Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods

Previous reports have left unresolved discrepancies between human sperm cryopreservation methods developed using theoretical optimization approaches and those developed empirically. This study was designed to investigate possible reasons for the discrepancies. Human spermatozoa were exposed to 1 mol/l glycerol, 1 mol/l dimethyl sulphoxide (DMSO), 1 mol/l propylene glycol (PG) or 2 mol/l ethylene glycol (EG) at 22, 11 and 0 degrees C, then returned to isosmotic media while changes in cell volume were monitored. Activation energies (E(a)) of the hydraulic conductivity (L(p)) in the presence of cryoprotective agents (CPA) (L(p)(CPA)) were 22.2 (DMSO), 11.9 (glycerol), 15.8 (PG), and 7.8 (EG) kcal/mol. The E(a) values of the membrane permeability to CPA (P(CPA)) were 12.1 (DMSO), 10.4 (glycerol), 8.6 (PG) and 8.0 (EG) kcal/mol. These data indicated that even at low temperatures, EG permeates fastest. The high L(p)(CPA) in the presence of EG and low associated E(a) would allow spermatozoa to remain closer to equilibrium with the extracellular solution during slow cooling in the presence of ice. Collectively, these data suggest that the increase of the E(a) of L(p) in the presence of CPA at low temperature is the likely reason for the observed discrepancy between theoretical predictions of spermatozoa freezing response and empirical data.     

Cryopreservation of fractionated, highly motile human spermatozoa: effect on membrane phosphatidylserine externalization and lipid peroxidation

INTRODUCTION: This study investigated lipid peroxidation (LPO) and membrane integrity following cryopreservation-thawing. METHODS: Infertile men (study group) and donors (control group) were examined. Purified populations of highly motile spermatozoa were cryopreserved using TEST-yolk buffer and glycerol (TYB-G) followed by quick thaw. LPO was measured by a spectrophotometric assay, with and without a ferrous ion promoter. Annexin V binding was used to assess membrane translocation of phosphatidylserine (PS). RESULTS: Pre-freeze LPO was significantly higher in the study than in the control group (P = 0.03). In both groups, LPO measurements after thawing were significantly higher than the pre-freeze samples not exposed to TYB-G (P = 0.002 and P = 0.001 respectively). However, when the pre-freeze samples with TYB-G were compared with the post-thaw samples (all exposed to TYB-G), these differences were not significant. There was a significant increase in PS externalization following cryopreservation in both groups (P = 0.02 and P = 0.003 respectively). In donors, pre-freeze LPO concentrations had a significant positive correlation with thawed spermatozoa depicting PS externalization (r = 0.77, P = 0.04). CONCLUSION: Although patients had higher basal LPO than donors, LPO did not differ between fresh and cryopreserved-thawed fractionated motile spermatozoa. Freezing-thawing was associated with translocation of PS to the external membrane leaflet.     

The yield, motility and performance in the hamster egg test of human spermatozoa prepared from cryopreserved semen by four different methods.

Different procedures were investigated for the dilution of human cryopreserved semen and the preparation of an enriched population of motile spermatozoa for assisted reproduction. The dilution of a 0.25 ml straw of cryopreserved human semen by addition of 2.0 ml Ham's F-10 buffer in one step caused a large decrease in the proportion of motile spermatozoa. This was due to osmotic stress because many of the diluted spermatozoa exhibited swollen tails. To a large extent the damage could be avoided by adding the buffer in 0.10-ml aliquots at 30-s intervals. Spermatozoa obtained after such dilution of cryopreserved human semen were subjected to the swim-up procedure, to centrifugation on two-step gradients of Nycodenz or Percoll, or to filtration through glass fibre paper and compared with respect to yield, motility parameters and penetrating ability in the hamster egg test. The swim-up procedure yielded spermatozoa with excellent motility but only 12% of the available motile spermatozoa were recovered. On both Nycodenz and Percoll gradients, greater than 40% of the available motile spermatozoa were recovered and the average velocity of the spermatozoa was not significantly less than for the swim-up technique. When A23187 was used to promote acrosome reactions in the hamster egg test, Percoll-prepared spermatozoa achieved an average of 8.6 decondensed sperm heads/egg compared to 1.9 for Nycodenz and 1.3 for the swim-up procedure. The yield from glass fibre paper filtration was only 12% and the velocity of the spermatozoa and their performance in the hamster egg test was significantly poorer than in all the other methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of chilling on membrane lipid phase transition in human oocytes and zygotes

