Characterization and in vitro culture of male germ cells from developing bovine testis

The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. In this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stem cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stem cell potential in vitro.     

Pluripotent cell derivation from male germline cells by suppression of Dmrt1 and Trp53

Diploid germ cells are thought to have pluripotency potential. We recently described a method to derive pluripotent stem cells (PSCs) from cultured spermatogonial stem cells (SSCs) by depleting Trp53 and Dmrt1, both of which are known suppressors of teratomas. In this study, we used this technique to analyze the effect of this protocol in deriving PSCs from the male germline at different developmental stages. We collected primordial germ cells (PGCs), gonocytes and spermatogonia, and the cells were transduced with lentiviruses expressing short hairpin RNA against Dmrt1 and/or Trp53. We found that PGCs are highly susceptible to reprogramming induction and that only Trp53 depletion was sufficient to induce pluripotency. In contrast, gonocytes and spermatogonia were resistant to reprogramming by double knockdown of Dmrt1 and Trp53. PSCs derived from PGCs contributed to chimeras produced by blastocyst injection, but some of the embryos showed placenta-only phenotypes suggestive of epigenetic abnormalities of PGC-derived PSCs. These results show that PGCs and gonocytes/spermatogonia have distinct reprogramming potential and also suggest that fresh and cultured SSCs do not necessarily have the same properties.     

Biallele Expression of PEG10 Gene in Primordial Germ Cells Derived from Day 27 Porcine Fetuses

Primordial germ cells (PGCs) from day 27 porcine fetuses have often been isolated to establish pluripotent embryonic germ (EG) cell lines, but little is known regarding their imprinted gene status. In our study, we attempted to detect the imprinted gene expression of cloned embryos and EG cells derived from individual PGC of day 27 and day 35, using single nucleotide polymorphism (SNP) analysis of the paternally expression gene 10 (PEG10) as a sign of parental‐origin‐specific expression. The results showed biallelic gene expression of the SNP that occurred in EG cell colonies and almost all of the cloned blastocysts, demonstrating that aberrant imprinted gene expression of PEG10 occurs in the day 27 porcine PGCs, whereas monoallelic expression of the PEG10 gene occurs in all the PGC clones derived from day 35 PGCs. In addition, the same imprinted gene status was observed for blastocysts derived from both male and female PGCs, indicating that the parental genomic imprinting is erased in male and female germlines.     

Manipulation of fish germ cell: visualization, cryopreservation and transplantation

Germ-cell transplantation has many applications in biology and animal husbandry, including investigating the complex processes of germ-cell development and differentiation, producing transgenic animals by genetically modifying germline cells, and creating broodstock systems in which a target species can be produced from a surrogate parent. The germ-cell transplantation technique was initially established in chickens using primordial germ cells (PGCs), and was subsequently extended to mice using spermatogonial stem cells. Recently, we developed the first germ-cell transplantation system in lower vertebrates using fish PGCs and spermatogonia. During mammalian germ-cell transplantation, donor spermatogonial stem cells are introduced into the seminiferous tubules of the recipient testes. By contrast, in the fish germ-cell transplantation system, donor cells are microinjected into the peritoneal cavities of newly hatched embryos; this allows the donor germ cells to migrate towards, and subsequently colonize, the recipient genital ridges. The recipient embryos have immature immune systems, so the donor germ cells can survive and even differentiate into mature gametes in their allogeneic gonads, ultimately leading to the production of normal offspring. In addition, implanted spermatogonia can successfully differentiate into sperm and eggs, respectively, in male and female recipients. The results of transplantation studies in fish are improving our understanding of the development of germ-cell systems during vertebrate evolution.     

