Spermatogonial stem cells: unlimited potential.

Recent reports have demonstrated that adult cells can be reprogrammed to pluripotency, but mostly with genes delivered using retroviruses. Some of the genes are cancer causing; thus, these adult-derived embryonic stem (ES)-like cells cannot be used for therapy to cure human diseases. Remarkably, it has also been demonstrated recently by several groups that, in mice, spermatogonial stem cells (SSCs) can be reprogrammed to ES-like cells without the necessity of exogenously added genes. SSCs constitute one of the most important stem cell systems in the body, not only because they produce spermatozoa that transmit genetic information from generation to generation, but also because of the recent studies showing their remarkable plasticity. Very little is known about SSCs in humans, except for the earlier work of Clermont and colleagues who demonstrated that there are A(dark) and A(pale) spermatogonia, with the A(dark) referred to as the reserve stem cells and the A(pale) being the renewing stem cells. We now demonstrate that G protein-coupled receptor 125 (GPR125) may be a marker for human SSCs. Putative human SSCs can also be reprogrammed to pluripotency. We were able to achieve this result without the addition of genes, suggesting that human SSCs have considerable potential for cell-based, autologous organ regeneration therapy for various diseases.

Expression of Notch pathway components in spermatogonia and Sertoli cells of neonatal mice.

Members of the Notch gene family have been shown to play an important role in the control of cell fate in many developmental systems. We hypothesized that the fate of the male germ line stem cells may also be mediated through the Notch signaling pathway. We therefore sought to determine whether the components of the Notch pathway are expressed in the mouse testis. Western blot analysis revealed the expression of three Notch receptors (Notch 1, Notch 2, and Notch 3), Notch ligands (Jagged 1, Jagged 2, and Delta 1), and presenilin 1 (PS1) in neonatal mouse testis. We then examined their cellular localization by immunohistochemical analysis of cocultures of spermatogonia and Sertoli cells. The 3 Notch receptors were found to be expressed in spermatogonia. Sertoli cells expressed only Notch 2 receptor. Among the Notch ligands, Delta 1 and Jagged 1 were localized exclusively in spermatogonia and Sertoli cells, respectively. PS1 was apparent in both spermatogonia and Sertoli cells. The presence of Notch receptors and Notch ligands in spermatogonia and Sertoli cells indicates that these cells are capable of responding to and eliciting Notch signaling during the process of spermatogenesis. Key words: Cell fate, delta, jagged, presenilin, spermatogenesis.

Androgen depletion activates telomerase in the prostate of the nonhuman primate, Macaca mulatta.

BACKGROUND: The activity of telomerase, an enzyme that synthesizes telomeric repeats at the ends of chromosomes, is not detectable in normal human prostate. However, the majority of human prostate cancers exhibit telomerase activity. Since androgens play a major role in prostate tumorigenesis, we investigated the effect of androgen-depletion on the expression of telomerase activity in the prostate. METHODS: Adult male rhesus monkeys were either bilaterally castrated or subjected to sham surgery (n = 5 each). Approximately 6 weeks later, the animals were killed and the different regions of the prostate gland were removed and frozen immediately. Telomerase activity was assayed using the telomeric repeat amplification protocol. RESULTS: All five regions of the prostate from sham operated control animals failed to exhibit telomerase activity. In the castrated monkey, all regions of the prostate, except for the anterior lobe, expressed high levels of telomerase activity. CONCLUSIONS: Our results indicate that in monkeys, androgen-ablation leads to up-regulation of telomerase activity. The negative-regulation of telomerase activity by androgens is probably lost during prostate tumorigenesis.

Direct toxic effects of clinical doses of chloroquine on transferrin secretion in immature rat sertoli cells in vitro.

