Acrosomal Cap Research Articles

Spermicidal efficacy of VRP, a synthetic cationic antimicrobial peptide, inducing apoptosis and membrane disruption.

Presently available contraceptives are mostly hormonal or detergent in nature with numerous side effects like irritation, lesion, inflammation in vagina, alteration of body homeostasis, etc. Antimicrobial peptides with spermicidal activity but without adverse effects may be suitable alternatives. In the present study, spermicidal activity of a cationic antimicrobial peptide VRP on human spermatozoa has been elucidated. Progressive forward motility of human spermatozoa was instantly stopped after 100 μM VRP treatment and at 350 μM, all kinds of sperm motility ceased within 20 s as assessed by the Sander-Cramer assay. The spermicidal effect was confirmed by eosin-nigrosin assay and HOS test. VRP treatment (100 μM) in human spermatozoa induced both the intrinsic and extrinsic pathways of apoptosis. TUNEL assay showed VRP treatment significantly disrupted the DNA integrity and changed the mitochondrial membrane permeability as evident from MPTP assay. AFM and SEM results depicted ultra structural changes including disruption of the acrosomal cap and plasma membrane of the head and midpiece region after treatment with 350 μM VRP. MTT assay showed after treatments with 100 and 350 μM of VRP for 24 hr, a substantial amount of Lactobacillus acidophilus (about 90% and 75%, respectively) remained viable. Hence, VRP being a small synthetic peptide with antimicrobial and spermicidal activity but tolerable to normal vaginal microflora, may be a suitable target for elucidating its contraceptive potentiality.

Loss of the Na+/H+ exchanger NHE8 causes male infertility in mice by disrupting acrosome formation

Mammalian sperm feature a specialized secretory organelle on the anterior part of the sperm nucleus, the acrosome, which is essential for male fertility. It is formed by a fusion of Golgi-derived vesicles. We show here that the predominantly Golgi-resident Na+/H+ exchanger NHE8 localizes to the developing acrosome of spermatids. Similar to wild-type mice, Nhe8-/- mice generated Golgi-derived vesicles positive for acrosomal markers and attached to nuclei, but these vesicles failed to form large acrosomal granules and the acrosomal cap. Spermatozoa from Nhe8-/- mice completely lacked acrosomes, were round-headed, exhibited abnormal mitochondrial distribution, and displayed decreased motility, resulting in selective male infertility. Of note, similar features are also found in globozoospermia, one of the causes of male infertility in humans. Germ cell-specific, but not Sertoli cell-specific Nhe8 disruption recapitulated the globozoospermia phenotype, demonstrating that NHE8's role in spermiogenesis is germ cell-intrinsic. Our work has uncovered a crucial role of NHE8 in acrosome biogenesis and suggests that some forms of human globozoospermia might be caused by a loss of function of this Na+/H+ exchanger. It points to NHE8 as a candidate gene for human globozoospermia and a possible drug target for male contraception.

Systematic characterization of human testis-specific actin capping protein β3 as a possible biomarker for male infertility.

