First Wave Of Spermatogenesis Research Articles

BAX and Tumor Suppressor TRP53 Are Important in Regulating Mutagenesis in Spermatogenic Cells in Mice1

During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

TACE/ADAM17 is involved in germ cell apoptosis during rat spermatogenesis

The pathways leading to male germ cell apoptosis in vivo are poorly understood, but are highly relevant for the comprehension of sperm production regulation by the testis. In this work, we show the evidence of a mechanism where germ cell apoptosis is induced through the inactivation and shedding of the extracellular domain of KIT (c-kit) by the protease TACE/a disintegrin and metalloprotease 17 (ADAM17) during the first wave of spermatogenesis in the rat. We show that germ cells undergoing apoptosis lacked the extracellular domain of the KIT receptor. TACE/ADAM17, a membrane-bound metalloprotease, was highly expressed in germ cells undergoing apoptosis as well. On the contrary, cell surface presence of ADAM10, a closely related metalloprotease isoform, was not associated with apoptotic germ cells. Pharmacological inhibition of TACE/ADAM17, but not ADAM10, significantly prevented germ cell apoptosis in the male pubertal rat. Induction of TACE/ADAM17 by the phorbol-ester phorbol 12-myristate 13-acetate (PMA) induced germ cell apoptosis, which was prevented when an inhibitor of TACE/ADAM17 was present in the assay. Ex-vivo rat testis culture showed that PMA induced the cleavage of the KIT extracellular domain. Isolation of apoptotic germ cells showed that even though protein levels of TACE/ADAM17 were higher in apoptotic germ cells than in nonapoptotic cells, the contrary was observed for ADAM10. These results suggest that TACE/ADAM17 is one of the elements triggering physiological germ cell apoptosis during the first wave of spermatogenesis.

Expression of prothymosin alpha in meiotic and post-meiotic germ cells during the first wave of rat spermatogenesis

Prothymosin alpha (PTMA) is a highly acidic small polypeptide, that is, widely distributed and conserved among mammals. Its possible involvement in male gametogenesis has been mentioned but not clarified yet; in particular, it has been suggested that, in non-mammalian vertebrates, it could play a role during GC meiosis and differentiation. In the present work we investigated the possible association between PTMA and meiotic and post-meiotic phases of mammalian spermatogenesis. Three different time points during postnatal development of rat testis were analyzed, that is, 27 dpp (completed meiosis), 35 dpp (occurring spermiogenesis), and 60 dpp (first wave of spermatogenesis definitely ended). RT-PCR and Western blot analyses showed that the expression levels of both Ptma mRNA and corresponding protein decrease in total extracts from 27 to 60 dpp. The in situ hybridization localized the transcript in interstitial Leydig cells, peritubular myoid cells and, inside the tubules, in germ cells from pachytene spermatocytes to newly formed haploid spermatids. The immunohistochemistry analysis localized the protein in the same cell types at 27 dpp, while at 35 and 60 dpp the haploid cells remain the only germ cells that still express it. In particular, PTMA specific localization in the heads of spermatids and epididymal spermatozoa, associated with the acrosome system, supports for the first time the hypothesis of a direct function in male germ cells.

Single administration of di(n-butyl) phthalate delays spermatogenesis in prepubertal rats

Morphological alterations in seminiferous tubules caused by single administration of di(n-butyl) phthalate (DBP) in 3-week-old rats were investigated throughout the first wave of spermatogenesis. Single administration of DBP (500 mg/kg) showed progressive detachment and displacement of spermatogenic cells and disappearance of tubular lumen at 3 h after treatment, and then showed thin seminiferous epithelia and wide tubular lumen at day 1 (D1). At D1, quite significant numbers of apoptotic spermatogenic cells were detected, and then they gradually decreased in accordance with the passage of time. In contrast, the testes revealed lower weight gain, even after completion of first wave of spermatogenesis in the DBP-treated group, compared to the control. In order to clarify whether spermatogenic cells differentiate into mature spermatids in the DBP-treated rats, immunohistochemical staining for Hsc 70t, a specific marker for elongate spermatids, was carried out. As a result, the decrease in mature spermatids in the DBP-treated testes, compared to the control, was demonstrated. For example, at D20 (41-day-old) after treatment, the most advanced spermatids in the tubules from rats in the DBP-treated groups were steps 2–4, while those of the control were steps 12–13. Moreover, in some tubules, pachytene spermatocytes were the most advanced spermatogenic cell. At D30 (51-day-old) after treatment, maturation of spermatogenic cells in the DBP-treated rats proceeded further, and the most advanced spermatids in tubules were steps 8–9, while those of the control were steps 15–19. These results lead us to the postulation that a single administration of DBP to prepubertal rats delays maturation of spermatogenic cells, even after completion of first wave of spermatogenesis.

