United States

Background and aim: Polycystic ovary syndrome (PCOS) is associated with obesity, and white adipose tissue (WAT) and brown adipose tissue (BAT) dysregulation. However, the molecular mechanisms that mediate WAT and BAT derangements in PCOS are poorly understood. Subcutaneous (SC) WAT (SC-WAT) can transition to a beige/brite adipose tissue phenotype (browning) under altered thermogenic conditions. MicroRNAs play critical functions in brown adipocyte differentiation and maintenance. We aim to study the role of microRNA-21 (miR-21) in androgen-mediated browning and beiging derangements in both SC-WAT and BAT. Methods: Three week-old miR-21 knockout (miR21KO) or wild type (WT) female mice were treated with dihydrotestosterone (DHT, 8 mg/silastic tube) or vehicle for 90 days (n=12/grp). Body composition was measured by EchoMRI. Energy expenditure (EE), oxygen consumption (VO2), and carbon dioxide production (VCO2) were measured by indirect calorimetry. Glucose homeostasis was measured by oral glucose tolerance test (OGTT). HOMA-IR index was calculated from fasting serum glucose and insulin levels. Gene expression for browning (UCP1, Cox7a1, Elov3, Dio2 and Cidea) and beiging (Hspb7 and Txb1) markers was quantified by RT-qPCR in SC-WAT and BAT. Results: DHT increased body weight (25.07 ± 0.52 vs 21.79 ± 0.47 g, p&lt;0.05) and fat mass (4.60 ± 0.46 vs 1.98 ± 0.12 g, p&lt;0.05), impaired OGTT (186.10 ± 5.99 vs 250.70 ± 14.76 mg.min/dL, p&lt;0.05), and did not significantly change EE, VO2 or VCO2 in WT mice. All browning markers were downregulated by DHT in SC-WAT; however, only iodothyronine deiodinase 2 (Dio2) downregulation reached significance in both SC-WAT and BAT (by 53 and 40%, respectively) compared with the vehicle-treated mice. Beiging markers were significantly upregulated in SC-WAT and did not change in BAT. DHT-treated miR21KO mice showed attenuated DHT-mediated increase in body weight (23.84 ± 0.99 vs 25.07 ± 0.52 g, p&lt;0.05) compared with WT mice. MiR-21 ablation did not modify DHT-mediated increase in fat mass or OGTT but worsened insulin resistance as calculated by the HOMA-IR index. Additionally, DHT-treated miR21KO mice showed a trend to reduced EE, VO2 and VCO2 values compared with DHT-treated WT. Gene expression analysis showed an exacerbation in DHT-mediated reduction in browning markers expression in the SC-WAT. Additionally, the induction in the adaptive beiging response was abolished in SC-WAT. Conclusion and significance: These findings suggest that adipose tissue miR-21 may have a protective role in PCOS and ameliorate the DHT-mediated decrease in energy expenditure. Adipose tissue-specific modulation of miR-21 levels could be a novel therapeutic approach for the treatment of PCOS-associated metabolic derangements. (Supported by NIH grants NIGMS P20GM121334 to LLYC and DGR, NIDDK R21DK113500 to DGR, NIGMS P20GM104357 and NHLBI P01HL51971)?? 

ENDO 2021

MicroRNA-21 Modulates White Adipose Tissue Browning and Altered Thermogenesis in a Mouse Model of Polycystic Ovary Syndrome

