Spermatogonial stem cells are self-renewal and differentiate into sperm in post-pubertal mammals. There exists a balance between the self-renewal and differentiation in the testes. Spermatogonial stem cells make up only 0.03% of testicular cells in adult mice. These cells maintain sperm production by differentiating after puberty. Therefore, analyzing the expression of genes associated with spermatogenesis is critical for understanding differentiation. The present study aimed to establish the postnatal period of cells in relation to spermatogenesis. To study the expression of differentiated and undifferentiated marker genes in enriched spermatogonial stem cells, in vitro culture was performed and cells from pup (6–8-day-old) and adult (4-months-old) testicular tissues were isolated. As a result, undifferentiated genes, Pax7, Plzf, GFRa1, Etv5 and Bcl6b , were highly increased in cultured spermaotogonial stem cells compared with pup and adult testicular cells. On the other hands, differentiated gene, c-kit was highly increased in adult testicular cells, Also Stra8 gene was highly increased in pup and adult testicular cells. This study provides a better understanding of spermatogenesis-associated gene expression during postnatal periods.

As a preclinical study, many researchers have been attempted to convert the porcine PSCs into several differentiated cells with transplantation of the differentiated cells into the pigs. Here, we attempted to derive neuronal progenitor cells from pig embryonic germ cells (EGCs). As a result, neuronal progenitor cells could be derived directly from pig embryonic germ cells through the serum-free floating culture of EB-like aggregates (SFEB) method. Treating retinoic acid was more efficient for inducing neuronal lineages from EGCs rather than inhibiting SMAD signaling. The differentiated cells expressed neuronal markers such as PAX6, NESTIN, and SOX1 as determined by qRT-PCR and immunostaining. These data indicated that pig EGCs could provide valid models for human therapy. Finally, it is suggested that developing transgenic pig for disease models as well as differentiation methods will provide basic preclinical data for human regenerative medicine and lead to the success of stem cell therapy.

Mammalian fetal ovaries contains numerous primordial germ cells, however fewer ones can yield mature oocytes due to apoptosis and follicle atresia. Successful in vitro reconstitution of primordial germ cells has recently had a significant effect in the field of assisted reproductive technologies. However, the regulatory mechanisms underlying oogenesis remain unknown and recapitulation of oogenesis in vitro remains unachieved. Therefore, development of methods for obtaining mature oocytes by culturing the fetal ovaries in vitro could contribute to clarify these mechanisms. We adapt an in vitro system for culturing mouse fetal ovaries that support successful follicle assembly and improve oocyte growth and maturation. Ovarian tissues from 12.5 days postcoitum (dpc) fetal mice were cultured in vitro and the matured oocytes were differentiated from primordial germ cells after a 31 days culture period. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with artificial ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro. Taken together, this in vitro culture system is expected to aid in the development of new strategies to identify the reasons behind failure of follicle assembly and offer a platform for innovative research into preservation of female germ cells and conservation of endangered species.

The deleted in azoospermia like (DAZL) gene has been identified in many vertebrate species. DAZL shows high homology with deleted in azoospermia (DAZ) genes that identified only in humans, great apes and Old World monkeys, and boule homolog (BOLL) that identified in many vertebrate species. These genes encode RNA binding proteins (RBP), which regulate the post-transcriptional functions of several genes. In humans, DAZ copies are linked to Y chromosome, while DAZL and BOLL are linked to chromosomes 3 and 2, respectively. DAZ copies has been reported to express in prenatal and postnatal germ cells, particularly in the premeiotic spermatogonia. BOLL has been reported to express during the meiotic G2/M transition in germ cells. DAZL has been reported to express in all stages of germ cells. Compared to humans and mice, the detailed functionalities of DAZL is not clear in many vertebrate species. In our studies, we use chickens as an animal model to examine the expression profiling of DAZL gene in germ cells right from the early embryonic development to the adult. Also, we are studying the effects of small interfering RNA (siRNA) mediated knockdown of DAZL and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) mediated knockout of DAZL in the chicken primordial germ cells (PGCs). In the chicken, DAZL is linked to chromosome 2 (2p1.3-p1.2), and encodes a 289 amino acids protein. By in situ hybridization, we detected a strong expression of DAZL in the germ plasm of chicken oocytes. Later, the expression of DAZL was strongly detected in all stages of intrauterine development and post-ovipositional development especially in the PGC specifying cells. Moreover, the expression of DAZL was strong and constant in the male and female germ cells until adult stage. The siRNA mediated knockdown of DAZL significantly reduced the PGCs proliferation and increased the apoptosis in vitro. We examined the knockout efficiency of DAZL using CRISPR/Cas9 technique in chicken DF1 fibroblast cell line, prior to test in the PGCs. The results of T7 endonuclease I (T7E1) assay and subsequent sequencing indicates clear mutations on the DAZL gene in DF1 cells, and the method could be applicable to cause mutations on the DAZL gene in PGCs. In conclusion, chicken DAZL express in all stages of germ cells as a germ line marker, and alteration in the gene expression causes germ cells impairment.

