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MiR-202-5p is identified as the maker microRNA (miRNA) of germplasm in fish. However, the role of miR-202-5p in gonadal development is still unclear. Here we investigated the expression patterns of miR-202-5p in Japanese flounder (Paralichthys olivaceus). Real-time PCR displayed that miR-202-5p was specifically expressed in bisexual gonads, and its transcription in the testis was relatively abundant than that in the ovary. Moreover, it was gradually increased in testis from stage II to stage IV. Fluorescent in situ hybridization (FISH) illustrated that miR-202-5p was co-localized with vasa (a marker gene of germ cells) in testis, and it was predominately expressed in spermatogonia and spermatocytes, implying that miR-202-5p might participate in spermatogenesis. Considering the important role of retinoic acid (RA) on regulating meiosis during spermatogenesis, we treated Japanese flounders by injecting all-trans retinoic acid (ATRA). MiR-202-5p in testis was shown to be up-regulated, and ccnd1, a key cell cycle regulator, was found to be down-regulated after ATRA treatment. Furthermore, ccnd1 was demonstrated to be a direct regulatory target of miR-202-5p using Dual-luciferase assay. In the primary testis cell from Japanese flounder, we further confirmed that ccnd1 was down-regulated by miR-202-5p. The results suggest that miR-202-5p exerts a crucial role in gonadal development by inhibiting ccnd1 in Japanese flounder.

In mammals, Yin Yang 1 (YY1), a pervasively expressed transcription factor related to many biological processes as an activator or inhibitor of the transcription of various genes, plays a critical role in the development of male gonads and spermatogenesis. Although the role of YY1 on the development of male gonads and spermatogenesis in mammals has been reported, its function on chicken testis are yet to be clarified. In this study, we used immunofluorescence analysis to investigate the location of YY1 in chicken testis. In embryo testis, YY1 was detected in spermatogonia and Sertoli cells, while in adult testis, YY1 was shown to be expressed in spermatogenic cells and Sertoli cells, but not in spermatozoa. Furthermore, we investigated the regulatory functions of YY1 in chicken testicular Sertoli cells by combining overexpression with RNA-sequencing. Overexpression of YY1 in Sertoli cells revealed a total of 2955 differentially expressed genes involved in various biological processes, such as male gonad development and seminiferous tubule development. Overexpression of YY1 also caused significant differences in the expression of the androgen receptor gene and the inhibin βA gene, two major genes involved in the regulation of spermatogonia in Sertoli cells. These observations indicate that YY1 may regulate the development and function of the gonads by affecting the secretion of cytokines and hormones in Sertoli cells to mediate the production and differentiation of spermatogonia.

The establishment and survival of the population of species in their breeding ground can be attributed to the species’ reproductive characteristic and capacity. The Monopterus albus species is an introduced fish in the Philippines. It is a protogynous hermaphrodite which has a natural ability for sex reversal. This study describes the histomorphology of the different stages of gonadal maturation and determines the concentration levels of the steroid hormones 17ß-estradiol (E2) and testosterone (T) in the blood plasma. These aim to a further understanding on the gonadal development and sex differentiation of the rice paddy eel as a hermaphroditic fish. Fish samples were collected randomly from farm ponds located in three provinces within the island of Luzon, Philippines. The sexual identity of the fish was categorized based on the presence or absence of ovarian and testicular tissues. Histological analysis of the gonad showed that immature, maturing-matured female gonad exhibited ovarian tissues of varying stages. The intersex gonad showed the existence of both ovarian and testicular tissues. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration levels of gonadal steroid hormones. Differences in the concentration levels of the two hormones were evident across all the stages of gonad maturation. The functional role of these hormones in vitellogenesis and spermatogenesis leads to the development of this sex-changing gonads. This reproductive characteristic contributes to our understanding of the reproductive success of hermaphroditic fish.

Pacific bluefin tuna (PBT), Thunnus orientalis, is one of the most important species for aquaculture in Japan. Recently, the reduction in muscle fat content associated with sexual maturation in farmed PBT has become a serious problem. To develop technologies for inducing sterility, detailed and reliable data on gonadal development in PBT are needed. Here, we demonstrated the process of gonadal sex differentiation, and of early ovarian and testicular development during the immature stages in PBT. Gonadal sex differentiation was first characterized by the formation of the ovarian cavity in female and of the efferent ducts in male 57 days post hatching (dph). The gonads then differentiated into ovaries or testes according to the genotypic sex until 83 dph. During this period, primordial germ cells, oogonia, and type-A spermatogonia were solitarily distributed in the gonads, and the number of germ cells did not differ between sexes. After gonadal sex differentiation, gonads of PBTs developed in a sexually dimorphic manner: proliferation and differentiation of germ cells occurred earlier in the ovaries than in the testes. The oogonia in ovaries formed cysts at 185 dph, but the type-A spermatogonia were solitarily distributed in testes at this stage, and cysts of type-A spermatogonia were first observed at 247 dph. Moreover, the oogonia entered meiosis and differentiated into chromatin-nucleolus stage oocytes until 247 dph, and subsequently into peri-nucleolus stage oocytes until 285 dph, whereas the type-A spermatogonia differentiated into type-B spermatogonia, spermatocytes, spermatids, and spermatozoa from 446 dph onwards. We believe the results of this study provide the necessary basis for future studies on sterile PBT production.

Pearlscale angelfish Centropyge vrolikii is a kind of protogynous hermaphrodite fish with a natural sexual reversion. Under appropriate social conditions, a female fish can transform into a male fish spontaneously. It is an important prerequisite for artificial breeding to understand the process of its gonadal development and sexual reversion. Gonadal development is regulated by many sex-related genes. In this study, we used unreferenced RNA-Seq technology to sequence the ovary at the perinucleolus stage (OII), ovary at the yolk vesicle stage (OIV),IV and testis (T), respectively; screened the gonadal differential expression genes (DEGs); and analyzed the expression of these genes in different developmental stages of ovary and different sex gonads. The results showed that a total of 142,589 all-unigene samples were assembled, and gene annotation was performed by COG, GO, KEGG, KOG, Pfam, Swissprot, eggNOG, and NR functional database. Comparative analysis revealed that there were 1919 genes that were up-regulated and 1289 genes were down-regulated in comparison to OIV vs OII, while there were 3653 genes that were up-regulated and 2874 genes were down-regulated in comparison of OIV vs T, there were 3345 genes that were up-regulated and 2995 genes were down-regulated in comparison of the OII vs the T. At the same time, the results verified by RT-qPCR were consistent with the variation trend of transcriptome data. Among the results, amh, sox9b, dmrt1, dmrt2, cyp11a, cyp17a, and cyp19a were significantly expressed in the testes, while sox3, sox4, sox11, sox17, and hsd3b7 were significantly expressed in the ovaries. And, the expression of the amh, sox9b, dmrt2, and dmrt1 were low in the OII and OIV, while significantly increased during the ovotestis in the hermaphroditic period (OT), and finally reached the highest level in pure testis after sex reversal. The expression of sox3, sox4, hsd3b7, sox11, and sox17 was significantly reduced during the hermaphroditic period (OT). These results suggested that these genes may play an important role in the process of sex reversal. This study is helpful to further understand the molecular regulation mechanism of gonadal development and sexual reversion in Pearlscale angelfish and also provide important clues for future studies.

Ghrelin is a multifunctional peptide hormone which dominantly secreted in stomach and intestine. Ghrelin involves in growth control, food intake, energy balance, and reproductive process. Recent evidences suggested that ghrelin also interacts with hypothalamic–pituitary–gonadal axis (HPG axis) and controls gametes maturation. In addition, current clues showed that via regulating ghrelin system (acyl ghrelin: des acyl ghrelin ratio, GOAT, GHS-R1a), glucose and weight could be regulated promising a potential way for artificial controlling of gonadal development and gamete maturation. The ghrelin signaling system may be a new target for reproductive operation in fish artificial propagation and breeding. This review summarizes the recent studies of the function of ghrelin in fish reproduction and the regulatory methods for ghrelin system.

