Sperm Protein Research Articles

Step-wise reproductive toxicities of imidazolium- and pyridinium-based ionic liquids on Caenorhabditis elegans

Reproductive toxicities of imidazolium- ([EMIM]X) and pyridinium-based ([APYR]X) ionic liquids (ILs) are essential to fully assess their hazards. Presently, effects of five ILs on the intricate processes of reproduction, including sperm-oocyte interactions, were explored in Caenorhabditis elegans. Results showed that 1-ethylpyridinium bromide ([EPYR]Br) stimulated oocytes, zygotes and total reproduction. 1-Octylpyyridinium bromide ([OPYR]Br) inhibited oocytes and stimulated zygotes, it inhibited the initial and total reproduction. 1-Dodecylpyridinium bromide ([DPYR]Br) inhibited oocytes and zygotes, but stimulated the initial and total reproduction. 1-Ethyl-3-methylimidazolium bromide ([EMIM]Br) inhibited oocytes, but stimulated zygotes and reproduction. 1-Ethyl-3-methylimidazolium iodide ([EMIM]I) inhibited germcells and oocytes but stimulated zygotes and reproduction. Regarding hormones and lipid metabolism, the ILs commonly reduced main sperm protein, sperm transmembrane protein 9 and spermatocyte protein 8. The qRT-PCR results showed that the ILs commonly down-regulated the expressions of mpk-1, while up-regulated those of inx-14, with influences on the expressions of vab-1, unc-43 and rme-2. These biochemicals and genes were directly connected with the sperm-oocyte interactions. Transcriptomic analysis results supported the involvement of cAMP and cGMP-PKG signaling pathways underlying the effects on the sperm-oocyte interactions. The results also implied ILs’ impacts on neural and immune diseases and even cancers that should be considered in the future.

Cordycepin improves hyperactivation and acrosome reaction through adenosine receptors during human sperm capacitation in vitro.

Sperm capacitation is a prerequisite for natural or in vitro fertilization. After capacitation, sperm become hyperactivated and undergo an acrosome reaction, which helps them penetrate the oocyte. Cordycepin, a bioactive compound first isolated from Cordyceps militaris, is an adenosine analog with numerous physiological activities. However, its effects on sperm capacitation remain unclear. This study aims to elucidate the effects and mechanisms of cordycepin on human sperm capacitation. During in vitro capacitation culture, healthy human sperm were treated with cordycepin (20, 100, 500 µM). Sperm motility and hyperactivation were detected using a computer-assisted sperm analyzer. Sperm acrosome reaction was measured using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin. Sperm protein kinase A (PKA) activity was analyzed using an ELISA kit. The levels of sperm protein tyrosine phosphorylation were detected by western blotting. Sperm DNA damage was detected by a sperm chromatin dispersion assay. Reactive oxygen species (ROS) were measured using the fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate. The expression and localization of adenosine receptors were analyzed by western blotting and immunofluorescence. The specific inhibitors of adenosine receptors were used to confirm their effects on cordycepin-induced sperm capacitation. Finally, molecular docking was performed to analyze the interaction between cordycepin and adenosine receptors. Cordycepin improved hyperactivated sperm motility, acrosome reaction, PKA activity, and protein tyrosine phosphorylation during capacitation while having no obvious effects on sperm ROS or DNA damage. Four adenosine receptor subtypes were expressed in human sperm, but their localizations differed. Inhibition of adenosine receptors significantly decreased cordycepin-induced sperm hyperactivation and the acrosome reaction. Molecular docking showed that cordycepin can bind to the four subtypes of adenosine receptors. Cordycepin may promote human sperm capacitation through adenosine receptor-mediated signaling pathways. These findings may be useful for assisted reproductive technology and animal breeding.

Protamine 2 deficiency results in Septin 12 abnormalities.

