Dimorphic Expression Research Articles

Distribution of corticotropin-releasing factor receptor 1 in the developing mouse forebrain: A novel sex difference revealed in the rostral periventricular hypothalamus

Corticotropin-releasing factor (CRF) signaling through CRF receptor 1 (CRFR1) regulates autonomic, endocrine and behavioral responses to stress and has been implicated in the pathophysiology of several disorders including anxiety, depression, and addiction. Using a validated CRFR1 reporter mouse line (bacterial artificial chromosome identified by green fluorescence protein (BAC GFP-CRFR1)), we investigated the distribution of CRFR1 in the developing mouse forebrain. Distribution of CRFR1 was investigated at postnatal days (P) 0, 4, and 21 in male and female mice. CRFR1 increased with age in several regions including the medial amygdala, arcuate nucleus, paraventricular hypothalamus, medial septum, CA1 hippocampal area, and the lateral habenula. Regions showing decreased CRFR1 expression with increased age include the intermediate portion of the periventricular hypothalamic nucleus, and CA3 hippocampal area. We report a sexually dimorphic expression of CRFR1 within the rostral portion of the anteroventral periventricular nucleus of the hypothalamus (AVPV/PeN), a region known to regulate ovulation, reproductive and maternal behaviors. Females had a greater number of CRFR1-GFP-ir cells at all time points in the AVPV/PeN and CRFR1-GFP-ir was nearly absent in males by P21. Overall, alterations in CRFR1-GFP-ir distribution based on age and sex may contribute to observed age- and sex-dependent differences in stress regulation.

Expression pattern of foxl2 and dmrt1 in gonad of Amur sturgeon Acipenser schrenckii in relation to sex differentiation

The mechanisms that govern sex differentiation in sturgeon are poorly understood. This study aimed to examine sexual dimorphic expression of foxl2 and dmrt1 in Amur sturgeon Acipenser schrenckii during molecular and morphological sex differentiation. Histologically, ovaries were first observed at 9months after hatching (mah), reflected in an invaginated gonadal epithelium and the presence of germ cells just below the epithelium, whereas gonads with smooth epithelium and germ cells in the stroma were characterized as testis. Some fish showed undifferentiated gonads until 16mah, but all sampled fish had morphologically differentiated sex by 19mah. The full-length cDNAs of foxl2 and two types of dmrt1, dmrt1a and dmrt1b, of Amur sturgeon were isolated. The 2 types of dmrt1 cDNA had an identical structure from the 5′-UTR to the Y-rich domain, whereas the sequence downstream from this domain showed no homology and is considered to have resulted from alternative splicing. Foxl2 was principally expressed in the ovary and the 2 types of dmrt1 in the testis of 7-year-old Amur sturgeon, and much less so in various somatic tissues. Foxl2 expression was sexually dimorphic in morphologically differentiated gonads and was similarly dichotomous in the undifferentiated gonads of fish at 9mah. In contrast, sexually dimorphic expression of dmrt1a and dmrt1b was not observed in gonads of the juvenile Amur sturgeon, but these genes showed testis-dominant expression in the adults. We suggest that foxl2 expression is closely related to ovarian differentiation and that two types of dmrt1 play a role in testis development and/or sperm formation rather than testicular differentiation. Additionally, we suggest that mRNA levels of foxl2 are an appropriate marker for sexing of sturgeon at an early developmental stage.

A novel role of Krüppel-like factor 4 in Zhikong scallop Chlamys farreri during spermatogenesis.

Krüppel-like factor 4 (KLF4) is a kind of zinc finger transcription factor, which is involved in terminal differentiation of epithelial cells and reprogramming of somatic cells to induced pluripotent stem (iPS) cells in mammals. In the present study, we identified a full-length cDNA of Klf4 in Zhikong scallop Chamys farreri (Cf-Klf4) and found that Cf-Klf4 presented a sexual dimorphic expression characteristic in C. farreri gonads. Cf-Klf4 expression was significantly higher in testes than in ovaries from growing stage to mature stage detected by quantitative real-time PCR, and was located in male gametes, except for spermatozoa during spermatogenesis through in situ hybridization and immunohistochemistry, while no positive signal was visible in female gametes during oogenesis. Furthermore, the knockdown of Cf-Klf4 in testes by means of in vivo RNA interference led to an obviously developmental retardance, lower gonadosomatic index, less male gametes and more apoptotic spermatocytes. Interestingly, we found that two out of eight scallops showed a hermaphroditic phenotype characteristic of male-to-female sex reversal when the Klf4 mRNA and protein levels were knocked down in males. These results verified that Klf4 plays an important role in testis functional maintenance and is necessary in spermatogenesis of C. farreri.

Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a-eGFP zebrafish line

In zebrafish, there exists a clear need for new tools to study sex differentiation dynamic and its perturbation by endocrine disrupting chemicals. In this context, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a (gonadal aromatase) promoter. In most gonochoristic fish species including zebrafish, cyp19a1a, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the processes of reproduction and sexual differentiation. This novel cyp19a1a-eGFP transgenic line allowed a deeper characterization of expression and localization of cyp19a1a gene in zebrafish gonads both at the adult stage and during development. At the adult stage, GFP expression was higher in ovaries than in testis. We showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in gonads that was mainly localized in the cytoplasm of peri-follicular cells in the ovary and of Leydig and germ cells in the testis. During development, GFP was expressed in all immature gonads of 20 dpf-old zebrafish. Then, GFP expression increased in early differentiated female at 30 and 35dpf to reach a high GFP intensity in well-differentiated ovaries at 40dpf. On the contrary, males consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between both sexes. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40dpf) presented GFP levels similar to males or intermediate between females and males. In this transgenic line our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for the testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development of both sexes. Monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line will be a useful tool to study physiological processes such as reproduction and sexual differentiation, and their perturbations by EDCs.

Somatostatin-IRES-Cre Mice: Between Knockout and Wild-Type?

The neuropeptide somatostatin (SOM) is widely expressed in rodent brain and somatostatin-IRES-Cre (SOM-cre) mouse strains are increasingly used to unravel the physiology of SOM-containing neurons. However, while knock-in targeting strategy greatly improves Cre-Lox system accuracy, recent reports have shown that genomic insertion of Cre construct per se can markedly affect physiological function. We show that Cre transgene insertion into the 3′UTR of the somatostatin gene leads to the selective and massive depletion of endogenous SOM in all tested brain regions. It also strongly impacts SOM-related neuroendocrine responses in a similar manner to what has been reported for SST KO mice: increased corticosterone levels after 30-min restraint stress, decreased amplitude and regularity of ultradian growth hormone secretory patterns accompanied by changes in sexually dimorphic liver gene expression (serpina1, Cyp2b9, Cyp2a4, Cyp2d9, and Cyp7b1). In addition to demonstrating the need for examination of the consequences of Cre transgenesis, these results also reveal how this SOM-cre strain may be a useful tool in studying the functional consequences of moderate to low SOM levels as reported in neurological and psychiatric disorders.

Sexual dimorphic expression of TrkB, TrkB-T1, and BDNF in the medial preoptic area of the Syrian hamster

Neurotrophins regulate many aspects of neuronal function and activity. Specifically, the binding of Brain-derived neurotrophic factor (BDNF) to Tyrosine receptor kinase-B (TrkB) or its truncated version, TrkB-T1, can cause growth and differentiation or dominant inhibition of receptor signaling, respectively. There is evidence that these neurotropic effects on nervous tissue, in both the central and peripheral nervous system, behave differently between the sexes. This study used western blots to examine the expression of these neurotrophins in the medial preoptic area (MPOA), a sexually dimorphic region of the hamster brain that controls male sex behavior. We report that TrkB-FL and BDNF show greater expression in male MPOA tissue, when compared to female. On the contrary, TrkB-T1 is expressed in greater abundance in the female MPOA. Our results indicate a clear sexual dimorphism of neurotrophins in the MPOA of the Syrian hamster. Furthermore, the greater expression of TrkB-FL and BDNF in the male MPOA suggests that these neurotrophins could be promoting synaptic growth to facilitate male-typical copulation. In contrast, the greater TrkB-T1 expression in the female MPOA suggests a possible inhibition of synaptic growth, and may contribute to the lack of male-typical copulation. Altogether, our data suggests that neurotrophins may play a larger role sexual differentiation than previously thought.

