Identification of LH/hCG receptors in rabbit uterus

Luteinizing hormone (LH) is believed to act via specific receptors to control gonadal steroidogenesis and reproductive processes. Recently A. J. Ziecik, P. D. Stanchev, and J. E. Tilton (Endocrinology 119:1159, 1986) reported surprisingly that LH/hCG receptors were present in porcine uterus, a tissue not known to be a target for LH action. We report herein the identification of high-affinity LH receptors in the rabbit uterus. Uteri from adult New Zealand white rabbits were homogenized in Tris-HCl, 0.25 M sucrose. After filtration and sequential centrifugation, a partially purified pellet containing receptors was obtained. This preparation was incubated with a trace (1300 cpm) (50 pg) 125I-labeled chorionic gonadotropin and with various unlabeled protein hormones. Receptor bound was separated from free hormone by centrifugation at 1000 g. Affinity was estimated by Woolf plot analysis. Specific binding sites for LH/hCG were identified. The following Kd's were calculated: human LH, 1.6 X 10(-11); hCG, 0.5 X 10(-11); human TSH, 1.3 X 10(-9); and human FSH, 7.85 X 10(-9). The reaction of human FSH and TSH with the receptor is best explained by LH contamination of these hormones. A similar preparation of rat liver showed that no binding sites were present. Rabbit ovarian LH receptors had a Kd slightly higher at 4.1 X 10(-11) than that of the uterine LH receptors. Rabbit ovarian receptors were present at 2.27 X 10(-13) M/mg protein compared to uterine receptors at 4.65 X 10(-15) M/mg protein. We conclude specific- and high-affinity binding sites (receptors) for LH are present in the rabbit uterus. The function of these receptors remains unknown.     

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.     

Dependence of uterine cyclooxygenase2 expression on luteinizing hormone signaling

Several previous studies have demonstrated that uterine Cox2 (also known as Ptgs2) is required for implantation. Luteinizing hormone (LH) released from anterior pituitary gland and human chorionic gonadotropin released from placenta (hCG) can upregulate the uterine Cox2 gene expression. The Lhcgr knockout (herein designated LHRKO) animals have implantation failure even after estradiol and progesterone therapy. These findings led us to investigate the dependence of uterine Cox2 gene expression on LH signaling in LHRKO animals. The results revealed that, while Cox1 (also known as Ptgs1) mRNA levels were similar, Cox2 mRNA levels were lower in uterus of null animals than in wild-type siblings. Treatment with hCG did not increase Cox2 mRNA levels in null endometrial stromal or myometrial smooth-muscle cells unless gene therapy was performed to introduce native LHCGR. The Cox1 mRNA levels, on the other hand, did not change regardless of the introduction of native or activated Lhcgr or hCG treatment. The Cox2 mRNA increase paralleled the cAMP raise, suggesting that LH uses the cAMP second messenger system. Treating the wild-type uterine cells with hCG resulted in a Cox2 but not Cox1 mRNA increase. This increase became exaggerated when additional native LHCGR were introduced by gene therapy. In conclusion, deletion and reinsertion of Lhcgr further support that uterine Cox2 gene expression is dependent on LH signaling.     

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.     

A physiological increase in LH may influence vascular permeability in the rat testis

Adult male rats were injected with different doses of hCG, or with 2.5 micrograms ovine LH subcutaneously, and other rats were mated with oestrous females. The animals were examined 4 h after treatments. Treatment with hCG resulted in a dose-dependent increase in leucocyte concentration in testicular blood vessels and in the number of blood vessels which could be labelled with intravenously injected carbon particles. Carbon leakage was not observed in control testes. Treatment with a low dose of ovine LH or inducing an endogenous LH peak by mating also resulted in leucocyte accumulation and vascular leakage of carbon in the testis. The magnitude of the response was considerably lower than after high doses of hCG. The physiological relevance of the discrete response observed after physiological LH stimulation is unknown but LH-induced changes in testicular microcirculation could be of interest for the understanding of the physiology and pathophysiology of the testis.     

