Multiple sites of tumorigenesis in transgenic mice overproducing hCG

We have produced transgenic (TG) mice expressing under the ubiquitin C promoter either the glycoprotein hormone common alpha-subunit (C(alpha)) or human chorionic gonadotropin (hCG) beta-subunit. C(alpha) overexpression alone had no phenotypic effect, but the hCG(beta) expressing females, presenting with moderately elevated levels of bioactive LH/hCG, due to dimerization of the TG hCG(beta) with endogenous C(alpha), developed multiple gonadal and extragonadal neoplasias. Crosses of the C(alpha) and hCG(beta) mice (hCG(alpha)beta) had >1000-fold elevated hCG levels, due to ubiquitous transgene expression, and presented with more aggressive tumour formation. The ovaries displayed initially strong luteinisation of all somatic cell types, leading to formation of luteomas, and subsequently to germ cell tumours (teratomas). The pituitary glands of TG females were massively enlarged, up to >100 mg, developing macroprolactinomas with very high prolactin (PRL) production. This endocrine response probably induced breast cancers in the mice. In contrast to the females, similar high levels of hCG in male mice had only marginal effects in adulthood, with slight Leydig cell hyperplasia and atrophy in the seminiferous epithelium. However, clear Leydig cell adenomas were observed in postnatal mice, apparently originating from fetal Leydig cells. In conclusion, these studies demonstrate marked tumorigenic effects of supraphysiological hCG levels in female mice, but clear resistance to similar changes in males. The extragonadal tumours were induced by hCG stimulated aberrant ovarian endocrine function, rather than by direct hCG action, because gonadectomy prevented all extragonadal phenotypes despite persistent hCG elevation. The phenotypes of the TG mice apparently represent exaggerated responses to hCG/LH and/or gonadal steroids. It remains to be explored to what extent they simulate respective responses in humans to pathophysiological elevation of the same hormones.     

Human chorionic gonadotropin (hCG) up-regulates wnt5b and wnt7b in the mammary gland, and hCGbeta transgenic female mice present with mammary Gland tumors exhibiting characteristics of the Wnt/beta-catenin pathway activation

Transgenic (TG) mice expressing human chorionic gonadotropin (hCG) beta-subunit under the ubiquitin C promoter, presenting with a moderately elevated level of LH/hCG bioactivity develop multiple neoplasms secondary to the endocrine abnormalities, including mammary gland tumors after the age of 9 months. The increased levels of circulating estradiol, progesterone, and prolactin of the TG females after puberty boost the lobuloalveolar development in the mammary gland resulting ultimately in the formation of estrogen and progesterone receptor-negative, malignant tumors. These tumors have a similar histopathology with those observed in TG mice with activated wnt/beta-catenin pathway, showing increased expression of beta-catenin, also a common finding in human breast tumors. Transdifferentiation is observed in mammary tumors of the hCGbeta TG mice, accompanied by abnormal expression of the Wnt genes in the tumorous and nontumorous mammary gland tissue. Specifically we found increased expression of Wnt5b in the TG mammary glands at the age of 3 months and up-regulation of Wnt7b and -5b in the subsequently appearing tumors. Importantly, hCG was found to up-regulate these wnt ligands in mouse mammary gland, independent of the changes in ovarian steroidogenesis. Thus, the hCGbeta-overexpressing TG mice represent a novel model that links enhanced hCG action to dysregulated wnt signaling in the mammary gland, resulting in beta-catenin-stabilizing mammary tumorigenesis. The novel finding of hCG up-regulating wnt7b and wnt5b could contribute to pregnancy-induced breast cancer in humans.     

Effects of immunoneutralization of luteinizing hormone (LH)-releasing hormone on testicular prolactin and LH receptors in the golden hamster and on LH receptors in the Djungarian hamster

