Sperm Plasma Membrane Research Articles

Piceatannol Protects Sperm from Cryopreservation Damage by Modulating the Keap1-Nrf2/ARE Signaling Pathway.

The purpose of this study was to explore the mechanism of action of Piceatannol (PIC) in attenuating oxidative damage to sperm during cryopreservation. Semen samples were collected and homogenized into six equal parts for freeze-thawing experiments. Four different concentrations of PIC were utilized as cryoprotectants during the freeze-thawing process, maintaing a semen to PIC ratio of 1:1, while sperm motility after freezing and thawing was analyzed using computer-assisted sperm analysis (CASA). Sperm plasma membrane integrity was assessed via the hypo-osmotic swelling (HOS) test. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities, long with the ability to scavenge sperm malondialdehyde (MDA), were examined in sperm following the addition of PIC. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression levels of Keap1, Nrf2, GCLC, GCLM, and HMOX1 in sperm. The mechanism by which PIC protects sperm during cryopreservation from oxidative stress damage was further verified. Treatment with PIC at a dose of 5.0 μmol/L significantly improved both sperm motility and viability while effectively reducing ROS levels in frozen sperm. Additionally, the integrity of the sperm plasma membrane was significantly enhanced. Furthermore, the expression level of Keap1 was significantly reduced, whereas the expression levels of GCLC, GCLM, HMOX1, and Nrf2 were significantly increased (p < 0.05) after the addition of PIC. Notably, a significant attenuation of sperm motility and viability was observed in this treatment group when PIC treatment was accompanied by the addition of an Nrf2 inhibitor, resulting in a significant elevation of ROS levels. The finding that PIC modulates ROS in frozen sperm via the Keap1-Nrf2/ARE pathway thereby enhancing sperm viability levels after freezing and thawing provides a novel approach to optimize semen cryopreservation.

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.

Impact of semen cryopreservation season on invitro embryo production of prepubertal goat oocytes.

Goat production is affected by reproductive seasonality. Invitro embryo production (IVEP) could overcome this effect. This study aimed to evaluate the impact of the season of semen collection/freezing on IVEP of prepubertal goat oocytes and on sperm quality and functionality concerning capacitation. Semen from six fertile bucks was collected, pooled and cryopreserved in spring and autumn and used for IVEP of oocytes recovered during the breeding season. Oocytes were IVM in TCM-199 with hormones, EGF and cysteamine; fertilized and cultured in BO-IVF and BO-IVC media (IVF Bioscience, UK). Semen samples were assessed at 0 and 3 h after culture in capacitating (BO-IVF, CAP) and non-capacitating conditions for sperm plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), intracellular calcium and plasma membrane lipid disorder. Blastocyst production was higher with spring sperm compared to autumn (12.0% vs. 2.1%, respectively; p < .05). After CAP, acrosome reaction and intracellular calcium were higher (p < .05) in spring than autumn sperm. No differences were found in other sperm parameters. In conclusion, seasonal variations in the IVEP of prepubertal goats could be linked to differences in sperm ability to undergo invitro capacitation.

A review of antioxidant strategies to improve reproduction in aging male broiler breeders.

As only 10% of the broiler breeder flock is roosters, their fertility is very important. The rooster sperm plasma membrane has high concentrations of polyunsaturated fatty acids that are sensitive to oxidative stress. Lipid peroxidation can change membrane structure, permeability, and fluidity, adversely affecting the acrosome reaction and fertility. Aging roosters have decreases in sexual behavior, serum androgen concentrations, sperm quantity and quality, and fertility. Low fertility in aging roosters is attributed to an imbalanced testicular oxidant-antioxidant system, with increased reactive oxygen species (ROS) damaging spermatogenic epithelium. However, antioxidant components can enhance antioxidant defenses in aging broiler breeder roosters. Protection against oxidative damage, particularly in the testes, improves reproductive hormone concentrations, testicular histology, sperm membrane function, and mitochondrial activity and thereby improves semen volume, sperm concentration, viability, motility, and sperm polyunsaturated fatty acid content, sperm-egg penetration, fertility, and reproductive performance. This review summarizes antioxidants that could improve fertility and reproductive performance and delay or prevent age-related declines in broiler breeder roosters, with benefits for poultry production.

