The dynamic cytoskeleton of the developing male germ cell

Mammalian spermatogenesis is characterised by dramatic cellular change to transform the non-polar spermatogonium into a highly polarised and functional spermatozoon. The acquisition of cell polarity is a requisite step for formation of viable sperm. The polarity of the spermatozoon is clearly demonstrated by the acrosome at the apical pole of the cell and the flagellum at the opposite end. Spermatogenesis consists of three basic phases: mitosis, meiosis and spermiogenesis. The final phase represents the period of greatest cellular change where cell-type specific organelles such as the acrosome and the flagellum form, the nucleus migrates to the plasma membrane and elongates, chromatin condenses and residual cytoplasm is removed. An important feature of spermatogenesis is the change in the cytoskeleton that occurs throughout this pathway. In this review, the author will provide an overview of these transformations and provide insight into possible modes of regulation of these rearrangements during spermatogenesis. Although primary focus will be given to the microtubule cytoskeleton, the importance of actin filaments to the cellular transformation of the male germ cell will also be discussed.     

Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene

The full-length cDNA corresponding to Stra8, a novel gene inducible by retinoic acid (RA) in P19 embryonal carcinoma cells, has been isolated and shown to encode a 45-kD protein. Both Stra8 mRNA and protein were induced in cells treated by all-trans and 9-cis retinoic acids. Two- dimensional gel analysis and dephosphorylation experiments revealed that the two stereoisomers of RA differentially regulate the phosphorylation status of the Stra8 protein, which was shown to exist in differently phosphorylated forms. Subcellular fractionation and immunocytochemistry studies showed that the Stra8 protein is cytoplasmic. During mouse embryogenesis, Stra8 expression was restricted to the male developing gonads, and in adult mice, the expression of Stra8 was restricted to the premeiotic germ cells. Thus, Stra8 protein may play a role in the premeiotic phase of spermatogenesis.     

Testicular germ cell development in relation to 5alpha-androstenone levels in pubertal entire male pigs

Androstenone is a 16-androstene steroid pheromone produced in the Leydig cells in the testis, and considered to be one of the major compounds responsible for boar taint. In entire male pigs, progress of sexual maturation has been related to an increase in androstenone levels in fat. Onset of puberty and subsequent reproductive function involves genetic factors affected by the internal and external environment. In this study entire male cross-bred pigs were housed under two different light regimens in order to manipulate the onset of puberty. DNA flow cytometry (FCM) was used to study spermatogenesis and monitor the proportions of haploid (1n), diploid (2n), and tetraploid (4n) testicular cells, with conventional histological evaluation used as the reference technique. Agreement between these two methods was found to be good. The best fit model explained 34% of the variation in the androstenone concentrations. Sexual maturation in boars of 125-146 days of age, as assessed by DNA FCM, was not significantly associated with the variation in androstenone concentrations in adipose tissue when various independent variables (breed, age, light strategy, skatole concentrations in fat, and length of the bulbourethralis glands) were included in this model. These findings support the suggestion that selection against androstenone may be an option in the breeding of entire male pigs.     

Mouse model of male germ cell apoptosis in response to a lack of hormonal stimulation

As a prerequisite for studies using mutant mice, we established a mouse model for induction of male germ cell apoptosis after deprivation of gonadotropins and intratesticular testosterone (T). We employed a potent long acting gonadotropin-releasing hormone antagonist (GnRH-A), acyline, alone or in combination with an antiandrogen, flutamide for effective induction of germ cell apoptosis in mice. Combined treatment with continuous release of acyline (3 mg/kg BW/day) with flutamide (in the form of sc pellets of 25 mg) resulted in almost the same level of suppression of spermatogenesis, as judged by testis weight and by germ cell apoptotic index, in 2 weeks as that reported for rats after treatment with 1.25 mg/kg BW Nal-Glu GnRH-A for the same time period. Within the study paradigm, the maximum suppression of spermatogenesis occurred after a single sc injection of high (20 mg/kg BW) dose of acyline with flutamide. The combined treatment resulted in complete absence of elongated spermatids. Germ cell counts at stages VII-VIII showed a significant (P < 0.05) reduction in the number of preleptotene (27.1%) and pachytene spermatocytes (81.9%), and round spermatids (96.6%) in acyline + flutamide group in comparison with controls. In fact, treatment with a single high (20 mg/kg BW) dose of acyline combined with flutamide in mice achieved same or greater level of suppression, measured by germ cell counts at stages VII-VIII, in two weeks when compared with those reported after daily treatment with Nal-Glu GnRH-A for 4 weeks in rats. Both plasma and testicular T levels were markedly suppressed after administration of acyline alone either by miniosmotic pump or by a single sc injection. Addition of flutamide to acyline had no discernible effect on plasma or intratesticular T levels when compared with acyline alone. These results demonstrate that optimum suppression of spermatogenesis through increased germ cell death is only possible in mice if total abolition of androgen action is achieved and further emphasize the usefulness of acyline + flutamide treated mice as a suitable model system to study hormonal regulation of testicular germ cell apoptosis.     

