Induction Of Acrosome Reaction Research Articles

The effect of temperature shock on enhancing sperm motility: A new insight into molecular and cellular mechanisms through current procedures

Asthenozoospermia, characterized by reduced progressive motility (below 32%), is a leading cause of male infertility. Intrauterine Insemination (IUI) serves as the first-line treatment in sperm-related processes, aiming to increase sperm motility. Although Density Gradient Centrifugation (DGC) and swim-up procedures have become primary treatment options, considered the gold standard for IUI, there is a need to optimize these methods with additional factors to enhance motility, particularly for Asthenozoospermia patients.We hypothesize that a brief cold shock, lasting approximately 15–20 min at 17 °C, followed by incubation and sperm processing, can substantially enhance motility. For the first time, the hypothesis and evaluation of post-exposure incubation following a short-term cold shock, within the temperature range of 17–20 °C, were explored and discussed.This hypothesis appears valid and holds importance for several reasons. Firstly, there is a significant relationship between cold shock and its impact on intracellular components and the molecular organization of the cell membrane. Additionally, this process regulates various physiological cellular processes, including transbilayer distribution, acrosome reaction, and sperm motility. Moreover, lowering the temperature induces a molecular flux, facilitating calcium uptake by sperm. This process significantly impacts the quantity of calcium accessible to the sperm, particularly within the flagellum. Choosing the temperature below 20 °C temperature can lead to the activation of calcium-absorbing protein channels in the shortest possible time. Furthermore, a crucial reaction in IUI is sperm capacitation, which occurs when the protein tyrosine phosphorylation pathway, a key factor, is activated. Alternatively, the cold incubation of sperm reveals an increased level of tyrosine phosphorylation and a higher percentage of induced Acrosome Reaction (AR) compared to sperm incubated at normal temperatures. Hence, when sperm is cooled, it initiates a process called tyrosine phosphorylation, ultimately leading to sperm capacitation. Besides these logical reasons, there is limited empirical and incidental support in two cases that confirmed the hypothesis well

Induction of acrosome reaction by 4-Br-A23187 alters the glycoproteomic profile of boar spermatozoa

Protein glycosylation is a post-translational modification involved in wide range of biological processes. In mammalian spermatozoa this modification has been identified in numerous proteins, and membrane glycoproteins are involved in the fertilization process. The objective of the present study was to identify changes in protein glycosylation after acrosome reaction (AR) induction using the 4-Br-A23187 ionophore. Our results showed that treatment with 10 μM of 4-Br-A23187 for 20 min significantly increased the percentage of live acrosome-reacted spermatozoa compared to the control (69.8 ± 0.8 vs. 6.4 ± 0.5; mean % ± SEM, respectively). Also, we observed an increase in 32 kDa tyrosine-phosphorylated protein (p32) and a decrease in serine/threonine phosphorylation of the protein kinase A substrates (phospho-PKA-substrates) after ionophore treatment. Furthermore, changes in glycosylated proteins following AR induction were analyzed using different HRP-conjugated lectins (GNA, DSA, and SNA), revealing changes in mannose and sialic acid residues. Proteomic analysis of isolated proteins using GNA lectin revealed that 50 proteins exhibited significantly different abundance (q-value < 0.01). Subsequent analysis using Uniprot database identified 39 downregulated and 11 upregulated proteins in the presence of 4-Br-A23187. Notably, six of these proteins were classified as transmembrane proteins, namely LRRC37A/B like protein 1 C-terminal domain-containing protein, Membrane metalloendopeptidase like 1, VWFA domain-containing protein, Syndecan, Membrane spanning 4-domains A14 and Serine protease 54. This study shows a novel protocol to induce acrosome reaction in boar spermatozoa and identifies new transmembrane proteins containing mannose residues. Further work is needed to elucidate the role of these proteins in sperm-oocyte fusion.

