Background: Germ cells undergo towards male or female pathways to produce spermatozoa or oocyte respectively which is essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the potential of trans-differentiation to the multiple cell lineages. Methods: Herein, rat MSCs were isolated from bone marrow and characterized by their morphological features, expression of MSC surface markers, and in vitro differentiation capability. Results: Thereafter, we induced these cells only by retinoic acid supplementation in MSC medium and, could able to show that bone marrow derived MSCs are capable to trans-differentiate into male germ cell-like cells in vitro. We characterized these cells by morphological changes, the expressions of germ cell specific markers by immunophenotyping and molecular biology tools. Further, we quantified these differentiated cells. Conclusions: This study suggests that only Retinoic acid in culture medium could induce bone marrow MSCs to differentiate germ cell-like cells in vitro . This basic method of germ cell generation might be helpful in the prospective applications of this technology.

Testicular germ cell tumors (GCT) arise from embryonic or extraembryonic differentiated totipotential germ cells. The tumor cells can differentiate into germ cells as well as, other lineages including yolk sac tumors, choriocarcinomas, and teratomas. Mixed GCT is composed of more than one GCT component including one or more nonseminomatous elements in a tumor, accounting for one-third of GCT. Herein we report the case of a collision tumor with two distinct and separated GCTs in the testis, adjacent to each other. A 48-year-old, previously healthy man showed the hard swelling and heterogeneous enhancing mass in the right scrotum, and right orchiectomy was performed. Grossly, the lesion was two distinct and well-circumscribed masses in the testis. Microscopically, a larger tumor was immature teratoma (prepubertal type) and another smaller tumor was seminoma. These two tumors can be from a common precursor, germ cell neoplasia in situ (GCNIS); however, they are two distinct pathological entities. Given that teratomas can evolve from seminomas by additional genetic alterations, seminomas are also a precursor for postpubertal-type teratomas. Two distinct GCNISs may occur at different times. Because GCNIS is patchy distributed, a close gross examination must be performed in GCNIS or GCT to miss other GCTs with poor prognosis and to prevent under-treatment.

Endocrine system of hormones is the critical factor for the development of testes. The levels of hormones are orchestrated by a positive or negative feedback system controlled by the hyphothalamic-pituitary-gonad axis. The aim of this study was to investigate the effect of unbalanced endocrine system induced by the hemi-castration on testicular development in stallions. Four Thoroughbred stallions (age ranging from 3 to 5 yr) were used in this study. To disturb endocrine system, hemicastration has been performed on the stallions. Several parameters including testicular weight, volume, germ cell population on the cross-sections of round tubule, and the area of seminiferous tubules of stallion testes collected at the 1st hemi-castration and the 2nd hemi-castration (about 1 year after 1st hemi-castration) were compared. Testosterone levels were compared for 3 weeks before, after 1st castration, and before 2nd castration using enzyme-linked immunosorbent assay (ELISA) analysis. Immunohistochemistry (IHC) procedure was conducted to compare germ cell populations between after 1st and 2nd castration using VASA antibody. The VASA positive cell population per a cross section of round seminiferous tubule was obtained by monitoring 100 tubules. Interestingly, the weight of testes obtained at 2nd hemi-castration (384±14 g) were significantly higher compared to that of testes collected at the 1st hemi-castration (288±34 g). The volume of testes (306±34 ml) collected at the 2nd hemi-castration was higher than that of testes (169±18 ml) collected at the 1st castration. In contrast, VASA positive germ cell population on the cross section of round tubule (124.9±12.4 vs 142.9±21.6) and the area of round tubule (124±9.7 vs 122.9±1.7 mm2) were not different after 1st castration and 2nd castration. the testosterone levels in the blood collected before, after 1st castration, and before 2nd castration were not significantly different. In conclusion, the hemi-castration induces testicular development to maintain the normal reproductive systems in stallions.

