Successful formation of sperm cells from transplanted primordial germ cells in sterile interspecific avian recipients.

Primordial germ cells (PGCs) are stem cells, from which only gametes develop. In birds, the female sex is heterogametic, thus female gene conservation necessitates preservation of PGCs. PGC transplantation can generate germline chimeras in a host organism and develop into gametes. However, competition between host and transplanted PGCs hinder efficiency of germline chimera generation. We hypothezised that in sterile hybrid recipients with no germ cells of its own, transplanted donor PGCs may exclusively form gametes. Advantages of sterile hybrids as host for PGCs is compliant with many national regulations on genetically modified organisms and technically simpler procedure than the use of busulphan. Therefore, we investigated whether sterile interspecific hybrids may be suitable as recipients for supporting donor PGCs by injecting green fluorescent protein-labelled chicken PGCs into 3-day-old Guinea fowl and domestic fowl hybrid embryos and monitoring PGC development. The injected PGCs colonized almost 100% of the recipient gonads and produced mature spermatozoa after 44 weeks. However, gamete production in these hybrids was initiated much slower than in domestic fowls. This delay may be caused by suboptimal hormonal regulation of gametogenesis in the hybrids. Our results suggest that sterile interspecific hybrids may be suitable hosts for PGCs for efficient gamete production.

A dual ribosomal system in the zebrafish soma and germline.

Protein synthesis during vertebrate embryogenesis is driven by ribosomes of two distinct origins: maternal ribosomes synthesized during oogenesis and stored in the egg, and somatic ribosomes, produced by the developing embryo after zygotic genome activation (ZGA). In zebrafish, these two ribosome types are expressed from different genomic loci and also differ in their ribosomal RNA (rRNA) sequence. To characterize this dual ribosome system further, we examined the expression patterns of maternal and somatic rRNAs during embryogenesis and in adult tissues. We found that maternal rRNAs are not only expressed during oogenesis but are continuously produced in the zebrafish germline. Proteomic analyses of maternal and somatic ribosomes unveiled differences in core ribosomal protein composition. Most nucleotide differences between maternal and somatic rRNAs are located in the flexible, structurally not resolved expansion segments. Our in vivo data demonstrated that both maternal and somatic ribosomes can be translationally active in the embryo. Using transgenically tagged maternal or somatic ribosome subunits, we experimentally confirm the presence of hybrid 80S ribosomes composed of 40S and 60S subunits from both origins and demonstrate the preferential in vivo association of maternal ribosomes with germline-specific transcripts. Our study identifies a distinct type of ribosomes in the zebrafish germline and thus presents a foundation for future explorations into possible regulatory mechanisms and functional roles of heterogeneous ribosomes.

Initial formation of and sex differences in primordial germ cells in Japanese quail.

DEAD-box RNA helicase 4 (DDX4) is posited to be a key maternal germ cell factor regulating avian germ cell formation. We herein showed that the DDX4 gene product of zygotic genome activation associated with the nuclear localization of the cyclin D1 protein in presumptive primordial germ cells (PGCs) plays an essential role in the proliferation of PGCs using a CRISPR/Cas9 system approach combined with in vitro fertilization techniques in Japanese quail. A proteome analysis also revealed molecular-based differences in the features of early male and female PGCs.

Emerging Contributions of Pluripotent Stem Cells to Reproductive Technologies in Veterinary Medicine.

The generation of mature gametes and competent embryos in vitro from pluripotent stem cells has been successfully achieved in a few species, mainly in mice, with recent advances in humans and scarce preliminary reports in other domestic species. These biotechnologies are very attractive as they facilitate the understanding of developmental mechanisms and stages that are generally inaccessible during early embryogenesis, thus enabling advanced reproductive technologies and contributing to the generation of animals of high genetic merit in a short period. Studies on the production of in vitro embryos in pigs and cattle are currently used as study models for humans since they present more similar characteristics when compared to rodents in both the initial embryo development and adult life. This review discusses the most relevant biotechnologies used in veterinary medicine, focusing on the generation of germ-cell-like cells in vitro through the acquisition of totipotent status and the production of embryos in vitro from pluripotent stem cells, thus highlighting the main uses of pluripotent stem cells in livestock species and reproductive medicine.

