The Amoroso Lecture. The human spermatozoon--a cell in crisis?

A great deal of evidence has accumulated in recent years to suggest that there has been a gradual increase in male reproductive pathology over the past 30-40 years, as evidenced by increased rates of testicular cancer and declining semen quality. The hypothesis is advanced that this phenomenon is causally related to the ability of male germ cells to generate reactive oxygen metabolites. When produced in low levels, such metabolites are thought to enhance sperm function by stimulating DNA compaction and promoting a redox-regulated cAMP-mediated pathway that is central to the induction of sperm capacitation. When produced in excessive amounts, the same metabolites stimulate DNA fragmentation and a loss of sperm function associated with peroxidative damage to the sperm plasma membrane. Free radical-induced mutations in the male germ line may also be involved in the aetiology of childhood cancer and recent increases in the incidence of seminoma. In light of these considerations, establishing the mechanisms for free radical generation by the male germ line and determining the factors that influence this activity are important objectives for future research in this area.     

Efficient GFP‐labeling and analysis of spermatogenic cells using the IRG transgene and flow cytometry

Spermatogenesis is a highly ordered developmental program that produces haploid male germ cells. The study of male germ cell development in the mouse has provided unique perspectives into the molecular mechanisms that control cell development and differentiation in mammals, including tissue‐specific gene regulatory programs. An intrinsic challenge in spermatogenesis research is the heterogeneity of germ and somatic cell types present in the testis. Techniques to separate and isolate distinct mouse spermatogenic cell types have great potential to shed light on molecular mechanisms controlling mammalian cell development, while also providing new insights into cellular events important for human reproductive health. Here, we detail a versatile strategy that combines Cre‐lox technology to fluorescently label germ cells, with flow cytometry to discriminate and isolate germ cells in different stages of development for cellular and molecular analyses.     

Mammalian target of rapamycin (mTOR): a central regulator of male fertility?

Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolic phenotype and is involved in virtually all aspects of cellular function. It integrates not only nutrient and energy-sensing pathways but also actin cytoskeleton organization, in response to environmental cues including growth factors and cellular energy levels. These events are pivotal for spermatogenesis and determine the reproductive potential of males. Yet, the molecular mechanisms by which mTOR signaling acts in male reproductive system remain a matter of debate. Here, we review the current knowledge on physiological and molecular events mediated by mTOR in testis and testicular cells. In recent years, mTOR inhibition has been explored as a prime strategy to develop novel therapeutic approaches to treat cancer, cardiovascular disease, autoimmunity, and metabolic disorders. However, the physiological consequences of mTOR dysregulation and inhibition to male reproductive potential are still not fully understood. Compelling evidence suggests that mTOR is an arising regulator of male fertility and better understanding of this atypical protein kinase coordinated action in testis will provide insightful information concerning its biological significance in other tissues/organs. We also discuss why a new generation of mTOR inhibitors aiming to be used in clinical practice may also need to include an integrative view on the effects in male reproductive system.     

A unique combination of male germ cell miRNAs coordinates gonocyte differentiation

The last 100 years have seen a concerning decline in male reproductive health associated with decreased sperm production, sperm function and male fertility. Concomitantly, the incidence of defects in reproductive development, such as undescended testes, hypospadias and testicular cancer has increased. Indeed testicular cancer is now recognised as the most common malignancy in young men. Such cancers develop from the pre-invasive lesion Carcinoma in Situ (CIS), a dysfunctional precursor germ cell or gonocyte which has failed to successfully differentiate into a spermatogonium. It is therefore essential to understand the cellular transition from gonocytes to spermatogonia, in order to gain a better understanding of the aetiology of testicular germ cell tumours. MicroRNA (miRNA) are important regulators of gene expression in differentiation and development and thus highly likely to play a role in the differentiation of gonocytes. In this study we have examined the miRNA profiles of highly enriched populations of gonocytes and spermatogonia, using microarray technology. We identified seven differentially expressed miRNAs between gonocytes and spermatogonia (down-regulated: miR-293, 291a-5p, 290-5p and 294*, up-regulated: miR-136, 743a and 463*). Target prediction software identified many potential targets of several differentially expressed miRNA implicated in germ cell development, including members of the PTEN, and Wnt signalling pathways. These targets converge on the key downstream cell cycle regulator Cyclin D1, indicating that a unique combination of male germ cell miRNAs coordinate the differentiation and maintenance of pluripotency in germ cells.     

