Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses.

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I-XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

Hedgehog signalling promotes germ cell survival in the rat testis.

Hedgehog (Hh) signalling has a crucial role in testis development. Sertoli cell-derived desert hedgehog (DHH) guides the formation of testis cords and differentiation of foetal-type Leydig cells. Dhh mutant mice are infertile due to a block in germ cell differentiation, hypogonadism and hypoandrogenism. Hh signalling pathway components are also expressed in postnatal testis. In the rat testis the transcription factor of the Hh pathway, glioma-associated oncogene homologue (GLI1), is expressed by a wide variety of germ cells. This suggests that Hh signalling is involved in spermatogenesis at many different levels. Our data show that canonical Hh signalling is turned off in early condensing spermatids that strongly express the negative regulator of the pathway, suppressor of fused (SUFU). Most of the Hh pathway specific mRNAs display the highest values in stages II-VI of the rat seminiferous epithelial cycle. The key endocrine regulator of germ cell differentiation, FSH, down-regulates Dhh mRNA levels in vitro. Hh signalling inhibition in vitro leads to massive apoptosis of germ cells. In prepubertal rat testis imatinib mesylate-induced inhibition of tyrosine kinases impinges on Dhh transcript levels and Hh signalling. Our data indicate that Hh signalling is part of the paracrine signalling network in the rat testis. It promotes the survival of germ cells and is suppressed by FSH.

DAX-1 and SOX6 molecular interplay results in an antagonistic effect in pre-mRNA splicing.

DAX-1 (DSS-AHC Critical Region on the X Chromosome-1) is a member of the nuclear hormone receptor superfamily that has an important role in steroidogenesis and gonadogenesis. To investigate the role of DAX-1 in the testis, a yeast two-hybrid screen was performed and SOX6, member of the Sry box (SOX) protein family, was cloned as candidate. The interaction was confirmed biochemically and expression of SOX6 overlapped with that of DAX-1 in the developing gonad, as well as in Sertoli cells of the adult testis. We show here that DAX-1 is able to inhibit splicing in in vivo and in vitro splicing assays, and this inhibition is relieved by the addition of SOX6. SOX6 appears to inhibit the interaction between DAX-1 and the splicing machinery, thus providing a likely mechanism for functional interference. We conclude that DAX-1 and SOX6 proteins interact, have overlapped expression in the testis, and act antagonistically during pre-mRNA splicing. Developmental Dynamics 238:1595-1604, 2009. (c) 2009 Wiley-Liss, Inc.

Estrogen receptor beta selective ligand 5alpha-Androstane-3beta, 17beta-diol stimulates spermatogonial deoxyribonucleic acid synthesis in rat seminiferous epithelium in vitro.

Gonadotropins and testosterone are important regulators of spermatogenesis, even though gonadotropin receptors and the androgen receptor are not expressed by germ cells. However, a functional role for estrogens in connection with male reproduction has been postulated on the basis of the phenotypes of mice lacking estrogen receptor (ER) and cytochrome P-450 aromatase. This has further support by findings of ER expression in the testis, including that of ERbeta in spermatogonia. 5alpha-Androstane-3beta, 17beta-diol (3betaAdiol), a metabolite of testosterone produced via the intermediate potent androgen 5alpha-dihydrotestosterone (DHT), has been reported to selectively bind ERbeta rather than EpsilonRalpha, but not androgen receptor. Here, we have characterized the influence of 17beta-estradiol (E), the major physiological estrogen, 3betaAdiol, and DHT on DNA synthesis in vitro by segments of the seminiferous epithelium at different stages of the seminiferous epithelial cycle in the rat. E and 3betaAdiol exerted similar stimulatory effects on premitotic DNA synthesis in stage I segments, whereas other stages tested (V, VIIa, and XIII-IX) remained unresponsive. In contrast, DHT had no effect on this process. 5-bromo-2'-deoxyuridine labeling of stage I segments revealed a 30-fold higher labeling index in the presence than in the absence of E, and the labeled cells were identified as spermatogonia. Moreover, high levels of 3betaAdiol were found in the testis of intact rats as well as in primary cultures of rat Leydig cells in response to human chorionic gonadotropin. We suggest that 3betaAdiol may serve as a growth factor for germ cells stimulating premitotic DNA synthesis in connection with spermatogenesis via an ERbeta-dependent pathway.

Differential expression of members of the E2F family of transcription factors in rodent testes.

