Ovarian Differentiation Research Articles

Evaluation of a convolutional neural network for ovarian tumor differentiation based on magnetic resonance imaging.

There currently lacks a noninvasive and accurate method to distinguish benign and malignant ovarian lesion prior to treatment. This study developed a deep learning algorithm that distinguishes benign from malignant ovarian lesion by applying a convolutional neural network on routine MR imaging. Five hundred forty-five lesions (379 benign and 166 malignant) from 451 patients from a single institution were divided into training, validation, and testing set in a 7:2:1 ratio. Model performance was compared with four junior and three senior radiologists on the test set. Compared with junior radiologists averaged, the final ensemble model combining MR imaging and clinical variables had a higher test accuracy (0.87 vs 0.64, p < 0.001) and specificity (0.92 vs 0.64, p < 0.001) with comparable sensitivity (0.75 vs 0.63, p = 0.407). Against the senior radiologists averaged, the final ensemble model also had a higher test accuracy (0.87 vs 0.74, p = 0.033) and specificity (0.92 vs 0.70, p < 0.001) with comparable sensitivity (0.75 vs 0.83, p = 0.557). Assisted by the model's probabilities, the junior radiologists achieved a higher average test accuracy (0.77 vs 0.64, Δ = 0.13, p < 0.001) and specificity (0.81 vs 0.64, Δ = 0.17, p < 0.001) with unchanged sensitivity (0.69 vs 0.63, Δ = 0.06, p = 0.302). With the AI probabilities, the junior radiologists had higher specificity (0.81 vs 0.70, Δ = 0.11, p = 0.005) but similar accuracy (0.77 vs 0.74, Δ = 0.03, p = 0.409) and sensitivity (0.69 vs 0.83, Δ = -0.146, p = 0.097) when compared with the senior radiologists. These results demonstrate that artificial intelligence based on deep learning can assist radiologists in assessing the nature of ovarian lesions and improve their performance. • Artificial Intelligence based on deep learning can assess the nature of ovarian lesions on routine MRI with higher accuracy and specificity than radiologists. • Assisted by the deep learning model's probabilities, junior radiologists achieved better performance that matched those of senior radiologists.

Mammalian gonadal differentiation: the pig model

In mammals, testicular differentiation is initiated by SRY (the sex-determining region of the Y chromosome) gene expression in Sertoli cell precursors, followed by upregulation of the SOX9 gene (SRY-related HMG box gene 9). Subsequently, differentiated testis produces two hormones that induce sexual differentiation of the internal and external genital tract. Knowledge of the molecular mechanisms involved in gonadal differentiation has increased greatly over the past decade. Several genes are involved in genital ridge formation in both sexes, and others act specifically in testicular or ovarian developmental pathways. As for other mammals, relatively few data are available on the first steps of ovarian differentiation in pigs. In this review, the expression profiles of most genes known to be involved in gonadal differentiation in pigs will be presented and compared with those observed in mice. The main feature of gonadal differentiation in the pig is fetal steroidogenesis, especially cytochrome P450 aromatase gene organization and expression. Another specific feature of gonadal differentiation in pigs is the appearance of numerous cases of XX sex-reversed animals. This intersex condition occurs as early as day 50 after coitus, during embryogenesis, and appears to be triggered genetically. It leads to a wide range of phenotypes, strikingly similar to those observed in humans. Identification of the genes involved in this pathology will improve our knowledge of mammalian gonadal differentiation and may allow the eradication of this genetic disease in pigs.

