Sperm Migration Research Articles

Sperm Migration and Hyaluronic Acid Binding: Implications for Male Fertility Evaluation.

Mature, vital, and motile spermatozoa are essential for reaching the oocyte and binding to hyaluronic acid (HA) in the cumulus oophorus matrix. This study aims to determine the relationship between sperm-migration ability and HA-binding potential, as well as the relationship between sperm concentration and motility. Semen samples were collected from 702 men aged 20-56 years (median 34.8). We evaluated the sperm concentration and motility from basic semen analysis, the swim-up test (expressed as millions per mL and the migration efficiency percentage), and the hyaluronan-binding assay (HBA). A moderate positive correlation was found between the migration test results and HBA (R = 0.48). The highest correlation was observed between the concentration of motile spermatozoa and the migration test results (R = 0.85) and HBA (R = 0.4). The sperm migration efficiency strongly correlated with progressive motility (R = 0.6). Although significantly higher sperm migration was observed in patients with normal HBA results, the results of the functional tests were found to differ in some cases. For infertility treatment, the current diagnostic algorithm should be enhanced with more comprehensive seminological methods that assess the sperm-migration ability and HA-binding potential. We also recommend incorporating the swim-up method into the diagnostic protocol before planning assisted reproductive technology (ART) treatment.

Structural and network analysis of ADAM9 and ADAM10 and its transcript expression in the male reproductive tract of Bos Taurus

A disintegrin and metalloproteinases (ADAMs) are a family of proteins expressed as transmembrane and secreted proteins in tissues. This study investigated the expression of glycoproteins and transcripts of ADAM9 and ADAM10 in different regions of the bovine male reproductive tract (MRT). Fresh bovine samples were collected from the testis, epididymis, and vas deferens of MRT for this study. Special stains were utilized for identifying glycoproteins depending on the type of mucopolysaccharides in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. Quantitative real-time PCR (qPCR) was used to assess the mRNA concentrations of ADAMs9 and 10. Additionally, structural and network analysis was performed to investigate the similarity between human and bovine ADAM9 and ADAM10. Variable glycoprotein (acidic and neutral mucopolysaccharides) expression was observed in the seminiferous tubules of the testis and epithelial cells of the epididymis and vas deferens. ADAM9 and 10 mRNA were detected in the testis epididymis and vas deferens, with ADAM9 expression being significantly higher than ADAM10 mRNA expression in the testis and tail of epididymis. Significant similarity between the protein sequence of human and bovine ADAM9 (85.75%) and ADAM10 (96.66%) was observed, while the similarity between protein sequence of ADAM 9 and ADAM10 in humans and bovine was <26%. Bovine ADAM9 showed 4 binding sites, while bovine ADAM10, human ADAM9, and human ADAM10 showed 7 binding pockets, each with considerable variations in the affinity of specific antibody sequences to these binding pockets. Network analysis confirmed that ADAM9 and ADAM10 have critical roles in physiological processes involving cell migration, protease activity, and secretase-related functions. In addition, they have large numbers of hydrogen bonds to most other members of the ADAM family. Our study reports similarities in the expression of glycoproteins and ADAM9 and ADAM10 mRNA in the MRT of Bos Taurus. Structural and network analysis of ADAM9 and ADAM10 suggested functional similarities in these proteins, which are likely to be in facilitating spermatogenesis, transcervical sperm migration, egg-sperm fusion, and fertilization. Further, the similarity between human and bovine ADAM9 and ADAM10 proteins suggests that bovine sperms can be considered as a model system for comparative medicine studies.

Genital courtship and female-active roles in mating: sexual selection by mate choice in Caenorhabditis elegans.