Male germ cell development: turning on the apoptotic pathways

From the viewpoint of improving germ cell production and treatment of testicular cancers, understanding the control of testicular cell death is of great relevance. One of the prominent features of spermatogenesis is apoptosis of germ cells at different stages of differentiation, by which excess and unfit cells are discarded to maintain proper tissue homeostasis. A phase of heightened apoptosis known as the 'first wave of spermatogenesis' occurs when the gonocytes differentiate into spermatogonia. The germ cells use an extrinsic pathway of apoptosis involving the Fas/FasL molecules as well as the mitochondrial pathway of death using the Bcl-2 family of proteins. A comprehensive view of the involvement of the different pro- and anti-apoptotic molecules has been defined through the use of mutant and knockout mice and toxin-induced cell death models. In addition, hormones such as estrogens in the male are of great interest. The presence of estrogen receptors on germ cells makes these cells susceptible to environmental agents which can mimic estrogens and potentially cause functional impairment of the male gamete. Post-industrialization, an increase in testicular cancers has been recorded and carcinoma of germ cell origin is susceptible to platinum-based compounds that induce multiple apoptotic pathways. This review covers recent progress made on the above issues. The challenge is now to identify the precise signaling pathways and the mechanisms by which germ cells and germ cell tumors initiate cell death processes, and to utilize this information for improving reproductive health related issues.

Differential localization of α’ and β subunits of protein kinase CK2 during rat spermatogenesis

Protein kinase CK2 is a serine/threonine kinase expressed in organisms from yeast to human and is composed of a catalytic subunit (alpha or alpha') and a regulatory subunit (beta) forming a holoenzyme with the possible subunit combinations alpha(2)beta(2), alpha'(2)beta(2), or alphaalpha'beta(2). This kinase has been shown to be involved in embryonic development and gametogenesis. We have studied the expression of the CK2alpha' and CK2beta subunits during the first wave of spermatogenesis and in adult testis in the rat. Western blot analyses have demonstrated that both CK2alpha' and CK2beta are expressed in testes from birth to adulthood. A more detailed study of the protein localization of CK2alpha' and CK2beta by immunohistochemistry suggests that CK2alpha' and CK2beta are localized in the nuclei of Sertoli cells in 5-day-old rats, whereas they appear to have a cytoplasmic localization in older animals. In adult testes, CK2alpha' and CK2beta subunits are present in spermatocytes. Both subunits exhibit a similar expression pattern with the highest level in spermatocytes at stages VIII-XIV. Interestingly, CK2beta is highly expressed in spermatogonia, whereas CK2alpha' is barely detectable. Mature epididymal spermatozoa express CK2alpha' in the acrosome and CK2beta in the flagellum. This new evidence therefore indicates that protein kinase CK2 has a possible role at various stages during mammalian spermatogenesis, a process that involves proliferation, meiosis, apoptosis, and differentiation. CK2 might thus emerge as a new pivotal control enzyme at various levels in mammalian spermatogenesis.