Le cancer à cellules germinales du testicule, élément constitutif du Syndrome de Dysgénésie Testiculaire: rôle des facteurs environnementaux et de la susceptibilité génétique

The incidence of testicular germ cell cancer has been increasing over recent decades in many countries of the world. Many studies over recent years have reported adverse trends in other aspects of male reproductive health, such as high and possibly increasing frequencies of undescended testis and hypospadias, declining semen quality, and an apparently growing demand for assisted reproduction due to male infertility. This article summarises the available evidence supporting a new concept that these male reproductive abnormalities may be signs of a single underlying entity: testicular dysgenesis syndrome (TDS). This syndrome, caused by nonspecific delays and aberrations of early testicular development, may be increasingly common because of deteriorating environmental and life-style factors that impair gonadal development. Geographical and ethnic differences in the incidence of various forms of TDS could be explained either by differences in exposure to adverse factors or by differences in genetic susceptibility to these factors.     

大鼠睾丸曲细精管组织支持细胞/生殖细胞长期共培养与精子发生分析

睾丸体外生殖模型的发展为体外研究睾丸的精子发生分子机制和睾丸毒理学提供了实验工具。很多报道的模型都无法真正地模拟体内复杂的生化分子及功能性相互作用从而导致研究价值有限。该实验拟建立一个体外长期维持睾丸生殖细胞存在,并能持续产生精子细胞的支持细胞/生殖细胞共培养体系。体系中的支持细胞和生殖细胞均由曲细精管组织块迁移到培养皿上,在不添加任何生长因子的情况下维持体外精子发生至圆形精子细胞超过2个月。RT-PCR分析显示,共培养细胞稳定表达cdh1、scp3、tnp2;免疫荧光染色结果显示,CDH1、PLZF、SCP3以及SOX9阳性细胞存在。这些结果例证了体系中同时存在精原干细胞、精母细胞、精子细胞和支持细胞。简单高效的支持细胞/生殖细胞体外共培养体系可用于雄性生殖的分子机制和毒理学研究。     

Protective effects of melatonin on male fertility preservation and reproductive system

Melatonin is a ubiquitous indoleamine hormone synthesized primarily by the pineal gland. Diverse biological actions of melatonin involve quite complex mechanisms via its membrane receptors. More recently, studies have focused on the role of melatonin in male fertility preservation and male reproductive system. The protective effects of melatonin on immature testicular tissue freshness and activity maintenance and the preservation of sperm and spermatogonial stem cells (SSCs) have attracted considerable attention in recent years. Furthermore, since melatonin has strong antioxidant and anti-apoptotic properties, researchers have examined its potential role in male reproductive system. In this article, recent progress regarding melatonin's effects on male fertility preservation and its potential role is reviewed.     

Loss of Wnt5a disrupts primordial germ cell migration and male sexual development in mice

Disruptions in the regulatory pathways controlling sex determination and differentiation can cause disorders of sex development, often compromising reproductive function. Although extensive efforts have been channeled into elucidating the regulatory mechanisms controlling the many aspects of sexual differentiation, the majority of disorders of sex development phenotypes are still unexplained at the molecular level. In this study, we have analyzed the potential involvement of Wnt5a in sexual development and show in mice that Wnt5a is male-specifically upregulated within testicular interstitial cells at the onset of gonad differentiation. Homozygous deletion of Wnt5a affected sexual development in male mice, causing testicular hypoplasia and bilateral cryptorchidism despite the Leydig cells producing factors such as Hsd3b1 and Insl3. Additionally, Wnt5a-null embryos of both sexes showed a significant reduction in gonadal germ cell numbers, which was caused by aberrant primordial germ cell migration along the hindgut endoderm prior to gonadal colonization. Our results indicate multiple roles for Wnt5a during mammalian reproductive development and help to clarify further the etiology of Robinow syndrome (OMIM 268310), a disease previously linked to the WNT5A pathway.     