Effects of Butylated Hydroxytoluene and Sorbitol as Diluent Components on Structural and Surface Ultrastructural Changes of Gaga Chicken Sperm During Cryopreservation

The Gaga chicken is an indigenous Indonesian breed that is important to preserve using semen cryopreservation technology. The study was conducted to determine the effect of adding sorbitol and butylated hydroxytoluene (BHT) in the diluent on the structural and surface ultrastructure of cryopreserved Gaga chicken sperm during cryopreservation /frozen storage. The study aimed to assess how adding sorbitol and butylated hydroxytoluene (BHT) to the diluent affects the structure and surface ultrastructure of cryopreserved Gaga chicken sperm. A completely randomized design was employed with four treatments and 10 replications including egg yolk-lactate ringer diluent (EYLR) as the control group, EYLR diluent with 3 mM BHT, EYLR diluent with 2% sorbitol, and EYLR diluent with both 3 mM BHT and 2% sorbitol. Semen was collected using a massage technique from 4 male chickens aged approximately 10 months, pooled semen was diluted, packaged in 0.25 mL straws, equilibrated for 2 hours at 5 °C, pre-freeze for 10 minutes, frozen for 24 hours, and thawed for 30 seconds at 37 °C. The parameters evaluated were sperm plasma membrane integrity, acrosome integrity, DNA damage, mitochondrial functionality, and surface ultrastructure. The results showed that the treatment had a significant effect on plasma membrane integrity and post-thawing mitochondrial functionality compared to the control, but no effect was observed on acrosome integrity or DNA damage. The results showed that the combination treatment of BHT with sorbitol had a significant effect on plasma membrane integrity and post-thawing mitochondrial function, but did not affect acrosome integrity or DNA damage when compared to the control group. Ultrastructural observations indicated that cryopreservation caused damage to the head, middle, and tail of the sperm in the control groups. However, these changes were prevented by the diluent containing a combination of BHT and sorbitol. The addition of both components (BHT 3 mM + sorbitol 2%) effectively maintained plasma membrane integrity, mitochondrial functionality, and surface ultrastructure of Gaga chicken sperm during cryopreservation. Keywords: Butylated hydroxytoluene, Chicken sperm, Cryopreservation, Sorbito, Structure, Sperm ultrastructure

Sub-acute bisphenol A exposure induces proteomic alterations and impairs male reproductive health in mice.

Bisphenol A (BPA) is one of the most prevalent endocrine disrupting chemicals (EDCs) and there is widespread concern about the adverse effects of EDCs on human health. However, the exact mechanism of these toxicities has still not been fully deciphered. Additionally, studies have reported the toxicological effects at far low doses to the generally considered no-observed-adverse-effect level (NOAEL) dose. The present study investigates the effects of a sub-acute (28 days) exposure to BPA (10, 50 and 100 mg/kg/day) in adult male mice on various hormones levels, sperm motility, sperm count, functional integrity of sperm plasma membrane, testicular histological changes, oxidative stress markers and DNA damage. The key proteome signatures were quantified by LC-MS/MS analysis using Orbitrap Fusion Lumos Tribrid Mass Spectrometer equipped with nano-LC Easy-nLC 1200. Data suggest that the BPA exposure in all doses (below/above NOAEL dose) have greatly impacted the hormone levels, sperm parameters (sperm count, motility and membrane integrity) and testicular histology. Mass spectrometry-based proteomics data suggested for 1352 differentially expressed proteins (DEPs; 368 upregulated, 984 downregulated) affecting biological process, cellular component, and molecular functions. Specifically searched male reproductive function related proteins suggested a complex network where 46 potential proteins regulating spermatogenesis, sperm structure, activity and membrane integrity while tackling oxidative stress responses were downregulated. These potential biomarkers could shed some more light on our current understanding of the reproductive toxicological effects of BPA and may lead to exploration of novel interventions strategies against these targets for male infertility.

