Male-specific Phenotype Research Articles

Developmental thyroid hormone action on pro-opiomelanocortin-expressing cells programs hypothalamic BMPR1A depletion and brown fat activation.

Thyroid hormone excess secondary to global type 3 deiodinase (DIO3) deficiency leads to increased locomotor activity and reduced adiposity, but also to concurrent alterations in parameters of the leptin-melanocortin system that would predict obesity. To distinguish the underlying contributions to the energy balance phenotype of DIO3 deficiency, we generated mice with thyroid hormone excess targeted to pro-opiomelanocortin (POMC)-expressing cells via cell-specific DIO3 inactivation. These mice exhibit a male-specific phenotype of reduced hypothalamic Pomc expression, hyperphagia, and increased activity in brown adipose tissue, with adiposity and serum levels of leptin and thyroid hormones remained normal. These male mice also manifest a marked and widespread hypothalamic reduction in the expression of bone morphogenetic receptor 1a (BMPR1A), which has been shown to cause similar phenotypes when inactivated in POMC-expressing cells. Our results indicate that developmental overexposure to thyroid hormone in POMC-expressing cells programs energy balance mechanisms in a sexually dimorphic manner by suppressing adult hypothalamic BMPR1A expression.

Sexually dimorphic activation of innate antitumor immunity prevents adrenocortical carcinoma development.

Unlike most cancers, adrenocortical carcinomas (ACCs) are more frequent in women than in men, but the underlying mechanisms of this sexual dimorphism remain elusive. Here, we show that inactivation of Znrf3 in the mouse adrenal cortex, recapitulating the most frequent alteration in ACC patients, is associated with sexually dimorphic tumor progression. Although female knockouts develop metastatic carcinomas at 18 months, adrenal hyperplasia regresses in male knockouts. This male-specific phenotype is associated with androgen-dependent induction of senescence, recruitment, and differentiation of highly phagocytic macrophages that clear out senescent cells. In contrast, in females, macrophage recruitment is delayed and dampened, which allows for aggressive tumor progression. Consistently, analysis of TCGA-ACC data shows that phagocytic macrophages are more prominent in men and are associated with better prognosis. Together, these data show that phagocytic macrophages are key players in the sexual dimorphism of ACC that could be previously unidentified allies in the fight against this devastating cancer.

Maternal heme-enriched diet promotes a gut pro-oxidative status associated with microbiota alteration, gut leakiness and glucose intolerance in mice offspring

Maternal environment, including nutrition and microbiota, plays a critical role in determining offspring's risk of chronic diseases such as diabetes later in life. Heme iron requirement is amplified during pregnancy and lactation, while excessive dietary heme iron intake, compared to non-heme iron, has shown to trigger acute oxidative stress in the gut resulting from reactive aldehyde formation in conjunction with microbiota reshape. Given the immaturity of the antioxidant defense system in early life, we investigated the extent to which a maternal diet enriched with heme iron may have a lasting impact on gut homeostasis and glucose metabolism in 60-day-old C3H/HeN mice offspring.As hypothesized, the form of iron added to the maternal diet differentially governed the offspring's microbiota establishment despite identical fecal iron status in the offspring. Importantly, despite female offspring was unaffected, oxidative stress markers were however higher in the gut of male offspring from heme enriched-fed mothers, and were accompanied by increases in fecal lipocalin-2, intestinal para-cellular permeability and TNF-α expression. In addition, male mice displayed blood glucose intolerance resulting from impaired insulin secretion following oral glucose challenge. Using an integrated approach including an aldehydomic analysis, this male-specific phenotype was further characterized and revealed close covariations between unidentified putative reactive aldehydes and bacterial communities belonging to Bacteroidales and Lachnospirales orders.Our work highlights how the form of dietary iron in the maternal diet can dictate the oxidative status in gut offspring in a sex-dependent manner, and how a gut microbiota-driven oxidative challenge in early life can be associated with gut barrier defects and glucose metabolism disorders that may be predictive of diabetes development.