Detrimental effect of Bisphenol S in mouse germ cell cyst breakdown and primordial follicle assembly

The female reproductive lifespan is largely determined by the size of primordial follicle pool, which is established in early life. Bisphenol S (BPS), frequently present in plastic products used in daily life, has been demonstrated as an exogenous estrogen-like endocrine disrupting chemical interfering with the endocrine and reproductive systems. However, the molecular mechanisms of its reproductive toxicity remain to be determined. In the present study, we focused on the effect of BPS on the early ovarian folliculogenesis of mice. Our in vivo experiments showed that the treatment with BPS at 2 and 10 μg/kg body weight/day for 3 days induced abnormal germ cell cyst breakdown and primordial follicle assembly in the mouse ovary, further affecting later ovarian differentiation and reducing oocyte quality. In addition, our in vitro study demonstrated that BPS could interact with estrogen receptors (ERs) to induce phosphorylation of JNKs, which is responsible for reducing oocyte adhesion in cysts. Meanwhile, BPS exposure up-regulated Notch signaling pathway to increase the proliferation of granulosa cells precursors. Our study provided new evidence for the adverse effects of BPS on female reproduction, especially after perinatal exposure, and elucidated how it works.

Molecular and morphological sex differentiation in sablefish (Anoplopoma fimbria), a marine teleost with XX/XY sex determination.

Phenotypic sex of an organism is determined by molecular changes in the gonads, so-called molecular sex differentiation, which should precede the rise of cellular or anatomical sex-distinguishing features. This study characterized molecular and morphological sex differentiation in sablefish (Anoplopoma fimbria), a marine teleost with established XX/XY genotypic sex determination. Next generation sequencing was conducted on sablefish ovarian and testicular mRNAs to obtain sequences for transcripts associated with vertebrate sex determination and differentiation and early reproductive development. Gene-specific PCRs were developed to determine the distribution and ontogenetic gonadal expression of transcription, growth, steroidogenic and germline factors, as well as gonadotropin and steroid receptors. Molecular changes associated with sex differentiation were first apparent in both XY- and XX-genotype sablefish at~60mm in body length and prior to histological signs of sex differentiation. The earliest and most robust markers of testicular differentiation were gsdf, amh, dmrt1, cyp11b, star, sox9a, and fshr. Markedly elevated mRNA levels of several steroidogenesis-related genes and ar2 in differentiating testes suggested that androgens play a role in sablefish testicular differentiation. The earliest markers of ovarian differentiation were cyp19a1a, lhcgr, foxl2, nr0b1, and igf3. Other transcripts such as figla, zp3, and pou5f3 were expressed predominantly in XX-genotype fish and significantly increased with the first appearance and subsequent development of primary oocytes. This study provides valuable insight to the developmental sequence of events associated with gonadal sex differentiation in marine teleosts with XX/XY sex determination. It also implicates particular genes in processes of male and female development and establishes robust molecular markers for phenotypic sex in sablefish, useful for ongoing work related to sex control and reproductive sterilization.

Using naturalistic incubation temperatures to demonstrate how variation in the timing and continuity of heat wave exposure influences phenotype.

Most organisms are exposed to bouts of warm temperatures during development, yet we know little about how variation in the timing and continuity of heat exposure influences biological processes. If heat waves increase in frequency and duration as predicted, it is necessary to understand how these bouts could affect thermally sensitive species, including reptiles with temperature-dependent sex determination (TSD). In a multi-year study using fluctuating temperatures, we exposed Trachemys scripta embryos to cooler, male-producing temperatures interspersed with warmer, female-producing temperatures (heat waves) that varied in either timing during development or continuity and then analysed resulting sex ratios. We also quantified the expression of genes involved in testis differentiation (Dmrt1) and ovary differentiation (Cyp19A1) to determine how heat wave continuity affects the expression of genes involved in sexual differentiation. Heat waves applied during the middle of development produced significantly more females compared to heat waves that occurred just 7 days before or after this window, and even short gaps in the continuity of a heat wave decreased the production of females. Continuous heat exposure resulted in increased Cyp19A1 expression while discontinuous heat exposure failed to increase expression in either gene over a similar time course. We report that even small differences in the timing and continuity of heat waves can result in drastically different phenotypic outcomes. This strong effect of temperature occurred despite the fact that embryos were exposed to the same number of warm days during a short period of time, which highlights the need to study temperature effects under more ecologically relevant conditions where temperatures may be elevated for only a few days at a time. In the face of a changing climate, the finding that subtle shifts in temperature exposure result in substantial effects on embryonic development becomes even more critical.

