Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation

Although methods to assess testis cell populations are established in mice, the detailed validation of similar methods for bovine testis cells is necessary for the development of emerging technologies such as male germ cell transplantation. As young calves provide donor cells for germ cell transplantation, we characterized cell populations from three key pre-pubertal stages. Nine Angus bull calves were selected to represent three stages of testis development at ages (and testis weights) of 2–3 months (Stage 1, 10 g), 4–5 months (Stage 2, 35 g), and 6–7 months (Stage 3, 70 g). The proportion and absolute numbers of germ and somatic cells in fixed sections and from enzymatically dissociated seminiferous tubules were assessed. Germ cells were identified by DBA and PGP9.5 staining, and Sertoli cells by vimentin and GATA-4 staining. Staining of serial sections confirmed that DBA and PGP9.5 identified similar cells, which were complementary to those stained for vimentin and GATA-4. In fixed tubules, the proportion of cells within tubules that were positive for DBA and PGP9.5 increased nearly three-fold from Stage 1 to Stage 2 with no further increase at Stage 3. Absolute numbers of spermatogonia also increased between Stages 1 and 2. After enzymatic dissociation of tubules, three times more DBA- and PGP9.5-positive cells were isolated from Stage 3 testes than from either Stage 1 or 2 testes. A higher proportion of spermatogonia was observed after enzymatic isolation than were present in seminiferous tubules. These data should help to predict the yield and expected proportions of spermatogonia from three distinct stages of testis development in pre-pubertal bull calves.     

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.     

Immunolocalization of proliferating cell nuclear antigen (PCNA) in cynomolgus monkey (Macaca fascicularis) testes during postnatal development

The age-related distribution of proliferating cell nuclear antigen (PCNA) in the testes of cynomolgus monkeys (Macaca fascicularis) during postnatal development was detected using light-microscopic immunohistochemistry. In neonatal testes, some PCNA-positive spermatogonia, Sertoli cells, peritubular cells, and Leydig cells were detected. In early infantile testes, only a few of these cell types were positive. In late infantile testes, the numbers of positive cells were greater than in the earlier developmental stages. In pubertal testes, the numbers of positive spermatogonia, spermatocytes, Sertoli cells, peritubular cells, and Leydig cells were considerably higher. In adult testes, a larger percentage of spermatogonia and spermatocytes was positive, and peritubular cells and Leydig cells were occasionally positive; secondary spermatocytes, spermatids, and Sertoli cells were not positive. We concluded that immunolocalization of PCNA can serve as a tool for studying proliferation status in developing testes of cynomolgus monkeys. A relatively low proliferative activity in early infantile testes and a remarkable increase of proliferative activity in pubertal testes correlate with the fluctuations of steroidogenic functions during postnatal development in cynomolgus monkeys.     

UCHL-1 protein expression specifically marks spermatogonia in wild bovid testis

Spermatogonia are germ cells that initiate spermatogenesis in mammalian testis and they are the only cells in adult body capable of dividing both mitotically and meiotically. Therefore, isolation and preservation of spermatogonia can provide an alternative method for preservation of genetic pool of endangered animals. To achieve this objective, it is essential to identify markers that can specifically distinguish spermatogonia from other cells in the testis. In the present study, anti-ubiquitin C-terminal hydroxylase-1 (UCHL-1/PGP 9.5) antibody specifically recognized spermatogonia in the testis of wild and domestic bovid. The size of the UCHL-1 protein in various species of bovid testes was identical, and similar to that in mice. Furthermore, UCHL-1 staining could be utilized for the identification of spermatogonia in isolated testicular cells from wild bovids suggesting that UCHL-1 protein expression could be used as a specific marker for spermatogonia in bovid family.     

Immunoexpression of Tyro 3 family receptors--Tyro 3, Axl, and Mer--and their ligand Gas6 in postnatal developing mouse testis.

