Maturation-inducing Hormone Research Articles

Zinc modulates hypothalamo-pituitary-gonadal-liver axis to impair reproduction in female Mystus vittatus (Bloch, 1794).

The present study investigated the effects of zinc on the hypothalamo-pituitary-gonadal-liver (HPGL) axis of the bagrid catfish Mystus vittatus. Female fish (pre-ovulatory and ovulatory phases) were exposed to zinc sulphate at 1/10th of LC50 (5.62mg/L) for 60days and sacrificed at every 15-day interval to collect tissues. Zinc concentration in all tissues was significantly higher in the metal-exposed group at all exposure durations compared to control for both phases. Metallothionein (MT) levels increased in the brain, liver and ovary of fish from both phases with exposure duration. Reactive oxygen species (ROS) generation in the brain, liver and ovary tissues increased with exposure duration at both reproductive phases while serum cortisol levels in ovulatory fish increased significantly compared to pre-ovulatory. Condition factor, gonadosomatic index and hepatosomatic index decreased in Zn-exposed fish. Brain GnRH and kisspeptin levels decreased significantly in the Zn-exposed group for both phases. GnIH was significantly higher in Zn-exposed fish. Serum FSH levels in pre-ovulatory and LH levels in ovulatory fish decreased gradually with an increase in the duration of exposure. Zn exposure reduced vitellogenin (Vtg) and estradiol (E2) in the liver and ovary with an increase in duration from both phases. Ovary maturation-inducing hormone (MIH) levels showed a decrease with exposure duration in ovulatory fish. Moreover, Zn-exposed ovulatory fish showed a degenerated oocyte nucleus due to the disintegration of the nuclear membrane. It might be inferred that Zn altered the HPGL regulatory system of M. vittatus reproduction at both the pre-ovulatory and ovulatory phases.

Higher intake of β-glucan impairs reproduction in a female teleost, Tor putitora (Hamilton, 1822).

Although the immuno-modulatory and stress-relieving properties of β-glucan is well elucidated in humans and other animal models, including fish, its role as a dietary supplement on reproduction is extremely scarce. Therefore, in this study, adult female fish were fed one of four test diets having 0 (control), 0.5, 1, and 1.5% β-D-glucan for 130 days and its effect on reproductive performance, ovarian and liver histology, sex hormones, and transcript abundance of selected reproduction-related genes was assessed. Low dietary intake of β-glucan improved fertilization and hatching rates (p<0.05). The relative fecundity and percentage of spawning females were higher (non-significant) in 0.5% β-glucan-fed groups. Surprisingly, even after 130 days, spawning did not occur in 1.5% β-glucan-fed individuals. Irrespective of β-glucan intake, all the brooders recorded similar plasma 17β-estradiol and maturation-inducing hormone (p>0.05). Higher intake of β-glucan (1.5%) upregulated aromatase genes without a parallel increase in 17β-estradiol. However, plasma vitellogenin increased with increasing β-glucan up to 1.0% then declined at 1.5% (p<0.05). The fish that received control, 0.5, and 1.5% β-glucan recorded similar vitellogenin levels in their plasma. Significantly higher plasma cortisol was evidenced in 1.5% β-glucan fed brooders (p<0.05). Histologically, higher follicular atresia and leaking of yolk material was evidenced in 1.5% β-glucan-fed group. Liver histology revealed the highest nutrient/lipid accumulation in fish that received 1.0% and 1.5% β-glucan. This study demonstrated the stimulatory effect of β-glucan intake at a lower dose (0.5%) on reproduction. However, higher intake (1.5%) could perturb normal reproductive function in a fish model and caused an increased number of atretic follicles leading to spawning/reproductive failure.

