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.

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.

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.

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.

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.

DNA polymerase beta is critical for mouse meiotic synapsis.

We have shown earlier that DNA polymerase beta (Pol beta) localizes to the synaptonemal complex (SC) during Prophase I of meiosis in mice. Pol beta localizes to synapsed axes during zygonema and pachynema, and it associates with the ends of bivalents during late pachynema and diplonema. To test whether these localization patterns reflect a function for Pol beta in recombination and/or synapsis, we used conditional gene targeting to delete the PolB gene from germ cells. We find that Pol beta-deficient spermatocytes are defective in meiotic chromosome synapsis and undergo apoptosis during Prophase I. We also find that SPO11-dependent gammaH2AX persists on meiotic chromatin, indicating that Pol beta is critical for the repair of SPO11-induced double-strand breaks (DSBs). Pol beta-deficient spermatocytes yielded reduced steady-state levels of the SPO11-oligonucleotide complexes that are formed when SPO11 is removed from the ends of DSBs, and cytological experiments revealed that chromosome-associated foci of replication protein A (RPA), RAD51 and DMC1 are less abundant in Pol beta-deficient spermatocyte nuclei. Localization of Pol beta to meiotic chromosomes requires the formation of SPO11-dependent DSBs. Taken together, these findings strongly indicate that Pol beta is required at a very early step in the processing of meiotic DSBs, at or before the removal of SPO11 from DSB ends and the generation of the 3' single-stranded tails necessary for subsequent strand exchange. The chromosome synapsis defects and Prophase I apoptosis of Pol beta-deficient spermatocytes are likely a direct consequence of these recombination defects.

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 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.

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.

Polyunsaturated fatty acids are incorporated into maturating male mouse germ cells by lysophosphatidic acid acyltransferase 3.

Long-chain polyunsaturated fatty acids (PUFAs) accumulate in mammalian testis during puberty and are essential for fertility. To investigate whether lysophospholipid acyltransferases determine the PUFA composition of testicular phospholipids during pubertal development, we compared their mRNA expression, in vitro activity, and specificity with the lipidomic profile of major phospholipids. The accumulation of PUFAs in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine correlated with an induced lysophosphatidic acid acyltransferase (LPAAT)3 mRNA expression, increased microsomal LPAAT3 activity, and shift of LPAAT specificity to PUFA-coenzyme A. LPAAT3 was induced during germ cell maturation, as shown by immunofluorescence microscopy. Accordingly, differentiation of mouse GC-2spd(ts) spermatocytes into spermatides up-regulated LPAAT3 mRNA, increased the amount of polyunsaturated phospholipids, and shifted the specificity for the incorporation of deuterium-labeled docosahexaenoic acid toward phosphatidylcholine and phosphatidylethanolamine. Stable knockdown of LPAAT3 in GC-2spd(ts) cells significantly decreased microsomal LPAAT3 activity, reduced levels of polyunsaturated phosphatidylethanolamine species, and impaired cell proliferation/survival during geneticin selection. We conclude that the induction of LPAAT3 during germ cell development critically contributes to the accumulation of PUFAs in testicular phospholipids, thereby possibly affecting sperm cell production.

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.

Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells.

Sam68 plays an essential role in mouse spermatogenesis and male fertility. Herein, we report an interaction between Sam68 and the phosphorylated forms of the RNA polymerase II (RNAPII) in meiotic spermatocytes. RNase treatment decreased but did not abolish the interaction, consistently with in vitro binding of RNAPII to the Sam68 carboxyl-terminal region. Sam68 retention in the spermatocyte nucleus was dependent on the integrity of cellular RNAs, suggesting that the protein is recruited to transcriptionally active chromatin. Mouse knockout models characterized by stage-specific arrest of spermatogenesis and staining with the phosphorylated form of RNAPII documented that Sam68 expression is confined to the transcriptionally active stages of spermatogenesis. Furthermore, Sam68 associates with splicing regulators in germ cells and we report that alternative splicing of Sgce exon 8 is regulated in a Sam68-dependent manner during spermatogenesis. RNA and chromatin crosslink immunoprecipitation experiments showed that Sam68 binds in vivo to sequences surrounding the intron 7/exon 8 boundary, thereby affecting the recruitment of the phosphorylated RNAPII and of the general splicing factor U2AF65. These results suggest that Sam68 regulates alternative splicing at transcriptionally active sites in differentiating germ cells and provide new insights into the regulation of Sam68 expression during spermatogenesis.