Background and aim: Polycystic ovary syndrome (PCOS) is associated with obesity, and white adipose tissue (WAT) and brown adipose tissue (BAT) dysregulation. However, the molecular mechanisms that mediate WAT and BAT derangements in PCOS are poorly understood. Subcutaneous (SC) WAT (SC-WAT) can transition to a beige/brite adipose tissue phenotype (browning) under altered thermogenic conditions. MicroRNAs play critical functions in brown adipocyte differentiation and maintenance. We aim to study the role of microRNA-21 (miR-21) in androgen-mediated browning and beiging derangements in both SC-WAT and BAT. Methods: Three week-old miR-21 knockout (miR21KO) or wild type (WT) female mice were treated with dihydrotestosterone (DHT, 8 mg/silastic tube) or vehicle for 90 days (n=12/grp). Body composition was measured by EchoMRI. Energy expenditure (EE), oxygen consumption (VO2), and carbon dioxide production (VCO2) were measured by indirect calorimetry. Glucose homeostasis was measured by oral glucose tolerance test (OGTT). HOMA-IR index was calculated from fasting serum glucose and insulin levels. Gene expression for browning (UCP1, Cox7a1, Elov3, Dio2 and Cidea) and beiging (Hspb7 and Txb1) markers was quantified by RT-qPCR in SC-WAT and BAT. Results: DHT increased body weight (25.07 ± 0.52 vs 21.79 ± 0.47 g, p<0.05) and fat mass (4.60 ± 0.46 vs 1.98 ± 0.12 g, p<0.05), impaired OGTT (186.10 ± 5.99 vs 250.70 ± 14.76 mg.min/dL, p<0.05), and did not significantly change EE, VO2 or VCO2 in WT mice. All browning markers were downregulated by DHT in SC-WAT; however, only iodothyronine deiodinase 2 (Dio2) downregulation reached significance in both SC-WAT and BAT (by 53 and 40%, respectively) compared with the vehicle-treated mice. Beiging markers were significantly upregulated in SC-WAT and did not change in BAT. DHT-treated miR21KO mice showed attenuated DHT-mediated increase in body weight (23.84 ± 0.99 vs 25.07 ± 0.52 g, p<0.05) compared with WT mice. MiR-21 ablation did not modify DHT-mediated increase in fat mass or OGTT but worsened insulin resistance as calculated by the HOMA-IR index. Additionally, DHT-treated miR21KO mice showed a trend to reduced EE, VO2 and VCO2 values compared with DHT-treated WT. Gene expression analysis showed an exacerbation in DHT-mediated reduction in browning markers expression in the SC-WAT. Additionally, the induction in the adaptive beiging response was abolished in SC-WAT. Conclusion and significance: These findings suggest that adipose tissue miR-21 may have a protective role in PCOS and ameliorate the DHT-mediated decrease in energy expenditure. Adipose tissue-specific modulation of miR-21 levels could be a novel therapeutic approach for the treatment of PCOS-associated metabolic derangements. (Supported by NIH grants NIGMS P20GM121334 to LLYC and DGR, NIDDK R21DK113500 to DGR, NIGMS P20GM104357 and NHLBI P01HL51971)?? 

technical paper

We identified a stop-gain mutation in eIF4ENIF1 in a family in which multiple women developed primary ovarian insufficiency (POI) at approximately age 30 years. We hypothesized that the same mutation in a mouse model would replicate POI. Methods: The Eif4enif1 C57/Bl6 transgenic mouse model contains a floxed exon 10-19 cassette and a conditional knock-in cassette containing exon 10 with the c.1286C>G stop-gain mutation causing familial POI and WT exons 11-19 (Eif4enif1WT/flx). The hybrid offspring of CMV-Cre mice with Eif4enif1WT/flx mice were designated Eif4enif1WT/Δ for simplicity. Follicles were counted in fixed H&E stained ovaries from mice age days 1-5 (primordial and primary follicles), day 10, day 22 (first wave of growing follicles from small preantral to small antral follicles), week 20 (peak fertility), then every 2 months from 10 months to 26 months (follicle exhaustion). Litter frequency, pup number and genotype were recorded. Serum FSH levels were measured by the University of Virginia Ligand Assay and Analysis Core. Results: The heterozygotes have no outward or internal phenotypic differences compared to WT (Eif4enif1WT/flx), with the exception of reproductive organs in females and males. A subset of female heterozygotes (Eif4enif1WT/Δ) had no litters for 20 weeks (2 of 18; 11%). In those with litters, the average length of time between litters was not different but the final litter was earlier (5.6±2.7 vs. 10.5±0.7 months; p=0.02). Heterozygous breeding pair (Eif4enif1WT/Δ x Eif4enif1WT/Δ) litter size was 60% of WT litter size (3.9±2.3 vs. 7.2±2.1 pups/litter; 0<0.001). The genotypes were 35% Eif4enif1WT/flx and 65% Eif4enif1WT/Δ, with no homozygotes. The number of follicles in ovaries from Eif4enif1WT/Δ mice was lower starting at the primordial (499±290 vs. 1445±381) and primary follicle stage (1069±346 vs. 1450±193) on day 10 (p<0.05). The preantral follicle number was lower starting on day 21 (213±86 vs. 522±227; p<0.01) and the antral follicle count was lower starting on week 20 (78±38 vs. 119±18; p<0.01). The FSH level in 12-month old mice during estrus was higher in a heterozygote compared to WT (25.0 vs. 12.1 ng/mL). Conclusions: Heterozygous Eif4enif1 stop-gain mutants have follicle loss documented by day 10, decreased pup number with no homozygotes, earlier end of reproductive function and elevated FSH levels. These mice replicate the POI phenotype in women. eIF4ENIF1 regulates protein translation by binding and storing eIF4E bound mRNA. Therefore, the unique mouse model provides a platform to study temporal and spatial regulation of protein translation across oocyte and embryo development in mammals. Further studies will determine whether follicle loss results from premature protein translation in oocytes.?? 