Germ cells originate outside of the fetal gonads and migrate toward the genital ridges through the embryonic tissue. Germ cell is the most important and valuable cell in livestock because germ cell is the only cell type that can transfer the genetic information and content into the next generation. In this study, we established the primordial germ cell (PGC) lines derived from the fetal gonads of 6 day-old-embryonic chicks, and then cryopreserved for long-term storage. First, we determined each chick embryo sex by genomic PCR with DNA extracted from blood. After dissociation of the whole gonads from individual embryos, total gonadal cells were plated into the culture dish and cultured with 20% fetal bovine serum-contained culture media. PGC lines were derived from three different chicken strains; White Leghorn (WL), Korean Oge (KO), and a commercial line, Hyline. There was no significant difference between the efficiencies of the PGC line derivation according to the different chicken strains. Thus, PGC culture and long-term storage system could be applied to maintain the endangered avian species and also produce the offspring through germline chimera production system.

In the study for a differentiation and development of spermatogonial cells, the researchers should commonly require a simple, fast and reasonable method that could evaluate the developmental stage of male germ cells without any damage and also relentlessly culture them so far as a cell stage aiming at experimental applications. For developing the efficient method to identify the stage of sperm cells, the morphological characteristics of sperm cells were investigated by staining the cells with blue fluorescent dye Hoechst 33258, and a criterion for male germ cell classification was elicited from results of the previous investigation, then the efficiency of the criterion was verified by applying it to assort the germ cells recovered from male mice in age from 6 to 35 days. As morphological characteristics, spermatogonia significantly differed from spermatocytes in size, appearance and fluorescent patches of nucleus, and spermatids could also be distinguished from spermatozoa by making a difference in the volume and shape of nucleus and the shape and fluorescence of tail. Aforesaid criterion was applicable for classifying in vitro cultured sperm cells by verifying its efficiency and propriety for assorting the stages of testicular germ cells. However, the fluorescent staining showed that germ cells in mouse testis should be dramatically differentiated and developed at 21 days and 35 days of age, which were known as times of sexual puberty and maturity in male mice, respectively. In conclusion, the results indicated that this simple criterion for sperm cell classification using fluorescence staining with Hoechst 33258 may be highly efficient and reasonable for spermatogenesis study.

Phasianus colchicus is not only a beautiful bird but also a great value in science and under the threat of endanger. Hence, the aim of this study was to isolate the pheasant male germ cells (mGCs) and then induce them into elongated sperm-like cells in vitro. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the c-kit positive cells and c-kit negative cells examined by flow cytometry analysis (FCA) was 92.87% and 2.57%, respectively. Subsequently, the mGCs were induced for 48h in DMEM/F12 medium supplemented factors such as retinol acid, testosterone and bovine FSH, followed by 5 weeks in culture. We found that some elongated sperm-like cells appeared initially in vitro under inducement of stimulated factors. The elongated sperm-like cells showed in the expression of changed morphology and post-transcriptional marker such as spermatid associated (SPERT), spermatid perinuclear RNA binding protein (STRBP), round spermatid basic protein 1 (RSBN1) and SPER1L. Moreover, in DNA content identified assay, induced cells showed that the 1C DNA population markedly increased in differentiated group but it was not change in undifferentiated group. Successful in vitro differentiation of pheasant testicular germline cells into spermatids appears to offer extremely attractive potential for the conservation of endangered birds and treatment of male infertility.