Is actin capping protein (CP) β3 involved in human spermatogenesis and male infertility? Human CPβ3 (hCPβ3) is expressed in testis, changes its localization dynamically during spermatogenesis, and has some association with male infertility. The testis-specific α subunit of CP (CPα3) was previously identified in human, and mutations in the cpα3 gene in mouse were shown to induce malformation of the sperm head and male infertility. However, CPβ3, which is considered to be a heterodimeric counterpart of CPα3, has been neither characterized in human nor reported in association with male infertility. To confirm the existence of CPβ3 in human testis, fresh semen samples from proven fertile men were analyzed. To investigate protein expression during spermatogenesis, cryopreserved testis obtained from men with obstructive azoospermia were examined by immunofluorescent analysis. To assess the association of CP with male infertility, we compared protein expression of human CPα3 (hCPα3) and hCPβ3 using immunofluorescent analysis of cryopreserved sperm between men with normozoospermia (volunteers: Normo group, n = 20) and infertile men with oligozoospermia and/or asthenozoospermia (O + A group, n = 21). The tissue-specific expression of hCPβ3 was investigated by RT-PCR and Western blot analysis. To investigate whether hCPα3 and hCPβ3 form a heterodimer, a tandem expression vector containing hcpα3 tagged with monomeric red fluorescent protein 1 and hcpβ3 tagged with enhanced green fluorescent protein in a single plasmid was constructed and analyzed by co-immunoprecipitation (Co-IP) assay. The protein expression profiles of hCPα3 and hCPβ3 during spermatogenesis were examined by immunohistochemical analysis using human spermatogenic cells. The protein expressions of hCPα3 and hCPβ3 in sperm were compared between the Normo and O + A groups by immunohistochemical analysis. RT-PCR showed that mRNA of hcpβ3 was expressed exclusively in testis. Western blot analysis detected hCPβ3 with anti-bovine CPβ3 antibody. Co-IP assay with recombinant protein showed that hCPα3 and hCPβ3 form a protein complex. At each step during spermatogenesis, the cellular localization of hCPβ3 changed dynamically. In spermatogonia, hCPβ3 showed a slight signal in cytoplasm. hCPβ3 expression was conspicuous mainly from spermatocytes, and hCPβ3 localization dynamically migrated from cytoplasm to the acrosomal cap and acrosome. In mature spermatozoa, hCPβ3 accumulated in the postacrosomal region and less so at the midpiece of the tail. Double-staining analysis revealed that hCPα3 localization was identical to hCPβ3 at every step in the spermatogenic cells. Most spermatozoa from the Normo group were stained homogenously by both hCPα3 and hCPβ3. In contrast, significantly more spermatozoa in the O + A versus Normo group showed heterogeneous or lack of staining for either hCPα3 or hCPβ3 (abnormal staining) (P < 0.001). The percentage of abnormal staining was higher in the O + A group (52.4 ± 3.0%) than in the Normo group (31.2 ± 2.5%). Even by confining the observations to morphologically normal spermatozoa selected in accordance with David's criteria, the percentage of abnormal staining was still higher in the O + A group (39.9 ± 2.9%) versus the Normo group (22.5 ± 2.1%) (P < 0.001). hCPβ3 in conjunction with hCPα3 seemed to play an important role in spermatogenesis and may be associated with male infertility. Not applicable. Owing to the difficulty of collecting fresh samples of human testis, we used cryopreserved samples from testicular sperm extraction. To examine the interaction of spermatogenic cells or localization in seminiferous tubules, fresh testis sample of healthy males are ideal. The altered expression of hCPα3 and hCPβ3 may not only be a cause of male infertility but also a prognostic factor for the results of ART. They may be useful biomarkers to determine the fertilization ability of human sperm in ART. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JP16K20133). The authors declare no competing interests.

Translocation of Tektin 3 to the equatorial segment of heads in bull spermatozoa exposed to dibutyryl cAMP and calyculin A.

Tektins (TEKTs) are filamentous proteins associated with microtubules in cilia, flagella, basal bodies, and centrioles. Five TEKTs (TEKT1, -2, -3, -4, and -5) have been identified as components of mammalian sperm flagella. We previously reported that TKET1 and -3 are also present in the heads of rodent spermatozoa. The present study clearly demonstrates that TEKT2 is present at the acrosome cap whereas TEKT3 resides just beneath the plasma membrane of the post-acrosomal region of sperm heads in unactivated bull spermatozoa, and builds on the distributional differences of TEKT1, -2, and -3 on sperm heads. We also discovered that hyperactivation of bull spermatozoa by cell-permeable cAMP and calyculin A, a protein phosphatase inhibitor, promoted translocation of TEKT3 from the post-acrosomal region to the equatorial segment in sperm heads, and that TEKT3 accumulated at the equatorial segment is lost upon acrosome reaction. Thus, translocation of TEKT3 to the equatorial segment may be a capacitation- or hyperactivation-associated phenomenon in bull spermatozoa. Mol. Reprod. Dev. 84: 30-43, 2017. © 2016 Wiley Periodicals, Inc.

