CiteULike: altotor's spermatogonia

Establishment of a short-term in vitro assay for mouse spermatogonial stem cells.

JR Yeh — 2008-07-22T13:44:32-00:00

Biology of reproduction, Vol. 77, No. 5. (November 2007), pp. 897-904.

Spermatogonial stem cells (SSCs) are responsible for life-long, daily production of male gametes and for the transmission of genetic information to the next generation. Unequivocal detection of SSCs has relied on spermatogonial transplantation, in which functional SSCs are analyzed qualitatively and quantitatively based on their regenerative capacity. However, this technique has some significant limitations. For example, it is a time-consuming procedure, as data acquisition requires at least 8 weeks after transplantation. It is also laborious, requiring microinjection of target cells into the seminiferous tubules of individual testes. Donor-recipient immunocompatibility for successful transplantation and large variations in data obtained represent further limitations of this technique. In the present study, we provide evidence that a recently developed SSC culture system can be employed as a reliable, short-term in vitro assay for SSCs. In this system, donor cells generate three-dimensional structures of aggregated germ cells (clusters) in vitro within 6 days. We show that each cluster originates from a single cell. Thus, by counting the clusters, cluster-forming cells can be quantified. We observed a strong linear correlation between the numbers of clusters and SSCs over extended culture periods. Therefore, cluster numbers faithfully reflect SSC numbers. These results indicate that by simply counting the number of clusters, functional SSCs can be readily detected within 1 week in a semi-quantitative manner. The faithfulness of this in vitro assay to the transplantation assay was further confirmed under two experimental situations. This in vitro cluster formation assay provides a reliable short-term technique to detect SSCs.

Expression of the pluripotency marker UTF1 is restricted to a subpopulation of early A spermatogonia in rat testis.

MP van Bragt — 2008-07-22T12:14:15-00:00

Reproduction (Cambridge, England), Vol. 136, No. 1. (July 2008), pp. 33-40.

The population of early A spermatogonia includes stem cells that possess spermatogonial stem cell properties. Recent reports suggest that these cells have the ability to regain pluripotent properties. Here, we show that expression of the pluripotency marker undifferentiated embryonic cell transcription factor 1 (UTF1) is restricted to distinct germ cells within the testis. In embryonic and neonatal testes, all gonocytes were found to strongly express UTF1. During further testicular development, expression of UTF1 was restricted to a subset of A spermatogonia and with the increase in age the number of cells expressing UTF1 decreased even more. Ultimately, in the adult rat testis, only a small subset of the A spermatogonia expressed UTF1. Remarkably, even in testes of vitamin A-deficient rats, in which the early A spermatogonia (A(s), A(pr), and A(al)) are the only type of spermatogonia, only a subset of the spermatogonia expressed UTF1. In the adult rat testis, expression of UTF1 is restricted to a subpopulation of the ZBTB16 (PLZF)-positive early A spermatogonia. Furthermore, the observed distribution pattern of UTF1-expressing cells over the different stages of the cycle of the seminiferous epithelium suggests that the expression of UTF1 is restricted to those A(s), A(pr), and short chains of A(al) spermatogonia that are in the undifferentiated state and therefore maintain the ability to differentiate into A1 spermatogonia in the next round of the epithelial cycle or possibly even in other directions when they are taken out of their testicular niche.

Long-term culture of male germline stem cells from hamster testes.

M Kanatsu-Shinohara — 2008-06-10T09:31:41-00:00

Biology of reproduction, Vol. 78, No. 4. (April 2008), pp. 611-617.

Spermatogonial stem cells provide the foundation for spermatogenesis in male animals. We recently succeeded in culturing and genetically engineering mouse spermatogonial stem cells, but little is known regarding the culture and growth requirements of spermatogonial stem cells in other animal species. In this study, we report the successful long-term culture of spermatogonial stem cells from hamster testes. Spermatogonial stem cells were purified using an anti-ITGA6 antibody and cultured in the presence of glial cell line-derived neurotrophic factor. The cells continued to proliferate for at least 1 year. During this period, they were genetically modified using a lentivirus and underwent spermatogenesis after transplantation into the testes of immunodeficient nude mice. However, germ cells generated in the surrogate xenogeneic recipients did not differentiate beyond the spermatid stage, and these round spermatids could not produce offspring through in vitro microinsemination. These results suggest that the germ cells may not have acquired characteristics necessary for fertility in the xenogeneic microenvironment. Nevertheless, the successful establishment of culture conditions conducive for hamster spermatogonial stem cell growth and maintenance indicates that this technique can be extended to other animal species in which current genetic modification techniques are impossible or inefficient.

Identification of neuregulin as a factor required for formation of aligned spermatogonia.

FK Hamra — 2007-03-05T15:38:34-00:00

J Biol Chem, Vol. 282, No. 1. (5 January 2007), pp. 721-730.

In the absence of somatic cells, medium conditioned by the SNL fibroblast line (SNL-CM) is able to stimulate primary cultures of rat type-A single spermatogonia to develop into chains of aligned spermatogonia at the 8-, 16-, and 32-cell stages. By comparison, medium conditioned by an MSC-1 Sertoli cell line is ineffective. Glial cell line-derived neurotrophic factor (GDNF)-like molecules were identified in SNL-CM and recombinant forms of GDNF, neurturin, and artemin were shown to stimulate formation of aligned spermatogonia, but principally to only the 4- and 8-cell stages. Because SNL-CM and GDNF-like molecules stimulated the formation of spermatogonial chain length differently, we purified components of SNL-CM to identify the additional contributing factor(s). A fraction was isolated that was dependent on GDNF, but required for effective formation of 16- and 32-cell chain lengths. Sequence analysis identified the factor as mouse neuregulin-1. At picomolar concentrations, recombinant neuregulin-1 in combination with GDNF effectively stimulated formation of aligned spermatogonia up to the 32-cell stage. Neuregulin in the absence of GDNF was relatively ineffective. Soluble receptors for neuregulins blocked the effects of GDNF and SNL-CM, suggesting that both neuregulin and GDNF are required for effective formation of long spermatogonial chains. Addition of neuregulin-1 to cultures on MSC-1 feeder layers resulted in spermatogonial behavior similar to that seen on feeder layers of SNL fibroblasts. In fact, SNL cells were found to express 100-fold higher levels of neuregulin-1 transcripts than MSC-1 cells. Thus, we identify neuregulin as a factor required for spermatogonial amplification and differentiation in culture.

