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.     

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.     

红鳍东方鲀精原干细胞鉴定、分离及纯化

为获得高比例精原干细胞, 开展了不同月龄红鳍东方鲀精原干细胞分离纯化研究。采用组合酶消化法制备不同月龄的精巢单细胞悬液, 通过形态学观察和生殖细胞特异基因vasa免疫荧光鉴别精原干细胞, Percoll不连续梯度(10%、30%和50%)纯化精原干细胞。结果显示: 14月龄雄鱼精巢生殖细胞主要为A型精原细胞, 精原干细胞占比极显著性地高于22月龄(P<0.05)。纯化后精原干细胞主要分布在10%—30% Percoll梯度带中, 14月龄雄鱼生殖细胞主要分布在此层, 且纯化后精原干细胞占比远高于纯化前以及22月龄纯化前后。结果表明, 14月龄更适用组合酶消化分离, 并通过Percoll梯度离心法获得高比例的红鳍东方鲀精原干细胞。     

Spermatogonia-specific proteins expressed in prepubertal buffalo (Bubalus bubalis) testis and their utilization for isolation and in vitro cultivation of spermatogonia

Buffalo is an economically important livestock species in Asia. Little is known about male germ line technology owing to lack of sufficient understanding regarding expression of germ- and somatic-cell specific-proteins in the testis. In this study, we identified UCHL-1 (PGP 9.5) and lectin- Dolichos biflorus agglutinin (DBA) as specific markers for spermatogonia in buffalo testis. Expression of germ-cell and pluripotency-specific proteins such as DDX4 (VASA) and POU5F1 (OCT3/4) were also present in spermatogonia. Interestingly, the expression of somatic cell-specific proteins such as VIMENTIN and GATA4 were also detected in germ cells. Using two-step enzymatic digestion followed by differential plating and Percoll density-gradient centrifugation, an approximately 55% spermatogonia-enriched cell population could be obtained from the prepubertal buffalo testis. Isolated spermatogonia could survive and proliferate in vitro in DMEM/F12 medium containing 10% fetal bovine serum in the absence of any specific growth factors for a week. Cultured spermatogonia showed DBA affinity and expressed DDX4 and POU5F1. These results may help to establish a long-term culture system for buffalo spermatogonia.     

Magnetic activated cell sorting allows isolation of spermatogonia from adult primate testes and reveals distinct GFRa1‐positive subpopulations in men

Background Isolation of spermatogonial stem cells (SSCs) could enable in vitro approaches for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. SSC isolation from adult testes is difficult due to low cell numbers and lacking cell surface markers. Glial cell‐derived neurotrophic factor family receptor alpha‐1 (GFRα1) plays a crucial role for the maintenance of SSCs in rodents and is expressed in monkey spermatogonia. Methods Magnetic activated cell sorting was employed for the enrichment of GFRα1+ spermatogonia from adult primate testes. Results Magnetic activated cell sorting of monkey cells enriched GFRα1+ cells threefold. 11.4% of GFRα1+ cells were recovered. 42.9% of GFRα1+ cells were recovered in sorted fractions of human testicular cells, representing a fivefold enrichment. Interestingly, a high degree of morphological heterogeneity among the GFRα1+ cells from human testes was observed. Conclusions Magnetic activated cell sorting using anti‐GFRα1 antibodies provides an enrichment strategy for spermatogonia from monkey and human testes.     

Protein gene product 9.5 is a spermatogonia-specific marker in the pig testis: application to enrichment and culture of porcine spermatogonia

Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P < 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attached to the culture dish (fraction II) (P < 0.05) after differential plating. Cells in fraction II appeared to be superior for future experiments due to higher viability (>90%) than in fraction I ( approximately 50%). Velocity sedimentation plus differential plating achieved cell populations containing up to 70% spermatogonia with good viability (>80%). Enriched spermatogonia from 2wo and 10wo testes could be maintained in a simple culture medium without additional growth factors for at least 2 weeks and continued to express PGP 9.5. These data provide the basis for future studies aimed at refining conditions of germ cell culture and manipulation prior to germ cell transplantation in pigs.     

