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![Histological sections of gonads from wild-type and hot-homozygous fish at 6 months after hatching. The specimens are sections of the tissues shown in Fig. 1K stained with hematoxylin-eosin. (A) Wild-type ovary. (B) XX hot-homozygous ovary. The arrowhead in A (enlarged in A) indicates previtellogenic follicles, with which the immature follicles abundant in B share a similar appearance (enlarged in B). An oocyte at the yolk formation stage is indicated by an asterisk in A. (C) Wild-type testis. (C) Enlargement of the boxed area in C (arrow), where spermatogonia (sg), spermatocytes (sc), spermatids (st), and spermatozoa (sz) are labeled according to the morphological criteria (39). (D) Enlargement of the boxed area in D (arrow), showing sp, sc, st and sz. [Scale bars, 0.5 mm (A-D); 0.1 mm (A-D).]](profile/Takashi-Sasaki-9/publication/6299997/figure/fig2/AS:667873391439872@1536244833070/Histological-sections-of-gonads-from-wild-type-and-hot-homozygous-fish-at-6-months-after.png)
Histological sections of gonads from wild-type and hot-homozygous fish at 6 months after hatching. The specimens are sections of the tissues shown in Fig. 1K stained with hematoxylin-eosin. (A) Wild-type ovary. (B) XX hot-homozygous ovary. The arrowhead in A (enlarged in A) indicates previtellogenic follicles, with which the immature follicles abundant in B share a similar appearance (enlarged in B). An oocyte at the yolk formation stage is indicated by an asterisk in A. (C) Wild-type testis. (C) Enlargement of the boxed area in C (arrow), where spermatogonia (sg), spermatocytes (sc), spermatids (st), and spermatozoa (sz) are labeled according to the morphological criteria (39). (D) Enlargement of the boxed area in D (arrow), showing sp, sc, st and sz. [Scale bars, 0.5 mm (A-D); 0.1 mm (A-D).]
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We previously performed mutant screens in the medaka for defects in gonadal development and identified a mutant of interest in this regard, which was designated as hotei (hot). This mutant manifests a number of remarkable phenotypic abnormalities including: (i) excessive proliferation of germ cells that initiates at around the hatching stage regard...
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... mature ovaries during the spawning period of the medaka contain follicles at various developmental stages, from previtellogenic to fully grown ( Fig. 2 A and A). In contrast to wild-type, the hypertrophic ovaries of XX and XY female hot homozygotes are filled with small follicles ( Fig. 2 B and B). Thus, in hot homozygotes the germ cells continue to increase, whereas follicular development is arrested during early vitello- genesis, and these features can be observed even at 6 months after ...
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... mature ovaries during the spawning period of the medaka contain follicles at various developmental stages, from previtellogenic to fully grown ( Fig. 2 A and A). In contrast to wild-type, the hypertrophic ovaries of XX and XY female hot homozygotes are filled with small follicles ( Fig. 2 B and B). Thus, in hot homozygotes the germ cells continue to increase, whereas follicular development is arrested during early vitello- genesis, and these features can be observed even at 6 months after hatching. In wild-type testes, spermatogonia are located in the most peripheral regions of the lobule structures, and sper- matogenesis ...
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... continue to increase, whereas follicular development is arrested during early vitello- genesis, and these features can be observed even at 6 months after hatching. In wild-type testes, spermatogonia are located in the most peripheral regions of the lobule structures, and sper- matogenesis proceeds synchronously within the cysts in these lobules ( Fig. 2 C and C). In the male-type XY hot mutants, hypertrophic testes develop that are filled with germ cells and sperm at various developmental stages (Fig. 2 D and D). In a minor fraction of XY hot homozygotes, the gonads contained both testicular and ovarian components (see Fig. ...
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... hatching. In wild-type testes, spermatogonia are located in the most peripheral regions of the lobule structures, and sper- matogenesis proceeds synchronously within the cysts in these lobules ( Fig. 2 C and C). In the male-type XY hot mutants, hypertrophic testes develop that are filled with germ cells and sperm at various developmental stages (Fig. 2 D and D). In a minor fraction of XY hot homozygotes, the gonads contained both testicular and ovarian components (see Fig. ...