A new bridge between studies of sexual selection and the massive literature on Caenorhabditis elegans behaviourand nervous system properties promise to provide important new insights into both fields. This paper shows that mate choice likely occurs in hermaphrodite C. elegans on the basis of stimulation from the male genital spicules, making it possible to apply the toolkit of extensive background knowledge of C. elegans and powerful modern techniques to test in unprecedented detail the leading hypotheses regarding one of the most sweeping trends in all of animal evolution, the especially rapid divergence of genital morphology. The recognition that sexual selection by mate choice may also occur in other contexts in C. elegans suggests additional payoffs from exploring previously unrecognized possibilities that female-active hermaphrodite reproductive behaviours are triggered by male stimulation. These facultative behaviours include attracting males, fleeing from or otherwise resisting males, opening the vulva to allow intromission, guiding sperm migration, avoiding rapid oviposition following copulation that results in sperm loss, expelling recently received sperm, and increasing feeding rates following copulation.

O-020 Investigating the effect of advanced sperm preparation techniques on embryo developmental potential and clinical outcomes: a network meta-analysis of randomized controlled trials

Abstract Study question Are embryological and clinical outcomes affected when employing Magnetic-Activated Cell-Sorting (MACS), Density Gradient Centrifuge (DGC)-Zeta and Microfluidic Sperm Selection (MSS) instead of DGC and Swim-Up? Summary answer MACS and MSS enhance both laboratory and clinical outcomes, while DGC-Zeta seems to improve only clinical outcomes, in comparison to conventional sperm preparation techniques. What is known already Sperm preparation techniques have been developed to enhance sperm selection process towards improving IVF laboratory and clinical outcomes. Based on centrifugation and sperm migration, DGC and swim-up constitute the standard methods for improving sperm parameters, nonetheless disregarding molecular aspects. Certain techniques, namely MACS, DCG/Zeta and MSS have been proposed to effectively select non-apoptotic sperm with intact chromatin and high DNA integrity claiming their place in routine clinical practice. Concurring on the efficacy of these adjunct methods from the standpoint of embryo developmental dynamic and clinical outcomes is both pertinent and imperative. Study design, size, duration A systematic search of the literature was performed in the database Pubmed/Medline up to December 2023. Only full-text manuscripts published in English where randomization was performed on oocytes or patients were eligible. Studies reporting on semen samples obtained by surgical techniques were excluded. A total of 20 RCTs were included in this network meta-analysis. Other reported sperm preparation techniques were excluded due to the limited number of available Randomized Controlled Trials (RCTs). Participants/materials, setting, methods All RCTs providing results on the aforementioned sperm preparation techniques were included. The outcome measures were fertilization, cleavage, blastocyst formation, clinical pregnancy, and live-birth rates. To evaluate the network estimate both direct and indirect effects were assessed. When heterogeneity was I2=0% the fixed-effects model was preferred. In any other case the random-effects model was employed. To rank the sperm preparation techniques' efficiency, the frequentist method employing the P-score was opted for. Main results and the role of chance The 20 included RCTs presented with a total of 17 different designs and 8 different combinations of sperm preparation techniques. Seventeen studies reported on fertilization rate providing no statistically significant difference between the sperm preparation techniques albeit with high heterogeneity I2=69%. Regarding cleavage rate (n = 10 studies), only MACS/DGC presented with an improved outcome when compared to DGC (RR: 1.43; 95%CI: 1.16-1.76; I2=96%). MACS/DGC presented with the highest efficiency (P-Score=0.90), followed by combination of MACS/DGC/swim-up (P-Score=0.83). Both MSS and MACS/DGC presented with significantly higher blastocyst formation rate compared to DGC (MSS: RR:1.14;95%CI:1.02-1.29; MACS: RR: 1.37; 95%CI: 1.04-1.82; I2=0%). According to P-Score, MACS/DGC was presented as the most efficient technique (P-Score=0.89) followed by MSS (P-Score=0.44). Regarding clinical pregnancy rates, the 12 studies featured low heterogeneity (I2=0). MACS/DGC presented with higher clinical pregnancy rates compared to DGC (RR: 1.30; 95%CI: 1.09-1.36) and the combination of DGC/swim-up (RR: 1.55; 95%CI: 1.12-1.95). MSS presented with enhanced results compared to swim-up (RR: 1.37; 95%CI: 1.11-1.69) and DGC-Zeta presented with higher clinical pregnancy rates compared to DGC (RR: 1.76; 95%CI: 1.28-1.92). The most efficient technique was DGC-Zeta (P-Score=0.98), followed by MACS/DGC (P-Score=0.82). Regarding live-birth rates no statistically significant difference was observed, albeit high heterogeneity was observed (I2=51%). Limitations, reasons for caution The blastocyst formation rate and the live-birth rate outcomes should be interpreted with caution due to the limited number of RCTs. The indirect effect estimates may pose as an intrinsic limitation of the network meta-analysis, especially in cases with limited direct data. Wider implications of the findings This data demonstrates that the combination of MACS/DGC provided enhanced laboratory and clinical outcomes compared to conventional techniques. Investigating sperm selection techniques that assess advanced sperm characteristics rather than parameters alone is timely and essential. To concur on optimal practice further RCTs directly comparing MSS, MACS and DGC-Zeta are required. Trial registration number Not applicable

