Boar spermadhesin AWN: Novel insights in its binding behavior and localization on sperm

As a major spermadhesin first found in the seminal plasma of boars, AWN is described to fulfil a variety of reproduction related tasks. Although being the best investigated boar spermadhesin, information about its interaction with membranes is inconsistent. In this regard, previous reports locate AWN either inside or on the surface of sperm cells and at different regions, depending on the method and antibody used. Here, we localize native AWN in/on epididymal, ejaculated, capacitated and acrosome-reacted boar sperm using epifluorescence and electron microscopy, as well as an analysis of potential lipid binding partners of native and recombinant AWN. By applying a custom-made anti-AWN antibody, localization of AWN in the equatorial segment of ejaculated, capacitated and acrosome-reacted boar sperm was discovered. Electron microscopy showed that AWN is localized both on the sperm surface and on the cytoplasmic side of the plasma membrane, and in close vicinity to the nuclear and both acrosomal membranes of sperm. Analysis of epididymal sperm indicated migration of AWN from the retral postacrosomal part to the equatorial segment during the epididymal passage. In contrast to hypotheses claiming a specific association of AWN to phosphatidylethanolamine and in line with our previous study describing an interaction with phosphatidic acid, the current results show a rather electrostatically-driven binding mechanism of AWN to negative lipids. In conclusion, this work provides new insights into the arrangement of AWN in the equatorial segment that suggest a possible role in sperm-oocyte fusion.

Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm

Sperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a successful strategy to reduce cryodamage in sperm of several species, once allowing to stabilize the plasma membrane constituents. Therefore, this study intends to understand the effects of sugars in the plasma membrane, DNA integrity, and oxidative response during Portuguese oyster sperm cryopreservation. Three cryoprotectants solutions with an initial concentration of 20% dimethyl sulfoxide (DMSO) and 20% DMSO complemented with 0.9 M trehalose or sucrose in artificial seawater were employed. Sperm samples of mature males were individually collected and diluted 1:10 (v/v) in artificial seawater followed by addition of cryoprotectants [1:1 (v/v)]. Thereafter, sperm was loaded into 0.5 ml straws, maintained at 4°C for 10 min, frozen in a programmable biofreezer at −6°C/min from 0 to −70°C, and stored in liquid nitrogen. Samples were thawed in a 37°C bath for 10 s. Several techniques were performed to evaluate post-thaw quality. Sperm motility and DNA integrity were analyzed by using computer-assisted sperm analysis (CASA) software and comet assay. Flow cytometry was employed to determine membrane and acrosome integrity and to detect intracellular reactive oxygen species (ROS) and apoptosis activity. Lipid peroxidation was determined by malondialdehyde (MDA) detection by using spectrophotometry. Sperm antioxidant capacity was evaluated through glutathione peroxidase, glutathione reductase, and superoxide dismutase. Motility was not affected by the extenders containing sugars; these compounds did not reduce the DNA damage. However, both the trehalose and sucrose protected plasma membrane of cells by increasing cell viability and significantly reducing MDA content. The same finding was observed for the ROS, where live cells registered significantly lower levels of ROS in samples cryopreserved with sugars. The activity of antioxidant enzymes was higher in treatments supplemented with sugars, although not significant. In conclusion, the addition of sugars seems to play an important role in protecting the Crassostrea angulata sperm membrane during cryopreservation, showing potential to improve the post-thaw sperm quality and protect the cells from cryoinjuries.

Technology Updates in Male Infertility Management

Background: Technologies are replacing manpower many fields including medical field. Several devices have been marketed for replacing/reducing manpower in the medical field including male infertility. Here we reviewed several technologies that developed in male infertility. Review: Computer assisted sperm analysis (CASA), Automatic assessment of biochemical marker of seminal plasma, B-mode ultrasound, and automatic sperm cryopreservation can be applied routinely. Several updates i.e. automatic histopathology assessment, ultrasound strain elastography, magnetic resonance imaging (MRI) stronger than 3.0 T, Artificial intelligence for predicting the presence of sperm in azoospermia cases, automatic sperm selection, and automatic intracytoplasmic sperm injection (ICSI) need more studies before their application. Summary: Prudent choice based on valid studies is needed in order to give a comprehensive management to patient with male infertility without using useless technology.