In male Syrian hamsters, seasonal transitions between reproductive activity and quiescence are accompanied by major alterations in testicular binding of LH/hCG and PRL. These alterations are believed to be due to the photoperiod-induced changes in PRL secretion, but other adenohypophyseal hormones appear to be involved as well. To elucidate the role of LH and FSH in the control of testicular LH/hCG and PRL receptors, we have examined the consequences of selective suppression of gonadotropin release by immunoneutralization of endogenous LHRH. In adult Syrian hamsters, four weekly injections of LHRH antiserum produced significant reductions in plasma levels of LH, FSH, and testosterone and in the weights of the testes and the seminal vesicles; no change in plasma PRL; and a significant reduction in the total content (femtomoles per testis) of testicular LH/hCG and PRL receptors. The concentration of LH/hCG receptors (femtomoles per mg protein) was significantly increased. In adult males of another seasonally breeding species, the Djungarian hamster (Phodopus sungorus), four weekly injections of LHRH antiserum produced suppression of plasma LH and FSH, no change in plasma PRL, a reduction in the weights of the testes and the seminal vesicles, an increase in the concentration of testicular LH/hCG receptors, and a decrease in their total content. Alterations in testicular LH and PRL binding induced by treatment with LHRH antiserum in the present study were qualitatively similar to changes induced by exposure to short photoperiod or by treatment with bromocriptine. We conclude that gonadotropins normally participate in the regulation of testicular LH/hCG and PRL receptors in the Syrian hamster and LH/hCG receptors in the Djungarian hamster. Seasonal changes in the content of LH/hCG and PRL receptors in the testes appear to be due to photoperiod-induced alterations in the secretion of both PRL and gonadotropins.     

Reproductive disturbances,pituitary lactotrope adenomas,and mammary gland tumors in transgenic female mice producing high levels of human chorionic gonadotropin

To assess the consequences of prolonged exposure to elevated levels of LH/human chorionic gonadotropin (hCG) in the female, we developed a transgenic (TG) mouse model (hCGbeta+) that overexpresses the hCGbeta-subunit cDNA. Because of the promoter used, ubiquitin C, the transgene is expressed in multiple tissues, including the pituitary gland, in which coupling with the endogenous common alpha-subunit results in synthesis of high levels of bioactive hCG. The TG females presented with precocious puberty, infertility, enhanced ovarian steroidogenesis, and abnormal uterine structure. Pituitary enlargement was evident from the age of 2 months, which progressed to adenomas by the age of 10-12 months. Immunohistochemical studies and electron microscopy demonstrated lactotrope origin for the adenomas, associated with severe hyperprolactinemia. The mammary glands of TG females showed marked lobuloalveolar development followed by mammary tumors with characteristics of adenocarcinoma at the age of 9-12 months. More than 90% of penetrance and high frequency of metastasis (47%) was observed. Formation of the pituitary and mammary gland tumors was totally abolished by ovariectomy despite persistently elevated hCG levels. Taken together, these findings suggest that the hCG-induced aberrations of ovarian function are clearly responsible for the extragonadal tumors observed in these TG mice.     

Effect of administration of human chorionic gonadotrophin on criteria used to assess testosterone administration in athletes.

Abnormal ratios of testosterone to epitestosterone (T/E) and testosterone to LH (T/LH) in the urine of male athletes are indicative of testosterone administration. The T/E ratio has been adopted by the International Olympic Committee as the sole criterion used in the detection of testosterone administration. An athlete is usually considered to have failed a drug test if the urinary T/E ratio is greater than 6. Human chorionic gonadotrophin (hCG) has been used by some male athletes to stimulate testicular secretion of testosterone. The purpose of this investigation was to examine whether the urinary T/E ratio can remain unaffected by administration of hCG to normal adult males. Administration of hCG resulted in large increases in serum testosterone concentrations and urinary T/LH ratios but small changes in urinary T/E ratios of two subjects (maximum T/E values observed were 0.8 and 1.2 respectively). These observations suggest that the urinary T/LH ratio is a valuable indicator of hCG as well as of testosterone administration. This study is the first to measure urinary T/LH ratios using the technique of gas chromatography-mass spectrometry for quantification of testosterone, and highly specific monoclonal antibodies for the measurement of LH. An ultrafiltration method is proposed as part of a confirmatory procedure to be adopted in the measurement of urinary gonadotrophins for drug control in sport.     