Genomics of sex hormone receptor signaling in hepatic sexual dimorphism.

The liver plays a crucial role in a variety of physiological processes. Sexual dimorphism is markedly defined in liver disorders, such as fatty liver diseases and liver cancer, but barely addressed in the normal liver. Distinct sex hormone signaling between male and female livers is the major driving factor for hepatic sexual dimorphism. Over 6000 genes are differently expressed between male and female livers in mice. Here we address how sex hormone receptors, estrogen receptor alpha (ERα) and androgen receptor (AR), mediate sexually dimorphic gene expression in mouse livers. We identified 5192ERα target genes and 4154 AR target genes using ChIP-Seq. Using liver-specific ERα or AR knockout mice, we further identified direct and functional target genes of ERα (123 genes) and AR (151 genes) that contribute to hepatic sexual dimorphism. We also found that the most significant sexually dimorphic gene expression was initiated at birth by comparing hepatic gene expression data from the embryonic stage E10.5 to the postnatal stage P60 during liver development. Overall, our study indicates that sex hormone receptor signaling drives sexual dimorphism of hepatic gene expression throughout liver development.

Functional characterization of <italic>Cyp19a1</italic> in female sexual differentiation in <italic>Pelodiscus sinensis</italic>

Aromatase is the key enzyme for estrogen synthesis, and functions as a key regulator in early female sexual differentiation in non-mammalian vertebrates. In this study, the expression pattern of Cyp19a1 was examined in tissues and its functional role in ovarian differentiation was investigated through lentivirus-mediated RNA interference and over-expression systems. Results showed that Cyp19a1 was robustly expressed in adult ovary, and was not or weakly detected in testis, heart, liver, spleen, lung, kidney, muscle and intestine. Moreover, Cyp19a1 exhibited female sexual dimorphic expression pattern in embryonic gonads early at stage 18, preceding the onset time of gonadal differentiation (stage 19). Loss-of-function analysis revealed that ZW embryos with AI treatment or Cyp19a1 knockdown exhibited female to male sex reversal, characterized by obviously masculinized gonads with well-organized testis cord structure in the medulla and a degenerated cortex, an upregulation of testicular marker Sox9 and Amh , and a decline in ovarian marker Foxl2 . Gain-of-function analysis showed that ZZ gonads overexpressing Cyp19a1 were feminized, determined by a thickened outer cortex with a number of germ cells and a vacuolated medulla, as well as upregulation of Foxl2 and downregulation of Sox9 and Amh . In summary, Cyp19a1 is both necessary and sufficient to initiate ovarian development, thereby functioning as a key regulator of female sexual differentiation. This study provides a foundation for understanding the mechanism of sex determination in Pelodiscus sinensis.

Sexually dimorphic expression in developing and adult gonads shows an important role of gonadal soma-derived factor during sex differentiation in olive flounder (Paralichthys olivaceus).

Gonadal soma-derived factor (gsdf) is a new member of transforming growth factor beta (TGF-β) superfamily. As a teleost- and gonad-specific growth factor, gsdf has been indicated to play an important role in early germ cell development. However, little is known about its significance in germ cell development of olive flounder (Paralichthys olivaceus). In the present study, a 1338 bp gsdf gene was isolated from P. olivaceus for the first time. Bioinformatic analysis revealed that the genomic structure and synteny relationship of gsdf in teleosts were conserved. Quantitative real-time PCR (qRT-PCR) showed that gsdf expressed before sex gonadal differentiation, and the expression level increased rapidly after initiation of sex differentiation in males. In adult individuals, the expression of gsdf was higher in testis than that in ovary (P<0.01). In situ hybridization (ISH) indicated that gsdf mRNA was detected in the somatic cells of both males and females, and also in the cytoplasm of oocytes. These results suggested that gsdf might play an important role as initial switches to promote testis differentiation and participate in early germ cell development, such as proliferation and differentiation of spermatogonia and oogonia in P. olivaceus.