Inactivation of Insulin-Like Factor 6 Disrupts the Progression of Spermatogenesis at Late Meiotic Prophase

Insulin-like factor 6 (INSL6), a member of the insulin-like superfamily, is predominantly expressed in male germ cells. Expression of the Insl6 is first detected in mouse testis at postnatal d 15 when the first wave of spermatogenesis progresses to pachytene spermatocytes. To elucidate the role of INSL6 in germ cell development, we generated Insl6-deficient mice. The majority of the Insl6-deficient males on a hybrid genetic background exhibited impaired fertility, whereas females were fertile. The number of mature sperm and sperm motility were drastically reduced in the epididymis. The reduced sperm count could be due to apoptotic death of a significant number of developing germ cells. Analysis of germ cell development during the juvenile life showed an arrest of the first wave of spermatogenesis in late meiotic prophase. RNA analysis revealed a significant decrease in expression of late meiotic- and postmeiotic-specific marker genes, whereas expression of early meiotic-specific genes remains unaffected in the Insl6(-/-) testes. These results demonstrate that INSL6 is required for the progression of spermatogenesis.

Disruption of blood-testis barrier dynamics in ether-lipid-deficient mice

One of the major roles of Sertoli cells is to establish the blood-testis (Sertoli cell) barrier (BTB), which is permanently assembled and disassembled to accommodate the translocation of leptotene spermatocytes from the basal into the adluminal compartment of the seminiferous epithelium and to guarantee completion of meiosis and spermiogenesis. Recently, we have demonstrated spermatogenesis to be arrested before spermatid elongation in Gnpat-null mice with selective deficiency of ether lipids (ELs) whose functions are poorly understood. In this study, we have focused on the spatio-temporal expression of several BTB tight-junctional proteins in the first wave of spermatogenesis to obtain insights into the physiological role of ELs during BTB establishment and dynamics. Our data confirm the transient existence of Russell's intermediate or translocation compartment delineated by two separate claudin-3-positive luminal and basal tight junctions and reveal that EL deficiency blocks BTB remodeling. This block is associated with (1) downregulation and mistargeting of claudin-3 and (2) impaired BTB disassembly resulting in deficient sealing of the intermediate compartment as shown by increased BTB permeability to biotin. These results suggest that ELs are essential for cyclic BTB dynamics ensuring the sluice mechanism for leptotene translocation into the adluminal compartment.

Dynamics of INSL3 Peptide Expression in the Rodent Testis1

The Leydig cell-specific factor insulin-like peptide 3 (INSL3) is involved in testicular descent during embryo development, and has been suggested to regulate spermatogenesis and bone metabolism in the adult. Using a new, sensitive assay specific for rodent INSL3, we have mapped the secretion of INSL3 into peripheral blood in mice and during postnatal male rat development (in female rats, circulating INSL3 is at the level of detection). Maximum INSL3 is measured at Postnatal Day (PD) 40 in the rat and decreases to a significantly lower, stable value by PD60, indicating an "overshoot" effect in the establishment of Leydig cell functionality during the first wave of spermatogenesis. Aging rats ( approximately 24 mo) have markedly reduced circulating INSL3 levels, as do humans. Treatment of young adult rats with ethane dimethylsulfonate (EDS) leads to loss of mature Leydig cells and no detectable INSL3 in peripheral blood. INSL3 can be detected first at Day 27 after EDS treatment, returning to near normal levels by Day 37. Both primary rat Leydig cells and the mouse MA-10 tumor cell line secrete substantial amounts of INSL3 into the culture media in a constitutive manner, unregulated by common effectors, including hCG. Analysis of different testicular fluid compartments shows highest INSL3 concentration in the interstitial fluid (391.4 +/- 47.8 ng/ml). However, INSL3 evidently traverses the blood-testis barrier to enter the seminiferous compartment, rete testis, and epididymis in sufficient concentration to be able to address the specific INSL3 receptors (RXFP2) on post-meiotic germ cells and in the epididymis.

