The binding of oestradiol-17ß to human spermatozoa — an electron microscope autoradiographic study

The location of steroid binding sites with specificity for oestradiol-17ß at an ultrastructural level has been examined on human spermatozoa by electron microscope autoradiography. About 75% of the 438 grains produced by ³H-oestradiol-17ß, and counted on electron microscope autoradiographs, were found over the plasma membrane or within 1000 Å of it. This study provides evidence suggesting that the plasma membrane is a site of action of oestradiol-17β in human spermatozoa.     

Partial characterization of human spermatozoal phosphodiesterase and adenylate cyclase and the effect of steroids on their activities

In sonicated human spermatozoa, phosphodiesterase hydrolyzed adenosine 3':5'-monophosphate (cAMP) at 20.80 +/- 3.23 nmoles/10(8)sperm/20 min at 37 degrees C (50 microM cAMP, initially). In human semen, about 60% of the phosphodiesterase activity was in the spermatozoa. Both polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography indicated that there are at least 5 isozymes of phosphodiesterase. Various steroids were tested at a concentration of 2 micrograms/ml for their effects on phosphodiesterase activity in semen. None was found to have any significant effect. In sonicated human spermatozoa, adenylate cyclase synthesized cAMP at 0.02-2.11 nmoles/10(8)sperm/20 min at 37 degrees C (1 mM ATP, initially) depending on the availability of Mn2+ and caffeine in the assay mixture. Mn2+ was demonstrated to be a potent adenylate cyclase activator in human spermatozoa and its effect on human sperm adenylate cyclase was found to be dose-dependent. Cholera toxin, at a concentration of 20 micrograms/ml, had no effect on human sperm adenylate cyclase activity after it had been incubated with the intact spermatozoa for between 5 min and 5 h at 37 degrees C before the sperm were homogenized and the rate of cAMP formation assayed. In addition, human sperm adenylate cyclase decayed rapidly at 37 degrees C. Of various steroids tested at a concentration of 2 micrograms/ml for their effects on human sperm adenylate cyclase activity, only oestradiol-17 beta showed a significant effect, elevating the rate of cAMP formation by about 30%.     

Use of human zonae from cryopreserved oocytes in a test to assess the binding capacity of human spermatozoa

Aged unfertilized oocytes from an assisted conception programme were cryopreserved and then utilized after thawing in a zona-binding assay. Oocytes frozen using dimethyl sulphoxide (DMSO) showed poor survival post-thaw (2/40, 5%) compared to those frozen with propanediol (PROH) (63/134, 47%). When the zonae were exposed to spermatozoa from fertile donors, those frozen with DMSO showed a significantly higher number of bound spermatozoa than did those frozen with PROH (P< 0.002). In both groups, oocytes which failed to survive the freeze-thaw processes had greater numbers of bound spermatozoa than did those which survived (P<0.05). Oocytes from cases of failed fertilization showed no difference in their rate of sperm binding compared with oocytes from cases in which some fertilization had occurred. Zonae frozen in PROH but which were from oocytes which were not viable after thawing were used to assess the binding of spermatozoa from men who had failed previously to fertilize their partner's oocytes in vitvo and spermatozoa from men with poor quality semen and who had elected for treatment by micro-injection sperm transfer. The number of spermatozoa bound to zonae was reduced significantly in both groups compared to a fertile donor.     

Effects of castration, sex steroids, LHRH and glucocorticoids on LHRH binding in the anterior pituitary of male rats

In the present study we have examined the effects of gonadotrophin releasing hormone (LHRH), sex steroids and glucocorticoids on the binding of LHRH to receptors in the pituitary of male intact and castrated rats. In intact rats, LHRH (10 μg/day) treatment for 11 days caused a significant increase in LHRH binding, whereas testosterone (500 μg/day) or oestradiol (50 μg/day) were inhibitory. 17-hydroxyprogesterone and dexamethasone were without effects. In castrated rats, LHRH caused a marginal decrease in LHRH binding. Much greater inhibition was observed with testosterone and oestradiol. 17-hydroxyprogesterone reduced binding to that of intact controls, whereas dexamethasone was ineffective. When different doses of sex steroids were tested, both oestradiol, testosterone and 5α-dihydrotestosterone inhibited LHRH in a dose-dependent manner. The lowest doses of steroids causing significant inhibition of LHRH binding in castrated animals were 0.5, 50 and 500 μg/day for oestradiol, 5α-dihydrotestosterone and testosterone, respectively. The present study shows that pituitary receptors for LHRH are regulated both by sex steroids and LHRH itself.     