P-260 In Vitro Fertilization (IVF) using magnetotactic sperm cells and their prospects for assisted in vivo reproduction

Abstract Study question Are bovine Magnetotactic Sperm Cells (MSC) able to fertilize an oocyte in-vitro and Which would be the main requirements for translating this technology to in-vivo? Summary answer IVF using highly purified MSC result in embryo development until blastocyst stage. We demonstrate different alternatives to control multiple MSC, individually and as a swarm. What is known already Medical microrobots have appeared as the new generation of drug delivery vehicles. They are tiny devices in the order of a few micrometers, able to move in biological environments by different means, physical, chemical, or biohybrid. They are functionalized to deliver targeted therapies for drugs, cells, or other payloads. In assisted reproduction, sperm-hybrid microcarriers have been pioneered by our group, and various in vitro demonstrations have been done to assist sperm cells with motion deficiencies or low count and to deliver anticancer drugs for treating, for example, cervical and ovarian cancer, but so far in-vitro fertilization has never been shown. Study design, size, duration Experimental quantitative study design. A total of 520 bovine cumulus-oocyte complexes were used for in vitro fertilization. The analyzed sample groups were using bovine frozen sperm cells, MSC, or by evaluating parthenogenetic activation. A power of 0.98 with an a-error of 0.05 was used for the IVF experiments. Sperm samples in the control as well as the micromotor groups have been analyzed for different sperm parameters. Participants/materials, setting, methods Bovine MSC are created by thawing frozen sperm cells and adding polystyrene microparticles. Different sperm parameters were evaluated to check the effects of the particles in the cells. After developing a purification method for the MSC, bovine ovaries were collected from a slaughterhouse nearby and, after in vitro maturation, were used for the IVF experiments. Ultrasound, and photoacoustic imaging setups with a set of electromagnets were employed for real-time manipulation and visualization of MSC. Main results and the role of chance Sperm viability was significantly higher in the MSC group compared to the control group 6h after formation (72.3 ± 5.0% vs 35.0 ± 4.1%), and after 24 hours, both viabilities decreased drastically to 16.6 ± 3.9% and 7.7 ± 1.7% respectively, probably a possible local antioxidant effect by the microparticles through the formation of a protein corona. In the case of motility, the MSC group presented significantly higher motility up to 6h (64.0 ± 1.5%), and in all the measured times, the motility of the MSC group was higher than the control group. Purification of the micromotor sample was performed, but an average of 63 free sperm was still on the MSC sample. IVF experiments were performed, and blastocyst development was observed in IVF control (2x106 sperm/ml), medium concentration (same concentration as MSC group), and the MSC groups (275.000 sperm/ml). No blastocyst formation was observed in the parthenogenetic, parthenogenetic + particles, and low-concentration groups (number of free sperm). The MSC group was significantly different in cleavage and blastocyst rates compared to the low concentration groups, with p-values of 0.04552 and 0.005865, respectively, which suggest that fertilization may have occurred by the micromotors instead by the free sperm in the sample. Limitations, reasons for caution More studies of these MSC in the different gynecological tissues and on embryo development are needed to ensure the safety of future human translation. In addition, developing a detection system for micromotors in deep tissue is needed to perform in vivo experiments. Wider implications of the findings MSC show an alternative to efficiently guide efficiently sperm cells in different biological environments and can be a promising alternative method for assisted fertilization, and the treatment of a variety of gynecological diseases. They can also be used as exploratory tools to learn more about sperm migration in-vivo, among others Trial registration number European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program No. 853609

