TrkB-mediated neuroprotection in female hippocampal neurons is autonomous, estrogen receptor alpha-dependent, and eliminated by testosterone: a proposed model for sex differences in neonatal hippocampal neuronal injury.

BACKGROUND: Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of learning disabilities and memory deficits in children. In both human and animal studies, female neonate brains are less susceptible to HI than male brains. Phosphorylation of the nerve growth factor receptor TrkB has been shown to provide sex-specific neuroprotection following in vivo HI in female mice in an estrogen receptor alpha (ERα)-dependent manner. However, the molecular and cellular mechanisms conferring sex-specific neonatal neuroprotection remain incompletely understood. Here, we test whether female neonatal hippocampal neurons express autonomous neuroprotective properties and assess the ability of testosterone (T) to alter this phenotype. METHODS: We cultured sexed hippocampal neurons from ERα+/+ and ERα-/- mice and subjected them to 4 h oxygen glucose deprivation and 24 h reoxygenation (4-OGD/24-REOX). Sexed hippocampal neurons were treated either with vehicle control (VC) or the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following in vitro ischemia. End points at 24 h REOX were TrkB phosphorylation (p-TrkB) and neuronal survival assessed by immunohistochemistry. In addition, in vitro ischemia-mediated ERα gene expression in hippocampal neurons were investigated following testosterone (T) pre-treatment and TrkB antagonist therapy via q-RTPCR. Multifactorial analysis of variance was conducted to test for significant differences between experimental conditions. RESULTS: Under normoxic conditions, administration of 3 µM 7,8-DHF resulted an ERα-dependent increase in p-TrkB immunoexpression that was higher in female, as compared to male neurons. Following 4-OGD/24-REOX, p-TrkB expression increased 20% in both male and female ERα+/+ neurons. However, with 3 µM 7,8-DHF treatment p-TrkB expression increased further in female neurons by 2.81 ± 0.79-fold and was ERα dependent. 4-OGD/24-REOX resulted in a 56% increase in cell death, but only female cells were rescued with 3 µM 7,8-DHF, again in an ERα dependent manner. Following 4-OGD/3-REOX, ERα mRNA increased ~ 3 fold in female neurons. This increase was blocked with either the TrkB antagonist ANA-12 or pre-treatment with T. Pre-treatment with T also blocked the 7,8-DHF- dependent sex-specific neuronal survival in female neurons following 4-OGD/24-REOX. CONCLUSIONS: OGD/REOX results in sex-dependent TrkB phosphorylation in female neurons that increases further with 7,8-DHF treatment. TrkB phosphorylation by 7,8-DHF increased ERα mRNA expression and promoted cell survival preferentially in female hippocampal neurons. The sex-dependent neuroprotective actions of 7,8-DHF were blocked by either ANA-12 or by T pre-treatment. These results are consistent with a model for a female-specific neuroprotective pathway in hippocampal neurons in response to hypoxia. The pathway is activated by 7,8-DHF, mediated by TrkB phosphorylation, dependent on ERα and blocked by pre-exposure to T.

Penile erection and cardiovascular function: effects and pathophysiology.

Penile erection (PE) is a hemodynamic event that results from a neuroendocrine process, and it is influenced by the cardiovascular status of the patient. However, it may also modulate an individual's cardiovascular events. The present study provides the mechanisms involved in the association of PE and cardiovascular function. Erection upsurges the cardiac rate, blood pressure, and oxygen uptake. Sex-enhancing strategies, such as phosphodiesterase inhibitors, alprostadil, and testosterone also promote vasodilatation and cardiac performance, thus preventing myocardial infarction. More so, drugs that are used in the treatment of hypertensive heart diseases (such as angiotensin system inhibitors and ß-blockers) facilitate vasodilatation and PE. These associations have been linked with nitric oxide- and testosterone-dependent enhancing effects on the vascular endothelium. In addition, impaired cardiovascular function may negatively impact PE; therefore, impaired PE may be a pointer to cardiovascular pathology. Hence, evaluation of the cardiovascular status of an individual with erectile dysfunction (ED) is essential. Also, employing strategies that are used in maintaining optimal cardiac function may be useful in the management of ED.

Testosterone Supplementation Promotes Estrogen Synthesis of Buffalo Cumulus Cells Surrounding In Vitro-Matured Oocytes.