Exploratory Metabolomics and Lipidomics Profiling Contributes to Understanding How Curcumin Improves Quality of Goat Semen Stored at 16 °C in Tropical Areas.

Reactive oxygen species (ROS) exert a vital role in sperm quality during semen preservation, where excessive ROS leads to oxidative damage and undermines sperm integrity. Curcumin, a botanical extract, is capable of neutralizing ROS and enhancing the activity of antioxidant enzymes. This study was aimed at evaluating the effects of curcumin on sperm viability, acrosome integrity, and antioxidant levels, as well as metabolomic and lipidomic profiles. The results demonstrated that curcumin at 25 µmol/L significantly enhanced sperm motility, plasma membrane, and acrosome integrity, elevated the levels of antioxidant enzymes (T-AOC, CAT, SOD), and decreased ROS production (p < 0.05). Metabolomic analysis identified 93 distinct metabolites that showed significant differences between the control and curcumin-treated groups. KEGG pathways emphasized the participation of these metabolites in key metabolic processes such as the citric acid cycle, cholesterol metabolism, and fatty acid metabolism. Curcumin treatment brought about notable variations in lipid profiles, including increased levels of phosphatidylcholine, acylcarnitine, and triglyceride over the storage time, suggesting enhanced lipid anabolic activity. Overall, the supplementation of curcumin at 25 µmol/L effectively mitigates oxidative stress and prolongs the viability of semen storage at 16 °C by modulating specific metabolic and lipid profiles.

Potential Protective Effect of Hesperidin (Vitamin P) against Glyphosate-Induced Spermatogenesis Damage in Male Rats: Biochemical and Histopathological Findings on Reproductive Parameters.

The aim of this study was to investigate the effectiveness of hesperidin (HES) on testicular histopathological changes, biochemical changes, and semen characteristics in rats exposed to glyphosate (GLP). The control group was given a normal diet devoid of GLP and HES, the HES group was given 100 mg/kg/day HES with the normal diet, the GLP group was given GLP at the LD50/10 dose of normal feed, which was 787.85 mg/kg/day, and the GLP + HES group was given normal feed containing 787.85 mg/kg/day LD50/10 dose of GLP in addition to 100 mg/kg/day HES. GLP administration reduced sperm motility, sperm plasma membrane integrity, glutathione levels, and total antioxidant levels in the testicular tissues of rats. Moreover, it caused an increase in right testis and left epididymis weights, abnormal sperm counts, malondialdehyde levels, total oxidant status, and DNA damage. The HES treatment showed curative effects on these parameters. Furthermore, HES was effective in lessening the histopathological damage that was caused by GLP. The results showedthat HES protects spermatological parameters and DNA integrity, improves antioxidant defenses, and lowers the damage and lipid peroxidation caused by GLP in testicular tissue.

Effects of short-term oral letrozole on fresh semen parameters, endocrine balance, and prostate gland dimensions in domestic dogs

BackgroundAromatase inhibitors improve male fertility by modifying the hormonal control of spermatogenesis. The present study aimed to investigate the effects of oral administration of letrozole on testosterone and estradiol concentrations and their ratios in blood serum, seminal plasma, prostatic fluid, sperm quality in fresh semen, and prostate gland dimensions. Seven adult male intact mixed-breed dogs were selected. The animals received letrozole (72 µg/kg, PO) daily for four weeks. Blood samplings and semen collections were carried out on days 0 (control), 14 (treatment), 28 (treatment), and 42 (post-treatment).ResultsOur results showed that letrozole administration resulted in a 4.3 fold significant increase in serum, seminal plasma, and prostatic fluid testosterone levels after 14 days. This remained high until the end of the study. Serum and prostatic fluid estradiol levels did not change significantly over the study period. However, the seminal plasma estradiol level showed a significant increase on day 14. The estradiol: testosterone ratio was significantly reduced on day 14 in serum, seminal plasma, and prostatic fluid samples. Letrozole significantly improved the ejaculated spermatozoa viability and concentration after 28 days of oral administration. However, the sperm plasma membrane functional integrity and kinematic parameters were not significantly affected by the treatment. Transabdominal ultrasound examination revealed a significant increase in the height, width, and volume of the prostate gland after 28 days of treatment.ConclusionsAccording to the present research, oral administration of letrozole for 28 days affects local and systemic sex hormone balance leading to an improvement of the ejaculated canine spermatozoa viability and concentration concurrent with an increase in the prostate gland dimensions.