Functional morphology of the male reproductive system of the white shrimp Litopenaeus schmitti (Burkenroad, 1936) (Crustacea, Penaeidea) compared to other Litopenaeus

The male reproductive system of L. schmitti is described, including spermatozoon ultrastructure. The testis is composed of 8 to 14 lobules arranged as seminiferous tubules. The proximal vas deferens is short and filled with spermatozoa without spikes. The median vas deferens is larger than the other parts of the vas deferens and it shows two ducts separated by typhlosoles, the sperm and accessory ducts. The former is filled with sperm masses, while the accessory duct shows only secretions that are part of the spermatophore material. In this region, the spermatozoa have a spike, indicating the end of the spermatogenesis. The distal vas deferens is a short tube ending in the ampulla, composed of three parts. The spermatozoa have a round main body and an anterior spike associated with the acrosomal cap. The subacrosomal space has a centrosome-like structure above the nucleus. The features of the male reproductive system of L. schmitti share the pattern of penaeoidean shrimps with closed thelyca related to the median vas deferens and the ampulla. All the differences in spermatozoon morphology and vasa deferentia are discussed, and compared to other Litopenaeus species.

GSK3β is involved in the spermatogenesis of the Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853

Glycogen synthase kinase3β (GSK3β) is a Serine/Threonine kinase involved in metabolic reactions, and the Wnt, Akt, Hedgehog and BMP/Smad1 signalling pathways. It regulates cell metabolism, the cell cycle and cell fate through the phosphorylation of a diverse array of substrates. Its disorders lead to testicular degeneration, testicular cord disruption and many other reproductive diseases that often lead to infertility. In mammals, GSK3β plays an important role in the regulation of spermatogenesis. To explore functions of GSK3β during spermatogenesis inEriocheir sinensiswe cloned the full-lengthGSK3β cDNA sequence from the testis ofE. sinensis(EsGSK3β). The 1161-nucleotide open reading frame encodes a 386-amino-acid protein with a predicted molecular mass of 43 kDa. Additionally, we examined the expression pattern of EsGSK3β in different tissues and testis developmental stages using real-time PCR and Western blotting. Localization analysis ofEsGSK3β RNA and protein by in situ hybridization and immunofluorescence, respectively, showed that molecules translocated from the cytoplasm and nucleus to the regions of the acrosomal tubule and apical cap during spermatogenesis. The results indicated that EsGSK3β might play an important role duringE. sinensisspermatogenesis.

The IP3 R Binding Protein Released With Inositol 1,4,5-Trisphosphate Is Expressed in Rodent Reproductive Tissue and Spermatozoa.

Besides its capacity to inhibit the 1,4,5-trisphosphate (IP3) receptor, the regulatory protein IRBIT (IP3 receptor binding protein released with IP3) is also able to control the activity of numerous ion channels and electrolyte transporters and thereby creates an optimal electrolyte composition of various biological fluids. Since a reliable execution of spermatogenesis and sperm maturation critically depends on the establishment of an adequate microenvironment, the expression of IRBIT in male reproductive tissue was examined using immunohistochemical approaches combined with biochemical fractionation methods. The present study documents that IRBIT is expressed in Leydig and Sertoli cells. In addition, pronounced IRBIT expression was detected in sperm precursors during early stages of spermatogenesis as well as in spermatozoa. Analyzing tissue sections of rodent epididymides, IRBIT was found to co-localize with the proton pumping V-ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of narrow and clear cells. A similar co-localization of IRBIT with CFTR was also observed for Sertoli cells and developing germ cells. Remarkably, assaying caudal sperm in immunogold electron microscopy, IRBIT was found to localize to the acrosomal cap and the flagellum as well as to the sperm nucleus; moreover, a prominent oligomerization was observed for spermatozoa. The pronounced occurrence of IRBIT in the male reproductive system and mature spermatozoa indicates a potential role for IRBIT in establishing the essential luminal environment for a faithful execution of spermatogenesis and epididymal sperm maturation, and suggest a participation of IRBIT during maturation steps after ejaculation and/or the final fertilization process.

The acrosome of eutherian mammals.

The acrosome is not just a bag of enzymes, most of which, if not all, are singly non-essential for sperm-oocyte interaction. The Golgi-derived acrosomal cap reveals some extraordinary development and structure particularities. The acrosome of eutherian spermatozoa basically consists of two parts, the anterior and equatorial segments; the present review is devoted to the former, the initial actor in fertilization. Its occasional fanciful morphological changes during epididymal maturation are analyzed, together with its heterogeneous contents: enzymes, zona binding proteins, structural proteins (matrix) and yet to be chemically characterized crystalloids. The plasma and acrosomal membranes present stabilized ordered domains, whereas glycoprotein-free areas appear during capacitation and before fusion. Exocytosis, induced by the cumulus oophorus and/or the zona pellucida, may generally start proximally and progress anteriorly, resulting in the detachment of a hybrid membrane shroud, whose entity is probably maintained by the bound matrix. Immediately released soluble enzymes must be active during the first interactions of the gametes, whereas other lysins, bound to the matrix or stored as proenzymes, are only progressively released. Zona binding is probably achieved via the shroud and/or the IAM (depending on species). Penetration along an incurved slit through the stratified zona is allowed by the rigid and denuded head tip and flagellar hyperactivity, and assisted by the local proteolytic activity of proteasomes bound to the IAM, the unique essential zona lysin system.

