Follicle-stimulating hormone and luteinizing hormone mediate the androgenic pathway in Leydig cells of an evolutionary advanced teleost.

The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC(50) 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole.

How we almost discovered LH receptors, but didn't.

The metabolism of gonadotropins was unclear until the 1960s. The chief theory, utilization of gonadotropins by gonads, was unproven, but radioimmunoassay indicated that the levels of luteinizing hormone entering the ovary were higher than the levels in the ovarian veins. The availability of radiolabeled proteins opened the possibility of following the fate of gonadotropins in the end organ. Independently, two teams in Tel Aviv and Seattle researched the uptake of radiolabeled human chorionic gonadotropin by rodent ovary. Both concluded that the ovary bound gonadotropin; however, neither pursued the mechanism of the observation, gonadotropin receptors on ovarian cells. Had they done so, the course of discovery and study of cell surface receptors might have been altered.

Luteinizing hormone and follicle-stimulating hormone receptors and their transcribed genes (mRNA) are present in the lower urinary tract of intact male and female dogs.

In dogs, one of the side effects of neutering is the development of urinary incontinence. The relationship between neutering and urinary incontinence caused by acquired urethral sphincter mechanism incompetence (USMI) has been reported. Recently, GnRH analogue treatment that suppresses elevated plasma gonadotrophin concentrations post-spaying has been successfully used in incontinent bitches. These data and the fact that non-gonadal tissues may contain receptors for LH (LHR) and FSH (FSHR) suggest that there might be a functional relationship between gonadotrophins and the lower urinary tract in dogs. This study aimed to investigate the presence of LHR and FSHR in the lower urinary tract of intact male and female dogs. Four regions of the lower urinary tract, i.e. (i) body of the bladder, (ii) neck of the bladder, (iii) proximal urethra and (iv) distal urethra were collected from 10 healthy dogs (5 males and 5 anoestrous females). In situ hybridization and immunohistochemistry were performed to characterise the presence of receptor mRNA and receptor protein. Staining was rated semi-quantitatively, incorporating both the distribution and intensity of specific staining. The distribution of receptor expression in different tissue layers (epithelium, subepithelial stroma and muscle) in each region was statistically analyzed. Luteinizing hormone receptor and FSHR mRNA and protein were present in all four regions and in three tissue layers of males and females. Irrespective of region and layer, female dogs expressed significantly higher expression for LHR mRNA (P<0.001), LHR protein (P<0.05) and FSHR protein (P<0.001). The expression of LHR and FSHR mRNA and protein was not uniform and depended on region, tissue layer and gender. The expression of LHR mRNA was higher in the bladder, compared to the urethra (P<0.05). The FSHR mRNA significantly increased from the bladder to the urethra. Protein expression for LHR and FSHR was highest in the proximal urethra (P<0.05). The overall expression for LHR and FSHR at both mRNA and protein levels was highest in the epithelium, intermediate to low in the subepithelial stroma and muscle. A significant interaction between region and tissue layer showed that mRNA and protein expression for LHR and FSHR decreased from the bladder to the urethra in the epithelium and subepithelial stroma. In contrast, it gradually increased from the bladder to the urethra in the muscle. In conclusion, the present study showed that both mRNA and protein for LHR and FSHR were expressed in the canine lower urinary tract, and the expression levels varied between genders and among regions and tissue layers. The presence of these receptors suggests that gonadotrophins have a role in the physiology and/or pathology of the lower urinary tract function in the dog.

High-level expression of a functional single-chain human chorionic gonadotropin-luteinizing hormone receptor ectodomain complex in insect cells.

