Comparison of heterospermic and homospermic inseminations as measures of male fertility

This study attempted to determine a basis for the previously observed greater sensitivity of heterospermic tests when compared to homospermic tests for detecting differences in fertility between males. In theory, the results of heterospermic tests are an indication of the proportion of eggs fertilized per unit time whereas results of homospermic inseminations measure only the cumulative or final proportion of eggs fertilized. The fertilizing ability of sperm from males of CF1 and C57BL/6N strains of mice was compared homospermically using both relatively high and low concentrations of sperm and by measuring the proportion of eggs penetrated per unit of time. The fertilizing ability of sperm from these strains was also compared using heterospermic inseminations. When females were inseminated with a high concentration of sperm, males of both strains fertilized a high and indistinguishable percentage of eggs when examined after 30 hr. When females were inseminated with either a low concentration of sperm or when the proportion of eggs penetrated was measured at 5 hr, differences between strains of mice were distinguishable. Heterospermic insemination further magnified the observed difference between strains. The results of this study confirm that measuring the percentage of eggs fertilized per unit of time can enhance the magnitude of differences between males in fertility as compared to measuring only the final percentage of eggs fertilized. Measuring the percentage of eggs fertilized per unit of time does not, however, entirely account for the large differences observed between fertility of males when they are compared using heterospermic inseminations.     

Penetration of mouse eggs at various intervals after insemination as influenced by concentration of sperm and strain of male

This study was conducted to determine the extent to which the percentage of mouse eggs that were penetrated by sperm at the end of the period of sperm penetration was due to the proportion of eggs penetrated per unit of time and to the span of time of sperm penetration. Female mice of ICR strain were inseminated 1.5 hr after ovulation with 5 X 10(6) sperm/50 microliter from males of DBA/2N, CF1 or C57BL/6N strains to determine the effect of the male. To determine the effect of concentration of sperm ICR females were inseminated with 2, 4, 6, or 8 X 10(6) sperm/50 microliter from CF1 males. Females were killed at various intervals after insemination and the eggs were recovered and examined for evidence of penetration by a sperm. The time intervals from both insemination to the onset of egg penetration and from insemination to cessation of penetration were similar for the three strains of males. Throughout the period of penetration of eggs a constant percentage of eggs was penetrated per hour for a particular strain of male. The relative percentage penetrated per hour very closely approximated the relative percentage of eggs finally penetrated for each strain of male. The percentage of eggs penetrated per hour was linearly positively related to the concentration of sperm inseminated. The final percentage of eggs penetrated depended primarily on the rate at which the eggs were penetrated during the period of sperm penetration and not on the length of the period of egg penetration which was constant.     

In vitro fertilization of hamster and human oocytes by microinjection of human sperm

In this study, swollen sperm heads were obtained after the injection of human sperm into the perivitelline space of hamster oocytes. The number of injected sperm and the sperm concentration in the preincubation medium were found to have an influcnce on the rate of penetrated hamster oocytes. The optimal injected sperm number was always between five to 12 to obtain 8, 37, and 36% penetration for donors A, B, and C, respectively. The optimal sperm concentration in preincubation medium was between 6 and 22 × 10⁶ sperm/ ml to obtain 16, 47, and 43% penetration for donors A, B, and C, respectively. The rate of polyspermic oocytes was related to the injected sperm number (0, 55, and 100% for one to four, five to 12, and more than 12 injected sperm respectively). Ten human mature oocytes were injected with the sperm from six normal donors. Five fertilized eggs were obtained, and of these four cleaved in in vitro culture.     

Effect of dietary selenium deficiency on the in vitro fertilizing ability of mice spermatozoa

Selenium is an essential micronutrient for mammals, being integral part of antioxidant system. The aim of the study was to evaluate the effect of selenium deficiency on in vitro fertilization (IVF) capacity of spermatozoa and on oxidative stress in these cells. Male C57BL/6N mice were maintained on selenium-deficient or selenium-sufficient diets (0.02 or 0.2 ppm of selenium as selenomethionine, respectively) for 4 months. Liver glutathione peroxidase activity measurements were used to confirm selenium deficiency. Sperm quality and IVF capability among both groups were evaluated. To assess oxidative damage, lipid peroxidation as malondialdehyde production was determined in spermatozoa as well as the testes. Ultrastructural analyses of spermatozoa nuclei using transmission electron microscopy were also performed. The percentage of eggs fertilized with sperm from selenium-deficient mice was significantly decreased by approximately 67%. This reduced fertilization capacity was accompanied by increased levels of lipid peroxidation in both the testes and sperm, indicating that selenium deficiency induced oxidative stress. Consistent with this finding, spermatozoa from selenium-deficient animals exhibited altered chromatin condensation. Deficiency in dietary selenium decreases the reproductive potential of male mice and is associated with oxidative damage in spermatozoa.     