Contribution of environmental pollutants to male infertily: a working model of germ cell apoptosis induced by plasticizers

Bisphenol A [2,2-bis(4-hydroxyphenyl)propane] (BPA), 4-nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP), and its metabolite mono-2-ethylhexyl phthalate (MEHP) are chemicals found in plastics, which act as endocrine disruptors (EDs) in animals, including human. EDs act like hormones in the endocrine system, and disrupt the physiologic function of endogenous hormones. Most people are exposed to different endocrine disruptors and concern has been raised about their true effect on reproductive organs. In the testis, they seem to preferentially attack developing testis during puberty rather than adult organs. However, the lack of information about the molecular mechanism, and the apparently controversial effect observed in different models has hampered the understanding of their effects on mammalian spermatogenesis. In this review, we critically discuss the available information regarding the effect of BPA, NP and DEHP/ MEHP upon mammalian spermatogenesis, a major target of EDs. Germ cell sloughing, disruption of the blood-testis-barrier and germ cell apoptosis are the most common effects reported in the available literature. We propose a model at the molecular level to explain the effects at the cellular level, mainly focused on germ cell apoptosis.     

Aging and diethylstilbestrol-induced aneuploidy in male germ cells: a transgenic mouse model

The incidence of aneuploidy in male germ cells was evaluated by analyzing extra marker chromosome(s) signal(s) in round and/or hook spermatids of transgenic mice. Two types of transgenic mice were used as models. The inserted foreign DNA (λ-gt10LacZ shuttle vector and/or pSVc-myc plasmid) was located at the middle of the long arms of chromosome 2 (λ DNA) and/or chromosome 8 (c-myc). The number of marker chromosomes present could easily be detected after fluorescence in situ hybridization (FISH) in testicular cells. The frequency of spontaneous aneuploidy of chromosome 2 was similar in round spermatids of lambda and λ-myc mice. Differential involvement of chromosomes 2 and 8 was observed in both round and hook spermatids. The frequency of spontaneous aneuploidy in round spermatids was higher than that in hook spermatids. The frequency of aneuploidy of marker chromosomes was significantly higher in older mice (2 years old) than in younger ones. Diethylstilbestrol (DES)-induced aneuploidy was dose dependent, and was not influenced by the stage at which germ cells were treated with DES. These results demonstrate the usefulness of a transgenic mouse model for the study of aneuploidy in germ cells. Received: 5 August 1998 / Accepted: 27 August 1998     

A male contraceptive targeting germ cell adhesion

Throughout spermatogenesis, developing germ cells remain attached to Sertoli cells via testis-specific anchoring junctions. If adhesion between these cell types is compromised, germ cells detach from the seminiferous epithelium and infertility often results. Previously, we reported that Adjudin is capable of inducing germ cell loss from the epithelium. In a small subset of animals, however, oral administration of Adjudin (50 mg per kg body weight (b.w.) for 29 d) resulted in adverse effects such as liver inflammation and muscle atrophy. Here, we report a novel approach in which Adjudin is specifically targeted to the testis by conjugating Adjudin to a recombinant follicle-stimulating hormone (FSH) mutant, which serves as its 'carrier'. Using this approach, infertility was induced in adult rats when 0.5 microg Adjudin per kg b.w. was administered intraperitoneally, which was similar to results when 50 mg per kg b.w. was given orally. This represents a substantial increase in Adjudin's selectivity and efficacy as a male contraceptive.     

In vitro male germ cell cultures of zebrafish

Transgenic modification of sperm before fertilization has the advantages of a much shorter timeline for the production of transgenic animals. A culture system using primary cultures of zebrafish male germ cells, in which the differentiation of spermatogonia to functional sperm can occur in vitro, allows us to introduce foreign DNA into the cultured sperm and to produce transgenics from the sperm. This chapter describes methods for the co-culture of male germ cells and a Sertoli cell feeder layer and the introduction of foreign DNA with retroviruses. This male germ cell culture system should prove useful not only in producing genetically modified sperm, but also in analyzing the regulatory function of Sertoli cells for spermatogenesis in vertebrates.     

