Luteinizing Hormone Receptor Protein Research Articles

Influence of epidermal growth factor supplementation during in vitro maturation on nuclear status and gene expression of canine oocytes

This study evaluated the effect of epidermal growth factor (EGF) supplementation during in vitro maturation on the meiotic status and the expression of EGF receptor (EGFr), luteinizing hormone receptor (LHr) and gap junction protein α 5 (GJA5) in canine cumulus-oocyte-complexes (COCs). COCs of ⩾110 μm diameter, exhibiting dark pigmentation and completely surrounded by three or more layers of cumulus cells collected from anestrus stage ovaries in natural cycle were matured in TCM-199 supplemented with 10% fetal bovine serum, 0.57 mM cysteine, 10 μg/ml LH and FSH, and different concentrations of EGF (0, 10 and 30 ng/ml). Oocytes cultured for 72 h were fixed to assess the nuclear maturation. Expression of EGFr, LHr and GAJ5 was assessed by immunocytochemistry and real-time PCR. Proportion of metaphase II status of oocytes cultured in in vitro maturation (IVM) medium supplemented with 10 ng/ml EGF for 72 h was significantly ( P < 0.05) higher than 0 and 30 ng/ml EGF supplemented IVM medium (9.8% vs. 6.5% and 5.2%). In both cumulus cells and oocytes, EGFr protein was undetectable, LHr protein level of expression was low and a strong expression of GJA5 protein was observed. The relative abundance (RA) of EGFr transcript revealed low levels and the LHr expression decreased steadily with addition of EGF. However it did not vary among different concentrations of EGF supplementation. The RA of GJA5 transcript exhibited lower level at 10 ng/ml EGF supplementation. In conclusion, the supplementation of 10 ng/ml EGF in IVM media exerted a positive influence on the progression of maturation to MII phase and the expression level of GJA5 at 72 h, but did not demonstrate any stimulatory role on the expression of EGFr and LHr during the maturation of the canine IVM oocytes.

Melatonin regulates delayed embryonic development in the short-nosed fruit bat, Cynopterus sphinx

The aim of the present study was to evaluate the seasonal variation in serum melatonin levels and their relationship to the changes in the serum progesterone level, ovarian steroidogenesis, and embryonic development during two successive pregnancies of Cynopterus sphinx. Circulating melatonin concentrations showed two peaks; one coincided with the period of low progesterone synthesis and delayed embryonic development, whereas the second peak coincided with regressing corpus luteum. This finding suggests that increased serum melatonin level during November-December may be responsible for delayed embryonic development by suppressing progesterone synthesis. The study showed increased melatonin receptors (MTNR1A and MTNR1B) in the corpus luteum and in the utero-embryonic unit during the period of delayed embryonic development. The in vitro study showed that a high dose of melatonin suppressed progesterone synthesis, whereas a lower dose of melatonin increased progesterone synthesis by the ovary. The effects of melatonin on ovarian steroidogenesis are mediated through changes in the expression of peripheral-type benzodiazepine receptor, P450 side chain cleavage enzyme, and LH receptor proteins. This study further showed a suppressive impact of melatonin on the progesterone receptor (PGR) in the utero-embryonic unit; this effect might contribute to delayed embryonic development in C. sphinx. The results of the present study thus suggest that a high circulating melatonin level has a dual contribution in retarding embryonic development in C. sphinx by impairing progesterone synthesis as well as by inhibiting progesterone action by reducing expression of PGR in the utero-embryonic unit.

