Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3ß). GSK3α and GSK3ß are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3ß) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

ADAD1 is required for normal translation of nuclear pore and transport protein transcripts in spermatids of Mus musculus†.

ADAD1 is a testis-specific RNA-binding protein expressed in post-meiotic spermatids whose loss leads to defective sperm and male infertility. However, the drivers of the Adad1 phenotype remain unclear. Morphological and functional analysis of Adad1 mutant sperm showed defective DNA compaction, abnormal head shaping, and reduced motility. Mutant testes demonstrated minimal transcriptome changes; however, ribosome association of many transcripts was reduced, suggesting ADAD1 may be required for their translational activation. Further, immunofluorescence of proteins encoded by select transcripts showed delayed protein accumulation. Additional analyses demonstrated impaired subcellular localization of multiple proteins, suggesting protein transport is also abnormal in Adad1 mutants. To clarify the mechanism giving rise to this, the manchette, a protein transport microtubule network, and the LINC (linker of nucleoskeleton and cytoskeleton) complex, which connects the manchette to the nuclear lamin, were assessed across spermatid development. Proteins of both displayed delayed translation and/or localization in mutant spermatids implicating ADAD1 in their regulation, even in the absence of altered ribosome association. Finally, ADAD1's impact on the NPC (nuclear pore complex), a regulator of both the manchette and the LINC complex, was examined. Reduced ribosome association of NPC encoding transcripts and reduced NPC protein abundance along with abnormal localization in Adad1 mutants confirmed ADAD1's impact on translation is required for a NPC in post-meiotic germ cells. Together, these studies lead to a model whereby ADAD1's influence on nuclear transport leads to deregulation of the LINC complex and the manchette, ultimately generating the range of physiological defects observed in the Adad1 phenotype.

Effects of preconception exposure to phthalates on mouse sperm capacitation parameters.

BACKGROUND: Phthalates have been linked to adverse male reproductive health, including poor sperm quality and embryo quality as well as a longer time to pregnancy (months of unprotected intercourse before conception occurs). The present study aimed to evaluate the effect of preconception exposure to two ubiquitous phthalate chemicals, di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DBP), and their mixture on sperm function, fertilization, and embryo development in mice. MATERIALS AND METHODS: Adult male C57BL/6J mice aged 8-9 weeks were exposed to di(2-ethylhexyl) phthalate, di-n-butyl phthalate, or their mixture (di-n-butyl phthalate + di(2-ethylhexyl) phthalate) at 2.5 mg/kg/day or vehicle for 40 days (equivalent to one spermatogenic cycle) via surgically implanted osmotic pumps. Caudal epididymal spermatozoa were extracted and analyzed for motility using computer-assisted sperm analyses. Sperm phosphorylation of protein kinase A substrates and tyrosine phosphorylation, markers of early and late capacitation events, respectively, were analyzed by Western blots. In vitro fertilization was used to evaluate the sperm fertilizing capacity. RESULTS: While the study did not reveal any significant differences in sperm motility and fertilization potential, abnormal sperm morphology was observed in all phthalate exposures, particularly in the phthalate mixture group. In addition, the study revealed significant differences in sperm concentration between control and exposed groups. Moreover, protein phosphorylation of protein kinase A substrates was decreased in the di(2-ethylhexyl) phthalate and mixture exposure groups, while no significant changes in protein tyrosine phosphorylation were observed in any of the groups. Assessment of the reproductive functionality did not reveal significant effects on in vitro fertilization and early embryo development rates but showed wide variability in the phthalate mixture group. CONCLUSION: Our findings suggest that preconception phthalate exposure affects sperm numbers and phosphorylation of protein kinase A substrates involved in capacitation. Future research is warranted to examine the associations between phthalate exposure and capacitation in human spermatozoa.

Paternal factors in recurrent pregnancy loss: an insight through analysis of non-synonymous single-nucleotide polymorphism in human testis-specific chaperone HSPA2 gene.