Cumulus cells (CCs) synthesize estrogens that are essential for follicular development. However, the effects of androgen on estrogen production in buffalo CCs remain unknown. In the present study, the impacts of testosterone on estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes were investigated. The results showed that testosterone supplementation improved both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 17ß-HSD) and the secretion levels of estradiol in buffalo CCs surrounding in vitro-matured oocytes. Furthermore, testosterone treatment enhanced the sensitivity of buffalo CCs surrounding in vitro-matured oocytes to follicle-stimulating hormone (FSH). This study indicated that testosterone supplementation promoted the estrogen synthesis of buffalo CCs surrounding in vitro-matured oocytes mainly through strengthening the responsiveness of CCs to FSH. The present study serves as a foundation of acquiring high-quality recipient oocytes for buffalo somatic cell nuclear transfer.

Central MOTS-c infusion affects reproductive hormones in obese and non-obese rats.

MOTS-c, a mitochondrial-derived peptide, acts as a systemic hormone and MOTS-c level is inversely correlated with markers of obesity. Obesity is a risk factor for male reproductive physiology and is expressed as an important cause of infertility. In this study, we aimed to determine the effects of MOTS-c, which has been proven in the hypothalamus and testicles, on the actors involved in the reproductive axis. In the study, 80 male Wistar-Albino rats were divided into two main groups, obese and non-obese (n = 40). Rats in the first main group were fed with fatty diet feed and obesity was induced. The second main group was fed with normal diet feed. Each main group was divided into 4 subgroups (Control, Sham, 10 and 100 µM MOTS-c). The lateral ventricles of the animals in the treatment groups were infused with 10 and 100 µM MOTS-c (solvent in Sham group) for 14 days. At the end of the experiment, hypothalamic Gonadotropin-Releasing Hormone (GnRH) gene expression level, serum testosterone, Luteinizing hormone (LH) and Follicle stimulating hormone (FSH) levels were determined. MOTS-c infusion caused an increase in GnRH mRNA, protein expression levels and serum testosterone, LH and FSH levels in obese and non-obese rats (p < 0.05). MOTS-c administration more significantly upregulated hormone levels in non-obese rats (p < 0.05). MOTS-c administration increases these hormones, suggesting that MOTS-c may stimulate the reproductive axis. Our results reveal that MOTS-c plays a role in the central regulation of reproduction, as well as causes increased LH, FSH and testosterone release.

Androgenic steroids induce pathologic scarring in a preclinical porcine model via dysfunctional extracellular matrix deposition.

Hypertrophic scarring is a major source of morbidity. Sex hormones are not classically considered modulators of scarring. However, based on increased frequency of hypertrophic scarring in patients on testosterone, we hypothesized that androgenic steroids induce abnormal scarring and developed a preclinical porcine model to explore these effects. Mini-swine underwent castration, received no testosterone (noT) or biweekly testosterone therapy (+T), and underwent excisional wounding. To create a delayed wound healing model, a subset of wounds were re-excised at 2 weeks. Scars from postoperative day 42 (POD42) and delayed wounds (POD28) were harvested 6 weeks after initial wounding for analysis via histology, bulk RNA-seq, and mechanical testing. Histologic analysis of scars from +T animals showed increased mean fibrosis area (16 mm2noT, 28 mm2+T; p = .007) and thickness (0.246 mm2noT, 0.406 mm2+T; p < .001) compared to noT. XX+T and XY+T scars had greater tensile burst strength (p = .024 and p = .013, respectively) compared to noT swine. Color deconvolution analysis revealed greater deposition of type I and type III collagen as well as increased collagen type I:III ratio in +T scars. Dermatopathologist histology scoring showed that +T exposure was associated with worse overall scarring (p < .05). Gene ontology analysis found that testosterone exposure was associated with upregulation of cellular metabolism and immune response gene sets, while testosterone upregulated pathways related to keratinization and laminin formation on pathway analysis. In conclusion, we developed a preclinical porcine model to study the effects of the sex hormone testosterone on scarring. Testosterone induces increased scar tissue deposition and appears to increase physical strength of scars via supraphysiologic deposition of collagen and other ECM factors. The increased burst strength seen in both XX and XY animals suggests that hormone administration has a strong influence on scar mechanical properties independent of chromosomal sex. Anti-androgen topical therapies may be a promising future area of research.

