Role Of Sperm Research Articles

Male Factors: the Role of Sperm in Preimplantation Embryo Quality.

Few studies have been conducted on the paternal effects on embryogenesis as compared with studies on maternal effects. The fertility potential of sperm decreases with genomic material abnormalities. Damaged DNA of sperm has been correlated with poor fertilization, reduced implantation and pregnancy rates, and increased production of aneuploid embryos. Evidence suggests that the role of sperm in embryogenesis goes beyond genomic material transfer, and centrosomes, sperm-derived cytoplasmic factors, paternal mRNA, and small RNAs are essential for early embryonic development. Epigenetic factors like histone modification and DNA methylation participate in the regulation of gene expression in sperm. The etiology of sperm chromatin abnormalities is important in male fertility and may affect reproductive outcomes. Success in implantation depends on the quality of the fertilized sperm and oocyte as well as the type of assisted reproductive techniques. Therefore, male factors affecting development of embryo can play a role in the failure of assisted reproductive techniques. Further studies are needed to evaluate clinical aspects and the risks of transmitting genetic or epigenetic disorders to provide safe therapies for infertility.

Interaction of sperm with endometrium can regulate genes involved in endometrial receptivity pathway in mice: An experimental study.

BackgroundMany researchers consider implantation and endometrial receptivity as pertinent issues in reproductive science. Although, several experiments have been performed and their results evaluated, yet there is no confirmed evidence about the related factors and the role of sperm in endometrial receptivity.ObjectiveTo investigate the effect of the sperm-endometrium interaction in regulating genes involved in the endometrial receptivity pathway.Materials and Methods In this experimental study, 10 male and 30 female NMRI mice were included, and half of the male cases were vasectomized. The subjects were divided into two groups as follows; group 1 (case) comprised of 15 females mated with 5 non-vasectomized male mice, while group 2 (control) consisted of 15 females mated with 5 vasectomized males. Cases were sacrificed and assessed after 36 hr and the endometrial tissue was extracted and kept at -80°C until the next use. The expression of the endometrial receptivity pathway genes, including VEGF, HBEGF, FGF2, EGF, LIF, LIFR, HOXA10, MUC1, PGR, and CSF, was examined in both groups. For statistical analysis, an independent samples test (Mean SD) was used.ResultsThe mRNA levels of LIF (p = 0.045), LIFR (p = 0.040), MUC1 (p = 0.032), VEGF (p = 0.022), EFG (p = 0.035), and FGF2 (p = 0.040) were significantly upregulated in the case group compared with the control group.ConclusionFinally, seminal plasma was observed to be effective in expressing the involved genes in the successful implantation pathway, including LIF, LIFR, MUC1, VEGF, EGF, and FGF2.

Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice

Sperm-oocyte fusion is a critical event in mammalian fertilization, categorized by three indispensable proteins. Sperm membrane protein IZUMO1 and its counterpart oocyte membrane protein JUNO make a protein complex allowing sperm to interact with the oocyte, and subsequent sperm-oocyte fusion. Oocyte tetraspanin protein CD9 also contributes to sperm-oocyte fusion. However, the fusion process cannot be explained solely by these three essential factors. In this study, we focused on analyzing a testis-specific gene 4930451I11Rik and generated mutant mice using the CRISPR/Cas9 system. Although IZUMO1 remained in 4930451I11Rik knockout (KO) spermatozoa, the KO spermatozoa were unable to fuse with oocytes and the KO males were severely subfertile. 4930451I11Rik encodes two isoforms: a transmembrane (TM) form and a secreted form. Both CRISPR/Cas9-mediated TM deletion and transgenic (Tg) rescue with the TM form revealed that only the TM form plays a critical role in sperm-oocyte fusion. Thus, we renamed this TM form Fertilization Influencing Membrane Protein (FIMP). The mCherry-tagged FIMP TM form was localized to the sperm equatorial segment where the sperm-oocyte fusion event occurs. Thus, FIMP is a sperm-specific transmembrane protein that is necessary for the sperm-oocyte fusion process.

