Testicular Somatic Cells Research Articles

Cytokines and chemokines in testicular inflammation: A brief review

A wide spectrum of data in the literature shows the relevance of cytokines as paracrine regulators of spermatogenesis and steroidogenesis in the normal testis. In this brief review, we highlight the relevance of cytokines in the testis during inflammation. This phenomenon involves complex and multiple interactions among immune and germ cells generally resulting in the alteration of spermatogenesis. The complexity of these cell interactions is multiplied because Sertoli and Leydig cells are also producers of pro- and anti-inflammatory cytokines and chemokines. Also, cytokines are pleiotropic and they exert opposite and/or redundant effects in different conditions. However, in spite of this bidirectional immunoregulatory function of cytokines, the mass of the data, reported from experiments of acute testicular inflammation, shows upregulation of interleukin (IL)-1beta, IL-1alpha, IL-6, and tumor necrosis factor alpha (TNF-alpha), which induce adverse effects on germ cells. In autoimmune orchitis, a chronic testicular inflammation, chemokines such as CCL2, CCL3, and CCL4 induce attraction and extravasation of immune cells within the testicular interstitium. These cells alter the normal immunosuppressor microenvironment principally through the secretion of proinflammatory cytokines, interferon-gamma initially, and IL-6 and TNF-alpha thereafter. Germ cells expressing TNFR1, IL-6R, and Fas increase in number and undergo apoptosis, through the TNF-alpha/TNFR1, IL-6/IL-6R, and Fas/Fas L systems. The knowledge of immune-germ and somatic testicular cell interactions will contribute to the understanding of the mechanisms by which chronic inflammatory conditions of the testis can disrupt the process of spermatogenesis.

Dose‐dependent effects on cell proliferation, seminiferous tubules, and male germ cells in the fetal rat testis following exposure to di(n‐butyl) phthalate

Adult male rats gestationally exposed to di(n-butyl)phthalate (DBP) have dysgenetic testes characterized by seminiferous epithelial degeneration, clustering of Leydig cells, and decreased spermatogenesis. Cell proliferation and apoptosis are key processes regulating development of the testis, and alterations in these processes may underlie testicular dysgenesis. To determine whether gestational exposure to DBP affects cell proliferation and apoptosis in the developing rat testis. Pregnant dams were exposed to different dose levels of DBP in mid-gestation and cellular outcomes in fetal and early postnatal testes were assayed by histological and morphometric approaches. Gestational exposure to high dose DBP inhibited proliferation of fetal testicular somatic cells but did not affect apoptosis. Exposed fetal testes had a smaller volume and decreased cell numbers, with decreases in both the tubular and interstitial cell populations. A reduction was observed in the testis volume and altered seminiferous tubule morphometry at > or =50 mg/kg/d, and a decreased testicular cell number at > or =30 mg/kg/d DBP. The number of multinucleated gonocytes in DBP-exposed fetal testes increased after exposure to > or =100 mg/kg/d. The number of proliferating cells in the DBP-exposed testis rapidly rose after birth (when exposure stopped), and the testis volume and the total cell number was comparable to control by postnatal day 2. DBP reversibly inhibits proliferation of somatic cells in the fetal rat testis. Decreased proliferation, rather than increased apoptosis, is the underlying mechanism of altered fetal development of DBP-exposed seminiferous tubules contributing to testicular dysgenesis.

Proliferative Activity In Vitro and DNA Repair Indicate that Adult Mouse and Human Sertoli Cells Are Not Terminally Differentiated, Quiescent Cells1