Citations
... The TGF-β signaling pathway can regulate gonad differentiation by regulating the number of germ cells and expression levels of aromatase genes. In medaka, the Amhr2 male mutant exhibited remarkable phenotypic abnormalities, including sex reversal and proliferation of the germ cells [56]. The TGF-β signaling pathway is also essential for ovary development. ...
The East Asian common octopus (Octopus sinensis) is an economically important species among cephalopods. This species exhibits a strict dioecious and allogamous reproductive strategy, along with a phenotypic sexual dimorphism, where the third right arm differentiates into hectocotylus in males. However, our understanding of the molecular mechanisms that underlie sex determination and differentiation in this species remains limited. In the present study, we surveyed gene-expression profiles in the immature male and female gonads of O. sinensis based on the RNA-seq, and a total of 47.83 Gb of high-quality data were generated. Compared with the testis, we identified 8302 differentially expressed genes (DEGs) in the ovary, of which 4459 genes were up-regulated and 3843 genes were down-regulated. Based on the GO enrichment, many GO terms related to sex differentiation were identified, such as sex differentiation (GO: 0007548), sexual reproduction (GO: 0019953) and male sex differentiation (GO: 0046661). A KEGG classification analysis identified three conserved signaling pathways that related to sex differentiation, including the Wnt signaling pathway, TGF-β signaling pathway and Notch signaling pathway. Additionally, 21 sex-related DEGs were selected, of which 13 DEGs were male-biased, including Dmrt1, Foxn5, Foxj1, Sox30, etc., and 8 DEGs were female-biased, including Sox14, Nanos3, β-tubulin, Suh, etc. Ten DEGs were used to verify the expression patterns in the testis and ovary using the RT-qPCR method, and the results showed that the expression level shown by RT-qPCR was consistent with that from the RNA-seq, which confirmed the reliability of the transcriptome data. The results presented in this study will not only contribute to our understanding of sex-formation mechanisms in O. sinensis but also provide the foundational information for further investigating the molecular mechanisms that underline its gonadal development and facilitate the sustainable development of octopus artificial breeding.
... In mammals, it is required for the inhibition of Müllerian ducts (Josso, 1973). A primary role for AMH pathways in germ cell proliferation and sex determination became evident in studies of AMH and AMH receptor (amh-R) mutants in teleost species (Eshel et al., 2011;Hattori et al., 2012;Kamiya et al., 2012;Kikuchi & Hamaguchi, 2013;Lin et al., 2017;Liu et al., 2022;Morinaga et al., 2007;Mullen et al., 2019;Song et al., 2021;Yamaguchi & Kitano, 2023;Yan et al., 2019;Zhang et al., 2020). Y-linked amh paralog called amh-Y is the major male determinant in pejerrey (Odontesthes hatcheri; Hattori et al., 2012). ...
This is the first work using gonads from undifferentiated, genetically-sexed Siberian sturgeon describing expression changes in genes related to steroid synthesis and female and male sex differentiation. One factor identified as relevant for ovarian differentiation was the gene coding for the enzyme Hsd17b1, which converts estrone into estradiol-17β. hsd17b1 was highly activated in female gonads at 2.5 months of age, around the onset of sex differentiation, preceding activation of two other genes involved in estrogen production (cyp19a1 and foxl2). hsd17b1 was also strongly repressed in males. Two known foxl2 paralogs are found in Siberian sturgeon-foxl2 and foxl2l-but only foxl2 appeared to be associated with ovarian differentiation. With regard to the male pathway, neither 11-oxygenated androgens nor classic male genes (amh, dmrt1, sox9, and dhh) were found to be involved in male sex differentiation, leaving open the question of which genes participate in early male gonad development in this ancient fish. Taken together, these results indicate an estrogen-dependence of female sex differentiation and 11-oxygenated androgen-independence of male sex differentiation.
