Germ Cell Formation Research Articles

Inhibition of Autophagy Maintains ESC Pluripotency and Inhibits Primordial Germ Cell Formation in Chickens.

Autophagy plays an important role in the pluripotency and differentiation of stem cells. Transcriptome data showed that the autophagy genes MAP1LC3A and MAP1LC3B were significantly upregulated in primordial germ cells (PGCs). The Kyoto Encyclopedia of Genes and Genome (KEGG) results showed that the lysosome signaling pathway, which is related to autophagy, was significantly enriched in PGCs. Quantitative RT-PCR, western blotting, and transmission electron microscopy (TEM) results showed that autophagy was expressed in both embryonic stem cells (ESCs) and PGCs but was significantly activated in PGCs. To explore the role of autophagy in the differentiation of chicken ESCs into PGCs, autophagy was activated and inhibited using rapamycin and bafilomycin A1, respectively. Results of qRT-PCR, flow cytometry, and indirect immunofluorescence showed that the efficiency of PGC formation significantly decreased after autophagy inhibition. Our results showed, for the first time, that autophagy plays an indispensable role in the formation of chicken PGCs, which lays the foundation for studying the mechanism of autophagy in chicken PGCs and in bird gene editing and the rescue of endangered birds.

Assessment of Thyroid Functions and its Relationship With Semen Quality in Men

Introduction: In recent years, a partial relationship has been discovered between thyroid hormones and the formation of germ cells and the process of spermatogenesis. The current study aimed to assess thyroid hormone levels and the relationship between those levels and semen quality. Methods: Forty-seven infertile males, as the treatment group, and 25 healthy individuals, as the control group, were enrolled in this study. Thyroid-stimulating hormone (TSH), triiodothyronine (T3) hormone, and tetraiodothyronine (T4) hormone were calculated, and the parameters of seminal fluid (count, motility, and morphology) were assessed for semen quality. Results: The results demonstrated that sperm counts, sperm motility%, and normal morphology% were significantly lower in the infertile male group compared with the healthy group. The results further represented a highly significant level of TSH and total T3, while the total T4 was negligible in the infertile male group in comparison with the healthy group. The infertile male group was divided into subgroups based on sperm abnormalities, including asthenozoospermia, oligozoospermia, and azoospermia. Based on the findings, there was a significant reduction in TSH, T3, and T4 levels in oligozoospermia compared with the other groups. However, the levels of TSH, T3, and T4 were significantly higher in asthenozoospermia compared with the other groups, demonstrating the existence of a relationship between thyroid indicators and Asthenozoospermia. Conclusion: Overall, the mean serum levels of TSH, T3, and T4 were significantly lower in the infertile male group compared with the healthy group. Thus, they were negatively associated with sperm counts, motility%, and normal morphology%. Hence, these negative impacts on thyroid hormones were associated with different sperm abnormalities and semen quality in the infertile males group.

Formation, Application, and Significance of Chicken Primordial Germ Cells: A Review.

Chicken is one of the most widely consumed sources of protein globally. Primordial germ cells (PGCs) are the precursors for ova and sperm. One of the early embryogenesis events in most animals is the segregation of the somatic and germ lineages. PGC cultures occur in the germline, and PGCs are less studied in many species. It is relatively challenging to separate, cultivate, and genetically alter chicken without mutating the basic germline. The present study aims to gather previous research about chicken PGCs and provide a customized review of studies and developments in the field of PGCs, especially for avian species. Furthermore, we show that the propagation of chicken PGCs into embryonic germ cells that contribute to somatic tissues may be produced in vitro. Primordial germ cells offer an ideal system in developmental biology, as these cells play a vital role in the genetic modification and treatment of infertility. Cryopreservation helps to maintain genetic resources and sustainable production in the poultry industry. Keeping in mind the significance of cryopreservation for storage and gametogenesis, we discuss its role in the preservation of primordial germ cells. Transgenesis and genetic modifications in chicken lead to the development of various medicinal chicken varieties and aid in improving their production and quality for consumption purposes. Additionally, these characteristics open up new possibilities for modifying the chicken genome for agricultural and medical purposes.