Cold-induced damage in boar sperm cooled to 5°C in Lactose Egg Yolk (LEY) medium with different Glycerol concentrations

Boar sperm is highly susceptible to cold damage. When temperature drops to 5°C, the plasmatic membrane is destabilized. The freezing process causes a reduction of the fertility window because frozen/thawed boar sperm has less survivability. The aim of this work was to analyze the effect on sperm characteristics and response to capacitation stimuli of cooling to 5°C using a controlled protocol. Also, we evaluated if the addition of Glycerol 2% or 3% at 5°C was able to modify these parameters. For this purpose, we assessed motility, plasmatic membrane integrity and acrosomal membrane status. Capacitation was induced using Tyrode´s capacitating medium (TCM) and assessed by chlortetracycline stain and induction of acrosomal reaction with Progesterone. Motility patterns were analyzed using a CASA system. These tests were performed at three different points of the freezing curve: 37°C; 17°C and 5°C. Response to TCM vs TBM was only significant at 37°C. While at 37°C and 17°C capacitated sperm was below 20%, at 5°C reached 50% both in the TBM and TCM. CASA analysis showed that spermatozoa exposed to TCM had higher LIN and WOB than those in TBM. All parameters were similar in the Glycerol concentrations studied. These results suggest that the chilling process may be causing an effect similar to cryocapacitation along the cooling curve, starting subtle at 17°C and reaching 50% of the sperm population at 5°C, being independent of Glycerol concentration.

Zinc mitigates cadmium-induced sperm dysfunction through regulating Ca2+ and metallothionein expression in the freshwater crab Sinopotamon henanense

Cadmium (Cd) is a highly toxic heavy metal element that might adversely affect sperm function such as the acrosome reaction (AR). Although it is widely recognized that zinc (Zn) plays a crucial role in sperm quality, the complete elucidation of how Zn ameliorates Cd-induced sperm dysfunction is still unclear. In this study, we aimed to explore the protective effects of Zn against the sperm dysfunction induced by Cd in the freshwater crab Sinopotamon henanense. The results demonstrated that Cd exposure not only impaired the sperm ultrastructure, but also caused sperm dysfunction by decreasing the AR induction rate, acrosome enzyme activity, and Ca2+ content in sperm while elevating the activity and transcription expression of key Ca2+ signaling pathway-related proteins Calmodulin (CAM) and Ca2+-ATPase. However, the administration of Zn was found to alleviate Cd-induced sperm morphological and functional disorders by increasing the activity and transcription levels of CaM and Ca2+-ATPase, thereby regulating intracellular Ca2+ homeostasis and reversing the decrease in Ca2+ contents caused by Cd. Furthermore, this study was the first to investigate the distribution of metallothionein (MT) in the AR of S. henanense, and it was found that Zn can reduce the elevated levels of MT in crabs caused by Cd, demonstrating the significance of Zn in inducing MT to participate in the AR process and in metal detoxification in S. henanense. These findings offer novel perspectives and substantiation regarding the utilization of Zn as a protective agent against Cd-induced toxicity and hold significant practical implications for mitigating Cd-induced sperm dysfunction.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. N/A. This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Involvement of CATSPER 2 mutation in a familial context of unexplained infertility and fertilization failure associated with hearing loss: a case report

To explore the functional implications of a homozygous CATSPER 2 (cation channel for sperm) deletion within the acrosome reaction pathway during fertilization in 2 brothers, who have unexplained infertility and hearing loss. Case report. Two twin brothers aged 30 years with hearing loss and unexplained infertility. Molecular genetic diagnosis of deafness. Evaluation of the acrosome reaction and calcium mobilization assays after induction by progesterone and ionomycin on spermatozoa of the CATSPER 2-mutated patient and on fertile controls. Fertilization rate during conventional in vitro fertilization. Molecular genetic test. Percentage of acrosome-reacted spermatozoa with peanut agglutinin lectin staining. Recording of progesterone and ionomycin-induced intracellular calcium signals with a fluorescent probe. Mr. S and his brother have normal, conventional sperm parameters. Both brothers have had repeated intrauterine insemination failures and one fertilization failure after conventional in vitro fertilization. Mr. S obtained 2 healthy babies after intracytoplasmic sperm injection. Genetic analysis found a homozygote deletion of the STRC (stereocilin) gene (NM 153700: c.1-? 5328+?del) that removes the CATSPER 2 gene. Mutation of the STRC gene is known to be associated with hearing loss. Sperm functional tests revealed an inability of progesterone to activate intracellular calcium signaling and to induce acrosome reaction. We demonstrate the absence of a calcium signal and acrosome reaction after progesterone in our patient with a CATSPER 2 mutation. We emphasize the importance of the male medical interview and of the genetic investigation of hearing loss. We show that in vitro fertilization-intracytoplasmic sperm injection is necessary, even where normal sperm parameters are present.