ATM and RPA in meiotic chromosome synapsis and recombination.

ATM is a member of the phosphatidylinositol 3-kinase (PIK)-like kinases, some of which are active in regulating DNA damage-induced mitotic cell-cycle checkpoints. ATM also plays a role in meiosis. Spermatogenesis in Atm-/- male mice is disrupted, with chromosome fragmentation leading to meiotic arrest; in human patients with ataxia-telangiectasia (A-T), gonadal atrophy is common. Immuno-localization studies indicate that ATM is associated with sites along the synaptonemal complex (SC), the specialized structure along which meiotic recombination occurs. Recombination, preceded by pairing of homologous chromosomes, is thought to require heteroduplex formation between homologous DNA, followed by strand exchange. These early meiotic steps (entailing the formation and processing of meiotic recombination intermediates with DNA-strand interruptions) require ssDNA-binding proteins such as replication protein A (RPA; refs 5-7). In somatic cells, DNA damage induces ATM-dependent phosphorylation of RPA. We demonstrate here that ATM and RPA co-localize along synapsed meiotic chromosomes and at sites where interactions between ectopic homologous chromosome regions appear to initiate. In Atm-/- meiotic prophase spermatocytes, immuno-localization shows that RPA is present along synapsing chromosomes and at sites of fragmentation of the SC. These results suggest that RPA and ATM co-localize at sites where interhomologous-DNA interactions occur during meiotic prophase and where breaks associated with meiotic recombination take place after synapsis, implying a possible functional interaction between these two proteins.

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.

STK31(TDRD8) is dynamically regulated throughout mouse spermatogenesis and interacts with MIWI protein.

Tudor-domain-containing proteins (TDRDs) are suggested to be critical regulators of germinal granules assembly involved in Piwi-interacting RNAs (piRNAs)-mediated pathways, of which associated components and the underlying functional mechanisms, however, remain to be elucidated. We herein characterized the expression pattern of STK31, a member of TDRDs subfamily (also termed as TDRD8), throughout spermatogenesis during mouse postnatal development. RT-PCR and Western blot verified its preferential expression in testis, but not in any other somatic tissues, in addition to embryonic stem cells. Immunofluorescent staining demonstrated that STK31 was confined to granules-like structures in mid-to-late spermatocyte cytoplasm and to acrosomal cap starting at steps 7-8 of spermatids. Furthermore, STK31 retained its localization to equatorial segment of acrosome during epididymal maturation, capacitation, and acrosome reaction. Co-immunoprecipitation assay in vivo and in vitro confirmed MIWI is a bona fide partner of STK31 in mice testes, in combination with LC/MS identification. We also discovered a group of heat shock proteins specifically associated with STK31 in vivo. Our findings suggest mouse STK31 could be a potential nuage-associated protein in the cytoplasm of mid-to-late spermatocytes and play pivotal roles related to fertilization.

Expression and immunolocalization of 20ß-hydroxysteroid dehydrogenase during testicular cycle and after hCG induction, in vivo in the catfish, Clarias gariepinus.

The maturation inducing hormone, 17α,20ß-dihydroxy-4-pregnen-3-one (17α,20ß-DP) is required for the meiotic maturation and is produced from the precursor 17α-hydroxyprogesterone by the enzyme 20ß-hydroxysteroid dehydrogenase (20ß-HSD) in several teleosts. Central role of 20ß-HSD in ovarian cycle and final oocyte maturation is well studied when compared to spermatogenesis. In the present study, we investigated the localization and expression of 20ß-HSD in testicular cycle and gonadotropin induced sperm maturation. During testicular ontogeny, 20ß-HSD expression was detectable at 50 and 100 days post-hatch (dph), while the expression was high at 150 dph. In testicular cycle, highest levels of mRNA and protein of 20ß-HSD were observed during spawning phase. Intraperitoneal injection of human chorionic gonadotropin (hCG) to prespawning catfish elevated both 20ß-HSD transcripts and protein levels when compared to saline treated controls in a time-dependent manner. Serum 17α,20ß-DP levels, measured during different phases of testicular cycle as well as following the treatment of hCG, showed a positive correlation with the expression of 20ß-HSD. Immunolocalization revealed the presence of 20ß-HSD protein predominantly in interstitial cells and spermatogonia/spermatocytes while 20ß-HSD was undetectable in haploid cells (spermatids/sperm). These results together with high expression during spawning phase of testicular cycle and after hCG treatment in the prespawning catfish suggests a pivotal role for 20ß-HSD during testicular recrudescence leading to sperm maturation. Further studies using various fish models on testicular 20ß-HSD may provide interesting details to understand its importance in teleostean spermatogenesis.