Loss of Etv5 Decreases Proliferation and RET Levels in Neonatal Mouse Testicular Germ Cells and Causes an Abnormal First Wave of Spermatogenesis1

Mice that are ets variant gene 5 (ETV5) null (Etv5(-/-)) undergo the first wave of spermatogenesis but lose all spermatogonial stem cells (SSCs) during this time. The SSC loss in Etv5(-/-) mice begins during the neonatal period, suggesting a role for ETV5 in SSC self-renewal during this period. Herein, we show that Etv5 mRNA was present in perinatal mouse testis and that ETV5 was expressed in fetal Sertoli cells and by germ cells and Sertoli cells during the neonatal period. Transplantation of Etv5(-/-) germ cells failed to establish spermatogenesis in W/W(v) mice testes, indicating that germ cell ETV5 has a key role in establishment or self-renewal of transplanted SSCs. The SSC self-renewal is stimulated by glial cell-derived neurotrophic factor (GDNF) acting through the RET/GDNF family receptor alpha 1 (GFRA1) receptor complex in SSCs. Immunohistochemistry, quantitative PCR, and laser capture microdissection revealed decreased RET mRNA and protein expression in spermatogonia of neonatal Etv5(-/-) mice by Postnatal Days 4-8, indicating that disrupted GDNF/RET/GFRA1 signaling may occur before initial spermatogonial stem/progenitor cell decrease. Etv5(-/-) spermatogonia had reduced proliferation in vivo and in vitro. Decreased cell proliferation may cause the observed decreases in the number of type A spermatogonia (Postnatal Day 17) and daily sperm production (Postnatal Day 30) in Etv5(-/-) mice, indicating quantitative impairments in the first wave of spermatogenesis. In conclusion, ETV5 is expressed beginning in fetal Sertoli cells and can potentially have effects on neonatal Sertoli cells and germ cells. In addition, ETV5 has critical effects on neonatal spermatogonial proliferation, which may involve impaired signaling through the RET receptor.

Downstream Genes of Sox8 That Would Affect Adult Male Fertility

Sertoli cells provide nutritional and physical support to germ cells during spermatogenesis. Sox8 encodes a high mobility group transcription factor closely related to Sox9 and Sox10. Sertoli cells produceSOX8 protein, and its elimination results in an age-dependent deregulation of spermatogenesis resulting in male infertility. This suggests that Sox8 is a critical regulator of Sertoli cell function for the maintenance of male fertility beyond the first wave of spermatogenesis. To better understand the roles of Sox8 in testicular development and maintenance of male fertility, we have performed a detailed analysis to explore its downstream genes. We have used mRNA expression profiling to identify affected genes in Sertoli cells in the mutant testes of 2-month-old mice. Expression profiling of the heterozygous and homozygous Sox8 mutant testes indicates alterations in several important spermatogenesis and blood-testis barrier genes, providing insight into the molecular basis of the defects in Sox8^–/– testes beyond the first wave of spermatogenesis.

Exposure of the mouse perinatal testis to radiation leads to hypospermia at sexual maturity

The first round of mouse spermatogenesis begins from 3 to 4 days after birth through differentiation of gonocytes into spermatogonial-stem cells and type A spermatogonia. Consequently, this step of differentiation may determine generation of the original population of stem cells and the fertility potential of the adult mouse. We aimed to determine the effect of perinatal exposure to ionizing radiation on the testis at the end of the first wave of spermatogenesis and at sexual maturity. Our results show that, radiation sensitivity of the testis substantially decreases from late foetal life to the end of the first week after birth. In addition, partial or full recovery from radiation induced testicular weight loss occurred between the first round of spermatogenesis and sexual maturity, and this was associated with the stimulation of spermatogonial proliferation. Exposure of mice at 17.5 days after conception or at 1 day after birth to gamma-rays decreased the sperm counts at sexual maturity, while exposure of 8 day-old mice had no effect. This suggests that irradiation of late foetal or early neonatal testes has a direct impact on the generation of the neonatal spermatogonial-stem cell pool.