Testicular function in strains of mice selected for differences in gonadotrophin-induced ovulation

Mice were selected on the basis of ovulatory responses to injections of pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG). Various parameters of pituitary and gonadal function as well as responsiveness to exogenous gonadotrophins were examined in males from high induced-ovulation rate (HIOV) and low induced-ovulation rate (LIOV) lines. Testicular weight, seminal vesicle weight and plasma LH levels were lower in HIOV than in LIOV males, while plasma concentrations of FSH and testosterone did not differ. Binding of FSH, but not of LH, in the testes was significantly higher in HIOV mice. Twenty-four hours after administration of hCG, plasma testosterone levels were higher and testicular LH binding sites appeared more depleted in HIOV than in LIOV males. Production of testosterone by decapsulated testes in vitro was significantly higher in HIOV than in LIOV mice under basal conditions, as well as in the presence of LH, FSH, hCG or PMSG. It was concluded that selection for differences in gonadotrophin-induced ovulation rate produced correlated differences in the steroidogenic response of the testes to gonadotrophins and that these differences may be due to divergence in the number of gonadal FSH binding sites.     

cis-platinum-mediated decrease in serum testosterone is associated with depression of luteinizing hormone receptors and cytochrome P-450scc in rat testis

Previously we had shown that cis-platinum decreases testosterone levels in rat serum and that hCG reverses this effect. The purpose of these studies was to determine the biochemical basis of cis-platinum-mediated effects on testicular testosterone production. In the testis of rats treated with cis-platinum (7 mg/kg, iv), the mitochondrial P-450scc concentration and side-chain cleavage activity were depressed by 40%. Also, the microsomal 17 alpha-hydroxylase activity and cytochrome P-450 concentration were decreased. Testicular binding capacity (in vitro) for [125I]hCG was decreased by 75-80%. On the other hand, FSH binding to Sertoli cell membrane receptors was not appreciably changed. hCG (25 IU/100 g daily) in treated rats caused complete occupancy of the remaining 20-25% LH receptors and caused a 20- to 30-fold increase in serum and testicular testosterone, a 2-fold increase in mitochondrial P-450scc, and a 5-fold acceleration of side-chain cleavage activity. 17 alpha-Hydroxylase activity and microsomal cytochrome P-450 were not increased over the control values. In addition to testicular functions, pituitary glycoprotein hormone production was assessed. Treatment of rats with cis-platinum (7 mg/kg, iv) did not change serum LH or FSH, but caused a 50% decrease in serum and testicular testosterone levels. A GnRH challenge test (1.5 micrograms/100 g, in 30 min) of treated rats caused prompt increases of 10- to 15-fold in serum LH and resulted in increases in serum and testicular testosterone. Thus, there was little evidence for cis-platinum effects at the level of hypothalamus or pituitary that could account for the decreased testosterone production. Reversal of the cis-platinum effect on steroidogenesis by hCG or GnRH appears to be due to the induction of suprasaturating levels of LH with full occupancy of remaining Leydig cell LH receptors. This, in turn, would reverse the diminished levels of mitochondrial side-chain cleavage activity and cytochrome P-450scc. These data suggest that cis-platinum causes a depression in serum testosterone, mainly by decreasing the number of LH receptors and inhibiting side-chain cleavage activity.     

A novel transgenic model to characterize the specific effects of follicle-stimulating hormone on gonadal physiology in the absence of luteinizing hormone actions

Gonadal function is wholly reliant on the two pituitary-derived gonadotropins, FSH and LH. Identifying the specific effects of FSH has been difficult because of the intimate relationship between LH and FSH action and inherent limitations of classic research paradigms. We describe a novel transgenic model to characterize the definitive actions of FSH alone, distinct from LH effects, created by combining transgenic FSH expression with the gonadotropin-deficient background of the hypogonadal (hpg) mouse. A tandem transgene construct encoding each alpha- and beta-subunit of human FSH, under the rat insulin II promoter, expressed biologically active heterodimers at serum levels, by immunoassay, equivalent to circulating FSH concentrations in fertile humans (0.1-25 IU/liter). Transgenic mice were crossed into the hpg mouse genotype to obtain LH-deficient animals secreting FSH alone. Testis weights of adult FSHxhpg mice were increased up to 5-fold, relative to nontransgenic hpg controls (P < 0.001). However, only transgenic males with serum FSH levels more than 1 IU/liter showed testis weights increased relative to hpg controls, indicating a physiological FSH threshold for the testicular response. Histology of enlarged FSHxhpg testes revealed round spermatids and sparse numbers of elongated spermatids, demonstrating that the testosterone-independent FSH response targeting the Sertoli cell can facilitate completion of meiosis and minimal initiation, but not completion, of spermiogenesis. Transgenic FSH also induced inhibin B secretion in FSHxhpg mice, but showed a distinct sexual dimorphism with only females exhibiting a strong FSH dose-dependent increase in serum inhibin B levels (r(2) = 0.84). In addition, ovaries of FSHxhpg females were enlarged up to 10-fold (P < 0.001), characterized by increased follicular recruitment and development to type 7 antral follicles. Thus, these findings show that the transgenic FSHxhpg mouse provides a unique model for detailed investigations of the definitive in vivo actions of FSH alone.     

hCG suppression of LH receptors and responsiveness of testicular tissue to hCG.