Sertoli cells isolated from the adult mouse and human testis resume proliferation in culture. After 20 days of culture in Dulbecco modified Eagle medium/Ham F12 (DMEM/F12) medium containing 5%% fetal calf serum, about 36%% of the mouse Sertoli cells, identified by their immunohistochemical staining for the Sertoli cell marker vimentin, incorporated bromodeoxyuridine (BrdU). The renewed proliferation was associated with a 70%% decrease in expression of the cell cycle inhibitor CDKN1B (P27(kip1)) and a 2-fold increase in the levels of the proliferation inducer ID2. In vivo, the balance between cell cycle inhibitors and inducers probably is such that the cells remain quiescent, whereas in culture the balance is disturbed such that Sertoli cells start to proliferate again. The renewed proliferative activity of Sertoli cells in culture was further confirmed by double staining for BrdU and the Sertoli cell marker clusterin (CLU), showing about 25%% of the CLU-positive Sertoli cells to be also positive for BrdU after 13 days of culture. Radiobiologically, Sertoli cells are also different from other quiescent somatic cells in the testis because they express several DNA repair proteins (XRCC1, PARP1, and others). Indeed, a comet assay on irradiated Sertoli cells revealed a 70%% reduction in tail length and tail moment at 20 h after irradiation. Hence, Sertoli cells repair DNA damage, whereas other quiescent somatic testicular cells do not. This repair may be accomplished by nonhomologous end joining via XRCC1 and PARP1. In conclusion, cell kinetic and radiobiological data indicate that Sertoli cells more resemble arrested proliferating cells than the classic postmitotic and terminally differentiated somatic cells that they have always been assumed to be.

169. SERTOLI CELL-SPECIFIC DISRUPTION OF THE ANDROGEN RECEPTOR DNA-BINDING DOMAIN REVEALS DIFFERENTIAL TEMPORAL CONTROL OF DISTINCT ANDROGEN-REGULATED GENES

Androgen receptor (AR) actions are vital for spermatogenesis. However, in postnatal development male germ cells do not express AR, highlighting its key role in testicular somatic cells. We recently used a Cre-loxP strategy to determine the in vivo requirement of AR DNA-binding in Sertoli cell (SC) function. Transgenic (Tg) mice with Cre expression targeted by SC-specific AMH or Abp promoters were crossed with floxed-Ar (Arflox) mice for Cre-loxP inframe deletion of Ar exon 3, which encodes a zinc finger essential for the DNA-binding domain (DBD). SC-specific mutated ARΔex3 (SCARΔex3) produced infertile AMH.SCARΔex3 and Abp.SCARΔex3 males. Testes from adult homozygous TgCre(+/+) AMH.SCARΔex3 or Abp.SCARΔex3 males were 30% of normal size and exhibited meiotic arrest, whereas testes from hemizygous TgCre(+/–) Abp.SCARΔex3 males were larger (47% normal) with more postmeiotic germ cell development. Despite marked Leydig cell hypertrophy, testicular expression of the adult Leydig marker Hsd3b6 (RT-PCR) and normal intratesticular testosterone levels (LC-MS/MS) in SCARΔex3 males indicated the presence of morphologically distinct but functional adult Leydig cells. SC-specific mutated AR Δex3 was predicted to disrupt classical AR-regulated pathways via loss of direct DNA interaction. Androgen-repressed testicular Ngfr expression (known to be via non-classical AR pathways) was not upregulated in SCARΔex3 testes, suggesting maintenance of a non-classical mechanism independent of AR-DBD. In contrast, SC-specific Rhox5 and Eppin transcription, regulated by divergent or classical androgen-response elements respectively, were both decreased in postnatal SCARΔex3 vs. control testes, demonstrating SC-specific AR function as early as postnatal day 5. However, Rhox5 expression declined dose-dependently, whereas Eppin expression increased, in adult TgCre(+/−) and TgCre(+/+) SCARΔex3 testes, revealing differential temporal control for distinct AR-regulated transcripts. Thus, our SCARΔex3 paradigm displayed dose-dependent TgCre-disruption of meiotic competence and post-meiotic development as well as gene expression, and represents a unique model to selectively differentiate AR-regulated genes.