A role for Aurora C in the chromosomal passenger complex during human preimplantation embryo development.

BACKGROUND: Human embryos generated by IVF demonstrate a high incidence of chromosomal segregation errors during the cleavage divisions. To analyse underlying molecular mechanisms, we investigated the behaviour of the chromosomal passenger complex (CPC) in human oocytes and embryos. This important mitotic regulatory complex comprises the inner centromere protein (INCENP), survivin, borealin and Aurora B, or the meiotic kinase Aurora C. METHODS: We analysed mRNA expression by quantitative RT-PCR of all CPC members in human oocytes, tripronuclear (3PN) zygotes, 2-cell and 4-cell embryos developed from 3PN zygotes, plus good-quality cryopreserved 8-cell, morula and blastocyst stage embryos. Protein expression and localization of CPC members were investigated by immunofluorescence in oocytes and embryos arrested at prometaphase. Histone H3S10 phosphorylation was investigated as an indicator of a functional CPC. RESULTS: INCENP, survivin and borealin were detected at the inner centromere of prometaphase chromosomes in all stages investigated. Whereas Aurora B and C are both present in oocytes, Aurora C becomes the most prominent kinase in the CPC during the first three embryonic cell cycles. Moreover, Aurora C mRNA was up-regulated with Aurora B after activation of the embryonic genome and both proteins were detected in early Day 4 embryos. Subsequently, only Aurora B was detected in blastocysts. CONCLUSIONS: In contrast to somatic cells, our results point to a specific role for Aurora C in the CPC during human preimplantation embryo development. Although, the presence of Aurora C in itself may not explain the high chromosome segregation error rate, the data presented here provide novel information regarding possible mechanisms.

Localization of the chromatin remodelling protein, ATRX in the adult testis.

Mutations in ATRX (alpha-thalassaemia and mental retardation on the X-chromosome) can give rise to ambiguous or female genitalia in XY males, implying a role for ATRX in testicular development. Studies on ATRX have mainly focused on its crucial role in brain development and α-globin regulation; however, little is known about its function in sexual differentiation and its expression in the adult testis. Here we show that the ATRX protein is present in adult human and rat testis and is expressed in the somatic cells; Sertoli, Leydig, and peritubular myoid cells, and also in germ cells; spermatogonia and early meiotic spermatocytes. The granular pattern of ATRX staining is consistent with that observed in other cell-types and suggests a role in chromatin regulation. The findings suggest that ATRX in humans may play a role in adult spermatogenesis as well as in testicular development.

[Correlation analysis between meiotic recombination frequencies and age in human spermatocyte].

Faithful meiotic recombination is essential for the segregation of homologous chromosomes and the formation of normal haploid gametes. Little is known about the mechanism of meiotic recombination in human germ cells. MLHl (a DNA mismatch repair protein) foci on synaptonemal complexes (SCs) at prophase I of meiosis can be used to examine recombination frequency. In 10 fertile men, the mean number of MLH1 foci per cell in all donors was 49.4 with a range from 33 to 63. There was significant variation in the recombination frequency found among 10 normal individuals: the mean frequencies of chromosomal recombination foci ranged from 47 to 52.7. The bivalents without recombination focus were rare, with a frequency of only 0.4%. Thus, achiasmate chromosomes appeared to be rare in human male meiosis. Spearman correlation analysis between age and the frequencies of recombination foci failed to get any significantly statistical correlation, suggesting that aging contributes nothing to the variation among individuals.

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.