Fertility-related proteomic profiling bull spermatozoa separated by percoll

Infertility or subfertility of bovine spermatozoa may lead to disintegration of the breeding system and large economic losses. Recently, proteomics have identified candidates for the sperm fertility biomarkers, but no definite studies have clearly identified the relationship between the proteome and sperm fertility after proteomic study. Therefore, to determine the clinical value of the protein markers identified by proteomic study, we first compared the protein expression profiles of spermatozoa from high and low fertility bulls using 2-dimensional electrophoresis. We then investigated the relationship between protein expression and the fertility of individual bulls as assessed by Western blot analysis. Five proteins, enolase 1 (ENO1), ATP synthase H(+) transporting mitochondrial F1 complex beta subunit, apoptosis-stimulating of p53 protein 2, alpha-2-HS-glycoprotein, and phospholipid hydroperoxide glutathione peroxide, were more highly represented in high fertility bulls, whereas three proteins, voltage dependent anion channel 2 (VDAC2), ropporin-1, and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2), were more highly represented in low fertility bulls. Among those proteins, ENO1, VDAC2, and UQCRC2 were significantly correlated with individual fertility. Therefore, these results suggest that concurrent comparisons between protein expression and other fertility assays may represent a good in vitro assay to determine sperm fertility.     

Ras-related proteins (Rab) play significant roles in sperm motility and capacitation status

Rab proteins are widely known for their involvement in establishing Golgi apparatus and controlling Golgi trafficking in eukaryotic cells. Specifically, Rab proteins play significant roles in acrosome formation and exocytosis. Furthermore, mechanisms involved in the regulation of Rab proteins during capacitation have been identified. However, there has been no direct evaluation to assess the correlation between Rab proteins and sperm function. Consequently, this study was designed to analyze the correlation between Rab proteins and sperm functions. Individually, we analyzed the sperm motility patterns, motion kinematics, capacitation status, and Rab protein expression levels of sperm samples from 31 boars before and after capacitation. As a result, we discovered that Rab3A, Rab5, Rab11, Rab14, and Rab27A correlated with various sperm motility patterns, motion kinematics before capacitation. Rab3A, Rab5, Rab11, Rab14, and Rab34 correlated with various sperm motility patterns, motion kinematics after capacitation. Moreover, Rab4 and Rab34 were associated with capacitation status before capacitation, and Rab3A, 25, and 27A correlated with capacitation status after capacitation. This is the first study to analyze the correlation between Rab proteins and sperm functions. Collectively, our results indicate that specific sperm motility and kinematics, as well as the structural condition of the sperm head and capacitation status, regulate individual Rab protein. Therefore, we expect that the current findings will be used to identify the etiology of idiopathic male infertility patients and to diagnose male fertility and that Rab proteins will be employed as biomarkers to predict and analyze male fertility.     

An Epididymis-Specific Secretory Protein HongrES1 Critically Regulates Sperm Capacitation and Male Fertility

Mammalian sperm capacitation is an essential prerequisite to fertilizion. Although progress had been made in understanding the physiology and biochemistry of capacitation, little is known about the potential roles of epididymal proteins during this process. Here we report that HongrES1, a new member of the SERPIN (serine proteinase inhibitor) family exclusively expressed in the rat cauda epididymis and up-regulated by androgen, is secreted into the lumen and covers the sperm head. Co-culture of caudal sperms with HongrES1 antibody in vitro resulted in a significant increase in the percentage of capacitated spermatozoa. Furthermore, the percentage of capacitated spermatozoa clearly increased in rats when HongrES1 was down-regulated by RNAi in vivo. Remarkably, knockdown of HongrES1 in vivo led to reduced fertility accompanied with deformed appearance of fetuses and pups. These results identify HongrES1 as a novel and critical molecule in the regulation of sperm capacitation and male fertility.     

Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.     

