David R. Mann

Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis in adult male rats

We have examined the role of glucocorticoids in the stress-induced inhibition of testicular steroidogenesis. Immobilization (3 hr) reduced plasma testosterone (T) levels to 24% of control values but did not affect plasma LH levels. This reduction was partially reversed by in vivo injections of the antiglucocorticoid, RU486, prior to the stress session at a dose of 10 mg/kg BW, but not at 1.0 or 50 mg/kg BW. Stressed rats that were treated with 10 mg/kg BW RU486 had twofold higher plasma T levels than vehicle-treated stressed animals. Injections of RU486 did not affect plasma LH levels in control or stressed rats and did not affect T levels of unstressed rats. Stressed rats had eightfold higher plasma corticosterone levels than controls, and RU486 had no effect on control or stress levels of corticosterone. The possible role of glucocorticoids in mediating the effect of stress on testicular T production was investigated also in vitro by incubating testicular interstitial cells from unstressed rats for 3 hr with corticosterone (0, 0.01, 0.1, or 1.0 microM) or dexamethasone (0, 0.001, 0.01, or 0.1 microM), followed by an additional 2 hr with hCG (0, 25, 50, or 100 microIU). Both corticosterone and dexamethasone inhibited hCG-stimulated T production in a dose-dependent manner. Cells incubated with the highest concentration of either of the glucocorticoids showed significantly reduced responses to hCG stimulation. In the absence of hCG, in vitro T production was not affected by dexamethasone or 0.01 and 0.1 microM corticosterone. However, the highest dose of corticosterone (1.0 microM) produced a 63% elevation in basal T production. Coincubation of testicular interstitial cells with corticosterone (1.0 microM) or dexamethasone (0.1 microM) and RU486 (0.01, 0.1, and 1.0 microM) reversed the glucocorticoid-induced suppressions of T production in a dose-dependent manner. Our results suggest that during stress increases in plasma levels of glucocorticoids in male rats act via glucocorticoid receptors on testicular interstitial cells to suppress the testicular response to gonadotropins, and that the decline of testosterone production during immobilization stress is in part mediated by a direct action of glucocorticoids on the testis.

Leptin-Signaling Inhibition Results in Efficient Anti-Tumor Activity in Estrogen Receptor Positive or Negative Breast Cancer

IntroductionWe have shown previously that treatment with pegylated leptin peptide receptor antagonist 2 (PEG-LPrA2) reduced the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor type 2 (VEGFR2) and growth of 4T1-breast cancer (BC) in syngeneic mice. In this investigation, PEG-LPrA2 was used to evaluate whether the inhibition of leptin signaling has differential impact on the expression of pro-angiogenic and pro-proliferative molecules and growth of human estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) BC xenografts hosted by immunodeficient mice.MethodsTo test the contribution of leptin signaling to BC growth and expression of leptin-targeted molecules, PEG-LPrA2 treatment was applied to severe immunodeficient mice hosting established ER+ (MCF-7 cells; ovariectomized/supplemented with estradiol) and ER- (MDA-MB231 cells) BC xenografts. To further assess leptin and PEG-LPrA2 effects on ER+ and ER- BC, the expression of VEGF and VEGFR2 (protein and mRNA) was investigated in cell cultures.ResultsPEG-LPrA2 more effectively reduced the growth of ER+ (>40-fold) than ER- BC (twofold) and expression of pro-angiogenic (VEGF/VEGFR2, leptin/leptin receptor OB-R, and IL-1 receptor type I) and pro-proliferative molecules (proliferating cell nuclear antigen and cyclin D1) in ER+ than in ER- BC. Mouse tumor stroma in ER+ BC expressed high levels of VEGF and leptin that was induced by leptin signaling. Leptin upregulated the transcriptional expression of VEGF/VEGFR2 in MCF-7 and MDA-MB231 cells.ConclusionsThese results suggest that leptin signaling plays an important role in the growth of both ER+ and ER- BC that is associated with the leptin regulation of pro-angiogenic and pro-proliferative molecules. These data provide support for the potential use of leptin-signaling inhibition as a novel treatment for ER+ and ER- BC.