Germ cell-specific hyaluronidases such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5) are in part responsible for dispersal of the cumulus cell mass, which is a critical step in establishing fertilization in mammals. In this study, we identified two testis-hyaluronidases, SPAM1 and Hyal5, in hamster and rat. These two genes were expressed specifically in the testis. At the protein level, hamster SPAM1 and Hyal5 display 78.7% and 75.4% identity with mouse SPAM1 and Hyal5. Further, the activity of the enzymes with respect to cumulus cell dispersion did not differ, although we observed that the enzymatic activity differed in pH range. These studies suggest that different sperm hyaluronidases are capable of dispersing the cumulus cell mass despite differences in enzyme activity.

Epigenetic modification including genome-wide DNA demethylation is essential for normal embryonic development. Insufficient demethylation of somatic cell genome may cause various anomalies and prenatal loss in the development of nuclear transfer embryos. Hence, the source of nuclear donor often affects later development of nuclear transfer (NT) embryos. In this study, appropriateness of porcine embryonic germ (EG) cells as karyoplasts for NT with respect to epigenetic modification was investigated. These cells follow methylation status of primordial germ cells from which they originated, so that they may contain less methylated genome than somatic cells. This may be advantageous to the development of NT embryos commonly known to be highly methylated. The rates of blastocyst development were similar among embryos from EG cell nuclear transfer (EGCNT), somatic cell nuclear transfer (SCNT), and intracytoplasmic sperm injection (ICSI) (16/62, 25.8% vs. 56/274, 20.4% vs. 16/74, 21.6%). Genomic DNA samples from EG cells (n=3), fetal fibroblasts (n=4) and blastocysts from EGCNT (n=8), SCNT (n=14) and ICSI (n=6) were isolated and treated with sodium bisulfite. The satellite region (GenBank Z75640) that involves nine selected CpG sites was amplified by PCR, and the rates of DNA methylation in each site were measured by pyrosequencing technique. The average methylation degrees of CpG sites in EG cells, fetal fibroblasts and blastocysts from EGCNT, SCNT and ICSI were 17.9, 37.7, 4.1, 9.8 and 8.9%, respectively. The genome of porcine EG cells were less methylated than that of somatic cells (p<0.05), and DNA demethylation occurred in embryos from both EGCNT (p<0.05) and SCNT (p<0.01). Interestingly, the degree of DNA methylation in EGCNT embryos was approximately one half of SCNT (p<0.01) and ICSI (p<0.05) embryos, while SCNT and ICSI embryos contained demethylated genome with similar degrees. The present study demonstrates that porcine EG cell nuclear transfer resulted in hypomethylation of DNA in cloned embryos yet leading normal preimplantation development. Further studies are needed to investigate whether such modification affects long-term survival of cloned embryos.

Accumulating evidence suggests that chemotherapy can cause long‐term detrimental effects and alter the biology of the recipient environment. Therefore, a subsequent report claimed that the transplantation of female germline stem cells (FGSCs) into the ovaries of recipient mice that were pretreated with a high dose of busulfan and cyclophosphamide (B/C) resulted in the successful production of offspring. Therefore, this study was conducted to further clarify the impact of female germ cell transplantation on female ovaries after B/C treatment. RT‐PCR analysis showed that the period of germ cell depletion coincide with decreased Figla, Lhx8, Nobox, Kit, and Sox3 gene expression in the B/Ctreated ovary. However, depletion of female germ cells is mediated by a Fas/FasL‐, Kit/ Kitl‐, TNF‐, p53‐ and autophagy‐ independent pathway. Also, histological analysis is similar to that of Nobox null‐derived ovaries, indicating that follicle death after B/C treatment might be caused by down‐regulating of Nobox pathway. When female mice during 15 weeks after B/C treatment were checked for reproductive activity, B/C treated mice did not produce their pups. In addition, when 3×106 GFP positive primordial follicles were injected into B/C treated female mouse ovaries, donor follicle were not able to colonize into the ovaries of recipients. In conclusion, these data from a preclinical mouse model strongly suggested that female ovary until 15 weeks after B/C treatment could not support environment for maturing of exogenous FGSCs.

