Changes In Human Spermatozoa Research Articles

Mechanisms underlying human sperm cryodamage: the role of reactive oxygen species (ROS) and antioxidants

Sperm cryopreservation is an efficient procedure for male fertility preservation, although the freeze-thaw procedure causes irreversible structural and functional changes in human spermatozoa. Indeed, the procedure is responsible for harmful changes that may affect sperm biology. In mammalian cells, cryopreservation induces a shift of redox homeostasis towards increasing generation of reactive oxygen species (ROS). The characteristics of ROS and the cellular outcomes depend on the cell type. Supra-physiological ROS levels during cryopreservation severely impact sperm survival, reproductive potential and DNA integrity, the latter a fundamental factor for fertilisation and transmission of paternal genetic information to offspring. The aim of this review is to summarise current knowledge of the main molecular mechanisms underlying ROS generation during sperm cryopreservation and its subsequent effects. In addition, we report current experimental approaches based on the supplementation of cryopreservation media with enzymatic and non-enzymatic antioxidants with the aim of minimising the harmful effects of ROS, and thus improving post-thaw sperm quality. Current data indicate that the potential use of antioxidants as constituents of the sperm freezing solution in clinical settings would require considerable attention. KEY WORDS: Spermatozoa, cryopreservation, ROS, oxidative stress, antioxidants.

Lipidomics profiles of human spermatozoa: insights into capacitation and acrosome reaction using UPLC-MS-based approach.

Lipidomics elucidates the roles of lipids in both physiological and pathological processes, intersecting with many diseases and cellular functions. The maintenance of lipid homeostasis, essential for cell health, significantly influences the survival, maturation, and functionality of sperm during fertilization. While capacitation and the acrosome reaction, key processes before fertilization, involve substantial lipidomic alterations, a comprehensive understanding of the changes in human spermatozoa's lipidomic profiles during these processes remains unknown. This study aims to explicate global lipidomic changes during capacitation and the acrosome reaction in human sperm, employing an untargeted lipidomic strategy using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Twelve semen specimens, exceeding the WHO reference values for semen parameters, were collected. After discontinuous density gradient separation, sperm concentration was adjusted to 2 x 106 cells/ml and divided into three groups: uncapacitated, capacitated, and acrosome-reacted. UPLC-MS analysis was performed after lipid extraction from these groups. Spectral peak alignment and statistical analysis, using unsupervised principal component analysis (PCA), bidirectional orthogonal partial least squares discriminant analysis (O2PLS-DA) analysis, and supervised partial least-squares-latent structure discriminate analysis (PLS-DA), were employed to identify the most discriminative lipids. The 1176 lipid peaks overlapped across the twelve individuals in the uncapacitated, capacitated, and acrosome-reacted groups: 1180 peaks between the uncapacitated and capacitated groups, 1184 peaks between the uncapacitated and acrosome-reacted groups, and 1178 peaks between the capacitated and acrosome-reacted groups. The count of overlapping peaks varied among individuals, ranging from 739 to 963 across sperm samples. Moreover, 137 lipids had VIP values > 1.0 and twenty-two lipids had VIP > 1.5, based on the O2PLS-DA model. Furthermore, the identified twelve lipids encompassed increases in PI 44:10, LPS 20:4, LPA 20:5, and LPE 20:4, and decreases in 16-phenyl-tetranor-PGE2, PC 40:6, PS 35:4, PA 29:1, 20-carboxy-LTB4, and 2-oxo-4-methylthio-butanoic acid. This study has been the first time to investigate the lipidomics profiles associated with acrosome reaction and capacitation in human sperm, utilizing UPLC-MS in conjunction with multivariate data analysis. These findings corroborate earlier discoveries on lipids during the acrosome reaction and unveil new metabolites. Furthermore, this research highlights the effective utility of UPLC-MS-based lipidomics for exploring diverse physiological states in sperm. This study offers novel insights into lipidomic changes associated with capacitation and the acrosome reaction in human sperm, which are closely related to male reproduction.

Proteomic changes in human spermatozoa during in vitro capacitation and acrosome reaction in normozoospermia and asthenozoospermia.

