hCG Challenge Research Articles

Development and Validation of an Enzyme Immunoassay for Fecal Dehydroepiandrosterone Sulfate in Japanese Macaques (Macaca fuscata)

Measuring hormonal profiles is important in monitoring stress, physical fitness, and reproductive status in primates. Noninvasive methods have been used to measure several steroid hormones in primates without causing them stress. However, few studies have used feces or urine to measure dehydroepiandrosterone sulfate (DHEAS), an important precursor of sex steroids that has been studied as a biomarker of aging, pregnancy, and stress in humans and nonhuman primates. We developed an enzyme immunoassay to detect DHEAS in the feces of Japanese macaques (Macaca fuscata). Our subjects included eight singly housed Japanese macaques. To validate the assay, we administrated oral DHEA to one male and one female macaque, collected their feces, and measured DHEAS levels over time. Given that DHEAS is related to gonadal steroids and the stress response, we also measured DHEAS concentrations in response to adrenal (adrenocorticotropic hormone [ACTH]) and gonadal (human chorionic gonadotropin [hCG]) stimulation. Our assay successfully detected DHEAS in Japanese macaque feces, and levels of DHEAS were associated with the amount of DHEA ingested. Parallelism and accuracy tests revealed that fecal extracts were reliable measures of DHEAS. Neither ACTH nor hCG challenge appeared to affect DHEAS levels. The method we describe is less expensive than that using the commercially available kits and is applicable to investigations involving aging, stress, and reproduction in Japanese macaques.

Luteinizing Hormone Receptors are Confined in Mesoscale Plasma Membrane Microdomains Throughout Recovery from Receptor Desensitization

We examined the involvement of membrane microdomains during human luteinizing hormone (LH) receptor recovery from receptor desensitization after removal of bound hormone. Lateral motions of individual desensitized LH receptors expressed on the surface of Chinese hamster ovary cells and transient association of these receptors with detergent-resistant membrane (DRM) microdomains isolated using isopycnic sucrose gradient ultracentrifugation were assessed. Single particle tracking experiments showed untreated individual LH receptors to be confined within cell-surface membrane compartments with an average diameter of 199 ± 17 nm and associated with membrane fractions characteristic of bulk plasma membrane. After brief exposure to human chorionic gonadotropin (hCG), LH receptors remained for several hours desensitized to hCG challenge. Throughout this period, significantly increased numbers of LH receptors were confined within smaller diameter (<120 nm) membrane compartments and associated with DRM fragments of characteristically low density. By 5 h, when cells again produced cAMP in response to hCG, unoccupied LH receptors were found in larger 169 ± 22 nm diameter cell-surface membrane compartments and >90 % of LH receptors were again found in high-density membrane fragments characteristic of bulk plasma membrane. Taken together, these results suggest that, during recovery from LH receptor desensitization, LH receptors are both located with DRM lipid environments and confined within small, mesoscale (80-160 nm) cell-surface compartments. This may reflect hormone-driven translocation of receptors into DRM and formation there of protein aggregates too large or too rigid to permit effective signaling. Once bound hormone is removed, receptor structures would have to dissociate before receptors can again signal effectively in response to hormone challenge. Moreover, such larger protein complexes would be more easily constrained laterally by membrane structural elements and so appear resident in smaller cell-surface compartments.

Dehydroepiandrosterone sulfate levels reflect endogenous luteinizing hormone production and response to human chorionic gonadotropin challenge in older female macaque (Macaca fascicularis)