An Antisense Oligodeoxynucleotide to the LH Receptor Attenuates FSH-induced Oocyte Maturation in Mice

: It has been recently shown that expression of the LH receptor (LHR) in cumulus cells is related with FSH-induced meiotic resumption of mouse cumulus enclosed oocytes (CEOs). However, to date, it is still unclear whether LHR expression in cumulus cells plays a key role during FSH-induced oocyte maturation. The purpose of this study was to characterize the functional role of LHRs in cumulus cells. CEOs were isolated from eCG-primed preovulatory follicles and cultured in hypoxanthine (HX) arrested medium. LHR protein expression in cumulus cells was time-dependent increasing during the process of FSH-induced oocyte maturation. While the sense oligodeoxynucleotide (ODN) had no effect, antisense ODN inhibited FSH-induced LHR expression and meiotic resumption. Moreover, this antisense ODN against LHR could inhibit FSH-induced mitogen-activated protein kinase (MAPK) phosphorylation. This study suggested that LHR expression in cumulus cells is involved in FSH-induced oocyte meiotic resumption, which process is possibly regulated by MAPK cascade.

Gonadotropin-Releasing Hormone Stimulates Prolactin Release from Lactotrophs in Photoperiodic Species Through a Gonadotropin-Independent Mechanism

Previous studies have provided evidence for a paracrine interaction between pituitary gonadotrophs and lactotrophs. Here, we show that GnRH is able to stimulate prolactin (PRL) release in ovine primary pituitary cultures. This effect was observed during the breeding season (BS), but not during the nonbreeding season (NBS), and was abolished by the application of bromocriptine, a specific dopamine agonist. Interestingly, GnRH gained the ability to stimulate PRL release in NBS cultures following treatment with bromocriptine. In contrast, thyrotropin-releasing hormone, a potent secretagogue of PRL, stimulated PRL release during both the BS and NBS and significantly enhanced the PRL response to GnRH during the BS. These results provide evidence for a photoperiodically modulated functional interaction between the GnRH/gonadotropic and prolactin axes in the pituitary gland of a short day breeder. Moreover, the stimulation of PRL release by GnRH was shown not to be mediated by the gonadotropins, since immunocytochemical, Western blotting, and PCR studies failed to detect pituitary LH or FSH receptor protein and mRNA expressions. Similarly, no gonadotropin receptor expression was observed in the pituitary gland of the horse, a long day breeder. In contrast, S100 protein, a marker of folliculostellate cells, which are known to participate in paracrine mechanisms within this tissue, was detected throughout the pituitaries of both these seasonal breeders. Therefore, an alternative gonadotroph secretory product, a direct effect of GnRH on the lactotroph, or another cell type, such as the folliculostellate cell, may be involved in the PRL response to GnRH in these species.

Human fetal testis Leydig cell disruption by exposure to the pesticide dieldrin at low concentrations

Declining human reproductive health over the last 60 years has been proposed to be due to effects of environmental chemicals, especially endocrine disrupting compounds, on fetal development. We investigated whether a model pesticide, dieldrin, at concentrations within both maternal circulation and environmental ranges (1 pmol/l = 0.0004 p.p.b. = 380.9 pg/l), could disrupt the human fetal testis. Human fetal testes were collected during the second trimester, a critical period of male sexual differentiation (development and masculinization). Testis explants were cultured for 24 h in the presence and absence of LH (10-1000 IU LH/l) and dieldrin (1 pmol and 1 nmol/l). Endocrine, immunohistological and proteome characteristics of the tissues were investigated. Exposure to dieldrin reduced LH-induced testosterone secretion (P < 0.05) and tissue protein concentrations of LH receptor and steroid acute regulatory protein (P < 0.05). Dieldrin altered proteins associated with cancer, apoptosis, transcription and development. Wnt-2b was reduced 3-fold and immunolocalized to Leydig and Sertoli cells. Dieldrin also reversed some LH-induced changes in protein expression, supporting the conclusion that Leydig cell function is at risk from environmental chemicals. Our findings indicate that exposure to very low, biologically relevant, concentrations of environmental chemicals could affect the fetal human Leydig cell, reducing testosterone secretion and potentially leading to subtle dysregulation of reproductive development and adult fecundity.