The cellular and molecular mechanisms of the events that help spermatozoa acquire their fertilizing capability during capacitation and acrosome reaction are not completely understood. This study was performed with a postulation that the identification of sperm proteins and their changes during in vitro capacitation and acrosome reaction will unravel unknown molecular aspects of fertilization that impact male fertility. Spermatozoa collected from sequential conditions, that is, separation of ejaculated spermatozoa by Percoll gradient centrifugation, in vitro capacitation, and acrosome reaction were processed for tandem mass spectrometric analysis, followed by protein identification, label-free quantitation, and statistical analysis. Collectively, a total of 1088 sperm proteins were identified. In comparison to ejaculated spermatozoa, 44 and 141 proteins were differentially expressed in capacitated and acrosome reacted spermatozoa, respectively. A large number of proteins were found downregulated, including clusterin, pyruvate dehydrogenase E1 component, semenogelin-1 and 2, heat shock protein 90, beta-microseminoprotein, and keratin. It was expected as sperm-membrane-associated proteins are removed during capacitation. There were significant proteomic alterations in asthenozoospermia compared to normozoospermia; however, variation was more noticeable among proteins of acrosome reacted spermatozoa and those released during the acrosome reaction. The processes enriched among downregulated proteins in asthenozoospermia included acrosome assembly, binding of spermatozoa to zona pellucida, nucleosome assembly, flagellated sperm motility, protein folding, oxidative phosphorylation, tricarboxylic acid cycle, chromatin silencing, gluconeogenesis, glycolytic process, and glycolysis. The dynamic information generated about proteomic alterations in spermatozoa during capacitation and acrosome reaction and their variability in asthenozoospermia will contribute not only to enhancing our understanding of processes that prepare spermatozoa to acquire fertilization capability but also help in deciphering novel factors of male infertility.

Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling

Introduction: Spermatozoa cryopreservation is an important technique to preserve fertility for males. This study aimed at exploring the stability of epigenetics information in human spermatozoa, manipulated by two different technologies, freezing and vitrification. Methods: Spermatozoa samples were distributed into three groups: 1. Fresh spermatozoa (control group), 2. Frozen spermatozoa, 3. Vitrified spermatozoa. Epigenetic differences of fresh and cryopreserved spermatozoa were evaluated using high-throughput RNA sequencing. Results: Differentially expressed genes (DEGs) in frozen (1103 genes) and vitrified (333 genes) spermatozoa were evaluated. The bioinformatical analysis identified 8 and 15 significant pathways in groups of frozen and vitrified spermatozoa, respectively. The majority of these pathways are most relevant to immune and infectious diseases. The DEGs of the fertilization process are not detected during vitrification. The freezing process induces more down-regulation of genes and is relevant to apoptosis changes and immune response. Conclusion: Cryopreservation of human spermatozoa is an epigenetically safe method for male fertility preservation. Cryoprotectant-free vitrification can induce more minor biological changes in human spermatozoa, in comparison with conventional freezing.

Knockout serum replacement is an efficient serum substitute for cryopreservation of human spermatozoa

The freeze-thaw procedure causes irreversible structural and functional changes in human spermatozoa. In order to decrease the detrimental effects of cryopreservation and improve the quality of post-thawed spermatozoa, the constituents of the freezing solution attracted considerable attention. In this study, for the first time, we evaluated the efficacy of knockout serum replacement (KSR) as a substitute for human serum albumin (HSA) for cryopreservation of human spermatozoa. Twenty semen samples were collected from normozoospermic men and divided them into five equal groups. One of the aliquots was diluted with glycerol-based medium as a control group (CON). The other four aliquots were diluted with the sucrose solution containing 5% HSA (H5), 10% HSA (H10), 5% KSR (K5), and 10% KSR (K10). The diluted samples were frozen and preserved in liquid nitrogen. Post thawed sperm parameters including motion characteristics, viability, membrane integrity, mitochondrial activity, acrosome integrity and DNA intactness in all of the sucrose-based groups were comparable with glycerol-based medium. The replacement of HSA by 10% KSR in the freezing medium resulted in significantly higher post-thawed viability, acrosome integrity and DNA intactness compared with other sucrose-based groups. In conclusion, the addition of 10% KSR to the sucrose-based freezing solution improves the quality of post-thawed human spermatozoa and may have potential to develop chemically defined freezing medium.

