The dissection of a despotic society: exploration, dominance and hormonal traits.

Naked mole-rats (Heterocephalus glaber) live in large colonies with one breeding female (queen), one to three breeding males (BMs) and the remainder are non-reproductive subordinates. The animals have a linear dominance rank with the breeders at the top of the hierarchy. We investigated how dominance rank in naked mole-rats differs with exploration (the propensity to explore a novel environment) and related endocrine markers. Exploration behaviour, faecal progestagen metabolite (fPM), faecal glucocorticoid metabolite (fGCM), faecal androgen metabolite (fAM) and plasma prolactin concentrations were quantified in breeding, high-, middle- and low-ranked females and males from five naked mole-rat colonies. There were no significant differences between the dominance rank and exploration behaviour. Interestingly, the queens and high-ranking females had higher fGCM and fAM concentrations compared with middle- and low-ranked females. The queens had significantly higher fPM concentrations than all other ranked females, since they are responsible for procreation. In the males, the BMs had higher fGCM concentrations compared with high- and low-ranked males. In addition, BMs and middle-ranking males had overall higher prolactin levels than all other ranked males, which could be linked to cooperative care. Overall, the results suggest that physiological reproductive suppression is linked to high dominance rank.

Hyperandrogenic environment regulates the function of ovarian granulosa cells by modulating macrophage polarization in PCOS.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder characterized by oligo-anovulation, hyperandrogenism, and polycystic ovaries, with hyperandrogenism being the most prominent feature of PCOS patients. However, whether excessive androgens also exist in the ovarian microenvironment of patients with PCOS, and their modulatory role on ovarian immune homeostasis and ovarian function, is not clear. METHODS: Follicular fluid samples from patients participating in their first in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment were collected. Androgen concentration of follicular fluid was assayed by chemiluminescence, and the macrophage M1:M2 ratio was detected by flow cytometry. In an in vitro model, we examined the regulatory effects of different concentrations of androgen on macrophage differentiation and glucose metabolism levels using qRT-PCR, Simple Western and multi-factor flow cytometry assay. In a co-culture model, we assessed the effect of a hyperandrogenic environment in the presence or absence of macrophages on the function of granulosa cells using qRT-PCR, Simple Western, EdU assay, cell cycle assay, and multi-factor flow cytometry assay. RESULTS: The results showed that a significantly higher androgen level and M1:M2 ratio in the follicular fluid of PCOS patients with hyperandrogenism. The hyperandrogenic environment promoted the expression of pro-inflammatory and glycolysis-related molecules and inhibited the expression of anti-inflammatory and oxidative phosphorylation-related molecules in macrophages. In the presence of macrophages, a hyperandrogenic environment significantly downregulated the function of granulosa cells. CONCLUSION: There is a hyperandrogenic microenvironment in the ovary of PCOS patients with hyperandrogenism. Hyperandrogenic microenvironment can promote the activation of ovarian macrophages to M1, which may be associated with the reprogramming of macrophage glucose metabolism. The increased secretion of pro-inflammatory cytokines by macrophages in the hyperandrogenic microenvironment would impair the normal function of granulosa cells and interfere with normal ovarian follicle growth and development.

Androgen synthesis cell-specific CREBZF deficiency alters adrenal cortex steroid secretion and develops behavioral abnormalities in adult male mice.

The global challenge of male infertility is escalating, notably due to the decreased testosterone (T) synthesis in testicular Leydig cells under stress, underscoring the critical need for a more profound understanding of its regulatory mechanisms. CREBZF, a novel basic region-leucine zipper transcription factor, regulates testosterone synthesis in mouse Leydig cells in vitro; however, further validation through in vivo experiments is essential. Our study utilized Cyp17a1-Cre to knock out CREBZF in androgen-synthesis cells and explored the physiological roles of CREBZF in fertility, steroid hormone synthesis, and behaviors in adult male mice. Conditional knockout (cKO) CREBZF did not affect fertility and serum testosterone level in male mice. Primary Leydig cells isolated from CREBZF-cKO mice showed impaired testosterone secretion and decreased mRNA levels of Star, Cyp17a1, and Hsd3b1. Loss of CREBZF resulted in thickening of the adrenal cortex, especially X-zone, with elevated serum corticosterone and dehydroepiandrosterone levels and decreased serum dehydroepiandrosterone sulfate levels. Immunohistochemical staining revealed increased expression of StAR, Cyp11a1, and 17ß-Hsd3 in the adrenal cortex of CREBZF-cKO mice, while the expression of AR was significantly reduced. Along with the histological changes and abnormal steroid levels in the adrenal gland, CREBZF-cKO mice showed higher anxiety-like behavior and impaired memory in the elevated plus maze and Barnes maze, respectively. In summary, CREBZF is dispensable for fertility, and CREBZF deficiency in Leydig cells promotes adrenal function in adult male mice. These results shed light on the requirement of CREBZF for fertility, adrenal steroid synthesis, and stress response in adult male mice, and contribute to understanding the crosstalk between testes and adrenal glands.

Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

Sexual dimorphism in skin immunity is mediated by an androgen-ILC2-dendritic cell axis.

Males and females exhibit profound differences in immune responses and disease susceptibility. However, the factors responsible for sex differences in tissue immunity remain poorly understood. Here, we uncovered a dominant role for type 2 innate lymphoid cells (ILC2s) in shaping sexual immune dimorphism within the skin. Mechanistically, negative regulation of ILC2s by androgens leads to a reduction in dendritic cell accumulation and activation in males, along with reduced tissue immunity. Collectively, our results reveal a role for the androgen-ILC2-dendritic cell axis in controlling sexual immune dimorphism. Moreover, this work proposes that tissue immune set points are defined by the dual action of sex hormones and the microbiota, with sex hormones controlling the strength of local immunity and microbiota calibrating its tone.

Sex differences orchestrated by androgens at single-cell resolution.

Sex differences in mammalian complex traits are prevalent and are intimately associated with androgens1-7. However, a molecular and cellular profile of sex differences and their modulation by androgens is still lacking. Here we constructed a high-dimensional single-cell transcriptomic atlas comprising over 2.3 million cells from 17 tissues in Mus musculus and explored the effects of sex and androgens on the molecular programs and cellular populations. In particular, we found that sex-biased immune gene expression and immune cell populations, such as group 2 innate lymphoid cells, were modulated by androgens. Integration with the UK Biobank dataset revealed potential cellular targets and risk gene enrichment in antigen presentation for sex-biased diseases. This study lays the groundwork for understanding the sex differences orchestrated by androgens and provides important evidence for targeting the androgen pathway as a broad therapeutic strategy for sex-biased diseases.

RISING STARS: Androgens and immune cell function.

Androgens can modulate immune cell function and may contribute to differences in the prevalence and severity of common inflammatory conditions. Although most immune cells are androgen targets, our understanding of how changes in androgen bioavailability can affect immune responses is incomplete. Androgens alter immune cell composition, phenotype, and activation by modulating the expression and secretion of inflammatory mediators or by altering the development and maturation of immune cell precursors. Androgens are generally associated with having suppressive effects on the immune system, but their impacts are cell and tissue context-dependent and can be highly nuanced even within immune cell subsets. In response to androgens, innate immune cells such as neutrophils, monocytes, and macrophages increase the production of the anti-inflammatory cytokine IL-10 and decrease nitric oxide production. Androgens promote the differentiation of T cell subsets and reduce the production of inflammatory mediators, such as IFNG, IL-4 and IL-5. Additionally, androgens/androgen receptor can promote the maturation of B cells. Thus, androgens can be considered as immunomodulatory agents, but further work is required to understand the precise molecular pathways that are regulated at the intersection between endocrine and inflammatory signals. This narrative review focusses on summarising our current understanding of how androgens can alter immune cell function and how this might affect inflammatory responses in health and disease.

The antiviral potential of the antiandrogen enzalutamide and the viral-androgen signaling interplay in seasonal coronaviruses.

