The Histone H3K9 Demethylase Kdm3b Is Required for Somatic Growth and Female Reproductive Function

Kdm3b is a Jumonji C domain-containing protein that demethylates mono- and di-methylated lysine 9 of histone H3 (H3K9me1 and H3K9me2). Although the enzyme activity of Kdm3b is well characterized in vitro, its genetic and physiological function remains unknown. Herein, we generated Kdm3b knockout (Kdm3bKO) mice and observed restricted postnatal growth and female infertility in these mice. We found that Kdm3b ablation decreased IGFBP-3 expressed in the kidney by 53% and significantly reduced IGFBP-3 in the blood, which caused an accelerated degradation of IGF-1 and a 36% decrease in circulating IGF-1 concentration. We also found Kdm3b was highly expressed in the female reproductive organs including ovary, oviduct and uterus. Knockout of Kdm3b in female mice caused irregular estrous cycles, decreased 45% of the ovulation capability and 47% of the fertilization rate, and reduced 44% of the uterine decidual response, which were accompanied with a more than 50% decrease in the circulating levels of the 17beta-estradiol. Importantly, these female reproductive phenotypes were associated with significantly increased levels of H3K9me1/2/3 in the ovary and uterus. These results demonstrate that Kdm3b-mediated H3K9 demethylation plays essential roles in maintenance of the circulating IGF-1, postnatal somatic growth, circulating 17beta-estradiol, and female reproductive function.     

Kdm3a lysine demethylase is an Hsp90 client required for cytoskeletal rearrangements during spermatogenesis

The lysine demethylase Kdm3a (Jhdm2a, Jmjd1a) is required for male fertility, sex determination, and metabolic homeostasis through its nuclear role in chromatin remodeling. Many histone-modifying enzymes have additional nonhistone substrates, as well as nonenzymatic functions, contributing to the full spectrum of events underlying their biological roles. We present two Kdm3a mouse models that exhibit cytoplasmic defects that may account in part for the globozoospermia phenotype reported previously. Electron microscopy revealed abnormal acrosome and manchette and the absence of implantation fossa at the caudal end of the nucleus in mice without Kdm3a demethylase activity, which affected cytoplasmic structures required to elongate the sperm head. We describe an enzymatically active new Kdm3a isoform and show that subcellular distribution, protein levels, and lysine demethylation activity of Kdm3a depended on Hsp90. We show that Kdm3a localizes to cytoplasmic structures of maturing spermatids affected in Kdm3a mutant mice, which in turn display altered fractionation of β-actin and γ-tubulin. Kdm3a is therefore a multifunctional Hsp90 client protein that participates directly in the regulation of cytoskeletal components.     

Masculine Sexual Behavior Is Disrupted in Male and Female Mice Lacking a Functional Estrogen Receptor α Gene

Masculine sexual behavior is regulated by testosterone (T). However, T can be metabolized to form estrogens or other androgens, which then activate their own receptors. We used knockout mice lacking a functional estrogen receptor α (ERα) gene to test the hypothesis that, following aromatization, T acts via the ERα to activate normal masculine sexual behavior. After gonadectomy and T replacement, wild-type (WT) male and female mice displayed masculine behavior. However, given the same T treatment, little masculine behavior was displayed by mice of either sex that lack a normal copy of the ERα gene. In particular, the latency to display masculine sex behavior and the number of mount attempts per trial were significantly reduced in the ERα⁻mice compared to WT littermates (P< 0.05). In addition, we found that in both sexes, ERα⁻mice have a smaller cluster of androgen receptor immunoreactivity in the bed nucleus of the stria terminalis. Using adult ERα⁻mice we were unable to determine whether these genotypic differences are due to organizational or activational effects. However, it is clear that the ERα plays a key role in the expression of masculine sexual behavior and in the regulation of androgen receptors in a neuronal cell population involved in the display of motivated behaviors.     

Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice

Androgens are important regulators of bone mass but the relative importance of testosterone (T) versus dihydrotestosterone (DHT) for the activation of the androgen receptor (AR) in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2), encoded by separate genes (Srd5a1 and Srd5a2). 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1^−/− mice. Four-month-old male Srd5a1 ^−/− mice had reduced trabecular bone mineral density (−36%, p<0.05) and cortical bone mineral content (−15%, p<0.05) but unchanged serum androgen levels compared with wild type (WT) mice. The cortical bone dimensions were reduced in the male Srd5a1 ^−/− mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05) in orchidectomized WT mice but not in orchidectomized Srd5a1 ^−/− mice. Male Srd5a1 ^−/− mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05). Female Srd5a1 ^−/− mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1 ^−/− mice, is an indirect effect mediated by elevated circulating androgen levels.     