Addition of Cryoprotectant DMSO Reduces Damage to Spermatozoa of Yellow Catfish (Pelteobagrus fulvidraco) during Cryopreservation: Ultrastructural Damage, Oxidative Damage and DNA Damage.

Spermatozoa cryopreservation protocols have been established for yellow catfish (Pelteobagrus fulvidraco), but cryopreservation can still cause cellular damage and affect spermatozoa viability and fertility. Therefore, the aim of this paper was to evaluate the effects of adding or not adding cryoprotectants during low-temperature storage on the ultrastructural damage, oxidative damage, and DNA damage of thawed yellow catfish spermatozoa. The mixed semen of three male yellow catfish was divided into a fresh spermatozoa group, a frozen spermatozoa group (DMSO+) with a cryoprotectant (10% DMSO), and a frozen spermatozoa group without a cryoprotectant (DMSO-). Ultrastructural of the spermatozoa after thawing were observed under an electron microscope and the spermatozoa were assayed for SOD, MDA, and T-AOC enzyme activities, as well as for DNA integrity. In terms of movement parameters, compared with DMSO-, the addition of DMSO has significantly improved sperm motility, curve line velocity (VCL), and straight line velocity (VSL). The ultrastructural results showed that although thawed spermatozoa exhibited increased damage than fresh spermatozoa, 10% DMSO effectively reduced the damage to the plasma membrane, mitochondria, and flagellum of spermatozoa by cryopreservation, and most of the spermatozoa were preserved with intact structure. The results of oxidative damage showed that compared with frozen spermatozoa, 10% DMSO significantly increased the activities of SOD and T-AOC enzymes and clearly reduced the activity of the MDA enzyme. The antioxidant capacity of spermatozoa was improved, lipid peroxidation was reduced, and the oxidative damage caused by cryopreservation was mitigated. The DNA integrity of spermatozoa showed that 10% DMSO clearly reduced the DNA fragmentation rate. In conclusion, 10% DMSO can effectively reduce the ultrastructural damage, oxidative damage, and DNA damage of yellow catfish spermatozoa during cryopreservation; it can also further optimize the cryopreservation protocol for yellow catfish spermatozoa. Meanwhile, it also provides a theoretical basis for the future optimization of the cryopreservation protocols.

Delayed Reproduction, Injury, and Regeneration of Testes in Out-of-Season Breeding of Largemouth Bass (Micropterus nigricans).