Exposure to environmentally-relevant concentrations of hexavalent chromium does not induce ovarian toxicity in mice

Exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water (≥250 ppm) is reported to decrease ovarian follicle counts and increase follicular atresia in mice. To assess effects at lower concentrations, herein we exposed B6C3F1 mice to 0.1–150 ppm Cr(VI) in drinking water for 90 days in a GLP-compliant study. Ovarian follicular counts, differentiation, and degeneration were assessed from every 10th serial section (up to 14 sections per ovary). Ovarian follicular counts, differentiation, and rate of atresia were not altered in any exposure group. Gross and microscopic changes were not apparent in any of the evaluated reproductive or glandular organs. The no observable adverse effect level (NOAEL) for follicular effects was 150 ppm. In addition to these findings, published Cr(VI) studies examining follicles were scored using two methods for assessing study quality for use in risk assessment—including the Toxic Substance Control Act (TSCA) scoring method. Both methods revealed that studies reporting adverse effects on follicles generally received low scores. Overall, the current study indicates no/low potential for Cr(VI) to induce follicular toxicity in mice below 150 ppm Cr(VI) in drinking water (17.7 mg/kg bodyweight).

Transcriptomic analysis identifies early cellular and molecular events by which estrogen disrupts testis differentiation and causes feminization in Xenopus laevis

Extensive studies have shown that estrogenic endocrine-disrupting chemicals (EDCs) can disrupt testis differentiation and even cause feminization in vertebrates. However, little is known about the mechanisms by which estrogenic EDCs disrupt testis differentiation. Here, we employed Xenopus laevis, a model amphibian species sensitive to estrogenic EDCs, to explore the molecular and cellular events by which 17β-estradiol (E2) disrupts testis differentiation and causes feminization. Following waterborne exposure to E2 from stage 45/46, genetically male X. laevis were confirmed to undergo testis differentiation inhibition and ovary differentiation activation at stages 52 and 53, ultimately displaying gonadal feminization at stage 66. Using a time-course RNA sequencing approach, we then identified thousands of differentially expressed transcripts (DETs) in genetically male gonad-mesonephros complexes at stages 48, 50 and 52 (the window for testis differentiation) between E2 treatment and the control. Enrichment analysis suggests alterations in cell proliferation, extracellular matrix, and cell motility following E2 exposure. Further verification by multiple methods demonstrated that E2 inhibited cell proliferation, disrupted extracellular matrix, and altered cell motility in the genetically male gonads compared with controls, implying that these events together contributed to testis differentiation disruptions and feminization in X. laevis. This study for the first time uncovered some of the early molecular and cellular events by which estrogen disrupts testicular differentiation and causes feminization in X. laevis. These new findings improve our understanding of the mechanisms by which estrogenic EDCs disrupt testicular differentiation in vertebrates.

Reproductive development of the priapium fish Neosthetus lankesteri Regan, 1916 (Atheriniformes: Phallosthethidae) from Pranburi river estuary, Thailand using the histological approach

Reproduction in the family Phallostethidae is an area that has long been of interest especially in gonadal structure and gametogenesis. However, no published data concerning as mentioned above in Neostethus lankesteri Regan, 1916 from Thailand is noted. This fish is important for rural livelihood in the estuarine fisheries of Pranburi River estuary, Thailand. The construction of the female reproductive system of N. lankesteri composed of the ovary and short oviduct. The mature ovarian morphology is a single organ, which is histologically considered to be an asynchronous development ovary. The differentiating oocytes were observed and classified into oogonia proliferation, primary growth phase, secondary growth phase, post-ovulatory phase and atretic follicle phase. Under the ovarian differentiation, our observation indicated that the ovarian parenchyma of N. lankesteri during 1.8 – 2.4 cm in standard length (SL) was accompanied by the secondary growth phases. Identification of the post-ovulatory phase was detected during our sampling periods, indicating to entire life in the Pranburi River estuary as well as true resident species. In the male reproductive system of N. lankesteri, the mature testicular parenchyma was organized morphologically as a single organ and histologically appeared to be a restricted spermatogonial lobular testis type. The differentiation of the spermatogenetic stage was composed of three phases including spermatogonial, spermatocyte and spermiogenetic phases. It was to identify possible trends that the size at first maturity of the female fish was estimated at 1.8 cm SL. Certainly, results from this study clearly showed on the reproductive pattern of N. lankesteri, which could be applied to further studies (the reproductive cycle, spawning season and histopathology).