The compound YK 3-237 promotes pig sperm capacitation-related events

Before fertilization of the oocyte, the spermatozoa must undergo through a series of biochemical changes in the female reproductive tract named sperm capacitation. Spermatozoa regulates its functions by post-translational modifications, being historically the most studied protein phosphorylation. In addition to phosphorylation, recently, protein acetylation has been described as an important molecular mechanism with regulatory roles in several reproductive processes. However, its role on the mammal’s sperm capacitation process remains unraveled. Sirtuins are a deacetylase protein family with 7 members that regulate protein acetylation. Here, we investigated the possible role of SIRT1 on pig sperm capacitation-related events by using YK 3-237, a commercial SIRT1 activator drug. SIRT1 is localized in the midpiece of pig spermatozoa. Protein tyrosine phosphorylation (focused at p32) is an event associated to pig sperm capacitation that increases when spermatozoa are in vitro capacitated in presence of YK 3-237. Eventually, YK 3-237 induces acrosome reaction in capacitated spermatozoa: YK 3-237 treatment tripled (3.40 ± 0.40 fold increase) the percentage of acrosome-reacted spermatozoa compared to the control. In addition, YK 3-237 induces sperm intracellular pH alkalinization and raises the intracellular calcium levels through a CatSper independent mechanism. YK 3-237 was not able to bypass sAC inhibition by LRE1. In summary, YK 3-237 promotes pig sperm capacitation by a mechanism upstream of sAC activation and independent of CatSper calcium channel.

Capacitation and Acrosomal Reaction Parameters of Fresh and Cryopreserved Rooster Semen

Background: Semen cryopreservation induces alterations in spermatozoa and changes in the plasma membrane, limiting the acrosome reaction and fertilizing ability. The research aim was to study the sperm capacitation indicators and acrosome reaction capacity in rooster semen, comparing fresh semen and post-thaw semen. Methods: Fifty ejaculates were obtained from five roosters through dorsal-ventral massage. Semen was diluted and preserved fresh or cryopreserved in Beltsville Poultry Semen Extender. In both groups of preserved semen, basic seminal parameters, capacitation and acrosome reaction were assessed. The latter were evaluated both with and without a perivitelline layer as the acrosome reaction inducer, using chlortetracycline staining. Result: The results revealed a higher percentage of sperm progressive motility in fresh semen (85.2±1.5%) compared with thawed semen (44.8±1.7%; P less than 0.05). The percentages were similar when comparing sperm with an acrosome reaction in fresh semen (10.6±1.1%) to sperm incubated without a perivitelline layer (13.2±1.7%; P greater than 0.05). However, when incubated with a perivitelline layer for 40 minutes, fresh semen spermatozoa exhibited a higher percentage of acrosome reaction (59.2±2.1%) compared with thawed semen incubated with a perivitelline layer (40.6±2.3%; P less than 0.05).

IZUMO1 Receptor Localization during Hyaluronic Acid Selection in Human Spermatozoa.

IZUMO1 is an acrosome transmembrane protein implicated in the adhesion and fusion of gametes. This study aims to describe the distribution of IZUMO1 in human sperm under different physiological conditions: before capacitation (NCS), at one-hour capacitation (CS1), after a hyaluronic acid (HA) selection test (mature, MS1 and immature, IS1), and induced acrosome reaction from one-hour-capacitated sperm (ARS1). The data obtained in NCS, CS1, and MS1 significantly highlight dotted fluorescence in the acrosomal region (P1) as the major staining pattern (~70%). Moreover, we describe a new distribution pattern (P2) with a dotted acrosomal region and a labelled equatorial region that significantly increases in HA-bound spermatozoa, suggesting the onset of the migration of IZUMO1. In contrast, unbound spermatozoa presented an increase in P3 (equatorial region labelled) and P4 (not labelled). Finally, costaining to observe IZUMO1 distribution and acrosome status was performed in ARS1. Interestingly, we reported a variety of combinations between the IZUMO1 staining patterns and the acrosomal stages. In conclusion, these data show as a novelty the diffusion of the IZUMO1 protein during different physiological conditions that could contribute to the improvement in sperm selection techniques.

In vivo characterization of sAC null sperm.