Hepatitis B virus surface protein induces oxidative stress by increasing peroxides and inhibiting antioxidant defences in human spermatozoa.

Hepatitis B virus (HBV) infection may affect sperm motility in patients with HBV. HBV surface protein (HBs) decreases mitochondrial membrane potential, impairs motility and induces apoptotic-like changes in human spermatozoa. However, little is known about how human spermatozoa respond to reactive oxygen species (ROS; mainly peroxides) induced by HBs. In this study, HBs induced supraphysiological ROS levels in human spermatozoa and reduced the formation of 2-cell embryos (obtained from hamster oocytes and human spermatozoa). HBs induced a pre-apoptotic status in human spermatozoa, as well as antioxidant defences by increasing glutathione peroxidase 4 (GPX4) and peroxiredoxin 5 (PRDX5) levels. These results highlight the molecular mechanism responsible for the oxidative stress in human spermatozoa exposed to HBV and the antioxidant defence response involving GPX4 and PRDX5.

Diethylstilbestrol activates CatSper and disturbs progesterone actions in human spermatozoa.

Is diethylstilbestrol (DES), a prototypical endocrine-disrupting chemical (EDC), able to induce physiological changes in human spermatozoa and affect progesterone actions? DES promoted Ca2+ flux into human spermatozoa by activating the cation channel of sperm (CatSper) and suppressed progesterone-induced Ca2+ signaling, tyrosine phosphorylation and sperm functions. DES significantly impairs the male reproductive system both in fetal and postnatal exposure. Although various EDCs affect human spermatozoa in a non-genomic manner, the effect of DES on human spermatozoa remains unknown. Sperm samples from normozoospermic donors were exposed in vitro to a range of DES concentrations with or without progesterone at 37°C in a 5% CO2 incubator to mimic the putative exposure to this toxicant in seminal plasma and the female reproductive tract fluids. The incubation time varied according to the experimental protocols. All experiments were repeated at least five times using different individual sperm samples. Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a multimode plate reader following sperm loading with Ca2+ indicator Fluo-4 AM, and the whole-cell patch-clamp technique was performed to record CatSper and alkalinization-activated sperm K+ channel (KSper) currents. Sperm viability and motility parameters were assessed by an eosin-nigrosin staining kit and a computer-assisted semen analysis system, respectively. The ability of sperm to penetrate into viscous media was examined by penetration into 1% methylcellulose. The sperm acrosome reaction was measured using chlortetracycline staining. The level of tyrosine phosphorylation was determined by western blot assay. DES exposure rapidly increased human sperm [Ca2+]i dose dependently and even at an environmentally relevant concentration (100 pM). The elevation of [Ca2+]i was derived from extracellular Ca2+ influx and mainly mediated by CatSper. Although DES did not affect sperm viability, motility, penetration into viscous media, tyrosine phosphorylation or the acrosome reaction, it suppressed progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation. Consequently, DES (1-100 μM) significantly inhibited progesterone-induced human sperm penetration into viscous media and acrosome reaction. N/A. Although DES has been shown to disturb progesterone actions on human spermatozoa, this study was performed in vitro, and caution must be taken when extrapolating the results in practical applications. The present study revealed that DES interfered with progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation, ultimately inhibited progesterone-induced human sperm functions and, thereby, might impair sperm fertility. The non-genomic manner in which DES disturbs progesterone actions may be a potential mechanism for some estrogenic endocrine disruptors to affect human sperm function. National Natural Science Foundation of China (No. 31400996); Natural Science Foundation of Jiangxi, China (No. 20161BAB204167 and No. 20142BAB215050); open project of National Population and Family Planning Key Laboratory of Contraceptives and Devices Research (No. 2016KF07) to T. Luo; National Natural Science Foundation of China (No. 81300539) to L.P. Zheng. The authors have no conflicts of interest to declare.