We have examined the effects of increasing doses of chloroquine (CQ), on transferrin secretion in primary cultures of immature rat Sertoli cells (SC) grown on a reconstituted basement membrane (Matrigel) in bicameral chambers. SC cells were seeded in serum-free defined medium at a density of 3 x 10(6) cells/0.64cm2/well on Matrigel covered Millicell-HA filters. CQ at concentrations ranging from 0.04-1.0 microM was added to the basal compartment of the bicameral system from day 7 of the culture. The formation of the tight junction was monitored by the measurement of the transepithelial resistance (TER) at 24 hr intervals using an impedance meter TER in untreated controls was 50 Ohms/cm2 on day 1, and increased progressively to 80 Ohms/cm2 by day 7 and plateaued until day 12. On the seventh day of culture, CQ was introduced into the basal chamber During the 4 days of the experiment, the secretion of transferrin decreased with time. Maximal transferrin secretion by SC was detected during the initial 2 day collection period. During the subsequent collection period, CQ (1 microM) decreased significantly transferrin secretion by SC, while 0.04 microM CQ did not affect transferrin secretion. The polarized secretion of transferrin in response to CQ was also studied. During both collection periods there was no significant difference between controls and 0.04 microM CQ cultures in the ratio of apical to basal transferrin secretion. In the 1 microM culture medium, CQ diminished significantly the ratio of apical to basal transferrin secretion. These observations demonstrate the heterogenous effects of lower doses of CQ on immature rat SC in cultures.

Telomere length in male germ cells is inversely correlated with telomerase activity.

Telomeres, the noncoding sequences at the ends of chromosomes, progressively shorten with each cellular division. Spermatozoa have very long telomeres but they lack telomerase enzymatic activity that is necessary for de novo synthesis and addition of telomeres. We performed a telomere restriction fragment analysis to compare the telomere lengths in immature rat testis (containing type A spermatogonia) with adult rat testis (containing more differentiated germ cells). Mean telomere length in the immature testis was significantly shorter in comparison to adult testis, suggesting that type A spermatogonia probably have shorter telomeres than more differentiated germ cells. Then, we isolated type A spermatogonia from immature testis, and pachytene spermatocytes and round spermatids from adult testis. Pachytene spermatocytes exhibited longer telomeres compared to type A spermatogonia. Surprisingly, although statistically not significant, round spermatids showed a decrease in telomere length. Epididymal spermatozoa exhibited the longest mean telomere length. In marked contrast, telomerase activity, measured by the telomeric repeat amplification protocol was very high in type A spermatogonia, decreased in pachytene spermatocytes and round spermatids, and was totally absent in epididymal spermatozoa. In summary, these results indicate that telomere length increases during the development of male germ cells from spermatogonia to spermatozoa and is inversely correlated with the expression of telomerase activity.

Expression of the leptin receptor during germ cell development in the mouse testis.

Leptin, a recently identified hormonal product of the ob gene, is known to regulate appetite, body metabolism, and reproductive functions. We investigated the expression of the leptin receptor (Ob-R) in testes from different age groups. The messenger RNA for Ob-R was found in testes from all age groups using RT-PCR. Using immunohistochemistry, we observed age- and stage-dependent distribution of the Ob-R in mouse testis. In testis of 5-day-old mice, its expression was mainly in type A spermatogonia. In the 20- and 30-day-old testis, Ob-R expression was in the spermatocytes; in the adult testis, it was specific to spermatocytes in stages IX and X of the cycle of the seminiferous epithelium. Five main immunoreactive proteins were detected using Western blot (220, 120, 90, 66, and 46 kDa). The 120-kDa protein was evident only in 20-day-old and older testes, whereas the 90-kDa band was present only in the 5- and 10-day-old testis. Leptin treatment induced phosphorylation of signal transducer and activator of transcription-3 in cultured seminiferous tubules from adult and 5-day-old testes. Our results show for the first time age- and stage-specific localization of a functional Ob-R in testicular germ cells. We hypothesize a direct role for leptin, through phosphorylation of signal transducer and activator of transcription-3, in proliferation and differentiation of germ cells, which may partially explain the infertility observed in leptin-deficient mice.

Stem cell factor/c-kit up-regulates cyclin D3 and promotes cell cycle progression via the phosphoinositide 3-kinase/p70 S6 kinase pathway in spermatogonia.

Stem cell factor (SCF)/c-kit plays an important role in the regulation of hematopoiesis, melanogenesis, and spermatogenesis. In the testis, the SCF/c-kit system is believed to regulate germ cell proliferation, meiosis, and apoptosis. Studies with type A spermatogonia in vivo and in vitro have indicated that SCF induces DNA synthesis and proliferation. However, the signaling pathway for this function of SCF/c-kit has not been elucidated. We now demonstrate that SCF activates phosphoinositide 3-kinase (PI3-K) and p70 S6 kinase (p70S6K) and that rapamycin, a FRAP/mammalian target of rapamycin-dependent inhibitor of p70S6K, completely inhibited bromodeoxyuridine incorporation induced by SCF in primary cultures of spermatogonia. SCF induced cyclin D3 expression and phosphorylation of the retinoblastoma protein through a pathway that is sensitive to both wortmannin and rapamycin. Furthermore, AKT, but not protein kinase C-zeta, is used by SCF/c-kit/PI3-K to activate p70S6K. Dominant negative AKT-K179M completely abolished p70S6K phosphorylation induced by the constitutively active PI3-K catalytic subunit p110. Constitutively active v-AKT highly phosphorylated p70S6K, which was totally inhibited by rapamycin. Thus, SCF/c-kit uses a rapamycin-sensitive PI3-K/AKT/p70S6K/cyclin D3 pathway to promote spermatogonial cell proliferation.

