Basement Membrane Of Seminiferous Tubules Research Articles

164. Gonadal Expression of Foxo1, but Not Foxo3, Is Conserved in Diverse Mammalian Species.

The Foxo forkhead transcription factors are key effectors of the PI3K/Akt signaling pathway and regulate diverse physiologic processes. Two of these factors, Foxo1 and Foxo3, serve specific roles in reproduction in the mouse. Intriguingly, the functional requirement for each factor (revealed by analyses of knockout mice) mirrors the remarkably specific expression of the protein in a cell-autonomous manner. For example, Foxo3 is required for the maintenance of primordial oocytes in a quiescent state, and Foxo3 protein is highly expressed in primordial oocytes but not in other ovarian cell types. More recently, our laboratory demonstrated a specific role for Foxo1 in male fertility and the maintenance of spermatogonial stem cells in the mouse, and Foxo1 is highly expressed in spermatogonial stem cells but not in other testicular cell types. In the mouse ovary, Foxo1 is highly expressed in granulosa cells but not in oocytes or other cell types, suggesting an important functional role for Foxo1 in granulosa cells. Given that invertebrate model species such as C. elegans and D. melanogaster harbor a single ancestral Foxo homolog, these observations suggest that Foxo gene duplication conferred an evolutionary advantage by permitting the Foxos to adopt distinct but controlling roles in oogenesis and spermatogenesis. This study's objective is to determine if the remarkably specific expression patterns of Foxo1 and Foxo3 in mouse gonads (and by inference, Foxo function) are conserved in diverse mammalian species, including humans. The conservation of Foxo function in mammals would have significant implications for our understanding of reproduction and the causes of infertility in these diverse species. Western blotting was used to validate isoform-specific antibodies in mammalian species including rodents, companion animals, farm animals, non-human primates, and humans. Following validation of each antibody, immunohistochemical analysis was performed to ascertain Foxo1 and Foxo3 gonadal expression patterns. In the testis, Foxo1 expression was confined to a rare subpopulation of germ cells along the seminiferous tubule basement membrane in all species studied. In the ovary, Foxo1 expression was readily detected in the granulosa cells of developing follicles in all species. Foxo3 was highly expressed in primordial oocytes but absent by the secondary follicle stage in all rodent species. Interestingly, Foxo3 oocyte expression was not consistently conserved in non-rodent species. In some species, Foxo1 was expressed in primordial oocytes, but absent by the secondary follicle stage. In conclusion, while Foxo1 expression in spermatogonial stem cells and granulosa cells was conserved in all mammalian species evaluated, Foxo3 expression in oocytes was more variable. Our findings suggest that Foxo3 may not be uniquely required for primordial follicle maintenance in non-rodent species, and that other Foxos, particularly Foxo1, may contribute to oocyte maintenance in a functionally-redundant manner.

Postinflammation stage of autoimmune orchitis induced by immunization with syngeneic testicular germ cells alone in mice

We previously established an immunological infertility model, experimental autoimmune orchitis (EAO), which can be induced by two subcutaneous injections of viable syngeneic testicular germ cells on days 0 and 14 in mice without using any adjuvant. In this EAO model, CD4+ T-cell-dependent lymphocytic infiltration and immune deposits were found with spermatogenic disturbance on day 120. However, the late stage of EAO (= postactive inflammation stage on day 365) has not yet been investigated. Therefore, we investigated the histopathological characteristics of the late stage. The results revealed that the lymphocytic infiltration finally resolved; however, the seminiferous epithelium persistently showed maturation arrest and the Sertoli cell-only feature. In the seminiferous tubules showing maturation arrest, both proliferation and apoptosis of germ cells had occurred simultaneously. It was also noted that there were deposits of immunoglobulin G and the third component of complement on the thickened basement membrane of seminiferous tubules in the late stage of EAO. These results indicate that histopathology after active inflammation in EAO comprises persistent damage to the seminiferous epithelium and may resemble the histopathology of "idiopathic disturbance of spermatogenesis" in man.