Sexual fate of murine external genitalia development: Conserved transcriptional competency for male-biased genes in both sexes

Testicular androgen is a master endocrine factor in the establishment of external genital sex differences. The degree of androgenic exposure during development is well known to determine the fate of external genitalia on a spectrum of female- to male-specific phenotypes. However, the mechanisms of androgenic regulation underlying sex differentiation are poorly defined. Here, we show that the genomic environment for the expression of male-biased genes is conserved to acquire androgen responsiveness in both sexes. Histone H3 at lysine 27 acetylation (H3K27ac) and H3K4 monomethylation (H3K4me1) are enriched at the enhancer of male-biased genes in an androgen-independent manner. Specificity protein 1 (Sp1), acting as a collaborative transcription factor of androgen receptor, regulates H3K27ac enrichment to establish conserved transcriptional competency for male-biased genes in both sexes. Genetic manipulation of MafB, a key regulator of male-specific differentiation, and Sp1 regulatory MafB enhancer elements disrupts male-type urethral differentiation. Altogether, these findings demonstrate conservation of androgen responsiveness in both sexes, providing insights into the regulatory mechanisms underlying sexual fate during external genitalia development.

A novel knockout mouse for the small EDRK-rich factor 2 (Serf2) showing developmental and other deficits

The small EDRK-rich factor 2 (SERF2) is a highly conserved protein that modifies amyloid fibre assembly in vitro and promotes protein misfolding. However, the role of SERF2 in regulating age-related proteotoxicity remains largely unexplored due to a lack of in vivo models. Here, we report the generation of Serf2 knockout mice using an ES cell targeting approach, with Serf2 knockout alleles being bred onto different defined genetic backgrounds. We highlight phenotyping data from heterozygous Serf2+/− mice, including unexpected male-specific phenotypes in startle response and pre-pulse inhibition. We report embryonic lethality in Serf2−/− null animals when bred onto a C57BL/6 N background. However, homozygous null animals were viable on a mixed genetic background and, remarkably, developed without obvious abnormalities. The Serf2 knockout mice provide a powerful tool to further investigate the role of SERF2 protein in previously unexplored pathophysiological pathways in the context of a whole organism.

Transvection regulates the sex-biased expression of a fly X-linked gene.

Sexual dimorphism in animals results from sex-biased gene expression patterns. These patterns are controlled by genetic sex determination hierarchies that establish the sex of an individual. Here we show that the male-biased wing expression pattern of the Drosophila biarmipes gene yellow, located on the X chromosome, is independent of the fly sex determination hierarchy. Instead, we find that a regulatory interaction between yellow alleles on homologous chromosomes (a process known as transvection) silences the activity of a yellow enhancer functioning in the wing. Therefore, this enhancer can be active in males (XY) but not in females (XX). This transvection-dependent enhancer silencing requires the yellow intron and the chromatin architecture protein Mod(mdg4). Our results suggest that transvection can contribute more generally to the sex-biased expression of X-linked genes.

Enhancer-gene interactions drive split

Sexual Dimorphism In many species across the animal kingdom, male and female phenotypes differ. One such example is the wing spot seen in male Drosophila biarmipes flies but not the female flies. Galouzis and Prud'homme investigate the X-linked yellow gene and its enhancer. Investigating the genetics of the trait, they found evidence that the male-specific phenotype is caused by a trans interaction between the enhancer and gene that silences the gene when it is present in only one copy in the male (which only has a single X chromosome) versus the two copies found in the female. This gene thus appears to be regulated by differing genomic interactions in male and female flies and is an example of the phenomenon known as transvection. Science , this issue p. [396][1] [1]: /lookup/doi/10.1126/science.abc2745

CCDC42 Localizes to Manchette, HTCA and Tail and Interacts With ODF1 and ODF2 in the Formation of the Male Germ Cell Cytoskeleton.