Japanese medaka (Oryzias latipes) exposed via maternal transfer to the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), experience decreased fecundity and impaired oocyte maturation

Early life-stage exposure of fishes to endocrine disrupting chemicals can induce reproductive impairment at sexual maturity. Previously, we demonstrated decreased fecundity of Japanese medaka (Oryzias latipes) exposed via maternal transfer to the novel brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO). However, that study failed to identify the causative mechanism. In other studies we have shown that decreased fecundity of adult fish exposed to dietary TBCO is likely due to impaired oocyte maturation. The goal of the present study was to determine if impaired oocyte maturation is responsible for decreased fecundity of Japanese medaka exposed as embryos to TBCO, via maternal transfer. Sexually mature fish (F0) were fed either a control diet or a low (74.7 μg/g) or high (663 μg/g) diet containing TBCO for 21 days. Eggs (F1) were collected during the final week of exposure and reared to sexual maturity at which point fecundity was assessed using a 21-day reproduction assay. Upon termination of the assay, an ex vivo oocyte maturation assay was used to determine whether maturation inducing hormone (MIH) stimulated oocyte maturation was impaired. Additionally, concentrations of 17β -estradiol (E2) in blood plasma and expression of genes involved in vitellogenesis and oocyte maturation were quantified. The F1 generation females reared from the low or high F0 treatments experienced a 26.0 % and 56.8 % decrease in cumulative fecundity, respectively. Ex vivo MIH stimulated oocyte maturation from the low and high TBCO treatments were decreased by 23.4 % and 20.0 % respectively. There was no significant effect on concentrations of E2. Transcript abundance of vtgI was significantly decreased in a concentration dependent manner. Transcript abundance of mPRα, pgrmc1, pgrmc2, and igf3 were decreased but effects were not statistically significant. Overall, results suggest that impaired oocyte maturation causes decreased fecundity of Japanese medaka exposed to maternally deposited TBCO.

Impairment of oocyte maturation as a mechanism of decreased fecundity in Japanese medaka (Oryzias latipes) exposed to the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO)

Inhibition of oocyte maturation is an understudied mechanism by which chemical stressors can impair fecundity of female fishes. The primary objective of the present study was to develop an assay to assess oocyte maturation disruption by chemical stressors in Japanese medaka (Oryzias latipes). First, an in vitro assay to assess maturation inducing hormone (MIH)-stimulated oocyte maturation in zebrafish was validated for use with Japanese medaka. Next, using the brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), which previously was shown to decrease fecundity of Japanese medaka and inhibit oocyte maturation in zebrafish, effects on oocyte maturation were quantified using in vitro and in vivo exposure. Adaptation of the protocol for in vitro MIH-stimulated maturation of stage IV oocytes from zebrafish was successful in inducing greater than 80% of stage IX oocytes from female Japanese medaka to mature. To assess effects of in vitro exposure, stage IX oocytes were exposed to 0, 2, 20, and 200 μg/L of TBCO, followed by exposure to MIH. The in vitro exposure caused a significant decrease in maturation of oocytes exposed to 20 and 200 μg/L of TBCO. To assess effects of TBCO on fecundity and oocyte maturation following in vivo exposure, sexually mature fish were fed a control, 100 μg/g, or 1000 μg/g concentration of TBCO-spiked fish food for 21 days, where fecundity was measured daily, and following the exposure, stage IX oocytes were excised to assess MIH-stimulated maturation. Fecundity and oocyte maturation were significantly decreased at either concentration of TBCO. Plasma concentrations of 17β-estradiol (E2) and hepatic abundances of transcripts of vitellogenin (vtgI and vtgII) were quantified, but there were no significant differences between treatments. Results suggest that inhibition of oocyte maturation is a mechanism by which TBCO decreases fecundity, and that in vitro assays of oocyte maturation might be predictive of fecundity in this species.

The induction of oocyte maturation and ovulation in the European eel (Anguilla anguilla): in vitro and in vivo comparison of progesterone with 17α,20β-dihydroxy-4-pregnen-3-one.