Two spliced isoforms of the sex-determination gene fruitless in the Chinese mitten crab Eriocheir sinensis.

The fruitless (fru) gene plays an important role in the sex determination pathway and courtship behavior of Drosophila melanogaster. In the present study, two fru isoforms (Esfru1 and Esfru2) were identified from the Chinese mitten crab Eriocheir sinensis. Sequence analysis showed that Esfru1 and Esfru2 were encoded by the same genomic locus and generated by alternative splicing of pre-mRNA. Esfru1 had all introns completely spliced out, while Esfru2 had a longer exon1 with an additional 78bp sequence. They both contained a conserved BTB domain which was also found in D. melanogaster Fru isoforms. Analysis on temporal expression profiles of Esfru1-2 (a common region of Esfru1 and Esfru2) and Esfru2 (a specific region of Esfru2) showed that they expressed in similar patterns during embryonic development but in different patterns during larval development. The expression of Esfru1-2 decreased gradually from zoea III stage, however, Esfru2 increased from zoea IV stage and reached the peak at megalopa stage. Expression distribution in tissues and in situ hybridization analysis revealed that they showed sexually dimorphic expression in gonads, hepatopancreas and brains. Esfru1-2 showed significantly higher expression in female gonads and hepatopancreas than in males, but highly expressed in male brains than that in females. Interestingly, Esfru2 was displayed in a male-specific manner in each tissue, especially in gonads and hepatopancreas. Our results indicate that Esfru1 might be involved in both sexual brain neuronal structure development and related to female-specific character development. Esfru2 may participate in male-specific character development. This is the first report that characterizes two spliced variants of fru in crustaceans and provides basic information for further functional studies of the crab sex-determination mechanism.

Identification and expression of piwil2 in turbot Scophthalmus maximus, with implications of the involvement in embryonic and gonadal development.

Piwil2, a member of the Argonaute family, is involved in the biogenesis of PIWI-interacting RNAs (piRNAs) and plays an important role in regulating gametogenesis. In the present study, we identified turbot Scophthalmus maximus piwil2 gene, named Smpiwil2, which contained a PAZ domain and a PIWI domain. Sequence comparison, genomic structure and phylogenetic analyses showed that Smpiwil2 is homologous to that of teleosts and tetrapods. The Smpiwil2 transcript showed higher expression in the ovary than in the testis, demonstrating a sexually dimorphic gene expression pattern. In situ hybridization (ISH) showed that Smpiwil2 was expressed in the oogonia and all the stages of oocytes in the ovary as well as in spermatogonia and spermatocytes in the testis. Embryonic expression profile revealed that Smpiwil2 was maternally inherited, and its level was higher from the zygote to the blastula stage and subsequently decreased until hatching. Moreover, a CpG island was predicted to locate in the 5'-flanking region of Smpiwil2 gene, and its methylation levels detected by sodium bisulfite sequencing showed significant disparity between females and males, implying that the sexually dimorphic expression of Smpiwil2 might be regulated by methylation. These results indicated that Smpiwil2 had potentially vital functions in embryonic and gonadal development in this species. In addition, the temporal and sex differences in Smpiwil2 expression indicated that this gene may play different roles in gonadal development of different sexes.

Widespread patterns of sexually dimorphic gene expression in an avian hypothalamic\u2013pituitary\u2013gonadal (HPG) axis

The hypothalamic-pituitary-gonadal (HPG) axis is a key biological system required for reproduction and associated sexual behaviors to occur. In the avian reproductive model of the rock dove (Columba livia), we characterized the transcript community of each tissue of the HPG axis in both sexes, thereby significantly expanding our mechanistic insight into HPG activity. We report greater sex-biased differential expression in the pituitary as compared to the hypothalamus, with multiple genes more highly expressed in the male pituitary being related to secretory function, and multiple genes more highly expressed in the female pituitary being related to reproduction, growth, and development. We report tissue-specific and sex-biased expression in genes commonly investigated when studying reproduction, highlighting the need for sex parity in future studies. In addition, we uncover new targets of investigation in both sexes, which could potentially change our understanding of HPG function.