There is a well-established link between abnormal sperm chromatin states and poor motility, however, how these two processes are interdependent is unknown. Here, we identified a possible mechanistic insight by showing that Protamine 2, a nuclear DNA packaging protein in sperm, directly interacts with cytoskeletal protein Septin 12, which is associated with sperm motility. Septin 12 has several isoforms, and we show, that in the Prm2 -/- sperm, the short one (Mw 36kDa) is mis-localized, while two long isoforms (Mw 40 and 41kDa) are unexpectedly lost in Prm2 -/- sperm chromatin-bound protein fractions. Septin 12 co-immunoprecipitated with Protamine 2 in the testicular cell lysate of WT mice and with Lamin B1/2/3 in co-transfected HEK cells despite we did not observe changes in Lamin B2/B3 proteins or SUN4 expression in Prm2 -/- testes. Furthermore, the Prm2 -/- sperm have on average a smaller sperm nucleus and aberrant acrosome biogenesis. In humans, patients with low sperm motility (asthenozoospermia) have imbalanced histone-protamine 1/2 ratio, modified levels of cytoskeletal proteins and we detected retained Septin 12 isoforms (Mw 40 and 41kDa) in the sperm membrane, chromatin-bound and tubulin/mitochondria protein fractions. In conclusion, our findings present potential interaction between Septin 12 and Protamine 2 or Lamin B2/3 and describe a new connection between their expression and localization, contributing likely to low sperm motility and morphological abnormalities.

Pyrroroquinoline Quinone (PQQ) Improves the Quality of Holstein Bull Semen during Cryopreservation.

Cryopreserved semen is extensively utilized in the artificial insemination (AI) of domestic animals; however, suboptimal conception rates due to oxidative damage following AI continue to pose a challenge. The present study investigated the effects of Pyrroroquinoline Quinone (PQQ), a novel antioxidant, on the semen quality of Holstein bulls during cryopreservation, as well as its potential molecular mechanisms. Semen samples were diluted with varying concentrations of PQQ (0, 50 μmol/L, 100 μmol/L, 150 μmol/L) prior to cryopreservation. Following the freeze-thaw process, a comprehensive evaluation was conducted to assess sperm motility, plasma membrane integrity, acrosome integrity, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and adenosine triphosphate (ATP). Western blot analysis was employed to examine the levels of proteins including PGAM2, CAPZB, CAT, SOD1, and GPX1. Notably, the inclusion of 100 μmol/L PQQ significantly enhanced sperm motility, membrane integrity, and acrosome integrity post freeze-thawing (p < 0.05). Furthermore, the group treated with 100 μmol/L PQQ exhibited reduced levels of MDA and ROS (p < 0.05), while ATP levels were significantly elevated (p < 0.05). Interestingly, treatment with 100 μmol/L PQQ resulted in decreased consumption of PGAM2, CAPZB, CAT, SOD1, and GPX1 proteins in sperm after freeze-thawing, compared to the control group (p < 0.05). These findings indicate that PQQ treatment enhances the quality of bull semen, mitigates oxidative stress damage, and ultimately improves the efficacy of sperm cryopreservation.

Secretion of endoplasmic reticulum protein VAPB/ALS8 requires topological inversion

VAMP-associated protein (VAP) is a type IV integral transmembrane protein at the endoplasmic reticulum (ER). Mutations in human VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS). The N-terminal major sperm protein (MSP) domain of VAPB (Drosophila Vap33) is cleaved, secreted, and acts as a signaling ligand for several cell-surface receptors. Although extracellular functions of VAPB are beginning to be understood, it is unknown how the VAPB/Vap33 MSP domain facing the cytosol is secreted to the extracellular space. Here we show that Vap33 is transported to the plasma membrane, where the MSP domain is exposed extracellularly by topological inversion. The externalized MSP domain is cleaved by Matrix metalloproteinase 1/2 (Mmp1/2). Overexpression of Mmp1 restores decreased levels of extracellular MSP domain derived from ALS8-associated Vap33 mutants. We propose an unprecedented secretion mechanism for an ER-resident membrane protein, which may contribute to ALS8 pathogenesis.