Heterozygous Eif4nif1 Stop Gain Mice Replicate the Primary Ovarian Insufficiency Phenotype

Background: Polycystic ovary syndrome (PCOS) is a heterogeneous condition that affects 6-10% of women of reproductive age. PCOS is often characterized by a triad of ovulatory dysfunction, hyperandrogenism, and cardiometabolic dysfunction. Both ovarian-related and ovarian-independent factors have been implicated in the pathogenesis of PCOS, but it remains to be determined which are the inciting events and which are the secondary consequences. Studies of male relatives of women with PCOS have proposed a male counterpart of PCOS, which suggests that PCOS is not always a primary disorder of female reproduction, but rather can be, at least in part, a condition of cardiometabolic dysregulation and hyperandrogenism, with ovarian dysfunction as a secondary consequence.?Methods: To investigate a genetically defined male counterpart of PCOS, we optimized a polygenic risk score (PRS) algorithm for predicting PCOS based on 206,851 unrelated women of European ancestry in the UK Biobank, then used this algorithm to calculate PCOS PRS for 176,360 men in the UK Biobank. We used logistic regression to calculate odds ratios for dichotomous outcomes by comparing men with high and low PRS (testing a variety of percentile cutoffs) and ANCOVA to compare continuous outcomes across deciles of PRS. All analyses were adjusted for age, age2, assessment center, genotyping array, and the first 10 principal genetic components to account for ancestry.?Results: Men who carried a high PCOS PRS (top 20%) had a 17% increased risk of obesity defined as BMI ≥30 kg/m2 (OR 1.17, 95% confidence interval [CI] 1.14-1.20, p=1.3x10-30), 15% increased risk of type 2 diabetes mellitus (OR 1.15, 95% CI 1.09-1.20, p=5.3x10-8), 5% increased risk of coronary artery disease (OR 1.05, 95% CI 1.01-1.09, p=0.03), and 5% increased risk for androgenic alopecia (OR 1.05, 95% CI 1.01-1.08, p=0.01). BMI, hemoglobin A1c, triglycerides, and the free androgen index all increased across deciles of the PRS, while HDL and SHBG decreased across PRS deciles (p all <0.001). The relationship between the PCOS PRS and coronary artery disease, HDL, and triglycerides appeared to be mediated by BMI. In contrast, the associations between the PCOS PRS and type 2 diabetes mellitus and hemoglobin A1c remained significant after adjusting for BMI, suggesting independent mechanisms of pathogenesis.?Conclusions: By demonstrating associations between PCOS genetic risk factors and cardiometabolic dysfunction and androgenic conditions in men, we have shown that these genetic risk factors can act independently of ovarian function. Thus, at least in some cases, the reproductive dysfunction of PCOS in women may arise secondarily from disruption of biological pathways common to both men and women. Future dissection of these biological pathways will further inform efforts to identify pathological mechanisms underlying PCOS.?? 