Spermatogenic cell-specific type 1 hexokinase (HK1S) is essential for capacitation-associated increase in tyrosine phosphorylation and male fertility in mice.

Hexokinase (HK) catalyzes the first irreversible rate-limiting step in glycolysis that converts glucose to glucose-6-phosphate. HK1 is ubiquitously expressed in the brain, erythrocytes, and other tissues where glycolysis serves as the major source of ATP production. Spermatogenic cell-specific type 1 hexokinase (HK1S) is expressed in sperm but its physiological role in male mice is still unknown. In this study, we generate Hk1s knockout mice using the CRISPR/Cas9 system to study the gene function in vivo. Hk1s mRNA is exclusively expressed in testes starting from postnatal day 18 and continuing to adulthood. HK1S protein is specifically localized in the outer surface of the sperm fibrous sheath (FS). Depletion of Hk1s leads to infertility in male mice and reduces sperm glycolytic pathway activity, yet they have normal motile parameters and ATP levels. In addition, by using in vitro fertilization (IVF), Hk1s deficient sperms are unable to fertilize cumulus-intact or cumulus-free oocytes, but can normally fertilize zona pellucida-free oocytes. Moreover, Hk1s deficiency impairs sperm migration into the oviduct, reduces acrosome reaction, and prevents capacitation-associated increases in tyrosine phosphorylation, which are probable causes of infertility. Taken together, our results reveal that HK1S plays a critical role in sperm function and male fertility in mice.

Generation of Oviductal Glycoprotein 1 Cre Mouse Model for the Study of Secretory Epithelial Cells of the Oviduct.

The epithelial cell lining of the oviduct plays an important role in oocyte pickup, sperm migration, preimplantation embryo development, and embryo transport. The oviduct epithelial cell layer comprises ciliated and nonciliated secretory cells. The ciliary function has been shown to support gamete and embryo movement in the oviduct, yet secretory cell function has not been well characterized. Therefore, our goal was to generate a secretory cell-specific Cre recombinase mouse model to study the role of the oviductal secretory cells. A knock-in mouse model, Ovgp1Cre:eGFP, was created by expressing Cre from the endogenous Ovgp1 (oviductal glycoprotein 1) locus, with enhanced green fluorescent protein (eGFP) as a reporter. EGFP signals were strongly detected in the secretory epithelial cells of the oviducts at estrus in adult Ovgp1Cre:eGFP mice. Signals were also detected in the ovarian stroma, uterine stroma, vaginal epithelial cells, epididymal epithelial cells, and elongated spermatids. To validate recombinase activity, progesterone receptor (PGR) expression was ablated using the Ovgp1Cre:eGFP; Pgrf/f mouse model. Surprisingly, the deletion was restricted to the epithelial cells of the uterotubal junction (UTJ) region of Ovgp1Cre:eGFP; Pgrf/f oviducts. Deletion of Pgr in the epithelial cells of the UTJ region had no effect on female fecundity. In summary, we found that eGFP signals were likely specific to secretory epithelial cells in all regions of the oviduct. However, due to a potential target-specific Cre activity, validation of appropriate recombination and expression of the gene(s) of interest is absolutely required to confirm efficient deletion when generating conditional knockout mice using the Ovgp1Cre:eGFP line.

Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Pyrroloquinoline Quinone Improves Ram Sperm Quality through Its Antioxidative Ability during Storage at 4 °C.

Sperm motility is an important factor in the migration of sperm from the uterus to the oviduct. During sperm preservation in vitro, sperm generates excessive ROS that damages its function. This study aims to investigate whether the addition of pyrroloquinoline quinone (PQQ) to the diluted medium could improve chilled ram sperm quality, and then elucidates the mechanism. Ram semen was diluted with Tris-citric acid-glucose (TCG) medium containing different doses of PQQ (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM), and stored at 4 °C. Sperm motility patterns, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) levels, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and ATP levels were measured after preservation. Furthermore, the expressions of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) in sperm were also detected by western blotting. In addition, sperm capacitation and the ability of sperm to bind to the zona pellucina were also evaluated. It was observed that the addition of PQQ significantly (p < 0.05) improved ram sperm motility, membrane integrity, and acrosome integrity during preservation. The percentage of sperm with high mitochondrial membrane potential in the PQQ treatment group was much higher than that in the control. In addition, supplementation of PQQ also decreased the sperm MDA and ROS levels, while increasing ATP levels. Interestingly, the levels of MT-ND1 and MT-ND6 protein in sperm treated with PQQ were also higher than that of the control. Furthermore, the addition of 100 nM PQQ to the medium decreased ROS damage in MT-ND1 and MT-ND6 proteins. The addition of 100 nM PQQ significantly (p < 0.05) increased protein tyrosine phosphorylation in ram sperm after induced capacitation. Furthermore, the value of the sperm-zona pellucida binding capacity in the 100 nM PQQ treatment group was also much higher than that of the control. Overall, during chilled ram- sperm preservation, PQQ protected ram sperm quality by quenching the ROS levels to reduce ROS damage and maintain sperm mitochondrial function, and preserved the sperm's high ability of fertilization.

Multiple genes in the Pate5-13 genomic region contribute to ADAM3 processing†.

Sperm proteins undergo post-translational modifications during sperm transit through the epididymis to acquire fertilizing ability. We previously reported that the genomic region coding Pate family genes is key to the proteolytic processing of the sperm membrane protein ADAM3 and male fertility. This region contains nine Pate family genes (Pate5-13), and two protein-coding genes (Gm27235 and Gm5916), with a domain structure similar to Pate family genes. Therefore, in this study, we aimed to identify key factors by narrowing the genomic region. We generated three knockout (KO) mouse lines using CRISPR/Cas9: single KO mice of Pate10 expressed in the caput epididymis; deletion KO mice of six caput epididymis-enriched genes (Pate5-7, 13, Gm27235, and Gm5916) (Pate7-Gm5916 KO); and deletion KO mice of four genes expressed in the placenta and epididymis (Pate8, 9, 11, and 12) (Pate8-12 KO). We observed that the fertility of only Pate7-Gm5916 KO males was reduced, whereas the rest remained unaffected. Furthermore, when the caput epididymis-enriched genes, Pate8 and Pate10 remained in Pate7-Gm5916 KO mice were independently deleted, both KO males displayed more severe subfertility due to a decrease in mature ADAM3 and a defect in sperm migration to the oviduct. Thus, our data showed that multiple caput epididymis-enriched genes within the region coding Pate5-13 cooperatively function to ensure male fertility in mice.

Expression Analysis of Genes Corresponding to Mucins and Their Glycans from Cervical Tissue Using RNA Sequencing.