Identification and characterization of spermatogonial subtypes and their expansion in whole mounts and tissue sections from primate testes.

J Ehmcke — 2008-05-27T09:01:22-00:00

Methods in molecular biology (Clifton, N.J.), Vol. 450 (2008), pp. 109-118.

The different types of spermatogonia present in the testes of all mammalian species have a series of functions in the adult testis. Some cycle regularly to (1) maintain the spermatogonial population and (2) derive differentiating germ cells to maintain continuous spermatogenesis; other spermatogonia act as a functional reserve, proliferating only very rarely under healthy conditions but repopulating the depleted seminiferous tubules after gonadotoxic insult. The number, appearance, and function of different types of spermatogonia differ greatly between mammalian species, and therefore the precise number of mitotic steps and the number of identifiable stages in spermatogenesis, the sperma-togenic efficiency, and the histological appearance of the seminiferous epithelium show remarkable variation. To characterize spermatogonial phenotypes and their respective functions and to understand the kinetics of spermatogenesis in any given species, a series of methods can be combined for best results. Conventional (hema-toxylin or Periodic acid Schiff's reagent PAS/hematoxylin) staining on sections allows histological identification of the different types of spermatogonia and stages of spermatogenesis in the tissue. Immunohistochemical detection of the proliferation marker bromodeoxyuridine (BrdU) in sections and whole mounts of seminiferous tubules allows determination of which types of spermatogonia proliferate in which stage of spermatogenesis and determine the sizes of clones of proliferation spermatogonia in each stage. Combined, these methods allow the best possible characterization of spermatogenesis in any given mammalian species.

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

S Shikina — 2008-05-27T08:56:41-00:00

Molecular reproduction and development, Vol. 75, No. 3. (March 2008), pp. 529-537.

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.

High-resolution light microscopic characterization of spermatogonia.

H Chiarini-Garcia — 2008-05-27T07:07:58-00:00

Methods in molecular biology (Clifton, N.J.), Vol. 450 (2008), pp. 95-107.

It is possible to distinguish the morphological features of the spermatogonial nuclei and nucleoli and to further identify their distinct generations using an appropriate method to fix whole testes via vascular perfusion with glutaraldehyde, postfixation by immersion in reduced osmium, embedding in araldite, and staining of semithin tissue sections. A well-trained individual can distinguish each of the spermatogonial types in rodents (A(undiferentiated), A(1), A(2), A(3), A(4), In, and B) using this tissue preparation technique based on their morphological details and without correlation with the stages of the epithelium cycle or other parameters. The possibility of distinguishing each spermatogonial type by their morphological characteristics allows a more accurate evaluation of their kinetics during the spermatogenic cycle. Moreover, the understanding of spermatogonial behavior is a means to elucidate the functional control of the spermatogenesis, which consequently allows the determination of their effects on the fertility of humans and other animals.

Isolating highly pure rat spermatogonial stem cells in culture.

FK Hamra — 2008-05-27T07:04:15-00:00

Methods in molecular biology (Clifton, N.J.), Vol. 450 (2008), pp. 163-179.

Methods are detailed for isolating highly pure populations of spermatogonial stem cells from primary cultures of testis cells prepared from 22- to 24-day-old rats. The procedure is based on the principle that testicular somatic cells bind tightly to plastic and collagen matrices when cultured in serum-containing medium, whereas spermatogonia and spermatocytes do not bind to plastic or collagen when cultured in serum-containing medium. The collagen-non-binding testis cells obtained using these procedures are thus approx. 97% pure spermatogenic cells. Stem spermatogonia are then easily isolated from the purified spermatogenic population during a short incubation step in culture on laminin matrix. The spermatogenic cells that bind to laminin are more than 90% undifferentiated, type A spermatogonia and are greatly enriched in genetically modifiable stem cells that can develop into functional spermatozoa. This method does not require flow cytometry and can also be applied to obtain enriched cultures of mouse spermatogonial stem cells. The isolated spermatogonia provide a highly potent and effective source of stem cells that have been used to initiate in vitro and in vivo culture studies on spermatogenesis.

Spermatogenesis in testis xenografts grafted from pre-pubertal Holstein bulls is re-established by stem cell or early spermatogonia

Stephanie Huang — 2008-05-20T09:25:51-00:00

Animal Reproduction Science, Vol. 103, No. 1-2. (15 January 2008), pp. 1-12.

Xenografting of testis explants into recipient mice has resulted in successful restoration of spermatogenesis in several species. Most studies have utilized neonatal donor tissue, although a few have used prepubertal testes. In Holstein bulls, prepubertal development of the testis occurs between 16 and 32 weeks of age. The purpose of the present study was to determine the optimal age during prepubertal development of Holstein bulls for testis grafting. Explants of testis tissue from Holstein bulls between 12 and 32 weeks of age (2 bulls/age; 6 ages) were subcutaneously grafted into castrated or intact immunocompromised mice (n = 8/age), then recovered after 75 and 173 days (n = 4 mice/grafting period) and evaluated histologically for spermatogenic progression. Seminiferous tubules were assigned a score based on the most advanced type of germ cell present within the tubule and the average for all tubules scored (n = 25) within an explant was calculated. Scores for all explants per mouse (n = 6) were averaged to give a single spermatogenic progression score per mouse. No difference in spermatogenic progression of grafts between intact and castrated recipients was observed. Spermatocytes were observed in testis grafts from bulls of all ages 75 days post-grafting. At 173 days, the spermatogenic progression score for explants derived from 20 weeks bulls was greater than all ages except 12 weeks donors (p < 0.05), with 8% of tubules containing spermatids. Donor material from bulls older than 20 weeks had lesser spermatogenic progression scores largely attributed to the greater number of atrophic tubules in grafts from older donors. Grafts from 28 and 32 weeks donors showed signs of degeneration by 75 days post-grafting, with 30 and 55% atrophic tubules, respectively, and lesser spermatogenic efficiency scores. By 173 days post-grafting, 72% of tubules in explants from 32 weeks donors were atrophic. The results of the present study suggest that the early stages of prepubertal development are optimal for testis grafting while advanced spermatogenesis in the donor tissue prior to grafting had a negative effect on graft development. Spermatogenesis within the grafts apparently needs to be re-established by spermatogonial stem cells or early spermatogonia.

