Early Gonad Research Articles

Steroid hormone-deprived sex reversal in cyp11a1 mutant XX tilapia experiences an ovary-like stage at molecular level

Fish sex is largely influenced by steroid hormones, especially sex hormones. Here, we established a steroid hormone-free genetic model by mutation of cyp11a1 in Nile tilapia, which was confirmed by EIA assay. Gonadal phenotype and transcriptome analyses showed that the XX mutants displayed sex reversal from female to male but with defective spermatogenesis. Despite the sex reversal, the aromatase encoding gene cyp19a1a was continuously expressed in the gonads of the XX mutants, which might be caused by androgen deficiency. Whole-mount fluorescence in situ hybridization and transcriptome analysis showed that the gonads of the XX mutants firstly developed towards ovary but shifted to testis between 10 to 15 days after hatching. Detailed expression analysis of key sex differentiation pathway genes foxl3 and dmrt1 combined with apoptosis analysis revealed transdifferentiation of germ cells from female to male during sex reversal. Rescue experiments showed that both P5 and E2 treatment rescued the sex reversal of cyp11a1 mutant XX fish. Overall, our results revealed a transient ovary-like stage and transdifferentiation of germ cells from female to male in the early gonads of the steroid hormone-deprived cyp11a1 mutant XX fish.

Genome-wide analysis of the cytochrome P450 gene family in Pacific oyster Crassostrea gigas and their expression profiles during gonad development

The cytochrome P450 (CYP) gene superfamily plays a significant role in various physiological processes, producing different compounds such as hormones, fatty acids, and biomolecules. However, little information is known their roles during gonad development in Pacific oyster (Crassostrea gigas). In this study, total of 116 CgCYP (Crassostrea gigas cytochrome P450) genes were identified and their expression pattern was analyzed for the first time. The relative molecular weights of these CgCYP genes ranged from 63.52 to 113.41 kDa, and the length of encoded amino acids ranged from 103 to 993. And total 26 cis-acting elements of these CgCYP genes were identified. GO and KEGG enrichment analysis showed some CgCYP genes are essential for the metabolism of male and female sex hormones. Additionally, expression anslysis showed 69 CgCYP genes were over-expressed in early gonad development and triploid infertile individuals. More importantly, expression levels of CgCYP1, CgCYP15, CgCYP34, CgCYP46, CgCYP69, CgCYP87, CgCYP88, and CgCYP103, were found to be significantly higher in female gonad, suggesting their important roles in female gonad development. The results of this study will provide a better understanding of the CgCYP genes in the gonad development of Pacific oyster.

Trade-off between growth and reproduction in Argopecten purpuratus (L.) scallops exposed to medium-term hypoxia and acidification

Peruvian bays that are home to aquatic organisms of commercial interest are increasingly affected by hypoxia and low pH events. These stressors produce unfavorable conditions for the aquatic fauna, leading to mass mortality events. In this study, we evaluated the ecophysiological responses of the scallop Argopecten purpuratus exposed to moderate hypoxia (20% DO saturation) and low pH (OA, pH 7.4) using a 2 × 2 factorial design. We found that a 51-day exposure to low pH (OA treatment) had greater negative effects on A. purpuratus performances than hypoxia (lower survival and decreased clearance and calcification rates), but stimulated early gonad maturation. The survival rate was 1.3-fold higher under hypoxia than under acidic treatment. The interaction between hypoxia and low pH had an antagonistic effect on survival, since the combined treatment (HOA) resulted in lower scallop mortality than the single stress treatments. Calcification was negatively affected by all stress treatments, whereas hypoxia and OA both stimulated gametogenesis. OA treatment resulted in higher frequencies of previtellogenic and vitellogenic oocytes, greater gonad coverage area, and lower frequency of atretic oocytes, suggesting higher reproductive potential. HOA was positively related to oocyte development and high frequency of post-vitellogenic and atretic oocytes. These results suggest that, due to hypoxia and low pH, feeding is reduced and energy allocation prioritizes scallop gonad maturation. This trend would have negative effects on scallop growth and calcification, while increased reproduction under environmental stress could mitigate the effect on recruitment.

