Ovulation In Zebrafish Research Articles

Zebrafish prss59.1 is involved in chorion development

The prss59.1 gene was identified as one of 11 genes that were highly upregulated during the induction of ovulation in zebrafish by using an in vivo ovulation assay. Previously, we conducted biochemical characterization of Prss59.1 and revealed it to be a trypsin-like proteolytic enzyme. In this study, we established a prss59.1 gene knockout strain using the CRISPR/Cas9 system. Phenotypic analysis of prss59.1 knockout fish showed that prss59.1 is associated with chorion elevation, a prominent event in egg activation during fertilization. The chorions of heterozygous and homozygous prss59.1 mutant zebrafish were smaller than those of the wild type. The results suggested that Prss59.1 is necessary for chorion expansion. The homozygous prss59.1 mutant strain, with a small chorion, showed an extremely low survival rate. Fiber-supported knob-like structures (KS) on the chorion showed an abnormal structure in prss59.1 mutants. Prss59.1 was detected in the KS on the chorion. The pores on the chorion were smaller in the prss59.1 mutants than in the wild type. Transmission electron microscopy (TEM) observations of the cross sections of the chorions showed abnormalities in the chorion structure in prss59.1 mutants. These results demonstrated that Prss59.1 is involved in chorion elevation and in proper formation of the chorion, which is necessary for embryo development.

Nuclear Progestin Receptor-mediated Linkage of Blood Coagulation and Ovulation.

Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, ~3145-fold) and tissue factor (f3a, ~120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wild-type preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting-triggered ovulation in vertebrates.

Zebrafish stm is involved in the development of otoliths and of the fertilization envelope.

SummaryUsing an in vivo assay, we selected 11 genes that were highly upregulated during the induction of ovulation in zebrafish using microarray analysis and RNA sequencing. The starmaker gene (stm) was one of these genes. Although stm has been previously reported to be involved in otolith formation during the early development of zebrafish, we detected its expression in eggs and showed that stm was related to fertilization by establishing an stm gene knockout strain using the CRISPR/Cas9 system. Further phenotypic analysis of stm knockout fish was conducted in this study. With a higher nonfertilization rate, the stm mutant strain showed an extremely low survival rate. Otoliths of stm homozygous mutant zebrafish showed abnormal morphology in embryos and adult fish. However, fish did not show any abnormalities in swimming behaviour in either embryos or adults. Stm proteins were detected on the chorion of ovulated eggs before spawning. Fibre-supported knob-like structures on the fertilization envelope (FE) also showed abnormal structures in stm mutants. The Stm protein is necessary for otolith formation, and a lack of Stm causes abnormal otolith formation. The partial defect of otolith formation does not cause defects in swimming behaviour. The Stm protein is expressed in the chorion and is responsible for the formation of fibre-supported knob-like structures on the FE. It was suggested that a lack of Stm caused a lower fertilization rate due to inadequate formation of the FE.Lay summaryIn zebrafish, the protein Starmaker (Stm) was identified as having a role in ovulation. Stm is also known to be required for the formation of ear stones (otoliths) which are needed to keep the body in balance. Zebrafish lacking Stm were produced by genome editing. As expected, Stm-deficient fish formed abnormal otoliths. To investigate the role of Stm in ovulation, fertilization and early development, we tried mating of Stm mutants and observed their juveniles. Although no problem found in ovulation, we found low fertilization rate and abnormal structure of knob-like structure (small pit) on the egg membrane. Survival rate of embryos with abnormal egg membrane was extremely low. It was demonstrated that Stm protein is necessary to form the functional egg membrane to protect embryos from the outside environment.

The microRNA-200 cluster on chromosome 23 is required for oocyte maturation and ovulation in zebrafish†.