BACKGROUND: The E2F family of transcription factors is required for the activation or repression of differentially expressed gene programs during the cell cycle in normal and abnormal development of tissues. We previously determined that members of the retinoblastoma protein family that interacts with the E2F family are differentially expressed and localized in almost all the different cell types and tissues of the testis and in response to known endocrine disruptors. In this study, the cell-specific and stage-specific expression of members of the E2F proteins has been elucidated. METHODS: We used immunohistochemical (IHC) analysis of tissue sections and Western blot analysis of proteins, from whole testis and microdissected stages of seminiferous tubules to study the differential expression of the E2F proteins. RESULTS: For most of the five E2F family members studied, the localizations appear conserved in the two most commonly studied rodent models, mice and rats, with some notable differences. Comparisons between wild type and E2F-1 knockout mice revealed that the level of E2F-1 protein is stage-specific and most abundant in leptotene to early pachytene spermatocytes of stages IX to XI of mouse while strong staining of E2F-1 in some cells close to the basal lamina of rat tubules suggest that it may also be expressed in undifferentiated spermatogonia. The age-dependent development of a Sertoli-cell-only phenotype in seminiferous tubules of E2F-1 knockout males corroborates this, and indicates that E2F-1 is required for spermatogonial stem cell renewal. Interestingly, E2F-3 appears in both terminally differentiated Sertoli cells, as well as spermatogonial cells in the differentiative pathway, while the remaining member of the activating E2Fs, E2F-2 is most concentrated in spermatocytes of mid to late prophase of meiosis. Comparisons between wildtype and E2F-4 knockout mice demonstrated that the level of E2F-4 protein displays a distinct profile of stage-specificity compared to E2F-1, which is probably related to its prevalence and role in Sertoli cells. IHC of rat testis indicates that localization of E2F-5 is distinct from that of E2F-4 and overlaps those of E2F-1 and E2F-2. CONCLUSION: The E2F-1 represents the subfamily of transcription factors required during stages of DNA replication and gene expression for development of germ cells and the E2F-4 represents the subfamily of transcription factors that help maintain gene expression for a terminally differentiated state within the testis.

Inhibition of Aurora kinases perturbs chromosome alignment and spindle checkpoint signaling in rat spermatocytes.

In somatic cells, integrity of cell division is safeguarded by the spindle checkpoint, a signaling cascade that delays the separation of sister chromatids in the presence of misaligned chromosomes. Aurora kinases play important roles in this process by promoting centrosome maturation, chromosome bi-orientation, spindle checkpoint signaling, and cytokinesis. To investigate the functions of Aurora kinases in male meiosis, we applied a small molecule Aurora inhibitor, ZM447439, to seminiferous tubules in vitro. Primary and secondary spermatocytes exposed to ZM447439 exhibit defects in the spindle morphology and fail to align their chromosomes at the metaphase plate. Moreover, the treated spermatocytes undergo a forced exit from the meiotic M-phase without cytokinesis. These results suggest that the activities of Aurora kinases are required for normal spindle assembly as well as for establishment and maintenance of proper microtubule-kinetochore attachments and spindle checkpoint signaling in male mammalian meiosis.

Interplay of PIWI/Argonaute protein MIWI and kinesin KIF17b in chromatoid bodies of male germ cells.

Chromatoid bodies are thought to act as male-germ-cell-specific platforms for the storing and processing of haploid transcripts. The molecular mechanisms governing the formation and function of these germ-cell-specific structures have remained elusive. In this study, we show that the kinesin motor protein KIF17b, which is involved in the nucleocytoplasmic transport of RNA and of a transcriptional coactivator, localizes in chromatoid bodies. The chromatoid body moves actively and non-randomly in the cytoplasm of round spermatids, making frequent contacts with the nuclear envelope. The localization of KIF17b thereby offers a potential mechanism for microtubule-dependent mobility of chromatoid bodies, as well as for the transport of the specific components in and out of the chromatoid body. Interestingly, we demonstrate that KIF17b physically interacts with a testis-specific member of the PIWI/Argonaute family, MIWI, a component of chromatoid bodies implicated in RNA metabolism. A functional interplay between KIF17b and MIWI might be needed for the loading of haploid RNAs in the chromatoid body. Importantly, chromatoid bodies from round spermatids of miwi-null mice are not fully compacted and remain as a diffuse chromatoid material, revealing the essential role played by MIWI in the formation of chromatoid bodies. These results shed new light on the function of chromatoid bodies in the post-transcriptional regulation of gene expression in haploid germ cells.