Innovations in poultry reproduction using cryopreserved avian germ cells.

Meat and eggs from chicken are the major source of animal protein for the human population. The cryopreservation of poultry species is needed to guarantee sustainable production. Here, we describe the existing cryopreservation technologies for avian reproductive cells using embryonic germ cells, spermatozoa and ovarian tissues. We outline strategies to reconstitute chicken breeds from their cryopreserved embryonic germ cells using surrogate hosts and discuss the perspectives for genetic conservation and reconstitution of chicken and wild avian species using surrogate host animals.

Ten-eleven translocation 1 mediating DNA demethylation regulates the proliferation of chicken primordial germ cells through the activation of Wnt4/ß-catenin signaling pathway.

OBJECTIVE: The objective of this study was to investigate the regulation relationship of Teneleven translocation 1 (Tet1) in DNA demethylation and the proliferation of primordial germ cells (PGCs) in chickens. METHODS: siRNA targeting Tet1 was used to transiently knockdown the expression of Tet1 in chicken PGCs, and the genomic DNA methylation status was measured. The proliferation of chicken PGCs was detected by flow cytometry analysis and cell counting kit-8 assay when activation or inhibition of Wnt4/ß-catenin signaling pathway. And the level of DNA methylation and hisotne methylation was also tested. RESULTS: Results revealed that knockdown of Tet1 inhibited the proliferation of chicken PGCs and downregulated the mRNA expression of Cyclin D1 and cyclin-dependent kinase 6 (CDK6), as well as pluripotency-associated genes (Nanog, PouV, and Sox2). Flow cytometry analysis confirmed that the population of PGCs in Tet1 knockdown group displayed a significant decrease in the proportion of S and G2 phase cells, which meant that there were less PGCs entered the mitosis process than that of control. Furthermore, Tet1 knockdown delayed the entrance to G1/S phase and this inhibition was rescued by treated with BIO. Consistent with these findings, Wnt/ß-catenin signaling was inactivated in Tet1 knockdown PGCs, leading to aberrant proliferation. Further analysis showed that the methylation of the whole genome increased significantly after Tet1 downregulation, while hydroxymethylation obviously declined. Meanwhile, the level of H3K27me3 was upregulated and H3K9me2 was downregulated in Tet1 knockdown PGCs, which was achieved by regulating Wnt/ß-catenin signaling pathway. CONCLUSION: These results suggested that the self-renewal of chicken PGCs and the maintenance of their characteristics were regulated by Tet1 mediating DNA demethylation through the activation of Wnt4/ß-catenin signaling pathway.

Early gonadogenesis in Columba livia (birds: Columbiformes): Migration, colonization, and differentiation of germ cells.

In birds, primordial germ cells (PGCs) use the bloodstream to travel to a specific region, where the cells undergo extravasation followed by intrastromal migration to the gonadal crest for further colonization. Currently, DDX4, SSEA1, and Oct4 are used to identify germ cells. Other germline cell-associated molecules are N-cadherin, GnRHR, and 3ß hydroxysteroid dehydrogenase (3ßHSD), which have been used in mice and birds during gonadal development; however, its role in early gonadogenesis in birds is poorly described. This study aimed to evaluate the differential immunodetection of N-cadherin binding molecule, Oct4 pluripotency protein, GnRHR receptor, and 3ßHSD enzyme in Columba livia embryos during migration colonization of PGCs in the gonadal crest and early gonadogenesis. These markers were revealed by immunohistochemistry in histological preparations of C. livia corresponding to stages (S)15 to S40. Immunodetection of N-cadherin, Oct4, GnRHR, and 3ßHSD in the germ line of C. livia allowed the identification of PGCs in the yolk sac membrane at the level of the splanchnic mesoderm during migration to the genital crest and its colonization. In the same way, it was possible to characterize and localize PGCs during early gonadogenesis. This study in C. livia demonstrates that Oct4, N-cadherin, GNRHR, and 3ßHSD are immunodetected in PGCs and could be used as potential germline cell markers during cell migration out of blood vessels, colonization in the genital crest, and early gonadogenesis. Furthermore, this study could be used as a novel general model to understand the early gonadogenesis in altricial species.