The reproductive process is usually controlled by the hypothalamic-pituitary-gonad axis in vertebrates, while Kiss/gonadotropin-releasing hormone (GnRH) system in the hypothalamus is required for mammalian reproduction but dispensable for fish reproduction. The regulation of follicle stimulating hormone/luteinizing hormone (LH) expression in fish species is still unknown. Here, we identified miR-200s on chromosome 23 (chr23-miR-200s) as important regulators for female zebrafish reproduction. Knockout of chr23-miR-200s (chr23-miR-200s-KO) resulted in dysregulated expression of luteinizing hormone beta lhb (luteinizing hormone beta) and some hormone genes in the pituitary as revealed by comparative transcriptome profiling, leading to failure of oocyte maturation and ovulation as well as defects in reproductive duct development. Chr23-miR-200s mainly expressed in the pituitary and regulated lhb expression by targeting the transcription repressor wt1a. Injection of human chorionic gonadotropin (hCG) could rescue the defects of oocyte maturation in chr23-miR-200s-KO zebrafish, whereas GnRH or LHRH-A2 could not, suggesting that Chr23-miR-200s regulated lhb expression in a GnRH-independent pathway. It was remarkable that either injection of carp pituitary extraction, or co-injection of hCG with synthetic oxytocin and vasotocin could greatly rescue the defects of both oocyte maturation and ovulation in chr23-miR-200s-KO zebrafish. Altogether, our findings highlight an important function of chr23-miR-200s in controlling oocyte maturation by regulation LH expression, and oxytocin and vasotocin are potentially responsible for the ovulation in fish species.

Upregulation of adamts9 by gonadotropin in preovulatory follicles of zebrafish

Previously we had identified adamts9 as a downstream target of Pgr, which is essential for ovulation in zebrafish. The primary goal of this study is to determine whether human chorionic gonadotropin (hCG, LH analog) also regulate adamts9 expression prior to ovulation. The expression of adamts9 was induced by hCG in a dose and time dependent manner in zebrafish preovulatory follicles in vitro. Interestingly, the stimulatory effect of hCG on adamts9 expression was not blocked in pgr−/− follicles but blocked in lhcgr−/−. This effect of hCG was via Lhcgr and its associated cAMP and PKC signaling pathways. Reduced fecundity and reduced expression of adamts9 were also found in lhcgr−/− females in vivo. Therefore, we have provided the first evidence of gonadotropin (hCG) regulated adamts9 in zebrafish, which could be important for ovulation.

Investigating the role of prostaglandin receptor isoform EP4b in zebrafish ovulation

Prostaglandins (PGs) are a class of fatty acid-derived hormones that play an essential role in the regulation of ovulation of teleosts. This study investigated the various isoforms of ovarian PG receptors in the zebrafish ovary and their role in ovulation. Using real time qPCR, six PG receptor isoforms (ptger1a, ptger1b, ptger2a, ptger4a, ptger4b, and ptgfr) were shown to be expressed in the ovary. Only the PG receptor isoform ptger4b was upregulated at the time of ovulation in vivo, or following treatment in vivo with Ovaprim, which contains a gonadotropin releasing hormone analogue and a dopamine receptor antagonist and stimulates ovulation. Treatment of full-grown follicles with the maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) in vitro also induced expression of EP4b mRNA. Females ovulate in vivo after injection with Ovaprim, or injection with Ovaprim and inhibitors of EP1 (ONO-8130) or EP2 (TG4-155) function; they do not ovulate when injected with Ovaprim and an EP4 inhibitor (GW237368x). These findings suggest that the EP4 receptor, in particular the EP4b isoform, is essential for ovulation.

Dydrogesterone exposure induces zebrafish ovulation but leads to oocytes over-ripening: An integrated histological and metabolomics study

Dydrogesterone (DDG) is a synthetic progestin widely used in numerous gynecological diseases. DDG has been shown to disturb fish reproduction, however, the mechanism is still unclear. Here we studied the histological changes and differences of metabolome between exposed and control fish gonads after exposure of zebrafish (Danio rerio) embryos to 2.8, 27.6, and 289.8 ng/L DDG until sexual maturity for a total of 140 days. Dydrogesterone exposure led to male-biased zebrafish sex ratios. Histological examination revealed that DDG induced postovulatory follicles and atretic follicles in the ovary of the female fish. Postovulatory follicles indicated the occurrence of ovulation. DDG also increased spermatids and spermatozoa in the male fish testis, suggesting promotion of spermatogenesis. Ovarian metabolome showed that DDG increased the concentrations of free amino acids, urea, putrescine, free fatty acids, acylcarnitines, lysophospholipids, and other metabolites catabolized from phospholipids. Most of these metabolites are biodegradation products of proteins and lipids, suggesting the existence of ovulated oocytes over-ripening. Further, DDG upregulated arachidonic acid (AA) and its 5‑lipoxygenase (5-LOX) metabolites 5‑oxo‑6,8,11,14‑eicosatetraenoic acid (5-oxo-ETE) in the ovary, which could lead to suppression of AA cyclooxygenase (COX) metabolite prostaglandin F2α (PGF2α). It is believed that AA induced oocyte maturation, while 5-oxo-ETE and related metabolites in purinergic signaling promoted ovulation. Whereas, the suppression of PGF2α production might block spawning and damaged follicular tissue digestion, which explained the oocytes over-ripening and atretic follicles in the treated ovary. Overall, our results suggested that DDG exposure induced zebrafish oocyte maturation and ovulation but led to oocytes over-ripening via the AA metabolic pathway and purinergic signaling.