Ovulation in European eel is induced by injection of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) as the maturation-inducing hormone (MIH). Female eels need to ovulate within 18h after injection to release good quality eggs. Progesterone (P), as an upstream precursor of DHP, may promote endogenous DHP production and improve egg quality. The purpose of this study was therefore to compare treatment of P with DHP on batch level, in vitro, to determine dose-response effects, and in vivo, at a single dose. For the in vitro experiment, ovarian tissue was extracted and placed in culture plates containing hormone-free medium and media supplemented with the treatment: DHP at 1, 10 and 100ngmL-1, or P at 10, 100 and 1,000ngmL-1. At the start of incubation, the folliculated oocytes were sampled for histology, microscopy and qPCR. After incubation for 12 and 18h, the oocytes were sampled for microscopy and qPCR analysis. For the in vivo experiment, females were either injected with DHP or P at a dose of 2mgkg-1 to assess their effects on ovulation and reproductive success. At the moment of release, eggs were sampled for RNA sequencing to compare effects of DHP and P on the expression of genes involved in egg quality aspects. Remaining eggs were fertilized and larval viability was recorded. Both DHP and P were able to induce GVBD (DHP at 10 and 100ngmL-1, P at 100 and 1,000ngmL-1) in vitro. Expression of genes involved in oocyte maturation and ovulation was similar in vitro for both DHP and P treatments. Regarding the in vivo results, RNAseq results reflected similar DHP and P effects on the expression of genes involved in egg quality aspects. Females injected with either DHP or P ovulated, released eggs, and were equally able to produce larvae without any differences in reproductive success. Our results support the conclusion that DHP and P work equally well in vitro and in vivo. P is more attractive to apply as the price is 3,000 times lower than the price of DHP.

Relaxin-like Gonad-Stimulating Peptides in Asteroidea.

Starfish relaxin-like gonad-stimulating peptide (RGP) is the first identified peptide hormone with gonadotropin-like activity in invertebrates. RGP is a heterodimeric peptide, comprising A and B chains with disulfide cross-linkages. Although RGP had been named a gonad-stimulating substance (GSS), the purified peptide is a member of relaxin-type peptide family. Thus, GSS was renamed as RGP. The cDNA of RGP encodes not only the A and B chains, but also signal and C-peptides. After the rgp gene is translated as a precursor, mature RGP is produced by eliminating the signal and C-peptides. Hitherto, twenty-four RGP orthologs have been identified or predicted from starfish in the orders Valvatida, Forcipulatida, Paxillosida, Spinulosida, and Velatida. The molecular evolution of the RGP family is in good accordance with the phylogenetic taxonomy in Asteroidea. Recently, another relaxin-like peptide with gonadotropin-like activity, RLP2, was found in starfish. RGP is mainly present in the radial nerve cords and circumoral nerve rings, but also in the arm tips, the gonoducts, and the coelomocytes. RGP acts on ovarian follicle cells and testicular interstitial cells to induce the production of 1-methyladenine (1-MeAde), a starfish maturation-inducing hormone. RGP-induced 1-MeAde production is accompanied by an increase in intracellular cyclic AMP levels. This suggests that the receptor for RGP (RGPR) is a G protein-coupled receptor (GPCR). Two types of GPCRs, RGPR1 and RGPR2, have been postulated as candidates. Furthermore, 1-MeAde produced by RGP not only induces oocyte maturation, but also induces gamete shedding, possibly by stimulating the secretion of acetylcholine in the ovaries and testes. Thus, RGP plays an important role in starfish reproduction, but its secretion mechanism is still unknown. It has also been revealed that RGP is found in the peripheral adhesive papillae of the brachiolaria arms. However, gonads are not developed in the larvae before metamorphosis. It may be possible to discover new physiological functions of RGP other than gonadotropin-like activity.

Epigenetics and Probiotics Application toward the Modulation of Fish Reproductive Performance

