Undifferentiated Germ Cells Research Articles

Impaired Planar Germ Cell Division in the Testis, Caused by Dissociation of RHAMM from the Spindle, Results in Hypofertility and Seminoma

Hypofertility is a risk factor for the development of testicular germ cell tumors (TGCT), but the initiating event linking these pathologies is unknown. We hypothesized that excessive planar division of undifferentiated germ cells promotes their self-renewal and TGCT development. However, our results obtained from mouse models and seminoma patients demonstrated the opposite. Defective planar divisions of undifferentiated germ cells caused their premature exit from the seminiferous tubule niche, resulting in germ cell depletion, hypofertility, intratubular germ cell neoplasias, and seminoma development. Oriented divisions of germ cells, which determine their fate, were regulated by spindle-associated RHAMM-a function we found to be abolished in 96% of human seminomas. Mechanistically, RHAMM expression is regulated by the testis-specific polyadenylation protein CFIm25, which is downregulated in the human seminomas. These results suggested that spindle misorientation is oncogenic, not by promoting self-renewing germ cell divisions within the niche, but by prematurely displacing proliferating cells from their normal epithelial milieu. Furthermore, they suggested RHAMM loss-of-function and spindle misorientation as an initiating event underlying both hypofertility and TGCT initiation. These findings identify spindle-associated RHAMM as an intrinsic regulator of male germ cell fate and as a gatekeeper preventing initiation of TGCTs. Cancer Res; 76(21); 6382-95. ©2016 AACR.

Gonadogenesis analysis and sex differentiation in cultured turbot (Scophthalmus maximus).

As a flatfish, the turbot (Scophthalmus maximus) is one of the most important farmed fish species with great commercial value, which has a strong sexual dimorphism on growth rate and sexual maturity. In this study, using histology, the basic information on proliferation and migration of germ cells and early gonadal development during sex differentiation in turbot were described in detail. There were six to nine individual primordial germ cells (PGCs) with large nuclei until 15days post-hatching (dph). The PGCs located under the mesonephric ducts undergo migration along the dorsal mesentery toward the region of the genital ridge from 0 to 15dph. During migration, the number of PGCs was constant, and the expression of vasa had no significant changes. At 20dph, the aggregation of somatic cells at genital ridge indicated the formation of primary gonad. Furthermore, the number of PGCs was increased to 60 and the expression of vasa was upregulated for the first time. The undifferentiated gonads with no morphological indications of sex differentiation grew larger with the increase in germ cells and somatic cells number/size from 20 to 35dph. During 36-52dph, cytological gonadal differentiation was observed. In presumptive testes of type I gonadal tissue (with a lance shape), the number of germ cells increased steadily and the germ cells had the same characteristics as before. Meanwhile, in presumptive ovaries of type II gonadal tissue (with a club-like shape), the germ cells proliferated and induced in two different populations of germ cells. One type had the morphological characteristics as undifferentiated germ cells, while the other type of germ cells underwent mitosis exhibiting smaller size and mottled nuclei. At 60dph, ovarian cavity was present in the gonad of type II, which would develop into ovaries. However, spermatogonial cysts were not noticed in the gonad of type I until 90dph, which indicated the formation of the testes.

Effect of age on expression of spermatogonial markers in bovine testis and isolated cells

Spermatogonial stem cells (SSC) are the most undifferentiated germ cell present in adult male testes and, it is responsible to maintain the spermatogenesis. Age has a negative effect over stem cell, but the aging effect on SSC is not elucidated for bovine. The present study aim to evaluate the effect of age on the expression of undifferentiated spermatogonial markers in testis and in enriched testicular cells from prepubertal calves and adult bulls. In this matter, testicular parenchyma from calves (3–5 months) (n=5) and bulls with 3 years of age (n=5) were minced and, isolated cells were obtained after two enzymatic digestions. Differential platting was performed for two hours onto BSA coated dish. Cell viability was assessed by Trypan Blue solution exclusion method and testicular cells enriched for SSC was evaluated by expression of specific molecular markers by qRT-PCR (POU5F1, GDNF, CXCR4, UCHL1, ST3GAL, SELP, ICAM1 and ITGA6) and flow cytometry (GFRA1, CXCR4 and ITGA6). CXCR4 and UCHL1 expression was evaluated in fixated testes by immunohistochemistry. We observed that age just affected the expression of selective genes [SELP (Fold Change=5.61; p=0.0023) and UCHL1 (Fold Change=4.98; p=0.0127)]. By flow cytometry, age affected only the proportion of ITGA6+ cells (P<0.001), which was higher in prepubertal calves when compared to adult bulls. In situ, we observed an effect of age on the number of UCHL1+ (p=0.0006) and CXCR4+ (p=0.0139) cells per seminiferous tubule. At conclusion, age affects gene expression and the population of cells expressing specific spermatogonial markers in the bovine testis.