The sertoli cell--a hormonal target and 'super' nurse for germ cells that determines testicular size

The somatic Sertoli cell plays an essential role in embryonic determination of male somatic sex and in spermatogenesis during adult life. One individual Sertoli cell supplies a clone of developing germ cells with nutrients and growth factors and it is well established that the number of Sertoli cells present is closely correlated to both testicular size and sperm output. Sertoli cells continue to proliferate and differentiate until the beginning of puberty, when they cease dividing and start nursing the germ cells. At this point in time, the future capacity of the testis for sperm production has thus been determined. Prior to puberty the Sertoli cells are immature and differ considerably with respect to morphology and biochemical activity from the mature cell. The several investigations that have focused on hormonal and paracrine regulation of the functions of the mature cell are reviewed here, but the mechanisms underlying the maturation and general maintenance of well-functioning Sertoli cells remain obscure. An alarming decline in male reproductive health has been observed in several Western countries during recent decades. Disturbance of Sertoli cell differentiation is thought to be involved in the pathogenesis of both a poor sperm count and testicular cancer. It is speculated that environmental agents that disrupt the estrogenic/androgenic balance in the testis may play a role in this connection.     

Bile Acid Alters Male Mouse Fertility in Metabolic Syndrome Context

Bile acids have recently been demonstrated as molecules with endocrine activities controlling several physiological functions such as immunity and glucose homeostases. They act mainly through two receptors, the nuclear receptor Farnesol-X-Receptor alpha (FXRα) and the G-protein coupled receptor (TGR5). These recent studies have led to the idea that molecules derived from bile acids (BAs) and targeting their receptors must be good targets for treatment of metabolic diseases such as obesity or diabetes. Thus it might be important to decipher the potential long term impact of such treatment on different physiological functions. Indeed, BAs have recently been demonstrated to alter male fertility. Here we demonstrate that in mice with overweight induced by high fat diet, BA exposure leads to increased rate of male infertility. This is associated with the altered germ cell proliferation, default of testicular endocrine function and abnormalities in cell-cell interaction within the seminiferous epithelium. Even if the identification of the exact molecular mechanisms will need more studies, the present results suggest that both FXRα and TGR5 might be involved. We believed that this work is of particular interest regarding the potential consequences on future approaches for the treatment of metabolic diseases.     

Stem cell-based therapies for fertility preservation in males: Current status and future prospects

With the decline in male fertility in recent years, strategies for male fertility preservation have received increasing attention. In this study, by reviewing current treatments and recent publications, we describe research progress in and the future directions of stem cell-based therapies for male fertility preservation, focusing on the use of spermatogonial stem cells (SSCs), SSC niches, SSC-based testicular organoids, other stem cell types such as mesenchymal stem cells, and stem cell-derived extracellular vesicles. In conclusion, a more comprehensive understanding of the germ cell microenvironment, stem cell-derived extracellular vesicles, and testicular organoids will play an important role in achieving male fertility preservation.     

Differentiation of human umbilical cord Wharton's jelly‐derived mesenchymal stem cells into germ‐like cells in vitro

Recent studies have demonstrated that mesenchymal stem cells could differentiate into germ cells under appropriate conditions. We sought to determine whether human umbilical cord Wharton's jelly‐derived mesenchymal stem cells (HUMSCs) could form germ cells in vitro. HUMSCs were induced to differentiate into germ cells in all‐trans retinoic acid, testosterone and testicular‐cell‐conditioned medium prepared from newborn male mouse testes. HUMSCs formed “tadpole‐like” cells after induction with different reagents and showed both mRNA and protein expression of germ‐cell‐specific markers Oct4 (POUF5), Ckit, CD49_f (α6), Stella (DDPA3), and Vasa (DDX4). Our results may provide a new route for reproductive therapy involving HUMSCs and a novel in vitro model to investigate the molecular mechanisms that regulate the development of the mammalian germ lineage. J. Cell. Biochem. 109: 747–754, 2010. © 2010 Wiley‐Liss, Inc.     