A single injection of 75 IU of human chorionic gonadotropin (hCG) into adult male rats caused a dramatic reduction in the concentration of membrane receptors for luteinizing hormone (LH) in the testis. The mean receptor level reached a nadir which was 5--10% of that in the control testes, 3 days after the injection, after which it gradually returned toward normal. This cannot be due to increased competition caused by the injected hCG since no decrease was observed at a time when the circulating levels of hCG were at a maximum (2--24 h after injection). Furthermore, at a time when receptor levels had been maximally reduced, circulating hCG was at or below the level of detection. Reduction in the number of LH binding sites in the testis was associated with a decreased responsiveness of the testicular tissue to hCG as measured by hCG-stimulated testosterone production in vitro. This inhibitory effect of large quantities of LH on its own receptor is suggested as a possible explantation for the previously observed low concentrations of LH receptor in the testis of the testicular feminized male (tfm) rat. This syndrome is characterized by high endogenous levels of plasma LH (Sherins et al., 1971).     

Consequences of genetic manipulations of gonadotrophins and gonadotrophin receptors in mice

We have produced over the years several genetically modified mouse models (transgenic [TG], knockout [KO] and knockin [KI]) for the study of normal and aberrant functions of gonadotrophins and their receptors. We summarise in the present review some of our recent findings on these animal models. One is the cascade of extragonadal phenotypes triggered by ovarian hyperstimulation in TG mice overexpressing the human choriongonadotrophin (hCG) β-subunit and presenting with elevated levels of serum luteinising hormone (LH)/hCG bioactivity. Massively elevated levels of serum progesterone, rather than oestrogens, are responsible for the induction of pituitary prolactinomas and the subsequently elevated prolactin (PRL) levels. Along with normal oestradiol and elevated progesterone levels, the increased concentration of PRL induces lobuloalveolar development of the mammary gland, with ultimate formation of oestrogen and progesterone receptor-negative malignant tumours. Another TG mouse model expressing a constitutively activating mutant form of the follicle-stimulating hormone receptor (FSHR) presents with a strong ovarian phenotype inducing advanced follicular development and depletion, haemorrhagic follicles, teratomas and infertility. A third TG mouse model, coexpressing binding- and signalling-deficient mutants of LHCGR in the KO background for the same receptor (R) gene provided convincing evidence that functional complementation through homo-di/oligomerisation is a physiologically relevant mode of activation of class A G protein-coupled receptors (GPCR). Taken together, genetically modified mouse models provide powerful tools for the elucidation of normal and pathological functions of gonadotrophins and their R.     

Comparative in vitro and in vivo studies on the biological characteristics of recombinant human follicle-stimulating hormone

The in vitro and in vivo activities of recombinant human FSH (recFSH) produced by a Chinese hamster ovary cell line were studied and compared with those of natural FSH preparations. The specific FSH activities of recFSH established by immunoassay and in vivo bioassay were greater than 10,000 IU/mg protein and considerably higher than the activities of tested urinary FSH references, while the in vivo bio/immuno ratios of these preparations were not significantly different. Compared to a highly purified pituitary standard (IS 83/575), recFSH had a comparable high specific in vivo bioactivity, but the specific immunoreactivity of IS 83/575 was about 2 times lower. In receptor displacement and in vitro bioassay studies recFSH provided dose-response curves parallel to those of pituitary and urinary FSH references. When equal amounts of immunoreactivity FSH were tested, recFSH and urinary and pituitary FSH displayed comparable activities in both assays. The in vitro bioactivity of recFSH could be neutralized effectively by each of three monoclonal antibodies raised against recFSH (alpha-specific), urinary FSH (beta-specific), and pituitary FSH (alpha beta-specific), respectively. Moreover, 50% inhibition of comparable responses induced by recFSH, urinary "pure" FSH, or pituitary FSH was established by the same amount of monoclonal antibody. These results support the structural and functional similarity of recFSH and natural FSH. To test whether recFSH is capable of inducing LH-specific biological responses, the in vitro induction of testosterone production in mouse Leydig cells was assessed. At least 16 IU recFSH/ml incubate were needed to increase testosterone production, indicating that the intrinsic LH bioactivity of recFSH is negligible (less than 0.025 mIU LH/IU FSH). The in vivo efficacy of recFSH was examined by treating immature female hypophysectomized rats during 4 days with recFSH only or with recFSH supplemented with hCG. RecFSH only treatment increased ovarian weight and aromatase activity in a dose-dependent manner. When recFSH dosages providing submaximal responses were supplemented with 1 IU hCG, both ovarian weight and aromatase activity were largely augmented. Neither recFSH nor urinary pure FSH, administered in a high dose was able to increase plasma estradiol levels, while ovarian weight and aromatase activity were increased to the same extent. However, when recFSH was supplemented with only 0.1 IU hCG, a 3-fold increase in median plasma estradiol levels was obtained. These findings support the two-cell two-gonadotropin theory, holding that both FSH and LH are required for estrogen biosynthesis, but also reveal that only very small amounts of LH activity are sufficient to increase estrogen secretion up to measurable plasma levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fertility and spermatogenesis are altered in {alpha}1b-adrenergic receptor knockout male mice