Ras-related proteins (Rab) are key proteins related to male fertility following a unique activation mechanism

Ras-related protein Rab (Rab) proteins, member of Ras superfamily of monomeric G proteins, are well known key regulators of intracellular vesicular transport. Recently, it has been reported that Rab 2A and 3A are related to acrosomal exocytosis in spermatozoa and Rab 2A can be used to fertility-related biomarker in male. However, the role and mechanism of Rab proteins in spermatozoa has not been fully understood yet. Therefore, the study to analyze the expression and location of various Rab proteins in spermatozoa is required to understand the role and mechanism of Rab proteins in spermatozoa. In present study, to analyze the expression level and location of various Rab proteins (Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34) and Rab protein regulators (RabGAP, RabGDI, RabGEF) in spermatozoa following capacitation, immunofluorescence and western blot analysis were performed. All of 11 Rab proteins were expressed in acrosomal region and tail of spermatozoa. Furthermore, all Rab proteins and Rab protein regulators, except RabGAP, have decreased expression patterns after capacitation. Taken together, Rab proteins were located in sperm head and tail. In addition, expression patterns of Rab proteins in spermatozoa were altered following capacitation. Therefore, our results suggested that Rab proteins may be key proteins related with capacitation as well as playing important role with uniquely activation pathway for male fertility.     

Radicaux libres et spermatozoïdes humains: physiologie et physiopathologie

The role of reactive oxygen species in the physiopathology of human sperm function has been emphasized in recent years. Their production in semen has been associated with loss of motility, decreased capacity for spermoocyte fusion and loss of fertility. In semen preparations, there are two major sources of reactive oxygen species: leucocytes and spermatozoa themselve. It has been proposed that reactive oxygen species production by human spermatozoa was dependent upon a membrane-bound NADPH oxidase or a mitochondrial diaphorase. Hydrogen peroxide produced by the dismutation of superoxide anion has been recognized as the most toxic oxidizing species for human spermatozoa. Owing to their high content of polyunsaturated fatty acids, it has been proposed that lipid peroxidation of the sperm plasma membrane is largely responsible for defective sperm function. Reactive oxygen species also affect the sperm axoneme as a result of ATP depletion, inhibit mitochondrial functions, and synthesis of DNA, RNA and proteins, produce cytoskeletal modifications and inhibit sperm-oocyte fusion. Human spermatozoa possess enzymatic defence systems such as superoxide dismutase, glutathion peroxidas/reductase and catalase to counteract the toxic effects induced by reactive oxygen species. Correlations have been reported between their effectiveness and the duration of sperm motility. If the excessive production of reactive oxygen species is detrimental for human spermatozoa, they could also participate in the physiological function of the spermatozoa when present at low concentrations. Indeed, reactive oxygen species have been shown to be involved in the activation of several enzymes. Furthermore, sperm capacitation, acrosome reaction and sperm-zona interaction would be enhanced by reactive oxygen species.     

Expression of the gene for mouse lactate dehydrogenase C (Ldhc) is required for male fertility

The lactate dehydrogenase (LDH) protein family members characteristically are distributed in tissue- and cell type-specific patterns and serve as the terminal enzyme of glycolysis, catalyzing reversible oxidation reduction between pyruvate and lactate. They are present as tetramers, and one family member, LDHC, is abundant in spermatocytes, spermatids, and sperm, but also is found in modest amounts in oocytes. We disrupted the Ldhc gene to determine whether LDHC is required for spermatogenesis, oogenesis, and/or sperm and egg function. The targeted disruption of Ldhc severely impaired fertility in male Ldhc(-/-) mice but not in female Ldhc(-/-) mice. Testis and sperm morphology and sperm production appeared to be normal. However, total LDH enzymatic activity was considerably lower in Ldhc(-/-) sperm than in wild type sperm, indicating that the LDHC homotetramer (LDH-C(4)) is responsible for most of the LDH activity in sperm. Although initially motile when isolated, there was a more rapid reduction in the level of ATP and in motility in Ldhc(-)(/-) sperm than in wild-type sperm. Moreover, Ldhc(-/-) sperm did not acquire hyperactivated motility, were unable to penetrate the zona pellucida in vitro, and failed to undergo the phosphorylation events characteristic of capacitation. These studies showed that LDHC plays an essential role in maintenance of the processes of glycolysis and ATP production in the flagellum that are required for male fertility and sperm function.     