A Genetically Defined Male Counterpart of Polycystic Ovary Syndrome: Evidence for Ovarian-Independent Pathogenesis

Effect of Experimentally Induced Sleep Fragmentation and Hypoestrogenism on Fasting Nutrient Utilization in Pre-Menopausal Women Background: Both sleep disturbance and menopause have independently been associated with weight gain in women. Possible mechanisms contributing to this weight gain may be changes in resting energy expenditure (REE) and/or nutrient utilization. Therefore, in the current study we aimed to examine the effects of experimentally induced sleep fragmentation and pharmacologic estradiol (E2) withdrawal on REE and nutrient utilization in the fasted state. Design: We studied pre-menopausal women during 5-night inpatient studies repeated in the mid-to-late follicular phase (high-E2; n=21) and following leuprolide-induced hypoestrogenism (low-E2; n=9 completed second visit). During each admission there were two nights of unfragmented sleep [8-h time in bed (TIB)] and three nights of fragmented sleep [9-h TIB]. Sleep was fragmented using an auditory stimulus delivered every 15 minutes that sustained wake for 2 minutes, producing 1 hour of wake after sleep onset. Study diets consisted of 3 meals and a snack each day and were iso-caloric across the two visits. REE and nutrient utilization were assessed in the fasted state via indirect calorimetry and compared between E2 states following unfragmented and fragmented sleep using linear mixed models. Results: Sleep fragmentation in the high-E2 state increased the respiratory quotient (RQ; +3%; p=0.03) with an accompanying increase in carbohydrate oxidation (+20%; p=0.02) and decrease in fat oxidation (-16%; p=0.03). The same effect was observed in response to E2-withdrawl during unfragmented sleep [increased RQ (+5%; p=0.01) and carbohydrate oxidation (+33%; p=0.01), and decreased fat oxidation (-26%; p=0.01)]. There was no additive effect of sleep fragmentation on nutrient utilization in the low-E2 state suggesting a possible ceiling (RQ and carbohydrate oxidation) and floor (fat oxidation) effect. There was no effect of sleep fragmentation or E2 state on REE. Conclusion: Both sleep fragmentation and hypoestrogenism were shown to alter fasting nutrient utilization, but not REE, in a manner that may contribute to weight gain in menopausal women. These findings are important for understanding weight gain during menopause, which is characterized by estrogen withdrawal and often accompanied by sleep disturbances.?? 

Effect of Experimentally Induced Sleep Fragmentation and Hypoestrogenism on Fasting Nutrient Utilization in Pre-Menopausal Women

Background: Exogenous androgen treatment suppresses the hypothalamo-pituitary testicular (HPT) axis causing reduced serum LH, FSH and testosterone (T). Recovery of male reproductive endocrine function in past androgen abusers takes 9-18 months with persistent mild lowering of serum T. The natural history of recovery of HPT axis following prolonged injectable testosterone undecanoate (TU) treatment at standard dose is not known. Therefore, the Runoff Study investigated the rate and extent of reproductive hormone recovery over 12 months following cessation of 2 years of TU treatment in the Testosterone for Diabetes Mellitus (T4DM) Study, while men remain blinded to treatment allocation.Methods: T4DM participants without pathological hypogonadism (n=1007) were randomised to TU or Placebo (P) injections every 3 months for 2 years with 303 subsequently volunteering to enter the Runoff study at 12 weeks after last injection. Before T4DM study unblinding, they provided blood samples and validated sexual function questionnaires (PDQ, IIEF-15) at entry (3 months after last injection), 6, 12, 18, 24, 40 and 52 weeks later. Serum steroid profile (T, DHT, E2, E1) was measured batchwise by LCMS and serum LH, FSH and SHBG by immunoassays.Results: Runoff study participants in both groups were similar and did not differ from all T4DM participants. As expected, at entry to Runoff serum T was higher in TU-treated men but at all timepoints from 12 weeks onwards serum T and SHBG remained consistently 11% and 13%, respectively, lower in TU-treated than in P-treated men. Similarly, at entry sexual function scores were higher in TU-treated men but subsequently no different from P-treated men. Serum LH and FSH recovered slowly with the median time to reach their own pre-treatment baseline of serum LH was 51.1 weeks [95% CI 50.4 – 53.0 weeks] and for serum FSH was 52.7 weeks [51.0 – 60.9 weeks]. Conclusion: After stopping 2 years of standard dose injectable TU treatment in men without pathological hypogonadism, recovery of testicular endocrine function is eventually complete but slow with serum gonadotropin recovery taking on 12 months since the last dose. Persistent mild, proportionate reduction in serum SHBG and T reflects lasting exogenous T effects on hepatic SHBG secretion rather than signifying androgen deficiency. This suggests that recovery from androgen-induced HPT axis suppression depends primarily on time since cessation rather than dose or duration of androgen exposure.?? 

