Spermatogonial Cell Population Research Articles

The ubiquitin ligase c-CBL is expressed in undifferentiated marmoset monkey pluripotent stem cells but is not a general stem cell marker

The protein c-CBL is a ubiquitin ligase. It catalyzes the last step of the transfer of ubiquitin to target proteins. Upon completion of polyubiquitination, the target proteins are degraded. Clinically, it is important that c-CBL is mutated in a subset of patients who develop myeloid malignancies, which are diseases of the hematopoietic stem or progenitor cells. c-CBL has also been shown to be expressed by human spermatogonia. The whole spermatogonial cell population possesses a subset that comprises also the spermatogonial stem cells. Based on these findings we hypothesized that c-CBL might be a general stem cell marker. To test this, we first validated the antibody using marmoset bone marrow and adult testis. In both tissues, the expected staining pattern was observed. Western blot analysis revealed only one band of the expected size. Then, we examined the expression of c-CBL in marmoset monkey embryonic stem (ES) cells, induced pluripotent stem (iPS) cells and adult stem cells. We found that c-CBL is strongly expressed in undifferentiated marmoset iPS cells and ES cells. However, adult stem cells in the gut and the stomach did not express c-CBL, indicating that c-CBL is not a general stem cell marker. In summary, c-CBL is strongly expressed in pluripotent stem cells of the marmoset monkey as well as in selected adult stem cell types. Future studies will define the function of c-CBL in pluripotent stem cells.

The Lin28 Expression in Stallion Testes.

The molecular markers for specific germ cell stages can be utilized for identifying, monitoring, and separating a particular stage of germ cells. The RNA-binding protein Lin28 is expressed in gonocytes of human fetal testes. The Lin28 expression is restricted to a very small population of spermatogonial cells in human, mice, and monkey. The main objective of this study was to investigate the expression pattern of Lin28 in stallion testes at different reproductive stages. Based on the presence or absence of full spermatogenesis and lumina in seminiferous tubules, the testicular samples were categorized into two reproductive stages pre-pubertal and post-pubertal. We performed a reverse transcription polymerase chain reaction to confirm the presence of Lin28 mRNA in the testicular tissues and a western blot analysis to verify the cross-reactivity of rabbit Lin28 antibody with horse testicular tissue. For immunohistochemistry, Lin28 (rabbit anti-human), GATA4 (goat anti-human) or DAZL (goat anti-human) antibodies were used. The results of RT-PCR confirmed the expression of Lin28 mRNA in the stallion testes. The western blot analysis showed that the expression of 28 kDa Lin28 protein was localized in the cytoplasm of spermatogonia at both reproductive stages. The numbers of Lin28-positive germ cells per 1000 Sertoli cells in pre- and post-pubertal stages were 253 ± 8.66 and 29.67 ± 2.18, respectively. At both reproductive stages, all Lin28 positive cells showed no co-stained with GATA4 antibody, whereas only some of the Lin28-positive germ cells showed co-staining with DAZL antibody. The results from whole-mount staining showed that the Lin28 expression was limited to Asingle (As) and Apaired (Apr) spermatogonia. In conclusion, Lin28 might be utilized as a molecular marker for undifferentiated spermatogonial stem cells when used with DAZL antibody.

Production of fertile sperm from in vitro propagating enriched spermatogonial stem cells of farmed catfish, Clarias batrachus.

Spermatogenesis is a highly co-ordinated and complex process. In vitro propagation of spermatogonial stem cells (SSCs) could provide an avenue in which to undertake in vivo studies of spermatogenesis. Very little information is known about the SSC biology of teleosts. In this study, collagenase-treated testicular cells of farmed catfish (Clarias batrachus, popularly known as magur) were purified by Ficoll gradient centrifugation followed by magnetic activated cell sorting using Thy1.2 (CD90.2) antibody to enrich for the spermatogonial cell population. The sorted spermatogonial cells were counted and gave ~3 × 106 cells from 6 × 106 pre-sorted cells. The purified cells were cultured in vitro for >2 months in L-15 medium containing fetal bovine serum (10%), carp serum (1%) and other supplements. Microscopic observations depicted typical morphological SSC features, bearing a larger nuclear compartment (with visible perinuclear bodies) within a thin rim of cytoplasm. Cells proliferated in vitro forming clumps/colonies. mRNA expression profiling by qPCR documented that proliferating cells were Plzf + and Pou2+, indicative of stem cells. From 60 days onwards of cultivation, the self-renewing population differentiated to produce spermatids (~6 × 107 on day 75). In vitro-produced sperm (2260 sperm/SSC) were free swimming in medium and hence motile (non-progressive) in nature. Of those, 2% were capable of fertilizing and generated healthy diploid fingerlings. Our documented evidence provides the basis for producing fertile magur sperm in vitro from cultured magur SSCs. Our established techniques of SSC propagation and in vitro sperm production together should trigger future in vivo experiments towards basic and applied biology research.

