The group of Dr. Dagmar Wilhelm at Queensland University have found a gene that could be the potential target for Sry action during sex determination. They immunoprecipitated this fragment from E11.5 dpc gonads using anti-Sry antibody specific to mouse. The approach they have used is novel and there are other potential candidate genes in the list, which they will slowly be characterizing.

The Cerebellin 4 Precursor Gene Is a Direct Target of SRY and SOX9 in Mice
Bradford ST, Hiramatsu R, Maddugoda MP, Bernard P, Chaboissier MC, Sinclair A, Schedl A, Harley V, Kanai Y, Koopman P, Wilhelm D.
Biol Reprod. 2009 Feb 11. [Epub ahead of print]

In most mammals, the expression of SRY (sex-determining region on the Y chromosome) initiates the development of testes and thus determines the sex of the individual. However, despite the pivotal role of SRY, its mechanism of action remains elusive. One important missing piece of the puzzle is the identification of genes regulated by SRY. In this study we used chromatin immunoprecipitation to identify direct SRY target genes. Anti-mouse SRY antibody precipitated a region 7.5 kb upstream of the transcriptional start site of cerebellin 4 precursor (Cbln4), which encodes a secreted protein. Cbln4 is expressed in Sertoli cells in the developing gonad with a profile mimicking that of the testis-determining gene Sox9 SRY-box containing gene 9). In transgenic XY mouse embryos with reduced Sox9 xpression, Cbln4 expression also was reduced, whereas over-expression of Sox9 in XX mice caused an up-regulation of Cbln4 expression. Finally, ectopic up-regulation of SRY in vivo resulted in ectopic expression of Cbln4. Our findings suggest that both SRY and SOX9 contribute to the male-specific up-regulation of Cbln4 in the developing testis, and identified a direct in vivo target gene of SRY.

Article link: http://www.ncbi.nlm.nih.gov/pubmed/19211811?

Epigenetic programming of the germ line: effects of endocrine disruptors on the development of transgenerational disease.
Anway MD, Skinner MK. Reprod Biomed Online. 2008 Jan;16(1):23-5
School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4231, USA.

Epigenetic programming of the germ line occurs during embryonic development in a sex-specific manner. The male germ line becomes imprinted following sex determination. Environmental influences can alter this epigenetic programming and affect not only the developing offspring, but also potentially subsequent generations. Exposure to an endocrine disruptor (i.e. vinclozolin) during embryonic gonadal sex determination can alter the male germ-line epigenetics (e.g. DNA methylation). The epigenetic mechanism involves the alteration of DNA methylation in the germ line that appears to transmit transgenerational adult onset disease, including spermatogenic defects, prostate disease, kidney disease and cancer.

Article link: http://www.ncbi.nlm.nih.gov/pubmed/18252044

By Youngson NA, Whitelaw E.
Department of Population Studies and Human Genetics, Queensland Institute of Medical Research, Brisbane 4006, Australia; email: emma.whitelaw@qimr.edu.au.

Transgenerational epigenetic effects include all processes that have evolved to achieve the nongenetic determination of phenotype. There has been a long-standing interest in this area from evolutionary biologists, who refer to it as non-Mendelian inheritance. Transgenerational epigenetic effects include both the physiological and behavioral (intellectual) transfer of information across generations. Although in most cases the underlying molecular mechanisms are not understood, modifications of the chromosomes that pass to the next generation through gametes are sometimes involved, which is called transgenerational epigenetic inheritance. There is a trend for those outside the field of molecular biology to assume that most cases of transgenerational epigenetic effects are the result of transgenerational epigenetic inheritance, in part because of a misunderstanding of the terms. Unfortunately, this is likely to be far from the truth.

Article link: Full Text  PDF

Onset of meiosis in the chicken embryo; evidence of a role for retinoic acid. 
Smith CA, Roeszler KN, Bowles J, Koopman P, Sinclair AH. 
BMC Dev Biol. 2008 Sep 17; 8:85

Meiosis in higher vertebrates shows a dramatic sexual dimorphism: germ cells enter meiosis and arrest at prophase I during embryogenesis in females, whereas in males they enter mitotic arrest during embryogenesis and enter meiosis only after birth. Here we report the molecular analysis of meiosis onset in the chicken model and provide evidence for conserved regulation by retinoic acid. RESULTS: Meiosis in the chicken embryo is initiated late in embryogenesis (day 15.5), relative to gonadal sex differentiation (from day 6). Meiotic germ cells are first detectable only in female gonads from day 15.5, correlating with the expression of the meiosis marker, SCP3. Gonads isolated from day 10.5 female embryos and grown in serum-free medium could still initiate meiosis at day 16.5, suggesting that this process is controlled by an endogenous clock in the germ cells themselves, and/or that germ cells are already committed to meiosis at the time of explantation. Early commitment is supported by the analysis of chicken STRA8, a pre-meiotic marker shown to be essential for meiosis in mouse. Chicken STRA8 is expressed female-specifically from embryonic day 12.5, preceding morphological evidence of meiosis at day 15.5. Previous studies have shown that, in the mouse embryo, female-specific induction of STRA8 and meiosis are triggered by retinoic acid. A comprehensive analysis of genes regulating retinoic acid metabolism in chicken embryos reveals dynamic expression in the gonads. In particular, the retinoic acid-synthesising enzyme, RALDH2, is expressed in the left ovarian cortex at the time of STRA8 up-regulation, prior to meiosis. CONCLUSION: This study presents the first molecular analysis of meiosis onset in an avian embryo. Although aspects of avian meiosis differ from that of mammals, a role for retinoic acid may be conserved.