Methanol Extract of Azanza garckeana Fruit Pulps Protects against Formalin-Induced Reproductive Toxicity in Adult Albino Male Mice

Introduction: The aqueous extract of Azanza garckeana was recently reported of exhibiting ameliorative and pro-fertility properties however the protective effects on formalin testicular toxicity have not been studied. Objective: This study investigated the protective effect of methanol extract of Azanza garckeana on formalin-induced testicular toxicity. Methods: Forty male albino mice were randomly divided into 8 groups of 5. Animals in the first group (1) served as control and administered normal saline (1 ml/kg) by the oral route daily for 40 days. In similar manner, animal in groups 2 received formalin (10 mg/kg) by the IP route, while animals in groups 3; 4 and 5 concurrently received formalin (10 mg/kg IP) and extract at doses of 125; 250 and 500 mg/kg respectively by the oral route. Mice in groups 6; 7 and 8 received the extract at doses of 125; 250 and 500 mg/kg respectively. Phytochemical analysis was conducted for each constituent using specific methods. Gonadotropin and sperm analysis were carried out using standard methods. Result: Phytochemical screening revealed the presence of various constituents, but notably flavonoids. Induced-toxicity with formalin and concurrent treatment with extract at doses of 250 and 500 mg/kg from day 20 to 40 caused significant body weight increase compared to baseline (p < 0.05). Similarly, treatment with the extract alone at all doses caused significant increase in body weight from day 20 to 40 (p < 0.05). Treatment with the extract at 250 and 500 mg/kg, caused a significant increase in weight of testes and epididymis compared to control and untreated group (p < 0.05).The extract induced significant increase in gonadotropin levels of animals compared to control and the untreated group (p < 0.05).The extract at 125 mg/kg demonstrated the highest fecundity potential, but there was no any consistent relationship between GSI and fecundity. Conclusion: This investigation was able to establish the protective and pro-fertility potentials of methanol extract of Azanza garckeana.

Meta data analysis of conception rate in relation to sperm motility in Madura superior bulls

Madura bulls are Indonesian germplasm with a very high capacity to adapt to dry environments. Madura bulls come from a crossbreed between Zebu (Bos indicus) and banteng (Bos javanicus). One of the breeding strategies of Madura cattle is the use of artificial insemination (AI) with frozen semen. Regarding sperm motility as one of the standard parameters of good semen quality, it is good to know the reliability of sperm motility with the bull fertility rate. This study aimed to determine the conception rate percentage (%CR) relation to sperm motility in Superior Madura bulls. The frozen semen from eight Madura bulls belonging to the National Singosari and Lembang AI centre were used. They were classified based on the selected field reproductive efficiency data from the year 2018 until 2020. Sperm motility was evaluated using Computer Assisted Sperm Analysis (CASA). The data were analyzed using oneway ANOVA and Pearson correlation. The data showed that %CR was significantly higher (P<0.05) and positively correlated with sperm motility. It is proved that sperm motility represents good quality sperm as one of the fertility parameters in Madura bulls.

COVID-19 disease in clinical setting: impact on gonadal function, transmission risk, and sperm quality in young males

Objectives We want to evaluate the possible presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in semen samples and semen quality, looking for a possible relationship between the infectious disease and fertility. Methods In this prospective study, we enrolled 15 consecutive men (age 18–50 years) with positive oropharyngeal swab to SARS-CoV-2 and classified, according to WHO criteria, in mild to moderate disease. A semen sample was collected to detect SARS-CoV viral RNA by the automated Real-Time PCR ELITe InGenius® system and the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup, France). Analysis of semen characteristics was performed according to WHO laboratory manual 5th ed. for the examination and processing of human semen. Blood samples for the dosage of hormonal assay, procalcitonin, interleukin 6, C-reactive protein were obtained. Results SARS-CoV-2 RNA has not been detected in semen samples from any of the subjects analysed. Sperm analysis exhibited abnormal seminal values in 14 out of 15 patients (93.3%). Furthermore, no difference was detected regarding sperm quality between mild and moderate SARS-CoV-2 patients. No alteration in the inflammatory indices was observed in the studied population, as well gonadotropins and testosterone levels. Conclusions COVID patients studied exhibits alteration of the seminal fluid both in microscopic and macroscopic characteristics such as hypoposia and increased viscosity, which have not been detected in previous studies. The presence of viral RNA within the seminal fluid was excluded.