Cohabitation with receptive females under D2-type agonism in adulthood restores partner preference and brain dimorphism in the SDN-POA following neonatal gonadectomy in male rats.

Perinatal testosterone, or its metabolite estradiol, organize the brain toward a male phenotype. Male rodents with insufficient testosterone during this period fail to display sexual behavior and partner preference for receptive females in adulthood. However, cohabitation with non-reproductive conspecifics under the influence of a D2 agonist facilitates the expression of conditioned partner preference via Pavlovian learning in gonadally intact male rats. In the present experiment, three groups of neonatal PD1 males (N = 12/group) were either gonadectomized (GDX), sham-GDX, or left intact and evaluated for social preferences and sexual behaviors as adults. We then examined whether the effects of GDX could be reversed by conditioning the males via cohabitation with receptive females under the effects of the D2 agonist quinpirole (QNP) or saline, along with the size of some brain regions, such as the sexually dimorphic nucleus of the preoptic area (SDN-POA), suprachiasmatic nucleus (SCN), posterior dorsal medial amygdala (MeApd) and ventromedial hypothalamus (VMH). Results indicated that neonatal GDX resulted in the elimination of male-typical sexual behavior, an increase in same-sex social preference, and a reduction of the area of the SDN-POA. However, GDX-QNP males that underwent exposure to receptive females in adulthood increased their social preference for females and recovered the size in the SDN-POA. Although neonatal GDX impairs sexual behavior and disrupts partner preference and brain dimorphism in adult male rats, Pavlovian conditioning under enhanced D2 agonism ameliorates the effects on social preference and restores brain dimorphism in the SDN-POA without testosterone.

Neonatal inhibition of androgen activity alters the programming of body weight and orexinergic peptides differentially in male and female rats.

The involvement of androgens in the regulation of energy metabolism has been demonstrated. The main objective of the present research was to study the involvement of androgens in both the programming of energy metabolism and the regulatory peptides associated with feeding. For this purpose, androgen receptors and the main metabolic pathways of testosterone were inhibited during the first five days of postnatal life in male and female Wistar rats. Pups received a daily s.c. injection from the day of birth, postnatal day (P) 1, to P5 of Flutamide (a competitive inhibitor of androgen receptors), Letrozole (an aromatase inhibitor), Finasteride (a 5-alpha-reductase inhibitor) or vehicle. Body weight, food intake and fat pads were measured. Moreover, hypothalamic Agouti-related peptide (AgRP), neuropeptide Y (NPY), orexin, and proopiomelanocortin (POMC) were analyzed by quantitative real-time polymerase chain reaction assay. The inhibition of androgenic activity during the first five days of life produced a significant decrease in body weight in females at P90 but did not affect this parameter in males. Moreover, the inhibition of aromatase decreased hypothalamic AgRP mRNA levels in males while the inhibition of 5α-reductase decreased hypothalamic AgRP and orexin mRNA levels in female rats. Finally, food intake and visceral fat, but not subcutaneous fat, were affected in both males and females depending on which testosterone metabolic pathway was inhibited. Our results highlight the differential involvement of androgens in the programming of energy metabolism as well as the AgRP and orexin systems during development in male and female rats.

Long noncoding RNA XIST inhibition promotes Leydig cell apoptosis by acting as a competing endogenous RNA for microRNA-145a-5p that targets SIRT1 in late-onset hypogonadism.

