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Infertility is a global health problem affecting one in six couples, with 50% of cases attributed to male infertility. Spermatozoa are male gametes, specialized cells that can be divided into two parts: the head and the flagellum. The head contains a vesicle called the acrosome that undergoes exocytosis and the flagellum is a motility apparatus that propels the spermatozoa forward and can be divided into two components, axonemes and accessory structures. For spermatozoa to fertilize oocytes, the acrosome and flagellum must be formed correctly. In this Review, we describe comprehensively how functional spermatozoa develop in mammals during spermiogenesis, including the formation of acrosomes, axonemes and accessory structures by focusing on analyses of mouse models., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Scanning ion-conductance microscopy allowed us to document an external Ca 2+ dependent ATP driven volume increase (ATPVI) in capacitated human sperm heads. We examined the involvement of purinergic receptors (PRs) P2X2R and P2X4R in ATPVI using their co-agonists progesterone and Ivermectin (Iver), and Cu 2+ , which co-activates P2X2Rs and inhibits P2X4Rs. Iver enhanced ATPVI and Cu 2+ and 5BDBD inhibited it, indicating P2X4Rs contributed to this response. Moreover, Cu 2+ and 5BDBD inhibited the ATP-induced acrosome reaction (AR) which was enhanced by Iver. ATP increased the concentration of intracellular Ca 2+ ([Ca 2+ ] i ) in >45% of individual sperm, most of which underwent AR monitored using FM4-64. Our findings suggest that human sperm P2X4R activation by ATP increases [Ca 2+ ] i mainly due to Ca 2+ influx which leads to a sperm head volume increase, likely involving acrosomal swelling, and resulting in AR., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bacterial contamination in the semen deteriorates spermatozoa function. One mechanism through which this may occur is by inducing a premature form of the acrosome reaction (spontaneous acrosome reaction (sAR)) which has been shown to abrogate fertilization. To understand the mechanism by which bacteria affect sperm functions, we determined the effects of bacteria on sperm sAR and on other parameters involved in sperm capacitation. Sperm cells undergo biochemical changes in the female reproductive tract collectively called capacitation. Only capacitated sperm can undergo the physiological acrosomal exocytosis process near or on the oocyte, which allows the spermatozoon to penetrate and fertilize the egg. Bovine sperm incubated with the bacteria Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) or Pseudomonas aeruginosa (P. aeruginosa), revealed a sperm-bacteria interaction, however only E. coli and P. aeruginosa caused an increase in sperm sAR. This effect was seen only when the bacteria were present with the sperm during the full incubation under capacitation conditions but not when the bacteria were added to capacitated sperm. These results indicate that bacteria affect sperm during capacitation and not at the AR step. In addition, Ca 2+ influx, protein kinase A, and protein tyrosine phosphorylation activities, three essential processes that promote capacitation, were inhibited by the bacteria. Moreover, increasing intracellular cAMP, which also occur during sperm capacitation, caused significant reverse of sAR induced by the bacteria., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In somatic cells, this ratio indicates that autophagy has been activated and similar LC3 processing has been described in mammalian spermatozoa. The subcellular localization of autophagy-related proteins was assessed by immunofluorescence with specific antibodies that recognized Atg16, Beclin-1, and LC3. The colocalization of acrosomal membranes (PNA) and LC3 was studied by confocal microcopy, and the acrosome reacted cells were quantified by flow cytometry. The incubation of stallion sperm in capacitating conditions (BWW; 3 h) significantly increased LC3 processing. This increment was three to four times higher after the induction of the acrosome reaction in these cells. LC3 was mainly expressed in the head in mature ejaculated sperm showing a clear redistribution from the post-acrosomal region to the acrosome upon the incubation of sperm in capacitating conditions (BWW, 3 h). After the induction of the acrosome reaction, LC3 colocalized with the acrosome or the apical plasmalemma membranes in the head of the stallion spermatozoa. The inhibition or activation of autophagy-related pathways in the presence of autophagy activators (STF-62247) or inhibitors (E-64d, chloroquine) significantly increased LC3 processing and increased the percent of acrosome reacted cells, whereas 3-methyladenine almost completely inhibited LC3 processing and the acrosome reaction. In conclusion, we found that sperm capacitation and acrosome reaction could be regulated by autophagy components in sperm cells ex vivo by processes that might be independent of the intraluminal pH of the acrosome and dependent of LC3 lipidation. It can be speculated that, in stallion sperm, a form of noncanonical autophagy utilizes some components of autophagy machinery to facilitate the acrosome reaction.

The widely used porcine artificial insemination procedure involves the use of liquid-stored semen because it is difficult to control the quality of frozen-thawed porcine sperm. Therefore, there is a high demand for porcine semen. The control and enhancement of sperm function are required for the efficient reproduction of pigs. We previously reported that gamma-aminobutyric acid (GABA) enhanced sperm capacitation and acrosome reaction in mice. In this study, we demonstrated the presence of GABA A receptors in porcine sperm acrosome. Furthermore, we investigated the GABA effects on porcine sperm function. We did not detect any marked effect of GABA on sperm motility and tyrosine phosphorylation of sperm proteins. However, GABA promoted acrosome reaction, which was suppressed by a selective GABA A receptor antagonist. GABA binds to GABA A receptors, resulting in chloride ion influx. We found that treatment with 1 μM GABA increased the intracellular concentration of chloride ion in the sperm. In addition, the GABA concentration effective in the acrosome reaction was correlated with the porcine sperm concentration. These results indicate that GABA and its receptors can act as modulators of acrosome reaction. This study is the first to report the effects of GABA on porcine sperm function., (© 2022 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