Luteinizing hormone receptor mediates neuronal pregnenolone production via up‐regulation of steroidogenic acute regulatory protein expression

The functional consequences of luteinizing hormone/human chorionic gonadotropin signaling via neuronal luteinizing hormone/human chorionic gonadotropin receptors expressed throughout the brain remain unclear. A primary function of luteinizing hormone (LH) in the gonads is the stimulation of sex steroid production. As LH can cross the blood-brain barrier, present in cerebrospinal fluid and is expressed by neuronal cells, we tested whether LH might also modulate steroid synthesis in the brain. Treatment of differentiated rat primary hippocampal neurons and human M17 neuroblastoma cells with LH (100 mIU/mL) resulted in a twofold increase in pregnenolone secretion in both cell types, suggesting an increase in P450scc-mediated cleavage of cholesterol to pregnenolone and its secretion from neurons. To explore how LH might regulate the synthesis of pregnenolone, the precursor for steroid synthesis, we treated rat primary hippocampal neurons with LH (0, 10 and 100 mIU/mL) and measured changes in the expression of LH receptor and steroidogenic acute regulatory protein (StAR). LH induced a rapid (within 30 min) increase in the expression of StAR, but induced a dose-dependent decrease in LH receptor expression. Consistent with these results, the suppression of serum LH in young rats treated with leuprolide acetate for 4 months down-regulated StAR expression, but increased LH receptor expression in the brain. Taken together, these results indicate that LH induces neuronal pregnenolone production by modulating the expression of the LH receptor, increasing mitochondrial cholesterol transport and increasing P450scc-mediated cleavage of cholesterol for pregnenolone synthesis and secretion.

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin‐independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.

Expression of the Mature Luteinizing Hormone Receptor in Rodent Urogenital and Adrenal Tissues Is Developmentally Regulated at a Posttranslational Level

The LH receptor (LHR) is a G protein-coupled receptor involved in the regulation of ovarian and testicular functions. In this study we demonstrate novel and unexpected patterns of receptor expression and regulation in fetal and adult rodent urogenital and adrenal tissues. Two rat LHR promoter fragments (approximately 2 and 4 kb) were shown to direct expression of the lacZ reporter in transgenic mice to gonads, adrenal glands, and kidneys, starting at 14.5 d post coitum, and to genital tubercles, starting at 11.5 d post coitum. These tissues were also found to express the full-length LHR mRNA and protein during rat fetal development, but, importantly, only immature receptors carrying unprocessed N-linked glycans were detected. After birth, the receptor gene activity ceased, except in the gonads, which started to express the mature receptor carrying fully processed N-linked glycans. Surprisingly, both LHR mRNA and mature protein levels were up-regulated substantially in pregnant female adrenal glands and kidneys at a time that coincides with differentiation of fetal urogenital tissues. Taken together, these results indicate that the LHR protein is expressed constitutively in gonadal and nongonadal urogenital tissues as well in adrenal glands, but its final functional maturation at the posttranslational level appears to be developmentally and physiologically regulated.

Acquisition of luteolytic capacity involves differential regulation by prostaglandin F2α of genes involved in progesterone biosynthesis in the porcine corpus luteum

