Acrosome Reaction Research Articles

Microfluidic devices employing chemo- and thermotaxis for sperm selection can improve sperm parameters and function in patients with high DNA fragmentation.

Conventional sperm processing uses centrifugation has a negative effect on sperm parameters and DNA integrity. We designed and fabricated a novel microfluid device based on chemotaxis and thermotaxis, and compared it with the swim-up method. Twenty normal samples with high DNA fragmentation were included. Each sample was divided into four groups: Group 1, control, Group 2: sperm selection by thermotaxis, Group 3: sperm selection by chemotaxis, and Group 4: sperm selection with thermotaxis and chemotaxis. We used cumulus cells in a microfluid device to create chemotaxis, and, two warm stages to form a temperature gradient for thermotaxis. The spermatozoa were assessed based on the concentration, motility, and fine morphology using Motile Sperm Organelle Morphology Examination, mitochondrial membrane potential (MMP), acrosome reaction (AR), and sperm DNA fragmentation. Concentration (22.40 ± 5.39 vs. 66.50 ± 19.21; p < 0.001) and DNA fragmentation (12.30 ± 3.96% vs. 17.95 ± 2.89%; p < 0.001) after selection in the chemotaxis and thermotaxis microfluid device were significantly lower than control group. The progressive motility (93.75 ± 4.39% vs. 75.55 ± 5.86%, p < 0.001), normal morphology (15.45 ± 2.50% vs. 10.35 ± 3.36, p < 0.001), MMP (97.65 ± 1.81% vs. 94 ± 3.89%, p=0.02), and AR status (79.20 ± 5.28% vs. 31.20 ± 5.24%, p < 0.001) in the chemotaxis and thermotaxis microfluid device were significantly increased compared to control group. According to these findings, spermatozoa that have penetrated the cumulus oophorus have better morphology and motility, as well as acrosome reactivity and DNA integrity.

Role of antioxidants in fertility preservation of sperm - A narrative review.

Male fertility is affected by multiple endogenous stressors, including reactive oxygen species (ROS), which greatly deteriorate the fertility. However, physiological levels of ROS are required by sperm for the proper accomplishment of different cellular functions including proliferation, maturation, capacitation, acrosomal reaction, and fertilization. Excessive ROS production creates an imbalance between ROS production and neutralization resulting in oxidative stress (OS). OS causes male infertility by impairing sperm functions including reduced motility, deoxyribonucleic acid damage, morphological defects, and enhanced apoptosis. Several in-vivo and in-vitro studies have reported improvement in quality-related parameters of sperm following the use of different natural and synthetic antioxidants. In this review, we focus on the causes of OS, ROS production sources, mechanisms responsible for sperm damage, and the role of antioxidants in preserving sperm fertility.

Di-2-ethylhexyl phthalate (DEHP) exposure induces sperm quality and functional defects in mice

Di-2-ethylhexyl phthalate (DEHP) harms mammalian testis development, yet the specific mechanism of its effect on sperm quality and function is unclear. In this study, male mice were administrated DEHP (200 mg/kg/day) via intragastric (i.g.) injection for 35 days. The sperm quality and function of DEHP-exposed mice were evaluated. DEHP exposure reduced the relative testis weight and serum testosterone levels. In addition, sperm count and motility parameters decreased significantly, which led to reduced sperm fertility characterized by reduced acrosome reaction rate, sperm-egg binding capacity and blastocyte formation. DEHP exposure decreased anti-oxidant indicators and the expressions of Cat, Sod1, Prdx6 and Sirt1 in the testis. DEHP-exposure also resulted in decreased proliferating cell nuclear antigen (PCNA) expression in mice testis, as well as the dose-dependent inhibition of the proliferation of GC-1 and GC-2 cells. These phenotypes may be related to increased cell apoptosis characterized by BAX/BCL2 and P53 up-regulation. DEHP exposure resulted in the down-regulation of SIRT1 and p-AKT in mice testis and decreased levels of GC-1and GC-2 cells. DEHP co-incubation with sperm in vitro resulted in decreased tyrosine phosphorylation and progressive motility, as well as p-AKT expression in capacitated sperm. Differential sperm proteomics identified 495 differentially expressed proteins, including 257 proteins down-regulated in the DEHP-exposure group. Bioinformatics analysis showed that proteins involved in sperm-egg interaction and fertilization processes were significantly down-regulated. Pathway analysis demonstrated that the adhesion pathway was enriched in down-regulated proteins, while the pathway associated with ribosomes was enriched in up-regulated proteins. Conclusively, DEHP exposure impaired male fertility by affecting sperm quality and function, and a pathway mediating the DEHP-induced decline in sperm quality and function was identified. The study provides additional information for understanding the molecular mechanisms of DEHP exposure and its effects on male reproduction.