Melatonin prevents testicular damage in hyperlipidaemic mice

The damaging effect of hyperlipidaemia on testicular structure was determined, and the influence of melatonin was evaluated in testicular damage related to hyperlipidaemia. Hyperlipidaemia was induced in ApoE-knockout C57BL/6J male mice fed with high-fat diet alone (group A), or with high-fat diet and melatonin (group B). Six ApoE wild-type C57BL/6J male mice were fed with normal diet, served as controls. At the end of the experimental period, ultrastructural observations showed dramatically histopathological alterations in testicular tissues of group A. The basement membranes of seminiferous tubules were partially thickened and wavy-like in testes of mice with hyperlipidaemia, and vacuolar degeneration of mitochondria and dilation of endoplasmic reticulum were identified as well as the number of mitochondria and lipid droplets decreased significantly in Leydig cells and Sertoli cells. Electrondense deposits were observed in cytoplasms of germ cells. The testicular histostructure in group B treated with melatonin was similar to that of control. Apoptosis was determined by terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labelling. Apoptotic germ cells were significantly more numerous in group A than in group B and controls. The results suggest that melatonin may be potential to attenuate testicular damage by improving histopathological changes and reducing germ cell apoptosis in hyperlipidaemic mice.

CXCL12/CXCR4 Signaling is Required for the Maintenance of Mouse Spermatogonial Stem Cells

Continual spermatogenesis relies on the activities of a tissue-specific stem cell population referred to as spermatogonial stem cells (SSCs). Fate decisions of stem cells are influenced by their niche environments, a major component of which is soluble factors secreted by support cells. At present, the factors that constitute the SSC niche are undefined. We explored the role of chemokine (C-X-C motif) ligand 12 (CXCL12) signaling via its receptor C-X-C chemokine receptor type 4 (CXCR4) in regulation of mouse SSC fate decisions. Immunofluorescent staining for CXCL12 protein in cross sections of testes from both pup and adult mice revealed its localization at the basement membrane of seminiferous tubules. Within the undifferentiated spermatogonial population of mouse testes, a fraction of cells were found to express CXCR4 and possess stem cell capacity. Inhibition of CXCR4 signaling in primary cultures of mouse undifferentiated spermatogonia resulted in SSC loss, in part by reducing proliferation and increasing the transition to a progenitor state primed for differentiation upon stimulation by retinoic acid. In addition, CXCL12-CXCR4 signaling in mouse SSCs was found to be important for colonization of recipient testes following transplantation, possibly by influencing homing to establish stem-cell niches. Furthermore, inhibition of CXCR4 signaling in testes of adult mice impaired SSC maintenance, leading to loss of the germline. Collectively, these findings indicate that CXCL12 is an important component of the growth factor milieu of stem cells in mammalian testes and that it signals via the CXCR4 to regulate maintenance of the SSC pool.

Immunological Effects of Acute Testicular Torsion on the Contralateral Testis in Rats

The mechanism by which unilateral testicular torsion leads to injury of the contralateral testis is still controversial. The present study aimed to evaluate immunohistopathological changes in the contralateral testis of rats after acute testicular torsion. 40 pubertal rats were randomly divided into 3 groups. Group 1 (n=10) functioned as controls. Group 2 rats (n=10) underwent a sham operation, and Group 3 (n=20) underwent 720° torsion of the left spermatic cord (3 h) followed by detorsion (24 h). Immunofluorescence tests were done to investigate contralateral lesions in half of the animals in Group 3. Nitro blue tetrazolium (NBT) was injected in both testes to identify the presence of oxygen-derived free radicals. 24 h after reperfusion, bilateral samples of the testes were obtained for histological analysis. Fisher's exact test was used for statistical analysis with p-values <0.05 considered significant.Immunofluorescence tests were positive for the contralateral (right) testis but with a lower intensity than that of the testis submitted to torsion. NBT reaction was intense all over the left testis while a minor reaction was visible in the right testis. In Group 3 the left distorted testicle showed diffuse edema with cell elimination and extensive necrosis of the seminiferous tubules. Histological lesions were moderate to mild in the contralateral testis. An autoimmune IgG-mediated mechanism deposited on the basement membrane of seminiferous tubules seems to be involved in the genesis of contralateral testis injury.