Divergent Roles of KLF4 During Primordial Germ Cell Fate Induction from Human Embryonic Stem Cells.

As a core transcriptional factor regulating pluripotency, Krüppel-like factor 4 (KLF4) has gained much attention in the field of stem cells during the past decades. However, few research have focused on the function of KLF4 during human primordial germ cell (PGC) specification. Here, we induced human PGC-like cells (hPGCLCs) from human embryonic stem cells (hESCs) and the derived hPGCLCs upregulated PGC-related genes, like SOX17, BLIMP1, TFAP2C, NANOS3, and the naïve pluripotency gene KLF4. The KLF4-knockout hESCs formed typical multicellular colonies with clear borders, expressed pluripotency genes, such as NANOG, OCT4, and SOX2, and exhibited no differences in proliferation capacity compared with wild type hESCs. Notably, KLF4 deletion in hESCs did not influence the induction of PGCLCs in vitro. In contrast, overexpression of KLF4 during PGC induction process inhibited the efficiency of PGCLC formation from hESCs in vitro. Overexpression of KLF4 may regenerate the naïve ground state in hESCs and results in repression for PGC specification. Thus, KLF4 could be a downstream target of human PGC program and the upregulation of KLF4 is prepared for late stage of germline development.

Effects of Insulin on Proliferation, Apoptosis, and Ferroptosis in Primordial Germ Cells via PI3K-AKT-mTOR Signaling Pathway.

Primordial germ cells (PGCs) are essential for the genetic modification, resource conservation, and recovery of endangered breeds in chickens and need to remain viable and proliferative in vitro. Therefore, there is an urgent need to elucidate the functions of the influencing factors and their regulatory mechanisms. In this study, PGCs collected from Rugao yellow chicken embryonic eggs at Day 5.5 were cultured in media containing 0, 5, 10, 20, 50, and 100 µg/mL insulin. The results showed that insulin regulates cell proliferation in PGCs in a dose-dependent way, with an optimal dose of 10 µg/mL. Insulin mediates the mRNA expression of cell cycle-, apoptosis-, and ferroptosis-related genes. Insulin at 50 µg/mL and 100 µg/mL slowed down the proliferation with elevated ion content and GSH/oxidized glutathione (GSSG) in PGCs compared to 10 µg/mL. In addition, insulin activates the PI3K/AKT/mTOR pathway dose dependently. Collectively, this study demonstrates that insulin reduces apoptosis and ferroptosis and enhances cell proliferation in a dose-dependent manner via the PI3K-AKT-mTOR signaling pathway in PGCs, providing a new addition to the theory of the regulatory role of the growth and proliferation of PGC in vitro cultures.

Porcine Germ Cells Phenotype during Embryonic and Adult Development.

Primordial germ cells (PGCs) are the precursors of gametes. Due to their importance for the formation and reproduction of an organism, understanding the mechanisms and pathways of PGCs and the differences between males and females is essential. However, there is little research in domestic animals, e.g., swine, regarding the epigenetic and pluripotency profiles of PGCs during development. This study analyzed the expression of epigenetic and various pluripotent and germline markers associated with the development and differentiation of PGCs in porcine (pPGCs), aiming to understand the different gene expression profiles between the genders. The analysis of gonads at different gestational periods (from 24 to 35 days post fertilization (dpf) and in adults) was evaluated by immunofluorescence and RT-qPCR and showed phenotypic differences between the gonads of male and female embryos. In addition, the pPGCs were positive for OCT4 and VASA; some cells were H3k27me3 positive in male embryos and adult testes. In adults, the cells of the testes were positive for germline markers, as confirmed by gene expression analysis. The results may contribute to understanding the pPGC pathways during reproductive development, while also contributing to the knowledge needed to generate mature gametes in vitro.

Identification and Expressional Analysis of Putative PRDI-BF1 and RIZ Homology Domain-Containing Transcription Factors in Mulinia lateralis.