Experimental induction and mathematical modeling of Ca 2+ dynamics in rat round spermatids

ABSTRACT Cytosolic Ca2+ concentration ([Ca2+ ]) has an important role in spermatozoa and hence it regulates fertilization. In male germinal cells, there are indirect evidences that this ion could regulate physiological processes in spermatogenesis. Since little is known about Ca 2+ homeostasis in spermatogenic cells, in this work we propose a mathematical model that accounts for experimental [Ca2+ ] dynamics triggered by blockade of the SERCA transport ATPase with thapsigargin in round rat spermatids, without external Ca2+ and with different extracellular lactate concentrations. The model included three homogeneous calcium compartments and Ca2+-ATPase activities sensitive and insensitive to thapsigargin, and it adjusted satisfactorily the experimental calcium dynamic data. Moreover, an extended version of the model satisfactorily adjusted the stationary states of calcium modulated by extracellular lactate, which is consistent with the participation of a low affinity lactate transporter and further lactate metabolism in these cells. Further studies and modeling would be necessary to shed some light into the relation between Ca 2+-lactate-ATP homeostasis and cell–cell interactions in the seminiferous tubules that are expected to modulate Ca 2+ dynamics by hormonal factors or energetic substrates in meiotic and postmeiotic spermatogenic cells.

Protective Role of Peroxiredoxins against Reactive Oxygen Species in Neonatal Rat Testicular Gonocytes

Peroxiredoxins (PRDXs) are antioxidant enzymes that protect cells from oxidative stress and play a role in reactive oxygen species (ROS)-mediated signaling. We reported that PRDXs are critical for human fertility by maintaining sperm viability and regulating ROS levels during capacitation. Moreover, studies on Prdx6−/− mice revealed the essential role of PRDX6 in the viability, motility, and fertility competence of spermatozoa. Although PRDXs are abundant in the testis and spermatozoa, their potential role at different phases of spermatogenesis and in perinatal germ cells is unknown. Here, we examined the expression and role of PRDXs in isolated rat neonatal gonocytes, the precursors of spermatogonia, including spermatogonial stem cells. Gene array, qPCR analyses showed that PRDX1, 2, 3, 5, and 6 transcripts are among the most abundant antioxidant genes in postnatal day (PND) 3 gonocytes, while immunofluorescence confirmed the expression of PRDX1, 2, and 6 proteins. The role of PRDXs in gonocyte viability was examined using PRDX inhibitors, revealing that the 2-Cys PRDXs and PRDX6 peroxidases activities are critical for gonocytes viability in basal condition, likely preventing an excessive accumulation of endogenous ROS in the cells. In contrast to its crucial role in spermatozoa, PRDX6 independent phospholipase A2 (iPLA2) activity was not critical in gonocytes in basal conditions. However, under conditions of H2O2-induced oxidative stress, all these enzymatic activities were critical to maintain gonocyte viability. The inhibition of PRDXs promoted a two-fold increase in lipid peroxidation and prevented gonocyte differentiation. These results suggest that ROS are produced in neonatal gonocytes, where they are maintained by PRDXs at levels that are non-toxic and permissive for cell differentiation. These findings show that PRDXs play a major role in the antioxidant machinery of gonocytes, to maintain cell viability and allow for differentiation.

Role of spermatozoa in pH stability of caudal epididymis environment

Role of spermatozoa in pH stability of caudal epididymis environment

The Evolution of Centriole Structure: Heterochrony, Neoteny, and Hypermorphosis.

Centrioles are subcellular organelles that were present in the last eukaryotic common ancestor, where the centriole's ancestral role was to form cilia. Centrioles have maintained a remarkably conserved structure in eukaryotes that have cilia, while groups that lack cilia have lost their centrioles, highlighting the structure-function relationship that exists between the centriole and the cilium. In contrast, animal sperm cells, a ciliated cell, exhibit remarkable structural diversity in the centriole. Understanding how this structural diversity evolved may provide insight into centriole assembly and function, as well as their unique role in sperm. Here, we apply concepts used in the study of the evolution of animal morphology to gain insight into the evolution of centriole structure. We propose that centrioles with an atypical structure form because of changes in the timing of centriole assembly events, which can be described as centriolar "heterochrony." Atypical centrioles of insects and mammals appear to have evolved through different types of heterochrony. Here, we discuss two particular types of heterochrony: neoteny and hypermorphosis. The centriole assembly of insect sperm cells exhibits the retention of "juvenile" centriole structure, which can be described as centriolar "neoteny." Mammalian sperm cells have an extended centriole assembly program through the addition of novel steps such as centrosome reduction and centriole remodeling to form atypical centrioles, a form of centriole "hypermorphosis." Overall, centriole heterochrony appears to be a common mechanism for the development of the atypical centriole during the evolution of centriole assembly of various animals' sperm.

