Gonadotropin receptor complexes and free receptors in porcine Leydig cell cultures during recovery from hCG stimulation

Free and occupied gonadotropin receptors were studied in vitro in porcine Leydig cells culture maintained in chemically defined medium. Free receptors were evaluated by the binding capacity for 125I-hCG. hCG bound molecules (or hCG receptor complexes) were evaluated using immunocytochemical visualization on fixed cells. Exposure to hCG for 16 hours (.5 to 50 ng/ml) induced the disappearance of free receptors. After removal of the hormone, the return to control levels was observed at 48 and 72 hours. Visualization of hCG bound at the cell surface indicates that, following continuous exposure to gonadotropins for 48 hours, hCG molecules are still present on the cell. Following short-time exposure (1 h) to hCG and 48 hrs washing the number of stained cells is very close to the initial value suggesting that the occupied sites (at 48 hours) represent the initial hormone receptor complexes. These results indicate that, during prolonged incubation, hCG binding is not reversible, that the half-life of some of the complexes at the cell surface is very long and that the receptors recovery is slow and is probably the result of a de novo synthesis.     

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

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

Early hCG-induced desensitization in Leydig cells.

The early mechanism of hCG induced down regulation of its own receptor as well as steroidogenesis refractoriness of rat Leydig cells to gonadotropin stimulation have been investigated. A single injection of 5, 12, 25, 50 and 100 IU of hCG in rats induced within 8 hours, Leydig cells desensitization. However, apparent receptor loss was significantly lower only in the rats who received 50 and 100 IU of hCG. Cycloheximide inhibits hCG-induced receptors loss but had no effect on hCG-induced desensitization. The most likely explanation for desensitization in the presence of binding sites and a normal adenylate cyclase, is a defective coupling between the receptor sites and the catalytic subunit.     

In vivo pretreatment with human chorionic gonadotropin fails to reverse the dysfunction of isolated Leydig cells from chronically uremic rats

In order to further investigate the previously reported hypogonadal state of chronically uremic rats, we examined the effects of in vivo pretreatment with human chorionic gonadotropin (hCG) on in vivo and in vitro Leydig cell function, comparing paired intact rats with rats made chronically uremic by 5/6 nephrectomy. The in vitro testosterone (T) secretory responses to varying concentrations of hCG or dibutyryl cAMP and the number of gonadotropin receptors were determined following hemicastration. The rats were then treated with hCG for 3 days and the remaining testes were removed and studied as before. Compared with intact rats, the uremic rats had higher serum concentrations of urea nitrogen (P less than 0.001); serum T concentrations were lower in uremic rats before (P less than 0.001), but not after (P greater than 0.6) treatment. Treatment produced increases in serum T only in uremic rats (P less than 0.001). Serum LH was lower in uremic rats before treatment (P less than 0.001) and was reduced (P less than 0.001) to similar levels (P greater than 0.8) in both groups after treatment. Baseline in vitro T secretion was lower (P less than 0.001) from Leydig cells of uremic than intact rats both before and after treatment. Analysis of variance of dose-response curves showed pre- and post-treatment T secretory responses to hCG or dibutyryl cAMP in vitro to be less from Leydig cells of uremic rats (P less than 0.01). Before treatment, Leydig cell gonadotropin receptor number was lower in uremic than intact rats (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Type I IGF receptors of Leydig cells are upregulated by human chorionic gonadotropin

The effects of human chorionic gonadotropin (hCG) on type I insulin-like growth factor (IGF) receptors of purified Leydig cells were investigated. Sprague-Dawley rats (50 day-old) were treated with a single injection of hCG 10 units intraperitoneally, type I IGF receptors were then determined daily for 4 days. HCG caused a rapid increase in type I IGF receptors within 24 h, which returned to basal by 72 h. There was no significant change in binding affinity. Our present study indicates that type I IGF receptors of Leydig cells are up regulated by hCG, and this may be one mechanism by which hCG and IGF-I interact to enhance Leydig cell steroidogenesis.     