Transcriptome analysis reveals differentially expressed genes associated with germ cell and gonad development in the Southern bluefin tuna (Thunnus maccoyii).

BackgroundControlling and managing the breeding of bluefin tuna (Thunnus spp.) in captivity is an imperative step towards obtaining a sustainable supply of these fish in aquaculture production systems. Germ cell transplantation (GCT) is an innovative technology for the production of inter-species surrogates, by transplanting undifferentiated germ cells derived from a donor species into larvae of a host species. The transplanted surrogates will then grow and mature to produce donor-derived seed, thus providing a simpler alternative to maintaining large-bodied broodstock such as the bluefin tuna. Implementation of GCT for new species requires the development of molecular tools to follow the fate of the transplanted germ cells. These tools are based on key reproductive and germ cell-specific genes. RNA-Sequencing (RNA-Seq) provides a rapid, cost-effective method for high throughput gene identification in non-model species. This study utilized RNA-Seq to identify key genes expressed in the gonads of Southern bluefin tuna (Thunnus maccoyii, SBT) and their specific expression patterns in male and female gonad cells.ResultsKey genes involved in the reproductive molecular pathway and specifically, germ cell development in gonads, were identified using analysis of RNA-Seq transcriptomes of male and female SBT gonad cells. Expression profiles of transcripts from ovary and testis cells were compared, as well as testis germ cell-enriched fraction prepared with Percoll gradient, as used in GCT studies. Ovary cells demonstrated over-expression of genes related to stem cell maintenance, while in testis cells, transcripts encoding for reproduction-associated receptors, sex steroids and hormone synthesis and signaling genes were over-expressed. Within the testis cells, the Percoll-enriched fraction showed over-expression of genes that are related to post-meiosis germ cell populations.ConclusionsGonad development and germ cell related genes were identified from SBT gonads and their expression patterns in ovary and testis cells were determined. These expression patterns correlate with the reproductive developmental stage of the sampled fish. The majority of the genes described in this study were sequenced for the first time in T. maccoyii. The wealth of SBT gonadal and germ cell-related gene sequences made publicly available by this study provides an extensive resource for further GCT and reproductive molecular biology studies of this commercially valuable fish.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2397-8) contains supplementary material, which is available to authorized users.

Generation of Transparent Zebrafish with Fluorescent Ovaries: A Living Visible Model for Reproductive Biology.

The transparent zebrafish enables researchers to study the morphology and distribution of cells and tissues in vivo. To capture the dynamic processes of germ cell proliferation and juvenile ovarian development in zebrafish in vivo, we established transgenic (TG) lines to allow us to monitor the changes in the ovaries of living fish. The original transgenic line with ovarian fluorescence was occasionally established. Although the cDNA integrated in the strain was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka β-actin promoter, expression of EGFP is restricted to the oocytes and gills in adult fish. Mutant strains with transparent bodies, roy and ruby, were isolated in zebrafish. In this study, we crossed the TG strain with fluorescent ovary with transparent strains and established the TG (β-actin:EGFP);ruby strain. The strain is highly transparent, and the oocytes are easily observed in living fish. We identified a fluorescent tissue that might contain the undifferentiated germ cells close to the cloaca in the strain. This strain can be used for analysis of ovarian development in vivo.

The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations.

Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of 'protamine', a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells.

Safety and effectiveness of percutaneous cholecystostomy in critically ill children who are immune compromised.