The chromatoid body of male germ cells: similarity with processing bodies and presence of Dicer and microRNA pathway components.

The chromatoid body is a perinuclear, cytoplasmic cloud-like structure in male germ cells whose function has remained elusive. Here we show that the chromatoid body is related to the RNA-processing body of somatic cells. Dicer and components of microRNP complexes (including Ago proteins and microRNAs) are highly concentrated in chromatoid bodies. Furthermore, we show that Dicer interacts with a germ cell-specific chromatoid body component, the RNA helicase MVH (mouse VASA homolog). Thus, chromatoid bodies seem to operate as intracellular nerve centers of the microRNA pathway. Our findings underscore the importance of posttranscriptional gene regulation and of the microRNA pathway in the control of postmeiotic male germ cell differentiation.

The regulated expression of c-IAP1 and c-IAP2 during the rat seminiferous epithelial cycle plays a role in the protection of germ cells from Fas-mediated apoptosis.

The inhibitor of apoptosis proteins, c-IAP1 and c-IAP2, are highly expressed in rat testis and potentially play a regulatory role in testicular apoptosis. To better understand their functions during spermatogenesis, we have analyzed their spatio-temporal distribution in rat testis, how their expression is controlled by the paracrine stem-cell factor (SCF) and how they affect Fas-mediated apoptosis. Both c-IAP1 and c-IAP2 showed cycles of transcriptional expression, throughout the seminiferous epithelial cycle. c-IAP1 protein showed a diffuse nuclear distribution in type B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes. In pachytene spermatocytes, c-IAP1 colocalized with SUMO-1 in the XY-body. c-IAP2 protein was cytoplasmic in spermatocytes, from stage VI pachytene onwards, round spermatids, elongated spermatids and Leydig cells. Its expression was upregulated by SCF. Inhibition of IAP activity resulted in a greater sensitivity of germ cells to Fas-mediated apoptosis. These results suggest an important role for IAPs in the regulation of spermatogenic apoptosis.

Doxorubicin induces apoptosis in germ line stem cells in the immature rat testis and amifostine cannot protect against this cytotoxicity.

The underlying primary damage to the seminiferous epithelium caused by chemotherapeutic regimens at childhood is largely unknown. The present investigation was designed to identify acute cytotoxic events in the testis caused by a single dose of doxorubicin. Male rats at 6, 16, and 24 days of age were injected with doxorubicin (3 mg/kg, i.p.) or vehicle (saline) alone and 24 and 48 hours later, the germ cell types and apoptotic cells in the seminiferous epithelium were examined. As indicated by microscopy and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, an 8-fold increase in the number of apoptotic germ cells in the testes of 6-day-old rats was observed 48 hours after doxorubicin treatment. Spermatogonia migrating to the basement membrane were the primary cell type undergoing this induced apoptosis. A single dose of amifostine (200 mg/kg) administered i.p. 15 minutes before injection of doxorubicin provided no protection against this enhanced apoptosis. Under the same conditions, testicular levels of p53 and activated caspase 8 were elevated, whereas the level of murine double minute-2 was lowered. In contrast, doxorubicin treatment did not result in any significant change in the physiologic, stage-specific germ cell apoptosis occurring in the testes of 16- and 24-day-old rats. These observations suggest that the initiation phase of spermatogenesis is highly sensitive to doxorubicin-induced apoptosis. Gonocytes and early spermatogonia are the cell types that are vulnerable to this p53-trigged apoptosis, which results in a decrease in the size of the pool of germ-line stem cells. Amifostine fails to protect the germ cells against this cytotoxic insult.

The chromatoid body in spermatogenesis.

All germ cells throughout the animal kingdom contain cytoplasmic cloud-like accumulations of material called nuage. Polar bodies in Drosophila oocytes are probably the best known forms of nuage. In spermatogenic cells, the nuage is called chromatoid body (CB). In early spermatids of the rat, it has a diameter of 1-1.5 microm and a finely filamentous lobular structure. Typically, it is associated with a multitude of vesicles. It is first clearly seen in mid- and late pachytene spermatocytes as an intermitochondrial dense material. During early spermiogenesis it is seen near the Golgi complex and frequently connected by material continuities through nuclear pore complexes with intranuclear particles. In living cells, the CB moves around the Golgi complex and has frequent contacts with it. The CB also moves perpendicularly to the nuclear envelope, and even through cytoplasmic bridges to the neighbour spermatids. One of the major components of the CB is a DEAD-box RNA helicase VASA that belongs to a class of proteins thought to act as RNA chaperones. It is a general marker of all germ cells and best characterized in Drosophila. The mouse VASA homologue was recently used as a marker of sperm formation from embryonic stem cells. It becomes generally accepted that the CB with its associated structures constitute a mechanism of post-transcriptional processing and storage of several mRNA species that are shared between neighbour cells and used for translation when the genome of the spermatids becomes inactive.

