Toxic potential of dietary genistein isoflavone and beta-lapachone on capacitation and acrosome reaction of epididymal spermatozoa

We determined if acrosomal reaction was influenced by exposure of sperm cells to two dietary phytochemicals, genistein isoflavone and beta-lapachone, using the rat model. Spermatozoa were capacitated in capacitating medium with or without genistein isoflavone and beta-lapachone, and the percentage of posttreatment acrosome reaction compared with controls was assessed with two fluorescent probes, chlortetracycline (CTC) and fluorescein isothiocyanate- Pisum sativum ag-glutinin conjugate (FITC-PSA). Spermatozoa were permeabilized in ethanol and labeled with the FITC-PSA or CTC to determine the acrosome status. The results revealed that calcium ionophore could induce acrosome reaction in spermatozoa and that acrosome-reacted sperm cells showed obvious darkness in the head region, whereas acrosome-intact sperm displayed bright fluorescence over the entire sperm head. The basic response and pattern of acrosome reaction status were significantly similar in both CTC and FITC assays and in both treatment (genistein and beta-lapachone) groups. It was observed that higher doses of both genistein and beta-lapachone significantly suppressed acrosome reaction and that this inhibitory effect was both dose- and time-dependent. It was stipulated that the observed genistein inhibition of acrosome reaction could be due to suppression of protein kinase C, and that beta-lapachone could inhibit acrosome reaction through direct cytotoxic effects on sperm cell membrane at higher doses. However, light microscopic examination indicated that both phytochemicals had no significant effect on sperm morphology. It is concluded that, in view of the fact that acrosome reaction is a physiological prerequisite for fertilization of most mammalian eggs, both genistein and beta-lapachone could potentially suppress male fertility via suppression of acrosome reaction at higher doses, but could enhance fertility by promoting acrosome reaction at lower doses. This bimodal mode of action of both phytochemicals could offer a potentially new dimension in the search for causes of male infertility and possibly for male contraceptive development.     

Effects of hypothermic liquid storage and cryopreservation on basal and induced plasma membrane phospholipid disorder and acrosome exocytosis in boar spermatozoa

Flow cytometry was utilised to determine whether short-term (Day 1) or long-term hypothermic liquid storage (Day 5), or cryopreservation of boar spermatozoa (1) caused changes in plasma membrane phospholipid disorder (MPLD) and acrosome exocytosis (AE), indicative of an advanced stage of capacitation or acrosome status, and (2) facilitated or inhibited the induction of capacitation and the acrosome reaction. Merocyanine with Yo-Pro-1 and peanut agglutinin-fluorescein isothiocyanate with propidium iodide were used to identify MPLD and AE, respectively, in viable spermatozoa. The incidence of basal sperm MPLD and AE in fresh semen was very low (1.1 and 2.2%, respectively) and was increased (P < 0.05) only a small amount in Day 5 and cryopreserved semen (3-8%). Compared to no bicarbonate, incubation with bicarbonate increased MPLD, but the response was greatest (P < 0.05) in fresh sperm (52.3%) compared with Day 1 (36.6%), Day 5 (13.9%) and cryopreserved sperm (13.6%). Incubation with calcium ionophore A23187 increased AE in spermatozoa, but the response was less (P < 0.05) for fresh (34%) and cryopreserved (27%) semen than for Day 1 (45%) and Day 5 (57%) semen. In summary, hypothermic liquid storage and cryopreservation of boar spermatozoa did not advance capacitation or acrosome status in viable spermatozoa, but did alter their responses to induction of capacitation and the acrosome reaction.     

Plasma Membrane Cytoskeletal Complex of the Mammalian Spermatozoa

Human and ram sperm were incubated in vitro in Tris buffer with (1) calcium ions, (2) ionophore A23187, (3) calcium plus A23187, (4) without any substance, as described in [13], and were reincubated in situ with the protein S-1 of heavy meromyosin (1 mg/ml). These spermatozoa were examined with the electron microscope to study and characterize the cytoskeletal structures after negative staining, freeze fracturing, and thin sectioning. The formation of the cytoskeletal complex, associated with the plasma membrane in the postacrosome region, appeared to be triggered by S-1 in those sperm that were earlier treated with the ionophore. The dynamic nature of actin present in the postacrosomal dense matrix becomes evident with the formation of a cytoskeletal basket in that region. The cytoskeletal complex may play an important role during the exocytotic acrosome reaction, helping to retain the shape of the sperm head and protect the genetic material until fertilization.     