Capacitation-Induced Mitochondrial Activity Is Required for Sperm Fertilizing Ability in Mice by Modulating Hyperactivation

To become fully competent to fertilize an egg, mammalian sperm undergo a series of functional changes within the female tract, known as capacitation, that require an adequate supply and management of energy. However, the contribution of each ATP generating pathway to sustain the capacitation-associated changes remains unclear. Based on this, we investigated the role of mitochondrial activity in the acquisition of sperm fertilizing ability during capacitation in mice. For this purpose, the dynamics of the mitochondrial membrane potential (MMP) was studied by flow cytometry with the probe tetramethylrhodamine ethyl ester (TMRE). We observed a time-dependent increase in MMP only in capacitated sperm as well as a specific staining with the probe in the flagellar region where mitochondria are confined. The MMP rise was prevented when sperm were exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the protein kinase A (PKA) inhibitor H89 during capacitation, indicating that MMP increase is dependent on capacitation and H89-sensitive events. Results showed that whereas nearly all motile sperm were TMRE positive, immotile cells were mostly TMRE negative, supporting an association between high MMP and sperm motility. Furthermore, CCCP treatment during capacitation did not affect PKA substrate and tyrosine phosphorylations but produced a decrease in hyperactivation measured by computer assisted sperm analysis (CASA), similar to that observed after H89 exposure. In addition, CCCP inhibited the in vitro sperm fertilizing ability without affecting cumulus penetration and gamete fusion, indicating that the hyperactivation supported by mitochondrial function is needed mainly for zona pellucida penetration. Finally, complementary in vivo fertilization experiments further demonstrated the fundamental role of mitochondrial activity for sperm function. Altogether, our results show the physiological relevance of mitochondrial functionality for sperm fertilization competence.

Cyclic FEE Peptide Improves Human Sperm Movement Parameters without Modification of Their Energy Metabolism

Cyclic fertilin peptide (cFEE: phenylalanine, glutamic acid; glutamic acid) improves gamete interaction in humans. We investigate whether it could be via improvement of sperm movement parameters and their mitochondrial ATP production. Sperm movement parameters were studied using computer-assisted sperm analysis (CASA) in sperm samples from 38 patients with normal sperm in medium supplemented with cyclic fertilin against a control group. Sperm mitochondrial functions were studied using donor’s sperm, incubated or not with cFEE. It was evaluated by the measurement of their ATP production using bioluminescence, their respiration by high resolution oxygraphy, and of mitochondrial membrane potential (MMP) using potentiometric dyes and flow cytometry. cFEE significantly improved sperm movement parameters and percentage of hyperactivated sperm. Impact of inhibitors showed OXPHOS as the predominant energy source for sperm movement. However, cFEE had no significant impact on any of the analyzed mitochondrial bioenergetic parameters, suggesting that it could act via a more efficient use of its energy resources.

Cementing the relationship between conventional and advanced semen parameters

Background Affordable conventional semen analysis remains a fundamental procedure to be performed routinely during the diagnosis of male infertility. Advanced semen analyses provide valuable clinical insights in treatment-related decision-making, but these are highly expensive and lack universal standardization. This study aimed at determining the relationship between conventional semen parameters, measured with assistance of computer-aided sperm analysis (CASA), and a set of advanced semen tests. Basic semen analysis (n = 124) was performed according to the World Health Organization (WHO) guidelines. Sperm DNA fragmentation and intracellular superoxide (O2−•) levels were assessed by flow cytometry. Seminal plasma thiobarbituric acid reactive substances (TBARS) levels as well as superoxide dismutase (SOD) and catalase (CAT) activity were measured by spectrophotometry. Spearman’s rank correlation coefficient was used, with significance set at p < 0.05. Results Semen pH correlated negatively with TBARS (p < 0.01). The proportions of total and progressively motile as well as rapid spermatozoa correlated positively with CAT activity (p < 0.05). Sperm viability correlated negatively with both O2−• (p < 0.05) and DNA fragmentation (p = 0.01), while normal morphology correlated negatively with O2−• levels (p < 0.05) and positively with CAT activity (p < 0.05). Straight-line velocity (VCL) and average-path velocity (VAP) correlated negatively with both O2−• (p < 0.01) and TBARS (p < 0.01). Amplitude of lateral head displacement (ALH) correlated negatively with O2−• (p < 0.01) and DNA fragmentation (p < 0.01), while its correlation with SOD activity was positive (p < 0.05). Conclusion The results obtained from this study support the validity of some CASA parameters as sensitive indicators of changes in sperm oxidative status and DNA integrity. Predicting advanced from conventional parameters through the building of linear regression models should be considered for future studies.