A Potential Role for the Gsdf-eEF1α Complex in Inhibiting Germ Cell Proliferation: A Protein-Interaction Analysis in Medaka (Oryzias latipes) From a Proteomics Perspective.

Gonadal soma-derived factor (gsdf) has been demonstrated to be essential for testicular differentiation in medaka (Oryzias latipes). To understand the protein dynamics of Gsdf in spermatogenesis regulation, we used a His-tag "pull-down" assay coupled with shotgun LC-MS/MS to identify a group of potential interacting partners for Gsdf, which included cytoplasmic dynein light chain 2, eukaryotic polypeptide elongation factor 1 alpha (eEF1α), and actin filaments in the mature medaka testis. As for the interaction with transforming growth factor ß-dynein being critical for spermatogonial division in Drosophila melanogaster, the physical interactions of Gsdf-dynein and Gsdf-eEF1α were identified through a yeast 2-hybrid screening of an adult testis cDNA library using Gsdf as bait, which were verified by a paired yeast 2-hybrid assay. Coimmunoprecipitation of Gsdf and eEF1α was defined in adult testes as supporting the requirement of a Gsdf and eEF1α interaction in testis development. Proteomics analysis (data are available via ProteomeXchange with identifier PXD022153) and ultrastructural observations showed that Gsdf deficiency activated eEF1α-mediated protein synthesis and ribosomal biogenesis, which in turn led to the differentiation of undifferentiated germ cells. Thus, our results provide a framework and new insight into the coordination of a Gsdf (transforming growth factor ß) and eEF1α complex in the basic processes of germ cell proliferation, transcriptional and translational control of sexual RNA, which may be fundamentally conserved across the phyla during sexual differentiation.

Population genomics of an icefish reveals mechanisms of glacier-driven adaptive radiation in Antarctic notothenioids.

BACKGROUND: Antarctica harbors the bulk of the species diversity of the dominant teleost fish suborder-Notothenioidei. However, the forces that shape their evolution are still under debate. RESULTS: We sequenced the genome of an icefish, Chionodraco hamatus, and used population genomics and demographic modelling of sequenced genomes of 52 C. hamatus individuals collected mainly from two East Antarctic regions to investigate the factors driving speciation. Results revealed four icefish populations with clear reproduction separation were established 15 to 50 kya (kilo years ago) during the last glacial maxima (LGM). Selection sweeps in genes involving immune responses, cardiovascular development, and photoperception occurred differentially among the populations and were correlated with population-specific microbial communities and acquisition of distinct morphological features in the icefish taxa. Population and species-specific antifreeze glycoprotein gene expansion and glacial cycle-paced duplication/degeneration of the zona pellucida protein gene families indicated fluctuating thermal environments and periodic influence of glacial cycles on notothenioid divergence. CONCLUSIONS: We revealed a series of genomic evidence indicating differential adaptation of C. hamatus populations and notothenioid species divergence in the extreme and unique marine environment. We conclude that geographic separation and adaptation to heterogeneous pathogen, oxygen, and light conditions of local habitats, periodically shaped by the glacial cycles, were the key drivers propelling species diversity in Antarctica.

Loss of Gsdf leads to a dysregulation of Igf2bp3-mediated oocyte development in medaka.

Gonadal soma-derived factor (Gsdf) is a unique TGF-ß factor essential for both ovarian and testicular development in Hd-rR medaka (Oryzias latipes). However, the downstream genes regulated by Gsdf signaling remain unknown. Using a high-throughput proteomic approach, we identified a significant increase in the expression of the RNA-binding protein Igf2bp3 in gsdf-deficient ovaries. We verified this difference in transcription and protein expression against normal gonads using real-time PCR quantification and Western blotting. The genomic structure of igf2bp3 and the syntenic flanking segments are highly conserved across fish and mammals. igf2bp3 expression was correlated with oocyte development, which is consistent with the expression of the igf2bp3 ortholog Vg1-RBP/Vera in Xenopus. In contrast to the normal ovary, cysts of H3K27me3- and Igf2bp3-positive germ cells were dramatically increased in the one-month-old gsdf-deficient ovary, indicating that the gsdf depletion led to a dysregulation of Igf2bp3-mediated oocyte development. Our results provide novel insights into the Gsdf-Igf2bp3 signaling mechanisms that underlie the fundamental process of gametogenesis; these mechanisms may be well conserved across phyla.

Insights of sex determination and differentiation from medaka as a teleost model.

