Partial characterization of chorionic gonadotropin-like binding sites from the bacteria Xanthomonas maltophilia

The gram-negative bacterium, Xanthomonas maltophilia, has low- and high-affinity luteinizing hormone/chorionic gonadotropin (LH/CG)-binding sites, similar to the LH/CG receptor found in mammals. Although the low-affinity site binds both LH and human CG (hCG), the high-affinity site is specific for hCG. In the current investigation, these two binding sites were independently isolated from X. maltophilia for further characterization. To isolate functional binding sites, we developed a solubilization method using the detergent zwittergent 3,14 and high glycerol concentrations that allowed for the maintenance of ligand-binding integrity. Gel filtration experiments established molecular weights of 170 and 11.5 kDa for the two binding sites, which were supported by data from photoaffinity labeling and ultracentrifugation experiments. Gel filtration data also suggested the presence of a third binding site of 5.4 kDa. The 170-kDa site had a binding affinity of Kd = 12 x 10(-6) and bound both LH and hCG. The small molecular weight site had an affinity of Kd = 9.4 x 10(-8) and was CG specific. Collectively, these data demonstrate the presence of multiple hormone binding sites in X. maltophilia that differ in molecular size, binding affinity, and ligand specificity.     

Amino-terminal leucine-rich repeats in gonadotropin receptors determine hormone selectivity.

Recombinant expression of truncated receptors for luteinizing hormone/chorionic gonadotropin (LH/CG) revealed that the amino-terminal leucine-rich repeats 1-8 of the extracellular receptor domain bind human chorionic gonadotropin (hCG) with an affinity (Kd = 0.72 +/- 0.2 nM) similar to that of the native LH/CG receptor (Kd = 0.48 +/- 0.05 nM). LH/CG receptor leucine-rich repeats 1-8 were used to replace homologous sequences in the closely related receptor for follicle stimulating hormone (FSH). Cells expressing such chimeric LH/CG-FSH receptors bind hCG and show elevated cylic AMP levels when stimulated by hCG but not by recombinant human FSH (rhFSH). Similarly, a chimeric LH/CG receptor in which leucine-rich repeats 1-11 originated from the FSH receptor is activated by rhFSH but not by hCG. For this chimera, no residual [125I] hCG binding was observed in a range of 2 pM to 10 nM. Our results demonstrate that specificity of gonadotropin receptors is determined by a high affinity hormone binding site formed by the amino-terminal leucine-rich receptor repeats.     

The glycoprotein hormones: recent studies of structure-function relationships

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.     

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

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).     

Purification and partial characterization of rat ovarian lutropin receptor

Lutropin (LH) receptor was solubilized from pseudopregnant rat ovaries and purified by two cycles of affinity chromatography on human choriogonadotropin (hCG)-Affi-Gel 10. The purified receptor preparation contained a single class of high-affinity 125I-hCG binding sites with an equilibrium dissociation constant (Kd) of 5.1 X 10(-10) M (at 20 degrees C) and had a specific hormone binding capacity of 7920 pmol/mg of protein. The purified receptor migrated as a single 90-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both nonreducing and reducing conditions. Affinity cross-linking of the purified receptor to 125I-hCG produced a 130-kDa complex. Hormone-binding ability of the purified 90-kDa polypeptide was demonstrated also by ligand blotting. The purified receptor was electroblotted onto nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by incubation with 125I-hCG. Autoradiography revealed labeling of a 90-kDa band. This labeling was displaced by unlabeled hCG and human LH but not by human follitropin or rat prolactin. In addition, LH receptors of bovine corpora lutea and mouse Leydig tumor cells were shown by ligand blotting to contain a 90-kDa hormone binding unit, suggesting that LH receptor structure is well conserved among mammalian species. The purified rat ovarian LH receptor bound to immobilized wheat germ agglutinin, implying that the receptor is a glycoprotein. These results demonstrate that the hormone-binding unit of rat ovarian LH receptor is a 90-kDa membrane glycopolypeptide.     