Leydig cell (LCs) apoptosis is responsible for decreased serum testosterone levels during late-onset hypogonadism (LOH). Our study was designed to illustrate the regulatory effect of lncRNA XIST on LCs and to clarify its molecular mechanism of action in LOH. The Leydig cells (TM3) was treated by 300 µM H2O2 for 8 h to establish Leydig cell oxidative stress model in vitro. The expression levels of lncRNA XIST in the testicular tissues of patients with LOH were measured using fluorescence in situ hybridization (FISH). The interaction between lncRNA XIST/SIRT1 and miR-145a-5p was assessed using starBase and dual-luciferase reporter gene assays. Apoptotic cells and Caspase3 activity were determined by flow cytometry (FCM) assay. Testosterone concentration was determined by ELISA. Moreover, histological assessment of testicles in mice was performed by using HE staining and the TUNEL assay was used to determine apoptosis. We found that the lncRNA XIST was downregulated in the testicular tissues of LOH patients and mice and in H2O2-induced TM3 cells. XIST siRNA significantly promoted apoptosis, enhanced Caspase3 activity and reduced testosterone levels in H2O2-stimulated TM3 cells. Further studies showed that the miR-145a-5p inhibitor reversed the effect of XIST-siRNA on H2O2-induced Leydig cell apoptosis. MiR-145a-5p negatively regulated SIRT1 expression, and SIRT1-siRNA reversed the effects of the miR-145a-5p inhibitor on H2O2 stimulated TM3 cells. The in vivo experiments indicated that silencing of the lncRNA XIST aggravated LOH symptoms in mice. Inhibition of lncRNA XIST induces Leydig cell apoptosis through the miR-145a-5p/SIRT1 axis in the progression of LOH.

Ursonic acid attenuates spermatogenesis in oligozoospermia mice through inhibiting ferroptosis.

Ursonic acid (UNA) is a natural pentacyclic triterpene found in some medicinal plants and foods. The reproductive protective effect of UNA was evaluated in a mouse model of oligozoospermia induced by busulfan (BUS) at 30 mg/kg b.w.. The mice were initially divided into groups with UNA concentrations of 10, 30, 50, 100 mg/kg. Subsequently, based on sperm parameters, the optimal concentration of 50 mg/kg was identified. As a control, an additional group was supplemented with ursolic acid at a concentration of 50 mg/kg. The results indicated that BUS caused the loss of spermatogenic cells in testis, the decrease of sperm in epididymis, the disorder of testicular cytoskeleton, the decrease of serum sex hormones such as testosterone which induced an increase in feedback of androgen receptor and other testosterone-related proteins, the increase of malondialdehyde and reactive oxygen species levels and the increase of ferroptosis in testis while UNA successfully reversed these injuries. High-throughput sequencing revealed that UNA administration significantly upregulated the expression of genes associated with spermatogenesis, such as Tnp1, Tnp2, Prm1, among others. These proteins are crucial in the histone to protamine transition during sperm chromatin remodeling. Network pharmacology analysis reveals a close association between UNA and proteins related to the transformation of histones to protamine. Molecular docking studies reveal that UNA can interact with the ferroptosis-inhibiting gene SLC7A11, thereby modulating ferroptosis. Taken together, UNA alleviated BUS-induced oligozoospermia by regulating hormone secretion, mitigating oxidative stress and promoting recovery of spermatogenesis by inhibiting the ferroptosis.

Investigating the Effects of Sex Hormones on Macrophage Polarization.

Sex differences in the development and progression of cardiovascular disease are well established, but the effects of sex hormones on macrophage polarization and pro-atherogenic functions are not well described. We hypothesize that sex hormones directly modulate macrophage polarization, and thereby regulate the progression of atherosclerosis. Bone marrow-derived monocytes from adult male and female C57BL/6 mice were differentiated into macrophages using macrophage colony-stimulating factor (20 ng/mL) and pre-treated with either 17ß-estradiol (100 nM), testosterone (100 nM), or a vehicle control for 24 h. Macrophages were polarized into pro- or anti-inflammatory phenotypes and the effects of sex hormone supplementation on the gene expression of macrophage phenotypic markers were assessed using RT-qPCR. Inflammatory markers, including IL-1ß, were quantified using an addressable laser bead immunoassay. A transwell migration assay was used to determine changes in macrophage migration. Sex differences were observed in macrophage polarization, inflammatory responses, and migration. Pre-treatment with 17ß-estradiol significantly impaired the gene expression of inflammatory markers and the production of IL-1ß in inflammatory macrophages. In anti-inflammatory macrophages, 17ß-estradiol significantly upregulated the expression of anti-inflammatory markers and enhanced migration. Pre-treatment with testosterone enhanced anti-inflammatory mRNA expression and impaired the production of IL-1ß. Our observations suggest a protective role of 17ß-estradiol in atherogenesis that may contribute to the sexual dimorphisms in cardiovascular disease observed in human patients.

Testosterone administration decreases sensitivity to angry facial expressions in healthy males: A computational modeling approach.