Mollusca represents one of the ancient bilaterian groups with high morphological diversity, while the formation mechanisms of the precursors of all germ cells, primordial germ cells (PGCs), have not yet been clarified in mollusks. PRDI-BF1 and RIZ homology domain-containing proteins (PRDMs) are a group of transcriptional repressors, and PRDM1 (also known as BLIMP1) and PRDM14 have been reported to be essential for the formation of PGCs. In the present study, we performed a genome-wide retrieval in Mulinia lateralis and identified 11 putative PRDMs, all of which possessed an N-terminal PR domain. Expressional profiles revealed that all these prdm genes showed specifically high expression levels in the given stages, implying that all PRDMs played important roles during early development stages. Specifically, Ml-prdm1 was highly expressed at the gastrula stage, the key period when PGCs arise, and was specifically localized in the cytoplasm of two or three cells of blastula, gastrula, or trochophore larvae, matching the typical characteristics of PGCs. These results suggested that Ml-prdm1-positive cells may be PGCs and that Ml-prdm1 could be a candidate marker for tracing the formation of PGCs in M. lateralis. In addition, the expression profiles of Ml-prdm14 hinted that it may not be associated with PGCs of M. lateralis. The present study provides insights into the evolution of the PRDM family in mollusks and offers a better understanding of the formation of PGCs in mollusks.

Characterization of the Postnatal Naked Mole-Rat Ovary: From Primordial Germ Cells to Meiotic Prophase I Oocytes.

The mammalian reproductive cycle, including those of humans and mice, begins very early in development. In utero, the ovaries become populated with primordial germ cells (PGCs) that will generate the oogonia. First, these cells proliferate mitotically, and then they trigger the meiotic program and initiate meiotic prophase I. Since these processes happen during gestation, their study had been very limited and challenging. Recently, we reported that, in the naked mole-rat (Heterocephalus glaber) ovary, there is mitotic expansion of the PGCs, and the initiation of the meiotic program occurs postnatally. In this chapter, we present a comprehensive collection of protocols that permit the analysis of naked mole-rat germ cells, from PGCs to oocytes, in meiotic prophase I, using in vivo and in vitro approaches.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction.

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Insights into Early Ontogenesis of Salmo salar: RNA Extraction, Housekeeping Gene Validation and Transcriptional Expression of Important Primordial Germ Cell and Sex-Determination Genes.

The challenge in extracting high-quality RNA impedes the investigation of the transcriptome of developing salmonid embryos. Furthermore, the mRNA expression pattern of important PGC and SD genes during the initial embryonic development of Salmo salar is yet to be studied. So, in the present study, we aimed to isolate high-quality RNA from eggs and developing embryos to check vasa, dnd1, nanos3a, sdf1, gsdf, amh, cyp19a, dmrt1 and foxl2 expression by qPCR. Additionally, four HKGs (GAPDH, UB2L3, eEf1a and ß-actin) were validated to select the best internal control for qPCR. High-quality RNA was extracted, which was confirmed by spectrophotometer, agarose gel electrophoresis and Agilent TapeStation analysis. UB2L3 was chosen as a reference gene because it exhibited lower intra- and inter-sample variation. vasa transcripts were expressed in all the developmental stages, while dnd1 was expressed only up to 40 d°C. Nanos3a was expressed in later stages and remained at its peak for a shorter period, while sdf1 showed an irregular pattern of mRNA expression. The mRNA expression levels of SD genes were observed to be upregulated during the later stages of development, prior to hatching. This study presents a straightforward methodology for isolating high-quality RNA from salmon eggs, and the resulting transcript profiles of significant PGC and SD genes in S. salar could aid in improving our comprehension of reproductive development in this commercially important species.

Endocannabinoid system and epigenetics in spermatogenesis and testicular cancer.