Fish represent an excellent source of animal protein as well as a biomedical research model as a result of their evolutionary relatedness and similarity with the human genome. Commercial and ornamental fish culture has achieved popularity, but reproductive dysfunctions act as a limiting factor for quality fry production, interfering with the sustainability of the aquaculture industry. Fish reproduction is crucial for any species’ existence, and reproductive performance can potentially be improved through applications of epigenetics and probiotics. Epigenetics is a highly sensitive molecular approach that includes chromatin structure and function alteration, DNA methylation, and modification of non-coding RNA molecules for the transfer of desired information from parents to offspring. DNA methyltransferase improves reproductive cyp11a1, esr2b, and figla gene expression and feminizes zebrafish (Danio rerio). Moreover, epigenetics also contributes to genome stability, environmental plasticity, and embryonic development. However, methylation of specific genes can negatively affect sperm quality, resulting in poor fertilization. Probiotic administration is able to induce responsiveness of incompetent follicles to maturation-inducing hormones and can change oocyte chemical composition during vitellogenic development. The positive role of probiotics on testicular cells is validated by upregulating the transcription levels of leptin, bdnf, and dmrt1 genes facilitating the spermatogenesis. This review not only discusses the effects and mechanism of epigenetics and probiotics for improving fish reproduction, but also presents an overview of the causal factors and current techniques used to eradicate dysfunction. Moreover, key genes and hormones related to fish reproduction along with research gaps and future prospects are also considered. This review provides an overview of necessary information for students, scientists, researchers, and breeders to resolve fish reproduction-related problems to ensure profitable and sustainable aquaculture.

Inhibition of Oocyte Maturation by Malathion and Structurally Related Chemicals in Zebrafish (Danio rerio) After In Vitro and In Vivo Exposure.

Oogenesis is the process by which a primary oocyte develops into a fertilizable oocyte, making it critical to successful reproduction in fish. In zebrafish (Danio rerio), there are five stages of oogenesis. During the final step (oocyte maturation), the maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (MIH) activates the membrane progestin receptor, inducing germinal vesicle breakdown. Using in vitro assays, it has been shown that anthropogenic stressors can dysregulate MIH-induced oocyte maturation. However, it is unknown whether the in vitro assay is predictive of reproductive performance after in vivo exposure. We demonstrate that a known inhibitor of oocyte maturation, malathion, and a structurally related chemical, dimethoate, inhibit oocyte maturation. However, malaoxon and omethoate, which are metabolites of malathion and dimethoate, did not inhibit oocyte maturation. Malathion and dimethoate inhibited maturation to a similar magnitude when oocytes were exposed for 4 h in vitro or 10 days in vivo, suggesting that the in vitro zebrafish oocyte maturation assay might be predictive of alterations to reproductive performance. However, when adult zebrafish were exposed to malathion for 21 days, there was no alteration in fecundity or fertility in comparison with control fish. Our study supports the oocyte maturation assay as being predictive of the success of in vitro oocyte maturation after in vivo exposure, but it remains unclear whether inhibition of MIH-induced oocyte maturation in vitro correlates to decreases in reproductive performance. Environ Toxicol Chem 2022;41:1381-1389. © 2022 SETAC.

Starfish oocytes of A. pectinifera reveal marked differences in sperm-induced electrical and intracellular calcium changes during oocyte maturation and at fertilization.

Although changes in membrane potential and intracellular Ca2+ (Cai ) during fertilization in starfish oocytes have been known for long time, little is known precisely about how and what kind of channels are involved during oocyte maturation and in fertilization, and how the mechanisms of changes in Cai in oocytes develop during oocyte maturation. Since in starfish, oocyte maturation-inducing hormone, 1-methyladenine (1MA) is well known, we took advantage of it to investigate the developmental process of channel-function and changes in Cai in three different developmental stages using 1MA. Sperm-induced membrane current at voltage clamp and changes in Cai in starfish oocytes, Asterina pectinifera, were examined in stages of immature, partly mature (a state in 15-20 min after sufficient concentration, 1 µM of 1MA addition, or 30-40 min exposure to subthreshold concentration of 1MA), and mature oocytes (MO). We found some immature and many partly MOs showed fluctuating responses in membrane current, membrane potential, and corresponding changes in Cai , which are distinct from those in MOs. The responses in immature and partly MOs indicate physiologically characteristic responses of insufficient changes in Cai and its corresponding electrical responses at the partial developmental stage during maturation. Our data should shed light on the mechanism of egg activation and oocyte maturation in terms of examining membrane current and corresponding changes in Cai .

In vitro follicle culture shows that progestagens are the maturation-inducing hormones (MIH) and possible regulators of the ovulation-mediating hormone PGE2 in female Eurasian perch Perca fluviatilis.