Effects of human chorionic gonadotrophin on contractile activity of steroid-primed pig myometrium in vitro

We examined the effects of (a) oestrogen and progesterone on concentrations of luteinizing hormone/human chorionic gonadotrophin (LH/hCG) receptors in uterine smooth muscle in vivo and (b) hCG on spontaneous myometrial contractions in vitro. Ovariectomized gilts received 2 ml corn oil (control; n = 5), 2 mg oestradiol benzoate (n = 6) or 20 mg progesterone (n = 5) for 5 days. Gilts were hysterectomized 8 h after the last injection and longitudinal sections of myometrium were incubated in modified Krebs' solution with 0 or 10 i.u. of hCG (n = 10/gilt) for 4 h at 37 degrees C in 95% O2:5% CO2. After incubation, myometrial sections were placed in a tissue chamber perfused with Krebs' solution and mechanical activity was recorded for 30 min. Cell membrane fractions were prepared from myometrial tissue not used for in-vitro studies and analysed for LH/hCG receptors. Treatment with oestradiol benzoate increased (P less than 0.01) the number of LH/hCG-binding sites compared with gilts receiving corn oil or progesterone. Incubation of myometrium with hCG reduced (P less than 0.01) the frequency and amplitude of spontaneous uterine contractions in gilts treated with oestradiol benzoate. In contrast, hCG had no effect (P greater than 0.05) on the pattern of myometrial contractions in gilts given corn oil or progesterone. These results indicate that oestradiol promotes the synthesis of LH/hCG receptors in pig myometrium and incubation of oestrogen-primed tissue with hCG has a quiescent effect on myometrial contractility.     

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

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

Human chorionic villous macrophages as a fetal biological shield from maternal chorionic gonadotropin

In addition to its most well characterized biological role in the rescue and maintenance of corpus luteum function, human chorionic gonadotropin (hCG) also stimulates the onset of fetal gonadal steroidogenesis. However, excess hCG is teratogenic to fetal gonadal tissues, and therefore hCG must be tightly regulated. Although there is an anatomical barrier between the fetal vessels and maternal blood, other mechanisms may regulate hCG levels. In the present study, we investigated whether human chorionic villous macrophages degraded maternal hCG. Isolated human macrophages incorporated and degraded hCG in a time-dependent manner. Human placental villous macrophages and phorbol myristate acetate (PMA)-treated THP-1 cells expressed the gene encoding an exon 9-deleted form of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor; expression of the full-length receptor was not determined. While both PMA-treated or untreated THP-1 cells could uptake hCG into their cytoplasms, hCG degradation and excretion of its byproducts only progressed in PMA-treated THP-1 cells. In conclusion, hCG internalization and degradation are different processes in macrophages that protect fetal gonadogenesis from excess hCG. The exon 9-deleted LH/CG receptor, but not the full-length receptor, is involved in the degradation of cytoplasmic hCG by organ-specific, dominant–negative interactions.     

Uterine trauma and limb defects

The temporary clamping of the uterine blood vessels on one side of the uterus during late pregnancy in the rat (days 14-16) results in hemorrhage and tissue necrosis in the extremities of the fetuses from the experimental side and occasionally from the control side. A further series of experiments showed that similar fetal hemorrhage followed the temporary clamping (45 minutes) of the uterine wall or uterine fat, excluding major uterine vessels; handling the uterus for 5 minutes; and stretching of the uterine blood vessels. A low incidence of fetal hemorrhage was also associated with laparotomy alone, but the fetuses were unaffected by extensive handling of the uterus through the abdominal wall or by intraperitoneal anesthesia. Fetal hemorrhage was also induced by a short episode of severe maternal hyperthermia but not by a high dose of ethanol given by gavage. These results suggest that a range of uterine trauma may result in fetal hemorrhage, perhaps through a common mechanism.     

