AKAP3-mediated type I PKA signaling is required for mouse sperm hyperactivation and fertility†.

The protein kinase A (PKA) signaling pathway, which mediates protein phosphorylation, is important for sperm motility and male fertility. This process relies on A-kinase anchoring proteins that organize PKA and its signalosomes within specific subcellular compartments. Previously, it was found that the absence of A-kinase anchoring protein 3 (AKAP3) leads to multiple morphological abnormalities in mouse sperm. But how AKAP3 regulates sperm motility is yet to be elucidated. AKAP3 has two amphipathic domains, here named dual and RI, in its N-terminus. These domains are responsible for binding regulatory subunits I alpha (RIα) and II alpha (RIIα) of PKA and for RIα only, respectively. Here, we generated mutant mice lacking the dual and RI domains of AKAP3. It was found that the deletion of these domains caused male mouse infertile, accompanied by mild defects in the fibrous sheath of sperm tails. Additionally, the levels of serine/threonine phosphorylation of PKA substrates and tyrosine phosphorylation decreased in the mutant sperm, which exhibited a defect in hyperactivation under capacitation conditions. The protein levels of PKA subunits remained unchanged. But, interestingly, the regulatory subunit RIα was mis-localized from principal piece to midpiece of sperm tail, whereas this was not observed for RIIα. Further protein-protein interaction assays revealed a preference for AKAP3 to bind RIα over RIIα. Collectively, our findings suggest that AKAP3 is important for sperm hyperactivity by regulating type-I PKA signaling pathway mediated protein phosphorylation via its dual and RI domains.

Odorant Receptor OR2C1 Is an Essential Modulator of Boar Sperm Capacitation by Binding with Heparin.

Heparin, a class of glycosaminoglycans (GAGs), is widely used to induce sperm capacitation and fertilization. How heparin induces sperm capacitation remains unclear. Olfactory receptors (ORs) which are G protein-coupled receptors, have been proposed to be involved in sperm capacitation. However, the interaction between ORs and odor molecules and the molecular mechanism of ORs mediating sperm capacitation are still unclear. The present study aimed to explore the underlying interaction and mechanism between heparin and ORs in carrying out the boar sperm capacitation. The results showed that olfactory receptor 2C1 (OR2C1) is a compulsory unit which regulates the sperm capacitation by recognizing and binding with heparin, as determined by Dual-Glo Luciferase Assay and molecular docking. In addition, molecular dynamics (MD) simulation indicated that OR2C1 binds with heparin via a hydrophobic cavity comprises of Arg3, Ala6, Thr7, Asn171, Arg172, Arg173, and Pro287. Furthermore, we demonstrated that knocking down OR2C1 significantly inhibits sperm capacitation. In conclusion, we highlighted a novel olfactory receptor, OR2C1, in boar sperm and disclosed the potential binding of heparin to Pro287, a conserved residue in the transmembrane helices region 7 (TMH7). Our findings will benefit the further understanding of ORs involved in sperm capacitation and fertilization.

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.

GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility.

For a sperm to successfully fertilize an egg, it must first undergo capacitation in the female reproductive tract and later undergo acrosomal reaction (AR) upon encountering an egg surrounded by its vestment. How premature AR is avoided despite rapid surges in signaling cascades during capacitation remains unknown. Using a combination of conditional knockout (cKO) mice and cell-penetrating peptides, we show that GIV (CCDC88A), a guanine nucleotide-exchange modulator (GEM) for trimeric GTPases, is highly expressed in spermatocytes and is required for male fertility. GIV is rapidly phosphoregulated on key tyrosine and serine residues in human and murine spermatozoa. These phosphomodifications enable GIV-GEM to orchestrate two distinct compartmentalized signaling programs in the sperm tail and head; in the tail, GIV enhances PI3K→Akt signals, sperm motility and survival, whereas in the head it inhibits cAMP surge and premature AR. Furthermore, GIV transcripts are downregulated in the testis and semen of infertile men. These findings exemplify the spatiotemporally segregated signaling programs that support sperm capacitation and shed light on a hitherto unforeseen cause of infertility in men.

Phosphoproteomics and Bioinformatics Analyses Reveal Key Roles of GSK-3 and AKAP4 in Mouse Sperm Capacitation.