The binding patterns of antisera to sex steroids and human gonadotropins on human and rhesus monkey spermatozoa

The presence of different hormones on the surface of ejaculated spermatozoa was determined by immunofluorescence studies of the binding patterns of specific antisera to these hormones. There were striking similarities in the binding pattern of antisera to steroid hormones found on human and monkey spermatozoa. Assuming the intensity of fluorescence is proportional to the concentration of the hormone, concentrations of testosterone on the acrosomal and the postacrosomal regions were higher than levels of progesterone and estrogens. Spermatozoa with a "tapering head" had more hCG bound on the acrosomal and postacrosomal regions than spermatozoa with "normal head" (oval shaped). Correlating these findings to the functions of spermatozoa will require further studies.     

The effect of serum on motility of human spermatozoa in culture

The motility of spermatozoa was higher at 6-22 h in Ham's F10 culture medium supplemented with 20-30% human serum than with lower proportions of serum or with 1% human serum albumin. Heat treatment (56 degrees C, 1 h), charcoal extraction and dialysis (18,000 molecular weight cut-off) of the serum did not reduce sperm motility suggesting that high molecular weight components are responsible for maintenance of motility. Renewing the medium (Ham's F10 with 30% serum) at 7 h resulted in better sperm motility and velocity at 22 h. At 22 h the pO2, pCO2, pH and sodium concentrations were not different in replenished and control cultures, but the concentration of glucose was higher and that of potassium lower if the medium was changed. These results suggest that addition of 20-30% human serum and renewal of medium at intervals is beneficial for sperm culture and may be of use in in vitro fertilization.     

The relationship between morphology, motility and zona pellucida binding potential of human spermatozoa

Summary. Prediction of the fertilizing potential of human gametes under in vitro conditions has been a major field of interest of assisted reproductive programmes. However, sperm morphology has been regarded as a predictor of human in vitro fertilization rate. This paper prospectively evaluates the relationships among normal sperm morphology and (1) motion characteristics viz. curvilinear velocity (VCL), straight line velocity (VSL), and linearity (LIN) (n = 37) and (2) spermzona pellucida binding capacity under HZA conditions (n = 144) of two separate groups of infertile couples. Semen was evaluated for sperm concentration, percentage motility, forward progression, and percentage normal morphology (strict criteria). The motility characteristics were measured using a computerized Sperm Motility Quantifier (SMQ). The zona binding potential of sperm was evaluated using the hemizona assay. Firstly, the VCL significantly differred between the P-pattern and both the G (72.9 ± 7 vs. 86.3±16 μm s^?1; P = 0.04) and N patterns (72.9 ± 7 vs. 91.0 ± 15 μm s^?1; P = 0.002). The VSL differed only between the P and N patterns, being 19.7 ± 7 vs. 32.6±15 μm s^?1 (P = 0.02), respectively. No significant differences in LIN were noted between any of the three patterns. The sperm concentration differed significantly between the P and both the G (37.9±35 vs. 80.8 ± 9 × 10⁶ ml^?1; P = 0.03) and the N patterns (37.9 ± 35 vs. 89.7 ± 72 × 10⁶ ml^?1; P = 0.05). Significant differences were observed in the percentage motility between the P and both the G (38.0 ± 21% vs. 43.7 ±9%; P = 0.03) and the N patterns (38.0 ± 21% vs. 52.1±8%; P = 0.04). In the second study, the hemizona indices (HZI) differed significantly between the P and both the G (29.3 ± 26% vs. 57.6 ± 62%; P = 0.01) and the N patterns (29.3 ± 26% vs. 102.4 ± 80%; P < 0.001). The G and N patterns also differed significantly in their HZI (57.6 ± 62% vs. 102.4 ± 80%; P = 0.005). Sperm concentration differed between the P and both the G (32.8 ± 29 vs. 76.1±54 × 10⁶ ml^?1; P < 0.001) and the N patterns (32.8 ± 29 vs. 95.44 ± 61 × 10⁶ ml^?1; P < 0.001). The percentage motility differs significantly between the P pattern and both the G (41.2± 17% vs. 50.9±11%; P = 0.002) and the N patterns (41.2±17% vs. 53.4±11%; P = 0.001). Sperm morphology seems to be indicative of important functional characteristics of spermatozoa, for example motility and zona pellucida binding.     

