Leydig-cell agenesis: a cause of male pseudohermaphroditism.

We studied a 35-year-old patient with female external genitalia, primary amenorrhea and XY karytotype. Plasma testosterone was 10 ng per deciliter, which did not change after administration of human chorionic gonadotropin, increased to 22 ng per deciliter after ACTH, and decreased to 0.9 ng per deciliter after dexamethasone. Plasma delta 4-androstenedione, dehydroepiandrosterone and 17-hydroxyprogesterone were in the normal range. Plasma luteinizing hormone was high, but follicle-stimulating hormone normal (7.5 mlU per milliliter). There were two testes with epididymis and vas deferens, but no Mullerian structures. Microscopical examination showed hyalinization of tubules, which were lined by normal Sertoli cells and occasional immature germ cells. No Leydig cells were seen. After castration follicle-stimulating hormone increased to 43 mlU per milliliter. We conclude that this case of male pseudohermaphroditism was probably due to a Leydig-cell agenesis, that the epididymis and vas deferens can be developed in such a condition and the follicle-stimulating hormone secretion is regulated, at least in part, by a non-androgen substance secreted by Sertoli cells.     

枸杞多糖对精原干细胞体外增殖的影响

背景：精原干细胞移植对不育具有潜在的临床应用价值,体外建立精原干细胞的培养系统获得数量较多的精原干细胞,仍是目前研究中亟待解决的问题。 目的：观察枸杞多糖对精原干细胞体外增殖的影响。 方法：采用两步酶消化法获取出生4~6 d雄性C57BL/6小鼠睾丸Sertoli细胞与精原干细胞,将精原干细胞接种在Sertoli细胞饲养层上,再加入枸杞多糖或联合细胞因子添加到细胞培养液中。1周后以流式细胞仪检测细胞周期及细胞活性率,并检测各组精原干细胞GFRa-1、Thy-1、c-kit的阳性率。 结果与结论：单独加入枸杞多糖后精原干细胞数量明显增加,增殖明显,联合加入胶质细胞源性神经营养因子与白血病抑制因子精原干细胞增殖更加明显(P < 0.05)。并发现体外培养1周后的精原干细胞仍保持其睾丸组织内的精原干细胞特征,大多仍维持在未分化状态。表明在枸杞多糖或枸杞多糖联合胶质细胞源性神经营养因子及白血病抑制因子作用下,可促进精原干细胞体外增殖。     

Partial obstruction of the seminal path, a frequent cause of oligozoospermia in men

The objective of this clinical study was to determine the real frequency and clinical importance of partial obstruction of the seminal path in patients with oligozoospermia. We have designed a prospective clinical study including men with oligozoospermia seen at an andrological consultation in both private and institutional hospitals. A testicular biopsy was done on all patients under local anaesthesia. A complete study for sterility was also done [hormonal determinations: follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, testicular ultrasound, semen analysis, testicular Doppler ultrasound, etc.]. We have made a quantitative and qualitative evaluation of testicular biopsy (percentage of tubules with Sertoli cell only or with hyalinization; mean tubular diameter; number of spermatogonia, primary spermatocytes, young spermatids, mature spermatids and Sertoli cells; and evaluation of testicular interstitium: number of Leydig cell clusters, presence of angiectasis, presence of perivascular inflammation). Sixty one per cent of all oligozoospermia cases were obstructive. The principal cause of obstructive oligozoospermia was the presence of testicular varicocele. In obstructive oligozoospermia, the tubular diameter and number of mature spermatids are statistically significantly higher than in non- obstructive oligozoospermia. Obstructive oligozoospermia is a frequent condition caused by partial obstruction of seminal path. A quantitative analysis of the testicular biopsy is the only method of diagnosis.      

Reversion of the differentiated phenotype and maturation block in Sertoli cells in pathological human testis.