Protein phosphorylation can induce signal transduction to change sperm motility patterns during sperm capacitation. However, changes in the phosphorylation of sperm proteins in mice are still incompletely understood. Here, capacitation-related phosphorylation in mouse sperms were firstly investigated by label-free quantitative (LFQ) phosphoproteomics coupled with bioinformatics analysis using ingenuity pathway analysis (IPA) methods such as canonical pathway, upstream regulator, and network analysis. Among 1632 phosphopeptides identified at serine, threonine, and tyrosine residues, 1050 novel phosphosites, corresponding to 402 proteins, were reported. Gene heatmaps for IPA canonical pathways showed a novel role for GSK-3 in GP6 signaling pathways associated with capacitation for 60 min. At the same time, the reduction of the abundant isoform-specific GSK-3α expression was shown by western blot (WB) while the LFQ pY of this isoform slightly decreased and then increased. The combined results from WB and LFQ methods explain the less inhibitory phosphorylation of GSK-3α during capacitation and also support the predicted increases in its activity. In addition, pAKAP4 increased at the Y156 site but decreased at the Y811 site in a capacitated state, even though IPA network analysis and WB analysis for overall pAKAP revealed upregulated trends. The potential roles of GSK-3 and AKAP4 in fertility are discussed.

The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility.

Pachytene PIWI-interacting RNAs (piRNAs), which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like microRNAs, cleave targets like short interfering RNAs or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 (pi6) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6-/- fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving messenger RNAs encoding proteins required for sperm function. pi6 also participates in a network of piRNA-piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10, as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.

HVCN1 Channels Are Relevant for the Maintenance of Sperm Motility During In Vitro Capacitation of Pig Spermatozoa.

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca2+ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca2+ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca2+ entrance to the sperm head.

Capacitation-associated alkalization in human sperm is differentially controlled at the subcellular level.

Capacitation in mammalian sperm involves the accurate balance of intracellular pH (pHi), but the mechanisms controlling this process are not fully understood, particularly regarding the spatiotemporal regulation of the proteins involved in pHi modulation. Here, we employed an image-based flow cytometry technique combined with pharmacological approaches to study pHi dynamics at the subcellular level during capacitation. We found that, upon capacitation induction, sperm cells undergo intracellular alkalization in the head and principal piece regions. The observed localized pHi increases require the initial uptake of HCO3-, which is mediated by several proteins acting consistently with their subcellular localization. Hv1 proton channel (also known as HVCN1) and cAMP-activated protein kinase (protein kinase A, PKA) antagonists impair alkalization mainly in the principal piece. Na+/HCO3- cotransporter (NBC) and cystic fibrosis transmembrane regulator (CFTR) antagonists impair alkalization only mildly, predominantly in the head. Motility measurements indicate that inhibition of alkalization in the principal piece prevents the development of hyperactivated motility. Altogether, our findings shed light on the complex control mechanisms of pHi and underscore their importance during human sperm capacitation.This article has an associated First Person interview with the first author of the paper.

Mouse t-complex protein 11 is important for progressive motility in sperm†.

The t-complex is defined as naturally occurring variants of the proximal third of mouse chromosome 17 and has been studied by mouse geneticists for decades. This region contains many genes involved in processes from embryogenesis to sperm function. One such gene, t-complex protein 11 (Tcp11), was identified as a testis-specific gene whose protein is present in elongating spermatids. Later work on Tcp11 localized TCP11 to the sperm surface and acrosome cap and implicated TCP11 as important for sperm capacitation through the cyclic AMP/Protein Kinase A pathway. Here, we show that TCP11 is cytoplasmically localized to elongating spermatids and absent from sperm. In the absence of Tcp11, male mice have severely reduced fertility due to a significant decrease in progressively motile sperm; however, Tcp11-null sperm continues to undergo tyrosine phosphorylation, a hallmark of capacitation. Interestingly, null sperm displays reduced PKA activity, consistent with previous reports. Our work demonstrates that TCP11 functions in elongated spermatids to confer proper motility in mature sperm.

Freezing-Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation.

Mammalian sperm must undergo a set of structural and functional changes collectively termed as capacitation to ensure a successful oocyte fertilization. However, capacitation can be compromised by cryopreservation procedures, which alter the proteome and longevity of sperm. To date, how the protein changes induced by cryopreservation could affect the acquisition of sperm fertilizing potential remains unexplored. The present study investigated the protein profile of ram sperm during in vitro capacitation before and after cryopreservation to elucidate the impact of cryopreservation on sperm capacitation at a molecular level. Fresh and cryopreserved ram sperm were incubated under capacitating (CAP) and non-capacitating (NC) conditions for 240 min. The sperm proteome of these four treatments was analyzed and compared at different incubation times using reverse phase liquid chromatography coupled to mass spectrometry (RP-LC-MS/MS). The comparison between fresh and cryopreserved sperm suggested that cryopreservation facilitated an apoptosis-stress response and redox process, while the comparison between sperm incubated in CAP and NC conditions showed that capacitation increased those biological processes associated with signaling, metabolism, motility, and reproductive processes. In addition, 14 proteins related to mitochondrial activity, sperm motility, oocyte recognition, signaling, spermatogenesis, and the apoptosis-stress response underwent significant changes in abundance over time when fresh and cryopreserved sperm incubated in CAP and NC conditions were compared. Our results indicate that disturbances in a ram sperm proteome after cryopreservation may alter the quality of sperm and its specific machinery to sustain capacitation under in vitro conditions.