Gonad development is a highly regulated process that coordinates cell specification and morphogenesis to produce sex-specific organ structures that are required for fertility, such as testicular seminiferous tubules and ovarian follicles. While sex determination occurs within specialized gonadal supporting cells, sexual differentiation is evident throughout the entire organ, including within the interstitial compartment, which contains immune cells and vasculature. While immune and vascular cells have been traditionally appreciated for their supporting roles during tissue growth and homeostasis, an increasing body of evidence supports the idea that these cell types are critical drivers of sexually dimorphic morphogenesis of the gonad. Myeloid immune cells, such as macrophages, are essential for multiple aspects of gonadogenesis and fertility, including for forming and maintaining gonadal vasculature in both sexes at varying stages of life. While vasculature is long known for supporting organ growth and serving as an export mechanism for gonadal sex steroids in utero, it is also an important component of fetal testicular morphogenesis and differentiation; additionally, it is vital for ovarian corpus luteal function and maintenance of pregnancy. These findings point toward a new paradigm in which immune cells and blood vessels are integral components of sexual differentiation and organogenesis. In this review, we discuss the state of the field regarding the diverse roles of immune and vascular cells during organogenesis of the testis and ovary and highlight outstanding questions in the field that could stimulate new research into these previously underappreciated constituents of the gonad.

There is increasing pressure to develop alternative ecotoxicological risk assessment approaches that do not rely on expensive, time-consuming, and ethically questionable live animal testing. This study aimed to develop a comprehensive early life stage toxicity pathway model for the exposure of fish to estrogenic chemicals that is rooted in mechanistic toxicology. Embryo-larval fathead minnows (FHM; Pimephales promelas) were exposed to graded concentrations of 17α-ethinylestradiol (water control, 0.01% DMSO, 4, 20, and 100 ng/L) for 32 days. Fish were assessed for transcriptomic and proteomic responses at 4 days post-hatch (dph), and for histological and apical end points at 28 dph. Molecular analyses revealed core responses that were indicative of observed apical outcomes, including biological processes resulting in overproduction of vitellogenin and impairment of visual development. Histological observations indicated accumulation of proteinaceous fluid in liver and kidney tissues, energy depletion, and delayed or suppressed gonad development. Additionally, fish in the 100 ng/L treatment group were smaller than controls. Integration of omics data improved the interpretation of perturbations in early life stage FHM, providing evidence of conservation of toxicity pathways across levels of biological organization. Overall, the mechanism-based embryo-larval FHM model showed promise as a replacement for standard adult live animal tests.

Larkinia grandis (Broderip & G.B. Sowerby I, 1829), an important fishing resource for Mexican communities, is an Arcidae clam. It is also considered a species with aquaculture potential. In this work we investigated the gonadal phases and sexuality in a population of L. grandis in the Gulf of California. Our findings support the hypothesis that there is one male per female in the population studied. It also documents that the shape, position and color of the gonads of L. grandis are consistent with observations in other Arcidae species. Additionally, five gonadal phases are differentiated and described in males and females (development, mature, spawning, post-spawning and resting), with a noticeable presence of brown cells during post-spawning and the onset of the resting phase, suggesting that those cells are involved in the reabsorption of remnants. Additionally, asynchronous gametogenesis in males, synchronic gametogenesis in females and batch spawning are defined. The results of this contribution can be used in the efforts to protect this bivalve.

Giant prawns (Macrobrachium rosenbergii) need enough nutrients for the reproductive process. One way to give feed which contains high enough protein. Complete nutritional content, especially protein, can be found in natural food. The type of natural feed used for main feeds including squid, sea worms, trash fish. Complete nutrient content can affect the rate of gonadal development and fecundity. (Squid, sea worms, trash fish) on the level of gonadal maturity and density of broodstock parents (Macrobrachium rosenbergii). This research was conducted by a completely randomized design (CRD) trial design. This study used 4 replications of different types of feeding. The types of feed given are squid, sea worms, trash fish, and pellets at a dose of 5% from biomass/day. The parameters of observation include observation of the level of gonad resistance, fundamentality of broodstock parent, and measurement of water quality (temperature, pH, and dissolved oxygen). The results showed that the administration of different types of feed (sea worms, trash fish) had a very significant effect on the level of maturity and had a significant effect on the probability of prawns (M. rosenbergii). After analysis of variance, the results of F Calculations are greater than F Table 0.05 and F Table 0.01. The smallest significant difference test (LSD) was obtained in P1 (Squid feed) with the Gonad maturity rate ± 5- 6 days as much as 83 % parent and fecundity average results of 28,846 ± 4,925.3 thousand / 45 g.

Phenanthrene (PHE) as a tricyclic polycyclic aromatic hydrocarbon is one of the common pollutants in water and sediments, which can cause reproductive toxicity to aquatic organisms. In this study, enzyme-linked immunosorbent assay (ELISA) was used to detect the vitellogenin (VTG) of loach, and then to explore the estrogenic toxicity effect of PHE on loach. The results were as follows: (1) with the increase of PHE concentrations and the extension of exposure time, the gonadosomatic index (GSI) of males decreased significantly, while it increased in female loaches. In addition, males had more obvious changes than females and were more sensitive to PHE. (2) The increase of VTG contents in serum of males were stronger than that in females. Those results reveal that PHE has estrogenic effect, which can affect the generation of VTG, thus causing damage to the gonad development of loach.

The dynamics of physiological and biochemical parameters of blood and urine in the process of gonad maturation in females of the sterlet × beluga hybrid (Acipenser ruthenus L., 1758 × Huso huso L., 1758) grown using CWSI technology is studied. It is shown that, when this technology is applied, features in functional changes that accompany the gonadogenesis of female hybrids appear, distinguishing them from those of fish from natural populations. At the same time, the direction of the generative exchange aimed at the formation of gametes is generally the same as in females from natural populations. However, the evidence of this process, reflecting its intensity at different stages of gonad development, is not as distinct as in females migrating to spawn. Possible reasons for high levels of the studied physiological and biochemical parameters in blood of females of the hybrid after the end of their artificial wintering are discussed.

Background Gonad development and differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. However, the mechanisms underlying gonad development and gametogenesis remain unclear in Tridacna squamosa, a large-size bivalve of great ecological value. They are protandrous simultaneous hermaphrodites, with the male gonad maturing first, eventually followed by the female gonads. In this study, nine gonad libraries representing resting, male and hermaphrodite stages in T. squamosa were performed to identify the molecular mechanisms. Results Sixteen thousand four hundred ninety-one unigenes were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 5091 and 7328 unigenes were assigned to Gene Ontology categories and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database, respectively. A total of 4763 differentially expressed genes (DEGs) were identified by comparing male to resting gonads, consisting of 3499 which were comparatively upregulated in males and 1264 which were downregulated in males. Six hundred-ninteen DEGs between male and hermaphroditic gonads were identified, with 518 DEGs more strongly expressed in hermaphrodites and 101 more strongly expressed in males. GO (Gene Ontology) and KEGG pathway analyses revealed that various biological functions and processes, including functions related to the endocrine system, oocyte meiosis, carbon metabolism, and the cell cycle, were involved in regulating gonadal development and gametogenesis in T. squamosa. Testis-specific serine/threonine kinases 1 (TSSK1), TSSK4, TSSK5, Doublesex- and mab-3-related transcription factor 1 (DMRT1), SOX, Sperm surface protein 17 (SP17) and other genes were involved in male gonadal development in Tridacna squamosal. Both spermatogenesis- (TSSK4, spermatogenesis-associated protein 17, spermatogenesis-associated protein 8, sperm motility kinase X, SP17) and oogenesis-related genes (zona pellucida protein, Forkhead Box L2, Vitellogenin, Vitellogenin receptor, 5-hydroxytryptamine, 5-hydroxytryptamine receptor) were simultaneously highly expressed in the hermaphroditic gonad to maintain the hermaphroditism of T. squamosa. Conclusion All these results from our study will facilitate better understanding of the molecular mechanisms underlying giant clam gonad development and gametogenesis, which can provided a base on obtaining excellent gametes during the seed production process for giant clams.

