Whole-genome assembly and annotation for the little yellow croaker (Larimichthys polyactis) provide insights into the evolution of hermaphroditism and gonochorism.

The evolutionary direction of gonochorism and hermaphroditism is an intriguing mystery to be solved. The special transient hermaphroditic stage makes the little yellow croaker (Larimichthys polyactis) an appealing model for studying hermaphrodite formation. However, the origin and evolutionary relationship between of L. polyactis and Larimichthys crocea, the most famous commercial fish species in East Asia, remain unclear. Here, we report the sequence of the L. polyactis genome, which we found is ~706 Mb long (contig N50 = 1.21 Mb and scaffold N50 = 4.52 Mb) and contains 25,233 protein-coding genes. Phylogenomic analysis suggested that L. polyactis diverged from the common ancestor, L. crocea, approximately 25.4 million years ago. Our high-quality genome assembly enabled comparative genomic analysis, which revealed several within-chromosome rearrangements and translocations, without major chromosome fission or fusion events between the two species. The dmrt1 gene was identified as the male-specific gene in L. polyactis. Transcriptome analysis showed that the expression of dmrt1 and its upstream regulatory gene (rnf183) were both sexually dimorphic. Rnf183, unlike its two paralogues rnf223 and rnf225, is only present in Larimichthys and Lates but not in other teleost species, suggesting that it originated from lineage-specific duplication or was lost in other teleosts. Phylogenetic analysis shows that the hermaphrodite stage in male L. polyactis may be explained by the sequence evolution of dmrt1. Decoding the L. polyactis genome not only provides insight into the genetic underpinnings of hermaphrodite evolution, but also provides valuable information for enhancing fish aquaculture.

Growth and gonadal development retardations after long-term exposure to estradiol in little yellow croaker, Larimichthys polyactis.

Endocrine disrupting chemicals (EDCs) including 17ß-estradiol (E2) are widely distributed in the aquatic environment and are known to negatively affect the reproductive system of many animals, including fish. EDCs leading to feminization, altered sex ratio and reduced fecundity, it is possibly posing potential risks to the ecosystems. To investigate the potentially toxic effects of E2 exposure on little yellow croaker (Larimichthys polyactis, L. poliactis) who have a unique gonadal development pattern that males undergo a hermaphroditic stage. An experiment was set up where L. poliactis were maintained in tanks and exposed to E2 concentrations of 10 µg/L or no E2 exposure (the ethanol and control groups) from 30 to 90 days post-hatching (dph). After exposure, the E2 withdrawal and continual cultured to 150 and 365 dph. The morphological and histological analyses were used to compare the changes in the fish body and gonad under E2 exposure. The results showed that E2 exposure caused three major phenotypes at 30 and 60 days after treatment (dat), including ovary, ovotestis and gonadal development retardation compared with the control groups. The average ratio of these three phenotypes is 60.6%, 11.97% and 27.43%, respectively. The body length and weight of E2 exposure groups were repressed during the E2 exposure period, while it can recover after E2 withdrawal. However, the gonadal development (Gonadosomatic Index) of E2 exposure groups testis were retarded at 60 dat and doesn't recover until 365 dph. The sex determination/differentiation-related genes erα, erßI, erßII, fshß and cyp11b2 were significantly decreased in E2-exposure male fish. This research highlights the E2 leads to feminization, disrupts testis maturation and spermatogenesis, this effect persisted into the stage of sexual maturity. Collectively, our findings provide insights into the molecular mechanisms underlying E2 disturbance of a marine economic fish reproduction.

A Transient Hermaphroditic Stage in Early Male Gonadal Development in Little Yellow Croaker, Larimichthys polyactis.

