Plasminogen activator activity and fertilizing ability of human spermatozoa

Mature spermatozoa contain a number of proteases that are supposed to contribute to their fertilizing ability. The present study was directed at plasminogen activator (PA), a protease that belongs to the group of serine proteases and converts the zymogen plasminogen to the active broad-spectrum protease plasmin. To investigate the possible role of PA in the fertilization process, we have measured sperm-bound PA activity in 63 patients included in an in-vitro fertilization (IVF) programme and assessed their relationship to standard semen parameters and the rate of fertilization. PA activity was correlated significantly with the sperm count, as well as with sperm motility and morphology. Using logistic regression analysis, specific PA (pmol pNA 10^--⁶ cells minP²) was found to significantly influence the probability of fertilization. Other significantly predictive factors were motility and the percentage of spermatozoa with normal morphology. The sperm concentration (10⁶ cells ml^-1) did not significantly affect the outcome of IVF. We suggest that sperm-bound PA is involved in the fertilization process and may represent a potential indicator of sperm fertilizing capacity.     

Apoptosis and DNA damage in human spermatozoa

DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis, resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apoptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.     

ATP and ADP content of human ejaculated spermatozoa

ATP and ADP were measured by a bioluminescent method in human ejaculated spermatozoa from 31 normal donors and oligozoospermic patients. ATP and ADP expressed for the sperm total number were significantly correlated ( P < 0.001). ATP and ADP levels were found significantly correlated with the total motile spermatozoa and with the total viable cells (respectively P < 0.001 and P < 0.001 for ATP and P < 0.001 and P < 0.001 for ADP). ATP/ADP ratio was found significantly lower in subjects with sperm motility less than 50% ( P = 0.024) and total viable cells less than 80% ( P = 0.026).     

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.     

ATP and ADP content of human ejaculated spermatozoa

ATP and ADP content of ejaculated spermatozoa of 7 donors were measured by a bioluminescent method within 24 h. The semen samples were kept at room temperature and at prefixed time were tested for motility and viability and extracted for ATP and ADP measurement. ATP pattern in relation to the time was studied with a mathematical model and the resulting curve showed a very high significance ( P < 0.001). ADP decrease with time was not significant and was variable among our subjects. The relationship between ATP decrease (%) and the decrease (%) of viability and motility was not linear, and till now no mathematical model has been found to express this phenomenon.     

A possible haploid effect in acrosome malformations of human spermatozoa

Acrosome malformations of spermatids and spermatozoa in the testes of two infertile patients were investigated with the light and electron microscope. The first visible abnormalities appear at early spermatid stages. The detailed morphological analysis of the malformed spermatids shows that in most cases only the differentiation of the acrosome granule is interfered with. This may be the origin of the malformations of the mature spermatozoa. The fact that almost half the early spermatids lack the acrosome granule suggests that the original cause is genetic and that the genes are expressed in the haploid phase.     

Ultracytochemical localization of 5'-nucleotidase activity in human ejaculated spermatozoa

5'-nucleotidase, an adenosine producing enzyme with a glycosylphosphatidylinositol-anchored structure, was localized in human ejaculated spermatozoa. The poly-L-lysine-coated dish method was used to prepare the specimens, and the cerium method was employed for electron-microscopic enzyme localization. Precipitates indicating enzyme activity were detected on the outer side of the external plasma membrane of the acrosomal region. This enzyme may play a role in sperm motility and male infertility.     

Effect of pentoxifylline on experimentally induced lipid peroxidation in human spermatozoa

We demonstrated previously that pentoxifylline in millimolar concentrations can inhibit superoxide anion production by human spermatozoa. In the present study we have examined the effects of the same concentrations of pentoxifjrlline on experimentally induced lipid peroxidation, as measured by malondialdehyde formation in the thiobarbituric (TBA) assay. Under the experimental conditions used, preincubation of spermatozoa with pentoxifjdline led to a significant dose-dependent stimulation (p<0.005) of malondialdehyde production amounting to 10.77 ± 2.35%, 13.45 ±2.99% and 17.4 ± 1.99% (mean ± SEM) for 1.9, 3.7 and 11.2 mmol/l pentoxifylline, respectively. In the presence of 11.2 mmol/l pentoxifylline, an increase in iron-catalysed lipid peroxidation potential was detected in samples of spermatozoa from 29 infertile men, regardless of their initial levels of malondialdehyde. The results of this study indicate that pentoxifylline might further augment the ferrous ion-stimulated decomposition of pre-accumulation lipid hydroperoxides in the sperm plasma membrane and thus promote malondialdehyde generation in the TBA assay.
It is concluded that the stimulatory effect of pentoxifjdline on iron-induced lipid peroxidation may have an adverse effect on the quality of sperm suspensions prepared for in vitro fertilization, a possibility which should be investigated further.     

