The aim of this study was to investigate the relationship between apoptotic markers present in human spermatozoa, namely phosphatidylserine translocation (PST) from the inner to the outer layer of the cytomembrane and the active form of caspase-3 (c3) versus the fertilizing potential of male gametes in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) models. A total of 116 male patients treated with their partners for infertility underwent basic semen analysis and an assessment of the presence of PST and the active c3 in sperm using flow cytometry. Forty patients underwent IVF, group A, while 76 patients underwent ICSI, group B. The fertilizing potential of the gametes was measured as the percentage of oocytes with pronuclei present after either procedure. PST and active c3 were identified in vital gametes, mainly in the midpiece area. Concentration, motility, morphology, and viability of spermatozoa strongly negatively correlated with both markers. In group A, a negative correlation between both markers and the success rate of conventional IVF was observed (r = −0.4, p = 0.04 for PST; r = −0.4, p = 0.02 for active c3, respectively). In group B, the success rate of ICSI did not correlate with either marker (r = −0.2, p = 0.85 for PST and r = 0.1, p = 0.51 for active c3). The two apoptotic markers localized in the sperm midpiece area may affect their function not only by decreasing basic andrologic parameters but also by reducing the probability of conception. Therefore, analysis of PST and active c3 in the sperm of patients undergoing infertility treatment should be recommended.

BackgroundIt is assumed that spermatozoa are target cells for estrogens however, the mechanism of their action is not fully understood. The aim of this study was to investigate the influence of 17β-estradiol (E2) on the human spermatozoa mitochondrial function.MethodsThe effects on spermatozoa of E2 at final concentrations of 10−10, 10−8 and 10−6 M were studied regarding the following phenomena: (1) kinetics of intracellular free calcium ions changes (using Fluo-3), (2) mitochondrial membrane potential ΔΨm (using JC-1 fluorochrome), (3) production of superoxide anion in mitochondria (using MitoSOX RED dye), (4) spermatozoa vitality (propidium iodide staining) and (5) phosphatidylserine membrane translocation (staining with annexin V marked with fluorescein).ResultsE2 initiated rapid (within a few seconds) dose dependent increase of intracellular free calcium ions concentration. E2 was changing the mitochondrial membrane potential: 10−8 M initiated significant increase of percentage of high ΔΨm spermatozoa while the 10−6 M induced significant decrease of high ΔΨm cells. In spermatozoa stimulated with E2 10−6 M a significant increase of mitochondrial superoxide anion level was observed. 2 h incubation of spermatozoa with E2 did not alter cells vitality nor stimulated phosphatidylserine membrane translocation, for all three doses.Conclusions17β-estradiol affected the human spermatozoa mitochondrial function. E2 in low concentration improved while in high concentration might deteriorate mitochondrial function.

The complex structure of the human spermatozoa membrane comprises five topographic domains. Transmembrane asymmetry of the distribution of phospholipids including phosphatidylserine (PS) is considered a marker of cell activity. The objective of the study was to determine which cytomembrane domains of human spermatozoa are involved in PS membrane translocation and to identify the possible relationship of PS translocation with spermatozoa morphology and vitality. In normozoospermic semen of 35 donors, annexin-V labeling with fluorescein determined PS translocation. Propidium iodide staining distinguished between vital and dead spermatozoa. Three types of PS membrane translocation have been distinguished: (1) in the midpiece, (2) in the acrosomal part and (3) simultaneously in the midpiece and acrosomal part. In morphologically normal vital spermatozoa, PS translocation occurred in the midpiece but never in the equatorial region. In dead spermatozoa, simultaneous PS translocation in the midpiece and acrosomal part was most often observed. The difference between proportions of, respectively, vital and dead spermatozoa presenting PS translocation located in different domains was significant (P < 0.0001). In vital cells, there was no difference in PS translocation prevalence between morphologically normal and abnormal spermatozoa (P > 0.05). The strict relation of PS translocation to specific membrane domains indicates functional specificity. It seems doubtful to include this phenomenon in physiological mechanisms of elimination of abnormal spermatozoa.

Objectives: The aim of the study was to investigate the influence of 17β-estradiol (main endogenous estrogen) and selected xenoestrogens (genistein, bisphenol-A), individually and in combination, on the mitochondrial function of human spermatozoa. In natural environment, human beings are exposed to multiple xenoestrogens, so their impact is combined with endogenous steroids. Material and methods:The effects of ligands on human spermatozoa were assessed regarding the following phenomena: spermatozoa vitality (propidium iodide staining), phosphatidylserine membrane translocation (staining with annexin V marked with fluorescein), mitochondrial membrane potential (using JC-1 fluorochrome), and production of superoxide anion in mitochondria (using MitoSOX RED dye).Results: Two-hour incubation of spermatozoa with 17β-estradiol , genistein, and bisphenol-A neither altered cell vitality nor stimulated phosphatidylserine membrane translocation. Incubation of spermatozoa with 17β-estradiol or bisphenol-A separately, as well as incubation with the three ligands simultaneously, resulted in altered mitochondrial membrane potential. Spermatozoa incubation with the three ligands significantly increased the mitochondrial superoxide anion level. Conclusions:It seems safe to conclude that human spermatozoa mitochondria are target cell structures for both, 17β-estradiol and xenoestrogens. The reaction to the 17β-estradiol and xenoestrogens mixture suggests a synergistic mechanism of action. Xenoestrogens may increase the sensitivity of spermatozoa to 17β-estradiol.

