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.     

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.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Identification, isolation, and in vitro culture of porcine gonocytes

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

Functional analysis of the p53 gene in apoptosis induced by heat stress or loss of stem cell factor signaling in mouse male germ cells

Apoptosis plays an important role in controlling germ cell numbers and restricting abnormal cell proliferation during spermatogenesis. The tumor suppressor protein, p53, is highly expressed in the testis, and is known to be involved in apoptosis, which suggests that it is one of the major causes of germ cell loss in the testis. Mice that are c-kit/SCF mutant (Sl/Sld) and cryptorchid show similar testicular phenotypes; they carry undifferentiated spermatogonia and Sertoli cells in their seminiferous tubules. To investigate the role of p53-dependent apoptosis in infertile testes, we transplanted p53-deficient spermatogonia that were labeled with enhanced green fluorescence protein into cryptorchid and Sl/Sld testes. In cryptorchid testes, transplanted p53-deficient spermatogonia differentiated into spermatocytes, but not into haploid spermatids. In contrast, no differentiated germ cells were observed in Sl/Sld mutant testes. These results indicate that the mechanism of germ cell loss in the c-kit/SCF mutant is not dependent on p53, whereas the apoptotic mechanism in the cryptorchid testis is quite different (i.e., although the early stage of differentiation of spermatogonia and the meiotic prophase is dependent on p53-mediated apoptosis, the later stage of spermatids is not).     

Clinical aspects of male germ cell apoptosis during testis development and spermatogenesis

Apoptosis appears to have an essential role in the control of germ cell number in testes. During spermatogenesis germ cell deletion has been estimated to result in the loss of up to 75% of the potential number of mature sperm cells. At least three factors seem to determine the onset of apoptosis in male germ cells: (1) lack of hormones, especially gonadotropins and androgens; (2) the specific stage in the spermatogenic cycle; (3) and the developmental stage of the animal. Although male germ cell apoptosis has been well characterized in various animal models, few studies are presently available regarding germ cell apoptosis in the human testis. The first part of this review is focused on germ cell apoptosis in testes of prepubertal boys, with special emphasis on apoptosis in normal and cryptorchid testes. A higher percentage of apoptotic spermatogonia was seen in the cryptorchid testes than in the scrotal testes. The hCG-treatment increased the number of apoptotic spermatogonia. The hCG-treatment-induced apoptosis in spermatogonia had severe long-term consequences in reproductive functions in adulthood. Increased apoptosis after hCG-treatment was associated with subnormal testis volumes, subnormal sperm density and pathologically elevated serum FSH. This finding indicates that increased apoptosis in spermatogonia in prepuberty leads to disruption of testis development. To evaluate the role of apoptosis in human adult testes, apoptosis was induced in seminiferous tubules that were incubated under serum-free conditions in the absence or presence of testosterone. Most frequently apoptosis was identified in spermatocytes. Occasionally some spermatids also showed signs of apoptosis. In short term incubations apoptosis was suppressed by testosterone. Our findings lead to the conclusion that apoptosis is a normal, hormonally controlled phenomenon in the human testis. The role of apoptosis in disorders of spermatogenesis remains to be established.     

Autoantigenic germ cells exist outside the blood testis barrier

Preleptotene spermatocytes and spermatogonia are germ cells located outside the blood-testis barrier provided by the Sertoli cells. These cells have been found to express autoantigens accessible to circulating antibodies. Mice immunized with syngeneic testis with or without bacterial adjuvant had detectable IgG on cells at the periphery of seminiferous tubules. Sera from orchiectomized but not from testes-intact mice immunized with testis and adjuvants readily transferred similar IgG deposits to testes of normal recipients. When testis-specific antisera from orchiectomized mice and testis-intact mice were compared for their reactivity on prepuberal testicular cells, serum from orchiectomized donors had significantly higher reactivity. Ig was eluted from IgG-positive testes with acid buffer and was shown to be highly enriched in antibody to prepuberal testicular cells, confirming the Ag-specific nature of the IgG deposits. The testis IgG deposits reacted with antisera to IgG1 and IgG3 but not IgG2a or IgG2b. This finding can explain lack of association of C3 in the deposits. Only 30 to 40% of seminiferous tubules had IgG deposits and they coincided with stages 7 to 12 of the spermatogenic cycle. Thus, the expression of the autoantigens is stage specific. The in situ formation of immune complexes by circulating autoantibodies demonstrates conclusively that testis autoantigens are not completely sequestered, and the blood-testis barrier as an immunologic barrier is incomplete.     