A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis.

S Yoshida — 2008-03-26T13:29:56-00:00

Science, Vol. 317, No. 5845. (21 September 2007), pp. 1722-1726.

Mammalian spermatogenesis produces numerous sperm for a long period based on a highly potent stem cell system, which relies on a special microenvironment, or niche, that has not yet been identified. In this study, using time-lapse imaging of green fluorescent protein-labeled undifferentiated spermatogonia (A(undiff)) and three-dimensional reconstitution, we revealed a biased localization of A(undiff) to the vascular network and accompanying Leydig and other interstitial cells, in intact testes. Differentiating spermatogonia left these niche regions and dispersed throughout the basal compartment of the seminiferous epithelium. Moreover, rearrangement of A(undiff) accompanied the vasculature alteration. We propose that the mammalian germline niche is established as a consequence of vasculature pattern formation. This is different from what is observed in Drosophila or Caenorhabditis elegans, which display developmentally specified niche structures within polarized gonads.

High-resolution light microscopic characterization of mouse spermatogonia.

H Chiarini-Garcia — 2008-03-25T10:12:16-00:00

Biol Reprod, Vol. 65, No. 4. (October 2001), pp. 1170-1178.

Characteristics of spermatogonia were determined in the C57BL/6J strain mouse using high-resolution light microscopy of plastic-embedded tissues and identifying cells during stages of the spermatogenic cycle. The frequency of expecting each spermatogonial cell type was a major factor in identifying and categorizing various cell types. Although numerous characteristics were described, several major differences were noted in spermatogonial cell types. The group comprising A(s), A(pr), and A(al) spermatogonia could be differentiated based primarily on mottling of heterochromatin throughout the nucleus in the absence of heterochromatin lining the nuclear envelope. The A(1) cells displayed finely granular chromatin throughout the nucleus and virtually no flakes of heterochromatin along the nuclear membrane. The A(2) through A(4) spermatogonia contained progressively more heterochromatin rimming the nucleus. Intermediate-type spermatogonia displayed flaky or shallow heterochromatin that completely rimmed the nucleus. Type B spermatogonia showed rounded heterochromatin periodically along the nuclear envelope. Use of gray-scale histograms allowed objective quantification of nuclear characteristics and showed a logical shift in the gray scale to a narrower and darker profile, from four cell types leading to A(1) cells. The ability to differentiate spermatogonial types is a prerequisite to studying the behavior and kinetics of the earliest of the germ cell types in both normal and abnormal spermatogenesis.

Distribution of type A spermatogonia in the mouse is not random.

H Chiarini-Garcia — 2008-03-25T10:11:31-00:00

Biol Reprod, Vol. 65, No. 4. (October 2001), pp. 1179-1185.

The distribution of type A spermatogonia was studied using drawings of cross-sectioned tubules at various stages of the spermatogenic cycle of perfusion-fixed, epoxy-embedded mouse testis. Spermatogonia were classified as either positioned opposite the interstitium or opposite the region where two tubules make contact or in a defined, intermediate region at which the two tubules diverged. At stage V, the population of type A spermatogonia, comprised of A(s) through A(al) cells, is randomly positioned around the periphery of the seminiferous tubule. The A(s) through A(al) population becomes nonrandomly distributed beginning at stage VI, being located primarily in regions where the tubule opposes the interstitium, and remains nonrandom through stage III of the next cycle. The A(1) spermatogonia of stage VII, derived from most A(pr) and A(al) spermatogonia, and the A(2) spermatogonia of stage IX, derived from the A(1) spermatogonia, are also nonrandomly positioned opposing the interstitium. However, the A(3) population of stage XI becomes randomly distributed around the tubule. To our knowledge, these are the first data to show that the more primitive spermatogonial types (A(s) to A(al)) move to specific sites within the seminiferous tubule. Division of the regularly spaced, more primitive spermatogonia (A(s) to A(al)) leads to the spread of their progeny (A(1) to A(4)) laterally along the base of the seminiferous tubule. The lateral spread from more or less evenly spaced foci ensures that spermatogenesis is conducted uniformly around the entire tubule. The data also suggest that the position of a seminiferous tubule in the mouse is stabilized in relationship to other seminiferous tubules.

Non-random distribution of spermatogonia in rats: evidence of niches in the seminiferous tubules.

H Chiarini-Garcia — 2008-03-25T10:08:57-00:00

Reproduction, Vol. 126, No. 5. (November 2003), pp. 669-680.

The relationships and distribution of spermatogonia were studied as a function of the stage of the seminiferous epithelium cycle in rats. Primitive spermatogonia in the mouse are located along regions of the basal lamina that face the interstitium. Before studying the distribution of spermatogonia in rats, it was necessary to characterize the various types of spermatogonia, as recently performed for mice. The Strauss' linear index (Li) selectivity method was then used and spermatogonia of the A(single) (A(s)) to A(aligned) (A(al)) lineage were preferentially found to be located in regions opposing the interstitium at stages V, VII and IX of the spermatogenic cycle. Because relatively little tubule-to-tubule contact occurs in rats, the aim of this study was to determine whether tubule-to-tubule contact or tubule proximity (or alternatively, the amount of interstitium) was an important factor in spermatogonial position. In this regard, another method (tubule proximity) was devised to determine spermatogonial position that accounted for the presence of adjacent tubules. This method showed that the position of tubules, rather than tubule contact, was more accurate than the Li method in determining the location of spermatogonia in the rat. The results also showed a non-random distribution of spermatogonia resembling that of the mouse, and that tubule-to-tubule contact is not essential for the positioning of spermatogonia. In conclusion, the results of this study strongly indicate that the most primitive type A spermatogonia (A(s), A(paired) and A(al)) in rats are present in niches located in those areas of the seminiferous tubules that border the interstitial tissue.

