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

BACKGROUND: Exposure 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. RESULTS: Lead 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. CONCLUSIONS: Pb2+ 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.

Subtle membrane changes in cryopreserved bull spermatozoa when modified temperature drop rates are used during the first phase of freezing.

BACKGROUND: Cryopreservation of spermatozoa involves reduction of temperature to a subzero level, leading to increased longevity. However, temperature reduction has a significant effect on sperm membranes. OBJECTIVE: To evaluate the impact of the rate of temperature drop during the first phase of freezing on subtle membrane changes in cryopreserved bull spermatozoa. MATERIALS AND METHODS: Thirty-two ejaculates from four bulls (eight ejaculates/bull) were collected using artificial vagina while keeping a 3 to 4 days gap between two collections. Diluted semen samples were equilibrated at 5 degree C for 4 hours. The samples were then placed in a pre-programmed semen freezer. The first phase of freezing, that is, 5 degree C till -10 degree C was subjected to three different temperature drop rates: accelerated (F1), moderate (F2), and slow (F3), at 20 degree C per min, 10 degree C per min and 5 degree C per min, respectively. After thawing, spermatozoa were assessed for percentage live, plasma, and acrosomal membrane integrity, along with the external appearance of phosphatidyl serine, indicating apoptosis. RESULTS: A significant difference (p < 0.05) in viability, plasma membrane integrity (HOS test), and acrosome membrane integrity (PSA test) was observed between F3 and the other groups. However, the parameters did not significantly differ between F1 and F2. The annexin V-PI assay (AN/PI) categorized four types of sperm populations: non-apoptotic and viable (AN-/PI-), apoptotic and viable (AN+/PI-), non-apoptotic and non-viable (AN-/PI+), and apoptotic and non-viable (AN+/PI+). The proportion of spermatozoa with (AN-/PI-) and (AN+/PI+) differed significantly (p < 0.05) between F3 and the other groups. The values for apoptotic and viable (AN+/PI-) and non-apoptotic and non-viable (AN-/PI+) sperm were not significantly different among all freezing categories. CONCLUSION: A slower temperature drop rate (freezing rate) during the first phase of freezing results in less damaging, subtle membrane changes. Doi.org/10.54680/fr24410110312.

Enhancement of Frozen-Thawed Human Sperm Quality with Zinc as a Cryoprotective Additive.

BACKGROUND Cryopreservation preserves male fertility, crucial in oncology, advanced age, and infertility. However, it damages sperm motility, membrane, and DNA. Zinc (Zn), an antioxidant, shows promise in improving sperm quality after thawing, highlighting its potential as a cryoprotectant in reproductive medicine. MATERIAL AND METHODS Gradient concentration of ZnSO4 (0, 12.5, 25, 50, and 100 µM) was added in the Glycerol-egg yolk-citrate (GEYC) cryopreservative medium as an extender. Alterations in sperm viability and motility parameters after cryopreservation were detected in each group. Sperm plasma membrane integrity (PMI), acrosome integrity (ACR), DNA fragment index (DFI), and changes in sperm mitochondrial function were examined, including: mitochondrial potential (MMP), sperm reactive oxygen species (ROS), and sperm ATP. RESULTS We found that 50 µM ZnSO4 was the most effective for the curvilinear velocity (VCL) and the average path velocity (VAP) of sperm after cryo-resuscitation. Compared to the Zn-free group, sperm plasma membrane integrity (PMI) was increased, DNA fragmentation index (DFI) was decreased, reactive oxygen species (ROS) was reduced, and mitochondrial membrane potential (MMP) was increased after cryorevival in the presence of 50 µM ZnSO4. CONCLUSIONS Zn ion is one of the antioxidants in the cell. The results of our current clinical study are sufficient to demonstrate that Zn can improve preserves sperm quality during cryopreservation when added to GEYC. The addition of 50 µM ZnSO4 increased curve velocity, mean path velocity, sperm survival (or plasma membrane integrity), and mitochondrial membrane potential while reducing ROS production and DNA breaks compared to GEYC thawed without ZnSO4.