DNA sequencing in the classroom: complete genome sequence of two earwig (Dermaptera; Insecta) species

BackgroundDespite representing the largest fraction of animal life, the number of insect species whose genome has been sequenced is barely in the hundreds. The order Dermaptera (the earwigs) suffers from a lack of genomic information despite its unique position as one of the basally derived insect groups and its importance in agroecosystems. As part of a national educational and outreach program in genomics, a plan was formulated to engage the participation of high school students in a genome sequencing project. Students from twelve schools across Chile were instructed to capture earwig specimens in their geographical area, to identify them and to provide material for genome sequencing to be carried out by themselves in their schools.ResultsThe school students collected specimens from two cosmopolitan earwig species: Euborellia annulipes (Fam. Anisolabididae) and Forficula auricularia (Fam. Forficulidae). Genomic DNA was extracted and, with the help of scientific teams that traveled to the schools, was sequenced using nanopore sequencers. The sequence data obtained for both species was assembled and annotated. We obtained genome sizes of 1.18 Gb (F. auricularia) and 0.94 Gb (E. annulipes) with the number of predicted protein coding genes being 31,800 and 40,000, respectively. Our analysis showed that we were able to capture a high percentage (≥ 93%) of conserved proteins indicating genomes that are useful for comparative and functional analysis. We were also able to characterize structural elements such as repetitive sequences and non-coding RNA genes. Finally, functional categories of genes that are overrepresented in each species suggest important differences in the process underlying the formation of germ cells, and modes of reproduction between them, features that are one of the distinguishing biological properties that characterize these two distant families of Dermaptera.ConclusionsThis work represents an unprecedented instance where the scientific and lay community have come together to collaborate in a genome sequencing project. The versatility and accessibility of nanopore sequencers was key to the success of the initiative. We were able to obtain full genome sequences of two important and widely distributed species of insects which had not been analyzed at this level previously. The data made available by the project should illuminate future studies on the Dermaptera.

Oocyte Arrested at Metaphase II Stage were Derived from Human Pluripotent Stem Cells in vitro

Initiation of meiosis is the most difficult aspect of inducing competent oocytes differentiation from human stem cells in vitro. Human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) were cultured with follicle fluid, cytokines and small molecule to induced oocyte-like cells (OLCs) formation through a three-step induction procedure. Expression of surface markers and differentiation potential of germ cells were analyzed in vitro by flow cytometry, gene expression, immunocytochemistry, western blotting and RNA Sequencing. To induce the differentiation of hiPSCs into OLCs, cells were firstly cultured with a primordial germ cell medium for 10 days. The cells exhibited similar morphological features to primordial germ cells (PGCs), high expressing of germ cell markers and primordial follicle development associated genes. The induced PGCs were then cultured with the primordial follicle-like cell medium for 5 days to form the induced follicle-like structures (iFLs), which retained both primordial oocytes-like cells and granulosa-like cells. In the third step, the detached iFLs were harvested and transferred to the OLC-medium for additional 10 days. The cultured cells developed cumulus-oocyte-complexes (COCs) structures and OLCs with different sizes (50–150 μm diameter) and a zona pellucida. The in vitro matured OLCs had polar bodies and were arrested at metaphase II (MII) stage. Some OLCs were self-activated and spontaneously developed into multiple-cell structures similar to preimplantation embryos, indicating that OLCs were parthenogenetically activated though in vitro fertilization potential of OLCs are yet to be proved. in vitro maturation of OLCs derived from hiPSCs provides a new means to study human germ cell formation and oogenesis.Graphical

Prime editing in chicken fibroblasts and primordial germ cells.

CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. However, there has been no report of taking advantage of the PE system for gene editing in primordial germ cells (PGCs) thus far. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and PGCs. By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chicken fibroblasts and PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.

Identification and Functional Analysis of MAPKAPK2 in Hyriopsis cumingii.