We propose that the adrenal gland of an older higher primate female animal model will respond to human chorionic gonadotropin (hCG) hormone challenge by secreting additional dehydroepiandrosterone sulfate (DHEAS). Such a response in surgically and chemically castrated animals will provide proof of concept and a validated animal model for future studies to explore the rise in DHEAS during the menopausal transition of women. Twenty-four 18- to 26-year-old female cynomolgus monkeys were screened for ovarian function and then either ovariectomized (n = 4) or treated with a gonadotropin-releasing hormone agonist (GnRHa; n = 20) to block ovarian steroid production. After a recovery period from surgical procedure or down-regulation, a single-dose challenge (1,000 IU/animal, IM) of hCG was then administered to determine if luteinizing hormone (LH)/chorionic gonadotropin could accelerate circulating DHEAS production. Serum DHEAS, bioactive LH, and urinary metabolites of ovarian sex steroids were monitored before, during, and after these treatments. Circulating LH bioactivity and immunoreactive DHEAS concentrations were suppressed in all animals 14 days postadministration of GnRHa. Urinary metabolites of estradiol and progesterone remained low after the surgical procedure or a flare reaction to GnRHa. Circulating DHEAS levels were increased after hCG administration, and the increase in individual animals was proportional to the pretreatment DHEAS at baseline. Circulating DHEAS concentrations were positively correlated to endogenous LH bioactive concentrations prior to hCG challenge and were subsequently further elevated by the hCG challenge while no concomitant change in ovarian steroid hormone excretion was observed. These data demonstrate a positive adrenal androgen response to LH/chorionic gonadotropin in older female higher primates and suggest a mechanism for the rise in adrenal androgen production during the menopausal transition in women. These results also illustrate that the nonhuman primate animal model can be effectively used to investigate this phenomenon.

Studies on reactivation of regressing bonnet monkey corpus luteum on day 1 of menses: A pilot study

Studies on functional characteristics of the regressing primate corpus luteum (CL) to luteotrophic stimulus on day 1 of the non-fertile menstrual cycle are scarce. Recombinant human luteinizing hormone (rhLH) (20 IU/Kg BW; n = 10) or human chorionic gonadotropin (hCG) (180 IU; n = 6) were administered intravenously to female bonnet monkeys on day 1 of menses. Exogenous treatment of rhLH or hCG caused a significant increase in circulating progesterone (P4) levels 2-4 hours post treatment (P < 0.05). Lutectomy prior to onset of menses confirmed that CL is the site of the increased P4 concentrations. Increased levels of phosphorylated P44/42 MAPK, MKK3/6 activation and concomitant histological changes were observed within 4 hours in CL of monkeys receiving hCG treatment. The results from this study demonstrate the acute progesterone synthesizing capacity of regressing monkey CL after LH or hCG challenge. This has potential implications for interpreting the steroidogenic response after gonadotropin stimulation tests in the early follicular phase of the normal ovulatory and anovulatory women undergoing controlled ovarian stimulation protocols as part of assisted reproductive technology (ART) and in women with polycystic ovarian syndrome.

The Effects of Anti‐TNF‐α Antibody on Hyperprolactinemia‐Related Suppression of hCG‐Induced Testosterone Release in Male Rats

Hyperprolactinemia (hyperPRL)-related hypogonadism or suppression of human chorionic gonadotropin (hCG)-induced testosterone (T) release is hypothesized to be mediated by a testicular interstitial macrophage and tumor necrosis factor alpha (TNF-α)-involved blockage. To test if the lower T response after hCG challenge in the hyperPRL rats is reversed by administrating anti-TNF-α antibody (Ab). HyperPRL was induced by allografting two anterior pituitary (AP) glands per rat. Control rats were grafted with similar amount of cerebral cortex. The testicular interstitial cells (TIC) were isolated from the testis 6 weeks after grafting. TIC was treated with anti-TNF-α Ab with or without hCG. The other groups of rats received intra-testicular or intra-muscular anti-TNF-α Ab 7 days before in vitro study. The TIC isolated from each testis was incubated and T release with or without hCG challenge were measured. Prolactin (PRL) and T were measured by radioimmunoassay. TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). When low dose of anti-TNF-α Ab was administered to the TIC incubation, the effects of PRL-related suppression of hCG-stimulated T release were not significant. While a higher dose of anti-TNF-α Ab almost abolished the suppressive effects of PRL to hCG-stimulated T release. Prior intra-testicular or intra-muscular administration of anti-TNF-α Ab reversed the suppressive effects of AP grafting on TIC's T release. This was demonstrated in groups with anti-TNF-α Ab injection both 7 and 1 day prior to TIC incubations. The data support the hypothesis that the suppression of hCG-induced T release associated with hyperPRL is through a TNF-α-mediated mechanism to suppress the Leydig cells. The effect of anti-TNF-α Ab is durable for at least 7 days. Besides intra-testicular injection, there might be other ways available for administrating Ab. Anti-TNF-α Ab has a potential therapeutic application on hyperPRL-induced hypogonadism or suppression of hCG-induced T release.