Terminal differentiation of male germ cells into functional spermatozoa requires shaping and condensation of the nucleus as well as the formation of sperm-specific structures. A transient microtubular structure, the manchette, is mandatory for sperm head shaping and the development of the connecting piece and the sperm tail. The connecting piece or head-to-tail coupling apparatus (HTCA) mediates the tight linkage of sperm head and tail causing decapitation and infertility when faulty. Using mice as the experimental model, several proteins have already been identified affecting the linkage complex, manchette or tail formation when missing. However, our current knowledge is far too rudimentary to even draft an interacting protein network. Depletion of the major outer dense fiber protein 1 (ODF1) mainly caused decapitation and male infertility but validated binding partners collaborating in the formation of sperm-specific structures are largely unknown. Amongst all candidate proteins affecting the HTCA when missing, the structural protein CCDC42 attracted our attention. The coiled-coil domain containing 42 (CCDC42) is important for HTCA and sperm tail formation but is otherwise largely uncharacterized. We show here that CCDC42 is expressed in spermatids and localizes to the manchette, the connecting piece and the tail. Beyond that, we show that CCDC42 is not restricted to male germ cells but is also expressed in somatic cells in which it localizes to the centrosome. Although centrosomal and sperm tail location seems to be irrespective of ODF1 we asked whether both proteins may form an interacting network in the male germ cell. We additionally considered ODF2, a prevalent protein involved in the formation of spermatid-specific cytoskeletal structures, as a putative binding partner. Our data depict for the first time the subcellular location of CCDC42 in spermatids and deepen our knowledge about the composition of the spermatid/sperm-specific structures. The presence of CCDC42 in the centrosome of somatic cells together with the obvious restricted male-specific phenotype when missing strongly argues for a compensatory function by other still unknown proteins most likely of the same family.

Rapid functional divergence after small-scale gene duplication in grasses

BackgroundGene duplication has played an important role in the evolution and domestication of flowering plants. Yet little is known about how plant duplicate genes evolve and are retained over long timescales, particularly those arising from small-scale duplication (SSD) rather than whole-genome duplication (WGD) events.ResultsWe address this question in the Poaceae (grass) family by analyzing gene expression data from nine tissues of Brachypodium distachyon, Oryza sativa japonica (rice), and Sorghum bicolor (sorghum). Consistent with theoretical predictions, expression profiles of most grass genes are conserved after SSD, suggesting that functional conservation is the primary outcome of SSD in grasses. However, we also uncover support for widespread functional divergence, much of which occurs asymmetrically via the process of neofunctionalization. Moreover, neofunctionalization preferentially targets younger (child) duplicate gene copies, is associated with RNA-mediated duplication, and occurs quickly after duplication. Further analysis reveals that functional divergence of SSD-derived genes is positively correlated with both sequence divergence and tissue specificity in all three grass species, and particularly with anther expression in B. distachyon.ConclusionsOur results suggest that SSD-derived grass genes often undergo rapid functional divergence that may be driven by natural selection on male-specific phenotypes. These observations are consistent with those in several animal species, suggesting that duplicate genes take similar evolutionary trajectories in plants and animals.

Sex Differences in Androgen Receptor Enhancer Activity and Expression during Mus musculus Embryonic Development

Study objectiveThe objective of this study is to compare the sex‐specific enhancer function of a cis‐regulatory element during mammary gland development to the expression of the androgen receptor (AR) in mammary tissues.IntroductionConserved noncoding elements regulate gene expression during mammalian development with remarkable precision. AR is critical for the development of male specific phenotypes in many tissues, yet little is known regarding the tissue‐specific regulation of AR expression. We have previously identified a 7.7kb sequence near the AR gene that activates lacZ reporter gene expression in prenatal and postnatal mice. Embryologically, the enhancer drives consistent lacZ expression in developing hair follicles and the genital tubercle. However, expression in the mammary ridge/glands occurs in only half of the transgenic embryos produced. Each of these structures are known sites of expression of AR or are affected by androgens during growth and development. We hypothesize that the variability in this enhancer function during mammary gland development may be sex dependent.MethodsWe utilized a previously established transgenic mouse line containing the 7.7kb AR enhancer mouse sequence linked to a basal promoter driving lacZ. Mice were collected and stained during different stages of embryonic development to visualize localization of AR enhancer activity. The sex of the embryos were identified by PCR amplification of the Sry gene. We compared enhancer driven lacZ expression to endogenous AR expression determined by in situ hybridization (ISH) in mouse embryos.ResultsWe have surveyed lacZ expression at E15.5 (embryonic day) and E16.5. Despite consistent expression in other tissues, mammary gland expression of lacZ occurs only in female embryos. No mammary associated lacZ expression is visible in males. These expression results are consistent with AR gene expression as detected by ISH.ConclusionWe show that there are sex differences in enhancer activity of AR during mouse development. AR is crucial for inhibiting the development of female‐specific traits as well as promoting the male phenotype. The lack of lacZ expression in male embryos suggests the inhibitory effects of androgens have been sufficient to either eliminate the mammary associated cell types that normally express AR or the expression of an upstream trans‐acting factor that activates this AR enhancer. This mouse line will be useful for tracing developmental progression of AR expressing tissues and signalling during mammalian integumentary development as well as the development of sexually dimorphic characteristics.Support or Funding InformationFunding provided by NSF IOS‐1656315 & NSF BCS‐1638812 and PCOM's Center for Chronic Disorders of Aging.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SNP variation in male pattern hair loss in Russians with different dihydrotestosterone levels

