Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF.

BACKGROUND: The zona pellucida (ZP) has multiple roles in reproductive processes, including oocyte maturation, fertilization and implantation. We used, for the first time, a genetic approach to study whether human ZP genes possess structural alterations in women with unsuccessful IVF trials. In theory, this may result in gradual reduction of sperm-zona interaction and eventually in total fertilization failure (TFF). METHODS: Eighteen infertile women (TFFs) whose IVF did not result in any fertilized oocytes, whereas fertilization by ICSI was successful, were screened for mutations in ZP genes by means of conformation-sensitive gel electrophoresis. Twenty-three fertilizers in IVF (FIVFs) and 68 women with proven fertility (WPFs) constituted the two control groups. RESULTS: Altogether, 20 sequence variations were found in the ZP genes. Two variations in ZP3, one in the regulatory region (c. 1-87 T --> G) and one in exon 6 [c. 894 G --> A (p. K298)] existed more frequently in TFFs than in FIVF and WPF groups (P-values 0.027 and 0.008, respectively). CONCLUSIONS: Our study on ZP genes of infertile women revealed a high degree of sequence variations. This may reflect gradual reduction of fertility among TFFs, but the putative roles and influences of single variations can only be hypothesized.

Distribution of collagen XII and XIV in the bladder wall of the male rat with outlet obstruction.

PURPOSE: Our previous studies indicate that neonatal estrogenization with diethylstilbestrol (neoDES) of male mice and rats causes partial outlet obstruction. In the present study, type XII and XIV collagens were localized in the bladder to study their role in the development of obstruction. MATERIALS AND METHODS: The bladder sections immunostained with smooth muscle specific a-actin antibody were double labeled either with collagen type XII or type XIV antibodies. The specimens were then analyzed with conventional and confocal fluorescence microscope. RESULTS: Type XII and XIV collagens were not evenly distributed in the bladder. Further, in neonatally estrogenized rats collagen XIV appeared inside smooth muscle fascicles. CONCLUSIONS: Non-overlapping distributions of collagen XII and XIV suggest their different roles in the urinary bladder. Penetration of collagen XIV inside smooth muscle fascicles may have a role in the development of DES-induced partial outlet obstruction.

A novel component of epidermal cell-matrix and cell-cell contacts: transmembrane protein type XIII collagen.

Type XIII collagen is a short chain collagen which has recently been shown to be a transmembrane protein. The purpose of this study was to elucidate the presence and localization of type XIII collagen in normal human skin and cultured keratinocytes. Expression of type XIII collagen was demonstrated in normal human skin and epidermis at the RNA level using reverse transcription followed by polymerase chain reaction and at the protein level using western blotting and indirect immunofluorescence labeling. Immunolabeling of epidermis revealed type XIII collagen both in the cell-cell contact sites and in the dermal-epidermal junction. In cultured keratinocytes type XIII collagen epitopes were detected in focal contacts and in intercellular contacts. The results of this study show very little colocalization of type XIII collagen and desmosomal components at the light microscopic level. Thus, these results suggest that type XIII collagen is unlikely to be a component of desmosomes. Instead, the punctate labeling pattern of type XIII collagen at the cell-cell contact sites and high degree of colocalization with E-cadherin suggests that type XIII collagen is very likely to be closely associated with adherens type junctions, and may, in fact, be a component of these junctions.

Characterization of molecular and catalytic properties of intact and truncated human 17beta-hydroxysteroid dehydrogenase type 2 enzymes: intracellular localization of the wild-type enzyme in the endoplasmic reticulum.