Gonadotropin-inhibitory hormone (GnIH) plays a critical role in regulating gonadotropin-releasing hormone (GnRH), gonadotropin hormone (GtH), and steroidogenesis. The Lpxrfa (the piscine ortholog of GnIH) system has been found to regulate fish reproduction. To gain insight into the role of Lpxrfa in the regulation of spotted scat (Scatophagus argus) reproduction, spotted scat Lpxrfa (ssLpxrfa), and its receptor (ssLpxrfa-r) were cloned and analyzed. Tissue distribution and expression patterns at the hypothalamo-pituitary-gonadal axis (HPG axis) of sslpxrfa and sslpxrfa-r mRNA were also investigated during gonadal development of spotted scat. The open reading frame (ORF) of the sslpxrfa was 606 bp encoding 201 amino acids and includes a putative signal peptide and two mature ssLpxrfa peptides with LPXRFamide motif at their C-terminus. The sslpxrfa-r ORF was 1449 bp encoding 482 amino acids and contracted a seven-hydrophobic transmembrane (TM) domain structure. The tissue distribution showe d that the sslpxrfa was highly expressed in hypothalami, gill, and the gonads. In addition, sslpxrfa-r was highly expressed in hypothalami, pituitaries, and the gonads. Quantitative real-time polymerase chain reaction (qPCR) revealed that sslpxrfa had the highest expression in the hypothalami and pituitaries, and the lowest expression in the gonads in stage V. During gonadal development, the expression of sslpxrfa-r was gradually increased in the hypothalami but reduced in the gonads. However, no obvious trend was observed in the pituitaries. The expression of sslpxrfa and sslpxrfa-r decreased significantly after injection with 17β-estradiol (E2). However, the expression of both sslpxrfa and sslpxrfa-r was not changed after injection with 17α-methyltestosterone(17α-MT) in the hypothalami. In addition, no changes were observed in the expression of fshβ and lhβ in the pituitaries after injecting ssLpxrfa-1. However, ssLpxrfa-2 could downregulate the expression of sbgnrh and fshβ in the hypothalami and pituitaries, respectively. Taken together, these findings suggested that ssLpxrfa may participate in E2 feedback in reproduction and regulate the reproductive axis of spotted scat.

The P-element induced wimpy testis (Piwi) proteins, which are associated with PIWI-interacting RNAs (piRNAs), play important roles in meiosis, germ cell division, and germline maintenance. In this study, we identified and characterized the Paralichthys olivaceus piwil2 gene, a constituent factor of the piRNA pathways involved in the biogenesis of reproductive development. The biological analysis indicated that piwil2, which contains PAZ and PIWI domains, was highly conserved between teleosts and tetrapods. The piwil2 distribution profile in different tissues confirmed a sexually dimorphic expression pattern, with a higher expression level in testis. In situ hybridization demonstrated that piwil2 was expressed in the oogonia and oocytes of the ovaries as well as in the Sertoli cells and spermatocytes of the testes. Gene piwil2 showed a maternally inherited expression pattern during embryonic development, and was highly expressed during the early embryonic development. Different luciferase reporters were constructed to determine the transcriptional regulatory mechanisms of piwil2. The piwil2 core promoter region was located at −360 bp to −60bp. Furthermore, some representative sex hormones, including human chorionic gonadotropin, 17α-methyltestosterone, and estradiol-17β had distinct regulatory effects on piwil2. In a summery, these results indicate that piwil2, regulated by sex hormones and transcriptional elements, has vital functions in the reproductive cycle and gonadal development.

The present study was carried out to compare gonad development, oocyte maturation and fecundity of the spotted snakehead (Channa punctatus) fish during January to November 2015 in different water bodies (such as pond, Joghati baor and Bhairab river) in Jashore, Bangladesh. Mean GSI (gonadosomatic index) values of the female C. punctatus during January, March, May, July, September and November were significantly highest in pond, followed by those in Joghati baor and the lowest in Bhairab river. Absolute fecundity during the month of July in pond, baor and river was 30,006±2,027, 23,629±2,356 and 16,659±2,486, respectively, and the relative fecundity was 5064±183, 4865±106 and 4641±138 eggs, respectively. Mean ova diameter in pond, baor and river in March was 0.25±0.04 mm, 0.16±0.02 mm and 0.21±0.03 mm; in May was 0.36±0.03 mm, 0.29±0.03 mm and 0.23±0.04 mm; in July was 1.06±0.06 mm, 0.95±0.05 mm and 0.87±0.03 mm; and in September was 0.84±0.04 mm, 0.82±0.03 mm and 0.72±0.05 mm, respectively. Mean GSI values of the male C. punctatus during January, March, May, July, September and November were significantly higher in pond than those in baor and river, respectively. The mean highest GSI for female was found to be 6.06±0.11 in pond during the month of July, while the lowest GSI of 0.30±0.08 was in Bhairab river during November. The highest GSI value for male was obtained to be 0.70±0.16 in pond during the month of July, while the lowest GSI of 0.12±0.03 was found in Bhairab river during January. However, significant differences (P

In recent years, the increasing use of environmental endocrine disruptors has caused serious environmental pollution and hurt aquatic organisms. It is still risky for aquatic species and humans exposed to 17α-methyltestosterone (MT), however, the harmful effect of MT on fish is still poorly understood. The main purpose of this study is to evaluate the influence of MT on Pseudorasbora parva at multi-levels. We analyzed gonadal histology, the sex steroid hormones, steroidogenic genes expression, and transcriptome profiling of gonads in response to MT in adult P. parva. Through this study, we found MT could inhibit the gonadal development of P. parva, and the growth and development of fish could be delayed by exposure to MT at 200 ng/L. MT could produce disruption effects on fish from multiple pathways, while its interference to the HPGL axis happens primarily through the steroidogenic pathway, e.g., disturbing the expression of crucial genes and sex steroids synthesis. Besides, we constructed 4 RNAseq libraries and obtained 7758 and 11,543 DEGs in females and males, respectively. Interestingly, we found MT had more obvious disruption effects on males than the females, mainly reflected in the immune system. Interestingly, we found three common pathways in both sexes after MT exposure, i.e., cell adhesion molecules, cytokine-cytokine receptor interaction, and neuroactive ligand-receptor interaction. These results confirm the suitability of P. parva as a model fish for aquatic toxicological study and provide us a multidimensional sight for the disruption effects of MT on fish.

Background The rete testis connects seminiferous tubules of the testis with efferent ducts having a mesonephric origin. The development of the rete testis is insufficiently studied, but there is evidence suggesting that it originates from gonadal cells. Here, the formation of the rete testis was investigated from E11.5 to E16.5 using immunofluorescent staining and 3D-modeling. Results The rete testis became visible by SOX9 and PAX8 staining starting from E12.5. It was located in the mesonephros but connected with testis cords formed by Sertoli cells expressing SOX9, AMH, DMRT1. Between E13.5 and E14.5, AMH+ network of testis cords at the mesonephric side began to disintegrate in a gradient-dependent manner along the anterior-posterior axis of the gonad and connections between testis cords gradually lost AMH becoming a part of the rete. Cells combining features of Sertoli and rete cells (PAX8+ AMH+ and DMRT1+ AMH- cells) were detected starting from E14.5, suggesting that some rete cells originated from Sertoli cells. The rete ovarii, a female counterpart of the rete testis, developed in a similar way as the rete testis until E13.5. Conclusions A part of the rete testis originates from connections between testis cords. Evidence that Sertoli cells contribute to rete cells is provided.