To examine whether norepinephrine, through activation of alpha1b-adrenergic receptor, regulates male fertility and testicular functions, we used alpha1b-adrenergic receptor knockout (alpha1b-AR-KO) mice. In the adult stage (3-8 months), 73% of the homozygous males were hypofertile with relatively preserved spermatogenesis. Of the remaining males, 27% exhibited a complete infertility with a drastic reduction in testicular weight and spermatogenesis defect with germ cells entering a cell death pathway at meiotic stage. In both phenotypes, circulating levels of testosterone were highly reduced (-55 and -81% in hypofertile and infertile males respectively versus wild-type males). Consequently, circulating levels of LH were significantly elevated in alpha1b-AR-KO infertile mice. When incubated in vitro, the whole testes from infertile KO mice released significantly lower levels of testosterone (-40%). This, together with the fact that the mean absolute volume of Leydig cells per testis was not changed, suggests a compromised steroidogenic capacity of Leydig cells in infertile animals. In addition, RNA in situ hybridization study indicated an apparent higher expression of inhibin alpha- and betaB-subunits in Sertoli cells of infertile alpha1b-AR-KO mice. This was correlated with a higher intra-testicular content of inhibin B (+220% above wild-type mice). Using specific primers, mRNA encoding alpha1b-AR was localized in early spermatocytes of wild-type testes. Our results indicate, for the first time, that alpha 1b-AR signaling plays a critical role in the control of male fertility, spermatogenesis, and steroidogenic capacityof Leydig cells. It is thus hypothesized that the absence of alpha1b-AR alters either directly germ cells or indirectly Sertoli cell/Leydig cell communications in infertile alpha1b-AR-KO mice.     

Stimulation of ovarian follicular maturation with pure follicle-stimulating hormone in women with gonadotropin deficiency

According to the 2-cell theory, ovarian steroidogenesis requires the coordinate action of both FSH and LH. To evaluate the relative importance of these hormones in follicular maturation, a randomized cross-over study was performed in 10 women with complete gonadotropin deficiency (absence of pulsatile LH secretion and no LH response to LHRH). Five women were treated with highly purified FSH (LH bioactivity, 0.09%) and 3 months later with human menopausal gonadotropin (hMG; LH bioactivity, 65%), each given for 10 days at a daily dose of 225 IU FSH, im. The sequence was reversed in the other 5 women. hCG (5000 IU) was administered im 24 h after the last injection of FSH or hMG. Plasma estradiol (E2), estrone (E1), androstenedione (A), testosterone, LH, and FSH concentrations and urinary LH and FSH were measured daily by RIA. Ultrasonography was performed during each treatment and 2 days after each hCG injection. After FSH treatment, mean plasma and urinary FSH levels increased, mean plasma LH did not change, and urinary LH increased slightly but not significantly from 91 +/- 32 (SE) to 164 +/- 55 mIU/24 h (10(-3) IU/24 h). After hMG treatment, mean plasma and urinary LH and FSH levels increased accordingly. The mean basal plasma E2 [11 +/- 1 pg/mL (40 +/- 4 pmol/L)] and E1 [14 +/- 4 pg/mL (52 +/- 15 pmol/L)] levels increased after FSH treatment to 207 +/- 69 pg/mL (760 +/- 253 pmol/L) and 82 +/- 21 pg/mL (303 +/- 78 pmol/L), respectively (P less than 0.01), but plasma A did not change. In response to hMG, the mean plasma E2, E1, A, and testosterone levels increased more than during FSH treatment. Ultrasonography revealed multiple preovulatory follicles (greater than or equal to 16 mm) in 2 women after hMG and 1 woman after FSH treatment; therefore, hCG was not administered. In 3 women given FSH, hCG did not induce ovulation. hCG induced ovulation in 8 women given hMG and in 6 women given FSH, based on ultrasonography and plasma progesterone levels. Thus, in the presence of profound gonadotropin deficiency pharmacological doses of FSH, with minute LH contamination, are capable of stimulating ovarian follicular maturation, underlining the key role of FSH in folliculogenesis.     