Human 17beta-hydroxysteroid dehydrogenase (17HSD) type 2 is a widely distributed enzyme that primarily converts the highly active 17beta-hydroxysteroids to their inactive keto forms. In the present study, full-length human 17HSD type 2 was localized in the endoplasmic reticulum using a double immunofluorescence labeling technique. As a consequence of its strong membrane interaction, full-length human 17HSD type 2 could not be solubilized as a biologically active form in vitro. However, by deleting the first 29 amino acids from the N-terminus, we were able to purify a catalytically active enzyme from the cytosolic fraction of Sf9 insect cells. Biochemical and catalytic properties of the purified truncated human 17HSD type 2 protein confirm its suitability for structure-function analyses of the enzyme. Both intact and truncated 17HSD type 2 enzymes efficiently catalyzed the oxidation of estradiol, testosterone, dihydrotestosterone, androstenediol, and 20alpha-dihydroprogesterone. The oxidation of estradiol brought about by human 17HSD type 2 was effectively inhibited by several other steroidal compounds, such as 2-hydroxyestradiol, 5beta-androstan-3alpha,17beta-diol, 5alpha-androstan-3alpha,17beta-diol, and 5alpha-androstan-3beta,17beta-diol. The broad substrate specificity of human 17HSD type 2 together with its predominant oxidative activity and intracellular location, as observed in this study, indicate the physiological role of the enzyme to be primarily an inactivator of highly active 17beta-hydroxysteroids.

Expression of the elastin promoter in novel tissue sites in transgenic mouse embryos.

We have previously shown in a transgenic mouse line, in which 5.2 kb of the elastin promoter was linked to the reporter enzyme chloramphenicol acetyltransferase (CAT), that the highest levels of expression were found in embryonic lungs and aorta, while lower levels were detected in other elastin-containing tissues. Furthermore, in general, expression of the transgene showed developmental regulation similar to that of the endogenous gene. However, the precise location of cellular expression could not be determined in this model. To overcome this limitation, we have developed a similar model, but replaced CAT with the reporter enzyme beta-galactosidase. Enzyme activity was readily detected in the transgenic mouse embryos in expected regions of tissue forming elastic fibers, including the dermis and elastic cartilage. Of considerable interest, however, was the novel finding of expression in specific areas of neuroepithelium of the brain and in the perichondrium surrounding areas destined to form hyaline cartilage in endochondral bone formation. These latter areas included all the bones of the limbs, the spine and rib cage. It appeared that these segments of elastin expression demarcated the border between the developing cartilage and the surrounding mesenchymal tissue. Elastin promoter expression was also found in developing somites, in the mesenchymal layer of the forming cornea of the eye, in the genital tubercle and in the epithelium destined to form the olfactory epithelium. These findings indicate that the elastin promoter is activated during embryonic development in a variety of tissues, suggesting that elastin gene expression may play a role in organizing cutaneous, skeletal and neural structures.

Upregulation of tumor suppressor protein neurofibromin in normal human wound healing and in vitro evidence for platelet derived growth factor (PDGF) and transforming growth factor-beta1 (TGF-beta1) elicited increase in neurofibromin mRNA steady-state levels in dermal fibroblasts.

We first studied expression of neurofibromin by immunohistochemistry in scars obtained from operations involving areas of healing wounds. The results demonstrated increased immunoreactivity for neurofibromin in the fibroblastic cell population of the lesions when compared with fibroblasts of apparently healthy perilesional skin, or those of intact control skin. Furthermore, dermal fibroblasts of 19 and 34 wk-old fetuses displayed a clearly detectable immunosignal for neurofibromin. In vitro studies were designed to investigate the potential effects of selected growth factors--known to be operative in wound healing--on neurofibromin mRNA steady-state levels in cultured fibroblasts. Northern transfer analyses revealed that different isoforms of platelet derived growth factor (PDGF) exerted selective effects on the neurofibromin mRNA levels: PDGF isoform AB elevated neurofibromin mRNA levels in a concentration-dependent manner when concentrations of 0.1, 1, 10, and 30 ng per ml were used. The maximal upregulatory effect of PDGF BB was reached at a concentration of 1 ng per ml. In contrast, PDGF AA did not alter the steady-state levels of neurofibromin mRNA. As estimated by RNase protection assay, transforming growth factor-beta1 (TGF-beta1) upregulated neurofibromin gene expression when concentrations of 0.5 and 5 ng per ml were used. Reverse transcription followed by polymerase chain reaction did not detect apparent alterations in the ratio of type I/type II neurofibromin isoforms in PDGF- or TGF-beta1-treated cultures. Taken together, our results suggest that expression of tumor suppressor protein neurofibromin is upregulated in response to skin injury, and that this upregulation can be mediated through PDGF and TGF-beta.