Adverse effects associated with persistent stimulation of Leydig cells with hCG in vitro

The detrimental effects of persistent stimulation with hCG were investigated in rat Leydig cells in vitro. Significant rise in lipid peroxidation and reactive oxygen species (ROS) with concomitant attenuation in the activities of antioxidant enzymes: superoxide dismutase, catalase, and glutathione-S-transferase was observed. Transcripts for catalase and superoxide dismutase were also depleted. Subsequent to each hCG challenge, the total antioxidant capacity in the target cells also declined significantly (P < 0.05). There was an increase in cell apoptosis (23%), which was associated with a rise in caspase-3 activity, PARP cleavage, and Fas, FasL, caspase-8 expression. While Bax and Caspase-9 expression remained unchanged, Bcl-2 demonstrated a marked decline. Taken together, the above data indicate that persistent hCG stimulation of Leydig cells induced adverse effects leading to oxidative stress and apoptosis which was channeled primarily through the extrinsic pathway.

Effects of bromodichloromethane on ex vivo and in vitro luteal function and bromodichloromethane tissue dosimetry in the pregnant F344 rat

Bromodichloromethane (BDCM), a drinking water disinfection by-product, causes pregnancy loss, i.e. full-litter resorption, in F344 rats when treated during the luteinizing hormone (LH)-dependent period. This effect is associated with reduced maternal serum progesterone (P) and LH levels, suggesting that BDCM disrupts secretion of LH. To test the hypothesis that BDCM also affects luteal responsiveness to LH, we used ex vivo and in vitro approaches. For the ex vivo study (i.e., in vivo exposure followed by in vitro assessment), dams were dosed by gavage on gestation days (GD) 6–9 (plug day = GD 0) at 0 or 100 mg/kg/d. One hour after the GD-9 dose, rats were killed, blood was collected, and tissue concentrations of BDCM were assessed. Corpora lutea (CL) were incubated with or without hCG, an LH agonist, to stimulate P secretion. For the in vitro study, CL were pooled from untreated F344 rats on GD 9 and cultured with BDCM at 0, 0.01, 0.10 or 3.0 mM. BDCM was found at highest concentrations in adrenal, ovarian, adipose, and hypothalamic tissues. BDCM treatment decreased serum P and LH levels in vivo. Ex vivo, however, BDCM-exposed CL showed >2-fold increases in P secretion relative to controls. Both control and BDCM-exposed CL displayed a 2.4-fold increase in P secretion in response to hCG challenge. In contrast, in vitro exposures reduced CL responsiveness in a dose-related fashion while baseline levels were unaffected. It is unclear if the ex vivo ‘rebound’ reflects the removal of the CL from a possible direct inhibitory influence of BDCM, or a response to diminished LH stimulation in vivo. Thus, these data suggest that BDCM disrupts pregnancy in F344 rats via two modes: disruption of LH secretion, and disruption of the CL’s ability to respond to LH.

TNF-α Antibody and TNF-α Antagonist Entanercept Counteracts the Detrimental Effects of Prolactin on Testosterone Release of the Leydig cells From Hyperprolactinemic Rats

Hyperprolactinemia (hyperPRL) related hypogonadism is caused by either a suppressed hypothalamic function by PRL or by a direct inhibitory influence of PRL on the Leydig cells. However, the phenomenon is associated with the interaction of the testicular interstitial macrophages. PRL challenges the macrophages to release TNF-α, which consequently intervenes the function of Leydig cells and their testosterone (T) secretion. This study is to test the hypothesis that adding TNF-α antibody or TNF-α antagonist can counteract the detrimental effects of PRL on T release by the Leydig cells under the status of hyperPRL. Animal studies and in vitro cell cultures. HyperPRL was induced by transplantation of 2 ectopic anterior pituitary glands to the space under the renal capsule. The control groups were transplanted with an equal amount of brain cortex. Six weeks after the surgery, the male Spraque-Dawley rats were sacrificed and the testicular interstitial cells (TICs) were isolated. The TICs were treated with human chorionic gonadotropin (hCG, 0.05 IU/ml) and ovine PRL (o-PRL) with or without the presence of TNF-α antibody or TNF-α antagonist Etanercept. The incubating time was 1h and the medium was collected for T measurement by radioimmunoassay. Under the challenge of hCG, the more o-PRL added, the more significantly the suppression of T release from the TICs. After the TNF-α antibody or TNF-α antagonist Etanercept were added, the phenomenon of suppression of T release was reversed. It showed a dose-dependent relationship. At this circumstance, o-PRL becomes a boosting aid on T release to hCG challenge. While incubating the Leydig cells alone (with the macrophages removed) with o-PRL, the inhibitory effects of PRL to T release were replaced with stimulatory effects. The results indicate that the detrimental effects of hyperPRL on testicular T release are due to the influence of testicular interstitial macrophages through the action of TNF-α that in term hampering the Leydig cells’ response to hCG. The effects are potentially reversible by manipulating the actions of TNF-α.