Caste-, sex-, and age-dependent expression of immune-related genes in a Japanese subterranean termite, Reticulitermes speratus.

Insects protect themselves from microbial infections through innate immune responses, including pathogen recognition, phagocytosis, the activation of proteolytic cascades, and the synthesis of antimicrobial peptides. Termites, eusocial insects inhabiting microbe-rich wood, live in closely-related family groups that are susceptible to shared pathogen infections. To resist pathogenic infection, termite families have evolved diverse immune adaptations at both individual and societal levels, and a strategy of trade-offs between reproduction and immunity has been suggested. Although termite immune-inducible genes have been identified, few studies have investigated the differential expression of these genes between reproductive and neuter castes, and between sexes in each caste. In this study, we compared the expression levels of immune-related genes among castes, sexes, and ages in a Japanese subterranean termite, Reticulitermes speratus. Using RNA-seq, we found 197 immune-related genes, including 40 pattern recognition proteins, 97 signalling proteins, 60 effectors. Among these genes, 174 showed differential expression among castes. Comparing expression levels between males and females in each caste, we found sexually dimorphic expression of immune-related genes not only in reproductive castes, but also in neuter castes. Moreover, we identified age-related differential expression of 162 genes in male and/or female reproductives. In addition, although R. speratus is known to use the antibacterial peptide C-type lysozyme as an egg recognition pheromone, we determined that R. speratus has not only C-type, but also P-type and I-type lysozymes, as well as other termite species. Our transcriptomic analyses revealed immune response plasticity among all castes, and sex-biased expression of immune genes even in neuter castes, suggesting a sexual division of labor in the immune system of R. speratus. This study heightens the understanding of the evolution of antimicrobial strategies in eusocial insects, and of sexual roles in insect societies as a whole.

The elusive MAESTRO gene: Its human reproductive tissue-specific expression pattern.

The encoded transcript of the Maestro—Male-specific Transcription in the developing Reproductive Organs (MRO) gene exhibits sexual dimorphic expression during murine gonadal development. The gene has no homology to any known gene and its expression pattern, protein function or structure are still unknown. Previously, studying gene expression in human ovarian cumulus cells, we found increased expression of MRO in lean-type Polycystic Ovarian Syndrome (PCOS) subjects, as compared to controls. In this study, we examined the MRO splice variants and protein expression pattern in various human tissues and cells. We found a differential expression pattern of the MRO 5’-UTR region in luteinized granulosa-cumulus cells and in testicular tissues as compared to non-gonadal tissues. Our study also shows a punctate nuclear expression pattern and disperse cytoplasmic expression pattern of the MRO protein in human granulosa-cumulus cells and in testicular germ cells, which was later validated by western blotting. The tentative and unique features of the protein hampered our efforts to gain more insight about this elusive protein. A better understanding of the tissue-specific MRO isoforms expression patterns and the unique structure of the protein may provide important insights into the function of this gene and possibly to the pathophysiology of PCOS.

Sexual dimorphic expression of dnd in germ cells during sex reversal and its requirement for primordial germ cell survival in protogynous hermaphroditic grouper

Dead end (dnd), vertebrate-specific germ cell marker, had been demonstrated to be essential for primordial germ cell (PGC) migration and survival, and the link between PGC number and sex change had been revealed in some teleost species, but little is known about dnd in hermaphroditic vertebrates. In the present study, a protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides) dnd homologue (Ecdnd) was identified and characterized. Quantitative real-time PCR and in situ hybridization analysis revealed a dynamic and sexually dimorphic expression pattern in PGCs and germ cells of gonads. During sex changing, the Ecdnd transcript sharply increased in early transitional gonad, reached the highest level at late transitional gonad stage, and decreased after testis maturation. Visualization of zebrafish PGCs by injecting with RFP-Ecdnd-3'UTR RNA and GFP-zfnanos3-3'UTR RNA confirmed importance of Ecdnd 3'UTR for the PGC distribution. In addition, knockdown of EcDnd by using antisense morpholinos (MO) caused the ablation of PGCs in orange-spotted grouper. Therefore, the current data indicate that Ecdnd is essential for PGCs survival and may serve as a useful germ cell marker during gametogenesis in hermaphroditic grouper.

