Cross-talk between free and bound spermatozoa to modulate initial sperm:egg ratios at the site of fertilization in the mammalian oviduct

This essay proposes that highly localized communication between free and bound spermatozoa in the caudal portion of the oviduct acts to regulate the numbers detaching from the epithelium and progressing to the site of fertilization close to the time of ovulation. Low initial sperm:egg ratios are essential for monospermic fertilization. Liberation of surface macromolecules and metabolic prompting from activated spermatozoa, together with altered patterns of sperm movement and dynamic differences in intracellular Ca²⁺ ion status between neighboring sperm cells, would influence the progressive release of spermatozoa from the reservoir in the oviduct isthmus. Different intensities of preovulatory epithelial binding, reflecting a range of states in the sperm surface membranes and associated proteins, would provide a further explanation for a chronologically staggered periovulatory detachment of spermatozoa. Intimate sperm–sperm interactions within the confines of the oviduct isthmus offer a sensitive means of fine-tuning the vanguard of competent male gametes reaching the isthmo-ampullary junction.     

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa

Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca²⁺-dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca²⁺ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca²⁺ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca²⁺ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca²⁺-dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca²⁺-dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.     

Functional Diversity and Evolution of the Drosophila Sperm Proteome

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here, we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and we provide the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. We also identified almost one-half of known Drosophila ribosomal proteins in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. We show that a significant fraction of ‘sperm-associated Sfps’ are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, our results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.     

Endoplasmic reticulum protein 29 (ERp29), a protein related to sperm maturation is involved in sperm-oocyte fusion in mouse

Background  

Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29), which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process.     

How do sperm swim? Molecular mechanisms underlying sperm motility.

Motility is a characteristic function of the male gamete, which allows spermatozoa to actively reach and penetrate the female gamete in organisms with internal and external fertilization. Sperm motility is acquired under the control of many extrinsic and intrinsic factors and is based on the specialized structure of the sperm flagellum. After a brief overview of how the sperm flagellum is organized and works to support cell motility, the present review focuses on the molecular mechanisms and factors involved in the development and maintenance of sperm motility. Data obtained both in organisms with external fertilization, such as fishes and sea urchin, and with internal fertilization, such as Mammals, are critically analyzed. In particular, a great attention has been put on the ionic mechanisms and on the involvement of protein kinases and phosphatases in regulation of sperm motility. A brief overview of the pharmacological and physiological molecules which have been studied for their possible application as therapeutic molecules for in vitro treatment of defects of sperm motility in asthenozoospermic human subjects, is presented. Moreover, we show some preliminary data obtained in our laboratory on the involvement of the phosphatydilinositol 3-kinase and the A kinase anchoring protein (AKAP3) in regulation of motility in human spermatozoa. The last section is dedicated to hyperactivation, a peculiar pattern of motility which is developed in association with capacitation occurring during sperm transit through the female genital tract and which can also be obtained in vitro by incubation in defined media.     

Haprin‐deficient spermatozoa are incapable of in vitro fertilization

Haprin (TRIM36) is a ubiquitin‐protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. It is expressed in the testes in both mice and humans and is thought to be involved in spermiogenesis, the acrosome reaction, and fertilization. However, the functional role of Haprin is poorly understood. The aim of this study was to investigate the physiological role of Haprin in fertility. Homozygous haprin‐deficient mice were generated and these mice, and their spermatozoa, were analyzed to detect morphological and fertility‐related abnormalities. In these models, normal spermatogenesis was observed but sperm quality was reduced with haprin‐deficient mice having poorer sperm morphology and motility than wild‐type mice. Interestingly, haprin‐deficient mice showed normal in vivo fertility but could not fertilize oocytes under standard in vitro fertilization conditions. In conclusion, this study demonstrated that Haprin deficiency causes morphological abnormalities in spermatozoa, indicating that Haprin is involved in spermiogenesis.     

Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa.

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to produce fertilization of the oocyte in vivo and in vitro. Although this process results from a poorly understood series of morphological and molecular events, protein tyrosine phosphorylation has been associated with sperm capacitation in several mammalian species, but it still remains to be demonstrated in ram spermatozoa. Studies of capacitation in ram spermatozoa are of great interest, since several reports have suggested that the reduced fertility of cryopreserved spermatozoa is due to their premature capacitation. In this work, we report for the first time, to our knowledge, that tyrosine phosphorylation of ram sperm membrane proteins is related to the capacitation state of these cells. Capacitation induced tyrosine phosphorylation of some plasma membrane proteins of ram spermatozoa freed from seminal plasma by a dextran/swim-up procedure. It has also been proved that cold-shock induces protein tyrosine phosphorylation as well as a decrease in plasma membrane integrity. Addition of seminal plasma proteins prior to cold-shock not only improved sperm survival but also promoted a decrease in protein tyrosine phosphorylation.     

