Increased Testosterone Production Research Articles

Testosterone: More Is Not Always Better

Having conducted population-based studies on the relationship of sex steroid levels to bone mass in Rochester, Minnesota for many years, I have been struck by just how often male members of the medical staff at our institution who participate in these studies inquire about their testosterone level. Discovering that they happen to be in the top 10th percentile is often a cause for celebration; conversely, being told that they happen to be “average” or, most distressingly, “below average,” generally leads to at least temporary anguish. Not surprisingly, despite the numerous publications from our group and others regarding the key role for estrogen in bone metabolism in men (summarized in Ref. 1), none of my colleagues inquire or seem to care one way or another about their estradiol level. At least based on this anecdotal experience, it seems that even in the minds of well-informed male medical professionals, more testosterone is clearly “better,” whereas estrogen is largely irrelevant. It is in this context that men with modest testosterone levels should perhaps have their chance to celebrate based on the results of the study by Burnett-Bowie et al. (2) published in this issue of JCEM. This carefully conducted clinical trial clearly demonstrates that at least for bone, more testosterone is not necessarily better, especially if it comes at the price of lower estrogen production. The trial is based on the premise that because male aging is associated with declining testosterone levels and with increased body fat, decreased muscle mass, strength, and bone mineral density (BMD) (1), replacing testosterone in elderly men should be beneficial. However, rather than administering exogenous testosterone, which requires topical or parenteral dosing and significantly increases estrogen levels (via peripheral aromatization) and which may, in turn, result in side effects such as gynecomastia, the authors chose to test the effects of increasing endogenous testosterone production by blocking aromatization of testosterone to estradiol using a potent orally administered aromatase inhibitor, anastrozole (2). By removing negative feedback signals, the anastrozole-induced decrease in estradiol increases GnRH and gonadotropin production at the hypothalamus and pituitary, respectively, thereby increasing testosterone production (3). The Burnett-Bowie et al. (2) paper reports on 69 men, aged 60 and older, who completed the 1-yr, double-blind, randomized, placebo-controlled trial. Anastrozole had the desired effects on serum testosterone levels, with mean values increasing from 319 ng/dl (the lower end of the normal range) at baseline to 524 ng/dl at 3 months and a slight decline to 474 ng/dl at 1 yr, an overall increase of approximately 50%. As expected, estradiol levels decreased (by 20%), from 15 to 12 pg/ml at 3 months, and remained stable at this level thereafter. However, despite the substantial increase in serum testosterone levels, posterior-anterior (PA) spine BMD decreased by 1.7% in the anastrozole group while increasing by 0.8% in the placebo group (P 0.0014 for the difference between groups), with similar trends for differences between the groups for femoral neck, total hip, and total body BMD. The inescapable conclusion from these data is that the 50% increase in testosterone levels was trumped by the much more modest 20% reduction in estradiol levels, leading to negative skeletal effects of anastrozole therapy in aging men. To some extent, these findings are not surprising and are perhaps predictable, based on the now extensive body of evidence for a critical role for estrogen in regulating the male skeleton (1). Although previous studies in men had shown that serum estradiol levels were related to bone density (4) and that estrogen regulated bone turnover (5,

Effects of hypoxia on testosterone release in rat Leydig cells

The aim of this study was to explore the effect and action mechanisms of intermittent hypoxia on the production of testosterone both in vivo and in vitro. Male rats were housed in a hypoxic chamber (12% O(2) + 88% N(2), 1.5 l/ml) 8 h/day for 4 days. Normoxic rats were used as control. In an in vivo experiment, hypoxic and normoxic rats were euthanized and the blood samples collected. In the in vitro experiment, the enzymatically dispersed rat Leydig cells were prepared and challenged with forskolin (an adenylyl cyclase activator, 10(-4) M), 8-Br-cAMP (a membrane-permeable analog of cAMP, 10(-4) M), hCG (0.05 IU), the precursors of the biosynthesis testosterone, including 25-OH-C (10(-5) M), pregnenolone (10(-7) M), progesterone (10(-7) M), 17-OH-progesterone (10(-7) M), and androstendione (10(-7)-10(-5) M), nifedipine (L-type Ca(2+) channel blocker, 10(-6)-10(-4) M), nimodipine (L-type Ca(2+) channel blocker, 10(-5) M), tetrandrine (L-type Ca(2+) channel blocker, 10(-5) M), and NAADP (calcium-signaling messenger causing release of calcium from intracellular stores, 10(-6)-10(-4) M). The concentrations of testosterone in plasma and medium were measured by radioimmunoassay. The level of plasma testosterone in hypoxic rats was higher than that in normoxic rats. Enhanced testosterone production was observed in rat Leydig cells treated with hCG, 8-Br-cAMP, or forskolin in both normoxic and hypoxic conditions. Intermittent hypoxia resulted in a further increase of testosterone production in response to the testosterone precursors. The activity of 17β-hydroxysteroid dehydrogenase was stimulated by the treatment of intermittent hypoxia in vitro. The intermittent hypoxia-induced higher production of testosterone was accompanied with the influx of calcium via L-type calcium channel and the increase of intracellular calcium via the mechanism of calcium mobilization. These results suggested that the intermittent hypoxia stimulated the secretion of testosterone at least in part via stimulatory actions on the activities of adenylyl cyclase, cAMP, L-type calcium channel, and steroidogenic enzymes.