Percoll密度梯度离心法分离大鼠肺Ⅱ型上皮细胞

目的建立大鼠肺泡Ⅱ型上皮细胞（AT-Ⅱ）的改良体外原代培养法。方法采用循环冲洗、支气管灌洗、胰蛋白酶消化、滤网过滤并用轻比重及重比重PercolL密度梯度离心,分离AT-Ⅱ。原代培养后通过碱性磷酸酶（alkaline phosphatase,AKP）法和电镜鉴定AT-Ⅱ。结果经AKP染色和电镜鉴定所得的AT-Ⅱ纯度为（80．2±6．3）％,每只大鼠可分离出1×10^7AT-Ⅱ。结论采用Percoll密度梯度离心法能分离出较高纯度的AT-Ⅱ。     

Phenotypic changes in germ cells during gonadal development of the common carp (Cyprinus carpio)

A procedure is described in which large early spermatogonia were isolated from carp testes and purified from an initial 4–5% recovery up to 60–70% using equilibrium density centrifugation on a continuous Percoll gradient. Mice were immunized with the spermatogonia via the intrasplenic route. Six hybridoma cultures, producing monoclonal antibodies (MAbs) reacting selectively with germ cells, were selected and further analysed. Reactivity with five of these MAbs was observed on primordial germ cells (PGCs) in the developing indifferent gonads at the onset of proliferation, i.e. the age of 7 weeks. One MAb, encoded WCG 6, appeared to define a new surface marker on PGCs being gradually expressed on the surface membrane between the age of 2 and 4 weeks, concomitantly with an increase in size of these mitotically silent cells. The reactivity of germ cells with five of the MAbs disappeared completely (WCG 7, 12, 15, 21) or nearly completely (WCG 6) during spermatogenesis, providing a striking difference from patterns obtained with MAbs raised previously against carp spermatozoa. Differences between male and female germ cells were not observed with the WCG-MAbs during gonad development, indicating that a common set of surface antigens is shared between germ cells of both sexes up to and including spermatogonia and oogonia.Abbreviation WCG Wageningen carp spermatogonia antibody     

Enrichment of transplantable germ cells in salmonids using a novel monoclonal antibody by magnetic‐activated cell sorting

In the fish germ cell transplantation system, only type A spermatogonia (ASGs) and oogonia are known to be incorporated into the recipient genital ridges, where they undergo gametogenesis. Therefore, high colonization efficiency can be achieved by enriching undifferentiated germ cells out of whole testicular cells. In this study, we used magnetic‐activated cell sorting (MACS) for enriching undifferentiated germ cells of rainbow trout using a monoclonal antibody that recognizes a specific antigen located on the germ cell membrane. We screened the antibodies to be used for MACS by performing immunohistochemistry on rainbow trout gonads. Two antibodies, nos. 172 and 189, showed strong signals for ASGs and oogonia. Next, we performed MACS with antibody no. 172 using gonadal cells isolated from vasa‐gfp rainbow trout showing GFP in undifferentiated germ cells. We found that GFP‐positive cells are highly enriched in antibody no. 172‐positive fractions. Finally, to examine the transplantability of MACS‐enriched cells, we intraperitoneally transplanted sorted or unsorted cells into recipient larvae. We observed that transplantability of sorted cells, particularly ovarian cells, were significantly higher than that of unsorted cells. Therefore, MACS with antibody no. 172 could enrich ASGs and oogonia and become a powerful tool to improve transplantation efficiency in salmonids.     