Testis-specific sulfoglycolipid, seminolipid, is essential for germ cell function in spermatogenesis

More than 90% of the glycolipid in mammalian testis consists of a unique sulfated glyceroglycolipid, seminolipid. The sulfation of the molecule is catalyzed by a Golgi membrane-associated sulfotransferase, cerebroside sulfotransferase (CST). Disruption of the Cst gene in mice results in male infertility due to the arrest of spermatogenesis prior to the metaphase of the first meiosis. However, the issue of which side of the cell function-germ cells or Sertoli cells-is deteriorated in this mutant mouse remains unknown. Our findings show that the defect is in the germ cell side, as evidenced by a transplantation analysis, in which wild-type spermatogonia expressing the green fluorescent protein were injected into the seminiferous tubules of CST-null testis. The transplanted GFP-positive cells generated colonies and spermatogenesis proceeded over meiosis in the mutant testis. The findings also clearly show that the seminolipid is expressed on the plasma membranes of spermatogonia, spermatocytes, spermatids, and spermatozoa, as evidenced by the immunostaining of wild-type testes using an anti-sulfogalactolipid antibody, Sulph-1 in comparison with CST-null testes as a negative control, and that seminolipid appears as early as day 8 of age, when Type B spermatogonia emerge.     

Seasonal spermatogenic cycle and morphology of germ cells in the viviparous lizard Mabuya brachypoda (Squamata,Scincidae)

We describe seasonal variations of the histology of the seminiferous tubules and efferent ducts of the tropical, viviparous skink, Mabuya brachypoda, throughout the year. The specimens were collected monthly, in Nacajuca, Tabasco state, Mexico. The results revealed strong annual variations in testicular volume, stages of the germ cells, and diameter and height of the epithelia of seminiferous tubules and efferent ducts. Recrudescence was detected from November to December, when initial mitotic activity of spermatogonia in the seminiferous tubules were observed, coinciding with the decrease of temperature, photoperiod and rainy season. From January to February, early spermatogenesis continued and early primary and secondary spermatocytes were developing within the seminiferous epithelium. From March through April, numerous spermatids in metamorphosis were observed. Spermiogenesis was completed from May through July, which coincided with an increase in temperature, photoperiod, and rainfall. Regression occurred from August through September when testicular volume and spermatogenic activity decreased. During this time, the seminiferous epithelium decreased in thickness, and germ cell recruitment ceased, only Sertoli cells and spermatogonia were present in the epithelium. Throughout testicular regression spermatocytes and spermatids disappeared and the presence of cellular debris, and scattered spermatozoa were observed in the lumen. The regressed testes presented the total suspension of spermatogenesis. During October, the seminiferous tubules contained only spermatogonia and Sertoli cells, and the size of the lumen was reduced, giving the appearance that it was occluded. In concert with testis development, the efferent ducts were packed with spermatozoa from May through August. The epididymis was devoid of spermatozoa by September. M. brachypoda exhibited a prenuptial pattern, in which spermatogenesis preceded the mating season. The seasonal cycle variations of spermatogenesis in M. brachypoda are the result of a single extended spermiation event, which is characteristic of reptilian species. J. Morphol. © 2012 Wiley Periodicals, Inc.     

Successful intra- and interspecific male germ cell transplantation in the rat

The lumen of the seminiferous tubules has hitherto been regarded as an immunologically privileged site. We report here the birth of young following transplantation of stem spermatogonia from Long-Evans rats to the seminiferous tubules of Sprague-Dawley rats after treatment with the immunosuppressive agent cyclosporin. Follicle-stimulating hormone was also given to stimulate Sertoli cell proliferation, and testosterone to stimulate the recovery of spermatogenesis. Donor germ cells underwent normal spermatogenesis, and progeny were repeatedly produced from the donor germ cells as demonstrated by microsatellite paternity analysis. In addition, donor germ cells from the cryptorchid testes of LacZ mice were also able to colonize the seminiferous tubules of Sprague-Dawley rats using this protocol. Morphologically normal rat and mouse spermatozoa were present in the epididymis and vas deferens of the recipient rats. This highlights the potential for transplantation of male germ cells between different species.     

Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.     

Real-time observation of transplanted 'green germ cells': proliferation and differentiation of stem cells

To elucidate the mechanism of proliferation and differentiation of testicular germ cells, donor testicular germ cells labeled with enhanced green fluorescent protein (eGFP) were transplanted to recipient seminiferous tubules. The kinetics of colonization as well as of differentiation of the donor cells was followed in the same transplanted tubules (alive) under ultraviolet light. One week after transplantation, clusters of fluorescent cells were randomly spread as dots in the recipient seminiferous tubule, whereas non-homed cells flowed out from the testis to the epididymis. By 4 weeks after transplantation, green germ cells were observed with weak and moderate fluorescence along the recipient seminiferous tubule. By 8 weeks, proliferation and differentiation of the germ cells occurred, resulting in strong fluorescence in the middle part of the seminiferous tubule but in weak and moderate fluorescence at both terminals. The length of the fluorescent positive seminiferous tubule became longer. Detailed histological analyses of the recipient tubules indicated that the portions of the seminiferous tubule in weak, moderate, and strong fluorescence contained the spermatogonia, spermatogonia with spermatocytes, and all types of germ cells including spermatids, respectively. Thus, testicular stem cells colonized first as dots within 1 week, and then proliferated along the basement membrane of the seminiferous tubules followed by differentiation.     

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.     

Seminiferous tubule involution in elderly men

The observation of different types of seminiferous tubules (from tubules with normal spermatogenesis to sclerosed tubules) in aging human testes points to the progressive stages of tubular involution in elderly men. The tubules with hypospermatogonesis (reduced number of elongated spermatids) show numerous morphological anomalies in the germ cells, including multinucleated cells. Abnormal germ cells degenerate, causing Steroli cell vacuolation. These vacuoles correspond to dilations of the extracellular spaces resulting from the premature exfoliation of germ cells. Degenerating cells that are phagocytized by Sertoli cells lead to an accumulation of lipid droplets in the Sertoli cell cytoplasm. The loss of germ cells begins with spermatids, but progressively affects the preceding germ cell types, and tubules with maturation arrested at the level of spermatocytes or spermatogonia are observed. Simultaneously, an enlargement of the tunica propria occurs. This leads to the formation of sclerosed tubules, some of which display a low seminiferous epithelium consisting of a few cells--including lipid-loaded Sertoli cells and both Ap and Ad spermatogonia--and others, showing complete sclerosis, are devoid of seminiferous epithelium. The development of tubular involution is similar to that reported after experimental ischemia, which also seems to cause nonspecific effects on the testis such as multinucleate cells, vacuoles, and increased lipids in Sertoli cells.     

Morphological and histochemical characterization of the seminiferous epithelial and Leydig cells of the turkey

Unlike mammals, there is little fundamental information about spermatogenesis in birds. This study was undertaken to clarify the morphology, histochemistry, and lectin affinity of the seminiferous epithelial cells and Leydig cells in pre-pubertal (8- to 15-week old) and adult (40- to 44-week old) domestic turkeys. In adult turkeys, three types of spermatogonia were defined based on their chromatin distribution and nuclear morphology: the dark type A (A(d)); the pale type A (A(p)); and the type B. The A(d) is the least numerous and least conspicuous and consequently difficult to locate. Based on its spatial distribution and overall morphology, type A(d) spermatogonia were postulated to be the spermatogonia stem cells in the turkey. Antibodies to c-kit were localized to spermatogonia in the pre-pubertal and to a lesser extent in adult males. Peanut agglutinin (PNA) was specific for spermatocytes in the pre-pubertal males and spermatogonia and early spermatocytes in adult males. Wheat-germ agglutinin (WGA) highlighted Sertoli cells in both age groups. Bandeiraea simplicifolia I, soybean agglutinin, and winged-pea agglutinin staining were limited to the wall of the seminiferous tubule and some extra-tubular cell types. Concanavalin A staining was diffuse and not cell-specific and, therefore, could not be used to selectively identify a particular cell type. It was concluded that WGA and PNA could aid in identifying specific cell types in the seminiferous epithelium of testis from pre-pubertal and mature turkeys. Only Leydig cells were alkaline phosphatase reactive in the mature turkey testes. The information from this study is being used to adapt techniques for the isolation and partial purification developed for mammalian spermatogonia to avian spermatogonia and other specific cell types in the testes.     