Reproductive capacity in primates is dependent on the high-affinity binding of the glycoprotein hormones LH and human (h)CG to the large ectodomain (ECD) of their common receptor (LHR). Our understanding of the precise molecular determinants of hormone binding is limited, because there are no structural data for any of the glycoprotein hormone receptors. Overexpression of the ECD of the receptor has been attempted in various expression systems. Prokaryotic expression does not yield properly folded ECD. Eukaryotic expression, on the other hand, results in mostly heterogeneous, intracellularly trapped protein, but the secreted ECD is completely folded. Accordingly, we have tethered the single-chain hormone, yoked hCG, to the N terminus of LHR-ECD (yoked hormone-extracellular domain). Yoked hCG is secreted at high levels; binds LHR with high affinity; and, when tethered to the N terminus of full-length LHR, it binds and constitutively activates the receptor. Using recombinant baculovirus, yoked hormone-extracellular domain is secreted from insect cells at levels greater than 1 microg/ml, nearly 20-fold higher than that previously reported in eukaryotic expression systems. The protein was purified and binds exogenous (125)I-hCG with high affinity but, significantly, only after protease treatment to remove the tethered hormone. Thus, the fusion protein seems to form a functional hormone-receptor complex that is expressed at levels sufficient for its biophysical characterization.

Purification and structural analysis of a soluble human chorionogonadotropin hormone-receptor complex.

Receptors for the luteotropin/human chorionogonadotropin hormone belong to the G-protein-coupled receptor family by their membrane-anchoring domains. They also possess a large extracellular domain (ECD) responsible for most of the hormone-receptor interactions. Structure-function studies identified several contacts between hormone and receptor ECD, but the precise topology of the complex is still unknown because of the lack of suitable heterologous expression means. Receptor ECDs exhibit leucine repeats and have been modelized on the basis of the three-dimensional structure of the porcine ribonuclease inhibitor, the first structurally known leucine-rich repeats protein. Here we report overexpression (up to 20 mg per liter) and purification to homogeneity of a soluble human chorionogonadotropin-ECD receptor complex secreted by stably cotransfected Chinese hamster ovary cells. Biochemical analysis and surface plasmon resonance data were in favor of a unique dimer with a 1:1 ligand-receptor stoichiometry. Immunopurified complex was submitted to circular dichroism characterization; CD spectra deconvolution indicated more than 25% alpha helices contributed by the receptor, in agreement with the porcine ribonuclease inhibitor-based modelization.

Posttranslational modifications of the lutropin receptor: mass spectrometric analysis.

Our present knowledge of the lutropin (LH/hCG) receptor structure derives from deductions made from its amino acid sequence as established by studying the cDNA. To obtain direct experimental information, luteinizing hormone (LH) receptor expressed in L cells was immunopurified in sufficient amounts to warrant analysis by mass spectrometry and microsequencing. The mature receptor, complexed to human chorionic gonadotropin (hCG), was purified by using monoclonal antibodies recognizing the hormone, whereas the mannose-rich non-hormone-binding precursor was purified by use of antireceptor antibodies. Determination of the N-terminus showed that (2)/(3) of protein molecules started at Thr24 whereas (1)/(3) started at Ala28. All these molecules bound hCG, suggesting that the most N-terminal region of the receptor does not participate in hormone binding. Six N-glycosylation sites have been predicted from the amino acid sequence. One of them (Asn299) was found to be nonglycosylated in both the precursor and the mature protein. The most heavily glycosylated residue was Asn291, followed by Asn195 and Asn99. These three sites accounted for 82% and 97% of carbohydrate moieties in the mature receptor and in the mannose-rich precursor, respectively. The presence of some receptor molecules nonglycosylated at sites 99, 174, and 195 in hormone-receptor complexes dismisses a direct role of these glycosylation sites in hormone binding or in the correct folding of the protein. The mature carbohydrate chains were homogeneous at position 174, 195, and 313 (absence of Golgi mannosidase II activity at positions 174 and 313, absence of GlcNAc tranferases III and IV activity at position 195). Heterologous carbohydrates were present at sites 99 and 291. The latter, which is highly variable in carbohydrate chains, is unlikely to participate in a direct interaction with hormone. Site 313 thus remains as the main candidate for a role in hormone binding.

Murine oocytes suppress expression of luteinizing hormone receptor messenger ribonucleic acid by granulosa cells.