Sexual development of mouse germ cells: Nanos2 promotes the male germ cell fate by suppressing the female pathway

Much research has been conducted in recent years to elucidate the mechanisms underlying the crucial developmental process of sex determination. It has now been shown that somatic sex is principally determined by the chromosomal sex and the molecular mechanisms involved in this process have become relatively well understood in both human and mouse. However, the pathways involved in the sex determination of the germ cells remain largely unknown except for the fact that the somatic cues surrounding these cells play a significant role. Moreover, which sexual pathway of the germ cells is induced or suppressed has long been a subject of some dispute. Recent findings indicate that the key molecule that influences this choice is retinoic acid. In addition, the Nanos protein has been shown to play a critical role in promoting male germ cell differentiation. In this review, the possible mechanisms underlying these events, which have been brought to light by recent findings, are summarized to provide a better and more precise understanding of our current knowledge of the sex determination and subsequent differentiation of germ cells.     

Effect of metformin on testicular expression and localization of leptin receptor and levels of leptin in the diabetic mice

Diabetes mellitus impairs testicular activity and leads to infertility. Leptin is one of the endogenous regulators of the male reproductive functions, but the role of leptin and its receptor (LEPR/Ob‐R) in the control of testosterone production and testicular proliferation has not been investigated so far, especially in the Type 1 diabetes mellitus (DM1). Metformin is an anti‐hyperglycemic drug which is beneficial for treating the both DM2 and DM1. The aim of this work was to study the possible role of leptin and Ob‐R in the regulation of steroidogenesis and proliferation in the testes of mice with streptozotocin‐induced DM1 (75 mg/kg/day, 4 days) and to estimate the restoring effect of metformin treatment (500 mg/kg, 2 weeks) on the diabetic testes. In the diabetic testes, the plasma and intratesticular leptin levels and plasma testosterone levels were reduced and completely restored by metformin treatment. Metformin also restored the expression of the steroidogenic transport protein steroidogenic acute regulatory protein reduced in DM1. In the diabetic testes, the expression of Ob‐R was downregulated and the immunolocalization of Ob‐R showed weak staining in the Leydig cells, the primary spermatocytes and the round spermatids. The germ cell proliferation was also reduced in DM1, as noticed with proliferating cell nuclear antigen (PCNA) expression. Metformin increased the Ob‐R expression and immunostaining in the different cell types and improved the PCNA expression. Thus, DM1 impairs the testicular steroidogenesis and proliferation by inhibiting the leptin signaling, causing a decrease in leptin levels and Ob‐R expression in the testes of diabetic mice, while metformin improves the leptin signaling and restores testosterone production and testicular proliferation.     

Notch-1, c-kit and GFRalpha-1 are developmentally regulated markers for premeiotic germ cells

Culture, transfection and immortalization of mouse germ line stem cells, germ cell transplantation and grafting of testicular tissue are milestones of recent biotechnological breakthroughs. Alone and in combination they offer new pathways to explore the cellular mechanisms responsible for pluripotency and the requirements of cells to enter the germ line. Efficient markers, isolation and culture systems as well as transfection approaches are developed to elucidate the molecular and cellular mechanisms leading to the development of male germ line cells. Here, we describe the localization pattern of c-kit, Notch-1 and GFRalpha-1 in postnatal, immature and adult testes. All three proteins are potentially useful markers for spermatogonial characterization and enrichment. First attempts and various future perspectives to use spermatogonial stem cells as pathway for the introduction of transgenes are discussed.     

Impact of methoxyacetic acid on mouse Leydig cell gene expression

Background  

Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function.     

内质网应激与哺乳动物雄性生殖

内质网应激 (endoplasmic reticulum stress,ERS) 激活未折叠蛋白反应,维持哺乳动物细胞的胞内稳态,过度持续的ERS导致细胞凋亡。最新研究表明,ERS对哺乳动物雄性生殖有重要的调节作用,包括对精母细胞、睾丸结构及精子发生的影响。ERS是研究生殖细胞生存和凋亡的新通路。雄性不育可能是由过度ERS引起的。本文通过简述ERS的最新研究进展,分析雄性生殖与ERS的关系,并从ERS调控雄性生殖角度提出新的理解和展望。     