Diabetes alters the mRNA expression of insulin-like growth factors and their receptors in the mouse fallopian tube and uterus during the preimplantation stages

The possible role of insulin-like growth factors (IGFs) and their receptors (IGFRs) in the pathogenesis of diabetic embryopathy was investigated. Sexually mature female ICR mice of 6-8 weeks old were made diabetic by a single intraperitoneal injection with 200 mg/kg streptozotocin ten days prior to mating. Fallopian tubes and uterine tissues were obtained from the superovulated diabetic and normal mice 48, 72 and 96 hours following human chorionic gonadotropin (hCG) injection. The mRNA expression of IGF-1 and IGF-2 as well as their receptors was determined in the tissues using Real-time Polymerase Chain Reaction (Real-time PCR). The mRNA expression of IGF-1 in the fallopian tube and uterus of the diabetic mice was significantly lower 72 and 96 hours after hCG treatment, respectively, as compared to the controls. The mRNA expression of IGF-1R at 96 hours post-hCG treatment was significantly higher in the fallopian tube and lower in the uterus of the diabetic mice as compared to the controls. The mRNA expression IGF-2 in the fallopian tube was significantly higher 48 and 96 hours after hCG treatment, but was lower in the uterus of diabetic mice 96 hours after hCG treatment as compared to controls. The mRNA expression of IGF-2R in the diabetic mice was significantly higher 48 and 96 hours (the fallopian tube) and 48 hours (uterus) after hCG treatments as compared to the controls. In conclusion, an alteration in mRNA expression of IGFs and their receptors in the diabetic mice as observed in this study could possibly result in diabetic embryopathy.     

Internalization and recycling of lutropin receptors upon stimulation by porcine lutropin and human choriogonadotropin in porcine Leydig cells

Porcine lutropin (pLH) and human choriogonadotropin (hCG) recognize the same hormonal receptor and elicit the same steroidogenic response in porcine Leydig cells in primary culture. We compared the variation in the number of occupied and free receptors present on the cell surface under short-term stimulation by the two hormones at 35 degrees C. Both hormones produced a rapid dose-dependent decrease in the total number of the gonadotropin receptors present on the cell surface with a half-life of 8-10 min. This decrease was reversible upon hormone removal and receptors were recovered on the cell surface with the same half-life of 8-10 min. With pLH, the receptors were recycled in a free state, but in the presence of hCG the receptors were recycled in an occupied state. This difference could be related to the higher affinity of hCG for the receptor in 150 mM NaCl buffer (Ka = 1.6 X 10(9) for hCG and 1.5 X 10(7) for pLH) and higher stability to acid pH of the hCG-receptor complex (dissociation pK = 3.7 for hCG and 4.5 for pLH).     

Primary cultures of Leydig cells from rat, mouse and pig: advantages of porcine cells for the study of gonadotropin regulation of Leydig cell function

Primary cultures of interstitial cells were prepared from the testis of mice, rats, and pigs. The cells were grown in a defined medium supplemented with low (0.1%) serum and insulin, transferrin and epidermal growth factor. Comparisons of the interstitial cell cultures from the three species were made for plating efficiency, cell survival, maintenance of hCG receptors and maintenance of steroidogenic responsiveness to hCG. The porcine cultures had a higher plating efficiency and higher hCG receptor levels per cell than Leydig cells from either rodent. Additionally, the porcine cells showed an increase in testosterone (T) production with hCG stimulation throughout their lifespan in culture while the rodent cultures showed a decrease in T stimulation with time with no stimulation by day 6 in culture. These data indicate that species differences exist in hCG receptor concentrations per cell, the maintenance of hCG receptors and steroidogenic response in culture. The initial high survival, purity and continued functional response of porcine interstitial cell cultures make them a superior system for the study of gonadotropin regulation of Leydig cell function.     

Gonadotropin-induced changes in the luteinizing hormone receptors of cultured Leydig cells. Evidence of up-regulation in vitro

Previous in vivo studies have demonstrated that gonadotropic desensitization of luteinizing hormone/human chorionic gonadotropin (hCG) receptors and steroid responses is preceded by an early phase of receptor up-regulation. Hormonal desensitization has been recently reproduced in an in vitro Leydig cell culture, which has now been applied to studies on the early up-regulation of receptors. We performed comparative studies on the binding of 125I-hCG in isolated Leydig cells in plated culture and in suspension. In plated cells the total binding was up to 200% higher than that measured in suspension. This difference was not due to differential internalization. Preincubation with hCG in plated culture for 2 to 6 h increased the number of binding sites measured in suspension. The kinetics of the binding of labeled hCG to plated cells showed a secondary increase which reached its maximum after 3 h of incubation. This increase in hCG binding was not prevented by preincubation with inhibitors of protein synthesis and steroidogenesis or of microtubule or microfilament function. The sensitivities of the testosterone and cAMP responses to hCG in the plated cells were lower than those observed in suspension. These differences were maintained in the presence of a phosphodiesterase inhibitor. These results demonstrated that the cell interaction with a solid substratum is required for the acute up-regulation of the luteinizing hormone receptor and can induce changes in the Leydig cell responsiveness to gonadotropin stimulation.     