Proteolytic cleavage of ALF into alpha- and beta-subunits that form homologous and heterologous complexes with somatic TFIIA and TRF2 in male germ cells.

Male germ cells specifically express paralogues of components of the general transcription apparatus including ALF a paralogue of TFIIAalpha/beta. We show that endogenous ALF is proteolytically cleaved to give alpha- and beta-subunits and we map the proteolytic cleavage site by mass spectrometry. Immunoprecipitations show that ALFalpha- and beta-subunits form a series of homologous and heterologous complexes with somatic TFIIA which is coexpressed in male germ cells. In addition, we show that ALF is coexpressed in late pachytene spermatocytes and in haploid round spermatids with transcription factor TRF2, and that these proteins form stable complexes in testis extracts. Our observations highlight how cleavage of ALF and coexpression with TFIIA and TRF2 increases the combinatorial possibilities for gene regulation at different developmental stages of spermatogenesis.

Polar nuclear localization of H1T2, a histone H1 variant, required for spermatid elongation and DNA condensation during spermiogenesis.

Spermiogenesis entails a major biochemical and morphological restructuring of the germ cell involving replacement of the somatic histones by protamines packing the DNA into the condensed spermatid nucleus and elimination of the cytoplasm during the elongation phase. We describe H1T2, an histone H1 variant selectively and transiently expressed in male haploid germ cells during spermiogenesis. In round and elongating spermatids, H1T2 specifically localizes to a chromatin domain at the apical pole, revealing a polarity in the spermatid nucleus. Inactivation by homologous recombination shows that H1T2 is critical for spermiogenesis as male H1t2(-/-) mice have greatly reduced fertility. Analysis of spermiogenesis in H1t2 mutant mice shows delayed nuclear condensation and aberrant elongation. As a result, mutant spermatids are characterized by the presence of residual cytoplasm, acrosome detachment, and fragmented DNA. Hence, H1T2 is a protein required for proper cell restructuring and DNA condensation during the elongation phase of spermiogenesis.

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death.

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.

NuMA in rat testis--evidence for roles in proliferative activity and meiotic cell division.

NuMA is a well-characterized organizer of the mitotic spindle, which is believed to play a structural role in interphase nucleus. We studied the expression of NuMA in rat seminiferous epithelium in detail. Different stages of the cycle of the seminiferous epithelium were identified using transillumination. Corresponding areas were microdissected and analysed using immunofluorescence, immunohistochemistry, or immunoblotting. NuMA was expressed in Sertoli cells, proliferating type A and B spermatogonia, and early spermatids but it was absent in late spermatids and mature spermatozoa. Interestingly, NuMA-positive primary spermatocytes lost their nuclear NuMA at the beginning of long-lasting prophase of the first meiotic division. A strong expression was again observed at the end of the prophase and finally, a redistribution of NuMA into pole regions of the meiotic spindle was observed in first and second meiotic divisions. In immunoblotting, a single 250-kDa protein present in all stages of the rat seminiferous epithelial cycle was detected. Our results show that NuMA is not essential for the organization of nuclear structure in all cell types and suggest that its presence is more likely connected to the proliferation phase of the cells. They also suggest that NuMA may play an important role in meiotic cell division.

Survivin expression in rat testis is upregulated by stem-cell factor.

The inhibitor of apoptosis protein BIRC-5/survivin plays roles in both apoptosis and the regulation of chromosome-segregation/cytokinesis during mitosis. As the population dynamics of male germ cells are regulated by both proliferation (mitosis and meiosis) and apoptotic culling, we hypothesized that BIRC-5/survivin could be central to the regulation of spermatogenesis. We have analyzed BIRC-5/survivin expression throughout the seminiferous epithelial cycle of the rat. BIRC-5/survivin RNA and protein exhibit rhythms of expression throughout the seminiferous epithelial cycle. The highest levels of expression were found, by immunohistochemistry and in situ hybridization, to occur during the long first meiotic prophase of spermatocytes. Cytoplasmic abundance declined at metaphase and reappeared at anaphase. Some BIRC-5/survivin expression was also found to occur in interstitial Leydig cells. BIRC-5/survivin protein levels were up-regulated in vitro by the paracrine, Stem-Cell Factor, that is known to regulate both proliferation and apoptosis of germ cells and Leydig cells.