Genomic reprogramming factors in the GV cytoplasm improved cloning efficiency in mice through the pre‐exposure of somatic cell nuclei to a GV cytoplasmic extract prior to nuclear transfer. To overcome difficulties in preparing mice oocyte extract, a pig GV oocyte extract (pGV extract) was developed to investigate the epigenetic reprogramming events in treated somatic cell nuclei. The pGV extract promoted colony formation concomitant with the expression of stem cell markers and repression of differentiated cell markers in treated cells. Using fibroblasts transfected with human Oct‐4 promoter‐ driven enhanced green fluorescent protein (Oct4‐EGFP), pGV extract treatment induced the reactivation of the Oct‐4 promoter in Oct4‐EGFP cells by 10 days post‐treatment. Interestingly, reconstructed embryos with pGV extract‐treated Oct4‐EGFP fibroblast nuclei showed prolonged expression of Oct4 in the ICM of embryos. Using donor nuclei treated with pGV extract, increase the number of high‐quality blastocysts that expressed Me‐H3‐K9, Oct4 and Nanog at levels comparable to in vitro fertilized embryos. The pGV extracttreated fibroblast cells can differentiated into neuronal, pancreas, cardiac, and endothelial lineages that were confirmed by antibodies against specific marker proteins. These data provide evidence for the generation of stem‐like cells from differentiated somatic cells by treatment with GV oocyte extracts in pig. Next, we identified germ line stem cells that supported oogenesis. female germ line stem cells (FGSC) from neonatal pig was established and cultured for more than 6 months. After long‐term culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. To further study developmental potential of oocyte‐like cells generated from FGSCs, these cells were aggregated with granulosa cells collected from neonatal pig ovaries. Interestingly after overnight culture in hanging drops, oocyte‐like cells aggregated with granulosa cells and formed structures very similar to primordial follicles containing the oocyte‐like cell in the middle and a layer of granulosa cells around it. Our results demonstrate the presence of a population of germ line stem cells in postnatal pig ovary with the ability to self‐renew and differentiate to oocyte‐like cells that might be useful for follicle engineering and assisted reproductive technologies. However, the functionality of FGSC‐derived oocytes us-ing in vitro maturation, fertilization and embryo development as well as ovarian transplantation is currently under investigation. In conclusion, gene manipulation of FGSCs or iPS cells is a rapid and efficient method of animal transgenesis and may serve as a powerful tool for biomedical science and biotechnology.

Testes‐derived unipotent male germ‐line stem (GS) cells can acquire multipotency under appropriate culture conditions to become mGS cells which can contribute to all three germ‐layers. This study was designed to investigate the epigenetic characteristics of mGS cells derived from adult mouse testes (maGS cells). The GS cells were isolated from 4 6 week DBA mouse and were cultured in Dulbecco’s modified Eagle Medium supplemented with 15% (v/v) fetal bovine serum, 1,000 U/ml LIF, 4 ng/ml GDNF at 37℃ in an humidified atmosphere of 5% CO2 in air to derive the maGS cells. The multipotency of maGS cells were verified by morphological and gene expression analyses, teratoma formation upon transplantation into nude mouse and in vitro differentiation ability. Bisulfite genomic sequencing revealed that GS cells had androgenetic DNA methylation pattern at the Igf2‐H19, Gnas‐Nespas , and Dlk1‐Dio3 imprinted gene clusters which changed to hemi‐zygotic embryonic stem (ES)‐cell like pattern in the maGS cells. Western blot analysis, using modification‐ and residue‐specific antibodies, revealed that both maGS and ES cells had similar level of histone di‐methylation at 4th and 27th lysine residue of histone 3 (H3K4me2 and H3K27me2) which represent “bivalent domain” for regulating self‐renewal and differentiation of mouse ES cells. Both maGS and ES cells also shared similar hisone modification for H3K9me2, H3K79me2, H3K9ac and H3K18ac. However, maGS cells had higher level of H3K- 36me2 and H3S10p. These data suggest that maGS and ES cells share several epigenetic characteristics but they also have their own unique epigenetic marks that may be useful as a molecular marker for their identification.