INTRODUCTION In rodent, molecular markers of spermatogonia, spermatocyte, spermatid and sperm have been identified. It has been reported that DBA, PGP 9.5 and NanpG can be the markers for spermatogonia in pig. For further understanding the spermatogenesis of the pig on morphological and molecular level, we report identification of testicular cells in neonatal and pubertal pig testis, and a putative marker for spermatogonia and spermatid in pig testis. MATERIALS AND METHODS Neonatal (3 day) and pubertal testis (150 day) was cut and fixed in Bouin’s solution for immunohistochemistry (IHC). Gonocytes were isolated from neonatal testis for immunocytochemistry (ICC). Based on references (Phillips et al., 2010), thirteen antibodies (VASA, Oct4, NanoG, PGP 9.5, DAZL, SCF, GFR-alpha 1, PLZF, c-kit, integrin-beta1, Thy1, Sohlh1 and CD9) were used for IHC and ICC. Paraffin section was performed for IHC. Gonocytes were attached to the APS-coated slides for ICC. HRP-conjugated and florescent-labeled secondary antibody was used for IHC and ICC, respectively. RESULTS In histological analysis, spermatogonia and Sertoli cells, which are enclosed by seminiferous tubule and connective tissue, were observed in neonatal testis. However, complete spermiogenesis, including spermatocyte, spermatid and spermatozoa, was not observed in neonatal testis. Spermatocyte, spermatid and elongated spermatid were observed in pubertal testis but matured spermatozoa were not observed. As a result, two antibodies (PGP 9.5 and GFR-alpha1) of thirteen antibodies were available for IHC and ICC. As reported in other studies, PGP 9.5 protein was detected in spermatogonia of ne-onatal in IHC. In addition, it was observed in spermatogonia of pubertal testis. GFR- alpha1 protein was detected in spermatogonia of neonatal testis and spermatid of pubertal testis. In ICC, PGP 9.5 protein was detected in gonocytes as reported in other studies. GFR-alpha1 was also observed in gonocyte isolated from pig testis. In this study, we found that 1) only spermatogonia exist in neonatal pig testis (3 day), 2) GFR-alpha1 is a new marker for spermatogenesis in pig testis.

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.

본 연구는 돼지에 있어서 정원줄기세포를 포함하는 정소세포를 recipient 돼지의 정소 내로 이식할 수 있는 기법을 개발하기 위하여 시행되었다. 공여세포는 10~14일령의 돼지로부터 채취된 정소에서 효소처리법을 이용하여 회수하였고, recipient의 정소 내로 이식하기 전 형광 마커(PHK26)로 표지하였다. 외과적 수술을 통하여 recipient 돼지부터 정소를 꺼낸 후 초음파 기기와 이식 장치를 이용하여 형광표지된 공여세포를 recipient 정소의 세정관 내로 이식하였다. 14주령의 recipient 정소에 5~7ml 의 공여 세포부유액을 주입하여 정소 내 50% 이상의 세정관 내로 새포부유액의 주입이 가능하였고, 세포부유액이 주입된 세정관 내에서 형광표지된 정소세포들이 고루 이식되어짐이 관찰되었다. 본 연구에서 개발한 이식 기법을 이용하여 효율적인 정소세포의 이식이 가능함에 따라 향후 돼지 정원줄기세포의 연구 및 활용법 개발에 획기적인 돌파구가 마련될 것으로 기대된다.

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of differentiation both in vitro and in vivo have been established. Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide inexhaustible source of karyoplasts in nuclear transfer (NT). In this study the efficiencies of NT using porcine EG and fetal fibroblast cells were compared. Two different techniques were used to perform NT. With conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes, the rates of development to the blastocyst stage in EG and somatic cell NT were 16.8% (59/351) and 14.5% (98/677), respectively. In piezo-driven microinjection (Honolulu method) of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 9.4% (9/96), respectively. Regardless of NT methods used in this study, EG cell NT gave rise to comparable rate of blastocyst development to somatic cell NT. Overall, EG cells can be used as karyoplast donor in NT procedure, and embryos can be produced by EG cell NT that may be used as an alternative to conventional somatic cell NT.