The role of miR-128-3p through MAPK14 activation in the apoptosis of GC2 spermatocyte cell line following heat stress.

BACKGROUND: MicroRNAs play a crucial role in the regulation of spermatogenesis. For example, miR-128-3p expression is known to decrease significantly after testicular hyperthermia, but the regulatory effect of this change on the spermatogenesis damage caused by heat stress remains unclear. OBJECTIVES: This study aimed to verify whether the target gene of miR-128-3p is MAPK14, which affects spermatogenic cell proliferation and apoptosis under testicular hyperthermia. MATERIALS AND METHODS: Mouse testis and GC2 spermatocyte cell line heat stress models were established. miR-128-3p expression before and after heat stress was analyzed by reverse transcription polymerase chain reaction. MAPK14 and p-MAPK14 expression was detected by Western blot, and cell apoptosis was analyzed by Annexin V-FITC/PI. Subsequently, miR-128-3p inhibitors and mimics were used to interfere with spermatocytes before and after heat stress, respectively, for correlation detection. RESULTS: Compared with the control group, the heat stress group showed decreased miR-128-3p expression, increased p-MAPK14 expression, and decreased cell proliferation activity. In the GC2-spd cell line in vitro, miR-128-3p inhibitors were found to upregulate p-MAPK14 expression, reduce cell proliferation activity, and increase apoptosis, consistent with the results obtained in the heat treatment alone. Furthermore, miR-128-3p mimics transfected in the GC2 cells after heat stress reduced p-MAPK14 expression, alleviated the decrease in cell proliferation, and decreased the apoptosis level. CONCLUSIONS: The downregulation of miR-128-3p expression plays an important role in spermatogenesis damages after testicular hyperthermia, which is probably attributable to the activation of the MAPK signaling pathway. Downregulated miR-128-3p expression induces the apoptosis and inhibits the proliferation of spermatogenic cells by promoting MAPK14 phosphorylation.

Elevated Temperatures Cause Transposon-Associated DNA Damage in C. elegans Spermatocytes.

Sexually reproducing organisms use meiosis to generate haploid gametes and faithfully transmit their genome to the next generation. In comparison to oogenesis in many organisms, spermatogenesis is particularly sensitive to small temperature fluctuations, and spermatocytes must develop within a very narrow isotherm [1-4]. Although failure to thermoregulate spermatogenetic tissue and prolonged exposure to elevated temperatures are linked to male infertility in several organisms, the mechanisms of temperature-induced male infertility have not been fully elucidated [5]. Here, we show that upon exposure to a brief 2°C temperature increase, Caenorhabditis elegans spermatocytes exhibit up to a 25-fold increase in double-strand DNA breaks (DSBs) throughout meiotic prophase I and a concurrent reduction in male fertility. We demonstrate that these heat-induced DSBs in spermatocytes are independent of the endonuclease SPO-11. Further, we find that the production of these heat-induced DSBs in spermatocytes correlate with heat-induced mobilization of Tc1/mariner transposable elements, which are known to cause DSBs and alter genome integrity [6, 7]. Moreover, we define the specific sequences and regions of the male genome that preferentially experience these heat-induced de novo Tc1 insertions. In contrast, oocytes do not exhibit changes in DSB formation or Tc1 transposon mobility upon temperature increases. Taken together, our data suggest spermatocytes are less tolerant of higher temperatures because of an inability to effectively repress the movement of specific mobile DNA elements that cause excessive DNA damage and genome alterations, which can impair fertility.

Dual histone methyl reader ZCWPW1 facilitates repair of meiotic double strand breaks in male mice.