The proto-oncogene Ret is required for male foetal germ cell survival

The spermatogenic and oogenic lineages originate from bipotential primordial germ cells in response to signalling in the foetal testis or ovary, respectively. The signals required for male germ cell commitment and their entry into mitotic arrest remain largely unknown. Recent data show that the ligand GDNF is up regulated in the foetal testis indicating that it may be involved in male germ cell development. In this study genetic analysis of GDNF-RET signalling shows that RET is required for germ cell survival. Affected germ cells in Ret-/- mice lose expression of key germ cell markers, abnormally express cell cycle markers and undergo apoptosis. Surprisingly, a similar phenotype was not detected in Gdnf-/- mice indicating that either redundancy with a Gdnf related gene might compensate for its loss, or that RET operates in a GDNF independent manner in mouse foetal germ cells. Either way, this study identifies the proto-oncogene RET as a novel component of the foetal male germ cell development pathway.     

Stage-dependent variation in the radiosensitivity of DNA in developing male germ cells

The induction and rejoining of gamma-ray-induced DNA single-strand breaks (SSBs) were measured in the spermatogenic cells of mice using the alkaline elution technique. The animals were injected with [3H]thymidine and sacrificed on subsequent days to examine selectively cohorts of radiolabeled cells in the successive stages of maturation. A significantly increased frequency of SSB was observed in the unirradiated early spermatocytes and late spermatids, associated with genetic recombination and chromatin compaction, respectively. The frequency of SSBs induced by irradiation of animals in vivo remained constant from the early spermatocyte through mid-spermatid stages and decreased significantly only after the cells matured to the late spermatid stage. The frequency of SSBs after in vitro irradiation of testicular cell suspensions also decreased as round spermatids matured to late spermatids. Such decreases for both modes of irradiation may result from maturation-dependent alterations in chromatin in late spermatids, such as condensation and replacement of histones with protamines, rather than from changes in oxygen tension. Rejoining of SSBs in vivo was efficient in the spermatocytes and early spermatids but declined in late spermatids. Possible reasons for the discrepancy between the greater number of unrepaired lesions and lower susceptibility to mutation induction in late spermatids than in round spermatids are discussed.     

Transgenic mice produced by retroviral transduction of male germ line stem cells in vivo

Spermatogonial stem cells are the only stem cells in the postnatal body that can transmit parental genetic information to the offspring, making them an attractive target cell population for animal transgenesis. Although transgenic mice and rats were recently produced by retrovirus transduction of these cells in vitro, with transplantation of the transduced cells into infertile recipients, the difficulty of restoring fertility and preparing recipients using spermatogonial transplantation limits practical application of the technique. In this article, we describe a novel approach for producing transgenic animals by transducing spermatogonial stem cells in vivo using a retrovirus vector. Microinjection of retrovirus into immature seminiferous tubules resulted in the direct transduction of spermatogonial stem cells in situ, and the animals produced transgenic offspring after mating with females. Transgenic mice were produced in C57BL/6, BALB/C, A, and C3H backgrounds, with an average efficiency of 2.8%. The transgene was transmitted stably and expressed in the next generation. The technique overcomes the drawback of the in vitro-transduction approach, and will be useful as a novel method for producing transgenic animals as well as providing a means for analyzing the self-renewal and differentiation processes of spermatogonial stem cells in vivo.     

CYP26B1 promotes male germ cell differentiation by suppressing STRA8-dependent meiotic and STRA8-independent mitotic pathways

Germ cell sex is defined by factors derived from somatic cells. CYP26B1 is known to be a male sex-promoting factor that inactivates retinoic acid (RA) in somatic cells. In CYP26B1-null XY gonads, germ cells are exposed to a higher level of RA than in normal XY gonads and this activates Stra8 to induce meiosis while male-specific gene expression is suppressed. However, it is unknown whether meiotic entry by an elevated level of RA is responsible for the suppression of male-type gene expression. To address this question, we have generated Cyp26b1/Stra8 double knockout (dKO) embryos. We successfully suppressed the induction of meiosis in CYP26B1-null XY germ cells by removing the Stra8 gene. Concomitantly, we found that the male genetic program represented by the expression of NANOS2 and DNMT3L was totally rescued in about half of dKO germ cells, indicating that meiotic entry causes the suppression of male differentiation. However, half of the germ cells still failed to enter the appropriate male pathway in the dKO condition. Using microarray analyses together with immunohistochemistry, we found that KIT expression was accompanied by mitotic activation, but was canceled by inhibition of the RA signaling pathway. Taken together, we conclude that inhibition of RA is one of the essential factors to promote male germ cell differentiation, and that CYP26B1 suppresses two distinct genetic programs induced by RA: a Stra8-dependent meiotic pathway, and a Stra8-independent mitotic pathway.     

