The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing

A firm biophysical basis for the cryopreservation of human spermatozoa is limited by a lack of knowledge regarding the water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPA). Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape and their small dimensions which are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPA. Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 micrometer and a radius of 0.42 micrometer with an osmotically inactive cell volume, V(b), of 0.23V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for human sperm cells in modified media are: L(pg) = 2. 4x10(-14) m(3)/Ns (0.14 micrometer/min-atm) and E(Lp) = 357.7 kJ/mol (85. 5 kcal/mol) (R(2) = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L(pg)[cpa] = 0.67x10(-14) m(3)/Ns (0.04 micrometer/min-atm) and E(Lp)[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R(2) = 0.98). These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPA. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates (<100 degrees C/min) and the numerically predicted optimal cooling rates (>7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.     

Fertilization and early embryology: A cytosolic sperm factor triggers calcium oscillations and membrane hyperpolarizations in human oocytes

Intracellular free Ca2+ concentrations ([Ca2+]₁) and membranepotentials were measured in mature human oocytes. Injectionof cytosolic extracts made from human or hamster spermatozoatriggered oscillations in [Ca2+]₁ in human oocytes similar tothose described previously in mouse and hamster oocytes. Incontrast, injection of carrier buffer caused no [Ca2+]₁ increaseand injection of Ca2+-containing solutions caused only a single[Ca2+]₁ transient. Injection of human sperm extracts also triggered[Ca2+]₁ oscillations in mature mouse oocytes. The [Ca2+]₁ oscillationsin human oocytes were accompained by hyperpolarizations in membranepotential. Perfusing oocytes with the sulphydry1 reagent thimerosalalso caused oscillations in the free [Ca2+]₁ concentration simultaneouslywith membrane potential hyperpolarizations. These data suggestthat human oocytes possess a similar mechanism for generating[Ca2+]₁ oscillations to those described in other mammalian oocytesand a membrane potential response similar to that seen previouslyspecifically in hamster oocytes. The data also support the viewthat human oocytes are activated at fertilization by diffusionof a protein from the spermatozoa into the ooplasm after gametemembrane fusion.     

In-vitro effects of anti-sperm antibodies on human sperm movement

The present study was designed to investigate whether autoantibodlesto external domains of the sperm plasma membrane affect themovement of normal motile spermatozoa. Eight sera and 20 semInalplasma samples containing high levels of anti-sperm antibodiesas well as antibodies eluted from the sperm fraction of 19 autounmuneejaculates were incubated with donor's motile spermatozoa, obtainedby swim-up migration in Tyrode's solution. Sperm movement wasanalysed using is exposure microphotography when >70% ofthe spermatozoa were coated with antibodies (after 30–90mi of incubation). At least 50 tracks of progressively motilespermatozoa were analysed in order to obtain the mean valuesof the amplitude of lateral head displacement (ALH) and thevelocity of progression (VSL). Serum antibodies and sperm elutedantibodies had quite consistent but opposite effects on spermmovement; serum antibodies Increased ALH and decreased VSL whereaseluted antibodies decreased ALH and increased VSL. Seminal antibodiesdid not affect these two parameters significantly. Furthermore,seminal antibodies and sperm eluted antibodies obtained fromthe same ejaculates had distinct effects on ALH and/or VSL.This diversity was apparently not linked to antibody isotypeor localization on the sperm membrane; it might be due to differencesin the composition of the extracellular media. These resultssuggest a dynamic effect of anti-sperm antibodies on sperm movement,a possibility that merits further investigation.     

Effects of cryopreservation on progesterone-induced ion fluxes and acrosome reaction in human spermatozoa

The present study evaluated the effects of cryopreservation on progesterone-induced variations of calcium ion concentration [Ca(2+)](i), plasma membrane potential and acrosome reaction in human spermatozoa. Spermatozoa from 10 fertile donors were divided in two equivalent aliquots, one used as control (fresh spermatozoa) and the other used after freezing-thawing. Measurement of spermatozoa [Ca(2+)](i) before and after freezing-thawing showed a significant reduction of basal [Ca(2+)](i) in thawed spermatozoa (P < 0.01). Progesterone induced a rise of [Ca(2+)](i) both in fresh and thawed spermatozoa with a significant reduction after freezing-thawing (P < 0.01). The monitoring of sperm plasma membrane potential demonstrated that progesterone induced plasma membrane depolarization in fresh spermatozoa that was absent in thawed spermatozoa. The inhibitory effects of freezing-thawing on progesterone induced [Ca(2+)](i) and plasma membrane potential variations in human spermatozoa were closely related to the inhibition of the acrosome reaction. In conclusion the present study demonstrates that freezing-thawing procedures reduce the responsiveness of human spermatozoa to progesterone in terms of [Ca(2+)](i) rise and completely inhibit its effects on plasma membrane potential variations, thus supporting the hypothesis that freezing-thawing procedures may differently modify the plasma membrane receptors for progesterone in human spermatozoa which are known to express at least two receptors for this steroid in their plasma membrane.     