BACKGROUND: Chilling injury occurs when the cell membrane undergoes a transition from the liquid state to the gel state. Human oocytes and single-cell zygotes are of similar shape and size but the post-thawing survival rate of oocytes is poorer. We set out to investigate the possible difference in membrane lipid phase transition (LPT) temperature between the two cell types. METHODS: The LPT temperature was measured with a Fourier Transform Infrared analyser, which detects the change in the vibration frequency of the CH2 bond stretches of the membrane lipid molecules during temperature change. The LPT temperatures of unfertilized human oocytes, in vitro-matured oocytes, and immature germinal vesicle (GV) stage oocytes were compared with that of abnormally fertilized human zygotes. RESULTS: The LPT temperatures of zygotes and of mature and immature GV oocytes differ significantly from each other (10.0 +/- 1.2, 16.9 +/- 0.9 and 24.4 +/- 1.6 degrees C respectively; P < 0.05). CONCLUSIONS: Zygotes show a higher resistance to chilling injury compared to oocytes at different developmental stages; this might explain the relatively poor survival rates of cryopreserved human oocytes and indicates the necessity to adjust the cryopreservation protocols in order to minimize cryoinjury.     

The effect of intracytoplasmic sperm injection and semen parameters on blastocyst development in vitro

The present study compares the development and quality of blastocysts derived from conventional oocyte insemination with those derived from intracytoplasmic sperm injection (ICSI). Oocytes were collected from patients undergoing ovarian stimulation with human menopausal gonadotrophins for IVF. Patients with normal semen were assigned to conventional oocyte insemination while those with progressive motility <20% and/or normal sperm morphology < or =4% were assigned to ICSI. Resulting embryos were cultured for up to 6 days. The mean number and percentage of embryos reaching the blastocyst stage and the mean number and percentage of blastocysts of high quality on days 5-6 were assessed for both treatment groups and compared. The influence of paternal factors (sperm concentration, motility, progressive motility, morphology) on blastocyst development and quality were assessed by regression analyses. Significantly more ICSI-derived embryos arrested at the 5- to 8-cell stage (P = 0.024) concomitant with the activation of the paternal genome than those derived from conventional oocyte insemination. Significantly fewer ICSI-derived embryos reached the blastocyst stage on days 5-6 (P<0.001) and significantly fewer ICSI-derived embryos were of high quality (P = 0.002) compared with conventional oocyte insemination. When treatment groups were combined and evaluated by regression analysis, progressive motility and sperm morphology were significantly correlated with diminished blastocyst development and quality (P < 0.05). From these data, we conclude that paternal factors and/or performing ICSI in cases of severe male factor infertility may have a detrimental effect on blastocyst development and their quality.     

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.     

A detailed study of the effect of videoframe rates of 25, 30 and 60 Hertz on human sperm movement characteristics

A comparison was made of the movement characteristics of humanspermatozoa analysed at three videoframe rates (25, 30 and 60Hz) using two computerized motility analysers from Hamilton-ThornResearch (the HTM-2030 and the IVOS) operating at 25 and 30Hz respectively. Analysis at 30 and 60 Hz was performed on theIVOS. The use of uncapacitated, capacitated and pentoxifylline-stimulatedspermatozoa ensured a full range of movement characteristicswas analysed. The velocity parameters curvilinear velocity andaverage path velocity were highly frame-rate dependent, andmean values increased with videoframe rate. An interaction offraming rate and time of data collection resulted in an increasein straight-line velocity with framing rate. Mean lateral headdisplacement and linearity were similar at 25 or 30 Hz but significantlydepressed at 60 Hz. Beat-cross frequency increased by 74% whenanalysed at 60 rather than 30 Hz. The following criteria: curvilinearvelocity >100 µ/s, linearity >65% and lateral headdisplacement >7.5 µm, were used to define hyperactivatedspermatozoa. Significantly more hyperacti-vated cells were identifiedat 30 Hz than 25 Hz (1–10%) but not at 60 Hz. A differentpopulation of cells is likely to have been identified as hyperactivatedat 60 Hz due to alterations in component movement parametersfrom which the definition of hyperactivation was derived. Inconclusion, direct comparisons should not be drawn between dataanalysed at 25 and 30 Hz. Analysis at 60 Hz introduced complexalterations which made simple comparisons with 30 Hz data invalid.     