Previous research indicates that higher testosterone levels are related to increased aggressive and dominant behaviors, particularly in males. One possible mechanism for these hormone-behavior associations could involve threat perception. However, the causal influence of testosterone on men's recognition of threatening facial expressions remains unknown. Here, we tested the causal effect of exogenous testosterone on men's sensitivity to facial threat by combining a psychophysical task with computational modeling. We administered a single dose (150 mg) of testosterone or placebo gel to healthy young men (n = 120) in a double-blind, placebo-controlled, between-participant design. Participants were presented with morphed emotional faces mixing anger/fear and neutral expressions and made judgments about the emotional expression. Across typical regression analysis, signal detection analysis, and drift diffusion modeling, our results consistently showed that individuals who received testosterone (versus placebo) exhibited a lower perceived sensitivity to angry facial expressions. But we observed no significant effects of testosterone administration on fearful facial expressions. The findings indicate that testosterone attenuates sensitivity to facial threat, especially angry facial expressions, which could lead to a misestimation of others' dominance and an increase in one's own aggressive and dominant behaviors.

Hormonal basis of sex differences in anesthetic sensitivity.

General anesthesia-a pharmacologically induced reversible state of unconsciousness-enables millions of life-saving procedures. Anesthetics induce unconsciousness in part by impinging upon sexually dimorphic and hormonally sensitive hypothalamic circuits regulating sleep and wakefulness. Thus, we hypothesized that anesthetic sensitivity should be sex-dependent and modulated by sex hormones. Using distinct behavioral measures, we show that at identical brain anesthetic concentrations, female mice are more resistant to volatile anesthetics than males. Anesthetic sensitivity is bidirectionally modulated by testosterone. Castration increases anesthetic resistance. Conversely, testosterone administration acutely increases anesthetic sensitivity. Conversion of testosterone to estradiol by aromatase is partially responsible for this effect. In contrast, oophorectomy has no effect. To identify the neuronal circuits underlying sex differences, we performed whole brain c-Fos activity mapping under anesthesia in male and female mice. Consistent with a key role of the hypothalamus, we found fewer active neurons in the ventral hypothalamic sleep-promoting regions in females than in males. In humans, we demonstrate that females regain consciousness and recover cognition faster than males after identical anesthetic exposures. Remarkably, while behavioral and neurocognitive measures in mice and humans point to increased anesthetic resistance in females, cortical activity fails to show sex differences under anesthesia in either species. Cumulatively, we demonstrate that sex differences in anesthetic sensitivity are evolutionarily conserved and not reflected in conventional electroencephalographic-based measures of anesthetic depth. This covert resistance to anesthesia may explain the higher incidence of unintended awareness under general anesthesia in females.

Testosterone Treatment and Fractures in Men with Hypogonadism.

BACKGROUND: Testosterone treatment in men with hypogonadism improves bone density and quality, but trials with a sufficiently large sample and a sufficiently long duration to determine the effect of testosterone on the incidence of fractures are needed. METHODS: In a subtrial of a double-blind, randomized, placebo-controlled trial that assessed the cardiovascular safety of testosterone treatment in middle-aged and older men with hypogonadism, we examined the risk of clinical fracture in a time-to-event analysis. Eligible men were 45 to 80 years of age with preexisting, or high risk of, cardiovascular disease; one or more symptoms of hypogonadism; and two morning testosterone concentrations of less than 300 ng per deciliter (10.4 nmol per liter), in fasting plasma samples obtained at least 48 hours apart. Participants were randomly assigned to apply a testosterone or placebo gel daily. At every visit, participants were asked if they had had a fracture since the previous visit. If they had, medical records were obtained and adjudicated. RESULTS: The full-analysis population included 5204 participants (2601 in the testosterone group and 2603 in the placebo group). After a median follow-up of 3.19 years, a clinical fracture had occurred in 91 participants (3.50%) in the testosterone group and 64 participants (2.46%) in the placebo group (hazard ratio, 1.43; 95% confidence interval, 1.04 to 1.97). The fracture incidence also appeared to be higher in the testosterone group for all other fracture end points. CONCLUSIONS: Among middle-aged and older men with hypogonadism, testosterone treatment did not result in a lower incidence of clinical fracture than placebo. The fracture incidence was numerically higher among men who received testosterone than among those who received placebo. (Funded by AbbVie and others; TRAVERSE ClinicalTrials.gov number, NCT03518034.).