Human OVGP1 enhances tyrosine phosphorylation of proteins in the fibrous sheath involving AKAP3 and increases sperm-zona binding.

PURPOSE: To investigate if the recombinant human oviduct-specific glycoprotein (rHuOVGP1)-enhanced tyrosine-phosphorylated (pY) proteins are components of specific structure(s) of the sperm tail and if rHuOVGP1 binds to the oocyte and enhances sperm-egg binding. METHODS: Immunofluorescent staining and confocal microscopy were performed to examine the localization of pY proteins, outer dense fiber (ODF), and A-Kinase Associated Protein 3 (AKAP3) in human sperm during capacitation. Western blot and immunoprecipitation were employed to analyze protein levels of pY proteins and AKAP3. Immunofluorescent staining was performed to examine the binding of rHuOVGP1 to human oocytes. The effect of rHuOVGP1 on enhancing sperm-zona binding was examined using hemizona assay. RESULTS: pY proteins were detected mainly in the fibrous sheath (FS) surrounding the ODF with a relatively weak immunoreaction in the neck and mid-piece. Western blot analysis revealed co-migration of the pY 105 kDa protein with AKAP3, which was further confirmed by immunoprecipitation correlating immunofluorescent results of co-localization of pY proteins with AKAP3 in the sperm tail. rHuOVGP1 binds specifically to the zona pellucida (ZP) of human oocytes. Prior incubation of sperm and/or ZP with rHuOVGP1 increased sperm-egg binding. CONCLUSIONS: The present study revealed that one of the major rHuOVGP1-enhanced pY proteins could be AKAP3 of the FS and that rHuOVGP1 is capable of binding to human ZP and its presence in the medium results in an increase in sperm-zona binding. Supplement of rHuOVGP1 in in vitro fertilization media could be beneficial for enhancement of the fertilizing ability of human sperm.

Improvement in blastocyst quality by neurotensin signaling via its receptors in bovine spermatozoa during in vitro fertilization.

Previously, we reported that neurotensin (NT), which is expressed in the uterus and oviduct, enhanced bovine sperm capacitation and acrosome reactions. As NT mRNA expression in bovine oviducts increases dramatically in the follicular phase, we hypothesized that NT modulates fertilization and subsequent conception in cattle. The objective of this study was to evaluate the effect of NT on embryo development and blastocyst quality. The rate of embryo cleavage was significantly increased by the addition of NT to the fertilization medium. Furthermore, the total number of cells and numbers of cells in the inner cell mass of blastocysts were significantly increased by NT during in vitro fertilization (IVF). These results suggested that NT enhanced the efficiency of early bovine embryo development and blastocyst quality. The expression of NT receptors (NTRs) in sperm, testes, oocytes, and cumulus cells was evaluated to determine whether NT acted via NTRs in sperm alone or in both male and female reproductive cells during IVF. Immunocytochemistry and reverse transcription polymerase chain reaction revealed that NTR1 and NTR2 were expressed in sperm and testes, but not in oocytes and cumulus cells. We propose that NT selectively acts upon sperm via NTR1 and NTR2 during IVF to improve the cleavage rate and quality of blastocysts, which are important determinants of sperm quality for successful conception. This research supports our hypothesis that NT acts as a key modulator of fertilization and conception in cattle. Further studies are necessary to apply our findings to the industrial framework of bovine reproduction.

Deficiency of TPPP2, a factor linked to oligoasthenozoospermia, causes subfertility in male mice.