Immunocytochemical detection of mitochondrial dihydroorotate dehydrogenase in human spermatozoa

In mammalian cells the requirement for pyrimidines is met by uridine phosphate (UMP) de novo synthesis and, to a greater or lesser extent, by salvage of free nucleosides. The fourth enzyme of the de novo synthesis, the mitochondrially bound dihydroorotate dehydrogenase (DHODH) was the focus of the present study. Rabbit anti-DHODH IgG, which was generated using an immunization protocol with truncated recombinant human DHODH protein and purified by an immunosorbent method, was used for immunocytochemical detection and localization of this enzyme in ejaculated human spermatozoa. The presence of DHODH protein was demonstrated by Western blotting of solubilized membrane fractions with peroxidase conjugated anti-rabbit IgG in combination with chemiluminescence detection. Indirect immunofluorescence microscopy, using Cy3-conjugated anti-rabbit IgG, revealed specific binding in the midpiece of spermatozoa. As these cells no longer have a demand for de novo biosynthesis of pyrimidines, we hypothesize that the pathway could serve a specialized function in nitrogen or zinc metabolism during the process of spermiogenesis and/or epididymal maturation.     

不育症患者精子头部及尾部超微结构的研究

目的研究不育男性精子超微结构的形态特征。方法利用透射电镜对8例不育男性新鲜精液标本中的精子头及尾部超微结构进行观察。结果在电镜下不育男性精子存在多种形态超微结构异常,有以下几种类型：(1)顶体异常精子,包括顶体膜受损,顶体发育不良、缺失,顶体内形成包涵体精子。(2)头部异常精子,包括尖头精子、圆头精子、头部含空泡精子。(3)尾部异常精子,①尾部形态异常精子,包括无尾精子、短尾精子、卷尾精子、体尾胞质残余。②尾部结构异常精子,包括线粒体缺失精子、尾部线粒体多种形态和结构异常。结论不育男性精子存在顶体、头部、尾部线粒体、微管多种形态和结构异常。     

Development of the oocyte-penetrating capacity of spermatozoa in the human epididymis

The development of the penetrating capacity of human epididymal spermatozoa has been assessed in vitro using zona-free hamster oocytes. Spermatozoa were recovered from epididymal tissue of men undergoing vasectomy or epididymovasostomy. The results suggest that human spermatozoa first develop the potential to penetrate oocytes in the proximal corpus region. Spermatozoa recovered from more proximal sites of the excurrent duct failed to bind or fuse with zona-free hamster eggs although a small proportion (3%) in the caput region were capable of progressive motility. The data are discussed in relation to biochemical changes to human spermatozoa during maturation.     

Effect of glycerol and cryopreservation on oocyte penetration by human spermatozoa

A poor penetration rate of glycerol-treated, cryopreserved human spermatozoa as compared to untreated fresh control, was observed in the zona-free hamster oocyte test. Similarly, glycerol treatment of freshly ejaculated spermatozoa depressed the penetration rate unless the culture medium also contained glycerol. Immediately after thawing, glycerol-treated, cryopreserved spermatozoa possessed adequate progressive motility, but their incubation in glycerol-free culture medium caused a severe reduction in motility. Even if the same number of progressively motile, cryopreserved, glycerol-treated spermatozoa as unfrozen spermatozoa were added to the eggs, a much lower penetration rate was obtained by the treated spermatozoa. It is concluded that spermatozoa develop a glycerol dependence and that removal of glycerol from the surrounding medium, as most likely occurs when spermatozoa pass through the cervix, reduces both the motility and the ability of spermatozoa to become capacitated and fuse with oocytes. Thus, glycerol is not an optimal cryopreservative agent. Further, the decreased oocyte penetration rate of glycerol-treated, cryopreserved spermatozoa is due to other factors besides the decrease in sperm motility.     

Ultrastructural studies in Morphological assessment of human spermatozoa

The fine morphology of the regional structure of ejaculated spermatozoa which was observed by light microscope, scanning electron microscope and transmission electron microscope was the basis for the classification of various morphological characteristics. A quantitative analysis of the frequencies of these morphological characteristics in the fertile and suspected infertile populations was conducted. A discriminant analysis of these data revealed that 6 morphological characteristics out of 30 examined by light microscope, 9 out of 42 examined by scanning electron microscope and 8 out of 35 examined by transmission electron microscope were discriminatory. In addition, the discriminant analysis enabled obtaining a morphological score for each semen sample which was calculated from the weights of the different discriminatory characteristics for the 3 microscopic techniques. It was possible to separate the fertile from the infertile population using these morphological scores. The fertile man obtained positive scores while the suspected infertile men obtained negative scores. The practical use of the different morphological scores as diagnostic tools for male infertility clinics is discussed.     