Design, Synthesis, and Pharmacological Evaluation of Second-Generation Soluble Adenylyl Cyclase (sAC, ADCY10) Inhibitors with Slow Dissociation Rates.

Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.

Impaired fertility in 4930590J08Rik mutant male mice is associated with defective sperm energy metabolism.

Testis-specifically expressed genes are important for male reproduction according to their unique expression patterns. However, the functions of most of these genes in reproduction are unclear. Here, we showed that mouse 4930590J08Rik was a testis-specifically expressed gene. 4930590J08Rik knockout mice exhibited a delay in the first wave of spermatogenesis and a reduction of cauda epididymal sperm. Furthermore, knockout spermatozoa exhibited defective acrosome reactions and decreased progressive motility, which led to impaired in vivo fertilization. Transcriptome analysis of testes revealed that most of the differentially expressed genes in knockout testes were associated with metabolic processes. 4930590J08Rik knockout sperm exhibited oxidative phosphorylation deficiency and were highly dependent on increased anaerobic glycolysis to compensate for ATP demands. Taken together, the 4930590J08Rik-disrupted mouse partially mimics the phenotypes of human asthenospermia and oligozoospermia, which provides a new model for further understanding the pathogenesis of idiopathic male infertility.

Lactoferrin affects in vitro and in vivo fertilization and implantationin rats.

Lactoferrin (LF) is present in the oviduct, reduces in vitro gamete interaction, and affects sperm capacitation parameters in humans. Our aim was to investigate LF actions on further stages of the reproductive process in the Wistar rat model. Motile sperm were obtained from cauda epididymis to assess LF binding by direct immunofluorescence and LF effect on acrosome reaction (AR) using a Coomassie blue staining. After ovarian hyperstimulation of female rats, oocytes were surgically recovered and coincubated with motile sperm and different doses of LF to estimate the in vitro fertilization (IVF) rate. To evaluate the LF effect on pregnancy and embryo implantation, female rats (80days old) were placed with males and received daily intraperitoneal injections of LF during one complete estrous cycle (pregnancy experiments) or during the first 8 gestational days (implantation experiments). The number of pregnant females and live born pups was recorded after labor. Moreover, the number of implantation sites was registered during the implantation period. LF was able to bind to the sperm head, midpiece, and tail. 10 and 100μg/ml LF stimulated the AR but reduced the IVF rate. The administration of 100 and 200mg/kg LF significantly decreased the number of implantation sites and the litter size, whereas 100mg/kg LF declined the pregnancy rate. The results suggest that LF might interfere with the reproductive process, possibly interfering with gamete interaction or inducing a premature AR; nevertheless, the mechanisms involved are yet to be elucidated.

N-acetyl-l-cysteine and alpha lipoic acid are protective supplement on human sperm parameters in cryopreservation of asthenoteratozoospermia patients.

The aim of this study is to evaluate the beneficial effects of N-acetyl-cysteine (NAC), alpha lipoic acid (ALA) and combination of NAC + ALA supplement in freezing medium on Sperm structural and functional in asthenoteratozoospermia patients. Thirty freshly ejaculated semen samples were cryopreserved with sperm freezing medium (SFM) as control group and three group that SFM supplemented with NAC, ALA and their combination NAC+ ALA. The sperm samples were analysed according to WHO. Mitochondrial membrane potential (MMP), acrosome reaction (AR), antioxidant enzymes and DNA fragmentation were assessed using by Rhodamine123, PSA- FITC ELISA and TUNEL staining respectively. Expression level of NRF2 was assessed by real-time PCR assay. NAC and ALA alone significantly improved sperm motility, viability and DNA fragmentation (p< 0.05). MMP increased in NAC and ALA separately (p< 0.05). While did not affect the amount of sperm morphology and AR (p> 0.05). Antioxidant enzymes significantly difference in NAC and ALA groups (p< 0.05). In addition, NAC and ALA groups showed a significantly higher expression of NRF2 gene compared with other groups (p< 0.05). Our results revealed that the ALA and NAC supplements had a protective effect in cryopreservation process on the structural and functional characteristics of sperm.