237 Diethylstilbestrol Activates Catsper and Disturbs Progesterone Actions in Human Sperm

Is the prototypical endocrine-disrupting chemicals (EDCs), diethylstilbestrol (DES), able to evoke physiological changes in human spermatozoa and affects progesterone actions? DES promoted Ca2+ flux into human sperm by activating CatSper channel and suppressed progesterone-induced Ca2+ signaling, protein tyrosine phosphorylation and human sperm functions. Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a plate reader following sperm loading with Ca2+ indicator Fluo-4 AM and further whole-cell patch-clamp technique was performed to record CatSper currents. Sperm viability and motility parameters were assessed by eosin-nigrosin staining kit and a computer-assisted semen analysis (CASA) system, respectively. The ability of sperm to penetrate into viscous media was detected by 1% methylcellulose. Sperm acrosome reaction was measured using chlortetracycline (CTC) staining. The level of protein tyrosine phosphorylation was determine by western blot analysis. DES rapidly increased human sperm [Ca2+]i even at environmentally relevant concentrations (100 pM). The elevation of [Ca2+]i was derived from extracellular Ca2+ influx and can be significantly inhibited by the CatSper blocker mibefradil, suggesting CatSper participate in this process. In fact, whole-cell patch-clamp recordings confirmed that DES could increase CatSper currents. DES did not affect sperm viability, motility, penetrate into viscous media, protein tyrosine phosphorylation and the acrosome reaction. However, it suppressed progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation. Consequently, DES (1-100 μM) significantly inhibited progesterone-induced human sperm penetration into viscous medium and acrosome reaction.

Identification of protein changes in human spermatozoa throughout the cryopreservation process.

Cryoinjury is a consequence of cryopreservation and may have a negative impact on sperm quality regarding motility, morphology, and viability. This study was designed to identify potential proteomic changes in human sperm cells throughout the cryopreservation process. Comparisons made within this study included the detection of the sperm proteomic changes induced by incubation of the sperm cells with a protein-free cryoprotectant (with and without CryoSperm), and the proteomic changes induced by freezing, thawing, and subsequent after-thawing incubation at two different temperatures (0°C vs. 23°C). Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS was used for protein quantification. LC-MS/MS resulted in the identification of 769 quantifiable proteins. The abundance of 105 proteins was altered upon CryoSperm incubation. Freezing and thawing also induced substantial protein changes. However, fewer changes were observed when semen was thawed and then maintained after-thawing at approximately 0°C than when it was maintained after-thawing at 23°C, with 60 and 99 differential proteins detected, respectively, as compared to unfrozen semen incubated in CryoSperm. Collectively, these differences indicate that substantial changes occur in the sperm proteome at every stage of the cryopreservation process which may ultimately impair the sperm fertilizing capability. This is the first study to compare protein levels in fresh and cryopreserved semen using the TMT technology coupled to LC-MS/MS.

Protective effects of exogenous gangliosides on ROS-induced changes in human spermatozoa.

This article summarizes the available evidence on the efficacy of gangliosides to reduce the degree of reactive oxygen species (ROS)-mediated damage. The antioxidative efficacy of exogenous gangliosides in protecting different cells encouraged us to examine their ability to protect human spermatozoa. Gangliosides are sialic acid-containing glycosphingolipids with strong amphiphilic character due to the bulky headgroup made of several sugar rings with sialic acid residues and the double-tailed hydrophobic lipid moiety. The amphiphilicity of gangliosides allows them to exist as micelles in aqueous media when they are present at a concentration above their critical micellar concentration. The protective effect of ganglioside micelles on spermatozoa is believed to stem from their ability to scavenge free radicals and prevent their damaging effects. In our study, we particularly focused our attention on the protective effect of ganglioside micelles on DNA in human spermatozoa exposed to cryopreservation. The results indicate that ganglioside micelles can modulate the hydrophobic properties of the sperm membrane to increase tolerance to DNA fragmentation, thus protecting the DNA from cryopreservation-induced damage. Further actions of ganglioside micelles, which were documented by biochemical and biophysical studies, included (i) the modulation of superoxide anion generation by increasing the diffusion barrier for membrane events responsible for signal translocation to the interior of the cell; (ii) the inhibition of iron-catalysed hydroxyl radical formation due to the iron chelation potential of gangliosides; and (iii) inhibition of hydrogen peroxide diffusion across the sperm membrane.