In mammals, male germ cell development starts during fetal life and is carried out in postnatal life with the formation of sperms. Spermatogenesis is the complex and highly orderly process during which a group of germ stem cells is set at birth, starts to differentiate at puberty. It proceeds through several stages: proliferation, differentiation, and morphogenesis and it is strictly regulated by a complex network of hormonal, autocrine and paracrine factors and it is associated with a unique epigenetic program. Altered epigenetic mechanisms or inability to respond to these factors can impair the correct process of germ development leading to reproductive disorders and/or testicular germ cell cancer. Among factors regulating spermatogenesis an emerging role is played by the endocannabinoid system (ECS). ECS is a complex system comprising endogenous cannabinoids (eCBs), their synthetic and degrading enzymes, and cannabinoid receptors. Mammalian male germ cells have a complete and active ECS which is modulated during spermatogenesis and that crucially regulates processes such as germ cell differentiation and sperm functions. Recently, cannabinoid receptor signaling has been reported to induce epigenetic modifications such as DNA methylation, histone modifications and miRNA expression. Epigenetic modifications may also affect the expression and function of ECS elements, highlighting the establishment of a complex mutual interaction. Here, we describe the developmental origin and differentiation of male germ cells and testicular germ cell tumors (TGCTs) focusing on the interplay between ECS and epigenetic mechanisms involved in these processes.

Infertility control of transgenic fluorescent zebrafish with targeted mutagenesis of the dnd1 gene by CRISPR/Cas9 genome editing.

Transgenic technology and selective breeding have great potential for the genetic breeding in both edible fish and ornamental fish. The development of infertility control technologies in transgenic fish and farmed fish is the critical issue to prevent the gene flow with wild relatives. In this study, we report the genome editing of the dead end (dnd1) gene in the zebrafish model, using the CRISPR/Cas9 technology to achieve a loss-of-function mutation in both wild-type zebrafish and transgenic fluorescent zebrafish to develop complete infertility control technology of farmed fish and transgenic fish. We effectively performed targeted mutagenesis in the dnd1 gene of zebrafish with a single gRNA, which resulted in a small deletion (-7 bp) or insertion (+41 bp) in exon 2, leading to a null mutation. Heterozygotes and homozygotes of dnd1-knockout zebrafish were both selected by genotyping in the F 1 and F 2 generations. Based on a comparison of histological sections of the gonads between wild-type, heterozygous, and homozygous dnd1 zebrafish mutants, the dnd1 homozygous mutation (aa) resulted in the loss of germ cells. Still, there was no difference between the wild-type (AA) and dnd1 heterozygous (Aa) zebrafish. The homozygous dnd1 mutants of adult zebrafish and transgenic fluorescent zebrafish became all male, which had normal courtship behavior to induce wild-type female zebrafish spawning. However, they both had no sperm to fertilize the spawned eggs from wild-type females. Thus, all the unfertilized eggs died within 10 h. The targeted mutagenesis of the dnd1 gene using the CRISPR/Cas9 technology is stably heritable by crossing of fertile heterozygous mutants to obtain sterile homozygous mutants. It can be applied in the infertility control of transgenic fluorescent fish and genetically improved farmed fish by selective breeding to promote ecologically responsible aquaculture.

Germline engineering of the chicken genome using CRISPR/Cas9 by in vivo transfection of PGCs.

Development of simple and readily adoptable methods to mediate germline engineering of the chicken genome will have many applications in research, agriculture and industrial biotechnology. We report germline targeting of the endogenous chicken Interferon Alpha and Beta Receptor Subunit 1 (IFNAR1) gene by in vivo transgenic expression of the high-fidelity Cas9 (Cas9-HF1) and guide RNAs (gRNAs) in chickens. First, we developed a Tol2 transposon vector carrying Cas9-HF1, IFNAR1-gRNAs (IF-gRNAs) and green fluorescent protein (GFP) transgenes (pTgRCG) and validated in chicken fibroblast DF1 cells. Next, the pTgRCG plasmid was directly injected into the dorsal aorta of embryonic day (ED) 2.5 chicken embryos targeting the circulating primordial germ cells (PGCs). The resulting chimera roosters generated a fully transgenic generation 1 (G1) hen with constitutive expression of Cas9-HF1 and IF-gRNAs (G1_Tol2-Cas9/IF-gRNA). We detected a spectrum of indels at gRNA-targeted loci in the G1_Tol2-Cas9/IF-gRNA hen and the indels were stably inherited by the G2 progeny. Breeding of the G1_Tol2-Cas9/IF-gRNA hen resulted in up to 10% transgene-free heterozygote IFNAR1 mutants, following null-segregation of the Tol2 insert. The method described here will provide new opportunities for genome editing in chicken and other avian species that lack PGC culture.