Developmental regulation of NF1 tumor suppressor gene in human peripheral nerve.

Mutations of the NF1 tumor suppressor gene cause type 1 neurofibromatosis, characterized by multiple tumors of the peripheral nerves, as well as other tumor types. The NF1 protein, neurofibromin, is intricately linked to the cell growth regulatory signalling pathways, e.g. by possessing RAS-GTPase activity. The regulation and role of neurofibromin are not known in normal human development. We addressed this issue by studying the regulation of neurofibromin in normal human peripheral nerves, from early fetal development to adulthood. The barely detectable neurofibromin immunosignal in peripheral nerves during the first trimester of gestation contrasted dramatically to its increase in Schwann cells, perineurial cells, and axons during the second and third trimesters. Interestingly, the type I and II isoforms of neurofibromin, differing in their RAS oncoprotein inactivation capacity, displayed clearly different expression profiles throughout these periods. This suggests distinct cellular functions for these neurofibromin isoforms. The results also revealed distinct species-specific differences in neurofibromin expression, potentially bearing relevance to the lack of human neurofibromatosis-like disorders in other species.

Citric acid increases viable epidermal thickness and glycosaminoglycan content of sun-damaged skin.

BACKGROUND: Recently, there has been an exponential increase in the use of alpha-hydroxy acids in dermatologic practice. Their inclusion in a myriad of cosmetic preparations underscores their popularity. Among the clinical effects of alpha-hydroxy acids are their ability to prevent the atropy resulting from potent topical corticosteroids, improve the appearance of photoaged skin, and correct disorders of keratinization. Despite this range of desirable effects, very little is known about the specific changes produced by various alpha-hydroxy acid preparations in the epidermis and dermal extracellular matrix. Previous work by others has demonstrated the ability of another alpha-hydroxy acid to increase viable epidermal thickness, and dermal glycosaminoglycans. OBJECTIVE: In this study, we examined the effect of 20% citric acid lotion, as compared with vehicle alone, on skin thickness, viable epidermal thickness, and dermal glycosaminoglycan content. Biopsy samples were harvested after 3 months of treatment. RESULTS: Image analysis of biopsy sections revealed increases in viable epidermal thickness and dermal glycosaminoglycans in treated skin. CONCLUSIONS: Topical citric acid produces changes similar to those observed in response to glycolic acid, ammonium lactate, and retinoic acid including increases in epidermal and dermal glycosaminoglycans and viable epidermal thickness. Further studies of citric acid and other alpha-hydroxy acids are warranted to clarify their clinical effects and mechanisms of action.

Quantitative histochemical analysis of mast cells and sensory nerves in psoriatic skin.

To study the elements of neurogenic inflammation in psoriatic skin, morphological contacts were examined between mast cells and sensory nerves containing the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP) or vasoactive intestinal polypeptide (VIP). Because mast cells in psoriatic lesions appear in great numbers at the basement membrane (BM) zone, neuropeptide-mast cell contacts with the BM were also counted. A double stain for active mast cell tryptase and the neuropeptides was applied and the contacts were quantitated morphometrically. Sensory nerve-mast cell contacts were also studied three-dimensionally with a confocal laser scanning microscope. Increases in the contact values of SP and CGRP with mast cells, as well as with the BM, were obtained in developing (1-3 weeks) lesions when compared with their non-lesional controls. This increase reached statistical significance in mature lesions. In contrast, the corresponding contact values for VIP were decreased. By confocal microscopy, a close association between mast cells and sensory nerves was observed in the lesional dermis. Since tryptase is known to degrade CGRP but not SP, neurogenic stimuli, mainly via SP, can result in degranulation of mast cells, which release substances to enhance inflammation. At the BM zone in psoriatic lesions, the numerous mast cells loaded with tryptase can promote degradation of BM components and allow entry of various mediators to interact with keratinocytes.