In the female reproductive tract, mucin proteins are the main component of mucus secreted by cervical goblet cells and play an essential role in many biological functions. They act as a medium for lubrication and mainting a cervical mucosal barrier against ascending pathogens from the vagina while also allowing sperm migration. The expression of mucin genes as well as the levels of O-glycosylation changes across the oestrous cycle. Detection and characterization of mucins and their glycans is important to understand the interface between the external and the internal environment, as the cervical epithelium represents the first line of defense against infections of the upper reproductive tract. Advances in the field of molecular biology have made possible to study differences in mucin and glycan gene expression which can help to understand impeded sperm transport as well as variation in the susceptibility to infection. This chapter discusses procedures relevant for both animals and humans on how to recover cervical tissue and perform a gene expression analysis of genes corresponding to mucins and their glycans using RNA sequencing.

Spermbots: A Promising Futuristic Innovation in Assisted Reproductive Technology

Spermbots are robotic sperms formed out of sperm cells conjugated to artificial microstructures, having potential applications ranging from biomedical processes, drug delivery systems, in situ real time imagery and assisted reproduction. The robotic sperm can act as an exploratory microdevice in biological networks. Incorporation of a biological entity like sperm into microstructures under the environment of magnetic or electric fields helps in shape templating and carrying chemotherapeutic agents to target sites. Besides its role in drug delivery systems, spermbots can potentially contribute in combating infertility, especially in oligo-zoospermia and necro-zoospermia in males. Numerous checkpoints may impede sperm cells to reach the oocyte in vivo. Spermbots bypass these sites and carry sperm to oocyte. Targeted delivery always requires interventions of natural functional aspects of living systems. Sperm flagellum, being a biological motor in nature, can be harnessed as a driving force in spermbots to ensure delivery of fertile sperm cells with impaired motile machinery to the target. Moreover, the technology has a potential to unravel sperm migration patterns and understanding the processes in vivo. After a review of documented literature on possible use of spermbot technology in assisted reproduction, we hereby discuss the application of this new and innovative technology in humans and animals. The paper also highlights certain shortfalls in the widespread application of this cargo delivery technology in assisted reproduction.

SPSL1 is essential for spermatophore formation and sperm activation in Spodoptera frugiperda.

The reproductive process in various species has undergone evolutionary adaptations at both the physiological and molecular levels, playing a significant role in maintaining their populations. In lepidopteran insects, the spermatophore is a unique structure formed in the female reproductive system, in which sperm storage and activation take place. It is known that the formation of the spermatophore is regulated by seminal fluid proteins derived from males. However, studies investigating the genetic mechanisms behind spermatophore formation in lepidopterans have been limited. In this study, our focus was on SPSL1, a gene that encodes a trypsin-type seminal fluid protein in Spodoptera frugiperda, a pest species with global invasive tendencies. Our findings revealed that SPSL1 expression was predominantly observed in the male reproductive tracts, and the disruption of this gene resulted in male sterility. Surprisingly, fluorescence analysis indicated that the absence of SPSL1 did not affect spermatogenesis or sperm migration within the male reproductive system. However, when females mated with SPSL1-mutant males, several defects were observed. These included disruptions in spermatophore formation, sperm activation in the copulatory bursae, and sperm migration into the spermathecae. Additionally, mass spectrometry analysis highlighted reduced levels of energy-related metabolites, suggesting that SPSL1 plays an essential role in promoting hydrolysis reactions during copulation. Consequently, our study demonstrates that SPSL1 is crucial for male fertility due to its functions in spermatophore formation and sperm activation. This research provides valuable insights into the genetic factors underlying reproductive processes in lepidopteran insects and sheds light on potential strategies for controlling invasive pest populations.

Is glycerol a good cryoprotectant for sperm cells? New exploration of its toxicity using avian model

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation.

Uterine defects and estradiol-dependent development of oviductal diverticula in mice lacking the SMAD4 C-terminal Mad homology 2 domain.