Effet immobilisant des spermatozoïdes par les extraits des feuilles du Cestrum parqui

The leaves extracts of Cestrum parqui were reported to have spermicidal activity. The current investigation identified the spermicidal component of the extracts and evaluated its spermicidal potential in vitro, particularly the effects on sperm motility and vitality.Sperms were prepared by discontinuous buoyant density gradient centrifugation and incubated with varying concentrations of extract from C. parqui (40-250 μg/ml) at 37°C and 5% CO(2). The mode of spermicidal action was evaluated by sperm motility and vitality at different intervals ranging from 5 to 240 minutes. Morphological changes in human spermatozoa after exposure to the extract were evaluated under transmission electron microscope.A dose- and time-dependent effect of this extract on sperm motility and viability was observed. The mean effective concentration of extracts that induced irreversible immobilization was 250 μg/ml. Transmission electron microscope revealed a significant damage to sperm membrane in head and acrosomal membranes, notable swelling and disruption.Our study indicates that this natural extract has potential spermicidal effect in vitro. It can adequately replace nonoxynol-9 in vaginal contraceptives to make them more vaginally safe and ecofriendly.

Identification of diabetes- and obesity-associated proteomic changes in human spermatozoa by difference gel electrophoresis

Difference gel electrophoresis (DIGE) of fluorescently labelled human sperm proteins was used to identify diabetes- and obesity-associated changes of the sperm proteome. Semen samples from type 1 diabetics, non-diabetic obese individuals and a reference group of clinically healthy fertile donors were evaluated in a comparative study. The adaptation of a general protein extraction procedure to the solubilization of proteins from isolated progressively motile human spermatozoa resulted in the detection of approximately 2700 fluorescent protein spots in the DIGE images. Comparison of the patients' sperm proteomes with those of the reference group allowed the identification of 20 spots containing proteins that were present in the sperm lysates at significantly increased or decreased concentrations. In detail, eight of these spots were apparently related to type 1 diabetes while 12 spots were apparently related to obesity. Tryptic digestion of the spot proteins and mass spectrometric analysis of the corresponding peptides identified seven sperm proteins apparently associated with type 1 diabetes and nine sperm proteins apparently associated with obesity, three of which existing in multiple molecular forms. The established proteomic approach is expected to function as a non-invasive experimental tool in the diagnosis of male infertility and in monitoring any fertility-restoring therapy.

Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation

To investigate whether early apoptotic changes in spermatozoa can be significant markers for sperm quality. Two early apoptotic changes in the semen of 56 men were assessed using Annexin V (AN)/propidium iodide (PI) staining for phosphatidylserine externalization and JC-1 staining for mitochondrial membrane potential (MMP). The results were compared with conventional semen parameters and DNA fragmentation identified using the TUNEL assay. The different labeling patterns in the bivariate Annexin V/PI analysis identified four distinctive spermatozoa populations. The percentage of AN(-)/PI(-) spermatozoa positively correlated with conventional semen parameters and MMP, but negatively correlated with TUNEL (+) spermatozoa. As for the AN(-)/PI(+) fraction, we found an opposite result in comparison to AN(-)/PI(-) spermatozoa. The level of early apoptotic AN(+)/PI(+) spermatozoa negatively correlated with MMP and sperm motility. The level of late apoptotic AN+/PI+ spermatozoa negatively correlated with conventional semen parameters and MMP, and positively correlated with TUNEL (+) spermatozoa. MMP positively correlated with conventional semen parameters, but negatively correlated with TUNEL (+) spermatozoa. Although early apoptotic AN+/PI(-) spermatozoa only negatively correlates with sperm motility, the differences in proportion of each subpopulation of spermatozoa (especially, the percentage of AN(-)/PI(-) spermatozoa), and decreased MMP might be significant markers for diagnosing male infertility. They possibly bring additional information to predict the outcome of in vitro fertilization.