Age- and substrain-dependent sperm abnormalities in BALB/c mice and functional assessment of abnormal sperm by ICSI

Male BALB/c mice produce a high proportion of morphologically abnormal sperm. Although the BALB/c male may be a useful model of human male infertility, it remains unclear whether the sperm abnormality rate (SAR) is affected by age or the BALB/c substrain. SARs (head shape) were assessed in three BALB/c substrains (A, AnN, ByJ) at 7 and 9 weeks and 6-10 months of age (c.100 sperm/male). The functional ability of abnormal sperm produced from 7-week-old and 6-10-month-old males was determined in BALB/c AnN mice by ICSI. The SAR (quasi-normal plus abnormal sperm) was lower in BALB/c A than in the other two strains (P < 0.05). Further, the SARs of BALB/c AnN and ByJ strains at 7 weeks old were high and decreased rapidly by 9 weeks, suggesting that early spermatogenesis (i.e. the first wave of spermatogenesis) produced low-quality sperm. ICSI experiments indicated that 2-cell stage embryos which developed from morphologically abnormal sperm from both the first wave of spermatogenesis and the older mice (6-10 months) were similar to those from morphologically normal sperm in terms of producing progeny, although the number of 2-cell embryos was slightly lower (P < 0.05, chi(2) test) than with normal sperm. Although the SARs of BALB/c mice are affected by both age and substrain, the embryos that developed from morphologically abnormal sperm have normal genetic potential in terms of production of progeny.

TNF Alpha-Mediated Disruption of Spermatogenesis in Response to Sertoli Cell Injury in Rodents Is Partially Regulated by MMP21

Mono-(2-ethylhexyl) phthalate (MEHP)-induced Sertoli cell injury in peripubertal rodents results in the stimulation of germ cell apoptosis through an interaction of FAS/FASL between these two cell types. During this peripubertal period, an early spike in the incidence of germ cell apoptosis occurs during the first wave of spermatogenesis and is essential for the development of functional spermatogenesis in adults. Our previous observations revealed that soluble tumor necrosis factor alpha (sTNFA) released by germ cells after MEHP exposure consequently resulted in a robust induction of FASL by Sertoli cells. Metalloproteinases (MPs) are essential for processing the TNFA precursor to its soluble form and its ability to bind to TNFRSF1A. The activity of MPs is regulated by the tissue inhibitors of MPs (TIMPs) family. Herein we report that TIMP2 is predominately expressed in Sertoli cells and that protein levels decrease in a time-dependent manner after MEHP exposure. The secretion of matrix MP 2 (MMP2) in primary rat Sertoli cell-germ cell cocultures is induced after MEHP exposure, and its activity increases in a time-dependent manner. The addition of SB-3CT, a specific gelatinase inhibitor, decreases the activity of MMP2 and significantly reduces MEHP-enhanced sTNFA production in primary cocultures. In vivo challenges with SB-3CT decrease sTNFA and reduce MEHP-induced testicular germ cell apoptosis. In primary cocultures, MEHP exposure causes a 9.46-fold increase in sTNFA, while the addition of recombinant MMP2 protein results in a 5.4-fold increase in sTNFA, suggesting that MEHP-induced MMP2 is in part responsible for the activation of TNFA in the testis. Taken together, these observations indicate the distinct role of specific MPs in response to toxicant-induced Sertoli cell injury, providing further insights into the mechanism by which Sertoli cells control the sensitivity of germ cells to undergo apoptosis.

Functional in vitro model to examine cancer therapy cytotoxicity in maturing rat testis

Childhood cancer treatment can lead to infertility. Organ culture of early postnatal testicular tissue might provide a valuable approach to the study of acute testicular toxicity. The aim of the present study was to develop a functional in vitro organ culture method, in order to identify sensitive target cells to doxorubicin-induced cytotoxicity in immature rat testis during germ cell migration prior initiation of the first wave of spermatogenesis. Testicular tissue fragments from 5-day-old Sprague–Dawley rats were cultured in the absence or presence of doxorubicin (40 and 100 ng/ml) and morphology, apoptosis, proliferation and testosterone secretion was analyzed. Postnatal testicular development proceeded normally in control samples for 48 h in vitro. In these untreated culture conditions germ and Sertoli cell numbers and germ cell migration were comparable to in vivo. Germ cells were the primary, most sensitive targets for in vitro-induced doxorubicin (100 ng/ml) toxicity and their death was not associated with any morphological defects in the Sertoli cells. Organ culture which reduces the need of animal experimentation can be used to study the cytotoxic effects of doxorubicin on the immature testis.