Recovery of Male Reproductive Endocrine Function Following Prolonged Injectable Testosterone Undecanoate Treatment

Erythrocytosis in Trans Men Using Testosterone

The Hypothalmic-Pituitary-Testicular Axis in Men with Exceptional Longevity

Introduction: As life expectancy continues to increase and more men reach the extremes of age, it is important to understand the physiology of the aging hypothalamic-pituitary-testicular (HPT) axis and its role in health. While prior studies primarily focused on men younger than age ninety, we studied a unique cohort enriched for men with exceptional longevity to characterize the age-related trends in male sex-hormones, the etiology of the observed changes in the HPT axis, and its relationship with metabolic dysfunction and survival at the extremes of age. Methods: This is a cross-sectional study of community-dwelling Ashkenazi Jewish men (n = 427), age range 50-106 years. Longitudinal follow-up for vital status was conducted for men age ≥ 88 at enrollment (n = 86). Measurements included serum total testosterone (TT) by LC/MS, LH, SHBG, lipids, glucose and BMI. Free testosterone (FT) was calculated according to Vermeulen et. al. A change-point linear regression model was applied to describe the age trend of TT. Multivariable linear regression adjusted for comorbidities tested the associations between metabolic parameters and TT. The association between survival and TT was evaluated with the age-adjusted Cox proportional hazards model. Age-specific cutoffs for TT and LH were used to define primary and secondary hypogonadism. Results: The change point model was a significantly better fit for the data compared to the straight-line model (p = 0.004), indicating that TT significantly declines after age 88 years. Men age < 88 years had higher average TT (401 ± 162 vs. 278 ± 178 ng/dL, p < 0.001), FT (6.3 ± 2.0 vs. 3.3± 2.1 pg/mL, p < 0.001), and lower LH (4.3 [3.0 - 6.1] vs. 14.6 [7.2 - 25.5] mIU/mL, p < 0.001), compared to men age ≥ 88 years. The prevalence of primary and secondary hypogonadism was 2% and 11%, respectively, in men age < 88 years, and 30% and 11%, among men age ≥ 88 years (p < 0.001). A multivariable linear regression analysis revealed interactions between age, dichotomized at the change-point of 88 years, and metabolic parameters. Models stratified at age 88 demonstrated an inverse association between TT and BMI (p = 0.02), serum triglycerides (p = 0.007), and random glucose levels (p = 0.02) among men age < 88; whereas a positive association was noted between TT and HDL cholesterol (p = 0.009) in this group. In men age ≥ 88 years, TT was not associated with any of the metabolic parameters or overall survival. Conclusions: Low testosterone in men with exceptional longevity is largely a result of primary testicular failure that occurs around age 88 and is accompanied by preserved hypothalamic-pituitary response with no associated metabolic dysfunction or effect on survival. This is in contrast to younger men, whose low T typically results from hypothalamic-pituitary dysfunction and is associated with metabolic derangements.?? 