The male-specific phenotype termed androgenetic alopecia or male pattern hair loss (MPH) often affects quality of life. The development of this pattern is mainly caused by a wide spectrum of genomic peculiarities involving metabolism, follicle development, cell signaling, autophagy, and epigenetic regulation. In this study the prediction model proposed by Marcinska et al. (2015) is based on the variability of 5 single-nucleotide polymorphisms (SNPs) that are supposed to be strongly associated with MPHL development has been applied to Russians. The 5 SNPs, namely rs5919324 (upstream of the AR gene region), rs1998076 (20p11 locus), rs929626 (EBF1 gene), rs12565727 (TARDBP gene), and rs756853 (HDAC9 gene), were analyzed in group with MPHL phenotype, following subdivision based on dihydrotestosterone (DHT) level, and healthy control group. Significant differences were not found between separate allele and genotype frequencies at the MPHL group and healthy individuals, but the application of artificial neural network to the dataset allows distinguishing the group variability patterns. Prediction modeling was implemented using the multilayer perceptron neural networks and model fitness was assessed using receiver-operating characteristic analytical curves. The DHT level subgroups were compared for the first time, revealing the association between AR genomic region (SNP rs5919324) and MPHL patients with high DHT level. The split of the MPHL group into subgroups according to DHT level and their comparison with the controls led to an increase in the AUC of the multilayer perceptron network to 0.85, revealing the increase of the effect of genomic variability on MPHL development under normal DHT levels.

Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

Spermatogenesis is a dynamic process involving self-renewal and differentiation of spermatogonial stem cells, meiosis, and ultimately, the differentiation of haploid spermatids into sperm. Centrosomal protein 55 kDa (CEP55) is necessary for somatic cell abscission during cytokinesis. It facilitates equal segregation of cytoplasmic contents between daughter cells by recruiting endosomal sorting complex required for transport machinery (ESCRT) at the midbody. In germ cells, CEP55, in partnership with testes expressed-14 (TEX14) protein, has also been shown to be an integral component of intercellular bridge before meiosis. Various in vitro studies have demonstrated a role for CEP55 in multiple cancers and other diseases. However, its oncogenic potential in vivo remains elusive. To investigate, we generated ubiquitously overexpressing Cep55 transgenic ( Cep55Tg/Tg) mice aiming to characterize its oncogenic role in cancer. Unexpectedly, we found that Cep55Tg/Tg male mice were sterile and had severe and progressive defects in spermatogenesis related to spermatogenic arrest and lack of spermatids in the testes. In this study, we characterized this male-specific phenotype and showed that excessively high levels of Cep55 results in hyperactivation of PI3K/protein kinase B (Akt) signaling in testis. In line with this finding, we observed increased phosphorylation of forkhead box protein O1 (FoxO1), and suppression of its nuclear retention, along with the relative enrichment of promyelocytic leukemia zinc finger (PLZF) -positive cells. Independently, we observed that Cep55 amplification favored upregulation of ret ( Ret) proto-oncogene and glial-derived neurotrophic factor family receptor α-1 ( Gfra1). Consistent with these data, we observed selective down-regulation of genes associated with germ cell differentiation in Cep55-overexpressing testes at postnatal day 10, including early growth response-4 ( Egr4) and spermatogenesis and oogenesis specific basic helix-loop-helix-1 ( Sohlh1). Thus, Cep55 amplification leads to a shift toward the initial maintenance of undifferentiated spermatogonia and ultimately results in progressive germ cell loss. Collectively, our findings demonstrate that Cep55 overexpression causes change in germ cell proportions and manifests as a Sertoli cell only tubule phenotype, similar to that seen in many azoospermic men.-Sinha, D., Kalimutho, M., Bowles, J., Chan, A.-L., Merriner, D. J., Bain, A. L., Simmons, J. L., Freire, R., Lopez, J. A., Hobbs, R. M., O'Bryan, M. K., Khanna, K. K. Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

Enduring effects of perinatal nicotine exposure on murine sleep in adulthood.