This study tested the hypothesis that murine oocytes participate in the establishment of granulosa cell phenotypic heterogeneity in preovulatory follicles. In these follicles, mural granulosa cells express LH receptors (LHR) and LHR mRNA, but expression of these molecules is low or undetectable in cumulus cells. Thus, the expression of LHR mRNA is a marker of the mural granulosa cell phenotype in preovulatory follicles. Cumulus cells expressed elevated steady-state levels of LHR mRNA when oocytes were microsurgically removed from oocyte-cumulus cell complexes, and this was prevented by paracrine factor(s) secreted by isolated oocytes. These factors also suppressed FSH-induced elevation of the level of LHR mRNA expression by mural granulosa cells isolated from small antral follicles, even when expression was augmented by culturing granulosa cells on components of basal lamina. Moreover, factor(s) secreted by oocytes suppressed the expression of LHR mRNA in mural granulosa cells isolated from preovulatory follicles already expressing elevated levels of these transcripts. The ability of oocytes to suppress the LHR mRNA expression by granulosa cells was developmentally regulated. Oocytes from preantral follicles and mature (metaphase II arrested) oocytes were less effective in suppressing expression than fully grown, germinal vesicle (GV)-stage oocytes. Furthermore, two-cell-stage embryos did not suppress LHR mRNA levels. Coculture of isolated oocytes with granulosa cells affected the synthesis of very few granulosa cell proteins detected by fluorography of two-dimensional gels after 35S-methionine labeling. Thus, oocyte suppression of FSH-induced LHR mRNA expression is specific in both the suppressing cell type and the effects on granulosa cells. It is concluded that the default pathway of granulosa cell differentiation produces the mural granulosa cell phenotype, as represented by the expression of LHR mRNA. This pathway is abrogated by oocytes. Thus, oocytes play a dominant role in establishing the fundamental heterogeneity of the granulosa cell population of preovulatory follicles.

Immunization of male rabbits with sheep luteal receptor to LH results in production of antibodies exhibiting hormone-agonistic and -antagonistic activities.

Antibodies to LH/chorionic gonadotrophin receptor (LH/CG-R; molecular weight 67 000), isolated in a homogenous state (established by SDS-PAGE and ligand blotting) from sheep luteal membrane using human CG (hCG)-Sepharose affinity chromatography, were raised in three adult male rabbits (R-I, R-II and R-III). Each of the rabbits received 20-30 micrograms of the purified receptor in Freund's complete adjuvant at a time. Primary immunization was followed by booster injection at intervals. Production of receptor antibodies was monitored by (1) determining the dilution of the serum (IgG fraction) that could specifically bind 50% of 125I-LH/CG-R added and (2) analysing sera for any change in testosterone levels. Following primary immunization and the first booster, all three rabbits exhibited a 2.5- to 6.0-fold increase in serum testosterone over basal levels and this effect was spread over a period of time (approximately 40 days) coinciding with the rise and fall of receptor antibodies. The maximal antibody titre (ED50) produced at this time ranged from 1:350 to 1:100 to below detectable limits for R-I, R-II and R-III respectively. Subsequent immunizations followed by the second booster resulted in a substantial increase in antibody titre (ED50 of 1:5000) in R-I, but this was not accompanied by any change in serum testosterone over preimmune levels, suggesting that with the progress of immunization the character of the antibody produced had also changed. Two pools of antisera from R-I collected 10 days following the booster (at day 70 (bleed I) and day 290 (bleed II)) were used in further experiments. IgG isolated from bleed I but not from bleed II antiserum showed a dose-dependent stimulation of testosterone production by mouse Leydig cells in vitro, thus confirming the in vivo hormone-mimicking activity of antibodies generated during the early immunization phase. The IgG fractions from both bleeds were, however, capable of inhibiting (1) 125I-hCG binding to crude sheep luteal membrane (EC50 of 1:70 and 1:350 for bleed I and II antisera respectively) and (2) ovine LH-stimulated testosterone production by mouse Leydig cells in vitro, indicating the presence of antagonistic antibodies irrespective of the period of time during which the rabbits were immunized. The fact that bleed I-stimulated testosterone production could be inhibited in a dose-dependent manner by the addition of IgG from bleed II to the mouse Leydig cell in vitro assay system showed that the agonistic activity is intrinsic to the bleed I antibody. The receptor antibody (bleed II) was also capable of blocking LH action in vivo, as rabbits passively (for 24 h with LH/CG-R antiserum) as well as actively (for 430 days) immunized against LH/CG-R failed to respond to a bolus injection of LH (50 micrograms). At no time, however, was the serum testosterone reduced below the basal level. This study clearly shows that, unlike with LH antibody, attempts to achieve an LH deficiency effect in vivo by resorting to immunization with holo LH receptor is difficult, as receptor antibodies exhibit both hormone-mimicking (agonistic) as well as hormone-blocking (antagonistic) activities.