Luteolytic capacity is defined as the ability of corpora lutea (CL) to undergo luteolysis after prostaglandin (PG) F2α treatment. The mechanisms causing acquisition of luteolytic capacity are not yet identified but CL without luteolytic capacity have PGF2α receptors and respond to PGF2α with some changes in gene expression. Inhibition of progesterone biosynthesis is a key feature of luteolysis and therefore we postulated that genes involved in progesterone biosynthesis would be regulated by PGF2α differently in CL with or without luteolytic capacity. Gilts on day 9 after estrus (lack luteolytic capacity) or day 17 of pseudopregnancy (with luteolytic capacity) were treated with saline or a PGF2α analog (cloprostenol) and CL were collected 0.5 (Experiment I) or 10 h (Experiment II) later. In Experiment III, large luteal cells from CL on day 9 or 17 were cultured for 1, 12 and 24 h with or without PGF2α. PGF2α decreased LDL receptor mRNA (27%), steroidogenic acute regulatory protein (StAR) mRNA (41%), StAR protein (75%), LH receptor mRNA (55%), and LH receptor protein (45%) at 10 h after treatment in day 17 but not day 9 CL. PGF2α increased DAX-1 mRNA at 0.5 h (43%) and 10 h (46%) after PGF2α in day 17 but not day 9 CL but decreased 3βHSD mRNA (∼20% at 10 h) in both days 9 and 17 CL. In vitro, PGF2α decreased StAR mRNA at 12 h only in day 17 luteal cells; however, continuous treatment with PGF2α for 24 h decreased StAR mRNA in both days 9 and 17 luteal cells. Thus, luteolytic capacity involves a critical change in responsiveness of DAX-1, StAR, and LH receptor to PGF2α that results in inhibition of luteal progesterone biosynthesis.

Adrenocortical tumorigenesis in transgenic mice expressing the inhibin alpha-subunit promoter/simian virus 40 T-antigen transgene: relationship between ectopic expression of luteinizing hormone receptor and transcription factor GATA-4.

We have analyzed the ontogeny and putative mechanisms of transregulation of LH receptor (LHR) and transcription factor GATA-4, coexpressed during the adrenocortical tumorigenesis of prepubertally gonadectomized transgenic (TG) mice expressing the inhibin alpha-subunit promoter/simian virus 40 T-antigen (inhalpha/Tag) transgene. The onset of adrenal LHR mRNA and protein expression coincided with that of GATA-4 at the age of 4 months and preceded the appearance of discernible adrenal tumors at about 6 months. In situ hybridization and double-immunohistochemistry demonstrated colocalization of the LHR and GATA-4 messages and proteins in the adrenal cortex. A GATA-4 expression plasmid cotransfected with a murine LHR promoter-driven luciferase reporter plasmid, containing a consensus GATA-binding site, induced a dose-dependent significant transactivation of the LHR promoter in nonsteroidogenic human embryonic kidney 293, steroidogenic murine mLTC-1 Leydig cells and in murine adrenal Y-1 cells. The Calpha1 cells derived from an Inhalpha/Tag adrenal tumor did not show this response, apparently due to their high endogenous GATA-4 expression. However, an additional link between GATA-4 and LHR in Calpha1 cells was provided upon the LH/human chorionic gonadotropin stimulation of LHR promoter activity; mutations or deletion of the consensus GATA-4 binding site of the LHR promoter abolished this transactivation. EMSAs further proved GATA-4 binding to the putative consensus GATA recognition site. Our results demonstrate direct interrelationship between LHR and GATA-4 expression during adrenocortical tumorigenesis of the inhalpha/Tag mice. There is apparently a positive and reciprocal feed-forward amplification link between LHR and GATA-4 expression. This mechanism gradually and in synergy with Tag expression leads to formation of the LH-dependent adrenocortical tumors.

Inhibition of luteinizing hormone receptor expression during the development of caprine corpora lutea by administration of gonadotropin-releasing hormone antagonist.

The present study was conducted to examine effects of a potent GnRH antagonist (GA), which suppresses release of luteinizing hormone (LH), on LH receptor expression during the development of the caprine corpus luteum (CL). Goats were divided into control and GA-treated groups. The goats were treated with saline or GA (50 microg/kg, s.c.) on days 0 (day of ovulation), 4 and 8 (control only), and CL collected on a subset of goats (n = 3 for each day) on days 0 (no saline), 4, 8, or 14 (control only). Ribonuclease protection assay and [(125)I]-hCG binding assay were performed to quantitate mRNA and protein of the LH receptor in the CL, respectively. On day 4, CL weight, levels of LH receptor mRNA and protein in the GA-treated group were similar to those of the control group. By day 8, CL weight and levels of LH receptor mRNA and protein in the GA-treated group were reduced relative to those of the control group (P < 0.05). There was no difference of affinity of the LH receptor between both groups on day 8. These results suggest that the treatment with GA inhibits gene and protein expressions of the LH receptor during the development of CL in the goat, and thus, support an idea that endogenous LH participates in the increase of its own receptor.