A female pseudohermaphrodite Holstein heifer with gonadal mosaicism

An 11-month-old Holstein heifer diagnosed as a female pseudohermaphrodite (PH) was subjected to clinical, hormonal, histological, and cytogenetic examinations. The urogenital sinus and external genitalia of the heifer were virilized and no vulval orifice was present. A male urethra-like tubular structure was palpable in the perineal area; urination occurred through a urethral fossa at the distal end of the urethra (cranial aspect of the mammary tissue). Bilateral ovaries and uterine horns, ovarian follicular development, ovulation and CL formation (with increasing blood P4 concentration) were similar to a normal heifer. Testosterone (T) concentrations of all samples collected both before and after hCG challenge were <0.1 ng/mL (absence of a responsive endogenous androgen source). Histologically, the ovary, uterus, oviduct and vagina resembled the normal female reproductive tract. Although the corpus cavernosum was present, the penile urethra and corpus spongiosum penis were hypoplastic. A normal female karyotype (60, XX) was detected in metaphase spreads obtained from cultured peripheral lymphocytes. Moreover, Y-chromosome-specific sequences were not detected in genomic DNA from lymphocytes. By PCR analysis of genomic DNA (from the tissues of urogenital organs), Y-chromosome-specific sequences (both SRY and Amelogenin (Amel) locus) were detected. In conclusion, this was a female PH heifer due to gonadal mosaicism, with normal female genitalia and ovarian activity. This is the first report of an SRY-positive, mosaic female PH heifer.

Effects of prenatal treatment with antiandrogens on luteinizing hormone secretion and sex steroid concentrations in adult spotted hyenas, Crocuta crocuta.

Prenatal androgen treatment can alter LH secretion in female offspring, often with adverse effects on ovulatory function. However, female spotted hyenas (Crocuta crocuta), renowned for their highly masculinized genitalia, are naturally exposed to high androgen levels in utero. To determine whether LH secretion in spotted hyenas is affected by prenatal androgens, we treated pregnant hyenas with antiandrogens (flutamide and finasteride). Later, adult offspring of the antiandrogen-treated (AA) mothers underwent a GnRH challenge to identify sex differences in the LH response and to assess the effects of prenatal antiandrogen treatment. We further considered the effects of blocking prenatal androgens on plasma sex steroid concentrations. To account for potential differences in the reproductive state of females, we suppressed endogenous hormone levels with a long-acting GnRH agonist (GnRHa) and then measured plasma androgens after an hCG challenge. Plasma concentrations of LH were sexually dimorphic in spotted hyenas, with females displaying higher levels than males. Prenatal antiandrogen treatment also significantly altered the LH response to GnRH. Plasma estradiol concentration was higher in AA-females, whereas testosterone and androstenedione levels tended to be lower. This trend toward lower androgen levels disappeared after GnRHa suppression and hCG challenge. In males, prenatal antiandrogen treatment had long-lasting effects on circulating androgens: AA-males had lower T levels than control males. The sex differences and effects of prenatal antiandrogens on LH secretion suggest that the anterior pituitary gland of the female spotted hyena is partially masculinized by the high androgen levels that normally occur during development, without adverse effects on ovulatory function.

The luteinizing hormone receptor activates phospholipase C via preferential coupling to Gi2.