In European aquaculture, Eurasian perch, Perca fluviatilis L., is perceived as one of the most highly valuable freshwater fish species and a strong candidate for the development of freshwater aquaculture. In the pursuit of improving the quality of reproduction in this domesticated species, investigating the hormones mediating the final oocyte maturation (FOM) is therefore indispensable. But, the exact nature of the maturation-inducing hormone (MIH) in Eurasian perch is unknown. To further validate the existence of a maturation-inducing activity behind potential hormonal candidates in this species, we in vitro tested a group of nine hormones: cortisol (Co), 11-deoxycortisol (11-D), corticosterone (coS), 11-deoxycorticosterone (DOC), 17α,20βdihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21 trihydroxy-4-pregnen-3-one (THP), prostaglandin E2 (PGE2), estradiol-17β (E2) and testosterone (T), in their ability to trigger FOM advancement and the production of sex steroids potentially involved in FOM. Using mature female perch, two in vitro experiments were conducted with oocytes at the start of the FOM. The follicles were incubated for 62 h in Cortland media with and without human chorionic gonadotropin (hCG). By the end of the incubation, only DHP and THP triggered the full advancement in FOM even at low doses with the effect of DHP being in vivo validated. However, the de novo productions of E2 and DHP were not shown to be regulated by either of the MIH candidates. Progestagens are hence more credible candidates as MIH than corticosteroids in Eurasian perch. Our in vitro study also revealed that both PGE2 and DHP are strongly associated with ovulation and that PGE2 might have slightly contributed to such DHP activity.

Vasotocin stimulates maturation-inducing hormone, oocyte maturation and ovulation in the catfish Heteropneustes fossilis: Evidence for a preferential calcium involvement

Arginine vasotocin (VT) is the basic neurohypophysial nonapeptide hormone in teleosts. VT is also distributed in the ovary of the catfish Heteropneustes fossilis and induces final oocyte maturation (FOM) and ovulation by stimulating the maturation-inducing hormone (MIH). The present study reports the effects of cAMP (0.5 mM), phosphodiesterase inhibitors (IBMX -0.5 mM and theophylline- 0.5 mM), the inositol triphosphate (IP3) receptor inhibitor heparin (10 μg/mL) and the Ca2+ chelator BAPTA-AM (25 μM) on VT (100 nM) - induced progestin stimulation, FOM and ovulation. Incubation of post-vitellogenic follicles with cAMP, IBMX and theophylline for 0, 8, 16 and 24 h stimulated basal secretion of progesterone (P4), 17-hydroxyprogesterone (17-P) and 17, 20β-dihydroxy-4-pregnen-3-one (MIH) in a duration-dependent manner. The incubation of the follicles with heparin stimulated P4 modestly, and 17-P and MIH levels in a duration-dependent manner. The incubation of the follicles with BAPTA-AM stimulated P4 and MIH levels marginally and 17-P robustly. The stimulation was in the order cAMP > IBMX > theophylline > heparin > BAPTA-AM. The incubation of the follicles with VT stimulated P4, 17-P, MIH, GVBD and ovulation in a duration-dependent manner. The co-incubations with VT and the test compounds inhibited the VT-induced stimulation of P4, 17-P and MIH levels in a time-dependent manner in the order heparin > BAPTA-AM > cAMP > IBMX > theophylline. Concurrently, the VT-induced stimulation of GVBD and ovulation were also inhibited by the test compounds in the same order. The results show that VT induces FOM and ovulation preferentially acting through Ca2+ pathway and a crosstalk between Ca2+ and cAMP signaling pathways seems to integrate the processes.

