FUNCTIONS OF G-PROTEIN COUPLED ESTROGEN RECEPTOR (GPER) IN FEMALE ZEBRAFISH REPRODUCTION

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Marcus J.J Williams (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: "Abstract Steroid hormones such as estrogen depend on receptors like Estrogen Receptor alpha (ER?) and Estrogen Receptor (ERß) to mediate their slow ""genomic€ functions , and G-Protein coupled Estrogen Receptor (GPER) to exert their rapid ""non-genomic€ functions. The integration of the genomic and the less studied non-genomic functions of estrogen are crucial to proper reproductive timing and function. The effects of estrogen in reproduction via genomic signaling has been extensively studied. Conversely , the functions of estrogen in reproduction via non-genomic GPER signaling remains poorly understood. The most characterized function of GPER (mammals) in reproduction is the regulation oocyte maturation (the resumption of meiosis). Gper has previously been shown to mediate the estrogen inhibition of oocyte maturation in vitro in teleosts using numerous experimental approaches , such as the silencing of both gper (zebrafish) and Estrogen Receptors (ERs) , and in-vitro pharmacological tools. Despite this , no labs have used gper knockouts to investigate this phenomenon in teleosts. Furthermore , the effects of gper in other aspects of reproduction such as fertility and oocyte development remain largely unstudied in zebrafish. However , knockout studies in mice models indicate that GPER may be dispensable in mediating the various effects of estrogen in reproduction , including fertility and reproduction. To investigate the role of gper in fertility , oocyte development , and oocyte maturation in zebrafish , we generated two gper zebrafish mutant lines using CRISPR/Cas9 , causing a -8 or a -10 nucleotide deletion , resulting in frameshift mutations in the third exon of gper. The shift in the reading frame resulted in the truncation of the gper mRNA leading to a non-functional product. We then examined reproductive function in homozygous mutant gper8nt zebrafish , assaying their overall fertility , oocyte development , and oocyte maturation. In contrast to previous studies in mice and other zebrafish models , we conclude that gper is essential for reproduction. gper mutant females exhibit a buildup of stage III oocytes compared to wild-type , possibly indicating dysfunction in oocyte development. Surprisingly , we found no significant difference in oocyte maturation in gper mutants compared to wild-type in an in vitro germinal vesicle breakdown (GVBD) assay. Interestingly , we observed gene expression changes in the hypothalamic-pituitary-gonadal axis (HPG axis) which could indicate gper exerts an indirect effect on genes important for reproduction. In the brain , ER? (esr1) and ERß (esr2a/esr2b) were upregulated in gper mutants. In the ovary , genes encoding steroid hormone synthesis enzymes were downregulated , including 3ß-hydroxysteroid dehydrogenase (hsd3b1) , which is essential for progestin synthesis , and 17ß-hydroxysteroid-dehydrogenase (17ßhsd) , which is crucial for testosterone synthesis. Our results suggest an important role of gper in fertility , oocyte development , and female reproduction. Our data has shown that gper is necessary for numerous reproductive processes , but further experimentation is needed to explain the molecular mechanisms which gper effects fertility , oocyte development , and oocyte maturation."

Additional Information

Publication

Thesis

Language: English

Date: 2019

Keywords

GPER, Endocrinology, CRISPR, Molecular Biology, Zebrafish, Reproduction, Oocyte Maturation

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