Tyro 3 family receptors contain three members-Tyro 3, Axl, and Mer-that are essential regulators of mammalian spermatogenesis. However, their exact expression patterns in testis are unclear. In this study, we examined the localizations of Tyro 3, Axl, Mer, and their ligand Gas6 in postnatal mouse testes by immunohistochemistry. All three members and their ligand were continuously expressed in different testicular cells during postnatal development. Tyro 3 was expressed only in Sertoli cells with a varied distribution during testis development. At day 3 postnatal, Tyro 3 was distributed in overall cytoplasmic membrane and cytoplasm of Sertoli cells. From day 14 to day 35 postnatal, Tyro 3 appeared on Sertoli cell processes toward the adlumenal compartment of seminiferous tubules. A stage-dependent Tyro 3 immunoexpression in Sertoli cells was shown by adulthood testis at day 56 postnatal with higher expression at stages I-VII and lower level at stages IX-XII. Axl showed a similar expression pattern to Tyro 3, except for some immunopositive Leydig cells detected in mature testis. In contrast, immunostaining of Mer was detected mainly in primitive spermatogonia and Leydig cells, whereas a relative weak signal was found in Sertoli cells. Gas6 was strongly expressed in Leydig cells, and a relative weak staining signal was seen in primitive spermatogonia and Sertoli cells. These immunoexpression patterns of Tyro 3 family receptors and ligand in testis provide a basis to further study their functions and mechanisms in regulating mammalian spermatogenesis.     

Expression of Insulin-like Growth Factor Binding Protein-3 and Regulation of the Insulin-like Growth Factor-I Axis in Pig Testis

The stem cell niche is a complex unit comprising key components, such as the extracellular matrix and various paracrine factors, which regulate the differentiation of adult stem cells. In our previous study, we established pig spermatogonial stem cells (pSSCs) in culture and identified the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in pSSCs. The present study investigated not only the expression of IGFBP-3, but also its possible role in pSSCs. In this study, IGFBP-3-expressing cells responded positively to protein gene product 9.5 (PGP9.5), which is a marker for pig spermatogonia. IGFBP-3 expression was significantly increased in 60-dayold pig testes. Additionally, the expression levels of insulinlike growth factor I (IGF-I) and its receptor (IGF-IR) were observed in pSSCs and pig Sertoli cells (pSCs). Furthermore, IGF-I treatment enhanced the proliferation of pSCs and pSSCs when they were co-cultured. Blocking the IGF-I pathway using a specific IGF-IR inhibitor dramatically reduced the proliferation of pSCs. In addition, when heparan sulfate was used to sequester IGFBP-3 from IGF-I binding, a significant increase in the proliferation of pSCs was observed. Exogenous IGF-I treatment also increased the expression level of IGFBP-3 in cultured pSSCs. Furthermore, pSSCs grew well in IGF-I-treated pSC conditioned media. In summary, IGF-I and IGF-IR signaling are important for the proliferation of pSCs, and the germ cell-derived IGFBP-3 had an inhibitory effect on the mitotic activity of IGF-I in pSCs.     

Expression pattern of acetylated α‐tubulin in porcine spermatogonia

Mammalian spermatogonial stem cells reside on the basement membrane of the seminiferous tubules. The mechanisms responsible for maintenance of spermatogonia at the basement membrane are unclear. Since acetylated α‐tubulin (Ac‐α‐Tu) is a component of long‐lived, stable microtubules and deacetylation of α‐tubulin enhances cell motility, we hypothesized that acetylation of α‐tubulin might be associated with positioning of spermatogonia at the basement membrane. The expression pattern of Ac‐α‐Tu at different stages of testis development was characterized by immunohistochemistry for Ac‐α‐Tu and spermatogonia‐specific proteins (PGP 9.5, DAZL). In immature pig testes, Ac‐α‐Tu was present exclusively in gonocytes at 1 week of age, and in a subset of spermatogonia at 10 weeks of age. At this age, spermatogonia are migrating toward the tubule periphery and Ac‐α‐Tu appeared polarized toward the basement membrane. In adult pig testes, Ac‐α‐Tu was detected in few single or paired spermatogonia at the basement membrane as well as in spermatids and spermatozoa. Only undifferentiated (DAZL?), proliferating (determined by BrdU incorporation) spermatogonia expressed high levels of Ac‐α‐Tu. Comparison with the expression pattern of β‐tubulin and tyrosinated α‐tubulin confirmed that only Ac‐α‐Tu is specific to germ cells. The unique pattern of Ac‐α‐Tu in undifferentiated germ cells during postnatal development suggests that posttranslational modifications of microtubules may play an important role in recruiting and anchoring spermatogonia at the basement membrane. Mol. Reprod. Dev. 77: 348–352, 2010. © 2009 Wiley‐Liss, Inc.     