Timing of Prenatal Androgen Exposure: Anatomical and Endocrine Effects on Juvenile Male and Female Rhesus Monkeys

Prenatal androgen shapes genital differentiation. In humans, genital anatomy determines sex of rearing and subsequent behavioral development. Rhesus monkey genital anatomy and neuroendocrine function are sexually differentiated, and behavioral development occurs in a complex social environment. We investigated prenatal hormonal influences on sexual differentiation by suppressing or increasing androgens in male and female rhesus monkeys. Pregnant multiparous female rhesus monkeys received 35-40 days of testosterone enanthate (TE) treatment, androgen antagonist (flutamide, FL) treatment, or vehicle starting on gestation day (GD) 35 or 40 (early) or GD 110 or 115 (late). Exogenous androgen increased neonatal LH secretion in females when given early and altered female genital differentiation when administered either early or late. TE treatment, early or late in gestation, had no measurable effects on male genital differentiation or neuroendocrine function. Early FL treatment, however, radically altered male genital differentiation, producing in two cases males with a urethral opening separate from the glans. In females, early FL treatment produced detectable alterations in genitalia consistent with a reduced exposure to prenatal androgen, suggesting that female rhesus monkeys are naturally exposed prenatally to meaningful levels of T. Late FL treatment reduced male penis size and increased neonatal T secretion, but had no effect in females. This is the first study to block endogenous prenatal testosterone in rhesus monkeys, thereby altering sexual differentiation. These findings illustrate the complexity of prenatal influences on anatomical and neuroendocrine development. The relationship between the anatomical changes reported here and sex differences in behavior is currently under investigation.

Folliculogenesis Is Impaired and Granulosa Cell Apoptosis Is Increased in Leptin-Deficient Mice

Abstract Leptin purportedly plays an important role in pubertal development in a number of mammalian species. Adult leptin-deficient (ob/ob) female mice are infertile, but the mechanisms responsible for the reproductive failure have not been fully elucidated. The major objective of the current study was to assess the effects of a leptin deficiency on ovarian folliculogenesis and apoptosis. Beginning at 4 wk of age, control (n = 8) and ob/ob (n = 7) mice were weighed and examined daily for vaginal opening. After 3 wk the mice were killed, and the reproductive organs were weighed. Ovaries were paraffin-embedded for hematoxylin and eosin histology, TUNEL assay, and immunohistochemistry for Fas, Fas ligand (FasL), and proliferating cell nuclear antigen (PCNA). Vaginal opening was delayed, uteri were smaller, and the number of primordial follicles and total number of ovarian follicles were subnormal in ob/ob animals. Leptin-deficient animals also had a higher number of atretic follicles than controls. Granulosa cells (predominantly in preantral and early antral follicles) of ob/ob mice exhibited increased apoptotic activity as documented by TUNEL assay and elevated expression of the apoptotic markers Fas and FasL, compared with that in control animals. Ovarian expression of PCNA, a marker of DNA replication, repair, or both, did not differ between ob/ob and control mice. The data suggest that a leptin deficiency in mice is associated with impaired folliculogenesis, which results in increased follicular atresia. This impairment may be one of the causative components of infertility in leptin-deficient animals.

Effects of restraint stress on plasma LH and testosterone concentrations, Leydig cell LH/HCG receptors, and in vitro testicular steroidogenesis in adult rats

We examined the effect of restraint stress (3 hr) on plasma LH and testosterone levels, on the Leydig cell LH/hCG receptor, and on the activity of enzymes in the testicular steroidogenic pathway of the adult rat. Restraint stress caused a 47% reduction in plasma testosterone concentrations, but had no effect on plasma LH levels. The binding capacity and affinity of Leydig cell LH/hCG receptors were not affected by restraint. Stress did not affect the testicular activity of 20,22 desmolase or 3 beta-hydroxysteroid dehydrogenase, but testicular interstitial cells of stressed rats incubated in vitro with progesterone as a substrate produced more 17 alpha-hydroxyprogesterone but less testosterone than control cells, and when incubated with 17 alpha-hydroxypregnenolone, produced 39% less androstenedione and 40% less testosterone than control cells. These results suggest that restraint stress inhibited 17,20 desmolase but not 17 alpha-hydroxylase activity. When the delta 4 pathway was blocked with cyanoketone (3 beta-HSD inhibitor), stress did not alter the production of pregnenolone or 17 alpha-hydroxypregnenolone, but the production of dehydroepiandrosterone by cells from stressed rats was subnormal, suggesting again a reduction of 17,20 desmolase activity. The data suggest that a major site of the inhibitory action of restraint stress on testicular steroidogenesis is the 17,20 desmolase step. The disruption of androgen production by restraint appears to be LH independent since stress did not affect plasma LH levels, the binding capacity or affinity of LH/hCG receptors, or the activity of 20,22 desmolase.