Expression of antizyme inhibitor 2 in male haploid germinal cells suggests a role in spermiogenesis

Recently, we have found that the antizyme inhibitor 2, a novel member of the antizyme binding proteins related to polyamine metabolism, was expressed mainly in the adult testes, although its function in testicular physiology is completely unknown. Therefore, in the present work, the spatial and temporal expression of antizyme inhibitor 2, and other genes related to polyamine metabolism were studied in the mouse testis, in an attempt to understand the role of antizyme inhibitor 2 in testicular functions. For that purpose, the temporal expression of different genes, during the first wave of spermatogenesis in postnatal mice, was studied by real-time RT-PCR, and the spatial distribution of transcripts and protein in the adult testis was examined by both RNA in situ hybridization and immunocytochemistry. The results indicated that antizyme inhibitor 2 was specifically expressed in the haploid germinal cells, similarly to antizyme 3, the testis specific antizyme. Conversely, ornithine decarboxylase mRNA was mainly found in the outer part of the seminiferous tubules where spermatogonia and spermatocytes are located. Functional transfection assays and co-immunoprecipitation experiments corroborated that antizyme inhibitor 2 counteracts the negative action of antizyme 3 on polyamine biosynthesis and uptake. All these results indicate that the expression of antizyme inhibitor 2 is postnatally regulated and strongly suggest that antizyme inhibitor 2 may have a role in spermiogenesis.

Leydig Cells Express Follicle-Stimulating Hormone Receptors in African Catfish

This report aimed to establish, using African catfish, Clarias gariepinus, as model species, a basis for understanding a well-known, although not yet clarified, feature of male fish reproductive physiology: the strong steroidogenic activity of FSHs. Assays with gonadotropin receptor-expressing cell lines showed that FSH activated its cognate receptor (FSHR) with an at least 1000-fold lower EC50 than when challenging the LH receptor (LHR), whereas LH stimulated both receptors with similar EC50s. In androgen release bioassays, FSH elicited a significant response at lower concentrations than those required to cross-activate of the LHR, indicating that FSH stimulated steroid release via FSHR-dependent mechanisms. LHR/FSHR-mediated stimulation of androgen release was completely abolished by H-89, a specific protein kinase A inhibitor, pointing to the cAMP/protein kinase A pathway as the main route for both LH- and FSH-stimulated steroid release. Localization studies showed that intratubular Sertoli cells express FSHR mRNA, whereas, as reported for the first time in a vertebrate, catfish Leydig cells express both LHR and FSHR mRNA. Testicular FSHR and LHR mRNA expression increased gradually during pubertal development. FSHR, but not LHR, transcript levels continued to rise between completion of the first wave of spermatogenesis at about 7 months and full maturity at about 12 months of age, which was associated with a previously recorded approximately 3-fold increase in the steroid production capacity per unit testis weight. Taken together, our data strongly suggest that the steroidogenic potency of FSH can be explained by its direct trophic action on FSHR-expressing Leydig cells.

Computational identification of transcription frameworks of early committed spermatogenic cells

It is known that transcription factors (TFs) work in cooperation with each other to govern gene expression and thus single TF studies may not always reflect the underlying biology. Using microarray data obtained from two independent studies of the first wave of spermatogenesis, we tested the hypothesis that co-expressed spermatogenic genes in cells committed to differentiation are regulated by a set of distinct combinations of TF modules. A computational approach was designed to identify over-represented module combinations in the promoter regions of genes associated with transcripts that either increase or decrease in abundance between the first two major spermatogenic cell types: spermatogonia and spermatocytes. We identified five TFs constituting four module combinations that were correlated with expression and repression of similarly regulated genes. These modules were biologically assessed in the context that they represent the key transcriptional mediators in the developmental transition from the spermatogonia to spermatocyte.