Immunolocalization of inhibin and activin alpha and betaB subunits and expression of corresponding messenger RNAs in the human adult testis

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an alpha and a beta(B) subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin alpha and inhibin/activin beta(B) subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin alpha subunit and expressed inhibin alpha subunit mRNA. Using inhibin beta(B) subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin beta(B) subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin beta(B) subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas beta(B) subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.     

Change of cyclin D2 mRNA expression during murine testis development detected by fragmented cDNA subtraction method

Using subtractive hybridization and polymerase chain reaction, we developed a differential cloning system, the fragmented cDNA subtraction method, that requires only small amounts of materials. The cloning system was used to isolate several cDNA fragments expressed more abundantly in the premeiotic day 3 post-natal mouse testis than in the adult mouse testis. The isolated cDNA fragments included cDNA encoding the murine cyclin D2. Northern blot and in situ hybridization analyses revealed that, during testis development, cyclin D2 expression was most abundant in the neonatal proliferating Sertoli cells. Those type A spermatogonia that were thought to divide mitotically also expressed cyclin D2 mRNA. Other spermatogenic cells, such as mitotically arrested gonocytes in neonatal testis and meiotically dividing germ cells in adult testis as well as adult Sertoli cells, were negative for the cyclin D2 signal. Adult W/W ^v mutant mice lacking germ cells expressed cyclin D2 mRNA in terminally differentiated Sertoli cells. Elimination of germ cells other than the undifferentiated type A spermatogonia by treating wild-type mice with an anti-c- kit monoclonal antibody did not result in the expression of cyclin D2 in Sertoli cells. These results demonstrate that there are lineage- and developmental-specific expression patterns of cyclin D2 mRNA during mouse testis development. At the same time, it is suggested that primitive type A spermatogonia affect the cyclin D2 expression of Sertoli cells.     

Differentiation of Secondary Spermatogonia to Primary Spermatocytes by Mammalian Follicle-Stimulating Hormone in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster

In order to elucidate essential factors responsible for the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments, consisting of somatic cells and germ cells almost exclusively secondary spermatogonia, were cultured in control medium for three weeks, most of the testicular cysts still contained only secondary spermatogonia. On the other hand, in the medium supplemented with various kinds of hormones and vitamins primary spermatocytes (zygotene-pachytene) appeared in about 60% of the cysts by the second week. Selective removal of specific hormones and vitamins revealed that follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the differentiation of secondary spermatogonia to primary spermatocytes. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5α-dihydrotestosterone) to the control medium stimulated differentiation. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH. Since our ultrastructural studies revealed a major loss of contact between spermatogonia and Sertoli cells following exposure to FSH, we suggest that FSH triggers differentiation of spermatogonia by acting on Sertoli cells which in turn act on spermatogonia.     