Unraveling Androgen Action in Muscle: Genetic Tools Probing Cellular Mechanisms

For the largest tissue in the body and with functions relevant to virtually every medical discipline (although central to none), muscle physiology is surprisingly neglected in medical research. Despite profound discoveries in subcellular muscle structure and function winning Nobel prize recognition, understanding the physiological regulation of muscle as a tissue remains a mostly unexplored territory with few reliable landmarks. Perhaps in a world in which research remains most strongly propelled by discipline-based quests for knowledge, muscle as an orphan lacking a disciplinary home with the necessity and priority to drive deeper discovery has, accordingly, been relatively overlooked. Among the most salient biological clues to regulation of muscle function is the gender dimorphism in muscle mass and strength appearing after puberty and attributable to dramatic differences in endogenous androgen exposure. Considering the nearly 30-fold increase in testosterone production experienced across male puberty, men’s larger and stronger muscles testify to marked androgen dependence, providing an opportunity for pharmaceutical exploitation over the 7 decades since testosterone started being used clinically. Yet exploitation for therapeutic benefit has progressed slowly, due in part to the pharmaceutical industry’s failed, and then abandoned, quest for a pure anabolic (i.e. nonandrogenic) steroid in the postwar decades, the golden age of steroid pharmacology. By contrast, unscrupulous illicit androgen abuse has flourished on the fact of increased muscle mass and strength, which sufficed without requiring explanation (1). Only since decades of denial that exogenous androgens enhanced muscular mass and strength in eugonadal men (2) was overturned by the studies of Bhasin et al. (3) in the mid-1990s have androgens been clearly acknowledged as having the most potent known anabolic and ergogenic effects on skeletal muscle, a fact reflected by androgens continuing to be the most widely abused class of drugs used in sports doping as well as the wider community for nonmedical purposes (4). Rigorous, placebocontrolled studies show testosterone has dose-response effects on muscle mass (fiber hypertrophy) and strength from below to well above the physiological range in both young (5) and older (6) men. Furthermore, high-quality clinical studies show androgens increase muscle mass in various chronic diseases such as HIV wasting (7), chronic obstructive pulmonary disease (8), chronic renal disease (9), and burns (10) and possibly the frailty, falls, and fractures that diminish quality of life during ageing (11). Yet despite proven increases in muscle mass in a few small studies, androgens are not in routine clinical use for these indications because of both limited evidence of sufficient functional benefits and risks of adverse off-target effects on cardiovascular disease and the liver (for 17-alkylated androgens) as well as prostate disease in men and virilization or hormone-dependent cancers in women. Further pharmaceutical development coupled with well-controlled clinical studies still appears to have great, untapped potential for androgens as adjunctive therapy for ageing and chronic disease states. By modern biochemical definition, androgens act by binding to the androgen receptor (AR), a ligand-dependent nuclear transcription factor (12). Yet androgen action is complex, involving prereceptor enzymatic activation and postreceptor coregulator mechanisms (13) as well as cell and tissue specificity within target organs. Although androgens are classically considered reproductive hormones, AR is expressed almost ubiquitously (14) including in nonreproductive target tissues like skeletal muscle. Prereceptor activation of androgen action involves either amplification of testosterone to the more potent androgen dihydrotestosterone (15) or diversification by aromatization to estradiol with effects mediated via the estrogen receptor (ER)and (16). An additional mechanism of androgen action, involving nongenomic actions without apparently requiring DNA binding, remains ill defined and of unknown physiological importance (17). The pharmaceutical goal of separating androgenic (reproductive system, somatic virilization) from the anabolic (enhancing muscle and bone growth and function) actions of androgens has been revived after invention of the first nonsteroidal andro-