To study the relationship between abnormal Sertoli cell differentiation and spermatogenic impairment, we examined the expression of Sertoli cell markers normally lost at puberty, cytokeratin 18 (CK18), anti-Müllerian hormone (AMH) and M2A antigen, in three children (aged 1-2 years), 50 adults (aged 19-45 years) with obstructive or non-obstructive azoospermia or oligozoospermia, and six patients (aged 1-18 years) with 5 alpha-reductase deficiency. There was CK18 and/or AMH expression, but never M2A antigen expression, associated with spermatogonial arrest or Sertoli cell-only (SCO) syndrome in infertile men. Loss of M2A antigen suggests the transition of Sertoli cells to an adult phenotype, while CK18 and/or AMH expression may be a manifestation of de-differentiation of Sertoli cells. In 5 alpha-reductase deficiency, there was a sequential loss of CK18, M2A antigen and AMH around puberty, associated with partial spermatogenesis. The persistence of immature Sertoli cells expressing M2A antigen was associated with prepubertal seminiferous cords and SCO syndrome. Therefore, 5 alpha-reductase deficiency may prevent the maturation of Sertoli cells, resulting in impairment of spermatogenesis, and loss of M2A antigen expression coincides with a critical step in the Sertoli cell maturation. High follicle stimulating hormone concentrations due to failure of normal Sertoli cell differentiation indicate a normal development pattern of the hypothalamic-pituitary-gonadal axis.     

Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes

Basal and follicle-stimulating hormone (FSH)-stimulated cyclic AMP (cAMP) productions by seminiferous tubular segments from irradiated adult rats were investigated at defined stages of the epithelial cycle when specific spermatogenic cells were low in number. Seven days post-irradiation, depletion of spermatogonia did not influence the basal cAMP production, but FSH response increased in stages II-VIII. Seventeen days post-irradiation when spermatocytes were low in number, there was a small increase in basal cAMP level in stages VII-VIII and FSH-stimulated cAMP production increased in stages VII-XII and XIII-I. At 38 days when pachytene spermatocytes and round spermatids (steps 1-6) were low in number, a decreased basal cAMP production was measured in stages II-VI and IX-XII. FSH-stimulated cAMP output increased in stages VII-XII but decreased in stages II-VI. At 52 days when all spermatids were low in number, basal cAMP levels decreased in all stages of the cycle, whereas FSH response was elevated only in stages VII-XII. All spermatogenic cell types seem to have an effect on cAMP production by the seminiferous tubule in a stage-specific fashion. Germ cells appear to regulate Sertoli cell FSH response in a paracrine way, and a part of cAMP may originate from spermatids stimulated by an unknown FSH-dependent Sertoli cell factor. The FSH-dependent functions may control such phenomena as spermatogonial proliferation, final maturation of spermatids, and onset of meiosis.     

大鼠Sertoli细胞与精原细胞的分离及共培养

目的 分离纯化14 d大鼠Sertoli细胞与精原细胞,用Sertoli细胞作为饲养层对大鼠精原细胞进行体外培养研究.方法 酶消化法制备大鼠睾丸组织单细胞悬液,采用牛血清白蛋白(BSA)不连续密度梯度沉降法分离Sertoli细胞和精原细胞.结果 经分离的Sertoli细胞与精原细胞的纯度分别达到92.73%和78.36%.Sertoli细胞与精原细胞共培养,可见精原细胞发生分裂增殖等行为.结论 在添加EGF、bFGF和GDNF等细胞因子的10%胎牛血清DMEM/F12培养基中,精原细胞能够存活一定时间;而Sertoli细胞表现为旺盛的生长状态,并可促进精原细胞的分裂与增殖.     

Postnatal testis development, Sertoli cell proliferation and number of different spermatogonial types in C57BL/6J mice made transiently hypo- and hyperthyroidic during the neonatal period

The role of thyroid hormones in testis structure and function has been fairly well studied in laboratory rodents. However, there are no comprehensive data in the literature for mice regarding the effects of transiently induced neonatal hypo‐ and hyperthyroidism on testis and spermatogonial cell development from birth to adulthood. Our goals were to evaluate the effects of propylthiouracil (PTU) and triidothyronine (T3) on Sertoli cell proliferation/differentiation and to correlate these events with the evolution of the spermatogenic process, tubular lumen formation, blood vessel volume density, and size and number of different spermatogonial types. Although Sertoli cell maturation was accelerated or delayed, respectively, in T3‐ and PTU‐treated mice, the pace of the germ cell maturation was only slightly altered before puberty and the period of Sertoli cell proliferation was apparently not affected by the treatments. However, compared with controls, the total number of Sertoli cells per testis from 10 days of age to adulthood was significantly increased and decreased in PTU‐ and T3‐treated mice, respectively. In comparison to all other spermatogonia, type A₂ was the largest cell in all ages and groups investigated. The PTU‐treated mice had a significantly increased total number of undifferentiated spermatogonia as well as volume and percentage of vessels/capillaries, probably due to the higher number of Sertoli cells, particularly at 10 days of age. Taken together, our results suggest that neonatal hypothyroidism may be a valuable tool for studying spermatogonial biology as well as a means for providing more spermatogonial stem cells that could potentially be used for spermatogonial transplantation, thereby optimizing the efficiency of this technique when young mice are used as donors.     