Dog sperm cryopreservation using cryovials and different dilution steps.

BACKGROUND: The conventional sperm freezing method for dog sperm is with straws and includes two-step dilution and a long equilibration time. OBJECTIVE: To develop a more efficient freezing method using cryovials. MATERIALS AND METHODS: Three freezing protocols using cryovials (0.5 mL) were conducted with dog spermatozoa at 1 x 108 sperm/mL: Group 1 spermatozoa were cooled in cryovials and extender 1 (E1) and extender 2 (E1 +1 M glycerol) at 4 degree C for 50 min and then frozen over LN2 for 20 min; Group 2 sperm was cooled and frozen in cryovials with a mixture of E1 and E2 (1:1) in a deep freezer (-80 degree C) for 30 min; Group 3 sperm in cryovials and E1 were cooled at 4 degree C for 20 min, cooled for an additional 20 min after addition of E2 (E1:E2, 1:1), and then frozen using LN2/ vapour for 20 min. The control (Group 4) consisted of spermatozoa in straws being frozen using the conventional freezing method using two-step dilution. All groups were plunged and stored in LN2 after freezing and their functional performance and gene expression determined. RESULTS: Progressive motility and acrosome integrity were highest (P < 0.05) in Groups 2, 3 and 4 (only acrosome integrity). Viability in Group 3 was significantly better that in the other Groups, and the reactive oxygen species (ROS) level and phosphatidylserine (PS) translocation index were significantly lower in Group 2 than the other Groups. The expression of sperm mitochondria-associated cysteine-rich protein (SMCP) and anti-apoptotic B-cell lymphoma 2 (BCL2) genes was highest (P < 0.05) in Group 2 and the expression of pro-apoptotic Bcl2-associated X protein (BAX) was lowest (P < 0.05) in Group 4. CONCLUSION: The sperm frozen using cryovials, one step dilution and the deep freezer (Group 2) proved to be a simple and suitable cryopreservation method for dog sperm. https://doi.org/10.54680/fr24110110312.

Deletion of ACTRT1 is associated with male infertility as sperm acrosomal ultrastructural defects and fertilization failure in human.

STUDY QUESTION: Could actin-related protein T1 (ACTRT1) deficiency be a potential pathogenic factor of human male infertility? SUMMARY ANSWER: A 110-kb microdeletion of the X chromosome, only including the ACTRT1 gene, was identified as responsible for infertility in two Chinese males with sperm showing acrosomal ultrastructural defects and fertilization failure. WHAT IS KNOWN ALREADY: The actin-related proteins (e.g. ACTRT1, ACTRT2, ACTL7A, and ACTL9) interact with each other to form a multimeric complex in the subacrosomal region of spermatids, which is crucial for the acrosome-nucleus junction. Actrt1-knockout (KO) mice are severely subfertile owing to malformed sperm heads with detached acrosomes and partial fertilization failure. There are currently no reports on the association between ACTRT1 deletion and male infertility in humans. STUDY DESIGN, SIZE, DURATION: We recruited a cohort of 120 infertile males with sperm head deformations at a large tertiary hospital from August 2019 to August 2023. Genomic DNA extracted from the affected individuals underwent whole exome sequencing (WES), and in silico analyses were performed to identify genetic variants. Morphological analysis, functional assays, and ART were performed in 2022 and 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ACTRT1 deficiency was identified by WES and confirmed by whole genome sequencing, PCR, and quantitative PCR. Genomic DNA of all family members was collected to define the hereditary mode. Papanicolaou staining and electronic microscopy were performed to reveal sperm morphological changes. Western blotting and immunostaining were performed to explore the pathological mechanism of ACTRT1 deficiency. ICSI combined with artificial oocyte activation (AOA) was applied for one proband. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a whole-gene deletion variant of ACTRT1 in two infertile males, which was inherited from their mothers, respectively. The probands exhibited sperm head deformations owing to acrosomal detachment, which is consistent with our previous observations on Actrt1-KO mice. Decreased expression and ectopic distribution of ACTL7A and phospholipase C zeta were observed in sperm samples from the probands. ICSI combined with AOA effectively solved the fertilization problem in Actrt1-KO mice and in one of the two probands. LIMITATIONS, REASONS FOR CAUTION: Additional cases are needed to further confirm the genetic contribution of ACTRT1 variants to male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal a gene-disease relation between the ACTRT1 deletion described here and human male infertility owing to acrosomal detachment and fertilization failure. This report also describes a good reproductive outcome of ART with ICSI-AOA for a proband. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Chongqing medical scientific research project (Joint project of Chongqing Health Commission and Science and Technology Bureau, 2023MSXM008 and 2023MSXM054). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Morphology of the male reproductive system and sperm of Leptoglossus zonatus (Dallas, 1852) (Heteroptera: Coreidae).

