Upregulation of Profilin 2 on HDAC6 overexpression in mouse GC-1 cells and its putative role in germ cell migration in the testis.

Previous reports from this laboratory have demonstrated the involvement of histone deacetylase 6 (HDAC6) in sperm motility. As the presence of HDAC6 has also been reported in the earlier stage germ cells, studies were undertaken to explore its role during these stages of spermatogenesis. HDAC6 was overexpressed in GC-1spg cells, which represent the stage between type B spermatogonia and primary spermatocyte, and its effect on germ cell transcriptome was investigated by microarray. Among the many transcripts that were differentially regulated, Profilin 2, reported previously as a neuronal specific isoform, was observed as one of the genes highly upregulated at the transcript level, which was further confirmed by real-time PCR, and the protein confirmed by indirect immunofluorescence (IIF). Profilin 2 colocalized with HDAC6, as seen both in GC-1 cells and sperm. On the sperm, the presence of Profilin 2 was detected throughout the flagella with its colocalization with HDAC6 seen conspicuously in the mid-piece region of the flagella. Co-immunoprecipitation studies confirmed Profilin 2 interaction with HDAC6. Docking studies using Z dock suggested the interaction of 8 residues of HDAC6 with 6 residues of Profilin 2. The novel observation of Profilin 2 in spermatogonial cells, its significant upregulation on HDAC6 overexpression and its interaction with HDAC6 suggests that HDAC6 in collaboration with Profilin 2 may play a role in regulating the movement of germ cells from one stage/compartment to the next.

N-Formyl-L-aspartate mediates chemotaxis in sperm via the beta-2-adrenergic receptor.

Chemotaxis is a highly conserved physiological event required for directed sperm movement during fertilization. Recently, studies from our laboratory have identified N-formyl-L-aspartate (NFA) as a sperm chemoattractant. NFA is a known agonist for the beta-2-adrenergic receptor (ß-2-AR) that regulates cAMP production and Ca2+ mobilization in somatic cells. As these downstream signaling molecules are also reported to be involved in sperm chemotaxis, in the present study we investigated the putative mechanism/s by which NFA may mediate chemotaxis. Toward this, the expression and localization of ß-2-AR in sperm were studied by Western blot and indirect immunofluorescence, respectively. The responses of sperm to various concentration gradients of NFA and ICI-118,551, a ß-2-AR specific antagonist, were evaluated using the microfluidics device-based chemotaxis assay. The intracellular concentration of Ca2+, on exposure to NFA, was analyzed using FURA-2 AM-based fluorimetric assay. Furthermore, the effect of NFA on sperm capacitation and acrosome reaction was evaluated using Western blot and immunofluorescence. NFA exhibited a bell-shaped dose-response curve typical of chemotaxis, with maximum response observed at 0.01M NFA, beyond which it was inhibitory; ß-2-AR localization was seen on the sperm head and the mid-piece region of the flagella. Inhibition of sperm chemotaxis by ICI-118,551 confirms that sperm respond chemotactically to NFA via ß-2-AR. Interestingly, at the concentration used for chemotaxis, NFA induced an increase in the intracellular Ca2+ but decreased cAMP in capacitating sperm. However, NFA per se did not induce capacitation as seen from the lack of effect on tyrosine phosphorylation and membrane potential of uncapacitated sperm. Acrosome evaluation of NFA-treated sperm using PSA-FITC staining showed no effect on the acrosome structure. Our data thus provide evidence indicating that NFA induces sperm chemotaxis and the chemotactic response of sperm to NFA from the ovulatory phase of oviductal fluid is mediated through the ß-2-AR on sperm possibly via non-canonical signaling.

Proteomic and genetic dissection of testis-specific histone 2B in infertile men reveals its contribution to meiosis and sperm motility.