LH receptor defects.

In this article the role of LH receptor gene mutations in patients with aberrant sex differentiation is discussed. In a dominant autosomal familial form of precocious puberty in boys (familial male-limited precocious puberty) LH receptor gene mutations have been identified. These single amino acid changes, mostly found in the sixth transmembrane helix and the third intracellular loop of the transmembrane domain of the LH receptor, cause constitutive activation of LH receptor protein without the hormone present, resulting in precocious production of testosterone by the testicular Leydig cells. The large number of activating LH receptor mutations have allowed more precise molecular modeling of the LH receptor protein. In a rare hereditary form of 46,XY male pseudohermaphroditism known as Leydig cell hypoplasia, LH receptor gene mutations have been identified that completely or partially inactivate the LH receptor protein. Large gene deletions cause complete absence of the LH receptor protein, whereas other, more subtle missense mutations prevent the receptor from assuming an active conformation.

Mutant Luteinizing Hormone Receptors in a Compound Heterozygous Patient with Complete Leydig Cell Hypoplasia: Abnormal Processing Causes Signaling Deficiency

Over the past 5 yr several inactivating mutations in the LH receptor gene have been demonstrated to cause Leydig cell hypoplasia, a rare autosomal recessive form of male pseudohermaphroditism. Here, we report the identification of two new LH receptor mutations in a compound heterozygous case of complete Leydig hypoplasia and determine the cause of the signaling deficiency at a molecular level. On the paternal allele of the patient we identified in codon 343 a T to A transversion that changes a conserved cysteine in the hinge region of the receptor to serine (C343S); on the maternal allele a T to C transition causes another conserved cysteine at codon 543 in trans-membrane segment 5 to be altered to arginine (C543R). Both of these mutant receptors are completely devoid of hormone-induced cAMP reporter gene activation. Using Western blotting of expressed LH receptor protein with a hemagglutinin tag, we further show that despite complete absence of total and cell surface hormone binding, protein levels of both mutant LH receptors are only moderately affected. The expression and study of enhanced green fluorescent protein-tagged receptors confirmed this view and further indicated that initial translocation to the endoplasmic reticulum of these mutant receptors is normal. After that, however, translocation is halted or misrouted, and as a result, neither mutant ever reaches the cell surface, and they cannot bind hormone. This lack of processing is also indicated by reduced presence of an 80-kDa protein, the only N-linked glycosylated protein in the LH receptor protein profile. Thus, complete lack of signaling by the identified mutant LH receptors is caused by insufficient processing from the endoplasmic reticulum to the cell surface and results in complete Leydig cell hypoplasia in this patient.

Actions of gonadotrophins on the uterus.

Binding sites for LH/hCG are found in the uterus of several species, including humans. In cattle and pigs, the LH receptor, its mRNA and LH receptor protein are present in the uterus throughout the oestrous cycle, and maximum expression occurs at the luteal phase. GnRH receptor is also expressed maximally in the human endometrium at the luteal phase. LH activates both the adenylate cyclase and phospholipase C pathways and increases the concentrations of cyclooxygenase and its products. Activation of LH receptors in the endometrium is associated with PGF production. In contrast, bovine uterine vein LH receptor mRNA and LH receptor concentrations are greatest during pro-oestrus-oestrus and LH increases the production of both PGE and PGF. FSH receptor and its mRNA are present in the bovine cervix and the concentrations are greatest at the time of the FSH peak value in the blood, indicating a physiological role for FSH in the relaxation and opening of the cervix. The presence of gonadotrophin and releasing factor receptors with a dynamic pattern in the endometrium, myometrium, oviduct and cervix of different species provides evidence that gonadotrophins and GnRH play a substantial role as molecular autocrine-paracrine regulators of the oestrous cycle and implantation.