Genomic and non-genomic actions of sex steroids in the growth plate

Sex steroids, and particularly estrogens, are important regulators of bone growth and bone mass accrual. For a long time, it was thought that these effects were mainly caused by their modulatory effects on the somatotrophic axis. Data gathered in the past years have challenged this view and it is now widely accepted that many of the effects of sex steroids on growth and bone mass accrual are caused by direct effects on target cells in the growth plate and bone. This review summarizes and discusses some of our latest findings on the expression of sex steroid receptors in the growth plate, the source of the ligands activating these receptors, and their putatitive mechanism of action predominantly focusing on observations in the rat.This work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

Annexin V binding to plasma membrane predicts the quality of human cryopreserved spermatozoa

Cryopreservation-induced stress may result in membrane injury with consequent decrease of sperm motility and fertilizing capacity. This study has investigated the relationship between human spermatozoa tolerance to cryopreservation and the loss of plasma membrane asymmetry, especially translocation of phosphatidylserine (PS) from the inner to the outer leaflet. The prospective study was performed on semen samples from 31 men. Conventional characteristics of 20 semen were analysed, before and after cryopreservation as well as externalization of PS assessed by annexin V-staining in combination with the propidium iodide which stains dead cells. The fertilizing capacity was evaluated by a zona free hamster egg penetration test in 11 freeze/thaw spermatozoa samples. The percentages of vital annexin V-negative and annexin V-positive spermatozoa in post-thaw samples were significantly lower than in pre-freeze ones (10 +/- 3 vs. 25 +/- 5% and 22 +/- 3 vs. 34 +/- 4% respectively), while the percentages of dead spermatozoa annexin V-negative and annexin V-positive had increased (42 +/- 4 vs. 23 +/- 4% and 23 +/- 4 vs. 16 +/- 2% respectively). The percentages of progressive and total motile spermatozoa were significantly correlated with the percentage of vital annexin V-negative spermatozoa before as well as after cryopreservation. Furthermore, recovery of motile spermatozoa after freeze/thaw was strongly correlated (p < 0.002) with the proportion of vital annexin V-negative spermatozoa in fresh semen. The percentage of penetration of zona-free hamster eggs was correlated (p < 0.02) with the percentage of live annexin V-negative cryopreserved spermatozoa capacitated for 3 h. These findings provide evidence that annexinV-binding staining in combination with PI brings additional information to predict the outcome of cryopreserved ejaculated sperm and may be used as a novel and reliable marker to study the freeze/thaw process.     

Distribution of vimentin in abnormal human spermatozoa

Summary. Vimentin was immunocytochemically detected using a monoclonal anti-vimentin antibody and indirect immunogold electron microscopy. In abnormal human spermatozoa, vimentin was visualized at the level of whole acrosome, post-acrosomal region, neck and initial segment of the mid piece. The distribution of vimentin immunoreactivity was related to a spectrum of structural defects such as large cytoplasmic droplets, binucleated spermatozoa and mitochondrial disassembly in the mid piece.     