Acalculous cholecystitis is known to develop in critically ill patients without cystic duct obstruction. In the past, treatment for acalculous cholecystitis has been cholecystectomy; however, many children who are critically ill are Percutaneous cholecystostomy is likely the procedure of choice in this subgroup of patients. To assess the safety and effectiveness of percutaneous cholecystostomy in critically ill and immune-compromised children with acalculous cholecystitis. Retrospective review of immune-compromised and critically ill children who underwent percutaneous cholecystostomy between 2006 and 2013. Diagnostic imaging performed included ultrasound, CT and hepatobiliary scintigraphy. Every percutaneous cholecystostomy was performed using imaging guidance. Ten critically ill and immune-compromised children with acalculous cholecystitis underwent percutaneous cholecystostomy. Seven boys and 3 girls, ranging in age from 10months to 15years 8months, were treated. Six of the immune-compromised children had received a bone marrow transplant for leukemia (5 children) or severe combined immunodeficiency (SCID) (1 child), and ranged from 18 to 307days post bone marrow transplant at the time of their percutaneous cholecystostomy. Of the remaining four immune-compromised children with acalculous cholecystitis who underwent percutaneous cholecystostomy, two had leukemia, one had SCID and lymphoma, and the fourth was undergoing treatment for undifferentiated germ cell tumor. The 10 percutaneous gallbladder drains were placed using a transhepatic approach, except one unintentional transperitoneal approach. There were no complications. Three gallbladder drains were removed in Interventional Radiology. Those three patients had a return to normal gallbladder function and didn't require cholecystectomy. Two drains were removed during cholecystectomy and another as an outpatient. Four patients died in the hospital due to multiorgan system failure, with indwelling gallbladder drains. Percutaneous cholecystostomy is a safe procedure in immune-compromised and critically ill children with acalculous cholecystitis. Percutaneous cholecystostomy may obviate the need for future cholecystectomy.

Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment.

Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.

Hypermethylation of genes in testicular embryonal carcinomas.

Background:Testicular embryonal carcinoma (EC) is a major subtype of non-seminomatous germ cell tumours in males. Embryonal carcinomas are pluripotent, undifferentiated germ cell tumours believed to originate from primordial germ cells. Epigenetic changes during testicular EC tumorigenesis require better elucidation.Methods:To identify epigenetic changes during testicular neoplastic transformation, we profiled DNA methylation of six ECs. These samples represent different stages (stage I and stage III) of divergent invasiveness. Non-cancerous testicular tissues were included. Expression of a number of hypermethylated genes were examined by quantitative RT-PCR and immunohistochemistry (IHC).Results:A total of 1167 tumour-hypermethylated differentially methylated regions (DMRs) were identified across the genome. Among them, 40 genes/ncRNAs were found to have hypermethylated promoters. Quantitative RT-PCR confirmed downregulation of 8 out of 9 of the genes. Among the confirmed genes, five were sex-linked genes, including X-linked genes STAG2, SPANXD/E and MIR1184, and Y-linked genes RBMY1A1/1B/1D and FAM197Y2P. RBMY1A is a testis-specific gene for spermatogenesis. RNF168 and USP13 are potential tumour suppressors. Expression of RBMY1A was lost in EC and seminoma as documented in the Protein Atlas. We confirmed downregulation of USP13 in EC by IHC.Conclusions:Our genome-wide analysis of testicular EC identified methylation changes in several previously unknown genes. This may provide insight of crosstalk between normal germ cell development and carcinogenesis.

Potential roles of gonadotropins to control pulsatile retinoic acid signaling during spermatogenesis

Spermatogenesis is a highly regulated process that takes place in the seminiferous tubules of testis. This process initiates at puberty with differentiation of spermatogonia and their meiotic entry. The initiation of spermatogenesis depends on gonadotropins secreted by the pituitary gland; i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In the absence of FSH and LH only premeiotic germ cells are present in the testis. The prepubertal development phase in juvenile testis is characterized by a protracted hypogonadotropic state, which only consists of Sertoli and undifferentiated germ cells in the seminiferous epithelium. All germ cells in the juvenile testis are undifferentiated spermatogonia, which are proliferating in a relatively gonadotropin-independent manner. It has been revealed that vitamin A deficient (VAD) animals are also infertile, and only premeiotic germ cells (undifferentiated spermatogonia) are present in their seminiferous tubules. The developmental block in VAD animal can be removed by administration of retinol and germ cell differentiation reinitiates in a synchronous manner. Recent studies have revealed that the biologically active form of vitamin A, retinoic acid (RA), regulates germ cell differentiation and lead to the generation of the cycle of the seminiferous epithelium and normal spermatogenic wave. Recent study has shown that synchronous spermatogenesis at neonatal mouse, but not after initiation of meiosis, can be induced by treating vitamin A sufficient males with RA. The treatment of neonatal males at different ages with exogenous RA has revealed that although RA is sufficient to induce differentiation of spermatogonial at 4dpp and earlier, it fails to alter asynchrony and it does not irreversibly cause a spermatogonial differentiation. These observations led us to suggest that gonadotropins trigger differentiation of spermatogonia and spermatogenesis through regulation of RA signaling in the seminiferous epithelium of the adult testis.