Taxonomic data on Coreidae have been fragmented over time and need to be revised. Likewise, data related to the development of germ cells and the features of the male reproductive system, including sperm, will contribute to understanding the biological mechanisms of reproduction and the systematics of its representatives. Aiming to provide these data, we describe the morphology of the male reproductive system and spermatozoa of Leptoglossus zonatus using light and transmission electron microscopies, respectively. Each of the two testes is surrounded by a bright red-pigmented sheath and formed by seven follicles arranged side by side. The two vasa deferentia are filled with individualized sperm, especially in their final portion, which is dilated and curved. After dilation, the vasa deferentia receive the ducts of the accessory glands of mesodermal origin. The other unpaired accessory gland is of ectodermal origin and opens into the ejaculatory duct. Both glandular types are densely coiled and have lumens filled with secreted material. Testicular follicles contain cysts with germ cells at different stages of spermatogenesis, indicating continuous production of gametes throughout adult life. Mature sperm measure around 310 µm long, with a nucleus of 36 µm and a flagellum formed only by an axoneme of 9 + 9 + 2 microtubules and two symmetrical mitochondrial derivatives. Like the sperm of other Heteroptera, the acrosome has a single structure (without perforatorium), there are no accessory bodies in the flagella, and the mitochondrial derivatives are connected to the axonemes, supporting the synapomorphic condition of these characteristics for this suborder of bedbugs. RESEARCH HIGHLIGHTS: The Leptoglossus zonatus sperm are slender and long, about 310 µm in length, and a nucleus 36 µm long. Spermatogenesis occurs throughout adult life and equally in the seven testicular follicles. The centriole adjunct in L. zonatus sperm does not give rise to accessory bodies. The ectodermal gland produces a filamentous secretion, whereas in the ectodermal sac, the secretion is globular.

Loss of ACTL7A causes small head sperm by defective acrosome-acroplaxome-manchette complex.