The influence of a mineral oil overlay on the zona pellucida binding potential of human spermatozoa

Summary.  The objective of the study was to evaluate the effect of mineral oil on zona pellucida binding potential of human spermatozoa. The study compared zona binding using micro volume droplets under mineral oil as apposed to micro droplets in cryopreservation straws. Spermatozoa from eight proven fertile sperm donors were used. One hundred and fifty five matched hemizonae in 50 μl, 100 μl and 200 μl insemination sperm droplets were co-incubated; (i) under mineral oil and (ii) 0.5 ml plastic cryopreservation straws. The results were analysed to determine the number of the zona bound spermatozoa during each experiment. Microvolumes with an oil overlay had a decrease in sperm bound per hemizona of 38% (mean±SD; 563±415 vs. 921±597), 51% (mean±SD; 392±359 vs. 800±566 sperm) and 18% (mean±SD; 502±369 vs. 618±445) in 200 μl, 100 μl and 50 μl respectively, compared to microvolumes in cryopreservation straws. It was concluded that mineral oil may have some detrimental factors which interfere with zona binding of spermatozoa.     

The influence of sperm density on the motility characteristics of washed human spermatozoa

To study the effects of sperm density on the results of computer-assisted semen analysis (CASA), 10 washed semen samples were diluted and measured with the CellTrak/S CASA system in a concentration range of 10–180×10⁶ spermatozoa/ ml. All sperm motility parameters were influenced to some extent by sperm density. The motility percentage was influenced significantly in 5 samples ( P< 0.005), the straight line velocity in all samples (P<0.0005 in 7 samples), the curvilinear velocity in 3 samples ( P< 0.005), the linearity in 9 samples (P<0.0005 in 6 samples) and the lateral head displacement in 9 samples (P< 0.005 in 6 samples). In general, the CellTrak/S data are influenced significantly if sperm density exceeds 50 × 10⁶ spermatozoa/ml.
The influence of sperm density on the motility parameters can be explained both by the accuracy of the CASA system and by actual changes in the motility of the spermatozoa. In the light of other published studies, it is concluded that sperm motility measurements with CASA systems should be assessed using 25–50 × 10⁶ spermatozoa/ml, especially in studies concerning lateral head displacement and the linearity, as in sperm hyperactivation studies.     

Flow cytometric determination of binding of monoclonal antibodies to human spermatozoa

Summary. Although several monoclonal antibodies to sperm antigens are available, only few systematical reports on the appearance of distinct antibody binding structures in spermatozoa of infertile patients are available. We studied the binding frequency of six monoclonal antibodies (TÜS-1, -2, -10, -17, -19, -20) by means of a flow cytometer in different semen samples and their possible relationship to other sperm parameters, in particular to the motility parameters as determined by computer-assisted semen analysis. The percentage of vital spermatozoa binding the different antibodies was 21.5% (TÜS-2) to 52.1% (TÜS-20). The percentage binding was higher in samples with normozoospermia than in those with oligo-asthenoteratozoospermia. A significant correlation occurred between sperm concentration and the percentage of spermatozoa stained by TÜS-2 and TÜS-17; between motile spermatozoa and those binding TÜS-17 and TÜS-20; the velocity average path and those binding TÜS-17; an inverse correlation occurred between the morphologically-abnormal spermatozoa and those binding TÜS-20; an inverse correlation between TÜS-17 and acrosin. We conclude from our results that although the antigens of the antibodies concerned are not yet characterized, the determination of antibody binding will give additional information on the functional status during capacitation and acrosome reaction of spermatozoa.     

Comparative study of the kinetics of the acrosome reaction and survival of human spermatozoa in various media

The in-vitro kinetics of the acrosome reaction and the survival of human spermatozoa were studied under different capacitating conditions. Human preovulatory follicular fluid (FF), isotonic BWW (N-BWW) and hypertonic BWW (H-BWW) were tested. Motile sperm selected by migration in these media were examined after 1, 3, 5 and 22 h of incubation under 5% CO2. The kinetics of the reaction in the population of live, morphologically normal sperm was dependent on both the culture medium and time of incubation. In the first hour, the mean percentage of acrosome-reacted sperm in H-BWW and FF was significantly greater than in N-BWW. The proportion of reacted cells increased significantly after 3 h in N-BWW (P = 0.001), after 5 h in FF (P = 0.03) and after 22 h in H-BWW (P = 0.01). A significant decrease in sperm viability was registered at 3 and 22 h of incubation (P less than 0.002) in all media. These results demonstrate that both H-BWW and FF stimulate the acrosome reaction while survival is optimal in the latter.     

