Acrosomal Region Research Articles

Effect of silver nanoparticles on donkey sperm parameters and ultrastructure.

The aim of this study was to determine the effect of silver nanoparticles (AgNPs) on donkey sperm parameters and ultrastructure. AgNPs were synthesized, purified and resuspended in the extender. Nine frozen-thawed donkey sperm samples were exposed to different concentrations of AgNPs (0, 1.25, 2.5, 5, 12.5, 25 and 50 μg/mL). Sperm parameters: total (TMOT, %) and progressive (PMOT, %) sperm motility, plasma (LIVE, %) and acrosomal membrane integrity (AIS, %), and sperm morphology (MORF, %) were evaluated immediately after AgNPs exposure (T0) and after 2 h of incubation (T2). The interaction beween AgNPs and spermatozoa was visualized by transmission electron microscopy (TEM). At T0, sperm motility and AIS were reduced (p < .05) when using concentrations ≥50 and ≥25 μg/mL, respectively. At T2, sperm motility and LIVE were significantly decreased (p < .05) in concentrations ≥25 and ≥50 μg/mL, respectively. TEM analysis revealed nanoparticle adhesion to the acrosomal region of the plasma membrane. In conclusion, AgNPs at concentrations ≥25 μg/mL impair motility, acrosome and plasma membrane integrity of donkey sperm, which may be mediated by adhesion to the acrosomal region of the sperm surface membrane.

Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the “metabolism” and the “metabolism of lipids” pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction.

The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

The mammalian sperm factor phospholipase C zeta is critical for early embryo division and pregnancy in humans and mice.

The testicular form of angiotensin converting enzyme as a marker for human sperm quality assessment.

Spermatozoa are rapidly changing cellular structures that are highly dependent on their interaction with the environment. These interactions cause fundamental changes in the spermatozoa's cells and membrane.

Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human.

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

IZUMO1 Receptor Localization during Hyaluronic Acid Selection in Human Spermatozoa.

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

Regional abundances of binder of sperm (BSP) proteins are negatively associated with the quality of frozen-thawed bovine spermatozoa.

The localization and abundance of the sperm BSP proteins correlate with in vitro fertility in domestic bulls used in artificial insemination service. Binder of sperm (BSP) proteins, secreted mainly by the accessory sex glands, are the major protein family present in bovine seminal plasma and on the sperm surface after ejaculation. In vivo, BSP proteins facilitate sperm capacitation and sperm reservoir formation; however, their impact on sperm function within the in vitro systems is less clear. Therefore, this biomarker-based study aimed to characterize the localization and abundance of BSP proteins from in vitro processed frozen-thawed bovine spermatozoa. Using image-based flow cytometry and Western blotting, BSP protein localization, abundance, membrane and acrosomal integrity were investigated in the supernatant (nonmotile) and pellet (motile) fractions of gradient-separated bull spermatozoa. Spermatozoa from the supernatant fraction had high enrichment of all BSP proteins investigated (BSP1, BSP3, BSP5; P < 0.05) when compared to the pellet fraction. In the pellet fraction, BSP1 and BSP3 bound predominately to the acrosomal region, whereas BSP5 had a high affinity for the midpiece. However, in the supernatant fraction, BSP proteins predominately coated the entire sperm surface resulting in the loss of regional specificity. High BSP protein abundance in the spermatozoa also correlated with acrosome and membrane damage. Whereas a high abundance of BSP5 correlated with low embryo cleavage rates, high abundance of BSP1 on the sperm head coincided with a high blastocyst rate. Therefore, changes in the quantity and localization of specific BSP proteins could act as potential biomarkers of sperm quality and fertility.

Cryopreservation, in addition to protein tyrosine phosphorylation, alters the distribution of phosphatidyl inositol bisphosphate and the localization of cytoskeletal and signaling proteins (gelsolin, tyrosine kinase c-SRC and phospholipase C-ζ) in the perinuclear theca of boar sperm