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Phosphatidylserine membrane translocation (PST) is considered to be a marker of apoptosis; however, numerous studies have reported on its role in processes not related to cell death. The purpose of the study was to investigate: (1) what is the impact of PST on the motility of spermatozoa, and (2) does the swim-up isolation involve the percentage of cells presenting PST? Semen of 28 normozoospermic men (WHO criteria) was analyzed. High motility spermatozoa were isolated by the swim-up technique. The percentage of spermatozoa with PST in neat semen and after swim-up isolation was assessed with Annexin-V labeled with fluorescein, using flow cytometry technique. The spermatozoas’ motility was measured with a computer-assisted analysis system. The kinetic subpopulations of spermatozoa were identified with dedicated software and analyzed regarding PST. Vital spermatozoa with PST demonstrated progressive movement. The motion analysis system revealed a very strong positive correlation between the percentage of vital spermatozoa with PST and the percentage of spermatozoa belonging to the slow subpopulation (r = 0.83; p < 0.05), as well as a very strong negative correlation between the percentage of vital spermatozoa with PST and the percentage of spermatozoa belonging to the rapid subpopulation (r = −0.86; p < 0.05). After the swim-up isolation, the percentage of vital spermatozoa presenting PST significantly decreased (2.4 ± 2.1% vs. 5.2 ± 2.4%; p < 0.05). Spermatozoa with PST present progressive movement; however, their motility is decreased. Isolation of spermatozoa with the swim-up technique eliminates the cells with PST.

Górski R, Kotwicka M, Skibińska I, Jendraszak M, Wosiński S. Effect of low-frequency electric field screening on motility of human sperm. Ann Agric Environ Med. AbstractIntroduction. The human body is constantly exposed to an extremely low electromagnetic field (ELF-EMF), in particular at 50 Hz, emitted by power lines, domestic distribution lines, electrical appliances, etc. It is assumed that the increase in electromagnetic exposure may cause adverse effects upon human health, as well as raising concerns regarding the impact on human fertility. Objective. The aim of this in vitro study was to investigate the influence of ELF-EMF with a frequency of 50 Hz on the motility of human sperm. At the same time, the effectiveness of the dielectric screen constructed by ADR Technology ® in absorbing the emitted radiation was examined. Materials and method. Semen samples of 20 patients were exposed to the influence of an extremely low electromagnetic field. After 5, 15 and 30 min., spermatozoa motility was analysed using a computer-assisted spermatozoa motility analysis system. The following sperm motility parameters were examined: 1) velocity straight linear motility; 2) cross-beat frequency; 3) lateral head displacement; 4) homogeneity of progressive motility velocity. Results. It was found that the ELF-EMF presented a negative effect on the motility of human spermatozoa. A significant decrease in spermatozoa motility speed and a significant increase in lateral head deviation values were observed under the influence of the electromagnetic field. ELF-EMF did not show an effect on either lateral head displacement or homogeneity of progressive motility velocity. Conclusions.A positive effect of the dielectric screen ADR Technology® was found. This effect compensated spermatozoa motility changes induced with ELF-EMF

Sperm cells are target cells for both estrogens and xenoestrogens. Due to the specific structure of spermatozoa, these hormonal compounds may act on sperm in a non-genomic mechanism only. However, the ESR-mediated signaling pathways are still poorly understood. In this study, we obtained 119 samples from male participants of Caucasian descent who donated semen for standard analysis. We analyzed gene expression of estrogen receptors (ESR1 and ESR2) and their coregulators—proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), and cellular kinase c-Src (SRC). RNA level was established using reverse-transcribed RNA as a template, followed by a polymerase chain reaction. Proteins’ presence was confirmed by western blot and immunocytochemistry techniques. “Normal” values of semen parameters were defined as follows: > 32% sperm with progressive motility, > 4% sperm cells with normal morphology, > 15 × 106 sperm per mL, > 58% live spermatozoa and leukocyte amount < 106 cells per mL, according to WHO 2010 reference. Semen parameters that deviated from these “normal” values were labeled as “abnormal”. Gene expression ratios revealed significant, moderate, and negative correlations for ESR1/ESR2 and weak, negative ESR2/PELP1 correlations in the subgroup of patients with abnormal values of semen parameters. In addition, SRC/PELP1 was moderately and positively correlated in the subgroup with parameters within the reference values established by WHO 2010. Our study showed that both PELP1 scaffolding protein and SRC kinase might influence semen quality via ESRs. It seems that not the expression of a single gene may affect the sperm quality, but more gene-to-gene mutual ratio. Characterization of estrogen-signaling pathway-related genes’ modulated expression in sperm cells could aid in better understanding sperm biology and quality.