Effect of albumin on fertilization of mouse ova in vitro

Serum albumin is an obligatory component of the incubation medium for the fertilization of mouse ova. Normal, untreated bovine serum albumin supports high rates of fertilization of cumulus-free ova both with and without their zonae pellucidae. Heat-treated or trichloroacetic acid-extracted bovine serum albumin is unable to support the fertilization of a majority of zona-intact ova but fertilization of zona-free ova is unimpaired. Spermatozoa incubated in medium containing heated bovine serum albumin fertilize zona-intact ova when 2 mM caffeine is present but the progress of sperm head decondensation is delayed when compared to normal controls. Trichloroacetic acid extracted BSA preferentially and irreversibly inhibits zona penetration by spermatozoa, but this effect is not mediated by an inhibition of spermatozoal motility or zona-binding ability. This effect occurs after only a 10-min preincubation of the spermatozoa in the extracted BSA or when the medium contains only a 10% (v/v) proportion of this albumin. It is estimated that mouse spermatozoa under the conditions used take 2 hr to penetrate the zonae pellucidae of 50% of ova and effect fertilization.     

Influence of genetic factors on the fertilization of mouse ova in vitro

In-vitro fertilization was studied in F1 hybrids, 4 inbred and outbred strains of mice. Experiments were performed with intact ova (Series I) and denuded ova (Series II). In Series I the highest percentages of fertilization were obtained with gametes of F1 hybrids (86.7%) and of the CBA strain (87.5%). The KP, KE, and particularly the C57 strains, which in vivo give less than 100% fertilization, also gave low indices in vitro (42.9, 26.0 and 8.6% respectively). Testing gametes of these inbred strains with F1 gametes showed that spermatozoa and ova are responsible for the low percentages of fertilization. The rate of fertilization was mainly dependent on the genotype of the spermatozoon. In Series II, high percentages of fertilization (97-100%) were regularly obtained in all groups, indicating that differences between strains pertained mainly to binding of spermatozoa with the zona pellucida. The incidence of polyspermy, which did not exceed 5% in Series I and reached 58% in Series II, was dependent on the genotype of both gametes.     

The effect of zinc ions on fertilization of mouse ova in vitro

The fertilization rate of mouse gametes in a Krebs-Ringer bicarbonate medium was 60%. The rate fell to 0% when 250 micron-ZnCl2 was added, but MgCl2, CaCl2, MnCl2, FeCl2, and CuCl2 (each at 250 micron) had no effect. The inhibitory effect of Zn2+ was not evident if the spermatozoa or eggs were preincubated with Zn2+ or if previously capacitated spermatozoa were used. The Zn2+ is thought to act by interference with capacitation.     

In vitro fertilization of mouse ova by spermatozoa exposed isothermally to radio-frequency radiation

Mouse spermatozoa were exposed in vitro for 1 h to 27- or 2,450-MHz CW RF radiation at SARs of 0 to 90 W/kg under isothermal (37 +/- 0.2 degrees C) conditions. Exposure at either frequency to RF radiation at SARs of 50 W/kg or greater resulted in a statistically significant reduction in the ability of irradiated sperm to fertilize mouse ova in vitro (P less than .05). Over the range of SARs there was no apparent difference in the effects of 27- vs. 2,450-MHz RF radiation. There were no readily detectable exposure effects on spermatozoan morphology, ultrastructure, or capacitation. The reduction of in vitro fertilization is attributed to a direct effect of RF radiation on spermatozoa rather than to heating.     

Interactions of aged gametes: In vitro fertilization using in vitro-aged sperm and in vivo-aged ova in the mouse

A study of varying combinations of in vitro-aged sperm and in vivo-aged ova at 3 hr intervals from 0–24 hr resulted in failures at different steps of the fertilization process during in vitro fertilization of mouse ova. Significant decreases caused by sperm aging, ova aging, and sperm × ova aging interaction were found in sperm penetration. Pronuclear formation was not affected by sperm aging and was enhanced by ova aging, and there was a significant effect of sperm × ova aging interaction. Sperm aging significantly influenced the prometaphase stage of the fertilization process. Therefore, it is suggested that the detrimental fertilization effects resulting from aging gametes are due to different mechanisms in sperm and ova, that these mechanisms are affected at different times, and that they affect different steps in the fertilization process.     