Antireproductive effect of the calcium channel blocker amlodipine in male rats.

The purpose of the present study was to investigate whether treatment of male rats with the calcium antagonist amlodipine, used in the treatment of hypertension and angina, interferes with the reproductive function of male rats. Amlodipine treatment (0.04 mg amlodipine besylate/rat/day for 30 days) decreased plasma follicle-stimulating hormone and testosterone but not luteinizing hormone or prolactin concentrations (measured by double-antibody radioimmuno-assay). A significant reduction (23%) was observed in sperm density (sperm suspension collected from the cauda epididymidis) as well as in the amount of mature spermatids (14%) and Sertoli cells (9%) counted in seminiferous tubule cross-sections (400 x magnification). The results reveal the deleterious effects of subacute amlodipine treatment on the reproductive function of male rats.     

Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured monkey Sertoli cells

Sertoli cells play a central role in the control and maintenanceof spermatogenesis. Isolated Sertoli cells of mouse and rattestes have been shown to secrete plasminogen activator (PA)and a plasminogen activator inhibitor type-1 (PAI-1) in culture.In this study, we have investigated the hormonal regulationof PA and PAI-1 activities in cultured monkey Sertoli cells.Sertoli cells (5x10⁵ cells/well) isolated from infant rhesusmonkey testes were preincubated at 35°C for 16 h in 24-wellplates precoated with poly(D-lysine) (5 µg/cm²) in 0.5ml McCoy's 5a medium containing 5% of fetal calf serum and furtherincubated for 48 h in 0.5 ml serum-free medium with or withoutvarious hormones or other compounds. PA as well as PAI-1 activitiesin the conditioned media were assayed by fibrin overlay andreverse fibrin autography techniques respectively. The Sertolicells in vitro secreted only tissue-type PA (tPA), no detectableamount of urokinase-type PA (uPA) could be observed. MonkeySertoli cells were also capable of secreting PAI-1. Immunocytochemicalstudies indicated that both tPA and PAI-1 positive staininglocalized in the Sertoli cells, spermatids and residual bodiesof the seminiferous epithelium; Northern blot analysis furtherconfirmed the presence of both tPA and PAI-1 mRNA in monkeySertoli cells. Addition of follicle-stimulating hormone (FSH)or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generatingagents and gonadotrophin-releasing hormone (GnRH) agonist orphorbol ester (PMA) to the cell culture significantly increasedtPA activity. PAI-1 activity in the culture was also enhancedby these reagents except 8-bromo-dibutyryl-cAMP, forskolin and3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPAactivity, whereas decreased PAI-1 activity, implying that neutralizationof PAI-1 activity by the high level of tPA in the conditionedmedia may occur. These data suggest that increased intracellularsignals which activate protein kinase A (PKA), or protein kinaseC (PKC) can modulate Sertoli cell tPA and PAI-1 activities.The concomitant induction of PA and PAI-1 by the same reagentsin the Sertoli cells may reflect a finely tuned regulatory mechanismin which PAI-1 could limit the excession of the proteolysis. plasminogen activator inhibitor type-1/Rhesus monkey/Sertoli cells/tissue-type plasminogen activator     

