Morphological Sex Differentiation Research Articles

Histological observations and transcriptome analyses reveal the dynamic changes in the gonads of the blotched snakehead (Channa maculata) during sex differentiation and gametogenesis

BackgroundBlotched snakehead (Channa maculata) displays significant sexual dimorphism, with males exhibiting faster growth rates and larger body sizes compared to females. The cultivation of the all-male population of snakeheads holds substantial economic and ecological value. Nonetheless, the intricate processes governing the development of bipotential gonads into either testis or ovary in C. maculata remain inadequately elucidated. Therefore, it is necessary to determine the critical time window of sex differentiation in C. maculata, providing a theoretical basis for sex control in production practices.MethodsThe body length and weight of male and female C. maculata were measured at different developmental stages to reveal when sexual dimorphism in growth initially appears. Histological observations and spatiotemporal comparative transcriptome analyses were performed on ovaries and testes across various developmental stages to determine the crucial time windows for sex differentiation in each sex and the sex-related genes. Additionally, qPCR and MG2C were utilized to validate and locate sex-related genes, and levels of E2 and T were quantified to understand sex steroid synthesis.ResultsSexual dimorphism in growth became evident starting from 90 dpf. Histological observations revealed that morphological sex differentiation in females and males occurred between 20 and 25 dpf or earlier and 30–35 dpf or earlier, respectively, corresponding to the appearance of the ovarian cavity or efferent duct anlage. Transcriptome analyses revealed divergent gene expression patterns in testes and ovaries after 30 dpf. The periods of 40–60 dpf and 60–90 dpf marked the initiation of molecular sex differentiation in females and males, respectively. Male-biased genes (Sox11a, Dmrt1, Amh, Amhr2, Gsdf, Ar, Cyp17a2) likely play crucial roles in male sex differentiation and spermatogenesis, while female-biased genes (Foxl2, Cyp19a1a, Bmp15, Figla, Er) could be pivotal in ovarian differentiation and development. Numerous biological pathways linked to sex differentiation and gametogenesis were also identified. Additionally, E2 and T exhibited sexual dimorphism during sex differentiation and gonadal development. Based on these results, it is hypothesized that in C. maculata, the potential male sex differentiation pathway, Sox11a–Dmrt1–Sox9b, activates downstream sex-related genes (Amh, Amhr2, Gsdf, Ar, Cyp17a2) for testicular development, while the antagonistic pathway, Foxl2/Cyp19a1a, activates downstream sex-related genes (Bmp15, Figla, Er) for ovarian development.ConclusionsThis study provides a comprehensive overview of gonadal dynamic changes during sex differentiation and gametogenesis in C. maculata, establishing a scientific foundation for sex control in this species.

DNA methylation plays a critical role in testicular maintenance, but not in sex determination of male tilapia Oreochromis niloticus

The significance of DNA methylation in fish sex determination and differentiation is increasingly being recognized. Exploring the role of DNA methylation in sex determination and differentiation is advantageous for the advancement of sex control techniques in aquaculture. In this study, the global DNA methylation levels of XX and XY gonads at different developmental stages of tilapia (Oreochromis niloticus) was assessed using an anti-5-methylcytosin (5-mC) antibody. The results indicated that the difference of global DNA methylation levels between XX and XY gonads was established after morphological sex differentiation had occurred at 30 dah (days after hatching). The bisulfite sequencing PCR (BSP) analysis revealed a significant disparity in the DNA methylation level of cyp19a1a between the XX and XY gonads after 30 dah, rather than before. The Dnmts (DNA methyltransferases) inhibitor 5-aza-2′-deoxycytidine (5-aza-dC, 20 μg/g diet), the aromatase inhibitor letrozole (AI, 150 μg/g diet), and 17β-estradiol (E2, 150 μg/g diet) were employed for treating XX and XY tilapia in order to investigate the involvement of DNA methylation in sex determination and differentiation. Treatment of XX and XY fish with 5-aza-dC from 5 to 50 dah did not result in any sex reversal, either female-to-male or male-to-female. For XX tilapia, both the use of AI alone and its combination with 5-aza-dC resulted in sex reversal from female-to-male when treated from 30 to 60 dah. This indicates that the addition of 5-aza-dC did not prevent the sex reversal from female-to-male induced by AI treatment, which is different from the results observed in zebrafish (Danio rerio). Intriguingly, treatment of XY fish from 30 to 90 dah with either E2 or 5-aza-dC alone resulted in 6% and 0% sex reversal, respectively, while simultaneous treatment with 5-aza-dC and E2 resulted in significant decrease of cyp19a1a methylation level and increase of cyp19a1a expression and 65% sex reversal. Taken together, these results suggest that DNA methylation plays a critical role in gonadal maintenance, but not in sex determination in tilapia. This study deepens our understanding of the role of epigenetic modifications in fish sex differentiation and maintenance, providing an approach to achieve sex reversal in testis-differentiated male fish and improving the available methods for sex control in aquaculture.