Changes in the sialylglycoconjugate distribution on the human sperm surface during in-vitro capacitation: partial purification of a 20 kDa sialylglycoprotein of capacitated spermatozoa

Changes in the distribution of sialylglycoconjugates on thesurface of uncapacitated and in-vitro capacitated human spermatozoawere studied by means of two sialic acid specific lectins [Maackiaamurensis agglutinin and Sambucus nigra agglutinin). On theuncapacitated sperm surface, sialylglycoconjugates were foundbe localized from the post-acrosomal region of the sperm headto the tail middle piece, whereas after invitro capacitationthese molecules were only found in a small area of the post-acrosomalregion. The surface of capacitated human spermatozoa was alsoinvestigated by specifically radiolabelling its terminal sialicacid residues. A 20 kDa glycoprotein, which was partially purifiedby anion-exchange chromatography, was the main component ofthe sialylglycoconjugate pattern after in-vitro capacitation.     

Membrane permeability of secondary hydatid cysts of Echinococcus granulosus. Determination of the water diffusional and osmotic permeability coffficients thorugh a syncytial membrane

Diffusional (P_w) and osmotic (P_f) water permeability coefficients were determined for the syncytial epithelium of larval Echinococcus granulosus. P_w was calculated from simultaneous influx measurements of tritiated water and $\text{n-[}{}^{14}\text{C}\text{]}\text{butanol}$ through the hydatid cyst wall. The total diffusional water permeability coefficient, P′_w, was found to be 2.2 × 10^?4cms^?1; which is similar to that previously reported by Rotunno et al. (1974, J. Parasitol. 60, 13–620). Nevertheless, when P′_f is corrected for the unstirred water layer effects, a P_w value of 6.2 × 10^?4cms^?1 is obtained. Thus, the unstirred water layer effects have a very important contribution to P′_w.Total steady state osmotic permeability coefficient, P′_f, was bound to be about 15 × 10^?4 cm s^?1 and it is scarcely affected by those mechanisms that tend to distort the evaluation of P_f. The experimentally determined osmotic coefficient differs from the corrected P_f by only 6%. The P_f/P_w ratio was found to be 2.4.The present study clearly confirms that syncytial membranes can be highly permeable to water, in spite of the fact that they lack tight junctions. Thus, water permeability through epithelial syncytium must be exclusively controlled by the permeability of the apical and/or basocellular membranes.     

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.     

Andrology: The influence of sperm morphology and the acrosome reaction on fertilization outcome after sub-zonal injection (SZI) of human spermatozoa

The usefulness of sub-zonal injection (SZI) for the treatmentof severe male factor infertility has been restricted by lowand unpredictable fertilization rates and the high risk of polyspermyafter the injection of multiple spermatozoa. In this prospectivestudy, we have evaluated whether sperm morphology and the percentageof acrosome-reacted spermatozoa at the time of injection canbe used to predict SZI fertilization outcomes. Populations ofmotile spermatozoa equivalent to those injected were collectedfrom the medium/oil interface immediately after SZI of eachcohort of oocytes. Morphology was assessed using the World HealthOrganization 1987 criteria and the acrosomal status of spermatozoawas determined after staining with rhodamine-conjugated Pisumsativum agglutinin. A fertilization index (FI) was calculatedto express the actual fertilizing potential of the spermatozoainjected. In all, 67 patients underwent 72 SZI cycles. The overallfertilization and polyspermy rates were 36 and 47% respectively,and a clinical pregnancy rate per transfer of 22% was achieved.Linear regression analysis demonstrated a statistically significantrelationship between morphology and the FI (r = 0.506, P <0.0001). Patients with <10% normal morphology always hada FI < 10%, and this was reflected by low fertilization andpolyspermy rates and the high number (32%) of cycles with completefailure of fertilization in this group. In patients with >10% normal morphology, there were two patterns: low (10% FI)or high (>10% FI) fertility. This was evident in the fertilization(23 and 85%, respectively) and polyspermy (25 and 68%, respectively)rates of these two patient sub-groups. While the percentageof acrosome-reacted spermatozoa at the time of injection wasweakly correlated with the FI (r = 0.292, P < 0.05), it couldnot be used to predict differences in fertilization potentialbetween patient sub-groups. We conclude that sperm morphologyand acrosomal status at the time of injection are of limiteduse in predicting SZI fertilization outcomes, although patientswith poor morphology ( 10% normal) have lower fertilizationand polyspermy rates.     