Estrogen Receptors α and β in Human Testis: Both Isoforms are Expressed

Currently, clinical and experimental evidence point to an essential role of estrogens and estrogen receptors in male fertility. The expression of estrogen receptor α (ERα) and β (ERβ) in human testis has been described. However, some studies were unable to detect ERα, while others report the expression of both isoforms, with ERβ presenting a wide distribution within somatic and germinal testicular cells. This has suggested that estrogens may exert their testicular effects exclusively through ERβ. The present work aims to study the expression of ERα and ERβ in testicular biopsies of men with conserved and disrupted spermatogenesis, in order to better clarify the positive cell populations. Human testicular tissue was obtained from 10 men undergoing testicular biopsy for infertility relief due to azoospermia: two patients had secondary obstructive azoospermia with conserved spermatogenesis, five had Sertoli cell-only syndrome, two had hypospermatogenesis and one had meiotic arrest. Reverse-transcription polymerase chain reaction (RT-PCR) allowed the detection of both ERα and ERβ mRNAs in all samples. Immunohistochemistry revealed that ERα was present in Leydig cells, Sertoli cells, spermatogonia, spermatocytes, round spermatids and elongated spermatids/spermatozoa, while ERβ was present in the same cell types except spermatogonia and Sertoli cells. This study demonstrates ERα mRNA expression in human testis and describes its localization in somatic and germ cell subtypes. These findings suggest that both ER isoforms are involved in the control of testicular function.

3640 Unique EST Clusters from the Medaka Testis and Their Potential Use for Identifying Conserved Testicular Gene Expression in Fish and Mammals

BackgroundThe fish medaka is the first vertebrate capable of full spermatogenesis in vitro from self-renewing spermatogonial stem cells to motile test-tube sperm. Precise staging and molecular dissection of this process has been hampered by the lack of suitable molecular markers.Methodology and Principal FindingsWe have generated a normalized medaka testis cDNA library and obtained 7040 high quality sequences representing 3641 unique gene clusters. Among these, 1197 unique clusters are homologous to known genes, and 2444 appear to be novel genes. Ontology analysis shows that the 1197 gene products are implicated in diverse molecular and cellular processes. These genes include markers for all major types of testicular somatic and germ cells. Furthermore, markers were identified for major spermatogenic stages ranging from spermatogonial stem cell self-renewal to meiosis entry, progression and completion. Intriguingly, the medaka testis expresses at least 13 homologs of the 33 mouse X-chromosomal genes that are enriched in the testis. More importantly, we show that key components of several signaling pathways known to be important for testicular function in mammals are well represented in the medaka testicular EST collection.Conclusions/SignificanceMedaka exhibits a considerable similarity in testicular gene expression to mammals. The medaka testicular EST collection we obtained has wide range coverage and will not only consolidate our knowledge on the comparative analysis of known genes' functions in the testis but also provide a rich resource to dissect molecular events and mechanism of spermatogenesis in vivo and in vitro in medaka as an excellent vertebrate model.

Effects of Different Temperatures on Testis Structure and Function, with Emphasis on Somatic Cells, in Sexually Mature Nile Tilapias (Oreochromis niloticus)1

The Nile tilapia (Oreochromis niloticus) is economically one of the most important freshwater fish and is an excellent model for studies under laboratory conditions. Temperature is considered a very important modulator of reproductive activity in fish, although few studies have specifically addressed the effects of this key factor on morphological and functional aspects of teleost testes. Therefore, our main objectives in the present study were to analyze the effects of different temperatures (20, 25, 30, and 35 degrees C) on testicular somatic and germ cells in sexually mature Nile tilapias. Compared with fish kept at other temperatures, tilapias maintained at 20 degrees C demonstrated increased (P < 0.05) Sertoli cell and Leydig cell proliferation, volume density and frequency of most type B spermatogonia, and germ cell apoptosis. Conversely, tubular fluid secretion was decreased (P < 0.05) in the same animals. Although not significant, type A spermatogonia proliferation followed the pattern established for Sertoli cell and Leydig cell mitotic activity, suggesting that they preferentially would proliferate at lower temperatures. Based on most results found in our study and considering that tilapias are nonseasonal breeders, we suggest a model for temperature action on tilapia testes in which lower temperature (20 degrees C) would favor type A spermatogonial renewal, Sertoli cell and Leydig cell proliferation, and germ cell apoptosis, whereas higher temperatures (30-35 degrees C) would trigger rapid germ cell differentiation. Thus, tilapias could potentially be utilized in studies involving hormones and factors related to Sertoli cell and Leydig cell proliferation and spermatogonial self-renewal or differentiation.

Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa‐GFP transgenic rainbow trout (Oncorhynchus mykiss)

The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.

Maturation of Testicular Tissue from Infant Monkeys after Xenografting into Mice

In juvenile monkeys, precocious puberty can be induced by administration of gonadotropins resulting in testicular somatic cell maturation and germ cell differentiation. It is, however, unknown whether testicular maturation can also be induced in younger monkeys. Here we used testis tissue xenografting to investigate whether infant monkey testis tissue will undergo somatic cell maturation and/or spermatogenesis in response to endogenous adult mouse gonadotropins or exogenous gonadotropins. Testicular tissue pieces from 3- and 6-month-old rhesus monkeys were grafted to immunodeficient, castrated mice. Recipient mice were either left untreated or treated with pregnant mare serum gonadotropin and/or human chorionic gonadotropin twice weekly and were killed 28 weeks after grafting. Testicular maturation in grafted tissue was assessed based on morphology and the most advanced germ cell type present and by immunohistochemistry for expression of proliferating cell nuclear antigen, Mullerian-inhibiting substance, and androgen receptor. Testis grafts, irrespective of donor age or treatment, contained fewer germ cells than donor tissue. Grafts from 6-month-old donors showed tubular expansion with increased seminiferous tubule diameter and lumen formation, whereas those harvested from gonadotropin-treated mice contained elongated spermatids. Grafts from 3-month-old donors recovered from gonadotropin-treated mice contained pachytene spermatocytes, whereas those recovered from untreated mice showed only slight tubular expansion. Immunohistochemistry revealed that exposure to exogenous gonadotropins supported Sertoli cell maturation, irrespective of donor age. These results indicate that sustained gonadotropin stimulation of immature (<12 months old) monkey testis supports Sertoli cell maturation, thereby terminating the unresponsive phase of the germinal epithelium and allowing complete spermatogenesis in testis tissue from infant rhesus monkeys.

Sertoli Cell-Specific Modification of The Androgen Receptor DNA-Binding Domain Disrupts Sertoli Cell Function and Completion of Spermatogenesis.