Expression of p63 in the mouse primordial germ cells

Proteins encoded by p63 gene a have structural similarity with tumor suppressor p53, and were thought to induce cell cycle arrest and apoptosis during development. The p63 proteins are also expressed in the basal cells of many epithelial tissues in the adult, and supposed to play important roles in maintaining the epidermal stem cells. Previously, we reported the p63 expression in the testis of mouse embryos, suggesting their involvement in the growth arrest and apoptosis of testicular germ cells (Nakamuta and Kobayashi, J. Vet. Med. Sci. 65:853-856). In this study, we investigated the timing of this p63 expression in the germ cells during migration and colonization to the gonads. Immunohistochemical analysis of mice from embryonic day (E) 7.5 to E12.5 demonstrated that p63 positive reactivity was seen as early as E8.5 when the founder cells of germ cells, primordial germ cells (PGCs), were located in the hind gut epithelium, but PGCs were negative for p63 at E7.5 when they first appeared. p63 is expressed as six isoforms, resulting from alternative splicing at C-terminus and by the use of two promoters that generate variations at N-terminal end. RT-PCR analyses suggested that different types of p63 mRNAs were likely to be expressed in PGCs during development. These results imply that p63 may be involved in the regulation of PGC development by controlling the gene expression required for their migration and colonization to the gonads.     

Attempt to produce nuclear transferred primordial germ cells using electrofusion in domestic chicken

As a step to develop a somatic nuclear transfer technique for avian species, an attempt to produce somatic nuclear transferred primordial germ cells (PGC) in the domestic chicken was carried out. Primordial germ cells and embryonic blood cells (EBC) were collected from 2‐day‐old embryos and the nuclei were transferred from EBC into PGC by electrofusion. The most efficient pearl chain was developed when a 350‐V/cm AC field was applied for 60 s. Cell fusion between PGC and EBC was most effective when 4‐kV/cm DC pulses, 60 µs pulse width, were applied three times to a cell suspension dispersed in 0.2 or 0.25 mol/L saccharose solution. The present results provide basic information for the production of somatic cell nuclear transferred chickens using PGC as the nuclear recipient.     

Isolation,Identification and Enrichment of Type A Spermatogonia from the Testis of Chinese Cross‐Bred Buffaloes (Swamp × River)

The proportion of type A spermatogonia in the isolated testis cells is a prerequisite for conducting experiments and the manipulation of these germ cells. Thus, this study was designed to examine the wide range of strategies for the isolation, identification and enrichment of type A spermatogonia in pre‐pubertal buffalo calves (3–6 months). Histological findings revealed the presence of maximum number of type A spermatogonia at 5 months, which was further confirmed by DBA immunohistochemistry. In a newly modified strategy for the isolation of testis tissues, mincing followed by trituration and two rounds of digestion with collagenase, hyaluronidase and DNase yielded more than 95% testis cell population. Differential plating with laminin, poly‐l ‐lysine and gelatin significantly (p < 0.05) affected the purity of type A spermatogonia. Among these extracellular matrix (ECMs) molecules, laminin and gelatin performed well and reached at a purity of 39.38 ± 1.21% and 32.15 ± 1.60%, respectively. In addition, combination of laminin and gelatin followed by Percoll centrifugation performed the best and yielded >90% type A spermatogonial purity. Moreover, viability of the cells was not affected (p > 0.05) irrespective of different enrichment methods. In conclusion, type A spermatogonia isolation and enrichment system was developed using different ECM molecules in buffaloes, which will aid in solving wide range of problems especially fertility‐related problems and transgenic animal production in buffaloes.     

Innovative approaches to genome editing in avian species

The tools available for genome engineering have significantly improved over the last 5 years, allowing scientist to make precise edits to the genome. Along with the development of these new genome editing tools has come advancements in technologies used to deliver them. In mammals genome engineering tools are typically delivered into in vitro fertilized single cell embryos which are subsequently cultured and then implanted into a recipient animal.In avian species this is not possible, so other methods have been developed for genome engineering in birds. The most common involves in vitro culturing of primordial germ cells(PGCs), which are cells that migrate through the embryonic circulatory system to the developing gonad and colonize the gonad, eventually differentiating into the gonadocytes which produce either sperm or ova. While in culture the PGCs can be modified to carry novel transgenes or gene edits, the population can be screened and enriched, and then transferred into a recipient embryo. The largest drawback of PGC culture is that culture methods do not transfer well across avian species, thus there are reliable culture methods for only a few species including the chicken. Two newer technologies that appear to be more easily adapted in a wider range of avian species are direct injection and sperm transfection assisted gene editing(STAGE).The direct injection method involves injecting genome engineering tools into the circulatory system of the developing embryo just prior to the developmental time point when the PGCs are migrating to the gonads. The genome engineering tools are complexed with transfection reagents, allowing for in vivo transfection of the PGCs. STAGE utilizes sperm transfection to deliver genome engineering tools directly to the newly fertilized embryo. Preliminary evidence indicates that both methodologies have the potential to be adapted for use in birds species other than the chicken, however further work is needed in this area.     