Effects of Cell Concentration During Cryopreservation On the Post-thaw Quality of Santa Inês Ram Sperm

BACKGROUND: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose because the ewe's cervix is convoluted and often cannot be traversed with an insemination gun resulting in deposition of the sperm at the os cervix. OBJECTIVE: To compare a range of sperm concentrations when cryopreserving semen from Santa Inês rams and determine the effects of this on post-thaw quality. MATERIALS AND METHODS: One ejaculate from each ram (n = 10) was diluted to four sperm concentrations to obtain the following groups: G – 400, G – 800, G – 1200, and G – 1600 x 10 6 sperm/mL. The semen samples were packaged in 0.25 mL straws, cooled to 5 ºC, cryopreserved in liquid nitrogen vapor, thawed in a water bath (40 ºC/20 s), and were analyzed for computerized kinetics, capacitation and acrosome integrity, and plasma membrane integrity of sperm. RESULTS: The G – 400 treatment resulted in samples with the highest linearity and progressive motion (P &lt; 0.05) and had significantly greater plasma membrane integrity, and lower capacitation and acrosome reaction rates compared to G-1600 (P &lt; 0.05). Overall, use of the G – 400 treatment resulted in better kinetics, less plasma membrane damage and less early capacitation. However, despite reducing the ejaculate yield and increasing the costs of the semen freezing process, the G – 800 and G – 1200 treatments make a greater absolute number of sperm with good kinetics, plasma membrane integrity and capacitation status available. CONCLUSION: Ram sperm concentration impacts cryopreservation, and higher concentrations may be advantageous if a single rtificial insemination protocol is desirable.

The molecular chaperone cysteine string protein is required for monomeric SNARE proteins to assemble in trans-complexes during human sperm acrosomal exocytosis†.

Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility.

Cortisol modulates Ca2+ signaling and acrosome reaction in human sperm.

Stress and elevated cortisol levels have negative effects on fertility, although there is controversy about the effect of cortisol on human sperm. One study reported that hydrocortisone (HC), the synthetic form of cortisol, does not activate CatSper channel but is able to inhibit its activation by progesterone (Pg). However, subsequent reports showed that HC has an agonist effect on CatSper, producing intracellular Ca2+ ([Ca2+ ]i ) increases. These studies were performed using only electrophysiological techniques and fluorometric measurements of Ca2+ . Therefore, it is important to evaluate the effects of different HC concentrations on human sperm physiology using other approaches and techniques. To analyze the effects of different HC concentrations on human sperm physiology and to evaluate the association of perceived stress, anxiety and depression with acrosome reaction (AR), and semen quality. In capacitated human sperm, we tested the effects of HC on [Ca2+ ]i mobilization and AR. Furthermore, the perceived stress and symptoms of anxiety and depression were evaluated with standardized psychometric tests. HC induces a low increase in [Ca2+ ]i compared to Pg response while, at higher concentrations, HC inhibits the Pg-induced Ca2+ response. Also, low concentrations of HC induce AR and when co-incubating it with Pg, the percentages of AR are decreased. Finally, the anxiety symptoms are negatively correlated with Pg induced AR and semen quality. HC has an agonist effect on [Ca2+ ]i mobilization and AR. However, our data also suggest that HC may compete for the binding site of Pg. Additionally, the association between anxiety and decreased AR could be explained by high cortisol levels. Our data showed that HC interferes with the Pg action. Therefore, the negative correlation between anxiety symptoms and Pg-induced AR is due to the accompanying high cortisol levels of this condition and its competitive action with Pg.

Effect of cholesterol-loaded cyclodextrin treatment of bovine sperm on capacitation timing.

Pre-loading bovine sperm with cholesterol prior to freezing is known to increase cryosurvival, though the timing of capacitation in these sperm has not been evaluated. The objective of this study was to determine if there is a potential delay in capacitation timing in these sperm due to the increased cholesterol content. Flow cytometric evaluation was utilized to assess viability, and stain technology to assess acrosome intactness (Propidium Iodide/FITC-PNA), intracellular calcium levels (Propidium Iodide/FLUO 3-AM) and membrane fluidity (Merocyanine 540/YO-PRO-1). Cholesterol-loaded cyclodextrin (CLC) (2mg/mL) improved post-thaw viability to 61% from 45% in control sperm (p<.05). The addition of ionomycin (0.05 mM) induced capacitation in sperm by 1 h, resulting in increased intracellular calcium and increased acrosome reaction, and consequently viability loss by 3 h. Treatment with CLC significantly decreased membrane fluidity in sperm (p<.05). In conclusion, CLC-treated sperm required 1 h more to capacitate when compared with non-treated sperm based on percentage of live cells with high membrane disorder (p<.05). Increased cryosurvival and viability over time was observed, but longer time to capacitate may hinder fertilization capacity and/or require adjustments to timing of in vitro fertilization.