E2F1 controls germ cell apoptosis during the first wave of spermatogenesis.

SummaryCell cycle control during spermatogenesis is a highly complex process owing to the control of the mitotic expansion of the spermatogonial cell population and following meiosis, induction of DNA breaks during meiosis and the high levels of physiological germ‐cell apoptosis. We set out to study how E2F1, a key controller of cell cycle, apoptosis, and DNA damage responses, functions in the developing and adult testis. We first analyzed the expression pattern of E2f1 during post‐natal testis development using RNA in situ hybridization, which showed a differential expression pattern of E2f1 in the adult and juvenile mouse testes. To study the function of E2f1, we took advantage of the E2F1−/− mouse line, which was back‐crossed to C57Bl/6J genetic background. E2f1 loss led to a severe progressive testicular atrophy beginning at the age of 20 days. Spermatogonial apoptosis during the first wave of spermatogenesis was decreased. However, already in the first wave of spermatogenesis an extensive apoptosis of spermatocytes was observed. In the adult E2F1−/− testes, the atrophy due to loss of spermatocytes was further exacerbated by loss of spermatogonial stem cells. Surprisingly, only subtle changes in global gene expression array profiling were observed in E2F1−/− testis at PND20. To dissect the changes in each testicular cell type, an additional comparative analysis of the array data was performed making use of previously published data on transcriptomes of the individual testicular cell types. Taken together, our data indicate that E2F1 has a differential role during first wave of spermatogenesis and in the adult testis, which emphasizes the complex nature of cell cycle control in the developing testis.

Increased human AP endonuclease 1 level confers protection against the paternal age effect in mice

Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease 1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells.

Pharmacological stimulation of the regenerative capacity of rat testes damaged by paclitaxel.

Productivity of spermatogenesis and the population of spermatogonial cells were substantially reduced in male Wistar rats 3 months after administration of cytostatic drug paclitaxel, damaging stem spermatogonia. However, signs of reparative regeneration appeared in the testicular tissue. In animals receiving paclitaxel in combination with granulocyte CSF, the same period of observation, the number of spermatogonia and the efficiency of spermatogenesis at the same terms of the study did not differ from those in intact animals. Intensive recovery processes started 1 month earlier than after administration of paclitaxel alone. These data attest to pronounced potentiating effect of granulocyte CSF on reparative regeneration of the testicular tissue.

Mechanisms of reparative regeneration of rat testis after injection of paclitaxel.

Population of spermatogonia was reduced in 2, 3, and 6 months after single intravenous injection of antitumor drug paclitaxel in maximum tolerated dose (MTD). The count of Sertoli cell increased in 3 months after the start of the experiment. The maturity of the seminiferous tubule epithelium was lower than in intact rats. Spermatogenesis productivity did not differ from that in intact animals 6 months after start of the experiment. These data indicate that regeneration of the spermatogenous tissue after paclitaxel treatment is realized via renewal of the spermatogenic epithelium, but considering the amount of spermatogonial cell population, the recovery rate would be low.

Very Small Embryonic-Like Stem Cells: Are They Real?