An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis

In 27% DeBoer's saline (DBS), which yields maximum fertility rates, Xenopus eggs fertilized in vitro are monospermic, regardless of sperm concentration. One block to polyspermy (the “slow” block), described previously, occurs at the fertilization envelope that is elevated in response to the cortical reaction. This paper describes properties of an earlier, “fast” block at the plasma membrane and evaluates the functional significance of the two blocks at physiological sperm concentrations in natural mating conditions. Unfertilized eggs have a resting membrane potential of ?19 mV in 27% DBS. Fertilization triggers a rapid depolarization to +8 mV (the fertilization potential, FP); the potential remains positive for ca. 15 min. Activation of eggs with the ionophore, A23187, produces a slower but similar depolarization (the activation potential, AP). As in other amphibian eggs, the FP appears to result from a net efflux of Cl^?, since the peak of the FP (or the AP in ionophore-activated eggs) decreases as the concentration of chloride salts in the medium is increased. In 67% DBS no FP or AP is observed; eggs fertilized in 67% DBS become polyspermic and average 2 sperm entry sites per egg. In the 5–37 mM range, I^? and Br^?, but not F^?, are more effective than Cl^? in producing polyspermy. In 20 mM NaI the plasma membrane hyperpolarizes in response to sperm or ionophore; 100% levels of polyspermy and an average of 14 sperm entry sites per egg are observed. NaI does not inhibit or retard elevation of the fertilization envelope; the cortical reaction and fertilization envelope are normal in transmission electron micrographs. In 67% DBS, which also inhibits the fast block, the slow block was estimated to become functional 6–8 min after insemination. Eggs fertilized by natural mating in 20 mM NaI exhibit polyspermy levels of 50–90% and average 5 sperm entry sites per egg. Since eggs become polyspermic when fertilized by natural mating under conditions that inhibit the fast, but not the slow, block to polyspermy, we conclude that the fast block is essential to the prevention of polyspermy at the sperm concentrations normally encountered by the egg.     

Effects of dietary astaxanthin supplementation on reproductive characteristics of rainbow trout (Oncorhynchus mykiss)

The effect of dietary astaxanthin supplementation on reproductive characteristics was investigated in five groups of female rainbow trout broodstock fed diets containing either 0.07, 12.46, 33.33, 65.06 or 92.91 mg astaxanthin kg^?1, respectively, and two groups of male rainbow trout broodstock fed diets supplemented with 0.07 and 33.33 mg astaxanthin kg^?1, respectively, for 6 months in an artificial photoperiod system until sexual maturation. The eggs from each group of female broodstock were divided into two equal batches. One batch was fertilized with homogenized sperm of four males fed diets with 0.07 mg astaxanthin kg^?1 and the other portion with sperm of four males fed diets with 33.3 mg astaxanthin kg^?1. The females produced eggs with astaxanthin concentrations ranging from 2.03 to 29.79 mg kg^?1. Dietary astaxanthin supplementation had positive effects on investigated reproductive traits. Significant differences in rate of fertilization, percentage of eyed and hatched eggs, and mortality of eyed eggs were found between treatments (P < 0.05), but no significant difference was found on percentage of mortality before hatching (P > 0.05). A significant difference (P < 0.05) in fertilization rate was found for male groups fed 0.07 and 33.3 mg astaxanthin kg^?1. The astaxanthin content in the eggs and fertilization rate, eyed‐egg percentage and percentage hatch were significantly correlated (P < 0.05). It is concluded that dietary supplements of astaxanthin are required for optimum reproduction in rainbow trout.     