The effect of osmotic stress on the metaphase II spindle of human oocytes, and the relevance to cryopreservation

BACKGROUND: Knowing osmotic tolerance limits is important in the design of optimal cryopreservation procedures for cells. METHODS: Mature human oocytes were exposed to anisosmotic sucrose solutions at concentrations of 35, 75, 150, 600, 1200, or 2400 (+/-5) milliosmolal (mOsm) at 37 degrees C. A control treatment at 290 mOsm was also utilized. Oocytes were randomly allocated to each experimental treatment. After the treatment, the oocytes were cultured for 1 h, then fixed in cold methanol. Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II (MII) spindle. Logistic regression was used to determine if media osmolality had a significant effect on spindle structure. RESULTS: Osmolality was a significant predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150 mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles, respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted in 44, 44, and 100% of the spindles with abnormal structure, respectively. CONCLUSIONS: Anisosmotic conditions lead to disruption of the MII spindle in human oocytes. Applying this fundamental knowledge to human oocyte cryopreservation should result in increased numbers of cells maintaining viability.     

The effect of antisecretory factor on the permeability of nerve cell membrane to chloride ion

The antisecretory factor (ASF) is a hormone-like protein (m.w. 60,000) that most effectively counteracts hypersecretion in vivo in the small intestine of pigs and rats. The present report demonstrate that 10^–13 moles of ASF inhibits significantly the³⁶Cl^– permeation through the isolated neuronal plasma membrane of Deiters' cells in rabbits. This effect was enhanced by 0.2 mM -aminobutyric acid (GABA), and quenched by the addition of anti-ASF immunoglobulins; pretreatment of the neuronal membrane with nipecotic acid (10^–6 M) or with bicuculline (10^–3 M) abolished the ASF action whilst picrotoxin (10^–4 M) pretreatment left the inhibitory effect of ASF unaffected. The results suggest that ASF blocks chloride channels in neuronal membranes, including those channels activated by GABA.Abbreviations ASF antisecretory factor - GABA -aminobutyric acid - SEM standard error of the mean     

The effect of human immunodeficiency virus on sperm parameters and the outcome of intrauterine insemination following sperm washing

BACKGROUND: This is the first study to assess the outcome of sperm washing and intrauterine insemination (IUI) cycles in human immunodeficiency virus-positive (HIV(+)) men to determine any predictors of success, as well as evaluating the effect of HIV on sperm parameters. METHODS: Semen characteristics were evaluated in 106 HIV(+) men and a control group of 234 HIV(-) men, and the effect of markers of HIV disease assessed. Age, stimulation regime, sperm parameters, markers of HIV disease and the use of anti-retrovirals were assessed as predictors of the outcome of sperm washing/IUI cycles in the HIV(+) men. RESULTS: Ejaculate volume, sperm concentration, total count, progressive motility and normal morphology were all significantly higher in the control group compared to the HIV(+) men (P<0.05). A significant positive correlation was observed between CD4 count and sperm concentration, total count, motility, progressive motility type 'a'+'b' and post-preparation concentration and a significant negative correlation with normal sperm morphology of both raw and post-preparation samples. No correlation was observed between viral load (VL), years since diagnosis, use of anti-retrovirals or duration of use and any sperm parameter. The only factors that significantly improved IUI outcome were a VL <1000 copies/ml and the use of anti-retrovirals. CONCLUSIONS: These data demonstrate that sperm parameters are significantly impaired by the presence of HIV infection and in particular correlate with CD4 count. Undetectable VL and the use of anti-retrovirals improve the outcome of IUI/sperm washing in HIV(+) men.     