Immunocytochemical localization of receptor human chorionic gonadotropin complexes in rat leydig cells

Localization of receptor-bound human chorionic gonadotropin (hCG) in rat testis was studied by the peroxidase-antiperoxidase (PAP) complex method. The rats were injected with a single intravenous dose (1000 IU) of hCG. Three, 6, 12, and 24 hr after injection the testes were removed for localization of the hormone. The hormone localized to the periphery of the Leydig cells at all observation points. The intensity of the staining varied between the cells, suggesting that the number of receptors or the accessibility of the receptors to the circulating hormone varies from one cell to another. The staining surrounded the Leydig cells unevenly, but no progressive patching or capping was found. This observation suggests that hCG binds preferentially to the cell surface areas directed toward the capillaries. Compatible results were obtained with anti-hCG serum and with antisera against the hCG subunits. These results are consistent with previous observations that the luteinizing hormone (hCG) receptors accessible to the circulating hormone are located at the surface of the Leydig cells.     

The aging Leydig cell: 1. Testosterone and adenosine 3',5'-monophosphate responses to gonadotropin stimulation in rats

Plasma testosterone levels before and after a single injection of hCG were significantly lower in 24-month old rats than 60--90 day old animals (p less than 0.001). Even with repeated hCG administration for three weeks, plasma testosterone levels of old rats could not be restored to levels present in unstimulated young rats. In response to in vitro LH and 8-bromo-cyclic AMP stimulation, purified young Leydig cells produced significantly higher amounts of testosterone than Leydig cells from old rats. Maximal testosterone formation of the young Leydig cells in response to LH was 42.0 +/- 6.88 ng/10(6) cells, while cells from old rats produced only 16.8 +/- 3.69 ng/10(6) cells (p less than 0.01). However, the dose of LH at which one half maximal response (ED50) occurred was 0.1 mIU/ml for young Leydig cells and 0.05 mIU/ml for old Leydig cells. Basal and 1.0 mIU LH-stimulated cyclic AMP formation were comparable in both groups, but cyclic AMP formation in response to 10 mIU of LH was significantly less in the old rats (p less than 0.05). Present results demonstrate impaired steroidogenic capacity of old rats both in vivo and in vitro. Decreased testosterone response in old rats most likely is the consequence of understimulation of Leydig cells by gonadotropin; however, there appear to be additional intrinsic defects in old Leydig cells.     

Testicular steroidogenesis after human chorionic gonadotropin desensitization in rats.

When a single injection of 500 I.U. of human chorionic gonadotropin (hCG) is given to rats there is an initial acute rise of plasma testosterone and of testicular content for both cyclic AMP and testosterone. This response correlates with an increase in both lyase and 17 alpha-hydroxylase activities. Thereafter both plasma and testicular testosterone decline and do not increase after a second injection of hCG. During this period of desensitization, isolated Leydig cells were insensitive to the steroidogenic stimulatory effect of both hCG and dibutyryl cyclic AMP. The post-cyclic AMP block is not due to an alteration of the cyclic AMP-dependent protein kinase but it is correlated with a decrease in both lyase and 17 alpha-hydroxylase activities of the Leydig cell's microsomes. This decrease is not caused by the absence of the recently described cytosol activator of this enzyme because its addition did not restore the enzymatic activity. Within 60 to 96 h after hCG injection there was a spontaneous increase of both plasma and testicular testosterone and this parallels the recovery of lyase and 17 alpha-hydroxylase activities. These results suggest that both enzymatic activities are regulated, directly or indirectly, by hCG, and that this is partly responsible for the hCG-induced steroidogenic refractoriness of Leydig cells.     

125I-LH binding to rat testes at various ages and posthypophysectomy.