The relationship between sperm concentration and fertilization in vitro of rabbit ova

Different concentrations of in utero incubated rabbit sperm (1.5 × 10⁴-120 × 10⁴ /ml) were tested to determine whether there is a relationship between sperm concentration and level of fertilization achieved “in vitro” of rabbit ova. While low concentrations (1.5 × 10⁴-4.5 × 10⁴ /ml) resulted in relatively low fertilization (23–36%), those in the range of 13 × 10⁴?120 × 10⁴ /ml gave fertilization rates of 65–83%. Consistently high results were obtained with sperm counts above 40 × 10⁴ /ml. This is in agreement with the concentration of spermatozoa found in vivo in the Fallopian tubes around the time of fertilization (50 × 10⁴ /ml).     

Invitro fertilization of cow ova

Embryos were recovered from superovulated heifers and transferred non-surgically through the cervix of heifers via an ureteral catheter. The ureteral catheter was passed into the anterior portion of the uterine horn ipsilateral to the corpus luteum through a series of concentric stainless steel tubes. One morula or blastocyst was transferred to each of 95 heifers.Eighteen heifers (19%) were pregnant 55 to 60 days later. The pregnancy rate was not significantly influenced by treatment of the vagina with antibiotics before transfer; whether a morula or blastocyst was transferred; or whether ovulation occurred from the left or right ovary. There were no significant differences among the three technicians. Fifty of the 77 recipients that were not pregnant at 60 days of gestation were observed in estrus after 18–23.5 or 37–44.5 days, representing normal or double the normal estrous cycle lengths. However, 11 showed a shortened return to estrus (9–26 days), and 16 had a delayed return to estrus (25–33 or 48–55 days). We conclude that this technique results in premature luteolysis in some recipients and probably in more early embryonic death than normal.     

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.     

Development of vitrified mouse oocytes after in vitro fertilization

Mouse oocytes were cryopreserved by the vitrification method using vitrification solution (VSI) and the effects of dilution methods were examined on the rate of in vitro and in vivo development. Eighty-three percent and 75% of vitrified oocytes exhibited normal morphology when diluted in glycerol + sucrose and sucrose alone, respectively. In contrast, only 35% of the oocytes diluted by a stepwise method exhibited a normal appearance. A high proportion of vitrified oocytes was fertilized in vitro (84-94%), 80 to 87% of which were normal. Of the later embryos, 69 to 78% developed to blastocysts after 4 days of culture. Thirty-six live young (51%) were obtained when vitrified oocytes were transferred to recipient females. The overall rate of development to live young was 25% when vitrified oocytes were diluted with glycerol + sucrose solution. These results indicate that the simple and rapid procedure of vitrification and glycerol + sucrose dilution is suitable for the cryopreservation of mouse oocytes.     

Protein metabolism in preimplanted mouse ova

Intracellular specific radioactivity of an amino acid (leucine) was measured in preimplantation mouse ova. The measured specific activity along with leucine incorporation rates allow calculation of the protein synthetic rate in mouse ova. Unfertilized ova, fertilized ova, two-cell ova, and blastocysts convert amino acid to protein at the rate of 8.60, 7.36, 6.92, and 56.68 × 10^?13 moles of amino acid per hour per ovum, respectively. The specific activity measurements also allowed the calculation of intracellular leucine pool size. For the unfertilized, fertilized, two-cell, and blastocyst stage, the endogenous leucine pool was 11.9, 15.9, 6.4, and 62.4 × 10^?14 moles of leucine, respectively. Most of the increase in pool size in the blastocyst probably results from the increase in total volume of cells. Protein degradation measurements indicated a marked difference between protein turnover in the two-cell and blastocyst stage. Approximately 10% of the protein at the two-cell stage turns over with a half-life of 18.3 hr and 35% of the protein at the blastocyst stage turns over with a half-life of 11.2 hr. The large remaining fraction of protein turns over much more slowly.