A testis-specific gene doublesex is involved in spermatogenesis and early sex differentiation of the Chinese mitten crab Eriocheir sinensis

The doublesex (Dsx) is a DM domain gene involved in sex determination and/or differentiation in insects. In this study, a novel sex-specific Dsx homolog, designated as EsDsx-like, was identified from the Chinese mitten crab Eriocheir sinensis. The full-length cDNA of EsDsx-like was 921 bp, encoding a protein of 215 amino acids. The EsDsx-like mRNA and protein were detected exclusively in the testis. During spermatogenesis, the EsDsx-like mRNA were continuously expressed in spermatogonium, spermatocyte, spermatid and spermatozoon. In situ hybridization analysis showed that the EsDsx-like mRNA was mainly detected in the nucleus of spermatogonium and spermatocyte, and then concentrated in a dot on the edge of the nucleus in spermatid. In spermatozoa, the hybridization signal was localized to the acrosomal tubule. Immunofluorescence analysis revealed that the EsDsx-like protein was detected mainly in the cytoplasm around the nucleus in spermatocyte and spermatid, and then appeared as a dot immuno-signal in the acrosomal tubule and distributed in the cup-shaped nucleus of the spermatozoa. Together, these results suggest a potential role for EsDsx-like in the formation of acrosome complex and nuclear morphogenesis during sperm maturation. In the early development, similar with the male sex regulator, insulin-like androgenic gland hormone (EsIAG), the EsDsx-like mRNA was specifically detected in all male individuals tested at juvenile III stage just before the first appearance of the petasma in morphological sex differentiation at juvenile IV. Further, RNAi knockdown of EsDsx-like significantly downregulated the expression of EsIAG in adult and juvenile-III males. The direct interaction between the EsDsx-like protein and EsIAG promoter was confirmed by dual-luciferase reporter assays, indicating that EsDsx-like could act as an upstream regulatory gene of EsIAG in sex differentiation. This study provided valuable insights into the role of sex-specific gene associated with morphological sex differentiation and might contribute to development of sex control techniques in the Chinese mitten crab.

Dynamic transcriptome analysis reveals the gene network of gonadal development from the early history life stages in dwarf surfclam Mulinia lateralis

BackgroundGonadal development is driven by a complex genetic cascade in vertebrates. However, related information remains limited in molluscs owing to the long generation time and the difficulty in maintaining whole life cycle in the lab. The dwarf surfclam Mulinia lateralis is considered an ideal bivalve model due to the short generation time and ease to breed in the lab.ResultsTo gain a comprehensive understanding of gonadal development in M. lateralis, we conducted a combined morphological and molecular analysis on the gonads of 30 to 60 dpf. Morphological analysis showed that gonad formation and sex differentiation occur at 35 and 40–45 dpf, respectively; then the gonads go through gametogenic cycle. Gene co-expression network analysis on 40 transcriptomes of 35–60 dpf gonads identifies seven gonadal development-related modules, including two gonad-forming modules (M6, M7), three sex-specific modules (M14, M12, M11), and two sexually shared modules (M15, M13). The modules participate in different biological processes, such as cell communication, glycan biosynthesis, cell cycle, and ribosome biogenesis. Several hub transcription factors including SOX2, FOXZ, HSFY, FOXL2 and HES1 are identified. The expression of top hub genes from sex-specific modules suggests molecular sex differentiation (35 dpf) occurs earlier than morphological sex differentiation (40–45 dpf).ConclusionThis study provides a deep insight into the molecular basis of gonad formation, sex differentiation and gametogenesis in M. lateralis, which will contribute to a comprehensive understanding of the reproductive regulation network in molluscs.