Quantification of Mitochondrial Genome Transcription in Male Reproductive Cells by Real-Time PCR.

Mitochondria are organelles that play a key role in the regulation of cell energy metabolism, biosynthesis, and cell death. Mitochondria are also involved in important physiological processes, such as the three-carboxylic acid cycle, the oxidation of fatty acids and amino acids, and calcium ion homeostasis. The energy demands of male germ cells during mitosis and meiosis are higher than those of somatic cells, suggesting that mitochondria play a critical role in sperm. Mitochondria are nanotoxicity targets in male germ cells. The level of mitochondrial genome transcription is reflective of mitochondrial function and can therefore be used as a quantitative index for evaluating nanotoxicity. This study describes how to use real-time PCR to evaluate the effects of nanomaterials on mitochondrial genome transcription in male reproductive cells.

RNA in Human Sperm and Some Problems of Male Fertility

The progress of genomic technologies has led to an increased understanding of the role of spermatozoa and spermioplasm RNA complex which is associated with male fertility. In this review, information about functional role of spermatozoal RNA such as messenger RNA and different types of noncoding RNAs is discussed. Data on the relationship between specific noncoding sperm RNAs and male infertility, oocyte fertilization, and early embryo development are discussed. The potential role of sperm RNAs as a predictor of outcome of assisted reproductive technologies (ART) is highlighted. Accumulation and processing of data on a unique RNA pattern in human spermatozoa will improve the algorithm for examining infertile couples, make it possible to increase the efficiency of ART, and help to choose the best way to overcome infertility.

The Role of Phospholipase in Sperm Physiology and its Therapeutic Potential in Male Infertility

In mammals, the role of sperm in the process of fertilization is complex, and several well-defined steps must occur for the sperm to fertilize with the oocyte. Phospholipases (PLs), which regulate the lipid composition of the sperm by hydrolyzation of the phospholipids, are involved in the regulation of the flagellar beat, capacitation, and the acrosomal reaction, as well as in the triggering of the calcium oscillations in the oocyte that lead to oocyte activation. The emerging important role of phospholipases is also revealed by the fact that alterations of sperm lipids can lead to infertility. Phospholipases, especially PLCζ, could represent therapeutic targets to overcome male infertility.

Cyclic AMP and glycogen synthase kinase 3 form a regulatory loop in spermatozoa.

The multifaceted glycogen synthase kinase (GSK3) has an essential role in sperm and male fertility. Since cyclic AMP (cAMP) plays an important role in sperm function, we investigated whether GSK3 and cAMP pathways may be interrelated. We used GSK3 and soluble adenylyl cyclase (sAC) knockout mice and pharmacological modulators to examine this relationship. Intracellular cAMP levels were found to be significantly lower in sperm lacking GSK3α or GSK3β. A similar outcome was observed when sperm cells were treated with SB216763, a GSK3 inhibitor. This reduction of cAMP levels was not due to an effect on sperm adenylyl cyclase but was caused by elevated phosphodiesterase (PDE) activity. The PDE4 inhibitor RS25344 or the general PDE inhibitor IBMX could restore cAMP levels in sperm lacking GSK3α or β-isoform. PDE activity assay also showed that hyperactivated PDE4 contributes in lowering of cAMP levels in GSK3α null sperm suggesting that in wild-type sperm PDE4 activity is kept in check by GSK3. Conversely, PKA being triggered by cAMP, affected GSK3 activity through increasing its phosphorylation. Increased GSK3 phosphorylation also occurred by inhibition of sperm specific protein phosphatase type 1, PP1γ2. The relationship between cAMP, GSK3, and PP1γ2 activities was also confirmed in sperm from sAC null mice. Pull-down assay using recombinant PP1γ2 indicated that PKA, GSK3, and PP1γ2 could exist as a complex. Pharmacological inhibition of GSK3 in mature spermatozoa resulted in significantly reduced fertilization of eggs in vitro. Our results show that cAMP, PKA, and GSK3 are interrelated in regulation of sperm function.