OBJECTIVE: To investigate testis-specific histone 2B (TSH2B) and its gene anomalies in infertile men. DESIGN: Case-control study. SETTING: Basic science laboratory. PATIENT(S): Fertile and infertile men. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): The histone and protamine status of sperm was studied by aniline blue and chromomycin A3 staining, respectively. Testis-specific histone 2B, total H2B, and phosphorylated TSH2B (pTSH2B) were estimated by Western blot analysis. The frequency of genetic polymorphisms and rare variants in H2BC1 was studied by Sanger sequencing. Phosphosites on TSH2B in sperm were identified by reverse-phase high-performance liquid chromatography purification of TSH2B followed by mass spectrometric analysis. RESULT(S): Aniline blue and chromomycin A3 staining revealed significantly higher histone retention and low protamine in sperm of infertile men. Sperm TSH2B and total H2B levels were significantly lower in oligozoospermic and oligoasthenozoospermic men (in both groups). The TSH2B levels were comparable in asthenozoospermic men; however, the pTSH2B level was significantly low. The H2BC1 gene sequencing identified 6 variants, of which 2 are rare variants (rs368672899 and rs544942090) and 4 (rs4711096, rs4712959, rs4712960 and rs4712961) are single nucleotide polymorphisms. Minor allele frequency of 5'-untranslated region variant rs4711096 was significantly lower in infertile men (OR = 0.65). The rare nonsynonymous variant, rs368672899, p.Ser5Pro was seen in 1 oligoasthenoteratozoospermic individual. Interestingly, mass spectrometric analysis identified a site on TSH2B to bear a phosphate group in the sperm of fertile men. CONCLUSION(S): Our study reveals a defect in the replacement of somatic histones with testis-specific variants in infertile men. Chromatin compaction positively correlates with sperm motility, which is suggestive of its utility in diagnostic semen analysis of infertile individuals. Our observations with TSH2B and its cognate gene in sperm of infertile men indicate an essential role for TSH2B in meiosis and its phosphorylation in sperm motility, respectively.

Heterogeneity in the Epigenetic Landscape of Murine Testis-Specific Histone Variants TH2A and TH2B Sharing the Same Bi-Directional Promoter.

Testis-specific histone variants are crucial to promote open chromatin structure to enable nucleosome disassembly in the final stages of spermiogenesis. However, even after histone replacement, mature sperm retain a proportion of these variants, the function of which is unknown. The present study aimed to understand the functional relevance of the retained H2B and H2A variants, TH2B and TH2A. While no literature is available on the phenotype of TH2A knockouts, TH2B/TH2A double knockout male mice are reported to be infertile. In this study, ChIP-seq analysis was done for TH2B and TH2A to understand the epigenomics of the retained TH2B and TH2A, using murine caudal sperm. Distribution across genomic partitions revealed ∼35% of the TH2B peaks within ±5 kb of TSS whereas TH2A peaks distribution was sparse at TSS. Gene Ontology revealed embryo development as the most significant term associated with TH2B. Also, based on genomic regions, TH2B was observed to be associated with spindle assembly and various meiosis-specific genes, which is an important finding as TH2A/TH2B DKO mice have been reported to have defective cohesin release. A comparison of mouse and human TH2B-linked chromatin revealed 26% overlap between murine and human TH2B-associated genes. This overlap included genes crucial for embryogenesis. Most importantly, heterogeneity in the epigenetic landscape of TH2A and TH2B was seen, which is intriguing as TH2B and TH2A are well reported to be present in the same nucleosomes to promote open chromatin. Additionally, unlike TH2B, TH2A was enriched on the mitochondrial chromosome. TH2A was found to be associated with Nuclear insertion of Mitochondrial DNA sequences (NUMTs) in sperm. A comprehensive analysis of these observations indicates novel functions for the sperm-retained TH2B and TH2A.

Revisiting the Characteristics of Testicular Germ Cell Lines GC-1(spg) and GC-2(spd)ts.