COUP-TFII regulates early bipotential gonad signaling and commitment to ovarian progenitors

BackgroundThe absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored.ResultsBy differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells.ConclusionsOur findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.

EIF4E1b is a non-canonical eIF4E protecting maternal dormant mRNAs.

Maternal mRNAs are essential for protein synthesis during oogenesis and early embryogenesis. To adapt translation to specific needs during development, maternal mRNAs are translationally repressed by shortening the polyA tails. While mRNA deadenylation is associated with decapping and degradation in somatic cells, maternal mRNAs with short polyA tails are stable. Here we report that the germline-specific eIF4E paralog, eIF4E1b, is essential for zebrafish oogenesis. eIF4E1b localizes to P-bodies in zebrafish embryos and binds to mRNAs with reported short or no polyA tails, including histone mRNAs. Loss of eIF4E1b results in reduced histone mRNA levels in early gonads, consistent with a role in mRNA storage. Using mouse and human eIF4E1Bs (in vitro) and zebrafish eIF4E1b (in vivo), we show that unlike canonical eIF4Es, eIF4E1b does not interact with eIF4G to initiate translation. Instead, eIF4E1b interacts with the translational repressor eIF4ENIF1, which is required for eIF4E1b localization to P-bodies. Our study is consistent with an important role of eIF4E1b in regulating mRNA dormancy and provides new insights into fundamental post-transcriptional regulatory principles governing early vertebrate development.

Insights into the early gonad development of the protogynous ballan wrasse (Labrus bergylta)

The reproductive biology of ballan wrasse, a popular cleaner fish species now bred in captivity, is not well understood particularly the early stages of development and their accompanying endocrine changes. The protogynous nature of this species has been well documented in the wild, although it is still unclear if it follows a monandric or diandric hermaphroditism. In this study, we investigated the gonad development of hatchery produced ballan wrasse (n = 102) between 3 and 185 g body weight and 5–19 cm total length. About 90 % of all fish examined showed gonads undergoing primary oocyte growth, exhibiting chromatin-nucleolar stage, perinucleolar stage, and early cortical alveolar stage. Mean size of fish that entered primary oocyte growth (i.e., chromatin-nucleolar stage) is 26.2 ± 1.6 g and 10.9 ± 0.2 cm. The remaining 10 % were undifferentiated and constituted the smallest fish examined. The gonadosomatic index and circulating levels of 17β-Estradiol and Testosterone increased as fish progressed through the ovarian development. Expression levels of selected gonadal genes including cyp19a1a, figla, bmp15, dmrt1, amh, fshr, and lhr were also quantified. To the best of our knowledge, this paper is the first to investigate the early reproductive development of ballan wrasse, providing important insights into histological and endocrine changes. Results from this study provide basis for further research on the reproductive physiology of ballan wrasse as well as for the development of broodstock management techniques in this economically important cleaner fish.

The Effect of Habitat Manipulation on Early Gonad Maturation of Channa striata in Captive Condition

Background: With remarkable growth in aquaculture, the need for system diversification and species diversification has been demanded by the producers to gain fruitful output in terms of profit. Striped murrel, Channa striata has been identified as a suitable species for modern aquaculture systems in terms of species diversification due to their fast growth rate, air-breathing nature, higher market price and consumer acceptance. The major bottlenecks in seed production of murrel are captive maturation and short spawning window. To overcome these problems, an attempt was made to achieve early gonadal maturity of striped murrel in captive conditions by intervening in the brooder rearing habitat. Methods: In this regard, four habitats were tested in duplicates, namely T1 - Tank with clean reservoir water without macrophytes and soil base, T2 - Tanks provided with floating (Water hyacinth, Eichhornia sp) and submerged (Waterthyme, Hydrilla sp) aquatic macrophytes without any soil base, T3- Tanks provided with only soil base (15-20 cm) without any macrophytes and T4 - Tanks provided with both soil base and macrophytes. Result: At the end of the three-months rearing phase, the fishes provided with macrophytes and soil base (T4) resulted in early maturation characterized by an egg size of around 1.29±0.21 mm and the majority of late granular stage oocytes and spermatozoa in ovary and testes, respectively. The results concluded that providing a bottom soil base of 15-20 cm and covering the 20-30% water spread area using aquatic macrophytes in the brood rearing tank helped in the gonadal maturation of striped murrel during pre-monsoon in captive conditions.