Targeted disruption of the soluble adenylyl cyclase (ADCY10; sAC) gene results in male-specific sterility without affecting spermatogenesis, mating behavior, or spermatozoa morphology and count; however, it dramatically impairs sperm motility and prevents capacitation. These phenotypes were identified in sperm from sAC null mice surgically extracted from the epididymis and studied in vitro. Epididymal sperm are dormant, and never exposed to physiological activators in semen or the female reproductive tract. To study sAC null sperm under conditions which more closely resemble natural fertilization, we explored phenotypes of ejaculated sAC null sperm in vivo post-coitally as well as ex vivo, collected from the female reproductive tract. Ex vivo ejaculated sAC null sperm behaved similarly to epididymal sAC null sperm, except with respect to the physiologically induced acrosome reaction. These studies suggest there is a sAC-independent regulation of acrosome responsiveness induced upon ejaculation or exposure to factors in the female reproductive tract. We also studied the behavior of sAC null sperm in vivo post-coitally by taking advantage of transgenes with fluorescently labelled sperm. Transgenes expressing GFP in the acrosome and DsRed2 in the mitochondria located in the midpiece of sperm (DsRed2/Acr3-EGFP) allow visualization of sperm migration through the female reproductive tract after copulation. As previously reported, sperm from wild type (WT) double transgenic mice migrated from the uterus through the uterotubular junction (UTJ) into the oviduct within an hour post-copulation. In contrast, sperm from sAC null double transgenic mice were only found in the uterus. There were no sAC null sperm in the oviduct, even 8h after copulation. These results demonstrate that sAC KO males are infertile because their sperm do not migrate to the fertilization site.

A selective inhibitor of the sperm-specific potassium channel SLO3 impairs human sperm function.

To fertilize an oocyte, the membrane potential of both mouse and human sperm must hyperpolarize (become more negative inside). Determining the molecular mechanisms underlying this hyperpolarization is vital for developing new contraceptive methods and detecting causes of idiopathic male infertility. In mouse sperm, hyperpolarization is caused by activation of the sperm-specific potassium (K+) channel SLO3 [C. M. Santietal.,FEBS Lett. 584, 1041-1046 (2010)]. In human sperm, it has long been unclear whether hyperpolarization depends on SLO3 or the ubiquitous K+ channel SLO1 [N. Mannowetz, N. M. Naidoo, S. A. S. Choo, J. F. Smith, P. V. Lishko, Elife2, e01009 (2013), C. Brenkeret al.,Elife 3, e01438 (2014), andS. A. Mansell, S. J. Publicover, C. L. R. Barratt, S. M. Wilson,Mol. Hum. Reprod. 20, 392-408 (2014)]. In this work, we identified the first selective inhibitor for human SLO3-VU0546110-and showed that it completely blocked heterologous SLO3 currents and endogenous K+ currents in human sperm. This compound also prevented sperm from hyperpolarizing and undergoing hyperactivated motility and induced acrosome reaction, which are necessary to fertilize an egg. We conclude that SLO3 is the sole K+ channel responsible for hyperpolarization and significantly contributes to the fertilizing ability of human sperm. Moreover, SLO3 is a good candidate for contraceptive development, and mutation of this gene is a possible cause of idiopathic male infertility.

Identification and Validation of Yak (Bos grunniens) Frozen-Thawed Sperm Proteins Associated with Capacitation and the Acrosome Reaction.

To achieve fertilization, mammalian spermatozoa must undergo capacitation and the acrosome reaction (AR) within the female reproductive tract. However, the effects of cryopreservation on sperm maturation and fertilizing potential have yet to be established. To gain insight into changes in protein levels within sperm cells prepared for use in the context of fertilization, a comprehensive quantitative proteomic profiling approach was used to analyze frozen-thawed Ashidan yak spermatozoa under three sequential conditions: density gradient centrifugation-based purification, incubation in a capacitation medium, and treatment with the calcium ionophore A23187 to facilitate AR induction. In total, 3280 proteins were detected in these yak sperm samples, of which 3074 were quantified, with 68 and 32 being significantly altered following sperm capacitation and AR induction. Differentially abundant capacitation-related proteins were enriched in the metabolism and PPAR signaling pathways, while differentially abundant AR-related proteins were enriched in the AMPK signaling pathway. These data confirmed a role for superoxide dismutase 1 (SOD1) as a regulator of sperm capacitation while also offering indirect evidence that heat shock protein 90 alpha (HSP90AA1) regulates the AR. Together, these findings offer a means whereby sperm fertility-related marker proteins can be effectively identified. Data are available via Proteome Xchange with identifier PXD035038.