Spermatogenesis is a multifaceted and meticulously orchestrated process involving meiosis, chromatin build up, transcriptional and translational hushing, and spermiogenesis. Male germ cell lines GC-1spg (GC-1) and GC-2(spd)ts (GC-2) provide a useful resource to comprehend the molecular events occurring during such a tightly regulated process. Using cDNA microarray, expression profiling of GC-1 and GC-2 cell lines was done to precisely understand their characteristics and uniqueness. Our observations indicate that whilst both the cell lines are indeed of testicular origin, GC-2 is not haploid as was originally thought. Data analysis of the 23,351 transcripts detected in GC-1 and 20,992 in GC-2 cell lines demonstrates an 80% transcript overlap between GC-1 and GC-2 cells and ~ 40% similarity of both with the primary spermatocyte transcriptome. 3152 and 793 transcripts exclusive to GC-1 and GC-2, respectively, were identified. The presence of transcripts for 36 genes was validated in these cell lines including those showing testis-specific expression, as well as genes not reported previously. Overall, this study provides the transcriptome database of GC-1 and GC-2 cells. Analysis of the data demonstrates the transcriptomic transitions between GC-1 and GC-2 thus providing a glimpse to the process of germ cell differentiation from type B spermatogonium into preleptotene spermatocyte.

Epigenetic landscape of testis specific histone H2B variant and its influence on sperm function.

BACKGROUND: Biological relevance of the major testis specific histone H2B variant (TH2B) in sperm is not fully understood. Studies in TH2A/TH2B double knockout male mice indicate its role in chromatin compaction and male fertility. Additionally, the presence of TH2B and TH2A reportedly generates more dynamic nucleosomes, leading to an open chromatin structure characteristic of transcriptionally active genome. Given that mature human sperm are transcriptionally and translationally inactive, the presence of TH2B in mature sperm is intriguing. To address its role in sperm, we investigated the genome-wide localization of TH2B in sperm of fertile men. RESULTS: We have identified the genomic loci associated with TH2B in fertile human sperm by ChIP-seq analysis. Bioinformatic analysis revealed ~ 5% sperm genome and 5527 genes to be associated with TH2B. Out of these 105 (1.9%) and 144 (2.6%) genes showed direct involvement in sperm function and early embryogenesis, respectively. Chromosome wide analysis for TH2B distribution indicated its least distribution on X and Y chromosomes and varied distribution on autosomes. TH2B showed relatively higher percentage of gene association on chromosome 4, 18, 3 and 2. TH2B enrichment was more in promoter and gene body region. Gene Ontology (GO) analysis revealed signal transduction and associated kinase activity as the most enriched biological and molecular function, respectively. We also observed the enrichment of TH2B at developmentally important loci, such as HOXA and HOXD and on genes required for normal sperm function, few of which were validated by ChIP-qPCR. The relative expression of these genes was altered in particular subgroup of infertile men showing abnormal chromatin packaging. Chromatin compaction positively correlated with sperm- motility, concentration, viability and with transcript levels of PRKAG2 and CATSPER B. CONCLUSION: ChIP-seq analysis of TH2B revealed a putative role of TH2B in sperm function and embryo development. Altered expression of TH2B associated genes in infertile individuals with sperm chromatin compaction defects indicates involvement of TH2B in transcriptional regulation of these genes in post meiotic male germ cells. This altered transcriptome may be a consequence or cause of abnormal nuclear remodeling during spermiogenesis.

N-formyl-l-aspartate: A novel sperm chemoattractant identified in ovulatory phase oviductal fluid using a microfluidic chip.

BACKGROUND: Chemotaxis, as a mechanism for sperm guidance although known, has been difficult to demonstrate in vitro. Consequently, very few chemoattractants have been identified till date. OBJECTIVES: To investigate sperm motility behavior in response to ovulatory (OV) and preovulatory (preOV) oviductal fluid (OF) and identify potential chemotactic metabolites. MATERIALS AND METHODS: Intracellular calcium ([Ca2+ ]I ) influx in capacitating sperm was determined by spectrofluorimetry. The chemotactic response of rat caudal sperm to OF from the preOV- and OV- phases of normally cycling female rats was assessed in a microfluidic device developed by us. Hydrophilic metabolites extracted from the OF of both the phases were resolved and identified by LC-MS/MS, followed by data analysis using XCMS and MetaboAnalyst software, and chemotactic potential of the most promising compound was validated using the microfluidic device. RESULTS: Spectrofluorimetric analysis depicts a significant increase in sperm [Ca2+ ]I in response to OV-OF. With the microfluidic chemotaxis assay, sperm population shows a significantly increased directionality and velocity to an ascending gradient of 0.06 µg/µl OV-OF compared to preOV-OF. LC-MS/MS of the OFs demonstrates five and four metabolites to be exclusive to the OV-OF and preOV-OF, respectively, and 25 metabolites common to both, of which 14 metabolites, including N-formyl-l-aspartate (NFA), are increased in OV-OF; NFA was tested for its ability to influence sperm movement, and shows chemotaxis potential. DISCUSSION AND CONCLUSION(S): This is the first study that has systematically demonstrated sperm chemotaxis with OV phase rat OF, identified NFA present in this fluid as a novel chemoattractant to sperm, and proven the utility of the device to test putative chemoattractants. It remains to be seen whether NFA is present in the follicular fluid (FF) of infertile women, and whether it may likely be a reason for the failure of natural conception in idiopathic infertile women.