Out-of-season breeding is an effective method for addressing seasonal shortages of fry in aquaculture species such as largemouth bass (LMB) for year-round production. Off-season breeding of LMB can be achieved by subjecting breeding LMB to prolonged low-temperature conditions; however, this can alter reproductive rhythms, affecting the quality of their sperm and leading to a decrease in reproductive efficiency. Therefore, it is crucial to investigate issues such as the damage to the testes and the related mechanisms caused by low-temperature stress during out-of-season breeding. In this experiment, we assessed the changes in the testes during this time in LMB by comparing reproductive rhythms, testicular histomorphology, ultrastructure, antioxidant capacity and apoptosis. We synthesized measurements of LMB from three identically treated cement ponds and fish exposed to water temperatures of 13-16 °C to assess the changes in the testes. The results showed that (1) out-of-season reproduction delayed sperm production and promoted sperm redevelopment in LMB, various hormone levels have changed over time (e.g., LH, FSH, and T). (2) The head plasma membrane of LMB spermatozoa was separated, and the middle mitochondria were swollen. (3) The expression levels of antioxidant enzymes (cat, sod, and gpx) were upregulated, and oxidative stress occurred in LMB. (4) The expression levels of apoptosis genes (e.g., bax, bcl2, and caspase3) were upregulated, and apoptosis occurred in LMB due to off-season breeding. Moreover, important genes of the mitochondrial apoptosis pathway (bid, CYT-C) were upregulated, indicating that spermatozoan apoptosis in LMB was probably achieved through the mitochondrial apoptosis pathway. These results suggest the delays, damage, and regeneration of LMB testes. Our findings provide new insights into the molecular mechanisms that trigger changes in sperm quality during out-of-season breeding in fish.

Long-term cryopreservation of bull semen as a main strategy for the ex situ conservation of endangered Polish Red cattle

Abstract Regular verification of the quality of cryopreserved semen derived from native cattle is one of the tasks performed at the bank as recommended by the Food and Agriculture Organization. The purpose of the present study was to evaluate the quality of semen from PR bulls stored for 40, 50 and 60 years in the BMB using standard evaluation parameters such as sperm motility as well as structural-functional parameters such as plasma membrane integrity, transmembrane mitochondrial potential and sperm chromatin damage. Semen pellets from 27 PR bulls (3 ejaculates/bull) were tested. The data were analysed by one-way and two-way ANOVA, and the significance of the difference (P≤ 0.01) between the means was determined using Duncan’s test. Our study results revealed that the long-term storage of semen had no effect on sperm characteristics after thawing. However, statistically significant differences (P≤0.01) in sperm plasma membrane integrity and transmembrane mitochondrial potential, following storage in liquid nitrogen were noted between bulls at all time points. However, there were no significant differences (P&gt;0.01) in sperm chromatin damage between breeds or between different storage times, and the degree of DNA fragmentation ranged from 0.4 to 0.8%.

L-carnitine supplementation in conventional slow and ultra-rapid freezing media improves motility, membrane integrity, and fertilizing ability of dog epididymal sperm

This study aimed to assess the impact of L-carnitine (LC) supplementation in conventional-slow (CS) and ultra-rapid (UR) freezing media on post-thaw quality and fertilizing ability of dog epididymal spermatozoa. Sperm samples were collected from 60 epididymides obtained from 30 adult orchiectomized dogs via retrograde flushing. Twenty pooled sperm samples were then created (3 epididymal samples/pool). Four treatments were established according to the freezing method (CS and UR) and LC supplementation (5 and 0 mM [control, Co]): CS-LC5, CS-Co, UR-LC5, and UR-Co. The CS freezing involved exposing 0.25 mL straw to liquid nitrogen vapors (LN2), while UR freezing submerged 30-µL drops of sperm samples directly into LN2. Sperm kinematics, membrane integrity, and fertilizing ability (by heterologous in vitro fertilization using bovine oocytes) were evaluated for all treatments. Post-thaw results revealed that the CS freezing treatments resulted in significantly higher values (P < 0.05) of curvilinear and average-path velocities, and beat-cross frequency compared to the UR freezing treatments, regardless of LC supplementation. The CS-LC5 and UR-LC5 treatments cryoprotected the sperm by increasing (P < 0.05) the percentage of ‘live-sperm/intact-acrosome’ compared to their controls treatments CS-Co and UR-Co. Regarding fertilizing ability, the CS-LC5 treatment yielded a higher percentage (P < 0.05) of pronuclei formation compared to both UR treatments. The UR-LC5 treatment, however, obtained greater percentage (P < 0.05) than their control UR-Co. In conclusion, supplementation with L-carnitine in conventional-slow and ultra-rapid freezing improved sperm motility, plasma, and acrosome membranes integrity and fertilizing ability of dog epididymal spermatozoa.