Elevated and Sustained Intracellular Calcium Signalling Is Necessary for Efficacious Induction of the Human Sperm Acrosome Reaction.

Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH4Cl, alone or in combinations). AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success.

Symposium 1-4 - Immunological infertility caused by gamete immunity

1. Infertility caused by anti-sperm antibodies (ASA) 1) Female infertility caused by sperm-immobilizing antibodies in the sera In some women, ASA are produced as allogenic immunity, which causes infertility. Various methods for the antibody detection have been developed, but sperm-immobilization antibodies (SI-Ab) detected by the sperm immobilization test (SIT) have the greatest influence on the development of infertility. Sperm-immobilizing antibodies are present in the female genital tract, and also in the follicle fluid.SI-Ab cause a post-coital test (PCT) negative or impaired sperm transport from the uterine cavity to the Fallopian tubes, and fertilization disorders. There is a method to quantitatively measure SI-Ab titer, SI50 unit, and treatment methods are decided based on this SI 50 value. In cases with a SI50 value of 10 or more, it is difficult to conceive with AIH, and IVF is effective after preventing antibody contamination into the culture medium. As a mechanism for producing sperm-immobilizing antibodies, it has been speculated that women with disease-specific genes may be exposed to enough amount of sperm through sexual intercourse and develop symptoms triggered by such as chlamydia infection. CD52 is considered to be a promising antigen against sperm-immobilization antibodies, and is expected to be applied for the development of contraceptive vaccines. 2) Male infertility caused by ASA that bind to ejaculated sperm In some men, ASA are produced as autoantibodies, causing infertility. In men, it is not useful for measuring serum antibodies, therefore antibodies that bind to ejaculated sperm (sperm-binding antibodies) should be detected with ImmunoSpheres○R. However, since there are antibodies that are not related to the development of infertility, the treatment strategy can be determined by confirming whether the antibody has a biological effect. Inhibitory effect on cervical sperm penetration is confirmed by PCT and fertilization disorder is examined by the hemizona assay (HZA). If HZA cannot be performed, ICSI is recommended because it is likely to be accompanied by fertilization disorder in cases where the ratio of ASA-bound sperm to total motile sperm is 80% or more. 2. Female infertility caused by anti-zona pellucida (ZP) antibodies The ZP plays an important role in terms of egg formation, fertilization, protection of the embryo. These include the recognition of sperm species of animals, the induction of acrosome reaction, and the prevention of polysperm fertilization. On the other hand, since there is a strong antibody production ability as an antigen, if an anti-ZP antibodies are produced, the function of the ZP is impaired by the antibody, and there is a possibility of developing infertility. It was reported that anti-ZP antibodies were detected at a high rate in the sera of infertile women of unknown cause by the anti-ZP antibody detection method using porcine ZP, followed by controversial reports by another investigators. Therefore, we developed a detection method using human ZP. Anti-ZP antibodies disturb the follicular development, inhibit sperm-ZP binding and hatching. For the purpose of appropriate treatments, it was shown that ICSI is effective for fertilization disorder and assisted hatching (AHA) is effective for hatching disorder, respectively.

Label-Free Microfluidic Impedance Cytometry for Acrosome Integrity Assessment of Boar Spermatozoa.

Microfluidics and lab-on-chip technologies have been used in a wide range of biomedical applications. They are known as versatile, rapid, and low-cost alternatives for expensive equipment and time-intensive processing. The veterinary industry and human fertility clinics could greatly benefit from label-free and standardized methods for semen analysis. We developed a tool to determine the acrosome integrity of spermatozoa using microfluidic impedance cytometry. Spermatozoa from boars were treated with the calcium ionophore A23187 to induce acrosome reaction. The magnitude, phase and opacity of individual treated and non-treated (control) spermatozoa were analyzed and compared to conventional staining for acrosome integrity. The results show that the opacity at 19 MHz over 0.5 MHz is associated with acrosome integrity with a cut-off threshold at 0.86 (sensitivity 98%, specificity 97%). In short, we have demonstrated that acrosome integrity can be determined using opacity, illustrating that microfluidic impedance cytometers have the potential to become a versatile and efficient alternative in semen analysis and for fertility treatments in the veterinary industry and human fertility clinics.

Testis-specific serine protease PRSS54 regulates acrosomal granule localization and sperm head morphogenesis in mice†.