Actions of gonadotrophins on the uterus

Binding sites for LH/hCG are found in the uterus of several species, including humans. In cattle and pigs, the LH receptor, its mRNA and LH receptor protein are present in the uterus throughout the oestrous cycle, and maximum expression occurs at the luteal phase. GnRH receptor is also expressed maximally in the human endometrium at the luteal phase. LH activates both the adenylate cyclase and phospholipase C pathways and increases the concentrations of cyclooxygenase and its products. Activation of LH receptors in the endometrium is associated with PGF production. In contrast, bovine uterine vein LH receptor mRNA and LH receptor concentrations are greatest during pro-oestrus-oestrus and LH increases the production of both PGE and PGF. FSH receptor and its mRNA are present in the bovine cervix and the concentrations are greatest at the time of the FSH peak value in the blood, indicating a physiological role for FSH in the relaxation and opening of the cervix. The presence of gonadotrophin and releasing factor receptors with a dynamic pattern in the endometrium, myometrium, oviduct and cervix of different species provides evidence that gonadotrophins and GnRH play a substantial role as molecular autocrine-paracrine regulators of the oestrous cycle and implantation.

Functional importance of bovine myometrial and vascular LH receptors and cervical FSH receptors.

Bovine myometrium and cervix contain luteinizing hormone/human chorionic gonadotropin (LH/hCG) binding sites, LH receptor (LH-R) messenger RNA (mRNA), and LH-R protein. Expression of LH-R is dependent on the stage of the cycle. LH-R expression is high during the luteal phase but weak during the follicular phase. In both myometrium and cervix, LH activates both the adenylate cyclase and phospholipase C pathways, and the effect of LH on both pathways at each stage of the cycle is correlated with the amount of LH-R present in the tissue. Because activation of cyclic AMP (cAMP) is associated with myometrial quiescence, we suggest that LH activation of uterine cAMP could serve to keep the uterus quiescent during the luteal phase. On the other hand, in the uterine vein LH-R mRNA and LH-R are maximal during preestrus/estrus as opposed to the luteal phase. In the uterine vein, LH increases the expression of cyclooxygenase and production of both prostaglandin E2 (PGE2) and PGF2 alpha. Because PGF2 alpha is the physiological luteolytic signal in the cow, we suggest that this increase in prostaglandin production by the uterine vein is part of the physiological events leading to luteolysis. In addition to uterine LH-R, the bovine cervix at preestrus/estrus has high levels of follicle-stimulating hormone receptor (FSH-R) and its corresponding mRNA. As with LH-R, activation of FSH-R by FSH is associated with activation of a G protein-coupled receptor family that mediates the cAMP and inositol phosphate signaling pathways. Activation of these signaling pathways is associated with an increase in the expression of cyclooxygenase and production of PGE2. Because expression of the FSH receptor was maximal at the time of the FSH peak in the blood, we suggest a physiological role for FSH in the cervix relaxation and opening at estrus.

Role of Luteinizing Hormone in Control of Oviduct Function

ContentsThe Fallopian tube plays a critical role in reproduction by being the site of fertilization and early embryonic development. Recently it was shown that the oviduct is a novel target for luteinizing hormone (LH). LH receptor mRNA is present in the porcine oviduct. LH/hCG receptors were detected in the epithelium of the tubal mucosa and smooth muscle cells of the pig myosalpinx. Western blotting revealed that the porcine oviduct contains mainly 75 kDa immunoreactive LH receptor protein. Treatment with LH in vitro caused relaxation of the oviduct, especially during the peri‐ovulatory stage of the oestrous cycle. There is also evidence that combined estradiol and progesterone priming is necessary for LH‐induced relaxation of the oviduct in ovariectomized pigs. The results of these investigation indicate that LH should be added to the list of factors affecting oviduct function and it is suggested that LH, in addition to causing the rupture of follicles and luteinization of granulosa and theca cells, also plays an important role in controlling oviductal contractions.