Gonadotropin binding and Leydig cell activation in the rat testis in vivo

Testicular LH receptor occupancy and steroidogenic responses were measured in adult male rats after intracardiac injections of [125I]iodo-hCG (0.5--5 x 10(6) cpm) mixed with known amounts of nonradioactive hCG to yield doses ranging from 10 ng to 300 micrograms. Uptake of the hormone by the testis was measured in the whole tissue or the 20,000 x g homogenate, with correction for nonspecific binding in animals injected with a 100-fold excess of unlabeled hCG. The steroidogenic response to hCG was followed by measurements of serum and testicular testosterone. Maximum specific uptake of [125I]iodo-hCG by the testes was observed 4--6 h after hormone injection. Of the specific counts, 80% were recovered in the 20,000 x g pellet of the tissue homogenate. The testicular contents of hCG-binding sites were similar when measured by in vivo occupancy of the receptors and by the in vitro receptor assay, indicating the physiological validity of the receptor measurements in tissue homogenates. Serum and testicular testosterone levels reached a maximum at 1 h, independent of the hCG dose used. When receptor occupancy in vitro after injection of hCG was compared with stimulation of steroidogenesis, a significant (P less than 0.05) 3-fold elevation of serum testosterone was seen when only 0.05% of the receptors were occupied. The maximal testosterone response was reached with 0.8% receptor occupancy. It is concluded that the same number of testicular LH receptors can be occupied by the circulating hormone in vivo and in tissue homogenates in vitro. The spare receptor concept also applied to the in vivo situation, since stimulation of steroidogenesis in the intact animal requires occupancy of only a few receptors per Leydig cell. This may be a general feature of hormonal activation of endocrine target cells in vivo.     

Testicular endocrine function in GH receptor gene disrupted mice.

The consequences of disruption of GH receptor gene in GH receptor knockout mice on testicular function were evaluated. Adult male GH receptor knockout mice and their normal siblings were divided in to two subgroups and treated with either saline or ovine LH (0.3 microg/g BW) in saline. One hour after saline or LH administration, blood was obtained via heart puncture. Plasma IGF-I, LH, FSH, PRL, androstenedione, and testosterone levels were measured by RIAs. Testicular LH and PRL receptor numbers as well as pituitary LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were measured. Also, testicular morphometric analysis was performed. Unlike in normal, wild-type mice, the circulating IGF-I was undetectable in GH receptor knockout mice. The plasma PRL levels were (P<0.01) higher in GH receptor knockout mice than in their normal siblings. The basal LH secretion was similar in normal and GH receptor knockout mice. However, the circulating FSH levels were lower (P<0.001) in GH receptor gene disrupted mice. Administration of LH resulted in a significant (P<0.001) increase in plasma testosterone levels in both GH receptor knockout and normal mice. However, this testosterone response was attenuated (P < 0.01) in GH receptor knockout mice. Plasma androstenedione responses were similar in both GH receptor knockout and normal mice. Testicular LH and PRL receptor numbers were significantly decreased in GH receptor knockout mice. The results of the morphometric analysis of the testis revealed that the Leydig cell volume per testis was reduced in mice with GH receptor gene disruption. The steady-state of LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were not different in GH receptor knockout mice relative to their normal siblings. The present in vivo study demonstrates that in GH receptor knockout mice, LH action on the testis in terms of testosterone secretion is significantly attenuated and suggests that this is due to a decrease in the number of testicular LH receptors. The reduced number of PRL receptors may contribute to the diminished responsiveness of testicular steroidogenesis to LH by decreased ability to convert androstenedione to testosterone. These changes are most likely due to the absence of circulating IGF-I. These findings provide evidence that systemic IGF-I plays a major modulatory role in testicular endocrine function.     