The sex disparity in COVID-19 outcomes with males generally faring worse than females has been associated with the androgen-regulated expression of the protease TMPRSS2 and the cell receptor ACE2 in the lung and fueled interest in antiandrogens as potential antivirals. In this study, we explored enzalutamide, an antiandrogen used commonly to treat prostate cancer, as a potential antiviral against the human coronaviruses which cause seasonal respiratory infections (HCoV-NL63, -229E, and -OC43). Using lentivirus-pseudotyped and authentic HCoV, we report that enzalutamide reduced 229E and NL63 entry and infection in both TMPRSS2- and nonexpressing immortalized cells, suggesting a TMPRSS2-independent mechanism. However, no effect was observed against OC43. To decipher this distinction, we performed RNA-sequencing analysis on 229E- and OC43-infected primary human airway cells. Our results show a significant induction of androgen-responsive genes by 229E compared to OC43 at 24 and 72 h postinfection. The virus-mediated effect on AR-signaling was further confirmed with a consensus androgen response element-driven luciferase assay in androgen-depleted MRC-5 cells. Specifically, 229E induced luciferase-reporter activity in the presence and absence of the synthetic androgen mibolerone, while OC43 inhibited induction. These findings highlight a complex interplay between viral infections and androgen-signaling, offering insights for disparities in viral outcomes and antiviral interventions.

Inhibition of the glucocorticoid-activating enzyme 11ß-hydroxysteroid dehydrogenase type 1 drives concurrent 11-oxygenated androgen excess.

Aldo-keto reductase 1C3 (AKR1C3) is a key enzyme in the activation of both classic and 11-oxygenated androgens. In adipose tissue, AKR1C3 is co-expressed with 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1), which catalyzes not only the local activation of glucocorticoids but also the inactivation of 11-oxygenated androgens, and thus has the potential to counteract AKR1C3. Using a combination of in vitro assays and in silico modeling we show that HSD11B1 attenuates the biosynthesis of the potent 11-oxygenated androgen, 11-ketotestosterone (11KT), by AKR1C3. Employing ex vivo incubations of human female adipose tissue samples we show that inhibition of HSD11B1 results in the increased peripheral biosynthesis of 11KT. Moreover, circulating 11KT increased 2-3 fold in individuals with type 2 diabetes after receiving the selective oral HSD11B1 inhibitor AZD4017 for 35 days, thus confirming that HSD11B1 inhibition results in systemic increases in 11KT concentrations. Our findings show that HSD11B1 protects against excess 11KT production by adipose tissue, a finding of particular significance when considering the evidence for adverse metabolic effects of androgens in women. Therefore, when targeting glucocorticoid activation by HSD11B1 inhibitor treatment in women, the consequently increased generation of 11KT may offset beneficial effects of decreased glucocorticoid activation.

Aberrant activated Notch1 promotes prostate enlargement driven by androgen signaling via disrupting mitochondrial function in mouse.

The prostate is a vital accessory gonad in the mammalian male reproductive system. With the ever-increasing proportion of the population over 60 years of age worldwide, the incidence of prostate diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa), is on the rise and is gradually becoming a significant medical problem globally. The notch signaling pathway is essential in regulating prostate early development. However, the potential regulatory mechanism of Notch signaling in prostatic enlargement and hyperplasia remains unclear. In this study, we proved that overactivation of Notch1 signaling in mouse prostatic epithelial cells (OEx) led to prostatic enlargement via enhancing proliferation and inhibiting apoptosis of prostatic epithelial cells. Further study showed that N1ICD/RBPJ directly up-regulated the androgen receptor (AR) and enhanced prostatic sensitivity to androgens. Hyper-proliferation was not found in orchidectomized OEx mice without androgen supply but was observed after Dihydrotestosterone (DHT) supplementation. Our data showed that the number of mitochondrion in prostatic epithelial cells of OEx mice was increased, but the mitochondrial function was impaired, and the essential activity of the mitochondrial respiratory electron transport chain was significantly weakened. Disordered mitochondrial number and metabolic function further resulted in excessive accumulation of reactive oxygen species (ROS). Importantly, anti-oxidant N-Acetyl-L-Cysteine (NAC) therapy could alleviate prostatic hyperplasia caused by the over-activation of Notch1 signaling. Furthermore, we observed the incremental Notch signaling activity in progenitor-like club cells in the scRNA-seq data set of human BPH patients. Moreover, the increased number of TROP2+ progenitors and Club cells was also confirmed in our OEx mice. In conclusion, our study revealed that over-activated Notch1 signaling induces prostatic enlargement by increasing androgen receptor sensitivity, disrupting cellular mitochondrial metabolism, increasing ROS, and a higher number of progenitor cells, all of which can be effectively rescued by NAC treatment.