Transcriptome analysis of genes related to gonad differentiation and development in Muscovy ducks

BackgroundSex-related genes play a crucial role in gonadal differentiation into testes or ovaries. However, the genetic control of gonadal differentiation in Muscovy ducks remains unknown. Therefore, the objective of our study was to screen new candidate genes associated with ovarian and testicular development.ResultsIn this study, 24 males before gonadal differentiation (MB), 24 females before gonadal differentiation (FB), 24 males after gonadal differentiation (MA) and 24 females after gonadal differentiation (FA) were selected from Putian Muscovy ducks, forming 4 groups. RNA-Seq revealed 101.76 Gb of clean reads and 2800 differentially expressed genes (DEGs), including 46 in MB vs FB, 609 in MA vs FA, 1027 in FA vs FB, and 1118 in MA vs MB. A total of 146 signalling pathways were enriched by KEGG analysis, among which 20, 108, 108 and 116 signalling pathways were obtained in MB vs FB, MA vs MB, MA vs FA and FA vs FB, respectively. In further GO and KEGG analyses, a total of 21 candidate genes related to gonad differentiation and development in Muscovy ducks were screened. Among these, 9 genes were involved in the differentiation and development of the testes, and 12 genes were involved in the differentiation and development of the ovaries. In addition, RNA-Seq data revealed 2744 novel genes.ConclusionsRNA-Seq data revealed 21 genes related to gonadal differentiation and development in Muscovy ducks. We further identified 12 genes, namely, WNT5B, HTRA3, RSPO3, BMP3, HNRNPK, NIPBL, CREB3L4, DKK3, UBE2R2, UBPL3KCMF1, ANXA2, and OSR1, involved in the differentiation and development of ovaries. Moreover, 9 genes, namely, TTN, ATP5A1, DMRT1, DMRT3, AMH, MAP3K1, PIK3R1, AGT and ADAMTSL1, were related to the differentiation and development of testes. Moreover, after gonadal differentiation, DMRT3, AMH, PIK3R1, ADAMTSL1, AGT and TTN were specifically highly expressed in males. WNT5B, ANXA2 and OSR1 were specifically highly expressed in females. These results provide valuable information for studies on the sex control of Muscovy ducks and reveal novel candidate genes for the differentiation and development of testes and ovaries.

Sox8 and Sox9 act redundantly for ovarian-to-testicular fate reprogramming in the absence of R-spondin1 in mouse sex reversals.

In mammals, testicular differentiation is initiated by transcription factors SRY and SOX9 in XY gonads, and ovarian differentiation involves R-spondin1 (RSPO1) mediated activation of WNT/β-catenin signaling in XX gonads. Accordingly, the absence of RSPO1/Rspo1 in XX humans and mice leads to testicular differentiation and female-to-male sex reversal in a manner that does not requireSry or Sox9 in mice. Here we show that an alternate testis-differentiating factor exists and that this factor is Sox8. Specifically, genetic ablation of Sox8 and Sox9 prevents ovarian-to-testicular reprogramming observed in XX Rspo1 loss-of-function mice. Consequently, Rspo1 Sox8 Sox9 triple mutant gonads developed as atrophied ovaries. Thus, SOX8 alone can compensate for the loss of SOX9 for Sertoli cell differentiation during female-to-male sex reversal.