Differential effects of insulin sensitivity on androgens in obese women with polycystic ovary syndrome or normal ovulation

The differential effects of insulin sensitivity and adiposity on androgen concentrations in women with polycystic ovary syndrome (PCOS) are unclear. To address this issue, we divided 43 overweight women into 4 groups based on both their clinical classification (PCOS or normal) and whether they were insulin resistant (IR) or insulin sensitive (IS) by their steady-state plasma glucose concentrations. Total testosterone concentrations were significantly increased as a function of both clinical classification (PCOS vs normal, P < .0001) and steady-state plasma glucose concentration (IR vs IS, P = .002). Mean testosterone concentrations were higher in PCOS-IR compared with PCOS-IS, normal-IR, or normal-IS women ( P < .005). In addition, there was a statistically significant interaction ( P = .03) between clinical classification (PCOS vs normal) and insulin sensitivity (IR vs IS) for testosterone concentrations. In contrast, androstenedione concentrations were higher in women with PCOS ( P = .001), irrespective of whether they were IR or IS ( P = .31); and no interaction between clinical classification and insulin sensitivity was discerned ( P = .34). These results indicate that both PCOS and insulin resistance independently contributed to increased total testosterone concentrations within a group of overweight/obese women. These findings are consistent with the hypothesis that the ovaries of women with PCOS are hypersensitive to the ability of insulin to increase testosterone production and that the more insulin resistant the patient, the higher the testosterone concentration. In contrast, androstenedione concentrations seem to be independent of differences in insulin resistance. Our findings emphasize the need to increase understanding of the factors that modulate ovarian androgen secretion.

Cytoprotective effects ofMorinda officinalisagainst hydrogen peroxide-induced oxidative stress in Leydig TM3 cells

To investigate the antioxidant effects of Morinda officinalis (Morindae radix, MR) on H(2)O(2)-induced oxidative stress in cultured mouse TM3 Leydig cells. We carried out 2,2-diphenyl-1-picrylhydrazyl free radical scavenging, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lipid peroxidation, testosterone enzyme immunoassay, superoxide dismutase (SOD), and catalase (CAT) assays in Leydig TM3 cells. MR showed a 47.8% 2,2-diphenyl-1-picrylhydrazyl radical scavenging effect in TM3 cells with no significant cytotoxicity. Oxidative stress was induced in TM3 cells with 100 micromol H(2)O(2), and treatment of the cells with 250 microg/mL MR showed the most significant protective effect (64%, P < 0.001) in the cell viability assay with a decreased lipid peroxidation level (1.75 nmol/mg protein, P < 0.05), increased testosterone production (43.5 pg/mL), and improvements in SOD activity (7.49 units of SOD/mg protein, P < 0.001) and CAT activity (74.6 units of CAT/mg protein, P < 0.001). These findings indicate that MR, as an antioxidant, protects functions of cultured mouse TM3 Leydig cells from H(2)O(2)-induced oxidative stress.

Effect of Glutathione Depletion on Leydig Cell Steroidogenesis in Young and Old Brown Norway Rats

Changes in the oxidant/antioxidant environment of aging Leydig cells have been shown to be correlated with the reduced ability of these cells to produce testosterone. With this in mind, we hypothesized that the experimental depletion of glutathione (GSH), an abundant Leydig cell intracellular antioxidant, might result in reduced testosterone production. Incubation of Leydig cells isolated from the testes of adult Brown Norway rats with buthionine sulfoximine (BSO) reduced GSH content by more than 70% and testosterone production by about 40%. The antioxidants vitamin E, N-tert-butyl-alpha-phenylnitrone and Trolox countered BSO's effect on steroidogenesis but not on GSH depletion. Together, BSO and glutathione ethyl ester maintained intracellular GSH and also testosterone production, whereas 1,2-dithiole-3-thione, which increases intracellular GSH, increased testosterone production. In vivo studies also were conducted. Young (4 month old) and old (24 month old) rats were injected with BSO twice a day for 7 d, after which Leydig cells were isolated and analyzed in vitro. BSO treatment reduced Leydig cell GSH content by 70% and the ability of the Leydig cells to produce testosterone by more than 50%. As with aging, decreases were seen in LH-stimulated cAMP production, steroidogenic acute regulatory protein, cholesterol side-chain cleavage, 3beta-hydroxysteroid dehydrogenase, and 17alpha-hydroxylase/17,20-lyase. The results of these studies, taken together, are consistent with the hypothesis that alteration in the oxidant/antioxidant environment may play a significant, causative role in the age-related reduced ability of Leydig cells to produce testosterone.