In female mammals, the oviduct and uterus are essential sites for female and male gamete transport, fertilization, implantation, and maintenance of a successful pregnancy. To delineate the reproductive function of Mothers against decapentaplegic homolog 4 (Smad4), we specifically inactivated Smad4 in ovarian granulosa cells and, oviduct and uterine mesenchymal cells using the Amhr2-cre mouse line. Deletion of exon 8 of Smad4 results in the production of an MH2-truncated SMAD4 protein. These mutant mice are infertile due to the development of oviductal diverticula and defects during the implantation process. The ovaries are fully functional as demonstrated in an ovary transfer experiment. The development of oviductal diverticula occurs shortly after puberty and is dependent on estradiol. The diverticula interfere with sperm migration and embryo transit to the uterus, reducing the number of implantation sites. Analysis of the uterus shows that, even if implantation occurs, decidualization and vascularization are defective resulting in embryo resorption as early as the seventh day of pregnancy. Thus, Smad4 plays an important function in female reproduction by controlling the structural and functional integrity of the oviduct and uterus.

O-166 Distinct molecular signatures of successful sperm in situ

Abstract Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that spermatozoa past the utero-tubal junction possess the intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

Biphasic Chemokinesis of Mammalian Sperm.

The female reproductive tract (FRT) continuously modulates mammalian sperm motion by releasing various clues as sperm migrate toward the fertilization site. An existing gap in our understanding of sperm migration within the FRT is a quantitative picture of how sperm respond to and navigate the biochemical clues within the FRT. In this experimental study, we have found that in response to biochemical clues, mammalian sperm display two distinct chemokinetic behaviors which are dependent upon the rheological properties of the media: chiral, characterized by swimming in circles; and hyperactive, characterized by random reorientation events. We used minimal theoretical modeling, along with statistical characterization of the chiral and hyperactive trajectories, to show that the effective diffusivity of these motion phases decreases with increasing concentration of chemical stimulant. In the context of navigation this concentration dependent chemokinesis suggests that the chiral or hyperactive motion refines the sperm search area within different FRT functional regions. Further, the ability to switch between phases indicates that sperm may use various stochastic navigational strategies, such as run and tumble or intermittent search, within the fluctuating and spatially heterogeneous environment of the FRT.

Membrane Cholesterol Inhibits Progesterone-Mediated Sperm Function through the Possible Involvement of ABHD2.

Abhydrolase domain containing 2-acylglycerol lipase (ABHD2) was recently claimed as the membrane receptor of progesterone (P4) in sperm cells, mediating cell processes such as sperm chemotaxis and acrosome reaction. Here, we investigated the role of membrane cholesterol (Chol) on ABHD2-mediated human sperm chemotaxis. Human sperm cells were obtained from twelve normozoospemic healthy donors. ABHD2-Chol interaction was modelled by computational molecular-modelling (MM). Sperm membrane Chol content was depleted by incubating cells with cyclodextrin (CD) or augmented by the incubation with the complex between CD and Chol (CD:Chol). Cell Chol levels were quantified by liquid chromatography-mass spectrometry. Sperm migration upon P4 gradient was evaluated through the accumulation assay in a specific migration device. Motility parameters were evaluated by sperm class analyzer, whilst intracellular calcium concentration, acrosome reaction and mitochondrial membrane potential were evaluated with calcium orange, FITC-conjugated anti-CD46 antibody and JC-1 fluorescent probes, respectively. MM analysis showed the possible stable binding Chol to ABHD2, resulting in to major impact on the protein backbone flexibility. The treatment with CD was associated with a dose-dependent increase in sperm migration in a 160 nM P4 gradient, together with increase in sperm motility parameters and levels of acrosome reaction. The treatment with CD:Chol was associated with essentially opposite effects. Chol was, thus, suggested to inhibit P4-mediated sperm function through the possible inhibition of ABHD2.

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.