Effect of seminal plasma cholesterol and triacylglycerides concentrations and sperm morphology on semen freezability in domestic cats (Felis silvestris catus)

There is scarce information about the effect of sperm morphology and seminal plasma composition on cat semen freezability. Thus, this study aims to assess the effect of cat sperm morphology and seminal plasma cholesterol (CHOL) and triacylglyceride (TAG) concentrations on sperm post-thaw survival. Ejaculates (n = 49) were evaluated, and seminal plasma was separated and frozen until CHOL and TAG concentrations were measured. The sperm pellet was diluted in a tris-based egg yolk extender, frozen (n = 38), or processed for sperm ultrastructure study (n = 11). Abnormalities recorded were abnormal head shape and size, detached heads, knobbed or ruffled acrosomes, eccentric mid-piece insertion, proximal and distal cytoplasmic droplets, folded and coiled tails, and Dag defect. Ultramicroscopic evaluation detected several sperm abnormalities in fresh semen and some sperm damage in frozen semen. Seminal plasma lipids components were positively correlated with post-thaw motility and acrosome integrity. Higher freezability indices for motility and acrosome integrity were observed in frozen-thawed semen with high seminal plasma CHOL and TAG concentrations. No freezability differences were observed between teratozoospermic and normozoospermic ejaculates. Our results showed that even when seminal plasma was removed before cryopreservation, sperm survival after thawing was significantly higher in samples with high seminal plasma CHOL and TAG concentrations, indicating a rapid adherence to these compounds to the sperm plasma membrane, protecting sperm cells from temperature changes. Nevertheless, there were no differences in sperm freezability by sperm morphology.

Effect of tyrode albumin lactate pyruvate and modified Krebs Ringers broth media on in vitro capacitation of HD-K75 boar spermatozoa at different period of incubation.

In vitro capacitation allows for a greater understanding of the mechanisms underlying fertilization and the development of improved reproductive techniques for improving fertility rates in porcine. Tyrodes albumin lactate pyruvate (TALP) and modified Krebs Ringers Broth (m-KRB) are two medias that are commonly used in research experiments to induce capacitation in boar spermatozoa (Cañón-Beltrán etal., Theriogenology, 198, 2023 and 231; Oberlender etal., Archivos de Medicina Veterinaria, 44, 2012 and 201; Sahoo etal., International Journal of Biological Macromolecules, 241, 2023 and 124502). Moreover, understanding the morphological and functional changes in boar spermatozoa at different hours of capacitation periods might aid in the development of novel techniques for improving sperm quality and increasing the litter size. This study was carried out to investigate the effect of Tyrode albumin lactate pyruvate and modified Krebs Ringers Broth media on invitro capacitation of HD-K75 boar spermatozoa at three different periods of incubation. A total of 24 ejaculate from four clinically healthy, 10-12 months aged HD-K75 boars, maintained at ICAR-All India Coordinated Research Project (AICRP) on pig were selected. Semen was collected by 'Simple fist' method using a portable dummy. The semen samples having 200 mL volume, 103 × 106 spermatozoa/ml concentration and 70% initial motility were selected and split into two parts and suspended in TALP and m-KRB media, respectively, and incubated for 5 h at 37°C. Seminal parameters viz. sperm viability, plasma membrane integrity and acrosomal integrity were estimated in the samples at 0, 3 and 5 h of incubation. This study revealed that there was significant variation between media in live acrosome-reacted (p < .05) and HOST-reacted (p < .01) spermatozoa, while between capacitation periods significant (p < .01) variation was observed in hyperactivated spermatozoa, live acrosome-reacted spermatozoa, HOST-reacted spermatozoa, FITC-labelled PSA, extracellular protein and sperm cholesterol. Non-significant variation was observed in total phospholipid. TALP showed overall better consequence on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. From this study, it could be concluded that both TALP and m-KRB media were virtuous to induce capacitation in HD-K75 boar spermatozoa. TALP media, however, had a better effect on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. Out of the three different periods, 3 h capacitation period resulted in significantly (p < .01) higher incidence of sperm viability, plasma membrane and acrosomal integrity in HD-K75 boar spermatozoa.