Heat shock protein A2 (HSPA2) is a testis-specific molecular chaperone of the 70 kDa heat shock protein (HSP70) family and reported to play a key role in spermatogenesis as well as in the remodelling of the sperm surface during capacitation. It is established that mice lacking HSPA2 gene are infertile and spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. However, its role in post fertilization events is not fully understood. Owing to the importance of HSPA2 in male reproduction, the present study is undertaken to reveal the association between genetic mutation and phenotypic variation in recurrent pregnancy loss (RPL) patients through an in silico prediction analysis. In this study, we used different computational tools and servers such as SIFT, PolyPhen2, PROVEAN, nsSNPAnalyzer, and SNPs & GO to analyse the functional consequences of the nsSNPs in human HSPA2 gene. The most damaging amino acid variants generated were subjected to I-Mutant 2.0 and ConSurf. Post-translational modifications such as phosphorylation mediated by these deleterious nsSNPs were analysed using NetPhos 2.0, and gene-gene interaction study was conducted using GeneMANIA. Finally, in-depth studies of the nsSNPs were studied through Project HOPE. The findings of the study revealed 18 nsSNPs to be deleterious using a combinatorial bioinformatic approach. Further functional analysis suggests that screening of nsSNP variants of HSPA2 that tend to be conserved and has potential to undergo phosphorylation at critical positions (rs764410231, rs200951589, rs756852956) may be useful for predicting outcome in altered reproductive outcome. The physicochemical alterations and its impact on the structural and functional conformity were determined by Project HOPE. Gene-gene interaction depicts its close association with antioxidant enzyme (SOD1) strongly supporting an inefficient oxidative scavenging regulatory mechanism in the spermatozoa of RPL patients as reported earlier. The present study has thus identified high-risk deleterious nsSNPs of HSPA2 gene and would be beneficial in the diagnosis and prognosis of the paternal effects in RPL patients.

Proteomic Signatures in Spermatozoa Reveal the Role of Paternal Factors in Recurrent Pregnancy Loss.

PURPOSE: To identify the paternal factors responsible for aberrant embryo development leading to loss of foetus in recurrent pregnancy loss (RPL) through proteomic analysis of ejaculated spermatozoa. MATERIALS AND METHODS: This prospective study consisted of male partners of RPL patients (n=16) experienced with two or more consecutive unexplained miscarriages and with no female factor abnormality as revealed by gynaecologic investigation including karyotyping and age matched fertile healthy volunteers (n=20). All samples were collected during 2013 to 2015 after getting institutional ethical approval and written consent from the participants. Seminal ejaculates were collected by masturbation after 2 to 3 days of sexual abstinence and analyzed according to World Health Organization 5th criteria 2010. Two-dimensional difference gel electrophoresis followed by mass spectrophotometric analysis was used to identify differentially expressed proteins (DEPs). Western blotting was used for validation of the key proteins. RESULTS: The data identified 36 protein spots to be differentially expressed by more than 2-fold change with p<0.05 considered as significant. Matrix-assisted laser desorption/ionization time of flight/mass spectrometry identified GPx4, JIP4, ZN248 to be overexpressed while HSPA2, GSTM5, TF3C1, CC74A was underexpressed in RPL group. Western blot analysis confirmed the differential expression of key redox associated proteins GPx4 and HSPA2 in the RPL group. Functional analysis revealed the involvement of key biological processes that includes spermatogenesis, response to oxidative stress, protein folding and metabolic process. CONCLUSIONS: The present study provides a snapshot of the altered protein expression levels consistent with the potential involvement of the sperm chromatin landscape in early embryonic development.

Quantitative proteomics decodes clusterin as a critical regulator of paternal factors responsible for impaired compensatory metabolic reprogramming in recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a perplexing problem experienced with two or more consecutive miscarriages wherein the cause remains unexplained in >50% of cases. However, despite several evidences of involvement of paternal factors on early embryogenesis and placental development, its contribution towards RPL has been largely unexplored. There is augmented lipid peroxidation, protein carbonylation, thionylation and enhanced histone retention in spermatozoa of RPL patients. Differentially expressed proteins in the spermatozoa of RPL patients may contribute towards aberrant embryo development and pregnancy loss. The present study comprised of male partners of RPL patients (n = 16) with the absence of any female factor abnormality and age-matched fertile healthy donors (n = 20). Pooled sperm samples from each group were subjected to high-throughput liquid chromatography-tandem mass spectrophotometry (LC-MS/MS) and subsequent bioinformatic analysis that identifies key proteins to be differentially expressed (DEPs). A total of 23 DEPs were identified with ≥2.0 fold change were considered to be significant. A key finding of the study was clusterin (CLUS), a predominant oxidative stress protein that takes part in an array of pre- and post-fertilisation molecular processes, found to be underexpressed as it was confirmed by Western blot analysis. This pilot study supports contributions of paternal oxidative predominance in RPL and encourages further investigation.