Higher abundance of DLD protein in buffalo bull spermatozoa causes elevated ROS production leading to early sperm capacitation and reduction in fertilizing ability

BackgroudBefore fertilization, spermatozoa undergo a crucial maturation step called capacitation, which is a unique event regulates the sperm’s ability for successful fertilization. The capacitation process takes place as the spermatozoa pass through the female reproductive tract (FRT). Dihydrolipoamide dehydrogenase (DLD) protein is a post-pyruvate metabolic enzyme, exhibiting reactive oxygen species (ROS) production which causes capacitation. Additionally, other vital functions of DLD in buffalo spermatozoa are hyperactivation and acrosome reaction. DLD produces the optimum amount of ROS required to induce capacitation process in FRT. Depending on physiological or pathophysiological conditions, DLD can either enhance or attenuate the production of reactive oxygen species (ROS). Aim of this study was to investigate whether changes in the production of ROS in sperm cells can impact their ability to fertilize by triggering the capacitation and acrosome reaction.ResultsIn this study, abundance of DLD protein was quantified between high (n = 5) and low fertile bull (n = 5) spermatozoa. It was found that compared to high-fertile (HF) bulls, low-fertile (LF) bulls exhibited significantly (P < 0.05) higher DLD abundances. Herein, we optimised the MICA concentration to inhibit DLD function, spermatozoa were treated with MICA in time (0, 1, 2, 3, 4, and 5 h) and concentrations (1, 2.5, 5, and 10 mmol/L) dependent manner. Maximum DLD inhibition was found to be at 4 h in 10 mmol/L MICA concentration, which was used for further experimentation in HF and LF. Based on DLD inhibition it was seen that LF bull spermatozoa exhibited significantly (P < 0.05) higher ROS production and acrosome reaction in comparison to the HF bull spermatozoa. The kinematic parameters of the spermatozoa such as percent total motility, velocity parameters (VCL, VSL, and VAP) and other parameters (BCF, STR, and LIN) were also decreased in MICA treated spermatozoa in comparison to the control (capacitated) spermatozoa.ConclusionsThe present study provides an initial evidence explaining the buffalo bull spermatozoa with higher DLD abundance undergo early capacitation, which subsequently reduces their capacity to fertilize.

The Sperm-Associated Antigen 11A (Spag11a) Knockout Mice Display Sub-Fertility and Perturbations in the Sperm Proteome.

Spermatogenesis and sperm maturation that occur in the testis and epididymis respectively are crucial for fertility. Factors secreted by the testicular and epididymal epithelial cells govern the processes of gametogenesis and maturation. Proteins encoded by the sperm-associated antigen 11a (Spag11a) gene are implicated as having a possible role in sperm maturation. However, studies that demonstrate their definite role in fertility and sperm function using knockout models have not yet reported. In this study, Spag11a knockout mice were generated, genotyped and the reproductive parameters (fecundity, sperm count, capacitation, and acrosome reaction) and sperm proteome were determined. Litter size and sperm count were decreased in the Spag11a knockout mice when compared to the wild-type controls. Spermatozoa from the knockout mice were able to undergo capacitation. However, acrosome reaction did not occur in sperm obtained from knockout mice. Structural abnormalities in the head and tail structures were evident in the spermatozoa of knockout mice. Perturbations in the expression of sperm proteins that are involved in gametogenesis were evident. The subfertility observed in Spag11a knockout mice could be a manifestation of lower sperm count, impaired acrosome reactions, and disturbances in the sperm proteome. The results of this study lend further support to the role of Spag11a gene in male gamete function.

Bottom-up approach to deciphering the targets of the ubiquitin-proteasome system in porcine sperm capacitation