Egg multivesicular bodies elicit an LC3-associated phagocytosis-like pathway to degrade paternal mitochondria after fertilization

Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila, we show that special egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.

The contribution of different sperm parameters to better explain ram semen cryoresistance

In order to determine an effective procedure for explaining ram sperm cryoresistance and develop a new model for breeders classification, a retrospective study was conducted using sperm analysis data obtained over two consecutive years from a total of 82 sessions of ram semen cryopreservation. In each session, fresh ejaculates from eight males were collected via artificial vagina, pooled and frozen in liquid nitrogen vapors. After thawing, a total of 19,084 sperm tracks and 11,319 morphometric measurements were analysed. Clustering analyses were applied to establish motile and morphometric sperm subpopulations. Additionally, plasma and acrosome membrane integrity, as well mitochondrial activity using flow cytometry immediately after sperm thawing and following hypoosmotic shock test (HOST) was assessed. To develop a Ram Sperm Cryoresistance Index, Principal Component Analyses (PCA) using 22 variables were conducted. In the first PCA, the parameters that best explain cryoresistance include total motility (TM), motile subpopulation 2 (motSP2, which groups slow, very linear spermatozoa with low lateral head displacement), morphometric subpopulation 1 (morphSP1, grouping spermatozoa with the smallest head size and lowest shape values), sperm plasma membrane integrity immediately after thawing and following hypoosmotic shock test. These parameters collectively account for 77.34 % of the accumulated variance. To emphasize their importance, a second PCA was performed, revealing significant higher weighting coefficients for the quantity (TM) and quality (motSP2) of sperm movement after thawing, compared to the head size and shape of the thawed sperm (morphSP1). Furthermore, HOST Viability played a more decisive role than what was observed under isotonic conditions.

P-035 Catecholamine neuroreceptors are differentially located in functional regions of the human sperm

Abstract Study question How are the expression and distribution of Adrenergic and Dopamine receptors in human sperm cells? Summary answer Using ultra-high-resolution microscopy techniques, we identified that each type of catecholamine receptor is expressed in a different functional region of the human sperm. What is known already Human sperm present complex cell signalling complexes with several neuroreceptors in their membrane. The spermatozoa is a terminal differentiated cell showing specialized regions optimized for physiological processes like motility, acrosome reaction and membrane fusion during fertilization. Although indirect methods have determined the presence of catecholamine receptors in human sperm, there is no direct evidence nor description of the exact localisation of these receptors. Catecholamine receptors are the direct target of medications used to treat very frequent health problems like hypertension (beta blockers), asthma (bronchodilators) and mental health disorders (antipsychotics and antidepressants). Study design, size, duration We design the study by developing new techniques that allow us to use high-resolution microscopes (i.e. Laser Scanning Confocal Microscopy LSCM and Field Emission Scanning Electron Microscopy or FE-SEM) to identify the localisation and distribution of catecholamine receptors. We determined sperm functional regions and quantified the relative amount of the different receptors on each one of those regions to perform a statistical analysis. Participants/materials, setting, methods After informed consent and ethical approval, sperm from young, healthy donors (n = 20) were included in the study. We used the neuroblastoma cell line SH-SY5Y as a positive control. Immunolabelling techniques used specific primary antibodies for each receptor, followed by the gold-conjugated secondary antibodies for FE-SEM. Negative controls were performed by omitting the primary antibody. In FE-SEM images, we quantify the number of gold nanoparticles in the sperm cells and the background. Main results and the role of chance We confirm the differential localisation of the different receptors studied. Dopamine D2 receptors are mainly expressed on the equatorial band of the acrosome, while beta-adrenergic receptors are restricted to the terminal piece of the flagellum. In both cases, the mean number of gold particles showed statistical differences (p &amp;lt; 0,001) between the region of expression and the rest of the regions and the background. Limitations, reasons for caution The present study only shows the presence of the receptors on the sperm plasma membrane. The activity of those receptors should be investigated by developing bioassays based on the use of agonists and/or antagonists. A more reliable genetic overexpression or knockout approach was impossible in human sperm. Wider implications of the findings The presence of receptors on restricted regions of the sperm suggests their participation in critical physiological events, like regulation of sperm motility hyperactivation or acrosome reaction. It may also suggest a possible side effect of beta-blockers or bronchodilators with the male fertility potential that needs to be investigated. Trial registration number not applicable