Study on Transplanting Germ Like Cells Differentiated from Embryonic Stem Cell into Mouse Seminiferous Tubules

Objective To investigate whether germ like cells isolated from embryoid body formed by mouse embryonic stem cells could survive and initiate spermatogenesis in seminiferous tubules of adult mice. Methods SSEA-1+ cells were isolated from embryoid bodies prepared from mouse EGFP-ES cells, after retinoic acid treatment, the cells were detected for the expression of alkaline phosphatase, Rnh2, Stella, fragilis, Texl4, Sry, Hsp90-α, Stra8 and inte grin a6, and then, the cells were transplanted into seminiferous tubules of busulfan-treated adult mice. Results Six days after retinoic acid treatment, alkaline phosphatase expressing cells could still be found in embryoid body (EB) derived cells, indicating the existence of retinoic acid-resistant primordial germ cells. When the SSEA-1+ cells isolated from embryoid bodies were stimulated with retinoic acid for 6 days, some of these cells expressed cell markers of Hsp90-α, Stra8 and inte grin α6, resembling the expression profile of spermatogonial stem cells. Forty-five days after cell transplantation, a little amount of GFP-expressing cells attached to the basement membrane of seminiferous tubule and formed small colonies; Three months later, these cells started amplification in the form of cell chains with varied length, and moving towards the lumen of the seminiferous tubules. Five months after the transplantation, multilayered cell mass was found in seminiferous tubules of two, out of four recipient mice. There was no GFP-expressing cells existed in non-cell-transplanted seminiferous tubules. Conclusion In our study, although full-termed spermatogenesis was not observed in all of the recipients, the results did indicate that the embryoid body contains germ like cells, and these cells can survive and initiate amplification in seminiferous tubules of adult mouse.

THE POSSIBLE PROTECTIVE ROLE OF ANTIOXIDANTS (SELENIUM, VITAMIN E) IN REDUCING SMOKING EFFECTS ON TESTES OF ALBINO RATS الدور الوقائي المحتمل لمضادات األکسدة (السيلينيوم و فيتاميـن ھـ) في تقليل آثار التدخيـن على خصية الجرذان البيضاء

The main objective of the present work was to study the possible role of some antioxidant (Vitamin E and Selenium Se) in protecting the testicular tissue from degenerative changes evoked by exposure to cigarette smoking. The present work showed that exposure to cigarette smoking resulted in a significant decrease in body weight of animals where a significant increase of body weight was observed in animals supplemented with antioxidants while exposed to cigarette smoking. Animals given antioxidants alone did not show any differences in body weight compared to control groups. Histological changes of testicular tissue of the group exposed to the smoke showed thickening of testicular capsule, widening of intertubular spaces with a decrease in interstitial tissue elements, vascular congestion, odema and thickened basement membrane of seminiferous tubules. Some of the seminiferous tubules showed distorted germinal epithelial cell layers, vacuolation and abnormal spermatids with ring shaped arrangement of peripheral chromatin.Giant multinucleated cells were also observed in some tubules. In some specimens there was an increase in the amount of degenerated tubules. Concerning the histochemical changes, the basal lamina, spermtides, Sertoli cells and interstitial tissue gave a very strong reaction with Schiff’s reagent but spermatogonia given a weak reaction. The present results showed that giving Vit. and Se as antioxidant substances to cigarette smoking exposed animals, resulted in a decrease in the above histological and histochemical changes; the affected testicular tissues seemed to be more or less similar to controls. In conclusion, the present study showed that although using of certain antioxidants cannot completely prevent the drastic effects of cigarette smoking on living tissue, it prevented to some extent the degenerative changes observed in testicular tissues.

Pathogenic effect of bunt wheat on male reproductive system in Mices.

This study was carried out to study the toxic effect of smut wheat on male reproductive system of mice. The experimental study was performed in the laboratory using 20 mice, which divided into two groups, one group feed on normal diet, the other fed on a mixture of 50% normal diet with 50% smut wheat. A histopathological study was done on the testes of Mice fed on smut wheat for 30 days. The histopathological study revealed thickness of basement membrane of semiriferous tubule of mice, and desquamation of the sertoli cells and spermatogonium from the basement membrane of seminiferous tubule and has adversely effect on male fertility.