Immunocytochemical staining of isolated rat sertoli cells for anti-FSHβ

Sertoli cells are a primary target for the action of follicle-stimulating hormone (FSH) in the testis. The purpose of this investigation was to verify ultrastructurally that FSH binds to receptors on the plasma membrane of isolated rat Sertoli cells. A reltaively pure aliquot of Sertoli cells was obtained by first dissociating testicular tissue from immature rats with collagenase and then centrifuging the suspension in Percoll density gradients. Pre-embedding staining with the peroxidase anti-peroxidase (PAP) complex technique using anti-FSHβ as the primary antiserum localized endogenous receptor-bound FSH on the plasma membrane of isolated Sertoli cells. Staining was considered to be specific since membranes of Sertoli cells derived from hypophysectomized rats were not stained when subjected to the same procedure. Cytoplasmic vesicles in Sertoli cells from experimental, control, and hypophysectomized groups also stained with PAP. Staining of these structures appeared to be specific since it was obliterated by preabsorption of anti-FSHβ with FSH. Preabsorption with luteinizing hormone (LH) did not affect the staining of cytoplasmic vesicles. The results of this investigation provide the first evidence for ultrastructural localization of specific binding sites for anti-FSHβ on the cell membrane of isolated Sertoli cells using an unlabeled antibody technique, and they further support the contention that Sertoli cells are a primary target for the action of FSH.     

A morphological and histochemical study of sertoli cells in normal and XX sex-reversed mice

Morphological and histochemical studies on the testes of mice with XX sex-reversal demonstrates several profound differences in the ultracytology and oxidative enzyme activity of Sertoli cells as compared to the normal. Membranous structures, cytoplasmic inclusions, and certain dehydrogenases in Sertoli cells of normal mice are described in relation to a possible role in steroid synthesis. In XX sex-reversed mice, two types of Sertoli cells are observed in the seminiferous tubules which are devoid of germ cells. One type is the randomly dispersed cells, which reveal the following ultrastructural alterations when compared with the normal cells: large spherical nuclei with prominent nucleoli, reduced smooth endoplasmic reticulum, pleomorphic mitochondria with no apparent cristae, and decreased lipid droplets. The other type is clustered cells which appear similar to immature Sertoli cells. These cells have small nuclei with poorly developed nucleoli, small profiles of rough endoplasmic reticulum, pleomorphic mitochondria, decreased lipid droplets, and an absence of specialized inter-Sertoli cell junctions. The results of this study suggest that functional and ultrastructural alterations of Sertoli cells in XX sex-reversed mice may result from degeneration or absence of germinal cells, and that steroidogenesis by Sertoli cells in mice may be associated with the development of germinal cells. It is also suggested that Sertoli cells may secrete a steroid which controls follicle-stimulating hormone secretion.     

Modulation of basal and FSH-dependent cyclic AMP production in rat seminiferous tubules staged by an improved transillumination technique

The stage-dependent action of follicle-stimulating hormone (FSH) in the rat seminiferous epithelium was investigated in microdissected 1 mm tubule segments, where the precise stage of the cycle was identified by a rapid screening method of live cell squash preparations. For distinction of stages I and II and the substages of VII, new criteria were used. The step 16 spermatids with rapid assembly of outer dense fibers leading to marked increase of flagellar thickness were used for distinction of stages I and II. The form and density of the cytoplasmic lobes of step 19 spermatids was used for recognition of substages of VII. Highest basal production of cyclic AMP (cAMP, measured by radioimmunoassay) was found in stage II of the cycle and stages XIV-I-VI had higher values than did stages VII-XIII. A decline occurred during stage VII and an increase at stage XIV. When stimulated with FSH, highest cAMP secretion was found in stage IV of the cycle; again, stages XIV-I-VI had higher values than did other stages. A small but significant (P less than .01) stimulation was found at substage VIId. FSH-stimulated and basal cAMP productions of different stages were compared, highest values were found at stages IV and XIII, and lowest, at stages VIIa-c and IX of the cycle. Since the FSH-dependent cAMP production is confined to Sertoli cells, and the number of these cells is constant per unit length of seminiferous tubules, the Sertoli cells are obviously under a stage-specific paracrine control by the surrounding spermatogenic cells. Specific steps in cell differentiation, such as spermatogonial proliferation, final maturation of the spermatids (stages I-VII), onset of meiosis (substage VIId), and completion of meiotic divisions (stage XIV) may be involved in this interaction.     