Immunology: Identification of human sperm surface glycoproteins by sperm membrane-specific autoantibodies

In order to identify the surface antigens of human spermatozoarecognized by the sera of immune infertile men, sperm membrane-specificantibodies were obtained from three serum samples exhibitinghigh titres of antibodies with different sperm-binding patterns.Serum antibodies were adsorbed onto normal motile spermatozoaand subsequently eluted from the sperm membrane. Using the immunoblottingtechnique under renaturating conditions, the sperm-eluted antibodieswere tested against an electro-phoretically fractionated spermmembrane preparation. A total of 15 protein bands ranging from110 to 16 kDa were defined, but the immunoblot profiles differedquantitatively and qualitatively from one serum to another.Only two polypeptides were recognized by the three sperm membrane-specificantibody preparations; one of 90 kDa and another of 110 kDa.Blots were also used for the affinodetection of specific oligosaccharideside chains. Three lectins were tested (concanavalin A, Pisumsativum, and wheat germ agglutinin). Of the 15 protein zonesrecognized by the antibodies, 11 bound at least one lectin andshould contain glycopeptides with oligosaccharides of four differenttypes: N-linked biantennary complex type, N-linked fucosylatedcomplex type, N-linked lactosaminyl complex type with terminalsialic acid and polysialyl type oligosaccharides. Further analysisof these glycoproteins will be pursued with sperm-associatedantibodies eluted from the ejaculates of infertile men in orderto define those with a potential role in the fertilization process.     

Fertilization and early embryology: Direct assessment of cryopreservation of human spermatozoa using a cryomicroscope and computer-aided sperm analysis

Use of a cryostage has enabled direct observation of human spermatozoaas they are cryopreserved and thawed. Crystallization and recrystallizationevents are readily observed. In combination with computer-aidedsemen analysis (CASA) equipment it was possible to determinethe consequence of altering the cooling, freezing and thawingrates of a temperature-rate profile on sperm motility. Increasingthe cooling rate to 50°C/min resulted in significantly lowerpre-freeze to post-thaw ratios for average path velocity (VAP,13%), mean straight line velocity (VSL, 35%), mean linearity(LIN, 28%) and straightness (STR, 24%), while the ratio of thenumber of cells crossing the field of view (NCF) significantlyincreased (30%) compared to a standard freeze-thaw temperaturerate profile. The NCF pre-freeze to post-thaw ratio was associatedwith the percentage of cell recovery after cryopreservation.Faster thaw rates resulted in better survival of the cells,perhaps due to the shorter time during which recrystallizationoccurred. The NCF ratios were significantly higher (33 and 30%for thaw rates of 50 and 100°C/min respectively) than forthe standard profile samples. Previous studies on cell survivalhave shown a link between the cooling and thaw rates. The cryostageshould prove invaluable in future studies to identify the causesof cryodamage to spermatozoa. When used in combination withCASA, changes to sperm function during cryopreservation canbe accurately measured.     

The effects of human skin fibroblast monolayers on human sperm motility and mouse zygote development.

A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified.     

Andrology: The variable effects of 2'-deoxyadenosine on human sperm motility and hyperactivation in vitro

The response of human sperm motility and hyperactivation tothe stimulant 2'-deoxyadenosine (2'-DEA) was studied in vitrousing computer-assisted sperm motion analysis. A total of 20randomly selected individuals with normal sperm counts as definedby the World Health Organization were chosen and their migration-separatedspermatozoa exposed to a range (0.1–10.0 mM) of concentrationsof 2'DEA. The straight line velocity (VSL) was increased abovecontrol values only at 0.1 mM, while the curvilinear velocity(VCL) and lateral head displacement (ALH) were increased significantlyat all concentrations. Linearity of progression (LIN), on theother hand, declined with increasing concentration of 2'-DEA.These changes were related to a significant increase in thenumber of spermatozoa exhibiting hyperactive-like motion. Therewas, however, considerable intra-individual variability in theresponse to 2'-DEA. In some individuals VCL and ALH exhibitedlittle or no response to 2'-DEA, whilst in others an increaseabove the control of 50–55% occurred. The maximum responsefor VCL and ALH occurred at 2.5 mM 2'-DEA. Individuals showedgreater variability in the percentage of spermatozoa exhibitinghyperactivity in response to 2'-DEA, with increases rangingfrom 76 to 948% of the control value, although the maximum responsewas also most commonly seen at 2.5 mM 2'-DEA. The diversityof response to 2'-DEA emphasizes the importance of tailoringdoses to the individual rather than employing one concentrationfor all. Further tests on a subgroup of the individuals examinedthe longevity of spermatozoa in response to 24 h of continuedexposure to 2'-DEA. Prolonged exposure to 0.1 mM 2'-DEA continuedto enhance VSL, while higher concentrations produced detrimentaleffects on all other motion characteristics including hyperactivation.     