Androgen receptor (AR) actions are vital for spermatogenesis, but male germ cells do not express AR, highlighting its key role in testicular somatic cells. Recent genetic models with targeted Sertoli cell (SC) AR disruption confirmed the vital role for Sertoli AR-activated pathways in spermatogenesis. However, complete loss of functional AR in these SC AR knockout (SCARKO) models excludes selective analysis of classical (via direct AR DNA binding) versus potential indirect non-classical or non-genomic AR actions. We have used a Cre-loxP strategy to selectively study the in vivo requirement of AR DNA-binding in SC function. Transgenic (Tg) mice with SC-specific Cre were crossed with mice carrying a floxed-AR gene (Arflox) to target exon 3. Cre-mediated inframe excision of exon 3, encoding a zinc finger essential for DNA-binding, was predicted to generate mutant ARΔex3 to selectively disrupt AR-regulated gene transcription via direct DNA interaction. We established two unique SC-specific ARΔex3 (SCARΔex3) models: i) AMH.SCARΔex3 derived from [Tg antimullerian hormone (AMH) promoter-driven Cre mice] X [Arflox mice], and ii) Abp.SCARΔex3 derived from [Tg androgen-binding protein (Abp) promoter-driven Cre mice] X [Arflox mice]. ROSA-LacZ reporter mice showed that the Abp promoter directs SC-specific Tg expression consistent with our past work, with testis Abp.Cre expressed as early as day 17 postcoitum. RT-PCR detected testis ArΔex3 RNA in both SCARΔex3 models. Using hemizygous TgCre mice, Abp.SCARΔex3 (49.3 ± 1.8 mg) testes were significantly larger (p < 0.001) than AMH.SCARΔex3 testes (29.9 ± 0.9 mg), and both were smaller (p < 0.001) than age-matched 9 week old controls (105.4 ± 4.4 mg). Homozygous TgCre mice were used to increase Cre expression levels. Testis size was further reduced in homozygous Abp.SCARΔex3 (31.6 ± 1.9 mg, p < 0.001) but not homozygous AMH.SCARΔex3 (32.1 ± 1.5 mg) mice relative to corresponding hemizygous TgCre SCARΔex3 testes, suggesting a maximal effect for SC Ar exon 3 disruption was achieved using Tg homozygous Abp.Cre or hemizygous AMH.Cre mice. Immunohistochemistry revealed SC nuclear AR expression was retained in SCARΔex3 testes. Despite histological detection of Leydig cell hypertrophy in SCARΔex3 testes, serum testosterone levels were equivalent to normal (22.8 ± 5.0 nM) in Abp.SCARΔex3 (15.7 ± 8.0 nM) and AMH.SCARΔex3 (16.6 ± 7.0 nM) 9 week old males, unlike the elevated or reduced androgen levels reported in SCARKO models with complete SC AR loss. Stereology revealed total SC numbers were normal in SCARΔex3 testes, whereas spermatogenesis in both SCARΔex3 models showed predominant meiotic arrest, noting larger Abp.SCARΔex3 testes using hemizygous Tg Abp.Cre mice exhibited more post-meiotic development. The testicular phenotype of these unique SCARΔex3 models demonstrates that despite nuclear ARΔex3 localisation, a functional AR DNA-binding domain is vital for Sertoli cell function and full spermatogenic development. The SCARΔex3 testes resemble SCARKO testes with complete SC AR loss, suggesting that non-genomic or pathways independent of direct AR-DNA binding play no major role in SC function. In combination, our novel SCARΔex3 models provide unique opportunity to further determine dose-dependent effects of AR activity in Sertoli cells. This research was supported by NHMRC funding.

Pattern of expression of immune-relevant genes in the gonad of a teleost, the gilthead seabream ( Sparus aurata L.)

Immune responses in the testis are regulated in a way that provides protection for the developing male germ cells, while permitting qualitatively normal inflammatory responses and protection against infection. In addition, germ cells are potent targets for the growth factors and cytokines which regulate the reproductive process. Our study analyzes for the first time the pattern of expression of several immune-relevant genes in the gonad of a seasonal breeding teleost fish. The immune molecules analyzed include (i) inflammatory molecules, such as interleukin-1b ( il1b), il6, tumor necrosis factor-a ( tnfa), cyclooxygenase-2 ( cox2) and the NADPH oxidase subunit p40 phox ( ncf4 gene); (ii) the anti-inflammatory cytokine transforming growth factor-b1 ( tgfb1) and its type 2 receptor tgfbr2; (iii) innate immune receptors, including toll-like receptor 9 ( tlr9), tlr5, tlr22 and macrophage-colony stimulating factor receptor ( mcsfr); (iv) lymphocyte receptors, such as the β subunit of T-cell receptor ( Tcrb) and the heavy chain of immunoglobulin M ( ighm); (v) the anti-bacterial molecules lysozyme ( lyz), hepcidin ( hamp) and complement component 3 ( c3); (vi) the anti-viral molecule myxovirus (influenza) resistance protein ( mx); and (vii) molecules related to leukocyte infiltration, including the CC chemokine ccl4, the CXC chemokine il8 and the leukocyte adhesion molecule E-selectin (S ele). Notably, all of them show a pattern of expression that depends on the reproductive stage of the first two reproductive cycles when the fish develop and function as males. Furthermore, we demonstrate that some of these immune-relevant molecules, such as Il1b and Mcsfr, are produced by germ cells (Il1b) and ovarian and testicular somatic cells (Mcsfr). These data suggest that, as occurs in mammals, there is a critical balance between immune molecules and that these may play an essential role in the orchestration of gametogenesis and the maintenance of gonad tissue homeostasis in fish.