In vitro methods of male germ cell specification and differentiation

Germ line specification is an early cell fate decision essential for the transmission of totipotency over generations. Two types of germ line stem cells populate the male gonads in mammals. Primordial germ cells (PGCs) are the germ line founders only present during prenatal life. Spermatogonial stem cells (SSCs) appear a few days after birth and divide asymmetrically to give rise to one stem cell and one spermatogonia that initiates differentiation to produce spermatozoa. Germ cell specification and differentiation involve specific environmental stimuli and a sequential order of maturing phases required for gamete function. Spatio-temporal controls similarly dictate the erasure of somatic methylation marks and the subsequent acquisition of sex-specific marks at imprinted genes in gametes. We review here the recent advancements in male germ cell derivation from ES cells and discuss the limits of these in vitro methods in providing a kinetics and a microenvironment suitable for the programming of a proper gametic and parental identity.     

Impact of environmental pollutants on the male: effects on germ cell differentiation

A variety of so-called innocuous chemicals can have insidious and long lasting effects on the developing male reproductive system. Developmental exposures of male rabbits to common industrial contaminants in drinking water (a mixture of arsenic, chromium, lead, benzene, chloroform, phenol, and trichloroethylene); alkyl phenols (e.g. octylphenol); water disinfection by-products (e.g. dibromoacetic acid); anti-androgenic pesticides (e.g. p,p'-DDT and vinclozolin); and plasticizers (e.g. dibutyl phthalate) produce testicular dysgenesis. The lesions include testicular carcinoma in situ, also called intratubular germ cell neoplasia--the precursor lesion of germ cell tumors in men, and acrosomal dysgenesis--characterized by sharing of a dysplastic acrosome by two or more spermatids resulting in characteristic sperm acrosomal-nuclear malformations. Certain manifestations of testicular dysgenesis arch across environmental agents, and sequelae of intentional developmental exposures of rabbits duplicate what has been encountered in deer, horses, and humans for which the etiology is uncertain.     

Trypanosoma cruzi infection from the view of CD8+ T cell immunity--an infection model for developing T cell vaccine

Chagas' disease is caused by Trypanosoma cruzi (T. cruzi) which was once prevalent in Central and South America. Although the recent success in Triatoma vector control has made the disease being possibly extinct in the near future, the development of effective preventive and therapeutic vaccines is still necessary to prevent the resurgence of the neglected infection. In addition to the importance for containing the disease, T. cruzi infection presents unique features for elucidating hosts' immune responses against intracellular infectious agents. Due to its biological capacity for invading into principally any types of cells and for causing systemic infection which damages particularly muscle and neural cells, T cell immunity is critical for resolving its infection. Although T cell-mediated immune responses have been, so far, extensively investigated in viral and bacterial infections, parasitic infection such as malaria has presented epoch-making discovery in T cell immunity. Recent advances in the analyses of T cell-mediated immune responses against T. cruzi infection now make this infectious disease potentially more suitable for detecting subtle immunological changes in hosts' immune defense upon modifying immune system. The current review focuses on the usefulness of T. cruzi infection as a model for developing effective CD8(+) T cell-mediated vaccine against intracellular infectious agents.     

Juvenile spermatogonial depletion (jsd): a genetic defect of germ cell proliferation of male mice

Adult C57BL/6J male mice homozygous for the mutant gene, juvenile spermatogonial depletion (jsd/jsd), show azoosper4ia and testes reduced to one-third normal size, but are otherwise phenotypically normal. In contrast, adult jsd/jsd females are fully fertile. This feature facilitated mapping the jsd gene to the centromeric end of chromosome 1; the gene order is jsd-Isocitrate dehydrogenase-1 (Idh-1)-Peptidase-3 (Pep-3). Analysis of testicular histology from jsd/jsd mice aged 3-10 wk revealed that these mutant mice experience one wave of spermatogenesis, but fail to continue mitotic proliferation of type A spermatogonial cells at the basement membrane. As a consequence, histological sections of testes from mutant mice aged 8-52 wk showed tubules populated by modest numbers of Sertoli cells, with only an occasional spermatogonial cell. Some sperm with normal morphology and motility were observed in epididymides of 6.5- but not in 8-wk or older mutants. Treatment with retinol failed to alter the loss of spermatogenesis in jsd/jsd mice. Analyses of serum hormones of jsd/jsd males showed that testosterone levels were normal at all ages--a finding corroborated by normal seminal vesicle and vas deferens weights, whereas serum follicle-stimulating hormone levels were significantly elevated in mutant mice from 4 to 20 wk of age. We hypothesize the jsd/jsd male may be deficient in proliferative signals from Sertoli cells that are needed for spermatogenesis.