The influence of sperm/zona pellucida collision rates on zona binding

Summary. The satisfying success rates reported with intra-cytoplasmic sperm injection (ICSI) urged clinicians and scientists to re-address the emphasis in the management of the male factor patient towards gamete manipulation in order to circumvent the underlying problem causing fertilization failure. We have designed a study to (i) calculate the collision rate of a sperm population with the human zona pellucida, using a mathematical hypothesis and (ii) use the calculated collision rate to evaluate subsequent zona binding results obtained under hemizona assay conditions. Microdroplets were used to co-incubate sperm and human oocytes in order to evaluate zona binding. Using microvolumes, the track followed by sperm, as well as the maximum distance travelled were employed to calculate the collision rate of sperm and zona pellucida. The sperm concentrations of swim up samples were adjusted to 4 × 10⁶ and 0.8 × 10⁶ sperm ml⁻¹. Five separate droplets each of 20 μl containing 4 × 10⁶ sperm ml⁻¹ (80000 motile sperm) and 200 μl containing 0.8 × 10⁶ sperm ml⁻¹ (160000 motile sperm), respectively, were prepared. Both volumes were incubated for 18 h at 37°C. The mean (±SD) number of spermatozoa tightly bound to hemizona, incubated in 20 μl and 200 μl sperm droplets, was 2444±612 and 548±315, respectively ( P = 0.0001). The results can be used as a guideline to calculate the optimum insemination concentration needed for a specific sperm population to ensure the maximum collision rates with the oocyte.     

Use of acridine orange to evaluate chromatin integrity of human spermatozoa in different groups of infertile men

To study the sperm chromatin compactness various methods, such as acidic aniline blue or acridine orange staining, have been applied. Due to its metachromatic properties, acridine orange dye fluoresces green with double- and red with single-stranded DNA. Samples (n = 181) were evaluated and grouped as follows: group I, normal recently fertile; group II, male having female partner with repeated early pregnancy loss; group III, male with varicocele; and group IV in-vitro fertilization and intrauterine insemination failures. Routine semen analyses were carried out in all the cases. Amorphous particulate matter as observed under phase contrast microscope was graded on the scale of nil to +4. Fixed smears were stained with an aqueous solution of acridine orange and viewed under a fluorescence microscope. Two hundred cells were counted and the percentage of fluorescence calculated. Groups II, III and IV exhibited significantly low green fluorescence compared with the control group. The study also indicates that increased amorphous particulate matter (indicating infection) might be one of the contributing factors to lower acridine orange stainability. Thus acridine orange staining can be used to evaluate the integrity of the nucleus, disorders of which can cause unexplained infertility or lower fertilization potential that may go undetected by routine analysis.     

人增生性前列腺间质细胞体外培养及对性激素的反应性

为了了解前列腺间质细胞对性激素的反应性，用体外细胞培养技术分离培养人增生性前列腺间质细胞，观察了细胞纯度和由性激素诱导的细胞增殖反应。结果显示：间质细胞纯度９７０％，上皮细胞未构成污染；随传代数增加，细胞对性激素的反应性逐渐消失；较之对照组（１００％），雄激素（１７３％±１５％）及雌激素（１６４％±１０％）分别导致了特异性细胞增殖反应（Ｐ＜００５）。体外条件下雄、雌激素联合对间质细胞无协同作用，但使细胞增殖的激素浓度却仅为单纯雄或雌激素组的１％。人增生性前列腺间质细胞体外培养中，雄、雌激素各自对间质细胞的增殖发挥了其生物学作用。体外条件下二者合用敏化了细胞对性激素的反应性，从而导致了间质细胞的增殖，提示了ＢＰＨ发生发展中性激素作用的可能途径。     

Factors affecting sperm motility VIII. Velocity and survival of human spermatozoa as related to temperatures above zero