... 15,[28][29][30][31] Even in fish, where a renewing population of oocytes exists in the ovary, 14,15,30 the embryonic endowment of GCs is directly linked to sex determination: larger numbers of GCs induce female sex determination and, conversely, depletion of GCs leads to male sex determination. 16,32 It is important to note that this study does not address the mechanism by which temperature affects GC number; future studies will address whether the difference in GC number results from differences in GC specification, migration, or rate of proliferation/apoptosis. In this study we found no difference in GC number between the cool and intermediate incubation temperatures, suggesting that there is an optimal incubation temperature that leads to higher GC numbers at the time of hatching. ...
In many reptile species, gonadal sex is affected by environmental temperature during a critical period of embryonic development-a process known as temperature-dependent sex determination (TSD).1 The oviparous red-eared slider turtle, Trachemys scripta, has a warm-female/cool-male TSD system and is among the best-studied members of this group.2 When incubated at low temperatures, the somatic cells of the bipotential gonad differentiate into Sertoli cells, the support cells of the testis, whereas at high temperatures, they differentiate into granulosa cells, the support cells of the ovary.3 Here, we report the unexpected finding that temperature independently affects the number of primordial germ cells (GCs) in the embryonic gonad at a time before somatic cell differentiation has initiated. Specifically, embryos incubated at higher, female-inducing temperatures have more GCs than those incubated at the male-inducing temperature. Furthermore, elimination of GCs in embryos incubating at intermediate temperatures results in a strong shift toward male-biased sex ratios. This is the first evidence that temperature affects GC number and the first evidence that GC number influences sex determination in amniotes. This observation has two important implications. First, it supports a new model in which temperature can impact sex determination in incremental ways through multiple cell types. Second, the findings have important implications for a major unresolved question in the fields of ecology and evolutionary biology-the adaptive significance of TSD. We suggest that linking high GC number with female development improves female reproductive potential and provides an adaptive advantage for TSD.
... To maintain continuous spermatogenesis throughout the male reproductive life, a specialized microenvironment of the testes, known as SSCs niche, regulates the properties of self-renewal, quiescence, size, or the ability of the SSCs to differentiate and proliferate (Chiarini- Garcia et al. 2001;de Rooij 2017;Kitadate and Kobayashi 2010;La and Hobbs 2019;Losick et al. 2011;Mäkelä and Hobbs 2019;McIntyre and Nance 2020;Nishimura et al. 2016;Nóbrega et al. 2010). Moreover, the number of germ cells is maintained by a fine-tuned balance between proliferation and apoptosis, whose regulation takes place by genes such as amh (anti-müllerian hormone) or ndrg1b (n-myc downstream regulated gene 1b), and p53, respectively (Arias Padilla et al. 2021;Morinaga et al. 2007;Ohta et al. 2003;Rodríguez-Marí et al. 2010). This ability of the testes to maintain the homeostasis in SSCs and the decision among selfrenewal, differentiation, or apoptosis is what allows them to survive under adverse conditions (La and Hobbs 2019). ...
... It was observed that the number of spermatogonia (SGs: type A undifferentiated spermatogonia and type A differentiated spermatogonia as SGa; type B spermatogonia as SGb) as well as the number of cysts with SGs did not change during treatment (Fig. 1B, C). Moreover, the inhibition of proliferation of SGa and SGb was supported by the upregulation of amh, a well-known regulator of germ cell proliferation (Morinaga et al. 2007;Ohta et al. 2003;Rodríguez-Marí et al. 2010), at 10 days of heat treatment (threefold compared to NT) (Fig. 1D). ...
... In this regard, our results support a strong link between the activation of the Notch pathway and the inhibition of spermatogenesis by increasing temperature. Moreover, the inhibition of SGa proliferation was observed at a cellular level by the loss of the sperm lineage, and also molecularly by the increase of genes related to the inhibition of germ cell proliferation in fish, such as amh (Morinaga et al. 2007). Moreover, an initial increase in spermatocyte proliferation is observed with increasing temperature, which is corroborated by a decrease in the cystic proliferation inhibitor ndrg1b (Arias Padilla et al. 2021). ...