Neonatal Inhibition of DNA Methylation Alters Cell Phenotype in Sexually Dimorphic Regions of the Mouse Brain.

Many of the best-studied neural sex differences relate to differences in cell number and are due to the hormonal control of developmental cell death. However, several prominent neural sex differences persist even if cell death is eliminated. We hypothesized that these may reflect cell phenotype "decisions" that depend on epigenetic mechanisms, such as DNA methylation. To test this, we treated newborn mice with the DNA methyltransferase (DNMT) inhibitor zebularine, or vehicle, and examined two sexually dimorphic markers at weaning. As expected, control males had more cells immunoreactive for calbindin-D28k (CALB) in the medial preoptic area (mPOA) and fewer cells immunoreactive for estrogen receptor α (ERα) in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMHvl) and the mPOA than did females. Neonatal DNMT inhibition markedly increased CALB cell number in both sexes and ERα cell density in males; as a result, the sex differences in ERα in the VMHvl and mPOA were completely eliminated in zebularine-treated animals. Zebularine treatment did not affect developmental cell death or the total density of Nissl-stained cells at weaning. Thus, a neonatal disruption of DNA methylation apparently has long-term effects on the proportion of cells expressing CALB and ERα, and some of these effects are sex specific. We also found that sex differences in CALB in the mPOA and ERα in the VMHvl persist in mice with a neuron-specific depletion of either Dnmt1 or Dnmt3b, indicating that neither DNMT alone is likely to be required for the sexually dimorphic expression of these markers.

Sequential, Divergent, and Cooperative Requirements of Foxl2a and Foxl2b in Ovary Development and Maintenance of Zebrafish.

Foxl2 is essential for mammalian ovary maintenance. Although sexually dimorphic expression of foxl2 was observed in many teleosts, its role and regulative mechanism in fish remained largely unclear. In this study, we first identified two transcript variants of foxl2a and its homologous gene foxl2b in zebrafish, and revealed their specific expression in follicular layer cells in a sequential and divergent fashion during ovary differentiation, maturation, and maintenance. Then, homozygous foxl2a mutants (foxl2a-/-) and foxl2b mutants (foxl2b-/-) were constructed and detailed comparisons, such as sex ratio, gonadal histological structure, transcriptome profiling, and dynamic expression of gonadal development-related genes, were carried out. Initial ovarian differentiation and oocyte development occur normally both in foxl2a-/- and foxl2b-/- mutants, but foxl2a and foxl2b disruptions result in premature ovarian failure and partial sex reversal, respectively, in adult females. In foxl2a-/- female mutants, sox9a-amh/cyp19a1a signaling was upregulated at 150 days postfertilization (dpf) and subsequently oocyte apoptosis was triggered after 180 dpf. In contrast, dmrt1 expression was greater at 105 dpf and increased several 100-fold in foxl2b-/- mutated ovaries at 270 dpf, along with other testis-related genes. Finally, homozygous foxl2a-/-/foxl2b-/- double mutants were constructed in which complete sex reversal occurs early and testis-differentiation genes robustly increase at 60 dpf. Given mutual compensation between foxl2a and foxl2b in foxl2b-/- and foxl2a-/- mutants, we proposed a model in which foxl2a and foxl2b cooperate to regulate zebrafish ovary development and maintenance, with foxl2b potentially having a dominant role in preventing the ovary from differentiating as testis, as compared to foxl2a.