Cryopreservation of boar spermatozoa affects the perinuclear theca (PT) and involves several proteins and molecules that play important roles during capacitation and the acrosomal reaction. The objective of the present study was to evaluate whether the deleterious effects of cryopreservation in addition to protein tyrosine phosphorylation are accompanied by changes in the distribution of phosphatidyl inositol bisphosphate (PIP2) and the localization of cytoskeletal and signaling proteins in the perinuclear theca of cryopreserved boar spermatozoa. For this purpose, by immunocytochemistry (IC) the changes in localization of phosphorylated proteins in tyrosine residues, gelsolin, c-SRC kinase and PLC-ζ, as well as in the distribution of phosphatidyl inositol bisphosphate were analyzed in thawed spermatozoa (T) non capacitated (NC), capacitated (C) and in those with acrosomal reaction (AR) and compared with fresh spermatozoa (F) under the same physiological status. Western blotting (WB) and co-immunoprecipitation were performed to confirm the presence of these proteins in PT and to determine the interaction between these molecules. IC showed that immunostaining for phosphorylated proteins significantly increased in the acrosomal region and flagellum in TNC spermatozoa (p < 0.05). The proportion of cells displaying immunolabeling for gelsolin in the acrosomal region decreased after capacitation in cryopreserved spermatozoa; the same change was found (p < 0.05) in the proportion of spermatozoa immunoreactive to PIP2 in the sperm head. c-SRC was observed in the equatorial segment and acrosomal region, subdomains that coincide with the site where phosphorylated proteins were detected. PLC-ζ immunolocalization in fresh spermatozoa underwent changes after capacitation and acrosomal reaction, with a significant increase in the equatorial segment and post-acrosomal region in cryopreserved spermatozoa (p < 0.05). WB analysis indicated the presence of gelsolin, c-SRC and PLC-ζ in PT; besides, we confirmed that gelsolin co-immunoprecipitated with c-SRC and PLC-ζ, which changes according to the physiological state of spermatozoa. As a conclusion, cryopreservation together with increased immunodetection of tyrosine phosphorylated proteins decreases the detection of PIP2 and alters the immunolocalization patterns of gelsolin, c-SRC and PLC-ζ in the PT in boar spermatozoa.

Expression of Testis-specific Serine/Threonine Kinases during the Reproductive and Nonreproductive Seasons and Their Localization in Mature Spermatozoa of Tree Shrews (Tupaia belangeri).

Tree shrews display obvious reproductive cycles, and sexually mature male tree shrews produce little or no sperm with extremely low motility during the nonreproductive season; the mechanism underlying this phenomenon remains unknown. Because testis-specific serine/threonine kinases (TSSK) are specifically expressed in the testis and male germ cells of mammals, we hypothesized that they may have an important role in spermatogenesis or sperm function regulation in tree shrews. In addition, the expression, distribution, subcellular localization, and dynamic changes of TSSK in tree shrew sperm are unclear. Here we show that during the reproductive season, the seminiferous tubules were significantly larger as compared with the nonreproductive season and contained mature sperm and other germ cells. The mRNA expression of Tssk genes in testis was significantly higher than that in other tissues, and the mRNA level in the testis during the reproductive season was significantly higher than that in nonreproductive season. In addition, the mRNA level of Tssk3 in the testis and sperm was significantly higher than that of other members. Specifically, Tssk1 mRNA was distributed in the acrosome and throughout the flagellum of tree shrew sperm, Tssk2 was present in the acrosome, Tssk3 was localized to postacrosomal region and relocated to the main part of the flagellum after capacitation, and Tssk6 was distributed in the acrosome and postacrosomal region. These results indicate that the TSSK are important regulating reproductive function in tree shrews.

Metformin improves sheep sperm cryopreservation via vitalizing the AMPK pathway

Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) is a key regulator of sperm function and physiological metabolism. Metformin, an inexpensive and effective antioxidant, is known to play an important role in the activation of AMPK. Therefore metformin has potential to improve sperm cryopreservation. The aim of this study was to investigate the effect of metformin during semen cryopreservation of sheep and to find the most effective concentration in freezing extender. Semen were cryopreserved with extender containing different concentrations of metformin (0, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L). Sperm motility, acrosome integrity and plasma membrane integrity were measured after semen freezing and thawing. All results showed that sperm quality was significantly increased in the 1.0 mmol/L metformin-treated group compared with the control group (P < 0.05). In addition, the study showed that metformin effectively reduced the content of malondialdehyde (MDA) and reactive oxygen species (ROS), and increased the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total antioxidant capacity (T-AOC) of freeze-thawed sperm (P < 0.05). The optimal concentration of metformin was 1.0 mmol/L. Moreover, the results showed that AMPK was localized in the acrosome region, junction and midsection of sperm, and p-AMPK was distributed in the post-acrosomal region, junction and midsection. Western blot analysis indicated that 1.0 mmol/L metformin stimulated the phosphorylation of AMPK in sperm. Further results showed that 1.0 mmol/L metformin significantly increased the mitochondrial membrane potential (ΔΨm), ATP content, glucose uptake and lactate efflux of post-thawed sperm through the AMPK pathway, improved sperm quality, and increased the cleavage rate of in vitro fertilization (P < 0.05).