Mating strategy and sexual dimorphism of morphological traits of the leucosiid crabPyrhila pisumwere elucidated by analysing relative growth patterns of chelipeds and abdomen, and gonad development patterns. Male adults had long chelipeds compared with juvenile males and females. Among male adults, two phases with different slopes could be found in the regression lines; their chelipeds growth pattern changed from negative allometry to positive and longer chelipeds developed in large adults. The growth is more markedly expressed in the merus than in the propodus. Female adults had wider abdominal segments and a thicker body compared with juvenile females and males. Abdomen of females was greatly enlarged by a puberty moult. Male adults had well-developed gonads similar to female adults, and the weights of male gonads were often larger than those of females of the same body size. For adult males, a negative correlation was detected between carapace width and the weight ratio of their gonads, but no significant relationship was detected for adult females. Females had large and well-bloated seminal receptacles, whose weight was nearly equal to gonad weight. There was little difference in the amount of seminal receptacles regardless of the body size of females. There is a trade-off relationship in the development between chelipeds and gonads in adult males. Probably young adult males compensate for the disadvantageous condition of guarding by increasing the number of spermatozoids, and old adult males invest more of their energy to their chelae for guarding while decreasing investment in sperm production.

Suppressive regulatory T cells (Treg cells) play a vital role in preventing autoimmunity and restraining excessive immune response to both self- and non-self-antigens. Studies on humans and mice show that the Forkhead box p3 (Foxp3) is a key regulatory gene for the development and function of Treg cells. In zebrafish, Treg cells have been identified by using foxp3a as a reliable marker. However, little is known about the function of foxp3a and Treg cells in gonadal development and sex differentiation. Here, we show that foxp3a is essential for maintaining immune homeostasis in zebrafish testis development. We found that foxp3a was specifically expressed in a subset of T cells in zebrafish testis, while knockout of foxp3a led to deficiency of foxp3a-positive Treg cells in the testis. More than 80% of foxp3a-/- mutants developed as subfertile males, and the rest of the mutants developed as fertile females with decreased ovulation. Further study revealed that foxp3a-/- mutants had a delayed juvenile ovary-to-testis transition in definite males and sex reversal in about half of the definite females, which led to a dominance of later male development. Owing to the absence of foxp3a-positive Treg cells in the differentiating testis of foxp3a-/- mutants, abundant T cells and macrophages expand to disrupt an immunosuppressive milieu, resulting in defective development of germ cells and gonadal somatic cells and leading to development of infertile males. Therefore, our study reveals that foxp3a-positive Treg cells play an essential role in the orchestration of gonadal development and sex differentiation in zebrafish.

Monosex breeding technology has received enormous attention for the Scylla paramamosain, an economic crustacean species with distinctive sexual dimorphism. However, the molecular mechanism of gonadal development and sexual differentiation still remain inconclusive. In this study, we reported the cloning, characterization of a FoxL2-like gene with male-biased expression in the mud crab (SpFoxL2). The SpFoxL2 encoded a putative forkhead-box protein with 352 amino acids. Phylogenetic tree revealed the SpFoxL2 was clustered with the invertebrates FoxL2 homologues, most closed to E. sinensis. By quantitative real-time PCR, it was shown that the SpFoxL2 was expressed in intestine, ovary and testis, with the highest expression level in testis. During gonadal development, the expression level of SpFoxL2 in ovary was lower than that in testis at all stages. The expression level of SpFoxL2 constantly declined from stage I to stage V in ovary. While, there is a rising tendency from stage I to stage IV in testis. Notably, this male-biased expression pattern could be detected as early as the juvenile crab stage I (CI stage) before morphological sexual differentiation in the mud crab. By fluorescence in-situ hybridization, SpFoxL2 mRNA was found located in both somatic and germ cells in gonads. In testis, it was most strongly localized in the meiosis germ cells (spermatocytes). While in ovary, the signal became weaker in the meiosis germ cells (oocytes). The results imply that the SpFoxl2 may play an important role in male differentiation and development, laying a foundation for future research on sexual differentiation of the species.

This study analyzed the reproductive cycle of Chiton virgulatus and its relationship with air temperature (AT) sea and surface temperature (SST), photoperiod, and tidal cycle (TC) in Bahía de La Paz, in the coast of the Gulf of California. Monthly samples were collected from September 2008 to September 2009. The parameters recorded included total length, total weight, and gonad weight of each organism; the gonad was processed using a histological technique, and the gonadal index and length at first maturity (L50) were estimated. C. virgulatus displays partial spawning with a reproductive peak in summer and autumn (July to December). The frequency of ripe organisms was associated with rises in SST (30.6 ºC), AT (29.9 ºC), and TC (68 cm, high tidal), while photoperiod (longer days) regulated the frequency of developing organisms. The length at first maturity was 29.7 mm. Chiton virgulatus is a polyplacophoran species in which high temperatures and high tides influence reproductive timing, while an increase in daylight boosts gonad development.

To date, the molecular mechanisms of the unique gonadal development mode known as protandric simultaneous hermaphroditism (PSH) are unclear in crustaceans. In this study, cDNA of a gonad-inhibiting hormone (Lv-GIH1) was isolated from the PSH peppermint shrimp Lysmata vittata, and its expression was exclusively found in the eyestalk ganglion. Real-time quantitative polymerase chain reaction (qRT-PCR) revealed that the expression of Lv-GIH1 increased during gonadal development of the functional male stages but decreased significantly at subsequent simultaneous hermaphroditism stage. Further in vitro experiment showed that recombinant GIH1 protein (rGIH1) effectively inhibited Vg expression in the cultured hepatopancreas tissues while the short-term injection of GIH1-dsRNA resulted in reduced expression of Lv-GIH1 and upregulated expression of Vg in the hepatopancreas. Moreover, long-term rGIH1 injection led to significantly reduced expression of Lv-Vg, Lv-VgR, and Lv-CFSH1, subdued growth of oocytes, and feathery setae as a secondary sexual characteristic in females. Interestingly, while germ cells in testicular part were suppressed by rGIH1 injection, the expression of Lv-IAGs showed no significant difference; and long-term GIH1-dsRNA injection results were contrary to those of rGIH1 injection. Taken together, the results of this study indicate that Lv-GIH1 is involved in gonadal development and might also participate in controlling secondary sexual characteristic development in L. vittata by inhibiting Lv-CFSH1 expression.

This study was carried out to obtain information on the developmental biology and the management of biological resources of the abalone Haliotis gigantea in Korea. The sex ratio (female:male) in the present study was 1:1.7 and the proportion of females was 36.6% (n=106/290). Their gonadal structures displayed definitive seasonal changes which were similar in pattern to the changes in the gonad index (GI). The GI showed a pattern of definitive seasonal changes in both males and females it was high in the fall and low in the spring. The reproductive cycle could be categorized into the following six stages: inactive, early active, late active, ripe, spent, and degenerative stage. Based on the monthly changes in GI and stages of gonadal development, October to November was determined to be the main spawning period for H. gigantea on Jeju Island, Korea.

In mice, male sex determination depends on FGF9 signalling via FGFR2c in the bipotential gonads to maintain the expression of the key testis gene SOX9. In humans, however, while FGFR2 mutations have been linked to 46,XY disorders of sex development (DSD), the role of FGF9 is unresolved. The only reported pathogenic mutations in human FGF9, FGF9S99N and FGF9R62G, are dominant and result in craniosynostosis (fusion of cranial sutures) or multiple synostoses (fusion of limb joints). Whether these synostosis-causing FGF9 mutations impact upon gonadal development and DSD etiology has not been explored. We therefore examined embryonic gonads in the well-characterized Fgf9 missense mouse mutants, Fgf9S99N and Fgf9N143T, which phenocopy the skeletal defects of FGF9S99N and FGF9R62G variants, respectively. XY Fgf9S99N/S99N and XY Fgf9N143T/N143T fetal mouse gonads showed severely disorganized testis cords and partial XY sex reversal at 12.5 days post coitum (dpc), suggesting loss of FGF9 function. By 15.5 dpc, testis development in both mutants had partly recovered. Mitotic analysis in vivo and in vitro suggested that the testicular phenotypes in these mutants arise in part through reduced proliferation of the gonadal supporting cells. These data raise the possibility that human FGF9 mutations causative for dominant skeletal conditions can also lead to loss of FGF9 function in the developing testis, at least in mice. Our data suggest that, in humans, testis development is largely tolerant of deleterious FGF9 mutations which lead to skeletal defects, thus offering an explanation as to why XY DSDs are rare in patients with pathogenic FGF9 variants.