Computerized Microscopy Research on Structural Biology of the Seminiferous Tubules and Interstitial Gland at the Cocks 90-120 Days Old

From the age of 90 days is clear that an end point of pseudostratified seminal epithelium, but the cell types and Sertoli cells are still prevails spermatogoniums and take the other two age as described above. There is a very intense reaction P.A.S. Take the positive step of tubular basement of the seminiferous membrane. There are many cells in semen Sertoli epithelium, in peritubulary are located myofibroblastes. From the age of 120 days in the seminal epithelium are present all cells of the seminal line. Basement membrane of seminiferous tubules is evident, and intertubular connective tissue is P.A.S. positive. Sertoli cells (cells sustentacular) from cells support undifferentiated of the gonads of prepubertal. These cells are active from point of the mitotic, contains large amounts of rough endoplasmic reticulum and produce paramesonephrotic hormone, a glycoprotein that inhibits development of female to male genitalia. At the age of 90 days it observe a quantitative reduction of intertubular. Interstitial endocrinocytes have different size, some are fusiforme, others have cytoplasm vacuolar, being involved in hormonegenesis

Microscopic studies of the seminiferous tubules and interstitial gland at the cocks 90-120 days old.

From the age of 90 days is clear that an end point of pseudostratified seminal epithelium, but the cell types and Sertoli cells are still prevails spermatogoniums and take the other two age as described above. There is a very intense reaction P.A.S. Take the positive step of tubular basement of the seminiferous membrane. There are many cells in semen Sertoli epithelium, in peritubulary are located myofibroblastes. From the age of 120 days in the seminal epithelium are present all cells of the seminal line. Basement membrane of seminiferous tubules is evident, and intertubular connective tissue is P.A.S. positive. Sertoli cells (cells sustentacular) from cells support undifferentiated of the gonads of prepubertal. These cells are active from point of the mitotic, contains large amounts of rough endoplasmic reticulum and produce paramesonephrotic hormone, a glycoprotein that inhibits development of female to male genitalia. At the age of 90 days it observe a quantitative reduction of intertubular. Interstitial endocrinocytes have different size, some are fusiforme, others have cytoplasm vacuolar, being involved in hormonegenesis.

Temporal and Spatial Differential Expression of Chicken Germline-specific Proteins cDAZL, CDH and CVH During Gametogenesis

The Deleted in Azoospermia-Like (DAZL) protein coded by Dazl gene is a germline-specific RNA-binding protein essential for gametogenesis in vertebrates, and the chicken Dazl gene has also been identified in primordial germ cells (PGCs). However, the temporal and spatial expression of chicken DAZL (cDAZL) and its molecular role in germ cell development remain enigmatic. Here, we investigated the subcellular distribution and expression of cDAZL at the various stages by using a polyclonal antibody raised against its C-terminal region and compared them with those of additional germline-specific proteins chicken vasa homologue (CVH) and chicken dead end homologue (CDH). Western blot analysis for cDAZL revealed a single band in the embryonic gonads and premature chicken testis, whereas no band was detected in the premature chicken ovary. Fluorescent immunohistochemistry revealed that cDAZL was present in the nucleus and cytoplasm of circulating PGCs. Cells positive for cDAZL and CVH coexisted in the embryonic gonads and premature chicken testis, in which they were distributed near the basement membrane of seminiferous tubules. Of interest, cDAZL was not found in the premature chicken ovary, whereas CVH and CDH were present in germ cells. Collectively, three germline-specific proteins are expressed in chicken germ cells, but their patterns of expression are temporally and spatially distinct.

Antenatal Exposure of Cyclophosphamide Affects Cell Proliferation in Seminiferous Tubules (Gonocytes and Sertoli Cells) in Neonatal Rat Testis

The present study demonstrates the changes in the distribution and number of cellular contents of seminiferous tubules (Sertoli cells and gonocytes) in rat testis after exposure to different doses of cyclophosphamide (2, 10 or 20 mg/kg body wt) on day 12, 15 and 18 of gestation. The treatment caused significant decrease of these parameters. Besides, morphological changes such as discontinuity of epithelial lining and basement membrane of seminiferous tubules, and stunting of Sertoli cells and gonocytes were also observed in the pups in a dose-dependent manner. Reduction (p<0.001) in the number of gonocytes was observed on day 15 of gestation and Sertoli cells on day 18. These days of embryonic development / gestation may be considered as critical periods for cell proliferation during the differentiation of male gonad. Overall, our data suggest that antenatal exposure to cyclophosphamide may affect the normal process of testicular development.