The C. elegans adult male germline: Stem cells and sexual dimorphism

The hermaphrodite Caenorhabditis elegans germline has become a classic model for stem cell regulation, but the male C. elegans germline has been largely neglected. This work provides a cellular analysis of the adult C. elegans male germline, focusing on its predicted stem cell region in the distal gonad. The goals of this study were two-fold: to establish the C. elegans male germline as a stem cell model and to identify sex-specific traits of potential relevance to the sperm/oocyte decision. Our results support two major conclusions. First, adult males do indeed possess a population of germline stem cells (GSCs) with properties similar to those of hermaphrodite GSCs (lack of cell cycle quiescence and lack of reproducibly oriented divisions). Second, germ cells in the mitotic region, including those most distal within the niche, exhibit sex-specific behaviors (e.g. cell cycle length) and therefore have acquired sexual identity. Previous studies demonstrated that some germ cells are not committed to a sperm or oocyte cell fate, even in adults. We propose that germ cells can acquire sexual identity without being committed to a sperm or oocyte cell fate.     

Disruption of Ttll5/Stamp Gene (Tubulin Tyrosine Ligase-like Protein 5/SRC-1 and TIF2-associated Modulatory Protein Gene) in Male Mice Causes Sperm Malformation and Infertility

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp^tm/tm) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp^tm/tm sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp^tm/tm males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.     

Search for an Endogenous Bombesin-Like Receptor 3 (BRS-3) Ligand Using Parabiotic Mice

Bombesin-like receptor 3 (BRS-3) is an X-linked G protein-coupled receptor involved in the regulation of energy homeostasis. Brs3 null (Brs3 ^-/y) mice become obese. To date, no high affinity endogenous ligand has been identified. In an effort to detect a circulating endogenous BRS-3 ligand, we generated parabiotic pairs of mice between Brs3 ^-/y and wild type (WT) mice or between WT controls. Successful parabiosis was demonstrated by circulatory dye exchange. The Brs3 ^-/y-WT and WT-WT pairs lost similar weight immediately after surgery. After 9 weeks on a high fat diet, the Brs3 ^-/y-WT pairs weighed more than the WT-WT pairs. Within the Brs3 ^-/y-WT pairs, the Brs3 ^-/y mice had greater adiposity than the WT mice, but comparable lean and liver weights. Compared to WT mice in WT-WT pairs, Brs3 ^-/y and WT mice in Brs3 ^-/y-WT pairs each had greater lean mass, and the Brs3 ^-/y mice also had greater adiposity. These results contrast to those reported for parabiotic pairs of leptin receptor null (Lepr ^db/db) and WT mice, where high leptin levels in the Lepr ^db/db mice cause the WT parabiotic partners to lose weight. Our data demonstrate that a circulating endogenous BRS-3 ligand, if present, is not sufficient to reduce adiposity in parabiotic partners of Brs3 ^-/y mice.     

Regulation of Male Sexual Behavior by Progesterone Receptor, Sexual Experience, and Androgen

Recent studies have demonstrated that physiological doses of progesterone may facilitate the androgen-dependent display of male sexual behavior in laboratory rats and three species of lizard. We used mice with a targeted disruption of the progesterone receptor to investigate whether such interactions exist in male mice and whether they may be modified by sexual experience. We found that naive intact male progesterone receptor knockout (PRKO) mice exhibit reduced mount frequencies compared to wild-type (WT) mice. Also unlike WT mice, sexually experienced PRKO males show profound losses in many measures of sexual behavior following castration. In a second experiment, we tested whether male mice heterozygous for a null mutation at the progesterone receptor locus were responsive to testosterone and progesterone treatment. We found that heterozygous males showed a reduced response to testosterone. The data are consistent with experiments indicating that the progesterone receptor is able to facilitate male-typical sex behaviors in other species and suggest novel mechanisms underlying the interaction of androgens and experience.     