Oligoasthenozoospermia is a major cause of male infertility; however, its etiology and pathogenesis are unclear and may be associated with specific gene abnormalities. This study focused on Tppp2 (tubulin polymerization promoting protein family member 2), whose encoded protein localizes in elongating spermatids at stages IV-VIII of the seminiferous epithelial cycle in testis and in mature sperm in the epididymis. In human and mouse sperm, in vitro inhibition of TPPP2 caused significantly decreased motility and ATP content. Studies on Tppp2 knockout (KO) mice demonstrated that deletion of TPPP2 resulted in male subfertility with a significantly decreased sperm count and motility. In Tppp2-/- mice, increased irregular mitochondria lacking lamellar cristae, abnormal expression of electron transfer chain molecules, lower ATP levels, decreased mitochondrial membrane potential and increased apoptotic index were observed in sperm, which could be the potential causes for its oligoasthenozoospermia phenotype. Moreover, we identified a potential TPPP2-interactive protein, eEf1b (eukaryotic translation elongation factor 1 beta), which plays an important role in protein translation extension. Thus, TPPP2 is probably a potential pathogenic factor in oligoasthenozoospermia. Deficiency of TPPP2 might affect the translation of specific proteins, altering the structure and function of sperm mitochondria, and resulting in decreased sperm count, motility and fertility.

Morbid obesity-related changes in the expression of lipid receptors, transporters, and HSL in human sperm.

OBJECTIVE: To study the location and expression of receptors (SR-BI/CLA-1, SR-BII, and LDLr) and transporter (ABCA1) involved in uptake and efflux of cholesterol in human spermatozoa and assess whether obesity alters its location/expression and whether this could be related to infertility. DESIGN: Observational study. SETTING: None PATIENT(S): Ten controls and 20 obese patients. INTERVENTION(S): Anthropometric parameters. Serum and semen samples were collected. MAIN OUTCOME MEASURE(S): Spermatozoon concentration, immunolocalization, and protein expression in semen. RESULTS: Spermatozoon concentration and motility was decreased in morbidly obese patients. SR-BI/CLA-1, SR-BII, LDLr, and ABCA1 are located in the spermatozoon cell membrane and the localization does not change between obese patients and controls. Control spermatozoa showed high SR-BI expression, and less expression for the rest of the receptors analyzed, indicating that SR-BI/CLA-1 is relevant in human spermatozoon cholesterol uptake/efflux. On the contrary, spermatozoa of obese patients showed less SR-BI/CLA-1 expression than controls, and more intense positive staining for SR-BII, LDLr, and ABCA1. Finally, human sperm expresses the 130- and 82-kDa hormone-sensitive lipase (HSL) isoforms. The 130-kDa isoform is expressed in the control sperm, and the expression disappears in the obese patients. CONCLUSION(S): The presence of lipid receptors/transporters and HSL in human spermatozoa suggests their role in the process of maturation/capacitation. The changes in the expression of lipid receptors/transporters and the lack of the 130-kDa HSL isoform in obese patients prevent the hydrolysis of cholesterol esters internalized by these receptors, and favor their accumulation in the cytoplasm of the spermatozoa that could contribute to lipotoxicity and infertility.

Fms-like tyrosine kinase 3 is a key factor of male fertility.

Fms-like tyrosine kinase 3 (FLT3) is a type III kinase that is highly expressed in seminal plasma of infertile men. FLT3 activation can be blocked by inhibition of its phosphorylation using the nontoxic and selective inhibitor, quizartinib. We investigated the function of FLT3 and the corresponding effects of quizartinib in mouse spermatozoa. Spermatozoa were treated with different concentrations (0.1, 1, 10, 20, and 30 µM) of quizartinib for 90 min at 37 °C in 5% CO2 in air. FLT3 was detected in capacitated and non-capacitated spermatozoa. While the level of FLT3 was unaffected, the levels of phospho-FLT3 were significantly altered in spermatozoa by quizartinib. Exposure of spermatozoa to higher concentrations of quizartinib significantly altered sperm viability, motility, motion kinematics, levels of intracellular ATP, and capacitation status. Fertilization and early embryonic development were suppressed by quizartinib. This may have occurred as a consequence of decreased protein kinase A (PKA) activity and tyrosine phosphorylation. The inhibition of FLT3 by quizartinib may affect the fertilization and embryonic development by reducing tyrosine phosphorylation through a PKA-dependent pathway. Our data implicate FLT3 as a biomarker for diagnosis and prognosis of male fertility. In addition, quizartinib has potential for development as a new contraceptive agent.

Sperm solute carrier family 9 regulator 1 is correlated with boar fertility.