Gamete production is a fundamental process for reproduction; however, exposure to stress, such as increased environmental temperature, can decrease or even interrupt this process, affecting fertility. Thus, the survival of spermatogonial stem cells (SSCs) is crucial for the recovery of spermatogenesis upon stressful situations. Here, we show that the Notch pathway is implicated in such survival, by protecting the SSCs against thermal stress. First, we corroborated the impairment of spermatogenesis under heat stress in medaka, observing an arrest in metaphase I at 10 days of heat treatment, an increase in the number of spermatocytes, and downregulation of ndrg1b and sycp3. In addition, at 30 days of treatment, an interruption of spermatogenesis was observed with a strong loss of spermatocytes and spermatids. Then, the exposure of adult males to thermal stress condition induced apoptosis mainly in spermatogenic and supporting somatic cells, with the exception of the germinal region, where SSCs are located. Concomitantly, the Notch pathway–related genes were upregulated, including the ligands (dll4, jag1-2) and receptors (notch1a-3). Moreover, during thermal stress presenilin enhancer-2 (pen-2), the catalytic subunit of γ-secretase complex of the Notch pathway was restricted to the germinal region of the medaka testis, observed in somatic cells surrounding type A spermatogonia (SGa). The importance of Notch pathway was further supported by an ex vivo approach, in which the inhibition of this pathway activity induced a loss of SSCs. Overall, this study supports the importance of Notch pathways for the protection of SSCs under chronic thermal stress.
... The fact that Amh signaling to be linked to sexual differentiation in fish was observed in studies carried out on teleost fish medaka. Studies on the medaka mutant hotei showed an over-proliferation of germ cells and 50% of male-to-female sex reversal in the hotei homozygous [102]. The hotei phenotypes is caused by a mutation of the amhr2. ...
... Amh was found to be an endogenous ovarian reversal factor in rats (Vigier et al., 1989), and Mullerian derivatives also appeared in Amh mutant male mice, which caused the reproductive defects (Behringer et al., 1994). The first investigation into the role of Amh in non-mammalian vertebrates showed that it is essential for the development of germ cells and sex differentiation in the medaka (Morinaga et al., 2007). The studies on sex differentiation of Paralichthys olivaceus, O. niloticus and O. mykiss found that Amh has a sex differential expression pattern (Ijiri et al., 2008;Vizziano et al., 2008;Yoshinaga et al., 2004), and the expression of Amh in the testis was higher than that in ovaries in adult fish (Kluver et al., 2007). ...
In vertebrates, anti‐Mullerian hormone (Amh) secreted by Sertoli cells (SC) performs a pivotal function in male sex differentiation. Compared with that of higher vertebrates, the expression pattern of Amh is more diversified in fish. In this study, the full‐length complementary DNA (cDNA) of Amh in Centropyge vrolikii (Cv‐Amh) was cloned and analysed, which was 2,470 bp, including a 238 bp 5'UTR, a 1,602 bp ORF and a 633 bp 3'UTR; the similarity of Amh between Cv‐Amh and other fish is relatively high. The quantitative real‐time PCR (qRT‐PCR) results of healthy tissues and gonads at sex reversal stages in C. vrolikii showed that the expression level of Amh in the testis was significantly higher than that in other tissues (P < 0.05). Amh was weakly expressed in the vitellogenic stage ovary and perinucleolus stage ovary, but its expression significantly increased in the gonads at the hermaphroditic stage, and finally reached the highest in the pure testis after sexual reversal. The results of in situ hybridization indicated that the positive signal of Amh was strongly concentrated in SCs of testis. After Amh knockdown in the gonads, the effect on sex‐related genes was tested using qRT‐PCR. Among these, the expression of Dmrt1, Cyp11a, Hsd11b2, Sox8 and Sox9 significantly decreased, whereas that of Cyp19a, Sox4, Foxl2 and Sox3 increased. These results suggested that Amh could be the pivotal gene in reproductive regulation in C. vrolikii, and the data will contribute to sex‐related research of C. vrolikii in the future.