Identification and Characterization of C-Mos in Pearl Mussel Hyriopsis cumingii and Its Role in Gonadal Development

C-Mos, a proto-oncogene, regulates oocyte maturation by activating the classical MAPK pathway in cells. To examine the function of C-Mos in Hyriopsis cumingii, C-Mos was identified in this study. The full-length cDNA of C-Mos was 2213 bp, including 144 bp in the 5′ UTR, 923 bp in 3′ the UTR, and 1146 bp in the open reading frame (ORF) region. During early gonad development, the expression of C-Mos from 4 to 6 months of age in H. cumingii was significantly higher than that in other months, with the highest expression in 6-month-old H. cumingii, suggesting that C-Mos may be involved in early gonadal development in H. cumingii. Clear hybridization signals were found by in situ hybridization in the oocytes, oocyte nucleus and oogonium, and a small number of hybridization signals were found in the follicular wall of the male gonads. In addition, the C-Mos RNA interference (RNAi) assay results showed that the knockdown of C-Mos caused a down-regulation of ERK and P90rsk. In summary, these results indicate that C-Mos has a crucial part to play in gonadal development in H. cumingii.

Gonad Ontogeny and Sex Differentiation in a Poeciliid, Gambusia holbrooki: Transition from a Bi- to a Mono-Lobed Organ.

Despite their uniqueness, the ontogeny and differentiation of the single-lobed gonads in the poeciliids are very poorly understood. To address this, we employed both cellular and molecular approaches to systematically map the development of the testes and ovary in Gambusia holbrooki from pre-parturition to adulthood, encompassing well over 19 developmental stages. The results show that putative gonads form prior to the completion of somitogenesis in this species, a comparatively early occurrence among teleosts. Remarkably, the species recapitulates the typical bi-lobed origin of the gonads during early development that later undergoes steric metamorphosis to form a single-lobed organ. Thereafter, the germ cells undergo mitotic proliferation in a sex-dependent manner before the acquisition of the sexual phenotype. The differentiation of the ovary preceded that of the testes, which occurred before parturition, where the genetic females developed meiotic primary oocytes stage I, indicating ovarian differentiation. However, genetic males showed gonial stem cells in nests with slow mitotic proliferation at the same developmental stage. Indeed, the first signs of male differentiation were obvious only post-parturition. The expression pattern of the gonadosoma markers foxl2, cyp19a1a, amh and dmrt1 in pre- and post-natal developmental stages were consistent with morphological changes in early gonad; they were activated during embryogenesis, followed by the onset of gonad formation, and a sex-dimorphic expression pattern concurrent with sex differentiation of the ovary (foxl2, cyp19a1a) and testes (amh and dmrt1). In conclusion, this study documents for the first time the underlying events of gonad formation in G. holbrooki and shows that this occurs relatively earlier than those previously described for ovi- and viviparous fish species, which may contribute to its reproductive and invasive prowess.

De Novo Transcriptome Analysis of the Early Hybrid Triploid Loach (Misgurnus anguillicaudatus) Provides Novel Insights into Fertility Mechanism

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.

Declines in Reproductive Condition of Male Largemouth Bass (Micropterus salmoides) Following Seasonal Exposure to Estrogenic Endocrine-Disrupting Compounds.

Reproductive abnormalities, that could lead to possible effects at the population level, have been observed in wild fish throughout the United States, with high prevalence in largemouth bass (LMB; Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Estrone (E1) and atrazine (ATR) are common environmental contaminants often associated with agricultural land use. 17alpha-ethinylestradiol (EE2) is a contaminant associated with wastewater treatment effluent, and a representative, well-studied estrogen commonly used for fish toxicity testing. Our objective was to assess whether early gonad recrudescence in adult fish was a period of sensitivity for alterations in reproductive condition and function. Adult male LMB were exposed from post-spawning to early gonad recrudescence to either a mixture of E1 (47.9 ng/L) + ATR (5.4 µg/L), or EE2 (2.4 ng/L) in outdoor experimental ponds. Gonad samples were collected from fish just prior to the start of exposure (July), at the end of the exposure period (December), the following spring just prior to spawning (April), and post spawning (May). Gonadosomatic index (GSI) was significantly reduced in E1 + ATR-exposed and EE2-exposed males compared to control at every post-exposure time point. Reduced sperm count and sperm motility were observed in the mixture treatment (E1 + ATR) compared to the control. Sperm motility was also reduced in the EE2 treatment. These data together indicate that estrogenic endocrine-disrupting compounds can lessen the reproductive condition of adult male LMB, and that effects of exposure during early gonad recrudescence can persist at least through the subsequent spawning cycle.