Serine proteases (PRSS) constitute nearly one-third of all proteases, and many of them have been identified to be testis-specific and play significant roles during sperm development and male reproduction. PRSS54 is one of the testis-specific PRSS in mouse and human but its physiological function remains largely unclear. In the present study, we demonstrate in detail that PRSS54 exists not only in testis but also in mature sperm, exhibiting a change in protein size from 50kDa in testis to 42kDa in sperm. Loss of PRSS54 in mice results in male subfertility, acrosome deformation, defective sperm-zona penetration, and phenotypes of male subfertility and acrosome deformation can be rescued by Prss54 transgene. Ultrastructure analyses by transmission electronic microscopy further reveal various morphological abnormalities of Prss54-/- spermatids during spermiogenesis, including unfused vacuoles in acrosome, detachment and eccentrical localization of the acrosomal granules, and asymmetrical elongation of the nucleus. Subcellular localization of PRSS54 display that it appears in the acrosomal granule at the early phase of acrosome biogenesis, then extends along the inner acrosomal membrane, and ultimately presents in the acrosome region of the mature sperm. PRSS54 interacts with acrosomal proteins ZPBP1, ZPBP2, ACRBP, and ZP3R, and loss of PRSS54 affects the distribution of these proteins in testis and sperm, although their protein levels are largely unaffected. Moreover, Prss54-/- sperm are more sensitive to acrosome reaction inducers.

The semenogelin I-derived peptide SgI-52 in seminal plasma participates in sperm selection and clearance by macrophages

BackgroundMacrophages can phagocytose sperm, especially damaged spermatozoa, in the female genital tract. The semenogelin I-derived peptide SgI-52 in seminal plasma exhibits seminal plasma motility inhibitor (SPMI) activity and can inhibit sperm motility. This raises the question of the role played by SPMIs in macrophage-mediated phagocytosis of sperm. We speculated that SgI-52 promotes sperm clearance by macrophages. Therefore, we investigated the phagocytosis of sperm in different states using this peptide. MethodsSgI-52 was fluorescently labeled, and its binding site for sperm was observed. The ability of macrophages to phagocytose sperm was observed using fluorescence confocal microscopy. Spermatozoa from different sources were co-cultured with SgI-52 in BWW medium for 4 and 22 h to compare the differences in their phagocytosis by macrophages. Sperm motility, induced acrosome reaction, mitochondrial membrane potential, and ATP content were examined after incubation with SgI-52. ResultsSgI-52 could bind to spermatozoa in different states, mainly to the tail, and then spread to the acrosome. This effect was more pronounced in demembranated spermatozoa. SgI-52 promoted phagocytosis of spermatozoa by macrophages, decreased the mitochondrial membrane potential, and increased the average ATP content of spermatozoa (P < 0.05). ConclusionsWe found for the first time that SgI-52 can bind to spermatozoa in different states and promote their phagocytosis by macrophages. Therefore, we speculate that SgI-52 is involved in the screening of sperm in the female reproductive tract and has potential value in improving assisted reproductive technology.

Two Male-Specific Antimicrobial Peptides SCY2 and Scyreprocin as Crucial Molecules Participated in the Sperm Acrosome Reaction of Mud Crab Scylla paramamosain.

Antimicrobial peptides (AMPs) identified in the reproductive system of animals have been widely studied for their antimicrobial activity, but only a few studies have focused on their physiological roles. Our previous studies have revealed the in vitro antimicrobial activity of two male gonadal AMPs, SCY2 and scyreprocin, from mud crab Scylla paramamosain. Their physiological functions, however, remain a mystery. In this study, the two AMPs were found co-localized on the sperm apical cap. Meanwhile, progesterone was confirmed to induce acrosome reaction (AR) of mud crab sperm in vitro, which intrigued us to explore the roles of the AMPs and progesterone in AR. Results showed that the specific antibody blockade of scyreprocin inhibited the progesterone-induced AR without affecting intracellular Ca2+ homeostasis, while the blockade of SCY2 hindered the influx of Ca2+. We further showed that SCY2 could directly bind to Ca2+. Moreover, progesterone failed to induce AR when either scyreprocin or SCY2 function was deprived. Taken together, scyreprocin and SCY2 played a dual role in reproductive immunity and sperm AR. To our knowledge, this is the first report on the direct involvement of AMPs in sperm AR, which would expand the current understanding of the roles of AMPs in reproduction.