Structure-function correlations of human pituitary gonadotroph adenomas in vitro

Two pituitary gonadotroph adenomas were studied in vitro to characterize structure-function correlations. Both tumors were from men, aged 63 and 69 years, who had elevated blood levels of follicle-stimulating hormone (FSH) and normal blood luteinizing hormone (LH) and testosterone values. The surgically resected adenomas contained diffuse immunohistochemical positivity for beta-FSH, beta-LH, and alpha-subunit of glycoprotein hormones; by electron microscopy they were composed of well-differentiated gonadotrophs. In vitro, both tumors released FSH, LH, and alpha-subunit. Morphometric studies were performed on surgically resected and cultured adenoma cells. Compared with the surgical specimens, the cultured cells had decreased cytoplasmic volume densities of endoplasmic reticulum and Golgi apparatus and slightly increased cytoplasmic volume densities of secretory granules. Incubation with gonadotropin-releasing hormone (GnRH) for 2 and 24 hours increased FSH, LH, and alpha-subunit release by both tumors; morphometry after 2 consecutive days of exposure confirmed significant increases in cytoplasmic volume densities of endoplasmic reticulum and Golgi regions and marked decreases in that of secretory granules. There was no significant change in cell size, nuclear/cytoplasmic ratio, or secretory granule diameter. The two tumors differed in their response to gonadal steroids. Estradiol, testosterone, and progesterone stimulated release of FSH, LH, and alpha-subunit by one tumor and the morphologic changes paralleled the biochemical response; addition of testosterone suppressed the secretory and morphologic response to GnRH. The other tumor showed no significant response to estradiol or testosterone and addition of these steroids did not alter the response to GnRH. The results are consistent with the interpretation that GnRH stimulates not only release but also synthesis of gonadotropins by gonadotroph adenomas of men. The data also indicate variable sensitivity of these tumors to gonadal steroids with paradoxical stimulation alone and inhibition of response to GnRH. The structural changes correlate with the hormone release response in vitro.     

Caspase activity inhibition delays programmed spermatogenic cell death in vitro

Programmed cell death or apoptosis was analyzed in rat Sertoli-spermatogonial cell cocultures prepared from 2-9 day old rats using time-lapse video microscopy, a cell viability fluorescence microscopy assay, immunocytochemical markers, and cell-permeable caspase inhibitory peptides with reversible and irreversible effects. We show that apoptosis can initially affect a single member of a spermatogonial cell cohort and that single non-viable spermatogonial cells can remain conjoined to viable spermatogonial cells. The integrity of the cytoskeletal F-actin network and the presence on Bcl-2 immunoreactivity are valuable markers of spermatogonial cell viability. Apoptotic bodies released into the culture medium are generally eliminated after culture medium replenishment; however, spermatogonial apoptotic cell remnants can be taken up by Sertoli cells, which are known to represent a phagocytic somatic population within the seminiferous epithelium. Cell permeable caspase-1 and caspase-4 inhibitory peptides with reversible and irreversible action were supplemented to a serum-free hormone-growth factor-supplemented medium. In the absence of the caspase inhibitory peptide, the viability of spermatogonial cells decreases gradually with time in coculture. However, the addition of caspase inhibitory peptides causes a significant accumulation of spermatogenic cells per unit surface area. Although inhibition of caspases, the executors of spermatogonial cell death, results in a substantial increase of spermatogonial cells in the cocultures, it remains to be determined what the differentiation potential of caspase-inhibited spermatogonial cell cohorts is.     

In vitro culture of testicular germ cells: regulatory factors and limitations

Spermatogenesis is regulated mainly by endocrine factors and also by testicular paracrine/autocrine growth factors. These factors are produced by Sertoli cells, germ cells, peritubular cells and interstitial cells, mainly Leydig cells and macrophages. The interactions and the ratio between Sertoli and germ cells in the seminiferous tubules ensure successful spermatogenesis. In order to culture spermatogonial stem cells (SSCs) in vitro, researchers tried to overcome some of the obstacles -- such as the low number of stem cells in the testis, absence of specific markers to identify SSCs -- in addition to difficulties in keeping the SSCs alive in culture. Recently, some growth factors important for the proliferation and differentiation of SSCs were identified, such as glial cell line derived neurotrophic factor (GDNF), stem cell factor (SCF) and leukemia inhibitory factor (LIF); also, markers for SSCs at different stages were reported. Therefore, some groups succeeded in culturing SSCs (under limitations), or more differentiated cells and even were able to produce in vitro germ cells from embryonic stem cells. Thus, success in culturing SSCs is dependent on understanding the molecular mechanisms behind self-renewal and differentiation. Culture of SSCs should be a good tool for discovering new therapeutic avenue for some infertile men or for patients undergoing chemotherapy/radiotherapy (pre-puberty or post-puberty).     