Meiotic crossovers result from homology-directed repair of DNA double-strand breaks (DSBs). Unlike yeast and plants, where DSBs are generated near gene promoters, in many vertebrates DSBs are enriched at hotspots determined by the DNA binding activity of the rapidly evolving zinc finger array of PRDM9 (PR domain zinc finger protein 9). PRDM9 subsequently catalyzes tri-methylation of lysine 4 and lysine 36 of Histone H3 in nearby nucleosomes. Here, we identify the dual histone methylation reader ZCWPW1, which is tightly co-expressed during spermatogenesis with Prdm9, as an essential meiotic recombination factor required for efficient repair of PRDM9-dependent DSBs and for pairing of homologous chromosomes in male mice. In sum, our results indicate that the evolution of a dual histone methylation writer/reader (PRDM9/ZCWPW1) system in vertebrates remodeled genetic recombination hotspot selection from an ancestral static pattern near genes towards a flexible pattern controlled by the rapidly evolving DNA binding activity of PRDM9.

The histone modification reader ZCWPW1 links histone methylation to PRDM9-induced double-strand break repair.

The histone modification writer Prdm9 has been shown to deposit H3K4me3 and H3K36me3 at future double-strand break (DSB) sites during the very early stages of meiosis, but the reader of these marks remains unclear. Here, we demonstrate that Zcwpw1 is an H3K4me3 reader that is required for DSB repair and synapsis in mouse testes. We generated H3K4me3 reader-dead Zcwpw1 mutant mice and found that their spermatocytes were arrested at the pachytene-like stage, which phenocopies the Zcwpw1 knock-out mice. Based on various ChIP-seq and immunofluorescence analyses using several mutants, we found that Zcwpw1's occupancy on chromatin is strongly promoted by the histone-modification activity of PRDM9. Zcwpw1 localizes to DMC1-labelled hotspots in a largely Prdm9-dependent manner, where it facilitates completion of synapsis by mediating the DSB repair process. In sum, our study demonstrates the function of ZCWPW1 that acts as part of the selection system for epigenetics-based recombination hotspots in mammals.

PHB regulates meiotic recombination via JAK2-mediated histone modifications in spermatogenesis.

Previously, we have shown that human sperm Prohibitin (PHB) expression is significantly negatively correlated with mitochondrial ROS levels but positively correlated with mitochondrial membrane potential and motility. However, the possible role of PHB in mammalian spermatogenesis has not been investigated. Here we document the presence of PHB in spermatocytes and its functional roles in meiosis by generating the first male germ cell-specific Phb-cKO mouse. Loss of PHB in spermatocytes resulted in complete male infertility, associated with not only meiotic pachytene arrest with accompanying apoptosis, but also apoptosis resulting from mitochondrial morphology and function impairment. Our mechanistic studies show that PHB in spermatocytes regulates the expression of STAG3, a key component of the meiotic cohesin complex, via a non-canonical JAK/STAT pathway, and consequently promotes meiotic DSB repair and homologous recombination. Furthermore, the PHB/JAK2 axis was found as a novel mechanism in the maintenance of stabilization of meiotic STAG3 cohesin complex and the modulation of heterochromatin formation in spermatocytes during meiosis. The observed JAK2-mediated epigenetic changes in histone modifications, reflected in a reduction of histone 3 tyrosine 41 phosphorylation (H3Y41ph) and a retention of H3K9me3 at the Stag3 locus, could be responsible for Stag3 dysregulation in spermatocytes with the loss of PHB.

UHRF1-repressed 5'-hydroxymethylcytosine is essential for the male meiotic prophase I.

5'-hydroxymethylcytosine (5hmC), an important 5'-cytosine modification, is altered highly in order in male meiotic prophase. However, the regulatory mechanism of this dynamic change and the function of 5hmC in meiosis remain largely unknown. Using a knockout mouse model, we showed that UHRF1 regulated male meiosis. UHRF1 deficiency led to failure of meiosis and male infertility. Mechanistically, the deficiency of UHRF1 altered significantly the meiotic gene profile of spermatocytes. Uhrf1 knockout induced an increase of the global 5hmC level. The enrichment of hyper-5hmC at transcriptional start sites (TSSs) was highly associated with gene downregulation. In addition, the elevated level of the TET1 enzyme might have contributed to the higher 5hmC level in the Uhrf1 knockout spermatocytes. Finally, we reported Uhrf1, a key gene in male meiosis, repressed hyper-5hmC by downregulating TET1. Furthermore, UHRF1 facilitated RNA polymerase II (RNA-pol2) loading to promote gene transcription. Thus our study demonstrated a potential regulatory mechanism of 5hmC dynamic change and its involvement in epigenetic regulation in male meiosis.