Loss-of-function mutations in IQCN cause male infertility in humans and mice owing to total fertilization failure.

Fertilization failure is a significant manifestation of unexplained male infertility. Previous work has suggested a genetic origin. In this study, we report on a man with unexplained infertility from a large consanguineous marriage family. Whole-exome sequencing and Sanger sequencing identified a homozygous frameshift variation of the IQ motif containing N (IQCN; GenBank: NM_001145304.1; c.1061_1062delAT; p.Y354Sfs*13) in the proband and one of his two brothers, who also remained infertile. Analyses of spermatozoa by quantitative RT-PCR indicated that the level of IQCN mRNA was significantly reduced compared to fertile men and the protein could not be detected by western blotting and immunofluorescent staining in the proband. Immunofluorescent staining of spermatozoa from fertile men showed that IQCN was located in the acrosomal region and translocated to the equatorial segment after the acrosome reaction. The proband spermatozoa had abnormal morphology and function. Finally, the proband couple underwent IVF with donor sperm and a healthy baby was born. Furthermore, we developed an Iqcn-KO mouse model using the CRISPR/Cas9 technique. Sperm quality, except for sperm motility, and the fertility of male Iqcn-/- mice were consistent with those of the proband. In conclusion, the findings in humans and mice demonstrate that the homozygous frameshift variant of IQCN causes male infertility owing to autosomal-recessive fertilization failure.

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm.

Na+/H+ exchangers (NHEs) are a family of ion transporters that regulate the pH of various cell compartments across an array of cell types. In eukaryotes, NHEs are encoded by the SLC9 gene family comprising 13 genes. SLC9C2, which encodes the NHE11 protein, is the only one of the SLC9 genes that is essentially uncharacterized. Here, we show that SLC9C2 exhibits testis/sperm-restricted expression in rats and humans, akin to its paralog SLC9C1 (NHE10). Similar to NHE10, NHE11 is predicted to contain an NHE domain, a voltage sensing domain, and finally an intracellular cyclic nucleotide binding domain. An immunofluorescence analysis of testis sections reveals that NHE11 localizes with developing acrosomal granules in spermiogenic cells in both rat and human testes. Most interestingly, NHE11 localizes to the sperm head, likely the plasma membrane overlaying the acrosome, in mature sperm from rats and humans. Therefore, NHE11 is the only known NHE to localize to the acrosomal region of the head in mature sperm cells. The physiological role of NHE11 has yet to be demonstrated but its predicted functional domains and unique localization suggests that it could modulate intracellular pH of the sperm head in response to changes in membrane potential and cyclic nucleotide concentrations that are a result of sperm capacitation events. If NHE11 is shown to be important for male fertility, it will be an attractive target for male contraceptive drugs due to its exclusive testis/sperm-specific expression.