The effect of smoking and varicocele on human sperm acrosin activity and acrosome reaction

Smoking and varicocele are frequent findings in the medicalhistory and physical examination of patients attending and rologicaloutpatient departments. However, data about their influenceon human semen parameters, such as sperm concentration and motility,are contradictory. Therefore, the purpose of this study wasto examine sperm function (acrosin activity and induction ofthe acrosome reaction) in smokers (n = 130) and varicocele patients(n = 30)compared with normal fertile donors (n = 20). The acrosomereaction was detected by triple staining after 3 h ofincubationat 37°C, followed by treatment with 0.1%dimethyl sulphoxide(spontaneous acrosome reaction) and 10 µM calcium ionophoreA23187 (induced acrosome reaction) for 1 h at 37°C. Acrosinactivity was measured by gelatinolysis. The diameters aroundthe sperm heads after gelatinolysis and the percentages of spermatozoashowinghalo formations were evaluated. The inducibility of theacrosome reaction was significantly lower in semen samples fromsmokers than in those from the fertile group (7.1 ±3.2versus 11.2 ± 4.0%, P < 0.01), whereas no statisticallysignificant difference was demonstrated in spermatozoa frompatients with varicocele (9.3 ± 4.3%). Both the percentagesof spermatozoa with halo formation (53.3 ±20.0 versus76.6 ± 13.6%, P < 0.05) and the halo diameters (16.1± 6.6 versus 31.0 ± 14.5 urn, P < 0.001) weresignificantly lower in the varicocele group than in thesamplesfrom fertile men. These data suggest that smoking and varicoceleaffect sperm function, and that the standard semen parametersalone are insufficient to evaluate the influence of both factorson human male fertility.     

Expression of Hsp60 and Grp78 in the human endometrium and oviduct, and their effect on sperm functions

BACKGROUND: Within the female genital tract, spermatozoa undergo a series of membranous and intracellular transformations to become competent at fertilizing the oocyte. In the bovine, previous studies have shown that two oviductal proteins, heat shock protein 60 (Hsp60) and glucose regulated protein 78 (Grp78), bind to spermatozoa and may be involved in this acquisition of fertilizing competence. METHODS: Immunohistochemical studies were performed on human endometrial and oviduct tissues to localize these two chaperones in the female genital tract. Human spermatozoa were incubated under capacitating conditions in the presence or absence of recombinant Hsp60 or Grp78. Following a 4-h incubation, the effects of these proteins were evaluated on sperm acrosomal integrity, motility, protein phosphotyrosine content and free intracellular calcium concentrations. RESULTS: Both chaperones were present in the uterus and oviduct epithelial cells and were shown to bind to human spermatozoa. Incubation with either exogenous Hsp60 or Grp78 did not affect sperm viability, motility or acrosomal integrity. Hsp60 partially prevented the increase in p81 phosphotyrosine content induced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and both chaperones significantly increased the sperm intracellular calcium concentration. Moreover, the progesterone-induced increase in intracellular calcium was higher when sperm were pre-treated with either Hsp60 or Grp78. CONCLUSIONS: Our study suggests that these two proteins may affect human sperm intracellular signalling pathways and capacitation.     

Scanning electron microscopy analysis of the human zona pellucida: influence of maturity and fertilization on morphology and sperm binding pattern

Human oocytes from the same as well as from different patients have an extremely heterogeneous morphology of the zona pellucida surface as shown by scanning electron microscopy. For years it has been believed that this heterogeneous morphology plays an important part in the sperm-oocyte interaction. It was the aim of this investigation to analyse the morphology and the sperm binding patterns of the human zona pellucida. Oocytes were divided into four categories: mature, immature, fertilized and unfertilized. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between zona type and oocyte maturity or zona type and achieved fertilization. However, fertilized (polyploid) oocytes had a more compact and smooth zona surface than unfertilized ones. The analysis of the number and distribution patterns of bound spermatozoa on the zona pellucida revealed extremely variable patterns regardless of the zona morphology. Significant differences between mature and immature oocytes did not appear. In both groups there were oocytes with either no or numerous bound spermatozoa on the zona pellucida. Oocytes overloaded with spermatozoa could only be found in the mature group. Unfertilized oocytes had fewer bound spermatozoa on average than polyploid zygotes.     