性成熟前小鼠生精细胞的发育过程

用光镜、电镜观察了生后 1～ 1 8d昆明白小鼠的生精上皮。结果显示 ,生后 1～ 3 d,曲细精管内只有生殖母细胞和支持细胞 2类形态结构截然不同的细胞 ,前者位于管中部 ;生后 4～ 5d,少数生殖母细胞已附着在基膜上 ;生后 6～ 7d,原始 A型精原细胞大量出现并附着在基膜上 ;生后 8d,A型精原干细胞大量出现 ,B型精原细胞开始出现 ;生后 1 0 d,B型精原细胞大量出现 ;生后 1 2～ 1 3 d,前初级精母细胞出现 ,少数曲细精管的管腔开始出现 ;生后 1 4～ 1 5d,多数曲细精管管腔基本形成 ,前初级精母细胞大量出现。本试验的结果表明 ,7～ 8d小鼠的睾丸最适于分离精原干细胞     

水牛PGC和前精原细胞的体外培养

分别对取自50~95日龄水牛胎儿的原生殖细胞和前精原细胞进行体外培养,观察其生物学行为,并检测其碱性磷酸酶(AP)活性和Oct-4蛋白特性,探讨利用这些生殖细胞建立干细胞系的可行性和检测方法。结果表明水牛原生殖细胞及前精原细胞分别在体外培养时,均能形成细胞克隆;克隆与周围细胞分界明显,但克隆中细胞相互间界限不清;部分克隆有分隔现象,形如多个克隆共同组成一个大克隆;细胞克隆均至少能培养4代以上;原生殖细胞和前精原细胞及其来源的细胞克隆均呈AP阴性和Oct-4蛋白阴性,其中部分克隆表现为AP假阳性。研究结果显示水牛原生殖细胞和前精原细胞均可用于建立干细胞系;体外培养时,AP活性和Oct-4蛋白不适宜用来检测这些细胞及其来源的细胞克隆。     

Localization of Insulin‐Like Growth Factor‐I (IGF‐I) and IGF‐I Receptor (IGF‐IR) in Equine Testes

The insulin‐like growth factor‐I (IGF‐I) is a key regulator of reproductive functions. IGF‐I actions are primarily mediated by IGF‐IR. The main objective of this research was to evaluate the presence of IGF‐I and IGF‐I Receptor (IGF‐IR) in stallion testicular tissue. The hypotheses of this study were (i) IGF‐I and IGF‐IR are present in stallion testicular cells including Leydig, Sertoli, and developing germ cells, and (ii) the immunolabelling of IGF‐I and IGF‐IR varies with age. Testicular tissues from groups of 4 stallions in different developmental ages were used. Rabbit anti‐human polyclonal antibodies against IGF‐I and IGF‐IR were used as primary antibodies for immunohistochemistry and Western blot. At the pre‐pubertal and pubertal stages, IGF‐I immunolabelling was present in spermatogonia and Leydig cells. At post‐pubertal, adult and aged stages, immunolabelling of IGF‐I was observed in spermatogenic cells (spermatogonia, spermatocyte, spermatid, and spermatozoa) and Leydig cells. Immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at the pre‐pubertal stage. The immunolabelling becomes stronger as the age of animals advance through the post‐pubertal stage. Strong immunolabelling of IGF‐IR was observed in spermatogonia and Leydig cells at post‐puberty, adult and aged stallions; and faint labelling was seen in spermatocytes at these ages. Immunolabelling of IGF‐I and IGF‐IR was not observed in Sertoli cells. In conclusion, IGF‐I is localized in equine spermatogenic and Leydig cells, and IGF‐IR is present in spermatogonia, spermatocytes and Leydig cells, suggesting that the IGF‐I may be involved in equine spermatogenesis and Leydig cell function as a paracrine/autocrine factor.     