Effect of human cervical mucus on human sperm motion and hyperactivation in vitro.

The aim of our experiment was to examine the effect of exposure to human cervical mucus on quantitative sperm motility with specific reference to hyperactivated sperm motility. Human spermatozoa were allowed to penetrate cervical mucus for 20 min before swimming into Earle's balanced salt solution tissue culture medium for 25 min. The sperm motion characteristics were compared to those which had been obtained from a direct swim-up for 45 min. Spermatozoa treated with mucus were more 'active' than the control group. Multivariate statistical analysis indicated that cervical mucus promotes hyperactivated motility and that sperm sub-populations exposed to cervical mucus are very heterogeneous, as indicated by the numbers and motility characteristics of spermatozoa.     

Comparison of four fluorochromes for the detection of the inner mitochondrial membrane potential in human spermatozoa and their correlation with sperm motility

BACKGROUND: Sperm motility evaluation is associated with fertility in IVF programmes. The visual estimation of sperm motility is extremely subjective. Hence, alternative methods are required. Among them, determination of mitochondrial membrane potential (Deltapsi(m)) changes of spermatozoa using potentiometric dyes may be a reliable test to determine sperm quality. However, the use of the potentiometric dyes in sperm samples has not been compared. METHODS: We have studied sperm samples from 28 infertile patients enrolled in an IVF programme in flow cytometry after staining of spermatozoa with four commonly used potentiometric dyes. Sperm motility was evaluated visually. RESULTS: As expected, JC-1 seems to detect specifically Deltapsi(m) changes, CMX-Ros, DiOC(6)(3) and TMRE fluorescence is easily analysed and the latter three fluorochromes are particularly suitable for multiparametric staining. Irrespective of the Deltapsi(m)-dependent fluorochromes used to stain spermatozoa, a positive correlation was found between the percentage of Deltapsi(m)(high) cells and forward motility and also with high fertilization rates after IVF. CONCLUSION: The four fluorochromes may be useful for evaluation of sperm samples from infertile patients. The choice of the potentiometric dyes will depend on their fluorescence characteristics in order to use them in combination with other fluorescent markers.     

Andrology: Effects of pentoxifylline and progesterone on human sperm capacitation and acrosome reaction

This study was designed to test the effects of pentoxifyllineand progesterone upon capacitation of fresh human spermatozoa.Capacitation and acrosomal integrity were assessed using thefluorescent probe chlortetracycline on spermatozoa co-stainedwith a supravital fluorescent dye, Hoechst 33258. Hyperactivatedmotility was measured using computer-assisted movement analysis.After exposure to pentoxifylline (1 mg/ml; 30 min), the fluorescent‘B’ pattern, characteristic of capacitated, acrosome-intactcells, increased significantly (P < 0.01), though no increasein ‘AR’ pattern, characteristic of acrosome-reactedcells, was detected. There was a significant increase in hyperactivemotility (P < 0.001). Exposure to progesterone (1µg/ml;60 min) resulted in a significant increase in ‘B’pattern (P < 0.05) and ‘AR’ pattern (P < 0.005),though no effect on the expression of hyperactivation was detected.No effect upon hyperactivation was detected on exposure of freshor cryopreserved spermatozoa to a physiological range of progesteroneconcentrations (0.1–1000 ng/ml). Sequential exposure topentoxifylline then progesterone resulted in a significant increasein ‘B’ pattern, acrosome loss and hyperactivation.Sperm viability was not affected in any treatment group. Theseobservations suggest that pentoxifylline and progesterone affectcapacitation through independent mechanisms. Stimulation ofboth capacitation and acrosome reaction resulted from sequentialexposure to pentoxifylline and progesterone. This may have implicationsfor sperm handling for assisted reproductive techniques.     

Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.

The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C.     

Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa

The objectives of this cross-sectional observational study were: (i) to detect DNA damage and plasma membrane translocation of phosphatidylserine in purified sperm populations of high and low motility, and (ii) to analyse their relationship with the endogenous generation of reactive oxygen species. Ejaculates from infertile men were examined following gradient centrifugation. The main outcome measures were: sperm motion parameters (assessed with a computer analyser), generation of reactive oxygen species (measured by chemiluminescence), DNA damage (detected by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling and monoclonal antibody labelling of single-stranded DNA) and translocation of membrane phosphatidylserine (examined with annexin V staining). DNA fragmentation and membrane translocation of phosphatidyl-serine were observed in the fractions with low and high sperm motility in all patients. The fractions with low sperm motility had significantly higher proportion of cells with DNA damage and production of reactive oxygen species than the fractions with high sperm motility (P < 0.005). DNA fragmentation was significantly and positively correlated with the generation of reactive oxygen species (r = 0.42; P = 0.02). In conclusion: (i) spermatozoa from infertile men display translocation of membrane phosphatidylserine as diagnosed by annexin V positive staining; (ii) DNA damage (fragmentation and presence of single-stranded DNA) can be detected in ejaculated spermatozoa from infertile men in fractions with low and high sperm motility, and (iii) there is a relationship between DNA damage and oxidative stress.     

Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters

BACKGROUND: Detection of apoptosis in sperm samples may help evaluate sperm quality. Recently, it has been suggested that in some ejaculated sperm populations, apoptosis is caspase dependent. The aim of this study was to investigate the presence of activated caspases and examine possible correlations with apoptosis and sperm parameters in semen samples prepared for IVF. METHODS: To detect activated caspases, neat semen from infertile patients and sperm prepared by PureSperm gradient were stained with the fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) and analysed by flow cytometry. Cell death was determined by DNA fragmentation (TUNEL) and mitochondrial membrane potential. Sperm parameters were studied by conventional microscopy. RESULTS: FITC-VAD-fmk stained sperm cells in situ and the subcellular labeling pattern was compatible with the known localization of caspases. A significant correlation was found between the frequency of FITC-VAD-fmk stained cells and cell death markers. In both prepared sperm and neat semen a negative correlation was found between the percentage of FITC-VAD-fmk positive cells and standard parameters (concentration/motility). FITC-VAD-fmk positive cells negatively correlated with high fertilization rates after IVF. CONCLUSIONS: Labelling of sperm cells with the activated caspases-reacting fluorochrome provides a sensitive assay for detection of sperm apoptosis. This cytometric assay can be helpful to test sperm before IVF.     

The effects of cryopreservation on sperm morphology, motility and mitochondrial function

BACKGROUND: The effects of cryoinjury were determined simultaneously on the mitochondrial function, motility, morphology and viability of ejaculated human sperm. METHOD: Rhodamine 123 (R123) uptake (% of sperm) and stain intensity were used to determine sperm mitochondrial activity before and after cryopreservation from the semen of 50 men attending for infertility investigation. Morphology was assessed using Tygerberg's strict criteria and viability was assessed by eosin Y. Sperm motility was measured using computer-assisted semen analysis (CASA). RESULTS: Freeze-thawing caused a 37% (P = 0.001) reduction in normal morphological forms of sperm. All CASA sperm motility parameters except amplitude of lateral head displacement were similarly reduced. R123 uptake and intensity within sperm mitochondria decreased by 36 and 47% respectively (both P = 0.001). In addition, there was a similar significant decrease (31%, P = 0.001) in the viability of the sperm. CONCLUSIONS: Sperm morphology, motility, mitochondrial activities and viability are equally susceptible to cryopreservation-induced damage. R123 intensity is a novel and robust indicator of mitochondrial function before and after such trauma.     

Effects of hypercholesterolaemia on Leydig and Sertoli cell secretory function and the overall sperm fertilizing capacity in the rabbit.

The effects of hypercholesterolaemia on testicular endocrine and exocrine function were evaluated. The influence of hypercholesterolaemia on sperm quality, quantity, and fertilizing potential was also determined. Ten mature rabbits (group A) were fed chow containing 3% cholesterol for 12 weeks. Ten control rabbits (group B) were fed normal chow for the same period. At the end of the experimental period testosterone profiles and sperm parameters were evaluated and the sperm reproductive potential was assessed by in vitro fertilization (IVF) techniques. Peripheral serum testosterone responses to testicular stimulation with human chorionic gonadotrophin, androgen-binding protein activity in testicular cytosols, sperm concentration, sperm motility, length of sperm midpiece, and IVF outcome were all significantly lower in group A than in group B. In contrast, serum cholesterol concentrations were significantly higher in group A. There were no significant differences in either testicular versus intra-abdominal temperature differences or cholesterol concentrations in seminal plasma or testicular tissue between groups A and B. The results suggest that hypercholesterolaemia has a detrimental effect on Leydig and Sertoli cell secretory function, spermatogenesis, epididymal sperm maturation process, and the overall sperm fertilizing capacity.     

