Knockout of the Glucocorticoid Receptor Impairs Reproduction in Female Zebrafish

Abstract

The pleiotropic effects of glucocorticoids in metabolic, developmental, immune and stress response processes have been extensively investigated; conversely, their roles in reproduction are still less documented. It is well known that stress or long-lasting therapies can cause a strong increase in these hormones, negatively affecting reproduction. Moreover, the need of glucocorticoid (GC) homeostatic levels is highlighted by the reduced fertility reported in the zebrafish glucocorticoid receptor mutant (nr3c1ia30/ia30) line (hereafter named gr−/−). Starting from such evidence, in this study, we have investigated the role of glucocorticoid receptor (Gr) in the reproduction of female zebrafish. Key signals orchestrating the reproductive process at the brain, liver, and ovarian levels were analyzed using a multidisciplinary approach. An impairment of the kiss-GnRH system was observed at the central level in (gr−/−) mutants as compared to wild-type (wt) females while, in the liver, vitellogenin (vtg) mRNA transcription was not affected. Changes were instead observed in the ovary, particularly in maturing and fully grown follicles (classes III and IV), as documented by the mRNA levels of signals involved in oocyte maturation and ovulation. Follicles isolated from gr−/− females displayed a decreased level of signals involved in the acquisition of competence and maturation, causing a reduction in ovulation with respect to wt females. Fourier transform infrared imaging (FTIRI) analysis of gr−/− follicle cytoplasm showed major changes in macromolecule abundance and distribution with a clear alteration of oocyte composition. Finally, differences in the molecular structure of the zona radiata layer of gr−/− follicles are likely to contribute to the reduced fertilization rate observed in mutants.

Probiotic Administration Mitigates Bisphenol A Reproductive Toxicity in Zebrafish

“…Testis and ovaries were dissected out and divided as follows: 5 samples were fixed in Bouin's fixative for histology. The remaining 5 testis were stored at −80 • C, while ovaries were teased into separate follicles using transfer pipettes (Semco Scientific Corp., San Diego, CA, USA) without trypsinization; thereafter, follicles were separated into different maturation stages according to their diameters, as previously described [61,62] and class III and IV follicles were collected and stored at −80 • C for molecular analysis.…”

“…For each experimental group, total RNA was extracted from 5 female livers and 5 testis, and from 3, classes III and IV pools, containing 50 follicles each, using RNAeasy ® Minikit (Qiagen, Milano, Italy). RNA quality assessment and cDNA synthesis were performed as previously described [61].…”

Glucocorticoid-Mediated Developmental Programming of Vertebrate Stress Responsivity

“…Several zebrafish mutants lacking functional GR have been produced (reviewed in Dinarello et al, 2020), which have the advantage of being viable throughout adulthood, in contrast to mammalian systems wherein loss of the receptor results in neonatal death through mechanisms previously described. Unsurprisingly however, lower survival has been noted in mutants at zygotic/embryonic (Gans et al, 2020;Maradonna et al, 2020) and post-larval stages (Facchinello et al, 2017). In 2012, a GR mutant, s357, was identified in a behavioral screen for depressive-like behavior (Ziv et al, 2013).…”

Zebrafish Mutant Lines Reveal the Interplay between nr3c1 and nr3c2 in the GC-Dependent Regulation of Gene Transcription

“…The nr3c1 ia30 (herein called gr ia30/ia30 ) line has been generated with CRISPR/Cas9 technology and is characterized by an insertion of 5 nucleotides in the second exon of the nr3c1 gene. This mutation leads to a frameshift and a premature stop codon, and the mutation affects the response to stress stimuli, the feeding entrainment of zebrafish circadian clock and reproduction [ 14 , 17 , 18 ]. On the other hand, the nr3c1 s357 mutant line, generated with N-ethyl-N-nitrosourea (ENU) mutagenesis, has a point mutation that prevents GR interaction with the DNA [ 19 ].…”