Parp1-Xrcc1 and the repair of DNA double strand breaks in male germ cells and Sertoli cells

In male germ cells the repair of DNA double strand breaks (DSBs) differs from that described for somatic cell lines due to differences in the expression or even the absence of some DNA repair proteins in particular germ cells. For example, Ku70 is not expressed in early spermatocytes, DNA–PKcs is absent in spermatids, and 53bp1 and mdc1 are differentially expressed. Besides their role in base excision repair and single strand repair, Parp1 and Xrcc1, together with DNA ligase 3, were reported to be involved in the repair of DNA DSB in the absence of the Ku proteins. We now have studied the expression Xrcc1 and Parp1 in the irradiated and non irradiated mouse testis. Xrcc1 and Parp1 are highly expressed in spermatogonia, which also express other DNA repair proteins but not Mdc1. In these cells Xrcc1 and Parp1 may provide an additional repair pathway. In spermatocytes, Xrcc1 was found on the axial elements and sex chromosomes and in late spermatocytes after irradiation it formed foci that did not overlap with γ-H2AX foci. Round spermatids expressed both Parp1 and Xrcc1. We previously showed that in round spermatids DNA DSB repair does take place. However, in these haploid cells homologous recombination is not possible, and DNA–PKcs is not expressed making classical non-homologous end joining (NHEJ) impossible. In round spermatids the Xrcc1/Parp1 dependent repair pathway may be responsible for the observed DNA DSB repair. Finally, both Xrcc1 and Parp1 are expressed in early elongating spermatids and are probably involved in the extensive chromatin remodeling in these cells. In contrast to other testicular somatic cells, Sertoli cells were positive for Xrcc1 and Parp1, before and after IR. Sertoli cells were reported to express ku70, ku86 and p53 already before irradiation and pAtm in response to irradiation. Furthermore, after irradiation, 53BP1 foci are formed in Sertoli cells that diminish with time (Ahmed et al 2007, DNA Repair). Using the comet assay we now show that Sertoli cells can repair irradiation induced DNA damage. Taking together, Sertoli cells do not behave as normal terminally differentiated somatic cells, in that they are still able to repair DSBs. In conclusion, NHEJ repair of DNA DSBs via the Parp1/Xrcc1 pathway may occur in germ cells and Sertoli cells when homologous recombination is not possible and/or one of the main components of classical NHEJ is absent.

Reversible Changes in the Antifertility Induced by Aegle marmelos in Male Albino Rats

To study the effects of Aegle marmelos on the testicular reproductive system, a 50% ethanolic extract of Aegle marmelos leaves (AMLEt) was fed orally to male albino rats at the dose levels of 200 and 300 mg/kg body wt./day for 60 days. Recovery was assessed for an additional 120 days. Oral administration of AMLEt did not cause body weight loss. The motility and sperm concentration were significantly reduced along with complete inhibition of fertility at a dose of 300 mg/kg. The level of serum testosterone also declined and spermatogenesis was impaired. The number of normal tubules and the height of epithelial cells of the caput and cauda were reduced significantly. The cross sectional surface area of Sertoli cells and mature Leydig cells was reduced along with a dose dependent reduction of preleptotene and pachytene spermatocytes. Thus the antifertility effects of Aegle marmelos seemed to be mediated by disturbances in structure and function in testicular somatic cells including Leydig and Sertoli cells resulting in an alteration in physio-morphological events of spermatogenesis. However, complete recovery was observed after a 120 day withdrawal.