Immunolocalization of antioxidant enzymes in testis of rams submitted to long-term heat stress.

Long-term heat stress (HS) induced by testicular insulation generates oxidative stress (OS) on the testicular environment; consequently activating antioxidant enzymes such as superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPx). The aim of this work was to immunolocalize antioxidant enzymes present in different cells within the seminiferous tubule when rams were submitted to HS. Rams were divided into control (n = 6) and treated group (n = 6), comprising rams subjected to testicular insulation for 240 h. After the testicular insulation period, rams were subjected to orchiectomy. Testicular fragments were submitted to immunohistochemistry for staining against SOD, GR and GPx enzymes. We observed immunolocalization of GPx in more cell types of the testis after HS and when compared with other enzymes. In conclusion, GPx is the main antioxidant enzyme identified in testicular cells in an attempt to maintain oxidative balance when HS occurs.

Meiosis I progression in spermatogenesis requires a type of testis-specific 20S core proteasome.

Spermatogenesis is tightly regulated by ubiquitination and proteasomal degradation, especially during spermiogenesis, in which histones are replaced by protamine. However, the functions of proteasomal activity in meiosis I and II remain elusive. Here, we show that PSMA8-associated proteasomes are essential for the degradation of meiotic proteins and the progression of meiosis I during spermatogenesis. PSMA8 is expressed in spermatocytes from the pachytene stage, and assembles a type of testis-specific core proteasome. Deletion of PSMA8 decreases the abundance of proteasome in testes. Meiotic proteins that are normally degraded at late prophase I, such as RAD51 and RPA1, remain stable in PSMA8-deleted spermatocytes. Moreover, PSMA8-null spermatocytes exhibit delayed M-phase entry and are finally arrested at this stage, resulting in male infertility. However, PSMA8 is neither expressed nor required for female meiotic progression. Thus, meiosis I progression in spermatogenesis, particularly entry into and exit from M-phase, requires the proteasomal activity of PSMA8-associated proteasomes.

Characterization and expression profiles of cyp19a1a in the schizothoracine fish Schizothorax prenanti.

Aromatase plays a central role in ovarian differentiation and development in teleosts. In the present study, we identified a cyp19a1a homologue from the ovary of Schizothorax prenanti and analysed its expression at both the mRNA and protein levels. Cyp19a1a of S. prenanti showed high homology with that of other teleosts, especially S. kozlovi. The ovary and testis were the main sites of cyp19a1a expression in S. prenanti, and cyp19a1a transcript levels peaked in the mid-vitellogenic (MVG)-stage ovary and the mid-spermatogenic (MS)-stage testis. Signals of Cyp19a1a immunopositivity were detected in the spermatocytes and follicular cells of cortical alveolar-stage and MVG oocytes but not in spermatogonia or spermatids. Taken together, these findings indicate that Cyp19a1a may play an important role in oocyte vitellogenesis as well as spermatocyte development in S. prenanti.

Novel localization of Aurora A kinase in mouse testis suggests multiple roles in spermatogenesis.