We studied the effect of extracellular vesicles of the follicular fluid on morphofunctional characteristics of human spermatozoa using CASA (computer-assisted sperm analysis) analytical system. The vesicles were obtained by sequential centrifugation at different rotational speeds and frozen at -80°C in the Sydney IVF Gamete Buffer medium. The sperm fraction was isolated from the seminal fluid of 21 patients aged 27-36 years by differential centrifugation in a density gradient. The precipitate was suspended in Sydney IVF Gamete Buffer to a concentration of 10 6 /ml and incubated with vesicles (1:2) at 37°C in a CO 2 incubator for 30 min and 1 h. Sperm fraction incubated without vesicles served as the control. After incubation, some sperm samples were centrifuged at 700g for 5 min and fixed in 2.5% glutaraldehyde in 0.1 M buffer for transmission electron microscopy. After 30-min and 1-h incubation, the progressive and total sperm motility improved, the curvilinear and linear velocity of spermatozoa did not change significantly. Incubation with vesicles significantly changed the trajectory of sperm movement, which can attest to an increase in their hyperactivation and, probably, fertilizing capacity. Analysis of the effect of extracellular vesicles of follicular fluid on sperm motility will help to improve the effectiveness of assisted reproductive technology programs with male infertility factor by improving sperm characteristics in patients with asthenozoospermia and increasing the fertilizing ability of the sperm., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Sexual reproduction requires the fusion of two gametes in a multistep and multifactorial process termed fertilization. One of the main steps that ensures successful fertilization is acrosome reaction. The acrosome, a special kind of organelle with a cap-like structure that covers the anterior portion of sperm head, plays a key role in the process. Acrosome biogenesis begins with the initial stage of spermatid development, and it is typically divided into four successive phases: the Golgi phase, cap phase, acrosome phase, and maturation phase. The run smoothly of above processes needs an active and specific coordination between the all kinds of organelles (endoplasmic reticulum, trans-Golgi network, and nucleus) and cytoplasmic structures (acroplaxome and manchette). During the past two decades, an increasing number of genes have been discovered to be involved in modulating acrosome formation. Most of these proteins interact with each other and show a complicated molecular regulatory mechanism to facilitate the occurrence of this event. This review focuses on the progresses of studying acrosome biogenesis using gene-manipulated mice and highlights an emerging molecular basis of mammalian acrosome formation., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ruminant testes are ~2-6 °C below body temperature; increased testicular temperature reduces sperm motility and morphology. Our objective was to serially monitor scrotal subcutaneous temperatures during testicular heat stress and relate those to sperm quality. Two experiments were conducted, with temperature sensors surgically implanted in scrotal subcutaneous tissues recording temperatures every 15 min and semen collected and evaluated weekly. After an initial control interval, testicular temperature was increased. In Experiment 1, in two Angus bulls, whole-scrotum insulation for 96 h increased scrotal subcutaneous temperatures by ~2.0-2.5 °C (P < 0.05). Total and progressive motility decreased (P < 0.05) and reached a nadir at Week 3 (~20 and 10%, respectively). Furthermore, morphologically normal sperm and acrosome integrity also decreased significantly, reaching nadirs at Weeks 3 (15%) and 4 (34%). In Experiment 2, 10 Dorset rams were allocated randomly into two equal groups and either: 1) exposed to 28 °C ambient temperature and 30-34% RH for 8 h/d for 4 d; or 2) subjected to scrotal neck insulation that was applied and removed at the same time as the start and completion, respectively, of heat exposures in the other rams. Scrotal subcutaneous temperatures (monitored in three rams per group) were increased in response to whole-body heating (~0.8 °C, P < 0.05), but not significantly changed by scrotal neck insulation. Decreases in sperm quality were generally similar between treatments, with the most profound changes evident 4 wk after heat stress, with ~10 percentage point reductions in both total and progressive motility and ~10 and 20 percentage point decreases in morphologically normal sperm in rams with whole-body heating versus scrotal neck insulation, respectively. In conclusion, scrotal subcutaneous temperature was significantly increased by scrotal insulation or whole-body heating, but not by scrotal neck insulation; however, all three heat-stress models decreased sperm motility and morphology in bulls and rams., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Deciphering sperm transcriptome is the key to understanding the molecular mechanisms governing peri-fertilization, embryonic development, and pregnancy establishment. This study aimed to profile sperm transcriptome to identify signature transcripts regulating male fertility. Semen samples were collected from 47 bulls with varied fertility rates. The sperm total RNA was isolated (n = 8) and subjected to transcriptome sequencing. Based on the expression pattern obtained from RNA profiling, the bulls were grouped (p = 0.03) into high-fertile and sub-fertile, and signature transcripts controlling sperm functions and fertility were identified. The results were validated using the OMIM database, qPCR, and sperm function tests. The sperm contains 1100 to 1700 intact transcripts, of which BCL2L11 and CAPZA3 were abundant and associated (p < 0.05) with spermatogenesis and post-embryonic organ morphogenesis. The upregulated genes in the acrosome integrity and functional membrane integrity groups had a close association with the fertility rate. The biological functions of these upregulated genes (p < 0.05) in the high-fertile bulls were associated with spermatogenesis (AFF4 and BRIP1), sperm motility (AK6 and ATP6V1G3), capacitation and zona binding (AGFG1), embryo development (TCF7 and AKIRIN2), and placental development (KRT19). The transcripts involved in pathways regulating embryonic development such as translation (EEF1B2 and MTIF3, p = 8.87E-05) and nonsense-mediated decay (RPL23 and RPL7A, p = 5.01E-27) were upregulated in high-fertile bulls. The identified transcripts may significantly impact oocyte function, embryogenesis, trophectoderm development, and pregnancy establishment. In addition, the study also reveals that the genes governing sperm functional membrane integrity and acrosome integrity have a prospective effect on male fertility., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In boar sperm, we have previously shown that capacitation is associated with the appearance of the p32 tyrosine phosphoprotein complex. The principal tyrosine phosphoprotein involved in this complex is the acrosin-binding protein (ACRBP), which regulates the autoconversion of proacrosin to intermediate forms of acrosin in both boar and mouse sperm. However, the complete biological role of ACRBP has not yet been elucidated. In this study, we tested the hypothesis that tyrosine phophorylation and the presence of the ACRBP in the sperm head are largely necessary to induce capacitation, the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding, all of which are necessary steps for fertilization. In vitro fertilization (IVF) was performed using matured porcine oocytes and pre-capacitated boar sperm cultured with anti-phosphotyrosine antibodies or antibodies against ACRBP. Anti-ACRBP antibodies reduced capacitation and spontaneous AR (P<0.05). Sperm-ZP binding declined in the presence of anti-phosphotyrosine or anti-ACRBP antibodies. The localisation of anti-ACRBP antibodies on the sperm head, reduced the ability of the sperm to undergo the AR in response to solubilized ZP or by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase. These results support our hypothesis that tyrosine phosphorylated proteins and ACRBP are present upon the sperm surface in order to participate in sperm-ZP binding, and that ACRBP upon the surface of the sperm head facilitates capacitation and the AR in the porcine., Competing Interests: The authors have declared that no competing interests exist.

The encounter of oocyte and sperm is the key event initiating embryonic development in mammals. Crucial functions of this existential interaction are determined by proteolytic enzymes, such as acrosin, carried in the sperm head acrosome, and ovastacin, stored in the oocyte cortical granules. Ovastacin is released upon fertilisation to cleave the zona pellucida, a glycoprotein matrix surrounding the oocyte. This limited proteolysis hardens the oocyte envelope, and thereby provides a definitive block against polyspermy and protects the developing embryo. On the other hand, acrosin, the renowned and most abundant acrosomal protease, has been thought to enable sperm to penetrate the oocyte envelope. Depending on the species, proteolytic cleavage of the zona pellucida by acrosin is either essential or conducive for fertilisation. However, the specific target cleavage sites and the resulting physiological consequences of this proteolysis remained obscure. Here, we treated native mouse zonae pellucidae with active acrosin and identified two cleavage sites in zona pellucida protein 1 (ZP1), five in ZP2 and one in ZP3 by mass spectrometry. Several of these sites are highly conserved in mammals. Remarkably, limited proteolysis by acrosin leads to zona pellucida remodelling rather than degradation. Thus, acrosin affects both sperm binding and mechanical resilience of the zona pellucida, as assessed by microscopy and nanoindentation measurements, respectively. Furthermore, we ascertained potential regulatory effects of acrosin, via activation of latent pro-ovastacin and inactivation of fetuin-B, a tight binding inhibitor of ovastacin. These results offer novel insights into the complex proteolytic network modifying the extracellular matrix of the mouse oocyte, which might apply also to other species., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Little data exist about the physiological role of ion channels during the freeze-thaw process in mammalian sperm. Herein, we determined the relevance of potassium channels, including SLO1, and of voltage-gated proton channels (HVCN1) during mammalian sperm cryopreservation, using the pig as a model and through the addition of specific blockers (TEA: tetraethyl ammonium chloride, PAX: paxilline or 2-GBI: 2-guanidino benzimidazole) to the cryoprotective media at either 15 °C or 5 °C. Sperm quality of the control and blocked samples was performed at 30- and 240-min post-thaw, by assessing sperm motility and kinematics, plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and intracellular O 2 - ⁻ and H 2 O 2 levels. General blockade of K + channels by TEA and specific blockade of SLO1 channels by PAX did not result in alterations in sperm quality after thawing as compared to control samples. In contrast, HVCN1-blocking with 2-GBI led to a significant decrease in post-thaw sperm quality as compared to the control, despite intracellular O 2 - ⁻ and H 2 O 2 levels in 2-GBI blocked samples being lower than in the control and in TEA- and PAX-blocked samples. We can thus conclude that HVCN1 channels are related to mammalian sperm cryotolerance and have an essential role during cryopreservation. In contrast, potassium channels do not seem to play such an instrumental role.

The acrosome reaction (AR) is a strictly-regulated, synchronous exocytosis that is required for sperm to penetrate ova. This all-or-nothing process occurs only once in the sperm lifecycle through a sequence of signaling pathways. Spontaneous, premature AR therefore compromises fertilization potential. Although protein kinase A (PKA) pathways play a central role in AR across species, the signaling network used for AR induction is poorly understood in birds. Mechanistic studies of mammalian sperm AR demonstrate that PKA activity is downstreamly regulated by Src family kinases (SFKs). Using SFK inhibitors, our study shows that in chicken sperm, SFKs play a role in the regulation of PKA activity and spontaneous AR without affecting motility. Furthermore, we examined the nature of SFK phosphorylation using PKA and protein tyrosine phosphatase inhibitors, which demonstrated that unlike in mammals, SFK phosphorylation in birds does not occur downstream of PKA and is primarily regulated by calcium-dependent tyrosine phosphatase activity. Functional characterization of SFKs in chicken sperm showed that SFK activation modulates the membrane potential and plays a role in inhibiting spontaneous AR. Employing biochemical isolation, we also found that membrane rafts are involved in the regulation of SFK phosphorylation. This study demonstrates a unique mechanism for regulating AR induction inherent to avian sperm that ensure fertilization potential despite prolonged storage., Competing Interests: The authors have declared that no competing interests exist.

Male fertility disorders often have their origin in disturbed spermatogenesis, which can be induced by genetic factors. In this study, we used interspecific recombinant congenic mouse strains (IRCS) to identify genes responsible for male infertility. Using ultrasonography, in vivo and in vitro fertilization (IVF) and electron microscopy, the phenotyping of several IRCS carrying mouse chromosome 1 segments of Mus spretus origin revealed a decrease in the ability of sperm to fertilize. This teratozoospermia included the abnormal anchoring of the acrosome to the nucleus and a persistence of residual bodies at the level of epididymal sperm midpiece. We identified a quantitative trait locus (QTL) responsible for these phenotypes and we have proposed a short list of candidate genes specifically expressed in spermatids. The future functional validation of candidate genes should allow the identification of new genes and mechanisms involved in male infertility.