Long-term sun exposure alters the collagen of the papillary dermis. Comparison of sun-protected and photoaged skin by northern analysis, immunohistochemical staining, and confocal laser scanning microscopy.

BACKGROUND: Long-term solar irradiation produces both morphologic and functional changes in affected skin. Because collagen is the major structural component of skin, any alteration in its production or degradation could have profound effects on cutaneous functional integrity. OBJECTIVE: Our purpose was to investigate alterations in the production and morphology of collagen fibers brought about by long-term sun exposure. METHODS: We compared collagen and collagenase gene expression and collagen immunohistochemical staining and used confocal laser scanning microscopy for morphologic examination of dermal collagen fibers in photodamaged compared with sun-protected skin from the same persons. RESULTS: Despite a large increase in elastin messenger RNA in sun-damaged skin, collagen and collagenase gene expression remained essentially unchanged. However, striking alterations in the papillary dermis of photoaged skin were found, which revealed large, abnormally clumped elastic fibers and deformed collagen fibers of various diameters, replacing the normal architecture of the papillary dermis. CONCLUSION: Our data provide evidence for normal collagen gene expression in sun-damaged skin and suggest that degradation and remodeling of collagen take place in the papillary dermis accompanied by deposition of other matrix components, predominantly abnormal elastic fibers.

Neurofibromin: expression by normal human keratinocytes in vivo and in vitro and in epidermal malignancies.

BACKGROUND: Neurofibromin is the product of the NF1 gene, the mutations of which have been linked with type 1 neurofibromatosis. The expression of neurofibromin in human skin has not been analyzed in detail. EXPERIMENTAL DESIGN: Polyclonal Ab were raised against synthetic peptides corresponding to three different sites of neurofibromin. One of the Ab selectively recognized type II neurofibromin. The localization of neurofibromin was first studied in normal human skin. Further studies concentrated on neurofibromin expression in basal cell and squamous cell carcinomas. Reverse transcription-PCR (RT-PCR) and molecular hybridizations and immunocytochemistry were used to characterize the expression of neurofibromin in cultured keratinocytes. RESULTS: All neurofibromin-specific Ab immunolabeled the epidermis. The basal keratinocytes displayed the most prominent immunosignal for type II neurofibromin. RT-PCR demonstrated the presence of both type I and II neurofibromin mRNA transcripts in cultured keratinocytes. Keratinocytes induced to differentiate and to arrest division by a high (1.4 mM) Ca2+ concentration of the culture medium displayed a down-regulation of neurofibromin expression at the mRNA and protein levels. This was most strikingly demonstrated by a reduction of immunoreactivity for type II neurofibromin. Basal cell carcinomas displayed a weak immunosignal for type II neurofibromin. In contrast, particularly the central areas of squamous cell carcinoma, islands were intensely immunolabeled. CONCLUSIONS: The results suggest that neurofibromin acts as a regulator of the basal keratinocytes in normal skin and that cultured keratinocytes offer a human model for studies aimed to elucidate the regulation of neurofibromin gene expression. Furthermore, aberrations in neurofibromin expression may play a role in the pathogenesis of epidermal cancers.

Processing of the LH/CG receptor and bound hormone in rat luteal cells after hCG-induced down-regulation as studied by a double immunofluorescence technique in conjunction with confocal laser scanning microscopy.