The effect of temperature on motility and survival of ejaculated human spermatozoa was investigated with the aid of the multiple exposure photography (MEP) method for objective sperm motility determination. Fresh specimens from healthy donors were analyzed while being heated or cooled gradually, or during their storage at various temperatures from 0 to 48°C. Sperm velocity increased steadily from zero to 50.4 nm/sec between freezing point and body temperature. Thereafter, their activity dropped dramatically and total immobilization occurred at 45°C. The induced immobilization was reversible providing exposure to those extreme temperatures was short enough to prevent permanent damage. Sperm survived up to 24–48 h when stored at 23°C, while at body temperature, their survival in vitro was much shorter and rarely extended beyond 12 h. Their longevity was still shorter at higher or lower temperatures, especially when approaching 48°C. With the aid of the combined supravital staining and MEP methods it was found that temperatures of extreme levels induced mainly immobilization rather than a spermicidic effect. The possible mechanism of thermal effect on sperm motility and some of its practical implications are discussed.     

透明质酸受体精子的质量特性研究

目的:分析具有透明质酸受体精子的质量特征,寻找精子质量评估新指标。方法:用透明质酸包被载玻片检测活动精子-透明质酸结合率,分析其与精液常规、精子膜功能、精子受精功能和精子成熟障碍指标的关系。结果:头部具有透明质酸结合位点的精子,顶体完整性[(95.4±3.9)%]、线粒体膜高电位率[(97.8±2.1)%]明显高于原始活动精子[(68.8±6.2)%和(72.8±7.4)%,P均0.01];精子-透明质酸结合率与多项精液常规参数呈弱相关性(r=0.195~0.268,P均0.05),与诱发顶体反应率和精子正常形态率呈正相关(r=0.666、0.417,P均0.01),与精子核蛋白不成熟度、DNA碎片率和过量残留胞质率呈负相关(r=-0.266、-0.308、-0.218,P0.01、0.01、0.05)。结论:头部存在透明质酸受体的精子,其质膜结构、受精潜能、成熟度俱佳;用于检测精子透明质酸受体的精子-透明质酸结合试验,是独立的精子多重质量评价新指标。     

Activity of testis angiotensin converting enzyme (ACE) in ejaculated human spermatozoa

Testicular angiotensin converting enzyme (ACE) isozyme is likely to play important functional roles in male reproduction. Several studies have shown that ACE is released from human spermatozoa during capacitation and that ACE is associated with reduced sperm motility. Recently, we established an assay to detect testicular ACE activity in human spermatozoa. The purpose of this study was to determine if testicular ACE activity is related to sperm motility in human ejaculates. Semen samples were collected from 80 infertile patients. According to the semen characteristics, they were divided into four (WHO) categories. Enzyme activities of ACE in spermatozoa (testicular ACE) and seminal plasma (somatic ACE) were spectrophotometrically determined. Total testicular ACE activity in spermatozoa was measured by solubilization of spermatozoa with Triton X-100. Membrane testicular ACE activity was measured in a sperm : PBS suspension. Sperm concentration and sperm motility were 136.6 +/- 154.1 x 10(6)/mL and 58.6 +/- 23.4%, respectively (mean +/- SD). Enzyme activities of membrane testicular ACE, total testicular ACE and somatic ACE were 0.273 +/- 1.219 microU/10(6) spermatozoa, 0.35 +/- 1.34 microU/10(6) spermatozoa and 684.7 +/- 226.6 mU/mL, respectively. A negative correlation was observed between sperm motility and membrane testicular ACE activity (p < 0.05). Membrane testicular ACE activity in 44 normal semen samples was 0.04 +/- 0.02 microU/10(6) spermatozoa, whilst that in 36 abnormal semen samples was 0.24 +/- 0.42 microU/10(6) spermatozoa. There was a significant difference between these two groups (p < 0.01). Membrane testicular ACE in sperm samples from normozoospermic men was significantly lower than that from oligoasthenozoospermic men (p < 0.05). These findings suggest that testicular ACE is released from normal functional spermatozoa for them to have fertilizing ability.