Developmental changes in gene expression and gonad morphology during sex differentiation in Atlantic salmon (Salmo salar)

The Atlantic salmon (Salmo salar) is a globally important species for its value in fisheries and aquaculture, and as a research model. In order to characterise aspects of sex differentiation at the morphological and mRNA level in this species, the present study examined developmental changes in gonad morphology and gene expression in males and females between 0 and 79 days post hatch (dph). Morphological differentiation of the ovary (indicated by the formation of germ cell cysts) became apparent from 52 dph. By 79 dph, ovarian phenotype was evident in 100% of genotypic females. Testes remained in an undifferentiated-like state throughout the experiment, containing germ cells dispersed singularly within the gonadal region distal to the mesentery. There were no significant sex-related differences in gonad cross-section size, germ cell number or germ cell diameter during the experiment. The expression of genes involved in teleost sex differentiation (anti-müllerian hormone (amh), cytochrome P450, family 19, subfamily A, polypeptide 1a (cyp19a1a), forkhead box L2a (foxl2a), gonadal soma-derived factor (gsdf), r-spondin 1 (rspo1), sexually dimorphic on the Y chromosome (sdY)), retinoic acid-signalling (aldehyde dehydrogenase 1a2 (aldh1a2), cytochrome P450 family 26 a1 (cyp26a1), cytochrome P450 family 26 b1 (cyp26b1), t-box transcription factor 1 (tbx1a)) and neuroestrogen production (cytochrome P450, family 19, subfamily A, polypeptide 1b (cyp19a1b)) was investigated. Significant sex-related differences were observed only for the expression of amh, cyp19a1a, gsdf and sdY. In males, amh, gsdf and sdY were upregulated from 34, 59 and 44 dph respectively. In females, cyp19a1a was upregulated from 66 dph. Independent of sex, foxl2a expression was highest at 0 dph and had reduced ∼ 47-fold by the time of morphological sex differentiation at 52 dph. This study provides new insights into the timing and sequence of some physiological changes associated with sex differentiation in Atlantic salmon. These findings also reveal that some aspects of the mRNA sex differentiation pathways in Atlantic salmon are unique compared to other teleost fishes, including other salmonids.

Molecular and morphological changes in Nile tilapia ( Oreochromis niloticus ) gonads during high‐temperature‐induced masculinization

Genetic sex of Nile tilapia (Oreochromis niloticus) is determined by genotype (genetic sex determination). However, these fish are temperature-sensitive, and high-temperature-induced sex reversal is a common practice in research and tilapia farming. In this study, we characterized the molecular and morphological sex differentiation of Nile tilapia during the process of sex reversal induced by high temperatures. The offspring of parental pairs of XX and XY Nile tilapia underwent high-temperature treatment for 20 d beginning at 5 dpf (days post fertilization). Gonads were isolated from Nile tilapia at seven time points between 10–180 dpf. The histology results showed that gonads of high-temperature-induced XX pseudomales exhibited morphological differences from XX females during 25–35 dpf. qRT-PCR analysis revealed that the expression levels of cyp19a1a and foxl2 were temperature-sensitive, and were lowly expressed in the gonads of pseudomales from 35 dpf onward. Such findings were also consistent with the histological observations of gonadal morphological differentiation. The expression of the TGFβ superfamily member, gsdf, was increased in the gonads of pseudomales from 12 dpf onward. The transcription of kdm6bb was elevated by high-temperature treatment at 10–25 dpf and displayed sexual dimorphism from 35 dpf onward. Dmrt1 was specifically expressed in phenotypic males from 12 dpf onward. Collectively, this study identified the roles of specific genes involved in the process of high-temperature-induced masculinization in Nile tilapia and established robust molecular markers of phenotypic sex in Nile tilapia that will be useful for ongoing research related to sex control.