A Novel Mouse Enolase 1-like Gene is Expressed Specifically in Spermatogenic Cells and Encodes a Protein Present in the Principal Piece Region of the Sperm Flagellum.

The main source of ATP for sperm motility in the mouse is glycolysis and several spermatogenic cell-specific glycolytic enzymes are confined to the principal piece region of the flagellum. During a proteomics study using MALDI/MS to analyze non-ionic detergent-soluble sperm proteins, peptide sequences similar to those present in the glycolytic enzyme enolase 1 (alpha enolase) were identified. Other investigators reported that an enolase sperm-specific isozyme (ENOS) is present in the flagellum of spermatozoa of several mammalian species that is unlike other enolase family members (ENO1, ENO2 and ENO3) in electrophoretic mobility and enzymatic properties. However, the peptide and nucleotide sequences of this novel enolase have not been reported. Database mining identified multiple Eno1-like sequences in the mouse genome, but EST expression profiles and deduced protein sequence analyses strongly suggested that a gene located on chromosome 19 is the Enos gene. The use of PCR and 5 RACE determined that there are two Enos transcript variants expressed in mouse testis (Enos_v1 and Enos_v2), each with a 5 sequence different from those present in transcripts of Eno1 or of other enolase family members. Northern analysis using a probe from the 3′ region of the Enos transcript recognized mRNA only from testis. Northern analysis of RNA from testes of 10- to 35-day-old juvenile mice first detected Enos transcripts on day 14, corresponding to when pachytene spermatocytes appear in the first wave of spermatogenesis. In immunoblotting assays, a pan-enolase antibody reacted with a protein in 1 % Triton X-100, 1 % NP-40 and 0.1 % SDS lysates of testes and sperm, while enolase 2 or 3 were not detected in lysates of testis and sperm with antibodies specific to these enzymes. Immunofluorescence microscopy with this antibody recognized a protein localized to the principal piece region of sperm flagellum, strongly suggesting that the protein recognized by the pan-enolase antibody is ENOS. Extraction of sperm with NP-40 and then 6M urea and immunoblotting assays determined that ENOS is partially extracted by NP-40 and the remainder is extracted by 6M urea. These results suggest that ENOS is not an integral protein of the fibrous sheath like GAPDHS, AKAP4 and GSTM5, but is loosely associated like HK1S and PFKM. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

Pathways involved in testicular germ cell apoptosis in immature rats after FSH suppression

FSH is a key regulator of testis function, required for the establishment of full complements of Sertoli and germ cells during postnatal testis development and for the maintenance of spermatogenesis in the adult. FSH plays an important role in germ cell survival rather than proliferation, in the window between 14 and 18 days of testicular development, which coincides with the cessation of Sertoli cell proliferation and the onset of germ cell meiosis during the first wave of spermatogenesis. This study aimed to identify the pathway(s) of apoptosis regulated by changes in FSH levels in 14 - to 18-day-old rats, using a model of in vivo FSH suppression by passive immunoneutralization with a rat anti-FSH antibody. Apoptotic pathways were identified by immunohistochemistry using pathway-specific proteins as markers of the intrinsic (activated caspase 9) and extrinsic (activated caspase 8) pathways, followed by quantification of cell numbers using stereological techniques. In addition, RT-PCR was used to assess the expression of pathway-specific genes. We previously reported a 2.5-fold increase in spermatogonial apoptosis in these samples after 4 days of FSH suppression, and now show that this increase correlates with a 9.8-fold (P<0.001) increase in the frequency of caspase 9-positive spermatogonia in the absence of caspase 8 immunoreactivity. By contrast, spermatocytes exhibited both increased caspase 9 (7.5-fold; P<0.001) and caspase 8 (5.7 fold; P<0.001) immunoreactivities after 4 days of FSH suppression. No significant change in the transcription levels of candidate genes required for either pathway was detected. This study demonstrates that, in the seminiferous tubules, FSH suppression induces spermatogonial apoptosis predominantly via the intrinsic pathway, while spermatocyte apoptosis occurs via both the intrinsic and extrinsic pathways.