Transgenic mice as a model to study the regulation of human transferrin expression in Sertoli cells

BACKGROUND: Understanding the regulation of proteins secreted by human Sertoli cells is important for identifying the causes of infertility in men. However, experiments with Sertoli cells purified from healthy testes are difficult to perform, for obvious ethical reasons. Therefore, experiments with transgenic mouse models could provide an alternative approach to study the function and regulation of a human gene in Sertoli cells. METHODS: To validate this approach, transgenic mice were generated using phage P1 containing an 80 kbp insert encompassing the complete human transferrin (hTf) gene. The expression pattern of hTf in the mouse background was analysed by isolating Sertoli cells from transgenic mice and comparing the regulation of the human and mouse Tf genes by hormones, retinoids and a cytokine in vitro. RESULTS: The hTf gene in transgenic mice shows a tissue-specific expression pattern that mimics the pattern observed in the human. In Sertoli cell cultures, FSH, insulin, retinol or tumour necrosis factor-alpha (TNF-alpha) stimulated hTf secretion, while testosterone alone had no effect. A combination of FSH, insulin, retinol and testosterone or a combination of TNF-alpha and retinol stimulated hTf secretion, but no additive effect was observed. CONCLUSION: Besides their well-known advantages, transgenic mice seem to be useful models to recapitulate the normal regulation of a human gene.     

Removal of spermatogonial stem cells (SSCs) from in vitro culture modulates testosterone synthesis in bovine

Niche cells, encompassing spermatogonial stem cells (SSCs) and controlling their development are believed to have a reciprocal communication with SSCs. The present study was conducted to evaluate the impact of the elimination of SSCs on testosterone production and genes contributing to testosterone synthesis. Following isolation, bovine testicular cells were cultured for 12 days on extracellular matrix-coated plates. In the germ cell-removed group, the SSCs were removed from the in vitro culture using differential plating; however, in the control group, no intervention in the culture was performed. Testosterone concentration and the gene expression of luteinizing hormone (LH) receptor (LHR), CYP17A1, and steroidogenic acute regulatory protein (StAR), were assessed on days 6 and 12. The concentration of testosterone was lower in the germ cell-removed than control group (P?<?0.05). Moreover, quantitative real-time PCR revealed lower gene expression of LHR, CYP17A1, and StAR in the germ cell-removed group as compared with the control group (P?<?0.05). In conclusion, the present study showed that elimination of SSCs from in vitro culture could diminish testosterone production via downregulation of factors contributing to testosterone synthesis. Additionally, this study provided evidence for the reciprocal relationship between SSCs and their niche cells.     

A tumorigenic murine Sertoli cell line that is temperature-sensitive for differentiation.

The Sertoli cell is the epithelial cell within the seminiferous tubule responsible for supporting germ cells. Most current in vitro studies of Sertoli cell function use primary cultures because of the limited number of available Sertoli cell lines. In addition, few in vivo models of Sertoli cell malignancy have been described. In this study, a tumorigenic Sertoli cell line was developed by infection of isolated murine Sertoli cells by simian virus 40 tsA255; the ts mutation causes the inactivation of the large T antigen at elevated temperatures. A cloned Sertoli cell line, called S14-1, demonstrated temperature-dependent growth in soft agar and formed tumors in nude mice. Electron microscopy of the S14-1-derived tumor revealed extensive basal intercellular junctions and tubulobulbarlike processes supporting its Sertoli cell origin. Cytogenetic analysis showed that S14-1 cells were aneuploid with an average of 70 chromosomes per cell. At the nonpermissive (40 C) temperature, S14-1 cells in vitro demonstrated a reduced growth rate, enhanced secretion of transferrin, and increased expression of sulfated glycoprotein-2 messenger RNA, indicating the cells manifested increased differentiation following large T antigen inactivation. The murine S14-1 Sertoli cell line should be useful for both in vitro studies of Sertoli cell function and in vivo studies of Sertoli cell malignancy.     