Identification and expression of cytochrome P450 genes in the Chinese giant salamander Andrias davidianus

Cytochrome P450 (cyp) genes play vital roles in biological processes, including in metabolism of endogenous and exogenous compounds. Exogenous hormone influences on gene expression that leads to disruption of gonad development have been studied in several species, but whether exogenous hormones affect cyp genes that show sexually dimorphic expression remains to be determined. Here, we identified and characterized cyp genes from the widely-cultured Chinese giant salamander Andrias davidianus. We obtained 20 cyp genes including 11 genes with complete sequences. Phylogenetic analyses supported the classification of cyp genes similar to other vertebrates. Expression profile of female and male salamanders showed multiple cyp genes to exhibit higher expression in ovary than in testis, including cyp26a, cyp19a, cyp1a1, cyp4v2, cyp3a24, cyp2c20, cyp2d14, cyp2d15, and cyp4b; while cyp11a, cyp2b11, cyp11b1, cyp4f22, cyp2j6, cyp2k1, cyp2k4, cyp1a5 was higher in testis than in ovary. Seven sex-biased genes were detected after aromatase inhibitor injection and with exposure to high water temperatures. Cyp2k1, cyp11b1, and cyp2j6 expression were increased, while cyp26a, cyp2c20, cyp4b genes were decreased with aromatase inhibition. In ovary, cyp26a and cyp2c20 expression were significantly reduced; whereas cyp2k1, cyp2j6, and cyp2k4 expression were significantly elevated with no changes in cyp11a and cyp11b1 expression after temperature treatment. These findings provide valuable information for further study of sex differentiation mechanisms and cyp gene evolution.

Mating Changes Sexually Dimorphic Gene Expression in the Seed Beetle Callosobruchus maculatus.

Sexually dimorphic phenotypes arise largely from sex-specific gene expression, which has mainly been characterized in sexually naïve adults. However, we expect sexual dimorphism in transcription to be dynamic and dependent on factors such as reproductive status. Mating induces many behavioral and physiological changes distinct to each sex and is therefore expected to activate regulatory changes in many sex-biased genes. Here, we first characterized sexual dimorphism in gene expression in Callosobruchus maculatus seed beetles. We then examined how females and males respond to mating and how it affects sex-biased expression, both in sex-limited (abdomen) and sex-shared (head and thorax) tissues. Mating responses were largely sex-specific and, as expected, females showed more genes responding compared with males (∼2,000 vs. ∼300 genes in the abdomen, ∼500 vs. ∼400 in the head and thorax, respectively). Of the sex-biased genes present in virgins, 16% (1,041 genes) in the abdomen and 17% (243 genes) in the head and thorax altered their relative expression between the sexes as a result of mating. Sex-bias status changed in 2% of the genes in the abdomen and 4% in the head and thorax following mating. Mating responses involved de-feminization of females and, to a lesser extent, de-masculinization of males relative to their virgin state: mating decreased rather than increased dimorphic expression of sex-biased genes. The fact that regulatory changes of both types of sex-biased genes occurred in both sexes suggests that male- and female-specific selection is not restricted to male- and female-biased genes, respectively, as is sometimes assumed.

HO1 and Wnt expression is independently regulated in female mice brains following permanent ischemic brain injury

A gender difference in stroke is observed throughout epidemiologic studies, pathophysiology, treatment and outcomes. We investigated the neuroprotective role of hemeoxygenase (HO) enzyme, which catabolizes free heme to bilirubin, carbon monoxide and biliverdin in the female brain after permanent ischemia. We have previously reported in male mice that genetic deletion of HO1 exacerbates the brain damage after permanent ischemia, and the mechanism of neuroprotection is dependent on the HO1/Wnt pathway; however, the role of HO1/Wnt mediated neuroprotection in the female brain is yet to be investigated. We subjected ovary intact female mice, HO1−/− intact, HO1 inhibitor tin mesoporphyrin (SnMP) treated intact and/or ovariectomized female mice to permanent ischemia (pMCAO), and the animals were sacrificed after 7days. The SnMP treatment for 7days significantly reduced the HO1 enzyme activity as compared to that of vehicle treated group. Infarct volume analysis showed significantly lower infarct in intact, HO1−/− intact, and SnMP treated group as compared to the OVX group, suggesting the role of estrogen in neuroprotection. However, there were no differences in infarct volume observed between the intact, HO1−/− and SnMP treated group, suggesting a sexually dimorphic role of HO1 neuroprotection. Western blot analysis on intact and SnMP-treated groups subjected to pMCAO suggested no significant differences in Wnt expression. Together, these results suggest that HO1 neuroprotection is sexually dimorphic and Wnt expression is independently regulated in the female brain following permanent ischemia.