Kdm6a suppresses the alternative activation of macrophages and impairs energy expenditure in obesity

Histone lysine demethylase 6a (Kdm6a) mediates the removal of repressive trimethylation from histone H3 lysine 27 (H3K27me3) to activate target gene expression. Obesity is associated with metabolic inflammation, and adipose tissue macrophages (ATMs) are key players orchestrating metabolic inflammation. However, it is still unclear whether the Kdm6a pathway in ATMs regulates energy homeostasis. Here, we identified Kdm6a as a critical epigenetic switch that modulates macrophage polarisation and further disrupts energy balance. Myeloid-specific Kdm6a knockout in Kdm6a^F/Y;Lyz2-Cre mice significantly reversed the high-fat diet (HFD)-induced M1–M2 imbalance in white adipose tissue (WAT) and blocked HFD-induced obesity. The brown adipose tissue (BAT) activity, WAT browning and energy expenditure were significantly increased in Kdm6a^F/Y;Lyz2-Cre mice. Furthermore, Kdm6a regulated the Ire1α expression in a demethylase activity-dependent manner and augmented the M2 polarisation of macrophages. Macrophage with higher Kdm6a significantly promotes adipogenesis in white adipocyte and inhibits thermogenesis in beige adipocytes. These results suggest that the Kdm6a in macrophages drives obesity and metabolic syndrome by impairing BAT activity and WAT differentiation.Subject terms: Interleukins, Epigenetics     

Deletion of STAT5a/b in Vascular Smooth Muscle Abrogates the Male Bias in Hypoxic Pulmonary Hypertension in Mice: Implications in the Human Disease

Chronic hypoxia typically elicits pulmonary hypertension (PH) in mice with a male-dominant phenotype. There is an opposite-sex bias in human PH, with a higher prevalence in women, but greater survival (the “estrogen paradox”). We investigated the involvement of the STAT5a/b species, previously established to mediate sexual dimorphism in other contexts, in the sex bias in PH. Mice with heterozygous or homozygous deletions of the STAT5a/b locus in vascular smooth muscle cells (SMCs) were generated in crosses between STAT5a/b^fl/fl and transgelin (SM22α)-Cre^+/+ parents. Wild-type (wt ) males subjected to chronic hypoxia showed significant PH and pulmonary arterial remodeling, with wt females showing minimal changes (a male-dominant phenotype). However, in conditional STAT5^+/− or STAT5^−/− mice, hypoxic females showed the severest manifestations of PH (a female-dominant phenotype). Immunofluorescence studies on human lung sections showed that obliterative pulmonary arterial lesions in patients with idiopathic pulmonary arterial hypertension (IPAH) or hereditary pulmonary arterial hypertension (HPAH), both male and female, overall had reduced STAT5a/b, reduced PY-STAT5 and reduced endoplasmic reticulum (ER) GTPase atlastin-3 (ATL3). Studies of SMCs and endothelial cell (EC) lines derived from vessels isolated from lungs of male and female IPAH patients and controls revealed instances of coordinate reductions in STAT5a, STAT5b and ATL3 in IPAH-derived cells, including SMCs and ECs from the same patient. Taken together, these data provide the first definitive evidence for a contribution of STAT5a/b to the sex bias in PH in the hypoxic mouse and implicate reduced STAT5 in the pathogenesis of the human disease.     

Phospholipase Cγ2 Is Required for Luminal Expansion of the Epididymal Duct during Postnatal Development in Mice

Phospholipase Cγ2 (PLCγ2)-deficient mice exhibit misconnections of blood and lymphatic vessels, and male infertility. However, the cell type responsible for vascular partitioning and the mechanism for male infertility remain unknown. Accordingly, we generated a mouse line that conditionally expresses endogenous Plcg2 in a Cre/loxP recombination-dependent manner, and found that Tie2-Cre- or Pf4-Cre-driven reactivation of Plcg2 rescues PLCγ2-deficient mice from the vascular phenotype. By contrast, male mice rescued from the vascular phenotype exhibited epididymal sperm granulomas. As judged from immunostaining, PLCγ2 was expressed in clear cells in the epididymis. PLCγ2 deficiency did not compromise differentiation of epididymal epithelial cells, including clear cells, and tube formation at postnatal week 2. However, luminal expansion of the epididymal duct was impaired during the prepubertal period, regardless of epithelial cell polarity and tube architecture. These results suggest that PLCγ2-deficient clear cells cause impaired luminal expansion, stenosis of the epididymal duct, attenuation of luminal flow, and subsequent sperm granulomas. Clear cell-mediated luminal expansion is also supported by the observation that PLCγ2-deficient males were rescued from infertility by epididymal epithelium-specific reactivation of Plcg2, although the edematous and hemorrhagic phenotype associated with PLCγ2 deficiency also caused spontaneous epididymal sperm granulomas in aging males. Collectively, our findings demonstrate that PLCγ2 in clear cells plays an essential role in luminal expansion of the epididymis during the prepubertal period in mice, and reveal an unexpected link between PLCγ2, clear cells, and epididymal development.     