A stem cell niche shows self-restraint

![Figure][1] Compared with a wild-type germarium (left), a germarium whose escort cells lack Tkv (right) displays extensive Dpp signaling activity (green), and a large number of undifferentiated germ cells marked by a spherical structure containing α-Spectrin (red). DNA is labeled blue. [

Ovary ultrastructure and oogenesis in Propappus volki Michaelsen, 1916 (Annelida: Clitellata)

The paired ovaries of Propappus volki are small, conically shaped structures with a narrow end that is connected to the intersegmental septum, while the wide end extends into the coelom. Ultrastructural studies showed that the ovaries are composed of interconnected germ cells that form syncytia and accompanying somatic cells. Each germ cell in a cyst is connected with the central, anuclear cytoplasmic core, the cytophore, via one cytoplasmic bridge. The somatic cells form both a thin ligament that joins the gonad with the septum and also surround the germ cells, thus, forming a thin ovary envelope. The somatic cells that are in immediate contact with growing oocytes, i.e., follicular cells, are more voluminous than other somatic cells. There is a clear gradient in the development of germ cells along the long ovary axis. As a consequence, three zones can be distinguished. In zone I all of the germ cells are in the prophase I of meiosis, while zone II contains undifferentiated germ cells with a similar morphology. In zone III there are two morphologically distinct categories of germ cells – the more numerous but smaller germ cells that are regarded here as nurse cells and several growing oocytes. The growing oocytes gradually accumulate cytoplasm and yolk material and protrude from the ovary into the segment cavity. The late vitellogenic oocytes, which are filled with yolk spheres, lose contact with the gonad and float within the coelom.The comparison of the results that were obtained with the ovary structure and oogenesis in other Clitellata shows that: (1) the pattern of germ-line cyst organization in P. volki and other Clitellata is the same; (2) the course of oogenesis in P. volki is similar to other oligochaetous clitellates and (3) the ovary organization in P. volki is broadly similar to that which has been described in tubificins, brachiobdellids and lumbriculids but which differs significantly from that found in Enchytraeus albidus.

Developmental expression patterns of chemokines CXCL11, CXCL12 and their receptor CXCR7 in testes of common marmoset and human.

The chemokine receptor CXCR7 interacts with the chemokines CXCL11 and CXCL12. During development, this ligand receptor system (C-X-C) provokes cell-type-specific responses in terms of migration, adhesion or ligand sequestration. It is active in zebrafish and rodents but no data are available for its presence or function in primate testes. Real-time quantitative polymerase chain reaction was performed in monkeys to detect CXCL11, CXCL12 and CXCR7. At the protein level, CXCL12 and CXCR7 were localized in the testes of the marmoset (Callitrix jacchus) whereas CXCR7 patterns were determined for various stages in human testes. Morphometry and flow cytometry were applied to quantify CXCR7-positive cells in monkeys. Transcript levels and protein expression of CXCR7 were detectable throughout testicular development. In both species, CXCR7 protein expression was restricted to premeiotic germ cells. In immature marmoset testes, 69.9% ± 9% of the total germ cell population were labelled for CXCR7, whereas in the adult, 4.7% ± 2.7% were positive for CXCR7. CXCL12 mRNA was detectable in all developmental stages in marmosets. The CXCL12 protein was exclusively localized to Sertoli cells. This pattern of CXCL12/CXCR7 indicates their involvement in regulatory processes that possibly orchestrate the interaction between undifferentiated germ cells and Sertoli cells.

The majority of early primordial germ cells acquire pluripotency by AKT activation.

Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. Here, we examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused ∼60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency.

Spermatogonial Stem Cell Quest: nanos2, Marker of a Subpopulation of Undifferentiated A Spermatogonia in Trout Testis1