Testing hypotheses is usually considered the driving force that advances science; however, there is another force that not only advances science, it can also make science fun and engaging to watch: that driving force is controversy. The history of science is riddled with controversies. Perhaps, the most immediate that comes to mind is the controversial future use of induced pluripotent stem cells [1]. However, in the late 1980s and early ‘90s, one of the more overwhelmingly heated controversies dealt with how cells moved, by membrane flow or actin-based, focal adhesion. It was so heated, I remember sitting in the audience at a regional cell biology meeting, where insults were flung between rival laboratories during question and answer periods. Interestingly, as of 2013, what has emerged from that area of research is a general overarching theme that neither set of ideas was wrong. In fact, there are many ways of accounting for cell shapes and steady motion [2]. That was then, this is now.In this volume of SCD, fuel for a new generation of controversy resides in the evolution of very small embryonic-like stem cells (VSELs) [3]. Overall, the data the authors put forth for the existence of these cells are compelling. In fact, it is difficult to shake the feeling that the authors are on to something here. In Anand et al., the work revolves around efforts for restoring spermatogenesis to male cancer patients. The most striking data they show is the ability of distinct bone marrow cells to regenerate spermatogenesis within testes depleted of spermatogonia as a result of chemical ablation. However, before those data are presented, the hypothesis that VSELs are at the core of this regeneration is tested. Anand et al. clearly show that routine busulfan treatment virtually obliterates the proliferating spermatogonial cell population, but left behind within seminiferous tubules is a small subset of cells that express Sca-1 and Oct4A (the nuclear form). They then use cell sorting flow cytometry to characterize these cells by size and marker identification, first in mice and then in humans who are azoospermic survivors of childhood cancers. These latter sets of data are what really begin to raise eyebrows. The fact that a clinical similarity between the mouse model and a relatively equivalent set of human patients adds fuel to the controversy that VSELs may actually exist, furthering the need for more detailed analyses. It is the mesenchymal cell transplant data that really take us beyond raising eyebrows and more toward opening eyes. Histological sections of busulfan-treated testes that were transplanted with bone marrow cells two months earlier show striking examples of sperm production. The sperm appear to be morphologically normal and motile. How does the VSEL story fit in with the transplant data? The answer put forth is that the somatic cell transplants reengage spermatogenesis by replenishing the seminiferous tubule and somatic (ie, Leydig cells) compartment niches with a source of growth factors and cytokines. So where is the controversy? It comes from Irv Weismann’s laboratory, which has cast doubt on the existence of VSELs. An article from NBC News health (www.nbcnews. com/health/bioethicist-failed-search-controversial-formstem-cells-shows-danger-mixing-6C10776643) illustrated Dr. Weismann’s point of view. In essence, using a good bank of experiments, Weismann’s group could not confirm the existence of VSELs [4]. Specifically, they determined that most events within the VSEL flow cytometry gate had little DNA and the cells corresponding to these events could not form spheres. They also determined that these cells did not express Oct4 and could not differentiate into blood cells. The NBC News health story also delved into ethical issues between the adult stem cell and embryonic stem cell circles with respect to the potential of VSELs; however, the topic could turn this short editorial into a dissertation. So, I shall save that discussion for another day. The controversy, though, remains impassioned (as of July 24th 2013) as the blog article from SCOPE supported by Stanford University Medicine, http://scopeblog.stanford.edu/ 2013/07/24/very-small-embryonic-like-stem-cells-may-notexist-say-stanford-researchers/, enables members from both laboratories to rebut each other’s points about the existence or not of VSELs. So far the debate remains meaningful and civil with both parties making sound points. Also, Anand et al. in this volume of SCD make further comments on the NBC News health article and the work by Miyanishi et al. (2013) [4]. From an outsider’s point of view, these types of polarized points can keep science and the scientific community enthralled in a ‘‘which way is the science going to take us’’ storyline. Is one set of ideas going to triumph about the existence of VSELs or will there eventually be a resolution where both sides’ hypotheses contain aspects that are

219 ISOLATION AND CHARACTERIZATION OF DOMESTIC CAT SPERMATOGONIAL CELLS

Spermatogonial stem cells (SSC) have the capacity for self-renewal and the potential of producing progenitor spermatogonia that will differentiate into spermatozoa. SSC transplantation may be a valuable alternative for the propagation of genetically important males and preservation of endangered wild felids, as recently demonstrated by the production of ocelot spermatozoa after the xeno-transplantation of a mixed germ cell population into the testis of a domestic cat (Silva et al. 2011 J. Androl.). SSC are in low numbers in the testis and have been isolated in different mammalian species by using specific cell surface markers; however, the expression of SSC-surface markers in feline species has not been characterised. In the present study, testes of domestic cats were obtained from veterinary clinics. The selected testes (n = 4) ranged in size from 1.3 to 2.0 cm in length. To obtain a suspension of a mixed population of spermatogonial cells, seminiferous tubules were enzymatically dissociated using two digestion steps followed by dual filtration through 100-μm and 40-μm nylon mesh filters and a final separation over a 5-layer Percoll™ density gradient (35, 30, 27.5, 25 and 20%). Spermatogonial cells were morphologically identified by their characteristic large round nucleus with a homogenous appearance in the bands formed at the 30%, 27.5% and 25% layers. The mean number of cells/testis collected was ∼13 × 106 ± 12.2, with an overall percentage of live cells of 97%. After isolation, cells were fixed with 4% PFA, blocked overnight and stained with primary antibodies specific for SSC-surface markers (CD49f, CD9, C-Kit, GFRα1, GPR125 and Thy-1) and pluripotent stage-specific embryonic antigen markers (SSEA-1 and SSEA-4). Fluorescence microscopy showed a positive expression of GFRα1, GPR125 and C-Kit, but not for CD49f, CD9, or Thy-1. It also revealed that a low number of cells were positive for SSEA-1 and SSEA-4. For further characterisation, molecular detection of the pluripotent gene Oct-4 and the germ cell-specific genes BOLL, DAZL and VASA was performed in germ cells isolated from one testis of four individuals. For RT-qPCR, the Cells-to-cDNA™ II kit (Ambion) was used to produce cDNA from an aliquot of ∼30 000 cells directly after isolation. RT-qPCR showed that none had detectable levels of Oct-4 within the range of the standard. Three of the four testes expressed all three germ cell-specific genes, BOLL, DAZL and VASA, while only VASA was detected in the remaining testis. These results suggest that cat SSCs and spermatogonial cells express some of the SSC markers tested. However, the positive expression of SSEA-1 and SSEA-4 in a low number of cells further supports the stem cell-like state of cat SSCs and that these markers can be used in dual staining for purifying cat SSCs from a mixed germ cell population by fluorescence-activated cell sorting.