Deletion of CDKAL1 affects mitochondrial ATP generation and first-phase insulin exocytosis

Background

A variant of the CDKAL1 gene was reported to be associated with type 2 diabetes and reduced insulin release in humans; however, the role of CDKAL1 in β cells is largely unknown. Therefore, to determine the role of CDKAL1 in insulin release from β cells, we studied insulin release profiles in CDKAL1 gene knockout (CDKAL1 KO) mice.

Principal Findings

Total internal reflection fluorescence imaging of CDKAL1 KO β cells showed that the number of fusion events during first-phase insulin release was reduced. However, there was no significant difference in the number of fusion events during second-phase release or high K⁺-induced release between WT and KO cells. CDKAL1 deletion resulted in a delayed and slow increase in cytosolic free Ca²⁺ concentration during high glucose stimulation. Patch-clamp experiments revealed that the responsiveness of ATP-sensitive K⁺ (K_ATP) channels to glucose was blunted in KO cells. In addition, glucose-induced ATP generation was impaired. Although CDKAL1 is homologous to cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1, there was no difference in the kinase activity of CDK5 between WT and CDKAL1 KO islets.

Conclusions/Significance

We provide the first report describing the function of CDKAL1 in β cells. Our results indicate that CDKAL1 controls first-phase insulin exocytosis in β cells by facilitating ATP generation, K_ATP channel responsiveness and the subsequent activity of Ca²⁺ channels through pathways other than CDK5-mediated regulation.     

Insemination efficiency of two alternative male mating tactics in the guppy Poecilia reticulata

In this study we compared the insemination efficiency of two alternative mating tactics (courtship and sneak mating) in the guppy Poecilia reticulata by quantifying the number of sperm delivered during a copulation. During a single copulation, guppies delivered between zero and 92% of the sperm available, as determined by mechanically stripping the males'' sperm reserve at rest. The absolute number of sperm delivered after courtship was three times larger than that delivered through sneak mating; nonetheless, the variance was large with both tactics and the two distributions largely overlapped. The number of sperm available at rest increased with male size. With both tactics, the number of sperm delivered was positively correlated with the sperm available. Contrary to courtship copulations, in sneak copulations there was no correlation between the number of sperm delivered and male size. However, once the data were standardized for sperm reserve, small males delivered a larger proportion of their available sperm during sneak copulation. The rate of sexual acts (sigmoid and thrust rate) before copulation was not correlated with the number of sperm available. After the occurrence of a copulation in both the courtship and sneak copulation groups, the sexual activity of the male decreased in proportion to the amount of sperm he previously inseminated.     

乙醛脱氢酶基因ALDH2对雄性小鼠生育能力的影响

本研究使用乙醛脱氢酶2(ALDH2)基因全敲除的雄性C57BL/6(B6)小鼠（Musmusculus domesticus）,通过分析不同周龄小鼠的睾丸脏器系数、睾丸组织细胞形态、精子运动参数、配种后母鼠产仔数及子代雄雌比等生育指标,探讨ALDH2基因敲除对雄性小鼠生育能力的影响。结果表明,与野生型（WT）小鼠相比,5、7、10周龄ALDH2基因敲除型（KO）雄性小鼠睾丸脏器系数显著降低（P <0.05）;睾丸组织细胞间质变大,精子活率显著降低（P <0.05）;产仔数和雄雌比显著降低（P <0.05）。本研究为揭示乙醛脱氢酶ALDH2基因在雄性小鼠生殖中的作用提供了一定的基础。     

Male mice ultrasonic vocalizations enhance female sexual approach and hypothalamic kisspeptin neuron activity