The mechanisms of sex determination and differentiation in fish are highly divergent with a broad range of gonadal differentiation types from hermaphroditism to gonochorism. Multiple triggers regulate the process of sexual differentiation including genetic or environmental factors (temperature, light, hormones and/or pH value, etc.). In recent years, with the advances of molecular technologies and genetic engineering approaches, there are significant breakthroughs in identifying the master genes of vertebrate sex determination and differentiation. In this review, we explore the fundamental and molecular mechanisms underlying the sexual differentiation in teleost fish, using medaka (Oryzias latipes) as a model. We focus on the male pathways and factors, particularly on dmrt1, gsdf and amh genes involved in testicular differentiation, sexual reversal and plasticity. It is anticipated that new techniques will likely be developed in the field of sex manipulations and monosex breeding for fish aquaculture in the future.

Identification of gonadal soma-derived factor involvement in Monopterus albus (protogynous rice field eel) sex change.

We studied molecular events and potential mechanisms underlying the process of female-to-male sex transformation in the rice field eel (Monopterus albus), a protogynous hermaphrodite fish in which the gonad is initially a female ovary and transforms into male testes. We cloned and identified a novel gonadal soma derived factor (GSDF), which encodes a member of the transforming growth factor-beta superfamily. gsdf expression was measured in gonads of female, intersex and male with reverse transcription-PCR and gsdf's role in sex transformation was studied with qPCR, histological analysis and dual-color in situ hybridization assays and compared to other sex-related genes. gsdf was correlated to Sertoli cell differentiation, indicating involvement in testicular differentiation and sex transformation from female to male in this species. A unique expression pattern reveals a potential role of gsdf essential for the sex transformation of rice field eels.

Leptin A deficiency affecting the mitochondrial dynamics of aged oocytes in medaka (Oryzias latipes).

Mitochondrial dysfunction and metabolic disorder have been associated to age-related subfertility, however, the precise molecular mechanism controlling the development of fertile oocytes in aging females remains elusive. Leptin plays an important role in the maintenance of energy homeostasis, as both excessive or insufficient levels can affect the body weight and fertility of mice. Here, we report that leptin A deficiency affects growth and shortens reproductive lifespan by reducing fertility in medaka (Oryzias latipes). Targeted disruption of lepa (lepa-/-) females reduced their egg laying and fertility compared to normal 3-month-old females (lepa+/+ sexual maturity), with symptoms worsening progressively at the age of 6 months and beyond. Transcriptomic analysis showed that differentially expressed genes involved in metabolic and mitochondrial pathways were significantly altered in lepa-/- ovaries compared with the normal ovaries at over 6 months old. The expression levels of the autophagy-promoting genes ulk1a, atg7 and atg12 were significantly differentiated between normal and lepa-/- ovaries, which were further confirmed by quantitative polymerase chain reaction analysis, indicating abnormal autophagy activation and mitochondrial dysfunction in oocyte development lacking lepa. Transmission electron microscopy observations further confirmed these mitochondrial disorders in lepa-deficient oocytes. In summary, these research findings provide novel insights into how leptin influences female fertility through mitochondrial-mediated oocyte development.

Ol4E-T, a eukaryotic translation initiation factor 4E-binding protein of medaka fish (Oryzias latipes), can interact with nanos3 and vasa in vitro.

Maternal factors have essential roles in the specification and development of germ cells in metazoans. In Drosophila, a number of genes such as oskar, vasa, nanos, and tudor are required for specific steps in pole cell formation and further germline development. Drosophila cup, another maternal factor, is confirmed as a main factor in normal oogenesis, maintenance, and survival of female germ-line stem cells by interaction with Nanos. Through searching for the homolog of Drosophila cup in the medaka, the homolog of eukaryotic translation initiation factor 4E (eIF4E)-transporter, named Ol4E-T, was identified. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed that Ol4E-T is maternally deposited in the embryo and Ol4E-T expression is maintained throughout embryogenesis. Ol4E-T is predominantly expressed in the adult gonads. In the testes, Ol4E-T is expressed in the same regions where medaka vasa, named olvas is expressed. In the ovary, expression of Ol4E-T conforms to that of nanos3 and olvas. Ol4E-T harbors a well-conserved eIF4E-binding motif, YTKEELL, by which Ol4E-T interacts with eIF4E in medaka. Additionally, Ol4E-T can interact with medaka Nanos3 and Olvas, as shown by yeast two hybridization. The spatial expression and interactions between Ol4E-T with germ cell markers Olvas and Nanos3 suggest a role for Ol4E-T in germ-line development in medaka.

The effect of letrozole overlapped with gonadotropin on IVF outcomes in women with DOR or aged over 40 years old with repeated cycles.