Redox regulation & sperm function: A proteomic insight.

Infertility affects nearly 15 per cent of all couples within the reproductive age worldwide, with about 50 per cent being exhibited in the male, called male factor infertility. Successful reproduction is dependent on sperm chromatin integrity. Spermatozoa are highly specialized cells that aim to transmit the paternal genomic blueprint to the oocyte. The spermatozoon is regulated by redox mechanisms during its epididymal transit to acquire fertilizing ability. While, at physiological levels, the production of reactive oxygen species (ROS) supports the spermatozoon to acquire its fertilizing ability, at high concentrations, it affects sperm function leading to infertility. Emerging proteomic technologies provide an opportunity to address these key issues that may solve many fertility-associated problems resulting from oxidative stress (OS). This review highlights the need for an efficient therapeutic approach to male infertility with the application of high-throughput OS-mediated proteomic technology, and also addresses the question as to whether targeting these altered sperm-specific proteins may help in designing an efficient and reversible male contraceptive.

Histone retention, protein carbonylation, and lipid peroxidation in spermatozoa: Possible role in recurrent pregnancy loss.

Contribution from a defective paternal genome has been attributed to be an important cause for spontaneous recurrent pregnancy loss (RPL). Increased oxidative stress results in decreased detoxification and is a cause for damage to chromatin, proteins, and membrane lipids. The present study aimed to explore if there is a significant relationship between retained histones due to defective packaging of DNA in spermatozoa and oxidative stress. RPL patients (n=16) with a history of ≥2 embryo losses before the 20th week of gestation and no female factor abnormality, and fertile healthy volunteers (n=20) as controls were included in the study. A significant difference in the levels of protein carbonylation and lipid peroxidation together with an increased retention of histones in the experimental groups was noticed. Histone carrying sites for oxidative modification such as arginine and lysine might be responsible for disturbing the paternal epigenomic control during early stages of embryonic differentiation leading to abortion.

Oxidative phosphorylation versus glycolysis: what fuel do spermatozoa use?

Spermatozoa are highly specialized cells. Adenosine triphosphate (ATP), which provides the energy for supporting the key functions of the spermatozoa, is formed by 2 metabolic pathways, namely glycolysis and oxidative phosphorylation (OXPHOS). It is produced in the mitochondria through OXPHOS as well as in the head and principal piece of the flagellum through glycolysis. However, there is a great discrepancy as to which method of ATP production is primarily utilized by the spermatozoa for successful fertilization. Mitochondrial respiration is considered to be a more efficient metabolic process for ATP synthesis in comparison to glycolysis. However, studies have shown that the diffusion potential of ATP from the mitochondria to the distal end of the flagellum is not sufficient to support sperm motility, suggesting that glycolysis in the tail region is the preferred pathway for energy production. It is suggested by many investigators that although glycolysis forms the major source of ATP along the flagellum, energy required for sperm motility is mainly produced during mitochondrial respiration. Nevertheless, some studies have shown that when glycolysis is inhibited, proper functioning and motility of spermatozoa remains intact although it is unclear whether such motility can be sustained for prolonged periods of time, or is sufficiently vigorous to achieve optimal fertilization. The purpose of this article is to provide an overview of mammalian sperm energy metabolism and identify the preferred metabolic pathway for ATP generation which forms the basis of energy production in human spermatozoa during fertilization.

Sperm Proteome: What Is on the Horizon?

As the mammalian spermatozoa transcends from the testis to the end of the epididymal tubule, the functionally incompetent spermatozoa acquires its fertilizing capability. Molecular changes in the spermatozoa at the posttesticular level concern qualitative and quantitative modifications of proteins along with their sugar moieties and membranous lipids mostly associated with motility, egg binding, and penetration processes. Proteomic studies have identified numerous sperm-specific proteins, and recent reports have provided a further understanding of their function with respect to male fertility. High-throughput techniques such as mass spectrometry have shown drastic potential for the identification and study of sperm proteins. In fact, compelling evidence has provided that proteins are critically important in cellular remodeling event and that aberrant expression is associated with pronounced defects in sperm function. This review highlights the posttesticular functional transformation in the epididymis and female reproductive tract with due emphasis on proteomics.

Proteomic analysis of seminal fluid from men exhibiting oxidative stress.