Isolation of rat ventral prostate basal and luminal epithelial cells by the STAPUT technique.

BACKGROUND: The prostatic epithelium consists principally of basal epithelial cells, luminal epithelial cells, and neuroendocrine cells. Several studies support the concept that among basal cells, a subpopulation of stem cells resides which is capable of giving rise to other stem cells, basal epithelial cells, and also luminal epithelial cells and neuroendocrine cells. Other investigators suggest that luminal epithelial cells can also regenerate prostatic epithelium. Availability of pure populations of basal and luminal epithelial cells will aid in studies on defining the cellular pathways of differentiation during normal and pathological conditions. This study was designed to isolate and characterize pure populations of basal and luminal epithelial cells from adult rat ventral prostates. METHODS: Sequential enzymatic digestion and differential plating permitted the separation of glandular epithelial cells from stromal cells. The glandular epithelial cells were subjected to the STAPUT technique. RESULTS: Two types of cell populations, a large single-cell population and a small single-cell population, were obtained and characterized as basal and luminal epithelial cells by immunostaining for cytokeratin 5 and cytokeratin 8, respectively. CONCLUSIONS: Our results indicate that purified populations of prostatic basal and luminal epithelial cells can be isolated by the STAPUT technique.

Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM.

Spermatogenesis is initiated with the divisions of the type A spermatogonial stem cells; however, the regulation of this stem cell population remains unknown. In order to obtain a better understanding of the biology of these cells, type A spermatogonia were isolated from 80-day-old pig testes by sedimentation velocity at unit gravity. The cells were cultured for up to 120 h in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12) or a potassium-rich medium derived by the simplex optimization method (KSOM). At the end of the 120-h culture period, 30-50% of the spermatogonia were viable in KSOM, whereas in DMEM/F12 very few cells survived. Using KSOM as the culture medium, the effects of stem cell factor (SCF) and granulocyte macrophage-colony stimulating factor (GM-CSF) were studied. SCF significantly enhanced the percentage of cell survival at 100 ng/ml but not at lower concentrations. In comparison, GM-CSF promoted survival at relatively low concentrations (0.01, 0.1, and 1 ng/ml). At a higher dose (10 ng/ml), a significant reduction in percentage of cell survival was observed. The combination of SCF with GM-CSF had no significant effect on the percentage survival of type A spermatogonial cells. These data indicate that SCF and GM-CSF play a role in the regulation of survival and/or proliferation of type A spermatogonia.

Electrophysiological effects of chilotoquine on tight junctions of immature rat Sertoli cells in vitro.

We investigated the effect of CQ, an antimalarial drug with antiprotease activity, and NH4Cl, a related amines on the development of intercellular tight junctions in cultured immature rat Sertoli cells. Sertoli cells were seeded in serum-free defined medium at a density of 3 x 10(6) cells/0.64 cm2/well on Matrigel-covered Millicell-HA filters. CQ (1 microM and 2 microM) or NH4Cl (6.25 mM and 12 mM) was added to the outer (basal) compartment of the bicameral system either on day 1 or day 7 of the culture. Formation of tight junctions was monitored by measurement of the transepithelial resistance (TER) at 24 hr intervals using an impedance meter. TER in untreated controls was 50 omega/cm2 on day 1, increased progressively to 80 omega/cm2 by day 7 and plateaued until day 12. The cells treated from day 1 with CQ showed dose-dependent progressive increase in TER until day 12, reaching 191 omega/cm2 in cells treated with 1 microM concentration. In cells treated with CQ starting from day 7 of culture onwards, TER patterns were similar to those noted following exposure to chloroquine from day 1. Also in cultures containing NH4Cl, in comparison to the control, the increase in TER was significantly higher. These observations demonstrate that CQ and HN4Cl promote tight junction formation between immature rat Sertoli cells invitro suggesting that antiproteases may be involved in the formation of blood-testis barrier.