Evaluation of Sycp3, Plzf and Cyclin B3 expression and suitability as spermatogonia and spermatocyte markers in zebrafish

Recent studies in mammals have revealed the heterogeneity of spermatogonial populations which contain differentiated and undifferentiated cells that further divide into actual stem cells and potential stem cells. In fish however, there are no functional definitions, and very few molecular markers, for germ cells. In our present study, specific antibodies were raised against Sycp3, Plzf and Cyclin B3 in zebrafish and then used to determine the localization of these proteins in the testis. We wished to confirm whether these molecules were potential markers for spermatocytes and spermatogonia. Immunohistochemical observations revealed that Sycp3 is specifically localized in spermatocytes in typical nuclear patterns at each meiotic stage. Plzf was found to be localized in the nucleus of both type A and type B spermatogonia until the 8-cell clone, similar to the pattern in Plzf-positive A(single)-A(aligned) undifferentiated spermatogonia in rodents. In addition to Plzf, the localization of Cyclin B3 was predominantly detected in the nuclei of type A and early type B spermatogonia until the 16-cell clone. Additionally, Cyclin B3 protein signals were detected in germ cells in large cysts, possibly corresponding to spermatocytes at the preleptotene stage. Our present data thus show that these molecules have properties that will enable their use as markers of spermatocytes and early spermatogonia in zebrafish.     

Identification, isolation, and in vitro culture of porcine gonocytes

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.     

Primary culture of germ cells that portray stem cell characteristics and recipient preparation for autologous transplantation in the rhesus monkey

Fertility preservation for prepubertal cancer patients prior to oncologic treatment is an emerging issue, and non‐human primates are considered to constitute suitable models due to the limited availability of human testicular tissues. However, the feasibility of spermatogonial stem cell (SSC) propagation in vitro and autologous testicular germ cell transplantation in vivo requires further exploration in monkeys. Herein, we characterized germ cells in macaque testes at 6 months (M), 18 M and 60 M of age, and effectively isolated the spermatogenic cells (including the spermatogonia) from macaque testes with high purity (over 80%) using combined approaches of STA‐PUT separation, Percoll gradients and differential plating. We also generated recipient monkey testes with ablated endogenous spermatogenesis using the alkylating agent busulfan in six macaques, and successfully mimicked autologous cell transplantation in the testes under ultrasonographic guidance. The use of trypan blue led to successful intratubular injection in 4 of 4 testes. Although SSCs in culture showed no significant propagation, we were able to maintain monkey testicular germ cells with stem cell characteristics for up to 3 weeks. Collectively, these data provided meaningful information for future fertility preservation and SSC studies on both non‐human primates and humans.     

Germ cell–specific expression of Venus by Tol2-mediated transgenesis in endangered endemic cyprinid Honmoroko (Gnathopogon caerulescens)

Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.     

The interstitial origin of germinal cells in the testis of the stickleback

The brook stickleback, Culaea inconstans (Kirtland), in common with other bony fishes, lacks a germinal epithelium in the tubules of the testis, and the tubule wall is composed of a thin, discontinuous layer of myoid cells and collagenous fibers. Labelling of germ cells with tritiated thymidine has shown that the germ cells are derived from clumps of spermatogonia in the interstitial area. Large companion cells within the lumina of the tubules extend their processes to engulf spermatogonia from the interstitium which then enter the lumen of the tubule. Subsequent development of the germ cells takes place within individual compartments formed by folds of the plasma membrane of a companion cell. The companion cell, together with its complement of germ cells, constitutes a cyst. A companion cell may surround spermatogonia in the interstitium and at the same time encompass residual sperm of the previous season within the lumen. The plasma membranes of the germ cells and the companion cells remain discrete. Mature sperm are released into the lumen of the tubule and the companion cell again extends its processes into the interstitium and engulfs more spermatogonia for the following year. Companion cells may be homologous to the Sertoli cells of higher vertebrates although their processes penetrate the interstitium during the initial stages of spermatogenesis and they do not contain a permanent stock of spermatogonia.     

Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture

Objectives: This study has aimed to repopulate ‘primitive’ cells from late‐passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. Materials and methods: Effects of low density culture compared t high density culture on late‐passage bone marrow (BM)‐derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. Results and conclusions: We repopulated ‘primitive’ cells by replating late‐passage MSCs at low density (17 cells/cm²) regardless of donor age. Repopulated MSCs from low‐density culture were smaller cells with spindle shaped morphology compared to MSCs from high‐density culture. The latter had enhanced colony‐forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic‐related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late‐passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct‐4), Osterix and Msx2 reverted to levels of early‐passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony‐forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of ‘primitive’ cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2.     

