell fractions

In order to determine the significance of carbohydtrate residue of human chorionic gonadotropin )hCG) in receptor interaction and signal transduction leading to steriodogenesis, the effect of deglycosylated hCG (DG-hCH) was studied in vitro with two different hCG-responsive purified testicular interstitial cell fractions. Fractions I light cells, previously found to bind 125I-labeled hCG with high affinity witout producing testosterone, also bound 125I-labeled DG-hCG with high affinity ( K d 7.2·10 −1) M) wihtout stimulating testosterone production. Fraction IV heavier cells, which produced testosterone in response to hCG without detectable high-affinity hCG-binding sites, neither bound DG-hCG nor sufficiently produced cAMP and testosterone in response. With the addition of intact hCG, DG-hCG inhibited cAMP levels, although not sufficiently to inhibit testosterone production. This observation was contrary to previous studied in which DG-hCG was shown to be antagonist to hCG action. We conclude that: (a) DG-hCG retains its binding activity in light cells and this high-affinity binding is unrelated to steroidogenesis; (b) DG-hCG does not bind to heavier cells with high affinity and loses its biological activity as results of deglycosylation; (c) DG-hCG actions in this study strengthen the concept of two different hCG-responsive cells in the rat interstitium which, if not separated, will yield misleading data supporting the coexistence of hCG high-affinity binding and biological responses in the same cell; and (d) DG-hCG partially anatagonizes the activation of adenylate cyclase but does not block testosterone production, thus questioning the usefulness of this analogue in antagonizing the action of native hCG in rat testis.

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. The concentration of 125I-labelled hCG-binding sites was roughly 50-fold higher in particulate than in supernatant fractions of pFF. However, 30-40% of the total 125I-labelled hCG-binding activity in pFF was present in the supernatant fraction of commercial batches of pFF. 125I-Labelled oFSH binding to pFF membranes was markedly higher than to supernatant fractions. Binding of 125I-labelled hCG and 125I-labelled oFSH to granulosa cells and supernatants of pFF showed a time-dependent variation in response to hormone priming. The results suggest that gonadotrophin-binding sites in the supernatant fraction of pFF have properties similar to those of their membrane-bound counterparts. 125I-Labelled hCG-binding activity in the supernatant fraction of pFF was shown to be more stable than detergent-solubilized LH/hCG receptors, even in glycerol-preserved preparations. Based on a number of criteria, we have speculated that pFF may have components which may be similar in structure to the extracellular domain of the LH/hCG receptor.

Comparison of the Luteinizing Hormone-Sensitive Adenylyl Cyclase of the Pig Ovarian Follicle and Corpus Luteum and Its Susceptibility toin VitroHormone-Dependent Desensitization*

The hormone responsiveness of the adenylyl cyclase of pig ovarian follicles or corpora lutea was examined. Adenylyl cyclase activity was assayed in 10,000 x g membrane fractions that had been prepared with or without (control) a urea extraction. In control luteal membranes there was little stimulation (less than 2-fold) or adenylyl cyclase by saturating ovine (o) LH, hCG, or (-)isoproterenol in the absence or presence of 10 microM GTP. However, in urea-treated luteal membranes, a 2- to 3-fold stimulation of adenylyl cyclase was caused by saturating oLH or hCG, and a 4- to 5-fold stimulation by (-)isoproterenol; the marked stimulation by the gonadotropins was only observed if 10 microM GTP was added. In follicular membranes, a 3- to 4-fold stimulation of adenylyl cyclase by gonadotropins was observed regardless of whether GTP was added or the membranes had been urea extracted. Stimulation of adenylyl cyclase by (-)isoproterenol was always less than 2-fold in follicular membranes. The binding affinity for [125I]hCG was similar in control follicular and luteal membranes, but there were approximately 10-fold more [125I]hCG-binding sites in follicular compared with luteal membranes. The binding affinities and number of receptor sites were not significantly changed by urea treatment. The ED50 values for hCG or (-)isoproterenol were the same in follicular and luteal membranes and were uneffected by the addition of 10 microM GTP, but the ED50 for oLH was 3-fold lower in follicular than in luteal membranes. GTP caused a dose-dependent increase in adenylyl cyclase activity in luteal and follicular membranes, and both tissues had the same ED50. A saturating hormone concentration resulted in an approximately 2-fold decrease in the ED50 for GTP. In vitro hCG-induced desensitization of the hCG-responsive adenylyl cyclase was 31% in follicular membranes, but only 11-15% in luteal membranes. Hormone-induced desensitization was not increased in incubations of luteal homogenate or membranes plus cytosol. These results establish the existence of a LH/hCG-sensitive adenylyl cyclase in the pig corpus luteum and indicate that the G-protein and catalytic moieties of the follicular and luteal adenylyl cyclase complex are functionally the same, but some difference exists in the way the LH/hCG-receptor in the two tissues interacts with the G-protein/catalytic complex.