Figla gene roles in the proliferation of oocytes in the olive flounder Paralichthys olivaceus

Factor in the germ line alpha (a basic helix-loop-helix transcription factor, FIGLA) gene specifically expresses in female germ cells and is essential for ovarian differentiation and development in vertebrates. However, research on the roles in fish gonadal differentiation and development is limited. In this study, we cloned and characterized a figla gene from the olive flounder, Paralichthys olivaceus, an important marine cultured fish in China, Japan, and Korea. The full-length cDNA of the Po-figla is 1316 bp along with a 609 bp open reading frame. Real-time quantitative polymerase chain reaction analysis (qPCR) revealed that it was abundantly expressed in the ovary and female brain. During stages I-V of the ovary, the level of Po-figla mRNA reached the peak at stage II, and then gradually decreased, while Po-figla mRNA was slightly expressed along with the testicular development. Further, one color and two-color in situ hybridization results showed that the gene was mainly located in oocytes. Knocking down Po-figla in the ovary using RNAi led to its down-regulated expression and the reduction of oocyte number. In addition, qPCR results presented the differential expression patterns of Po-figla during gonadal differentiation period. In the exogenous 17β-estradiol treatment group (5 ppm), Po-figla expression levels constitutively increased in the differentiating and differentiated ovaries, while in the testosterone (5 ppm) treatment group, it was lower expressed in the testis during testicular differentiation period. Our findings provide evidence that Po-figla is involved in P. olivaceus ovarian differentiation and development, especially might have roles in the proliferation of fish oocytes. These findings provide the basic data for fish reproduction and gonadal development regulation in aquaculture.

Comparison and analysis of lincRNAs expression profile in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows

The normal estrus in weaned primiparous sows has a great impact on pig production and abnormal estrus is the main reason for the elimination of primiparous sows. In this study, we studied the long intergenic noncoding RNAs (lincRNAs) in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows. These long intergenic noncoding RNAs (lincRNAs) were screened and compared through RNA-seq analysis. The expression profiles of lincRNAs were obtained and their characteristics and functions were preliminarily analyzed. There are 3519 novel lincRNAs identified in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows. Compared with estrous primiparous sows, 17 differentially expressed lincRNAs were indentified, including 12 up-regulated lincRNAs and 5 down-regulated lincRNAs (FC≥2, P<0.05). The four lincRNA transcripts obtained through selection were verified by qRT-PCR, which are consistent with the RNA-seq results. The GO, KEGG pathway, and lincRNA-mRNA co-expression network analysis of these 17 lincRNAs revealed that these lincRNAs were mainly involved in reproductive activities, such as oocyte meiosis mature, ovarian cells differentiation and granulosa cells apoptosis. The results enriched the data resources of pig lincRNAs and provided useful information for further research about the reproductive performance of primiparous sows.

Transcriptomic analysis of female and male gonads in juvenile snakeskin gourami (Trichopodus pectoralis)

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 < 0.001) between the results of RNA-seq and qRT-PCR. Therefore, RNA-seq analysis in our study identified global genes that were associated with ovary and testis differentiation in the juvenile phase of the snakeskin gourami. Our findings provide valuable transcriptomic bioinformation for further investigation of reproductive biology and applications of sex manipulation.

Morphological characterization of pirarucu Arapaima gigas (Schinz, 1822) gonadal differentiation.

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.

Spatial and temporal expression pattern of sex-related genes in ovo-testis of the self-fertilizing mangrove killifish (Kryptolebias marmoratus).

In vertebrates, sex determination and differentiation comprehend a fine balance between female and male factors, leading the bipotential anlage to develop towards ovary or testis, respectively. Nevertheless, the mangrove killifish, (Kryptolebias marmoratus) a simultaneous hermaphroditic species, could overcome those antagonistic pathways and evolved to develop and maintain reproductively active ovarian and testicular tissues in the same organ. Morphological and mRNA localization analyzes of developing and adult gonads demonstrate that genes related to testis (dmrt1 and amh) and ovary differentiation (foxl2 and sox9a) follow the same expression pattern observed in gonochoristic species, thus functioning as two independent organs. In addition, Amh expression patterns make it a strong candidate for initiation of the formation and maintenance of the testicular tissue in the hermaphroditic gonad. Differently from described so far, foxl3 seems to have an important role in oogenesis as well as spermatogenesis and gonadal structure.

FOXL2 is a Progesterone Target Gene in the Endometrium of Ruminants.