RFX2 Is a Major Transcriptional Regulator of Spermiogenesis.

Spermatogenesis consists broadly of three phases: proliferation of diploid germ cells, meiosis, and finally extensive differentiation of the haploid cells into effective delivery vehicles for the paternal genome. Despite detailed characterization of many haploid developmental steps leading to sperm, only fragmentary information exists on the control of gene expression underlying these processes. Here we report that the RFX2 transcription factor is a master regulator of genes required for the haploid phase. A targeted mutation of Rfx2 was created in mice. Rfx2-/- mice are perfectly viable but show complete male sterility. Spermatogenesis appears to progress unperturbed through meiosis. However, haploid cells undergo a complete arrest in spermatid development just prior to spermatid elongation. Arrested cells show altered Golgi apparatus organization, leading to a deficit in the generation of a spreading acrosomal cap from proacrosomal vesicles. Arrested cells ultimately merge to form giant multinucleated cells released to the epididymis. Spermatids also completely fail to form the flagellar axoneme. RNA-Seq analysis and ChIP-Seq analysis identified 139 genes directly controlled by RFX2 during spermiogenesis. Gene ontology analysis revealed that genes required for cilium function are specifically enriched in down- and upregulated genes showing that RFX2 allows precise temporal expression of ciliary genes. Several genes required for cell adhesion and cytoskeleton remodeling are also downregulated. Comparison of RFX2-regulated genes with those controlled by other major transcriptional regulators of spermiogenesis showed that each controls independent gene sets. Altogether, these observations show that RFX2 plays a major and specific function in spermiogenesis.

Morphological characterization and conservation of bovine spermatogenic cells by refrigeration at 4 °C and freezing using different cryoprotective molecules

The objectives of this study were study a practical method to characterize bovine spermatogenic cells and test the efficiency cells conservation by refrigeration at 4°C and cryopreservation in different solutions using two cooling curves. Cellular identification was performing by analysis of shape, size and morphology, associated with nucleus positioning and nuclear-cytoplasm ratio (NCR). Cellular samples were kept at 4°C for a period of 96h in refrigeration solution and every 24h plasma membrane and DNA integrity were evaluated. Cryopreservation of cells was carried out using solutions containing 10% Dimethyl sulfoxide, 5% Dimethylformamide, 7% Glycerol and 7% Ethylene glycol, using a controlled and non-controlled cooling curve. Results of cellular characterization demonstrated that spermatocytes II presented a cylindrical shape, NCR of 1:1.5 and diameter ranging from 14.5 to 17.5μm. Round spermatids presented diameter ranging from 7.6 to 13.4μm, acrosomal cap and NCR of 1:2. Elongation and elongated spermatids showed to marked divergence in shape. There was a daily significant loss of viability of cooled cells until third day of storage, however they presented 72.77±5.16% viability after 4days of storage at 4°C. There was no difference among the cryoprotectant solutions and cooling curves. In conclusion we demonstrated that association of microscopes and staining was a practical method to identify bovine spermatogenic cells. Furthermore, refrigeration at 4°C is an important strategy to preserve over 70% of viable cells after 4days and cryopreservation, regardless of cryoprotectant solution or cooling curve used, can maintain over 50% of cells viable.

Redistribution of the intra-acrosomal EGFP before acrosomal exocytosis in mouse spermatozoa.