Photoaffinity labeling of the lutropin receptor with synthetic peptide for carboxyl terminus of the human choriogonadotropin alpha subunit.

Human choriogonadotropin (hCG) consists of an alpha subunit and a beta subunit. The existing evidence from various studies using truncation, substitution, synthetic hormone peptides, and hCG crystals suggests that the C-terminal region of the alpha subunit contacts the luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is involved in receptor activation. Despite a deluge of the speculation and the important role of the alpha C-terminal region, direct evidence for its interaction with the receptor has been elusive. Because of the significant biological activity, it is imperative to prove the interaction of the alpha C-terminal region. For this purpose, decamer peptides corresponding to the alpha subunit sequence from His83 to Ser92 (alpha 83-92) were derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoylglycine (ABG) and radioiodinated. The resulting ABG-125I-alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, UV-sensitive ABG-125I-alpha 83-92 exclusively photoaffinity-labeled an approximately of 86-kDa molecule. This labeled molecule was shown to be the LH/CG receptor by various methods including immunoprecipitation by anti-LH/CG receptor antiserum. In addition, evidence is presented that the amino group of alpha Lys91 of alpha 83-92 is in such close proximity to a carboxyl group of the receptor that this pair is cross-linked to form an amide, a zero length cross-link. This low affinity contact of alpha 83-92 and the receptor is sufficient for receptor activation and is crucial for the full understanding of the mechanistics of the receptor activation steps.

Identification of amino acid residues in transmembrane helices VI and VII of the lutropin/choriogonadotropin receptor involved in signaling.

The lutropin/chroriogonadotropin receptor (LH/CG-R) is a member of the G protein-coupled receptor superfamily containing a relatively large extracellular domain responsible for high affinity ligand binding. There is a paucity of information on the mechanism of ligand-mediated transmembrane signaling via the seven transmembrane helices (TMH) of these receptors. In the present study we have used site-directed mutagenesis to replace each of nine conserved amino acid residues in the TMHs of rat LH/CG-R. COS-7 cells were transiently transfected with the eukaryotic expression vector pSVL-LH/CG-R, wild-type and mutants, followed by measurements of human CG binding and human CG-mediated cAMP production. Three point mutants of LH/CG-R were prepared that diminished signaling but not binding: Pro-562-Leu (TMH VI), Pro-591-Leu(TMH VII), and Tyr-601-Ala (TMH VII). Two point mutants of LH/CG-R, Pro-479-Leu(TMH IV) and Pro-598-Leu(TMH VII), resulted in impaired localization, and four receptor mutants, Thr-424-Ala (TMH III), Ser-562-Ala (TMH VI), Met-560-Leu (TMH VI), and Tyr-590-Ala (TMH VII), were similar to wild-type LH/CG-R. In summary, these findings indicate a critical role of Tyr-601 in transmembrane signaling of LH/CG-R since an Ala replacement results in almost total abolition of cAMP production in response to human CG; prolines 562 and 591 also appear to be important for full signaling.

Functional reconstitution of detergent-solubilized bovine calf testis luteinizing hormone/chorionic gonadotropin receptor into phospholipid vesicles.

