Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa-GFP transgenic rainbow trout (Oncorhynchus mykiss)

The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.     

Flow-cytometric isolation of testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions

There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A-E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay.     

Non-sex specific genes associated with the secondary mitotic period of primordial germ cell proliferation in the gonads of embryonic rainbow trout (Oncorhynchus mykiss)

The purposes of this study were to quantify the secondary proliferation of primordial germ cells (PGCs) in both sexes of rainbow trout, determine if a sex difference in the timing of PGC proliferation and eventual pre‐meiotic number exists, and use microarray data collected during this period to identify genes that are associated with PGC mitosis. The experiments used vasa‐green fluorescent protein (vasa‐GFP) transgenic rainbow trout of known genetic sex that allowed for the identification and collection of PGCs in vivo. An increase was observed in the number of PGCs counted in the gonads of both female and male embryonic vasa‐GFP rainbow trout, from 300 to 700° days (water temperature in °C × days post‐fertilization). For both sexes, a statistically significant (P < 0.05) increase in the PGC number was first noted at either 350 or 400° days of development. By 700° days, a 20–50‐fold increase in germ cell number was apparent. No sex‐specific differences in the timing of PGC proliferation or number were notable in any of the families until 700° days. In conjunction, a custom microarray based on cDNA libraries from embryonic rainbow trout gonads was used to identify genes involved in PGC mitosis. Five genes were discovered: guanine nucleotide binding protein, integral membrane protein 2B, transmembrane protein 47, C‐src tyrosine‐protein kinase, and the decorin precursor protein. All the genes identified have not been previously associated with germ cell mitosis, but are known to be involved with the cell plasma membrane and/or cell signaling pathways. Mol. Reprod. Dev. 78:181–187, 2011. © 2011 Wiley‐Liss, Inc.     

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.     

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.     

CD9 is expressed on human male germ cells that have a long-term repopulation potential after transplantation into mouse testes

Human spermatogonial stem cells (SSCs) play critical roles in lifelong maintenance of male fertility and regeneration of spermatogenesis. These cells are expected to provide an important resource for male fertility preservation and restoration. A basic strategy has been proposed that would involve harvesting testis biopsy specimens from a cancer patient prior to cancer therapies, and transplanting them back to the patient at a later time; then, SSCs included in the specimens would regenerate spermatogenesis. To clinically apply this strategy, isolating live human SSCs is important. In this study, we investigated whether CD9, a known rodent SSC marker, is expressed on human male germ cells that can repopulate recipient mouse testes upon transplantation. Testicular tissues were obtained from men with obstructive azoospermia. Using immunohistochemistry, we found that CD9 was expressed in human male germ cells in the basal compartment of the seminiferous epithelium. Following immunomagnetic cell sorting, CD9-positive cells were enriched for germ cells expressing MAGEA4, which is expressed by spermatogonia and some early spermatocytes, compared with unsorted cells. We then transplanted CD9-positive cells into nude mouse testes and detected an approximately 3- to 4-fold enrichment of human germ cells that repopulated mouse testes for at least 4 mo after transplantation, compared with unsorted cells. We also observed that some cell turnover occurred in human germ cell colonies in recipient testes. These results demonstrate that CD9 identifies human male germ cells with capability of long-term survival and cell turnover in the xenogeneic testis environment.     

Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter

Mass isolation of live primordial germ cells (PGCs) was demonstrated for the first time in ectothermal vertebrates. To establish a stem cell-mediated gene transfer system in fish, a stem cell line that retains the ability to develop into gametes is necessary. PGCs are well suited for use as the initial material for such a stem cell line. We established transgenic rainbow trout (Oncorhynchus mykiss) strains carrying the green fluorescent protein (GFP) gene driven by a rainbow trout vasa-like gene (RtVLG) promoter/enhancer. Because GFP expression was specific to the PGCs, PGCs were successfully visualized in all developmental stages examined. Isolated genital ridges containing GFP-labeled PGCs were enzymatically dissociated. To isolate PGCs from the complex pools of dissociated genital ridges, GFP-labeled cells were sorted by flow cytometry. The sorted GFP-positive cells were large and round with a large nucleus, typical characters of PGC morphology. The expression of RtVLG was detected only in the GFP-positive cell population, confirming that these cells were PGCs. This simple and efficient technique to purify a large number of viable PGCs opens the way for establishing a stem cell line, which can differentiate into the germline. The purified PGCs would also be a novel tool for cellular and molecular study of vertebrate germline stem cells.     

Hypothermic storage of isolated spermatogonia and oogonia from rainbow trout (Oncorhynchus mykiss)

A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss, and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l-glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies.     