Male germ cells are transformed from undifferentiated stem cells into spermatozoa through a series of highly regulated steps together termed spermatogenesis. Spermatogonial stem cells undergo mitosis and differentiation followed by two rounds of meiotic division and then proceed through a series of dramatic cell shape changes to form highly differentiated spermatozoa. Using indirect immunofluorescence, we investigated a role for the mitotic kinase, Aurora A (AURKA), in these events through localization of this protein in mouse testis and spermatozoa. AURKA is expressed in several cell types in the testis. Spermatogonia and spermatocytes express AURKA as expected based on the known role of this kinase in cell division. Surprisingly, we also found AURKA localized to spermatids and the flagellum of spermatozoa. Total AURKA and activated AURKA are expressed in different compartments of the sperm flagellum with total AURKA found in the principal piece and its phosphorylated and activated form found in the sperm midpiece. In addition, active AURKA is enriched in the flagellum of motile sperm isolated from cauda epididymis. These results provide evidence for a unique role for AURKA in spermatogenesis and sperm motility. Defining the signaling mechanisms that govern spermatogenesis and sperm cell function is crucial to understanding and treating male infertility as well as for development of new contraceptive strategies.

Stk33 is required for spermatid differentiation and male fertility in mice.

Spermiogenesis is the final phase during sperm cell development in which round spermatids undergo dramatic morphological changes to generate spermatozoa. Here we report that the serine/threonine kinase Stk33 is essential for the differentiation of round spermatids into functional sperm cells and male fertility. Constitutive Stk33 deletion in mice results in severely malformed and immotile spermatozoa that are particularly characterized by disordered structural tail elements. Stk33 expression first appears in primary spermatocytes, and targeted deletion of Stk33 in these cells recapitulates the defects observed in constitutive knockout mice, confirming a germ cell-intrinsic function. Stk33 protein resides in the cytoplasm and partially co-localizes with the caudal end of the manchette, a transient structure that guides tail elongation, in elongating spermatids, and loss of Stk33 leads to the appearance of a tight, straight and elongated manchette. Together, these results identify Stk33 as an essential regulator of spermatid differentiation and male fertility.

Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa.

In mammals, adenosine 3', 5'-cyclic monophosphate (cAMP) is known to play highly important roles in sperm motility and acrosomal exocytosis. It is known to act through protein phosphorylation via PRKA and through the activation of guanine nucleotide exchange factors like EPAC. Sperm intracellular cAMP levels depend on the activity of adenylyl cyclases, mostly SACY, though transmembrane-containing adenylyl cyclases are also present, and on the activity of cyclic nucleotide phosphodiesterases (PDE) whose role is to degrade cAMP into 5'-AMP. The PDE superfamily is subdivided into 11 families (PDE1 to 11), which act on either cAMP or cGMP, or on both cAMP and cGMP although with different enzymatic properties. PDE10, which is more effective on cAMP than cGMP, has been known for almost 15 years and is mostly studied in the brain where it is associated with neurological disorders. Although a high level of PDE10A gene expression is observed in the testis, information on the identity of the isoforms or on the cell type that express the PDE10 protein is lacking. The objective of this study was to identify the PDE10A isoforms expressed in the testis and germ cells, and to determine the presence and localization of PDE10A in mature spermatozoa. As a sub-objective, since PDE10A transcript variants were reported strictly through analyses of bovine genomic sequence, we also wanted to determine the nucleotide and amino acid sequences by experimental evidence. Using RT-PCR, 5'- and 3'-RACE approaches we clearly show that PDE10A transcript variants X3 and X5 are expressed in bovine testis as well as in primary spermatocytes and spermatids. We also reveal using a combination of immunological techniques and proteomics analytical tools that the PDE10A isoform X4 is present in the area of the developing acrosome of spermatids and of the acrosome of mature spermatozoa.

Effect of 50 Hz Extremely Low-Frequency Electromagnetic Fields on the DNA Methylation and DNA Methyltransferases in Mouse Spermatocyte-Derived Cell Line GC-2.