Binding of 125I-LH by the rat testes was investigated during various stages of sexual maturation and in mature animals following hypophysectomy. Hormone binding per mg testicular tissue increased with age and was shown to be due to larger receptor concentration rather than greater binding affinity. This observation cannot be accounted for by changes in the relative number of Leydig cells and suggests, therefore, that Leydig cells acquire additional receptors during sexual maturation. Binding of 125I-LH to mature testes declined after hypophysectomy. Three days following pituitary removal the LH-receptor concentration decreased to one half of normal control value, then remained unchanged until the 37th post-operation day. Replacement therapy with LH, FSH or testosterone propionate failed to maintain 125I-LH binding at prehypophysectomy level.     

Gonadotropin-induced loss of hormone receptors and desensitization of adenylate cyclase in the ovary.

Gonadotropin receptor sites and adenylate cyclase activity were analyzed in luteinized rat ovaries following injection of human chorionic gonadotropin (hCG). Gonadotropin binding capacity and hormonal stimulation of adenylate cyclase declined rapidly to a minimum at 6 to 12 h, remained depressed for 4 days, and returned to the control level between 5 and 7 days. Total adenylate cyclase activity measured in the presence of fluoride fell by 50% within a few hours but returned to normal by 24 h. A close correlation was observed between the number of gonadotropin receptors and the ability of adenylate cyclase to be stimulated by hormone. Assay of tissue-bound hormone showed that the initial loss of hormone sensitivity and binding capacity was associated with occupancy of luteinizing hormone receptor sites, but that the prolonged changes in these activities were not attributable to receptor occupancy. These studies have demonstrated that induction of a refractory or desensitized state in ovarian adenylate cyclase by gonadotropin results from the loss of specific hormone receptor sites.     

Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells.

A study into the binding of 125I-human chorionic gonadotropin (hCG) to the lutropin (LH) receptor in rat testis Leydig cells, and subsequent internalization of the hormone-receptor complex, has been carried out. The results show that there is rapid internalization of the hormone-receptor complex; 240 receptors/cell (from a total of approx. 4000 receptors/cell) were internalized each minute in the first hour after exposure to hCG. Radioactivity was released from the cell 1 h after internalization and was found to be associated with highly degraded hCG. The endocytic process was found to have two temperature-sensitive steps. At 4 degrees C, movement of the hormone-receptor complex inside the cell did not occur, and at 21 degrees C hormone accumulated within the cytoplasm but was not degraded or released from the cell. At 34 degrees C, internalization, degradation and loss of the degraded hormone from the cell occurred. These processes appeared to reach a steady state after 2 h. Even though there is rapid internalization of the hormone-receptor complex following exposure to hCG, the binding sites on the cell surface were maintained for at least 4 h. The number of binding sites on the cell surface was not decreased by a protein synthesis inhibitor but was reduced to undetectable levels by monensin. This compound inhibits acidification of endocytic vesicles, which is known to be an important prerequisite to receptor cycling. It is concluded that, in the rat testis Leydig cells, following binding of hCG to the LH receptor there is rapid internalization of the complex and that recycling of the receptor occurs to the cell surface. This process may be essential in maintaining the capacity of the Leydig cell to bind fresh hormone.     

Effects of PRL and FSH on LH binding and number of Leydig cells in hypophysectomized mice

The effects of PRL and FSH on testicular LH receptors were studied in hypophysectomized (hypox) mice. From the eleventh day after hypophysectomy, they were given 100 micrograms ovine (o) PRL and/or 2 micrograms oFSH in two injections per day for 10 days. Hypophysectomy reduced the weight of testis, epididymides and seminal vesicles, and the LH binding to the testis. Treatment with oPRL and/or oFSH in hypox mice resulted in an increase in the weight of testis and epididymides, and the LH binding per testis. There was no difference between the testicular LH binding in oFSH- and oPRL-treated mice. Histological examination showed that oPRL and/or oFSH treatment in hypox mice restored normal spermatogenesis. Administration of oFSH to hypox mice led to an increase in the number of typical Leydig cells, whereas oPRL was not effective. These results suggest that either PRL or FSH stimulates the LH binding to the testis, but that the action of PRL and FSH on the increase in testicular LH binding is different.     