To date, sperm morphometric studies have assessed whole sperm populations without considering sperm function. The aim of this study was to evaluate the relationship of sperm membrane and acrosomal integrity with sperm morphometry in liquid semen samples collected from bulls. To this end, sperm morphometry was performed on cryopreserved semen samples from 16 bulls by a combination of fluorescent dyes, including Hoechst 33343, carboxyfluorescein diacetate, and propidium iodide. This allowed discrimination of different subpopulations on the basis of sperm membrane and acrosomal integrity and analysis of the morphometrics of the sperm head, nucleus, and acrosome using a specific plug-in module created on ImageJ. Acrosomal integrity was related to sperm morphometry as the heads of spermatozoa with a damaged acrosome were significantly smaller than those with a normal acrosome (P < 0.001). In the case of spermatozoa with an intact acrosome, those with a damaged plasma membrane had a larger sperm head and acrosome than spermatozoa with an intact plasma membrane (P < 0.001). No significant differences in the sperm head size were observed between sperm subpopulations without an acrosome or in the nuclear sperm morphometry of the different subpopulations. There was a positive correlation between the sperm motility values of the samples and the morphometric parameters for intact spermatozoa. These correlations were particularly strong for the morphometric parameters of the sperm acrosome. We conclude that there are clear differences in the sperm morphometry depending on the status of the sperm membrane and acrosome and this should be considered when performing this kind of analysis., Competing Interests: None

Bovine antisperm antibodies (ASAs) have been associated with teratospermia and asthenospermia. It was hypothesized here that scrotal insulation induces the formation of ASAs and deterioration of sperm function. Scrotal insulation bags were placed in 10 bulls for 8 days. Semen was collected on days -29, -22 and -2, twice weekly from days 5 to 54, and thereafter weekly until day 96 (day 0 = first day of scrotal insulation). On each collection day, scrotal circumference, sperm motility, morphology, membrane integrity, acrosome integrity, apoptosis, lipid peroxidation, mitochondrial membrane potential, ASA binding and DNA integrity were evaluated. The percentage of IgG- and IgA-bound sperm increased between days 12 and 96 (P < 0.0001), in association with poor motility (days 19-30, P < 0.005) and morphology (days 8-40, P < 0.0001). Mean scrotal circumference decreased between days 15 and 75 (P < 0.0001). There was also a deterioration in sperm membrane integrity (days 19-40, P < 0.0001), acrosome integrity (days 26-89, P < 0.0001), lipid peroxidation (days 5-12, P < 0.0001), and mitochondrial membrane potential (days 12-96, P = 0.001). In contrast, a decrease in apoptotic cells (days 37-83, P = 0.0002) and lipid peroxidation (days 19-96, P < 0.0001) was noticed. Most bulls recovered normospermia by day 96. However, the persistence of ASAs, acrosomal damage and dysfunctional mitochondria suggest a long term effect of scrotal insulation on sperm function and the homeostasis of the reproductive immune system.

Previously we demonstrated that multidrug resistance-associated protein 4 transporter (MRP4) mediates cAMP efflux in bovine spermatozoa and that extracellular cAMP (ecAMP) triggers events associated to capacitation. Here, we deepen the study of the role of MRP4 in bovine sperm function by using MK571, an MRP4 inhibitor. The incubation of spermatozoa with MK571 during 45 min inhibited capacitation-associated events. MRP4 was localized in post-acrosomal region and mid-piece at 15 min capacitation, while at 45 min it was mainly located in the acrosome. After 15 min, MK571 decreased total sperm motility (TM), progressive motility (PM) and several kinematic parameters. The addition of ecAMP rescued MK571 effect and ecAMP alone increased the percentage of motile sperm and kinematics parameters. Since actin cytoskeleton plays essential roles in the regulation of sperm motility, we investigated if MRP4 activity might affect actin polymerization. After 15 min capacitation, an increase in F-actin was observed, which was inhibited by MK571. This effect was reverted by the addition of ecAMP. Furthermore, ecAMP alone increased F-actin levels while no F-actin was detected with ecAMP in the presence of PKA inhibitors. Our results support the importance of cAMP efflux through MRP4 in sperm capacitation and suggest its involvement in the regulation of actin polymerization and motility.

Saimiri collinsi is used as an animal model in biotechnology research for conservation of species from the genus Saimiri. However, the development of biotechnologies depends on a proper knowledge of the sperm morphology to understand the basic aspects of sperm physiology, as potential male fertility depends on different cellular sperm structures. With this purpose, this study characterized the micromorphological and ultrastructural characteristics of squirrel monkeys (Saimiri collinsi) sperm using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM electromyography revealed that a normal Saimiri collinsi sperm measures 71.7 ± 0.7 μm with lateral tail insertion, a paddle-shaped flattened head and an acrosome occupying most of the head. TEM also showed that the middle piece is characterized by a central 9 + 2 microtubule axoneme surrounded by nine dense fibres, and that the mitochondria were juxtaposed, forming the mitochondrial sheath. Here we provide the first micromorphological and ultrastructure description of S. collinsi sperm.

The present study investigates the efficacy of dimehtlyformamide (DMF) as a permeable cryoprotectant and its effect on quality and fertility of Indian red jungle fowl sperm. Semen was collected from eight mature roosters, pooled, divided into five aliquots and diluted with red fowl extender having DMF (0%, 4%, 6%, 8% and 10%). Diluted semen samples were cooled from 37 °C to 4 °C, 20% glycerol added to control (0% DMF), equilibrated for 10 min and filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Sperm motility, plasma membrane functionality, viability and acrosome integrity were assessed at post dilution, cooling, equilibration and freeze-thawing stage of cryopreservation. Cryopreservation stages had negative effects (P < 0.05) on semen quality parameters. Percentages of sperm motility, plasma membrane functionality, viability and acrosome integrity were recorded highest in extender having 8% DMF at post-dilution, cooling, equilibration and freeze-thawing stage. Fertility results after artificial insemination were recorded higher (P < 0.05) with 8% DMF compared to 20% glycerol. Dimehtlyformamide (8%) in red fowl extender improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Species preservation depends on the success of fertilization. Sperm are uniquely equipped to fulfill this task, and, although several mechanisms are conserved among species, striking functional differences have evolved to contend with particular sperm-egg environmental characteristics. This review highlights similarities and differences in sperm strategies, with examples within internal and external fertilizers, pointing out unresolved issues.