The long-term consequences of early life nicotine exposure are poorly defined. Approximately 8-10% of women report smoking during pregnancy, and this may promote aberrant development in the offspring. To this end, we investigated potential enduring effects of perinatal nicotine exposure on murine sleep and affective behaviors in adulthood (~13-15 wk of age) in C57Bl6j mice. Mothers received a water bottle containing 200 µg/ml nicotine bitartrate dihydrate in 2% wt/vol saccharin or pH-matched 2% saccharin with 0.2% (vol/vol) tartaric acid throughout pregnancy and before weaning. Upon reaching adulthood, offspring were tested in the open field and elevated plus maze, as well as the forced swim and sucrose anhedonia tests. Nicotine-exposed male (but not female) mice had reduced mobility in the open field, but no differences were observed in anxiety-like or depressive-like responses. Upon observing this male-specific phenotype, we further assessed sleep-wake states via wireless EEG/EMG telemetry. Following baseline recording, we assessed whether mice exposed to nicotine altered their homeostatic response to 5 h of total sleep deprivation and whether nicotine influenced responses to a powerful somnogen [i.e., lipopolysaccharides (LPS)]. Males exposed to perinatal nicotine decreased the percent time spent awake and increased time in non-rapid eye movement (NREM) sleep, without changes to REM sleep. Nicotine-exposed males also displayed exaggerated responses (increased time asleep and NREM spectral power) to sleep deprivation. Nicotine-exposed animals additionally had blunted EEG slow-wave responses to LPS administration. Together, our data suggest that perinatal nicotine exposure has long-lasting effects on normal sleep and homeostatic sleep processes into adulthood.

Ehlers–Danlos syndrome with lethal cardiac valvular dystrophy in males carrying a novel splice mutation in FLNA

Filamin A is an X-linked, ubiquitous actin-binding protein whose mutations are associated to multiple disorders with limited genotype-phenotype correlations. While gain-of-function mutations cause various bone dysplasias, loss-of-function variants are the most common cause of periventricular nodular heterotopias with variable soft connective tissue involvement, as well as X-linked cardiac valvular dystrophy (XCVD). The term "Ehlers-Danlos syndrome (EDS) with periventricular heterotopias" has been used in females with neurological, cardiovascular, integument and joint manifestations, but this nosology is still a matter of debate. We report the clinical and molecular update of an Italian family with an X-linked recessive soft connective tissue disorder and which was described, in 1975, as the first example of EDS type V of the Berlin nosology. The cutaneous phenotype of the index patient was close to classical EDS and all males died for a lethal cardiac valvular dystrophy. Whole exome sequencing identified the novel c.1829-1G>C splice variation in FLNA in two affected cousins. The nucleotide change was predicted to abolish the canonical splice acceptor site of exon 13 and to activate a cryptic acceptor site 15 bp downstream, leading to in frame deletion of five amino acid residues (p.Phe611_Gly615del). The predicted in frame deletion clusters with all the mutations previously identified in XCVD and falls within the N-terminus rod 1 domain of filamin A. Our findings expand the male-specific phenotype of FLNA mutations that now includes classical-like EDS with lethal cardiac valvular dystrophy, and offer further insights for the genotype-phenotype correlations within this spectrum. © 2016 Wiley Periodicals, Inc.

Abstract T P324: Sex Differences in Ischemic Outcomes and Inflammatory Responses in Neonatal Stroke