Toll-like Receptor 2 is Involved in Calcium Influx and Acrosome Reaction to Facilitate Sperm Penetration to Oocytes During in vitro Fertilization in Cattle.

Cumulus cells of ovulated cumulus-oocyte complexes (COCs) express Toll-like receptor 2 (TLR2), pathogen recognition receptors, to recognize and react to sperm signals during fertilization. Sperm also express TLR2, but its contribution to the sperm-oocytes crosstalk is still unclear. Here, we adapted the in vitro fertilization (IVF) model to characterize the potential relevance of sperm TLR2 in sperm-oocytes interactions during fertilization in bovine. The IVF results showed that the ligation of sperm TLR2 with its specific antagonist/agonist resulted in down/up-regulation of the cleavage and blastocyst rates either in COCs or cumulus-free oocytes, but not in zona pellucida (ZP)-free oocytes. The computer-assisted sperm analysis (CASA) system revealed that sperm motility parameters were not affected in TLR2 antagonist/agonist-treated sperm. However, fluorescence imaging of sperm-ZP interactions revealed that the blockage or activation of the TLR2 system in sperm reduced or enhanced both binding and penetration abilities of sperm to ZP compared to control, respectively. Flow cytometrical analysis of acrosome reaction (AR) demonstrated that the TLR2 system adjusted the occurrence of AR in ZP-attached sperm, suggesting that sperm TLR2 plays physiological impacts on the sperm-oocyte crosstalk via regulating ZP-triggered AR in sperm. Given that calcium (Ca2+) influx is a pre-requisite step for the induction of AR, we investigated the impact of the TLR2 system on the ionophore A23187-induced Ca2+ influx into sperm. Notably, the exposure of sperm to TLR2 antagonist/agonist reduced/increased the intracellular Ca2+ level in sperm. Together, these findings shed new light that the TLR2 system is involved in sperm AR induction which enables sperm to penetrate and fertilize oocytes during the fertilization, at least in vitro, in cows. This suggests that sperm possibly developed a quite flexible sensing mechanism simultaneously against pathogens as well as COCs toward fertilization with the same TLR2 of the innate immune system.

New Approaches to Define The Functional Competency of Human Sperm Subpopulations and Its Relationship to Semen Quality.

BackgroundThis study aimed at comparing a comprehensive set of functional and structural sperm characteristics be- tween sperm motility fractions and correlating results to the standard semen parameters. By grouping related variables, our objective was to establish the predictive power of semen parameters and whether they accurately reflect the function- ality of sperm motility fractions or merely a small set of parameters within individual fractions.Materials and MethodsIn this non-invasive experimental study, donor semen samples (n=55) were separated via double density gradient centrifugation, isolating a high (HM) and low motile (LM) sperm fraction. Fractions were evaluated for percentage vitality, chromatin integrity, mature spermatozoa, motility and kinematic parameters, hyper- activation, positive reactive oxygen species, intact mitochondrial membrane potential (MMP) and acrosome reaction.ResultsHM fractions had significantly (P<0.001) enhanced percentages of induced acrosome reaction (HM, 55.6 ± 14.3%, LM, 25.0 ± 16.5%), motility and kinematic parameters, hyperactivation, vitality (HM, 70.4 ± 9.7%, LM, 47.9 ± 10.3%), mitochondrial membrane intactness (HM, 67.2 ± 10.4%, LM, 44.7 ± 15.0%) and mature spermatozoa (HM, 83.4 ± 10.0%, LM, 64.6 ± 8.2%) with intact chromatin (HM, 80.5 ± 8.1%, LM, 71.3 ± 8.0%). Various sperm mor- phology abnormalities correlated with LM fractions’ grouped motility parameters (range, 0.46 to 0.51; range -0.4 to -0.75), whereas combined semen traits of total motility, progressive motility, viscosity and mucus penetration (MPT) correlated with HM fractions’ grouped motility parameters (range, 0.44 to 0.84).ConclusionCollectively, total and progressive motility, viscosity and MPT may represent a reliable grouping of semen parameters for predicting the quality of HM sperm fractions. Separating the same donor semen samples into two signifi- cantly diverse motility sperm fractions could be a potential model in mimicking the qualities of fertile and sub-fertile males’ sperm populations and used for future research on the improvement of sperm subpopulations from males with different fertility statuses.