Acetylation/deacetylation and microtubule associated proteins influence flagellar axonemal stability and sperm motility.

PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl transferase (αTAT1) and histone deacetylase 6 (HDAC6) in animal models and cell lines illustrate disparity in correlating tubulin acetylation status with stability of MT. Our earlier studies showed reduced acetyl tubulin in sperm of asthenozoospermic individuals. Our studies on rat sperm showed that on inhibition of HDAC6 activity, although tubulin acetylation increased, sperm motility was reduced. Studies were therefore undertaken to investigate the influence of tubulin acetylation/deacetylation on MT dynamicity in sperm flagella using rat and human sperm. Our data on rat sperm revealed that HDAC6 specific inhibitor Tubastatin A (T) inhibited sperm motility and neutralized the depolymerizing and motility debilitating effect of Nocodazole. The effect on polymerization was further confirmed in vitro using pure MT and recHDAC6. Also polymerized axoneme was less in sperm of asthenozoosperm compared to normozoosperm. Deacetylase activity was reduced in sperm lysates and axonemes exposed to T and N+T but not in axonemes of sperm treated similarly suggesting that HDAC6 is associated with sperm axonemes or MT. Deacetylase activity was less in asthenozoosperm. Intriguingly, the expression of MDP3 physiologically known to bind to HDAC6 and inhibit its deacetylase activity remained unchanged. However, expression of acetyl α-tubulin, HDAC6 and microtubule stabilizing protein SAXO1 was less in asthenozoosperm. These observations suggest that MAPs and threshold levels of MT acetylation/deacetylation are important for MT dynamicity in sperm and may play a role in regulating sperm motility.

Membrane-bound Glucose regulated protein 78 interacts with alpha-2-macroglobulin to promote actin reorganization in sperm during epididymal maturation.

Glucose regulated protein 78 (GRP78) is expressed on cell surface in exceptional conditions as seen in cancer cells and macrophages. We have reported its membrane localization in sperm. The functional significance of its surface localization in sperm is an enigma. Alpha-2-macroglobulin (α2M) reportedly binds surface GRP78, regulating macrophage motility. Additionally, seminal plasma α2M levels are low in cases of asthenozoospermia. We investigated the functional relevance of sperm surface GRP78 and α2M crosstalk using testicular sperm (Tsp; immature) and caudal sperm (Cdsp; mature) from adult Holtzman rats. α2M colocalized and interacted with GRP78 and was significantly higher in Cdsp. Cofilin pathway proteins were detected in Tsp and Cdsp, however the pathway was highly active in Cdsp. Tsp surface GRP78 tyrosine phosphorylation and [Ca2+]i levels increased significantly on exposure to activated α2M (α2M*). This binding activated Rac/Cdc42, and consequently PAK, leading to LIMK and cofilin phosphorylation and thus promoting actin reorganization. Cofilin translocation from the sperm tail to the head in the presence of α2M* possibly prevented F-actin depolymerization in the tail. Thus, profiles observed with Cdsp could be re-created upon exposure of Tsp with α2M*. We conclude that α2M secreted into seminiferous tubule fluid by Sertoli cells, may be activated by proteinases in the epididymis and may bind to sperm surface GRP78 during epididymal transit, thereby facilitating sperm motility via actin reorganization. As F-actin is required for maintaining structural integrity and hyperactivated motility in sperm, our finding has significant implications in light of our previous reports of reduced GRP78 phosphorylation and the actin-based motility pathway being significantly altered in asthenozoospermia.