Capacitation is an essential post-testicular maturation event endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. By using a human-relevant large animal model, the domestic boar, this study focuses on furthering our understanding of the involvement of the ubiquitin-proteasome system (UPS) in sperm capacitation. The UPS is a universal, evolutionarily conserved, cellular proteome-wide degradation and recycling machinery, that has been shown to play a significant role in reproduction during the past two decades. Herein, we have used a bottom-up proteomic approach to (i) monitor the capacitation-related changes in the sperm protein levels, and (ii) identify the targets of UPS regulation during sperm capacitation. Spermatozoa were capacitated under proteasomal activity-permissive and inhibiting conditions and extracted sperm proteins were subjected to high-resolution mass spectrometry. We report that 401 individual proteins differed at least two-fold in abundance (P < 0.05) after in vitro capacitation (IVC) and 13 proteins were found significantly different (P < 0.05) between capacitated spermatozoa with proteasomal inhibition compared to the vehicle control. These proteins were associated with biological processes including sperm capacitation, sperm motility, metabolism, binding to zona pellucida, and proteasome-mediated catabolism. Changes in RAB2A, CFAP161, and TTR during IVC were phenotyped by immunocytochemistry, image-based flow cytometry, and Western blotting. We conclude that (i) the sperm proteome is subjected to extensive remodeling during sperm capacitation, and (ii) the UPS has a narrow range of distinct protein substrates during capacitation. This knowledge highlights the importance of the UPS in sperm capacitation and offers opportunities to identify novel pharmacological targets to modulate sperm fertilizing ability for the benefit of human reproductive health, assisted reproductive therapy, and contraception, as well as reproductive management in food animal agriculture.

Inhibition of forward and reverse transport of Ca2+ via Na+/Ca2+ exchangers (NCX) prevents sperm capacitation

BackgroundWhile calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated.ResultsPartial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation.ConclusionsInhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.

Next-Generation Electrochemical Biosensing Based on Ternary Nanocomposites of nMoS2@Rgo@MWCNT for Label-Free Cancer Biomarker Detection in Undiluted Human Serum

Sperm protein-17 (Sp17) is a cancer-testicular antigen that is expressed in various types of cancers and has been proposed as a potential biomarker for cancer. However, the detection of Sp17 in human serum is challenging due to its low concentration and the lack of specific and sensitive methods. Hence, there is an urgent need to develop fast, accurate, reliable, and cost-effective disposable biosensors for Sp17 detection. Herein, we report sensing of Sp17 with a disposable carbon cloth-based electrode (CC) modified with a ternary nanocomposite comprising molybdenum disulphide (MoS2), reduced graphene oxide (rGO), and multi-walled carbon nanotube (MWCNT). The nanocomposite was synthesized by a facile hydrothermal method and was well-characterized in terms of structural, morphological, and chemical attributes using X-ray diffraction, transmission electron microscopy, scanning electron microscopy, Raman spectroscopy spectral techniques, and electrochemical studies to prove the robust formation of the electroactive ternary nanocomposite and its suitability for Sp17 detection. The nanocomposite exhibited excellent electrocatalytic activity, biocompatibility, and stability. We immobilized a Sp17-specific antibody on the surface of the nanocomposite-modified electrode and used it as a biosensor for Sp17 detection. The biosensor showed a linear response range from 0.01 to 11 ng mL-1 and a limit of detection (LOD) and limit of quantification (LOQ) of 0.23 ng mL-1 and 0.79 ng mL-1, respectively having sensitivity of 9.97 mA ng mL-1 cm-2. The biosensor was successfully applied to detect Sp17 in human serum samples and showed good accuracy, reproducibility, and selectivity, making it helpful for use in translational medicine research and clinical applications. The biosensor also demonstrated a high correlation with a commercial enzyme-linked immunosorbent assay (ELISA) kit. Our results suggest that the proposed biosensor is a promising tool for Sp17 detection in human serum and could be useful for the diagnosis and prognosis of ovarian cancer and other Sp17-related cancers. Keywords: Carbon cloth; Biosensor; Electrochemistry; Sperm protein 17; Cancer biomarker; Nanocomposite

Comparative Study on the Sperm Proteomes of Horses and Donkeys.