Comparative study of the effect of human cervical mucus and a cervical mucus substitute, Healonid, on capacitation and the acrosome reaction of human spermatozoa in vitro

The present study was designed to investigate the effect ofhuman cervical mucus on capacitation and the acrosome reactionof human spermatozoa and compare its effect to that of a cervicalmucus substitute, sodium hyaluronate (Healonid). Spermatozoafrom donors of proven fertility were isolated from semen usingcervical mucus, Healonid or a direct swim-up (acting as thecontrol). Sperm capacitation and the acrosome reaction weremonitored by the chlortetracycline assay. In the mucus-treatedgroup, there was a significantly higher percentage of capacitatedspermatozoa, but a low incidence of spontaneous and A23187-inducedacrosome reactions compared to the control. The use of Healonidduring sperm isolation mimicked the effect of mucus relativelysuccessfully. Since mucus and Healonid show very little chemicalsimilarity, this finding would imply that cervical mucus exertsa physical effect during its interaction with spermatozoa, althougha chemical effect cannot be completely dismissed. In conclusion,this study confirms early reports describing the ability ofcervical mucus to capacitate spermatozoa but at the same timeconserve sperm function. The finding that Healonid exerts analmost identical effect on spermatozoa would lend support toits use as a cervical mucus substitute during in-vitro fertilityassessments and research studies.     

Study of the water structure in poly(methyl methacrylate-block-2-hydroxyethyl methacrylate) and its relationship to platelet adhesion on the copolymer surface

The water structure and platelet compatibility of poly(methyl methacrylate (MMA)-block-2-hydroxyethyl methacrylate (HEMA)) were investigated. The molecular weight (Mn) of the polyHEMA segment was kept constant (average: 9600), while the Mn of the polyMMA segment was varied from 1340 to 7390. The equilibrium water content of the copolymers was found to be mainly governed by the HEMA content. The water structure in the copolymers was characterized in terms of the amounts of non-freezing and freezing water (abbreviated as W_nf and W_fz, respectively) using differential scanning calorimetry. It was found that the W_nf for the copolymers were higher than those estimated from the W_nf for the HEMA and MMA homopolymers and that the amount of excess non-freezing water depended on the polyMMA segment length. In addition, X-ray diffraction analysis revealed that some of the copolymers had cold-crystallizable water. These facts suggested that the polyMMA segments were involved in determining the water structures in the copolymers. Furthermore, the platelet compatibility of the copolymers was improved as compared to that of the HEMA homopolymer. It was therefore concluded that the platelet compatibility of the copolymer was related to the amount of excess non-freezing water.     

Fertilization and early embryology: The effect of equilibration temperature and time on the outcome of ultrarapid freezing of 1-cell mouse embryos

Mouse 1-cell embryos were frozen ultrarapidly at a rate of 2500°C/minin solutions containing 0.25 M sucrose, 0.5% (w/v) bovine serumalbumin (BSA) and 3 or 4.5 M dimethyl sulphoxide (DMSO) or 3or 4.5 M 1, 2-propanediol (PROH) in HEPES-buffered modifiedEarle's medium. We investigated the effect of pre-freeze equilibrationfor 1, 3, 5 or 10 min at 22°C and for 1, 3, 5, 10, 15 or20 min at 4°C. After thawing in a 22°C water bath ata rate of 2500°C/min and dilution in 1 M sucrose in HEPES-bufferedmodified Earle's medium, embryos were cultured in vitro in bicarbonate-bufferedmodified Earle‘s medium with 0.5% (w/v) crystalline BSA.Embryo viability was expressed as the percentage of hatchingor hatched blastocysts resulting from the initial number offrozen-thawed 1-cell embryos. To determine the toxicity of thefreezing solutions, embryo viability was evaluated after equilibrationwithout freezing. Our results demonstrated that the concentration,the equilibration temperature and time are very important factorsin ultrarapid freezing of mouse 1-cell embryos. Optimal viabilitywas found when equilibration was done in 4.5 M DMSO for 3–5min at 22°C and in 4.5 M PROH for 3–5 min at 4°C.The results with regard to exposure to the freezing solutionsindicated that the loss of viability beyond an optimum is notdue solely to cryoprotectant toxicity, in particular not at4°C and not for DMSO. It is suggested that the temperatureand time of equilibration influence the degree of cryoprotectantpermeation and subsequent rehydration, which play a role indetermining freezing susceptibility in terms of ice formation.We conclude that both DMSO and, in contradiction to previousreports, PROH can be used perfectly adequately for ultrarapidfreezing on condition that concentration, and the temperatureand time of equilibration are controlled.