Morphological analysis of transportation pathways of microspheres after their introduction into the uterine horn cavity in cyclic pigs

Countercurrent transfer is thought to be one of the most important mechanisms involved in the transfer of substances between the uterus and oviduct. The present study was aimed at recognizing other putative transportation pathways from the uterine cavity through the oviduct onto the surface of and into internal ovarian structures. Microspheres (latex beads, 0.8 m in diameter) were introduced into the uterine horn cavity of pigs, for 30 min, at various days of the estrous cycle. The transportation pathways of the beads were then analyzed by light and electron microscopy. The transport of microspheres through the oviduct canal into ovarian tissues took place on each day of the estrous cycle. The largest numbers of microspheres passed through the tunica albuginea to the corpora lutea. Some of microspheres also reached the surface of the uterine ligament through the oviduct canal, where they attained the lumen of blood and lymphatic vessels, mainly of the vascular subovarian (VSP) and paraovarian lymphatic plexus (PLP), via the lymphatic stomata pathway. Transport of microspheres also took place simultaneously through the uterine and oviduct walls and from particular organs through blood and lymphatic vessels. Although the present results do not exclude the participation of countercurrent transfer between venous, lymphatic, and arterial vessels, they provide morphological evidence for the presence of direct transportation pathways of substances, released into the uterine lumen, into ovarian tissues through the oviduct canal.     

Dynamics of Eosinophils in the Uterus after Oestrogen Administration

To investigase the role of the eosinophil leukocytes in the early oestrogenic responses in the uterus, the kinetics of oestrogen-induced uterine eosinophilia and other parameters of oestrogen stimulation were studied at very early times. Uterine eosinophils increase as early as 5 min after an intravenous injection of oestradiol to immature rats, much earlier than several other changes in the early parameters of oestrogen stimulation. Large number of uterine eosinophils are found attached to the wall of small uterine blood vessels at early times. To elucidate the mechanisms involved in the specific attraction of eosinophils to the uterus in the presence of oestrogens, the in vivo localisation of oestrogens in the rat uterus at early times was studied using a radioautographic technique. Oestrogen receptors were found in the surface of eosinophils and in the wall of small uterine blood vessels. This simultaneous presence of both oestrogen receptors is proposed to explain the specific attachment of eosinophils to uterine blood vessels in the presence of oestrogens, which is the initial step toward eosinophil penetration into the uterus.     

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.     

Effects of mifepristone (RU486) treatment on the development of uterine adenomyosis induced by pituitary grafting in mice

To evaluate the effects of mifepristone (RU486) on the development of uterine adenomyosis induced by pituitary grafting (PG), 3 groups of mice receiving pituitary grafts at 7 weeks of age were given RU486 in food (20 mg/kg chow) from 3-14 (RU486-3 group) or 10-14 (RU486-10 group) weeks of age, or were given no further treatment (PG control group), respectively. All the mice were killed at 14 weeks of age. The uterine weight was significantly decreased in both RU486-treated groups compared with the PG control group. The incidence of adenomyosis was also decreased significantly in both the RU486-3 group (0/10 mice) and RU486-10 group (2/10 mice) compared with the PG control group (7/9 mice). To look for vascular changes in the uterine tissues, which have been reported to be related to the development of adenomyosis, immunohistochemical staining of von Willebrand factor in the blood vessels was performed. The mean surface area and minor axis of blood vessels in the uterus were thereby found to be significantly decreased in the RU486-10 group compared to the PG control group. The results clearly indicated that RU486, a potent antiprogestin, could inhibit the genesis of uterine adenomyosis in mice, and at the same time caused shrinkage of the vascular system. As in humans, progesterone as well as the vascular system therefore appear to be important factors in the pathogenesis of uterine adenomyosis in this mouse model.     