Characterization of the sex differentiation and gonadal development in small yellow croaker (Larimichthys polyactis) and its hybrid (L. polyactis ♀ × L. crocea ♂).

Interspecific hybridization has been considered as a possible approach to improve biological traits and has been applied in aquaculture practices. In the present study, artificial hybridization was carried out in the small yellow croaker (SYC; Larimichthys polyactis) ♀ × large yellow croaker (LYC; L. crocea) ♂ by artificial insemination, and the processes of sex differentiation and gonadal development in SYC and its hybrid were investigated under controlled conditions. Histological analysis of SYC larvae showed that migrating primordial germ cells (PGCs) were observed at 4days post-hatching (dph), a genital ridge was formed on the dorsal side of the peritoneum at 6 dph, and a pair of primary gonads was first observed at 10 dph. Signs of the differentiated ovary and ovarian cavity were observed at 45 dph. However, some presumptive testes showed alterations in morphology, including an increase in the number of oocytes and an enhanced basophilia at 50 dph. These presumptive testes seemed to alter again, and numerous gonial cells were arranged in cyst-like groups with several degenerating oocytes that developed into residual body-like structures during 60-90 dph. Compared with SYC, the hybrid had a lower number of PGCs and showed retarded gonadal development at the early stage. Ovarian differentiation in the hybrid was observed at 50 dph, while testicular differentiation occurred at 60 dph. The presence of vitellogenic oocytes and spermatozoa at 360 dph in the hybrids suggested that hybrid individuals can undergo successful gametogenesis in females and males, respectively. Overall, the present results suggest that morphological sex differentiation occurred at 40 and 50 dph in SYC and its hybrid, respectively, both of which have normal gametogenesis. Moreover, some level of heterosis (hybrid vigor) occurred in the growth of the hybrid (total length and body weight) compared with that in the growth of SCY over time. Gonadal development of the hybrid was also found to be advanced at 360 dph. The present information will contribute to the potential use and management of these fish for aquaculture.

Sexual Development of the Hermaphroditic Scallop Argopecten irradians Revealed by Morphological, Endocrine and Molecular Analysis.

Simultaneous or functional hermaphrodites possessing both ovary and testis at the same time are good materials for studying sexual development. However, previous research on sex determination and differentiation was mainly conducted in gonochoristic species and studies on simultaneous hermaphrodites are still limited. In this study, we conducted a combined morphological, endocrine and molecular study on the gonadal development of a hermaphroditic scallop Argopecten irradians aged 2–10 month old. Morphological analysis showed that sex differentiation occurred at 6 months of age. By examining the dynamic changes of progesterone, testosterone and estradiol, we found testosterone and estradiol were significantly different between the ovaries and testes almost throughout the whole process, suggesting the two hormones may be involved in scallop sex differentiation. In addition, we identified two critical sex-related genes FoxL2 and Dmrt1L, and investigated their spatiotemporal expression patterns. Results showed that FoxL2 and Dmrt1L were female- and male-biased, respectively, and mainly localized in the germ cells and follicular cells, indicating their feasibility as molecular markers for early identification of sex. Further analysis on the changes of FoxL2 and Dmrt1L expression in juveniles showed that significant sexual dimorphic expression of FoxL2 occurred at 2 months of age, earlier than that of Dmrt1L. Moreover, FoxL2 expression was significantly correlated with estradiol/testosterone ratio (E2/T). All these results indicated that molecular sex differentiation occurs earlier than morphological sex differentiation, and FoxL2 may be a key driver that functions through regulating sex steroid hormones in the scallop. This study will deepen our understanding of the molecular mechanism underlying sex differentiation and development in spiralians.

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.

Identification and Sexually Dimorphic Expression of a FoxL2-like Gene in Mud Crab (Scylla paramamosain): Potential Roles in Male Differentiation and Development

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

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.

Expression of Vasa, Nanos2 and Sox9 during initial testicular development in Nile tilapia (Oreochromis niloticus) submitted to sex reversal.