Chemotactic behavior of spermatozoa captured using a microfluidic chip.

Chemotaxis, as a mechanism for sperm guidance in vivo, is an enigma which has been difficult to demonstrate. To address this issue, various devices have been designed to study sperm chemotaxis in vitro. Limitations of traditional chemotaxis devices were related to the inability to maintain a stable concentration gradient as well as track single sperm over long times. Microfluidics technology, which provides superior control over fluid flow, has been recently used to generate stable concentration gradients for investigating the chemotactic behavior of several cell types including spermatozoa. However, the chemotactic behavior of sperm has not been unequivocally demonstrated even in these studies due to the inability to distinguish it from rheotaxis, thermotaxis, and chemokinesis. For instance, the presence of fluid flow in the microchannels not only destabilizes the concentration gradient but also elicits a rheotactic response from sperm. In this work, we have designed a microfluidic device which can be used to establish both, a uniform concentration and a uniform concentration gradient in a stationary fluid. By facilitating measurement of sperm response in ascending, descending ,and uniform chemoattractant concentration, the assay could isolate sperm chemotactic response from rheotaxis and chemokinesis. The device was validated using acetylcholine, a known chemoattractant and further tested with rat oviductal fluid from the estrus phase.

Correction to: HDAC6 deacetylates alpha tubulin in sperm and modulates sperm motility in Holtzman rat.

The published online version contains mistake. The chimeric peptide should read as 'DPSVLYVSLHRYGGYMNEGELRV'. It was inadvertently written as 'DPSVLYVSLYVSLHRYGGYMNEGELR' a mistake which we missed during proof reading.

Developmental Testicular Expression, Cloning, and Characterization of Rat HDAC6 In Silico.

We had previously reported presence of histone deacetylase 6 (HDAC6) in sperm and demonstrated its tubulin deacetylase activity and role in sperm motility in rat. In the present study we report its abundant expression in testis, epididymis, accessory sex organs, brain, and adrenal. In the testis, HDAC6 transcript and protein were observed throughout development. We therefore cloned the gene from rat testis using primers for hdac6 (accession number XM_228753.8) in order to determine the role of acetylation/deacetylation in spermatogenesis. The cloned rat hdac6 gene is ~3.5 kb with 28 exons and 1152 amino acids. We noted 4 single nucleotide polymorphisms (SNPs) on exons 2 (G/A), 5 (A/G), 7 (T/C), and 26 (G/T), respectively, in this sequence when compared to XM_228753.8. These were further validated at both cDNA and gene level. These SNPs resulted in 2 amino acids changes, namely, glycine → arginine and valine → phenylalanine at protein level. Cloned hdac6 overexpressed in HEK293T cells demonstrated significant overexpression by IIF. Alpha-tubulin acetylation analysis of the overexpressed cell lysate demonstrated that the protein was bioactive. This is the first study showing the ontogenic expression in the testis and reporting experimentally validated sequence of rat HDAC6 and its structural and functional annotation in silico. This sequence has been submitted to GenBank (Accession number Rattus KY009929.1).

Tubulin acetylation: A novel functional avenue for CDYL in sperm.