The Hypothalamic-Pituitary-Testicular Axis in Exceptionally Old Men

Renal sodium reabsorption is important for blood pressure homeostasis and is physiologically regulated by aldosterone; glucocorticoids may also contribute. Abnormal steroid hormone activity within the kidney contributes to hypertension but the mechanisms are not fully defined. Molecular profiling of receptors and metabolising enzymes indicates that steroid hormone action is compartmentalised within the kidney. Ambient steroid concentrations are a critical factor governing bioactivity at a cellular level, but this is largely unknown, and the kidney remains a “black box”. Mass spectrometry imaging (MSI) was applied recently to localise steroids in brain and testes, and here is applied to kidney. Image reconstruction permits characterisation and co-registration of kidney histological regions based on regional markers detectable by MSI. Our aim was to map and quantify glucocorticoids and aldosterone in different histological zones (cortex, medulla) of murine kidneys, using an optimised MSI method. This approach has the potential to map steroids within functional zones of the kidney, providing fundamental new information relevant to hormone action in health and in disease. Cryosections of male C57BL6 mouse kidneys (age 12 weeks, n=6) were subject to MSI following derivatisation using Girard T reagent and α-cyano-4-hydroxycinnamic acid matrix application. Images were reconstructed, and methods optimised to enhance signal and limit diffusion of analytes of interest. Matrix assisted laser desorption/ionisation (MALDI) was used as a sampling method, coupled to Fourier Transform Ion cyclotron mass spectrometry.Ions with m/z 458.3010, 460.3166 and 474.2957 were detected, using MALDI, in renal sections, close to the predicted masses of 458.3013 (Δppm=0.65), 460.3169 (Δppm=0.65), and 474.2962 (Δppm=1.05), for derivatives of 11-dehydrocorticosterone, corticosterone and aldosterone respectively. Untargeted evaluation of ions was conducted to find regional markers that would allow definition of kidney histological zones. The Heat maps generated indicated that corticosterone intensity was higher in the inner cortex area close to the corticomedullary junction than the rest of the kidney. In contrast 11-dehydrocorticosterone was detected mainly in medulla and aldosterone signal was equally strong in medulla and outer cortex. Thus, MSI can be used map the sites where glucocorticoid and mineralocorticoids are most active in regulating renal tubular function. Co-localisation of steroids of interest with zonal markers by MSI permits steroid mapping in functional renal zones of the kidney. This approach provides fundamental new insights into the physiological control of sodium transport by steroids and opens doors to understanding changes in disorders of blood pressure. The project was supported and funded by Kidney Research UK.?? 

Mapping of Corticosteroids in Murine Kidneys Using Mass Spectrometry Imaging

Excessive exposure to glucocorticoids (GCs), either from endogenous overproduction of cortisol, or exogenous pharmacological GC treatment, potentiates the development of diabetes and obesity in a fat depot-specific manner. Undesirable metabolic side effects resulting from the activation of the glucocorticoid receptor (GR) remain a key limitation to the long-term therapeutic use of GCs as immunosuppressants. GC treatment disrupts the thermogenic function of brown adipose tissue (BAT) and enhances futile cycling within white adipose tissue (WAT). Mice lacking the liver X receptors (LXRs) were previously shown to have smaller AT depots with enhanced BAT activity compared to wildtype (WT) mice. We previously demonstrated that LXRβ is required to mediate the side effects of GCs in the liver but not the beneficial anti-inflammatory effects of GCs. The discovery of this GC/LXRβ cross-talk led to the hypothesis that LXRβ antagonism may be therapeutically beneficial to prevent GC-induced dysfunction in AT. To test this idea, LXRα-/- mice were treated for 5 days with vehicle, 5 mg/kg dexamethasone (Dex, a synthetic GC agonist), and/or 40 mg/kg GSK2033 (GSK, non-selective LXR antagonist). As expected, Dex-treated mice showed significant accumulation of lipids in BAT and were unable to maintain their body temperature when exposed to cold, yet GSK was able to protect against these effects. Dex increased body fat mass and caused adipocyte enlargement in WAT which was not observed in mice co-treated with GSK. At the transcriptional level, GSK attenuated Dex-mediated downregulation of thermogenic genes in BAT, and upregulation of lipogenic genes in WAT. Similar beneficial changes were confirmed in WT mice. The protection afforded by GSK against Dex-induced fat accumulation was confirmed to be cell-autonomous from studies in adipose-specific LXRβ-/- mice (AdβKO). Dex-dependent increases in lipolysis in gonadal WAT and plasma free fatty acids (FFA) were also attenuated by GSK co-treatment. With GSK co-treatment, Dex-induced liver steatosis was diminished suggesting that LXRβ antagonism attenuated FFA shuttling to the liver. The lipolytic and lipotoxic effects of Dex in AT and liver were largely abrogated in AdβKO along with improved systemic insulin sensitivity. Overall, our data suggest that LXRβ antagonism prevents disruption of BAT and WAT (and indirectly liver) function caused by GC treatment in an in vivo model, highlighting the potential role of LXRβ antagonists in combating the negative effects of excessive GC exposure on the development of diabetes and obesity. The identification of this novel mechanism of interrupting GC adipose tissue action suggests therapeutic targeting of LXRβ with an antagonist could improve the health of patients currently taking GCs to control inflammation but suffer the detrimental side effects of drug treatment.?? 