Previous studies have shown that the male reproductive system is one of the most sensitive organs to electromagnetic radiation. However, the biological effects and molecular mechanism are largely unclear. Our study was designed to elucidate the epigenetic effects of 50 Hz ELF-EMF in vitro. Mouse spermatocyte-derived GC-2 cell line was exposed to 50 Hz ELF-EMF (5 min on and 10 min off) at magnetic field intensity of 1 mT, 2 mT, and 3 mT with an intermittent exposure for 72 h. We found that 50 Hz ELF-EMF exposure decreased genome-wide methylation at 1 mT, but global methylation was higher at 3 mT compared with the controls. The expression of DNMT1 and DNMT3b was decreased at 1 mT, and 50 Hz ELF-EMF can increase the expression of DNMT1 and DNMT3b of GC-2 cells at 3 mT. However, 50 Hz ELF-EMF had little influence on the expression of DNMT3a. Then, we established DNA methylation and gene expression profiling and validated some genes with aberrant DNA methylation and expression at different intensity of 50 Hz ELF-EMF. These results suggest that the alterations of genome-wide methylation and DNMTs expression may play an important role in the biological effects of 50 Hz ELF-EMF exposure.

Chronic intermittent stress-induced alterations in the spermatogenesis and antioxidant status of the testis are irreversible in albino rat.

The study was undertaken to find out whether or not chronic stress-induced alterations in spermatogenesis are accompanied by oxidative damage in the testis and reversibility of these effects. Adult male rats (n = 10) were subjected to restraint for 1 h and later after a gap of 4 h to forced swimming exercise for 15 min daily for 60 days and controls (n = 5) were maintained without disturbance. After treatment period, controls and 5 rats in stress group were killed and remaining rats in stress group were maintained without any treatment for 4 months and then autopsied to find out whether effects are reversible or not. The body and testicular weight, total sperm count, and mean number of type A spermatogonia, mid-pachytene spermatocytes, stage 7 spermatids, and elongated spermatids (cellular association in stage VII of spermatogenesis) showed a significant decrease whereas the abnormal sperm count and germ cell apoptosis were increased in stressed and recovery group rats compared to controls. Activities of testicular SOD, CAT, GPx, and GST were significantly decreased whereas MDA levels were significantly increased in stressed rats compared to controls. The SOD, GST, and CAT activities of recovery groups were significantly lower than controls, whereas MDA levels and GPx activity of these rats did not differ from controls. The results, for the first time, reveal that stress-induced loss of germ cells leading to decrease in sperm count may be due to oxidative damage caused by chronic stress and majority of these changes are not reversible.

Polo-like kinase is required for synaptonemal complex disassembly and phosphorylation in mouse spermatocytes.

During meiosis, accurate coordination of the completion of homologous recombination and synaptonemal complex (SC) disassembly during the prophase to metaphase I (G2/MI) transition is essential to avoid aneuploid gametes and infertility. Previous studies have shown that kinase activity is required to promote meiotic prophase exit. The first step of the G2/MI transition is the disassembly of the central element components of the SC; however, the kinase(s) required to trigger this process remains unknown. Here we assess roles of polo-like kinases (PLKs) in mouse spermatocytes, both in vivo and during prophase exit induced ex vivo by the phosphatase inhibitor okadaic acid. All four PLKs are expressed during the first wave of spermatogenesis. Only PLK1 (not PLK2-4) localizes to the SC during the G2/MI transition. The SC central element proteins SYCP1, TEX12 and SYCE1 are phosphorylated during the G2/MI transition. However, treatment of pachytene spermatocytes with the PLK inhibitor BI 2536 prevented the okadaic-acid-induced meiotic prophase exit and inhibited phosphorylation of the central element proteins as well as their removal from the SC. Phosphorylation assays in vitro demonstrated that PLK1, but not PLK2-4, phosphorylates central element proteins SYCP1 and TEX12. These findings provide mechanistic details of the first stage of SC disassembly in mammalian spermatocytes, and reveal that PLK-mediated phosphorylation of central element proteins is required for meiotic prophase exit.