The current study examined the impact of the supplementation of ginger and echinacea extract, as natural antioxidant agents, in freezing extender on the quality and fertility potential of ram epididymal spermatozoa after cryopreservation. Epididymal spermatozoa isolated from Forty testicles, obtained from 20 rams, with motility >80% and total morphological abnormalities <10% were pooled, divided into 7 aliquots and used for cryopreservation. The semen samples were re-suspended with basic Tris egg yolk diluent containing ginger and echinacea extracts (5, 10 and 20 mg/l). The control diluent comprised of only extender and lacked any antioxidant agent. For the determination of sperm quality, frozen straws were thawed after 7-10 days, and then the sperm characteristics were assessed. The supplementation of ginger at a concentration of 10 mg/l, as well as the addition of 10 and 20 mg/l echinacea extract significantly improved total motility and velocity parameters. The status of acrosome integrity and lipid peroxidation significantly improved in spermatozoa when supplemented with 10 mg/l ginger and 20 mg/l echinacea extract. Also, 5 mg/l ginger extract and 20 mg/l echinacea extract significantly improved mitochondrial activity. The highest ratio of the dispersion of sperm chromatin was observed in spermatozoa treated with 10 mg/l ginger extract. The cleavage rate was markedly higher in matured oocytes that were fertilized with frozen spermatozoa treated with 20 mg/l ginger extract and 10 mg/l echinacea. The application of ginger and echinacea extract resulted in improvement in the quality and fertility of frozen-thawed spermatozoa. However, future studies are wanted to elucidate how the active components in these extracts prevent cryo-damages in spermatozoa., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to -35 °C (40 °C/min), from -35 °C to -65 °C (17 °C/min), and then from -65 °C to -85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to -5 °C (4 °C/min), from -5 °C to -110 °C (25 °C/min), and then from -110 °C to -140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to -10 °C (5 °C/min), and then from -10 °C to -130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The study aimed at the analysis of the functional status of cryopreserved bovine sperm using multicolor flow cytometry. The value of sperm functional traits as predictors of bull fertility was further evaluated through a retrospective fertility study. For this purpose, 20 Holstein-Friesian bulls serving as mature sperm donors in an artificial insemination (AI) center were selected based on their annual 56-d non-return rate (%) after at least 1,000 AI, and were accordingly classified as high (HF; n HF = 10 bulls) or low fertility bulls (LF; n LF = 10 bulls). Four to 5 cryopreserved ejaculates per bull (91 ejaculates in total) were examined immediately after thawing (0 h) and after a 3-h incubation at 38°C (3 h). A panel of 5 fluorochromes including calcein violet, propidium iodide, pycoerythrin-conjugated lectin of Arachis hypogea, Fluo-4, and cyanine dye DiIC 1 (5) was configured by means of a 3-laser flow cytometer, to simultaneously assess sperm esterase activity, plasma membrane integrity, acrosomal status, intracellular Ca 2+ levels, and mitochondrial membrane potential, respectively. The % relative size of 18 sperm sub-populations showing 2 or more of a combination of the following features was determined: high esterase activity (C pos ), intact plasma membrane (PI neg ), unstained acrosome (PNA neg ), low intracellular Ca 2+ levels (F neg ), and high mitochondrial membrane potential (M pos ). In both fertility groups, M pos cells comprised more than 90 and 84% of PI neg PNA neg sperm at 0 and 3 h, respectively. The percentage of C pos PI neg PNA neg F neg M pos sperm did not differ between HF and LF ejaculates; however, the percentage of F neg cells within the PI neg PNA neg and PI neg M pos sperm populations at 0 h was higher in the HF than in the LF bulls. Applying the random forest ensemble learning method, approximately two-thirds of ejaculates could be correctly assigned to their fertility group. The fraction of F neg sperm within the PI neg M pos population at 0 h was the most important fertility predictor among the 18 defined sperm populations. In conclusion, multicolor flow cytometry offered an insight into the functional heterogeneity of cryopreserved bovine sperm. Indeed, the ability of viable sperm to retain low Ca 2+ levels differed between bulls of diverse fertility. A classifier based on selected sperm populations assessed through multicolor flow cytometry could contribute to the prognosis of bull fertility after AI., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the spatio-temporal expression and function of the end-directed KIFC1 (kinesin family member C1) protein during acrosomogenesis., Methods: The expression and location of KIFC1 were analyzed by flow separation and immunofluorescence, and the small interference RNA (RNAi1) of KIFC1 with high-interference efficiency was screened using in vitro GC2-spd cell lines. The KIFC1 RNAi1 mixed with 0.5% trypan blue solution was microinjected into the testicular seminiferous tubules and negative Con-RNAi1 into the contralateral testis of a 3-week-old Balb/c mouse, followed by morphological analysis of the sperm collected from the testis tail at 3 weeks after injection., Results: The expression of KIFC1 was observed mainly in the cytoplasm of the sperm cells in the early stage of sperm deformation, in the acrosomal vesicle and acrosome in the middle stage, and in the residual body in the late stage, but vanished during sperm maturation. The sperm head deformity rate was significantly higher in the RNAi1 than in the negative control group (［32.12 ± 0.25］% vs ［7.06 ± 1.25］%, P < 0.01)., Conclusions: The KIFC1 protein may play an important role in the formation of spermatozoa, mainly affecting acrosomogenesis.

Background: Voluntary control of fertility is of paramount importance to the modern society. But since the contraceptive methods available for women have their limitations such as urinary tract infections, allergies, cervical erosion and discomfort, a desperate need exists to develop safe methods. Vaginal contraceptives may be the answer to this problem, as these are the oldest ways of fertility regulation, practiced over the centuries. With minimal systemic involvement, these are also the safest. Natural substances blocking or impairing the sperm motility offer as valuable non-cytotoxic vaginal contraceptives. Antimicrobial peptides (AMPs) isolated from plants, animals and microorganisms are known to possess sperm immobilizing and spermicidal properties. Following this, in the quest for alternative means, we have cloned, over expressed and purified the recombinant sperm agglutinating factor (SAF) from Staphylococcus warneri, isolated from the cervix of a woman with unexplained infertility., Methods: Genomic library of Staphylococcus warneri was generated in Escherichia coli using pSMART vector and screened for sperm agglutinating factor (SAF). The insert in sperm agglutinating transformant was sequenced and was found to express ribonucleotide-diphosphate reductase-α sub unit. The ORF was sub-cloned in pET28a vector, expressed and purified. The effect of rSAF on motility, viability, morphology, Mg ++ -dependent ATPase activity and acrosome status of human sperms was analyzed in vitro and contraceptive efficacy was evaluated in vivo in female BALB/c mice., Results: The 80 kDa rSAF showed complete sperm agglutination, inhibited its Mg 2+ -ATPase activity, caused premature sperm acrosomal loss in vitro and mimicked the pattern in vivo showing 100% contraception in BALB/c mice resulting in prevention of pregnancy. The FITC labeled SAF was found to bind the entire surface of spermatozoa. Vaginal application and oral administration of rSAF to mice for 14 successive days did not demonstrate any significant change in vaginal cell morphology, organ weight and tissue histology of reproductive and non-reproductive organs and had no negative impact in the dermal and penile irritation tests., Conclusion: The Sperm Agglutinating Factor from Staphylococcus warneri, natural microflora of human cervix, showed extensive potential to be employed as a safe vaginal contraceptive.

Despite advances in vitrification techniques for sperm cryopreservation, cryo-damages of sperm caused by generation of reactive oxygen species (ROS) continue to impede implementation of this technique. This study analyses the effects of taurine and hypotaurine as anti-oxidants during vitrification of human sperms. The study was performed in two steps. In the first step, 20 normospermic semen samples were vitrified in the presence of varying concentrations of taurine and hypotaurine, and their effects as anti-oxidant agents on classical sperm parameters, hyaluronan-binding assay (HBA), lipid peroxidation (LPO) and acrosome reaction (AR) were studied. Statistical analyses showed that the sperm parameters in all vitrified groups decreased significantly (P < 0.05) compared to the fresh group. However, HBA and acrosome integrity in vitrified groups containing taurine and 50 mM of hypotaurine were better than in the control group (P < 0.05). The morphology of the vitrified group was good only in the group that contained 50 mM of hypotaurine (P < 0.05). Based on the results from the first step, 50 mM of hypotaurine was considered the ideal anti-oxidant formulation and further tests were carried out on 10 normospermic semen samples with this protecting agent. In addition to the mentioned parameters, the expression of heat shock proteinA2 (HSPA2) was studied in the vitrified group with 50 mM hypotaurine, warmed under two different warming temperatures 37 and 42 °C. 50 mM Hypotaurine was found to equally improve motility, morphology, HBA, and AR after warming at 37 °C and 42 °C (P < 0.05). However, at both warming temperatures, the expression of HSPA2 was reduced in all vitrified groups comparing to the fresh group (P < 0.05). In conclusion, taurine and hypotaurine antioxidants, especially 50 mM hypotaurine, are able to reduce deleterious cryo-injuries on morphology, acrosome and HBA and improve sperm recovery at both warming temperatures (37 and 42 °C). However, they do not have any protective action on expression of HSPA2., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sperm competition is a powerful selective force that has influenced many reproductive traits in males and females although additional evolutionary explanations may help to understand the diversity of mammalian reproduction. Sperm morphology varies considerably in mammals with extreme examples in several rodent lineages in which a wide range of sizes and complex head morphologies have been identified. Mammalian spermatozoa also differ in function, with swimming velocity and trajectory showing much divergence. Underlying processes mediating function have received little attention so far, but differences in timing and proportion of sperm undergoing capacitation or acrosomal exocytosis may be related to variation in signalling processes. Furthermore, energy required for sperm functions (such as motion, signalling and overall maintenance of cell integrity) can be produced and consumed, following different patterns among species and this could be the result of several selective forces. A more thorough understanding of the diversity in structure and function of sperm cells, and underlying selective forces, may help us develop better methods to assess them taking into account particulars and generalities of sperm form and performance. Such tests could then become more reliable in estimations of the impact of cryopreservation or effect of changes in the environment and their relevance for fertility., (© 2019 Blackwell Verlag GmbH.)