Mammalian spermatozoa must undergo complex physiological and morphological alterations within the female reproductive tract before they become fertilization competent. Two important alterations are capacitation and the acrosome reaction (AR), by which spermatozoa become capable of penetrating the zona pellucida (ZP) of the oocyte. Although various biochemical stimulants have been reported to induce the AR, the true physiological inducer in vivo remains to be identified. Previously, it has been reported that most fertilizing spermatozoa undergo the AR before contacting the ZP and that only a small fraction of in vitro-capacitated spermatozoa can penetrate the ZP. Therefore, it is important to identify which capacitating spermatozoa undergo the AR in response to potential AR inducers such as progesterone. Here we show that spermatozoa undergo a dynamic rearrangement of the acrosome during in vitro capacitation. This involves the rapid movement of an artificially introduced soluble component of the acrosome, enhanced green fluorescent protein (EGFP), from the acrosomal cap region to the equatorial segment (EQ) of the sperm head. Spermatozoa exhibiting the EQ pattern were more sensitive to progesterone than were those without it. We suggest that spermatozoa that are ready to undergo acrosomal exocytosis can be detected by real-time EGFP imaging. This offers a promising new method for identifying where spermatozoa undergo the AR in the female reproductive tract in vivo.

Characterization and immunolocalization of HBP, FA-1 and TIMP-2 like proteins in cattle bull semen: HBP modification in in vitro capacitated spermatozoa

Characterization and localization of HBP, FA-1 and TIMP-2 like proteins in spermatozoa and seminal plasma of cattle bulls was carried out by immunoblotting and immunofluoresence. Anti-HBP, anti-TIMP-2 and anti-FA-1 reacted with 55, 48, 45, 42, 35, 30, 24, 18, 16 kDa; 65, 45, 24, 16 kDa and 55, 48, 16 kDa sperm proteins on immunoblots. Immunofluoresence indicated that HBP/TIMP-2 are localized mainly on acrosomal cap, whereas, FA-1 predominantly on post acrosomal cap. Among the bulls, positive for 60, 45, 16 kDa FA-1 like proteins in sperm extracts and 11/16 kDa in SP; 7, 8, 6 and 7 bulls also showed higher rate of in vitro AR. Number of bulls positive for 65, 24 kDa-TIMP-2 and with higher rate of AR was more as compared to other anti-TIMP-2 reactive sperm/SP proteins. Therefore, 60, 45 and 16 kDA-FA-1, 65 and 24 kDa-TIMP-2 like proteins may serve as indicators of higher rate of in vitro AR vis a vis fertility of cattle bulls. Immunoblotting of cpapacitatd and un-capacitated spermatozoa with anti-HBP suggested that removal of 55, 48, 45, 40, 37 and 30 kDa HBP from in vitro acrosome reacted cattle bull spermatozoa allowed heparin to mediate in vitro AR and an increase in intensity of 110, 90 kDa and exposure of 65, 60, 26 and 16 kDa HBP after AR may be important for binding of sperm to ovum, penetration and fertilization.

Mutual interaction of dog sperm LDHC4, PH-20, actin and tubulin proteins and their immunocontraceptive potential in bitches

In this paper, interaction of LDHC4, PH-20, α-actin and β-tubulin on dog's sperm surface and their immunocontraceptive effect in bitches has been elucidate. Anti spam -1 and anti LDHC4 recognized 46/32 and 36/30/28 kDa proteins in dog sperm extracts on immunoblots. Whereas, proteins of 43, 36 kDa and 62, 46, 43, 30 and 28 kDa were detected on immunoblots with anti-α-actin and anti-β-tubulin. In MALDI-TOF analysis, sequence of 46, 30, 28 and 43, 36 kDa antigens matched twice to β-tubulin and α-actin respectively. Therefore, one sub unit each of LDHC4 and α-actin had same mol wt of 36 kDa and two other sub units each of LDHC4 and β-tubulin shared the same mol wt of 30/28 kDa. Reaction of 46 kDa protein both with anti-spam-1 and β-tubulin indicated similarity in their mol wts. Similarlythereaction of 43 kDa protein both with antiα-actin and anti-β-tubulin revealed some similarity between both. Sequence analysis revealed 19.49%, 24.28%, 21.42% and 18.75% similarity of LDHC4/α-actin, PH-20/α-actin, LDHC4/β-tubulin and LDHC4/tubulin respectively. Immunofluoresence staining of dog sperm smears also indicated similarity in localization of these four proteins. Localization of PH-20 and β-tubulin was mainly on the entire head region. Whereas, LDHC4 is uniformly distributed on the whole sperm and actin is more concentrated on the post acrosomal cap. Purified native proteins of 46/32 and 36/30/28 kDa were injected to a group of two bitches respectively through i/m route to elucidate their immunocontraceptive potential. Immunization of bitches in both the groups resulted in suppressed heat, and inhibited natural mating. On being subjected to AI, ultrasonography of four immunized and two unimmunized (control) bitches 35 days after insemination confirmed the presence of 0 and 3–4 fetuses respectively. Therefore, it seems that LDHC4/PH-20 and α-actin/tubulin share their molecular weights or interact with each other or actin/β-tubulin superimpose LDHC4/PH-20 on sperm surface. In view of observed contraceptive effect, it can be concluded that LDHC4 and PH-20 collectively with α-actin and β-tubulin affect estrus, process of natural mating and ultimately the fertility in bitches.