Sterility of males determined by functional features of the mouse spermatozoa bearing t-complex

The mechanisms underlying normal spermatogenesis and its pathology expressed as male sterility determined by t-complex located on chromosome 17 in mice are considered in this review. t-Complex is a very convenient model with diverse markers of expression of the genes involved in development of the functional features of the spermatozoa bearing t-complex. These features include defects of mobility, capacitation, and acrosome reactions, which determine full or partial male sterility. It has been proposed that the defects of capacitation are also inherent in humans and affect male fertility. This homology is confirmed by the presence of the male gene Tcp11 in humans and demonstration of the fact that the protein TCP11 plays a leading role in modulation of the capacitation of murine spermatozoa. Hence it follows that the defects of human genes leading to incomplete binding of the fertilization promoting peptide could play a certain role in a decreased male fertility. All this is essential not only for deeper understanding of the biology of spermatozoa, but also for development of new therapeutic methods of finding and treating the semen pathology.     

Inhibiting Sperm Pyruvate Dehydrogenase Complex and Its E3 Subunit,Dihydrolipoamide Dehydrogenase Affects Fertilization in Syrian Hamsters

Background/Aims

The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.

Methodology and Principal Findings

Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2^nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization.

Conclusions

This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.     

Localization and significance of molecular chaperones, heat shock protein 1, and tumor rejection antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction

Although the molecular basis of sperm-oocyte interaction is unclear, recent studies have implicated two chaperone proteins, heat shock protein 1 (HSPD1; previously known as heat shock protein 60) and tumor rejection antigen gp96 (TRA1; previously known as endoplasmin), in the formation of a functional zona-receptor complex on the surface of mammalian spermatozoa. The current study was undertaken to investigate the expression of these chaperones during the ontogeny of male germ cells through spermatogenesis, epididymal sperm maturation, capacitation, and acrosomal exocytosis. In testicular sections, both HSPD1 and TRA1 were closely associated with the mitochondria of spermatogonia and primary spermatocytes. However, this labeling pattern disappeared from the male germ line during spermiogenesis to become undetectable in testicular spermatozoa. Subsequently, these chaperones could be detected in epididymal spermatozoa and in previously unreported "dense bodies" in the epididymal lumen. The latter appeared in the precise region of the epididymis (proximal corpus), where spermatozoa acquire the capacity to recognize and bind to the zona pellucida, implicating these structures in the functional remodeling of the sperm surface during epididymal maturation. Both HSPD1 and TRA1 were subsequently found to become coexpressed on the surface of live mouse spermatozoa following capacitation in vitro and were lost once these cells had undergone the acrosome reaction, as would be expected of cell surface molecules involved in sperm-egg interaction. These data reinforce the notion that these chaperones are intimately involved in the mechanisms by which mammalian spermatozoa both acquire and express their ability to recognize the zona pellucida.     

Sterility of Males Determined by Functional Features of the Mouse Spermatozoa Bearing t-Complex

The mechanisms underlying normal spermatogenesis and its pathology expressed as male sterility determined by t-complex located on chromosome 17 in mice are considered in this review. t-Complex is a very convenient model with diverse markers of expression of the genes involved in development of the functional features of the spermatozoa bearing t-complex. These features include defects of mobility, capacitation, and acrosome reactions, which determine full or partial male sterility. It has been proposed that the defects of capacitation are also inherent in humans and affect male fertility. This homology is confirmed by the presence of the male gene Tcp11 in humans and demonstration of the fact that the protein TCP11 plays a leading role in modulation of the capacitation of murine spermatozoa. Hence it follows that the defects of human genes leading to incomplete binding of the fertilization promoting peptide could play a certain role in a decreased male fertility. All this is essential not only for deeper understanding of the biology of spermatozoa, but also for development of new therapeutic methods of finding and treating the pathology of spermatozoa.     