Predicting male fertility is extremely important for artificial insemination and profitable farm management. Conventional semen assessment together with computer-assisted sperm analysis is widely used to predict male fertility under field conditions. However, the clinical validation and sensitivity of these methods remain unclear. Therefore, a new approach is needed to predict male fertility. Here, we investigated the use of a transcriptomic marker (solute carrier family 9, subfamily A, member 3, regulator 1; SLC9A3R1) together with sperm motility parameters and capacitation status to predict fertility/infertility in boars at the commercial level. Our data showed that among motility parameters and the capacitation status, hyperactivation (HYP) differed between high- and low-litter size boars. HYP showed a significant positive correlation (R = 0.468) with boar litter size. Simultaneously, the expression of SLC9A3R1, a gene important in sperm ion channel regulation, was significantly negatively correlated (R = -0.523) with boar litter size. Quality assessment revealed that both HYP and SLC9A3R1 showed considerable sensitivity (71.43 vs. 100%), specificity (100 vs. 71.43%), and overall accuracy (90%) for predicting male fertility. Interestingly, the potential of SLC9A3R1 expression to increase the average piglet number per breeding was higher (0.7 piglets) than that of HYP (0.5 piglets). Thus, measuring SLC9A3R1 expression in spermatozoa may be a more accurate marker for evaluating male fertility/infertility than conventionally used motility parameters and capacitation status.

Deficiency of MTMR14 impairs male fertility in Mus musculus.

Calcium signalling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility and acrosome reactions are all mediated by increases in intracellular Ca2+. Our previous reports have shown that deficiency of MTMR14, a novel phosphoinositide phosphatase, induces a muscle disorder by disrupting Ca2+ homeostasis. Recently, we found that MTMR14 is also expressed in the testes; however, whether deficiency of MTMR14 in the testes also alters the Ca2+ concentration and impairs male fertility remains entirely unknown. In the present study, we found that MTMR14 is also expressed in the testes and mature sperm cells, suggesting that deficiency of MTMR14 might have some effect on male fertility. Both in vivo fertility and in vitro fertilization tests were then performed, and we found that MTMR14-/- male mice showed decreased fertility. A series of experiments were then arranged to test the testis and sperm parameters; we found that MTMR14 deficiency caused small size of the testes, small numbers of both total and immotile sperm, expanded membrane of sperm tail, a decreased proportion of acrosome reaction, and in contrast, an increased proportion of abnormal sperm and augmented apoptosis, etc. Further study also found that the muscle force of the vas deferens decreased significantly in KO mice. Intracellular calcium homeostasis in the testes and epididymis was impaired by MTMR14 deletion; moreover, the relative mRNA expression levels of Itpr1, Itpr2, and Ryr3 were dramatically decreased in MTMR14 KO mice. Thus, MTMR14 deletion impairs male fertility by causing decreased muscle force of the vas deferens and intracellular calcium imbalance.

High throughput small RNA and transcriptome sequencing reveal capacitation-related microRNAs and mRNA in boar sperm.

BACKGROUND: Capacitation, a prerequisite for oocyte fertilization, is a complex process involving series of structural and functional changes in sperms such as membrane modifications, modulation of enzyme activities, and protein phosphorylation. In order to penetrate and fertilize an oocyte, mammalian sperms must undergo capacitation. Nevertheless, the process of sperm capacitation remains poorly understood and requires further elucidation. In the current study, via high throughput sequencing, we identified and explored the differentially expressed microRNAs (miRNAs) and mRNAs involved in boar sperm capacitation. RESULTS: We identified a total of 5342 mRNAs and 204 miRNAs that were differentially expressed in fresh and capacitated boar sperms. From these, 12 miRNAs (8 known and 4 newly identified miRNAs) and their differentially expressed target mRNAs were found to be involved in sperm capacitation-related PI3K-Akt, MAPK, cAMP-PKA and Ca2+signaling pathways. CONCLUSIONS: Our study is first to provide the complete miRNA and transcriptome profiles of boar sperm. Our findings provide important insights for the understanding of the RNA profile in boar sperm and future elucidation of the underlying molecular mechanism relevant to mammalian sperm capacitation.

The functional anatomy of the human spermatozoon: relating ultrastructure and function.

The Internet, magazine articles, and even biomedical journal articles, are full of cartoons of spermatozoa that bear minimal resemblance to real spermatozoa, especially human spermatozoa, and this had led to many misconceptions about what spermatozoa look like and how they are constituted. This review summarizes the historical and current state of knowledge of mammalian sperm ultrastructure, with particular emphasis on and relevance to human spermatozoa, combining information obtained from a variety of electron microscopic (EM) techniques. Available information on the composition and configuration of the various ultrastructural components of the spermatozoon has been related to their mechanistic purpose and roles in the primary aspects of sperm function and fertilization: motility, hyperactivation, capacitation, the acrosome reaction and sperm-oocyte fusion.