MP30-20 HUMAN PAPILLOMAVIRUS SEMINAL CARRIAGE, SPERMOGRAM ABNORMALITIES AND MALE INFERTILY: A FRENCH COHORT STUDY

You have accessJournal of UrologyCME1 Apr 2023MP30-20 HUMAN PAPILLOMAVIRUS SEMINAL CARRIAGE, SPERMOGRAM ABNORMALITIES AND MALE INFERTILY: A FRENCH COHORT STUDY Armin Priam, Amiens, France, Antoine Le Bozec, Kremlin Bicetre, France, Vasco Dias Meireles, Fabien Saint, Rosalie Cabry, Moncef Benkhalifa, Baptiste Demey, and Dorian Bosquet Armin PriamArmin Priam More articles by this author , Amiens Amiens More articles by this author , France France More articles by this author , Antoine Le BozecAntoine Le Bozec More articles by this author , Kremlin BicetreKremlin Bicetre More articles by this author , France France More articles by this author , Vasco Dias MeirelesVasco Dias Meireles More articles by this author , Fabien SaintFabien Saint More articles by this author , Rosalie CabryRosalie Cabry More articles by this author , Moncef BenkhalifaMoncef Benkhalifa More articles by this author , Baptiste DemeyBaptiste Demey More articles by this author , and Dorian BosquetDorian Bosquet More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003258.20AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The impact of HPV on the male genital tract remains largely underestimated and underexplored, compared to the female side. Epidemiological data in men are scarce, and few studies have investigated the prevalence of HPV in the male population and spermogram abnormalities. The objective was to study the prevalence of Human papillomavirus seminal carriage in male genital tract and its association with the spermogram and spermocytogram abnormalities. METHODS: From May to October 2021, patients were recruited in the SPERM-VIR study and provided semen samples. A total of 461 patients evaluated for fertility disorders was included. Individual informed consent was obtained to use biological, demographic, health, and lifestyle data. The spermogram, spermocytogram and sperm migration assays were carried out by the local Department of Medicine and Reproductive Biology. Then, overages of semen were sent to the local Virology Department for Human Papillomavirus (HPV) screening. Semen characteristics (semen volume, sperm concentration, total sperm count, sperm motility, sperm morphology, spermatic denaturation, spermatic DNA fragmentation, sperm migration test results), demographic, health, and lifestyle data of patients carrying or not HPV in the sperm were compared using univariate and multivariate analyses with a logistic regression model. RESULTS: HPV was detected in 22.34% of sperm samples. Multivariate analysis showed that positive HPV status was significantly associated with at least one spermogram abnormality, according to WHO 2021 standards (oligozoospermia and/or asthenozoospermia and/or teratozoospermia) with an adjusted odds ratio of 4.10 [95% CI 2.32-7.25]. This statistical association was also found for the type of infertility (OR: 1.61 [95% CI 1.00-2.57]), the notion of varicocele (OR: 3.99 [95% CI 1.48-10.71]), Cryptorchidism/Testicular Ectopy/Monorchidism antecedent (OR: 3.54 [95% CI 1.07-11.66]). Either mono or pluri-HPV infection were significantly associated with at least 1 spermogram abnormality (OR: 3.93 [95% CI 2.08-7.41] and 4.11 [95% CI 1.58-10.68], respectively), independently of HPV oncogenic risk. The association between HPV spermatic infection and the risk of infertility was significant in multivariate analysis (OR: 5.63 [95% CI 3.16-10.01]) and by adjusting to propensity score (OR: 6.10 [95% CI 3.33-11.21]). CONCLUSIONS: Our study is an additional argument supporting the impact of seminal HPV carrying on male infertility, especially in cases labelled as idiopathic. The determination of spermatic HPV could therefore be a part of the exploration of an infertile man Source of Funding: No external funding was received for this project. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e399 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Armin Priam More articles by this author Amiens More articles by this author France More articles by this author Antoine Le Bozec More articles by this author Kremlin Bicetre More articles by this author France More articles by this author Vasco Dias Meireles More articles by this author Fabien Saint More articles by this author Rosalie Cabry More articles by this author Moncef Benkhalifa More articles by this author Baptiste Demey More articles by this author Dorian Bosquet More articles by this author Expand All Advertisement PDF downloadLoading ...