In vitro Capacitation of Ejaculated Rabbit Spermatozoa

Prior to fertilization mammalian spermatozoa undergo physiological changes in the female reproductive tract. These changes are collectively known as capacitation. In essence capacitation is a further differentiation that the sperm cell must acquire beyond the maturational changes that it undergoes in the epididymis. Although capacitation in vitro has been easily achieved in rodents, its accomplishment in the rabbit is inconsistent and difficult. We report here successful in vitro capacitation of ejaculated rabbit sperm, assessed by in vitro and in vivo fertilization of rabbit ova. Sperm were used from pooled ejaculates collected from bucks of proven fertility since sperm collected from individual bucks resulted in significant differences in fertilization levels. Conditions favoring in vitro capacitation were: (1) extended incubation time of 12 h, (2) addition of 20% heated rabbit serum to incubation medium, and (3) an atmosphere of 5% CO2, 8% O2, and 87% N2 during incubation. In vitro capacitation of sperm under these conditions resulted in 67% fertilization compared to 89% for control sperm capacitated for 13 h in the uterus.     

Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activity and sperm motility in mice

During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that thioredoxin-glutathione reductase (TXNRD3), a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male fertility. Using Txnrd3^−/− mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We find that mitochondria develop more defined cristae during capacitation in wildtype sperm. Furthermore, we show that absence of TXNRD3 alters thiol redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization.     

A differential mechanism is involved during heparin- and cryopreservation-induced capacitation of bovine spermatozoa

After ejaculation, mammalian spermatozoa must undergo capacitation to fertilize. Capacitation of bovine spermatozoa occurs in vitro in medium supplemented with heparin. Semen cryopreservation is an important tool for assisted reproduction, although the fertility of frozen-thawed spermatozoa is reduced, possibly due to precocious capacitation-like changes that are known to occur. Our purpose was to clarify the mechanisms involved in bull sperm cryocapacitation induced by cryopreservation. Our general hypothesis is that the signaling pathways that lead to capacitation are triggered by the cryopreservation procedure. Ejaculated bovine semen was divided into two aliquots and diluted in extender; one was then kept fresh, whereas the second was cryopreserved. Western blots of extracted sperm proteins with anti-phosphotyrosine antibody showed that capacitation, induced by either heparin in fresh sperm or cryopreservation (cryocapacitation), is associated with a differential profile of phosphotyrosine-containing proteins. Immunolocalization of phosphotyrosine-containing proteins in the fresh and cryopreserved spermatozoa showed that, after thawing, cryocapacitated sperm displayed labeling over the acrosomal region, whereas for fresh sperm, this labeling appeared after 5-h incubation with heparin. The chlortetracycline assay and the ability of the sperm to undergo the lysophosphatidylcholine-induced acrosome reaction were used to confirm that a subpopulation of cryopreserved sperm is capacitated at thawing, irrespective of heparin inclusion. Since glucose is known to inhibit heparin-induced capacitation, the semen extender was modified to include glucose as a means of inhibiting cryocapacitation; however, cryocapacitation was not prevented according to the chlortetracycline assay and profile of phosphotyrosine-containing sperm proteins.     