BACKGROUND: Seminal plasma serves as a natural reservoir of antioxidants. It helps to remove excessive formation of reactive oxygen species (ROS) and consequently, reduce oxidative stress. Proteomic profiling of seminal plasma proteins is important to understand the molecular mechanisms underlying oxidative stress and sperm dysfunction in infertile men. METHODS: This prospective study consisted of 52 subjects: 32 infertile men and 20 healthy donors. Once semen and oxidative stress parameters were assessed (ROS, antioxidant concentration and DNA damage), the subjects were categorized into ROS positive (ROS+) or ROS negative (ROS-). Seminal plasma from each group was pooled and subjected to proteomics analysis. In-solution digestion and protein identification with liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by bioinformatics analyses was used to identify and characterize potential biomarker proteins. RESULTS: A total of 14 proteins were identified in this analysis with 7 of these common and unique proteins were identified in both the ROS+ and ROS- groups through MASCOT and SEQUEST analyses, respectively. Prolactin-induced protein was found to be more abundantly present in men with increased levels of ROS. Gene ontology annotations showed extracellular distribution of proteins with a major role in antioxidative activity and regulatory processes. CONCLUSIONS: We have identified proteins that help protect against oxidative stress and are uniquely present in the seminal plasma of the ROS- men. Men exhibiting high levels of ROS in their seminal ejaculate are likely to exhibit proteins that are either downregulated or oxidatively modified, and these could potentially contribute to male infertility.

Proteomic analysis of human spermatozoa proteins with oxidative stress.

BACKGROUND: Oxidative stress plays a key role in the etiology of male infertility. Significant alterations in the sperm proteome are associated with poor semen quality. The aim of the present study was to examine if elevated levels of reactive oxygen species cause an alteration in the proteomic profile of spermatozoa. METHODS: This prospective study consisted of 52 subjects: 32 infertile men and 20 normal donors. Seminal ejaculates were classified as ROS+ or ROS- and evaluated for their proteomic profile. Samples were pooled and subjected to LC-MS/MS analysis through in-solution digestion of proteins for peptide characterization. The expression profile of proteins present in human spermatozoa was examined using proteomic and bioinformatic analysis to elucidate the regulatory pathways of oxidative stress. RESULTS: Of the 74 proteins identified, 10 proteins with a 2-fold difference were overexpressed and 5 were underexpressed in the ROS+ group; energy metabolism and regulation, carbohydrate metabolic processes such as gluconeogenesis and glycolysis, protein modifications and oxidative stress regulation were some of the metabolic processes affected in ROS+ group. CONCLUSIONS: We have identified proteins involved in a variety of functions associated with response and management of oxidative stress. In the present study we focused on proteins that showed a high degree of differential expression and thus, have a greater impact on the fertilizing potential of the spermatozoa. While proteomic analyses identified the potential biomarkers, further studies through Western Blot are necessary to validate the biomarker status of the proteins in pathological conditions.

Functional proteomic analysis of seminal plasma proteins in men with various semen parameters.

BACKGROUND: Alterations at the molecular level in spermatozoa and seminal plasma can affect male fertility. The objective of this study was to determine if analysis of differential expression of proteins in varying semen parameters can serve as potential biomarkers for male infertility. METHODS: The differential expression of proteins in the seminal plasma of men based on sperm count and morphology were examined utilizing proteomic tools. Subjects were categorized based on sperm concentration and morphology into 4 groups: 1) normal sperm count and normal morphology (NN); 2) normal sperm count and abnormal morphology (NA); 3) oligozoospermia and normal morphology (ON); and 4) oligozoospermia and abnormal morphology (OA). Proteomic analysis was performed by LC-MS/MS followed by functional bioinformatics analysis. Protein distribution in the NA, ON and OA groups was compared with that of the NN group. RESULTS: Twenty proteins were differentially expressed among the 4 groups. Among the unique proteins identified, 3 were downregulated in the NA group, 1 in the ON group and 1 in the OA group while 2 were upregulated in the ON and OA groups. The functional analysis 1) identified biological regulation as the major processes affected and 2) determined that most of the identified proteins were of extracellular origin. CONCLUSIONS: We have identified proteins that are over-or underexpressed in the seminal plasma of men with poor sperm quality. The distinct presence of some of the proteins may serve as potential biomarkers and provide insight into the mechanistic role played by these proteins in male infertility. Further studies using Western Blot analysis are required to validate these findings.