Sexual differentiation and early gonadal development are critical events in vertebrate reproduction. In this study, the initial testis development and expression of the Vasa, Nanos2 and Sox9 proteins were examined in Nile tilapia Oreochromis niloticus submitted to induced sex reversal. To that end, 150O. niloticus larvae at 5 days post-hatching (dph) were kept in nurseries with no hormonal addition (control group) and 150 larvae were kept with feed containing 17α-methyltestosterone to induce male sex reversal (treated group). Morphological sexual differentiation of Nile tilapia occurred between 21 and 25 dph and sex reversal resulted in 94% males, whereas the control group presented 53% males. During sexual differentiation, gonocytes (Gon) were the predominant germ cells, which decreased and disappeared after that stage in both groups. Undifferentiated spermatogonia (Aund) were identified at 21 dph in the control group and at 23 dph in the treated group. Differentiated spermatogonia (Adiff) were found at 23 dph in both groups. Vasa and Nanos2 occurred in Gon, Aund and Adiff and there were no significant differences between groups. Vasa-labelled Adiff increased at 50 dph in both groups and Nanos2 presented a high proportion of labelled germ cells during sampling. Sertoli cells expressed Sox9 throughout the experiment and its expression was significantly greater during sexual differentiation in the control group. The results indicate that hormonal treatment did not alter initial testis development and expression of Vasa and Nanos2 in Nile tilapia, although lower expression of Sox9 and a delay in sexual differentiation was detected in the treated group.

FOXL2 and DMRT1L Are Yin and Yang Genes for Determining Timing of Sex Differentiation in the Bivalve Mollusk Patinopecten yessoensis.

Sex determination and differentiation have long been a research hotspot in metazoans. However, little is known about when and how sex differentiation occurs in most mollusks. In this study, we conducted a combined morphological and molecular study on sex differentiation in the Yesso scallop Patinopecten yessoensis. Histological examination on gonads from 5- to 13-month-old juveniles revealed that the morphological sex differentiation occurred at 10 months of age. To determine the onset of molecular sex differentiation, molecular markers were screened for early identification of sex. The gonadal expression profiles of eight candidate genes for sex determination or differentiation showed that only two genes displayed sexually dimorphic expression, with FOXL2 being abundant in ovaries and DMRT1L in testes. In situ hybridization revealed that both of them were detected in germ cells and follicle cells. We therefore developed LOG10(DMRT1L/FOXL2) for scallop sex identification and confirmed its feasibility in differentiated individuals. By tracing its changes in 5- to 13-month-old juveniles, molecular sex differentiation time was determined: some scallops differentiate early in September when they are 7 months old, and some do late in December when they are 10 months old. Two kinds of coexpression patterns were found between FOXL2 and DMRT1L: expected antagonism after differentiation and unexpected coordination before differentiation. Our results revealed that scallop sex differentiation co-occurs with the formation of follicles, and molecular sex differentiation is established prior to morphological sex differentiation. Our study will assist in a better understanding of the molecular mechanism underlying bivalve sex differentiation.

Expression pattern of foxl2 and dmrt1 in gonad of Amur sturgeon Acipenser schrenckii in relation to sex differentiation

The mechanisms that govern sex differentiation in sturgeon are poorly understood. This study aimed to examine sexual dimorphic expression of foxl2 and dmrt1 in Amur sturgeon Acipenser schrenckii during molecular and morphological sex differentiation. Histologically, ovaries were first observed at 9months after hatching (mah), reflected in an invaginated gonadal epithelium and the presence of germ cells just below the epithelium, whereas gonads with smooth epithelium and germ cells in the stroma were characterized as testis. Some fish showed undifferentiated gonads until 16mah, but all sampled fish had morphologically differentiated sex by 19mah. The full-length cDNAs of foxl2 and two types of dmrt1, dmrt1a and dmrt1b, of Amur sturgeon were isolated. The 2 types of dmrt1 cDNA had an identical structure from the 5′-UTR to the Y-rich domain, whereas the sequence downstream from this domain showed no homology and is considered to have resulted from alternative splicing. Foxl2 was principally expressed in the ovary and the 2 types of dmrt1 in the testis of 7-year-old Amur sturgeon, and much less so in various somatic tissues. Foxl2 expression was sexually dimorphic in morphologically differentiated gonads and was similarly dichotomous in the undifferentiated gonads of fish at 9mah. In contrast, sexually dimorphic expression of dmrt1a and dmrt1b was not observed in gonads of the juvenile Amur sturgeon, but these genes showed testis-dominant expression in the adults. We suggest that foxl2 expression is closely related to ovarian differentiation and that two types of dmrt1 play a role in testis development and/or sperm formation rather than testicular differentiation. Additionally, we suggest that mRNA levels of foxl2 are an appropriate marker for sexing of sturgeon at an early developmental stage.