Fucoidin inhibits the zona pellucida-induced acrosome reaction in human spermatozoa

We recently reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits tight binding of human sperm to the human zona pellucida in vitro and that several oligosaccharides obtained after acid hydrolysis possess sperm-zona pellucida binding inhibitory activity equal to the original fucoidin. This inhibition may be specific to sperm-zona interactions or may be the consequence of the interruption of capacitation, a series of biochemical and physiological events leading to final sperm maturation, that must occur for successful fertilization. Completion of capacitation is most often determined by assessing two end-points of the process: acquisition of hyperactivated motility and ability to complete the acrosome reaction. Here, we examined the effects of fucoidin on these two end-points of capacitation in vitro. Fucoidin did not affect the proportion of sperm with hyperstimulated motility. Neither did fucoidin cause an increase in sperm that had spontaneously acrosome-reacted at 4.5 hours compared to controls as evaluated by indirect immunofluorescence using the acrosomal marker, monoclonal antibody, T-6. Comparable percentages of sperm had completed the acrosome reaction when exogenously stimulated by calcium ionophore A23187 with and without the addition of fucoidin. However, in the presence of fucoidin, stimulation of the acrosome reaction by acid solubilized human zonae pellucidae was significantly inhibited. These data indicate that fucoidin does not impede the normal progression of capacitation. These results provide strong evidence to support the hypothesis is that the inhibitory effect of fucoidin is at the level of the sperm membrane since inhibition can be bypassed by increasing intracellular calcium directly with a calcium ionophore.     

Acrosome stability in the spermatozoa of dasyurid marsupials

The spermatozoa of most marsupials lack nuclear stabilising disulfide-bonded protamines found in eutherian mammals. However, disulfide stabilisation has been observed in the acrosome of macropodid (Macropus eugenii) and phalangerid (Trichosurus vulpecula) marsupials. As a result this organelle, which is normally fragile in eutherian mammals, is robust and able to withstand physical and chemical challenge in these marsupials. The present study examined acrosomal characteristics of the spermatozoa of three dasyurid marsupials; the fat-tailed dunnart (Sminthopsis crassicaudata), eastern quoll (Dasyurus viverrinus) and northern quoll (Dasyurus hallucatus). In all species examined Bryan's staining demonstrated that significant acrosomal loss occurred following physical challenge with osmotic stress, cryopreservation without cryoprotectant and exposure to detergent (Triton-X). Bromobimane staining indicated that the acrosomes of dasyurids lacked stabilising disulfide bonds. As reported for the wallaby and possum, calcium ionophore (A23187) did not induce the acrosome reaction-like exocytosis in dasyurid spermatozoa but treatment with diacylglycerol (DiC8) caused significant acrosome loss at concentrations similar to those effective for other marsupials. The present study found that the spermatozoa of dasyurids are more sensitive to physical challenge than the previously-studied marsupials and we suggest that this is due to the absence of acrosomal stabilising disulfide bonds.     

Freezability of boar spermatozoa is improved by exposure to 2-hydroxypropyl-beta-cyclodextrin

The influence of 2-hydroxypropyl-beta-cyclodextrin (HBCD) exposure on post-thaw spermatozoa prior to freezing using acrosome integrity and the parameters of motility was studied. Acrosomal status was monitored by means of FITC-labelled peanut agglutinin, and the motility parameters were assessed using a computer-assisted sperm motility analysis (CASA) system. The spermatozoa were exposed to HBCD over a period of 3 h, during which the cells were slowly cooled from 25 to 5 degrees C, and then frozen into pellets. The percentage of frozen thawed spermatozoa with intact acrosomes in 40 mM HBCD group was approximately three-fold higher than that of the control. The motility and progressive motility values of the frozen-thawed spermatozoa were found to increase significantly with increased HBCD concentrations. On the other hand, further addition of cholesterol-3-sulfate to the BF5 extender containing 20 mM HBCD resulted in a drastic decrease in the percentage of spermatozoa with intact acrosomes, and decreased motility and progressive motility, suggesting that cholesterol-sulfate probably counter-acted the protective action of HBCD. In conclusion, the results of the present study indicate that HBCD protected boar spermatozoa against freeze-thaw damage, possibly by means of stimulating the efflux of membrane cholesterol.     

Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection

This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after i.v.f. or intracytoplasmatic sperm injection (i.c.s.i.). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL(-1) SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39 degrees C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators' observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For i.v.f., frozen-thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For i.c.s.i., semen derived from the same culture systems as that for i.v.f. was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the i.c.s.i. system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.     

Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws

Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to 4 degrees C in citrate-, Tris- and lactose-based diluents (Experiment 1) and as pellets in 0.25- and 0.5-mL straws on either dry ice or over liquid nitrogen vapour (Experiment 2) to determine the effects diluents and packaging on their motility and acrosome integrity. In Experiment 1, sperm motility was higher after cooling to 4 degrees C and after freeze-thawing (0 but not 3 h post-thaw) for spermatozoa extended in the lactose- than the citrate- or Tris-based diluent (P < 0.05). Post-thaw acrosome integrity after cooling to 4 degrees C and post-thaw (0 h) was reduced for spermatozoa frozen in citrate- compared with lactose- or Tris-based diluents, but was similar for all groups 3 h after thawing. In Experiment 2, sperm motility immediately after thawing was higher for pellet freezing than for 0.25- or 0.5-mL straws on dry ice or liquid nitrogen vapour (P < 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P > 0.05). Acrosome integrity was similar for all groups immediately after thawing and 3 h post-thaw. Cryopreservation of epididymal alpaca spermatozoa is feasible, with retained motility and acrosome integrity post-thaw. Freezing as pellets in a lactose-based diluent is recommended.     

Reproductive effects after exposure of male mice to vincristine and to a combination of X-rays and vincristine

This study was performed to investigate the effects of exposure to vincristine (VCR) alone (1 mg kg(-1) or 2 mg kg(-1)) and in combination with X-rays (0.25 Gy + 1 mg kg(-1) VCR or 1.00 Gy + 2 mg kg(-1) VCR) on the quality and quantity of spermatozoa, and the offspring of exposed Pzh : Sfis male mice. Both VCR and X-rays plus VCR reduced testis weight and sperm count, caused deterioration with respect to sperm morphology, and caused a slight increase in DNA damage. Exposure of some stages of male germ cells to high doses of VCR either alone or in combination with X-rays reduced the rate of pregnant females and the fertility of males. Such treatments reduced the number of total and live implantations, and induced dominant lethal mutations. The results of this study demonstrated the reproductive genotoxicity of VCR with or without X-rays, but did not unequivocally confirm their ability to cause external malformations in offspring.     

分段射精法收集少精子症患者精液质量比较

目的:探讨分段射精法在精子优选过程中的有效性。方法:采用分段射精法收集少精子症患者前后两部分精液,分别检测前后两部分精液的精子参数。结果:前部分精液的精子密度、精子活动率、精子形态和精子顶体完整性均显著高于后部分,而精子存活率、精子膜的完整性前后两部分无显著差异(P>0.05)。结论:对于少精子症患者,采用分段射精法优选精子,能有效改进精子的质量参数,提高精液中精子密度、精子活动率、正常形态精子和正常顶体精子比例。     

Effect of Ace-Inhibitors,Calmodulin Antagonists,Acetylcholine Receptor Blocking,and Alpha Receptor Blocking Agents on Motility of Human Sperm

The purpose of this study was to investigate the influence of several pharmacological compounds on the motility and velocity of washed human spermatozoa. Results were evaluated by multiple exposure photography and computer-aided picture analysis. The motility-inhibiting effect of the antifertility drug gossypol was confirmed. Gossypol proved to be a potent inhibitor of the angiotensin converting enzyme (ACE) detectable in high concentrations in seminal plasma. However, human sperm motility was not inhibited during incubation with two other specific ACE-inhibitors (captopril, enalapril). On the contrary, high concentrations of captopril even showed a slight motility-stimulating effect. These results indicate no direct involvement of ACE in the regulation of sperm motility but suggest a direct interaction of gossypol with the plasma membrane of spermatozoa. To clarify whether or not gossypol blocks membranous ion transport, the effect of well-defined ion transport blocking agents on sperm motility was investigated. It was determined that the acetylcholine receptor blocker alpha-bungarotoxin and trifluoperazin, a specific calmodulin antagonist, inhibit sperm motility completely. Since stimulation of sperm motility by captopril may be due to an alpha-mimetic action of this compound, the influence of two alpha receptor blockers (bromocriptine, lisuride) on sperm motility was studied. Although lisuride inhibited sperm motility completely, bromocriptine revealed no influence. A temporary and reversible intervention with membrane transport processes could be a suitable way to regulate human sperm motility and male fertility.     