Derivation and morphological characterization of mouse spermatogonial stem cell lines.

T Ogawa — 2008-03-25T10:07:25-00:00

Arch Histol Cytol, Vol. 67, No. 4. (November 2004), pp. 297-306.

Spermatogonial stem cells (SSCs), having yet to possess decisive markers, can only be detected retrospectively by transplantation assay. It was reported recently that mouse gonocytes collected from DBA/2 and ICR neonates propagated in vitro. This cultured germ cell, named the germline stem cell (GS cell), produced functional sperm to make progeny when transplanted into recipient mouse testes. Here we show that GS cell lines can be established not only from neonatal testes but also from the testis of adult mice. We also confirmed that GS cells once transplanted into a host testis can be recovered to resume in vitro expansion, indicating that they are convertible mutually with SSCs in adult testes. Confocal laser microscopic examination showed GS cells resemble undifferentiated spermatogonia in the adult testis. This unique cell line could be useful for research in germ cell biology and applicable as a new tool for the genetic engineering of animals.

Involvement of GnRH neuron in the spermatogonial proliferation of the scallop, Patinopecten yessoensiss.

S Nakamura — 2008-03-19T14:47:50-00:00

Mol Reprod Dev, Vol. 74, No. 1. (January 2007), pp. 108-115.

The aim of this study was to quantitatively analyze a pattern of proliferation of gonial cells and to demonstrate neural involvement in spermatogonial proliferation of the scallop by the in vitro experiment. Immunocytochemistry for incorporated BrdU was used to identify mitotically active gonial cells. The pattern of proliferation of gonial cells was divided into two phases: phase I; oogonia and spermatogonia slowly proliferate through the growing stage: phase II; oogonia develop into oocytes and spermatogonia start to proliferate rapidly from the mature stage through the spawning stage. The neurons detected with anti-mammalian (m)GnRH antibody were distributed sparsely in the pedal ganglion and predominantly in the cerebral ganglion of both sexes at the growing stage. The extracts from the cerebral and pedal ganglion (CPG) of both sexes collected at the growing stage promoted proliferation of spermatogonia in the in vitro culture of the testicular tissue as well as mGnRH. However, CPG extract had no effect on oogonial proliferation. The increased mitotic activity induced by CPG and mGnRH was abolished by the addition of mGnRH antagonists and anti-mGnRH antibody, suggesting that the spermatogonial proliferation is regulated by GnRH-like peptide in CPG of the scallop. The same mitotic activity as CPG extract and mGnRH was observed in the hemocyte lysate, but not in the serum. These findings suggest that the spermatogonial proliferation at phase II in the scallop may be under the neuroendocrine control by GnRH neuron in CPG.

Characterization of mouse spermatogonia by transmission electron microscopy

H Chiarini-Garcia — 2008-03-18T17:13:09-00:00

Reproduction, Vol. 123, No. 4. (1 April 2002), pp. 567-577.

Characteristics of the various type A, intermediate (In) and B spermatogonia were determined in C57BL/6J mice using transmission electron microscopy. Spermatogonia were photographed at all stages of the cycle of the seminiferous epithelium. Over 450 images were taken. Spermatogonia could be classified into As, Apr, Aal, A1 cells, A2 cells, A3 cells, A4 cells, intermediate type and type B cells primarily on the basis of nuclear and nucleolar characteristics. The most primitive spermatogonia (As, Apr, Aal) had mottled chromatin; A1 cells contained homogeneously finely granular chromatin throughout the nucleus; A2, A3, A4 and intermediate type spermatogonia had progressively increasing amounts of chromatin encrusting the nuclear envelope; type B spermatogonia had less heterochromatin along the nuclear envelope, although the particles were more dense and rounded than in intermediate type spermatogonia. Mitochondrial size and position of Golgi complexes varied in different types of spermatogonia. These data show that types of spermatogonia can be differentiated such that these characteristics can be used in functional studies. 10.1530/rep.0.1230567

Sohlh2 Knockout Mice are Male Sterile Due to Degeneration of Differentiating Type-A Spermatogonia.

Jing Hao — 2008-03-18T07:31:41-00:00

Stem Cells (13 March 2008)

The Spermatogenesis and Oogenesis-specific transcription factor, Sohlh2, is normally expressed only in pre-meiotic germ cells. In this study, Sohlh2, plus several other germ cell transcripts were found to be induced in mouse embryonic stem cells when cultured on a feeder cell line that over-expresses BMP4. To study the function of Sohlh2 in germ cells, we generated mice harboring null alleles of Sohlh2. Male Sohlh2-deficient mice were infertile due to a block in spermatogenesis. Though normal prior to birth, Sohlh2-null mice had reduced numbers of intermediate and type-B spermatogonia by postnatal day 7. By day 10, development to the pre-leptotene spermatocyte stage was severely disrupted, rendering seminiferous tubules with only Sertoli cells, undifferentiated spermatogonia and degenerating colonies of differentiating spermatogonia. Degenerating cells resembled type-A2 spermatogonia, and accumulated in M-phase prior to death. A similar phenotype was observed in Sohlh2-null mice on postnatal days 14, 21, 35, 49, 68 and 151. In adult Sohlh2-mutant mice, the ratio of undifferentiated type-A spermatogonia (DAZL+/PLZF+) to differentiating type-A spermatogonia (DAZL+/PLZF-) was twice normal levels. In culture, undifferentiated type-A spermatogonia isolated from Sohlh2-null mice proliferated normally, but linked the mutant phenotype to aberrant cell surface expression of the receptor-tyrosine kinase, cKit. Thus, Sohlh2 is required for progression of differentiating type-A spermatogonia into type-B spermatogonia. One conclusion originating from these studies would be that testicular factors normally regulate the viability of differentiating spermatogonia by signaling through Sohlh2. This would provide a crucial check-point to optimize the numbers of spermatocytes entering meiosis during each cycle of spermatogenesis.