Continuous or cyclic production of spermatozoa throughout life in adult male vertebrates depends on a subpopulation of undifferentiated germ cells acting as spermatogonial stem cells (SSCs). What makes these cells self-renew or differentiate is barely understood, in particular in nonmammalian species, including fish. In the highly seasonal rainbow trout, at the end of the annual spermatogenetic cycle, tubules of the spawning testis contain only spermatozoa, with the exception of scarce undifferentiated spermatogonia that remain on the tubular wall and that will support the next round of spermatogenesis. Taking advantage of this model, we identified putative SSCs in fish testis using morphological, molecular, and functional approaches. In all stages, large spermatogonia with ultrastructural characteristics of germinal stem cells were found, isolated or in doublet. Trout homologues of SSC and/or immature progenitor markers in mammals-nanos2 and nanos3, pou2, plzf, and piwil2-were preferentially expressed in the prepubertal testis and in the undifferentiated A spermatogonia populations purified by centrifugal elutriation. This expression profile strongly suggests that these genes are functionally conserved between fish and mammals. Moreover, transplantation into embryonic recipients of the undifferentiated spermatogonial cells demonstrated their high "stemness" efficiency in terms of migration into gonads and the ability to give functional gametes. Interestingly, we show that nanos2 expression was restricted to a subpopulation of undifferentiated spermatogonia (less than 20%) present as isolated cells or in doublet in the juvenile and in the maturing trout testis. In contrast, nanos2 transcript was detected in all the undifferentiated spermatogonia remaining in the spawning testis. Plzf was also immunodetected in A-Spg from spawning testis, reinforcing the idea that these cells are stem cells. From those results, we hypothesize that the subset of undifferentiated A spermatogonia expressing nanos2 transcript are putative SSC in trout.

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.

A DTC Niche Plexus Surrounds the Germline Stem Cell Pool in Caenorhabditis elegans

The mesenchymal distal tip cell (DTC) provides the niche for Caenorhabditis elegans germline stem cells (GSCs). The DTC has a complex cellular architecture: its cell body caps the distal gonadal end and contacts germ cells extensively, but it also includes multiple cellular processes that extend along the germline tube and intercalate between germ cells. Here we use the lag-2 DTC promoter to drive expression of myristoylated GFP, which highlights DTC membranes and permits a more detailed view of DTC architecture. We find that short processes intercalating between germ cells contact more germ cells than seen previously. We define this region of extensive niche contact with germ cells as the DTC plexus. The extent of the DTC plexus corresponds well with the previously determined extent of the GSC pool. Moreover, expression of a differentiation marker increases as germ cells move out of the plexus. Maintenance of this DTC plexus depends on the presence of undifferentiated germ cells, suggesting that germ cell state can influence niche architecture. The roles of this DTC architecture remain an open question. One idea is that the DTC plexus delivers Notch signaling to the cluster of germ cells comprising the GSC pool; another idea is that the plexus anchors GSCs at the distal end.

Cryopreservation of porcine spermatogonial stem cells by slow-freezing testis tissue in trehalose1

Spermatogonial stem cells provide the foundation for continued adult spermatogenesis and their manipulation can facilitate assisted reproductive technologies or the development of transgenic animals. Because the pig is an important agricultural and biomedical research animal, the development of practical application techniques to manipulate the pig Spermatogonial stem cell is needed. The ability to preserve porcine Spermatogonial stem cell or testis tissue long term is one of these fundamental techniques. The objective of this study was to optimize methods to cryopreserve porcine Spermatogonial stem cell when freezing testis cells or testis tissue. To identify the most efficient cryopreservation technique, porcine testis cells (cell freezing) or testis tissue (tissue freezing) were frozen in medium containing dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) or DMSO, FBS, and various concentrations of trehalose (50, 100, or 200 mM). After thawing, undifferentiated germ cells were enriched and treatments were evaluated for cryopreservation efficiency. The tissue freezing method resulted in significantly greater germ cell recovery (P = 0.041) and proliferation capacity (P < 0.001) compared to the cell freezing treatment. Regardless of freezing method (cell vs. tissue), addition of 200 mM trehalose to freezing medium increased germ cell recovery and proliferation capacity compared to cells frozen using the same freezing method without trehalose. Interestingly, addition of trehalose to the tissue freezing medium significantly increased germ cell recovery (P = 0.012) and proliferation capacity (P = 0.004) compared to the cell freezing treatment supplemented with trehalose. To confirm that cryopreservation in trehalose improves the survival of Spermatogonial stem cell, testis cells enriched for undifferentiated germ cells were xenotransplanted into recipient mouse testes. Germ cells recovered from tissue frozen with 200 mM trehalose generated significantly more (P < 0.001) donor derived colonies than tissue frozen without trehalose. Regardless of cryopreservation medium or freezing method, testis cell recovery, viability, and proliferation capacity of germ cells after thawing were significantly lower compared to those of untreated fresh control. Nevertheless, these data demonstrate that undifferentiated porcine germ cells can be efficiently cryopreserved in the presence of 200 mM trehalose.