Comparison of different sperm cryopreservation procedures on post-thaw quality and heterologous in vitro fertilisation success in the ocelot (Leopardus pardalis)

Cryopreservation of spermatozoa from free-living ocelots (Leopardus pardalis) could benefit their conservation by facilitating gene flow between in situ and ex situ populations without requiring removal of additional cats from the wild. The objective of this study was to investigate three different methods of ocelot sperm cryopreservation to identify the most appropriate technique for use in a field environment. Male ocelots (n = 10), housed in North American zoos, were anaesthetised with tiletamine-zolazepam (7 mg kg(-1) bodyweight; i.m.) and subjected to a regimented electroejaculation procedure. Recovered semen was evaluated for sperm concentration, motility and morphology and processed for cryopreservation by three methods: (1) pelleting on dry ice, (2) freezing in straws over liquid nitrogen vapour; and (3) freezing in straws in a dry shipper. Frozen samples were thawed and assessed for post-thaw acrosome status, viability, motility over time and ability to fertilize viable domestic cat oocytes. Although several post-thaw sperm parameters varied (P < 0.05) among freezing methods, frozen-thawed ocelot spermatozoa from all treatments showed a similar (P > 0.05) capacity to bind, penetrate and fertilize viable domestic cat oocytes. These findings suggest that spermatozoa collected from male ocelots under field conditions may be frozen in straws either using liquid nitrogen alone or in a charged dry shipper to retain adequate functional competence after thawing for use with assisted reproductive procedures.     

In vitro characteristics of fresh and frozen-thawed ram spermatozoa after sex sorting and re-freezing

The in vitro function of sex-sorted, frozen-thawed ram spermatozoa derived from fresh or frozen semen was investigated. Sorted, frozen-thawed spermatozoa had higher (P < 0.05) motility, viability, acrosome integrity and mitochondrial activity than non-sorted, frozen-thawed controls immediately following thawing and after incubation at 37 degrees C for 3 and 6 h. Similarly, frozen-thawed, sorted, re-frozen-thawed spermatozoa outperformed (P < 0.05) non-sorted controls upon thawing (mitochondrial activity) and following a 3-h incubation (motility, viability/acrosome integrity and mitochondrial activity), but there were no differences after incubation for 6 h (P > 0.05). Velocity characteristics (computer assisted sperm assessment 0-6 h post-thaw) of sorted spermatozoa derived from either fresh or frozen semen remained inferior (P < 0.05) to non-sorted spermatozoa, as did their ability to penetrate artificial cervical mucus after thawing. Direct comparison of cryopreserved spermatozoa derived from either fresh or frozen semen revealed that frozen-thawed, sorted, re-frozen-thawed spermatozoa had comparable (P > 0.05) motility, viability/acrosome integrity, mitochondrial activity, average path velocity and oviducal binding capacity immediately post-thaw, but reduced (P < 0.05) quality after 3 and 6 h of incubation. These findings indicate that, under the tested in vitro conditions, sex-sorted spermatozoa derived from fresh semen are superior in some respects to those derived from frozen semen. Further, that the use of either technique, while reducing velocity characteristics and cervical mucus penetration, results in comparable, if not enhanced motility, membrane and mitochondrial function in the post-thaw population of spermatozoa when compared with non-sorted, frozen-thawed controls.     