Sperm Proteome Maturation in the Mouse Epididymis

In mammals, transit through the epididymis, which involves the acquisition, loss and modification of proteins, is required to confer motility and fertilization competency to sperm. The overall dynamics of maturation is poorly understood, and a systems level understanding of the complex maturation process will provide valuable new information about changes occurring during epididymal transport. We report the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. This study identified 765 proteins that are present in sperm obtained from all three segments. We identified 1766 proteins that are potentially added (732) or removed (1034) from sperm during epididymal transit. Phenotypic analyses of the caput, corpus and cauda sperm proteomes identified 60 proteins that have known sperm phenotypes when mutated, or absent from sperm. Our analysis indicates that as much as one-third of proteins with known sperm phenotypes are added to sperm during epididymal transit. GO analyses revealed that cauda sperm are enriched for specific functions including sperm-egg recognition and motility, consistent with the observation that sperm acquire motility and fertilization competency during transit through the epididymis. In addition, GO analyses revealed that the immunity protein profile of sperm changes during sperm maturation. Finally, we identified components of the 26S proteasome, the immunoproteasome, and a proteasome activator in mature sperm.     

Characterizing disease-associated changes in post-translational modifications by mass spectrometry

Introduction: Exploring post-translational modifications (PTMs) with the use of mass spectrometry (PTMomics) is a rapidly developing area, with methods for discovery/quantification being developed and advanced on a regular basis. PTMs are highly important for the regulation of protein function, interaction and activity, both in physiological and disease states. Changes in PTMs can either cause, or be the result of a disease, making them central for biomarker studies and studies of disease pathogenesis. Recently, it became possible to study multiple PTMs simultaneously from low amount of sample material, thereby increasing coverage of the PTMome obtainable from a single sample. Thus, quantitative PTMomics holds great potential to discover biomarkers from tissue and body fluids as well as elucidating disease mechanisms through characterization of signaling pathways.

Areas covered: Recent mass spectrometry-based methods for assessment of the PTMome, with focus on the most studied PTMs, are highlighted. Furthermore, both data dependent and data independent acquisition methods are evaluated. Finally, current challenges in the field are discussed.

Expert commentary: PTMomics holds great potential for clinical and biomedical research, especially with the generation of spectral libraries of peptides and PTMs from individual patients (permanent PTM maps) for use in personalized medicine.     

A carbohydrate-antioxidant hybrid polymer reduces oxidative damage in spermatozoa and enhances fertility

Gamete-gamete interactions are critically modulated by carbohydrate-protein interactions that rely on the carbohydrate-selective recognition of polyvalent carbohydrate structures. A galactose-binding protein has been identified in mammalian spermatozoa that has similarity to the well-characterized hepatic asialoglycoprotein receptor. With the aim of exploiting the ability of this class of proteins to bind and internalize macromolecules displaying galactose, we designed hybrid carbohydrate-antioxidant polymers to deliver antioxidant vitamin E (alpha-tocopherol) to porcine spermatozoa. Treatment of sperm cells with one hybrid polymer in particular produced large increases in intracellular sperm levels of alpha-tocopherol and greatly reduced endogenous fatty acid degradation under oxidative stress. The polymer-treated spermatozoa had enhanced physiological properties and longer half-lives, which resulted in enhanced fertilization rates. Our results indicate that hybrid polymer delivery systems can prolong the functional viability of mammalian spermatozoa and improve fertility rates, and that our functionally guided optimization strategy can be applied to the discovery of active glycoconjugate ligands.     

Assessment of human sperm protein tyrosine phosphorylation by immunocytochemistry in a clinical andrology laboratory. Preliminary data

Abstract

We propose that evaluation of protein tyrosine phosphorylation (TP) status in ejaculated spermatozoa under capacitating conditions in an experiment that mimics “in vitro” the physiology of sperm from ejaculation through the female genital tract could potentially be used as a prognostic test for functional competence of sperm in fertilization. Our purpose was to elucidate whether there is a relation between conventional sperm parameters, occurrence of TP and pregnancy outcome obtained from intrauterine insemination (IUI). Semen samples were analyzed according to WHO criteria. TP levels were determined by immunocytochemistry under four different conditions: 1) ejaculated sperm, 2) postselection sperm, 3) postselection sperm incubated 5 h at 37° C and 5% CO₂, and 4) postselection sperm incubated overnight at 37° C and 5% CO₂. Data on sperm tyrosine phosphorylated proteins did not correlate with sperm concentration, progressive motility or normal sperm morphology. TP increased under capacitating conditions and showed a time dependent pattern except for five outlier cases. IUI was performed in 12 selected couples who had neither female nor male infertility factors. The three pregnancies had a time dependent pattern for TP. Of the unsuccessful cases, one had an outlier TP pattern. It appears that a TP time dependent pattern is necessary for fertilization.     