RAB5C is a TBC1D21 interactor involved in sperm morphology formation

&lt;p&gt;背景與目的: 不孕是重要的公眾健康議題，造成不孕的因素其中一半與男性相關。其中造成男性不孕的一項重要病因是畸型精子症。在先前的研究中，我們以cDNA微晶片比較造精功能正常與異常的睪丸組織中各別基因的表現量。其中發現一個與不孕相關Rab GTPase激活蛋白，名為TBC1D21蛋白。TBC1D21特異的表現於減數分裂後期精細胞中。而於Tbc1d21基因剔除小鼠模式中發現嚴重精子尾部的中節區域損壞。進一步我們以蛋白質質體學方法鑑定出與TBC1D21結合蛋白。於本研究中我們聚焦於其中一個TBC1D21結合蛋白RAB5C。研究方法與結果: RAB5C蛋白表現於小鼠睪丸組織切片中的精子細胞的頂體與尾部。於成熟精子中，RAB5C蛋白濃縮於頭部頂體與散落於尾部中節區域。其中有趣的是精子經過頂體反應後，於頂體的RAB5C蛋白量明顯下降消失。最後我們利用共同免疫染色法確認RAB5C與TBC1D21共同散落於成熟精子尾部中節區域。結論:基於以上結果，我們發現TBC1D21 交互作用蛋白RAB5C參與於精子頭部頂體與尾部中節的形成。&lt;/p&gt; &lt;p&gt;&amp;nbsp;&lt;/p&gt;&lt;p&gt;Background and purpose: Infertility is a critical public health issue, and half of its causes are related to male factors. Teratozoospermia is one of major cause of male infertility. In our previous studies, we identified the TBC1 domain family member 21 (TBC1D21) gene, a Rab GTPase-activating protein (GAP), as a sterility-related gene in cDNA microarray compared to normal and spermatogenic testicular tissues. TBC1D21 is specifically expressed in post-meiotic male germ cells. Furthermore, the loss of Tbc1d21 allele in mice results in severe defects in the midpiece of the sperm&amp;rsquo;s tail. Additionally, TBC1D21 interactors were selected using proteomic assays. Herein, we focused on RAB5C, which is a TBC1D21 interactor. Methods and Results: RAB5C was localized in the acrosomal region and elongating tail in spermatids of murine testicular sections. In a mature sperm cell, RAB5C signals were concentrated in the acrosome and were scattered around the midpiece. Interestingly, RAB5C pro-tein levels decreased in the acrosome after triggering acrosomal reaction. Finally, we confirmed that the interspersed RAB5C signals were co-localized with TBC1D21 within the midpieces of the mature sperm cells. Conclusion: Based on these results, we suggest RAB5C, a TBC1D21 interactor, was involved in acrosomal and midpiece formation.&lt;/p&gt; &lt;p&gt;&amp;nbsp;&lt;/p&gt;

Identification, characterization and expression analysis of rLcn13, an epididymal lipocalin in rats.

As the essential tissue for sperm maturation and storage, the epididymis secretes a number of tissue-specific proteins to exert its functions. Among these proteins, epididymal lipocalins have been intensively studied because of their epididymis-specific expression pattern and clustered genomic organization. In this study, rLcn13, a member of the rat epididymal lipocalin family, is identified and elaborately characterized. The cDNA sequence of rLcn13 consists of 719 nucleotides and encodes a 176 amino-acid protein with a predicted N-terminal signal peptide of 19 amino acids. rLcn13 shares a similar genomic structure and predicted 3D protein structure with other lipocalin family members. A recombinant rLCN13 mature peptide of 157 amino acids is expressed and purified, which is used to raise a polyclonal antibody against rLCN13 with high specificity and sensitivity. Northern blot, western blot, and immunohistochemistry assays reveal that rLcn13 is an epididymis-specific gene which is expressed predominantly in the initial segment and proximal caput epididymis and influenced by androgen. The rLCN13 protein is modified by N-glycosylation and secreted into the epididymal lumen, and then binds to the acrosome region of the sperm. Our data demonstrate that rLcn13 exhibits a specific temporospatial expression pattern and androgen dependence, indicating its potential roles in sperm maturation.

A comprehensive investigation of human endogenous retroviral syncytin proteins and their receptors in men with normozoospermia and impaired semen quality.

The study aims to investigate first the presence of Syncytin 2 and its receptor, MFSD2, in human sperm, and second whether the expressions of Syncytin 1, Syncytin 2, and their receptors, SLC1A5 and MFSD2, differ between normozoospermic, asthenozoospermic, oligozoospermic, and oligoasthenozoospermic human sperm samples. The localization patterns and expression levels of syncytins and their receptors were evaluated in normozoospermic (concentration = 88.9 ± 5.5 × 106, motility = 79.2 ± 3.15%, n = 30), asthenozoospermic (concentration = 51.7 ± 7.18 × 106, motility = 24.0 ± 3.12%, n = 15), mild oligozoospermic (concentration = 13.5 ± 2.17 × 106, motility = 72.1 ± 6.5%, n = 15), moderate oligozoospermic (concentration = 8.4 ± 3.21 × 106, motility = 65.1 ± 8.9%, n = 15), severe oligozoospermic (concentration = 2.1 ± 1.01 × 106, motility = 67.5 ± 3.2%, n = 15), and oligoasthenozoospermic (concentration = 5.5 ± 3.21 × 106, motility = 18.5 ± 1.2%, n = 15) samples by immunofluorescence staining and western blot. Syncytins and their receptors visualized by immunofluorescence showed similar staining patterns with slight staining of the tail in all spermatozoa regardless of normozoospermia, asthenozoospermia, oligozoospermia, or oligoasthenozoospermia. The localization patterns were categorized as equatorial segment, midpiece region, acrosome, and post-acrosomal areas. The combined staining patterns were also detected as acrosomal cap plus post acrosomal region, the midpiece plus equatorial segment, and midpiece plus acrosomal region. However, some sperm cells were categorized as non-stained. Both syncytin proteins were most intensely localized in the midpiece region, while their receptors were predominantly present in the midpiece plus acrosomal region. Conspicuously, syncytins and their receptors showed decreased expression in asthenozospermic, oligozoospermic, and oligoasthenozoospermic samples compared to normozoospermic samples. The expression patterns of HERV-derived syncytins and their receptors were identical regardless of the spermatozoa in men with normozoospermia versus impaired semen quality. Further, asthenozoospermia, oligozoospermia, and oligoasthenozoospermia as male fertility issues are associated with decreased expression of both syncytins and their receptors.