O ρόλος των ενεργοποιών του πλασμινογόνου και της πλασμίνης στην αναπαραγωγή των θηλαστικών

The current knowledge of the role of local and directed fibrinolysis controlled by plasminogen activators (PAs) and regulated by plasminogen activator inhibitors (PAls) in reproduction is summarized. The PA system has been found to play an important role in spermatogenesis in testis and modulation of sperm maturation in epididymis while a lot of studies indicate a role for sperm or seminal plasma PAs in sperm hyperactivation and/or capacitation. Hormoneinduced expression of tissue-type PA (tPA) and PAI-1 in the ovary is involved in the processes of ovulation and luteal regression; increases of urokinase-type PA (uPA) and PAI-1 in the early stage of luteinized follicles may be responsible for ovarian tissue remodeling and angiogenesis. The targeted proteolytic activity plays an essential role in the processes of the cyclic uterine angiogenesis, implantation and placentation as well as in the parturition. As the PA system is involved in multiple phases of mammalian fertilization specific regulatory molecules of this system provide opportunities for pharmacological intervention.

Sperm Affects Head Sensory Neuron in Temperature Tolerance of Caenorhabditis elegans.

Tolerance to environmental temperature change is essential for the survival and proliferation of animals. The process is controlled by various body tissues, but the orchestration of activity within the tissue network has not been elucidated in detail. Here, we show that sperm affects the activity of temperature-sensing neurons (ASJ) that control cold tolerance in Caenorhabditis elegans. Genetic impairment of sperm caused abnormal cold tolerance, which was unexpectedly restored by impairment of temperature signaling in ASJ neurons. Calcium imaging revealed that ASJ neuronal activity in response to temperature was decreased in sperm mutant gsp-4 with impaired protein phosphatase 1 and rescued by expressing gsp-4 in sperm. Genetic analysis revealed a feedback network in which ASJ neuronal activity regulates the intestine through insulin and a steroid hormone, which then affects sperm and, in turn, controls ASJ neuronal activity. Thus, we propose that feedback between sperm and a sensory neuron mediating temperature tolerance.

Oviductosome-Sperm Membrane Interaction in Cargo Delivery: DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM)

Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca(2+) efflux pump, plasma membrane Ca(2+)ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4-64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the αv integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving αvβ3 and α5β1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes.

Identification of ADAM10 and ADAM17 with potential roles in the spermatogenesis of the Chinese mitten crab, Eriocheir sinensis

The ADAM (a disintegrin and metalloprotease) family plays an important role in sperm and egg fusion, development, inflammation, adhesion and migration. ADAM10 and ADAM17 are involved in the spermatogenesis. To better understand the role of ADAM10 and ADAM17 in the Chinese mitten crab, Eriocheir sinensis, the full-length cDNAs of ADAM10 and ADAM17 were cloned, and named Es-ADAM10 and Es-ADAM17, respectively. Sequence and structural analysis showed that Es-ADAM10 and Es-ADAM17 have the typical structure of the ADAM family. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that Es-ADAM10 and Es-ADAM17 mRNAs were distributed in the heart, hepatopancreas, intestines, brain, muscle, thoracic ganglia, hemolymph, stomach, testis, ovary, gill and accessory gland. Both mRNAs were highly expressed in the muscles, and relatively high in the testis, ovary and accessory gland. In addition, the Es-ADAM17 mRNA level was detected in every stage of testis development, being relatively high from July to September, the lowest during October and November, increasing from December to January, and reached a peak in January. By contrast, the expression of Es-ADAM10 mRNA was constant during testis development. Immunofluorescence further showed that Es-ADAM10 and Es-ADAM17 proteins were present in the cytoplasm and cytomembrane of spermatocytes, and both detected in the sperm. Furthermore, etoposide induced upregulation of Es-ADAM17 and Es-ADAM10 at both the mRNA and protein levels. This study first showed that Es-ADAM10 and Es-ADAM17 were also involved in the spermatogenesis and mainly participated in the later germ cell apoptosis in E. sinensis.

Sperm-Impairing Escherichia coli, Alcohol and Male Infertility: An In Vivo Study

Purpose: Establishment of a male Balb/c mouse model to study the role of sperm immobilizing E. coli with or without alcohol on mouse reproductive potential. Materials and Methods: The mice in the experimental groups were intraperitoneally administered with different doses (104, 106 or 108 cfu) of sperm immobilizing E. coli alone or in combination with alcohol and as control group alcohol alone or PBS for 10 consecutive days. The groups were evaluated for change in body weight, seminal parameters, bacterial load and histopathological changes. Results: Weight gain was observed only in mice receiving PBS, whereas there was decrease in weight in all the groups except the group receiving 104 cfu where weight remained constant. No significant change in tissue somatic index was observed in any group. Reduced motility and viability was noticed in all groups except group receiving PBS. Decapitation was observed in groups instilled with 104 cfu and 104 cfu along with alcohol. Seminal parameters could not be evaluated due to non-specific aggregation of sperms in group administered with 106 and 108 cfu alone as well as with alcohol. Histopathological studies of testes showed very few mature spermatozoa and maturation arrest at later stage in all groups except PBS group. Conclusion: Intraperitoneal inoculation with sperm immobilizing E. coli with or without alcohol consumption could lead to alteration of semen parameters, induction of decapitation in spermatozoa, thereby decreasing the reproductive potential of male mice.