An LH/CG receptor-enriched fraction was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testicular homogenates and solubilized with Triton X-100. To confirm the functional nature of the detergent-solubilized LH/CG receptor, the extract was first incorporated by lipid hydration into phospholipid vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. LH/CG receptor incorporation was then determined by measurement of specific binding of [125I]hCG. Specific binding of [125I]hCG by the reconstituted receptor was saturable, time-dependent, and thermally stable at room temperature. Scatchard analysis of competitive binding data indicated the presence of a single class of high-affinity (6.9 x 10(10)M-1), low-capacity (17.5 fmol hCG/mg protein) binding sites. The reconstituted receptor was functionally coupled to adenylyl cyclase and responded to both LH and NaF with increased cyclic AMP (cAMP) production. Stimulation of LH/CG receptor-enriched proteoliposomes with LH resulted in concentration-dependent uptake of external calcium (as 45Ca2+), which was hormone-specific, saturable, and sensitive to blockade by voltage-dependent and voltage-independent calcium channel antagonists. Similar uptake could not be induced by sodium fluoride, (Bu)2 cAMP, GTP-gamma-S, cholera toxin, or pertussis toxin. These results indicate that the reconstituted LH/CG receptor, as is the membrane-associated receptor, is functionally coupled to signal transduction pathways involving both adenylyl cyclase activation and calcium mobilization, and is a reliable working model that will facilitate further examination of the molecular mechanisms of LH action.

Identification of two amino acid residues on the extracellular domain of the lutropin/choriogonadotropin receptor important in signaling.

The lutropin/choriogonadotropin receptor (LH/CG-R) is a G protein-coupled receptor with a relatively large extracellular domain. The cDNAs of LH/CG-R wild type and 15 point and double mutants, which encoded residues of opposite charge to that of wild type, were transiently transfected into COS-7 cells. Human choriogonadotropin (hCG) binding was determined, as was hCG-mediated cAMP production. Most of the replacements resulted in no substantive effect on the binding affinity of hCG to LH/CG-R or on hCG-stimulated cAMP production, although the mutants expressed at a lower level than LH/CG-R wild type. The most interesting observation was noted with two point mutants of LH/CG-R, Glu332-->Lys and Asp333-->Lys, which bound hCG but failed to give increased cAMP production. Several of the mutant forms of LH/CG-R that expressed at low levels were further analyzed by soluble binding assays and Western blots. There was no evidence of any significant degree of intracellular trapping of hCG-binding mutant receptors. The expected major (93 kDa) and minor (78 kDa) forms were found for LH/CG-R wild type and several of the mutants. The Lys235-->Asp and Asp333-->Lys mutants exhibited primarily the lower M(r) form, indicating that receptor processing was impaired or that the mutant higher M(r) form was more rapidly degraded than LH/CG-R wild type. These results demonstrate that Glu332 and Asp333, which are located near the first transmembrane helix, are important in receptor activation, while other conserved ionizable residues of LH/CG-R appear important in cell surface expression or stability but not in binding or signaling.

Identification of luteinizing hormone receptor binding inhibitor in bovine corpora lutea.

A 7000 g supernatant, obtained during the purification of luteinizing hormone (LH) receptor from bovine corpora lutea homogenate, was concentrated by ultrafiltration. The filtrate, containing < 50,000 molecular weight material, exhibited LH receptor binding inhibitor (LH-RBI) activity. The filtrate was ultrafiltered sequentially through Amicon PM-10, PM-30 and UM-2 filters to yield a LH-RBI-containing fraction in the higher molecular weight range of 30,000-10,000 and a LH-RBI-containing fraction in the lower molecular weight range of 10,000-1000. The higher molecular weight LH-RBI fraction was purified on Sephadex G-25 and the lower molecular weight LH-RBI fraction was purified on Sephadex G-50. Both the high- and the low-molecular-weight LH-RBI species inhibited the binding of 125I-labeled human chorionic gonadotropin (hCG) to bovine corpora lutea and to rat Leydig cell membrane receptors. Similarly, the production of testosterone by hCG-stimulated rat Leydig cells was inhibited in a dose-response manner by both the high- and the low-molecular-weight LH-RBI species. The LH-RBI activity in the low-molecular-weight species was stable at 4 degrees C for up to 6 months and at temperatures up to 90 degrees C for 15 mins, whereas the LH-RBI activity of the high-molecular-weight species was stable at 4 degrees C for 15 months and unstable at 60 degrees C after 15 min. The 7000 g supernatant provided a much-needed source to obtain larger than previously reported quantities of LH-RBI for isolation as well as for structure and function studies.