Binding of lutropin/choriogonadotropin (LH/CG) to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. This divergent signaling of the LH receptor is based on the independent activation of distinct G protein subfamilies, i.e. , Gs, Gi, and potentially also Gq. To examine the selectivity of LH receptor coupling to phospholipase C beta-activating G proteins, we used an in vivo reconstitution system based on the coexpression of the LH receptor and different G proteins in baculovirus-infected insect cells. In this paper, we describe a refined expression strategy for the LH receptor in insect cells. The receptor protein was inserted into the cell membrane at an expression level of 0.8 pmol/mg of membrane protein. Sf9 cells expressing the LH receptor responded to hCG challenge with a concentration-dependent accumulation of intracellular cAMP (EC50 = 630 nM) but not of inositol phosphates, whereas stimulation of the histamine H1 receptor in Sf9 cells led to increased phospholipase C (PLC) activity. Immunoblotting experiments using G protein-specific antisera revealed the absence of quantitative amounts of alpha i in Sf9 cells, whereas alpha s and alpha q/11 were detected. We therefore attempted to restore the hCG-dependent PLC activation by infection of Sf9 cells with viruses encoding the LH receptor and different G protein alpha subunits. HCG stimulation of cells coexpressing the LH receptor and exogenous alpha i2 resulted in stimulation of PLC activity. In cells coinfected with an alpha i3-baculovirus, hCG challenge led to a minor activation of PLC, whereas no hCG-dependent PLC stimulation was observed in cells coexpressing alpha i1. Most notably, coinfection with baculoviruses encoding alpha q or alpha 11 did not reproduce the PLC activation by the LH receptor. Thus, the murine LH receptor activates adenylyl cyclase via Gs and PLC via selective coupling to Gi2.

Hormonal and photoperiodic modulation of testicular mRNAs coding for inhibin/activin subunits and follistatin in Clethrionomys glareolus, Schreber.

Photoperiodic and hormonal modulation of mRNAs for testicular inhibin/activin subunits and follistatin were studied in a seasonally breeding rodent, the bank vole (Clethrionomys glareolus). Photoperiod-induced testicular regression had no effect on the relatively low steady-state levels of follistatin mRNA. Inhibin alpha (I alpha) and beta B (I beta B) mRNA levels were significantly higher in regressed than in active gonads, but inhibin beta A was undetectable. The effect of gonadotropin administration on testicular weight and mRNA concentrations differed between the sexually active and quiescent voles. Neither FSH (1.2 U/kg; s.c. for 5 days) nor hCG (600 IU/kg; s.c. for 5 days) affected testicular weight in sexually active voles, whereas both gonadotropins significantly increased testicular weight in photo-regressed individuals. FSH had no effect on I alpha or I beta B mRNA concentrations in the active testes, whereas excessive hCG challenge induced a decrease in the steady-state levels of these mRNAs. FSH induced an increase in I alpha mRNA concentrations in the regressed gonad, whereas both gonadotropins concomitantly down-regulated I beta B mRNA levels. In conclusion, the high expression of I alpha and I beta B mRNA in the regressed testis imply autocrine and paracrine roles for inhibin/activin in the quiescent gonad of seasonal breeders. Inhibin alpha-subunit expression is at least partly under the control of FSH in the bank vole testis.

Comparison of luteal function between ewe lambs and ewes during breeding and non breeding season

Reproductive performance of ewe lambs is lower than that of adult ewes (Quirke. 1979). This is mainly the result of preimplantation losses which can exceed 50% in ewe lambs as compared with 20-30% in ewes (Quirke et al.,1981). Previous evidence from this laboratory suggests that these losses may be associated with inadequate luteal function in ewe lambs (Davies and Beck, 1993). Inadequate luteal function may not only delay the embryo development but also impair the ability of the embryo to communicate its presence to the maternal system. There is also a possibility that inadequate corpora lutea may be refractory to luteotrophic factors produced by the developing embryo. The present study was designed to investigate whether the luteal function varies between ewe lambs and ewes. Another objective of this study was to compare the luteal response to hCG challenge between ewe lambs and ewes and its variation with season.