The morphogenic effect of gonadotropic hormones on the Leydig cells of the boar testis. II. Effects of administration of chorionic gonadotropin after hypophysectomy. Effects in vivo and in organ culture

The longer ago the hypophysectomy has been performed, the more marked is Leydig cell atrophy in the testis. The effects of HCG on cellular morphology have been observed in vivo and in organ culture; qualitative quantitative and ultrastructural aspects were studied. In vivo, the effects of a daily injection of gonadotropin on the testis of 2 boars hypophysectomized 3 1/2 months ago are shown. Markedly atrophied cells are strongly stimulated by HCG during the 15 first days (the cell and nucleus recover nearly to standard size, with the typical histological and ultrastructural appearance with all the cell organelles which characterize a functional steroid cell). Then after 1 1/2 month injection it decreases again to the initial state (very small size cytoplasm strongly reduced with very low organelle content). The number of the Leydig cells is maintained during the first 15 days, then it progressively decreases. The effects of HCG on the testicular tissue of 4 boars were studied in organ culture. Interstitial tissue with a greater or lesser degree of atrophy was examined experimentally (1 month, 3 months and 4 months after hypophysectomy) in order to prove a possible irreversibility of the effects of hypophysectomy. In each case, cell changes were studied according to the duration of the culture. Control cultures without HCG in the medium were set up simultaneously. 1 month and/or 3 months after hypophysectomy, the Leydig cells in culture progressively recover the size and the histological and ultrastructural appearances of a typical Leydig cell. After 16 days of culture, the stimulation is highest, as in vivo. The number of Leydig cells is maintained. From the 17th day stimulation decreases and the cell enters a new atrophy phase. In the anhormonal control medium the atrophy continues as long as the culture is maintained, and the number of Leydig cells decreases. 4 months after hypophysectomy, stimulation in culture is still possible during the first 10 days (proved by the same tests); however the size of the cell remains small compared to the normal; then it atrophies again quickly. In this case the hormone does not maintain the number of the Leydig cells. In the control cultures, slight response of the cell is observed, but this effect is limited and disappears a few days later; the number of the cells rapidly decreases. It has been shown that markedly atrophied Leydig cells can highly be stimulated during the first 2 weeks under the influence of HCG as well in vivo as in organ culture. The lability of the effect is not yet explained. 4 months after hypophysectomy, stimulation is not so effective.     

Rapid Leydig cell proliferation and luteinizing hormone receptor replenishment in the neonatal rat testis after a single injection of human chorionic gonadotropin

Two-day-old rats were stimulated with a single dose of human chorionic gonadotropin (hCG). Changes in the Leydig cell number, mitotic activity, cell size, and number of luteinizing hormone (LH) receptors were studied. The Leydig cell number of the hCG-treated animals was 1.8 times that of the control on Day 1 and remained elevated for the rest of the 5-day experiment (p less than 0.0001). On Day 1 the number of Leydig cell mitoses in the hCG group was greater (p less than 0.05) than in the controls. The Leydig cell size increased transiently to two times that of the control (p less than 0.01) within the first day after the treatment and returned to control size by Day 5. The number of LH receptors per testis decreased 81% in 1 day (p less than 0.01), but returned to control level by Day 3. Since Leydig cell numbers were constant after Day 1, the rapid receptor recovery was obviously due to restoration of the binding sites rather than increased cell number. The present results demonstrate a rapid proliferative response and rapid LH receptor replenishment in the fetal-neonatal Leydig cells after gonadotropic stimulation. These responses of fetal-type Leydig cells are in clear contrast to those observed in adult testes after a similar stimulation.     

Systematic shut-off of the hormone receptors in intraspecific adrenal x Leydig cell hybrids

The mouse Y1 adrenal cell line was fused with mouse Leydig cells in primary culture. The selected hybrids were examined for their response to gonadotropin (hCG) and ACTH. None of them bound specifically [125I]hCG, nor did they augment their cAMP production in response to gonadotropin or ACTH stimulation, whereas their adenylate cyclase remained responsive to forskolin and cholera toxin, thus indicating a repression of hCG receptor synthesis and probably a loss of ACTH receptors, rather than a lesion of the coupling between the hormone receptor complex and the adenylate cyclase. Basal pregnenolone production in 17 hybrids was close to that of Leydig and Y1 cells and was enhanced after 8-bromo adenosine 3',5'-monophosphate (8-Br-cAMP) stimulation in 11 of them. Therefore, the negative control leading to the extinction of both parental functions acts preferentially at the first step of steroidogenesis, i.e., the gene(s) coding for the hormone receptors.