Effect of estradiol and progesterone on oviductal LH-receptors and LH-dependent relaxation of the porcine oviduct

We have previously shown that the porcine oviduct possesses immunoreactive and functional LH receptors and that LH causes relaxation of the oviduct, especially during the periovulatory stage of estrous cycle. The current studies were undertaken to investigate the effects of estradiol and progesterone on LH receptor protein and LH-stimulated motility of the oviduct in steroid-primed ovariectomized gilts. Twenty-one cross-bred gilts were ovariectomized at 6 mo of age. Four weeks later gilts received daily intramuscular injection of either 2 mL corn oil (control n=4), estradiol benzoate (EB) 1.5 mg (n=6), progesterone 50 mg (n=5), or 1.5 mg EB plus 50 mg progesterone (n=6) for 4 consecutive days. The gilts were slaughtered on Day 5 after the first injection of steroids or vehicle. Rings of isthmus and ampulla were collected from each oviduct and placed in a tissue chamber perfused with Kreb's solution for 60 min. The mechanical activity was recorded for 30 min after LH treatment. Immunoreactivity of LHR in the Fallopian tube sections were detected in the epithelium of the tubal mucosa, smooth muscle cells and the blood vessel endothelium. Western blotting showed that porcine oviducts contain 75, 48 and 45 kDa immunoreactive LH receptor proteins, like the corpus luteum (CL). The lowest receptor expression was found in controls and in gilts treated with estradiol or progesterone. Combined treatment with estradiol and progesterone resulted in a significant increase of LH receptor protein concentrations when compared with control animals. In vitro LH treatment affected oviduct contractility of combined estradiol and progesterone treated gilts but not the oviduct of the remaining groups. It also caused a decrease in amplitude, frequency and areas under the curve (AUC) of ampulla (P<0.05) and the amplitude and AUC of isthmus (P<0.001) in combined estradiol and progesterone-primed gilts. These results indicate that estradiol and progesterone together, but not separately, increase LH receptor protein in the porcine oviduct and that combined estradiol and progesterone priming is necessary for LH-induced relaxation of the porcine oviduct.

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

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

Luteinising hormone receptor kinetic and LH-induced prostaglandin production throughout the oestrous cycle in porcine endometrium.

The present studies were performed to determine the LH/hCG receptor concentration and to evaluate the LH effect on prostaglandin production in porcine endometrium throughout the oestrous cycle. LH/hCG receptors in cell membrane preparations of the endometrium were found from days 12-14 and 15-16 of the oestrous cycle but not in preparations from days 6-7 and 18-20 using the ligand radioreceptor assay. Western blot analysis revealed, however, that the endometrium from all stages of the oestrous cycle contains a 75-kDa immunoreactive LH receptor protein similar to corpora lutea. The incubation of endometrial explants with LH (0, 1, 10 and 100 ng x mL(-1)) resulted in an increase of 13,14-dihydro-15-keto-PGF2alpha accumulation in a dose-dependent manner on days 5, 10, 14 and 16 of the oestrous cycle. The most effective dose was 10 ng LH x mL(-1) on days 5-16, but the strongest effect was found on days 14 and 16 (7.40 +/- 0.14 versus 12.75 +/- 1.40 and 5.67 +/- 0.35 versus 9.4 +/- 1.25 ng x 100 mg(-1) tissue/6 h, respectively; P < 0.01). It was also observed that 10 and 100 ng x mL(-1) of LH significantly increased cyclo-oxygenase expression to 135.2 and 123.5% respectively, above the control value (P < 0.01) on day 16 of the oestrous cycle. Our data suggest that LH receptors are of physiological significance in the porcine endometrium, since LH induces cyclooxygenase synthesis and increases prostaglandin production.