Comparison of luteal function between ewe lambs and ewes during breeding and non breeding season

Reproductive performance of ewe lambs is lower than that of adult ewes (Quirke. 1979). This is mainly the result of preimplantation losses which can exceed 50% in ewe lambs as compared with 20-30% in ewes (Quirke et al.,1981). Previous evidence from this laboratory suggests that these losses may be associated with inadequate luteal function in ewe lambs (Davies and Beck, 1993). Inadequate luteal function may not only delay the embryo development but also impair the ability of the embryo to communicate its presence to the maternal system. There is also a possibility that inadequate corpora lutea may be refractory to luteotrophic factors produced by the developing embryo. The present study was designed to investigate whether the luteal function varies between ewe lambs and ewes. Another objective of this study was to compare the luteal response to hCG challenge between ewe lambs and ewes and its variation with season.

Mechanism for Species-Specific Induction of Leydig Cell Tumors in Rats by Lansoprazole

Mechanism for Species-Specific Induction of Leydig Cell Tumors in Rats by Lansoprazole. Fort, F. L., Miyajima, H., Ando, T., Suzuki, T., Yamamoto, M., Hamashima, T., Sato, S., Kitazaki, T., Mahony, M. C., and Hodgen, G. D. (1995). Fundam. Appl. Toxicol. 26, 191-202. Lansoprazole is a substituted benzimidazole which inhibits gastric acid secretion by inhibiting the hydrogen-potassium ATPase (proton pump) in the parietal cell. The finding of Leydig cell hyperplasia and Leydig cell tumors in 2-year oral studies in Sprague-Dawley rats but not in CD-1 mice prompted investigative studies to determine the mechanism for the Leydig cell changes. hCG challenge studies in Sprague-Dawley rats revealed decreased testosterone responsiveness in rats treated orally for 1 or 2 weeks with lansoprazole. After 4 weeks of daily oral treatment increases in serum LH and decreases in serum testosterone were detected within a few hours after dosing. In a study where 9-month-old male F344 rats were given testosterone supplementation via Silastic implants and then treated with lansoprazole for 6 months, a high incidence of Leydig cell tumors was seen in lansoprazole-treated, unsupplemented rats, whereas no Leydig cell tumors were seen in testosterone supplemented rats. This implied that reduction of the normal feedback inhibition at the level of the hypothalamus and/or pituitary due to reduced testosterone levels, thus giving rise to elevated levels of LH, was involved in the induction of Leydig cell tumors by lansoprazole. In vitro studies with Leydig cells from rats using various stimulators and precursors of testosterone biosynthesis demonstrated that the most sensitive site for inhibition of testosterone synthesis by lansoprazole is the transport of cholesterol to the cholesterol side chain cleavage enzyme. The IC50s for inhibition of LH or hCG-stimulated testosterone synthesis in Leydig cells from rats, mice, and monkeys were 11-12, 8, and 27.4 μg/ml, respectively. In vitro studies with metabolites of lansoprazole revealed that three metabolites were more potent inhibitors of testosterone synthesis than the parent drug, two of them being at least 10 times more potent. These metabolites are present in rats at substantial levels but are undetectable in humans. The lack of induction of Leydig cell tumors in mice, lower sensitivity of primate Leydig cells, and the absence of testosterone synthesis-inhibiting metabolites in man suggest that Leydig cell tumors found in rats represent a species-specific sensitivity and does not imply a risk for clinical use in man.

Phenotypic feminization in a genetic male dog caused by nonfunctional androgen receptors

A dog raised as a female pup did not have utero-ovarian structures when ovariohysterectomy was attempted at 6 mo of age. Three months later, the dog exhibited male-like behavior, and 2 symmetrical testicular-shaped structures arose bilateral to the vulva. Intersex cases such as this one may involve chromosomal abnormalities, defects in testicular secretions or androgen insensitivity in tissues of the sex accessories. Serum concentrations of testosterone and dihydrotestosterone were measured in the intersex and in intact normal male (control; n=7) dogs. In addition, 5α-reductase enzyme activity was determined in the gonadal tissue of the intersex dog and control dogs. Androgen receptors were studied in cultured fibroblasts from the genital skin of the intersex and control dogs. The cytogenetic studies identified the animal's karyotype to be 78 XY. All complete cells from the gonadal tissue showed an XY sex chromosome complement. Serum testosterone concentrations before and after challenge with hCG were not different between the intersex and control dogs (6.2 vs 5.72 ± 1.8 ng/ml and 9.5 vs 8.69 ± 2.39 ng/ml before and after hCG challenge, respectively). Similarly serum dihydrotestosterone concentrations were not different between the intersex and control dogs before and after challenge with hCG (158 vs 162 ± 1.4 pg/ml and 270 vs 278.71 ± 45.98 pg/ml before and after hCG challenge, respectively). The 5α-reductase enzyme activity of the gonads was not different between the intersex and control dogs (105 pmol/h/mg protein vs 110 ± 2.4 pmol/h/mg protein). The Bmax values of binding in the control dogs fibroblast strains were 25 ± 3.5 fmol/mg protein. However, specific binding of dihydrotestosterone was virtually undetectable in the fibroblasts cultured from the intersex dog. These results exclude chromosomal abnormalities and deficient secretion of testosterone and dihydrotestosterone as causative factors. Because fibroblasts cultured from genital skin lacked any ability to specifically bind dihydrotestosterone, it is suggested that nonfunctional androgen receptors in some tissues of the sex accessories contributed to the feminine phenotype of this particular dog.