Vasopressin Effectively Suppresses Male Fertility

Arginine vasopressin (VP) is neurohypophysial hormone has been implicated in stimulating contractile activity of the male reproductive tract in the testis. Higher levels of VP decrease sperm count and motility. However, very little is known about the involvement of VP in controlling mammalian reproductive process. The goal of this study was to confirm that effect of VP receptor (AVPR2) on sperm function in capacitation condition. Deamino [Cys 1, D-ArgS] vasopressin (dDAVP), an AVPR2 agonist that operates only on AVPR2, was used. Also, Mouse spermatozoa were incubated with various concentrations of dDAVP (10⁻¹¹–10⁻⁵ M) and sperm motility, capacitation status, Protein Kinase A activity (PKA), tyrosine phosphorylation, fertilization, and embryo development were assessed using computer-assisted sperm analysis, Combined Hoechst 33258/chlortetracycline fluorescence, Western blotting, and in vitro fertilization, respectively. AVPR2 was placed on the acrosome region and mid-piece in cauda epididymal spermatozoa, but the caput epididymal spermatozoa was mid-piece only. The high dDAVP treatment (10⁻⁸ and 10⁻⁵ M) significantly decreased sperm motility, intracellular pH and PKA substrates (approximately 55 and 22 kDa) and increased Ca²⁺ concentration. The highest concentration treatment significantly decreased PKA substrate (approximately 23 kDa) and tyrosine phosphorylation (approximately 30 kDa). VP detrimentally affected capacitation, acrosome reaction, and embryo development. Treatment with the lowest concentration (10⁻¹¹ M) was not significantly different. Our data have shown that VP stimulates ion transport across sperm membrane through interactions with AVPR2. VP has a detrimental effect in sperm function, fertilization, and embryonic development, suggesting its critical role in the acquisition of fertilizing ability of mouse spermatozoa. These research findings will enable further study to determine molecular mechanism associated with fertility in capacitation and fertilization. It is also an important pivotal precondition to the progress of diagnostic test to identify infertility and to apply male contraception.     

Is sperm capacitation analogous to early phases of Ca2+‐triggered membrane fusion in somatic cells and viruses?

An important feature of male fertility is the physiological priming of spermatozoa by a multifaceted process collectively referred to as capacitation. The end point of this evasive process is the hyperactivated spermatozoa capable of binding to terminal sugar residues on the egg's extracellular coat, the zona pellucida (ZP), and undergoing acrosomal exocytosis (i.e., induction of the acrosome reaction). The hydrolytic action of acrosomal enzymes released at the site of zona binding, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoa, are important factors that regulate the penetration of ZP and fertilization of the egg. Despite many advances in identifying sperm components that promote capacitation, the mechanism underlying the calcium-triggered process remains elusive. The purpose of this review article is to focus on new advances that have enhanced our understanding of in vivo/in vitro capacitation, a prerequisite event resulting from a dramatic modification and reorganization of the sperm membrane molecules. Special emphasis has been laid on accumulating evidence suggesting potential similarities between the sperm capacitation and early phases of calcium-triggered membrane fusion (i.e., tethering and docking) during secretory and endocytotic pathways among eukaryotes.     

Precision Glycoproteomics Reveals Distinctive N-Glycosylation in Human Spermatozoa

Spermatozoon represents a very special cell type in human body, and glycosylation plays essential roles in its whole life including spermatogenesis, maturation, capacitation, sperm–egg recognition, and fertilization. In this study, by mapping the most comprehensive N-glycoproteome of human spermatozoa using our recently developed site-specific glycoproteomic approaches, we show that spermatozoa contain a number of distinctive glycoproteins, which are mainly involved in spermatogenesis, acrosome reaction and sperm:oocyte membrane binding, and fertilization. Heavy fucosylation is observed on 14 glycoproteins mostly located at extracellular and cell surface regions in spermatozoa but not in other tissues. Sialylation and Lewis epitopes are enriched in the biological process of immune response in spermatozoa, while bisected core structures and LacdiNAc structures are highly expressed in acrosome. These data deepen our knowledge about glycosylation in spermatozoa and lay the foundation for functional study of glycosylation and glycan structures in male infertility.     