Evaluation of sperm integrin α5β1 as a potential marker of fertility in humans.

Sperm selection for assisted reproduction techniques is generally based on basic parameters, while key aspects of sperm competence and its journey from the deposition site to the fertilization site are overlooked. Consequently, identifying molecular markers in spermatozoa that can efficiently predict the fertility of a semen sample could be of great interest, particularly in cases of idiopathic male infertility. When spermatozoa reach the female reproductive tract, it provides to them the cellular and molecular microenvironment needed to acquire fertilizing ability. In this sense, considering the role that integrin α5β1 of spermatozoa plays in reproduction-related events, we investigated the correlation between the subcellular localization of sperm integrin α5β1 and early embryo development outcome after in vitro fertilization (IVF) procedures in human. Twenty-four semen samples from normozoospermic men and metaphase II (MII) oocytes from healthy women aged under 38 years, from couples who underwent IVF cycles, were used in this work. Sperm α5β1 localization was evaluated by immunofluorescence assay using an antibody against integrin α5 subunit. Integrin α5β1 was mainly localized in the sperm acrosomal region (45.33±7.89%) or the equatorial segment (30.12±7.43%). The early embryo development rate (data obtained from the Fertility Center) correlated positively with the localization of α5β1 in the acrosomal region (number of usable embryos / inseminated oocytes: ρ = 0.75; p<0.01 and number of usable embryos/total number of two pronuclear zygotes: ρ = 0.80; p<0.01). However, this correlation was not significant when the equatorial segment mark was evaluated. In addition, human sperm released from co-culture with bovine oviductal epithelial cells (BOEC) showed a significant enrichment in the acrosomal localization pattern of α5β1 compared to those sperm that were not co-cultured with BOEC (85.20±5.35% vs 35.00±17.09%, respectively, p<0.05). In conclusion, the evaluation of sperm integrin α5β1 immunolocalization could be a useful tool to select sperm with fertilizing ability from human semen samples before IVF procedures.

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

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

Molecular Chaperone HSPA2 Distribution During Hyaluronic Acid Selection in Human Sperm

During fertilization, sperm hyaluronidase activity is essential for spermatozoa to successfully penetrate the hyaluronic acid-enriched extracellular matrix of the cumulus cells. Since molecular chaperones, as the heat shock protein A2, are typically involved in bringing hyaluronic acid receptors to the cell surface, here we evaluated the presence and spatial location of HSPA2 on human spermatozoa based on its hyaluronic acid binding capacity. This study included 16 normozoospermic sperm samples from volunteering donors. The location of HSPA2 was studied in cells before and after 1-h incubation under capacitating conditions, as well as in spermatozoa selected according to their ability of binding to hyaluronic acid. Our results showed no significant differences in HSPA2 immunofluorescent cells before and after 1 h of incubation in capacitating conditions. Nevertheless, after hyaluronic acid selection, the percentage of HSPA2-labelled cells increased significantly, indicating that the interaction with hyaluronic acid may induce the unmasking of HSPA2 epitopes. Furthermore, after swim-up and hyaluronic acid selection, spermatozoa presented a highly immunostained equatorial band with a homogeneous fluorescence throughout the acrosomal region. This distribution has been previously suggested to have important implications in male fertility. Noteworthy, a homogeneous fluorescence among the acrosomal region with a more intense labelling at the apical region was observed only in hyaluronic acid bound sperm cells, which may be associated with primary gamete recognition. Our findings suggest that the hyaluronic acid selection technique and HSPA2 biomarker should be considered candidates to complement the classic seminal analysis before recommending an appropriate assisted reproduction technique.