Reliability of neutrophilic nuclear appendages in morphological sex differentiation

Background: The inactive X-chromosome in neutrophils appears in one of the three forms. They are drumsticks, racquet forms, and sessile nodules. Objective: A correlative study based on the presence of “drumstick and other nuclear appendages” in polymorphonuclear neutrophils to determine the morphological sex. Materials and Methods: Sixty-eight randomly selected blood smears (34 males and 34 females) were stained with Leishman’s stain. One hundred well-stained neutrophils were double-blindly studied in the tail-end of the smears and classified into four groups based on Kosenow’s formula as drumstick (Form A), sessile nodule (Form B), and other pedunculated nuclear projections such as tag and hooks (Form C). Results: Significant correlation of neutrophilic nuclear appendage of Form A (P = 0.00001) and Form C (P = 0.00016) was obtained for females and males, respectively. Difference of A-C gives a positive value in females and a negative value in males. Conclusion: Neutrophilic nuclear appendages which include true drumsticks, sessile nodules, and racket structures form a useful tool for morphological sex differentiation.

북방전복 Haliotis discus hannai의 형태학적 성분화

This study was conducted to provide the reproductive biological information and basic data on the artificial sex control of Haliotis discus hannai. The morphological sex differentiation process of H. discus hannai could be classified into following five phases: 1) formation of gonad outer membrane (FGOM) (), 2) primordial germ cells (PGCs) appearance in the connective tissue between intestine and hepatopancreas (PAC), and formation of gonadal cavity (FGC) (SL ), 3) PGCs appearance in the epithelial layer of gonadal cavity (PAG) (SL ), 4) formation of gametogenic follicle and appearance of early oocytes and spermatogonia (FGOC) (SL ), 5) morphological sex differentiation (MSD) (). From histological analysis sex differentiation rate in SL 24.1-25.0 mm of H. discus hannai was 90.0% and sex ratio (female : male) was 1:0.8.

Histological observation of gonadal differentiation and effect of rearing temperature on sex differentiation in Amur sturgeon Acipenser schrenckii

Temperature-dependent sex determination has been demonstrated in some species of fish and tempera-ture during the period of sex differentiation typically produces male or female-dominant population.This research investigated the gonadal sex differentiation and effect of rearing temperature on the sex ratio in larval Amur stur-geon Acipenser schrenckii.In 1.5 days post-hatch,a PGC is visible along the dorsal epithelium of the body cavity,between the mesentery and the kidney.Later,in 3-7 days post-hatch,the numbers of the primordial germ cells(PGC) increased,and gathered into cell cable.At 17-31 dph,PGCs along the mesenteric migrate to below the kidney area and cells together to form the gonadal ridges.When 40-60 dph,the original gonadal development,groups of small cells from kidney area migrate to original gonad,original gonadal columnar epithelial cells.At 90-120 dph,germinal epithelium and cells appears in the original gonad,fat cells and blood vessels increase.Pu-tative ovaries with notches in the germinal epithelium and presumed testes with smooth germinal epithelium ap-peared in 180 days post-hatch,signs of juvenile Acipenser schrenckii already on the level of differentiation of go-nads in anatomy.Ovaries with proliferating oogonia and early meiotic oocytes clusters were observed in 210-day-old juveniles.In a temperature controlled experiment,the proportions of females were 50% at 21℃,43.3% at 15℃,43.3% at 18℃,50% at 24℃,and 46.7%(the same as 1:1 sex ratio) at 27℃.These results suggest that morphological sex differentiation in Amur sturgeon occurs at approximately 180 days after hatch,and though the high temperatures can not change the 1:1 sex ratio in this species,but it can help to promote gonadal devel-opment of juvenile Amur sturgeon.