Reduction of oxidative changes in human spermatozoa by exogenous gangliosides

The effect of exogenous gangliosides, the sialic acid-containing glycosphingolipids, on oxidative changes in human spermatozoa was investigated. The incorporation of disialogangliosides or trisialogangliosides (GD1b and GT1b, respectively) into the iron/ascorbate promoter system for induction of lipid peroxidation decreased the release of malondialdehyde (MDA) from peroxidizing spermatozoa. The application of monosialogangliosides and disialogangliosides (GM1 and GD1a, respectively) did not have any effect under identical experimental conditions. GT1b, at a micromolar concentration, significantly inhibited the production of MDA, a breakdown product of lipid peroxide decomposition in spermatozoa of normozoospermic infertile men (P < 0.001; n = 51). An enhanced generation of MDA exhibited by the sperm population from the low-density Percoll fraction containing defective and/or immature spermatozoa was significantly reduced in the presence of GT1b. These results and the experiments on the influence of iron-chelating agent ethylenediamine tetraacetic acid (EDTA) as well as ferrous ion concentration itself on lipid peroxidation support the hypothesis that the protective effect of ganglioside against MDA generation could be the result of its chelating activity. Furthermore, superoxide anion release of phorbol myristate acetate-stimulated spermatozoa was significantly reduced in the presence of 50 and 100 micromol l(-1) GD1b (P < 0.05) and GT1b (P < 0.005). The inhibitory effect of 100 micromol l(-1) GT1b on spermatozoa from infertile normozoospermic men was statistically significant (P < 0.001; n = 21) and did not depend on the initial superoxide anion production. In conclusion, the protective action of GD1b and GT1b could be related to both scavenging of free radicals and metal-chelating properties, which might have relevance in the protection against oxidation-induced processes in human spermatozoa.

Involvement of tyrosine kinase and cAMP-dependent kinase cross-talk in the regulation of human sperm motility.

Tyrosine phosphorylation and its upregulation by cAMP have been associated with capacitation and motility changes of spermatozoa. In the present study, washed spermatozoa were incubated for 6 h in protein-supplemented complete medium with or without kinase inhibitors to verify whether upstream activation of protein kinase A is indispensable for tyrosine phosphorylation and motility changes to occur in capacitating human spermatozoa. H89, a specific protein kinase A inhibitor, significantly inhibited the activity of sperm protein kinase A. However, this inhibition did not alter capacitation-related tyrosine kinase activation. Tyrosine phosphorylated proteins, motion parameters and the incidence of phosphotyrosine-immunoreactive spermatozoa were decreased only slightly. Conversely, genistein, a tyrosine kinase inhibitor which inhibited sperm tyrosine kinase but not protein kinase A, significantly reduced all the parameters studied. Spermatozoa incubated with cAMP and pentoxifylline showed a rapid enhancement of tyrosine phosphorylation and some of the sperm motion parameters, particularly hyperactivation. Inclusion of H89 reduced cAMP stimulation of tyrosine kinase, and tyrosine phosphorylation and motion parameters were reduced almost to basal values. Treatment with genistein reduced tyrosine kinase activity, especially in the soluble fraction of sperm extracts. A decrease in tyrosine phosphorylation of soluble proteins, 105, 81, 55 and 48 kDa, correlated with a significant reduction in sperm motion parameters. Hyperactivation was reduced by tenfold. Tyrosine phosphorylated proteins in the insoluble fraction and the incidence of tyrosine phosphorylated-positive spermatozoa were not reduced markedly. Upstream protein kinase A activation may be a facilitatory rather than an indispensable step in the capacitation-induced tyrosine phosphorylation mediating motility changes in human spermatozoa. Triton-x100 soluble tyrosine phosphorylated proteins, more than their insoluble counterparts, appear to be involved in the modulation of human sperm motion characteristics.