BRWD1 shows its gender bias

Protein necessary for fertility has distinct roles in sperm and eggs.

Role of zona pellucida glycoproteins during fertilization in humans

In the last decade, scientific investigations pertaining to the role of zona pellucida (ZP) glycoproteins during fertilization in humans have led to new insights. This has been achieved using purified native/recombinant human zona proteins and transgenic mice expressing human ZP glycoproteins. The proposed model in mice of ZP glycoprotein-3 (ZP3) acting as primary sperm receptor and ZP glycoprotein-2 (ZP2) as secondary sperm receptor has been modified for sperm–egg binding in humans. ZP glycoprotein-1 (ZP1), ZP3, and ZP glycoprotein-4 (ZP4) have been shown to bind to the capacitated human sperm. ZP2 binds to the acrosome-reacted human spermatozoa. Further, the eggs obtained from transgenic mice expressing human ZP2 alone or in conjunction with other human instead of mouse zona proteins showed binding of human sperm, suggesting that ZP2 might also play a role in sperm–egg binding. This function has been mapped to a domain corresponding to amino acid residues 51–144 of ZP2. In contrast to mice, where ZP3 is the primary agonist for inducing the acrosome reaction, in humans, the acrosome reaction can be mediated by ZP1, ZP3, and ZP4. The effect of mutations in the genes encoding zona proteins on the ZP morphology and infertility has not been established. Further, the role of autoantibodies against ZP in women with ‘unexplained infertility’ leading to poor outcome of in vitro fertilization is currently controversial and needs further investigations. Understanding the role of ZP glycoproteins during human fertilization facilitates the development of new contraceptives and strategies to overcome the problem of infertility.

AB219. The use of anti-VDAC2 antibody for the combined assessment of human sperm acrosome integrity and ionophore A23187-induced acrosome reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca transmembrane transport.

The Calcium/CaMKKalpha/beta and the cAMP/PKA Pathways Are Essential Upstream Regulators of AMPK Activity in Boar Spermatozoa1

Spermatozoa successfully fertilize oocytes depending on cell energy-sensitive processes. We recently showed that the cell energy sensor, the AMP-activated protein kinase (AMPK), plays a relevant role in spermatozoa by regulating motility as well as plasma membrane organization and acrosomal integrity, and contributes to the maintenance of mitochondrial membrane potential. As the signaling pathways that control AMPK activity have been studied exclusively in somatic cells, our aim is to investigate the intracellular pathways that regulate AMPK phosphorylation at Thr(172) (activity) in male germ cells. Boar spermatozoa were incubated under different conditions in the presence or absence of Ca(2+), 8Br-cAMP, IBMX, PMA, the AMPK activator A769662, or inhibitors of PKA, PKC, or CaMKKalpha/beta. AMPK phosphorylation was evaluated by Western blot using anti-phospho-Thr(172)-AMPK antibody. Data show that AMPK phosphorylation in spermatozoa is potently stimulated by an elevation of cAMP levels through the activation of PKA, as the PKA inhibitor H89 blocks phospho-Thr(172)-AMPK. Another mechanism to potently activate AMPK is Ca(2+) that acts through two pathways, PKA (blocked by H89) and CaMKKalpha/beta (blocked by STO-609). Moreover, phospho-Thr(172)-AMPK levels greatly increased upon PKC activation induced by PMA, and the PKC inhibitor Ro-32-0432 inhibits TCM-induced AMPK activation. Different stimuli considered as cell stresses (rotenone, cyanide, sorbitol, and complete absence of intracellular Ca(2+) by BAPTA-AM) also cause AMPK phosphorylation in spermatozoa. In summary, AMPK activity in boar spermatozoa is regulated upstream by different kinases, such as PKA, CaMKKalpha/beta, and PKC, as well as by the essential intracellular messengers for spermatozoan function, Ca(2+) and cAMP levels.