Improving semen quality of rams fed with ration containing protected maggot oil.

The study aimed to to evaluate the effect of feeding protected maggot oil at different levels on the ram sperm quality. The study used 15 local rams with an age of approximately 10-12 months and an initial weight of 19.99 ± 3.97kg. The feeding rate was 4% of body weight per day. Feed was given 3 times a day, specifically in the morning (08.00 WIB), afternoon (12.00 WIB) and evening (16.00 WIB). Water was provided ad libitum. This study used 3 treatments and 5 groups as replicates. The treatments used concentrates with different levels of protected maggot oil: P0(0% protected maggot oil (control)), P1(4% protected maggot oil), and P2(8% protected maggot oil). The variables measured were nutrient consumption, blood cholesterol levels, scrotal circumference, and sperm quality. Blood cholesterol and scrotal circumference measured at the end of the experimental diet. Semen samples were collected and analysed before and at the end of the experimental diet. The data obtained were analysed using ANOVA, with further testing using Duncan's test for significant differences. The results showed that there were no significant differences in the consumption of dry matter, crude protein, crude fiber, scrotal circumference, volume, colour, pH of semen, sperm concentration, live percentage, abnormal percentage, plasma membrane, and acrosome integrity of spermatozoa. There were significantly (p < 0.05) produced higher consumption of oleic and palmitic acids in 8% protected maggot oil compared to 4% treatments, the treatments containing 4% and 8% protected maggot oil produced significantly (p < 0.05) higher consumption of lauric and myristic acids, blood cholesterol levels, and sperm motility than the control. The result indicates that protected maggot oil up to 8% in the ram diet have positive effect on improving the microscopic quality of ram sperm, i.e. increased sperm motility.

Potential use of soy lecithin or butylated hydroxytoluene as an alternative to powdered egg yolk for ram semen cryopreservation

The aim of this study was to assess the effect of replacing powdered egg yolk (PEY) with soybean lecithin (SL) or butylated hydroxytoluene (BHT) on ram sperm cryopreservation. Two ejaculates/male were collected via artificial vagina from 8 rams during breeding season. Ejaculates were pooled and washed twice by centrifugation. The pellet was divided into three aliquots, diluted in a Tris-based media with 5% glycerol containing PEY (15%), SL (1%) or BHT (0.6 mM) and cooled for 4 h at 5 °C before freezing. Sperm motility, plasma and acrosome membrane integrity and mitochondria activity as lipid peroxidation were assessed immediately after thawing and after 4 h of resilience incubation in a modified PBS at 38 ºC. After thawing, sperm extended in BHT showed the poorest quality compared to sperm extended in PEY and SL. Similar total and progressive motility were observed in sperm preserved in PEY and SL media. Plasma membrane integrity, however, was significantly higher in sperm extended in SL, although most of them with non-functional mitochondria. Acrosome damage was significant lower in SL sperm samples compared to PEY samples. Highest level of lipid peroxidation was found in sperm preserved in PEY. Resilience test had a negative effect (P &lt; 0.05) on plasma and acrosome membrane integrity in all samples, and on progressive motility only in sperm preserved in PEY. In conclusion, soy lecithin could be a potential alternative to PEY for ram cryopreservation, although its adverse effect on sperm mitochondria function has to be strongly considered.