Protection against vanadium-induced testicular toxicity by testosterone propionate in rats

Vanadium is a well recognized industrial hazard known to adversely affect male reproductive functions. The intricate mechanistic aspects of this metal and the role of oxidative stress in the deterioration of testicular functions are investigated in the current study. The experiment also focused on the effects of testosterone propionate in testicular and sperm functions in the rat intoxicated with vanadate. Vanadium exposure resulted in a more prominent spermatogenic arrest and consistently abolished the conversion of round to mature spermatids along with decreased epididymal sperm number and increased percentage of abnormal sperm. This is followed by a precipitous decline in the level of serum testosterone and gonadotropins and consequently the testicular steroidogenic and antioxidant enzymes were inhibited. Vanadium induces degeneration in the genital organs of rats and exhibits high indices of lipid oxidative damage. In response to exogenous testosterone propionate (TP) administration, spermatogonial cell populations remained suppressed, while the spermatogenesis was restored quantitatively. In contrast, the hormone treatment had no effect on the dramatically decreased serum FSH level after vanadate treatment. Moreover, TP could ameliorate the toxicity, as indicated by decreased testicular lipid peroxidation with marginal but significant increase in the activities of all the measured enzymes following vanadate-treatment. Taken together all these studies establish that vanadium is a testicular toxicant that perturbs the male reproductive system adversely. However, hormone replacement therapy by testosterone propionate may provide partial protection. The results suggest the feasibility of using endocrine regimens to impede deleterious effects of vanadium on the male reproductive system.

Inducible Male Infertility by Targeted Cell Ablation in Zebrafish Testis

To generate a zebrafish model of inducible male sterility, we expressed an Escherichia coli nitroreductase (Ntr) gene in the male germ line of zebrafish. The Ntr gene encodes an enzyme that can convert prodrugs such as metronidazole (Met) to cytotoxins. A fusion protein eGFP:Ntr (fusing Ntr to eGFP) under control of approximately 2 kb putative promoters of the zebrafish testis-specific genes, A-kinase anchoring protein-associated protein (Asp), outer dense fibers (Odf), and sperm acrosomal membrane-associated protein (Sam) was expressed in the male germ line. Three independent and four compound transgenic zebrafish lines expressing eGFP:Ntr were established. Female carriers were fertile, while males exhibited different levels of sterility and appeared normal, otherwise. Developmental analysis shows that germ cells survived and testes were normal before Met treatment, but that the testes of all male transgenic zebrafish exhibited variously depleted prospermatogonia after Met treatment. Particularly in a triple-transgenic line, Tg(AOS-eGFP:Ntr)[Tg(Asp-eGFP:Ntr; Odf-eGFP:Ntr; Sam-eGFP:Ntr)], the transgenic males had very small testes that were virtually devoid of germ cells, and the residual germ cells had almost completely disappeared after 2 weeks of Met treatment. These zebrafish transgenic lines show the complete testis specificity of inducible male sterility after Met treatment and reveal a period of the Ntr/Met ablation activity just prior to formation of the definitive adult spermatogonial cell population. This study demonstrates that combined genetic and pharmacological methods for developing an "infertile breeding technology" have practical application in controlling genetically modified (GM) fish breeding and meet the standards of biological and environment safety for other GM species.