Presence of 11-ketotestosterone in pre-differentiated male gonads of Odontesthes bonariensis

The involvement of androgens during sex differentiation period was investigated in the pejerrey Odontesthes bonariensis, by classic biochemical studies and gonadal histology. We studied in particular whether the enzyme activities involved in 11-oxygenated androgen production were active in a gonadal/peritoneum complex (GPC) of very small larvae exposed to masculinizing temperatures previous to morphological sex differentiation (5weeks post-hatching). The GPC was incubated with 17-hydroxyprogesterone ((3)H-17P), and the presence of 11-KT as major metabolite in early gonads undergoing masculine pathway after temperature treatment exposure is reported. 11-KT was identified by thin-layer chromatography and high-pressure liquid chromatography. The present results show that 11-KT is produced at very early stages of testis development in pejerrey, being this androgen one of the main mediators of the masculinization induced by temperature treatment at the gonad level.

Sex differentiation in Atlantic cod (Gadus morhua L.): morphological and gene expression studies

BackgroundIn differentiated gonochoristic species, a bipotential gonad develops into an ovary or testis during sex differentiation. Knowledge about this process is necessary to improve methods for masculinizing genetically female Atlantic cod for the subsequent purpose of producing all-female populations.MethodsGonads were examined histologically in juveniles from 14 to 39 mm total body length (TL). Number and size of germ cells were determined in a subset of the samples. Relevant genes were cloned, and mRNA levels determined by qPCR of amh, cyp19a1a; dax1 (nr0b2); shp (nr0b2a) and sox9b in a mixed-sex and an all-female population ranging from 12–49 mm TL.ResultsIndividuals between 14–20 mm TL could be separated in two subgroups based on gonad size and germ cell number. Ovarian cavity formation was observed in some individuals from 18–20 mm TL. The mixed sex population displayed bimodal expression patterns as regards cyp19a1a (starting at 12 mm TL) and amh (starting at 20 mm TL) mRNA levels. After approximately 30 mm TL, cyp19a1a and amh displayed a gradual increase in both sexes. No apparent, sex-dependent expression patterns were found for dax1, shp or sox9b transcripts. However, shp levels were high until the larvae reached around 35 mm TL and then dropped to low levels, while dax1 remained low until 35 mm TL, and then increased sharply.ConclusionsThe morphological sex differentiation in females commenced between 14–20 mm TL, and ovarian cavities were evident by 18–20 mm TL. Testis development occurred later, and was morphologically evident after 30 mm TL. This pattern was corroborated with sexually dimorphic expression patterns of cyp19a1a from 12–13 mm TL, and a male-specific increase in amh from 20 mm TL.

Heritability for growth traits in giant freshwater prawn, Macrobrachium rosenbergii (de Mann 1879) based on best linear unbiased prediction methodology

In this study, heritability was estimated for growth-related traits of giant freshwater prawn (Macrobrachium rosenbergii) before and after morphological sexual differentiation. Estimation was made on data from 16 full-sib and eight half-sib families. The variance estimation was performed using a univariate mixed linear animal model and variance components were analysed following an animal model using restricted maximum likelihood procedure using average information algorithm. Heritability estimates (h2) varied considerably with ages. At 2 months old, h2for carapace length (CL; 0.35±0.15) and body weight (BW; 0.26±0.13) were higher than those estimated at 5 months old, based on mixed sex data. However, when data were sorted by sex, h2 calculated from data of females were higher than those of males for CL (0.26±0.16 vs. 0.10±0.06), BW (0.28±0.17 vs. 0.12±0.08), body length (0.40±0.17 vs. 0.11±0.07), total length (0.47±0.18 vs. 0.11±0.07) and claw length (0.29±0.16 vs. 0.03±0.04). The same trend was observed for traits at 6 months old in both bulk and individual rearing.