MAPKAPK2 (MK2) is an important regulator of the p38 mitogen-activated protein kinase (p38 MAPK) pathway, which is involved in a plethora of cellular processes concluding the development of gamete cells in meiosis and resisting pathogenic bacterial infestation. Hyriopsis cumingii is a significant mussel resource in China and a good material for pearl breeding. To explore the role of MK2 in H. cumingii, MK2 was identified and cloned, whose full-length cDNA was 1568 bp, including 87 bp in 5' UTR, 398 bp in 3' UTR, and 1083 bp in the open reading frame (ORF) region, encoding 360 amino acids. The expression of MK2 was the highest in the gills. Meanwhile, there was a significant difference in the gonads. After Aeromonas hydrophila and Lipopolysaccharide (LPS) infestation, the transcript level of the MK2 was upregulated in the gills. It indicated that MK2 might be involved in the innate immune response of H. cumingii after a pathogenic attack. After quantifying H. cumingii of different ages, it was found that the expression of MK2 was highest at 1 year old. In situ hybridization (ISH) results showed that the blue-purple hybridization signal was very significant in the oocytes and egg membranes of the female gonads of H. cumingii. The expression of MK2 increased gradually at the age of 1 to 5 months and showed a downward trend at the age of 5 to 8 months. It was suggested that MK2 might play an important role in the formation of primitive germ cells in H. cumingii. To sum up, MK2 might not only be involved in the immune response against pathogenic bacterial infection but also might play an important role in the development of the gonads in H. cumingii.

In vitro germ cell induction from fertile and infertile monozygotic twin research participants

SummaryHuman induced pluripotent stem cells (hiPSCs) enable reproductive diseases to be studied when the reproductive health of the participant is known. In this study, monozygotic (MZ) monoamniotic (MA) twins discordant for primary ovarian insufficiency (POI) consent to research to address the hypothesis that discordant POI is due to a shared primordial germ cell (PGC) progenitor pool. If this is the case, reprogramming the twin’s skin cells to hiPSCs is expected to restore equivalent germ cell competency to the twins hiPSCs. Following reprogramming, the infertile MA twin's cells are capable of generating human PGC-like cells (hPGCLCs) and amniotic sac-like structures equivalent to her fertile twin sister. Using these hiPSCs together with genome sequencing, our data suggest that POI in the infertile twin is not due to a genetic barrier to amnion or germ cell formation and support the hypothesis that during gestation, amniotic PGCs are likely disproportionately allocated to the fertile twin with embryo splitting.

Short telomeres impede germ cell specification by upregulating MAPK and TGFβ signaling.

Functional telomeres protect chromosome ends and play important roles in stem cell maintenance and differentiation. Short telomeres negatively impact germ cell development and can contribute to age-associated infertility. Moreover, telomere syndrome resulting from mutations of telomerase or telomere-associated genes exhibits short telomeres and reduced fertility. It remains elusive whether and how telomere lengths affect germ cell specification. We report that functional telomere is required for the coordinated germ cell and somatic cell fate decisions. Using telomerase gene Terc deficient mice as a model, we show that short telomeres restrain germ cell specification of epiblast cells but promote differentiation towards somatic lineage. Short telomeres increase chromatin accessibility to elevate TGFβ and MAPK/ERK signaling for somatic cell differentiation. Notably, elevated Fst expression in TGFβ pathway represses the BMP4-pSmad signaling pathway, thus reducing germ cell formation. Re-elongation of telomeres by targeted knock-in of Terc restores normal chromatin accessibility to suppress TGFβ and MAPK signaling, thereby facilitating germ cell formation. Taken together, our data reveal that functional telomeres are required for germ cell specification by repressing TGFβ and MAPK signaling.

Sertoli cells require TDP-43 to support spermatogenesis†.

TAR DNA binding protein of 43 kD (TDP-43) is an evolutionarily conserved, ubiquitously expressed transcription factor and RNA-binding protein with major human health relevance. TDP-43 is present in Sertoli and germ cells of the testis and is aberrantly expressed in the sperm of infertile men. Sertoli cells play a key role in spermatogenesis by offering physical and nutritional support to male germ cells. The current study investigated the requirement of TDP-43 in Sertoli cells. Conditional knockout (cKO) of TDP-43 in mouse Sertoli cells caused failure of spermatogenesis and male subfertility. The cKO mice showed decreased testis weight, and low sperm count. Testis showed loss of germ cell layers, presence of vacuoles, and sloughing of round spermatids, suggesting loss of contact with Sertoli cells. Using a biotin tracer, we found that the blood-testis barrier (BTB) was disrupted as early as postnatal day 24 and worsened in adult cKO mice. We noted aberrant expression of the junction proteins connexin-43 (gap junction) and N-cadherin (ectoplasmic specialization). Oil Red O staining showed a decrease in lipid droplets (phagocytic function) in tubule cross-sections, Sertoli cells cytoplasm, and in the lumen of seminiferous tubules of cKO mice. Finally, qRT-PCR showed upregulation of genes involved in the formation and/or maintenance of Sertoli cell junctions as well as in the phagocytic pathway. Sertoli cells require TDP-43 for germ cell attachment, formation and maintenance of BTB, and phagocytic function, thus indicating an essential role for TDP-43 in the maintenance of spermatogenesis.