The reproductive performance of horse sperm and donkey sperm has been reported to differ. Sperm proteins play a crucial role in sperm viability and fertility. Although differences between species are known, no prior study has investigated disparities in the sperm proteome between horses and donkeys. Therefore, this study characterized and compared the sperm proteomes of horses and donkeys using 4D-DIA mass spectrometry technology. We identified 3436 proteins in horse sperm and 3404 proteins in donkey sperm. Of these, 3363 proteins were expressed in both horse and donkey sperm, with 73 proteins being specifically expressed in horse sperm, and 41 in donkey sperm. According to data analysis, donkeys exhibited a greater percentage of motility and progressive movement in straight-line sperm than horses, as well as lower percentages of static and slow sperm than horses. Joint analysis of the results from the horse and donkey sperm proteomes and their CEROS II-read parameters demonstrated a possible association between sperm proteins and their sperm viability patterns. These findings suggest that there are discrepancies in the expression levels and protein compositions of horse and donkey sperm and that certain specific proteins may be responsible for the differences in performance between these two species.

Cryoprotectant-specific alterations in the proteome of Siberian sturgeon spermatozoa induced by cryopreservation

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods—liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability.

Role of genetic variations and protein expression of β-Microsemino protein in intrauterine insemination outcome of unexplained infertile men: A case-control study.

Intrauterine insemination (IUI) is often the first-line treatment for unexplained infertility. -Microsemino protein (MSMB) is an abundant protein in seminal plasma that has an inhibitory effect on spontaneous acrosome reaction. The present study aimed to evaluate MSMB gene variations and protein expression on IUI success rate in unexplained infertile men. A case-control study was performed on 100 unexplained infertile Iranian men referred to the Royan Institute, Tehran, Iran for IUI (50 men with IUI positive result [IUI+], and 50 men with IUI negative result [IUI-]). Couples with female infertility factors (such as hormonal disorders, infrequent menstrual period, abnormality in uterus, fallopian tubes, or ovaries) and men with infections of the male accessory glands, hypogonadotropic hypogonadism, clinical varicocele, retractile testis, genital trauma, drug use, or concurrent hormonal treatment Y chromosome microdeletions, and abnormal karyotype were excluded from the study. The polymerase chain reaction sequencing was performed for the promoter and the coding regions of MSMB functional domains. To study the protein expression, the total protein of sperm was extracted, and sandwich enzyme-linked immunosorbent assay was performed. 4 variations were detected (rs12770171, rs10993994, rs2075894, and rs4517463). None of them showed significant differences between the IUI+ and IUI- groups. The mean value of protein expression did not show any differences between the groups. In conclusion, there is no association between genetic variations of promoter and coding regions of MSMB functional domains as well as its expression with IUI success in unexplained infertile men.

Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences

BackgroundExposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 μg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h.ResultsLead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria.ConclusionsPb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.Graphical

O-284 Evidence for gamete recognition mechanisms in humans

Abstract Egg-sperm binding and fusion are the central events of fertilization and are essential for the formation of a new individual. While decades of research have provided a thorough understanding of the cellular processes, the identity of molecules orchestrating these events, along with their mechanism of action, remain elusive. Often the interactions established by cell surface proteins are weak and transient, making them challenging to deal with using standard biochemical methods, like purifications that involve stringent washing and denaturing conditions. Therefore, our knowledge of membrane proteins required for the sperm-egg interaction remains scarce and the research is further complicated by the limited amount of biological material, especially the eggs. Thanks to experimental approaches specifically developed to investigate cell surface interactions, we identified the first binding pair essential for fertilization in mammals 1. The pair is made of the egg protein JUNO and the sperm protein IZUMO1. Recently another membrane protein, the Fc receptor-like 3 (MAIA), has been indicated as a binding partner of IZUMO1 and suggested to be required for human fertilization2, but the data needs to be further investigated. Indeed, the same biochemical methods used to identify the JUNO-IZUMO1 binding pair, found no direct evidence of binding between MAIA and IZUMO13. In the last decade more sperm proteins have been shown to be essential for sperm-egg interaction other than IZUMO1, and all findings have been validated using transgenic mouse models. Among these sperm proteins, Tmem95 was firstly discovered in bulls, it was later confirmed to be required for fertilization in mice, and new evidence suggests that its role is conserved in humans4,5. Notably, the role played by each of the novel sperm candidates remains elusive and little is known on whether and how these cell surface molecules interact with one another. Insights into the architecture of the mammalian egg-sperm fusion synapse are now emerging with the use of AlphaFold2, an artificial intelligence-based program capable of predicting proteins’ 3D structure. Remarkably, AlphaFold2 not only predicts the structure of extracellular proteins essential for fertilization, also suggests the formation of a pentameric complex composed of three sperm (IZUMO1, SPACA6 and TMEM81) and two egg (JUNO and CD9) proteins which is likely to be important for gamete recognition and fusion6,7. In a high throughput screening we have recently identified a novel binding partner of IZUMO1, expressed in human leukocytes, potentially linking the reproductive and immune system (unpublished).