Background and Purpose: Neonatal arterial ischemic stroke (NAIS) is an important cause of motor and cognitive impairment in children. Clinically, male infants are more vulnerable to ischemic insult and suffer more long-term deficits than female infants though the mechanisms remain elusive. Inflammatory processes are fundamental in the pathophysiology of ischemia as microglial activation initiates the inflammatory response after ischemia. Recent studies report a sexual dimorphism in microglia numbers and expression of activation markers in neonatal brains under normal conditions. How these basal sex differences in microglia affect NAIS remains largely unexplored. This study investigated sex differences in stroke phenotypes and inflammation triggered by NAIS. We hypothesize that ischemia induces sex-specific tissue injury in male and female neonates, which is related to differences in microglial activation and inflammatory responses. Methods: Male and female C57BL6 mice were subjected to 60-minute Rice-Vanucci Modeling at post-natal day 10 (P10) to induce NAIS. Stroke outcomes were measured at 24 hours, 72 hours and 7 days after stroke. Microglial activation and inflammatory responses were evaluated by flow cytometry, immunohistochemistry, and multiplex cytokine analysis. Results: At 24 hours no difference in infarct volumes (total infarct: male vs. female 46.6±7.2% vs. 43.2±9.3%, n=6/gp) and in Iba-1 staining of the ischemic brain were seen between male and female neonates. However, at 72 hours female neonates exhibited significantly smaller infarct size and improved behavior outcomes compared to males (total infarct: male vs. female 43.1±9.9% vs. 27.1±8.8%, n=6/gp, p <.05). Male animals demonstrated increased microglial activation and up-regulated inflammatory response compared to females at 72 hours. This male-specific phenotype was also seen at 7 days after injury. There was no difference in hormone levels at any of the three time points after stroke. Conclusions: Acute ischemia leads to an equivalent primary brain injury in male and female P10 mice. However, infarct damage worsens in males at sub-acute time points vs. females, as does the immune response. This sex difference independent of hormone levels exists in NAIS.

Androgen Receptor (AR) Physiological Roles in Male and Female Reproductive Systems: Lessons Learned from AR-Knockout Mice Lacking AR in Selective Cells1

Androgens/androgen receptor (AR) signaling is involved primarily in the development of male-specific phenotypes during embryogenesis, spermatogenesis, sexual behavior, and fertility during adult life. However, this signaling has also been shown to play an important role in development of female reproductive organs and their functions, such as ovarian folliculogenesis, embryonic implantation, and uterine and breast development. The establishment of the testicular feminization (Tfm) mouse model exploiting the X-linked Tfm mutation in mice has been a good in vivo tool for studying the human complete androgen insensitivity syndrome, but this mouse may not be the perfect in vivo model. Mouse models with various cell-specific AR knockout (ARKO) might allow us to study AR roles in individual types of cells in these male and female reproductive systems, although discrepancies are found in results between labs, probably due to using various Cre mice and/or knocking out AR in different AR domains. Nevertheless, no doubt exists that the continuous development of these ARKO mouse models and careful studies will provide information useful for understanding AR roles in reproductive systems of humans and may help us to develop more effective and more specific therapeutic approaches for reproductive system-related diseases.

Mitochondrial haplotype does not affect sperm velocity in the zebra finchTaeniopygia guttata

Mitochondrial DNA (mtDNA) variation has been suggested as a possible cause of variation in male fertility because sperm activity is tightly coupled to mitochondrial oxidative phosphorylation and ATP production, both of which are sensitive to mtDNA mutations. Since male-specific phenotypes such as sperm have no fitness consequences for mitochondria due to maternal mitochondrial (and mtDNA) inheritance, mtDNA mutations that are deleterious in males but which have negligible or no fitness effect in females can persist in populations. How often such mutations arise and persist is virtually unknown. To test whether there were associations between mtDNA variation and sperm performance, we haplotyped 250 zebra finches Taeniopygia guttata from a large pedigreed-population and measured sperm velocity using computer-assisted sperm analysis. Using quantitative genetic 'animal' models, we found no effect of mtDNA haplotype on sperm velocity. Therefore, there is no evidence that in this system mitochondrial mutations have asymmetric fitness effects on males and females, leading to genetic variation in male fertility that is blind to natural selection.

Loss of ERalpha-Mediated Estrogen Signaling Resulted in the Ovarian Expression of Male-Specific Gene.