Relationship between Concentrations of Immunoreactive Insulin-Like Growth Factor-I in Follicular Fluid and Various Biochemical Markers of Differentiation in Bovine Antral Follicles

Three experiments were conducted to determine the relationship between concentrations of insulin-like growth factor-I (IGF-I) in ovarian follicular fluid and various biochemical markers of follicular differentiation in bovine follicles. In Experiment I, ovaries were removed on Days 7, 14, 28, 42, or 56 after parturition from a total of 21 cows. In Experiment II, ovaries of 31 cows were removed between Days 20 and 30 postpartum after 48 or 96 h of either saline (0.9% NaCl, 5 ml) or luteinizing hormone-releasing hormone (LHRH, 500 ng/5 ml saline) injections given every 2 h via jugular cannulae. In Experiment III, ovaries of six cows were removed 48-50 h after a 35-mg injection of prostaglandin F2 alpha during the midluteal phase of an estrous cycle. In Experiments I and II, all follicles greater than or equal to 8.0 mm in diameter were removed from each ovary (n = 33 and 46, respectively). In Experiment III, fluid from all follicles greater than 4 mm in diameter were removed individually (n = 10), and fluid from follicles 1-4 mm in diameter were pooled for each cow. Follicles for each experiment were further categorized as either estrogen-active (E-A, concentration of estradiol greater than progesterone in follicular fluid) or estrogen-inactive (E-I, concentration of progesterone greater than estradiol in follicular fluid). Measurements of immunoreactive IGF-I (i-IGF-I) were made after separating IGFs from their binding proteins with an acid-ethanol extraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic treatment with bromocryptine during the oestrous cycle of the cow: Evidence that prolactin is not involved in preovulatory follicular growth

Nine heifers received an intramuscular injection of 15 mg of bromocryptine twice daily from Day 1 (the day of first standing oestrus defined as Day 0) until 50 h after the onset of luteal regression. At this time the ovaries were collected and the number and quality of follicles > 2 mm were determined. Binding of 125I-labelled hCG and 125I-labelled ovine prolactin to follicles > 5 mm and concentrations of progesterone, oestradiol and androstenedione in the follicular fluid were estimated. Plasma concentrations of prolactin, LH and oestradiol were studied from the onset of luteal regression until ovariectomy. Seven heifers served as a control group. Prolactin was effectively suppressed during bromocryptine treatment (0.21 ± 0.01 μg/ l, mean ± SEM; n = 9) as compared to control heifers (16.8 ± 3.6 μg/ l; n = 7). Concentrations of LH were not affected by the low level of prolactin (0.99 ± 0.08 μg/ l vs. 0.95 ± 0.11 μg/1). Both groups showed a similar time-course of the oestradiol concentration in the peripheral blood during the 50-h period. No specific binding of prolactin to granulosa cells and thecal tissue of follicles > 5 mm could be demonstrated, regardless of their quality or diameter. Preovulatory follicles of bromocryptine-treated and control heifers did not differ with regard to the number of specific binding sites for hCG and the concentrations of oestradiol, progesterone and androstenedione in the follicular fluid. It is concluded that prolactin is not involved in the growth and development of antral follicles during the normal oestrous cycle of the cow.