A study was conducted to establish a sustainable and effective manual freezing technique for cryopreservation of Bangladeshi ram semen. Three diluents and freezing techniques were tested, both as treatment combinations (diluent × freezing technique) and fixed effects (diluent or freezing technique) on post-thaw sperm motility (SM), viability (SV), plasma membrane integrity (SPMI) and acrosome integrity (SAI). Ten rams were selected, based on semen evaluation. Eight ejaculates were used for each treatment combination. Semen samples were diluted using a two-step protocol for home-made Tris-based egg yolk (20%, v/v) diluents: D1 (7% glycerol, v/v) and D2 (5% glycerol, v/v), and one-step for commercial diluent: D3 (Triladyl ® , consists of bi-distilled water, glycerol, tris, citric acid, fructose, spectinomycin, lincomycin, tylosin and gentamycin) at 35 °C. Fraction-A (without glycerol) was added at 35 °C, and following cooling of sample to 5 °C (-0.30 °C/min), Fraction-B (with glycerol) was added. The diluted semen samples were aspirated into 0.25 ml French straws, sealed, and equilibrated at 5 °C for 2 h. The straws were frozen in liquid nitrogen (LN) vapour, in a Styrofoam box. The freezing techniques were; One-step (F1): at -15.26 °C/min from +5 °C to -140 °C; Two-step (F2): at -11.33 °C/min from +5 °C to -80 °C, and -30 °C/min from -80 °C-140 °C; and Three-step (F3): at -11.33 °C/min from +5 °C to -80 °C, at -26.66 °C/min from to -80 °C to -120 °C, and at -13.33 °C/min from -120 °C to -140 °C. Two semen straws from each batch were evaluated before and after freezing. The group F3D3 exhibited significantly higher (p < 0.05) post-thaw SM 63.1 ± 2.5%, SV 79.0 ± 2.1% and SPMI 72.9 ± 1.7%, whereas SAI 72.9 ± 1.7% was significantly higher (p < 0.05) in group F3D2. The freezing technique F2 and F3 had significantly higher (p < 0.05) post-thaw sperm values compared to F1. The post-thaw SM and SV were above 50% and 65% with the freezing technique F2 and F3 but differed non-significant. The SPMI 67.6 ± 2.0% and SAI 76.1 ± 1.4% were significantly higher (p < 0.05) with F3. Likewise, the diluent D2 and D3 had significantly higher (p < 0.05) post-thaw sperm values compared to D1. The post-thaw SM, SV and SPMI were above 50%, 65% and 55% with the diluents D2 and D3 but differed non-significant. The SAI 76.1 ± 1.1% was significantly higher (p < 0.05) with D3. We concluded that the use of a simple home-made Tris-based diluent containing 20% (v/v) egg yolk and 5% glycerol (v/v), two-step dilution and a three-step freezing technique is a sustainable and effective method for freezing ram semen. For further validation, the fertility of ewes artificially inseminated with the frozen semen will be observed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The aim of this study was to determine if the achievement of the "in vitro" capacitation (IVC) status and subsequent progesterone-induced "in vitro" acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono- and bi-dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono-dimensional Western blot in non-capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi-dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non-capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm., (© 2019 Blackwell Verlag GmbH.)

Extracellular vesicles (EVs) were isolated by ultracentrifugation of vaginal luminal fluid (VLF) from superovulated mice and identified for the first time using transmission electron microscopy. Characterized by size and biochemical markers (CD9 and HSC70), EVs were shown to be both microvesicular and exosomal and were dubbed as "Vaginosomes" (VGS). Vaginal cross-sections were analyzed to visualize EVs in situ: EVs were present in the lumen and also embedded between squamous epithelial and keratinized cells, consistent with their endogenous origin. Western blots detected Plasma membrane Ca 2+ -ATPase 1 (PMCA1) and tyrosine-phosphorylated proteins in the VGS cargo and also in uterosomes. Flow cytometry revealed that following coincubation of caudal sperm and VLF for 30 min, the frequencies of cells with the highest Sperm adhesion molecule 1 (SPAM1), PMCA1/4, and PMCA1 levels increased 16.4-, 8.2-, and 27-fold, respectively; compared with control coincubated in phosphate buffered saline (PBS). Under identical conditions, sperm tyrosine-phosphorylated proteins were elevated ~3.3-fold, after VLF coincubation. Progesterone-induced acrosome reaction (AR) rates were significantly (p < 0.001) elevated in sperm coincubated with VGS for 10-30 min, compared with PBS. Sperm artificially deposited in the vaginas of superovulated females for these periods also showed significant (p < 0.01) increases in AR rates, compared with PBS. Thus in vitro and in vivo, sperm acquire from the vaginal environment factors that induce capacitation, explaining recent findings for their acrosomal status in the isthmus. Overall, VGS appear to deliver higher levels of proteins involved in preventing premature capacitation and AR than those promoting them. Our findings which have implications for humans open the possibility of new approaches to infertility treatment with exosome therapeutics., (© 2018 Wiley Periodicals, Inc.)

Polymyxin B (PMB) is beneficial for boar semen storage since it neutralizes the endotoxin of bacteria. However, the direct effect of PMB on boar spermatozoa has been unknown. This study aimed to examine the effect of PMB on acrosomal exocytosis, an essential process for successful fertilization in boar spermatozoa. Ejaculated spermatozoa stored with BTS extender at 17°C were washed and incubated with 0-100 μM PMB for 20 min and then examined for % total motililty, vigor grade and viability. None of the parameters was significantly different between 0 and 50 μM PMB with a gradual decline at higher concentrations. Thus the effect on acrosomal exocytosis was investigated at 0-50 μM of PMB. Spermatozoa were preincubated with PMB for 10 min, incubated for stimulation of acrosomal exocytosis with Ca 2+ and the calcium ionophore A23187 and then fixed with glutaraldehyde at 5, 10 and 15 min. Preincubation with PMB at 0.01-50 μM and 0.05-50 μM resulted in significant enhancement of acrosomal exocytosis at 10 min and 15 min of incubation, respectively. Preincubation with PMB followed by incubation without A23187 did not affect acrosomal exocytosis. These results suggest that PMB exerts effects on the acrosomal exocytosis triggered by Ca 2+ and A23187 in boar spermatozoa., (© 2019 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

This study aimed to improve the staining of frozen-thawed Japanese Black bull sperm acrosomes with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Spermatozoa were washed, fixed with 1-3% paraformaldehyde (PFA) in suspension for 10, 20, and 30 min, permeabilized with 0-2% Triton X-100 for 5 min, stained with FITC-PNA, and mounted with different antifade agents (0.22 M 1,4-diazabicyclo [2,2,2] octane (DABCO), SlowFade®, and ProLong®) in suspension (In-suspension) or on a smear (On-smear). The spermatozoa were categorized into seven pattern types either immediately or after storage for 24 hr. Experiment 1 showed that 1) the In-suspension method was better than the On-smear method; 2) if spermatozoa were stained using the In-suspension method and examined immediately, the best antifade agent was SlowFade®; 3) if samples were to be stored after staining using the On-smear method, DABCO should be avoided; 4) if spermatozoa were stained using the In-suspension method, storage of the stained samples was not recommended; and 5) if samples were to be stored after staining using the In-suspension method, ProLong® might be the best antifade agent. The results of experiment 2 showed that the concentration of Triton X-100 could be reduced to 0.1 from 1%. The results of experiment 3 showed that the paraformaldehyde concentration used for a 30 min fixation could be reduced from 3 to 2%. It is expected that the improved staining protocol will be useful to determine bull sperm acrosomal integrity.

Sperm quality in donkeys (Equus asinus) after freezing thawing is still considered lower than that from other animals, including horses. The aim of this study was to test a new freezing extender supplemented with jenny colostrum on donkey sperm. After thawing, we evaluated sperm motility by means of computer-assisted analysis, viability by SYBR-14 and propidium iodide (PI), membrane functional integrity by HOS-test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC-PNA) and PI. Ejaculates were collected from five fertile Donkeys. Sperm samples were pooled, diluted and cryopreserved into three experimental extender groups: BotuCrio ® , lactose-extender supplemented with egg yolk (20%) and lactose-extender supplemented with jenny colostrum (20%). The results demonstrated that lactose-jenny colostrum samples displayed significantly higher values in almost all parameters evaluated (p < 0.05) compared with the other two extenders after thawing (BotuCrio ® and lactose-egg yolk based extender, respectively) -Total Motility, Viability, HOS test, VCL, VSL and VAP. Acrosome status, LIN, STR and WOB despite showing lower values, none of them were statistically significant (p > 0.05). In conclusion, the extender containing jenny colostrum can be successfully used for donkey semen crypreservation and could effectively improve donkey sperm qualities after freezing -thawing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To explore the expression and regulatory function of sperm-associated antigen 6 (SPAG6) in the formation of the sperm acrosome in mice., Methods: The expression of SPAG6 during the first wave of spermatogenesis on postnatal days (PN) 8, 12, 16, 20, 24, 28, 30 and 35 was examined by Western blot and the localization of SPAG6 in the testicular germ cells was determined by immunofluorescence. The expression plasmids of SPAG6 and serine protease inhibitor Kazal-type 2 (SPINK2) were constructed, the interaction between SPAG6 and SPINK2 in the AH109 and CHO cells examined by yeast two-hybrid and co-localization assays, and the expression and localization of SPINK2 in the testicular germ cells of the SPAG6-knockout (SPAG6 KO) mice detected by immunofluorescence., Results: SPAG6 was highly expressed between PN 16 and 28 and localized in the acrosome of the round spermatids. Yeast two-hybrid assay showed the growth of SPAG6 and SPINK2 in the selective culture medium SD/-Leu/-Trp/-His, and the transfection of the CHO cells revealed the co-localization of SPAG6 and SPINK2 around the nuclei. The expression and acrosomal localization of SPINK2 were not found in the testicular germ cells of the SPAG6-KO mice., Conclusions: SPAG6 interacts with SPINK2 and probably participates in the formation of the sperm acrosome by stabilizing the expression of SPINK2 during spermatogenesis.