Isolation of highly pure and viable primordial germ cells from rainbow trout by GFP-dependent flow cytometry

A highly pure and viable primordial germ cell (PGC) population appears to be an essential tool for establishing a cell line that can differentiate into a germ cell lineage and for studying the molecular biology and biochemistry of fish PGCs. Therefore, the aim of the present study was to establish a flow cytometric method for isolating highly pure and viable PGCs. As the material for PGC isolation, we used transgenic rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa-gene regulatory sequences (pvasa-GFP). Four independent transgenic strains were subjected to fluorescence microscopy and GFP-dependent flow cytometric analyses. We found that some of the pvasa-GFP transgenic strains exhibited ectopic background green fluorescence in the somatic cells aside from strong fluorescence in PGCs. Although flow cytometric analysis of genital ridge somatic cells in the four pvasa-GFP transgenic strains revealed a wide range of GFP intensities, we proved that somatic cell contamination of the GFP-positive cell population was markedly reduced if transgenic strains without the ectopic background green fluorescence were used. In addition, the forward light-scattering (FS) property, which is an indication of relative cell size, and the side light-scattering (SS) property, which is determined by cell shape and granularity, were employed to remove non-PGC contaminants from the GFP-positive cell population. By isolating GFP-positive cells with high FS/SS values, we were able to effectively remove cell blebs and the apoptotic fraction. Consequently, the purities and survival rates of isolated PGCs were greatly improved compared with those using GFP intensity as a single indicator. Thus, our flow cytometric method, in combination with the selection of suitable transgenic strains without the ectopic background green fluorescence, is capable of isolating highly pure and viable PGCs from rainbow trout. By using this method in combination with cell-cryopreservation and cell transplantation techniques, the isolated PGCs may also be used for preserving the genetic resources of endangered fish species and domesticated fish strains carrying commercially valuable traits. Mol. Reprod. Dev. 67: 91-100, 2004.     

Enriched gonadal migration of donor-derived gonadal primordial germ cells by immunomagnetic cell sorting in birds

This study was conducted to evaluate whether immunomagnetic treatment could improve the retrieval and migration capacity of avian gonadal primordial germ cells (gPGCs) collected from gonads in 5.5-day-old chick and 5-day-old quail embryos, respectively. Collected gPGCs were loaded into a magnetic-activated cell sorter (MACS) after being conjugated with specific gPGC antibodies and either MACS-treated or non-treated cells in each species were subsequently transferred to the recipient embryos. MACS treatment significantly (P < 0.05) increased the population ratio of gPGCs in gonadal cells retrieved (0.74 to 33.4% in the chicken and 2.68 to 45.1% in the quail). This was due to decreased number of non-gPGCs in total cell population. MACS treatment further enhanced gonadal migration of gPGCs transferred in both species (10% vs. 80-85% in the chicken and 10-15% vs. 70-80% in the quail). Increase in the number of microinjected cells up to 600 cells/embryo did not eliminate such promoting effect. In conclusion, MACS treatment greatly increased the population ratio of avian gPGCs in gonadal cells, resulting improved gonadal migration in recipient embryos.     

Activity of the ovarian germinal epithelium in the freshwater catfish,Pimelodus maculatus (Teleostei: Ostariophysi: Siluriformes): Germline cysts,follicle formation and oocyte development

Distinct types of oogonia are found in the germinal epithelium that borders the ovarian lamellae of Pimelodus maculatus: A‐undifferentiated, A‐differentiated and B‐oogonia. This is similar to the situation observed for spermatogonia in the vertebrate testis. The single A‐undifferentiated oogonia divide by mitosis giving rise to A‐groups of single differentiated oogonia, each enclosed by epithelial cells that are prefollicle cells. Subsequently, the single A‐differentiated oogonia proliferate to generate B‐oogonia that are interconnected by cytoplasmic bridges, hence, forming germline cysts. The prefollicle cells associated with them also divide. Within the germline cysts, B‐oogonia enter meiosis becoming oocytes. Meiotic prophase and early folliculogenesis occur within the germline cysts. During folliculogenesis, prefollicle cells grow between the oocytes, encompassing and individualizing each of them. The intercellular bridges disappear, and the germline cysts are broken down. Next, a basement membrane begins to form around the nascent follicle, separating an oocyte and its associated prefollicle cells from the cell nest. Folliculogenesis is completed when the oocyte and the now follicle cells are totally encompassed by a basement membrane. Cells derived from the ovarian stroma encompass the newly‐formed ovarian follicle, and become the theca, thereby completing the formation of the follicle complex. Follicle complexes remain attached to the germinal epithelium as they share a portion of basement membrane. This attachment site is where the oocyte is released during ovulation. The postovulatory follicle complex is continuous with the germinal epithelium as both are supported by a continuous basement membrane. The findings in P. maculatus reinforce the hypothesis that ovarian follicle formation represents a conserved process throughout vertebrate evolution. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and α1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinase-type plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes arise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.     

Isolation and characterization of the CD133+ precursors from the ventricular zone of human fetal brain by magnetic affinity cell sorting

A fast and effective method to enrich large number of neural precursors from the ventricular zone of human fetus by magnetic affinity cell sorting (MACS) is reported. After incubation with phycoerythrin (PE)-conjugated anti-CD133 antibodies and anti-PE magnetic beads followed by one cycle of MACS, CD133(+) cells were harvested at 85% purity as confirmed by flow-cytometry and immunocytochemistry. In contrast to CD133(-) cells, these CD133(+) cells initiated primary and secondary neurospheres in culture, and the progeny of sorted cells could be differentiated into both neurons and glia, indicating that these highly enriched cells are capable of self-renewal and multi-lineage potential.     