192 IDENTIFICATION AND CHARACTERIZATION OF Oct4-EGFP EXPRESSING CELLS IN TRANSGENIC PIG TESTIS

We have produced germ line transgenic pigs carrying the entire 18-kb genomic sequence of the murine Oct4 gene fused to the enhanced green fluorescent protein (EGFP) cDNA (OG2 construct; Nowak-Imialek et al., 2011 Stem Cells Dev.). Expression of the EGFP reporter construct is confined to germ line cells, the inner cell mass, and trophectoderm of blastocysts, and testicular germ cells, including putative spermatogonial stem cells (SSC). SSC are unique among stem cells because they can both self-renew and differentiate into spermatozoa. In-depth knowledge on porcine SSC has been hampered by the inability to isolate these cells from the complex cell population of the testis. In the Oct4-EGFP transgenic mouse, SSC are the only adult stem cells that express Oct4. Fluorescence microscopy of testicular tissue isolated from transgenic piglets revealed minimum numbers of EGFP-positive cells, whereas testicular tissue isolated from adult transgenic boars contained a high amount of EGFP fluorescent cells. Northern blot analysis confirmed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. Time course and the signal intensity of EGFP expression in Oct4-EGFP testis paralleled mRNA expression of the endogenous Oct4 gene. Here, we used adult Oct4-EGFP transgenic pigs as a model for fluorescence-activated cell sorting (FACS)-based isolation of EGFP-expressing cells from testes. To obtain a single-cell suspension, the testes were enzymatically dissociated using two digestion steps. Thereafter, FACS based on EGFP expression was successfully used to purify specific testicular cell populations. Two cell populations, i.e. EGFP+ (14%) and EGFP– (45%) could be isolated. Subsequently, qualitative PCR analyses were performed on EGFP+, EGFP–, and unsorted cell populations using marker genes specific for pluripotency and undifferentiated germ cells (OCT4, FGFR3, UTF1, PGP9.5, GFRα1, CD90, SALL4), differentiating germ cells (c-KIT), meiosis (BOLL), spermatids (PRM2), and somatic cells (VIM, LHCGR). All of the genes, including OCT4, UTF1, FGFR3, PGP9.5, CD90, SALL4, and GFRα1 were expressed at least 3-fold and up to 12-fold greater in the EGFP-positive population. Vimentin, which is mainly expressed in Sertoli cells and LHCGR, which is mainly expressed in Leydig cells, were expressed in unsorted and EGFP– cell populations and at very low level in EGFP+ cells. Moreover, expression of the c-KIT and PRM2 markers were detected also in EGFP+ cell population, indicating that these cells contain also differentiating spermatogonia. To explore the characteristics of the Oct4-EGFP expressing cells in greater detail, localization in the porcine testis sections and analysis of co-expression with germ cell markers using immunohistochemistry is currently underway.

Global proteomic profiling of the membrane compartment of bovine testis cell populations

Spermatogonial stem cells hold enormous potential in mammalian reproductive medicine through the preservation of gametes, the restoration of fertility, enhancement of germ-lineage genetic manipulation and the improvement in our understanding of stem cell biology. Here we describe the protein profiles of the membrane compartment of bovine testicular cell isolates which were enriched for germ cells using differential plating. The isolated cells were characterised with antibodies to UCHL1 (previously known as PGP9.5) for type A spermatogonia; DDX4 (previously known as VASA) for germ cells and vimentin for Sertoli cells. Ultracentrifugation techniques were used to specifically isolate cell membranes, with membrane protein identifications significantly increased when compared to whole cell lysates. We utilised the filter-aided sample preparation protocol for improved digestion efficiency of membrane proteins. Using ESI-LC-MS/MS, we compared the proteins present in two cell populations. A total of 1,387 proteins were identified in bovine testis cell isolates, of which 39% were membrane-associated. A total of 64 proteins were differentially expressed in the non-adhered fraction (enriched for undifferentiated germ cells), of which 16 were unique to this cell population and the remaining 48 showed a two-fold change as judged by spectral counting. This analysis revealed a number of candidate germ cell markers including the known markers, DDX4 and UCHL1. The proteomic profiles generated in this study support and complement transcriptomics studies and reinforce the potential of proteomics in identifying and characterising the protein effectors of self-renewal and/or differentiation in stem cells. Normal 0 false false false EN-AU X-NONE X-NONE