Disruption of Adipose Tissue Metabolism by Glucocorticoids Is Attenuated With LXRβ Antagonism

The androgen receptor (AR) is a nuclear receptor with a main role in the development and maintenance of the male phenotype. To execute its role as transcription factor, the AR forms homodimers. Three dimerization modes have been described for the AR: one via the DNA binding domain, a second via the ligand binding domain (LBD) and a third via interactions between the LBD and the aminoterminus of the AR (N/C). Based exclusively on in vitro data, all three dimerization modes seem to contribute to full AR activity, albeit to a different extent. The in vivo role of the dimerization modes, however, remains unknown. To study the physiological relevance, we generated two mouse models using a CRISPR/Cas9 approach, in which either the N/C interaction (ARNoC) or LBD dimerization (ARLmon) was disrupted. Surprisingly, the male ARNoC mice have a normal phenotype, indicating that the N/C interaction is not crucial for male development. In contrast, ARLmon males have an external female phenotype with cryptorchid testes and high levels of circulating testosterone (T), androstenedione and luteinizing hormone (LH) (6-, 13- and 45-fold higher, respectively). They have no prostate, seminal vesicles or epididymis, illustrating the importance of LBD dimerization during male development. Phenotyping the ARLmon model furthermore provided evidence of a crucial role for the AR in bone homeostasis as well as steroidogenesis. The ARLmon males display a severe bone phenotype, similar to that of complete AR knockout (ARKO) mice. The bone phenotype of ARKO was postulated to be mainly due to lower estrogen levels. However, in contrast to ARKO mice, ARLmon mice have high circulating levels of T, which can still function as prohormone for estradiol and support bone function via the ERα. Immunohistological analysis of ARLmon testes showed hyperplasia of the Leydig cells and residual spermatogenesis. Analysis of the steroidogenic pathway revealed that while the expression of most genes is increased, the expression of Hsd17b3, encoding the enzyme responsible for conversion of androstenedione into T, is low in ARLmon testis. Reporter assays confirmed that the promotor of this gene is indeed upregulated by the AR itself. In conclusion, our work uncovers the physiological role of the N/C interaction and LBD dimerization of the AR. It furthermore demonstrates a direct role for AR in male bone development independent of T aromatization into estrogens. Finally, we show that the AR controls the final step in the synthesis of its own ligand. In contrast to the in vitro data, N/C interaction is not crucial for male development in vivo. The ARLmon model illustrates that LBD dimerization could be an excellent new therapeutic target for inhibiting AR activity for example in advanced prostate cancer that has developed resistance to the current AR-targeting therapies.?? 

MicroRNA-21 Modulates White Adipose Tissue Browning and Altered Thermogenesis in a Mouse Model of Polycystic Ovary Syndrome

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Heterozygous Eif4nif1 Stop Gain Mice Replicate the Primary Ovarian Insufficiency Phenotype

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