Animal taxa show remarkable variability in sexual reproduction, where separate sexes, or gonochorism, is thought to have evolved from hermaphroditism for most cases. Hermaphroditism accounts for 5% in animals, and sequential hermaphroditism has been found in teleost. In this study, we characterized a novel form of the transient hermaphroditic stage in little yellow croaker (Larimichthys polyactis) during early gonadal development. The ovary and testis were indistinguishable from 7 to 40 days post-hatching (dph). Morphological and histological examinations revealed an intersex stage of male gonads between 43 and 80 dph, which consist of germ cells, somatic cells, efferent duct, and early primary oocytes (EPOs). These EPOs in testis degenerate completely by 90 dph through apoptosis yet can be rescued by exogenous 17-ß-estradiol. Male germ cells enter the mitotic flourishing stage before meiosis is initiated at 180 dph, and they undergo normal spermatogenesis to produce functional sperms. This transient hermaphroditic stage is male-specific, and the ovary development appears to be normal in females. This developmental pattern is not found in the sister species Larimichthys crocea or any other closely related species. Further examinations of serum hormone levels indicate that the absence of 11-ketotestosterone and elevated levels of 17-ß-estradiol delineate the male intersex gonad stage, providing mechanistic insights on this unique phenomenon. Our research is the first report on male-specific transient hermaphroditism and will advance the current understanding of fish reproductive biology. This unique gonadal development pattern can serve as a useful model for studying the evolutionary relationship between hermaphroditism and gonochorism, as well as teleost sex determination and differentiation strategies.

A totally laparoscopic peritoneal free flap for reconstruction of hand.

Management of defects on the hand and foot with exposed tendons remains a major challenge for plastic surgeons. Here, we present a case of hand reconstruction with a totally laparoscopic peritoneal flap. The anterior rectus sheath was preserved in situ. The peritoneal free flap supplied by peritoneal branches of the deep inferior epigastric artery was retrieved by laparoscopy to cover the soft tissue defect of the hand. The defect of the dorsal hand was 17 cm ×12 cm. The peritoneal flap measuring 22 cm × 15 cm survived completely without any complications. A following split-thickness skin graft offered the suc- cessful wound closure. Motor and sensory function improved gradually within the first year follow-up. The totally laparoscopic peritoneal free flap is a good choice for reconstruction of the soft tissue de- fects accompanied by exposed tendons on the hand and foot.

Haploinsufficiency of SF-1 Causes Female to Male Sex Reversal in Nile Tilapia, Oreochromis niloticus.

Steroidogenic factor-1 (Sf-1) (officially designated nuclear receptor subfamily 5 group A member 1 [NR5A1]) is a master regulator of steroidogenesis and reproduction in mammals. However, its function remains unclear in nonmammalian vertebrates. In the present study, we used immunohistochemistry to detect expression of Sf-1 in the steroidogenic cells, the interstitial, granulosa, and theca cells of the ovary, and the Leydig cells of the testis, in Nile tilapia. Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (Cas9) cleavage of sf-1 resulted in a high mutation rate in the F0 generation and a phenotype of gonadal dysgenesis and reduced steroidogenic cells in XX and XY fish. Sf-1 deficiency also resulted in decreased cytochrome P450, family 19, subfamily A, polypeptide 1a, forkhead box L2 expression, and serum estradiol-17ß in XX fish. In XY fish, Sf-1 deficiency increased cytochrome P450, family 19, subfamily A, polypeptide 1a and forkhead box L2 expression but decreased cytochrome P450, family 11, subfamily B, polypeptide 2 expression and serum 11-ketotestosterone levels. 17α-methyltestosterone treatment successfully rescued the gonadal phenotype of Sf-1-deficient XY fish, as demonstrated by normal spermatogenesis and production of F1 mutants. In contrast, estradiol-17ß treatment only partially rescued the gonadal phenotype of Sf-1-deficient XX fish, as demonstrated by the appearance of phase II oocytes. Furthermore, both sf-1(+/-) F1 XX and XY mutants developed as fertile males, although spermatogenesis was delayed and efferent duct formation was disordered. Our data suggest that Sf-1 is a major regulator of steroidogenesis and reproduction in fish, as it is in mammals. Sf-1 deficiency resulted in gonadal dysgenesis and feminization of XY gonads. However, unlike in mammals, Sf-1 deficiency also resulted in female to male sex reversal in 8.1% of F0 and 92.1% of sf-1(+/-) F1 in XX fish.