From the Cover: Identifying genes important for spermatogonial stem cell self-renewal and survival

Jon Oatley — 2006-06-21T12:49:38-00:00

PNAS, Vol. 103, No. 25. (20 June 2006), pp. 9524-9529.

Spermatogonial stem cells (SSCs) are the foundation for spermatogenesis and, thus, preservation of a species. Because of stem cell rarity, studying their self-renewal is greatly facilitated by in vitro culture of enriched biologically active cell populations. A recently developed culture method identified glial cell line-derived neurotrophic factor (GDNF) as the essential growth factor that supports in vitro self-renewal of SSCs and results in an increase in their number. This system is a good model to study mechanisms of stem cell self-renewal because of the well defined culture conditions, enriched cell population, and available transplantation assay. By withdrawing and replacing GDNF in culture medium, we identified regulated expression of many genes by using microarray analysis. The expression levels of six of these genes were dramatically decreased by GDNF withdrawal and increased by GDNF replacement. To demonstrate the biological significance of the identified GDNF-regulated genes, we examined the importance of the most responsive of the six, bcl6b, a transcriptional repressor. By using siRNA to reduce transcript levels, Bcl6b was shown to be crucial for SSC maintenance in vitro. Moreover, evaluation of Bcl6b-null male testes revealed degeneration and/or absence of active spermatogenesis in 24 +/- 7% of seminiferous tubules. These data suggest that Bcl6b is an important molecule in SSC self-renewal and validate the biological relevance of the GDNF-regulated genes identified through microarray analysis. In addition, comparison of data generated in this study to other stem cell types suggests that self-renewal in SSCs is regulated by distinctly different molecular mechanisms. 10.1073/pnas.0603332103

Testis tissue explant culture supports survival and proliferation of bovine spermatogonial stem cells.

JM Oatley — 2008-03-15T20:42:17-00:00

Biol Reprod, Vol. 70, No. 3. (March 2004), pp. 625-631.

The present study was designed to evaluate the survival and proliferation of bovine spermatogonial stem cells in an explant culture system over a 2-wk period. Explants of calf testicular parenchyma were placed on 0.45-microm pore membranes in culture and maintained for 1-2 wk. Histological examinations of fresh (t0) and cultured tissues revealed morphologically normal seminiferous tubules. Germ cell numbers/tubule increased (P < or = 0.05) during culture when compared with t0, yet germ cell differentiation was not observed. Testosterone was present in medium throughout the culture period, indicating functional Leydig cells. Sertoli, spermatogonial, and spermatogonial stem cell viability was evaluated by reverse transcription-polymerase chain reaction for cell-specific gene expression of stem cell factor, protein gene product 9.5, and glial cell line-derived neurotrophic factor family receptor-alpha1, respectively. Results demonstrated the expression of all genes at t0, 1 wk, and 2 wk of culture. Single-cell suspensions were prepared from the testicular tissues at t0 and during culture and transplanted into nude mouse testes to investigate spermatogonial stem cell viability. One month after transplantation, colonies of round bovine cells were identified in all mouse testes analyzed, indicating survival of spermatogonial stem cells. The average number of resulting colonies in recipient testes was significantly (P < or = 0.05) higher following 1 wk of culture compared with t0 and was numerically higher at 2 wk of culture compared with t0. This increase in colony numbers over time in culture indicates spermatogonial stem cell proliferation in vitro. This explant culture system appears to provide an environment that supports survival and proliferation of bovine spermatogonial stem cells.

Potential role of Nanos3 in maintaining the undifferentiated spermatogonia population

Francesca Lolicato — 2008-03-15T20:28:34-00:00

Developmental Biology, Vol. 313, No. 2. (15 January 2008), pp. 725-738.

Nanos gene encodes for zinc-finger protein with putative RNA-binding activity which shows an evolutionary conserved function in germ cell development. In the mouse, three Nanos homologs have been identified: Nanos1, Nanos2 and Nanos3. The Nanos3 ortholog is expressed in both male and female gonads of early embryo and, after birth, it is found only in the testis. Nanos3 targeted disruption results in the complete loss of germ cells in both sexes; however the role of Nanos3 in the testis during the postnatal period has not been explored yet. In this study, we show that, in prepuberal testis, Nanos3 is expressed in undifferentiated spermatogonia and that its up-regulation causes accumulation of cells in the G1 phase, indicating that this protein is able to delay the cell cycle progression of spermatogonial cells. This is in line with the observation that the cell cycle length of the undifferentiated germ cells is longer than in differentiating spermatogonia. We also demonstrate a conserved mechanism of action of Nanos3, involving the interaction with the murine RNA-binding protein Pumilio2 and consisting of a potential translational repressor activity. According to the possible role of Nanos3 in inhibiting spermatogonia cell differentiation, we show that treatment with the differentiating factor all-trans retinoic acid induces a dramatic down-regulation of its expression. These results allow to conclude that, in the prepuberal testis, Nanos3 is important to maintain undifferentiated spermatogonia via the regulation of their cell cycle.

Complementary Deoxyribonucleic Acid Cloning of Spermatogonial Stem Cell Renewal Factor

Takeshi Miura — 2008-03-12T16:38:30-00:00

Endocrinology, Vol. 144, No. 12. (1 December 2003), pp. 5504-5510.

Spermatogonial mitosis can be subdivided into two processes: spermatogonial stem cell renewal and spermatogonial proliferation toward meiosis. Recently it has been indicated that estrogen, estradiol-17beta, is involved in regulating the renewal of spermatogonial stem cells in eel. To determine the genes that directly regulate this process, we used expression screening to identify genes whose expression is regulated by estradiol-17beta in testes. We detected a previously unidentified cDNA clone that is up-regulated by estradiol-17beta stimulation and named it eel spermatogenesis-related substances 34 (eSRS34) cDNA. Homology searching showed that eSRS34 shares amino acid sequence similarity with human platelet-derived endothelial cell growth factor. We examined the function of eSRS34 using several in vitro systems. Recombinant eSRS34 produced by a baculovirus system induced spermatogonial mitosis in testicular organ culture. Furthermore, the addition of an antibody specific for eSRS34 prevented spermatogonial mitosis induced by estradiol-17beta stimulation in a germ cell/somatic cell coculture system. We therefore conclude that eSRS34 is a "spermatogonial stem cell renewal factor." 10.1210/en.2003-0800

The spermatogonial stem cell: from basic knowledge to transgenic technology.