The Ras-ERK MAPK regulatory network controls dedifferentiation in Caenorhabditis elegans germline.

How a committed cell can be reverted to an undifferentiated state is a central question in stem cell biology. This process, called dedifferentiation, is likely to be important for replacing stem cells as they age or get damaged. Tremendous progress has been made in understanding this fundamental process, but its mechanisms are poorly understood. Here we demonstrate that the aberrant activation of Ras-ERK MAPK signaling promotes cellular dedifferentiation in the Caenorhabditis elegans germline. To activate signaling, we removed two negative regulators, the PUF-8 RNA-binding protein and LIP-1 dual specificity phosphatase. The removal of both of these two regulators caused secondary spermatocytes to dedifferentiate and begin mitotic divisions. Interestingly, reduction of Ras-ERK MAPK signaling, either by mutation or chemical inhibition, blocked the initiation of dedifferentiation. By RNAi screening, we identified RSKN-1/P90(RSK) as a downstream effector of MPK-1/ERK that is critical for dedifferentiation: rskn-1 RNAi suppressed spermatocyte dedifferentiation and instead induced meiotic divisions. These regulators are broadly conserved, suggesting that similar molecular circuitry may control cellular dedifferentiation in other organisms, including humans.

Focal adhesion kinase-Tyr407 and -Tyr397 exhibit antagonistic effects on blood-testis barrier dynamics in the rat.

Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, displays phosphorylation-dependent localization in the seminiferous epithelium of adult rat testes. FAK is an integrated component of the blood-testis barrier (BTB) involved in regulating Sertoli cell adhesion via its effects on the occludin-zonula occludens-1 complex. Herein, we report that p-FAK-Tyr(407) and p-FAK-Tyr(397) display restricted spatiotemporal and almost mutually exclusive localization in the epithelium, affecting BTB dynamics antagonistically, with the former promoting and the latter disrupting the Sertoli cell tight junction-permeability barrier function. Using primary cultured Sertoli cells as an in vitro model that mimics the BTB in vivo both functionally and ultrastructurally, effects of FAK phosphorylation on BTB function were studied by expressing nonphosphorylatable and phosphomimetic mutants, with tyrosine replaced by phenylalanine (F) and glutamate (E), respectively. Compared with WT FAK, Y407E and Y397F mutations each promoted barrier function, and the promoting effect of the Y407E mutant was abolished in the Y397E-Y407E double mutant, demonstrating antagonism between Tyr(407) and Tyr(397). Furthermore, Y407E mutation induced the recruitment of actin-related protein 3 to the Sertoli cell-cell interface, where it became more tightly associated with neuronal Wiskott-Aldrich syndrome protein, promoting actin-related protein 2/3 complex activity. Conversely, Y407F mutation reduced the rate of actin polymerization at the Sertoli cell BTB. In summary, FAK-Tyr(407) phosphorylation promotes BTB integrity by strengthening the actin filament-based cytoskeleton. FAK serves as a bifunctional molecular "switch" to direct the cyclical disassembly and reassembly of the BTB during the epithelial cycle of spermatogenesis, depending on its phosphorylation status, to facilitate the transit of preleptotene spermatocytes across the BTB.

The RNase III enzyme DROSHA is essential for microRNA production and spermatogenesis.