Membrane remodulation and hyperactivation are impaired in frozen-thawed sperm of low-fertility bulls

Bulls used in artificial insemination programmes worldwide undergo quality control checks, which are typically based on the evaluation of sperm motility and morphology. Despite this, some bulls can have lower than expected field fertility and the reasons for this remain to be elucidated. Here we hypothesised that sperm from bulls of varying fertility will differ in their ability to undergo capacitation-related events including an increase in membrane fluidity, protein tyrosine phosphorylation, hyperactivation and the acrosome reaction. Firstly, we used frozen-thawed semen from 10 high-fertility (HF) and 10 low-fertility (LF) bulls, and subjected them to in vitro capacitating conditions, following which sperm viability, membrane fluidity, acrosome integrity and protein tyrosine phosphorylation were assessed using flow cytometry. We then assessed the ability of sperm to undergo hyperactivation (induced using caffeine) utilising computer-assisted sperm analysis, and the acrosome reaction (induced using calcium ionophore) using flow cytometry. When sperm were incubated in capacitating conditions, a higher percentage of viable sperm from HF bulls exhibited high membrane fluidity when compared to LF bulls (8.8 ± 0.8% and 5.8 ± 1.2%, respectively; mean ± standard error; P < 0.05). There was no difference between fertility groups in the percentage of acrosome-reacted sperm following the incubation in in vitro capacitating conditions or following the induction of the acrosome reaction using calcium ionophore. However, more sperm from HF bulls became hyperactive in response to caffeine stimulation than sperm from LF bulls (21.6 ± 2.5% versus 14.1 ± 2.4%, respectively; mean ± standard error; P < 0.05). Taken together, sperm from LF bulls had an impaired ability to undergo membrane remodulation and to hyperactivate when induced in vitro. These are key events in the journey of sperm along the female reproductive tract and in the interaction with the oocyte and thus could explain the lower field fertility exhibited by some bulls.

In vitro versus cryo-induced capacitation of bovine spermatozoa, part 1: Structural, functional, and oxidative similarities and differences.

Low temperatures during cryopreservation activate a cascade of changes, which may lead into irreversible damage and reduction of the fertilization potential, including the process of premature capacitation. The aim of our study was to evaluate the range of cell damage following the cryopreservation process and possible activation of cryocapacitation in bovine spermatozoa. For the experiments semen samples were obtained from 30 sexually mature Holstein bulls. Within the analysed parameters, we focused on the functional activity, structural integrity, capacitation status and oxidative profile. The samples were divided into three experimental groups, control (CTRL), in vitro capacitated (CAP) and cryopreserved (CRYO). Based on the collected data, there was a significant decrease in the sperm motility, mitochondrial membrane potential and concentration of cyclic adenosine monophosphate in the CRYO group when compared to CAP and CTRL (P<0.0001). A significant decrease (P<0.01; P<0.0001) in the membrane and acrosome integrity as well as DNA fragmentation index and a significant increase (P<0.0001) of necrotic cells were observed in the CRYO group. Following capacitation, a significant increase (P<0.01; P<0.0001) was recorded in the number of cells which underwent the acrosome reaction in the CRYO group against CAP and CTRL. Changes in the oxidative profile of the CRYO group indicates an increase (P<0.0001) in the reactive oxygen species generation, except for the superoxide radical, which was significantly higher (P<0.0001; P<0.001) in the CAP group in comparison with CRYO and CTRL. In summary, premature capacitation may be considered a consequence of cryopreservation and the assessed parameters could serve as physical markers of cryogenic damage to bovine spermatozoa in the future.

Scutellarein stimulates human sperm function by increasing the levels of intracellular calcium and tyrosine phosphorylation.