An agonist for membrane progestin receptor (mPR) induces oocyte maturation and ovulation in zebrafish in vivo

The maturation and ovulation of fish oocytes are well-characterized biological processes induced by progestins via coordination of nongenomic actions and genomic actions. Previously, we established a procedure that enables the induction of oocyte maturation and ovulation in live zebrafish by simple administration of the natural teleost maturation-inducing hormone 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20β-DHP) into the surrounding water. By this in vivo assay, the potencies of chemicals in inducing or preventing oocyte maturation and ovulation can be evaluated. The potencies of compounds in inducing ovulation of zebrafish oocytes also can be evaluated in vivo with improved in vitro assays. Here, we attempted to evaluate the effect of Org OD 02–0 (Org OD 02), a selective agonist for membrane progestin receptor (mPR), on fish oocyte maturation and ovulation with in vitro and in vivo assays. As reported previously, Org OD 02 triggered oocyte maturation in vitro. The same Org OD 02 triggered oocyte maturation within several hours in vivo. Surprisingly, Org OD 02 even induced ovulation both in in vivo and in vitro. Eggs from Org OD 02-induced ovulation could be fertilized by artificial insemination. The juveniles developed normally. These results indicated that Org OD 02 triggered physiological ovulation in live zebrafish. In summary, we have demonstrated the effect of Org OD 02 on fish oocyte maturation and ovulation in vitro and in vivo. The results suggested that Org OD 02 acted as an agonist not only of mPR but also of nuclear progesterone receptor (nPR).

A G protein-coupled receptor mediates neuropeptide-induced oocyte maturation in the jellyfish Clytia.

The reproductive hormones that trigger oocyte meiotic maturation and release from the ovary vary greatly between animal species. Identification of receptors for these maturation-inducing hormones (MIHs) and understanding how they initiate the largely conserved maturation process remain important challenges. In hydrozoan cnidarians including the jellyfish Clytia hemisphaerica, MIH comprises neuropeptides released from somatic cells of the gonad. We identified the receptor (MIHR) for these MIH neuropeptides in Clytia using cell culture–based “deorphanization” of candidate oocyte-expressed G protein–coupled receptors (GPCRs). MIHR mutant jellyfish generated using CRISPR-Cas9 editing had severe defects in gamete development or in spawning both in males and females. Female gonads, or oocytes isolated from MIHR mutants, failed to respond to synthetic MIH. Treatment with the cAMP analogue Br-cAMP to mimic cAMP rise at maturation onset rescued meiotic maturation and spawning. Injection of inhibitory antibodies to the alpha subunit of the Gs heterodimeric protein (GαS) into wild-type oocytes phenocopied the MIHR mutants. These results provide the molecular links between MIH stimulation and meiotic maturation initiation in hydrozoan oocytes. Molecular phylogeny grouped Clytia MIHR with a subset of bilaterian neuropeptide receptors, including neuropeptide Y, gonadotropin inhibitory hormone (GnIH), pyroglutamylated RFamide, and luqin, all upstream regulators of sexual reproduction. This identification and functional characterization of a cnidarian peptide GPCR advances our understanding of oocyte maturation initiation and sheds light on the evolution of neuropeptide-hormone systems.

Induction of oocyte maturation and changes in the biochemical composition, physiology and molecular biology of oocytes during maturation and hydration in the orange-spotted grouper (Epinephelus coioides)

In aquaculture, oocyte maturation, ovulation and spawning are important for successful breeding and seed production. Most marine teleost fish can be induced to spawn regularly using intramuscular injections of human chorionic gonadotropin (HCG). In this study, an intra-ovarian injection method was developed in orange-spotted grouper to deal with the broodstocks unresponsive to HCG. Successful oocyte maturation and ovulation in the unilateral injected ovary was induced within 35 h of treatment. An in vitro method was then established to induce HCG-primed oocyte maturation. HCG or maturation-inducing hormones (MIHs) induced the maturation of oocytes with a diameter between 490 and 560 μm. Biochemical, physiological and molecular changes during oocyte maturation and hydration were also characterized. The concentration of free amino acids except the cysteine increased during oocyte maturation, whereas Na+ decreased steadily and K+ increased. The expression of genes encoding cathepsin B, D, L, and the subunits of Na+, K+-ATPase were upregulated during hydration. Specific inhibitors for cathepsin and Na+, K+-ATPase were used to further confirm their crucial roles in oocyte hydration both in vivo and in vitro. These results provide valuable data for inducing oocyte maturation and evaluating oocyte quality in marine teleost fish.