A comparative study was conducted on gonad development of triploid Salvelinus fontinalis, Salvelinus umbla, and Salmo trutta f. lacustris. Triploid males developed testes and had a reproductive cyc...

Background Endocrine disruptors are one of the major threats to aquatic animals affecting their development and physiology. Amphibians are very sensitive to aquatic contaminants as their skin is semi-permeable. Several contaminants easily enter into their body, act as endocrine disruptors, and interfere in sexual development and metamorphosis. Endocrine disruptors have diverse effects in different species mainly due to the variations in developmental patterns. In the present study, I evaluated the endocrine disrupting potential of tamoxifen (anti-estrogen) in the tadpoles of Indian skipper frog Euphlyctis cyanophlyctis with undifferentiated type of gonad differentiation (testis differentiates through an ovarian phase). Methods I exposed the tadpoles to four concentrations of tamoxifen (25, 50, 100, and 200 μg/l) during larval development (Gosner stages 25–42) and studied their metamorphosis, somatic and sexual development. Results Tamoxifen does not influence gonad differentiation and sex ratio (proportion of males and females). However, all the concentrations of tamoxifen stimulated gonad development resulted in testis maturation in males and increased oocytes size in females. Tamoxifen treatments delayed metamorphosis and stimulated somatic growth. Conclusion These results suggest that tamoxifen does not act as an anti-estrogen in E. cyanophlyctis while stimulate sexual development in both males and females through unknown mechanism. These results are useful to understand the mechanism of action of tamoxifen in lower vertebrates and develop E. cyanophlyctis as an indigenous amphibian model for endocrine disruption studies.

STUDY QUESTION Which transcriptional program triggers sex differentiation in bipotential gonads and downstream cellular events governing fetal testis and ovary development in humans? SUMMARY ANSWER The characterization of a dynamically regulated protein-coding and non-coding transcriptional landscape in developing human gonads of both sexes highlights a large number of potential key regulators that show an early sexually dimorphic expression pattern. WHAT IS KNOWN ALREADY Gonadal sex differentiation is orchestrated by a sexually dimorphic gene expression program in XX and XY developing fetal gonads. A comprehensive characterization of its non-coding counterpart offers promising perspectives for deciphering the molecular events underpinning gonad development and for a complete understanding of the etiology of disorders of sex development in humans. STUDY DESIGN, SIZE, DURATION To further investigate the protein-coding and non-coding transcriptional landscape during gonad differentiation, we used RNA-sequencing (RNA-seq) and characterized the RNA content of human fetal testis (N = 24) and ovaries (N = 24) from 6 to 17 postconceptional week (PCW), a key period in sex determination and gonad development. PARTICIPANTS/MATERIALS, SETTING, METHODS First trimester fetuses (6–12 PCW) and second trimester fetuses (13–14 and 17 PCW) were obtained from legally induced normally progressing terminations of pregnancy. Total RNA was extracted from whole human fetal gonads and sequenced as paired-end 2 × 50 base reads. Resulting sequences were mapped to the human genome, allowing for the assembly and quantification of corresponding transcripts. MAIN RESULTS AND THE ROLE OF CHANCE This RNA-seq analysis of human fetal testes and ovaries at seven key developmental stages led to the reconstruction of 22 080 transcripts differentially expressed during testicular and/or ovarian development. In addition to 8935 transcripts displaying sex-independent differential expression during gonad development, the comparison of testes and ovaries enabled the discrimination of 13 145 transcripts that show a sexually dimorphic expression profile. The latter include 1479 transcripts differentially expressed as early as 6 PCW, including 39 transcription factors, 40 long non-coding RNAs and 20 novel genes. Despite the use of stringent filtration criteria (expression cut-off of at least 1 fragment per kilobase of exon model per million reads mapped, fold change of at least 2 and false discovery rate adjusted P values of less than LARGE-SCALE DATA Raw data files (fastq) and a searchable table (.xlss) containing information on genomic features and expression data for all refined transcripts have been submitted to the NCBI GEO under accession number GSE116278. LIMITATIONS, REASONS FOR CAUTION The intrinsic nature of this bulk analysis, i.e. the sequencing of transcripts from whole gonads, does not allow direct identification of the cellular origin(s) of the transcripts characterized. Potential cellular dilution effects (e.g. as a result of distinct proliferation rates in XX and XY gonads) may account for a few of the expression profiles identified as being sexually dimorphic. Finally, transcriptome alterations that would result from exposure to pre-abortive drugs cannot be completely excluded. Although we demonstrated the high quality of the sorted cell populations used for experimental validations using quantitative RT-PCR, it cannot be totally excluded that some germline expression may correspond to cell contamination by, for example, macrophages. WIDER IMPLICATIONS OF THE FINDINGS For the first time, this study has led to the identification of 1000 protein-coding and non-coding candidate genes showing an early, sexually dimorphic, expression pattern that have not previously been associated with sex differentiation. Collectively, these results increase our understanding of gonad development in humans, and contribute significantly to the identification of new candidate genes involved in fetal gonad differentiation. The results also provide a unique resource that may improve our understanding of the fetal origin of testicular and ovarian dysgenesis syndromes, including cryptorchidism and testicular cancers. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the French National Institute of Health and Medical Research (Inserm), the University of Rennes 1, the French School of Public Health (EHESP), the Swiss National Science Foundation [SNF n° CRS115_171007 to B.J.], the French National Research Agency [ANR n° 16-CE14-0017-02 and n° 18-CE14-0038-02 to F.C.], the Medical Research Council [MR/L010011/1 to P.A.F.] and the European Community’s Seventh Framework Programme (FP7/2007-2013) [under grant agreement no 212885 to P.A.F.] and from the European Union’s Horizon 2020 Research and Innovation Programme [under grant agreement no 825100 to P.A.F. and S.M.G.]. There are no competing interests related to this study.

SummaryTransparentCasperzebrafish allow studies of vertebrate sexual maturation and gonad developmentin vivo.Caspergonad dynamics can be observed longitudinally over time and non-invasively. Gonad maturation and reproduction are complex processes subject to disruption by endocrine-disrupting chemicals (EDCs), such as diethylstilbestrol (DES). DES was used as a ‘proof of principle’ to ascertain the usefulness of theCaspermodel to determine EDC effects on gonad maturation. Puberty onset in control juvenileCasperzebrafish (N= 43) averaged 13.2 weeks post fertilization (WPF) for females and included increased vent size, while in males puberty occurred at 11.7 WPF along with maintenance of small vents. DES treatment for 6 days in early juveniles (N= 20) induced an average delay in puberty of 5 weeks in females and 10 weeks in males. DES induced loss of breeding tubercles and vent enlargement in post-pubescent males. Puberty in control fish was correlated with an average body length of 1.7 cm for males and 1.8 cm for females. Increased testes opacity, small vent and breeding tubercles denoted male puberty. Puberty in females was defined as ovarian follicle diameters reaching 400 µm with increasingly opaque follicles and by an increased vent size. These results are like those for wild-type zebrafish and indicate that theCaspermodel is a useful system for studying gonad dynamicsin vivo. Future use of transgenic reporter lines inCasperwill allow new avenues of investigation into the reproductive biology of this vertebrate model.