Motility in sperm is driven by the flagella, the principal component of which is the axoneme. The microtubules which make up the 9 + 2 axoneme are composed of heterodimers of alpha and beta tubulins and undergo several post-translational modifications. We have earlier reported that HDAC6 functions as tubulin deacetylase in sperm and has a role in sperm movement. While exploring the specific tubulin acetyltransferase (TAT) in sperm, we observed the presence of Chromodomain Y-Like (CDYL), on the principal piece of rat spermatozoa which compelled us to explore its function in sperm. CDYL was observed to be colocalized with acetylated alpha-tubulin (Ac α Tubulin) in sperm flagella. Sperm axonemal fraction showed the presence of CDYL protein indicating its strong association with flagellar microtubules. Sequence alignment of CDYL chromo domain and Alpha tubulin acetyltransferase (αTAT1) revealed that of the 10 residues of αTAT1 known to be involved in α-tubulin binding, 5 residues were identical and 1 was conserved between the two proteins. Docking of CDYL chromo domain and α-tubulin showed that 6 of the 11 important binding residues of α-tubulin showed an interaction with CDYL chromo domain. The putative CDYL chromodomain -α-tubulin interaction was further confirmed by Microscale Thermophoresis. We further asserted the ability of recombinant CDYL and Sperm CDYL to acetylate soluble tubulin and microtubules in vitro. Acetylation of tubulin was increased over twofold in cells overexpressing CDYL. Thus, our studies convincingly demonstrate the ability of CDYL to moonlight as a tubulin acetyltransferase.

Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation.

GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant differences observed in 2 out of 3 phosphoforms in asthenozoosperm suggest that GRP78 phosphorylation may have functional relevance in sperm with consequent clinical implications.

Monocyte Proteomics Reveals Involvement of Phosphorylated HSP27 in the Pathogenesis of Osteoporosis.

Peripheral monocytes, precursors of osteoclasts, have emerged as important candidates for identifying proteins relevant to osteoporosis, a condition characterized by low Bone Mineral Density (BMD) and increased susceptibility for fractures. We employed 4-plex iTRAQ (isobaric tags for relative and absolute quantification) coupled with LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) to identify differentially expressed monocyte proteins from premenopausal and postmenopausal women with low versus high BMD. Of 1801 proteins identified, 45 were differentially abundant in low versus high BMD, with heat shock protein 27 (HSP27) distinctly upregulated in low BMD condition in both premenopausal and postmenopausal categories. Validation in individual samples (n = 80) using intracellular ELISA confirmed that total HSP27 (tHSP27) as well as phosphorylated HSP27 (pHSP27) was elevated in low BMD condition in both categories (P < 0.05). Further, using transwell assays, pHSP27, when placed in the upper chamber, could increase monocyte migration (P < 0.0001) and this was additive in combination with RANKL (receptor activator of NFkB ligand) placed in the lower chamber (P = 0.05). Effect of pHSP27 in monocyte migration towards bone milieu can result in increased osteoclast formation and thus contribute to pathogenesis of osteoporosis. Overall, this study reveals for the first time a novel link between monocyte HSP27 and BMD.

HDAC6 deacetylates alpha tubulin in sperm and modulates sperm motility in Holtzman rat.

Histone deacetylase 6 (HDAC6) is an alpha (α)-tubulin deacetylase and its over-expression has been demonstrated to promote chemotactic cell movement. Motility in sperm is driven by the flagella, the cytoskeletal structure comprising the microtubules, which are heterodimers of α- and ß-tubulins. We have hypothesized that HDAC6, by virtue of being an α-tubulin deacetylase, might modulate sperm motility. However, the presence of HDAC6 on sperm has hitherto not been reported. In this study, we have demonstrated, for the first time, the presence of HDAC6 transcript and protein in the testicular and caudal sperm of rat. We have observed a significantly overlapping expression of HDAC6 with acetyl α-tubulin (Ac α-tubulin) in the mid-piece and principal piece of sperm flagella, and the co-precipitation of α-tubulin and Ac α-tubulin together with HDAC6 and vice versa in sperm lysates. This indicates that HDAC6 interacts with α-tubulin. The HDAC6 activity of sperm, sperm motility and status of Ac α-tubulin investigated in the presence of HDAC inhibitors Trichostatin A, Tubastatin A and sodium butyrate demonstrate that HDAC6 in sperm is catalytically active and that inhibitors of HDAC6 increase acetylation and restrict sperm motility. Thus, we show that (1) active HDAC6 enzyme is present in sperm, (2) HDAC6 in sperm is able to deacetylate α-tubulin, (3) inhibition of HDAC6 results in increased Ac α-tubulin expression and (4) HDAC6 inhibition affects sperm motility. This evidence suggests that HDAC6 is involved in modulating sperm movement.