... Similarly, compared with those of wild-type fish, higher GSI values were observed in female gsdf − /− medaka, zebrafish, and Nile tilapia (Guan et al., 2017;Yan et al., 2017;Jiang et al., 2022). In XY amhr2 hotei mutant female and male Japanese medaka, the gonads become hypertrophic, which might be caused by excessive germ cell proliferation (Morinaga et al., 2007;Nakamura et al., 2012). Germ cell proliferation in gsdf − /− female fish might be more active, resulting in larger ovaries. ...
Gonadal soma-derived factor (gsdf) belongs to the TGF-β superfamily and is critical for sex determination and differentiation in fish. XY gsdf − /− Nile tilapia (Oreochromis niloticus) show male-to-female sex reversal 1 month after hatching (mah) and are infertile at the adult age; however, the functions of gsdf in spermatogenesis in fish are unclear. Herein, XY gsdf − /− Nile tilapia were converted to males with testis by feeding 200 μg/g letrozole (LE), an aromatase inhibitor, from 1 to 4 mah. The testis somatic index of XY gsdf − /− LE fish was significantly lower than that of XY gsdf +/+ LE fish at 6 mah. The artificial fertilization ratio of XY gsdf − /− LE fish sperm (53.22 ± 4.35%) was significantly lower than that of XY gsdf +/+ LE fish (86.33 ± 1.53%), indicating that XY gsdf − /− LE male fish were sub-fertile. Testes of LE XY gsdf − /− and gsdf +/+ fish at 6 mah were used for a transcriptome analysis. Compared with the testis of XY gsdf +/+ LE fish, 1426 and 1654 genes were up-regulated and ownregulated in the testis of XY gsdf − /− LE individuals, respectively. A KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis revealed that the differentially expressed genes (DEGs) were engaged in 21 pathways, including cancer, cell growth and death, and endocrine system pathways. Genes related to steroidogenesis and meiosis, such as cyp19a1a, foxl2, and cyp17a1, were significantly up-regulated, whereas sycp3, dmc1, and aldh1a1 were significantly down-regulated in the testis. In addition, a serum steroid hormone analysis showed that 17β-estradiol and 11-ketotestosterone levels of XY gsdf − /− LE fish were significantly higher and lower than those of XY gsdf +/+ LE fish. These findings demonstrated that endocrine system disruption in XY gsdf − /− LE might explain the decrease in fertility. This research establishes the groundwork for future research on the function of gsdf in spermatogenesis in fish.
... Amh bound to Amhr2 was reported to induce the phosphorylation of type I receptors, transduce signals by phosphorylating Smad proteins, and regulate the transcription of downstream genes in mammals [34]. In medaka fish, a hotei mutant (amhr2deficient) showed male-to-female sex reversal [35]. The mutant analysis identified a mutation in the amhr2 gene, namely, an A-to-G mutation in exon 9, which is located in the receptor kinase domain. ...
... The knockout of amhy, amhΔ-y, and amhr2 using the CRISPR/Cas9 system caused male-to-female sex reversal. Meanwhile, amhr2-deficient XY medaka showed sex reversal at a rate of more than 50% [35]. In this study, the loss of Amhr2 function causes male-to-female sex reversal in Japanese flounder. ...
... Foxl2 is a factor involved in female sex determination, which directly activates the expression of cyp19a1 in vertebrates [10,40,41]. In medaka, cyp19a1 expression is up-regulated in amhr2-deficient XY fish [35]. Similar results have reported that the knockdown of amhy in XY Patagonian pejerrey (Odontesthes hatcheri) resulted in the upregulation of foxl2 and cyp19a1 expression [42]. ...
The anti-Müllerian hormone (Amh) is a protein belonging to the TGF-β superfamily, the function of which has been considered important for male sex differentiation in vertebrates. The Japanese flounder (Paralichthys olivaceus) is a teleost fish that has an XX/XY sex determination system and temperature-dependent sex determination. In this species, amh expression is up-regulated in genetic males and in temperature-induced masculinization during the sex differentiation period. However, to the best of our knowledge, no reports on the Amh receptor (Amhr2) in flounder have been published, and the details of Amh signaling remain unclear. In this study, we produced amhr2-deficient mutants using the CRISPR/Cas9 system and analyzed the gonadal phenotypes and sex-related genes. The results revealed that the gonads of genetically male amhr2 mutants featured typical ovaries, and the sex differentiation-related genes showed a female expression pattern. Thus, the loss of Amhr2 function causes male-to-female sex reversal in Japanese flounder. Moreover, the treatment of genetically male amhr2 mutants with an aromatase inhibitor fadrozole, which inhibits estrogen synthesis, resulted in testicular formation. These results strongly suggest that Amh/Amhr2 signaling causes masculinization by inhibiting estrogen synthesis during gonadal sex differentiation in the flounder.