In this study, 515 Farfantepenaeus aztecus specimens were investigated in the Gulf of Antalya and 304 (59,02%) specimens (167 females and 137 males) infested with Bopyrid isopod parasite Epipenaeon ingens were found. In this study, we aimed to define the rate of infestation and its impact on survival rate and growth rate. Relationship of length- weight and condition factor values were lower in infested specimens compared to non-infested ones. While gonadal development was observed in different stages of non-infested female specimens, no gonadal development was observed in any infested specimens. Moreover, darker color and harder shell structure were observed in parasitized shrimps. During this research, infestation onto P. semisulcatus and P. kerathurus had been seen rarely.Keywords: Gulf of Antalya; Epipenaeon ingens, Farfantepenaeus aztecus; growth; reproduction

Arapaima gigas is a giant air-breathing and bony tongue fish from the Amazon basin and a promising species for aquaculture. A. gigas farming industry is still not established because of the lack of information on its reproductive physiology. Reproduction in captivity cannot be manipulated or stimulated, and the identification of males and females in a broodstock is not easy. We aimed to reveal the morphological sex differentiation of pirarucu as studies involving gonad development are essential to understanding the reproductive physiology of any species. We performed histological analysis on the whole body and extracted the gonads of 150 juveniles. The first sign of ovary differentiation is the sex-specific rearrangement of the germ cells. In 9 cm total length females, the germ cells group into nests and are restricted to the lateral face of the gonad, in close contact with the abdomen wall. With further development, this region invaginates and that later develops into ovigerous lamellae. Meiosis starts soon after ovary differentiation. In males, the germ cells are scattered along the elongated differentiating testis at first, and later become more restricted to the central region where the spermatogonial cysts start to develop. Somatic and germ cells are jointly involved in the cellular reorganization during gonadal differentiation, specifically when the germ cells begin to establish new associations during the development of both the germinal epithelium and stroma. RESEARCH HIGHLIGHTS: In Arapaima gigas, the ovary differentiation occurs in 9 cm TL females and it is marked by the rearrangement of germ and somatic cells; and the germ cells entering meiosis with no formation of ovarian cavity; testis differentiation occurs later and meiosis does not start in males smaller than 80 cm TL.

Photoperiodism has been shown to be an important synchronizer of seasonal reproduction in many rodent species in the wild; it is a reliable cue as in the southern hemisphere it coincides with the onset of rainfall and hence the availability of food resources for maximal reproductive success. The photoperiodic effect on the reproductive status of the Namibian gerbil, Gerbilliscus cf. leucogaster from central Namibia was investigated. Twenty adult males were exposed to a long-day length (16L:8D), while further 20 adult males were subjected to a short-day length (SD:8L:16D); all for a period of 3 months. Testicular mass per gram body mass, testicular volume and seminiferous tubule diameters were used to assess the effect of photoperiod on gonadal development. Body mass did not significantly differ between the two photoperiodic regimes. The testicular mass per gram of body mass was significantly heavier for the males maintained on a long photoperiod compared to those on a short photoperiod. Similarly, testicular volume and seminiferous tubule diameter were greater in males maintained on a long-day cycle compared to those on the short-day cycle. These findings suggest that G. cf. leucogaster is photoresponsive to day length changes. Photoperiodic changes in the semi-arid habitats can be used to herald the onset of reproduction as it often acts in concert with other proximate cues in desert rodents, but is a constant environmental cue that does not change from year to year, unlike rainfall patterns.

Smad2, a receptor-activated Smad, plays a critical role in regulating gametogenesis. In this study, a smad2 homologue was identified and sequenced from ricefield eel ovary cDNA, and its mRNA and protein expression levels were analysed during oocyte development. The cDNA sequence of ricefield eel smad2 consisted of 1863 bp encoding a 467-amino acid protein that had high sequence homology with Smad proteins in other teleosts, especially in Poeciliopsis prolifica. The results of real-time quantitative PCR (RT-qPCR) analysis revealed that smad2 is expressed in the ovary during gonad development, increased continuously until the early vitellogenic stage in the ovaries, and then decreased with ovary maturation. Smad2 protein immunoreactivity was localized in the cytoplasm of follicular cells, oogonia, and primary growth stage oocytes. In vitro experiments revealed that follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) promoted smad2 expression in ovary tissue in a time- and dose-dependent manner, respectively. In summary, Smad2 plays a potentially vital role in ricefield eel ovary development.

The snakeskin gourami (Trichopodus pectoralis) exhibits sexual dimorphism, particularly in body size. Since the snakeskin gourami is usually marketed during sexual maturation, the sexual size dimorphism has become an economically important trait. Sex-biased gene expression plays a key role in phenotypic sexual dimorphism. Therefore, using high-throughput RNA sequencing (RNA-seq) technology, we aimed to explore the differentially expressed genes (DEGs) in ovary and testis during sex differentiation in juvenile snakeskin gourami. Our results revealed a number of DEGs were demonstrated to be overexpressed in ovary (11,625 unigenes) and testis (16,120 unigenes), and the top 10 female-biased (rdh7, dnajc25, ap1s3, zp4, polb, parp12, trim39, gucy2g, rtbs, and fdxr) and male-biased (vamp3, nbl1, dnah2, ccdc11, nr2e3, spats1, pih1d2, tekt3, fbxo36, and mybl2) DEGs were suggested to be mainly associated with ovary and testis differentiation, respectively. Additionally, using real-time reverse transcription polymerase chain reaction (qRT-PCR), validation of the differential expression of 21 genes that were previously shown to be related to gonad development was performed (ar, bHLH, cyp19a1, daz, dead-end, esrb, esrrg, gnrhr, gpa, gsg1l, hsd17B, mospd1, nanos-1, nanos-2, p53, piwi-1, piwi-2, rerg, rps6ka, tgf-beta, and VgR). The results showed a significantly positive correlation (0.84; P

Background and Objective: The accumulation of heavy metals such as cadmium and lead in mussels is very high compared to that in another marine biota. The mussels are sessile, widely distributed filter-feeding organisms, with the ability to sequester many lipophilic organic compounds, absorb anything in their surroundings. The very low mobility allows heavy metal bioaccumulation to occur and cannot avoid pollutants, which increase over time. This bioaccumulation can be toxic to mussels. This study aimed to evaluate the effect of different toxic chemicals and histological changes in green mussels. Materials and Methods: All archive gonad sample of green mussels in 2015 was fixed in paraformaldehyde 4% solution and were sliced by a rotary microtome at 8 μm thickness and finally, the slides were stained with a solution of hematoxylin-eosin. Results: The obtained results demonstrated that developmental disorders in the testes are characterized by the arrangement of follicle cells in a relatively less dense state and some follicles are not fully filled with spermatozoa. It means that the male gonad samples of green mussels in the port of Muara Angke undergoing toxicity and the process of gonad developmental was disrupted. Conclusion: The effects of toxicity of the male gonad of green mussels were more sensitive and were more susceptible than the female gonad of the green mussels.

One of the main problem in giant river prawn (GFP) culture is early gonadal maturation in female resulting a reduced growth performance. This problem cause economic losses. When GFP at gonadal maturation, somatic growth will be inhibited because energy is used for reproduction. A factorial experimental design using two factors, namely dopamine and medroxyprogesterone, with each factor consist of three levels was applied. Three dopamine levels were 0, 10-5 mol/shrimp, and 10-10 mol/shrimp, while the medroxyprogesterone levels were 0, 75 mg/1.5 mL/bodyweight, and 150 mg/3 mL/bodyweight with a density 15 individual/tank. The utilization of dopamine and medroxyprogesterone in GFP (initial bodyweight : 11.27 ± 0.97 g) through injection at the third periopod was done three times at week 0, 2nd, and 4th with two weeks interval. The results showed that hormone inhibitor treatments affected both growth and reproductive performances in female GFP. The treated individuals showed a lower gonadal maturity indicator values and faster growth rate than control. Gonadal maturity, as shown by gonad histology, in all treatments were lower (previtelogenic and vitellogenic stages) than that in control which is in mature stage. Estradiol concentration premix dopamine 10-10 mol/shrimp and medroxyprogesterone 150 mg/3 mL/bodyweight treatments are lower than control. In conclusion, dopamine and medroxyprogesterone administration could suppres GSI and gonad development, and also increase growth rate. Keywords: Macrobrachium rosenbergii, dopamine, medroxyprogesterone, gonad development, growth.