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.     

Evidence for nonmuscle myosin in bovine ejaculated spermatozoa

Mammalian spermatozoa contain nonmuscle actin and many of the components of regulatory systems thought to be involved in nonmuscle actin-myosin function. An actin-stimulated adenosine triphosphate hydrolase (ATPase) activity has been fractionated from bovine ejaculated spermatozoa by immobilized-actin affinity chromatography. The actinstimulated ATPase activity has a specific activity (0.04 ± 0.02 mM phosphate released/min/mg protein) similar to nonmuscle myosins from other mammalian cells and tissues, but it does not have appreciable K⁺-EDTA ATPase activity. The sperm actin-myosin may function in sperm morphogenesis in the seminiferous tubule, in capacitated spermatozoa undergoing an acrosome reaction, or in decondensation of the sperm nucleus after fertilization.     

Full-term development of rats from oocytes fertilized in vitro using cryopreserved ejaculated sperm

For preservation of rat spermatozoa, the general-purpose method requires that the male be sacrificed for collection of spermatozoa from the epididymides. However, it would be highly useful if the ejaculated spermatozoa could be successfully cryopreserved and the frozen–thawed spermatozoa used for in vitro fertilization, since this would allow the genetically valuable rats to be maintained alive rather than sacrificed. The aim of the present study was to clarify whether ejaculated rat spermatozoa could be successfully cryopreserved and fertilized in vitro. The motility and viability of frozen–thawed ejaculated spermatozoa were similar to those of frozen–thawed epididymal spermatozoa (around 10%). The percentage of acrosomal integrity in epididymal spermatozoa was significantly higher than that in ejaculated spermatozoa after freezing/thawing. The level of capacitation-associated protein tyrosine phosphorylation in frozen–thawed ejaculated sperm was slightly increased at 5 h. When the frozen–thawed ejaculated spermatozoa were used for in vitro fertilization, the percentages of fertilization, pronuclear formation, and development to the 2-cell stage (26.5%, 23.0%, and 91.0%, respectively) were similar to those of frozen–thawed epididymal spermatozoa (19.4%, 15.0%, and 84.1%, respectively). However, the rate of blastocyst formation in the ejaculated group was significantly lower than that in the epididymal group (12.0% vs 43.2%). Results from the embryo transfer experiment showed that the proportions of embryos developed to term were similar between the ejaculated (47.7%) and epididymal groups (53.7%). We showed here for the first time that ejaculated spermatozoa can be cryopreserved and the frozen–thawed sperm could be developed to term via in vitro fertilization in rats.     

Fertilization of cumulus-free,zona-intact mouse ova in vitro at high and low sperm concentrations

The effect of varying the sperm concentration between 2 × 10⁵ sperm/ml and 8 × 10⁶ sperm/ml on fertilization of cumulus-free, zona-intact F1 (CBA × C57BL) mouse ova by QS and F1 (CBA × C57BL) mouse spermatozoa was studied. The spermatozoa from both strains of mice exhibited optimal fertilization rates at 2 × 106 sperm/ml. However, at sperm concentrations greater than 4 × 106 sperm/ml and less than 1 × 106 sperm/ml, fertilization rates were significantly reduced. F1 spermatozoa were more susceptible to dilution than QS spermatozoa. A significant interaction between strain and sperm concentration indicated that the two strains produced different fertilization rates at different sperm densities. Extracts of epididymal fluid, medium from capacitated spermatozoa, or ampulla fluid did not improve the fertilization rate at 2 × 105 sperm/ml, but retaining the cumulus oophorus did. The decrease in fertilization rate at 8 × 106 sperm/ml can in part be attributed to a nondialysable inhibitor from the neat sperm preparation that appeared to be of epididymal origin.     

Post-translational quantitation by SRM/MRM: applications in cardiology

Introduction: Post-translational modifications (PTMs) have an important role in the regulation of protein function, localization, and interaction with other molecules. PTMs apply a dynamic control of proteins in both physiological and pathological conditions. The study of disease-specific PTMs allows identifying potential biomarkers and developing effective drugs. Enrichment techniques combined with high-resolution mass spectrometry (MS)/MS analysis provide attractive results on PTM characterization. Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) is a powerful targeted assay for the quantitation and validation of PTMs in complex biological samples.