Oocyte recovery, maturation and fertilization in vitro in the puma (Felis concolor)

Eight female pumas were treated i.m. with 1000 (N = 5) or 2000 (N = 3) i.u. PMSG followed 84 h later by 800 i.u. hCG. Eggs were recovered 24-26 h after hCG from ovarian follicles by using laparoscopy and transabdominal aspiration. Mature eggs were inseminated in vitro 4-6 h later whereas immature eggs were cultured for 24 h and then inseminated. Electroejaculates from 3 pumas were diluted with mKRB before insemination to evaluate the influence of sperm concentration on fertilization. Seven of 8 pumas responded with follicle development, and 140 eggs were recovered from 145 follicles (96.6%; 77 mature, 43 immature, 20 degenerate eggs; mean +/- s.e.m., 20.0 +/- 5.9 eggs/female). Overall fertilization rate was 43.5% (total eggs fertilized = 40) despite using inseminates containing 82-99% pleiomorphic spermatozoa. Of the 36 immature oocytes matured in vitro and inseminated, 12 were fertilized even though 50% of the inseminating spermatozoa contained an acrosomal defect. Fertilization rate of mature oocytes collected from follicles appeared unrelated (P greater than 0.05) to PMSG dose or number of spermatozoa/inseminate. This study demonstrates that a high proportion of follicular eggs can be recovered laparoscopically from adult pumas treated with PMSG and hCG. These gametes are capable of being fertilized in vitro (immediately or after maturation in vitro) even with low quality semen with a high incidence of sperm pleiomorphisms.     

Genetic influences on mouse sperm capacitation in vivo and in vitro

Intial in vivo studies were performed to observe the proportion of eggs fertillized at specific intervals after natural mating and ovulation in our research mouse colony. Proestrous females of the C57BL/10Wt, SJL/Wt inbred strains and the F₁ hybrid cross (B10 × SJL or reciprocals) were paired in the after-noon with males of their respective strain and examined for vaginal plugs at the midpoint of the dark period (2400 hours). Oviducts were periodically collected from mated females, and ovulation was first observed at 4, 5.2, and 3 hours after 2400 hours in the B10, SJL, and F₁ hyrid, respectively. The clutch of eggs from each ovulating female, was placed in culture, and cleavage oviduct lavage verifying female mating was placed in culture, and cleavage was used as the criterion for fertilizaition. Fifty percent of the eggs were fertilized 2.2, 5.0, and 2.5 hours after ovulation in B10, SJL, and F₁ hybrid females, respectively. Because twice the legth of time was required to fertilize a similar proportion of eggs from the SJL strain as the F₁ hybrid, these two strains were used for determining their rate of fertilization under more fully controlled conditions in vitro. Forty-nine percent of F₁ hybrid eggs were fertilized after 4 hours incubation with SJL epididymal sperm, whereas 53% fo SJL and 56% of F₁ hybrid eggs were fertilized after only 2 hours incubation with F₁ hybrid epididymal sperm. Thus, using sperm from these two mouse strains, the amount of time required to fertilize approximately 50% of the eggs within a clutch both in vivo and vitro was very similar. These observations demonstrte teh validity of using this in vitro system for fertilization studies and confirm that the temporal events in sperm capacitation and egg penetration are dependent on the genotype of the sperm. Similarities in fertilization rates at specific times after ovulation or insemination in vitro imply that the initiationof sperm capacitation in vivo occurs near the time of ovulation and several hours after mating. We tentatively suggest that follicular fluid may be required for completion of mouse sperm capacitaiton in vivo.     

Treatment of sperm with extracellular adenosine 5'-triphosphate improves the in vitro fertility rate of inbred and genetically modified mice with low fertility

In vitro fertilization (IVF) is one of the most important techniques used for assisted reproduction in mouse colony management. As with natural mating, where mice have varying fertility indices, fertility rates of genetically modified (GM) [transgenic (Tg), knock out (KO) and congenic (Cg)] mice are influenced by their genetic background. Lines of GM mice that have poor fertility have a concomitant poor IVF outcome. Treatment of mouse sperm with extracellular adenosine 5'-triphosphate (ATPe) enhanced in vitro fertilization rates in outbred and hybrid mice. The objective of this study was to analyze the effects of using extracellular adenosine 5'-triphosphate-treated sperm for IVF of inbred wild type, and genetically modified mouse lines, for which standard IVF did not work well. The IVF was performed using the GM mice on C57BL/10SnJ, C57BL/6J, BALB/cJ and NFS/N background strains and wild type (WT) mice such as C57BL/6N, BALB/cAnN, and B6129SF1 strains. Oocytes from superovulated females were fertilized in vitro with sperm from the same background strain, and either treated or not treated with ATPe. The ATPe treatment enhanced IVF outcome in most of the GM and some WT strains, as indicated by the percentage of embryos that progressed to the two-cell stage. There was no marked difference between ATPe treated and control groups for the development rate of two-cell embryos to blastocysts in culture, or in the number of pups born after transfer of two-cell embryos into recipient females. The observed improvement of the IVF results following ATPe treatment of transgenic and KO mouse sperm were a potential solution for improving the outcome of assisted reproduction techniques used for rederivation or for gamete banking.     