As a kind of flavonoid, scutellarein is widely used to protect against various human diseases. Although the protective effects of scutellarein have been well studied, its influence on human reproduction remains unknown. In this research, we evaluated the effect of scutellarein on human sperm functions in vitro. Three different concentrations of scutellarein (1, 10, 100 μM) were applied to ejaculated human sperm. Fertilisation-essential functions, as well as the intracellular calcium concentration ([Ca2+ ]i ) and protein-tyrosine phosphorylation, two factors which are vital for sperm function regulation, were evaluated. The results demonstrated that all concentrations of scutellarein utilised in this study could significantly increase sperm spontaneous capacitation and acrosome reaction through the enhancement of [Ca2+ ]i . Besides, the level of tyrosine phosphorylation of sperm could also be increased by scutellarein. Meanwhile, the sperm motility could be improved by 10 and 100 μM scutellarein, which also make a significant enhancement in sperm penetration ability and hyperactivation. This is one of the limited studies showing the regulation of scutellarein on human spermatozoa functions and is helpful to enrich its application.

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.

Localization Patterns of RAB3C Are Associated with Murine and Human Sperm Formation.

Background and Objectives: Septins (SEPTs) are highly conserved GTP-binding proteins and the fourth component of the cytoskeleton. Polymerization of SEPTs contributes to several critical cellular processes such as cytokinesis, cytoskeletal remodeling, and vesicle transportation. In our previous study, we found that SEPT14 mutations resulted in teratozoospermia with >87% sperm morphological defects. SEPT14 interactors were also identified through proteomic assays, and one of the peptides was mapped to RAB3B and RAB3C. Most studies on the RAB3 family have focused on RAB3A, which regulates the exocytosis of neurotransmitters and acrosome reactions. However, the general expression and patterns of the RAB3 family members during human spermatogenesis, and the association between RAB3 and teratozoospermia owing to a SEPT14 mutation, are largely unknown. Materials and Methods: Human sperm and murine male germ cells were collected in this study and immunofluorescence analysis was applied on the collected sperm. Results: In this study, we observed that the RAB3C transcripts were more abundant than those of RAB3A, 3B, and 3D in human testicular tissues. During human spermatogenesis, the RAB3C protein is mainly enriched in elongated spermatids, and RAB3B is undetectable. In mature human spermatozoa, RAB3C is concentrated in the postacrosomal region, neck, and midpiece. The RAB3C signals were delocalized within human spermatozoa harboring the SEPT14 mutation, and the decreased signals were accompanied by a defective head and tail, compared with the healthy controls. To determine whether RAB3C is involved in the morphological formation of the head and tail of the sperm, we separated murine testicular tissue and isolated elongated spermatids for further study. We found that RAB3C is particularly expressed in the manchette structure, which assists sperm head shaping at the spermatid head, and is also localized at the sperm tail. Conclusions: Based on these results, we suggest that the localization of RAB3C proteins in murine and human sperm is associated with SEPT14 mutation-induced morphological defects in sperm.

Quantitative phosphoproteomics analyses reveal the regulatory mechanisms related to frozen-thawed sperm capacitation and acrosome reaction in yak (Bos grunniens).

Mammalian spermatozoa are not mature after ejaculation and must undergo additional functional and structural changes within female reproductive tracts to achieve subsequent fertilization, including both capacitation and acrosome reaction (AR), which are dominated by post-translational modifications (PTMs), especially phosphorylation. However, the mechanism of protein phosphorylation during frozen-thawed sperm capacitation and AR has not been well studied. In this study, the phosphoproteomics approach was employed based on tandem mass tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy to analyze frozen-thawed sperm in Ashidan yak under three sequential conditions (density gradient centrifugation-based purification, incubation in the capacitation medium and induction of AR processes by the calcium ionophore A23187 treatment). The identification of 1,377 proteins with 5,509 phosphorylation sites revealed changes in phosphorylation levels of sperm-specific proteins involved in regulation of spermatogenesis, sperm motility, energy metabolism, cilium movement, capacitation and AR. Some phosphorylated proteins, such as AKAP3, AKAP4, SPA17, PDMD11, CABYR, PRKAR1A, and PRKAR2A were found to regulate yak sperm capacitation and AR though the cAMP/PKA signaling pathway cascades. Notably, the phosphorylation level of SPA17 at Y156 increased in capacitated sperm, suggesting that it is also a novel functional protein besides AKAPs during sperm capacitation. Furthermore, the results of this study suggested that the phosphorylation of PRKAR1A and PRKAR2A, and the dephosphorylation of CABYR both play key regulatory role in yak sperm AR process. Protein-protein interaction analysis revealed that differentially phosphorylated proteins (AKAP3, AKAP4, FSIP2, PSMD11, CABYR, and TPPP2) related to capacitation and AR process played a key role in protein kinase A binding, sperm motility, reproductive process, cytoskeleton and sperm flagella function. Taken together, these data provide not only a solid foundation for further exploring phosphoproteome of sperm in yak, but an efficient way to identify sperm fertility-related marker phosphorylated proteins.