Fetal Leydig cells: What we know and what we don't.

During male fetal development, testosterone plays an essential role in the differentiation and maturation of the male reproductive system. Deficient fetal testosterone production can result in variations of sex differentiation that may cause infertility and even increased tumor incidence later in life. Fetal Leydig cells in the fetal testis are the major androgen source in mammals. Although fetal and adult Leydig cells are similar in their functions, they are two distinct cell types, and therefore, the knowledge of adult Leydig cells cannot be directly applied to understanding fetal Leydig cells. This review summarizes our current knowledge of fetal Leydig cells regarding their cell biology, developmental biology, and androgen production regulation in rodents and human. Fetal Leydig cells are present in basement membrane-enclosed clusters in between testis cords. They originate from the mesonephros mesenchyme and the coelomic epithelium and start to differentiate upon receiving a Desert Hedgehog signal from Sertoli cells or being released from a NOTCH signal from endothelial cells. Mature fetal Leydig cells produce androgens. Human fetal Leydig cell steroidogenesis is LHCGR (Luteinizing Hormone Chronic Gonadotropin Receptor) dependent, while rodents are not, although other Gαs -protein coupled receptors might be involved in rodent steroidogenesis regulation. Fetal steroidogenesis ceases after sex differentiation is completed, and some fetal Leydig cells dedifferentiate to serve as stem cells for adult testicular cell types. Significant gaps are acknowledged: (1) Why are adult and fetal Leydig cells different? (2) What are bona fide progenitor and fetal Leydig cell markers? (3) Which signaling pathways and transcription factors regulate fetal Leydig cell steroidogenesis? It is critical to discover answers to these questions so that we can understand vulnerable targets in fetal Leydig cells and the mechanisms for androgen production that when disrupted, leads to variations in sex differentiation that range from subtle to complete sex reversal.

New insights into the all-testis differentiation in zebrafish with compromised endogenous androgen and estrogen synthesis.

The regulatory mechanism of gonadal sex differentiation, which is complex and regulated by multiple factors, remains poorly understood in teleosts. Recently, we have shown that compromised androgen and estrogen synthesis with increased progestin leads to all-male differentiation with proper testis development and spermatogenesis in cytochrome P450 17a1 (cyp17a1)-/- zebrafish. In the present study, the phenotypes of female-biased sex ratio were positively correlated with higher Fanconi anemia complementation group L (fancl) expression in the gonads of doublesex and mab-3 related transcription factor 1 (dmrt1)-/- and cyp17a1-/-;dmrt1-/- fish. The additional depletion of fancl in cyp17a1-/-;dmrt1-/- zebrafish reversed the gonadal sex differentiation from all-ovary to all-testis (in cyp17a1-/-;dmrt1-/-;fancl-/- fish). Luciferase assay revealed a synergistic inhibitory effect of Dmrt1 and androgen signaling on fancl transcription. Furthermore, an interaction between Fancl and the apoptotic factor Tumour protein p53 (Tp53) was found in vitro. The interaction between Fancl and Tp53 was observed via the WD repeat domain (WDR) and C-terminal domain (CTD) of Fancl and the DNA binding domain (DBD) of Tp53, leading to the K48-linked polyubiquitination degradation of Tp53 activated by the ubiquitin ligase, Fancl. Our results show that testis fate in cyp17a1-/- fish is determined by Dmrt1, which is thought to stabilize Tp53 by inhibiting fancl transcription during the critical stage of sexual fate determination in zebrafish.