Effects of caffeine,Ca++, capacitation time,and strain on in vitro fertilization in mice

Oocytes (N=2922) were collected from superovulated female C57B16/J X DBA2/J (B6D2F₁) mice and distributed among 48 treatments consisting of a 2×3×2×2×2 factorial design. The factors were strain of spermatozoa, B6D2F₁ or SJL/J; caffeine concentration in the fertilization medium, 0,2, or 6 mM; time oocytes were exposed to sperm, 1 or 2 hours; Ca⁺⁺ concentration in the capacitation medium, 0 or 1.8 mM; and capacitation time, 1 or 2 hr. Ova were observed 400 min after they were initially exposed to 10⁵ spermatozoa per ml. Ova with two or more pronuclei and a second polar body were considered fertilized, In vitro embryonic development was monitored for 5 days. B6D2F₁ spermatozoa resulted in consistently higher rates of fertilization than SJL spermatozoa, 77.5% vs 38.7% when averaged over other treatments. Caffeine concentrations of 0,2, and 6 mM resulted in respective mean fertilization rates of 50.1%, 58.8%, and 65.4% (P<0.005) when averaged over other factors. Fertilization rates of ova exposed 1 and 2 hr to sperm were 53.0% and 63.3% (P<0.005). B6D2F₁ spermatozoa capacitated in medium with 1.8 mM Ca⁺⁺ fertilized more ova (P<0.01), 83.1%, than when no Ca⁺⁺ was present, 71.9%; this effect was absent with SJL spermatozoa. The effect of capacitation time depended on strain. Fertilization rates with B6D2F₁ spermatozoa were higher, 80.1%, with a 2-hour capacitation time than with a 1-hour capacitation time, 75.0%. Exactly the opposite was true for the SJL spermatozoa; 43.4% for the 1-hour and 34.1% for 2-hour capacitation (P<.01). Development to the blastocyst stage was significantly greater (P<0.025) for ova fertilized by B6D2F₁ (26.8%) than by SJL spermatozoa (17.7%).     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.     

The block to sperm penetration in zonal-free mouse eggs

The rate of sperm penetration and the number of sperm penetrating zona-free mouse eggs were found to be dependent on sperm concentration. At the lowest sperm concentrations examined (10² cells/ml, sperm-egg ratios of approximately 1:1), most eggs were penetrated (75%), and polyspermy was low (19%) following 3 hr of incubation. The number of sperm penetrating the egg was logarithmically related to sperm concentration. All eggs showed a delay of at least 20 min between insemination and penetration, and penetration was complete in approximately 2 hr at 10⁴ sperm/ml; this penetration block was attributed to egg-related changes. The existence and timing of the egg plasma membrane block to polyspermy were evaluated by reinsemination experiments. In this approach, the block was triggered in zona-free eggs with a low concentration of capacitated epididymal sperm at time 0, and the eggs were subsequently challenged with high sperm concentrations. The presence or absence of a block was inferred from the degree of polyspermy observed in these eggs after 3 hr of incubation. Adjusting for sperm concentration-dependent delays between insemination and sperm penetration, a blocking time of approximately 40 min was obtained.     

Inhibition of In Vitro Fertilizing Capacity of Cryopreserved Mouse Sperm by Factors Released by Damaged Sperm,and Stimulation by Glutathione

Background

In vitro fertilization (IVF) of eggs by frozen and thawed C57BL/6J mouse sperm is inhibited by dead sperm and enhanced by preincubation of the sperm in calcium-free medium. In other species, the presence of sperm killed by freezing and thawing has been associated with the generation of hydrogen peroxide.