Areas covered: The most frequent PTMs are described in terms of biological role and analytical methods commonly used to detect them. The applications of SRM/MRM for the absolute quantitation of PTMs are reported, and a specific section is focused on PTM detection in proteins that are involved in the cardiovascular system and heart diseases.

Expert commentary: PTM characterization in relation to disease pathology is still in progress, but targeted proteomics by LC-MS/MS has significantly upgraded our knowledge in the last few years. Advances in enrichment strategies and software tools will facilitate the interpretation of high PTM complexity. Promising studies confirm the great potential of SRM/MRM to study PTMs in the cardiovascular field, and PTMomics could be very useful in the clinical perspective.     

The Sperm Proteome of the Echiuran Urechis unicinctus (Annelida,Echiura)

Sperm proteins presumably play critical roles in reproduction, but in many non‐model animals their identities are unknown. A total of 147 sperm proteins from the echiuran worm Urechis unicinctus, the first sperm proteome in the phylum Annelida, are reported. The echiuran sperm proteome can be classified into diverse functional groups: energy metabolism (31%), protein synthesis and degradation (18%), spermatogenesis and sperm motility (12%), signal pathway (11%), ion channel and transport proteins (6%), cytoskeleton (4%), immunity and stress responses (3%), and fertilization (1%). These results will facilitate studies of mechanisms of fertilization in echiurans, as well as comparative studies of reproduction and evolution across lophotrochozoans. Data are available via ProteomeXchange with identifier PXD009176.     

The Proteome of Pig Spermatozoa Is Remodeled During Ejaculation

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Highlights

• •Proteome of mature boar spermatozoa from cauda epididymal and ejaculated sources were analyzed by iTRAQ-based LC-MS/MS.
• •1,723 sperm proteins identified (974 of Sus scrofa taxonomy).
• •1,602 sperm proteins quantified (960 of Sus scrofa taxonomy).
• •32 Sus scrofa sperm proteins were differentially expressed among sperm sources.
• •The proteome of boar spermatozoa is remodelled during ejaculation.

     

TCP-11, the product of a mouse t-complex gene,plays a role in stimulation of capacitation and inhibition of the spontaneous acrosome reaction

Tcp-11 is a candidate for a distorter gene within the t-complex on mouse chromosome 17; although t-complex genes appear to affect sperm function, relatively little is known about mechanisms whereby these genes might play a specific physiological role. We present evidence that the protein TCP-11 is found on the surface of mature epididymal spermatozoa. Although detected on both the acrosomal cap region of the head and the flagellum of acrosome-intact cells, it is absent from the heads of acrosome-reacted cells. When epididymal spermatozoa were incubated in the presence of anti-TCP-11 IgG Fab fragments for a total of 120 min and assessed using chlortetracycline fluorescence, we observed a stimulation of capacitation and an inhibition of spontaneous acrosome loss, suggestive of enhanced fertility compared with untreated suspensions. In vitro fertilization experiments confirmed that Fab-treated suspensions became fertile more quickly and then maintained high fertility. Because these responses were remarkably similar to those obtained using the TRH-related peptide FPP (fertilization promoting peptide; pGlu-Glu-ProNH₂) and adenosine, we investigated responses to Fab fragments, FPP, and adenosine. Results indicated that the Fab fragments appear to work at the same extracellular site as FPP, one that is distinct from the adenosine site of action. Further evidence for this conclusion was obtained using pGlu-Gln-ProNH₂, an FPP-related tripeptide known to competitively inhibit responses to FPP; as with FPP, pGlu-Gln-ProNH₂ inhibited the stimulatory effect of Fab fragments in a concentration-dependent manner. From these results we suggest that TCP-11 may be the receptor for FPP and that the adenylate clyclase/cyclic AMP pathway may be the signal transduction pathway activated by interactions between extracellular effector molecules (e.g., Fab fragments or FPP acting as an agonist) and TCP-11. A mechanism such as this that promotes capacitation but inhibits spontaneous acrosome loss in vivo would play a very important role by helping to maximize the fertilizing potential of the few spermatozoa that reach the site of fertilization. The fact that there is a human homolog of Tcp-11 suggests that this gene could play an important role in regulation of human, as well as mouse, sperm function. Mol. Reprod. Dev. 48:375–382, 1997. © 1997 Wiley-Liss, Inc.     

Posttranslational modification of autophagy-related proteins in macroautophagy

Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.