Caspian roach (Rutilus rutilus caspicus), a spring spawning teleost, were subjected to various photoperiod and temperature regimes to study the feasibility of shifting the timing of spawning for artificial propagation purposes. A total of 650 female reproductively mature R. rutilus caspicus were subjected to different photoperiod and temperature regimes including four light regimes (natural light (NL), 16 hr of light (L):8 hr of darkness (D), 9L:15D, 11L:13D), each affected by three temperature regimes (14, 20 and 24°C) for 70 days. Five fish per tank were randomly sampled on Feb. 10, Feb. 20, March 28, April 15 and April 30 (natural spawning time). Ovarian tissue sections were studied using light microscope and transmission electron microscope (TEM). The levels of 17‐β estradiol (E2) and 17α‐hydroxyprogesterone (OHP) were also measured in the serum samples. In late winter (March 28th), the gonadal maturation and spawning were accelerated in fish treated with the long day length (16L/8D) and warm temperature (20°C). While, the maturation of oocytes and spawning delayed in fish exposed to low temperature (14°C) and short day length (9L/15D and 11L/13D). Photoperiod seems to play a more important role in the ovarian development of the R. rutilus caspicus compared to temperature; since even among the fish treated with the lowest temperature (14°C), those exposed to a longer day length (16L/8D), matured and spawned earlier than the others. Considering that the earliest spawning occurred in R. rutilus caspicus treated with 16L/8D at 20°C and the latest spawning occurred in fish exposed to low temperature and short photoperiod, it can be concluded that temperature and photoperiod play an important role in accelerating oocyte maturation and spawning.

Piwi proteins are essential for germline stem cell maintenance, gametogenesis and gonadal development in diverse organisms. In this study, two piwi homologues were identified and characterized in Dabry’s sturgeon Acipenser dabryanus, designated Adpiwil1 and Adpiwil2. Multiple sequence alignments showed that both AdPiwil1 and AdPiwil2 shared highly homologous PAZ and PIWI domains, the typical regions of Piwi orthologs. Phylogenetic analysis revealed that AdPiwil1 and AdPiwil2 were conservatively grouped with teleosts. We found that the Adpiwil1 transcripts were abundantly expressed in gonadal tissues and rarely present in brain tissues, whereas the Adpiwil2 transcripts were found to be specifically expressed in gonads by tissue distribution analysis. Fluorescence in situ hybridization also revealed that both Adpiwil1 and Adpiwil2 mRNAs were restricted exclusively to germ cells of both sexes. In addition, E2 (17β-estradiol) intraperitoneal injection was performed on female Dabry’s sturgeons, and its effects on Adpiwil1 and Adpiwil2 mRNA levels were evaluated by relative real-time PCR. The results showed that the transcription levels of Adpiwil1 and Adpiwil2 were significantly decreased in ovaries after E2 injection. Our findings will contribute to an understanding of the piwi function and provide a basis for further studies.

The genetic regulatory network governing vertebrate gonadal differentiation appears less conserved than previously thought. Here, we investigated the gonadal development of Astyanax mexicanus Pachón cavefish by looking at primordial germ cells (PGCs) migration and proliferation, gonad histology, and gene expression patterns. We showed that PGCs are first detected at the 80% epiboly stage and then reach the gonadal primordium at 1 day post-fertilization (dpf). However, in contrast to the generally described absence of PGCs proliferation during their migration phase, PGCs number in cavefish doubles between early neurula and 8–9 somites stages. Combining both gonadal histology and vasa (germ cell marker) expression patterns, we observed that ovarian and testicular differentiation occurs around 65 dpf in females and 90 dpf in males, respectively, with an important inter-individual variability. The expression patterns of dmrt1, gsdf, and amh revealed a conserved predominant male expression during cavefish gonadal development, but none of the ovarian differentiation genes, i. e., foxl2a, cyp19a1a, and wnt4b displayed an early sexually dimorphic expression, and surprisingly all these genes exhibited predominant expression in adult testes. Altogether, our results lay the foundation for further research on sex determination and differentiation in A. mexicanus and contribute to the emerging picture that the vertebrate sex differentiation downstream regulatory network is less conserved than previously thought, at least in teleost fishes.

Hexavalent chromium (Cr(VI)) is a harmful environmental pollutant, which is widely distributed in the marine environment. However, the mechanisms underlying Cr-induced oxidative stress and reproductive toxicity in clams remain largely unknown. The objective of this study was to explore the acute toxic effects of Cr(VI) and estimate the applicability of male clam Geloina coaxans as a bioindicator in mangrove ecosystems. In this study, G. coaxans were exposed to 4.34, 8,69, 17.38 and 34.76 mg/L Cr(VI) for 24, 48 and 72 h. The activities of catalase (CAT), glutathione S-transferase (GST) and the content of malondialdehyde (MDA) were determined. The gene expression of CAT, GST, autophagy-related gene 5 (ATG5), cyclin B3 and spermatogenesis associated protein 4 (SAP4) were measured by RT-qPCR. Furthermore, histological alteration of the gonad and sperm was observed by light microscopy and transmission electron microscopy. The results showed that Cr(VI) exposure induced the accumulation of MDA and activities of enzymes. Cr(VI) exposure also inhibited the mRNA expression of ATG5, cyclin B3 and SAP4 in a dose-dependent manner, which might suggest that Cr(VI) caused reproductive toxicity by affecting gene expression. Significant changes in the histological structure of gonad were observed. The results demonstrate that Cr(VI) significantly affected functional and molecular parameters in Geloina coaxans, and indicate that gonad represents the target of exposure to Cr(VI).

The objective of this study was to test the efficiency of an aromatase inhibitor, letrozole (LZ), to induce masculinization in blue drum (Nibea mitsukurii) and gynogenetic yellow drum (N. albiflora). Two experiments (Exp‐1 and Exp‐2) were performed to investigate the effect of LZ on the growth, sex ratio and gonad development in these two fishes. In Exp‐1, blue drum were treated with oral administration of letrozole at different doses (0, 1, 10, 100 mg/kg). In Exp‐2, gynogenetic yellow drum were orally administered different doses of LZ (0, 10, 100 mg/kg) and a dose of 10 mg/kg 17α‐methyltestosterone (MT). The treatments in both experiments were performed from 25 to 85 days post hatch (dph). As a result, all the LZ‐treated fish were phenotypic males despite of slower growth than the control during the treatment period. Free spermatozoa were observed from 150 dph onwards and the gonadosomatic index of the LZ‐treated fish did not differ significantly from that in the control at maturity. However, the sex‐reversed fish induced by MT exhibited a high proportion (33.4%) of testicular abnormalities. Our results showed that oral administration of LZ was efficient in inducing sex reversal in drums, and thereby facilitated their mono‐sex culture.

The extreme reproductive potential of the crown-of-thorns starfish (CoTS) Acanthaster sp. is a key trait used to explain their population outbreaks. Despite their highly defended morphology, sublethal predation is prevalent, as documented here for 2 outbreak populations in the Great Barrier Reef: Davies Reef and Lynch's Reef (arm damage 73.0 and 58.3%, respectively). It is not known how this trauma affects reproductive potential as a quantitative change in gonad production. We investigated the variability in gonad weight in the arms of uninjured CoTS. For CoTS with injured arms, we assessed the impact of arm injury and regeneration on gonad development at the level of the whole individual and among arms. The gonad index (GI) was lower in CoTS with 3 or more injured arms on Davies Reef, but not Lynch's Reef. At the level of individual arms, arm damage (25-100% of individual arm lost at Davies Reef and 10-100% at Lynch's Reef) resulted in a lower gonad weight compared to intact arms. Arms regenerating following total arm loss had a lower gonad weight than intact arms, even for arms that were 50-74% (Davies Reef) and 75-99% regenerated (Lynch's Reef). For uninjured CoTS, we show strong support for GI estimates based on extrapolation from one arm, and that inclusion of more than 4 arms resulted in marginal reduction in error. It is clear that sublethal injury can have a significant effect on gonad production in CoTS, with implications for population control given the reproductive potential of this species.