We developed a double immunofluorescence technique for detection of the rat luteinizing hormone/choriogonadotropin (LH/CG) receptor and bound hCG in the same rat ovarian section and used it in conjunction with confocal laser scanning microscopy (CLSM) to study the fate of the receptor-hormone complex in luteal cells during the hCG-induced down-regulation. Pseudopregnant immature females rats were perfusion-fixed before (0 hr) and 2, 6, 12, 24, or 36 hr after a down-regulating dose of hCG (500 IU IV). The cryosections were stained for the LH/CG receptor and bound hormone by sequential incubations with a polyclonal rabbit antiserum to purified rat LH/CG receptor and a mouse monoclonal antibody (MAb) to hCG, followed by sequential incubation with TRITC- and FITC-conjugated secondary antibodies to rabbit and mouse immunoglobulins, respectively. The results were semiquantitatively analyzed by a pseudo-three-dimensional (3D) plotting of the intensities of the receptor and hormone-specific fluorescence in luteal cells by CLSM. The analysis suggested that the majority of the LH/CG receptors are located on the luteal cells before induction of the down-regulation and that their content seem to vary not only among cells but also on the surface of single cells, thus supporting the previous concept of the functional heterogeneity among the cells and their functional compartmentation. At 2 hr after injection of the down-regulating dose of hCG, the LH/CG receptor-specific and hCG-specific fluorescences clearly co-localized on the luteal cells. Both the LH/CG receptor- and hCG-specific fluorescences disappeared from the luteal cell surfaces in a parallel fashion within 36 hr without a detectable accumulation of either fluorescence deep in the cell interior. These results suggest that the LH/CG receptor and bound hCG do not differ in their manner of in vivo processing in luteal cells. Therefore, the disappearance of the receptor and bound hormone occurs in a parallel fashion and without detectable internalization.

Regulation of intracellular free Ca2+ by the LH/CG receptor in an established cell line 293 expressing transfected rat receptor.

Luteinizing hormone (LH)/chorionic gonadotropin (CG) receptor (LHR) regulation of intracellular free Ca2+, [Ca2+]i, was studied by spectrofluorometric analysis and digital imaging of intracellularly trapped fura-2 fluorescence in a stable cell line 293 expressing transfected rat LHR. Exposure of the suspensions of 293-LHR cells to human CG (hCG) resulted in single, dose-dependent burst of elevated [Ca2+]i, the maximum being obtained at 0.1-1 microgram hCG/ml. The subconfluent individual 293-LHR cells responded to 1 microgram hCG/ml with either a single or oscillating [Ca2+]i bursts, the appearance of the oscillation being dependent upon the presence of extracellular Ca2+ ([Ca2+]e). 72% of the cells produced oscillation in response to hCG in the presence of [Ca2+]e and only 33% in the absence. Moreover, removal of [Ca2+]e from the incubation medium lowered the elevated basal [Ca2+]i level to or below the prestimulatory level and concomitantly damped out the oscillation, while its readdition without hCG was capable of re-elevating [Ca2+]i level and of gradually restoring the oscillation. The 293-LHR cells exposed to increasing doses of hCG also developed a dose-dependent desensitization of [Ca2+]i increase to renewed hormonal stimulation. The [Ca2+]i bursts within individual 293-LHR cells appeared in particular regions at the cell peripheries rather than distributed uniformly throughout the cytoplasm, pointing to compartmentation of the Ca2+ stores and to a local differences in receptor number in most cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A coupled one-step reverse transcription PCR procedure for generation of full-length open reading frames.

A one-step (all reactants added simultaneously) reverse transcription and polymerase chain reaction (RT-PCR) procedure for amplification of full-length open reading frames (ORFs) of relatively rare transcripts was developed. It was applied for cloning rat luteinizing hormone/chorionic gonadotropin receptor cDNA isoforms larger than two kb. In the procedure developed, manual work is minimized, thus large numbers of samples can be handled, since after denaturation of template RNA and the primers and addition of other reagents, no further manual steps are needed. No inhibitory effect of avian myeloblastosis virus (AMV) reverse transcriptase (RT) on Thermus aquaticus (Taq) DNA Polymerase was found. This was because, under the conditions described, Taq DNA Polymerase effectively amplified picogram amounts of plasmid DNA or template reverse transcribed from nanograms of total ovarian RNA in the presence of AMV-RT. Even a large excess of AMV-RT did not inhibit Taq DNA Polymerase. Thus, our coupled one-step RT-PCR procedure amplifies fast and reproducibly full-length ORFs from nanogram amounts of total RNA.