Development of an enzyme-functionalized immunosensor for measuring maturation-inducing hormone in fish

In modern aquaculture, simple and rapid methods of predicting fish ovulation are needed to produce seedlings in a stable and efficient manner. Ovulation time is variable due to environmental and physiological factors, while 17α,20β-dihydroxy-4-pregnen-3-one (DHP), an indicator of ovulation, increases its concentration rapidly just before spawning. In this paper, we developed a biosensor system in which immune and enzymatic reaction assays were combined to monitor DHP levels with low environmental loads. We attempted to quantify DHP by analyzing the decrease rate in the output current after the immunoreaction using the amperometric method as a transfer inhibition degree of the enzyme-generated electrons generated by the formation of the antigen-antibody complex. DHP standard samples were measured under investigated optimal conditions and a specific response with a good linear correlation was obtained between the current decrease rate of the sensor system and the DHP concentration in a range of 7.81∼500 pg ml−1. Further, DHP levels in goldfish (Carassius auratus) during the ovulation period obtained using the sensor system were compared with those of using the conventional method (ELISA) and a good correlation was obtained. These findings suggest that the proposed sensor system can detect DHP quickly and easily compared with the conventional method.

Identification of Novel MicroRNAs and Characterization of MicroRNA Expression Profiles in Zebrafish Ovarian Follicular Cells.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression primarily at the post-transcriptional levels and thereby play important roles in regulating many physiological and developmental processes. Oocyte maturation in fish is induced by hormones produced from the hypothalamus, pituitary, and ovary. Gonadotropin-releasing hormone (GnRH) stimulates the secretion of luteinizing hormone (LH), which in turn, induces the secretion of maturation-inducing hormone (MIH) from the ovary. It is documented that small early vitellogenic (or stage IIIa) follicles are unable to undergo oocyte maturation whereas oocytes in mid- to late vitellogenic (stage IIIb) follicles can be induced by LH and MIH to become mature. To determine whether miRNAs may be involved in the growth and acquisition of maturational competency of ovarian follicles, we determined the miRNA expression profiles in follicular cells collected from stage IIIa and IIIb follicles using next-generation sequencing. It was found that miRNAs are abundantly expressed in the follicular cells from both stages IIIa and IIIb follicles. Furthermore, bioinformatics analysis revealed the presence of 214 known, 31 conserved novel and 44 novel miRNAs in zebrafish vitellogenic ovarian follicular cells. Most mature miRNAs in follicular cells were found to be in the length of 22 nucleotides. Differential expression analysis revealed that 11 miRNAs were significantly up-regulated, and 13 miRNAs were significantly down-regulated in the stage IIIb follicular cells as compared with stage IIIa follicular cells. The expression of four of the significantly regulated miRNAs, dre-miR-22a-3p, dre-miR-16a, dre-miR-181a-3p, and dre-miR-29a, was validated by real-time PCR. Finally, gene enrichment and pathway analyses of the predicted targets of the significantly regulated miRNAs supported the involvement of several key signaling pathways in regulating ovarian function, including oocyte maturation. Taken together, this study identifies novel zebrafish miRNAs and characterizes miRNA expression profiles in somatic cells within the zebrafish ovarian follicles. The differential expression of miRNAs between stage IIIa and IIIb follicular cells suggests that these miRNAs are important regulators of zebrafish ovarian follicle development and/or oocyte maturation.

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.

Daily rhythms in the reproductive axis of Nile tilapia (Oreochromis niloticus): Plasma steroids and gene expression in brain, pituitary, gonad and egg