Transdifferentiation of differentiated ovary into functional testis by long-term treatment of aromatase inhibitor in Nile tilapia.

Females with differentiated ovary of a gonochoristic fish, Nile tilapia, were masculinized by long-term treatment with an aromatase inhibitor (Fadrozole) in the present study. The reversed gonads developed into functional testes with fertile sperm. The longer the fish experienced sex differentiation, the longer treatment time was needed for successful sex reversal. Furthermore, Fadrozole-induced sex reversal, designated as secondary sex reversal (SSR), was successfully rescued by supplement of exogenous 17ß-estradiol. Gonadal histology, immunohistochemistry, transcriptome, and serum steroid level were analyzed during SSR. The results indicated that spermatogonia were transformed from oogonia or germline stem cell-like cells distributed in germinal epithelium, whereas Leydig and Sertoli cells probably came from the interstitial cells and granulosa cells of the ovarian tissue, respectively. The transdifferentiation of somatic cells, as indicated by the appearance of doublesex- and Mab-3-related transcription factor 1 (pre-Sertoli cells) and cytochrome P450, family 11, subfamily B, polypeptide 2 (pre-Leydig cells)-positive cells in the ovary, provided microniche for the transdifferentiation of germ cells. Decrease of serum 17ß-estradiol was detected earlier than increase of serum 11-ketotestosterone, indicating that decrease of estrogen was the cause, whereas increase of androgen was the consequence of SSR. The sex-reversed gonad displayed more similarity in morphology and histology with a testis, whereas the global gene expression profiles remained closer to the female control. Detailed analysis indicated that transdifferentiation was driven by suppression of female pathway genes and activation of male pathway genes. In short, SSR provides a good model for study of sex reversal in teleosts and for understanding of sex determination and differentiation in nonmammalian vertebrates.

Antagonistic roles of Dmrt1 and Foxl2 in sex differentiation via estrogen production in tilapia as demonstrated by TALENs.

Transcription activator-like effector nucleases (TALENs) are a powerful approach for targeted genome editing and have been proved to be effective in several organisms. In this study, we reported that TALENs can induce somatic mutations in Nile tilapia, an important species for worldwide aquaculture, with reliably high efficiency. Six pairs of TALENs were constructed to target genes related to sex differentiation, including dmrt1, foxl2, cyp19a1a, gsdf, igf3, and nrob1b, and all resulted in indel mutations with maximum efficiencies of up to 81% at the targeted loci. Effects of dmrt1 and foxl2 mutation on gonadal phenotype, sex differentiation, and related gene expression were analyzed by histology, immunohistochemistry, and real-time PCR. In Dmrt1-deficient testes, phenotypes of significant testicular regression, including deformed efferent ducts, degenerated spermatogonia or even a complete loss of germ cells, and proliferation of steroidogenic cells, were observed. In addition, disruption of Dmrt1 in XY fish resulted in increased foxl2 and cyp19a1a expression and serum estradiol-17ß and 11-ketotestosterone levels. On the contrary, deficiency of Foxl2 in XX fish exhibited varying degrees of oocyte degeneration and significantly decreased aromatase gene expression and serum estradiol-17ß levels. Some Foxl2-deficient fish even exhibited complete sex reversal with high expression of Dmrt1 and Cyp11b2. Furthermore, disruption of Cyp19a1a in XX fish led to partial sex reversal with Dmrt1 and Cyp11b2 expression. Taken together, our data demonstrated that TALENs are an effective tool for targeted gene editing in tilapia genome. Foxl2 and Dmrt1 play antagonistic roles in sex differentiation in Nile tilapia via regulating cyp19a1a expression and estrogen production.