Human chorionic gonadotrophin (CG)-induced down-regulation of the rat luteal LH/CG receptor results in part from the down-regulation of its synthesis, involving increased alternative processing of the primary transcript.

To elucidate the molecular mechanisms involved in the homologous regulation of LH/chorionic gonadotrophin (CG) receptors, the receptor and its mRNA levels were analysed in the same pseudopregnant rat ovarian samples after human (h)CG-induced down-regulation using a binding assay, ligand blotting, immunoblotting and Northern blotting together with the polymerase chain reaction (PCR). Treatment of the animals with 500 IU hCG resulted in a loss of 125I-labelled hCG binding and the 90 kDa receptor on the ligand and immunoblots within 12 and 24 h respectively, followed by a transient partial recovery on days 4 and 5, while a distinct decline occurred only on day 7 in the controls. Northern blots of total ovarian RNA, as probed with a 293 bp AvaI/HindIII fragment from the extracellular domain of PCR-generated full-length rat LH/CG receptor cDNA, revealed six major mRNAs of 7.0, 4.2, 2.8, 2.0, 1.4 and 1.1 kb. The 4.2 kb mRNA, which was the most abundant, possibly encodes the 90 kDa receptor, while the smaller species probably represent alternatively spliced forms of the LH/CG receptor pre-mRNA, as also supported by the finding that PCR produced three cDNA bands of 2.1, 2.0 and 1.8 kb when oligomers derived from the N and C termini of rat LH/CG receptor cDNA were used as primers and rat ovarian total RNA as a template. Treatment with hCG led to the down-regulation of all six mRNAs in a fashion parallel to the changes in receptor protein. No smaller receptor components capable of binding radiolabelled hCG or receptor antibody appeared on the ligand or immunoblots prior to or during down-regulation or the subsequent transient period of up-regulation, suggesting that the smaller mRNA species are translated in minute amounts in vivo or are not translated at all. Laser densitometric analysis of the Northern blots revealed that the amounts of the four smallest mRNA species increased during the period of down-regulation in relation to the 4.2 kb mRNA, and correspondingly decreased during the subsequent period of up-regulation, indicating changes in the alternative splicing of the primary transcript. The data suggest that hCG-induced transient down-regulation of the LH/CG receptor results in part from down-regulation of its mRNA levels, and that changes in alternative processing of the receptor pre-mRNA may play a regulatory role in the expression of functional LH/CG receptor during down- and up-regulation.

Expression of the LH/CG receptor gene in rat ovarian tissue is regulated by an extensive alternative splicing of the primary transcript.

Luteinizing hormone/chorionic gonadotropin (LH/CG) receptor complementary DNA (cDNA) isoforms were amplified using pseudopregnant rat ovarian total RNA as a template and the primers reaching over the coding regions at both ends in a reverse transcriptase-polymerase chain reaction (RT-PCR). Agarose gel electrophoresis of the PCR products revealed three bands corresponding to about 2.1, 2.0 and 1.8 kilobases (kb). Subcloning of pooled PCR products into EcoRI site of pUCBM20 resulted in 167 clones, from which five different restriction patterns were obtained by digestion with EcoRI and HaeIII. One clone of each was further characterized. It could be predicted from the nucleotide sequences that the clone rLHR2100 encoded a full-length receptor (a 674 amino acid mature protein), the clone rLHR2075 lacked part of exon IX (nucleotides 693-717) and encoded a truncated 225 amino acid mature protein, the clone rLHR1950 lacked exons III and IV (nucleotides 246-395) and encoded a nearly full-length protein (a 624 amino acid mature protein), and the clones rLHR1834 and rLHR1759 lacked the same part of exon XI (nucleotides 960-1225), with exon V (nucleotides 396-470) also absent in the latter, the deletion in exon XI leading both these clones to premature termination. The clone rLHR1834 encoded a 316 amino acid mature protein and rLHR1759 a 291 amino acid mature protein, respectively. The sequence data suggest that all of these isoforms contain the putative signal sequence and are derived from a single copy gene via alternative splicing. These results point further to the fact that the expression of the 90 kDa LH/CG receptor is regulated via an extensive alternative splicing of the receptor gene primary transcript.