BACKGROUND: Evaluating the efficacy of letrozole overlapped with gonadotropin-modified letrozole protocol (mLP) for diminished ovarian reserve (DOR) or advanced-age women with repeated cycles. METHODS: This is a retrospectively registered, paired-match study including 243 women with DOR and 249 women aged over 40 years old who received in vitro fertilization (IVF) treatment. 123 women received stimulation with mLP (mLP group). GnRH agonist (GnRH-a) long, GnRH antagonist (GnRH-anta), and mild stimulation protocol were used as controls with 123 women in each group. We further analyzed 50 of 123 patients in the mLP group who have experienced more than one failed cycles with other ovarian stimulation protocols (non-mLP group). Clinical pregnancy rate (CPR), cumulative clinical pregnancy rate (CCPR), and live birth rate (LBR) were main outcomes. RESULTS: The CPR in the mLP group (38.46%) was significantly higher than mild stimulation (17.11%), but not significantly different from GnRH-a long (26.13%) and GnRH-anta (29.17%) group. The CCPR showed an increasing trend in the mLP group (33.33%) although without significance when compared with controls. The CCRP of GnRH-a long, GnRH-anta, mild stimulation group were 21.68%, 29.03%, and 13.04%, respectively. In women with repeated cycles, mLP achieved the higher available embryo rate (P < 0.05), the top-quality embryo rate, the CPR (P < 0.001), and the LBR (P < 0.001). Further study showed a positive correlation between testosterone and the number of oocytes retrieved in the mLP group (r = 0.395, P < 0.01). CONCLUSION: The mLP may be effective for aged or DOR women who have experienced previous cycle failure by improving the quality of embryos, the CPR, and the LBR. An increasing serum testosterone level may reflect follicular growth during ovarian stimulation.

Sex-specific meiosis responses to Gsdf in medaka (Oryzias latipes).

The meiotic entry of undifferentiated germ cells is sexually specific and strictly regulated by the testicular or ovarian environment. Germline stem cells with a set of abnormal sex chromosomes and associated autosomes undergo defective meiotic processes and are eventually eliminated by yet to be defined post-transcriptional modifications. Herein, we report the role of gsdf, a member of BMP/TGFß family uniquely found in teleost, in the regulation of meiotic entry in medaka (Oryzias latipes) via analyses of gametogenesis in gsdf-deficient XX and XY gonads in comparison with their wild-type siblings. Several differentially expressed genes, including the FKB506-binding protein 7 (fkbp7), were significantly upregulated in pubertal gsdf-deficient gonads. The increase in alternative pre-mRNA isoforms of meiotic synaptonemal complex gene sycp3 was visualized using Integrative Genomics Viewer and confirmed by real-time qPCR. Nevertheless, immunofluorescence analysis showed that Sycp3 protein products reduced significantly in gsdf-deficient XY oocytes. Transmission electron microscope observations showed that normal synchronous cysts were replaced by asynchronous cysts in gsdf-deficient testis. Breeding experiments showed that the sex ratio deviation of gsdf-/- XY gametes in a non-Mendelian manner might be due to the non-segregation of XY chromosomes. Taken together, our results suggest that gsdf plays a role in the proper execution of cytoplasmic and nuclear events through receptor Smad phosphorylation and Sycp3 dephosphorylation to coordinate medaka gametogenesis, including sex-specific mitotic divisions and meiotic recombination.

GATA4 is involved in the gonadal development and maturation of the teleost fish tilapia, Oreochromis niloticus.

GATA4, a member of the GATA family, is a well-known transcription factor implicated in the regulation of sex determination and sexual differentiation in mammals. However, little is known about the possible role of GATA4 in fish reproduction. In the present study, a full-length GATA4 cDNA from the tilapia was cloned and characterized. The tilapia GATA4 gene contained an open reading frame (ORF) of 1179 nucleotides encoding a protein of 392 amino acids. Sequence alignment revealed that the tilapia GATA4 protein shared higher homology (ranging from 63.1 to 74.6%) with other vertebrates. RT-PCR analysis indicated that the GATA4 gene is expressed in the ovary, testis, liver, intestine and heart in adult tilapia. In situ hybridization was performed to examine the temporal and spatial expression patterns of GATA4 during tilapia gonadal differentiation and development. In the undifferentiated gonad, GATA4 was expressed in the somatic cells of both sexes. Subsequently, GATA4 expression persisted in the differentiated, juvenile and adult ovary and testis in tilapia. Our data indicate for the first time that GATA4 is not only necessary for the onset of gonadal differentiation, but also important for gonadal development and maturation.