Developmental Changes of Serum Thymosin α1 and β4 in Male and Male Castrated Pigs: Modulation by Testosterone and Human Chorionic Gonadotropin1

Thymic secretory peptides thymosin beta 4 and alpha 1 have possible endocrine roles in both immune and reproductive systems; thus, they should respond to endocrine feedback control mechanisms consistent with gonadal function. In an initial experiment, male pigs (boars; n = 90; 10/time) were bled at 1, 3, 6, 12, 18, 24, 30, 36, and 96 wk of age before and 24 h after hCG stimulation. Thymosin beta 4 concentrations were significantly depressed 24 h after hCG challenge. Testosterone concentrations increased with age up to 36 wk and were further increased with hCG stimulation (p less than 0.01). In a subsequent experiment, boars (n = 12) and barrows (males castrated shortly after birth; n = 12) were blood-sampled, administered hCG, and sampled again 24 h later at 1, 3, 6, 12, 18, and 24 wk of age. Barrows (n = 12) were administered testosterone with the same protocol. Testosterone concentrations increased in boars with maturity and were further increased from the hCG stimulation (p less than 0.01). Thymosin beta 4 concentrations decreased with age in boars and barrows (p less than 0.01), and hCG challenge depressed thymosin alpha 1 and beta 4 concentrations in boars and thymosin beta 4 in barrows (p less than 0.01). Testosterone treatment of barrows also depressed thymosin beta 4 and alpha 1 in barrows (p less than 0.01). The depression of thymosins by hCG treatment points to a role for gonadotropins in altering circulating thymosin concentrations independent of, but in conjunction with, the effect of gonadal steroids.

Induction of leydig cell adenomas by ammonium perfluorooctanoate: A possible endocrine-related mechanism

Ammonium perfluorooctanoate (C8) produced an increased incidence of Leydig cell adenomas in Crl:CD BR (CD) rats fed 300 ppm for 2 years. A hormonal (nongenotoxic) mechanism was examined since C8 was negative in short-term tests for genotoxicity. Adult male CD rats were gavaged with either 0, 1, 10, 25, or 50 mg/kg C8 for 14 days. In addition, a control group was pair-fed to the 50 mg/kg C8 group. A dose-dependent decrease in body and relative accessory sex organ (ASO) weights was seen, with the relative ASO weights of the 50 mg/kg group significantly less than those of the pair-fed control. Serum estradiol levels were elevated in the 10, 25, and 50 mg/kg C8-treated animals. Estradiol levels in the 50 mg/kg C8 group were 2.7-fold greater than those in the pair-fed control. The increase in serum estradiol levels occurred at the same dose levels as the increase in hepatic β-oxidation activity. A statistically significant downward trend with dose was seen in serum testosterone levels when compared with the ad libitum control. However, when the 50 mg/kg C8-treated rats were compared with their pair-fed control, no significant differences were seen. Challenge experiments, which can identify the presence and location of a lesion in an endocrine axis, were undertaken to clarify the significance of this downward trend in serum testosterone following C8 exposure. In the challenge experiments, adult CD rats were gavaged with either 0 or 50 mg/kg C8 for 14 days. One hour before termination, rats received either a human chorionic gonadotropin (hCG), gonadotropin-releasing hormone (GnRH), or naloxone challenge. Following hCG challenge, serum testosterone levels in the 50 mg/kg C8 were significantly decreased (50%) from those in the ad libitum controls. Similar decreases, although not significant, were seen in serum testosterone following GnRH and naloxone challenge. The challenge experiments suggest that the decrease in serum testosterone following C8 exposure is due to a lesion at the level of the testis. In addition, progesterone, 17α-hydroxyprogesterone, and androstenedione were examined in the 50 mg/kg C8-treated males following hCG challenge. A 60% decrease was observed in androstenedione levels in the C8-treated animals from those in the ad libitum controls; no other differences were seen. These data suggest that the decrease in serum testosterone following hCG challenge may be due to a decrease in the conversion of 17α-hydroxyprogesterone to androstenedione. The observed effects described above can be attributed to the elevated serum estradiol levels. It also has been shown that estrogens can induce Leydig cell tumors in mice. Hence, the elevated estradiol levels in C8-treated rats may be responsible for the decreased relative accessory sex organ weight and serum testosterone levels seen in this study as well as the increased incidence of Leydig cell adenomas in the 2-year feeding study with C8.