V Olive — 2007-03-26T00:25:40-00:00

Int J Biochem Cell Biol, Vol. 37, No. 2. (February 2005), pp. 246-250.

Differentiation of germ cells in the testis originates from a constantly renewed small pool of stem cells. They give rise to the first differentiated spermatogenic cells (spermatogonia). These committed cells will then follow a strictly defined succession of steps, starting with six synchronized mitotic cycles before reaching the first meiotic stages. Following a first identification of the spermatogonial stem cells on morphological and cytological criteria, a functional assay was devised, based on their ability to repopulate the testis of a sterile recipient. Purification and characterization of the stem fraction is in progress. Fundamental knowledge of the biology of the germ line and preclinical studies in several important fields will benefit of these advances, while gene transfer prior to reimplantation opens a new approach in transgenic technology.

Proliferation and differentiation of spermatogonial stem cells.

DG de Rooij — 2008-03-12T16:11:46-00:00

Reproduction, Vol. 121, No. 3. (March 2001), pp. 347-354.

Spermatogonial stem cells (A(s) spermatogonia) are single cells that either renew themselves or produce A(pr) (paired) spermatogonia predestined to differentiate. In turn, the A(pr) divide into chains of A(al) (aligned) spermatogonia that also divide. The ratio between self-renewal and differentiation of the stem cells is regulated by glial cell line-derived neurotrophic factor produced by Sertoli cells, while the receptors are expressed in stem cells. A(s), A(pr) and A(al) spermatogonia proliferate during part of the epithelial cycle forming many A(al) spermatogonia. During epithelial stage VIII, almost all A(al) spermatogonia, few A(pr) and very few A(s) spermatogonia differentiate into A1 spermatogonia. A number of molecules are involved in this differentiation step including the stem cell factor-c-kit system, the Dazl RNA binding protein, cyclin D(2) and retinoic acid. There is no fine regulation of the density of spermatogonial stem cells and consequently, in some areas, many A1 and, in other areas, few A1 spermatogonia are formed. An equal density of spermatocytes is then obtained by the apoptosis of A2, A3 or A4 spermatogonia to remove the surplus cells. The Bcl-2 family members Bax and Bcl-x(L) are involved in this density regulation. Several mechanisms are available to cope with major or minor shortages in germ cell production. After severe cell loss, stem cell renewal is preferred above differentiation and the period of proliferation of A(s), A(pr) and A(al) spermatogonia is extended. Minor shortages are dealt with, at least in part, by less apoptosis among A2-A4 spermatogonia.

Spermatogonial stem cells.

DG de Rooij — 2008-03-12T16:10:29-00:00

Curr Opin Cell Biol, Vol. 10, No. 6. (December 1998), pp. 694-701.

The mammalian seminiferous epithelium consists of a highly complex yet well-organized cell population, with germ cells in mitosis and meiosis and postmeiotic cells undergoing transformation to become spermatozoa. To study the factors which control renewal and differentiation of spermatogonial stem cells, animal models are now available which allow for arrest and restart of spermatogonial differentiation. In addition, marked progress has been made in understanding the control of apoptosis and its role in spermatogonia. For the future, spermatogonial stem cell transplantation may have important practical applications.

Spermatogonial stem cells: characteristics and experimental possibilities

Pedro Aponte — 2006-02-18T12:23:40-00:00

Apmis, Vol. 113, No. 11-12. (November 2005), pp. 727-742.

All you wanted to know about spermatogonia but were afraid to ask.

DG de Rooij — 2008-03-12T15:01:04-00:00

J Androl, Vol. 21, No. 6. (c 2000), pp. 776-798.

Sohlh1 is essential for spermatogonial differentiation

D Ballow — 2008-03-12T12:43:54-00:00

Developmental Biology, Vol. 294, No. 1. (1 June 2006), pp. 161-167.

Spermatogonia are adult germline stem cells that can both self-renew and differentiate into spermatocytes. Little is known about factors necessary for spermatogonial differentiation. We identified a novel germ-cell-specific transcription factor that we named Sohlh1 (spermatogenesis and oogenesis specific basic helix-loop-helix (bHLH) transcription factor). In males, Sohlh1 is preferentially expressed in prespermatogonia and Type A spermatogonia. Loss of Sohlh1 causes infertility by disrupting spermatogonial differentiation into spermatocytes. Seven-day-old testes without Sohlh1 still express the testis-specific transcription factors Etv5, Taf4b, Zfp148, and Plzf, overexpress a novel Sohlh2 bHLH transcription factor, but lack LIM homeobox gene Lhx8 and show reduced expression of Ngn3. Sohlh1 represents the first testis-specific bHLH transcription factor that is essential for spermatogonial differentiation.

Sox3 expression in undifferentiated spermatogonia is required for the progression of spermatogenesis

Gerald Raverot — 2008-03-12T10:18:39-00:00

Developmental Biology, Vol. 283, No. 1. (1 July 2005), pp. 215-225.