Distinct p53 isoforms code for opposing transcriptional outcomes.

p53 genes are conserved transcriptional activators that respond to stress. These proteins can also downregulate genes, but the mechanisms are not understood and are generally assumed to be indirect. Here, we investigate synthetic and native cis-regulatory elements in Drosophila to examine opposing features of p53-mediated transcriptional control invivo. We show that transcriptional repression by p53 operates continuously through canonical DNA binding sites that confer p53-dependent transactivation at earlier developmental stages. p53 transrepression is correlated with local H3K9me3 chromatin marks and occurs without the need for stress or Chk2. In sufficiency tests, two p53 isoforms qualify as transrepressors and a third qualifies as a transcriptional activator. Targeted isoform-specific knockouts dissociate these opposing transcriptional activities, highlighting features that are dispensable for transactivation but critical for repression and for proper germ cell formation. Together, these results demonstrate that certain p53 isoforms function as constitutive tissue-specific repressors, raising important implications for tumor suppression by the human counterpart.

O-161 Using pluripotent stem cells for in vitro gametogenesis

Abstract Human reproduction depends on the formation of germ cells, the cell lineage responsible for making gametes. Gametes are the only cell types in the body capable of passing DNA from one generation to the next, therefore problems at any stage of germ cell development leading up to the formation of gametes can lead to reproductive challenges or even premature ovarian aging. In humans, germ cell development begins soon after embryo implantation with the specification of primordial germ cells (PGCs) from peri-implantation progenitor cells. Once PGC specification has finished, no other cell type in the body is capable of contributing to the germ cell lineage. Therefore, failure to specify PGCs will lead to certain infertility. In order to study PGC development, my laboratory animal models, stem cells and organoid technology. The advantage of using laboratory animal model is that PGC specification development and differentiation can be studied in vivo. The disadvantage is that human-specific mechanisms of PGC specification can be overlooked when not using human cells in research. To overcome this, my lab builds and uses stem cell models of peri-implantation human development in order to understand PGC specification as well as stem cell models to study in vitro gametogenesis. In this lecture, I will discuss latest advances in stem cell technology to understand fertility and infertility as well as human embryology.

P-543 The role of FMRP-associated microRNAs miR-125b and miR-132 and their target genes in the regulation of ovarian function