Culture conditions for maintaining the survival and mitotic activity of rainbow trout transplantable type A spermatogonia

Germ-cell transplantation is a powerful tool for studying gametogenesis in many species. We previously showed that spermatogonia transplanted into the peritoneal cavity of trout hatchlings were able to colonize recipient gonads, and produced fully functional sperm and eggs in synchrony with the germ cells of the recipient. An in vitro-culture system enabling spermatogonia to expand, when combined with transplantation, would be valuable in both basic and applied biology. To this end, we optimized culture conditions for type A spermatogonia in the present study using immature rainbow trout at 8-10 month of age. Spermatogonial survival and mitotic activity were improved during culture in Leibovitz's L-15 medium (pH 7.8) supplemented with 10% fetal bovine serum at 10 degrees C compared with culture under standard conditions for salmonids (Hank's MEM (pH 7.3) supplemented with 25 mM HEPES and 5% FBS, and culture at 20 degrees C). Elimination of testicular somatic cells promoted spermatogonial mitotic activity. In addition, insulin, trout embryonic extract, and basic fibroblast growth factor promoted the mitosis of purified spermatogonia in an additive manner. Mitotic activity increased nearly sevenfold over 19 days of culture compared with growth factor-free conditions and was maintained for >1 month. Furthermore, the cultured spermatogonia could colonize and proliferate in recipient gonads following transplantation. This study represents the first step towards establishing a cell line that can be transplanted for use in surrogate broodstock technology and cell-mediated gene-transfer systems.

Cross-platform gene expression signature of human spermatogenic failure reveals inflammatory-like response

The molecular basis of human testicular dysfunction is largely unknown. Global gene expression profiling of testicular biopsies might reveal an expression signature of spermatogenic failure in azoospermic men. Sixty-nine individual testicular biopsy samples were analysed on two microarray platforms; selected genes were validated by quantitative real-time PCR and immunohistochemistry. A minimum of 188 transcripts were significantly increased on both platforms. Their levels increased with the severity of spermatogenic damage and reached maximum levels in samples with Sertoli-cell-only appearance, pointing to genes expressed in somatic testicular cells. Over-represented functional annotation terms were steroid metabolism, innate defence and immune response, focal adhesion, antigen processing and presentation and mitogen-activated protein kinase K signalling pathway. For a considerable proportion of genes included in the expression signature, individual transcript levels were in keeping with the individual mast cell numbers of the biopsies. When tested on three disparate microarray data sets, the gene expression signature was able to clearly distinguish normal from defective spermatogenesis. More than 90% of biopsy samples clustered correctly into the corresponding category, emphasizing the robustness of our data. A gene expression signature of human spermatogenic failure was revealed which comprised well-studied examples of inflammation-related genes also increased in other pathologies, including autoimmune diseases.

The onset of spermatogenesis in fish.

Under cultivation conditions, male Japanese eels (Anguilla japonica) have immature testes containing only spermatogonia together with inactive testicular somatic cells, Leydig cells and Sertoli cells. Using a recently developed organ culture system for eel testes, we have shown that hormonal induction of spermatogenesis in eel testes involves gonadotropin stimulation of Leydig cells to produce 11-ketotestosterone, a potent androgen in fish. In turn, 11-ketotestosterone activates Sertoli cells to stimulate premitotic spermatogonia to complete spermatogenesis. Our current research focuses on the isolation and characterization of genes that show altered expression in eel testes during gonadotropin-induced spermatogenesis. One up-regulated and three down-regulated genes have been isolated. Northern blot analysis and in situ hybridization reveal that mRNA for activin B is absent in testes before gonadotropin injection and is abundant in Sertoli cells in testes injected with gonadotropin for one to six days after injection. This stimulation of activin B mRNA is accompanied by spermatogonial proliferation. Gonadotropin treatment also causes a rapid rise in the testicular concentrations of mRNA for 3 beta-hydroxysteroid dehydrogenase, the rate-limiting enzyme for gonadotropin-induced 11-ketotestosterone production. We have also obtained three down-regulated cDNAs which are abundant in testes before gonadotropin treatment and disappear almost completely in testes one day after gonadotropin injection.