Altered basement membrane synthesis in the testis after tissue injury.

The basement membrane plays an important role in maintaining the structural and functional integrity of tissues. Altered basement membrane structure has been associated with severe functional impairment of the testis in several conditions, including vasectomy, autoimmune orchitis, cryptorchidism, and following x-irradiation. We have used efferent duct ligation as a model to examine seminiferous tubular basement membrane morphology, synthesis, and gene expression to determine whether altered basement membrane synthesis is responsible for the aberrant structures noted after tissue injury. On days 2 and 3 after ligation, both the seminiferous epithelium and the basement membrane appeared normal, but 7 days after ligation, the seminiferous epithelium began to degenerate. The basement membrane appeared detached from the epithelium, and redundant patches of basement membrane were observed adjacent to the Sertoli cells at 14 and 21 days postligation. Immunoprecipitation indicated an increase in laminin protein synthesis in the ligated tubules at the same time. Northern blot analysis showed increases in transcript levels for laminin as well as collagen IV and heparan sulfate proteoglycan. These data show that new protein synthesis is responsible, at least in part, for the duplication of the basement membrane coincident with the tissue damage caused by efferent duct ligation.

Loss of telomerase activity during male germ cell differentiation.

Although the activity of telomerase, an enzyme which synthesizes telomeres de novo and stabilizes telomere length has been demonstrated in the testis, the precise expression of activity in different germ cell types is not known. We examined telomerase activity using a PCR-based telomeric repeat amplification protocol during development of the rat testis from birth to adulthood. Telomerase activity was relatively high from birth to the 4th week of age, and then low between the 5th to 10th week, suggesting that the type A spermatogonial stem cells may be the population which is expressing the highest levels of telomerase activity. To ascertain which germ cells expresses the telomerase activity, purified populations of type A spermatogonia from 9-day old rats, and pachytene spermatocytes, round spermatids and epididymal spermatozoa from adult rats were isolated. While type A spermatogonia expressed very strong telomerase activity, the fractions containing pachytene spermatocytes and round spermatids also expressed telomerase activity, but at comparatively lower levels. Telomerase activity was totally absent in epididymal spermatozoa. Thus, it appears that the telomerase activity is expressed at high levels in the type A spermatogonial stem cells, is down-regulated during spermatogenesis, and is absent in the differentiated spermatozoa.

Regulation of rat testis gonocyte proliferation by platelet-derived growth factor and estradiol: identification of signaling mechanisms involved.

To determine what factors regulate gonocyte proliferation in newborn rats, we first examined the expression of several signal transduction molecules by immunocytochemistry in 3-day-old rat testis sections. We found that gonocytes specifically expressed the iota and zeta isoforms of protein kinase (PK) C (PKC) and the phosphatidylinositol 3-kinase (PI3-K). Because both the zeta PKC and PI 3-K have been shown to play a role in platelet-derived growth factor (PDGF)-induced cell proliferation, we examined the effects of PDGF on gonocytes. For this, we developed a method to obtain highly purified and viable gonocytes in culture. After enzymatic digestion, differential adhesion, and two successive gradient fractionations, the gonocyte suspension obtained was over 90% pure, as assessed by light microscopy. The viability of cultured gonocytes exceeded 90% after 48 h in the presence of 2.5% FBS used as a survival factor. Immunodetection studies showed that isolated gonocytes expressed zeta PKC, PI 3-K, and the PDGF receptor. Treatment with 10 ng/ml PDGF induced a 4-fold increase of bromodeoxyuridine incorporation into gonocytes (from 5% proliferative gonocytes under basal conditions to 20% in the presence of PDGF). Because neonatal Sertoli cells secrete high levels of the growth promoting steroid, 17 beta-estradiol, we also tested its effect and found that it induced gonocyte proliferation at a level comparable with that of PDGF and that this effect was blocked by the estrogen receptor antagonist, ICI 164384. The combination of PDGF and estradiol, however, was not additive, suggesting that their effects were mediated by common molecular target(s). These results demonstrate that PDGF and estradiol activate gonocyte proliferation in vitro, suggesting that they may act as the physiological regulators of gonocyte development in vivo.

Anatomical variant of the lateral pectoral nerve innervating the anterior portion of the deltoid muscle: a case report.