Establishment of gonadotropin-responsive murine leydig tumor cell line

Several clonal Leydig tumor cell lines have been established by adapting the transplantable Leydig tumor, M548OP, to culture. One of these cell line, MLTC-1, has been characterized with regard to the gonadotropin-responsive adenylate cyclase system. The binding of 125I-labeled human chorionic gonadotropin (hCG) was blocked by excess unlabeled hCG and lutropin (LH) but not by follitropin, thyrotropin, or insulin, indicating the presence of specific receptors for hCG and LH. Based on the specific binding of hCG to isolated MLTC-1 membranes, the calculated dissociation constant was 1.0 +/- 0.2 X 10(-10) M. The receptors appeared identical to those from normal murine Leydig cells when analyzed by SDS PAGE and sucrose density gradient centrifugation. The molecular weight and sedimentation coefficient were 95,000 daltons and 8.5 S, respectively. MLTC-1 cells responded to hCG by accumulating cyclic AMP and producing progesterone. Cyclic AMP accumulation was time- and dose-dependent with a maximal accumulation occurring at approximately 0.2 nM hCG. At saturating levels of hCG, cAMP levels reached a maximum by 30 min and then declined very slowly. Adenylate cyclase activity in membranes prepared from MLTC-1 cells was stimulated by hCG, LH, NaF, cholera toxin, and guanyl-5'-ylimidodiphosphate, Additionally, choleragen was found to ADP-ribosylate a membrane protein of 54,000 daltons. This protein resembles the proposed guanine nucleotide regulatory component in both size and choleragen-dependent reactivity. These data suggest that MLTC-1 cells possess a gonadotropin-responsive adenylate cyclase system consisting of a specific hormone receptor, a regulatory component, and a catalytic subunit.     

A molecular dissection of the glycoprotein hormone receptors

In glycoprotein hormone receptors, a subfamily of rhodopsin-like G protein-coupled receptors, the recognition and activation steps are carried out by separate domains of the proteins. Specificity of recognition of the hormones thyrotropin (TSH), lutropin (LH), human chorionic gonadotropin (hCG) and follitropin (FSH) involves leucine-rich repeats (LRRs) present in an N-terminal ectodomain, and can be associated with a limited number of residues at key positions of the LRRs. The mechanism by which binding of the hormones results in activation is proposed to involve switching of the ectodomain from a tethered inverse agonist to a full agonist of the serpentine, rhodopsin-like region of the receptor. Unexpectedly, the picture is complicated by the observation that promiscuous activation of one of the receptors (FSHr) by hCG or TSH can result from activating mutations affecting the serpentine region of the receptors.     

Clinical importance of vascular LH/hCG receptors--a review

It was believed for a long time that functional LH/hCG receptors were present only in gonads. Recent studies have demonstrated, however, that these receptors are also present in several nongonadal organs in the human body. Uterus is one of them. Besides two uterine layers, endothelial cells and smooth muscle of blood vessels in the uterus also contain these receptors. In vivo administration of hCG decreased vascular resistance in the human uterus and in vitro treatment increased vasodilatory and decreased vasoconstrictive eicosanoids in the vessels. These findings led us to investigate whether hCG administration to patients with signs of threatened abortion has any beneficial effect. Patients were treated with either magnesium or progesterone and/or hCG. The results showed that the frequency of patients reaching second trimester was higher when hCG was used, which was paralleled by a significant decrease in uterine vascular resistance. Patients who reached term after treatment had decreased incidence of preterm delivery and intrauterine growth retardation. In conclusion, we suggest that uterine vascular LH/hCG receptors play an important role in the peri-implantation period by increasing uterine blood flow through vasodilatation and also perhaps through angiogenesis and trophoblast invasion, resulting in therapeutic benefit.     

Oxytocin receptors coupled to uterine contraction in estrogen-dominated rabbits

The present study investigated whether specific [3H]oxytocin binding sites previously demonstrated in estrogen-dominated rabbit uterus have properties expected of physiologic receptors coupled to uterine contraction. Microsomal membranes from estrogen-dominated rabbit uterus were found to contain high-affinity specific oxytocin binding sites with Kd = 2-3 nM. These sites were predominantly myometrial in locus. Specific oxytocin binding exhibited a pH optimum between 7.5 and 8.0. Mg2+ or Mn2+ was necessary for maximal specific [3H]oxytocin binding; in contrast, Ca2+ at submillimolar concentrations inhibited specific binding. Oxytocin binding sites were not detectable in microsomal membranes isolated from progesterone-dominated rabbit uterus. Relative binding and uterotonic activities of 10 synthetic neurohypophyseal hormone analogues were determined in estrogen-dominated rabbit uterus. A qualitative correlation was observed between binding and uterotonic responses. Angiotensin II and insulin did not compete with [3H]oxytocin for uterine binding sites. It is concluded that the specific high affinity [3H]oxytocin binding sites demonstrated in estrogen-dominated rabbit uterus have the selectivity for neurohypophyseal hormone analogues expected for physiologic receptors coupled to uterine contraction.     

Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125I-labeled human FSH to testis receptor.     

Interstitial cell heterogeneity in rat testes. II. Purification of cells by Percoll and metrizamide gradient centrifugation with preferential localization of gonadotropin binding sites in light cell fraction and hormone-induced steroidogenesis in heavier cell fraction

The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.     

Radioreceptor and autoradiographic analysis of FSH, hCG and prolactin binding sites in primary to antral hamster follicles during the periovulatory period

As measured by radioreceptor assays, binding sites for FSH and prolactin were present at 09:00 h on the day of pro-oestrus in Stage 1-10 follicles (primary to antral) with prolactin receptors 3-6 times higher than FSH sites in Stages 1-3 (3 layers of granulosa cells). Specific binding sites for hCG were present in Stage 1 and 2 follicles (2 layers of granulosa cells) but thereafter their distribution was erratic and they were not consistently detectable until Stage 5, when thecal cells first appeared. Using topical autoradiography, specific binding for FSH was evident in Stage 1-4 follicles (4 layers granulosa cells) whereas specific hCG-binding was not. After the preovulatory gonadotrophin surges, by 21:00 h on pro-oestrus, FSH receptors declined in Stages 5-10, prolactin receptors fell in Stages 8 and 10 (small and large antral follicles) and hCG receptors were reduced in Stages 7 (start of antral cavity) to 10. On the morning of oestrus, for follicles from Stage 4 onwards, receptor numbers usually returned to levels found at 09:00 h on pro-oestrus. At oestrus, the few remaining Stage 10 follicles were all atretic and contained significantly reduced FSH and prolactin receptors but numbers of hCG binding sites comparable to those at 09:00 h of pro-oestrus. These results provide evidence of gonadotrophin receptors in small primary and secondary follicles which is consistent with increased DNA synthesis in small hamster follicles on the afternoon of pro-oestrus and on the morning and afternoon of oestrus. Periovulatory changes in gonadotrophin concentrations may therefore affect early stages of folliculogenesis.     

Extragonadal luteinizing hormone receptors in the reproductive tract of domestic animals

Binding sites for LH/hCG and/or its mRNA are found in the uterus of several species, including human, primate, pig, cow, and turkey. Activation of LH receptors around Day 15 of the estrous cycle is associated with increased prostaglandin F(2alpha) production in the bovine, porcine, and ovine uterus. Activation of uterine LH receptors is also associated with increased levels of prostaglandins in human and primate. The presence of gonadotropin receptors with a dynamic pattern in the oviduct, endometrium, myometrium, and cervix of different species provides evidence that gonadotropins play a substantial role in molecular autocrine-paracrine regulation of the estrous cycle and implantation.     

Eleven amino acids (Lys-201 to Lys-211) and 9 amino acids (Gly-222 to Leu-230) in the human thyrotropin receptor are involved in ligand binding.

Our previous studies involving chimeric thyrotropin-lutropin/choriogonadotropin (TSH-LH/CG) receptors suggest that multiple segments spanning the entire extracellular domain of the human TSH receptor contribute to the TSH binding site. Nevertheless, the mid-region (segment C, amino acid residues 171-260) of the receptor extracellular domain is particularly important in TSH binding. In the present studies, we constructed seven new chimeric receptors in order to analyze segment C in further detail. Seven small segments spanning segment C of the TSH receptor were replaced with the counterpart of the rat LH/CG receptor. These mutant receptors were stably introduced into Chinese hamster ovary cells and were tested for hormone binding and cAMP responsiveness to hormone stimulation. The results indicate that 11 amino acids of the TSH receptor (Lys-201 to Lys-211) and the corresponding region of the LH/CG receptor (Thr-202 to Ile-212) are important for specific TSH and human CG binding, respectively. In addition, nine amino acids of the TSH receptor (Gly-222 to Leu-230) are also involved in TSH binding. A further conclusion from these data is that TSH and human CG bind to partially overlapping sites on their respective receptor molecules.     

Restricted lateral diffusion of luteinizing hormone receptors in membrane microdomains

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nm hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homotransfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.     

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

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.     