The binding of exogenous DNA to sperm is a key process for sperm-mediated gene transfer; however, the underlying molecular mechanisms have yet to be elucidated. In the present study, we aimed to identify the DNA binding proteins (DBPs) in rabbit sperm and to gain further understanding of the molecular mechanism of sperm and exogenous DNA interaction. Native polyacrylamide gel electrophoresis was used for separating free sperm proteins and complexes of DNA fragment/sperm proteins. A distinct band was found after Coomassie blue staining, and seven potential proteins were identified by mass spectrometry analysis. An analysis of the physical/chemical properties of the seven proteins revealed that the sperm inner acrosomal membrane protein IAM38 (IAM38) matched the features of the DBPs. Western blotting analysis showed that the IAM38 and CD4 were present in the sperm but not in the seminal plasma. Blocking of the IAM38 impaired the DNA-binding capacity of the sperm. Blocking the CD4 decreased the DNA-uptake capacity of the sperm but did not influence the DNA-binding capacity of the sperm. Moreover, the EGFP-positive embryos and EGFP-positive blastocysts were also decreased after IAM38 blocking or CD4 blocking in comparison with the control group. In conclusion, our results imply that foreign DNA first binds to the transmembrane IAM38 of the sperm plasma membrane and then forms the complex of DNA/IAM38/CD4 with CD4 to complete the transportation of exogenous DNA into the nucleus of sperm.

We report here the differences in sperm functional attributes and sperm-oviduct binding index in bulls with different field fertility ratings. Cryopreserved spermatozoa from Murrah buffalo bulls (n=9) with different fertility ratings were evaluated for membrane integrity, capacitation status, acrosome intactness and protein tyrosine phosphorylation status. Frozen--thawed spermatozoa were incubated with oviduct explants for 1h under 5% CO2, 38.5°C with 95% relative humidity and the number of spermatozoa bound to the unit area of oviduct explants (binding index; BI) was assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) fluorescent staining. The proportion of membrane-intact and acrosome-intact spermatozoa was significantly (P<0.05) higher and the proportion of capacitated spermatozoa was significantly (P<0.05) lower in high-fertile bulls compared with medium- and low-fertile bulls. The relationship between BI and bull fertility was significant and positive (r=0.69; P=0.04). BI was negatively and significantly (r=-0.83; P=0.01) related to membrane-compromised spermatozoa. It was concluded that the sperm-oviduct explant binding index was positively related to (1) the proportion of membrane-intact spermatozoa in a given semen sample and (2) invivo fertility of the buffalo bull, indicating the possibility of developing a fertility prediction tool using a sperm-oviduct explant binding model, once validated on a greater number of bulls.

Oviductal proteins play an important role in mammalian fertilization, as proteins from seminal fluid. However, in contrast with the latter, their phylogenetic evolution has been poorly studied. Our objective was to study in 16 mammals the evolution of 16 genes that encode oviductal proteins involved in at least one of the following steps: (1) sperm-oviduct interaction, (2) acrosome reaction, and/or (3) sperm-zona pellucida interaction. Most genes were present in all studied mammals. However, some genes were lost along the evolution of mammals and found as pseudogenes: annexin A5 (ANXA5) and deleted in malignant brain tumor 1 (DMBT1) in tarsier; oviductin (OVGP1) in megabat; and probably progestagen-associated endometrial protein (PAEP) in tarsier, mouse, rat, rabbit, dolphin, and megabat; prostaglandin D2 synthase (PTGDS) in microbat; and plasminogen (PLG) in megabat. Four genes [ANXA1, ANXA4, ANXA5, and heat shock 70 kDa protein 5 (HSPA5)] showed branch-site positive selection, whereas for seven genes [ANXA2, lactotransferrin (LTF), OVGP1, PLG, S100 calcium-binding protein A11 (S100A11), Sperm adhesion molecule 1 (SPAM1), and osteopontin (SPP1)] branch-site model and model-site positive selection were observed. These results strongly suggest that genes encoding oviductal proteins that are known to be important for gamete fertilization are subjected to positive selection during evolution, as numerous genes encoding proteins from mammalian seminal fluid. This suggests that such a rapid evolution may have as a consequence that two isolated populations become separate species more rapidly.

The aim of this study was to investigate the effect of varying hydrostatic pressure treatments (HP) on the boar semen quality during the modified cryopreservation. In Experiment I, combinations of pressure level (20/40/80 MPa) and duration of application (40/80/120 min) were used. Before freezing, only the magnitude but not the duration influenced the total (TM%) and progressive motilities (PM%). The 20/40 MPa levels yielded a significant (p < 0.05) improvement compared to control samples (atmospheric), but the 80 MPa was detrimental. The post-freezing-thawing (FT) motilities were influenced significantly by both the HP level and its duration. For TM%, the 40 MPa:120 min gave the highest post-FT result (54.8% ± 3.3%); however, the 40 MPa:80 min (41.0% ± 3.1%) application showed the largest and significant improvement (18.4% ± 3.1%) compared to its control (22.6% ± 3.1%) and compared to the improvement (12.9% ± 3.6%) achieved by 40 MPa:120 min. For PM%, the improvement with the 40 MPa:120 min application was slightly larger than with the 40 MPa:80 min one (15.2% ± 4.2% vs. 13.8% ± 3.3%); furthermore, the difference was not significant. In Experiment II, the 40 MPa:80 min combination was tested at four different stages of the semen handling. By pressurization after dilution with the freezing extender without glycerol, significantly higher post-FT values (TM%, intact acrosome% and head membrane%) were obtained. The two experiments demonstrated possible improvement in post-FT semen quality achievable through the appropriate application of HP to boar semen during cryopreservation., (© 2018 Blackwell Verlag GmbH.)

Two experiments were carried out to investigate the efficiency of supplementing sperm preservation medium with omega-3 polyunsaturated fatty acids on improving liquid-chilled storage and cryopreservation capacity of ram spermatozoa. Ejaculates (n = 100) were collected from five adult rams, Ovis aries, by an artificial vagina twice weekly throughout the period February-April, 2017. After initial evaluation, ejaculates of each collection session from the same males were pooled, diluted (1:10) with Tris-citric acid egg yolk extender, and were further split into five aliquots using a split-sample technique. The first aliquot served as control (omega-free), whereas the other four portions were supplemented with 0.1, 0.2, 0.3 or 0.4 mM omega-3, respectively (T 0 ). Thereafter, the diluted specimens were stored at 4°C for 48 hr, during which sperm physical and morphometric properties were evaluated along with oxidative stress indices (T 24 , T 48 ). Omega-3 levels that efficiently mitigated the detrimental effects of chilled preservation, and maintained preservation aptitude of spermatozoa were further investigated for sperm cryosurvival against control (untreated). Post-thaw physical and kinematic properties of spermatozoa, in all groups, were objectively evaluated by a computer-assisted sperm analysis (CASA) system. The results showed that, at 48 hr of chilled storage, supplementing preservation medium with 0.4 mM omega-3 was positively correlated (p < 0.01) with each of progressive motility, live sperm, intact acrosome and intact cell membrane (r = 0.83, 0.85, 0.85, 0.89, respectively). Furthermore, a positive correlation (p < 0.01) was observed between inclusion of omega-3 in cryopreservation medium and each of post-thaw total sperm motility, progressive motility, live sperm, normal sperm, intact acrosome, intact cell membrane, VCL, VSL, VAP, ALH and STR (r = 0.76, 0.84, 0.79, 0.90, 0.89, 0.91, 0.61, 0.73, 0.65, 0.78 and 0.60, respectively). These results accentuate efficiency of supplementing the diluent with omega-3 fatty acids on improving chilled and cryopreservation aptitude of ram spermatozoa., (© 2018 Blackwell Verlag GmbH.)