Methodology/Principal Findings

The proportion of eggs fertilized by cryopreserved C57BL/6J mouse sperm was increased significantly by increasing the volume of fertilization medium in which sperm and eggs were coincubated. Enhanced fertilization occurred even though the concentration of potentially fertile sperm was decreased fivefold. This suggested that if a putative soluble factor was inhibiting fertilization, dilution of that factor, but not the sperm, should increase the fertilization rate. This was achieved by coincubation of the gametes in cell culture inserts (Transwells^®) that during incubation were transferred progressively to wells containing fresh fertilization medium. Fertilization rates using inserts were high (66.6±2.4% versus 27.3%±2.8% in wells alone). On the assumption that the soluble factor could be H₂O₂, reduced glutathione was added to the fertilization medium. This enhanced fertilization rate significantly (76.6%±2.0% versus 21.2%±1.9%), while addition of oxidized glutathione did not (82.7%±6.5% with reduced glutathione; 44.5±8.8% with oxidized glutathione; 47.8%±12.1% with no glutathione). Positive effects of reduced glutathione on IVF were also seen with frozen 129S1, FVB, and C3H sperm, and sperm from two lines of genetically modified C57BL/6J mice.

Conclusions/Significance

IVF in cell culture inserts and addition of glutathione to fertilization medium significantly increased the proportion of eggs fertilized by cryopreserved mouse sperm from four inbred strains, suggesting that reactive oxygen species generated during fertilization inhibit fertilization. The modified IVF techniques developed here enhance the feasibility and efficiency of using cryopreserved sperm from genetically modified lines of inbred mice.     

THE FERTILIZING CAPACITY OF SEA URCHIN SPERM RAPIDLY DECREASES AFTER INDUCTION OF THE ACROSOME REACTION*

When immotile, flagella-less sperm were added to acid-dejellied eggs of Strongylocentrotus purpuratus 11% of the eggs fertilized. Addition of soluble egg jelly increased the percentage fertilization to 90.5. Over 50% of the sperm exposed to egg jelly had undergone the acrosome reaction compared to only 3–5% in the absence of jelly. Egg jelly was added to flagella-less sperm to induce the acrosome reaction and dejellied eggs added at various times thereafter. The fertilizing capacity of the sperm decreased with first order kinetics with 50% loss by 23 sec after induction of the acrosome reaction. Intact, motile sperm bind to formaldehyde-fixed eggs with maximum binding occurring 40 sec after sperm addition. After 40 sec the sperm begin to detach from the fixed eggs and by 240 sec none remain attached. Sperm detachment from fixed eggs and loss of fertilizing capacity after the acrosome reaction show a close temporal correlation.     

Penetration of zona-free hamster eggs by liposome-treated sperm from the bull, ram, stallion, and boar

Spermatozoa from each of four rams, four stallions, and three boars (six semen samples) were treated with dilauroylphosphatidylcholine (PC12) liposomes and compared with control bull sperm to induce the acrosome reaction (AR) and study possible penetration of the sperm into zona-free hamster eggs. Diluted sperm were incubated with several concentrations of PC12 for 7 min at 39 degrees C prior to insemination of the hamster eggs in vitro. The sperm from the bull were diluted to 10(6) cells/ml, as previously studied. Sperm from the ram, stallion, and boar were diluted to 6 X 10(6) and 20 X 10(6) cells/ml. After addition to the eggs, the sperm concentration was reduced by 75 percent. Inseminated eggs were incubated with sperm for 3 h at 39 degrees C prior to being fixed, stained, and observed for sperm penetration. At an initial concentration of 6 X 10(6) cells/ml, bull sperm treated with 36.7 microM PC12 achieved an egg penetration rate of 92%, whereas under nearly identical conditions stallion spermatozoa achieved only 54% egg penetration. Under similar conditions, ram spermatozoa failed to penetrate eggs, but when the initial sperm concentration was increased to 20 X 10(6) cells/ml, sperm incubated with 51.1 microM PC12 achieved 52% egg penetration. Boar spermatozoa treated with PC12 at either sperm concentration failed to exhibit an AR or penetrate hamster eggs. In general, as PC12 concentration increased the percentage of sperm with an AR increased and sperm motility decreased. It is concluded that 1) PC12 liposomes are effective in inducing the AR in sperm from the bull, ram, and stallion, but under conditions tested are ineffective with boar sperm;(ABSTRACT TRUNCATED AT 250 WORDS)     