Study of endogenous profile of hCG in male Brazilian athletes

In the world of performance enhancing drugs, human Chorionic Gonadotrophin (hCG) is increasingly used alone and also in combination with various Anabolic Androgenic Steroid cycles. In males, hCG mimics luteinizing hormone (LH) and helps increase testosterone production in the testes. As such, hCG is commonly used during and after steroid cycles to maintain and restore testicular size as well as endogenous testosterone production. Doping control for hCG requires special attention since a difference must be made between the endogenous and exogenous origin of both substances. In the absence of a clearer pattern of what would be an expected level of normality, the doping control laboratories have been applying cut off values between 5 and 25 mUI/mL. Because of the large differences in assay design and specificity for different forms of hCG, the concentrations measured are highly dependent on the assay used and population analyzed. The aim of the study is the establishment of a cut off value for the characterization of exogenous misuse by athletes obtained with the usage of Microparticles Enzyme Immunoassays.

A Prostaglandin D2 system in the human testis

As shown recently, cyclooxygenase 2 (COX2), the inducible key enzyme for the prostaglandin (PG) biosynthetic pathway, is abundantly present in interstitial cells of testes of men suffering from different forms of impaired spermatogenesis and sub- or infertility, but it is absent in human testes with normal spermatogenesis. Although the spectrum of the downstream products of COX2 action in testis, namely PGs, and their effects are not known, our results show that Prostaglandin D2 (PGD2) likely plays a role. We describe (a) PGD2 synthetases, as well as receptors for PGD2 (DP) in testicular interstitial cells of men suffering from spermatogenic damage and infertility, and report that (b) PGD2 is produced by and can affect Leydig cells of an animal model, which expresses testicular COX2 and DP.

Cyclooxygenases in Rat Leydig Cells: Effects of Luteinizing Hormone and Aging

Previous studies suggested that increased Leydig cell cyclooxygenase (COX)2 expression may be involved in the reduced testosterone production that characterizes aged Leydig cells. Our objective herein was to further elucidate the relationships among LH stimulation, Leydig cell COX2 and COX1 expression, aging, and testosterone production. Incubation of Leydig cells from young or aged rats with LH or dibutyryl cAMP resulted in increases in both intracellular COX2 protein expression and testosterone production. COX1 expression did not respond to LH or dibutyryl cAMP. Incubation of adult cells with a protein kinase A inhibitor suppressed the stimulatory effects of LH on COX2 and testosterone production. Short-term incubation of Leydig cells with TGF-alpha or IL-1beta also increased COX2 protein levels; IGF-I had no effect. In vivo, LH also was found to stimulate both COX2 and testosterone, but not COX1. As reported previously, COX2 expression was greater in old than in young cells, and old Leydig cells responded to inhibition of COX2 in vitro with increased testosterone production. However, the effects of the COX2 inhibitors were not restricted to old cells; young Leydig cells also responded to COX2 inhibition with increased testosterone production. This and the observation that the incubation of young or old cells with LH resulted in increased COX2 and testosterone production in both cases suggests that the relationship between COX2 and testosterone production is not unique to aged Leydig cells. Moreover, the close correlation between increases in COX2 and testosterone in LH-stimulated young and aged Leydig cells is difficult to reconcile with the contention that the increased expression of COX2 in aged cells is responsible for age-related suppression of Leydig cell testosterone production.

Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH)

To study structure–activity relationships and the role of equine gonadotropins in the normal and pathophysiology of equine reproduction, the availability of purified hormones is essential. Previous expression studies in transfected CHO cells showed inefficient assembly of the human and bovine α and β subunits, resulting in low levels of recombinant LH. The ability to express a single chain bearing genetically linked α and β subunits bypasses this rate-limiting assembly step. A chimera was constructed by overlap PCR in which the carboxy terminal end of the eLHβ subunit was genetically fused to the amino end of the α subunit. This gene was transfected into CHO cells and the recombinant product was purified through multiple steps, including a Fractogel resin separation. Serial dilutions of pituitary derived native eLH and the single chain reLH were compared in an eLH radioimmunoassay (RIA); the concentration curves between the single chain recombinant eLH and the native eLH standard were parallel. The biological activity of the analog was determined in vitro and in vivo using homologous equine models. Testicular tissue from five colts was processed for Leydig cell cultures. Increasing doses of reLH were incubated with equine Leydig cells for 24 h in vitro and testosterone production was determined by RIA. Recombinant eLH stimulated a greater than 15-fold increase in testosterone production in a dose-dependent manner. Quarter Horse breeding stallions were treated with either reLH ( n = 5) or saline ( n = 3) and plasma testosterone concentrations were measured by RIA. Recombinant eLH stimulated a four-fold increase in circulating testosterone concentrations compared to the saline control. Therefore, the single chain recombinant will be effective for a variety of structure–function analyses and for breeding management in the horse.

Correlated responses in growth, carcass, and meat quality traits to divergent selection for testosterone production in pigs1

The objective of this project was to characterize changes in growth, carcass yield, and meat quality traits in castrates and gilts in response to divergent selection for testosterone production. In generation 21, endogenous testosterone concentrations in Duroc boars of the high (HTL) and low (LTL) testosterone lines averaged 49.0 and 27.8 ng/mL (P < 0.01), respectively. Eight LTL and 10 HTL boars were used to sire 29 LTL and 33 HTL litters. To remove the effects of inbreeding, these same boars were mated to females of a Large White x Landrace composite (WC) to generate 11 WC by LTL litters (WLT) and 23 WC by HTL litters (WHT). Castrates and gilts were then allotted to LTL (n = 53), HTL (n = 61), WLT (n = 102), and WHT (n = 101) for testing. Growth and carcass traits analyzed included days to 114 kg (D114), ADG, backfat adjusted to 114 kg (ABF), LM area adjusted to 114 kg and predicted percent lean (PPL). Fat-O-Meater data collected were adjusted fat depth (AFD), adjusted loin depth, and percent lean. Meat quality traits characterized at 24 h postmortem included marbling score, percent lipid, pH, drip loss, color score, and Minolta L*, a*, and b*. Data were analyzed with a mixed model including fixed effects of line, mating type (purebred or crossbred), sex, and the random effect of sire nested within line. All possible interactions among fixed effects were tested. The HTL had fewer D114 (P < 0.05), greater ADG (P < 0.01), greater ABF (P < 0.01), and lower PPL (P < 0.01) than LTL. The WHT and WLT did not differ for D114, ADG, or ABF. The WHT had smaller LM area adjusted to 114 kg (P < 0.05) and greater drip loss (P < 0.05) than WLT. The WLT had lower adjusted loin depth (P < 0.05) than LTL and HTL. The LTL and HTL had greater subjective scores for marbling (P < 0.05) compared with WLT and WHT. The least squares mean for percent lipid for HTL and LTL was 4.00. The WHT had greater means for L*, a*, and b* (P < 0.05) than WLT. Pigs selected for increased testosterone production grew faster and produced fatter carcasses than pigs selected for decreased testosterone. Changes in growth, carcass yield, and meat quality traits were detected in castrates and gilts in response to divergent selection for testosterone production.

Gonadotropin regulation of testosterone production by primary cultured theca and granulosa cells of Atlantic croaker: I. Novel role of CaMKs and interactions between calcium- and adenylyl cyclase-dependent pathways