Effect of Immobilization Stress on Plasma Luteinizing Hormone, Testosterone, and Corticosterone Concentrations and on 3β-Hydroxysteroid Dehydrogenase Activity in the Testes of Adult Rats

Abstract We have examined the effect of 3 hr of immobilization stress on plasma luteinizing hormone, testosterone, and corticosterone levels, and on the activity of 3β-hydroxysteroid dehydrogenase (3β-HSD) in microsomal and mitochondrial fractions of the testis from adult rats. Immobilization for 3 hr increased plasma corticosterone and reduced plasma testosterone concentrations by 57%. Plasma luteinizing hormone levels were lower, although not significantly (P = 0.093) so, in stressed animals. Immobilization (3 hr) reduced the V max values of 3β-HSD in the mitochondria and in the microsomal fraction of the testis by 40% and 34%, respectively, but had no effect on the Km values of 3β-HSD in the two cellular compartments. These results suggest that the inhibition of the activity of 3β-HSD may be partially responsible for the disruption of testicular steroidogenesis during immobilization stress.

A Longitudinal Study of Leptin During Development in the Male Rhesus Monkey: The Effect of Body Composition and Season on Circulating Leptin Levels

Abstract The objective of this study was to examine longitudinal changes in serum leptin concentrations during development and to correlate those changes with sexual development in male rhesus monkeys housed under natural environmental conditions. Blood samples were drawn from 8 control animals approximately every other month from 10 to 30 mo of age and thereafter monthly through 80 mo of age. Leptin levels declined through the juvenile period until the onset of puberty and were negatively correlated with body weight. Seven of the eight animals became sexually mature during the breeding season of their fourth year of life. Puberty was delayed in the other animal until the subsequent breeding season. There were no significant fluctuations in leptin levels prior to or in association with the pubertal rise in LH and testosterone (T) secretion. During the peripubertal period, levels of leptin varied between 2 and 3 ng/ml. The animal that exhibited delayed puberty had the lowest body weight and highest leptin levels during this period. With the achievement of sexual maturity, leptin levels varied seasonally, with peak levels in the late winter (Jan–Mar) and a nadir in the late summer (Aug–Sept). A late winter rise in leptin was also evident in most of the animals during Years 2 and 3, but not during Year 4. In the fall of Years 5 and 6, the seasonal rise in leptin concentrations lagged 3–4 mo behind the seasonal increase in LH and T. In the fall of Year 5, but not thereafter, leptin levels were positively related to percent body fat and negatively correlated with lean body mass. The data do not support the hypothesis that increasing leptin concentrations trigger the onset of puberty in the male rhesus monkey. During the juvenile period and after sexual maturation, but not during the peripubertal period, leptin secretion varied with season in the animals; but the environmental factors that cue or drive this rhythm remain to be determined.

Fenofibrate attenuates tubulointerstitial fibrosis and inflammation through suppression of nuclear factor-κB and transforming growth factor-β1/Smad3 in diabetic nephropathy.

Fibrates, the ligands of peroxisome proliferator-activated receptor-α, have been shown to have a renal protective action in diabetic models of renal disease, but the mechanisms underlying this effect are unknown. In the present study, we sought to investigate in greater detail the effect of fenofibrate and its mechanism of action on renal inflammation and tubulointerstitial fibrosis in an animal model of type 2 diabetes mellitus. Twelve-week-old non-diabetic Zucker lean (ZL) and Zucker diabetic fatty (ZD) rats were treated with vehicle or fenofibrate for 10 weeks. mRNA and protein analyses were performed by real-time polymerase chain reaction, Western blot and immunostaining. The diabetic condition of ZD rats was associated with an increase in collagen and α-smooth muscle actin accumulation in the kidney, which was significantly reduced by fenofibrate. Chronic treatment of ZD rats with fenofibrate attenuated renal inflammation and tubular injury as evidenced by a decrease in mRNA and protein expression of secreted phosphoprotein-1, monocyte chemotactic protein-1 and kidney injury molecule-1 in the kidneys. Renal interstitial macrophage infiltration was also significantly reduced in the kidneys of fenofibrate-treated diabetic animals. Moreover, renal nuclear factor (NF)-κB DNA-binding activity, transforming growth factor (TGF)-β1 and phospho-Smad3 proteins were significantly higher in ZD animals compared with ZL ones. This increase in NF-κB activity, TGF-β1 expression and Smad3 phosphorylation was greatly attenuated by fenofibrate in the diabetic kidneys. Taken together, fenofibrate suppressed NF-κB and TGF-β1/Smad3 signaling pathways and reduced renal inflammation and tubulointerstitial fibrosis in diabetic ZD animals.