Two polyclonal antisera to rat luteal LH/CG receptor with different ligand binding inhibition and immunohistochemical receptor detection capabilities.

Polyclonal antisera to a SDS-denatured and partially renatured rat luteal 90 K LH/CG receptor were raised in rabbits, characterized, and their applicability for immunohistochemical location of the receptor examined. The LH/CG receptor was purified by hCG-affinity chromatography and subjected either to a preparative SDS-PAGE or Western blotting. Gel slices containing the SDS-denatured or nitrocellulose strips containing the renatured 90 K LH/CG receptor were used for immunization. The antisera, termed ARS-2 and ARS-3, respectively, possessed similar antibody titres. Both antisera were able to recognize the native, SDS-denatured, and SDS-denatured and reduced forms of the LH/CG receptor on dot blots, but only ARS-3 contained antibodies to the hormone binding site or a region near to it, as it was able to inhibit the hCG binding to the membrane-bound LH/CG receptor in a dilution-dependent manner. Both antisera recognized the receptor-hCG complex, but ARS-2 stained the complex with about 50% less intensity than the free receptor. ARS-3 located the LH/CG receptor distinctly on the luteal cell surfaces in immunohistochemical staining with peroxidase antiperoxidase complex method, but ARS-2, although it possessed similar antibody titre, revealed negligible staining. Thus, the antisera readily recognize the native receptor, but differ in their capability for inhibiting hormone binding. Only ARS-3, produced against the renatured receptor, contains sufficient amounts of antibodies capable of recognizing free and occupied receptors in immunohistochemistry.

Application of the immunofluorescence technique and confocal laser scanning microscopy for studying the distribution of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor on rat luteal cells.

We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.

Production and characterization of polyclonal antiserum to rat luteinizing hormone/chorionic gonadotropin receptor and its immunohistochemical application for studying receptor location and down-regulation.

A polyclonal antiserum against the affinity-purified nondenatured rat 90K LH/CG receptor polypeptide was raised in rabbits, characterized, and used to study the location of the LH/CG receptor in pseudopregnant rat luteal cells and the fate of the receptor-hCG complex together with the specific anti-hCG serum during hCG-induced down-regulation by immunochemical techniques. Even at a 1:3000 dilution, the antiserum recognized a single 90K polypeptide on Western blots of both the affinity-purified receptor and the initial detergent extract of the pseudopregnant rat ovarian membranes. It recognized sodium dodecyl sulfate-denatured and reduced, sodium dodecyl sulfate-denatured, and native forms of the receptor on dot blots; the immunoreaction was the most intense with the native receptor. The antiserum also contained antibodies that recognized the hormone-binding site, or a region near to it, and the occupied receptor. The majority of the LH/CG receptors were located on the luteal cells in pseudopregnant rat ovaries before the induction of down-regulation. The receptor content seemed to vary among the luteal cells, however, and on single cells, suggesting both functional heterogeneity and a functional polarization of the luteal cells. Upon induction of down-regulation with hCG both the receptors and the bound hormone disappeared from the luteal cell surfaces at a very slow rate, without any simultaneous appearance of receptor- or hCG-specific immunostaining in the luteal cell interior. No accumulation of receptor degradation products capable of [125I]iodo-hCG or antibody binding could be detected on Western blots of the tissue. The polyclonal LH/CG receptor antiserum described here is useful for studying the structure and function of this receptor, particularly for immunohistochemical investigations into receptor location and regulation.