The effects of levonorgestrel on various sperm functions

Two doses of 750-μg levonorgestrel at 12 h apart is one of the regimens for emergency contraception. The mechanism of action of this regimen is not fully known. We investigated whether levonorgestrel influences sperm functions and thereby, exerts contraceptive activity. The motility, acrosome reaction, zona binding capacity, and oocyte fusion capacity of human spermatozoa treated with 1, 10, and 100 ng/mL levonorgestrel for 3 h were evaluated. Levonorgestrel decreased the curvilinear velocity of the treated spermatozoa in a dose-dependent manner. A significant decrease in straight-line velocity, average path velocity and linearity were also found with 100 ng/mL levonorgestrel treatment. This concentration of levonorgestrel, but not others, also marginally decreased (p = 0.045) the zona binding capacity of the treated spermatozoa. The steroid had no effect on acrosome reaction but had a dose-dependent inhibition on spermatozoa-oocyte fusion. These data show that levonorgestrel affects sperm function only at high concentration and the contribution of these effects to emergency contraception is unlikely to be significant.     

人精子冷冻前后超微结构及受精能力的变化

目的了解人精子冷冻前后超微结构及受精能力的变化。方法对10名志愿者的精液分别在冷冻前、冷冻后进行人精子低渗膨胀试验(HOS)、活动率及穿卵率(SPA)测定,并利用电镜对精子进行微细结构观察。结果冷冻后SPA及精子活动率与冷冻前相比显著下降(P<0.05),精子微细结构的变化主要局限于头部顶体,而精子形态、生物膜及亚细胞结构基本完整。结论人精子冷冻后虽然微细结构及功能受到一定程度的损伤,但部分精子的顶体内膜和中纬段未丢失,精子尾部的动力装置未受到损害。     

醋酸乌利司他体外对人精子参数与功能影响的研究

目的：醋酸乌利司他（ulipristal acetate,UPA）是一种孕激素受体调节剂,目前作为紧急避孕药物广泛应用于临床。本研究探索在体外UPA对人精子参数与功能的影响。方法：选择15例精液参数正常的标本,液化后以G-IVF调节精子浓度为2×106/mL。每例标本再分为6份,每份0.5 mL,共6组（n=15）。密度梯度离心后,其中4组分别在含0.04、0.4、4、40 μmol/L UPA的培养液中孵育1 h;另2组为空白对照组、二甲基亚砜（DMSO）对照组。检测各组精子活力、存活率、形态、DNA完整性、顶体反应、精子超活化、精子内游离钙离子浓度。结果：不同浓度的UPA体外处理精子,精子存活率、活力、形态与对照组（空白+DMSO）比较差异无统计学意义（P＞0.05）。UPA浓度达到0.4 μmol/L时处理精子,精子损伤的比例和彗尾长度显著增加,精子超活化的比例降低（P＜0.01）;顶体反应被抑制,精子内钙离子浓度也显著降低（P＜0.01）。结论：UPA在体外可抑制精子的顶体反应和超活化,降低精子内钙离子浓度,可能与UPA致精子损伤有关。     

Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus)

Research was conducted to develop sperm sorting and novel sperm preservation methodologies for sex predetermination in the bottlenose dolphin (Tursiops truncatus) using artificial insemination. In Study 1, the effect of seminal plasma (SP), sperm concentration and freezing rate (FR) on in vitro sperm quality of liquid-stored, non-sorted spermatozoa was examined. There was no effect (P > 0.05) of prefreeze SP addition on post-thaw quality (progressive motility, kinetic rating, sperm motility index (SMI), viability and acrosome integrity). Post-thaw motility parameters and viability were higher (P < 0.05) for slow FR than fast FR samples. In Study 2 investigating the effects of liquid storage and sorting on sperm quality, motility and SMI after sorting and centrifugation were lower (P < 0.05) than those of the initial ejaculate. The sort rate for enrichment (91 +/- 4% purity) of X- and Y-bearing spermatozoa was 3400 +/- 850 spermatozoa sex(-1) s(-1). In Study 3, compared with a modified straw method, directional freezing resulted in enhanced in vitro quality of sorted and non-sorted spermatozoa derived from liquid-stored semen (P < 0.05). In Study 4, endoscopic insemination of three dolphins with sorted, frozen-thawed X-bearing spermatozoa resulted in one conception and the birth of a female calf. High-purity sorting of dolphin spermatozoa, derived from liquid-stored semen, can be achieved with minimal loss of in vitro sperm quality and samples are functional in vivo.     