BACKGROUND: Actin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. In this study, we aim to explore the additional functions of ACTL7A beyond the process of acrosome biogenesis and investigate the possible underlying mechanisms. METHODS: Nuclear morphology analysis was used to observe the sperm head shape of ACTL7A-mutated patients. Actl7a knock-out (KO) mouse model was generated. Immunofluorescence and transmission electron microscopy (TEM) were performed to analyze the structure of spermatids during spermiogenesis. Tandem mass tags labeling quantitative proteomics strategy was employed to explore the underlying molecular mechanisms. The expression levels of key proteins in the pathway were analyzed by western blotting. Intracytoplasmic sperm injection (ICSI)-artificial oocyte activation (AOA) technology was utilized to overcome fertilization failure in male mice with a complete knockout of Actl7a. RESULTS: The new phenotype of small head sperm associated with loss of ACTL7A in patients was discovered, and further confirmed in Actl7a-KO mice. Immunofluorescence and TEM analyses revealed that the deletion of ACTL7A damaged the formation of acrosome-acroplaxome-manchette complex, leading to abnormalities in the shaping of sperm heads. Moreover, a proteomic analysis of testes from WT and Actl7a-KO mice revealed that differentially expressed genes were notably enriched in PI3K/AKT/mTOR signaling pathway which is strongly associated with autophagy. Inhibition of autophagy via PI3K/AKT/mTOR signaling pathway activation leading to PDLIM1 accumulation might elucidate the hindered development of manchette in Actl7a-KO mice. Remarkably, AOA successfully overcame fertilization failure and allowed for the successful production of healthy offspring from the Actl7a complete knockout male mice. CONCLUSIONS: Loss of ACTL7A causes small head sperm as a result of defective acrosome-acroplaxome-manchette complex via autophagy inhibition. ICSI-AOA is an effective technique to rescue male infertility resulting from ACTL7A deletion. These findings provide essential evidence for the diagnosis and treatment of patients suffering from infertility.

Assessment of the stallion sperm acrosome in two different extenders: Spermac stain in comparison with PNA/PSA/PI triple-staining.

Since the stallion acrosome is very small compared to other species and cannot be properly assessed without additional staining, several labelling techniques were developed to facilitate its assessment. The aim of this study was to compare the Spermac stain (Minitüb GmbH) and a PNA/PSA/PI triple-staining detected by flow cytometry with regard to method agreement for detecting non-intact acrosomes within two different extenders. For this purpose, eighteen stallion ejaculates were split in half and diluted with the semen extenders EquiPlus or Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL, respectively. Subsequently, 126 semen samples were stained with both methods between 4 and 240 h (mean: 63.8 ± 48.9 h) after semen collection. Calculated Intraclass correlation coefficients revealed excellent correlations between both methods for EquiPlus (r = .77, p < .001) and fair correlations for Gent (r = .49, p < .001). Interestingly, flow cytometry detected more non-intact acrosomes in EquiPlus than in Gent (p < .001), whereas the Spermac stain showed no differences (p = .902) between extenders. The poorer method agreement in Gent could be caused by egg yolk artefacts, which made interpretation difficult, so flow cytometry might be preferred. The differences in detected non-intact acrosomes between extenders highlighted the importance of establishing adapted laboratory protocols for different extender types in order to generate comparable results.

ATP increases head volume in capacitated human sperm via a purinergic channel.

Scanning ion-conductance microscopy allowed us to document an external Ca2+ dependent ATP driven volume increase (ATPVI) in capacitated human sperm heads. We examined the involvement of purinergic receptors (PRs) P2X2R and P2X4R in ATPVI using their co-agonists progesterone and Ivermectin (Iver), and Cu2+, which co-activates P2X2Rs and inhibits P2X4Rs. Iver enhanced ATPVI and Cu2+ and 5BDBD inhibited it, indicating P2X4Rs contributed to this response. Moreover, Cu2+ and 5BDBD inhibited the ATP-induced acrosome reaction (AR) which was enhanced by Iver. ATP increased the concentration of intracellular Ca2+ ([Ca2+]i) in >45% of individual sperm, most of which underwent AR monitored using FM4-64. Our findings suggest that human sperm P2X4R activation by ATP increases [Ca2+]i mainly due to Ca2+ influx which leads to a sperm head volume increase, likely involving acrosomal swelling, and resulting in AR.

Characterization of acrosin and acrosin binding protein as novel CRISP2 interacting proteins in boar spermatozoa.