Gonadal androgens are associated with decreased type I interferon production by plasmacytoid dendritic cells and increased IgG titres to BNT162b2 following co-vaccination with live attenuated influenza vaccine in adolescents.

mRNA vaccine technologies introduced following the SARS-CoV-2 pandemic have highlighted the need to better understand the interaction of adjuvants and the early innate immune response. Type I interferon (IFN-I) is an integral part of this early innate response that primes several components of the adaptive immune response. Women are widely reported to respond better than men to tri- and quadrivalent influenza vaccines. Plasmacytoid dendritic cells (pDCs) are the primary cell type responsible for IFN-I production, and female pDCs produce more IFN-I than male pDCs since the upstream pattern recognition receptor Toll-like receptor 7 (TLR7) is encoded by X chromosome and is biallelically expressed by up to 30% of female immune cells. Additionally, the TLR7 promoter contains several putative androgen response elements, and androgens have been reported to suppress pDC IFN-I in vitro. Unexpectedly, therefore, we recently observed that male adolescents mount stronger antibody responses to the Pfizer BNT162b2 mRNA vaccine than female adolescents after controlling for natural SARS-CoV-2 infection. We here examined pDC behaviour in this same cohort to determine the impact of IFN-I on anti-spike and anti-receptor-binding domain IgG titres to BNT162b2. Through flow cytometry and least absolute shrinkage and selection operator (LASSO) modelling, we determined that serum-free testosterone was associated with reduced pDC IFN-I, but contrary to the well-described immunosuppressive role for androgens, the most bioactive androgen dihydrotestosterone was associated with increased IgG titres to BNT162b2. Also unexpectedly, we observed that co-vaccination with live attenuated influenza vaccine boosted the magnitude of IgG responses to BNT162b2. Together, these data support a model where systemic IFN-I increases vaccine-mediated immune responses, yet for vaccines with intracellular stages, modulation of the local IFN-I response may alter antigen longevity and consequently improve vaccine-driven immunity.

The effect of androgens on the risk of endometriosis sub-phenotypes and ovarian neoplasms: A Mendelian randomization study.

Endometriosis is a complex gynecological pathology with a broad spectrum of symptoms, affecting around 10% of reproductive-aged women. Ovarian cancer (OC) is a heterogeneous disease for which we lack effective diagnostic and therapeutic strategies. The etiology and pathogenesis of both diseases remain ambiguous. Androgens in endometriosis could have a distinct role beyond serving as estrogen sources, whereas in the case of serous OC could be important in the formation of precursor lesions which ultimately lead to tumor formation. Here we performed two-sample Mendelian randomization (MR) analysis to examine the causal relationship between the androgen precursor - dehydroepiandrosterone sulphate (DHEAS), bioactive androgen - testosterone (T), androgen metabolite - androsterone sulphate, steroid hormone binding globulin (SHBG) and albumin and the risk of endometrioses of various sub-phenotypes and ovarian neoplasms across the benign-borderline-malignant spectrum. Stringent quality control procedures were followed to select eligible instrumental variables that were strongly associated with the selected exposures, sensitivity analyses were performed to assess the heterogeneities, horizontal pleiotropy, and stabilities of SNPs in endometriosis and ovarian neoplasms. We discovered an inverse association between genetically predicted levels of all androgens and risk of endometriosis, the same trend was most evident in the ovarian sub-phenotype. Total T levels were also inversely associated with peritoneal sub-phenotype of endometriosis. Likewise, T was causally associated with decreased risk of clear-cell OC, an endometriosis-associated OC subtype, and with malignant serous OC of both low- and high-grade, but with higher risk of their counterpart of low malignant potential. These findings support further investigation of androgen's action at a molecular level in ovary-associated endometriotic lesions, clear cell ovarian tumors and serous precursor lesions.

1,3-Disubstituted-1,2,4-triazin-6-ones with potent activity against androgen receptor-dependent prostate cancer cells.