Loss of sperm in juvenile spermatogonial depletion (jsd) mutant mice is ascribed to a defect of intratubular environment to support germ cell differentiation.

C57BL/6(B6)-jsd/jsd mice are sterile due to the defective spermatogenesis in the testes. To know the cause of the deficient spermatogenesis in B6-jsd/jsd mice, we examined whether the problem is within or outside the seminiferous tubules by transplanting tubules from cryptorchid testes of B6- +/+ mice into B6-jsd/jsd testes or tubules from B6-jsd/jsd mice into testes of (WB x C57BL/6)F1-W/Wv (hereafter, WBB6F1-W/Wv) mice. Type A spermatogonia differentiated into spermatids in seminiferous tubules from cryptorchid testes transplanted into B6-jsd/jsd testes. In contrast, in B6-jsd/jsd tubules transplanted into WBB6F1-W/Wv testes, type A spermatogonia were stimulated to mitotic proliferation, but didn't proceed to any differentiated germ cells. The present results suggest that the cause of the deficient spermatogenesis in B6-jsd/jsd mice is a defect of intratubular environment to support germ cell differentiation.     

Temporal germ cell development strategy during spermatogenesis within the testis of the Ground Skink, Scincella lateralis (Sauria: Scincidae)

Ground Skink (Scincella lateralis) testes were examined histologically to determine the testicular organization and germ cell development strategy employed during spermatogenesis. Testicular tissues were collected from 19 ground skinks from Aiken County, South Carolina during the months of March-June, August, and October. The testes consisted of seminiferous tubules lined with germinal epithelia in which germ cells matured in close association with Sertoli cells. As germ cells matured, they migrated away from the basal lamina of the epithelia towards the lumina of the seminiferous tubules. The testes were spermatogenically active during the months of March, April, May, June, and October (largest seminiferous tubule diameters and epithelial heights), but entered a quiescent period in August (smallest seminiferous tubule diameter and epithelial height) where only spermatogonia type A and B and early spermatocytes were present in low numbers within the seminiferous epithelium. Although the testicular organization was similar to other amniotes, a temporal germ cell development strategy was employed during spermatogenesis within Ground Skinks, similar to that of anamniotes. Thus, this skink's germ cell development strategy, which also has been recently reported in all other major reptilian clades, may represent an evolutionary intermediate in terms of testicular organization between anamniotes and birds and mammals.     

Significant IgG-immunoreactivity of the spermatogonia of the germ cell-depleted testis after busulfan treatment

Busulfan kills spermatogonia with the exception of a few that are attached to the basal membrane of the seminiferous epithelium. In mice, these remaining spermatogonia reacted strongly to a goat anti-mouse IgG antibody. Spermatogonia in untreated testes rarely showed the same reactivity. Testicular IgG levels are normally minimal but increase markedly, 4 weeks after busulfan treatment before peaking at week 6. Laser scanning cytometry analysis of control and busulfan-treated testicular cells showed busulfan treatment increased the frequency of cells that were positive for not only IgG (from 0.67+/-0.29 to 16.5+/-3.8%) but also for alpha6-integrin, beta1-integrin, GFR(-1 and/or Ret. Thus, an enrichment in putative male stem cells correlates with appearance of IgG expression. Confocal microscopy revealed busulfan-treated cells contained both IgG and GFRalpha-1, and that the initial surface IgG became intracellular in the weeks following busulfan treatment. The basement membranes of the seminiferous tubules were compromised by busulfan treatment as the mRNA expression profiles of various adhesion molecules in the basement membranes were altered and electron microscopy revealed severe damage. Serum IgG levels increased in a manner corresponding with the increase in testicular IgG levels. Thus, it appears that in the busulfan-treated testis, small breaches of the blood-testis barrier leak IgG that is then taken up by a significant number of spermatogonia. When the busulfan-resistant germ cells were transferred into recipient germ cell-depleted testes, they settled and repopulated the recipient testes. Thus, the IgG-bearing cells observed after busulfan treatment may be putative spermatogonial stem cells.