Aquaporins and (in)fertility: More than just water transport

Aquaporins (AQPs) are a family of channel proteins that facilitate the transport of water and small solutes across biological membranes. They are widely distributed throughout the organism, having a number of key functions, some of them unexpected, both in health and disease. Among the various diseases in which AQPs are involved, infertility has been overlooked. According to the World Health Organization (WHO) infertility is a global public health problem with one third of the couples suffering from subfertility or even infertility due to male or female factors alone or combined. Thus, there is an urgent need to unveil the molecular mechanisms that control gametes production, maturation and fertilization-related events, to more specifically determine infertility causes. In addition, as more couples seek for fertility treatment through assisted reproductive technologies (ART), it is pivotal to understand how these techniques can be improved. AQPs are heterogeneously expressed throughout the male and female reproductive tracts, highlighting a possible regulatory role for these proteins in conception. In fact, their function, far beyond water transport, highlights potential intervention points to enhance ART. In this review we discuss AQPs distribution and structural organization, functions, and modulation throughout the male and female reproductive tracts and their relevance to the reproductive success. We also highlight the most recent advances and research trends regarding how the different AQPs are involved and regulated in specific mechanisms underlying (in)fertility. Finally, we discuss the involvement of AQPs in ART-related processes and how their handling can lead to improvement of infertility treatment.     

Effets d’une exposition à la Vinclozoline et à la Génistéine de la gestation à l’âge adulte sur la fonction de reproduction du ratWistar mâle: protocole et résultats préliminaires

Current evidence indicates that endocrine disrupters (EDs) can induce adverse effects on the male reproductive tract in various mammalian species. Recent reports indicate deterioration in male reproductive health in several human populations, but the evidence for a causal link with endocrine disruption is still weak. In addition, the experimental conditions of most of the reportedin vivo studies are not representative of environmental exposures (for example, high doses, short-term exposure, a single ED) and the mechanisms by which EDs disrupt the reproductive system are poorly understood. The objective of the present study is to develop an animal model to assess the reproductive effects and study the putative cellular and molecular mechanisms involved after exposure to genistein (phytoestrogen) and vinclozolin (fungicide with a known antiandrogenic potential) alone or in combination. The study will be performed in male Wistar rats, with administration of low and high doses of the compounds from conception to adulthood and a subset of the males in each treatment group will be mated with unexposed females. We plan to assess the level of sperm production, histology of the reproductive organs, motility and morphometry of spermatozoa and hormone levels, as well as DNA fragmentation of spermatozoa and determination of the number of germ cells, Sertoli cells and the diameters of seminiferous tubules. Estrogen, androgen, progesterone and FSH receptors will be detected and quantified and the level of testicular apoptosis and several apoptosis pathways will be studied to determine the putative cellular and molecular mechanisms involved. The preliminary results confirmed the developmental effects previously reported for high doses of vinclozolin. More interestingly, they indicated a number of deleterious effects for male rats exposed to low dosages alone or mixtures of low and high dosages compared to controls and rats exposed to high dosages alone. For example, a number of developmental anomalies of the genitalia were observed and a significant decrease of sperm motility and motion and fertilizing ability were observed. These preliminary results provide evidence that chronic exposure to environmental levels of EDs or mixtures of EDs have a detrimental impact on the male reproductive tract. The next step involves assessment of the anatomical disorders and the study of some of the cellular and molecular mechanisms possibly involved.     

Calpain inhibitors prevent p38 MAPK activation and germ cell apoptosis after heat stress in pubertal rat testes

Testicular injuries like torsion or cryptorchidism can cause massive germ cell death, which could have great impact on male reproductive health. In addition, it has been proposed that modern life style, in the form of underwear or sedentary work position, could increase the testicular temperature, induce germ cell apoptosis, reduce spermatozoa quality and promote male infertility. In this work we showed that a heat stress stimulus induced massive germ cells apoptosis, which was associated with p38 mitogen-activated protein kinase (MAPK) phosphorylation along with an increase in the levels of mRNA encoding calpain 2. Synthetic calpain inhibitors prevented heat stress-induced germ cell apoptosis through inhibition of p38 MAPK phosphorylation. Thus, our results indicate that the blockage of calpains suppresses p38 MAPK phosphorylation, and identifies calpain activation (most likely calpain 2) as an early event in heat stress-induced male germ cell apoptosis. J. Cell. Physiol. 221: 296–305, 2009. © 2009 Wiley-Liss, Inc.