Testosterone modulates vasodilation in mesenteric arteries of hypertensive rats.

AIMS: To evaluate the effects of testosterone on endothelium-dependent vasodilation and oxidative stress in mesenteric resistance arteries. MAIN METHODS: Spontaneously hypertensive rats (SHR), aged 8 to 10 weeks, were divided into four groups: intact (SHAM), intact treated with testosterone (TTO; 3 mg/kg/day) via subcutaneous route (s.c.), intact treated with testosterone and anastrozole [aromatase enzyme inhibitor (TTO + ANA; 0.1 mg/kg/day, s.c.)] and intact treated with testosterone and finasteride [5 α-reductase enzyme inhibitor (TTO + FIN; 5 mg/kg/day, s.c.)] for four weeks. Concentration-response curves to acetylcholine (ACh, 0.1 nmol/L - 10 µmol/L) were obtained in mesenteric resistance arteries previously contracted with phenylephrine (PE, 3 µmol/L), before and after the use of selective inhibitors. Reactive oxygen species (ROS) levels were assessed in the vessels and the endothelium analyzed by scanning electron microscopy. KEY FINDINGS: TTO group showed a lower participation of nitric oxide (NO), increased oxidative stress, and participation of prostanoids and endothelium-dependent hyperpolarization (EDH), possibly to maintain the vasodilator response. Lower participation of NO and prostanoids, combined to an increased participation of EDH, were observed in the TTO + ANA group, in addition to higher levels of ROS and altered endothelial morphology. The vasodilation to ACh was impaired in TTO + FIN, along increased participation of NO, reduction of prostanoids, and greater EDH-dependent vasodilation. SIGNIFICANCE: Testosterone contributes to endothelial vasodilation by enhancing EDH through an increased participation of epoxyeicosatrienoic acids. While the decrease in NO appears to involve the participation of dihydrotestosterone, 17 ß-estradiol seems to stimulate the action of the NO pathway and prostanoids.

Prolonged testosterone 17ß-cyclopentylpropionate exposition induces behavioral, ovarian, oviductal, uterine and reproductive disturbances in female mice.

Anabolic-androgenic steroids (AAS) abuse is often associated with metabolic disorders and infertility. However, the current evidence on AAS-induced reproductive toxicity is mainly based on male studies. Thus, AAS repercussions on female reproductive capacity remain poorly understood, despite scarce evidence that fertility determinants may be more severely impaired in females than males exposed to these drugs. Accordingly, this study used an integrated framework to investigate the impact of different testosterone 17ß-cyclopentylpropionate (TC) doses on pain sensitivity, aggressiveness, anxiety, sexual behavior, ovarian, oviductal, uterine and reproductive morphofunctional and molecular outcomes. These parameters were used to explore the reproductive capacity in female mice exposed to this synthetic testosterone ester. The animals were untreated or intraperitoneally treated with 5, 10 and 20 mg/kg TC every 48 h for 12 weeks. Our findings indicated that testosterone was upregulated while the hormones luteinizing, follicle-stimulating, estrogen and progesterone were down-regulated by TC. This AAS also exerted deleterious effects on anxiety, aggressivity, nociception, exploratory and sexual behavior in female mice. Concurrently, TC attenuated ovarian follicle maturation, interrupted the estrous cycle, induced oviductal and uterine hypotrophy. Estrous cyclicity was reestablished 60 days after AAS treatment. However, TC-treated mice still exhibited impaired reproductive capacity, a disturbance potentially related to deficiency in folliculogenesis, sex hormones production, and endometrial receptivity mediate by ER-α, PR, HOXA-10 and LIF down-regulation. Taken together, our findings indicated that in addition to female behavior, reproductive organs microstructure and function are markedly impaired by TC in a dose-dependent manner, whose time-dependent reversibility remains to be clarified.

Up to the maximum-testosterone dose-dependent effects on anabolic and androgen responsive tissues in orchiectomized rats.