An Orphan Gene Enhances Male Reproductive Success in Plutella xylostella.

Plutella xylostella exhibits exceptional reproduction ability, yet the genetic basis underlying the high reproductive capacity remains unknown. Here, we demonstrate that an orphan gene, lushu, which encodes a sperm protein, plays a crucial role in male reproductive success. Lushu is located on the Z chromosome and is prevalent across different P. xylostella populations worldwide. We subsequently generated lushu mutants using transgenic CRISPR/Cas9 system. Knockout of Lushu results in reduced male mating efficiency and accelerated death in adult males. Furthermore, our findings highlight that the deficiency of lushu reduced the transfer of sperms from males to females, potentially resulting in hindered sperm competition. Additionally, the knockout of Lushu results in disrupted gene expression in energy-related pathways and elevated insulin levels in adult males. Our findings reveal that male reproductive performance has evolved through the birth of a newly evolved, lineage-specific gene with enormous potentiality in fecundity success. These insights hold valuable implications for identifying the target for genetic control, particularly in relation to species-specific traits that are pivotal in determining high levels of fecundity.

O-197 Correlation of intracellular pH of non-normozoospermic men with fertilization rates in assisted reproduction procedures (Pilot study)

Abstract Study question Does measuring qualitative changes in the sperm pHi could predict the fertilizing potential of a non-normozoospermic patients under fertility treatment? Summary answer The increase of pHi correlates with fertilization rates. What is known already The intracellular pHi (pHi) is of great importance for a wide variety of sperm physiological processes, including its motility. The pHi regulates several key sperm proteins, such as the Ca2+ channel, CatSper (sperm cation channel) which is involved in sperm hyperactivation and the K+ channel SLO3.The latter participates in the regulation of membrane potential during capacitation. Interestingly, human sperm pHi has been found to positively correlate with both hyperactivated motility and in vitro fertilization (IVF) success in normozoospermic patients. Study design, size, duration This prospective study employed unidentified semen samples from 62 non-normozoospermic men (patients, age 38 ± 4 years old) undergoing fertility treatment from January to November 2023 at CITMER Reproductive Medicine, Mexico City. Frozen semen samples and procedures with less than 4 oocytes were excluded. The pHi of sperm samples was evaluated by time-lapse flow cytometry. For comparison, the pHi of 13 normozoospermic men (donors, age 28 + 6 years old) were also analyzed. Participants/materials, setting, methods Sperm were separated by density gradient and incubated in capacitating HTF media to be used for conventional IVF or ICSI. Cells were stained with vitality (propidium iodide) and pHi (BCECF-AM) sensitive fluorescent dyes. Fluorescence changes were evaluated using an AccuriC6 flow cytometer. For each pHi recording, 10 mM NH4Cl were used as an alkalinization positive control and for data normalization. Basal levels of sperm pHi and its correlation with fertilization rates, were performed using R. Main results and the role of chance The changes in pHi were recorded as changes in the BCECF fluorescence. The increase in fluorescence is proportional to an increase in pHi and vice versa. Interestingly, we observed that the increase in pHi in sperm used for ICSI but not for IVF procedures, which were paired with oocytes from female donors, are positively and significantly correlated with higher fertilization rates (R = 0.68; *p=0.02). This seems to be the same for ICSI treatment pairing sperm with oocyte from the female partner, although the correlation is not yet statistically significant (R = 0.44; p = 0.12). We did not find significant differences in the basal pHi values between sperm from donors and patients; however, we observed that sperm from patients, had higher pHi values (0.57 + 0.15 a.u.f) compared to sperm from donors (0.48 + 0.24 a.u.f). Our results suggest that sperm pHi may be an important regulator for the process of fusion and not only for fertilization. Qualitative sperm pHi values can contribute to predicting fertilization success in patients undergoing ICSI treatment. Limitations, reasons for caution The preliminary nature of the results implies a necessity to increase the number of analyzed semen samples. Physiological responses may vary even among the same patient or cell’s donor. Our population-based analysis could be underestimating such single cell variations. Wider implications of the findings Current protocols for sperm analysis fail to successfully predict the fertilizing capacity of semen samplest.Measuring pHi in capacitated sperm, could be a promising tool to help clinicians choose the best fertilization technique for each couple. This protocol is easy to perform in a laboratory that has a flow cytometer. Trial registration number Not applicable