Forkhead Box L2 (FOXL2) is a member of the FOXL class of transcription factors, which are essential for ovarian differentiation and function. In the endometrium, FOXL2 is also thought to be important in cattle; however, it is not clear how its expression is regulated. The maternal recognition of pregnancy signal in cattle, interferon-Tau, does not regulate FOXL2 expression. Therefore, in the present study, we examined whether the ovarian steroid hormones that orchestrate implantation regulate FOXL2 gene expression in ruminants. In sheep, we confirmed that FOXL2 mRNA and protein was expressed in the endometrium across the oestrous cycle (day 4 to day 15 post-oestrus). Similar to the bovine endometrium, ovine FOXL2 endometrial expression was low during the luteal phase of the oestrous cycle (4 to 12 days post-oestrus) and at implantation (15 days post-oestrus) while mRNA and protein expression significantly increased during the luteolytic phase (day 15 post-oestrus in cycle). In pregnant ewes, inhibition of progesterone production by trilostane during the day 5 to 16 period prevented the rise in progesterone concentrations and led to a significant increase of FOXL2 expression in caruncles compared with the control group (1.4-fold, p < 0.05). Ovariectomized ewes or cows that were supplemented with exogenous progesterone for 12 days or 6 days, respectively, had lower endometrial FOXL2 expression compared with control ovariectomized females (sheep, mRNA, 1.8-fold; protein, 2.4-fold; cattle; mRNA, 2.2-fold; p < 0.05). Exogenous oestradiol treatments for 12 days in sheep or 2 days in cattle did not affect FOXL2 endometrial expression compared with control ovariectomized females, except at the protein level in both endometrial areas in the sheep. Moreover, treating bovine endometrial explants with exogenous progesterone for 48h reduced FOXL2 expression. Using in vitro assays with COS7 cells we also demonstrated that progesterone regulates the FOXL2 promoter activity through the progesterone receptor. Collectively, our findings imply that endometrial FOXL2 is, as a direct target of progesterone, involved in early pregnancy and implantation.

Phenotypic traits and gonadal development of hybrids between Misgurnus anguillicaudatus and Paramisgurnus dabryanus

Misgurnus anguillicaudatus and Paramisgurnus dabryanus are two loach species widely sympatrically distributed in China. Hybrids of the two species have been detected in the wild and produced by artificial hybridization experiments, but their sexual development has not been studied. In this study, the phenotypic traits of purebred and hybrid groups were evaluated in the aspects of survival, growth, gonad development and sex-related genes (dmrt1a, dmrt1b, cyp19a1a and cyp19a1b) expression. Hybrids and purebreds showed the same high levels of fertilization, hatching success and early survival. Growth of hybrids was intermediate between those of parental species, showing hybrid heterosis compared to M. anguillicaudatus. Cyp19a1a and dmrt1s were expressed in the ovary and testis but expression levels were sexually dimorphic. Although the exact timing of dmrt1s and cyp19a1a expression during the gonad development varied between groups, in general their expressions were correlated with the differentiation and formation of testes and ovaries respectively. Histological analysis showed that in purebreds ovaries differentiate/form much earlier than testes: 15/35 and 35/80 days after hatching (dah) respectively. In hybrids, however, both differentiation and formation are simultaneous: 35 and 80 dah respectively. Adult hybrids possessed mature gametes, but they appeared to exhibit weaker reproductive ability than either of the parental species. Comprehensive gene expression and histological analysis demonstrate that gonad development in hybrids is retarded than in parental species.

Expression of genes involved in neurogenesis, and neuronal precursor cell proliferation and development: Novel pathways of human ovarian granulosa cell differentiation and transdifferentiation capability invitro.