Endocrinology and Paracrinology: Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured monkey Sertoli cells

Sertoli cells play a central role in the control and maintenanceof spermatogenesis. Isolated Sertoli cells of mouse and rattestes have been shown to secrete plasminogen activator (PA)and a plasminogen activator inhibitor type-1 (PAI-1) in culture.In this study, we have investigated the hormonal regulationof PA and PAI-1 activities in cultured monkey Sertoli cells.Sertoli cells (5x105 cells/well) isolated from infant rhesusmonkey testes were preincubated at 35°C for 16 h in 24-wellplates precoated with poly(D-lysine) (5 µg/cm²) in 0.5ml McCoy's 5a medium containing 5% of fetal calf serum and furtherincubated for 48 h in 0.5 ml serum-free medium with or withoutvarious hormones or other compounds. PA as well as PAI-1 activitiesin the conditioned media were assayed by fibrin overlay andreverse fibrin autography techniques respectively. The Sertolicells in vitro secreted only tissue-type PA (tPA), no detectableamount of urokinase-type PA (uPA) could be observed. MonkeySertoli cells were also capable of secreting PAI-1. Immunocytochemicalstudies indicated that both tPA and PAI-1 positive staininglocalized in the Sertoli cells, spermatids and residual bodiesof the seminiferous epithelium; Northern blot analysis furtherconfirmed the presence of both tPA and PAI-1 mRNA in monkeySertoli cells. Addition of follicle-stimulating hormone (FSH)or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generatingagents and gonadotrophin-releasing hormone (GnRH) agonist orphorbol ester (PMA) to the cell culture significantly increasedtPA activity. PAI-1 activity in the culture was also enhancedby these reagents except 8-bromo-dibutyryl-cAMP, forskolin and3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPAactivity, whereas decreased PAI-1 activity, implying that neutralizationof PAI-1 activity by the high level of tPA in the conditionedmedia may occur. These data suggest that increased intracellularsignals which activate protein kinase A (PKA), or protein kinaseC (PKC) can modulate Sertoli cell tPA and PAI-1 activities.The concomitant induction of PA and PAI-1 by the same reagentsin the Sertoli cells may reflect a finely tuned regulatory mechanismin which PAI-1 could limit the excession of the proteolysis.     

Spermatogonial stem cells: characteristics and experimental possibilities

The continuation of the spermatogenic process throughout life relies on a proper regulation of self-renewal and differentiation of the spermatogonial stem cells. These are single cells situated on the basal membrane of the seminiferous epithelium. Only 0.03% of all germ cells are spermatogonial stem cells. They are the only cell type that can repopulate and restore fertility to congenitally infertile recipient mice following transplantation. Although numerous expression markers have been helpful in isolating and enriching spermatogonial stem cells, such as expression of THY-1 and GFRalpha-1 and absence of c-kit, no specific marker for this cell type has yet been identified. Much effort has been put into developing a protocol for the maintenance of spermatogonial cells in vitro. Recently, coculture systems of testicular cells on various feeder cells have made it possible to culture spermatogonial stem cells for a long period of time, as was demonstrated by the transplantation assay. Even expansion of testicular cells, including the spermatogonial stem cells, has been achieved. In these culture systems, hormones and growth factors are investigated for their role in the process of proliferation of spermatogonial stem cells. At the moment the best culture system known still consists of a mixture of testicular cells with about 1.33% spermatogonial stem cells. Recently pure SV40 large T immortalized spermatogonial stem cell lines have been established. These c-kit-negative cell lines did not show any differentiation in vitro or in vivo. A telomerase immortalized c-kit-positive spermatogonial cell line has been established that was able to differentiate in vitro. Spermatocytes and even spermatids were formed. However, spermatogonial stem cell activity by means of the transplantation assay was not tested for this cell line. Both the primary long-term cultures and immortalized cell lines have represented a major step forward in investigating the regulation of spermatogonial self-renewal and differentiation, and will be useful for identifying specific molecular markers.