Acetylated α-tubulin is reduced in individuals with poor sperm motility.

OBJECTIVE: To determine the status of α-tubulin acetylation and of testis-specific acetylable α-tubulin isoforms in asthenozoospermia. DESIGN: Research study. SETTING: Research institute and an infertility clinic. PATIENT(S): 50 men with normal sperm parameters, and 50 men with asthenozoospermia. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Western blot analyses of α-tubulin, acetylated α-tubulin, and isoforms TUBA3C, TUBA4A, and TUBA8 in Percoll separated sperm and flow cytometry, real-time reverse-transcription polymerase chain reaction, and immunofluorescent localization. RESULT(S): A statistically significant decrease in the expression of acetylated α-tubulin in asthenozoosperm was seen with Western blot analysis, double immunostaining by direct immunofluorescence, and flow cytometric analysis. The transcript and protein of testis-specific acetylable α-tubulin isoform TUBA3C was decreased and TUBA4A was statistically significantly increased in asthenozoosperm as compared with normal spermatozoa. TUBA8 was reduced in asthenozoosperm. Similar observations were noted by indirect immunofluorescent localization. The potential transcription factors involved in the differential expression of TUBA4A and TUBA3C have been identified. CONCLUSION(S): Data suggest an association of α-tubulin acetylation with asthenozoospermia. Ours is the first report to demonstrate α-tubulin isoforms in sperm, implicating their role in motility. The differential expression of TUBA3C and TUBA4A suggests that tubulin acetylation may be governed by the isoform of α-tubulin that is expressed or silenced and that this in turn is transcriptionally controlled.

Proteomics in reproductive biology: beacon for unraveling the molecular complexities.

Proteomics, an interface of rapidly evolving advances in physics and biology, is rapidly developing and expanding its potential applications to molecular and cellular biology. Application of proteomics tools has contributed towards identification of relevant protein biomarkers that can potentially change the strategies for early diagnosis and treatment of several diseases. The emergence of powerful mass spectrometry-based proteomics technique has added a new dimension to the field of medical research in liver, heart diseases and certain forms of cancer. Most proteomics tools are also being used to study physiological and pathological events related to reproductive biology. There have been attempts to generate the proteomes of testes, sperm, seminal fluid, epididymis, oocyte, and endometrium from reproductive disease patients. Here, we have reviewed proteomics based investigations in humans over the last decade, which focus on delineating the mechanism underlying various reproductive events such as spermatogenesis, oogenesis, endometriosis, polycystic ovary syndrome, embryo development. The challenge is to harness new technologies like 2-DE, DIGE, MALDI-MS, SELDI-MS, MUDPIT, LC-MS etc., to a greater extent to develop widely applicable clinical tools in understanding molecular aspects of reproduction both in health and disease.

Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia.

Sperm motility is an important prerequisite for successful fertilization and is regulated by cyclic AMP activated protein kinase A which phosphorylates flagella proteins like axonemal dynein and initiates motility. Increase in calcium influx reverses this process by dephosphorylation that is mediated by calcineurin. Analyzing the phosphoenriched fractions of spermatozoa lysates from eight normozoospermic-, and asthenozoospermic-samples, respectively, by Nano UPLC-MS(E), the present study investigates the phosphoproteins involved in sperm motility in an attempt to identify the key pathways regulating sperm motility and likely to be altered in spermatozoa of asthenozoospermic individuals. 66 phosphoproteins were differentially regulated in asthenozoospermia. The deregulated proteins comprised predominantly the HSPs, cytoskeletal proteins, proteins associated with the fibrous sheath, and those associated with energy metabolism. EM analysis of these spermatozoa demonstrated significant defects in mitochondria, and fibrous sheath and these defects could be correlated with the altered proteome. Pathway analysis revealed that carbohydrate and energy metabolism, cAMP mediated PKA signaling, PI3K/AKT signaling and pathway regulating actin based motility by Rho were significantly altered indicating that motility in spermatozoa is regulated through the concerted effort of these pathways. The data identified signature molecules that have the potential as biomarkers for diagnosing etiology of asthenozoospermia.