Use of hcg stimulation test in women immunized with β-hCG vaccine

Eleven normally menstruating women, who had earlier been immunized with NII β-hCG vaccine but had no detectable anti-hCG antibody titres, were selected as controls for the hCG challenge test using 1000 2000 I.U. The test was repeated in five of them after a booster immunization, which raised antibody titres to 18–450 ng/ml. Stimulation of serum progesterone secretion was used as an index of corpus luteum (CL) response to the I.V. hCG. In the control group, the progesterone (P) secretory response following hCG stimulus showed peak levels which were significantly higher than basal levels in all except 2 subjects. The non-responsiveness in 2 subjects cannot be easily explained but may be dose-related. No significant difference was noted between the two dose levels. Length of luteal phase was increased by 4–5 days in 6 out of 20 cycles studied. The results of this test in 5 women before and after the vaccine boosters were encouraging as peak P levels appeared higher than basal levels in controls, but not so in the immunized group. However, these results could not be confirmed statistically. Nonetheless, this study is suggestive that the antibodies generated by this vaccine were capable of intercepting the effect of exogenous hCG in the human female. Further studies with more subjects and higher dosage of hCG are called for.

Serum and testicular testosterone and androgen binding protein profiles following subchronic treatment with carbendazim

While the general toxicity of the benzimidazole pesticides for mammals is low, one of these compounds, carbendazim (MBC), causes degeneration of testicular tissue and decreases spermatogenic activity at doses well below the LD 50 value. A study conducted by S. D. Carter, R. A. Hess, and J. W. Laskey (1987, Biol. Reprod. 37, 709–717) showed that treatment with 400 mg/kg/day MBC resulted in severe seminiferous tubular atrophy and infertility. Since spermatogenesis is an androgen-dependent process, we characterized the effects of MBC (0–400 mg/kg/day) on the endocrine function of the rat testes. Following subchronic (85 day) exposure, serum hormones (TSH, LH, FSH, and Prl) were measured as were androgen binding protein (ABP) and testosterone in testicular fluids (interstitial fluid and seminiferous tubule fluid). In addition, the functional capacity of the Leydig cell to secrete testosterone was assessed in vitro following and hCG challenge. Subchronic treatment with MBC at doses of 50–100 mg/kg/day had no effect on pituitary or testicular hormone concentrations; 200 mg/kg/day elevated the testosterone concentration in the seminiferous tubule fluid and the ABP concentration in both the interstitial fluid and the seminiferous tubule fluid without affecting serum testosterone or ABP concentrations. The 400 mg/kg/day dose resulted in increased concentration of both testosterone and ABP in the interstitial fluid and seminiferous tubule fluid and elevated serum ABP, with no change in serum testosterone. This endocrine profile is consistent with the testicular atrophy and “Sertoli cell-only” syndrome seen in these animals as reported by Gray et al. (1987, Toxicologist 7, 717). We conclude that seminiferous tubule fluid testosterone may be a result of two factors: (1) increased interstitial fluid testosterone concentrations and (2) decreased testosterone outflow from the testis to the general circulation. Also, increased ABP in the interstitial fluid may reflect a change in the relative secretion of ABP into the interstitial fluid and the seminiferous tubules.