Immunohistochemical study of osteopontin in boar testis

The expression of osteopontin (OPN) in boar testis was studied. Western blot analysis detected 66- and 32-kDa OPN immunopositive bands in the testes of adult boars. In postnatal piglets, the 66-kDa OPN band was detected in the testes, but not the 32-kDa band. In the newborn testis, OPN immunostaining was seen in gonocytes and in some supporting cells in the seminiferous tubules, as well as in interstitial Leydig cells. In the adult boar testis, OPN immunoreactivity was detected in seminiferous tubules with varying intensities. Intense OPN immunostaining was seen in the residual bodies and acrosomes in the spermatids while, occasionally, OPN immunostaining was seen in spermatogonia and various stage of spermatocytes but in few Sertoli cells in the seminiferous tubules. In addition, Leydig cells in adult boars were weakly immunostained with OPN. These findings suggest that OPN is detected in the majority of germ cells and is involved in spermatogenesis in boar testis.     

Histologic and immunohistochemical characterization of a testicular mixed germ cell sex cord-stromal tumor and a leydig cell tumor in a dog

Mixed germ cell sex cord-stromal tumors (MGSCTs) of the testis are rare in dogs. We describe the histopathology and immunohistochemical characteristics of an MGSCT associated with a Leydig cell tumor in a cryptorchid testis. Histologically, MGSCT consisted of two nodules of seminiferous tubules lined by germ cells and Sertoli cells in variable proportions. Germ cells had variable size and nuclear features, with frequent giant cells. Germ cells were evenly mixed with Sertoli cells or located in the center of tubules. Markers that labeled mainly germ cells and few or no Sertoli or Leydig cells were calretinin, KIT, and PGP 9.5. E-cadherin, GATA-4, inhibin-alpha (INH-alpha), and neuron-specific enolase (NSE) were predominantly detected in Sertoli cells, whereas melan A was particularly expressed in Leydig cells and vimentin in all three cell types. OCT3/4 was not detected in any cell type. Although more cases of canine MGSCT need to be examined, our results suggest that an immunohistochemical panel of E-cadherin, GATA-4, INH-alpha, KIT, NSE, PGP 9.5, and melan A will help distinguish the three main cell types in canine testicular germ cell and sex cord-stromal tumors.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

VASA (DDX4) is a Putative Marker for Spermatogonia,Spermatocytes and Round Spermatids in Stallions

Expression of the protein DDX4/MVH, or VASA, has been reported in germ cells of several species. The main objectives of this study were to (i) investigate VASA expression patterns in testicular cells of stallions at two different reproductive stages (pre‐pubertal and post‐pubertal) and (ii) evaluate the use of VASA antibody as a molecular marker for single germ cells from stallions. Testicular tissues were obtained from stallions and categorized as pre‐pubertal and post‐pubertal based on the formation of lumen and status of spermatogenesis on the cross section of seminiferous tubules. The results of Western blot showed a VASA protein band located at 76 kDa, indicating that the rabbit antibody has a cross‐reactivity with horse testicular tissues. The result of immunolabelling showed that VASA was expressed in the cytoplasm of spermatogonia at both reproductive stages and in spermatocytes and round spermatids at the post‐pubertal stage. GATA4‐positive Sertoli cells and Leydig cells located in the interstitial space were not immunolabelled with VASA. These results suggest that VASA can be utilized as a molecular marker for germ cells of stallions at pre‐pubertal and post‐pubertal stages. Interestingly, immunolabelling intensity was significantly higher in pachytene spermatocytes compared to spermatogonia and round spermatid. VASA antibody was also effective for staining of single germ cell preparations. In conclusion, VASA protein expression can be used as a marker for identification of spermatogonia, spermatocytes and round spermatids in testicular tissues of stallions.