Abstract Study question Do FMRP (Fragile-X-Mental Retardation 1 Protein)-associated miRNA miR-125b, miR-132 and their target genes participate in the FMR1/FMRP regulatory loop and result in altered ovarian response? Summary answer miR-125b and its target gene TP53 (Tumor Protein P53) are downregulated in follicular fluid and granulosa cells of poor ovarian responders, respectively. What is known already MicroRNAs are a class of endogenous non-coding RNAs that play an important role in ovarian function and follicular development. It was found that miRNAs directly interact with FMRP and are involved in translational repression. MicroRNAs miR-125b and miR-132 have been shown to associate with FMRP in neural cells, but their relationship in the human ovary has not yet been evaluated. TP53 and LIN28A (Lin-28 Homolog A) are targets of miR-125b, the former regulating cell cycle and apoptosis, and the latter involved in germ cell formation. Investigating these molecules more thoroughly may provide insight into the pathogenesis of poor ovarian response. Study design, size, duration RNA from granulosa cells (GCs) and follicular fluid (FF) from poor (POR, nGC = 26, nFF = 12) and normal (NOR, nGC = 31, nFF = 16) ovarian responders was extracted and reverse transcription was carried out. RT-PCR was then performed to compare the relative expression of the targets, with NOR serving as the control group. Sample collection is ongoing and began in 2013. Participants/materials, setting, methods 57 patients who underwent ovarian stimulation were recruited and divided into two groups based on their ovarian response according to the Bologna criteria. GCs (n = 57) and FF (n = 28) were collected during oocyte retrieval. RNA was extracted and specific or non-specific cDNA was synthesized depending on the target. RT-PCR was performed with TaqMan reagents. Statistical analysis was performed using the 2–ΔΔCt method, Student’s t-test and Pearson’s coefficient. Main results and the role of chance We found that miR-125b was significantly downregulated in FF of the POR group (p = 0,0190), with no similar effect observed in GCs. The miR-132 levels showed no difference between the NOR and POR groups. This finding suggests that miR-125b may play a role in the cell-to-cell communication, and its suppression may be related to poor ovarian response. We then investigated two potential miR-125b target genes, TP53 and LIN28A, and their relation to FMR1. While LIN28A is similarly expressed in both groups, TP53 is significantly downregulated in GCs of POR group (p = 0,0086). Interestingly, TP53 expression correlates moderately positively with FMR1> (Pearson’s r = 0,5) in the NOR group, while no significant correlation is evident in the POR group (Pearson’s r = 0,19). These results suggest the existence of regulatory mechanisms involving miR-125b, TP53, FMRP and FMR1 that may be disrupted in case of poor ovarian response. Limitations, reasons for caution More patient samples are needed to confirm the current results. A possible age-related influence cannot be excluded because of the existing difference between the groups. Wider implications of the findings For the first time, the role of miR-125b together with its target TP53 was investigated in the human ovary, suggesting that they are involved in the FMR1/FMRP regulatory loop and thus providing more insight into the etiology and pathogenesis of poor ovarian response. Trial registration number not applicable

Examination of the role of the forkhead box L2 (Foxl2) in gonadal and embryonic development in the boring giant clam Tridacna crocea

Tridacna crocea, the smallest and earliest sexually maturing giant clam species, harbours important ecological value in maintaining coral reefs and holds potential for development as an ornamental species. At present, the molecular mechanism of gonadal development of this species remains largely unclear, and this hinders its artificial breeding and subsequent resource proliferation. The transcription factor forkhead box L subfamily member 2 (FoxL2) is one of the most conserved genes involved in the process of ovarian differentiation and development in many vertebrates and invertebrates. In this study, the Foxl2 gene (Tc-Foxl2) was cloned and characterized from T. crocea for the first time. The ORF of Tc-Foxl2 cDNA encoded a putative protein of 384 amino acid residues with a deduced molecular mass of 43.39 kDa and a theoretical isoelectric point (pI) of 8.92. Phylogenetic analysis indicated that the amino acid sequence of Tc-Foxl2 contained a typical FH domain and clustered with molluscan Foxl2 homologues. Tc-Foxl2 was mainly expressed in the gonads of females and hermaphroditic individuals but was fairly low in the gonads of males and resting individuals. Quantitative RT–PCR revealed that the expression of Tc-Foxl2 mRNA was significantly upregulated during vitellogenesis, while in situ hybridization indicated that Tc-Foxl2 mRNA was mainly detectable in follicle cells in the ovary. Furthermore, RNAi-mediated Tc-Foxl2 knockdown impeded the further growth of oocytes on the 14th day postinjection in the process of early vitellogenesis. Whole-mount in situ hybridization and qRT–PCR analyses demonstrated that during embryonic development, Tc-Foxl2 mRNA had a conserved expression profile, which may be involved in the formation of primordial germ cells. These results collectively suggest that Tc-Foxl2 is a female-biased gene that is involved in the vitellogenesis and embryonic development in Tridacna crocea.