DROSHA is a nuclear RNase III enzyme responsible for cleaving primary microRNAs (miRNAs) into precursor miRNAs and thus is essential for the biogenesis of canonical miRNAs. DICER is a cytoplasmic RNase III enzyme that not only cleaves precursor miRNAs to produce mature miRNAs but also dissects naturally formed/synthetic double-stranded RNAs to generate small interfering RNAs (siRNAs). To investigate the role of canonical miRNA and/or endogenous siRNA production in spermatogenesis, we generated Drosha or Dicer conditional knock-out (cKO) mouse lines by inactivating Drosha or Dicer exclusively in spermatogenic cells in postnatal testes using the Cre-loxp strategy. Both Drosha and Dicer cKO males were infertile due to disrupted spermatogenesis characterized by depletion of spermatocytes and spermatids leading to oligoteratozoospermia or azoospermia. The developmental course of spermatogenic disruptions was similar at morphological levels between Drosha and Dicer cKO males, but Drosha cKO testes appeared to be more severe in spermatogenic disruptions than Dicer cKO testes. Microarray analyses revealed transcriptomic differences between Drosha- and Dicer-null pachytene spermatocytes or round spermatids. Although levels of sex-linked mRNAs were mildly elevated, meiotic sex chromosome inactivation appeared to have occurred normally. Our data demonstrate that unlike DICER, which is required for the biogenesis of several small RNA species, DROSHA is essential mainly for the canonical miRNA production, and DROSHA-mediated miRNA production is essential for normal spermatogenesis and male fertility.

Inactivation of Dicer1 has a severe cumulative impact on the formation of mature germ cells in mouse testes.

Dicer1, an RNase III endonuclease, is indispensable for the maturation of miRNA and siRNA, which control gene expression through the RNAi pathway. The diverse functions of miRNA involving multiple developmental processes have been elucidated, but the role of Dicer1 in spermatogenesis is just beginning to be revealed. Mice lacking Dicer1 were reported to be embryonic lethal at E7.5. In the present study, mice with a Dicer1 conditional allele were crossed with Vasa-cre transgenic mice to delete Dicer1 as early as the prospermatogonia stage (at E15). At P40, seminiferous tubules of Dicer1 deficient mice showed several aberrant phenotypes. A large number of apoptotic germ cells were detected by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, but several events in meiosis of spermatocytes appeared unaffected. The mutant mice were found to be sterile, likely due to the extensive decrease in number and morphological abnormalities of mature sperm in the epididymis, which, together with the numerous haploid cells in the testis, indicated a severely affected transition from round to functional elongated spermatozoa. Additionally, we found milder phenotypes when Dicer1 was inactivated in later stages of spermatogenesis in Stra8-cre and Pgk2-cre transgenic mice. In conclusion, our findings suggest that the loss of Dicer1 has a continuous and cumulative effect on the process of spermatogenesis and blocks the germ cells in the stage of round spermatids to a large extent, ultimately leading to the generation of abnormal sperm.

Roles of histone H3K9 methyltransferases during Drosophila spermatogenesis.

Epigenetic regulation of gene expression by covalent modification of histones is important for germ line cell development. In mammals, histone H3 lysine 9 (H3K9)-specific histone methyltransferases (HMTases), such as G9a, SETDB1, and SUV39H, play critical roles, but the contribution of H3K9-specific HMTases in Drosophila remains to be clarified, especially in male sperm. Here, we performed immunocytochemical analyses with a specific antibody to dG9a, Drosophila G9a ortholog, and demonstrated localization in the cytoplasm from the growth to elongation stages of spermatogenesis. In the subsequent early canoe stage, strong dG9a signals were detected exclusively in nuclei, suggesting a regulatory role. However, mono-, di-, and trimethylated H3K9 signals were not extensively decreased in a homozygous dG9a null mutant throughout these stages. In contrast, mono- and trimethylated H3K9 signals were extensively decreased in a heterozygous DmSetdb1 mutant during spermatogenesis, and similar reduction in monomethylated H3K9 signals was observed in a homozygous Su(var)3-9 mutant. Therefore, DmSETDB1 is likely to be mainly responsible for mono- and trimethylation of H3K9 and SU(VAR)3-9 for monomethylation of H3K9 during spermatogenesis. However, the reduced methylation of H3K9 in premeiotic spermatocytes did not influence X-Y chromosome disjunction in male meiosis, suggesting that it may not be critical for spermatogenesis in Drosophila.