Vocal communication in animals is important for ensuring reproductive success. Male mice emit song-like “ultrasonic vocalizations (USVs)” when they encounter female mice, and females show approach to the USVs. However, it is unclear whether USVs of male mice trigger female behavioral and endocrine responses in reproduction. In this study, we first investigated the relationship between the number of deliveries in breeding pairs for 4 months and USVs syllables emitted from those paired males during 3 min of sexual encounter with unfamiliar female mice. There was a positive correlation between these two indices, which suggests that breeding pairs in which males could emit USVs more frequently had more offspring. Further, we examined the effect of USVs of male mice on female sexual behavior. Female mice showed more approach behavior towards vocalizing males than devocalized males. Finally, to determine whether USVs of male mice could activate the neural system governing reproductive function in female mice, the activation of kisspeptin neurons, key neurons to drive gonadotropin-releasing hormone neurons in the hypothalamus, was examined using dual-label immunocytochemistry with cAMP response element-binding protein phosphorylation (pCREB). In the arcuate nucleus (Arc), the number of kisspeptin neurons expressing pCREB significantly increased after exposure to USVs of male as compared with noise exposure group. In conclusion, our results suggest that USVs of male mice promote fertility in female mice by activating both their approaching behavior and central kisspeptin neurons.     

Sexual partner preference requires a functional aromatase (cyp19) gene in male mice

Sexual motivation, sexual partner preference, and sexual performance represent three different aspects of sexual behavior that are critical in determining the reproductive success of a species. Although the display of sexual behavior is under strict hormonal control in both sexes, the relative roles of androgen and estrogen receptors in activating the various components of male sexual behavior are still largely unknown. A recently developed mouse model that is deficient in estradiol due to targeted disruption of exons 1 and 2 of the Cyp19 gene (aromatase knockout (ArKO) mice) was used here to analyze the role of estradiol in the control of all three aspects of male sexual behavior. When tested in a Y-maze providing volatile olfactory cues, male ArKO mice did not show a preference for the odors from an estrous female over those from an intact male, whereas wild-type (WT) and heterozygous (HET) males clearly preferred to sniff estrous odors. When provided with visual and olfactory cues, male ArKO mice also failed to show a preference for an estrous female when given a choice between an estrous female and an empty arm. However, sexual partner preferences of male ArKO mice were not sex-reversed: they did not prefer to investigate an intact male over an estrous female or empty arm. Thus, male ArKO mice seemed to have general deficits in discriminating between conspecifics by using olfactory and visual cues. Male coital behavior was also severely impaired in male ArKO mice: they displayed significantly fewer mounts, intromissions, and ejaculations than WT and HET males. Latencies to first mount or intromission were also significantly longer in ArKO males compared to WT and HET males, in addition to them showing less interest in investigating olfactory and visual cues in a Y-maze, suggesting that they were sexually less motivated. However, three out of seven male ArKO mice were capable of siring litters provided they were housed with a female for a prolonged period of time. In conclusion, aromatization of testosterone to estradiol appears to be essential for sexual motivation and sexual partner preference. By contrast, estradiol may play only a limited role in the expression of male coital behaviors.     

Soluble Receptor for Advanced Glycation End-products regulates age-associated Cardiac Fibrosis

Myocardial aging increases the cardiovascular risk in the elderly. The Receptor for Advanced Glycation End-products (RAGE) is involved in age-related disorders. The soluble isoform (sRAGE) acts as a scavenger blocking the membrane-bound receptor activation. This study aims at investigating RAGE contribution to age-related cardiac remodeling.We analyzed the cardiac function of three different age groups of female Rage-/- and C57BL/6N (WT) mice: 2.5- (Young), 12- (Middle-age, MA) and 21-months (Old) old. While aging, Rage-/- mice displayed an increase in left ventricle (LV) dimensions compared to age-matched WT animals, with the main differences observed in the MA groups. Rage-/- mice showed higher fibrosis and a larger number of α-Smooth Muscle Actin (SMA)+ cells with age, along with increased expression of pro-fibrotic Transforming Growth Factor (TGF)-β1 pathway components. RAGE isoforms were undetectable in LV of WT mice, nevertheless, circulating sRAGE declined with aging and inversely associated with LV diastolic dimensions. Human cardiac fibroblasts stimulated with sRAGE exhibited a reduction in proliferation, pro-fibrotic proteins and TGF-beta Receptor 1 (TGFbR1) expression and Smad2-3 activation. Finally, sRAGE administration to MA WT animals reduced cardiac fibrosis.Hence, our work shows that RAGE associates with age-dependent myocardial changes and indicates sRAGE as an inhibitor of cardiac fibroblasts differentiation and age-dependent cardiac fibrosis.