Integration of single-cell transcriptome and chromatin accessibility of early gonads development among goats, pigs, macaques, and humans.

The early gonads of mammals contain primordial germ cells (PGCs) and somatic cell precursors that are essential for sex determination and gametogenesis. Although it is extensively documented in mice, the development of early gonads in non-rodents remains to be delineated. Because molecular differences between mouse and human gonadal cells have been reported, it warrants the study of the key markers and regulatory features that are conserved or divergent between non-rodent species and human. Here, we integrate single-cell transcriptome and chromatin accessibility analysis to identify regulatory signatures of PGCs and somatic cells in the early gonads of goats, pigs, macaques, and humans. We identify the evolutionarily conserved and species-specific events, including genes expression, signaling pathways, and cell-cell interactions. We also uncover potential cis-regulatory elements and key transcription factors in PGCs and somatic cells. Our datasets provide important resources for better understanding the evolutionary programs of PGCs and gonadal somatic cell development in mammals.

PMON275 GONAD-on-CHIP to study early gonad development and DSD

Abstract During embryonic development, mutually antagonistic signaling cascades determine the fate of the bipotential gonad towards a testicular or ovarian identity. Errors in this process result in human Disorders of Sex Development (DSDs), where there is discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in-vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells towards gonadal progenitors. Transcriptomic analysis reveals that the in-vitro-derived murine gonadal cells are equivalent to E11.5 in-vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete AMH, migrate and form 3D tubular structures on a specially designed microfluidic device. The cells derived from a 46,XY DSD female hiPSCs, carrying a NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR/Cas9-mediated correction of the variant rescued the phenotype. This is a robust tool to understand mechanisms of sex-determination and model DSDs. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Metzincin metalloproteases in PGC migration and gonadal sex conversion

Development of a functional gonad includes migration of primordial germ cells (PGCs), differentiations of somatic and germ cells, formation of primary follicles or spermatogenic cysts with somatic gonadal cells, development and maturation of gametes, and subsequent releasing of mature germ cells. These processes require extensive cellular and tissue remodeling, as well as broad alterations of the surrounding extracellular matrix (ECM). Metalloproteases, including MMPs (matrix metalloproteases), ADAMs (a disintegrin and metalloproteinases), and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), are suggested to have critical roles in the remodeling of the ECM during gonad development. However, few research articles and reviews are available on the functions and mechanisms of metalloproteases in remodeling gonadal ECM, gonadal development, or gonadal differentiation. Moreover, most studies focused on the roles of transcription and growth factors in early gonad development and primary sex determination, leaving a significant knowledge gap on how differentially expressed metalloproteases exert effects on the ECM, cell migration, development, and survival of germ cells during the development and differentiation of ovaries or testes. We will review gonad development with focus on the evidence of metalloprotease involvements, and with an emphasis on zebrafish as a model for studying gonadal sex differentiation and metalloprotease functions.

Deletion of Wt1 during early gonadogenesis leads to differences of sex development in male and female adult mice.