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.

Background: The membrane and acrosomal integrity of sperm play a vital role in fertilization process; however they are compromised upon cryopreservation., Objective: To study the effect of cholesterol-loaded cyclodextrin (CLC) on membrane and acrosome status of Hariana bull sperm during cryopreservation., Materials and Methods: Semen samples collected from Hariana bulls with mass motility ≥ 3+ and individual progressive motility ≥ 70% were utilized in the study. Each ejaculate was split into two parts, one part being evaluated freshly for various seminal attributes and the other part being diluted in Tris diluent (without egg yolk and glycerol) to obtain a final concentration of 120×10 6 sperm/mL. The diluted semen was divided into four treatments: Group I, without CLC (control); Group II, with CLC at 0.5 mg per 120 million sperm; Group III, at 1.0 mg per 120 million sperm; Group IV, at 2.0 mg per 120 million sperm. All aliquots were incubated for 15 min at 37°C and each sample was diluted with Egg yolk-Tris-Glycerol (EYTG) extender up to 80×10 6 sperm/mL. The diluted semen samples were packed in French mini straws (0.25 mL), sealed and equilibrated at 4°C for 4 h followed by cryopreservation. The samples at pre-freeze and post-thaw stage were evaluated for membrane and acrosomal integrity, as well as primary, secondary and tertiary acrosomal damages., Results: The membrane and acrosomal integrity was significantly higher in group II as compared to groups I, III, and IV, at pre-freeze and post-thaw stage (P<0.05). The primary and secondary acrosomal damage were significantly reduced in group II compared to other groups (P<0.05). No significant difference in tertiary acrosomal damage was found among different groups., Conclusion: CLC improves the membrane and acrosomal integrity, and reduces primary and secondary acrosomal damages during cryopreservation of Hariana bull sperm.

The present investigation was carried out to study the effect of various levels of dissolved oxygen (DO) on reactive oxygen species (ROS) and cryocapacitation-like changes in bull sperm. Egg yolk-Tris-glycerol (EYTG) extender was split into four subextenders; viz., Extender I (control; no flushing with liquid nitrogen (LN 2 )), Extender II, Extender III and Extender IV were flushed with LN 2 for 40, 16 and 8 min, respectively. The DO levels were standardized to 11.7, 2, 4 and 8 ppm, respectively, in control (Extender I), Extender II, Extender III and Extender IV. Ejaculates with mass motility of ≥ 3+ were divided into group I (diluted with Extender I), group II (diluted with Extender II), group III (diluted with Extender III) and group IV (diluted with Extender IV) up to 80 × 10 6  sperm/ml. Extended semen samples were packed in French mini straws (0.25 ml), equilibrated and cryopreserved. Semen samples were evaluated at prefreeze and post-thaw stage for various parameters (DO, progressive motility (PM), viability (VIB), acrosomal integrity (AI), hypo-osmotic swelling (HOS) test, ROS, cholesterol (C) and phospholipid (P). The percentage of PM, VIB, AI, HOS test, cholesterol (C) and phospholipid (P) levels, and capacitated sperm were significantly (p < 0.05) higher in groups III and IV as compared to groups I and II. However, the acrosome-reacted sperm (%; pattern AR) were significantly (p < 0.05) decreased in group III as compared to all other groups. Besides the proportion of sperm displaying tyrosine-phosphorylated pattern, EA (fluorescence at both equatorial and anterior acrosomal regions, i.e. high capacitation level) was significantly (p < 0.05) reduced in group III compared to all other groups. In conclusion, varying DO levels in the extender significantly affect sperm quality, ROS production and capacitation-like changes in bulls., (© 2018 Blackwell Verlag GmbH.)

The objective was to assess the influence of pomegranate seed oil supplementation on the quality of fresh, cooled and frozen-thawed Arabian breed stallion semen. Eight stallions (n = 4 per group) received their normal diet (control group) or normal diet top dressed with 200 ml of pomegranate seed oil (PSO group). Semen was collected every fifteen days for 90 days. Stallions were reversed across the treatments after a sixty-day interval. In cooled and stored condition (2, 12 and 24 hr), spermatozoa motion characteristics, membrane integrity, viability, morphology and lipid peroxidation were analysed. In frozen-thawed semen, sperm dynamic characteristics were analysed by CASA, acrosome status and mitochondrial activity (evaluated by Flow cytometry) determined. The effects of treatment, time, semen type and their interactions were submitted to PROCMIX (SAS ® ), and means compared by the Tukey test. Also, collected semen samples were artificially inseminated to evaluate fertility and pregnancy rate after day 60 of the experiment. The results from fresh condition showed that semen volume, sperm concentration, abnormality and live sperm were not affected by dietary treatment (p > 0.05). In cooled condition, the higher value for sperm plasma membrane integrity and viability was observed in PSO group compared to control after 24 hr cooled and stored in 5°C. In postthawed condition, the higher value for CASA total motility and acrosome status was observed in PSO group compared to control group (p < 0.05). One hundred and twenty-six mares were artificially inseminated for fertility trial using control and PSO groups' fresh semen. The average pregnancy rates were not significantly different between control and treated group (62.88% and 65.90%, respectively) (p > 0.05). We concluded that under the conditions of this study, dietary supplementation of 200 ml pomegranate seed oil seems to relatively improved Arabian horse sperm quality during storage in cooled and frozen condition via increasing plasma membrane integrity, viability and acrosome status, but did not improve the pregnancy rates., (© 2018 Blackwell Verlag GmbH.)

Assisted reproductive techniques have been used on several domestic animals to preserve desirable traits in strains of high genetic and commercial value; however in equines its efficiency rate is relatively low. To increase the conception ratio in stallions, some research groups have used pharmacological treatments which promote sperm hyperactivation in order to increase male's fertility rates. In this way, our previous work suggests that serotonin (5-HT) could be a good pharmacological candidate that facilitates conception rate in domestic horses. 5-HT is a neurohormone involved in several reproductive processes, i.e., it enhances hyperactivation, motility, and promotes the acrosome reaction in mammalian sperm, but it has not been described in the stallion sperm yet. Therefore, using both immunofluorescence and western blot techniques, we searched for and found some serotonin markers such as 5-HT, 5-HT1B, 5-HT2A, 5-HT₃ receptors, both TPH1 and MAOA enzymes, and serotonin transporter (5-HTT) in stallion sperm. In addition, we found a non-neuroendocrine cell, V-MAT₁ transporter, which has not been previously reported in mammalian sperm. Our results suggest that serotoninergic system is present in stallion sperm, which could be a pharmacological target to increase the conception rates in domestic horses.

The aim of this study was to investigate the effect of initial cooling time at 5°C during semen cryopreservation on post-thaw quality and reproductive performance of rabbit semen. Pooled semen samples (n = 6) were divided into two subsamples and cooled at 5°C for 45 or 90 min. After cooling, the semen samples were diluted to a ratio of 1:1 (v:v) with a freezing extender composed of Tris-citrate-glucose (TCG) containing 16% of dimethylsulfoxide and 0.1 mol/L sucrose. The semen was subsequently loaded in 0.25 ml straws, equilibrated at 5°C and frozen in liquid nitrogen vapor. After thawing, sperm motility, viability, osmotic resistance, acrosome and DNA integrity were assessed. Our results indicate that the longer cooling time, that is, 90 min before cryopreservation significantly improves sperm post-thaw viability, motility and fertility. In fact, reproductive performances obtained with semen frozen after a 90 min cooling time were similar to those produced by fresh semen insemination. Hence, the present research provides an effective freezing protocol for rabbit semen that will allow for the creation of a sperm cryobank for the conservation of Italian rabbit genetic resources, as well as the use of frozen semen doses in commercial farms., (© 2018 Japanese Society of Animal Science.)