Production of germ-line chimeras in rainbow trout by blastomere transplantation

We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.     

Cell adhesive affinity does not dictate primitive endoderm segregation and positioning during murine embryoid body formation

The classical cell sorting experiments undertaken by Townes and Holtfreter described the intrinsic propensity of dissociated embryonic cells to self‐organize and reconcile into their original embryonic germ layers with characteristic histotypic positioning. Steinberg presented the differential adhesion hypothesis to explain these patterning phenomena. Here, we have reappraised these issues by implementing embryoid bodies to model the patterning of epiblast and primitive endoderm layers. We have used combinations of embryonic stem (ES) cells and their derivatives differentiated by retinoic acid treatment to model epiblast and endoderm cells, and wild‐type or E‐cadherin null cells to represent strongly or weakly adherent cells, respectively. One cell type was fluorescently labeled and reconstituted with another heterotypically to generate chimeric embryoid bodies, and cell sorting was tracked by time‐lapse video microscopy and confirmed by immunostaining. When undifferentiated wild‐type and E‐cadherin null ES cells were mixed, the resulting cell aggregates consisted of a core of wild‐type cells surrounded by loosely associated E‐cadherin null cells, consistent with the differential adhesion hypothesis. However, when mixed with undifferentiated ES cells, the differentiated primitive endoderm‐like cells sorted to the surface to form a primitive endoderm layer irrespective of cell‐adhesive strength, contradicting the differential adhesion hypothesis. We propose that the primitive endoderm cells reach the surface by random movement, and subsequently the cells generate an apical/basal polarity that prevents reentry. Thus, the ability to generate epithelial polarity, rather than adhesive affinity, determines the surface positioning of the primitive endoderm cells. genesis 47:579–589, 2009. © 2009 Wiley‐Liss, Inc.     

Flow-cytometric isolation and enrichment of teleost type A spermatogonia based on light-scattering properties

The transplantation of germ cells is a powerful tool both for studying their development and for reproductive biotechnology. An intraperitoneal germ cell transplantation system was recently developed for use in several teleost species. Donor germ cells transplanted into the peritoneal cavity of hatchlings migrated toward and were incorporated into the recipient's genital ridges, where they underwent gametogenesis. Among male germ cells, only type A spermatogonia were capable of colonizing the recipient gonads, unlike those at more advanced stages. The enrichment of type A spermatogonia is therefore important to achieve efficient donor-cell incorporation and subsequent donor-derived gametogenesis. Here we established a simple and rapid system of isolation and enrichment for fish type A spermatogonia, using flow cytometry. Type A spermatogonia were found to have distinctive forward and side light scatter properties compared to that with other types of testicular cell. Based on these characteristics, we were able to isolate and enrich type A spermatogonia by using flow cytometry. After intraperitoneal transplantation, the enriched type A spermatogonia could be successfully incorporated into the recipient genital ridges. This flow cytometry approach using forward and side light scatter was also found to be applicable to other salmonid and sciaenid species, suggesting that it could be a powerful tool for isolating and enriching transplantable type A spermatogonia in a wide range of teleosts. We expect this method to contribute significantly to germ cell biology and biotechnology.     

Culture conditions for maintaining the survival and mitotic activity of rainbow trout transplantable type A spermatogonia

Germ-cell transplantation is a powerful tool for studying gametogenesis in many species. We previously showed that spermatogonia transplanted into the peritoneal cavity of trout hatchlings were able to colonize recipient gonads, and produced fully functional sperm and eggs in synchrony with the germ cells of the recipient. An in vitro-culture system enabling spermatogonia to expand, when combined with transplantation, would be valuable in both basic and applied biology. To this end, we optimized culture conditions for type A spermatogonia in the present study using immature rainbow trout at 8-10 month of age. Spermatogonial survival and mitotic activity were improved during culture in Leibovitz's L-15 medium (pH 7.8) supplemented with 10% fetal bovine serum at 10 degrees C compared with culture under standard conditions for salmonids (Hank's MEM (pH 7.3) supplemented with 25 mM HEPES and 5% FBS, and culture at 20 degrees C). Elimination of testicular somatic cells promoted spermatogonial mitotic activity. In addition, insulin, trout embryonic extract, and basic fibroblast growth factor promoted the mitosis of purified spermatogonia in an additive manner. Mitotic activity increased nearly sevenfold over 19 days of culture compared with growth factor-free conditions and was maintained for >1 month. Furthermore, the cultured spermatogonia could colonize and proliferate in recipient gonads following transplantation. This study represents the first step towards establishing a cell line that can be transplanted for use in surrogate broodstock technology and cell-mediated gene-transfer systems.