Asymmetric Distribution of UCH‐L1 in Spermatogonia Is Associated With Maintenance and Differentiation of Spermatogonial Stem Cells

Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is expressed in type A (A(s), A(pr), and A(al)) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH-L1 was associated with self-renewal versus differentiation divisions of SSCs as defined by co-localization of UCH-L1(high) and PLZF, a known determinant of undifferentiated SSCs, versus co-localization of UCH-L1(low/-) with proteins expressed during SSC differentiation (DAZL, DDX4, c-KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH-L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH-L1 and PLZF in situ in seminiferous tubules. Expression level of UCH-L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH-L1-positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self-renewal and differentiation in mammalian spermatogenesis.

Phenotypic and molecular characterization of spermatogonial stem cells in adult primate testes

Knowledge about the identity and characteristics of spermatogonial stem cells (SSCs) in human is very limited. Here, Rhesus monkey was used as an animal model to investigate molecular and phenotypic characteristics of SSCs in the adult testes. A variety of immunohistological, molecular biological and functional assays were used to study different populations of SSCs in the adult testes. In adult primate testes, there are distinct populations of CD90+ CD49f+ CD117- (Triple Stained) cells and a small population of stage-specific embryonic antigen-4 (SSEA-4)+ cells which both localized at the basement membrane of seminiferous tubules. Both SSEA-4+ and Triple Stained cells express germ cell and SSC-specific markers and show high telomerase activity; however, only adult Rhesus monkey SSEA-4+ testis cells appear to contain functional and actively dividing SSCs that can repopulate recipient mouse testes following spermatogonial transplantation. DNA analysis of these populations showed that SSEA-4+ cells contain a DNA profile similar to the actively dividing cells, whereas Triple Stained cells showed an accumulated number of cells arrested in the S phase of the cell cycle. SSEA-4+ cells also showed significantly higher proliferation activity, as shown by proliferating cell nuclear antigen staining, than Triple Stained cells (P < 0.01). Interestingly, SSEA-4+ cells expressed a significantly higher level of promyelocytic leukemia zinc finger, a factor required for SSC self-renewal, than Triple Stained cells (P < 0.001). Our data indicate that Triple Stained cells may represent a quiescent population of SSCs, whereas SSEA-4 might be expressed on a subpopulation of actively dividing SSCs.

Regulation of adult human spermatogonial stem cells proliferation by estradiol in vitro

OBJECTIVE: Spermatogonial stem cells (SSCs), which are the only germline stem cells in adults, self-renew and produce a large number of differentiating germ cells. In the niche on the basement membrane of seminiferous tubules, SSCs are secluded from the outside world and affected by all kinds of nutrients, growth factors and hormones. Estrogens are now known to have profound effects on male reproductive systems and estrogen receptors (ERs) have been detected in adult human germ cells, thus the aim of the present study was to evaluate the role of 17β-estradiol, the main physiological estrogen, in SSCs proliferation in vitro.

Seminiferous Tubule Degeneration and Infertility in Mice with Sustained Activation of WNT/CTNNB1 Signaling in Sertoli Cells1

WNT/CTNNB1 signaling is involved in the regulation of multiple embryonic developmental processes, adult tissue homeostasis, abd cell fate determination and differentiation. Many WNTs and components of the WNT/CTNNB1 signaling pathway are expressed in the testis, but their physiological roles in this organ are largely unknown. To elucidate the role(s) of WNT/CTNNB1 signaling in the testis, transgenic Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were generated to obtain sustained activation of the WNT/CTNNB1 pathway in both Leydig and Sertoli cells. Male Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were sterile because of testicular atrophy starting at 5 wk of age, associated with degeneration of seminiferous tubules and the progressive loss of germ cells. Although Cre activity was expected in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ Leydig cells, no evidence of Cre-mediated recombination of the floxed allele or of WNT/CTNNB1 pathway activation could be obtained, and testosterone levels were comparable to age-matched controls, suggesting that genetic recombination was inefficient in Leydig cells. Conversely, sustained WNT/CTNNB1 pathway activation was obtained in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ Sertoli cells. The latter often exhibited morphological characteristics suggestive of incomplete differentiation that appeared in a manner coincident with germ cell loss, and this was accompanied by an increase in the expression of the immature Sertoli cell marker AMH. In addition, a poorly differentiated, WT1-positive somatic cell population accumulated in multilayered foci near the basement membrane of many seminiferous tubules. Together, these data suggest that the WNT/CTNNB1 pathway regulates Sertoli cell functions critical to their capacity to support spermatogenesis in the postnatal testis.