Igf3 serves as a mediator of luteinizing hormone in zebrafish ovulation.

Both oocyte maturation and ovulation is triggered by the luteinizing hormone (LH) surge in vertebrates, but exactly how these processes are regulated by LH remains to be fully elucidated. Previously, we found that Igf3, a fish-specific member of the igf family predominantly expressed in the gonads, could mediate the action of LH on oocyte maturation in zebrafish. Here, we further reveal the importance of Igf3 in mediating the action of LH on ovulation in zebrafish. All the four igf gene family members are expressed in the zebrafish ovary but only the igf3 transcript level is increased in hCG-induced ovulation in vivo. The expression of Igf3 protein in the follicles is also increased during ovulation. The actions of hCG on the expression of ovulatory enzymes and on ovulation itself could be largely mimicked by the recombinant zebrafish Igf3 protein. Intriguingly, the phosphorylation of Igf1r, the receptor for Igf3, could be activated by hCG in the follicular cells during ovulation. And inhibition of Igf3 signaling by Igf1r inhibitors and Igf3 antiserum could significantly attenuate the hCG-induced ovulation. Collectively, all these data support the notion that Igf3 serves as a mediator of LH action in zebrafish ovulation.

Ovulation is associated with the LH-dependent induction of pla2g4aa in zebrafish.

The effects of the preovulatory luteinizing hormone (LH) surge on the ovulatory process are mediated by prostaglandins (PGs), the synthesis of which involves prostaglandin synthetase and cytosolic phospholipase A2 (cPLA2). In our previous study, we systematically investigated the function of prostaglandin endoperoxide synthase (ptgs) genes on ovulation in zebrafish. However, the role of cPLA2 in ovulation was not determined in zebrafish. In this study, we investigated the function of cpla2α in PGs production and ovulation in periovulatory follicles. Our data showed that the expression of pla2g4aa increased during zebrafish folliculogenesis and the follicular layer was the primary region with expression of pla2g4aa. In addition, the expression of pla2g4aa was regulated by LH invitro and invivo. Furthermore, injection of AACOCF3, a specific inhibitor of cPLA2, significantly reduced ovarian PGs level and blocked hCG-induced ovulation. Collectively, these findings suggest that pla2g4aa is related to the ovulation process in zebrafish.

LH signaling induced ptgs2a expression is required for ovulation in zebrafish

It is well known that ovulation is induced by luteinizing hormone (LH) surge. However, the down-stream factors that mediating LH surge induced ovulation are less clear. The cyclooxygenases (also known as PTGS) as key enzymes for prostaglandins synthesis appear to be important for ovulation in mammals, but their functional roles and molecular mechanism in regulation of fish ovulation are largely unexplored. In this study, we have systematically investigated the expression, regulation and functional roles of cox genes during zebrafish ovulation. Three types of cox genes including ptgs1, ptgs2a and ptgs2b have been identified in zebrafish. The ptgs2a was dominantly expressed in the ovary with a maximal level at the maturation stage of the follicles. In addition, the ptgs2a expression is up-regulated by LH signaling in vitro and in vivo. Moreover, co-injection of a selective Ptgs2 inhibitor and non-selective Ptgs inhibitor with hCG could significantly block the stimulatory effect of hCG induced ovulation in vivo. Collectively, our findings indicate that LH signaling induced ptgs2a expression is required for ovulation in zebrafish.