In toto light sheet fluorescence microscopy live imaging datasets of Ceratitis capitata embryonic development

The Mediterranean fruit fly (medfly), Ceratitis capitata, is an important model organism in biology and agricultural research with high economic relevance. However, information about its embryonic development is still sparse. We share nine long-term live imaging datasets acquired with light sheet fluorescence microscopy (484.5 h total recording time, 373 995 images, 256 Gb) with the scientific community. Six datasets show the embryonic development in toto for about 60 hours at 30 minutes intervals along four directions in three spatial dimensions, covering approximately 97% of the entire embryonic development period. Three datasets focus on germ cell formation and head involution. All imaged embryos hatched morphologically intact. Based on these data, we suggest a two-level staging system that functions as a morphogenetic framework for upcoming studies on medfly. Our data supports research on wild-type or aberrant morphogenesis, quantitative analyses, comparative approaches to insect development as well as studies related to pest control. Further, they can be used to test advanced image processing approaches or to train machine learning algorithms and/or neuronal networks.

The Dynamic of PRAMEY Isoforms in Testis and Epididymis Suggests Their Involvement in Spermatozoa Maturation.

The preferentially expressed antigen in melanoma, Y-linked (PRAMEY) is a cancer/testis antigen expressed predominantly in bovine spermatogenic cells, playing an important role in germ cell formation. To better understand PRAMEY’s function during spermatogenesis, we studied the dynamics of PRAMEY isoforms by Western blotting (WB) with PRAMEY-specific antibodies. The PRAMEY protein was assessed in the bovine testicular and epididymal spermatozoa, fluid and tissues, and as well as in ejaculated semen. The protein was further examined, at a subcellular level in sperm head and tail, as well as in the subcellular components, including the cytosol, nucleus, membrane, and mitochondria. RNA expression of PRAMEY was also evaluated in testis and epididymal tissues. Our WB results confirmed the previously reported four isoforms of PRAMEY (58, 30, 26, and 13 kDa) in the bovine testis and spermatozoa. We found that testicular spermatozoa expressed the 58 and 30 kDa isoforms. As spermatozoa migrated to the epididymis, they expressed two additional isoforms, 26 and 13 kDa. Similarly, the 58 and 30 kDa isoforms were detected only in the testis fluid, while all four isoforms were detected in fluid from the cauda epididymis. Tissue evaluation indicated a significantly higher expression of the 58 and 13 kDa isoforms in the cauda tissue when compared to both the testis and caput tissue (p < 0.05). These results indicated that testis samples (spermatozoa, fluid, and tissue) expressed predominantly the 58 and 30 kDa PRAMEY isoforms, suggesting their involvement in spermatogenesis. In contrast, the 26 kDa isoform was specific to epididymal sperm and the 13 kDa isoform was marked in samples derived from the cauda epididymis, suggesting their involvement in sperm maturation. Results from the sperm head and tail experiments indicated that the 13 kDa isoform increased 4-fold in sperm tails from caput to cauda, suggesting this isoform may have a significant role in tail function. Additionally, the 13 kDa isoform increased significantly (p < 0.05) in the cytosol during epididymal passage and tended to increase in other subcellular components. The expression of PRAMEY in the sperm subcellular components during epididymal maturation suggests the involvement of PRAMEY, especially the 13 kDa isoform, in sperm motility.

Comparative Evaluation of the Parameters of Sperm Apoptosis of Young and Middle-Aged Men by Flow Cytometry.

A comparative analysis of the parameters characterizing sperm apoptosis of young (27-42 years) and middle-aged (44-51 years) men was performed by flow cytometry. Irrespective of age, activity of caspase-3 and p53-mediated controlling the transmission of apoptogenic signal transmission in gametes remained stable with the formation of germ cells with delayed (p<0.05) cell death according to the Annexin V-FITC+PI- criterion (predominantly in middle-aged men). Inhibition of the transmission of a proapoptogenic stimulus mediated by membrane cell death receptors (FAS) was also observed in this group. Comparison of indicators of sperm apoptosis showed age-related features of cell death, in particular, inhibition of membrane reception triggering FAS-dependent apoptosis, which is associated with insufficient phosphatidylserine production in middle-aged men, excessive life cycle duration, and aging of spermatozoa.

Culture supplementation of bFGF, GDNF, and LIF alters in vitro proliferation, colony formation, and pluripotency of neonatal porcine germ cells.