Possible Protective Role of Grape Seed Proanthocyanidin on Fluoride Induced Testicular Toxicity in Adult and Young Albino Rats

Increasing infertility, has become a serious issue. Sodium fluoride (NaF) is a wide spread natural pollutant and large number of the world population is exposed to high doses of fluoride. Grape seed proanthocyanidins (GSP), exerts a novel spectrum of biological, pharmacological, and therapeutic properties against oxidative stress , so it was of special concern to investigate the role of the naturally occurring antioxidant GSP on effect of fluoride on male reproduction . The study included 72 male rats, 36 adult and 36 young rats were divided into six groups (n =12, 6 adult & 6 young ). Group I: negative control. Group II: was given distilled water. Group III: was given GSP 75 mg / kg. Group IV: was given NaF 18 mg / kg. Group V: was given NaF 18 mg /kg and GSP 75 mg /kg. Group VI: was given NaF 18 mg /kg then it was stopped and rats were examined 30 days after its discontinuation. All treatments were given daily by oral gavage for 30 days. At the end of experimental and follow up periods, serum levels of testosterone, follicle stimulating (FSH) and Luteinizing (LH) hormones in addition to serum levels of malondialdhyde (MDA) and plasma nitrite were determined. Absolute and relative testes weights and histopathological examination of testes of all rats also performed. The results revealed that, fluoride induced significant decrease in plasma testosterone, FSH and LH levels in adult rats and non significant change of these hormones in young rats as compared to control groups. It also induced significant increase in serum MDA and nitrite levels in adult and young rats as compared to control groups. Absolute and relative testes weights of the adult and young rats of NaF group were significantly reduced compared to control groups. Section of adult rats testes of NaF treated group revealed arrest of spermatogenesis,sloughing of spermatogenic cells and disorganization of the epithelial lining of seminiferous tubules. While, testes of young rats revealed total spermatogenic atrophy and degeneration with thickening of the basement membrane of seminiferous tubules and interstitial fibrosis. Co-administration of NaF and GSP showed significant increase of plasma testosterone, FSH and LH levels in adult rats and decrease in serum MDA and nitrite levels compared to NaF alone or follow up group in adult and young rats. Also,

Characterization, Cryopreservation, and Ablation of Spermatogonial Stem Cells in Adult Rhesus Macaques

Spermatogonial stem cells (SSCs) are at the foundation of mammalian spermatogenesis. Whereas rare A(single) spermatogonia comprise the rodent SSC pool, primate spermatogenesis arises from more abundant A(dark) and A(pale) spermatogonia, and the identity of the stem cell is subject to debate. The fundamental differences between these models highlight the need to investigate the biology of primate SSCs, which have greater relevance to human physiology. The alkylating chemotherapeutic agent, busulfan, ablates spermatogenesis in rodents and causes infertility in humans. We treated adult rhesus macaques with busulfan to gain insights about its effects on SSCs and spermatogenesis. Busulfan treatment caused acute declines in testis volume and sperm counts, indicating a disruption of spermatogenesis. One year following high-dose busulfan treatment, sperm counts remained undetectable, and testes were depleted of germ cells. Similar to rodents, rhesus spermatogonia expressed markers of germ cells (VASA, DAZL) and stem/progenitor spermatogonia (PLZF and GFRalpha1), and cells expressing these markers were depleted following high-dose busulfan treatment. Furthermore, fresh or cryopreserved germ cells from normal rhesus testes produced colonies of spermatogonia, which persisted as chains on the basement membrane of mouse seminiferous tubules in the primate to nude mouse xenotransplant assay. In contrast, testis cells from animals that received high-dose busulfan produced no colonies. These studies provide basic information about rhesus SSC activity and the impact of busulfan on the stem cell pool. In addition, the germ cell-depleted testis model will enable autologous/homologous transplantation to study stem cell/niche interactions in nonhuman primate testes.