Fine selection of up-regulated genes during ovulation by in vivo induction of oocyte maturation and ovulation in zebrafish

BackgroundTwo essential processes, oocyte maturation and ovulation, are independently induced, but proceed cooperatively as the final step in oogenesis before oocytes become fertilizable. Although these two processes are induced by the same maturation-inducing steroid, 17α, 20β-dihydroxy-4-pregnen-3-one (17, 20β-DHP), in zebrafish, it has been suggested that the receptor, and thus the signal transduction pathway is different for each process. Although much progress has been made in understanding the molecular mechanisms underlying the induction of oocyte maturation, the mechanisms for inducing ovulation remain under investigation. In the present study, in vivo induction techniques that permit the induction of oocyte maturation and ovulation in living zebrafish (in vivo assays) were used to select highly up-regulated genes (genes associated with ovulation). Using an in vivo assay, ovarian tissues that induced only oocyte maturation could be obtained. This made it possible for the first time to distinguish maturation-inducing genes from ovulation-inducing genes. Using a genome-wide microarray of zebrafish sequences, the gene expression levels were compared among an ethanol (EtOH)-treated group (non-activated group), a diethylstilbestrol (DES)- or testosterone (Tes)-treated group (maturation-induced group), and a 17, 20β-DHP-treated group (maturation- and ovulation-induced group). Ovulation-specific up-regulated genes were selected. The mRNA expression levels of the selected genes were measured by quantitative polymerase chain reaction (qPCR).ResultsAmong 34 genes identified, three that showed ovulation-specific increases were selected as candidates potentially associated with ovulation. The ovulation-specific up-regulation of three candidates, slc37a4a, zgc:65811 and zgc:92184 was confirmed by qPCR.ConclusionOur in vivo assay provides a new approach to precisely select genes associated with ovulation.

Gene knockout of nuclear progesterone receptor provides insights into the regulation of ovulation by LH signaling in zebrafish.

It is well established that the luteinizing hormone surge triggers ovulation, a dynamic process leading to the release of the mature oocyte from the ovarian follicle. But how this process controlled by LH signaling remains largely unknown in non-mammalian species. In this study, we investigated the roles of nuclear progesterone receptor (npr) in LH-induced ovulation. Our results indicate that the nuclear progesterone receptor serves as an important mediator of LH action on ovulation. This conclusion is based on the following results: (1) the expression level of npr peaks at the full-grown stage of the follicles; (2) the expression of npr is stimulated by LH signaling in vitro and in vivo; and (3) the npr null females are infertile due to ovulation defects. Moreover, we further show that LH signaling could induce ptger4b expression in an npr-dependent manner, and blockage of Ptger4b could also block hCG-induced ovulation. Collectively, our results not only demonstrate that npr serves an indispensable role in mediating the action of LH on ovulation in zebrafish, but also provide insights into the molecular mechanisms of the regulation of ovulation in fish.

The role of eicosanoids in 17α, 20β-dihydroxy-4-pregnen-3-one-induced ovulation and spawning in Danio rerio

This study employed a hormone bioassay to characterize the eicosanoids involved in zebrafish ovulation and spawning, in particular the prostaglandin (PG) products of cyclooxygenase (COX) metabolism and the leukotriene (LT) products of lipoxygenase (LOX) metabolism. Exposure to the teleost progestogen 17α, 20β-dihydroxy-4-pregnen-3-one (17,20βP) induced ovulation, but not spawning, in solitary females and both ovulation and spawning in male–female pairs. Transcription of the eicosanoid-synthesizing enzymes cytosolic phospholipase A2 (cPLA2) and COX-2 increased and LTC4 synthase decreased in peri-ovulatory ovaries of 17,20βP-exposed fish. Ovarian PGF2α levels increased post-spawning in 17,20βP-exposed fish, but there was no difference in LTB4 or LTC4. Pre-exposure to cPLA2 or LOX inhibitors reduced 17,20βP-induced ovulation rates, while a COX inhibitor had no effect on ovulation or spawning. Collectively, these findings suggest that eicosanoids, in particular LOX metabolites, mediate 17,20βP-induced ovulation in zebrafish. COX metabolites also appear to be involved in ovulation and spawning but their role remains undefined.