Developmental hormonal profiles accompanying the neonatal hypothyroidism-induced increase in adult testicular size and sperm production in the rat.

Neonatal treatment with the reversible goitrogen 6-N-propyl-2-thiouracil (PTU) results in a near doubling of testicular size and a 25% increase in the efficiency of spermatogenesis, without affecting circulating testosterone (T) levels in adult rats. The objectives of the present study were to examine the effects of neonatal PTU treatment on the pattern of testicular growth and circulating levels of anterior pituitary (FSH, LH, PRL, GH, and TSH), gonadal [immunoreactive inhibin-alpha (irI alpha) and T], and thyroid (T3 and T4) hormones over the first 100 days of life. Treatment of rats with PTU from birth to 24 days of age significantly reduced testicular weights between 10 and 60 days of age. However, the duration of testicular growth was extended in treated males, resulting in a 68% increase at 100 days of age. Serum gonadotropin levels in treated males were reduced throughout the experimental period, typically remaining between 50-70% of control levels. The characteristic robust prepubertal FSH peak was absent in PTU-treated males. Initially high until 20 days of age, irI alpha levels characteristically declined to adult levels (200-300 pg/ml) in control males. In treated males, irI alpha levels were reduced during the period of hypothyroidism, increased between 30 and 60 days, and then declined, but remained significantly higher (1.7- to 2-fold greater) than those observed in control males. Serum T levels were similar in treated and control males. Control males demonstrated increased T levels beginning at 45 days of age, earlier than observed in treated males; however, similar peak T levels were observed in all males. PTU treatment significantly suppressed serum GH and PRL and led to a 14-fold increase in circulating TSH during the period of treatment. However, unlike the gonadotropins, these hormones returned to control levels after PTU treatment, suggesting that the reduced levels of FSH and LH observed are not due to a generalized reduction in pituitary function. Serum T4 and T3 levels returned to control levels within 15 days after the removal of PTU. These results demonstrate that the neonatal PTU treatment-induced increases in adult testicular size and sperm production were not due to increased levels of FSH at any point in development. On the contrary, the observed increases occur in spite of chronically reduced FSH levels.     

Specificity of cognate ligand-receptor interactions: fusion proteins of human chorionic gonadotropin and the heptahelical receptors for human luteinizing hormone,thyroid-stimulating hormone,and follicle-stimulating hormone

The family of glycoprotein hormones and their homologous heptahelical receptors represent an excellent system for comparative structure-function studies. We have engineered single chain molecules of human chorionic gonadotropin (hCG) fused to its cognate receptor, LH receptor (LHR), and to the noncognate receptors, TSH receptor (TSHR) and FSH receptor (FSHR; N-beta-alpha-receptor-C), to create the yoked (Y) complexes YCG/LHR, YCG/TSHR, and YCG/FSHR. The expression and bioactivity of these fusion proteins were examined in transiently transfected HEK 293 cells. Western blot analysis and antibody binding assays demonstrated that each of the proteins was expressed. In the case of YCG/LHR, minimal binding of exogenous hormone was observed due to the continued occupation of receptor by the fused ligand. The presence of hCG in the YCG/TSHR and YCG/FSHR, however, did not prevent binding of exogenous cognate ligand, presumably due to the lower affinity of hCG. The basal cAMP levels in cells expressing the YCG/LHR complex was approximately 20-fold higher than that in cells expressing LHR. Increases in basal cAMP production were also observed with YCG/TSHR and YCG/FSHR, e.g. 13- and 4-fold increases, respectively. Whereas the affinity and specificity of hCG for LHR are extraordinarily high, the hormone is capable of binding to and activating both TSHR and FSHR under these conditions that mimic high ligand concentrations. These findings were confirmed by adding high concentrations of hCG to cells expressing TSHR and FSHR. Although the functional interaction of hCG and TSHR has been recognized in gestational hyperthyroidism, there are no reports linking hCG to FSHR activation. This study, however, suggests that such a functional interaction is capable of occurring under conditions of high circulating levels of hCG, e.g. the first trimester of pregnancy and in patients with hCG-secreting tumors.     