A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F 1 hybrid mouse

In whole mounts of seminiferous tubules of C3H/101 F 1 hybrid mice, spermatogonia were counted in various stages of the epithelial cycle. Furthermore, the total number of Sertoli cells per testis was estimated using the disector method. Subsequently, estimates were made of the total numbers of the different spermatogonial cell populations per testis. The results of the cell counts indicate that the undifferentiated spermatogonia are actively proliferating from stage XI until stage IV. Three divisions of the undifferentiated spermatogonia are needed to obtain the number of A 1 plus undifferentiated spermatogonia produced each epithelial cycle. Around stage VIII almost two-thirds of the A pr and all of the A al spermatogonia differentiate into A 1 spermatogonia. It was estimated that there are 2.5 × 10 6 differentiating spermatogonia and 3.3 × 10 5 undifferentiated spermatogonia per testis. There are about 35,000 stem cells per testis, constituting about 0.03% of all germ cells in the testis. It is concluded that the undifferentiated spermatogonia, including the stem cells, actively proliferate during about 50% of the epithelial cycle.

Long-Term Effects of Iron Particle (HZE) Irradiation on Mouse Spermatogonia

High-energy (HZE) cosmic ray particles in space present an environmental hazard, especially to long-duration high altitude space travel. The energy released when these particles slow down and stop in living tissue causes deleterious biological effects, including death of cells. In order to study and quantitate the effects of this radiation, a number of cell systems could be used. The spermatogonial cell population in mouse testes has a rapidly dividing cell population, can be examined three days after irradiation, and is one of the most sensitive tissues to radiation. Therefore, it is ideally suited for quantitative studies of radiation effects. Previous work has shown that the various types of spermatogonial cells differ in their radiation sensitivity and that this can be very easily evaluated with the light/electron microscope. Using the system of staging established by Oak-berg and Huckins, the mixed or single cell population can be evaluated. Our work, using this method, has revealed cell sensitivity at doses as low as one rad. In these experiments, the animals were sacrificed three days post-irradiation because this is the time when maximum cell loss can be viewed. However, we are now reporting long term results of HZE particles using iron irradiation.

The response of a single spermatogonial cell type in mouse testes to HZE irradiation

It has been well documented that the spermatogonial cell population demonstrates a heterogeneous response to irradiation (1,5). Our previous work (3,4,5) has shown that when one observes cross sections of irradiated testis tissue at spermatogonial stage VI, the survival curve is different from that typical for a single cell response. Since we score all surviving spermatogonia in our cell counts, the results actually represent a response of several cell types. The plotted data as shown in Figure 1 shows a steeper slope at the beginning of the curve, a somewhat different slope between 20 and 80 rads, and a new increase at 80 rads. We have interpreted this to represent several cell types with different levels of sensitivity (5). This interpretation is consistent with the known increase in radiosensitivity of spermatogonia as they proceed through the cycle from stage I to stage VI. In this report, we will correlate the response of a single spermatogonial cell type with HZE particle irradiation dose and compare it to results using x rays.

The mutagenic effect of repeated small radiation doses to mouse spermatogonia: III. Does repeated irradiation reduce translocation yield from a large radiation dose?

Previous results had suggested that daily repeated doses of 10 rad X- or γ-rays to mouse spermatogonia decreased their sensitivity to translocation yield. This was tested by comparing the effects of a dose of 300 rad γ-rays given before, 24 h after, or 8 days after, 30 daily doses of 10 rad γ-rays to male mice. The yield of translocations per cell from those receiving the 300 rad dose 24 h after the repeated ones (7.3%) was significantly lower than that from the other two regimes (9.4 and 9.7% respectively). This was consistent with the explanation that the repeated irradiation had temporarily increased the resistance of the spermatogonial cell population to translocation yield. However, there must remain some doubt about this interpretation since the absolute values of translocations found were too high, and the yield from those which had received the 300-rad dose 24 h after the repeated ones was not significantly below the sum of those from a single dose and from repeated doses given separately.

Spermatogonial Cell Population Kinetics under Continuous Low Dose Rate Irradiation

Spermatogonial Cell Population Kinetics under Continuous Low Dose Rate Irradiation

Dose-response curve for the yield of translocations in mouse-spermatogonia after repeated small radiation doses

When male mice were given small doses of about 10.4 rad γ-rays at high dose rate (17–18 rad/min) on 5 days/week for 12 weeks the yield of translocations in spermatocytes irradiated as spermatogonia was less than after the same total dose (620 rad) given in a single exposure. A dose-response curve for the yields of translocations after 3, 6, 9 or 12 weeks of such daily repeated irradiation was linear but with intercept on the ordinate significantly different from the control. The interpretation was that the reduced effect of repeated small doses was due to change in susceptibility of the spermatogonial cell population after repeated irradiation.