The gonochoristic feature with environmental sex determination that occurs during the yellow stage in the eel provides an interesting model to investigate the mechanisms of gonadal development. We previously studied various sex-related genes during gonadal sex differentiation in Japanese eels. In the present study, the members of transforming growth factor beta (TGF-β) superfamily were investigated. Transcript levels of anti-Müllerian hormone, its receptor, gonadal soma-derived factor (amh, amhr2, and gsdf, respectively) measured by real-time polymerase chain reaction (qPCR) showed a strong sexual dimorphism. Transcripts were dominantly expressed in the testis, and their levels significantly increased with testicular differentiation. In contrast, the expressions of amh, amhr2, and gsdf transcripts were low in the ovary of E2-feminized female eels. In situ hybridization detected gsdf (but not amh) transcript signals in undifferentiated gonads. amh and gsdf signals were localized to Sertoli cells and had increased significantly with testicular differentiation. Weak gsdf and no amh signals were detected in early ovaries of E2-feminized female eels. Transcript levels of amh and gsdf (not amhr2) decreased during human chorionic gonadotropin (HCG)-induced spermatogenesis in males. This study suggests that amh, amhr2, and especially gsdf might be involved in the gene pathway regulating testicular differentiation of Japanese eels.

The rock bream (Oplegnathus fasciatus) is a typical fish with a unique multiple sex chromosome system. In this study, we investigated the gene expression profiling in the gonads and brains of both males and females using RNA-Seq to identify sex-related genes and pathways. In accordance with the dimorphic expression profiles, combined with Gene ontology and KEGG enrichment analyses, a number of potential genes and pathways associated with sex determination were obtained from transcriptional analysis, especially some sex-biased genes and pathways. Next, we selected 18 candidate genes and analyzed their expression in different tissues and developmental stages. We found that the expression levels of Amh, Dmrt1, Sox9, Dmrtb1, and Nanos2 were significantly higher in the testis than those in the ovary or other tissues, whereas the expression levels of ZP4, Bouncer, RNF208, FoxH1, and TOB were significantly higher in the ovary than those in the testis. Furthermore, the expression levels of these genes in different developmental stages of gonads also showed sexually dimorphic patterns, suggesting that they might play important roles during gonadal development. These genes are useful markers for investigating sex determination and differentiation in rock bream. The findings of this study can provide insights into the molecular mechanisms of sex determination and differentiation in fish with multiple sex chromosome systems.

17α-Methyltestosterone (MT) is a synthetic androgen. The objective of this study was to explore the effects of exogenous MT on the growth and gonadal development of female rare minnow Gobiocypris rarus. Female G. rarus groups were exposed to 25–100 ng/L of MT for 7 days. After exposure for 7 days, the total weight and body length were significantly decreased in the 50-ng/L MT groups. The major oocytes in the ovaries of the control group were vitellogenic oocytes (Voc) and cortical alveolus stage oocytes (Coc). In the MT exposure groups, some fish had mature ovaries with a relatively lower proportion of mature oocytes, and the diameter of the perinucleolar oocytes (Poc) was decreased compared with those of the control group. Ovarian VTG, FSH, LH, 11-KT, E2, and T were significantly increased after exposure to 50 ng/L of MT for 7 days. Unigenes (73,449), 24 known mature microRNAs (miRNAs), and 897 novel miRNAs in the gonads of G. rarus were found using high-throughput sequencing. Six mature miRNAs (miR-19, miR-183, miR-203, miR-204, miR-205, and miR-96) as well as six differentially expressed genes (fabp3, mfap4, abca1, foxo3, tgfb1, and zfp36l1) that may be associated with ovarian development and innate immune response were assayed using qPCR. Furthermore, the miR-183 cluster and miR-203 were differentially expressed in MT-exposed ovaries of the different G. rarus groups. This study provides some information about the role of miRNA–mRNA pairs in the regulation of ovarian development and innate immune system, which will facilitate future studies of the miRNA–RNA-associated regulation of teleost reproduction.

Animals must partition limited resources between their own growth and subsequent reproduction. Endocrine disruptors (ED) may cause maternal metabolic disorders that decrease successful reproduction and might be responsible for multi- and transgenerational effects in amphibians. We found that the frog Silurana (Xenopus) tropicalis, exposed to environmentally relevant concentrations of benzo[a]pyrene and triclosan throughout its life cycle, produced F1 females with delayed sexual maturity and decreased size and weight. These F1 females displayed a marked metabolic syndrome associated with decreased fasting plasma cholesterol and triglyceride concentrations and decreased gonadal development. F1 females from F0 exposed animals also had decreased reproductive investment highlighted by a decrease of oocyte lipid reserves associated with significant F2-tadpole mortality. F2 females from F0 exposed animals also displayed a marked metabolic syndrome but were able to correctly direct liver lipid metabolism to the constitution of fat bodies and oocyte yolk stores. In addition, the F2 females produced progeny that had normal mortality levels at 5 days post hatching compared to the controls suggesting a good reproductive investment. Our data confirmed that these ED, at concentrations often found in natural ponds, can induce multi- and transgenerational metabolic disorders in the progeny of amphibians that are not directly exposed. We present a hypothesis to explain the transmission of the metabolic syndrome across generations through modification of egg reserves. However, when high mortality occurred at the tadpole stage, surviving females were able to cope with metabolic costs and produce viable progeny through sufficient investment in the contents of oocyte reserves.

Fecundity (F) and Gonadosomatic index (GSI) of Cyprinus carpio (Common carp) at fish seed production center at Aurangabad was estimated for a period during June to September months (2013-2015). During the present study the fish samples was within the range of 30cm to 60cm in length and 400g to 750g in weight. Fecundity and its correlation with body weight of Cyprinus carpio upon administration of ovaprim, ovatide and pituitary gland extract, Average body wt ±SD and Average number of eggs ± SD, a±SD, b±SD, r, and Average relative fecundity±SD for both fecundity and gonadosomatic index were estimated. Fish body weight and weight of gonad gives the gonadosomatic index (G.S.I.). It is the ratio of fish gonad weight to body weight, it is particularly helpful in identifying days and seasons of spawning, as the ovaries of gravid females swiftly increase in size just prior to spawning. Gonadosomatic index of fish increases with maturation being maximum during peak period of maturity and abruptly declines after spawning. The development and growth of gonad simultaneously takes place in the fish and fish grows and the GSI is high. Present study ensured that the growth of fish, body weight and gonad development is correlated. Gonadosomatic index of fish is related to spawning and reproduction of fish. At maturity stage fish has maximum GSI value and after spawning GSI value declines. The GSI value also related to amount of food available to them in water and temperature of water also. So generally during the breeding season of such selected fish shows maximum GSI value and after spawning it is reduced. In this study we have carried out GSI of male and female fish simultaneously. When GSI of both sexes were we came to know that maximum values of GSI have occurred almost at the same time in both sexes and after spawning it is recorded that there was a great decrease in GSI values.

The kisspeptin system, which lies upstream of the hypothalamic-pituitary-gonadal (HPG) axis, is believed to function as a regulator of reproduction in teleosts. In this study, we isolated and characterized kiss2 and its receptor kissr2 in largemouth bass (Micropterus salmoides). The complete coding sequences of kiss2 and kissr2 were 375 and 1134 bp long and encoded precursor proteins 124 and 377 amino acid long, respectively. Real-time PCR showed that kiss2 and kissr2 were primarily expressed in the HPG axis. The expression profile of kiss2 and kissr2 varied with gonadal development, with the highest and lowest expression levels being detected during the immature and final maturation stages, respectively. Intraperitoneal injection of exogenous Kiss2-10 peptide increased the transcript levels of gnrh3, kissr2, fshβ, lhβ, ar, and er2 within 24 h (p 0.05), as well as plasma levels of 17β-estradiol and testosterone. Histological analysis indicated that chronic administration of exogenous Kiss2-10 peptide accelerated vitellogenesis in females and spermatogenesis in males. Further, in situ hybridization revealed that kiss2 is expressed in the ooplasm and vitelline envelope of oocytes and the spermatocytes of testes. In addition, experiments using gonad tissue primary cell cultures indicated that exogenous Kiss2-10 peptide stimulates the expression of reproduction-related genes. Collectively, our findings indicate that the kiss2/kissr2 system in largemouth bass is involved in regulating gonadal development through the HPG axis.