The pluripotency factor LIN28 marks undifferentiated spermatogonia in mouse

Background  

Life-long production of spermatozoa depends on spermatogonial stem cells. Spermatogonial stem cells exist among the most primitive population of germ cells – undifferentiated spermatogonia. Transplantation experiments have demonstrated the functional heterogeneity of undifferentiated spermatogonia. Although the undifferentiated spermatogonia can be topographically divided into A_s (single), A_pr (paired), and A_al (aligned) spermatogonia, subdivision of this primitive cell population using cytological markers would greatly facilitate characterization of their functions.     

Two states of active spermatogenesis switch between reproductive and non-reproductive seasons in the testes of the medaka, Oryzias latipes

Seasonal change in spermatogenesis was examined in the restricted spermatogonium‐type testes of a teleost, Oryzias latipes. Histological observation revealed that the number of each stage of germ cells during most of the non‐reproductive season, from October to January (O–J period) was nearly half of that during the reproductive season, from May to July (M–J period), except for type B spermatogonia (B‐gonia), which was actually equal. As a result, the ratio of primary spermatocytes (P‐cytes) to B‐gonia was remarkably small in the O–J period. Despite the differences between both time periods, the proliferative activity of type A spermatogonia (A‐gonia), B‐gonia, or P‐cytes was at a similar level in both periods. Moreover, in cultured testes treated with bromodeoxyuridine as a cell‐lineage tracer, P‐cytes differentiated to spermatids in 11–15 days in both M–J and O–J periods. These indicate that spermatogenesis is active in each period at a different state. In the spermatogenic testis, A‐gonial proliferation was maintained by human follicle stimulating hormone/luteinizing hormone in culture. Whereas cell death of B‐gonia and/or P‐cytes gradually increased in the M–J period in spite of those cells being constant in population sizes. In transition to the O–J period, A‐gonia and P‐cytes first decreased, which was accompanied by a decrease in proliferative activity of A‐gonia and relative increase of dead cells from B‐gonia and/or P‐cytes against live P‐cytes. These suggest that A‐gonial proliferation and cell death of B‐gonia and/or P‐cytes that is induced coordinately with B‐gonial differentiation are critical for the spermatogenic control.     

Recombinant human insulin-like growth factor I stimulates all stages of 11-ketotestosterone-induced spermatogenesis in the Japanese eel, Anguilla japonica, in vitro.

In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-ketotestosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type B spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.     

Veligers of an introduced bivalve,Limnoperna fortunei,are a new food resource that enhances growth of larval fish in the Paraná River (South America)

1. Larvae of ‘sábalo’, Prochilodus lineatus, whose adults represent over 60% of overall fish biomass in the Río de la Plata Catchment, have been observed to feed intensively on veligers of the exotic bivalve Limnoperna fortunei. 2. To assess the effects of this dietary shift on the growth of P. lineatus, 28‐day laboratory experiments were carried out feeding newly hatched P. lineatus larvae with three diets: zooplankton artificially enriched with L. fortunei veligers; natural zooplankton; and zooplankton artificially enriched with cladocerans and copepods. The average length, weight and gut contents of the fish larvae were assessed weekly and metabolic rates of fish larvae were measured. 3. Proportions of veligers in gut contents were always higher than those in the experimental diet: 100, 76 and 21% for veliger‐enriched, natural and low‐veliger diets, respectively. Larvae fed a veliger‐enriched diet grew to a significantly larger size than larvae fed the other two diets. In energetic balance comparisons using metabolic rates and prey energy content, all three diets were sufficient to support metabolism and growth. The greatest values of excess energy at the end of each week were in the veliger‐enriched experiments. 4. Feeding on veligers of L. fortunei significantly enhances the growth of P. lineatus larvae and supports the idea that this new and abundant resource is selectively preyed upon by P. lineatus during its larval stage. Higher growth rates may stem from the higher energy contents of veligers compared to crustaceans and/or from the lower energy costs of capturing slower prey.