Evaluation of human spermatozoa in cervical mucus: comparison of different microscopic and extraction techniques

This study was designed to describe an accurate and consistent microscopic technique for the assessment of sperm number and motility in sperm-cervical mucus samples, such as those of postcoital tests (PCTs), and to identify a suitable method to extract functional spermatozoa from cervical mucus (CM). Sperm-CM preparations containing various sperm concentrations were counted using three different microscopic illuminations. The dark field-Makler technique was compared with the more classical bright field-slide technique currently used by our clinicians. Several sperm extraction techniques were applied first to bovine (BCM) and then to human (HCM) cervical mucus. Dark field microscopic illumination provided accurate, fast, and easy sperm identification. Counting variability was significantly greater with bright field-slide than with dard field-Makler, while sperm motility was always higher with this latter methodology. A high degree of agreement (intraclass correlation coefficient = 0.965) among three raters, i.e., low interobserver variability, was obtained only with dark field-Makler. Extraction procedures based on "swim-out," Percoll, trypsin, an enzyme cocktail, and mercaptoethanol resulted in small sperm yields in BCM. Mercaptoethanol and trypsin also showed poor sperm recovery in HCM. Among the protocols with the largest yields, the mechanical technique had the largest amount of residual CM, and bromelain reduced sperm motility. The extraction with dithiothreitol (DTT) showed the best results with a mean sperm recovery of 76% and enhanced sperm motility. Sperm viability as well as spontaneous and induced acrosome reaction were conserved in all techniques. In conclusion, use of the dark field-Makler counting technique in combination with DTT extraction of spermatozoa from CM samples, such as those of PCTs, would allow accurate and functional assessment of spermatozoa for preliminary contraceptive efficacy or infertility evaluation.     

Effects of bull, sperm type and sperm pretreatment on male pronuclear formation after intracytoplasmic sperm injection in cattle

This study investigated the effects of the bull, sperm type (dead, immotile or motile) and sperm pretreatment (i.e. mechanical (tail-cutting or tail-scoring) or chemical (heparin, heparin + caffeine, calcium ionophore A23187 or dithiothreitol)) on male pronuclear formation after intracytoplasmic sperm injection (ICSI) in cattle. Three experiments were conducted. In Experiment 1, spermatozoa from three bulls (A, B and C) were used for both ICSI and in vitro fertilization (IVF). The results were that sperm from bull B yielded a higher penetration/male pronuclear formation rate than that of bull C when used for IVF (89.6% v 25.6%, P<0.01). However, when injected into oocytes by ICSI, sperm from bull C had a higher male pronuclear formation rate than that of bull B (34.6% v. 16.1%, P<0.05). The effects of sperm type and mechanical pretreatment were examined in Experiment 2. No significant difference was found in the male pronuclear formation rate when the three types of sperm were injected into oocytes. Tail-scored sperm achieved a higher male pronuclear rate than that of non-mechanically treated ones (38.2% v. 13.2%, P<0.005). In Experiment 3, chemical pretreatments were tested and compared. Higher male pronuclear rates, compared with the control, were obtained when sperm were pretreated with heparin + caffeine, calcium ionophore A23187 and dithiothreitol (48.2%, 62.5% and 64.5% v. 25.0%, P<0.05, 0.005 and 0.005, respectively). These results indicate that (1) there is a bull variation in male pronuclear formation with ICSI, and the male pronuclear rate by ICSI is not coincident with the results by IVF, (2) immobilization of a spermatozoon by tail-scoring before ICSI can improve the formation of the male pronucleus, and (3) an appropriate chemical pretreatment of spermatozoa is necessary to achieve a higher rate of male pronuclear formation.     

Quantitative Evaluation of the Human Spermatozoal Motility and Acrosome Reaction in Infertile Ollgozoospermic and Fertile Euspermic Men

Idiopathic oligozoospermia is one of the most important problems in Andrology, but up to now it is poorly understood because the often routine conventional semen parameters, unquestionably are not directly related to the evaluation of the morphological and functional integrity that determines the spermatozoan fertilizing capacity. A non complex strategy was designed to determine the presence of alterations in the functional integrity of the spermatozoa from infertile men with idiopathic oligozoospermia and from euspermic fertile men, by the quantitative analysis of the spermatozoan motility and the acrosome reaction. There was a lower percentage of acrosome reacted spermatozoa within the semen of the infertile men, accompanied with a significant decrease in the motility percentage, sperm velocity and motility index in comparison with semen from fertile men. These data strongly support a possible detrimental structural and functional integrity of the spermatozoa from the oligozoospermic men.