BACKGROUND: Despite the high number of synthetic androgenic-anabolic steroids, testosterone is still misused for doping in amateur and professional sports. However, only few studies investigated the dose-response effects of testosterone beyond its physiological concentrations and in over 90 years of research, no saturation dosage has been experimentally described for exogenous testosterone administration. OBJECTIVES: We want to elucidate the physiological and pathophysiological effects of supra-physiological testosterone application and close this gap in testosterone dose-response data. MATERIALS AND METHODS: Male orchiectomized rats were treated with different testosterone doses ranging from 0.1 to 50 mg/kg body weight for 3 weeks. Several physiological endpoints (e.g., body weight, organ and muscle weight, muscle strength, muscle fiber size) were examined during and after the termination of the treatment with an adjusted Hershberger assay, open-field-test, and (immuno-)histologic. RESULTS: The wet weights of androgen responsive organs (penis, prostate, seminal vesicle) showed a significant increase in a dose-dependent manner. Histological evaluation of the prostate showed a significant higher percentage of KI67 positive prostate nuclei in the highest dosage group and an increasing hyperplasia with increasing testosterone administered. A significant anabolic effect was only observed in Levator ani wet weight, and to minor degree for the cardiac muscle. Regarding other skeletal muscles (Musculus soleus and Musculus gastrognemicus), no significant testosterone effects were observed. We showed a significant increasing dosage-response effect for testosterone in androgen responsive organs with saturation at the two highest concentration of 10 and 50 mg/kg body weight. DISCUSSION AND CONCLUSION: The dose-dependent androgenic effects of testosterone were well observable and the anabolic effects on muscle tissue were visible although to a lesser degree, without the support of aerobic exercise and a protein rich diet. Future studies should investigate a combinatorial effect of testosterone and training. Nevertheless, with the chosen range of applied testosterone, we showed a saturation of testosterone effects in prostate, seminal vesicle, penis, and Levator ani.

Estrogen and testosterone supplementation improves tendon healing and functional recovery after rotator cuff repair.

Failure of healing after rotator cuff repair (RCR) is common. The purpose of the current study was to evaluate the effect of systemic estrogen or testosterone supplementation on tendon healing after RCR. Seventy-two adult male mice were utilized for all experiments. The supraspinatus tendon was transected and repaired with 6-0 Prolene suture on the left shoulder of 51 animals. Mice were segregated into three groups postoperative: (1) vehicle group (VG; n = 18), (2) estrogen group (EST; n = 17), and (3) testosterone group (TST; n = 16). An unrepaired control group (unrepaired, n = 21) did not have surgery. Utilizing these animals, histological analysis, activity testing, biomechanical testing and RNA sequencing (RNA-seq) was performed. At 8 weeks post-RCR, TST, and EST supplementation improved the overall histologic structure of the repaired enthesis site. No differences in ultimate failure loads or stiffness were detected between VG, EST, and TST groups after biomechanical testing. RCR caused a reduction in wheel activity compared to unrepaired controls and supplementation with TST restored wheel activity. RNA-seq analysis indicated that estrogen and testosterone regulated different pathways associated with enthesis healing, including a suppression of inflammatory signaling. Supplementation with sex hormones improved the structure of the repaired tendon enthesis and significantly regulated expression of diverse pathways regulating multiple biological processes. Testosterone administration following RCR restored wheel activity without having a detrimental impact on biomechanical strength. Future human studies of sex hormone supplementation after RCR are warranted as supplementation in an animal model may improve tendon enthesis healing.

Utility of vitamin C in ameliorating citalopram-induced testicular toxicity via modulating nitro-oxidative stress and apoptosis in mice.