Palmitoylation of luteinizing hormone/human choriogonadotropin receptors in transfected cells. Abolition of palmitoylation by mutation of Cys-621 and Cys-622 residues in the cytoplasmic tail increases ligand-induced internalization of the receptor.

We have examined whether the two cysteine residues (621 and 622) of the carboxyl-terminal cytoplasmic domain of the rat luteinizing hormone (LH/hCG) receptor are potential sites for palmitoylation. The full-length LH/hCG receptor cDNA was cloned into an expression vector (hCGR-pCMV4). A human embryonic kidney cell line expressing large T antigen (293T cells) was transiently transfected with hCGR-pCMV4 by the calcium phosphate precipitation technique. The functional expression of the receptor was confirmed by 35S-labeled cysteine incorporation into the receptor as well as by 125I-human chorionic gonadotropin (hCG) binding. The transfected cells were then labeled with [3H]palmitic acid, and the labeled receptors purified on hCG-Affi-Gel matrix and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The hCGR-pCMV4-transfected cells incorporated [3H]palmitic acid into a 92-kDa band corresponding to the mature form of the LH/hCG receptor; this band was absent in cells transfected with vector alone. Site-directed mutagenesis of either cysteine 621 or 622 to serine residue was partially inhibitory, whereas mutation of both cysteine residues (621 and 622) completely abolished palmitoylation. Scatchard analyses revealed that the mutant and wild type receptors have similar affinities for 125I-hCG. The biological function of palmitoylation was then examined in the transfected cells. The results showed that although the intracellular trafficking of the receptor and the ability to stimulate cyclic AMP production were unaffected, the rate of ligand-induced internalization of the receptor was higher in palmitoylation-deficient mutants compared to the wild type receptors. The first order rate constants of internalization of the mutant receptors were over 2-fold higher than the wild type. Intracellular degradation of the receptor-bound ligand was also higher in the mutants. These studies suggest that the native LH/hCG receptor is palmitoylated at cysteine residues 621 and 622, creating a membrane-anchoring site at the putative cytoplasmic domain. This palmitic acid-mediated anchoring decreases the ligand-induced receptor internalization thereby prolonging the retention of the ligand-bound receptor on the cell surface.

Uterine luteinizing hormone/human chorionic gonadotropin-binding sites in the early pregnant rat uterus: evidence for total occupancy in the periimplantation period.

We have investigated the presence of high affinity LH/hCG-binding sites (RLH) in crude membranes from early pregnant rats uteri. The uterine concentration of available RLH increased from day 1 to day 3 (1.3 +/- 0.2 vs. 2.8 +/- 0.4 fmol/mg protein) without a change in the affinity constant (approximately 5 x 10(10) M-1). However, unoccupied uterine RLH disappeared in the periimplantation period (days 4-6). To determine if the drop in available RLH was consecutive to their occupancy, uterine membranes were treated with acidified medium (25 mM Tris-HCl, and 5 mM MgCl2, pH 2.5) to remove endogenous ligand. The number and affinity of total (occupied plus available) RLH in acid-eluted membranes were estimated by Scatchard analysis of [125I]hCG binding and compared with those of available RLH in untreated membranes from the same uterine preparation. The uterine concentration of total RLH increased first between days 1 and 2 (2.2 +/- 0.5 vs. 4.2 +/- 0.8 fmol/mg protein), then between days 3 and 4 (4.2 +/- 0.6 vs. 6.5 +/- 0.8 fmol/mg protein), before plateauing until day 6. Thus, the reduction in the available uterine RLH in the periimplantation period is largely due to occupancy, rather than down-regulation, of RLH. The occupancy of uterine RLH 1) increased during early pregnancy (day 1, approximately 20%; days 2-3, approximately 40%; days 4-6, approximately 100%), 2) paralleled the increase in total RLH number, and 3) was probably due to pituitary LH only. However, the blastocyst itself seemed to influence uterine RLH occupancy, since available uterine RLH were detected on day 5 of pseudopregnancy. The increase in total uterine RLH as well as the perfect synchrony between their occupancy and the previously described pattern of uterine cAMP concentration during rat early pregnancy suggest that the response of uterine (and more precisely luminal epithelium) adenylate cyclase to LH (and/or related substance originating from embryo) may determine uterine receptivity for ovoimplantation and subsequent decidualization.