Assessing the role of the WT1 transcription factor (WT1) during early gonad differentiation and its impact on adult sex development has been difficult due to the complete gonadal agenesis and embryonic lethality exhibited by Wt1KO mouse models. Here, we generated Wt1LoxP/GFP;Wt1Cre mice, the first Wt1KO mouse model that reaches adulthood with a dramatically reduced Wt1 expression during early gonadogenesis. Wt1LoxP/GFP;Wt1Cre mice lacked mature gonads and displayed genital tracts containing both male and female genital structures and ambiguous genitalia. We found that WT1 is necessary for the activation of both male and female sex-determining pathways, as embryonic mutant gonads failed to upregulate the expression of the genes specific for each genetic programme. The gonads of Wt1LoxP/GFP;Wt1Cre mice showed a lack of production of Sertoli and pre-granulosa cells and a reduced number of germ cells. NR5A1 and the steroidogenic genes expression was modulated differently in XY and XX Wt1LoxP/GFP;Wt1Cre gonads, explaining the mutant phenotypes. Further studies of the XX Wt1LoxP/GFP;Wt1Cre gonads revealed that deletion of WT1 at an early stage impaired the differentiation of several cell types including somatic cells and the ovarian epithelium. Through the characterisation of this Wt1KO mouse model, we show that the deletion of Wt1 during early gonadogenesis produces dramatic defects in adult sex development.

A Shuttling Protein Kank1 Plays Important Role in the Sex Determination in Yellow Drum

Sex is one of the most important scientific topics, and the existence of sex dimorphism is a conserved feature in vertebrate taxa. The research on sex-determining genes and sex chromosome evolution is a hot topic in biology. The majority of sex-determining genes expressed in somatic cells, and reciprocal interactions between germ cells and somatic cells, are important for gonadal differentiation. However, the knowledge of how signals are transmitted between somatic cells and germ cells remains unclear. In this study, we confirmed the 36 dph was a sex determination critical stage in yellow drum through transcriptome analysis at the early development stages. We further found that Kank1 participated in the germ cells’ motility process in yellow drum through KEGG pathway analysis together with protein-protein interaction network. With the dual-luciferase reporter assay, we detected that Kank1 increased the transcription of dmrt1_Luc gene in CHO cells and 293T cells. Additionally, we observed that Kank1 was not only expressed in the cytoplasm but also in the nucleus in CHO cells through a fluorescence microscope. These results suggest that Kank1 acts as a shuttling protein as in humans and may play a difunctional role at the early gonad development stage in yellow drum, and it not only participated in the germ cells’ motility but also increased the expression of dmrt1 in yellow drum.

Fetal exposure of Aristolochic Acid I undermines ovarian reserve by disturbing primordial folliculogenesis

The primordial follicle pool established in early life determines the ovarian reserve in the female reproductive lifespan. Premature exhaustion of primordial follicles contributes to primary ovarian insufficiency (POI), that is dependent by the initial size of the primordial follicle pool and by the rate of its activation and depletion. AAI, a powerful nephrotoxin with carcinogenic potential, is present in the Aristolochiaceae species, which can release AAI into soil as a persistent pollutant. In order to assess the potential risk of Aristolochic Acid I (AAI) exposure on mammalian oogenesis, we uncovered its adverse effect on primordial folliculogenesis in the neonatal mouse ovary and its effect on female fertility in adulthood. Pregnant mice were orally administrated with doses of AAI without hepatic or renal toxicity during late-gestation. Ovaries from offspring of administered female displayed gross aberrations during primordial folliculogenesis. Also, unenclosed oocytes in germ-cell cysts showed increased DNA damage. Furthermore, several key factors, including NANOS3, SOX9, KLF4, that govern early gonad’s differentiation were abnormally expressed in the exposed ovary, while the follicle formation was partially restored by knockdown of Nanos3 or sox9. In adulthood, these aberrations evolved into a significant reduction in offspring number and impaired ovarian reserve. Together, our results show that AAI influences primordial folliculogenesis and, importantly, affected female fertility. This study shows that administration of drugs herbs or consumption of vegetables that contain AAs during pregnancy may adversely influence the fertility of offspring.

Gadd45g is required for timely Sry expression independently of RSPO1 activity.