Genetics: The use of epididymal and testicular spermatozoa for intracytoplasmic sperm injection: the genetic implications for male infertility

The results and rationale of using testicular and epididymalspermatozoa with intracytoplasmic sperm injection (ICSI) forsevere cases of male infertility are reviewed. A total of 72consecutive microsurgical epididymal sperm aspiration (MESA)cases were performed for congenital absence of the vas (CAV)and for irreparable obstructive azoospermia. ICSI was used toobtain normal embryos for transfer and fertilization in 90%of the cases. The overall fertilization rate was 46% with anormal cleavage rate of 68%. The pregnancy and delivery ratesper transfer were 58 and 37% respectively. The delivery rateper cycle was 33%. In many cases, no epididymal spermatozoawere available and so testicular sperm extraction (TESE) wasused for sperm retrieval. The transfer rate was lower with TESE(84 versus 96%) and the spermatozoa could not be frozen andsaved for use in future cycles. However, there was little differencein pregnancy rates using epidiymal or testicular spermatozoa.The results were not affected by whether the obstruction wascaused by CAV or failed vasoepididymostomy. Both fresh and frozenspermatozoa gave similar results; the only significant factorappeared to be the age of the female. Because of the consistentlygood results obtained using epididymal sperm with ICSI whencompared with conventional IVF, and the similarly good resultswith testicular tissue spermatozoa, ICSI is mandatory for allfuture MESA patients. All CAV patients and their partners shouldbe offered genetic screening for cystic fibrosis; hence pre-implantationembryo diagnosis should be available in any full service MESAprogramme. It is now clear that even with non-obstructive azoospermia,e.g. Sertoli-cell only, or maturation arrest, there are usuallysome small foci of spermatogenesis which allow TESE with ICSIto be carried out. This means that even in men with azoospermiadue to absence of spermatogenesis or to a block in meiosis,there are usually a few spermatozoa available in the testesthat are adequate for successful ICSI. Finally, it is likelythat some forms of severe male factor infertility are geneticallytransmitted and although ICSI offspring have been shown to becompletely normal, it is possible that the sons of these infertilecouples will also require ICSI when they grow up and wish tohave a family.     

Andrology: Effects of platelet activating factor on human sperm function in vitro

The direct effects of platelet activating factor (PAF) and thespecific PAF receptor antagonist, CV-3988, on the fertilizingability of human spermatozoa were investigated. PAF (10^–7–10^–11M) increased the human sperm penetration rates in a sperm penetrationassay at all doses >10^–11 M. In contrast, treatmentof the spermatozoa with 10^–5 CV-3988 caused a significantdecrease in human sperm penetration of zona-free hamster oocytesand adversely affected sperm motility after 24 h of incubation.This suppression was reversed by the addition of PAF. The acrosomereaction was also enhanced by PAF treatment of spermatozoa butthis effect was not observed in calcium-free medium. While 10^–5M CV-3988 decreased the acrosome reaction, the inhibition wasalso reversed by the addition of PAF. These results suggestthat PAF may have a direct role in the fertilizing capacityof human spermatozoa. These findings also suggest that PAF mayhave a clinical application in an in-vitro fertilization programme.     

Chemically and mechanically induced membrane fusion: non-activating methods for nuclear transfer in mature human oocytes

Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplasts. However, the application of electrofusion to human oocytes is hampered by the relative ease with which this procedure induces oocyte activation. Here we tested a previously described chemical fusion technique and an original mechanical fusion procedure in this application. Enucleated metaphase II oocytes were first agglutinated with karyoplasts originating from other metaphase II oocytes and then induced to fuse with the use of polyethylene glycol or by micromanipulation with an intracytoplasmic sperm injection (ICSI) micropipette. Both techniques yielded a high frequency of fusion and did not cause oocyte activation. Moreover, the reconstructed oocytes were easily activated by subsequent treatment with ionophore A23187 and 6-dimethylaminopurine. These techniques may be used in attempts to alleviate female infertility due to insufficiency of ooplasmic factors by nuclear transfer from patients' oocytes to enucleated donor oocyte cytoplasts. For eventual future use in human cloning, they would ensure prolonged exposure of transferred nuclei to metaphase promoting factor, which appears to be required for optimal nuclear reprogramming.