Solubilization of luteinizing hormone receptor from human corpora lutea in a stable form and identification of the hormone-binding unit by ligand blotting.

LH receptors were solubilized from human corpora lutea in phosphate-buffered saline containing 1% Triton X-100, 20% glycerol, and protease inhibitors. The presence of 20% (vol/vol) glycerol was necessary for quantitative preservation of [125I]hCG-binding activity in detergent solution. The solubilized receptors were stable for several weeks at -20 C and at -80 C and for at least 18 h at 4 C. Binding of [125I]hCG to the soluble LH receptors was time and temperature dependent and varied linearly with the amount of soluble protein. Equilibrium binding studies revealed a single class of high affinity [125I]hCG-binding sites with an equilibrium dissociation constant (Kd) of 4.3 x 10(-10) mol/L (at 20 C). The molecular size of the human LH receptors was analyzed by ligand blotting. Solubilized receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and transferred to nitrocellulose. Incubation of the protein blot with [125I]hCG to the 85/90K mol wt species was inhibited by unlabeled hCG. These results indicate that LH receptors can be solubilized in nonionic detergent while maintaining their hormone-binding activity and demonstrate that the receptors contain 85/90K hormone-binding species.

Chronic exposure of the developing corpus luteum in monkeys to chorionic gonadotropin: persistent progesterone production despite desensitization of adenylate cyclase.

The transient steroidogenic response of the macaque corpus luteum to chronic human CG (hCG) treatment beginning on days 9-10 of the luteal phase (i.e. stimulated early pregnancy) is associated with decreased numbers and affinity of available receptors for gonadotropin and homologous desensitization of adenylate cyclase. This study determined if similar changes in the receptor-adenylate cyclase system accompany the persistent steroidogenic response which occurs when hCG treatment begins earlier in the luteal phase. Female rhesus monkeys received increasing doses of hCG (15 up to 5760 LU) twice daily beginning 5-6 days after the midcycle LH surge. The levels of circulating progesterone increased (P less than 0.05) within 24 h of initial hCG exposure and did not decrease throughout the 10-day regimen. The corpus luteum was removed after 0 (n = 8), 6 (n = 4), or 10 (n = 4) days of hCG treatment. Whereas the numbers of available [125I]hCG binding sites in luteal particulates remained unchanged by 10 days of hCG exposure, the dissociation constant (Kd) for gonadotropin binding was greater than at day 0 (6.17 +/- 1.41 vs. 0.91 +/- 0.06 X 10(-10) M, P less than 0.05). Since the number of binding sites occupied by injected hCG increased with treatment (7.81 +/- 1.55 fmol/mg wet wt at day 10), the total number (available + occupied) of gonadotropin receptors was 3-fold greater (P less than 0.05) at day 10 than at day 0. Adenylate cyclase activity in luteal homogenates, assessed by conversion of [alpha-32P]ATP to [32P]cAMP, was stimulated on day 0 by hCG (2.7 +/- 0.7 X control, at 250 nM hCG), prostaglandin E2 (2.5 + 0.5 X control, at 0.5 mM), and prostaglandin I2 (2.3 +/- 0.5 X control at 0.5 mM) as well as forskolin (100 microM) and 5'-guanylyl-imidodiphosphate (50 microM). In contrast, cAMP production by day 6 of treatment was insensitive to hCG, but remained responsive to prostaglandin E2, prostaglandin I2, and nonhormonal activators. We conclude that CG treatment in the early luteal phase did not prevent the development of gonadotropin receptors to levels typically observed in the functional corpus luteum of the menstrual cycle. Also, many changes in the gonadotropin receptor-adenylate cyclase system in macaque luteal tissue were similar after CG treatment beginning on days 5-6 or days 9-10 of the luteal phase.(ABSTRACT TRUNCATED AT 400 WORDS)