Theca and granulosa cells for in vitro primary culture were obtained by enzymatic digestion of mature ovarian tissue from Atlantic croaker ( Micropogonias undulatus) and separation from the other cell types by Percoll density-gradient centrifugation. Histochemical staining and treatment with pregnenolone confirmed the presence in the cultured cells of enzymes involved in synthesizing the major sex steroids in croaker ovaries: testosterone, estradiol, and 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S). Croaker theca and granulosa cells maintained their steroidogenic response to gonadotropin when cultured with serum-supplemented media and produced high levels of testosterone for up to 5 days, although estradiol production was low. Multiple signal transduction pathways mediating gonadotropin stimulation of androgen production were identified in Atlantic croaker ovarian theca and granulosa cells in primary co-culture. Inhibitors of voltage-sensitive calcium channels (VSCCs) and calmodulin decreased the steroidogenic response to gonadotropin, whereas activators of adenylyl cyclase and protein kinase A (PKA) increased testosterone production, indicating that both calcium and PKA-dependent signaling pathways are involved in the regulation of follicular steroid production. In addition, the first evidence in vertebrates for an involvement of calcium/calmodulin-dependent protein kinases (CaMKs) in gonadal steroidogenesis was obtained, since the stimulatory effects of gonadotropin on testosterone media accumulation were attenuated by specific inhibitors of CaMKs. Some interactions among the signaling pathways were observed as demonstrated by the positive effect of elevated intracellular calcium on adenylyl cyclase activity and the reduction of forskolin- and dbcAMP-induced testosterone production by inhibitors of VSCCs, calmodulin, and CaMKs.

Effects of extracts from Hibiscus macranthus and Basella alba mixture on testosterone production in vitro in adult rat testes slices

To find an in vitro system for the measurement of the androgenic effects of different extracts of Hibiscus macranthus (Malvaceae) and Basella alba (Basellaceae). The production of testosterone from testes slices incubated in two media, either Krebs-Henseleit buffer containing 0.5% Bovine serum albumin (BSA) or Dubecco's Modified Eagle's medium-F12 Ham nutrient mixture (DME/Ham F12), under a mixture of 5% CO2 in 95% air was determined either in the presence or absence of cofactors and Hibiscus macranthus plus Basella alba (HMBA) extracts. The testosterone production was increased in testes slices incubated in DME/Ham F12 medium in response to the cofactors (49%) and aqueous extracts (34%-60% according to dilutions). Under the same atmospheric conditions, there was no positive response of the testes slices to either cofactor or HMBA extract stimulation in Krebs-Henseleit buffer containing 0.5% BSA. In further investigations related to the effect of HMBA, the DME/Ham F12 medium was used. The results obtained from the in vitro test showed that the activity was present mainly in methylene chloride and methanol, since these extracts induced an increase in testosterone production by testes slices. The testes slice system is suitable to be used for further in vitro investigations of the isolation of androgenic bioactive components of plants.

Effect of aromatase inhibition on bone metabolism in elderly hypogonadal men

Both estrogens and androgens play important roles in skeletal development and maintenance in men. The relative importance of estrogens and androgens in male bone metabolism, however, remains undefined. Anastrozole is an oral aromatase inhibitor that decreases estrogen production and increases androgen production in men. Currently, anastrozole is being investigated as a potential agent for the treatment of hypogonadism in aging men. Because anastrozole lowers estrogen levels and raises androgen levels, its effect on bone metabolism is difficult to predict. To assess the effects of anastrozole on bone turnover, we randomized 37 elderly (ages 62-74) mildly hypogonadal men (serum testosterone <350 ng/dl) to receive either anastrozole 1 mg daily (n=12), anastrozole 1 mg twice weekly (n=11), or daily placebo (n=14) for 12 weeks. Serum gonadal steroid levels, serum and urine biochemical markers of bone turnover, serum osteoprotegerin, and total body bone mineral density were measured at baseline and week 12. Mean serum levels of total and bioavailable testosterone increased substantially in both treated groups. Specifically, mean +/- SD bioavailable testosterone levels increased from 99+/-31 ng/dl to 207+/-65 ng/dl in the group receiving 1 mg of anastrozole daily and from 115+/-37 ng/dl to 178+/-55 ng/dl in the subjects receiving 1 mg of anastrozole twice weekly ( p <0.001 vs placebo for both groups). Serum estradiol levels decreased modestly in both treated groups (from 26+/-8 pg/ml to 17+/-6 pg/ml in the daily treatment group and from 27+/-8 pg/ml to 17+/-5 pg/ml in the twice-weekly treatment group, p <0.001 vs placebo for both groups). Despite these hormonal changes, no increases in biochemical markers of bone resorption were observed. Specifically, mean serum N-telopeptide and urinary deoxypyridinoline concentrations remained stable in both treated groups over the 12-week treatment period. Similarly, serum biochemical markers of bone formation (osteocalcin and amino-terminal propeptide of type 1 collagen), serum osteoprotegerin, and total body bone mineral density did not change. These data demonstrate that although short-term administration of anastrozole decreases serum estradiol levels in elderly men with mild hypogonadism, this intervention does not adversely affect bone metabolism over a 12-week period. This lack of an effect may be due to the concomitant increase in testosterone production, the relative modest effect on estradiol production, or a combination of both factors. These results suggest that anastrozole therapy is unlikely to have an adverse effect on bone metabolism when taken over extended periods and may prove to be a valuable method of normalizing testosterone production in older men.