The size of the mature membrane receptor for follicle-stimulating hormone is larger than that predicted from its cDNA.

A 240 kDa protein isolated from bovine calf testis has been shown to have properties characteristic of an FSH receptor. However, rat testis FSH receptor has, on the basis of cloning experiments, been found to have a much lower molecular mass of 75 kDa (peptide only). To examine this point, the size of the FSH receptor in membranes obtained from cultured Sertoli cells of immature rats was determined after polyacrylamide gel electrophoresis under non-reducing conditions, followed by transfer to polyvinylidene difluoride membranes and direct identification of the FSH receptor by ligand blot analysis utilizing radioiodinated human FSH. In this system, the rat Sertoli cell membrane FSH receptor also showed a molecular mass of 240 kDa. Bovine testis contains LH and FSH receptors. We compared the sizes of FSH and LH receptors present in the same bovine testis membrane preparation by ligand blot analysis. The FSH receptor again showed a molecular mass of 240 kDa, whereas the LH receptor showed a molecular mass of 90 kDa. The latter value is similar to that deduced by cloning techniques (75 kDa, peptide only). The evidence seems to suggest that, whereas the molecular mass deduced for the LH receptor on the basis of its cDNA is similar to that of the mature membrane receptor, the size of the FSH membrane receptor is considerably different from that deduced on the basis of its cDNA, presumably as a result of post-translational processing. The marked difference in size between mature FSH (240 kDa) and LH (90 kDa) receptors may reflect significant structural differences of importance with regard to mechanisms of signal transduction.

Developmental changes in levels of luteinizing hormone receptor and androgen receptor in rat Leydig cells.

To further assess the hormonal response capabilities of Leydig cell progenitors (PLC) from 21-day-old rats, their levels of LH and androgen receptors (LH-R and AR) were measured and compared to those of isolated immature (ILC) and adult Leydig cells (ALC) from 35- and 90-day-old rats, respectively. Levels of LH receptor were estimated by Scatchard analysis of binding to [125I]hCG, and levels of LH receptor mRNA were determined by Northern blot analysis using a rat LH receptor antisense RNA probe. The numbers of LH receptors per cell measured by the binding study were 2,623 +/- 1,110 in PLC, 9,024 +/- 1,992 in ILC, and 39,896 +/- 15,234 in ALC (mean +/- SEM of four replicate experiments; ALC significantly greater than either PLC or ILC at P less than 0.05). The Northern blotting revealed three major bands [6.7, 2.6, and 2.3 kilobases (kb)] that were present in Leydig cells at all three ages and were not detected in HepG2 cells. When the steady state levels of the predominant 6.7-kb species were normalized to actin mRNA, PLC were 6.3-fold lower than ILC and 1.7-fold lower than ALC (n = 3 replicate isolations of poly(A) RNA). The 2.6- and 2.3-kb species exhibited similar trends. Levels of AR were estimated by immunoblotting using a polyclonal antibody against a synthetic peptide of the receptor (residues 14-32) that detected a 110-kilodalton AR protein. Levels of AR mRNA were estimated by Northern blot analysis, using a rat AR antisense RNA probe that detected a single 10-kb AR mRNA. The relative levels of AR protein were 1.0, 1.5, and 0.5 in PLC, ILC, and ALC, respectively (n = 3). Similar trends were observed for AR mRNA (n = 3). The observation that both LH and AR levels were lower in PLC compared to ILC is consistent with the hypothesis that the former are progenitors of Leydig cells.