The influence of human follicular fluid on the acrosome reaction, fertilizing capacity and proteinase activity of human spermatozoa

The present study was designed to assess physiological and enzymatic changes in human spermatozoa following incubation in human follicular fluid (HFF). Initially, it was determined that infertility patients (n = 29) scored dramatically higher on the hamster egg penetration test (HEPT) when spermatozoa were incubated with HFF (22.9 +/- 4.4%) rather than the standard swim-up alone (4.6 +/- 1.1%). To further evaluate this effect, in experiment I, spermatozoa were obtained from proven fertile individuals and evaluated following exposure to three different HFF samples as well as control treatments (medium, cul de sac fluid and heparin). There were no significant differences in HEPT scores following sperm incubation in the three different follicular fluids although incubation in all the fluids significantly (P less than 0.01) enhanced sperm penetration (%PEN) when compared to the standard swim-up and other control treatments. The absence of an effect of cul de sac fluid on spermatozoa indicates that not all body fluids contain factors which stimulate sperm fertilizing capacity. The effect of HFF was demonstrated in a infertile patient population as well as in donors of proven fertility. In experiment II, the effect of HFF on the acrosome reaction (%AR), sperm fertilizing capacity and changes in sperm proteolytic enzymes were determined. There was no significant difference in the %AR between freshly ejaculated (7.9 +/- 3.1) and medium incubated (9.4 +/- 1.6) spermatozoa; however, in both of these treatments the %AR was less (P less than 0.01) than for spermatozoa treated with HFF (45.6 +/- 4.7). The %PEN following incubation of spermatozoa in HFF (52.2 +/- 6.8) was greatly increased (P +/- 0.01) compared to the standard swim-up (19 +/- 3.9).

Morphological changes in human spermatozoa as examined under scanning electron microscope after [formula omitted] exposure to saponins isolated from [formula omitted

Saponins isolated from Sapindus mukorossi have potent spermicidal activity. Morphological changes in human ejaculated spermatozoa after exposure to these saponins were evaluated under Scanning Electron Microscope. The minimum effective concentration (0.05% in spot test) did not affect the surface topography after exposure for 1 minute. However, incubation of spermatozoa for 10 minutes resulted in extensive vesiculation and disruption of plasma membrane in the head region. Higher concentrations (0.1%, 1.25%, 2.5% and 5.0%) caused more or less similar changes which included vesiculation, vacuolation, disruption or erosion of membranes in the head region. These findings suggest that the morphological changes observed are due to alterations in the glycoproteins associated with the lipid bilayer of plasma membrane of spermatozoa.

In vitro changes in human spermatozoa exposed to gastric juice: laboratory findings as a support for forensic practice.

Samples of complete human semen were incubated in gastric juice for different time periods at 37 degrees C, and by simulating the post-mortem temperature decrease of the human body. The changes in the spermatozoa were similar in both experiments. Short incubation specimens were examined directly with interference contrast microscopy and showed an almost immediate immobilization of spermatozoa when they were brought in contact with gastric juice. Specimens with longer incubation periods were stained with alcalic fuchsine and examined by immersion microscopy. There was a morphologically stable plateau for the heads of the spermatozoa for up to 6 h of incubation. The tails disappeared progressively in the first 45 min. After more than 6 h of incubation a progressive swelling and lysis of the heads was observed. Spermatozoa could be recognized for up to 7 days of incubation.

Metabolic Changes in Human Spermatozoa Related to Capacitation

Metabolic changes in human spermatozoa related to capacitation were investigated. Glycolysis, motility, oxygen uptake, survival time, and c hanges in tetracycline-binding capacity were studied in spermatozoa expo sed to the following possible capacitating agents: 1) human cervical mucus, 2) human serum, 3) hydroxalpinx fluid, and 4) follicular cyst fluid. Mucus and serum did not produce marked changes. Hydrosalpinx fluid produced an increase in oxygen uptake in the presence of citrate and succinate while follicular cyst fluid produced modifications that could be related to capacitation such as increase in oxygen uptake, increase in motility, and release of bound tetracycline. Similar changes were seen with cyclic adenosine monophosphate and adenosine triphosphate treatment but not with other related nucleotides. The parameters studied in this work are suggestive of functional capacitation.

Morphological changes in human spermatozoa following treatment of semen with certain dialkyldithiocarbamates

Morphological changes in human spermatozoa following treatment of semen with certain dialkyldithiocarbamates