Effects of Neonatal Testicular Suppression with a GnRH Antagonist on Social Behavior in Group-Living Juvenile Rhesus Monkeys

Twenty-four male and eight female 1-year-old rhesus monkeys (Macaca mulatta) were observed for social interaction with other yearlings and with their mothers. The males comprised three groups which differed in the level of neonatal androgen exposure. One group received the GnRH antagonist Antide during their first four neonatal months (Antide n = 8), which suppressed pituitary LH secretion resulting in peak neonatal T levels < 0.7 nmol/liter. A second group received Antide treatment combined with a long-lasting testosterone replacement (Ant/And n = 8), which resulted in peak neonatal T levels of 29.1 +/- 3.8 nmol/liter. The third group (Vehicle n = 8) received the Antide and androgen vehicles and had intermediate peak T levels of 5.2 +/- 1.0 nmol/liter. Behavior of males was compared to that of unmanipulated control females living in the same social group (Control Female n = 8) when androgen levels were uniformly low (< 0.7 nmol/liter) in all male groups. Subjects received 12 weekly 30-min focal observations by an observer blind to the neonatal treatments. Marked sex differences were found in several categories of sociosexual behavior. All three groups of males engaged in significantly more sexual and play behavior than females, with the exception of quiet solitary play, which females exhibited significantly more frequently In addition, females exhibited significantly more interest in infants than did any male group. There were no differences between groups in agonistic behavior or time spent in contact with other individuals, but females spent significantly more time than any male group in proximity to other animals. Both females and Antide males initiated proximity and followed animals significantly more frequently than Ant/And males, but not Vehicle males. Proximity durations with mothers initiated and terminated by yearlings were longer for females than for any male group and for Antide males than for Ant/and males. Antide males were groomed significantly longer than any other group. These results demonstrate effects of neonatal testosterone exposure on social behavior in yearling rhesus. Suppression of neonatal T did not affect sexually dimorphic patterns of play and sexual behavior, but altered the character of interactions with their mothers. Whether this reflects a delay in the development of maternal independence or a fundamental alteration in patterns of social interaction remains to be resolved.

Effect of restraint stress on gonadal proopiomelanocortin peptides and the pituitary-testicular axis in rats

We examined the effect of restraint on testicular interstitial fluid (TIF) concentrations of ACTH, beta-endorphin-lipotropin (beta-E-LI) and testosterone and correlated those changes with plasma concentrations of ACTH, beta-E-LI, corticosterone, LH and testosterone in adult rats. Animals were subjected to 1, 2, or 3 h of restraint and were killed immediately following the stress period. Plasma values of ACTH and beta-E-LI were elevated above control values after 1 and 2 h, but not after 3 h of restraint. Plasma corticosterone showed a similar response to restraint except that concentrations were also elevated after 3 h. Plasma testosterone concentrations were elevated after 1 h of restraint, but after 3 h of restraint had fallen below control values. Restraint reduced plasma testosterone concentrations without altering plasma LH concentrations. The decline in plasma testosterone during restraint was associated with a parallel decrease in testosterone in the TIF. Concentrations of ACTH and beta-E-LI were 6- and 3-fold greater in TIF than in the plasma. While 1 or 2 h of restraint did not affect ACTH and beta-E-LI in TIF, values of these hormones were elevated in rats exposed to 3 h of restraint. These data, coupled with recent reports that testicular proopiomelanocortin (POMC)-derived peptides may modulate testicular steroidogenesis, suggest that these factors may play an autocrine or paracrine role in mediating stressor-induced changes in testicular function.