Increased apoptosis occurring during the first wave of spermatogenesis is stage-specific and primarily affects midpachytene spermatocytes in the rat testis.

The physiological apoptosis that occurs in immature testis appears to be necessary for the maturation of this tissue. Thus, inhibition of the early apoptotic wave associated with the first round of spermatogenesis is followed by accumulation of spermatogonia and infertility later in life. To identify the cell types undergoing apoptosis in immature rat testis and to characterize the relationship between this apoptosis and progression of the first wave of spermatogenesis, sequential viable segments of seminiferous tubules from 8-, 18-, and 26-day-old rats were examined under a phase-contrast microscope. One novel observation was the existence of pronounced stage-specificity during the peak of apoptosis at the very early postnatal ages of 18 and 26 days. Increased apoptosis of pachytene spermatocytes in stages VII-VIII was the major feature that distinguished immature spermatogenesis from the corresponding adult process. The frequency of apoptosis among type A spermatogonia in immature stages IX-I was also elevated in comparison to the corresponding mature stages. The age-related peak of apoptosis was mediated by caspase 3; furthermore, stage-dependent expression of Bax in midpachytene spermatocytes was observed in the 18- and 26-day-old testis. These observations suggest that this Bax-regulated, caspase 3-mediated, increased apoptosis of midpachytene spermatocytes during the first wave of immature spermatogenesis represents a major difference in comparison to apoptosis occurring in the mature testis, and it may play an important regulatory role in establishing spermatogenesis in the rat testis.

Transcriptional control in male germ cells: general factor TFIIA participates in CREM-dependent gene activation.

Regulation of gene expression in haploid male germ cells follows a number of specific rules that differ from somatic cells. In this physiological context, transcriptional control mediated by the activator CREM (cAMP-responsive element modulator) represents an established paradigm. In somatic cells activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CBP (cAMP response element binding protein-binding protein). In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, ACT (activator of CREM in testis), which confers a powerful, phosphorylation-independent activation capacity. In addition to specialized transcription factors and coactivators, a variety of general factors of the basal transcriptional machinery, and their distinct tissue-specific isoforms, are highly expressed in testis, supporting the general notion that testis-specific gene expression requires specialized mechanisms. Here, we describe that CREM interacts with transcription factor IIA (TFIIA), a general transcription factor that stimulates RNA polymerase II-directed transcription. This association was identified by a two-hybrid screen, using a testis-derived cDNA library, and confirmed by coimmunoprecipitation. The interaction is restricted to the activator isoforms of CREM and does not require Ser117. Importantly, CREM does not interact with TFIIAtau-ALF, a testis-specific TFIIA homolog. CREM and TFIIA are expressed in a spatially and temporally coordinated fashion during the differentiation program of germ cells. The two proteins also colocalize intracellularly in spermatocyte and spermatid cells. These findings contribute to the understanding of the highly specialized rules of transcriptional regulation in haploid germ cells.

Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat: mechanisms of haploid gene product sharing.

Stable cytoplasmic bridges (or ring canals) connecting the clone of spermatids are assumed to facilitate the sharing of haploid gene products and synchronous development of the cells. We have visualized these cytoplasmic bridges under phase-contrast optics and recorded the sharing of cytoplasmic material between the spermatids by a digital time-lapse imaging system ex vivo. A multitude of small (ca. 0.5 microm) granules were seen to move continuously over the bridges, but only 28% of those entering the bridge were actually transported into other cell. The average speed of the granules decreased significantly during the passage. Immunocytochemistry revealed that some of the shared granules contained haploid cell-specific gene product TRA54. We also demonstrate the novel function for the Golgi complex in acrosome system formation by showing that TRA54 is processed in Golgi complex and is transported into acrosome system of neighboring spermatid. In addition, we propose an intercellular transport function for the male germ cell-specific organelle chromatoid body. This mRNA containing organelle, ca. 1.8 microm in diameter, was demonstrated to go over the cytoplasmic bridge from one spermatid to another. Microtubule inhibitors prevented all organelle movements through the bridges and caused a disintegration of the chromatoid body. This is the first direct demonstration of an organelle traffic through cytoplasmic bridges in mammalian spermatogenesis. Golgi-derived haploid gene products are shared between spermatids, and an active involvement of the chromatoid body in intercellular material transport between round spermatids is proposed.