In Vivo Induction of Oocyte Maturation and Ovulation in Zebrafish

The maturation of fish oocytes is a well-characterized system induced by progestins via non-genomic actions. In a previous study, we demonstrated that diethylstilbestrol (DES), a non-steroidal estrogen, induces fish oocyte maturation via the membrane progestin receptor (mPR). Here, we attempted to evaluate the effect of DES as an environmental endocrine disrupting chemical (EDC) upon fish oocyte maturation using live zebrafish. DES triggered oocyte maturation within several hours in vivo when administrated directly into the surrounding water. The natural teleost maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17,20beta-DHP) also induced oocyte maturation in vivo. Steroids such as testosterone, progesterone or 17alpha-hydroxyprogesterone were also effective in vivo. Further studies indicated that externally applied 17,20beta-DHP even induced ovulation. In contrast to 17,20beta -DHP, DES induced maturation but not ovulation. Theoretically this assay system provides a means to distinguish pathways involved in the induction of ovulation, which are known to be induced by genomic actions from the pathway normally involved in the induction of oocyte maturation, a typical non-genomic action-dependent pathway. In summary, we have demonstrated the effect of EDCs on fish oocyte maturation in vivo. To address the effects, we have explored a conceptually new approach to distinguish between the genomic and non-genomic actions induced by steroids. The assay can be applied to screens of progestin-like effects upon oocyte maturation and ovulation for small molecules of pharmacological agents or EDCs.

An investigation into the role of prostaglandins in zebrafish oocyte maturation and ovulation

This study explored the potential for ovarian-derived prostaglandins (PGs) to be involved in the regulation of oocyte maturation and ovulation in zebrafish. It was demonstrated that cultured vitellogenic follicles have the capacity to produce prostaglandin E 2 (PGE 2) and PGF 2α in response to arachidonic acid (AA) in a concentration-dependent manner, and that AA stimulates the in vitro production of 17β-estradiol (E 2). The production of AA-stimulated PGF 2α was significantly reduced by treatment with the non-selective cyclooxygenase (COX) inhibitor, indomethacin (INDO). Treatment of full-grown follicles with AA did not induce oocyte maturation as assessed by germinal vesicle breakdown, but INDO significantly decreased the rate of spontaneous maturation. Using Real-Time PCR, it was shown that follicles of different developmental size classes (primary growth and pre-vitellogenic, early-vitellogenic, and mid- to full-grown vitellogenic) express enzymes that release (cytosolic phospholipase A 2 (cPLA 2); phospholipase Cγ1) or metabolize (COX-1, COX-2, and prostaglandin synthase-2) AA to PG metabolites. The expression of cPLA 2 was found to be significantly greater in full-grown follicles compared to follicles of the pre- and early-vitellogenic stages. In vivo studies demonstrated that breeding groups of zebrafish exposed to 100 μg/L INDO exhibited reduced spawning rates and clutch sizes compared with control and 1 μg/L INDO exposed fish. In other studies, it was shown that naturally spawning groups of females exhibit increased ovarian levels of PGF 2α, E 2, and 17α,20β-dihydroxy-4-pregnen-3-one (a maturation-inducing hormone in zebrafish) near the time of ovulation compared with non-breeding females. Collectively, these experiments indicate that the AA pathway in zebrafish ovaries is involved in the regulation of oocyte maturation and ovulation and a non-selective inhibitor of COX disrupts these processes.

The biosynthesis of steroid glucuronides in the testis of the zebrafish, Brachydanio rerio, and their pheromonal function as ovulation inducers

In female zebrafish ovulation could be induced by male holding water, testis homogenates, and testis fractions containing steroid glucuronides. Deglucuronidation of these fractions led to a loss of ovulation-inducing potency, indicating steroid glucuronides as ovulation inducers. The chemical substances were perceived by the recipient females by means of olfaction. Incubation experiments showed the capacity of the testes to synthesize various C 19 and C 21 steroids and seven different steroid glucuronides, i.e., 17α,20β-dihydroxy-4-pregnen-3-one-, testosterone-, androsterone-, epiandrosterone-, 5α-androstane-3α,17β-diol-, and 5α-androstane-3β,17β-diol glucuronide. GC-MS analysis showed the presence of glucuronides of 5α-androstane-3α,17β-diol and cholesterol in male holding water, the latter probably originating from the liver. These compounds may be among the steroid glucuronides functioning as ovulation-inducing pheromones.