PROSPECTIVE IDENTIFICATION AND ISOLATION OF TYPE-A SPERMATOGONIA FROM JUVENILE RHESUS MACAQUE TESTES

Spermatogonial stem cells (SSCs) are undifferentiated germ cells that balance self-renewal and differentiation to support sperm production in the mammalian testis. Non-human primate testes contain two types of histologically distinguishable undifferentiated spermatogonia, designated Adark and Apale based on nuclear morphology. Classical and contemporary models suggest that Adark and Apale spermatogonia are reserve and renewing stem cells, respectively, although these designations are debatable. To gain insight into the biology of rhesus spermatogonia we prospectively identified and isolated two types of germ cells from juvenile rhesus testes (15–20 months of age). The only germ cells present at this stage of rhesus testis development are Adark and Apale spermatogonia. Putative germ cells were selected from heterogeneous juvenile testis cell suspensions using an inverted phase contrast microscope equipped with a micromanipulator and pulled-glass capillary pipette. Germ cells were distinguished from other testicular cell types based on their large size and characteristic color. hile both subpopulations of picked germ cells were relatively large (>12 μm), one was larger (large cells) than the other (small cells). Picking accuracy was determined by immunofluorescent staining for Vasa, which marks all germ cells in the juvenile rhesus testis. While 2.4% of testis cells in the heterogeneous suspensions expressed Vasa, each picked subpopulation was comprised of nearly pure germ cells (small=96.9%; large=97.3%). Hematoxylin staining was more intense in the nuclei of small germ cells than large germ cells, suggesting that they may represent Adark and Apale spermatogonia, respectively. To determine if either or both populations contain functional stem cells, we performed rhesus to nude mouse testis xenotransplantation. Two months after transplantation, recipient nude mouse testes were evaluated in whole mount by staining with an antibody that recognizes rhesus testis cells. Preliminary results indicated that the large germ cells produced chains of rhesus spermatogonia connected by intracytoplasmic bridges on the basement membrane of mouse seminiferous tubules. The small germ cells failed to produce similar foci of colonization. Thus, our data indicate that two phenotypically and functionally distinct germ cell types can be identified and isolated from juvenile rhesus testis cell suspensions, providing an opportunity to study their molecular characteristics. Supported by National Institutes of Health grants U54 HD08160 to TMP and KEO, R01 RR18500 to KEO, an institutional postdoctoral fellowship HD007332 to BPH, and the Magee-Womens Research Institute and Foundation. (poster)

Examination of basement membrane components associated with the bovine seminiferous tubule basal lamina

Immunohistology has been used to examine the distribution of certain components of the basement membrane (BM) associated with bovine spermatogonial germ cells that are located within the seminiferous tubules. Histology was performed on testis tissue from Brahman cattle (Bos indicus) of three different age groups: pre-pubescent (4-6 months), juvenile (8-10 months) and adult (18-24 months) animals. There were no major changes in the BM composition apparent between these three age groups, except for certain lectin staining. These data suggest that the predominant collagen type IV component may have an alpha3 and alpha4 composition, although other chains, including the alpha5 and alpha6 chains, were also present. Possibly the main laminin type present was laminin 121 (alpha1beta2gamma1), although other variants were also present. Both nidogen-1 and perlecan, which are normal BM components, were also found as part of the seminiferous tubule BM. Interstitial collagens, such as type I, III and VI collagens, were found in the peritubular space, but were not part of the BM itself, although type VI collagen was most visible in the peritubular zone adjacent to the tubules. Examination of the BM with a range of lectins gave strong staining for (glcNAc)(2) entities, weak positive staining for alpha-l-fuc, but little or no staining for alpha-galNAc and (glcNAc)(3) at all ages, whereas staining for alpha-gal, beta-gal(1-->3)galNAc and alpha-man showed developmental changes.