Oviductal fluid modulates the dynamics of tyrosine phosphorylation in cryopreserved boar spermatozoa during capacitation

Following insemination, spermatozoa are retained in the utero‐tubal junction and isthmic region of the oviduct, where essential steps of capacitation are coordinated. Although a majority of the spermatozoa is exposed to similar conditions in the oviduct, the speed of the response varies depending on the individual male and the state of the spermatozoa. The present study evaluated individual boar variations in terms of the ability of spermatozoa to undergo tyrosine phosphorylation in response to isthmic oviductal fluid (ODF). Cryopreserved spermatozoa from four boars were incubated with pre‐ and post‐ovulatory ODF for 6 hr. Sperm kinematics, global protein tyrosine phosphorylation, and dynamics of different phosphorylation patterns were analyzed at hourly interval. The percentage of phosphorylated spermatozoa in the pre‐ovulatory ODF‐treated group was significantly (P < 0.001) higher than in the other treatment groups. Motility, velocity, and protein tyrosine phosphorylation in spermatozoa in response to ODF and control media also showed differences between boars. Spermatozoa from all four boars showed strong expression of a 19‐kDa phosphoprotein while spermatozoa from two boars showed additionally strong expression of a 32‐kDa phosphoprotein when incubated with pre‐ovulatory ODF. While phosphorylation of proteins in the acrosome and the equatorial segment of the sperm were noticed at an early stage during incubation with ODF, tail phosphorylation appeared at a later stage of capacitation. The results indicate individual variation between boars in terms of sperm proteins, including different phosphorylation patterns, in response to ODF, which might be related to fertility.Mol. Reprod. Dev. 79: 525‐540, 2012. © 2012 Wiley Periodicals, Inc.     

Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress

Oxidative stress in the male germ line is known to be a key factor in both the etiology of male infertility and the high levels of DNA damage encountered in human spermatozoa. Because the latter has been associated with a variety of adverse clinical outcomes, including miscarriage and developmental abnormalities in the offspring, the mechanisms that spermatozoa use to defend themselves against oxidative stress are of great interest. In this context, the male germ line expresses three unique forms of thioredoxin, known as thioredoxin domain-containing proteins (Txndc2, Txndc3, and Txndc8). Two of these proteins, Txndc2 and Txndc3, retain association with the spermatozoa after spermiation and potentially play an important role in regulating the redox status of the mature gamete. To address this area, we have functionally deleted the sperm-specific thioredoxins from the male germ line of mice by either exon deletion (Txndc2) or mutation of the bioactive cysteines (Txndc3). The combined inactivation of these Txndc isoforms did not have an overall impact on spermatogenesis, epididymal sperm maturation, or fertility. However, Txndc deficiency in spermatozoa did lead to age-dependent changes in these cells as reflected by accelerated motility loss, high rates of DNA damage, increases in reactive oxygen species generation, enhanced formation of lipid aldehyde–protein adducts, and impaired protamination of the sperm chromatin. These results suggest that although there is considerable redundancy in the systems employed by spermatozoa to defend themselves against oxidative stress, the sperm-specific thioredoxins, Txndc2 and Txndc3, are critically important in protecting these cells against the increases in oxidative stress associated with paternal age.     

Fertilization by sperm injection in the rabbit

Whole rabbit spermatozoa and isolated sperm nuclei were microinjected directly into the ooplasm of hamster and rabbit ova. These injected sperm decondensed and formed male pronuclei during subsequent in-vitro culture. Injection of whole spermatozoa and sperm nuclei prepared by a protocol known to allow in-vitro capacitation of ejaculated spermatozoa yielded a significantly higher (P < 0.01) number of activated rabbit ova containing male pronuclei than did injection of uncapacitated epididymal sperm nuclei or ejaculated sperm nuclei. Rabbit ova fertilized by sperm injection were capable of undergoing normal-appearing cleavage division during 22 h of culture.