Octylphenol induces changes in glycosylation pattern, calcium level and ultrastructure of bank vole spermatozoa in vitro

Our previous studies revealed that in bank vole testicular cells octylphenol (OP) action is related either to its binding to estrogen receptors or increasing cAMP level that modulates spermatozoa motility and acrosome reaction (AR), (Kotula-Balak et al., 2011, 2014). To better understand the mechanisms underlying these changes, in the present study we aimed at evaluating the glycosylation pattern, calcium (Ca2+) level and ultrastructure of OP-treated vole spermatozoa. Glycans were recognized by lectins and localized for the first time on the surface of acrosomal cap and tail of vole spermatozoa. Expression and localization of glycans were determined histochemically and analyzed quantitatively. Following OP the expression of glycans markedly changed with concomitant increase of intracellular Ca2+ concentration. Altered Ca2+ signaling pathway and ultrastructural changes in sperm acrosome region were revealed by immunoenzymatic assay and electron microscope analysis together with hypo-osmotic swelling test, respectively. In detail, AR advancement reflected by the presence of small vesicles in the close vicinity to the sperm head was observed, while tail membrane integrity remained intact.Our study provides a new insight on OP direct effects on precocious AR of vole spermatozoa through modulation of the glycan expression and its distribution concomitantly with changes in Ca2+ signaling pathway and acrosome ultrastructure in vitro.

Fine structural traits of Crocidura shantungensis caudal epididymal spermatozoa

The structural and morphometric traits of spermatozoa reflect species-specific evolutionary and functional characters. The shrew, Crocidura shantungensis, which is an indigenous species of Ulleung-do, is dominant on this island and is suspected to have unique traits different from other shrews. Based on a previous sperm ultrastructural analysis, the structural and morphometric traits of caudal epididymal spermatozoa were evaluated by light, scanning, and transmission electron microscopy. Total length was 110.0 μm. The head was paddle shaped with a globular nucleus and was 16.0 μm in length. The acrosomal cap covered four-fifths of the nucleus, and the subacrosomal space had sawtooth-shaped edges. The number of mitochondrial gyres was 84–86. Nine segmented columns in the neck consisted of 12 knobs, and each column was fused with outer dense fiber. The outer dense fibers 1, 5, and 6 were thicker than the others. Outer fiber 1 was shaped like a horseshoe, whereas 5 and 6 were fused with each other. Numerous satellite fibers were scattered on the mid-piece between outer fibers 4 and 5, and 6 and 7. Two specific strong electron-dense fibers were found uniquely near the satellite fibers. In conclusion, our results demonstrate that the caudal sperm of Ulleung-do C. shantungensis has unique ultrastructural and morphometric traits based on common sperm characters of the subfamilies, suggesting unique physiological roles in reproduction.

The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.

Spermatozoal capacitation of pink shrimp Farfantepenaeus paulensis

The present study evaluated spermatozoal capacitation in Farfantepenaeus paulensis. This process has direct applications in aquaculture, and it consists of the ionic, biochemical and morphological changes during the period that the spermatophore is stored or adhered to the thelycum. These changes make the spermatozoa capable of fertilization. The morphological changes of spermatozoal capacitation have been previously investigated only in sicyoniids and open-thelycum penaeids. Thus, this study is the first morphological account of a closed-thelycum penaeid. F. paulensis broodstock were captured offshore in southern Brazil, and spermatophores were collected from the terminal ampoule and from the thelycum of sexually mature females that had naturally copulated. The ultrastructure of the spermatozoal capacitation was investigated via transmission electron microscopy. Spermatozoa of F. paulensis show the following changes related to capacitation: (1) the chromatin became less condensed; (2) the acrosomal region became more electron-dense (3) the acrosomal cap became less concave; and (4) the subacrosomal region became much more electron-dense. These results demonstrate that the morphological changes in capacitated spermatozoa of F. paulensis are similar to those previously reported for open-thelycum penaeids, i.e., Litopenaeus species. Further studies on the capacitation process are required, especially to evaluate the interaction between biochemical and morphological changes. Such research could be useful for developing biotechnologies that will allow spermatozoal induction without storing the spermatophore in the thelycum and therefore allow in vitro larval production.

Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB(+1), to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.

Localization of Tektin 1 at Both Acrosome and Flagella of Mouse and Bull Spermatozoa

Tektins (TEKTs) are constitutive filamentous proteins of microtubules in cilia, flagella, basal bodies, and centrioles. In mammals, five TEKTs (TEKT1, 2, 3, 4, and 5) have been identified in testis and spermatozoa. With the exception of TEKT1, these TEKTs have been reported to be present in spermatozoa with predominant localization at the peri-axoneme structures of flagella, i.e., mitochondria and outer dense fibers. In the present study, we produced an antibody against TEKT1 to examine the localization of TEKT1 in mouse, bull, and rat spermatozoa. By immunoblot analyses and immunofluorescence microscopy, we found TEKT1 to be present in sperm flagella and at the apical region of acrosome cap in spermatozoa of all these species. Acrosome-associated TEKT1 disappeared after in vitro acrosome reaction in mouse spermatozoa. These observations suggest another potential role for TEKT1 as a cytoskeletal element in the sperm head, or as a molecule involved in acrosome-related phenomena, such as acrosome reaction.

Epithelial cadherin is present in bovine oviduct epithelial cells and gametes, and is involved in fertilization-related events

Fertilization is a calcium-dependent process that involves sequential cell–cell adhesion events of spermatozoa with oviduct epithelial cells (OECs) and with cumulus-oocyte complexes (COCs). Epithelial cadherin (E-cadherin) participates in calcium-dependent somatic cell adhesion; the adaptor protein β-catenin binds to the E-cadherin cytoplasmic domain and links the adhesion protein to the cytoskeleton. The study was conducted to immunodetect E-cadherin and β-catenin in bovine gametes and oviduct (tissue sections and OEC monolayers), and to assess E-cadherin participation in fertilization-related events. Epithelial cadherin was found in spermatozoa, oocytes, cumulus cells, and OEC. In acrosome-intact noncapacitated spermatozoa, E-cadherin was mainly localized in the apical ridge and acrosomal cap (E1-pattern; 84 ± 9%; mean ± standard deviation of the mean). After sperm treatment with heparin to promote capacitation, the percentage of cells with E1-pattern (56 ± 12%) significantly decreased; concomitantly, the percentage of spermatozoa depicting an E-cadherin staining pattern similar to E1-pattern but showing a signal loss in the acrosomal cap (E2-pattern: 40 ± 11%) increased. After l-α-lysophosphatidylcholine–induced acrosome reaction, E-cadherin signal was mainly localized in the inner acrosomal membrane (E3-pattern: 67 ± 22%). In IVM COC, E-cadherin was immunodetected in the plasma membrane of cumulus cells and oocytes, but was absent in the polar body. The 120 KDa mature protein form was found in protein extracts from spermatozoa, oocytes, cumulus cells, and OEC. β-Catenin distribution followed E-cadherin's in all cells evaluated. Epithelial cadherin participation in cell–cell interaction was evaluated using specific blocking monoclonal antibody DECMA-1. Sperm incubation with DECMA-1 impaired sperm–OEC binding (the number of sperm bound to OEC: DECMA-1 = 6.7 ± 6.1 vs. control = 29.6 ± 20.1; P < 0.001), fertilization with COC (% fertilized COC: DECMA-1 = 68.8 ± 10.4 vs. control = 90.7 ± 3.1; P < 0.05) or denuded oocytes (% fertilized oocytes: DECMA-1 = 57.0 ± 15.2 vs. control = 89.2 ± 9.8; P < 0.05) and binding to the oolemma (the number of sperm bound to oolemma: DECMA-1 = 2.2 ± 1.1 vs. control = 11.1 ± 4.8; P < 0.05). This study describes, for the first time, the presence of E-cadherin in bovine spermatozoa, COC, and OEC, and shows evidence of its participation in sperm interaction with the oviduct and the oocyte during fertilization.