Apoptotic Extinction of Germ Cells in Testes of Cyp26b1 Knockout Mice

Cyp26b1 encodes a retinoic acid (RA) metabolizing cytochrome P450 enzyme that is expressed in embryonic tissues undergoing morphogenesis, including the testes. We have generated transgenic mice lacking Cyp26b1 and have observed increased RA levels in embryonic testes. Cyp26b1(-/-) germ cells prematurely enter meiosis at embryonic d 13.5 and appear to arrest at pachytene stage. Furthermore, after embryonic d 13.5, a rapid increase in apoptosis is observed in male germ cells derived from Cyp26b1(-/-) embryos; germ cells are essentially absent in mutant male neonates. In contrast, testicular somatic cells appear to develop normally in the absence of Cyp26b1. Moreover, ovarian germ and somatic cells appear unaffected by the lack of CYP26B1. We also show that the synthetic retinoid Am580, which is resistant to CYP26 metabolism, induces meiosis of male germ cells in cultured gonads, suggesting that abnormal development of germ cells in the Cyp26b1(-/-) testes results from excess RA rather than the absence of CYP26B1-generated metabolites of RA. These results provide evidence that CYP26B1 maintains low levels of RA in the developing testes that blocks entry into meiosis and acts as a survival factor to prevent apoptosis of male germ cells.

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.

P19Ink4d and p18Ink4c cyclin‐dependent kinase inhibitors in the male reproductive axis

The loss of the cyclin-dependent kinase inhibitors (CKIs) p18(Ink4c) and p19(Ink4d) leads to male reproductive defects (Franklin et al., 1998. Genes Dev 12: 2899-2911; Zindy et al., 2000. Mol Cell Biol 20: 372-378; Zindy et al., 2001. Mol Cell Biol 21: 3244-3255). In order to assess whether these inhibitors directly or indirectly affect male germ cell differentiation, we examined the expression of p18(Ink4c) and p19(Ink4d) in spermatogenic and supporting cells in the testis and in pituitary gonadotropes. Both p18(Ink4c) and p19(Ink4d) are most abundant in the testis after 18 days of age and are expressed in purified populations of spermatogenic and testicular somatic cells. Different p18(Ink4c) mRNAs are expressed in isolated spermatogenic and Leydig cells. Spermatogenic cells also express a novel p19(Ink4d) transcript that is distinct from the smaller transcript expressed in Sertoli cells, Leydig cells and in other tissues. Immunohistochemistry detected significant levels of p19(Ink4d) in preleptotene spermatocytes, pachytene spermatocytes, condensing spermatids, and Sertoli cells. Immunoprecipitation-Western analysis detected both CKI proteins in isolated pachytene spermatocytes and round spermatids. CDK4/6-CKI complexes were detected in germ cells by co-immunoprecipitation, although the composition differed by cell type. p19(Ink4d) was also identified in FSH+ gonadotrophs, suggesting that this CKI may be independently required in the pituitary. Possible cell autonomous and paracrine mechanisms for the spermatogenic defects in mice lacking p18(Ink4c) or p19(Ink4d) are supported by expression of these CKIs in spermatogenic cells and in somatic cells of the testis and pituitary.

Azoospermia in mice with targeted disruption of theBrek/Lmtk2(brain-enriched kinase/lemur tyrosine kinase 2) gene

Brek/Lmtk2 (brain-enriched kinase/lemur tyrosine kinase 2) is a member of the Aatyk family of kinases that comprises Aatyk1, Brek/Lmtk2/Aatyk2, and Aatyk3. Although several potential roles have been proposed for Brek and other Aatyk family members, the physiological functions of these kinases remain unclear. Here, we report that Brek(-/-) male mice are infertile, with azoospermia. Detailed histological analysis revealed that Brek(-/-) germ cells differentiated normally until the round-spermatid stage, but failed to undergo the normal change in morphology to become elongated spermatids. Testicular somatic cells appeared normal in these mice. Expression of Brek in testis was restricted to the germ cells, suggesting that the maturations of germ cells in Brek(-/-) mice are affected in a cell-autonomous manner. On the basis of these findings, we concluded that Brek is essential for a late stage of spermatogenesis. Further clarification of the mechanism by which Brek regulates spermatogenesis may help identify new targets for reproductive contraceptives and treatments against infertility.