Testicular Expression of NGF,TrkA and p75 During Seasonal Spermatogenesis of the Wild Ground Squirrel (Citellus Dauricus Brandt)

The nerve growth factor (NGF) not only has an essential effect on the nervous system, but also plays an important role in a variety of non-neuronal systems, such as the reproductive system. The aim of this study was to compare the quality and quantity in expression of NGF and its receptors (TrkA and p75) in testes of the wild ground squirrel during the breeding and nonbreeding seasons. Immunolocalization for NGF was detected mainly in Leydig cells and Sertoli cells in testes of the breeding and nonbreeding seasons. The immunoreactivity of TrkA was highest in the elongated spermatids, whereas p75 in spermatogonia and spermatocytes in testes of the breeding season. In the nonbreeding season testes, TrkA showed positive immunostainings in Leydig cells, spermatogonia and primary spermatocytes, while p75 showed positive signals in spermatogonia and primary spermatocytes. Consistent with the immunohistochemical results, the mean mRNA and protein level of NGF and TrkA were higher in the testes of the breeding season than in non-breeding season, and then decreased to a relatively low level in the nonbreeding season. In addition, the concentration of plasma gonadotropins and testosterone were assayed by radioimmunoassay (RIA), and the results showed a significant difference between the breeding and nonbreeding seasons with higher concentrations in breeding season. In conclusion, these results of this study provide the first evidence on the potential involvement of NGF and its receptor, TrkA and p75 in the seasonal spermatogenesis and testicular function change of the wild ground squirrel.Key words: NGF, p75, seasonal spermatogenesis, TrkA, wild ground squirrel     

Identification and In Vitro Derivation of Spermatogonia in Beagle Testis

Background

In vitro culture of spermatogonial stem cells (SSCs) is important for exploration of SSCs self-renewal, differentiation, and manipulation. There are several reports on rodent SSC cultures; however, data on SSC cultures in domestic animals are limited. To provide basic scientific information on canine SSC cultures, we report canine testes development, and the development of spermatogonia-derived colonies (SDCs) for in vitro cultures.

Methodology/Principal Findings

Testes from 2-, 3-, and 12-month-old beagles were used for histology, immunohistochemistry, in vitro culture, immunocytochemistry, and PCR. Protein gene product 9.5 (PGP9.5)-positive spermatogonia, both single and paired, were found to be abundant in the testes of 2-month-old beagles. stempro-34 and Dulbecco''s modified Eagle medium with 5% fetal bovine serum provided as useful substrates for culture of SDCs, and fibroblast growth factor (FGF) played a key role in colony formation. Colonies were positive for alkaline phosphatase and anti-PGP9.5 staining. The early spermatogonia and stem cell markers such as octamer binding protein 4 (Oct4), Nanog homeobox (Nanog), promyelocytic leukemia zinc finger (PLZF), PGP9.5, and GDNF family receptor alpha-1 (GFRα-1) were expressed in the colonies at higher levels than in the testis tissue.

Conclusions

Testes of the 2-month-old beagles had abundant single and paired spermatogonia, which can be used for derivation of SDCs, and FGF was important for colony formation.     

Localization of inhibin in testes of human, bonnet monkey, dog and rat by immunoperoxidase technique

Immunocytochemical study on the localization of inhibin in the testes of human, bonnet monkey, dog and rat was carried out using indirect immunoperoxidase technique, in order to investigate the cell types involved in inhibin production/storage. A positive reaction was observed in the testes of human, monkey and dog while it was negative in rat testis using specific antiserum to human testicular inhibin generated against homogeneous preparation of human testicular inhibin in our laboratory. Inhibin was found to be localized in Sertoli cells, spermatogonia and primary spermatocytes of human, monkey and dog testes. A weak positive reaction was observed in spermatids of human testis only. Interestingly, Leydig cells of human, monkey and dog testes showed positive reaction indicating presence of inhibin in these cells also.     

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.     

Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis

Heat shock proteins (HSPs) are molecular chaperones involved in protein folding, assembly and transport, and which play critical roles in the regulation of cell growth, survival and differentiation. We set out to test the hypothesis that HSP27 protein is expressed in the human testes and its expression varies with the state of spermatogenesis. HSP27 expression was examined in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) using immunofluorescent methods. The biopsies were obtained from patients undergoing investigations for infertility. The seminiferous epithelium of the human testes showing normal spermatogenesis had a cell type-specific expression of HSP27. HSP27 expression was strong in the cytoplasm of the Sertoli cells, spermatogonia, and Leydig cells. Alternatively, the expression was moderate in the spermatocytes, weak in the spermatids and absent in the spermatozoa. In testes showing maturation arrest, HSP27 expression was strong in the Sertoli cells, weak in the spermatogonia, and spermatocytes. It was absent in the spermatids and Leydig cells. In Sertoli cell only syndrome, HSP27 expression was strong in the Sertoli cells and absent in the Leydig cells. We report for the first time the expression patterns of HSP27 in the human testes and show differential expression during normal spermatogenesis, indicating a possible role in this process. The altered expression of this protein in testes showing abnormal spermatogenesis may be related to the pathogenesis of male infertility.     

A cystatin-related gene, testatin/cresp, shows male-specific expression in germ and somatic cells from the initial stage of murine gonadal sex-differentiation

Sex-differentiation in mammals initiates at mid-gestation when the differentiation of Sertoli cells is triggered by the expression of the testis-determining gene, Sry. However, little is known about the succeeding germ-soma interaction that directs the sex-differentiation of germ cells. We carried out subtraction and differential screening between male and female gonads at 13.5 dpc (days post coitum). A novel cystatin-related gene was identified and named cresp (cystatin-related expressed in Sertoli and spermatogonia), and has recently been reported independently under the name testatin (T?h?nen et al., 1998). The presumed amino acid sequence of testatin/cresp showed considerable homology to the cystatin family, but it lacked a few critical amino acid residues for the cysteine-protease inhibitory activity. A 0.7 kb RNA was detected by northern blotting specifically in the fetal and adult testes from 11.5 dpc and expression increased between 11.5 dpc and 12.5 dpc. Using RT-PCR analysis, the testatin/cresp mRNA was first detectable at 9.5 dpc in both male and female embryos but it was maintained only in the male. In females, the expression became weaker at 11.5 dpc and was undetectable after 12.0 dpc. In situ hybridization and immunohistochemical analyses, as well as single cell RT-PCR analysis, showed that the testatin/cresp mRNA was localized specifically in both the (pro)spermatogonia and Sertoli cells in the testis from 12.5 dpc to adult. Thus, expression of the testatin/cresp gene is upregulated in male gonads but downregulated in females immediately after the initiation of sex-differentiation, suggesting roles in the early developmental cascade of testis such as the germ-soma interaction.     

Gene expression alterations by conditional knockout of androgen receptor in adult Sertoli cells of Utp14b jsd/jsd (jsd) mice

Spermatogenesis is dependent primarily on testosterone action on the Sertoli cells, but the molecular mechanisms have not been identified. Attempts to identify testosterone-regulated target genes in Sertoli cells have used microarray analysis of gene expression in mice lacking the androgen receptor (AR) in Sertoli cells (SCARKO) and wild-type mice, but the analyses have been complicated both by alteration of germ cell composition of the testis when pubertal or adult mice were used and by differences in Sertoli-cell gene expression from the expression in adults when prepubertal mice were used. To overcome these limitations and identify AR-regulated genes in adult Sertoli cells, we compared gene expression in adult jsd (Utp14b jsd/jsd, juvenile spermatogonial depletion) mouse testes and with that in SCARKO-jsd mouse testes, since their cellular compositions are essentially identical, consisting of only type A spermatogonia and somatic cells. Microarray analysis identified 157 genes as downregulated and 197 genes as upregulated in the SCARKO-jsd mice compared to jsd mice. Some of the AR-regulated genes identified in the previous studies, including Rhox5, Drd4, and Fhod3, were also AR regulated in the jsd testes, but others, such as proteases and components of junctional complexes, were not AR regulated in our model. Surprisingly, a set of germ cell–specific genes preferentially expressed in differentiated spermatogonia and meiotic cells, including Meig1, Sycp3, and Ddx4, were all upregulated about 2-fold in SCARKO-jsd testes. AR-regulated genes in Sertoli cells must therefore be involved in the regulation of spermatogonial differentiation, although there was no significant differentiation to spermatocytes in SCARKO-jsd mice. Further gene ontogeny analysis revealed sets of genes whose changes in expression may be involved in the dislocation of Sertoli cell nuclei in SCARKO-jsd testes.     