速冻和缓慢冷冻法对精子运动特征的影响

目的了解冷冻方法对人精子运动特征的影响。方法精液标本进行速冻和缓慢冷冻保存,应用计算机精液分析仪进行精子运动特征分析。结果冷冻复温后精子运动能力与冷冻前精子运动能力比较明显下降(P<0.001,P<0.05);速冻与缓慢冷冻方法保存的精子运动参数相比较差异均无显著性(P>0.05)。结论冷冻保存易导致精子运动能力下降,速冻与缓慢冷冻方法对精子运动参数影响无明显差异。     

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.     

Headless spermatozoa in infertile men

Spermatozoa morphology, an important parameter in a semen specimen's potential fertility evaluation, is a significant factor for in vitro fertilisation in assisted reproductive technology. Eleven sterile men with headless spermatozoa, a type of human teratozoospermia, are presented. Their ejaculates’ headless spermatozoa percentages were high with rare normal spermatozoa forms. Additionally, abnormal morphology (e.g. round‐headed or microcephalic spermatozoa) was also found. Spermatozoa motility was somewhat affected, potentially because of the missing mitochondrial sheath at the sperm tail base. Patients who underwent assisted reproductive technology treatment experienced adverse pregnancy outcomes. Work types and corresponding environments seemed irrelevant, but specific family history may have prompted its genetic origin. Computer‐assisted semen analysis systems easily mistake headless spermatozoa as oligozoospermia because of nonrecognition of the loose head. However, morphological testing, especially with an electronic microscope, clearly identifies abnormal spermatozoa. Future exploration requires more methods investigating the frequency and percentage of this morphological abnormality in different populations with varied fertility levels. Such research would estimate the probable correlation of the abnormality with other semen parameters and examine the potential developmental or genetic origins. During clinical work, medical staff should detect these cases, avoid misdiagnosis and provide proper consultation about diagnosis and assisted reproductive technology treatment.     

Protective effect of Propolfenol® on induced oxidative stress in human spermatozoa

The propolis extract was shown to possess the capacity to protect sperm membrane from the deleterious action of oxidative attack. Oxidative stress can induce propagation of a lipid peroxidation (LPO) chain reaction because spermatozoa contain high concentration of unsaturated fatty acids. This study aimed at evaluating in vitro the possible toxicity and/or the antioxidant properties of Propolfenol^® in ejaculated human spermatozoa. A colorimetric assay determined the total flavonoid content by spectrophotometry and a high‐performance liquid chromatography–diode array detection analysis the quantity of galangin, pinocembrin and caffeic acid phenylethilic ester (CAPE). Sperm parameters such as motility, vitality and DNA integrity were assessed utilising optical microscopy. The antioxidant properties Propolfenol^® against LPO induced by tert‐Butyl Hydroperoxide were evaluated using the C11‐BODIPY581/591 probe. Chemical analysis of Propolfenol^® revealed low quantities of galangin, pinocembrin and CAPE; cyclic voltammetry experiments showed that Propolfenol^® may exert an antioxidant activity. A protective action of Propolfenol^® (20 and 100 μg/ml) on induced LPO in human spermatozoa was detected. Propolfenol^® may be proposed as the supplement in media for sperm preparation techniques or cryopreservation to counteract the increased presence of reactive oxygen species generated by these methods.     

Increased plasminogen activator activity and plasminogen activator inhibition in spermatozoa and seminal plasma of the ram after serum gonadotrophin (PMSG) administration. Correlation with the increased level of testosterone in the blood

After a single injection of serum gonadotrophin (PMSG) at the dose of 15 IU/kg, i.m. into rams testosterone in the plasma of blood showed a significant rise between 4th and 7th day post-injection. At the same time (4th-7th day) the plasminogen activator activity (PAA) in seminal plasma was found to be increased, but the plasminogen activator inhibition (PAI) expressed against t-PA (anti-t-PA) showed an increase between 32nd and 46th day. In spermatozoa a marked increase of PAA was revealed between 32nd and 46th day post-injection, while an increase of PAI (anti-t-PA) was exhibited on the 74th day. Plasmin inhibition (PI) in seminal plasma and spermatozoa showed no change compared to controls. A positive correlation has been found between increased concentrations of testosterone and PAA or PAI (anti-t-PA) in spermatozoa and seminal plasma. The induced increase of PAA in spermatozoa under the effect of testosterone might be of physiological importance, since PAA is localized to sperm membranes and might participate in the whole process of fertilization.