Control of reproduction is one of the key issues to extend tilapia aquaculture. In most fish species, reproduction is a rhythmic process which ensures that reproduction timing matches the most favorable environmental conditions to maximize the survival of offspring. Although the existence of daily spawning rhythms has been reported for a number of fish species, the daily rhythms in neuroendocrine factors along the BPG (brain-pituitary-gonadal) axis remains mostly unknown. The objective of this paper was to investigate the existence of daily rhythms in BPG gene expression and plasma sex steroids in tilapia (Oreochromis niloticus). To this end, 36 males and 36 females were kept in separate tanks at 29 ± 0.5 °C and in a 12 h:12 h light/dark cycle. After 4 weeks of acclimation, male and female tilapia were mixed into six groups at a female:male ratio of 6:6. Two weeks later, fish were anesthetized and sampled every 4 h during a 24-hour cycle, and blood, brain, pituitary, gonad and eggs were collected for later analyses. The expression levels of the genes from the brain (gnrh1, gnrh2, gnrh3, kiss2, gnih), pituitary (fshβ and lhβ), gonad (star, cyp11a, cyp17, cyp19a1a and amh) and eggs (zp3b and fyna) were analyzed by qPCR. ELISA was used to analyze testosterone and estradiol in male and female plasma, respectively, and maturation-inducing hormone (MIH) in plasma in both sexes. The results revealed timely-coordinated daily rhythms along the BPG axis of tilapia. The expression of the brain genes showed that their acrophase was distributed throughout the LD cycle. However, a coordinated and harmonious pattern of daily rhythms appeared on the following levels of the BPG axis. The peaks in expression of the pituitary genes were reached at night, while the gonadal genes and plasma sex steroids peaked in the light phase. These findings can be useful for improving breeding protocols for tilapia aquaculture and for defining optimum timing for reproduction in captivity.

Starfish Gonadotropic Hormone: From Gamete-Shedding Substance to Relaxin-Like Gonad-Stimulating Peptide.

The first report of a gonadotropic substance in an invertebrate hot-water extract of radial nerve cords from starfish Asterias forbesi that induced the shedding of gametes when injected into the coelomic cavity in a ripe individual occurred in 1959. The active substance was named gamete-shedding substance (GSS) or radial nerve factor. GSS is the primary mediator of oocyte maturation and ovulation in starfish. However, the effect of GSS is indirect. Resumption of meiosis in immature oocytes and release from the ovary are induced by a second mediator, maturation-inducing hormone, identified as 1-methyladenine (1-MeAde) in starfish. The role of GSS is to induce 1-MeAde production by ovarian follicle cells. Thus, GSS was redesignated as gonad-stimulating substance (also GSS). Although GSS has been characterized biochemically as a peptide hormone, identification of the chemical structure had to wait until 2009. Fifty years after the initial finding, GSS was purified from the radial nerve cords of starfish Patiria pectinifera (P. pectinifera). The purified hormone was a heterodimer composed of A- and B-chains, with disulfide cross-linkages. Based on its cysteine motif, GSS is classified as a member of the insulin/insulin-like growth factor (IGF)/relaxin superfamily. More specifically, phylogenetic sequence analysis revealed that P. pectinifera GSS is a member of the relaxin-type peptide family. Therefore, GSS in starfish has been redesignated as relaxin-like gonad-stimulating peptide (RGP). Subsequently, orthologs of P. pectinifera RGP have been identified in other starfish species, including Asterias amurensis (A. amurensis), and Aphelasterias japonica (A. japonica).

Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones.

Oocyte meiotic maturation is crucial for sexually reproducing animals, and its core cytoplasmic regulators are highly conserved between species. By contrast, the few known maturation-inducing hormones (MIHs) that act on oocytes to initiate this process are highly variable in their molecular nature. Using the hydrozoan jellyfish species Clytia and Cladonema, which undergo oocyte maturation in response to dark-light and light-dark transitions, respectively, we deduced amidated tetrapeptide sequences from gonad transcriptome data and found that synthetic peptides could induce maturation of isolated oocytes at nanomolar concentrations. Antibody preabsorption experiments conclusively demonstrated that these W/RPRPamide-related neuropeptides account for endogenous MIH activity produced by isolated gonads. We show that the MIH peptides are synthesised by neural-type cells in the gonad, are released following dark-light/light-dark transitions, and probably act on the oocyte surface. They are produced by male as well as female jellyfish and can trigger both sperm and egg release, suggesting a role in spawning coordination. We propose an evolutionary link between hydrozoan MIHs and the neuropeptide hormones that regulate reproduction upstream of MIHs in bilaterian species.