Effect of the W mutation, for white belly spot, on testicular germ cell differentiation in mice.

The effect of the mutation for white belly spot controlled by the dominant gene W on spermatogenesis in mice was examined by experimental cryptorchidism and its surgical reversal. The course of spermatogenesis from spermatogonia to spermatid was normal in intact testes of W/+ mice. In cryptorchid testes, there was no difference in the number and activity of Type A spermatogonia between the testes of W/+ and +/+ mice, in mitotic and labelling indices. Although surgical reversal of the cryptorchid testis resulted in regenerative differentiation of germ cells in both genotypes, the recovery of cell differentiation in the W/+ testis was slower than in the +/+ testis. There were fewer germ cells, such as intermediate-Type B spermatogonia or more advanced ones, in W/+ testes. On Day 17 after surgical reversal, cell associations in W/+ testes were abnormal and the numbers of intermediate-Type B spermatogonia, spermatocytes and spermatids were approximately 70, 50 and 15%, respectively, of those in +/+ testes. These results indicate that the W gene affects spermatogenic cell differentiation in adult mice.     

Expression of p57 in mouse and human testes

The expression of cyclin-dependent kinases inhibitors, p57kip2, was investigated during the postnatal development of mouse testis, and in adult human testis. Expression of p57kip2 mRNA was higher in immature than pubertal or adult mouse testes. In postnatal day 7 (PND7) testes, moderate p57kip2 immunoreactivity was found in spermatogonia, but signal was heterogeneous among the spermatogonia. In PND14 testes onward, strong immunoreactivity of p57kip2 was found in the nuclei of early spermatocytes but not in the late pachytene stage onward. In PND28 and PND50 testes, p57kip2 immunoreactivity was varying among the seminiferous tubules. There was no visible signal in late pachytene stage onward. In Leydig cells, heterogeneous immunoreactivity of p57kip2 was found in immature testis and the signal intensity was higher in adult testis than immature ones. In Sertoli cells, weak or negligible immunoreactivity of p57kip2 was found. In human seminiferous tubule, strong immunoreactivity of p57kip2 was found in the nucleus of early spermatocytes, but not in the late pachytene spermatocytes onward and Sertoli cells. These results suggest the possible role of p57kip2 in the regulation of early spermatogonial proliferation, meiotic progression of early spermatocytes and differentiation of Leydig cells in testis.     

Isolation and transplantation of spermatogonia in sheep

Studies in rodents show that spermatogonial transplantation is an excellent new tool for studying spermatogenesis and for preservation and dissemination of genetics. The aim of this study was to adapt the technique to rams. Two issues were addressed: purification of stem cell spermatogonia, and efficient injection of donor spermatogonia into the seminiferous tubules of rams. We compared differential plating and Percoll gradient methods for purifying donor spermatogonia from ram lamb testes. Spermatogonia were identified with an antibody against PGP 9.5, a ubiquitin C-terminal hydrolase. Both purity and total number of spermatogonia recovered were higher after purification by Percoll gradient than by differential plating. Four approaches for injecting cells into the seminiferous tubules of ram testes were compared ex vivo: insertion of a needle into the extra-testicular rete testis after reflection of the head of the epididymis ('surgical' approach), and ultrasound-guided insertion of a needle into the extra-testicular rete, and the proximal and distal parts of the intra-testicular rete testis. 'Surgical' and ultrasound-guided approaches into the extra-testicular rete resulted in highest success rates and best filling of the seminiferous tubules. Finally, the ultrasound guided approach into the extra-testicular rete testis was validated in vivo by transplanting purified spermatogonia previously labeled with a fluorescent molecule (CFDA-SE). In seven of eight testes injected, donor cells were identified within the seminiferous epithelium for up to 2wk after transplantation, indicating the integration of donor cells.