Proteomic Analysis of Murine Bone Marrow Very Small Embryonic-like Stem Cells at Steady-State Conditions and after In Vivo Stimulation by Nicotinamide and Follicle-Stimulating Factor Reflects their Germ-Lineage Origin and Multi Germ Layer Differentiation Potential.

Very small embryonic-like stem cells (VSELs) are a dormant population of development early stem cells deposited in adult tissues that as demonstrated contribute to tissue/organ repair and regeneration. We postulated developmental relationship of these cells to migrating primordial germ cells (PGCs) and explained the quiescent state of these cells by the erasure of differently methylated regions (DMRs) at some of the paternally imprinted genes involved in embryogenesis. Recently, we reported that VSELs began to proliferate and expand in vivo in murine bone marrow (BM) after exposure to nicotinamide (NAM) and selected pituitary and gonadal sex hormones. In the current report, we performed proteomic analysis of VSELs purified from murine bone marrow (BM) after repeated injections of NAM + Follicle-Stimulating Hormone (FSH) that in our previous studies turned out to be an effective combination to expand these cells. By employing the Gene Ontology (GO) resources, we have performed a combination of standard GO annotations (GO-CAM) to produce a network between BM steady-state conditions VSELs (SSC-VSELS) and FSH + NAM expanded VSELs (FSH + NAM VSELs). We have identified several GO biological processes regulating development, organogenesis, gene expression, signal transduction, Wnt signaling, insulin signaling, cytoskeleton organization, cell adhesion, inhibiting apoptosis, responses to extra- and intracellular stimuli, protein transport and stabilization, protein phosphorylation and ubiquitination, DNA repair, immune response, and regulation of circadian rhythm. We report that VSELs express a unique panel of proteins that only partially overlapped with the proteome of BM - derived hematopoietic stem cells (HSCs) and hematopoietic mononuclear cells (MNCs) and respond to FSH + NAM stimulation by expressing proteins involved in the development of all three germ layers. Thus, our current data supports further germ-lineage origin and multi germ layer differentiation potential of these cells.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Finer resolution analysis of transcriptional programming during the active migration of chicken primordial germ cells.

Primordial germ cells (PGCs) in chickens polarize and move passively toward the anterior region by the morphogenetic movement of the embryo. Further migration of PGCs towards the genital ridge via the germinal crescent region and blood vessels occurs actively through the chemoattractive signals. The mechanisms of initiation of PGCs migration, lodging the PGCs in the vascular system, and colonization of PGCs in the gonads are well-studied. However, transcriptome sequencing-based cues directing the migration of the PGCs towards gonads, some of the relevant molecules, biological processes, and transcription factors (TFs) are less studied in chickens. The current study comprehensively interprets the transcriptional programming of PGCs during their active migration (E2.5 to E8). Current results revealed several vital understandings, including a set of genes that upregulated male-specifically (XPA, GNG10, RPL17, RPS23, and NDUFS4) or female-specifically (HINTW, NIPBL, TERAL2, ATP5F1AW, and SMAD2W) in migrating PGCs, and transcriptionally distinct PGCs, particularly in the gonadal environment. We identified DNA methylation and histone modification-associated genes that are novel in chicken PGCs and show a time-dependent enrichment in migrating PGCs. We further identified a large number of differentially expressed genes (DEGs, including TFs) in blood PGCs (at E2.5) compared to gonadal PGCs (at E8) in both sexes; however, this difference was greater in males. We also revealed the enriched biological processes and signaling pathways of significant DEGs identified commonly, male-specifically, or female-specifically between the PGCs isolated at E2.5, E6, and E8. Collectively, these analyses provide molecular insights into chicken PGCs during their active migration phase.