P-188 The effect of follicular fluid extracellular vesicles on motility and hyperactivation of human spermatozoa and differential miRNA levels depend on the woman's age

Abstract Study question How does the woman’s reproductive age affect the ability of follicular fluid (FF) extracellular vesicles (EV) to change the motility and hyperactivation of spermatozoa and follicular fluid microRNA profiles? Summary answer The effect of FF EVs on sperm motility and hyperactivation decreases with a woman's age. MiR-134-5p, miR-21-5p expression levels increase in older age group. What is known already Aging reduces human fertility. FF is an important factor in attracting and activating spermatozoa in the oviduct during oocyte fertilization, enhancing the process of sperm capacitation and acrosomal reaction in various mammalian species, including humans. EVs from seminal plasma and oviduct fluid carry proteins and miRNAs playing a vital role in a multi-step process including sperm motility, capacitation, acrosomal reaction, further fertilization. Results of our previous experiments showed that FF EVs from young women significantly improve the indices of sperm motility and hyperactivation. Several studies have isolated some age associated differentially expressed miRNAs (hsa-miR-424, hsa-miR-21-5p, hsa-miR-134, hsa-miR-190b, hsa-miR-99b -3p). Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds, frozen at -80 °C. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 106/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Samples were centrifuged and fixed in 2.5% glutaraldehyde in 0.1 M buffer for TEM. Sperm motility were assessed using CASA. MicroRNAs isolation (miRNeasy Serum/Plasma Kit (Qiagen)). Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 18) aged 28-36 years without severe pathozoospermia. FF EVs were obtained from young (n = 4) aged 27-31 (EVs+) and older patients (n = 4) aged 41-46 with several IVF attempts (EVs-). The methods used in this work include sperm and EVs isolation, incubation, transmission electron microscopy, RT-PCR, statistical data analysis, computer-assisted semen analysis (CASA). Main results and the role of chance Our results showed that sperm incubation with EVs+ led to a significant increase in the number of progressively motile spermatozoa (paired Student's t test, p &amp;lt; 0.001), indicators of general mobility (p = 0.05) and hyperactivation of spermatozoa after 1 hour. Sperm incubation with EVs- slightly changed these parameters compared to the control (p = 0.171). Results from TEM showed that EVs- bind to spermatozoa worse than EVs+ after incubation. EVs- bind predominantly to the lateral membrane of the spermatozoon, rather than to the acrosomal region, compare to EVs+, which may reflect changes in the functional composition of FF EVs in older patients group and target areas of interaction with spermatozoa. This hypothesis is also supported by slight improvements in progressive sperm motility after incubation. Age-related changes are reflected in energy, metabolic and other important biological processes, which leads to a change in the composition and functions of FF (decrease in progesterone, glucose, increase in lactate and pyruvate, increase in reactive oxygen species) and affect the functional role of FF EVs. MiR-134-5p (p = 0.008), miR-21-5p (p = 0.008) expression levels were statistically significantly higher in EVs- group compared with EV + group with a successful first attempt. The expression of other microRNAs analyzed did not differ between groups. Limitations, reasons for caution Small amount of data. This study does not characterize isolated FF EVs in any way (specific markers, EVs concentration, size). To discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics, it is necessary to perform metabolomics analyses of FF EVs. Wider implications of the findings Further studies of the influence of FF EVs on sperm motility may help to improve of ART outcomes, associated with male and female infertility by improving sperm morphofunctional characteristics and increasing their fertilizing ability. The solutions to this problem could be the use of FF EVs from young fertile donors. Trial registration number not applicable

The Sperm Olfactory Receptor OLFR601 is Dispensable for Mouse Fertilization.

Fertilization involves the fusion of two gametes by means of yet unknown membrane binding and fusion events. Over the last years, many sperm proteins have been uncovered to play essential roles in sperm-egg fusion in mammals, but their precise role in fertilization remains unknown, being unclear how these proteins interact with each other or with other yet unknown sperm proteins. The aim of this study has been to identify possible sperm proteins interacting with TMEM95, a protein essential for fertilization located in the sperm membrane. A list of 41 sperm proteins that were pulled down with TMEM95 and identified by mass spectrometry did not include other sperm proteins known to play a role in fertilization, suggesting an independent role of TMEM95 in fertilization. Between these lists, OLFR601 is allocated to the acrosomal region and may mediate affinity for an odorant involved in fertilization. However, Olfr601 disruption did not impair the sperm fertilization ability, suggesting that its function may be redundant with that of other sperm proteins.