Article link: http://www.biomedcentral.com/1471-213X/8/85

Sex reversal of the amphibian, Xenopus tropicalis, following larval exposure to an aromatase inhibitor.
Olmstead AW, Kosian PA, Korte JJ, Holcombe GW, Woodis KK, Degitz SJ. 
Aquat Toxicol. 2008 Aug 15. [Epub ahead of print] 

U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Mid-Continent Ecology Division, 6201 Congdon Boulevard, Duluth, MN, United States.

Aromatase is a steroidogenic enzyme that catalyzes the conversion of androgens to estrogens in vertebrates. Modulation of this enzyme's activity by xenobiotic exposure has been shown to adversely affect gonad differentiation in a number of diverse species. We hypothesized that exposure to the aromatase inhibitor, fadrozole, during the larval development of the tropical clawed frog, Xenopus tropicalis, would result in masculinization of the developing female gonad. Tadpoles were exposed to fadrozole at nominal concentrations from 1 to 64mug/L in a flow-through system from <24h post-fertilization (Nieuwkoop Faber (NF) stage 15-20) to metamorphosis (NF stage 66). At metamorphosis, morphologically examined gonads indicated complete masculinization of all tadpoles at concentrations of 16mug/L and above and a significant bias in sex ratio towards males at concentrations of 1mug/L and above. No effects on time to metamorphosis, body mass, or body length were observed. A random subsample of frogs was raised to reproductive maturity (39 weeks post-fertilization) in control water. All frogs exposed as tadpoles to 16mug/L fadrozole or greater possessed testes at sexual maturity. Intersexed gonads characterized by the presence of both testicular and ovarian tissue were observed in 12% of frogs in the 4mug/L treatment. No differences in estradiol, testosterone, or vitellogenin plasma concentrations were observed in exposed males or females compared to controls. Females in the 4mug/L treatment possessed a significantly greater percentage of pre-vitellogenic oocytes than controls and were significantly smaller in body mass. No differences in sperm counts were observed in exposed males compared to controls. Results from this study demonstrate that larval exposure to an aromatase inhibitor can result in the complete masculinization of female gonads. These masculinized females are phenotypically indistinguishable from normal males at adulthood. Lower levels of aromatase inhibition resulted in intersexed gonads and possible female reproductive impairment at adulthood. These results indicate that exposure of amphibians to xenobiotics capable of inhibiting aromatase would result in adverse reproductive consequences.

Article link: http://www.ncbi.nlm.nih.gov/pubmed/18804292

Germ line control of female sex determination in zebrafish. 
Siegfried KR, Nüsslein-Volhard C. 
Dev Biol. 2008 Oct 7. [Epub ahead of print]

A major transition during development of the gonad is commitment from an undifferentiated "bi-potential" state to ovary or testis fate. In mammals, the oogonia of the developing ovary are known to be important for folliculogenesis. An additional role in promoting ovary fate or female sex determination has been suggested, however it remains unclear how the germ line might regulate this process. Here we show that the germ line is required for the ovary versus testis fate choice in zebrafish. When the germ line is absent, the gonad adopts testis fate. These germ line deficient testes have normal somatic structures indicating that the germ line influences fate determination of surrounding somatic tissues. In germ line deficient animals the expression of the ovary specific gene cyp19a1a fails to be maintained whereas the testis genes sox9a and amh remain expressed. Furthermore, we observed decreased levels of the ovary specific genes cyp19a1a and foxL2 in germ line deficient animals prior to morphological sex differentiation of the gonad. We propose that the germ line has a common role in female sex determination in fish and mammals. Additionally, we show that testis specification is sufficient for masculinization of the fish pointing to a direct role of hormone signaling from the gonad in directing sex differentiation of non-gonadal tissues.

Article link: http://www.ncbi.nlm.nih.gov/pubmed/18930041

A group of scientists in the University of the Ryukyus, Japan have shown that male wrasses, which do never change sex in their life, have potential to change sex. A brief estrogen treatment can cause those males to change into female. This finding cracks the code of sexual plasticity in wrasses and raises scientist`s eyebrows on developmental plasticity of lower vertebrates. Can other gonochoristic fish, such as salmon, trout, catfish, tilapias, etc. can change sex with such an exposure? Does a life-long exposure to such chemicals cause sex reversal in these species in a long run? Such issues are important in the current scenario of global environmental awareness.

Sex steroids are considered major regulators of sex change processes in fish. Estrogen depletion is shown to be crucial for female-male sex change initiation; however, its role in male-female sex change is largely unknown. In the present study, we examined the effects of estradiol-17beta (E2) treatments on testes of initial-phase (IP) males of the three-spot wrasse (Halichoeres trimaculatus), which naturally do not undergo male-female sex change. Sexually mature IP males were fed a diet containing E2 (low, 20 ug/g feed; high, 200 ug/g feed) for 6 or 12 weeks, and changes in gonadal structures were examined. Percentage of sex change varied with the dosage of E2 and the duration of treatment. All individuals treated with high-dose E2 for 6 weeks had ovaries with many immature oocytes; whereas 75% of individuals treated with low-dose of E2 for 6 weeks and sampled on the 12th week had ovaries with yolky oocytes and an ovarian cavity indicating a typical mature ovary. No testicular tissue was observed in sex-reversed gonads in both treatment groups. Contrary to the previous assumptions, present results suggest that IP male wrasses have the potential to undergo male-female sex change in response to exogenous estrogen. How the presence or absence of estrogen creates sexual plasticity in gonadal germ and somatic cells remains to be clarified.