Potent inhibitory activity of [D-Leu6, Des-Gly-NH2(10)]LHRH ethylamide on LH/hCG and PRL testicular receptor levels in the rat.

Injection of male rats with 40-200 ng of [D-Leu6, des-Gly-NH2(10)]LHRH ethylamide for 7 days caused a maximal 80% reduction of testicular LH/hCG receptor level with one injection per day being as efficient as 3 daily injections. A similar inhibitory effect was observed on testicular PRL receptors. Testis and seminal vesicle weight as well as plasma testosterone levels were also significantly reduced by this treatment. These data indicate that a LHRH agonist, when given at a relatively low dose, is capable of reducing testicular LH/hCG and PRL receptor levels as well as testicular function, the effect being probably mediated by increased endogenous gonadotropin secretion.     

Prolactin, growth hormone, luteinizing hormone receptors, and seasonal changes in testicular activity in the golden hamster.

In adult male hamsters, 2 months of exposure to a short photoperiod (5 h of light:19 h of darkness) caused testicular regression and a precipitous decline in plasma PRL, in agreement with earlier reports from other laboratories. Depressed release of PRL cannot be explained by a reduction in testicular steroidogenesis, because castration of males kept in a long photoperiod did not reduce PRL levels and administration of testosterone to males kept in a short photoperiod failed to reverse the decline in plasma PRL concentration. Treatment of such "regressed" animals with PRL, GH, or ectopic pituitary transplants stimulated growth of the testes and the accessory reproductive glands, increased the concentration of LH receptors in the testes, and elevated plasma testosterone levels. A single injection of 250 microgram PRL was sufficient to increase testicular LH binding, and chronic treatment with pituitary grafts completely reversed testicular regression. The effectiveness of exogenous PRL in stimulating testicular growth and LH receptors was significantly influenced by the timing of the injection. In some experiments, gonadotropin levels appeared elevated in animals injected with PRL, but these differences were not statistically significant. In hamsters with gonadal regression induced by exposure to a short photoperiod, daily administration of 20 microgram H and/or 150 microgram FSH had no apparent effect on testicular function. However, treatment with large doses of hCG and/or PMS gonadotropin resulted in significant stimulation of testicular growth and steroidogenesis. Chronic treatment of males maintained in a long photoperiod (14 h of light:10 h of darkness) with an inhibitor of PRL release, 2-Br-alpha-ergocryptine, resulted in a decreased weight of the testes and seminal vesicles. Administration of this inhibitor for a longer period (2 months) produced a significant increase in body weight but had little effect on testicular function. These results indicate that changes in the release of PRL (and possibly also GH) may plan an important role in mediating the effects of the photoperiod on testicular function in the golden hamster.     

Aberrant expression of human luteinizing hormone receptor by adrenocortical cells is sufficient to provoke both hyperplasia and Cushing's syndrome features

CONTEXT: Aberrant expression of LH/human chorionic gonadotropin (hCG) receptor has been suggested in several cases of bilateral macronodular adrenal hyperplasia with Cushing's syndrome. The cortisol production is then directly controlled by endogenous secretion of LH/hCG. However, the direct involvement of this aberrant LH/hCG receptor expression in the development of the hyperplasia has not been demonstrated. Moreover in most cases, whenever investigated, the aberrant expression of LH/hCG receptor has been associated with the ectopic expression of other G protein-coupled receptors such as gastric inhibitory polypeptide, serotonin, or vasopressin receptors. OBJECTIVE: The aim of this study was to explore the action of LH/hCG receptor on the development of adrenal hyperplasia. RESULTS: The ectopic expression of this single nonmutated gene transduced into bovine adrenocortical cells was sufficient to induce not only the aberrant cortisol secretion but also hyperproliferation and benign transformation. The cells were transplanted beneath the kidney capsule of adrenalectomized immunodeficient mice. Only the cells expressing the LH/hCG receptor gene formed an enlarged tissue with a high proliferation rate. The tissue expressing LH/hCG receptor was responsible for elevated plasma cortisol and decreased plasma ACTH levels in transplanted mice. These animals displayed physiological changes similar to those of patients with Cushing's syndrome, including muscle atrophy, thin skin, spleen atrophy, and hyperglycemia. CONCLUSIONS: These results demonstrate that a single genetic event such as the inappropriate expression of the nonmutated LH/hCG receptor gene is sufficient to initiate the phenotypic changes that cause the development of a benign adrenocortical tumor.