The process of neural tissue formation is associated primarily with the course of neurogenesis during embryonic life. The source of neural-like cells is stem cells, which, under the influence of appropriate differentiating factors, may differentiate/transdifferentiate towards a neural-like lineage. The present study suggested that, under long-term in vitro culture conditions, human ovarian granulosa cells (GCs), obtained from granulosa-rich follicular fluid, acquired new properties and expressed genes characteristic of the ontological groups ‘neurogenesis’ (GO:0022008), ‘neuronal precursor cell proliferation’ (GO:0061351) and ‘nervous system development’ (GO:0007399), which are closely related to the formation of neurons. The present study collected GCs from 20 women referred for the procedure of in vitro fertilization. Cells were maintained in long-term in vitro culture for 30 days, and RNA was isolated after 1, 7, 15 and 30 days of culture. The expression profile of individual genes was determined using the Affymetrix microarray method. The 131 genes with the highest expression change in relation to day 1 of culture were then selected; the 10 most affected genes found to be primarily involved in nerve cell formation processes were chosen for consideration in this study: CLDN11, OXTR, DFNA5, ATP8B1, ITGA3, CD9, FRY, NANOS1, CRIM1 and NTN4. The results of the present study revealed that these genes may be considered potential markers of the uninduced differentiation potential of GCs. In addition, it was suggested that GCs may be used to develop a cell line showing neuronal characteristics after 30 days of cultivation. In addition, due to their potential, these cells could possibly be used in the treatment of neurodegenerative diseases, not only in the form of ‘cultured neurons’ but also as producers of factors involved in the regeneration of the nervous system.

Common carp pentraxin gene: Evidence for its role in ovarian differentiation and growth

Pentraxins (PTX), belong to an evolutionarily conserved family, containing a PTX protein domain, having role in acute immunological responses and fertility in higher vertebrates. However, information regarding the action of ptx on reproduction is extremely limited in fish. To study this, ptx cDNA was cloned for downstream analysis. Tissue distribution and ontogeny expression analysis indicated the prevalence of ptx in ovary. Varied phase-wise expression during carp ovarian cycle and elevated ptx expression after human chorionic gonadotropin induction, in vitro and in vivo, indicated probable regulation of gonadotropin. In situ hybridization and immunohistochemistry revealed the presence of ptx transcript and protein in the follicular layer of stage-III/IV oocytes indicating a role in ovarian growth. To assess the functional significance of ptx, transient silencing was performed using follicular primary cell culture, in vitro and in common carp, in vivo, through ovary-targeted injection of PEI-siRNA. Transient silencing of ptx-siRNA reduced the expression of various genes/factors related to oogenesis such as transcription factors, several steroidogenic enzymes, and esrs genes. These alterations in expression suggested a plausible role for ptx in ovarian steroidogenesis either, directly or indirectly, which is evident from the changes in the serum estradiol-17β (E2) and 17α,20β-dihydroxyprogesterone levels. Furthermore, downregulation of aromatase activity was also noticed after transient silencing. Increased ptx expression after E2 induced sex reversal to juvenile carp showed the correlative role of ptx during ovarian differentiation and development. Taken together, these findings suggest that ptx exerts an important role during ovarian growth, maturation and/or recrudescence of common carp.

GATA4/6 regulate DHH transcription in rat adrenocortical autografts

Adrenal cortex autotransplantation with ACTH stimulation may be an alternative therapy for patients with bilateral adrenalectomy to avoid adrenal crisis, but its underlying mechanism has not been elucidated. Previously, we detected Dhh upregulation in rat adrenocortical autografts after transplantation. Here, we investigated potential regulators such as Gata4, Gata6, Sry and Sox9 which affect Dhh transcription in adrenocortical autografts with or without ACTH stimulation. In ACTH-stimulated autografts, Gata4 and Gata6 were downregulated compared to control autografts. This response was linked to rDhh repression. A reporter assay using the upstream region of rDhh and a GATA binding motif revealed that rDhh promoters were significantly upregulated by co-transfection with Gata4 or Gata6 or both. Sry and Sox9 expression in autografts with or without ACTH stimulation were verified by PCR and RNAscope analyses. The ovarian differentiation factors Foxl2 and Rspo1 were also upregulated in the autografts. Gata4 and Gata6 were found to be significant factors in the regulation of rDhh expression and could be associated with adrenocortical autograft maintenance. Gonadal primordia with bipotential testicular and ovarian functions may also be present in these autografts.