SPERM-ZONA BINDING INTERACTION: AN IN SILICO BLUEPRINT TO IDENTIFY MARKER MOLECULES FOR EFFECTIVE FERTILIZATION

Precise binding interaction between sperm surface-specific molecules and the ovum zona-pellucida is the key to a positive fertilization outcome. An in-silico approach was therefore adopted to understand the intricate molecular docking between a receptor molecule of the Zona-pellucida (ZP3) and effective sperm ligands selected viz., IZUMO1, ADAM2 and ERp57. The study aims at identifying the possible molecular biomarkers on the sperm plasma membrane which could efficiently initiate the acrosome reaction, recognize and bind to barrier membrane molecules and mediate fusion of sperm with ovum, thus culminating in successful fertilization. The in-silico identification of sperm-oocyte interaction biomarkers would serve a dual purpose in aiding to recognise potential targets for fertility regulation and infertility research.

G-Protein Coupled Receptors in Human Sperm: An In Silico Approach to Identify Potential Modulatory Targets.

G protein-coupled receptors (GPCRs) are involved in several physiological processes, and they represent the largest family of drug targets to date. However, the presence and function of these receptors are poorly described in human spermatozoa. Here, we aimed to identify and characterize the GPCRs present in human spermatozoa and perform an in silico analysis to understand their potential role in sperm functions. The human sperm proteome, including proteomic studies in which the criteria used for protein identification was set as <5% FDR and a minimum of 2 peptides match per protein, was crossed with the list of GPCRs retrieved from GLASS and GPCRdb databases. A total of 71 GPCRs were identified in human spermatozoa, of which 7 had selective expression in male tissues (epididymis, seminal vesicles, and testis), and 9 were associated with male infertility defects in mice. Additionally, ADRA2A, AGTR1, AGTR2, FZD3, and GLP1R were already associated with sperm-specific functions such as sperm capacitation, acrosome reaction, and motility, representing potential targets to modulate and improve sperm function. Finally, the protein-protein interaction network for the human sperm GPCRs revealed that 24 GPCRs interact with 49 proteins involved in crucial processes for sperm formation, maturation, and fertilization. This approach allowed the identification of 8 relevant GPCRs (ADGRE5, ADGRL2, GLP1R, AGTR2, CELSR2, FZD3, CELSR3, and GABBR1) present in human spermatozoa that can be the subject of further investigation to be used even as potential modulatory targets to treat male infertility or to develop new non-hormonal male contraceptives.

Analysis of reproductive damage in earthworms (Amynthas corticis) exposed to cypermethrin.

Cypermethrin contamination was a potential threat to soil organisms. In the present work, reproductive damage in earthworms (Amynthas corticis) exposed to cypermethrin was investigated. It was found that earthworms could absorb and accumulate residual cypermethrin in soil, and also earthworm activities helped accelerate the degradation of cypermethrin in soil. The accumulation of cypermethrin in earthworms induced sperm damage, and cypermethrin not only caused the imbalance of calcium homeostasis in earthworm sperm cells by inhibiting earthworm sperm Ca2+-ATP and Ca2+-Mg2+-ATP enzyme activities but also caused barriers in acrosome reaction. It also affected sperm energy supply of earthworms by inhibiting the activity of Na+-K+-ATPase and Mg2+-ATPase of earthworm sperm. Meanwhile, the inhibition of acrosome enzyme activity of earthworm sperm by cypermethrin led to hinder fertilization and reduced cocoon production of earthworms, and the damage of cypermethrin to sperm of earthworm was a significant cause of its reproductive toxicity. The results of the evaluation of IBR index showed that reproductive toxicity of cypermethrin to earthworms reduced with the increasing time. The decreased reproductive toxicity of cypermethrin to earthworms at the later stage of exposure (42-56 d) might be due to a combination of reduced absorption of cypermethrin in soil by earthworms, decreased accumulation of cypermethrin in the body, and improved sperm capacitation.