Evidence that constitutively active luteinizing hormone/human chorionic gonadotropin receptors are rapidly internalized.

The luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, which belongs to the family of G-protein coupled receptors, plays an important role in gonadal steroidogenesis. Substitution of aspartic acid 556 of the LH/hCG receptor with glycine (D556G) creates a constitutively active receptor that activates adenylyl cyclase in the absence of hormone. To examine receptor internalization, human embryonic kidney cells (293 T) expressing wild type (WT) or D556G mutant receptors were incubated with [125I]hCG and subsequently analyzed for cell surface bound and internalized radioactivity. Comparison of the rate constants of internalization of the D556G mutant and WT receptors revealed that the rate of internalization of the D556G mutant was five times greater than that of the WT receptor. Although the D556G receptor internalizes [125I]hCG rapidly, a corresponding increase in [125I]hCG degradation was not seen. The internalization of another constitutively active LH/hCG receptor (aspartic acid 556 to tyrosine) was also greater than that of the WT receptor. Internalization of receptor bound [125I]hCG was inhibited by a hypertonic sucrose solution, confirming that the ligand enters the cell by receptor-mediated endocytosis. Furthermore, the constitutively active D556G and D556Y LH/hCG receptors utilize the arrestin dependent internalization pathway. These results suggest that the active state conformation of the constitutively active receptor is conducive to rapid internalization.     

Changes of mRNA expression of vascular endothelial growth factor, angiopoietins and their receptors during the periovulatory period in eCG/hCG-treated immature female rats

Angiogenic factors can induce the perifollicular capillary network in the theca interna that shows marked changes in and around the preovulatory luteinizing hormone (LH) surge. To get more information on their functional crosstalk, the aim of the present study was to investigate the manner of mRNA expression of vascular endothelial growth factors (VEGFs) 120, 164, angiopoietin (Ang)-1, Ang-2 and their specific receptors during the periovulatory phase. We used an established equine and human chorionic gonadotropins (eCG/hCG)-derived experimental model capable of stimulating naturally occurring follicular maturation, ovulation and corpus luteum (CL) formation. On day 28 postpartum, immature female rats were administrated s.c. with 10 IU of eCG to promote follicular development, followed 48 hr later by i.p. administration of 20 IU of hCG. Ovaries were dissected at 0, 6, 12, 18 and 24 hr after hCG treatment, and were obtained on day 30 in the untreated control. After induction of follicular growth by the eCG treatment, each mRNA expression of VEGF 120, VEGF 164, Neuropilin-1 and Flt-1 significantly increased. The peaks in mRNA expressions of VEGF120 and VEGF164 were both found at 18 hr after hCG treatment. Flk-1 mRNA expression maintained up to 6 hr after hCG treatment, and then decreased at 12, 18 and 24 hr after hCG treatment. Ang-2 mRNA expression increased in the ovaries at 6 and 12 hr after hCG treatment. Tie-2 mRNA expression decreased at 24 hr after the treatment of gonadotropins. Our findings suggest that ovarian vascular formation during the periovulatory period including preovulatory follicles, ovulation and CL formation may develop via crosstalk of the VEGF-Flt-1 and Ang-Tie2 systems.     

鼠兔子宫血管铸型的光镜和扫描电镜观察

用光镜和扫描电镜观察了ＡＢＳ丁酮溶液灌注的达乌尔鼠兔子宫血管与微血管构筑情况。子宫大部分血液来自子宫动脉,小部分来自生殖动脉。各弓状动脉进入宫壁后,即在宫壁内分支形成３个血管层：浆膜层、大血管层和粘肌层。研究发现鼠兔子宫内膜血管呈树杆状或有轻度弯曲向腔面垂直穿行,直至浅层分支形成毛细血管网和较大的窦状毛细血管;其内膜血管形态与有月经的人子宫内膜螺旋动脉明显差异。文中还对子宫微血管构筑与月经产生机制