Gonocytes in the neonatal testis have male germline stem cell properties and as such have important potential applications in fertility preservation and regenerative medicine. Such applications require further studies aimed at increasing gonocyte numbers and evaluating their pluripotency in vitro. The objective of the present study was to test the effects of basic fibroblast growth factor (bFGF), glialcell line-derived neurotrophic factor (GDNF), and leukemia inhibitory factor (LIF) on in vitro propagation, colony formation, and expression of pluripotency markers of neonatal porcine gonocytes. Testis cells from 1-week-old piglets were cultured in basic media (DMEM + 15% FBS), supplemented with various concentrations of bFGF, GDNF, and LIF, either individually or in combinations, in a stepwise experimental design. Gonocytes and/or their colonies were evaluated every 7days and the gonocyte- (DBA) and pluripotency-specific markers (POU5F1, SSEA-1, E-cadherin, and NANOG) assessed on day 28. Greatest gonocyte numbers and largest colonies were found in media supplemented with 10ng/mL bFGF and 10ng/mL bFGF + 100ng/mL GDNF + 1500 U/mL LIF, respectively. The resultant gonocytes and colonies expressed both germ cell- and pluripotency-specific markers. These results shed light on thegrowth hormone requirements of porcine gonocytes for in vitro proliferation and colony formation.

Analysis of microRNA expression profiles during the differentiation of chicken embryonic stem cells into male germ cells

The differentiation of embryonic stem cells (ESCs) into germ cells in vitro could have very promising applications for infertility treatment and could provide an excellent model for uncovering the molecular mechanisms of germline generation. This study aimed to investigate the differentially expressed miRNAs (DEMs) during the differentiation of chicken ESCs (cESCs) into male germ cells and to establish a profile of the DEMs. Cells before and after induction were subjected to miRNA sequencing (miRNA-seq). A total of 113 DEMs were obtained, including 61 upregulated and 52 downregulated DEMs. GO and KEGG enrichment analyses showed that the target genes were enriched mainly in the MAPK signaling pathway, HTLV infection signaling pathway, cell adhesion molecule (CAM)-related pathways, viral myocarditis, Wnt signaling pathway, ABC transporters, TGF-β signaling pathways, Notch signaling pathways and insulin signaling pathway. The target genes of the miRNAs were related to cell binding, cell parts and biological regulatory processes. Six DEMs, let-7k-5p, miR-132c-5p, miR-193a-5p, miR-202-5p, miR-383-5p and miR-6553-3p, were assessed by qRT-PCR, and the results were consistent with the results of miRNA-seq. Based on qRT-PCR and western blot verification, miR-383-5p and its putative target gene STRN3 were selected to construct an STRN3 3'-UTR dual-luciferase gene reporter vector and its mutant vector. The double luciferase reporter activity of the cotransfected STRN3-WT + miR-383-5p mimics group was significantly lower (by approximately 46%) than that of the other five groups (p < 0.01). There was no significant difference in luciferase activity among the other 5 groups. This study establishes a DEM profile during the process of cESC differentiation into male germ cells; illustrates the mechanisms by which miRNAs regulate target genes; provides a theoretical basis for further research on the mechanisms of the formation and regulation of male germ cells; and provides an important strategy for gene editing, animal genetic resource protection and transgenic animal production.

Ultrastructure characteristics of primordial germ cells in stage X of pheasant (Phasianus colchicus) embryo.

Stage X is one of the formation stages in birds at which the blastoderm area is distinguished by two areas of area pellucida being responsible for formation of embryonic tissues and primordial germ cells, and area opaca forming the extra-embryonic tissues. Primordial germ cells are multi-potent stem cells giving rise to spermatogonia or oogonia. The present study was carried out to describe the characteristics of primordial germ cells in stage X of pheasants' embryo using a transmission electron microscope. The blastoderm was dissected out from embryos which were already incubated for 12 hr. Toluidine blue was used for staining semi-thin sections; lead citrate and uranyl acetate were also used to stain ultra-thin sections. Images of primordial germ cells elucidated that the nucleus was situated eccentrically and had a compact spherical structure. Moreover, the nucleolus appeared elongated and was located eccentrically. The cytoplasm was composed of yolk granules and glycogen particles. Mitochondria were observed as round structures in the cytoplasm. The most important finding was that the primordial germ cells contained yolk granules, mitochondria and small amount of glycogen at this stage.