Sex determination in mammals is controlled by the dominance of either pro-testis (SRY-SOX9-FGF9) or pro-ovary (RSPO1-WNT4-FOXL2) genetic pathways during early gonad development in XY and XX embryos, respectively. We have previously shown that early, robust expression of mouse Sry is dependent on the nuclear protein GADD45g. In the absence of GADD45g, XY gonadal sex reversal occurs, associated with a major reduction of Sry levels at 11.5 dpc. Here, we probe the relationship between Gadd45g and Sry further, using gain- and loss-of-function genetics. First, we show that transgenic Gadd45g overexpression can elevate Sry expression levels at 11.5 dpc in the B6.YPOS model of sex reversal, resulting in phenotypic rescue. We then show that the zygosity of pro-ovarian Rspo1 is critical for the degree of gonadal sex reversal observed in both B6.YPOS and Gadd45g-deficient XY gonads, in contrast to that of Foxl2. Phenotypic rescue of sex reversal is observed in XY gonads lacking both Gadd45g and Rspo1, but this is not associated with rescue of Sry expression levels at 11.5 dpc. Instead, Sox9 levels are rescued by around 12.5 dpc. We conclude that Gadd45g is absolutely required for timely expression of Sry in XY gonads, independently of RSPO1-mediated WNT signalling, and discuss these data in light of our understanding of antagonistic interactions between the pro-testis and pro-ovary pathways.

Development and gene expression analysis of gonad during 17α-methyltestosterone-induced sex reversal in mandarin fish (Siniperca chuatsi)

Mandarin fish (Siniperca chuatsi) is one of the most important commercial fish in aquaculture in the world. As females grow faster than males, the cultivation of all-female mandarin fish has great economic value and commercial prospects. However, little is known about the molecular mechanism of the early gonad development and sex reversal of mandarin fish. In this study, histological methods and transcriptome sequencing techniques were used to study the early gonadal development and gene expression pattern of genetically female (XX) mandarin fish during MT-induced masculinization. The histological results showed that the labile period of sex differentiation of XX mandarin fish was before 36 dpf and MT treatment before this period could induce complete sex reversal. The transcriptome results showed that the expression levels of a few female-related genes were down-regulated by MT during the morphological differentiation stage to cytological differentiation stage of XX gonad, such as cyp19a1a, foxl2, foxl3, hsd17b1, figla, gdf9, bmp15 and β-catenin, suggesting that the changes of these genes may determine the direction of sex differentiation in mandarin fish. In addition, MT also activated the expression of some male-related genes, such as dmrt1, hsd17b3, hsd11b2, cyp11a1, cyp11b, amh, amhr2, sox9 and gsdf. Some of these genes were activated during spermatogenesis stage, suggesting that the up-regulation of these male-related genes may be the result of sex differentiation rather than the cause. The qRT-PCR results of eight randomly selected differentially expressed genes were basically consistent with the transcriptome results, which proved that the transcriptome data were reliable. These results indicated that the expression of genes related to estrogen synthesis in early development stage may play a decisive role in the direction of sex differentiation in mandarin fish.

Polyubiquitin gene Ubb is required for upregulation of Piwi protein level during mouse testis development

Testis development, including early embryonic gonad formation and late postnatal spermatogenesis, is essential for the reproduction of higher metazoans to generate fertile gametes, called sperm. We have previously reported that the polyubiquitin gene Ubb is required for fertility in both male and female mice. In particular, the Ubb-null male mice showed an azoospermia phenotype due to arrest of spermatogenesis at the pachytene stage. Here, we analyzed the whole testis proteome at postnatal day 20 to define the molecular mediators of the male-infertility phenotype caused by Ubb knockout. From the identified proteome, 564 proteins were significantly and differentially expressed in Ubb-knockout testes and, among these, 36 downregulated proteins were involved at different stages of spermatogenesis. We also found that levels of piRNA metabolic process-related proteins, including Piwil2 and Tdrd1, were downregulated in Ubb-null testes through functional gene ontology analysis. Further, protein–protein interaction mapping revealed that 24 testis development-related proteins, including Hsp90aa1, Eef1a1, and Pabpc1, were directly influenced by the depletion of ubiquitin. In addition, the reduced mRNA levels of these proteins were observed in Ubb-knockout testes, which closely resembled the global downregulation of piRNA-metabolic gene expression at the transcriptional and post-transcriptional levels. Together with proteomic and transcriptional analyses, our data suggest that Ubb expression is essential for the maintenance of testicular RNA-binding regulators and piRNA-metabolic proteins to complete spermatogenesis in mice.