Oestrogen-dependent expression of LH/hCG receptors in pig Fallopian tube and their role in relaxation of the oviduct.

The current studies investigated the concentration and distribution of LH receptors in the oviduct of ovariectomized gilts at various times after administration of oestradiol benzoate (10 micrograms kg-1 body weight) to determine whether LH participates in the regulation of oviductal contractions. Polyclonal antibodies to the LH receptor were used in immunocytochemical and western blot analyses of oviductal tissues. The mechanical activity of the isthmus and ampullar segments of oviduct, collected from 16 cyclic gilts, was recorded for 30 min after LH or hCG treatment. In the oviduct, there was little competition for receptor occupancy between hCG and pig FSH, bovine thyroid-stimulating hormone (TSH), pig growth hormone (GH) and pig prolactin (1.2, 0.1, 0.01 and < 0.001%, respectively) but pig LH could completely inhibit the binding of [125I]hCG. Oestradiol benzoate increased (P < 0.01) the number of LH binding sites in oviduct 24, 48 and 72 h (0.60 +/- 0.08, 1.62 +/- 0.15, 2.48 +/- 0.35 fmol mg-1 protein; n = 4 per treatment, respectively) after injection compared with the control gilts treated with corn oil (0.20 +/- 0.04 fmol mg-1 protein; n = 4). The affinity of oviductal LH/hCG binding sites (Ka) varied from 4.0 to 8.5 x 10(10) l mol-1 and was similar to that of luteal cell binding sites (6.1 x 10(10) l mol-1). Oestradiol benzoate also resulted in more intense LH receptor immunostaining of the tubal mucosal epithelium, smooth muscle cells and blood vessels as compared with controls. Western blotting has revealed that the pig oviduct, similar to the corpus luteum, contains 75, 48 and 45 kDa immunoreactive LH receptor proteins. Treatment with LH in vitro (100 ng ml-1) affected the contractility of oviduct. During the peri-ovulatory stage of the oestrous cycle, the amplitude, frequency and area under curve(s) of the isthmus decreased (P < 0.05), as did the frequency and area under curve (P < 0.05 and P < 0.01, respectively) of the ampulla (n = 4). The frequency and area under curve of the oviductal contractions were also significantly reduced during the early follicular phase of the oestrous cycle (P < 0.05). There was no effect of LH (or hCG) on the frequency and area under curve of the oviductal contractions during luteal stages of the oestrous cycle (n = 8). These data indicate that (1) the pig oviduct possesses immunoreactive and functional LH receptor, (2) oestradiol promotes the synthesis of LH receptor in the epithelium and smooth muscles, and (3) LH causes the relaxation of oviduct, especially during the peri-ovulatory stage of the oestrous cycle. In summary, the results of the present study indicate that LH can control oviductal contractions directly and may be partially responsible for the relaxation of isthmus during fertilization in pigs.     

Temporal relationship of the prolactin-dependent LH-induced LH receptor to the LH stimulus

The time course for LH induction of luteinizing hormone (LH) receptors as reflected in binding of ¹²⁵l-labeled hCG was investigated in hypophysecto-mized adult male rats. A low dose of oLH (10 μg) was administered to hypophysectomized adult male rats following pretreatments with prolactin, follicle-stimulating hormone (FSH), growth hormone (GH), or saline. Testicular binding of hCG was determined at different times following the LH injection using Leydig cell membrane preparations from a testicular homogenate. Seven days after hypophysectomy, hCG binding was at a nadir of 19 ± 7% (mean ± SD) of control values. Pretreatment with prolactin (100 μg/day) for 7 days was associated with a nonsignificantly different hCG binding that was 30 ± 5% of control values. Prolactin pretreatment plus a single 10 μg LH i.p. injection increased ¹²⁵l hCG binding up to 56 ± 10% of control values within 30 minutes of the LH injection. Luteinizing hormone-induced hCG binding persisted at a high level (51 ± 4% of control values) for 2 hours but returned to hypophysectomized control levels 6 hours after the i.p. LH injection. Seven days pretreatment with FSH or GH at 100 μg/day plus 10-μg LH injections was also tested. Neither FSH nor GH had a statistically significant effect on hCG binding nor could they mimic the ability of prolactin to allow for LH induction of hCG binding in the hypophysectomized adult male rats. These studies suggest that the induction or “up-regulation” of Leydig cell hCG binding by ovine LH is rapid and specifically dependent upon pre-exposure to prolactin.