Variant forms of the pig lutropin/choriogonadotropin receptor.

The cloning and sequencing of porcine lutropin/choriogonadotropin (LH/hCG) receptor messenger RNAs have shown the presence of a full-length receptor (pLHR-A) and of shorter variants lacking either the transmembrane and the intracellular domains (pLHR-B and pLHR-C) or only the transmembrane domain (pLHR-D). Moreover, immunoblotting of testicular membrane extracts has detected 85-, 68-, and 45-48-kDa proteins reacting with antireceptor antibodies. Transfection experiments were performed to assign the protein species to the various messenger RNAs and to study the function of the various receptor species. COS-7 and L-cells transfected with an expression vector encoding full-length receptor pLHR-A yielded a protein of apparent molecular mass of 105 kDa. This corresponded to the complete receptor which had undergone a different glycosylation pattern to that found in testis, since after digestion with peptide N-glycosidase F both the 105-kDa COS-7 protein and the 85-kDa testicular glycoprotein yielded a holoprotein of approximately 63 kDa. Transfection with pLHR-A also yielded a high proportion of the 68-kDa glycoprotein which was shown by digestion with endoglycosidase H to be a high-mannose precursor of the full-length receptor. The existence of a large pool of precursor species in both transfected cells and Leydig cells evokes possible physiological regulations at the level of receptor maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormone glycosylation required for lutropin receptor recognition in sheep testis.

The high affinity binding sites for ovine pituitary lutropin (oLH) present in DLS-1 sheep testis recognized only the fully glycosylated ovine or bovine hormone (bLH) in receptor binding assays using 125I-labeled oLH. Chemically deglycosylated (DG-) oLH or bLH which were fully active with other lutropin receptors (rat/pig) were completely inert in the DLS-1 receptor assay. In the same membranes, the FSH (follitropin) receptor reacted well with both glycosylated FSH and DG-oFSH. In recombination studies, lutropin formed by glycosylated native alpha- and beta-subunits of the hormone was fully active but when one of the subunits was in the deglycosylated form, receptor binding activity was greatly reduced. The presence of glycosylated alpha-subunit in the recombined hormone gave rise to 5x more activity than DG-alpha + beta. All these preparations were fully active in the rat/pig receptor assays for LH. These results demonstrate that lutropin hormone glycosylation is essential for optimum receptor recognition in the sheep testis, further emphasizing the importance of correct glycosylation in oLH alpha hormone function.

Purification and characterization of the human ovarian LH/hCG receptor and comparison of the properties of mammalian LH/hCG receptors.

Methods previously published by us [Wimalasena et al., J Biol Chem 260: 10689-10697, 1985; Wimalasena et al., J Biol Chem 261: 9416-9420, 1986] were utilized to solubilize the human corpus luteal leuteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor with 3-[(3-cholamide-propyl)dimethylammonio]-1-propanesulfonate (CHAPS) and to purify the receptor by two steps of hCG-Sepharose affinity chromatography. The specific binding capacity (SBC) of the purified human receptor was 7510 pmol/mg protein, and KA = 2.2 x 10(9) M-1 when iodo hCG was competed by hCG; the yield was 4-7% of starting activity. When hLH was used in competition with hCG, specific binding capacity was 7900 pmol/mg protein and KA 1.0 x 10(9) M-1. Silver staining and autoradiography demonstrated a single protein of Mr 78,000 under reducing and Mr 58-62 x 10(3) under nonreducing conditions. Rat ovarian LH/hCG receptor was purified by similar methods and the KA of 3.5 x 10(10) M-1 for hCG was substantially different from the KA for hLH which was 2.1 x 10(9) M-1. Mr of the rat protein was 78-82 x 10(3) (reduced) and 58-62 x 10(3) (nonreduced) when analyzed by silver staining and autoradiography. For the first time, human LH/hCG receptor has been purified to apparent homogeneity, and its Mr of 78,000 was essentially identical to the Mr values of purified rat and porcine receptors.