Sex hormones play a critical role in the development as well as maintenance of reproductive organs and their reproductive characteristics. While it is well established that estrogen plays a critical regulatory role in the developing gonads, whether it has a sex-specific functional role in sustaining the gonadal functions is not well known. Recent gene knockout approach has found that loss of estrogen signaling by knocking either aromatase or estrogen receptors resulted in the formation of somniferous tubule-like structure in the ovary. This surprising finding led us to hypothesize that estrogen suppresses the genes that confer male-specific characteristics. To test the hypothesis, we examined whether deletion of ER gene induced male-reproductive track-specific gene expression in the ovary. For this purpose, we compared gene expression profiles in the presence or absence of ERα-mediated estrogen signaling using DNA microarray. Briefly, total RNA extracted from wild-type (WT) and ERα knockout (ERαKO) mouse ovaries was subjected to microarray, which identified a number of differentially expressed genes. Among those genes are epididymal protease inhibitor (Eppin), seminal vesicle secretion protein (SVS) 5, and SVS6, which are all known to be specifically expressed in male reproductive track. The expression of Eppin mRNA was 10 folds higher in ERαKO than the baseline Eppin mRNA content in the WT ovary, while SVS5 and SVS6 mRNA showed 60 and 80 folds higher in the ER αKO, respectively. Expression of Eppin is well documented in male reproductive system (testis, epididymis, and spermatozoa) and is currently being studied as a potential target of male contraceptives. SVS proteins are semen coagulating factors and are known to enhance sperm motility. As was expected, no expression of Eppin or SVS has been reported in female reproductive tissues. To detect protein expression in the ERαKO ovary, we employed immunohistochemistry. Interestingly, however, unlike the above-mentioned strong Eppin mRNA expression in the ERαKO ovary, the protein expression level was very low compared to the strong expression in the epididymal epithelial cells, testis Sertoli cells, and spermatozoa. We are currently investigating the potential damaging effect of Eppin expression in the ovary. In conclusion, this novel finding suggests that estrogen may contribute to the maintenance of functional and potentially structural integrity of female gonad by suppressing the expression of genes that induce male specific phenotypes if expressed. Supported by a COBRE grant from NIH NCRR: P20 RR15592. (poster)

A male‐specific effect of dominant‐negative Fos

The transcription factor Fos contains a basic DNA binding domain combined with a leucine zipper (bZip). Expression of a truncated form of Fos in Drosophila that contains only the bZip region (Fos bZip) elicits phenotypes resembling fos mutations. These effects presumably derive from competition between wild-type and truncated forms for dimerization partners, with the truncation acting in a dominant-negative manner. We found that expression of Fos bZip elicits male-specific phenotypes. Moreover, genetic interactions occur between Fos bZip and mutations in loci encoding the X chromosome dosage compensation complex. Fos bZip effects are correlated with aberrant male X chromosome structure and depressed signaling through the X-linked Notch locus. Unexpectedly, the male-specific effects are not reproduced with Fos RNAi, suggesting that Fos bZip can be neomorphic in nature. These results provide insight into how mutations in bZip proteins can exhibit gain of function activity.

Pulsatility of growth hormone (GH) signalling in liver cells: role of the JAK-STAT5b pathway in GH action.

The intracellular signalling molecule and transcriptional activator STAT5b is a key mediator of the effects of intermittent plasma growth hormone (GH) pulses on the male-specific pattern of liver gene expression and pubertal body growth rates in rodents. Experiments with Stat5b gene-knockout mice have revealed that these GH-regulated, male-specific phenotypes are a direct consequence of GH pulse-dependent STAT5b activation and that loss of function of STAT5b cannot be compensated for by the closely related signalling molecule STAT5a. Physiological plasma GH pulses are required to obtain the high levels of activated STAT5b seen in the livers of males, and down-regulation of the GH receptor (GHR)-JAK-STAT5b pathway in hepatocytes exposed to GH in a near-continuous fashion underlies the low level of liver STAT5b activity that is characteristic of adult female rats. Termination of nuclear STAT5b signalling occurs at the conclusion of a plasma GH pulse, with STAT5b deactivation catalysed by a tyrosine phosphatase. In males, termination of the intracellular signalling stimulated by a plasma GH pulse is proposed to be additionally facilitated by GH-STAT5b-inducible SOCS-CIS proteins, which block the further activation of STAT5b by binding to and inhibiting the action of the GHR-JAK2 complex via multiple mechanisms. In this manner, the liver cell is rendered temporarily unresponsive to further GH-signalling events. SOCS-CIS proteins synthesized in liver cells stimulated continuously with GH may also contribute to the apparent down-regulation of STAT5b signalling that is observed in the female rat liver.