There is a growing concern that antidepressant drugs impair sexual function and adversely impact spermatogenesis and male fertility. Vitamin C is a natural antioxidant that plays a vital role in the male reproductive system. The present study investigated the ameliorating potential of vitamin C against citalopram (CTL)-evoked testicular toxicity and spermatogenesis impairment in mice. Mice were randomly divided into six groups: control, CTL, vitamin C 100, vitamin C 200, CTL plus vitamin C 100, and CTL plus vitamin C 200. Adult male mice were intraperitoneally (ip) injected with 10 mg/kg of CTL for 35 days with or without vitamin C. At the end of the study, body and testes weight, sperm parameters, histopathology of testes, testosterone level, testicular levels of malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (TAC), and apoptosis (TUNEL assay) were evaluated. Our findings revealed that vitamin C restored spermatogenesis by improving sperm count, motility, viability, morphology, and chromatin integrity. Testosterone levels and testes histopathology were significantly improved in the vitamin C-administrated groups. Furthermore, vitamin C administration markedly alleviated CTL-induced nitro-oxidative damage, enhancing TAC levels, and reducing NO and MDA levels. Whilst CTL therapy induced a significant increase in the number of TUNEL-positive cells compared to the control, the administration of vitamin C significantly prevented the apoptotic effects of CTL. Together, vitamin C therapy protects against CTL-induced testicular damage via mitigating nitro-oxidative stress and apoptosis, which provides evidence for vitamin C as a beneficial therapy against antidepressant drug-associated reproductive toxicity and male sub/infertility.

Effects of testosterone enanthate on aggression, risk-taking, competition, mood, and other cognitive domains during 28 days of severe energy deprivation.

RATIONALE: Behavioral effects of testosterone depend on dose, acute versus sustained formulation, duration of administration, personality, genetics, and endogenous levels of testosterone. There are also considerable differences between effects of endogenous and exogenous testosterone. OBJECTIVES: This study was the secondary behavioral arm of a registered clinical trial designed to determine if testosterone protects against loss of lean body mass and lower-body muscle function induced by a severe energy deficit typical of sustained military operations. METHODS: Behavioral effects of repeated doses of testosterone on healthy young men whose testosterone was reduced by severe energy deficit were examined. This was a double-blind, placebo-controlled, between-group study. Effects of four weekly intramuscular injections of testosterone enanthate (200 mg/week, N = 24) or matching placebo (N = 26) were evaluated. Determination of sample size was based on changes in lean body mass. Tasks assessing aggression, risk-taking, competition, social cognition, vigilance, memory, executive function, and mood were repeatedly administered. RESULTS: During a period of artificially induced, low testosterone levels, consistent behavioral effects of administration of exogenous testosterone were not observed. CONCLUSIONS: Exogeneous testosterone enanthate (200 mg/week) during severe energy restriction did not reliably alter the measures of cognition. Study limitations include the relatively small sample size compared to many studies of acute testosterone administration. The findings are specific to healthy males experiencing severe energy deficit and should not be generalized to effects of other doses, formulations, or acute administration of endogenous testosterone or studies conducted with larger samples using tests of cognitive function designed to detect specific effects of testosterone.

Cholesterol Hydroperoxide Co-trafficking in Testosterone-generating Leydig Cells: GPx4 Inhibition of Cytotoxic and Anti-steroidogenic Effects.

Trafficking of intracellular cholesterol (Ch) to and into mitochondria of steroidogenic cells is required for steroid hormone biosynthesis. This trafficking is typically mediated by one or more proteins of the steroidogenic acute regulatory (StAR) family. Our previous studies revealed that 7-OOH, a redox-active cholesterol hydroperoxide, could be co-trafficked with Ch to/into mitochondria of MA-10 Leydig cells, thereby inducing membrane lipid peroxidation (LPO) which impaired progesterone biosynthesis. These negative effects of 7-OOH were inhibited by endogenous selenoperoxidase GPx4, indicating that this enzyme could protect against 7-OOH-induced oxidative damage/dysfunction. In the present study, we advanced our Leydig focus to cultured murine TM3 cells and then to primary cells from rat testis, both of which produce testosterone. Using a fluorescent probe, we found that extensive free radical-mediated LPO occurred in mitochondria of stimulated primary Leydig cells during treatment with liposomal Ch+7-OOH, resulting in a significant decline in testosterone output relative to that with Ch alone. Strong enhancement of LPO and testosterone shortfall by RSL3 (a GPx4 inhibitor) and reversal thereof by Ebselen (a GPx4 mimetic), suggested that endogenous GPx4 was playing a key antioxidant role. 7-OOH in increasing doses was also cytotoxic to these cells, RSL3 exacerbating this in Ebselen-reversable fashion. Moreover, GPx4 knockdown increased cell sensitivity to LPO with reduced testosterone output. These findings, particularly with primary Leydigs (which best represent cells in intact testis) suggest that GPx4 plays a key protective role against peroxidative damage/dysfunction induced by 7-OOH co-trafficking with Ch.