Sperm concentration influences fertilization and male pronuclear formation in vitro in pigs

To study the effect of sperm concentration on the results of pig in vitro fertilization (IVF), 313 oocytes recovered from oviducts of prepubertal gilts after induction of ovulation were used. After capacitation, the number of live spermatozoa in the fertilization dishes was adjusted to 3 x 10(5), 6 x 10(5) and 12 x 10(5) cell/ml. After 4 hours of co-culture in TCM-199, the oocytes were pippeted to remove cumulus cells and the excess spermatozoa around the zona pellucida, and were transferred to fresh TCM-199 for another 12 14 hours . The results showed that 6 x 10(5) spermatozoa/ml is the optimum concentration for this system; the percentage of fertilized ova (71.6%) was not different from the best (76.8%), that was obtained with the highest concentration, and the percentage of monospermy (62.3%) was not different from the best (68.1%), that was obtained with the lowest concentration. The percentage of spermatozoa that reached the pronuclear stage increased while sperm concentration was decreased. The percentage of spermatozoa at the decondensed stage was decreased when the sperm concentration increased.     

In vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs.

In vitro fertilization of rat and mouse eggs by ejaculated or epididymal spermatozoa in chemically defined media was studied. Penetration rates by ejaculated sperm was very low (0 to 8%) in the rat, but 11 to 41% of eggs were penetrated by ejaculated sperm in the mouse. The optimal concentration of sperm for in vitro fertilization appears to be similar whether ejaculated or epididymal sperm were used. The time of sperm penetration in the mouse eggs, however, was delayed for one-half to one hour when ejaculated sperm were used. The importance of sodium pyruvate, sodium lactate and glucose in the medium containing bovine serum albumin for in vitro fertilization of rat eggs was examined. When rat eggs in cumulus clot were exposed to epididymal sperm preincubated for five hours, the presence of sodium pyruvate, sodium lactate and glucose was found to play an important role. When exposed to non-incubated epididymal sperm sodium pyruvate could be omitted without much decline of the fertilization rate. When the denuded eggs were exposed to non-incubated sperm, penetration rates were very low (0 and 5%) in the absence of pyruvate. It appears that although lactate, pyruvate and glucose are all important for in vitro fertilization of rat eggs, pyruvate can be supplied by the follicular cells surrounding the eggs.     

Penetration of the zona-free or intact eggs by foreign spermatozoa and the fertilization of deer mouse eggs in vitro

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.     

Effect of concentration of spermatozoids on fertilization of oocytes and human embryo development in vitro

The correlation between sperm insemination concentrations, rates of normal and abnormal fertilization and embryo development was investigated. For male factor patients fertilization rates are significantly lower than for female factor. We have found the increased fertilization rate for male factor, if insemination concentration increased from 10 x 10(4) to 15 x 10(4) per 1 ml. In cases of severe male factor infertility the concentration of sperm of 30 x 10(4) per 1 ml had no effect. We have found no difference in abnormal rates of fertilization, when the number of sperm increased in male factor. The correlation between the frequency of polysperm zygote and slightly increased insemination concentration was observed in patients with normal sperm.     

Sperm binding and fertilization envelope formation in a cell surface complex isolated from sea urchin eggs

An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.     

Sperm factors related to failure of human in-vitro fertilization

Two groups of men were retrospectively selected according to their observed success in in-vitro fertilization. Seminal and post-migration sperm samples from a low fertilization rate group (less than or equal to 33% cleaved embryos) have been compared to results obtained from a high fertilization rate group (greater than or equal to 66%). It was found that a low mean value of the amplitude of lateral sperm head displacement and an increased percentage of abnormal acrosomes were related to in-vitro fertilization failure. None of the individual sperm factors studied was found to determine in-vitro fertilization success with certainty; only when they were considered in combination was it possible to predict the likelihood of successful in-vitro fertilization of human oocytes.     

A consistently successful procedure for in vitro fertilization of golden hamster eggs

Complete details are described for the first time of the procedures used in the author's laboratory for obtaining in vitro fertilization (IVF) of golden hamster eggs leading to the first cleavage division. These IVF procedures have been developed during the past 20 years and are very reproducible: IVF of at least 75% of eggs is routinely achieved, and on average 65% of inseminated eggs undergo the first cleavage division in vitro. These results can easily be obtained by inexperienced investigators. The ease and reproducibility of the hamster IVF procedures make them very suitable for studies of sperm:egg interaction and associated events. Studies in the author's laboratory have included analysis of sperm fertilizing ability under chemically defined conditions, the presence of sperm acrosome reaction stimulating factors in the egg investments, maturation of oocytes in vitro, the block to polyspermy, and the contribution of egg aging to fertilization anomalies. In addition, the motility of hamster sperm under chemically defined conditions is used in a routine screening protocol for detecting contaminants in the culture milieu. Golden hamster gametes of Ter several distinct advantages for IVF studies, including the large size of the sperm acrosome, the persistence of the very large sperm tail in the ooplasm for many hours following fertilization, and the translucence of the ooplasm, which facilitates observation of the sperm tail and pronuclei. The female golden hamster exhibits a regular 4 day estrous cycle, with distinctive indications of estrus and postestrus phases. Because of the advantages of using the golden hamster, the procedures described in this report may be useful to other investigators wishing to conduct research using IVF. Essentially the same IVF procedures can be used with monkey and bovine gametes.     

Chromosome studies in human in vitro fertilization

Summary The chromosome constitution of 22 human preimplantation embryos from donor oocytes fertilized in vitro by donor sperm was studied to assess the contribution of lethal chromosome anomalies to the high failure rate of implantation of in vitro fertilized embryos after embryo transfer in infertile women. Evidence was found of nondisjunction, resulting in trisomy, monosomy, and nullosomy; structural abnormalities; haploidy; and triploidy. Despite the lethality of their chromosome complements, these embryos could not be distinguished morphologically from those with normal chromosomes.