Synthesis and biological evaluation of a small, focused library of 1,3-disubstituted-1,2,4-triazin-6-ones for in vitro inhibitory activity against androgen-receptor-dependent (22Rv1) and androgen-receptor independent (PC3) castration-resistant prostate cancer (CRPC) cells led to highly active compounds with in vitro IC50 values against 22Rv1 cells of <200 nM, and with apparent selectivity for this cell type over PC3 cells. From metabolic/PK evaluations of these compounds, a 3-benzyl-1-(2,4-dichlorobenzyl) derivative had superior properties and showed considerably stronger activity, by nearly an order of magnitude, against AR-dependent LNCaP and C4-2B cells compared to AR-independent DU145 cells. This lead compound decreased AR expression in a dose and time dependent manner and displayed promising therapeutic effects in a 22Rv1 CRPC xenograft mouse model. Computational target prediction and subsequent docking studies suggested three potential known prostate cancer targets: p38a MAPK, TGF-ß1, and HGFR/c-Met, with the latter case of c-Met appearing stronger, owing to close structural similarity of the lead compound to known pyridazin-3-one derivatives with potent c-Met inhibitory activity. RNA-seq analysis showed dramatic reduction of AR signalling pathway and/or target genes by the lead compound, subsequently confirmed by quantitative PCR analysis. The lead compound was highly inhibitory against HGF, the c-Met ligand, which fitted well with the computational target prediction and docking studies. These results suggest that this compound could be a promising starting point for the development of an effective therapy for the treatment of CRPC.

Androgen receptor actions on AgRP neurons are not a major cause of reproductive and metabolic impairments in peripubertally androgenized mice.

Excess levels of circulating androgens during prenatal or peripubertal development are an important cause of polycystic ovary syndrome (PCOS), with the brain being a key target. Approximately half of the women diagnosed with PCOS also experience metabolic syndrome; common features including obesity, insulin resistance and hyperinsulinemia. Although a large amount of clinical and preclinical evidence has confirmed this relationship between androgens and the reproductive and metabolic features of PCOS, the mechanisms by which androgens cause this dysregulation are unknown. Neuron-specific androgen receptor knockout alleviates some PCOS-like features in a peripubertal dihydrotestosterone (DHT) mouse model, but the specific neuronal populations mediating these effects are undefined. A candidate population is the agouti-related peptide (AgRP)-expressing neurons, which are important for both reproductive and metabolic function. We used a well-characterised peripubertal androgenized mouse model and Cre-loxP transgenics to investigate whether deleting androgen receptors specifically from AgRP neurons can alleviate the induced reproductive and metabolic dysregulation. Androgen receptors were co-expressed in 66% of AgRP neurons in control mice, but only in <2% of AgRP neurons in knockout mice. The number of AgRP neurons was not altered by the treatments. Only 20% of androgen receptor knockout mice showed rescue of DHT-induced androgen-induced anovulation and acyclicity. Furthermore, androgen receptor knockout did not rescue metabolic dysfunction (body weight, adiposity or glucose and insulin tolerance). While we cannot rule out developmental compensation in our model, these results suggest peripubertal androgen excess does not markedly influence Agrp expression and does not dysregulate reproductive and metabolic function through direct actions of androgens onto AgRP neurons.

Activation of testosterone-androgen receptor mediates cerebrovascular protection by photobiomodulation treatment in photothrombosis-induced stroke rats.

RATIONALE: Numerous epidemiological studies have reported a link between low testosterone levels and an increased risk of cerebrovascular disease in men. However, there is ongoing controversy surrounding testosterone replacement therapy due to potential side effects. PBMT has been demonstrated to improve cerebrovascular function and promote testosterone synthesis in peripheral tissues. Despite this, the molecular mechanisms that could connect PBMT with testosterone and vascular function in the brain of photothrombosis (PT)-induced stroke rats remain largely unknown. METHODS: We measured behavioral performance, cerebral blood flow (CBF), vascular permeability, and the expression of vascular-associated and apoptotic proteins in PT-induced stroke rats treated with flutamide and seven consecutive days of PBM treatment (350 mW, 808 nM, 2 min/day). To gain further insights into the mechanism of PBM on testosterone synthesis, we used testosterone synthesis inhibitors to study their effects on bEND.3 cells. RESULTS: We showed that PT stroke caused a decrease in cerebrovascular testosterone concentration, which was significantly increased by 7-day PBMT (808 nm, 350 mW/cm2 , 42 J/cm2 ). Furthermore, PBMT significantly increased cerebral blood flow (CBF) and the expression of vascular-associated proteins, while inhibiting vascular permeability and reducing endothelial cell apoptosis. This ultimately mitigated behavioral deficits in PT stroke rats. Notably, treatment with the androgen receptor antagonist flutamide reversed the beneficial effects of PBMT. Cellular experiments confirmed that PBMT inhibited cell apoptosis and increased vascular-associated protein expression in brain endothelial cell line (bEnd.3) subjected to oxygen-glucose deprivation (OGD). However, these effects were inhibited by flutamide. Moreover, mechanistic studies revealed that PBMT-induced testosterone synthesis in bEnd.3 cells was partly mediated by 17ß-hydroxysteroid dehydrogenase 5 (17ß-HSD5). CONCLUSIONS: Our study provides evidence that PBMT attenuates cerebrovascular injury and behavioral deficits associated with testosterone/AR following ischemic stroke. Our findings suggest that PBMT may be a promising alternative approach for managing cerebrovascular diseases.