Characterization of expression patterns and dynamic relocation of Notch proteins during acrosome reaction of bull spermatozoa

Notch is a conserved cell-signaling pathway involved in spermatogenesis regulation. This study firstly evaluated the presence, localization patterns, acquisition origin and relation to acrosome reaction of Notch proteins in bull sperm. Western Blot analysis detected all Notch proteins in ejaculated bull sperm, and immunostaining described their specific sperm localization. Recovery of sperm from different segments showed that Notch proteins have testicular origin (NOTCH1, NOTCH2, DLL4), are sequentially acquired during sperm maturation along epididymal transit (NOTCH3, DLL3, JAGGED1-2), or post-ejaculation (DLL1, NOTCH4). Testis NOTCH2 is ubiquitously expressed in all germ-cell lines, whereas DLL4 is expressed in round and elongated spermatids during the Golgi, Cap, Acrosome and Maturation phases. In vitro spontaneous and induced sperm acrosome reaction induce consistent sperm regional relocation of NOTCH2, DLL4 and JAGGED1, and these relocation patterns are significantly associated to sperm acrosome status. NOTCH2 and JAGGED1 are relocated from the head apical to the post-equatorial regions, whereas DLL4 is lost along with the acrosome, evidencing that sperm spatial redistribution of NOTCH2 and JAGGED1 is linked to acrosome reaction onset, whereas DLL4 loss is linked to AR completion. Overall, results prompt for a relevant Notch role in bull sperm acrosome testicular development, epididymal maturation and acrosome reaction.

Dual role of valosin-containing protein (VCP/p97) in mouse sperm during capacitation.

Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.

A homozygous ARMC3 splicing variant causes asthenozoospermia and flagellar disorganization in a consanguineous family.

Male infertility due to asthenozoospermia is quite frequent, but its etiology is poorly understood. We recruited two infertile brothers, born to first-cousin parents from Pakistan, displaying idiopathic asthenozoospermia with mild stuttering disorder but no ciliary-related symptoms. Whole-exome sequencing identified a splicing variant (c.916+1G>A) in ARMC3, recessively co-segregating with asthenozoospermia in the family. The ARMC3 protein is evolutionarily highly conserved and is mostly expressed in the brain and testicular tissue of human. The ARMC3 splicing mutation leads to the exclusion of exon 8, resulting in a predicted truncated protein (p.Glu245_Asp305delfs*16). Quantitative real-time PCR revealed a significant decrease at mRNA level for ARMC3 and Western blot analysis did not detect ARMC3 protein in the patient's sperm. Individuals homozygous for the ARMC3 splicing variant displayed reduced sperm motility with frequent morphological abnormalities of sperm flagella. Transmission electron microscopy of the affected individual IV: 2 revealed vacuolation in sperm mitochondria at the midpiece and disrupted flagellar ultrastructure in the principal and end piece. Altogether, our results indicate that this novel homozygous ARMC3 splicing mutation destabilizes sperm flagella and leads to asthenozoospermia in our patients, providing a novel marker for genetic counseling and diagnosis of male infertility.