BACKGROUND: Previously, we reported that cysteine-rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine-rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning. OBJECTIVES: This study aimed to identify cysteine-rich secretory protein 2 interacting partners. These binding partner interactions were investigated under different conditions, namely, non-capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187-induced acrosome reaction. MATERIALS AND METHODS: The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC-MS for protein identification. The most prominent cystein-rich secretory protein 2 interacting proteins that appeared in both independent LC-MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein-rich secretory protein 2 in pig sperm. RESULTS: Blue native gel electrophoresis and native immunoblots revealed that cystein-rich secretory protein 2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of cystein-rich secretory-protein 2-immunoreactive bands from blue native gels as well as cystein-rich secretory protein 2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein-rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that cystein-rich secretory protein 2 interacted with pro-acrosin (∼53 kDa) and Aacrosin binding protein under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein-rich secretory protein 2 was assessed via in situ proximity ligation assays. The colocalization signal of cystein-rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein-rich secretory protein 2 and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post-acrosomal sheath region upon capacitation. DISCUSSION AND CONCLUSION: These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.

Cryopreservation effect on sperm viability, mitochondrial membrane potential, acrosome integrity and sperm capacitation of alpaca spermatozoa detected by imaging flow cytometry.

Eighty-five sperm samples were cryopreserved and SYBR14/PI, MitoTracker Deep Red FM, FITC-PSA/PI and chlortetracycline were used for imaging flow cytometry evaluation of sperm viability, mitochondrial membrane potential (MMP), acrosome integrity and sperm capacitation, respectively. Sperm motility was also registered. Sperm motility (46.1 ± 7.7 vs. 24.1% ± 6.5%), sperm viability (49.8 ± 11.5 vs. 32.3% ± 9.6%) and high MMP (49.8% ± 12.4% vs. 34.9% ± 9.9%) decreased significantly (p < .05) during cryopreservation process, in contrast to acrosome-reacted in viable spermatozoa (1.0% ± 1.6% vs. 1.0% ± 1.0%) and sperm capacitation (10.0 ± 9.8 vs. 8.2% ± 12.4%) that were similar (p > .05) before and after cryopreservation. Positive correlations were found between sperm motility versus high MMP (r = .63), sperm motility versus sperm viability (r = .67) and sperm viability versus high MMP (r = .88). In conclusion, cryopreservation of alpaca spermatozoa is related to a decrease in sperm motility, sperm viability and high MMP, meanwhile acrosome integrity and sperm capacitation are not affected.

F4-Neuroprostane Effects on Human Sperm.

Swim-up selected human sperm were incubated with 7 ng F4-neuroprostanes (F4-NeuroPs) for 2 and 4 h. Sperm motility and membrane mitochondrial potential (MMP) were evaluated. The percentage of reacted acrosome was assessed by pisum sativum agglutinin (PSA). Chromatin integrity was detected using the acridine orange (AO) assay and localization of the ryanodine receptor was performed by immunofluorescence analysis. Sperm progressive motility (p = 0.02) and the percentage of sperm showing a strong MMP signal (p = 0.012) significantly increased after 2 h F4-NeuroP incubation compared to control samples. The AO assay did not show differences in the percentage of sperm with dsDNA between treated or control samples. Meanwhile, a significantly higher number of sperm with reacted acrosomes was highlighted by PSA localization after 4 h F4-NeuroP incubation. Finally, using an anti-ryanodine antibody, the immunofluorescence signal was differentially distributed at 2 and 4 h: a strong signal was evident in the midpiece and postacrosomal sheath (70% of sperm) at 2 h, whereas a dotted one appeared at 4 h (53% of sperm). A defined concentration of F4-NeuroPs in seminal fluid may induce sperm capacitation via channel ions present in sperm cells, representing an aid during in vitro sperm preparation that may increase the positive outcome of assisted fertilization.

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

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

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

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

Role of the bovine PRAMEY protein in sperm function during in vitro fertilization (IVF).

Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen (CTA) that is predominantly expressed in normal male gonad tissues and a variety of tumors. PRAME proteins are present in the acrosome and sperm tail, but their role in sperm function is unknown. The objective of this study was to examine the function of the bovine Y-linked PRAME (PRAMEY) during spermatozoal capacitation, the acrosome reaction (AR), and fertilization. Freshly ejaculated spermatozoa were induced to capacitate and undergo AR in vitro. Western blotting results revealed a decrease in the PRAMEY protein in capacitated spermatozoa, and the release of the PRAMEY protein from the acrosome during the AR, suggesting its involvement in sperm capacitation and AR. IVF was performed using in vitro matured bovine oocytes and cauda epididymal spermatozoa either treated with PRAMEY antibody, rabbit IgG, or DPBS. Sperm-egg binding and early embryos were examined at 6 and 45 h post IVF, respectively. The number of spermatozoa that bound per oocyte was nearly two-fold greater in the PRAMEY antibody treatment group (34.4) when compared to both the rabbit IgG (17.6) and DPBS (18.1) controls (P < 0.01). Polyspermy rate in the antibody-treated group (18.9%) was three-fold greater than the rabbit IgG control (6.0%) (P < 0.01). The results indicate that PRAMEY may play a role in anti-polyspermy defense. This study thus provides the initial evidence for the involvement of the PRAME protein family in sperm function and fertilization.

Melatonin improves post-thaw sperm quality after mild testicular heat stress in rams.

The objective was to determine effects of slow-release melatonin on post-thaw sperm quality in rams exposed to mild testicular heat stress (HS; scrotal neck insulation). Twelve yearling Dorset rams were randomly and equally allocated to receive either 36 mg melatonin in 1 ml corn oil or 1 ml corn oil injected subcutaneously (SQ); 15 day later, all rams had HS for 96 h (start of HS = start of Week 0). Semen was collected before HS and once weekly from Weeks 1 to 7, extended in Steridyl CSS One Step, held at 5°C for ~3 h, loaded into 0.5 ml straws, held 5 cm above liquid nitrogen for 10 min and then plunged. Computer assisted semen analysis (CASA) was conducted on frozen-thawed sperm. There were group and week effects for total and progressive motility (p < .001), plus group and week effects and group*week interactions (p < .001) for post-thaw total abnormalities, acrosome integrity, post-thaw sperm DNA fragmentation index (DFI) and high mitochondrial membrane potential (HMMP). Post-thaw sperm total and progressive motility, acrosome integrity and HMMP were higher (p < .05) in melatonin versus control groups from Weeks 1 to 7, and the melatonin group reached baseline level (pre-heat stress) at Week 7 (75.79 ± 0.96, 65.48 ± 1.51, 75.00 ± 0.89 and 67.00 ± 1.06, respectively; mean ± SEM). Conversely, post-thaw sperm total abnormalities and DFI were lower (p < .05) in melatonin versus control, and both reached baseline at Week 7 in the melatonin group (26.00 ± 0.57 and 5.66 ± 0.17, respectively). Coiled tails, distal midpiece reflexes, distal cytoplasmic droplets, ruffled acrosomes, bowed midpieces, pyriform heads and knobbed acrosomes were the most common abnormalities in both groups, with lower percentages in melatonin-treated rams. Results supported our hypothesis that HS reduces post-thaw sperm quality, and that melatonin lessens those reductions, manifested by significantly better total and progressive motility, acrosome integrity and HMMP, and fewer sperm total abnormalities and DFI.

Morphology of male and female reproductive systems in the ground beetle Apotomus and the peculiar sperm ultrastructure of A. rufus (P. Rossi, 1790) (Coleoptera, Carabidae).