Evaluation of the role of FSH in regulation of Leydig cell function during different stages of its differentiation

The role of follicle stimulating hormone (FSH) in Leydig cell function was evaluated by a passive neutralization approach at different stages of Leydig cell development. Neutralization of endogenous FSH in neonatal rats (10-day-old) resulted in reduction of testes weight, however the testicular testosterone levels and in vitro testosterone production by purified Leydig cells were elevated. Administration of FSH antiserum to immature (25–28-day-old) and adult (90-day-old) rats did not have any effect on testes weight, serum testosterone and testicular testosterone. Interestingly, there was a significant reduction in testosterone production by isolated Leydig cells under hCG stimulated and 22-R-hydroxycholesterol (22-R-OH CHOL) saturated conditions. In support of this observation administration of recombinant FSH to immature and adult rats resulted in significant increase in testosterone production by Leydig cells following incubations in presence of hCG and saturating concentrations of 22-R-OH CHOL, although there was no change in serum and testicular testosterone levels. The role of FSH in immature rats was also confirmed employing FSH receptor antiserum which was raised against the unique domains of FSH receptor. RT-PCR analysis revealed a significant reduction in the mRNA levels of StAR and IGF-1 following blockade of FSH action by FSH receptor antiserum. The results of our studies suggest a stage specific function for FSH in regulation of Leydig cell development by modulating the LH responsiveness and steroidogenesis.

Ovarian steroid cell tumor and a contralateral ovarian thecoma in a postmenopausal woman with severe hyperandrogenism

A 49-year-old woman presented with rapidly progressing hirsutism, receding hairline, male-pattern baldness and deepening of voice, which had developed over the past 2 years. Hormonal evaluation showed amarkedly elevated serum testosterone level (418 ng/dl) and no evidence of increased production of cortisol, dehydroepiandrosterone, dehydroepiandrosterone-sulfate, androstenedione, or 17-hydroxyprogesterone.Transvaginal ultrasound examination suggested the presence of a small mass within the left ovary, but all other radiological studies, including adrenal and ovarian computed tomography, magnetic resonanceimaging, radio-labelled cholesterol scintigraphy and positron emission tomography, were negative. Subsequently, bilateral selective venous sampling showed a marked testosterone gradient in the right ovarianvein. Bilateral salpingo-oophorectomy was performed (the patient had had a previous vaginal hysterectomy), and histopathological examination revealed a 10-mm steroid cell tumor within the right ovary anda 15-mm thecal cell tumor within the left ovary. The postoperative serum testosterone level returned to normal and the patient showed a slow regression of clinical symptoms. The simultaneous occurrenceof a virilizing ovarian steroid cell tumor and an apparently non-functioning thecoma within the contralateral ovary emphasizes the potential pitfalls that may exist in the preoperative evaluation of patientswith markedly increased testosterone production.

The Biochemical Basis for Increased Testosterone Production in Theca Cells Propagated from Patients with Polycystic Ovary Syndrome

The Biochemical Basis for Increased Testosterone Production in Theca Cells Propagated from Patients with Polycystic Ovary Syndrome

The biochemical basis for increased testosterone production in theca cells propagated from patients with polycystic ovary syndrome.