BACKGROUND: The deltoid muscle is innervated by the axillary nerve. There is no collateral nerve supply described for this muscle. Palsy of the axillary nerve is common in shoulder trauma due to its close relationship to the surgical neck of humerus. METHODS: A dissection of the pectoral and axillary regions of two female cadavers was performed bilaterally for a detailed analysis of the innervation of the deltoid muscle. RESULTS: A branch of the lateral pectoral nerve provided supplemental innervation to the anterior portion of the deltoid muscle bilaterally in both cadavers. CONCLUSION: A branch of the lateral pectoral nerve could provide collateral nerve supply to the deltoid muscle. The frequency of this anatomical variation requires further exploration.

Characterization and cellular localization of PSG in rat testis.

In order to establish the rat testis as a model system for studying the human pregnancy-specific beta1-glycoprotein (PSG), expression and cellular distribution of PSG in rat testis were examined. Three partial PSG cDNAs, namely, rnCGM6, rnGCM7, and rnCGM8 were obtained when rat testis cDNA libraries were screened with a human placental PSG cDNA probe. Unlike the human PSGs, the rat PSGs show less nucleotide and amino acid sequence homology among family members. The rat PSGs also have multiple truncated leader sequences followed by immunoglobulin variable-like N domains while human PSGs have a single N domain. Examination of the testis, intestine, kidney, liver, lung, and muscle of male rats by reverse transcription-polymerase chain reaction (RT-PCR) with nested gene-specific primers showed that rnCGM6 was present only in the testis, while rnCGM8 was present in the testis, intestine and lung. On the other hand rnCMG7 was found in all tissues examined. Furthermore, rnCGM7 transcript was present in all somatic cells examined whereas rnCGM6 was predominantly in myoid cells and rnCMG8 in Leydig cells. These results suggest that there is cell-specificity in the expression of PSGs in the rat testis and that the rat testis is a good model for studying the biological activities of the PSGs.

Effect of chloroquine on the formation of tight junctions in cultured immature rat Sertoli cells.

Adjoining immature Sertoli cells in the seminiferous epithelium form a tight junctional complex leading to the development of the blood-testis barrier. Protease and antiprotease activities have been implicated in the process of formation of tight junctions. Here, we report the effect of chloroquine, an antimalarial drug with antiprotease activity, on the development of intercellular tight junctions in cultured immature rat Sertoli cells. For positive control, the classical lysosomotropic agent ammonium chloride was used. Sertoli cells were seeded in serum-free defined medium at a density of 3 x 10(6) cells/0.64-cm2 well on Matrigel-covered Millicell-HA filters. Chloroquine at concentrations ranging from 25 to 100 microM was added to the outer chamber of the bicameral system on either day 1 or 7 of the culture. The formation of the tight junction was monitored by the measurement of the transepithelial resistance (TER) at 24-hour intervals using an impedance meter. TER in untreated controls was 50 ohms/cm2 on day 1; it increased progressively to 80 ohms/cm2 by day 7 and plateaued until day 12. The cells treated from day 1 with chloroquine also showed a dose-dependent progressive increase in TER until day 9, reaching 225 ohms/cm2 in cells treated with the 100 microM concentration. In comparison to controls, the increase in TER was significantly higher. In cells treated with chloroquine starting from day 7 of culture onwards, there was no observable difference in TER from the untreated control. These observations demonstrate that chloroquine and ammonium chloride increase the TER of immature Sertoli cells in the bicameral chamber.

Regulation of c-fos mRNA expression in Sertoli cells by cyclic AMP, calcium, and protein kinase C mediated pathways.

The role of second messenger pathways, cyclic AMP, calcium, and protein kinase C (PKC) in the transcriptional regulation of c-fos protooncogene expression in rat Sertoli cells was investigated. c-fos expression was monitored by Northern blot analysis. Although the action of FSH on Sertoli cells is considered to be mediated by cAMP, dibutyryl cAMP (dbcAMP), a potent membrane permeable analog of cAMP, induced much less c-fos mRNA expression than FSH ( < 50%) suggesting that additional cAMP-independent mechanisms may mediate the effect of FSH on c-fos. Specific intracellular inhibitors of PKC decreased c-fos induction in response to FSH by more than 50%. Ionomycin, which increases intracellular free calcium concentration, induced c-fos expression significantly. These data demonstrate that Sertoli cell c-fos mRNA expression is under multifactorial regulation by cAMP, calcium, and PKC.