Sirt3 Regulates Proliferation and Progesterone Production in Leydig Cells via Suppression of Reactive Oxygen Species.

Sirt3 is a mitochondrial protein deacetylase functioning in energy metabolism, regulation of intracellular reactive oxygen species (ROS) levels, and aging. Although Sirt3 loss has negative effects on fertility of oocytes during in vitro fertilization and on progesterone production in granulosa cells, Sirt3's function in Leydig cells remains unclear. Therefore, we investigated Sirt3 activity in Leydig cells, focusing on androgen production. To do so, we performed immunohistochemistry to confirm Sirt3 localization in gonads and observed strong Sirt3 immunostaining in Leydig cells of human testes and of Sirt3+/+ and Sirt3+/- mouse testes, while Sirt3-/- mouse testis tissue was negative. In human ovary, hilus cells were strongly Sirt3-positive, theca cells showed weak positivity, and granulosa cells showed very weak or almost no immunostaining. Next, we used the murine Leydig tumor cell line MA-10 as a model. We overexpressed Sirt3 but observed no changes in proliferation, expression of Star, Cyp11a1 (p450scc gene), and Hsd3b, or progesterone production in MA-10 cells. Sirt3 knockdown significantly reduced proliferation, suppressed expressions of steroidogenic enzymes and of transcription factors Ad4bp (Sf-1 gene) and Gata4, and decreased progesterone production. Sirt3 knockdown in MA-10 cells also increased intracellular ROS levels based on CM-H2DCFDA fluorescence dye analysis and increased the proportion of both early and late apoptotic (necrotic) cells based on Annexin V/7AAD assays. These results indicate that Sirt3 has a potential function in androgen production in Leydig cells by regulating intracellular ROS levels.

CXCL12 Neutralizing Antibody Promotes Hair Growth in Androgenic Alopecia and Alopecia Areata.

We had previously investigated the expression and functional role of C-X-C Motif Chemokine Ligand 12 (CXCL12) during the hair cycle progression. CXCL12 was highly expressed in stromal cells such as dermal fibroblasts (DFs) and inhibition of CXCL12 increased hair growth. Therefore, we further investigated whether a CXCL12 neutralizing antibody (αCXCL12) is effective for androgenic alopecia (AGA) and alopecia areata (AA) and studied the underlying molecular mechanism for treating these diseases. In the AGA model, CXCL12 is highly expressed in DFs. Subcutaneous (s.c.) injection of αCXCL12 significantly induced hair growth in AGA mice, and treatment with αCXCL12 attenuated the androgen-induced hair damage in hair organ culture. Androgens increased the secretion of CXCL12 from DFs through the androgen receptor (AR). Secreted CXCL12 from DFs increased the expression of the AR and C-X-C Motif Chemokine Receptor 4 (CXCR4) in dermal papilla cells (DPCs), which induced hair loss in AGA. Likewise, CXCL12 expression is increased in AA mice, while s.c. injection of αCXCL12 significantly inhibited hair loss in AA mice and reduced the number of CD8+, MHC-I+, and MHC-II+ cells in the skin. In addition, injection of αCXCL12 also prevented the onset of AA and reduced the number of CD8+ cells. Interferon-γ (IFNγ) treatment increased the secretion of CXCL12 from DFs through the signal transducer and activator of transcription 3 (STAT3) pathway, and αCXCL12 treatment protected the hair follicle from IFNγ in hair organ culture. Collectively, these results indicate that CXCL12 is involved in the progression of AGA and AA and antibody therapy for CXCL12 is promising for hair loss treatment.