In contrast to other species, intracytoplasmic sperm injection (ICSI) in bovine remains inefficient, resulting in low embryo developmental rates. It is unclear whether such inefficiency is due to the poor response of bovine ooplasms to the injection stimulus, or to the inability of bull sperm to induce oocyte activation. In order to facilitate these events, two strategies were assessed: the use of high concentration of cysteamine [Cys] during IVM; and the selection of sperm attached to cumulus cells after incubation with COCs for ICSI. First, COCs were IVM with increasing [Cys] and subjected to IVF. Zygotes from all groups were cultured under different O 2 tensions and development to blastocyst was evaluated. In a second experiment, sperm were co-cultured for 3 h with COCs and acrosome reaction was studied. Afterwards, the best IVM and IVC conditions determined on Experiment 1 were used for ICSI assay. COCs were matured for 21 h with 1 (Cys 1) or 0.1 mM Cys (Cys 0.1 groups, standard condition). In addition, COCs were incubated for ≥3 h with 16 × 10 6 sperm/ml and only sperm attached to cumulus cells were selected for ICSI (ICSI + Co-cult groups). After chemical activation, embryos were cultured in SOF medium under low O 2 tension. Cleavage and blastocyst rates were evaluated at days 2 and 7 of IVC, respectively. Finally, the relative expression of eight genes indicators of embryo quality was compared between ICSI and IVF control blastocysts by qPCR. Cleavage rates were higher for Cys 0.1 ICSI + Co-cult and Cys 1 ICSI + Co-cult groups (n = 117, 92% and n = 116, 79%, respectively) compared to their controls (n = 132, 60% for Cys 0.1 ICSI and n = 108, 52% for Cys 1 ICSI) (p ≤ 0.05). Interestingly, the combined treatment (Cys 1 ICSI + Co-cult) showed higher blastocyst rates than all other ICSI groups (23 vs. 11, 18 and 14% for Cys 0.1 ICSI + Co-cult, Cys 1 ICSI, and Cys 0.1 ICSI, respectively) (p ≤ 0.05). Moreover, incubation with COCs increased the rates of live acrosome reacted sperm (p ≤ 0.05). The relative abundance of mRNAs coding for INFτ, CAT, DNMT1, OCT4, and HDAC3 did not differ between treatments (p ≤ 0.05). SOD2, HADC1 and HADC2 expression was higher for Cys 0.1 ICSI than for IVF embryos (p ≤ 0.05). Group Cys 1 ICSI did not differ from IVF for those three genes, neither did Cys 1 ICSI + Co-cult, except for HDAC1 (p ≤ 0.05). In conclusion, the use of 1 mM Cys during IVM and of sperm incubated with mature COCs might be a good strategy to improve ICSI outcomes in cattle., (Copyright © 2018. Published by Elsevier Inc.)

Inferior larval production rates of domesticated Penaeus monodon broodstock has been a major hurdle to the expansion of its aquaculture, so that a better understanding of basic male reproductive biology is critical to improve the reproductive performance of this commercially important penaeid species. Following our previous study of spermatogenesis in the species, this study explored the mechanism of spermatophore formation with regards to the contribution of the reproductive tract epithelium by light and transmission electron microscopy. Four types of epithelial secretions (S1-4) were observed contributing to the three layers of the spermatophore. The primary layer of spermatophore was composed of S1 and S2, which were released from the secretory epithelial cells of the proximal vas deferens (PVD) and the secretory epithelial cells of the sperm-bearing lumen of the mid vas deferens, respectively. The secondary layer of the spermatophore was composed of S3, the secretory product of epithelial cells in the accessory tubule lumen of the mid vas deferens. The outer layer of the spermatophore was formed from S4 which was secreted by the epithelial cells in the posterior mid vas deferens and the terminal ampulla. Unique folds of the vas deferens epithelium appeared to play an important role in the formation of the spermatophore, particularly in the formation of the laminated structure of the spermatophore appendage. With respect to acrosome maturation along the reproductive tract, most spermatozoa did not have a fully developed anterior spike and a subacrosomal region when in the PVD, whereas both structures were fully formed by the time the spermatozoa reached the mid vas deferens and increased electron density when in the terminal ampulla; this observation represents the first morphological evidence of post-testicular acrosome maturation in this taxon., (© 2018 Wiley Periodicals, Inc.)

Objective: To determine whether acrosome function scoring-including acrosomal enzyme (AE) levels and acrosome reaction (AR) results-can predict fertilization rate in vitro., Methods: We examined the predictive value of acrosomal enzymes (AE) determined by spectrophotometry/N-α-benzoyl-DL-arginine-p-nitroanilide for fertilization rate (FR) in vitro in a retrospective cohort study of 737 infertile couples undergoing IVF therapy. Additionally, a meta-analysis was done for prospective cohort or case-control studies; the following summary measures were reported to expand upon the findings: pooled spearman correlation coefficient (Rs), standardized mean difference (SMD), sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic score (DS), diagnostic odds ratio (DOR), and area under the summary receiver operating characteristic curve (AUC)., Results: Lower AE levels determined by spectrophotometry with a cut-off value of <25μIU/10 6 spermatozoa were predictive of total fertilization failure (TFF) with moderate SEN (88.23%) and low SPE (16.50%). On meta-analysis, a total of 44 unique articles were selected, but given the multiple techniques described there was a total of 67 total datasets extracted from these 44 articles, comprising 5356 infertile couples undergoing IVF therapy. The AE levels or induced AR% was positively correlated with FR (Rs = 0.38, SMD = 0.79; Rs = 0.40, SMD = 0.86, respectively). Lower AE levels or induced AR% was predictive of lower fertilization rate with moderate accuracy (AUC = 0.78, AUC = 0.84, respectively); this was accompanied by low SEN/moderate SPE (0.57/0.85), moderate SEN/moderate SPE (0.79/0.87), respectively. For AE assay, the diagnostic performance in Asia (Rs = 0.24, SMD = 0.50) was inferior to that in North America (Rs = 0.54, SMD = 0.81) and Europe (Rs = 0.46, SMD = 0.92). Cryopreserved spermatozoa (SMD = 0.20, P = 0.204) were inferior to fresh spermatozoa (SMD = 0.89, P <  0.001). Sperm preparation yielded inferior results as compared to no preparation; spermatozoa after swim up were weak relevant (Rs = 0.27, P = 0.044); and there was no correlation for spermatozoa after a discontinuous gradient (SMD = 1.07, P >  0.05). Lower AE levels determined by fluorometry or substrate assay were used for predicting lower FR with low sensitivity and high specificity; the spectrophotometry assay had an uncertain predictive value. For induced AR assay, the diagnostic performance in the other areas was inferior to that in Africa (Rs = 0.65, SMD = 1.86). No preparation or double preparation yielded inferior results as compared to one preparation (Rs = 0.41); discontinuous gradient (Rs = 0.17, SMD = 0.47) was inferior to swim up (Rs =0.65, SMD = 1.51). Nonphysiological triggers (SMD = 0.81) did not differ from physiological triggers (SMD = 0.95) in general; ZP (Rs = 0.63) or mannose (Rs = 0.59) was superior to other physiological or nonphysiological triggers; and there was no correlation for human follicle fluid, progesterone, cyclic adenosine 3'-5'-phosphate analogue and phorbol ester-BSA-GlcNAc Neoglycoproteins with N-acetylglucosamine residues. Lower induced AR% determined by indirect immunofluorescence, direct immunofluorescence with lection, or triple stain was used for predicting lower FR, with moderate sensitivity/high specificity, moderate sensitivity/high specificity, or high sensitivity/low specificity., Conclusions: Although the correlation between acrosome function scoring and FR was significant, the assays were neither highly sensitive nor specific. Additionally, the diagnostic performance showed regional effects as well as an effect of the sperm preparation or assay method. More studies of multicenter, large-scale, careful design and synthesizing multiple sperm functional assays and oocyte quality assays are still needed in clinical settings to better predict fertilization outcome in IVF.

Sperm samples used on fertilization strongly influence the in vitro production (IVP) rates. However, sperm traits behind this effect are not stated consistently until now. This study aimed to evaluate the isolated and combined effect of some sperm traits (MB: total motility before Percoll® gradient, MA: total motility after Percoll® gradient, AI: acrosome integrity, MI: membrane integrity, MP: mitochondrial membrane potential, and CR: chromatin resistance) on IVP rates. This is the first study focusing on the isolated effect of distinct traits. For this purpose, the experiment was divided in three steps. In first step, to study behavior of traits sperm samples (n = 63 batches) were analyzed and ranked based on each trait. In second step, samples ranked were selected from target ranks regions and allocated in groups of four to five batches, creating Higher and Lower groups, according to two different approaches. One aimed to form groups that differed to all sperm traits simultaneously (effect of combined traits). The other aimed to form groups that differed only to a single sperm trait while no differences were observed for the remaining traits (effect of each isolated trait). In third step, for each group successfully formed in step 2, sperm samples were individually and prospectively used for IVP. Cleavage, embryo development and blastocyst rates were recorded and compared between Higher and Lower of respective trait groups. Surprisingly, evaluation of isolated effects revealed that lower levels of MB, AI and MP resulted in higher embryo development and blastocyst rates (p<0.05), which was not observed on cleavage rate. We conclude that sperm traits strongly influence embryo development after in vitro fertilization (IVF), affecting the zygote competence to achieve blastocyst stage. Individually, levels of MB, AI or MP could be some of the key traits that may define IVP efficiency on current systems of embryo production., Competing Interests: The authors have declared that no competing interests exist.