Relatively few studies have focused on evolutionary losses of sexually selected male traits. We use light and electron microscopy to study the male and female reproductive anatomy of Apotomus ground beetles (Coleoptera, Carabidae), a lineage that we reconstruct as likely having lost sperm conjugation, a putative sexually selected trait. We pay particular attention to the structure of the testes and spermatheca. Both of these organs share a strikingly similar shape-consisting of long blind canals arranged into several concentric overlapping rings measuring approximately 18 mm and 19.5 mm in total length, respectively. The similarity of these structures suggests a positive evolutionary correlation between female and male genital organs. Males are characterized by unifollicular testes with numerous germ cysts, which contain 64 sperm cells each, and we record a novel occurrence of sperm cyst "looping", a spermatogenic innovation previously only known from some fruit fly and Tenebrionid beetle sperm. The sperm are very long (about 2.7 mm) and include an extraordinarily long helicoidal acrosome, a short nucleus, and a long flagellum. These findings confirm the structural peculiarity of sperm, testis, and female reproductive tract (FRT) of Apotomus species relative to other ground beetles, which could possibly be the result of shifts in sexual selection.

The Functions of Pt-DIC and Pt-Lamin B in Spermatogenesis of Portunus trituberculatus.

Cytoplasmic Dynein is a multiple-subunit macromolecular motor protein involved in the transport process of cells. The Dynein intermediate chain (DIC) is one of the subunits of Dynein-1. In our previous studies, we showed that Pt-DIC may play an important role in the nuclear deformation of spermiogenesis in Portunus trituberculatus. Lamin B is essential for maintaining nuclear structure and functions. Surprisingly, Pt-Lamin B was expressed not only in the perinucleus but also in the pro-acrosome during spermiogenesis in P. trituberculatus. Studies have also shown that Dynein-1 can mediate the transport of Lamin B in mammals. Thus, to study the relationship of Pt-DIC and Pt-Lamin B in the spermatogenesis of P. trituberculatus, we knocked down the Pt-DIC gene in P. trituberculatus by RNAi. The results showed that the distribution of Pt-DIC and Pt-Lamin B in spermiogenesis was abnormal, and the colocalization was weakened. Moreover, we verified the interaction of Pt-DIC and Pt-Lamin B via coimmunoprecipitation. Therefore, our results suggested that both Pt-DIC and Pt-Lamin B were involved in the spermatogenesis of P. trituberculatus, and one of the functions of Dynein-1 is to mediate the transport of Lamin B in the spermiogenesis of P. trituberculatus.

Improving the process of spermatogenesis in azoospermic mice using spermatogonial stem cells co-cultured with epididymosomes in three-dimensional culture system.

AIMS: This study aimed to explore the effect of epididymosomes on the proliferative efficiency of spermatogonial stem cells (SSCs) in vitro and the resumption of spermatogenesis in the azoospermic mice. MAIN METHODS: The epididymosomes were extracted from the epididymis and characterized. SSCs were cultured in 2D (two-dimensional) and hydrogel-based 3D culture in the presence of 20 µg/mL epididymosome or 10 ng/mL GDNF. After two weeks of culture, the proliferation and purity of the separated SSCs were evaluated using the MTT test and flow cytometry, respectively. qRT-PCR was used to analyze PLZF, caspase-3, TGF-ß, miR-10b, and miR-21 expression levels. Then, SSCs grown in the 3D culture system were labeled by DiI and transplanted into azoospermic mice via the efferent duct. After 2 weeks, tracing of DiI and cell homing were evaluated. Subsequently, histomorphometric studies and immunohistochemistry analysis were performed in testes after eight weeks of transplantation. KEY FINDINGS: The expression of PLZF, TGF-ß, miR-10b, and miR-21 increased significantly (*p < 0.05) in the 3D + GDNF and 3D + epididymosomes groups than in the 2D group. Transplanted SSCs migrated into the seminiferous tubules of recipient mice and the number of spermatogenic cells and protein expression of PLZF, SCP3 and ACRBP in the 3D + GDNF and 3D + epididymosomes groups were considerably higher (∗ ∗ ∗ p < 0.001) compared to the azoospermic group. SIGNIFICANCE: This finding indicates that culturing SSCs on decellularized testicular matrix (DTM) hydrogel with 10 ng/mL GDNF or 20 µg/mL epididymosomes could lead to an increase in SSCs proliferation which provides a sufficient number of SSCs for successful transplantation in azoospermic mice.

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

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