Ovarian theca cells propagated from patients with polycystic ovary syndrome (PCOS) convert steroid precursors into T more efficiently than normal theca cells. To identify the basis for increased T production by PCOS theca cells, we examined type I-V 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) isoform expression in long-term cultures of theca and granulosa cells isolated from normal and PCOS ovaries. RT-PCR analysis demonstrated that theca cells express type V 17 beta HSD a member of the aldo-keto reductase (AKR) superfamily (17 beta HSDV, AKR1C3), whereas expression of type I, II, and IV 17 beta HSD, which are members of the short-chain dehydrogenase/reductase superfamily, was limited to granulosa cells. Type III 17 beta HSD, the testicular isoform, was not detected in either granulosa or theca cells. Northern and real-time PCR analyses demonstrated that 17 beta HSDV transcripts were not significantly increased in PCOS theca cells compared with normal theca cells. RT-PCR analysis revealed that theca cells also express another AKR, 20 alpha HSD (AKR1C1). Both basal and forskolin-stimulated 20 alpha HSD mRNA levels were increased in PCOS theca cells compared with normal theca cells. However, 17 beta HSD enzyme activity per theca cell was not significantly increased in PCOS, suggesting that neither AKR1C3 nor AKR1C1 contributes to the formation of T in this condition. In contrast, 17 alpha-hydroxylase/C17,20 lyase and 3 beta HSD enzyme activities were elevated in PCOS theca cells, driving increased production of T precursors. These findings indicate that 1) increased T production in PCOS theca cells does not result from dysregulation of "androgenic" 17 beta HSD activity or altered expression of AKRs that may express 17 beta HSD activity; and 2) increased synthesis of T precursors is the primary factor driving enhanced T secretion in PCOS.

Association of selenoprotein P with testosterone production in cultured Leydig cells.

Selenoprotein P, a plasma selenoprotein, is thought to act as an antioxidant in the testis, similar to glutathione peroxidase. mRNA encoding selenoprotein P was selectively expressed by Leydig cells, suggesting participation in testosterone production. On the other hand, testosterone production has been linked to O2 toxicity in cultured Leydig cells. The authors, therefore, examined changes in selenoprotein P mRNA expression and testosterone production following stimulation by a stable analog cyclic adenosine 3',5'-monophosphate (cAMP) in cultured Leydig cells (MLTC-1 cells) under normal O2 concentrations. Selenoprotein P mRNA was analyzed by Northern blotting, while testosterone concentration in culture medium was measured by radioimmunoassay. When cAMP was added to cultures at 0, 0.01, 0.1, or 1 mM, selenoprotein P mRNA expression showed dose-dependent stimulation. cAMP was added at 0.1 mM to cultures, and the selenoprotein P mRNA expression and testosterone concentration were evaluated after incubation times of 2, 5, 9, 15, or 24 h. Selenoprotein P mRNA expression was maximal at 9 h. Testosterone concentration in the medium also increased, becoming maximal at 15 h. Selenoprotein P induced in Leydig cells following cAMP stimulation may counteract O2 toxicity from cAMP-mediated increases in testosterone production.

Rat Leydig cells use apolipoprotein E depleted high density lipoprotein to regulate testosterone production.

Rat HDL are known to increase testosterone production by cultured Leydig cells either following gonadotropin stimulation or cholesteryl ester depletion. However, rat HDL contain apolipoprotein E and have a high affinity for the members of the low density receptor family such as LDL receptor, LDL receptor related protein and VLDL receptor. In contrast with the adrenal cells, the contribution of apo A-I and apo E pathways in HDL cholesterol uptake has not been yet evidenced in rat Leydig cells. Recent data provided evidence that hCG stimulates scavenger receptor BI expression in testes. In order to investigate if testosterone production can be stimulated by apo E depleted HDL, we compared the level of testosterone stimulation by HDL with or without apo E first, in presence of saturating dose of hCG (1 IU/ml) and second, after depletion of cholesterol synthesis by pravastatin, an inhibitor of HMG-CoA reductase. In presence of hCG, HDL with or without apo E increased testosterone production respectively by 37 and 25%. Pravastatin at 100 microg/ml inhibited the cholesterol synthesis and the testosterone production by 25% and decreased the cholesteryl content by 25%. The addition of HDL with or without apo E (50 microg protein HDL/ ml) completely overcame the depletion of cellular cholesteryl esters and the inhibition of testosterone production induced by pravastatin. In the presence of heparin, apo E depleted HDL overcame the testosterone production induced by pravastatin, indicating that uptake of HDL without apo E via a secretion of apo E by the cells themselves was not involved. Therefore, in absence of apo E, it is suggested that rat Leydig cells used HDL to regulate steroidogenesis via an apolipoprotein A-I pathway.