Shannon N. Romano

Reproductive success relies on proper establishment and maintenance of biological sex. In many animals, including mammals, the primary gonad is initially ovary in character. We previously showed the RNA binding protein (RNAbp), Rbpms2, is required for ovary fate in zebrafish. Here, we identified Rbpms2 targets in oocytes (Rbpms2-bound oocyte RNAs; rboRNAs ). We identify Rbpms2 as a translational regulator of rboRNAs , which include testis factors and ribosome biogenesis factors. Further, genetic analyses indicate that Rbpms2 promotes nucleolar amplification via the mTorc1 signaling pathway, specifically through the mTorc1-activating Gap activity towards Rags 2 (Gator2) component, Missing oocyte (Mios). Cumulatively, our findings indicate that early gonocytes are in a dual poised, bipotential state in which Rbpms2 acts as a binary fate-switch. Specifically, Rbpms2 represses testis factors and promotes oocyte factors to promote oocyte progression through an essential Gator2-mediated checkpoint, thereby integrating regulation of sexual differentiation factors and nutritional availability pathways in zebrafish oogenesis.

Sex determination and differentiation is a complex process regulated by multiple factors, including factors from the germline or surrounding somatic tissue. In zebrafish, sex-determination involves establishment of a bipotential ovary that undergoes sex-specific differentiation and maintenance to form the functional adult gonad. However, the relationships among these factors are not fully understood. Here we identify potential Rbpms2 targets and apply genetic epistasis experiments to decipher the genetic hierarchy of regulators of sex-specific differentiation. We provide genetic evidence that the critical female factor, rbpms2 is epistatic to the male factor dmrt1 in terms of adult sex. Moreover, Rbpms2's role in promoting female fates extends beyond repression of Dmrt1, as Rbpms2 is essential for female differentiation even in the absence of Dmrt1. In contrast, female fates can be restored in mutants lacking cyp19a1a and dmrt1, and prolonged in bmp15 mutants in the absence of dmrt1. Taken together this work indicates that cyp19a1a-mediated suppression of dmrt1 establishes a bipotential ovary and initiates female fate acquisition. Then, after female fate specification, Cyp19a1a regulates subsequent oocyte maturation and sustains female fates independent of Dmrt1 repression.

Sex determination and differentiation is a complex process regulated by multiple factors, including factors from the germline or surrounding somatic tissue. In zebrafish, sex-determination begins with establishment of a bipotential gonad that undergoes sex-specific differentiation and maintenance to form the functional adult gonad. However, the relationships among these factors are not fully understood. Here we identify potential Rbpms2 targets and apply genetic epistasis experiments to decipher the genetic hierarchy of regulators of sex-specific differentiation. We provide evidence that the critical female factor, rbpms2 is epistatic to the male factor dmrt1 in terms of adult sex. Moreover, Rbpms2’s role in promoting female fates extends beyond repression of Dmrt1, as Rbpms2 is essential for female differentiation even in the absence of Dmrt1. In contrast, female fates can be restored in mutants lacking cyp19a1a in the absence of dmrt1. Taken together this work indicates that Cyp19a1a-mediated suppression of Dmrt1 is key to establish a bipotential gonad and initiate female fate acquisition, possibly by promoting rbpms2 . Then, after female fate specification, Cyp19a1a regulates subsequent oocyte maturation and sustains female fates independent of Dmrt1 repression. Author Summary: We show that cyp19a1a -mediated suppression of dmrt1 establishes a bipotential gonad and female fate acquisition, possibly through rbpms2 which is required for female fates, even in the absence of Dmrt1.

Estrogens regulate vertebrate development and function through binding to nuclear estrogen receptors α and β (ERα and ERβ) and the G protein–coupled estrogen receptor (GPER). Studies in mutant animal models demonstrated that ERα and ERβ are required for normal ovary development and function. However, the degree to which GPER signaling contributes to ovary development and function is less well understood. Previous studies using cultured fish oocytes found that estradiol inhibits oocyte maturation in a GPER-dependent manner, but whether GPER regulates oocyte maturation in vivo is not known. To test the hypothesis that GPER regulates oocyte maturation in vivo, we assayed ovary development and function in gper mutant zebrafish. We found that homozygous mutant gper embryos developed into male and female adults with normal sex ratios. Adult mutant fish exhibited normal secondary sex characteristics and fertility. Additionally, mutant ovaries were histologically normal. We observed no differences in the number of immature versus mature oocytes in mutant versus wild-type ovaries from both young and aged adults. Furthermore, expression of genes associated with sex determination and ovary function was normal in gper mutant ovaries compared with wild type. Our findings suggest that GPER is not required for sex determination, ovary development, or fertility in zebrafish.

Estrogens regulate vertebrate development and function through binding to nuclear estrogen receptors alpha and beta (ERα, ERβ) and the G protein-coupled estrogen receptor (GPER). Studies in mutant animal models demonstrated that ERα and ERβ are required for normal ovary development and function. However, the degree to which GPER signaling contributes to ovary development and function is less well understood. Previous studies using cultured fish oocytes found that estradiol inhibits oocyte maturation in a GPER-dependent manner, but whether GPER regulates oocyte maturation in vivo is not known. To test the hypothesis that GPER regulates oocyte maturation in vivo , we assayed ovary development and function in gper mutant zebrafish. We found that homozygous mutant gper embryos developed into male and female adults with normal sex ratios and fertility. Adult mutant fish exhibited normal secondary sex characteristics and fertility. Additionally, mutant ovaries were histologically normal. We observed no differences in the number of immature versus mature oocytes in mutant versus wild-type ovaries from both young and aged adults. Furthermore, expression of genes associated with sex determination and ovary function were normal in gper mutant ovaries compared to wild type. Our findings suggest that GPER is not required for sex determination, ovary development or fertility in zebrafish.

Estrogens act by binding to estrogen receptors alpha and beta (ERα, ERβ), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required for normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ERα and ERβ mutant embryos but not in GPER mutants. GPER mutant embryos exhibited reduced basal heart rate, while heart rate was normal in ERα and ERβ mutants. We detected gper transcript in discrete regions of the brain and pituitary but not in the heart, suggesting that GPER acts centrally to regulate heart rate. In the pituitary, we observed gper expression in cells that regulate levels of thyroid hormone triiodothyronine (T3), a hormone known to increase heart rate. Compared to wild type, GPER mutants had reduced levels of T3 and estrogens, suggesting pituitary abnormalities. Exposure to exogenous T3, but not estradiol, rescued the reduced heart rate phenotype in gper mutant embryos, demonstrating that T3 acts downstream of GPER to regulate heart rate. Using genetic and mass spectrometry approaches, we find that GPER regulates maternal estrogen levels, which are required for normal embryonic heart rate. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and suggest that GPER regulates embryonic heart rate by altering maternal estrogen levels and embryonic T3 levels. Estrogen hormones are important for the formation and function of the nervous, reproductive and cardiovascular systems. Here we report that acute exposure to estrogens increases heart rate, a previously unappreciated function of estrogens. Using zebrafish with mutations in genes that respond to estrogens, we found that heart rate is regulated not by the typical molecules that respond to estrogens–the nuclear estrogen receptors–but rather by a different molecule, the G protein-coupled estrogen receptor. We also show that estrogens increase heart rate by increasing levels of thyroid hormone. Our results reveal a new function for the G protein-coupled estrogen receptor and a new connection between estrogens and thyroid hormone. Environmental compounds that mimic estrogens can be harmful because they can influence gonad function. Our results suggest that endocrine disrupting compounds may also influence cardiac function.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.