... A gene that inhibits sperm formation has been isolated from the Japanese eel and was classified as an amh homologue [47]. At present, the homologous genes of amh have been cloned in many fish species such as Danio rerio [48], Oryzias Latipe [49], and Dicentrarchus labrax [50]. In these fish species, amh not only shows a conserved expression pattern, but also plays an important role in the regulation of their sex determination. ...
As a new freshwater aquaculture product, triploid loaches (Misgurnus anguillicaudatus) are characterized by fast growth, high-quality meat, high edibility, high resistance to disease, and sterility. In this study, a natural tetraploid loach (4n = 100) (♀) was crossed with a diploid loach (2n = 50) (♂), thus creating the hybrid triploid loach (3n = 75). The histological observations of triploid offspring and diploid controls at 4 days post-hatching (dph), 15dph, 22dph, and 50dph showed that most of the hybrid triploid loaches were abortive in the early gonad differentiation process. To explore its fertility mechanism, through transcriptome analyses of triploid offspring and diploid controls at four periods, 10 differentially expressed genes related to the early fertility mechanism were identified: amh, hormad1, rec8, h2b, plvap, zp3, h2a, nrb0b1, ddx4, and esr2. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs, two pathways were identified that are closely related to the early fertility mechanism at 50dph: the estrogen signaling pathway and steroid biosynthesis. The findings laid a foundation for further exploration of their molecular inhibition mechanism in hybrid triploid loaches.
... Alternative spliced variants of fshr may interfere with the development of gsdf-deficient oocytes, in addition to the canonical Smad3-dependent activation of Fshr promoter to increase the cell division rate of rat granulosa cells [46]. In fact, the irreversible synergistic effect of TGFβ and FSH in the expression of Fshr mRNA, as demonstrated in cultured rat granulosa cells [47], may explain the prevalence of 100% ovotestis development in Sissy individuals [20] by 10% compared to that of hotei (amhr2 mutant) [48]. ...
The meiotic entry of undifferentiated germ cells is sexually specific and strictly regulated by the testicular or ovarian environment. Germline stem cells with a set of abnormal sex chromosomes and associated autosomes undergo defective meiotic processes and are eventually eliminated by yet to be defined post‐transcriptional modifications. Herein, we report the role of gsdf, a member of BMP/TGFβ family uniquely found in teleost, in the regulation of meiotic entry in medaka (Oryzias latipes) via analyses of gametogenesis in gsdf‐deficient XX and XY gonads in comparison with their wild‐type siblings. Several differentially expressed genes, including the FKB506‐binding protein 7 (fkbp7), were significantly upregulated in pubertal gsdf‐deficient gonads. The increase in alternative pre‐mRNA isoforms of meiotic synaptonemal complex gene sycp3 was visualized using Integrative Genomics Viewer and confirmed by real‐time qPCR. Nevertheless, immunofluorescence analysis showed that Sycp3 protein products reduced significantly in gsdf‐deficient XY oocytes. Transmission electron microscope observations showed that normal synchronous cysts were replaced by asynchronous cysts in gsdf‐deficient testis. Breeding experiments showed that the sex ratio deviation of gsdf−/− XY gametes in a non‐Mendelian manner might be due to the non‐segregation of XY chromosomes. Taken together, our results suggest that gsdf plays a role in the proper execution of cytoplasmic and nuclear events through receptor Smad phosphorylation and Sycp3 dephosphorylation to coordinate medaka gametogenesis, including sex‐specific mitotic divisions and meiotic recombination.