Unstable Epigenetic reprogramming was DNA methylation, imprinting, RNA silencing, co-valent modifications of histones and remodelling by other chromatin-associated complexes. After fusion with an enucleated oocyte, a differentiated somatic cell can restructure its genetic program and acquire totipotent characteristics. However, these cases happen only with low frequency. primordial germ cells (PGC) was effectively remove of epigenetic modifications in the genetic totipotency which is necessary for the development. The present study was in vitro development of reconstruct PGC NT embryos compared with somatic cell NT embryos. The rate of cleavage did not differ between NT embryos from PGC and somatic cells (87.26 vs 91.36%). However, the rate of development to the blastocyst stage was significantly higher in PGC cell NT than somatic cell NT (31.03 vs 19.27%). PGC from a slightly younger stage of development have succeed to promote normal development of recipient eggs. This difference in results between germ cell and somatic cell nuclear transfers could only be a reflection of intimate differences in their reprograming. These results suggest that PGC NT embryos are significantly higher for the in vitro development. Furthermore, These study may represent an approach towards achieving better production of transgenic animal.

Some tissues retain extensive regeneration potential through out adult life and remain as active sites of cell production. Various cell types present in tissues are being produced through proliferation and progressive specialization from a pool of stem cells. In this regard, adult stem cells (ASCs) are multipotent progenitor cells with an ability to proliferate in vitro and undergo extensive self-renewal and differentiation into a wide range of cell types, including adipocytes, chondrocytes, osteocytes, myocytes, cardiomyocytes and neurons. In addition, recent studies showing the abilities of ASCs in generating oocytes-like cells (OLCs) present new perspectives to understand the specification and interaction during the germ cell formation and oogenesis. In the present study, ASCs were established from skin, adipose and ovarian tissues of minipigs. Isolated cells exhibited a fibroblast-like morphology with higher proliferation potential and stronger alkaline phosphatase (AP) activity. ASCs from all tissues expressed pluripotent transcriptional factors, such as Oct-3/4, Nanog and Sox-2 and phenotypic markers, including CD29, CD44, CD90 and vimentin. Further, ASCs were successfully dIfferentiated into osteocytes, adipocytes and neuron-like cells. Upon induction in oogenesis specific media, all ASCs were capable of differentiation into OLCs by exhibiting distinct morphological features. Generated OLCs expressed a range of germ cell specific markers, such as Vasa, deleted in Azoospermia-like (DAZL) factor, stella, c-kit, c-Mos, synaptonemal complex protein 3 (SCP-3), growth differentiation factor 9b (GDF- 9b), zona pellucida C (ZPC) and follicle stimulating hormone receptor (FSHR) at different time points of induction. Differentiated OLCs were also positive for the expression of Vasa and DAZL protein markers. Our findings showing that OLCs can be generated from ASCs of different tissue origin may offer pig as a suitable model for designing transgenic application strategies for reproductive tissue therapy. However, further studies are needed to understand the cellular and molecular mechanisms involved in germ cell differentiation from tissue specific stem cells.

To evaluate the biohazard properties of an extremely low frequency electromagnetic field (ELF-EMF), we explored the physical properties of the ELF-EMF that generates the electric current induction in the secondary coil from the chamber of a primary solenoid coil. We subsequently explored the biological effects of a strong alternating electromagnetic field (EMF), ranging from 730-960 Gauss, on the mouse testis. Mice were exposed to an alternating EMF field induced by a rectangular electric current at 1, 7, 20, 40, and 80 Hertz, for 1, 3, 5, and 7 hours. The mouse testes were examined for proliferative activity and apoptosis using the in situ terminal deoxynucleotidyl transferase (TdT) method and by immunostaining of proliferating cell nuclear antigen (PCNA), respectively. We found that the electric currentm induction increased in the 6-8 Hertz range, and that exposure to an ELF-EMF induced the apoptosis of mouse spermatocytes. In situ TdT staining was found to be most prominent in 7 Hertz group, and gradually reduced in the 20, 40, and 80 Hertz groups. These data suggest that a strong EMF can induce reproductive cell death within a short time, and the harmful effects of the EMF are maximal at low frequency alternating EMFs.