Sox3, a member of the high mobility group (HMG) family of transcription factors, is expressed in neural progenitor cells and in the gonads. Targeted deletion of Sox3 in mice causes abnormal development of the diencephalon and Rathke's pouch, the progenitor of the anterior pituitary gland. Male and female mice are also infertile and exhibit a primary defect in gametogenesis. In this study, we examined the expression and function of Sox3 in C57BL/6 mice to better understand its role in spermatogenesis. Testis development was normal during embryogenesis. However, spermatogenesis failed to progress during the postnatal period, with germ cell loss beginning at postnatal day 10 (P10). By P14, Sox3 null mice were nearly agametic, retaining only Sertoli cells and undifferentiated spermatogonia. Pituitary gonadotropin and testosterone levels were normal, suggesting a defect in Sertoli cell and/or germ cell function. Immunostaining revealed that Sox3 was expressed in a subpopulation of germ cells localized at the base of the seminiferous tubules. Sox3 expression was restricted to proliferating germ cells and colocalized with neurogenin 3 (Ngn3), a helix-loop-helix transcription factor implicated in spermatogonial differentiation. The absence of Sox3 decreased Ngn3 and increased expression of Oct4, a marker of undifferentiated spermatogonia. We conclude that Sox3 is expressed in As, Apr and Aal spermatogonia and is required for spermatogenesis through a pathway that involves Ngn3.

Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat.

BY Ryu — 2008-03-12T09:50:45-00:00

Proc Natl Acad Sci U S A, Vol. 102, No. 40. (4 October 2005), pp. 14302-14307.

Self-renewal of spermatogonial stem cells (SSCs) is the foundation for maintenance of spermatogenesis throughout life in males and for continuation of a species. The molecular mechanism underlying stem cell self-renewal is a fundamental question in stem cell biology. Recently, we identified growth factors necessary for self-renewal of mouse SSCs and established a serum-free culture system for their proliferation in vitro. To determine whether the stimulatory signals for SSC replication are conserved among different species, we extended the culture system to rat SSCs. Initially, a method to assess in vitro expansion of SSCs was developed by using flow cytometric analysis, and, subsequently, we found that a combination of glial cell line-derived neurotrophic factor, soluble glial cell line-derived neurotrophic factor-family receptor alpha-1 and basic fibroblast growth factor supports proliferation of rat SSCs. When cultured with the three factors, stem cells proliferated continuously for >7 months, and transplantation of the cultured SSCs to recipient rats generated donor stem cell-derived progeny, demonstrating that the cultured stem cells are normal. The growth factor requirement for replication of rat SSCs is identical to that of mouse; therefore, the signaling factors for SSC self-renewal are conserved in these two species. Because SSCs from many mammals, including human, can replicate in mouse seminiferous tubules after transplantation, the growth factors required for SSC self-renewal may be conserved among many different species. Furthermore, development of a long-term culture system for rat SSCs has established a foundation for germ-line modification of the rat by gene targeting technology.

Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia.

BH Schrans-Stassen — 2008-03-12T09:46:45-00:00

Endocrinology, Vol. 140, No. 12. (December 1999), pp. 5894-5900.

The proto-oncogene c-kit is encoded at the white-spotting locus and in the mouse mutations at this locus affect the precursor cells of melanocytes, hematopoietic cells, and germ cells. c-kit is expressed in type A spermatogonia, but whether or not c-kit is present both in undifferentiated and differentiating type A spermatogonia or only in the latter cell type is still a matter of debate. Using the vitamin A-deficient mouse model, we studied messenger RNA (mRNA) and protein expression in undifferentiated and differentiating type A spermatogonia. Furthermore, we quantified the immuno-positive type A spermatogonia in the epithelial stages VI, VII, IX/X, and XII in normal mice to correlate c-kit expression in type A spermatogonia with the differentiation of these cells. Our results show that in the VAD situation undifferentiated type A spermatogonia express little c-kit mRNA. The A spermatogonia with a larger nucleus expressed c-Kit protein, whereas the A spermatogonia with a smaller one did not. After induction of differentiation of these cells into type A1 spermatogonia, c-kit mRNA was enhanced. The percentage of A spermatogonia expressing c-Kit protein did not change during this process, suggesting that A spermatogonia, which are committed to differentiate express c-kit. Under normal circumstances in epithelial stage VI 16%+/-2% (mean +/- SD), in VII 45%+/-15%, in IX/X 78%+/-14% and in XII 90%+/-1.9% of the type A spermatogonia were c-kit positive, suggesting that Aaligned spermatogonia gradually change from c-Kit negative to c-Kit positive cells before their differentiation into A1 spermatogonia. It is concluded that c-kit can be used as a marker for differentiation of undifferentiated into differentiating type A spermatogonia.

Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells.

H Kubota — 2008-03-12T09:29:44-00:00

Proc Natl Acad Sci U S A, Vol. 101, No. 47. (23 November 2004), pp. 16489-16494.

Spermatogonial stem cells (SSCs) self-renew and produce large numbers of committed progenitors that are destined to differentiate into spermatozoa throughout life. However, the growth factors essential for self-renewal of SSCs remain unclear. In this study, a serum-free culture system and a transplantation assay for SSCs were used to identify exogenous soluble factors that promote proliferation of SSCs. Mouse pup testis cells were enriched for SSCs by selection with an anti-Thy-1 antibody and cultured on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) feeders in a serum-free defined medium. In the presence of glial cell line-derived neurotrophic factor (GDNF), SSCs from DBA/2J strain mice formed densely packed clumps of cells and continuously proliferated. However, other strains of mice required the addition of soluble GDNF-family receptor alpha-1 and basic fibroblast growth factor to support replication. The functional transplantation assay proved that the clump-forming cells are indeed SSCs. Thus, GDNF-induced cell signaling plays a central role in SSC self-renewal. The number of SSCs in culture doubled every 5.6 days, and the clump-forming cells strongly expressed Oct-4. Under these conditions, SSCs proliferated over 6 months, reconstituted long-term spermatogenesis after transplantation into recipient testes, and restored fertility to infertile recipients. The identification of exogenous factors that allow continuous proliferation of SSCs in vitro establishes the foundation to study the basic biology of SSCs and makes possible germ-line modification by sophisticated technologies. Moreover, the ability to recover, culture indefinitely, and transplant SSCs will make the germ-line of individual males available for periods extending beyond a normal lifetime.

Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells.

T Shinohara — 2008-03-11T17:41:31-00:00

Proc Natl Acad Sci U S A, Vol. 97, No. 15. (18 July 2000), pp. 8346-8351.