小鼠卵子在不同条件下的体外受精

本实验比较了小鼠精、卵细胞在不同生理状态下体外的受精能力。结果表明,体内受精率明显地高于体外(p<0.05),自发排出的卵子比超数排出的卵子受精率高(p<0.05),体外获能的附睾精子比体内获能的子宫精子受精率高(p<0.01)。唯独用超数排出的卵子和体外获能的附辜精子体外受精时,其受精率和体内相似。     

The effect of catecholamines and their antagonists on the fertilization of cumulus-free mouse ova in vitro at a suboptimal spermatozoal density

A suboptimal sperm concentration was used to assess the capacity of catecholamines to stimulate the fertilization of cumulus free F₁,(C57BL × CBA) mouse ova in vitro. At a concentration of 50 μM, (L) epinephrine significantly increased the proportion of ova fertilized at 2 × l0⁵ spermatozoa/ml. However, when (D, L) propranolol at an equimolar concentration was tested for inhibition of the (L) epinephrine effect, fertilization was inhibited in both the test and control dishes. At l0μM, propanolol by itself or in the presence of 50μM (L) epinephrine significantly increased the number of ova fertilized at 2 × l0⁵ sperm/ml. Norepinephrine (50 μM) and phentolamine (50 μM), either alone or together, were also slightly stimulatory. Some data are presented to suggest that propranolol may act in a nonadrenergic manner to precipitate the acrosome reaction and that the stimulatory effect is maximised when it is added to spermatozoa at the same time as ova addition. It was suggested that propranolol may act to trigger calcium influx by a nonspecific alteration in membrane function for example in (Ca + Mg) ATPase activity. It was concluded that spermatozoa at suboptimal densities are capable of achieving fertilization and that sperm concentration dependency in fertilization in vitro may be a reflection of the proportion of spermatozoa achieving capacitation.     

Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol

When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to -70 degrees C for solid CO2 and -70 to -196 degrees C for liquid nitrogen) as pellets, 18% raffinose provided the greatest protection to ICR mouse spermatozoa against cold-shock; sperm motility and fertilizing ability were 43% and 22.4%, respectively. A small proportion of spermatozoa frozen with 10% sucrose was motile but incapable of fertilizing ovulated oocytes. Glycerol and dimethylsulphoxide were less effective at any concentration examined. However, the fertilizing ability of frozen-thawed ICR spermatozoa was significantly improved (35.5%) by addition of glycerol (1.75% final concentration) to medium containing 18% raffinose. Spermatozoa from one outbred (ddY) and 5 inbred (C57BL/6N, C3H/HeN, DBA/2N, BALB/c and kk) strains of mice were successfully frozen in the presence of 18% raffinose and 1.75% glycerol, although the fertilization rates of frozen-thawed spermatozoa varied among strains (13% for C57BL/6N to 64% for DBA/2N). A small fraction of mouse eggs resulting from fertilization by frozen-thawed spermatozoa developed normally in vitro (37% in C57BL/6N to 71% in ICR) to the blastocyst stage and in vivo (19% for C57BL/6N spermatozoa and ddY oocytes) to Day 18 of gestation.     

Caffeine promotes in vitro fertilization of mouse ova within 15 minutes

Epididymal sperm were collected from C57Bl6/J X DBA2/J (B6D2) males and allowed to capacitate for 2 hr. When cumulus-free oocytes were exposed to sperm for 15 min in either the presence (6.0 mM) or absence of caffeine, fertilization did not occur. However, when cumulus cells were left intact, 23% of oocytes were fertilized in caffeine-free medium and 62% in caffeine-containing medium. When cumulus-free oocytes were incubated with sperm for 30 min, none was fertilized in the absence of caffeine, but 33% were fertilized when 6.0 mM caffeine was present (P less than .02). These effects of caffeine were on the sperm, as sperm exposed to caffeine and then coincubated with oocytes for 15 min in essentially caffeine-free media fertilized a similar percent of oocytes (93%) as when sperm and oocytes were exposed to caffeine during the fertilization period (86%). When sperm were capacitated in caffeine-containing medium, the percentage of ova fertilized was similar to capacitation without caffeine. We conclude that both cumulus cells and caffeine speed up the fertilization process with mouse gametes and that the effect of caffeine is on the sperm, but not due to more rapid capacitation.