The spermatogonial stem cell initiates and maintains spermatogenesis in the testis. To perform this role, the stem cell must self replicate as well as produce daughter cells that can expand and differentiate to form spermatozoa. Despite the central importance of the spermatogonial stem cell to male reproduction, little is known about its morphological or biochemical characteristics. This results, in part, from the fact that spermatogonial stem cells are an extremely rare cell population in the testis, and techniques for their enrichment are just beginning to be established. In this investigation, we used a multiparameter selection strategy, combining the in vivo cryptorchid testis model with in vitro fluorescence-activated cell sorting analysis. Cryptorchid testis cells were fractionated by fluorescence-activated cell sorting analysis based on light-scattering properties and expression of the cell surface molecules alpha6-integrin, alphav-integrin, and the c-kit receptor. Two important observations emerged from these analyses. First, spermatogonial stem cells from the adult cryptorchid testis express little or no c-kit. Second, the most effective enrichment strategy, in this study, selected cells with low side scatter light-scattering properties, positive staining for alpha6-integrin, and negative or low alphav-integrin expression, and resulted in a 166-fold enrichment of spermatogonial stem cells. Identification of these characteristics will allow further purification of these valuable cells and facilitate the investigation of molecular mechanisms governing spermatogonial stem cell self renewal and hierarchical differentiation.

Isolation and enrichment of murine spermatogonial stem cells using rhodamine 123 mitochondrial dye.

KC Lo — 2008-03-11T17:34:20-00:00

Biol Reprod, Vol. 72, No. 3. (March 2005), pp. 767-771.

Stem cells possess enormous therapeutic potential in tissue replacement. To study stem cells further, they must be isolated. Techniques are available for enrichment and study of hematopoietic stems cells, but thus far, techniques for purification of spermatogonial stem cells have not been described. Enrichment techniques for hematopoietic stem cells include the use of fluorescence-activated cell sorter analysis with Hoechst 33342 and rhodamine 123 (Rho) dyes. Use of Hoechst dye to isolate spermatogonial stem cells has been unsuccessful in our laboratory, and our results have conflicted with those from other laboratories. Taking advantage of the differential staining of the Rho dye, we report a novel method to enrich murine spermatogonial stem cells. Testicular cells are harvested from cryptorchid ROSA26 male mice. Populations of these cells are then stained with the Hoechst and Rho dyes, allowing them to be sorted by flow cytometry into a side population (SP) of Hoechst low-intensity cells and populations of low (Rho(low)) or high (Rho(hi)) fluorescent intensity. Sterile recipients, W/W(v) mice, with an intrinsic germ cell deficiency were transplanted with the Hoechst SP cells, Rho(low), Rho(hi), and nonsorted donor cells. No spermatogonial stem cell colonies were derived from the Hoechst SP cells. The number of spermatogonial stem cell colonies from transplanted Rho(low) cells showed a 17- and 20-fold enrichment over those of Rho(hi) and nonsorted cells, respectively.

'Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells.

B Lassalle — 2008-03-11T17:27:15-00:00

Development, Vol. 131, No. 2. (January 2004), pp. 479-487.

Stem cells in various somatic tissues (bone marrow, skeletal muscle) can be identified by the 'Side Population' marker based on Hoechst 33342 efflux. We show that mouse testicular cells also display a 'Side Population' that express Bcrp1 mRNA, the ABC transporter responsible for Hoechst efflux in hematopoietic cells. Inhibition of Hoechst efflux by specific BCRP1 inhibitor Ko143 show that germinal 'Side Population' phenotype is dependent on BCRP1 activity. Analysis of two well-defined models of altered spermatogenesis (W/Wv mutants and cryptorchid male mice) and RNA expression studies of differentiation markers demonstrate that germinal 'Side Population' contains spermatogonial cells. In addition, alpha 6-integrin and Stra8 germinal stem cell markers, are expressed in the 'Side Population'. In vivo repopulation assay clearly establishes that testis 'Side Population' in adult mice is highly enriched in male germ stem cells.

Development of a cryopreservation protocol for type A spermatogonia.

F Izadyar — 2008-03-05T15:54:43-00:00

J Androl, Vol. 23, No. 4. (g 2002), pp. 537-545.

The aim of this study was to develop a cryopreservation protocol for type A spermatogonia. Testes from 5- to 7-month-old calves were collected, and type A spermatogonia were isolated using two-step enzymatic digestion and Percoll separation. Cells were resuspended in minimum essential medium (MEM) supplemented with 1% bovine serum albumin (BSA) in a final concentration of 6 x 10(6) per mL, and the effects of different cryoprotectants and freezing protocols were tested. Cells frozen/thawed in medium containing 10% fetal calf serum (FCS) and 1.4 M glycerol or dimethyl sulfoxide (DMSO) had a significantly (P <.05) higher percentage of living cells compared to medium with only FCS, whereas DMSO gave a significantly better cell survival rate than glycerol did. An increase in the concentration of FCS in the DMSO-based medium to 20% had no effect on survival after freezing and thawing. Furthermore, inclusion of 0.07, 0.14, or 0.21 M sucrose in DMSO-based medium resulted in a significant improvement of cell survival, cell proliferation in culture, and colonization efficiency in recipient testes. A controlled slow-freezing rate (1 degrees C/min) resulted in significantly (P <.05) more viable cells than fast (5 degrees C/min) freezing. However, noncontrolled-rate freezing, with a comparably low cooling rate, gave even better results than the controlled-rate slow freezing. Cryopreservation in MEM-based medium containing 10% FCS, 10% DMSO, and 0.07 M sucrose using a non-controlled-rate freezing protocol appeared to be a simple and effective way to preserve type A spermatogonia, with a high yield of almost 70% living cells after thawing. Frozen/thawed spermatogonia survived in culture and retained the ability to proliferate as determined by colorimetric and bromodeoxyuridine incorporation assays. To test whether the stem cells among the A spermatogonia retained their ability to colonize the testis of a recipient mouse, bovine spermatogonia were transplanted. This resulted in colonization 2-3 months after transplantation. In conclusion, for the first time, a method specific for cryopreservation of type A spermatogonia, including spermatogonial stem cells was developed, which allows long-term preservation of these cells without apparent harmful effects to their function.