The Influence of X-Irradiation of Rabbit Spermatozoa on Fertilization and Early Cleavage

Abstract

Summary. Exposure of rabbit semen to X-irradiation did not reduce the fertilization rate until doses of >25,000 r were used, although motility was noticeably affected at 15,000 r. Evidence of a depressant effect of much lower doses of X-irradiation on the fertilizing ability of rabbit spermatozoa was obtained, however, by using a more sensitive system in which irradiated aliquots from an ejaculate were inseminated mixed together with an exactly equivalent volume of untreated semen from the same ejaculate. Under these competitive conditions the fertilizing ability of spermatozoa was found not to be affected by a dose of 10,000 r, but was significantly depressed (P<0\m=.\01)by 15,000 r, as judged by an increase in the proportion of normally developing to retarded ova, recovered 50 hr after an ovulation injection of hcg. It appears that this depressant effect is exerted at the site of fertilization rather than on sperm transport. X-irradiation of spermatozoa did not increase polyspermic or delayed fertilization, neither did irradiation have any significant effect on the formation of the male pronucleus. A dose response effect was seen at the time of the first cleavage division, for at 24 hr the cleavage rate of control ova (68\m=.\5%) was similar to that of ova fertilized by spermatozoa treated with 10,000 r (68%), but only 33% of ova fertilized by spermatozoa treated with 20,000 to 25,000 r had cleaved at this time compared with 78% of their controls. At 29 and 50 hr several ova fertilized by irradiated spermatozoa remained at the pronuclear stage or at metaphase of the first cleavage division, and even at 50 hr the great majority had only reached the 2-cell stage. Thus, although it is known that cleavage can occur in the absence of the male elements, the first cleavage division does not appear to follow activation of the egg automatically and independently since it has been shown that the state of the male elements introduced at fertilization can influence the rate and ultimate success of the changes which take place at this time.

Mechanisms of Selective Fertilization in the Rabbit: Sperm Transport and Viability

“…Following mixed insemination of equal volumes of X-irradiated and non-irradiated semen from 220 J. W. Overstreet and C. E. Adams the same ejaculate, Bedford & Hunter (1968) observed that the fertilizing ability of spermatozoa was not affected until doses of 15,000 R were adminis¬ tered. Determination of the source of the fertilizing spermatozoon was possible within only 48 to 50 hr following the ovulating injection since eggs fertilized by irradiated spermatozoa never cleaved more than twice whereas normally developing eggs had reached eight cells or more.…”

“…The effects of X-irradiation on rabbit spermatozoa have been studied on a number of occasions (Amoroso & Parkes, 1947; Chang, Hunt & Romanoff, 1957;Nuzhdin & Nizhnik, 1960;Bedford & Hunter, 1968). Following mixed insemination of equal volumes of X-irradiated and non-irradiated semen from 220 J. W. Overstreet and C. E. Adams the same ejaculate, Bedford & Hunter (1968) observed that the fertilizing ability of spermatozoa was not affected until doses of 15,000 R were adminis¬ tered.…”

A Method for Objective Evaluation of the Fertilizing Ability of Spermatozoa Irrespective of Genetic Character

“…In a previous study, Bedford & Hunter (1968) have reported that X-irradiation of rabbit spermatozoa at a dose level up to 10,000 rad has no effect on their fertilizing ability. Thus, when aliquots of spermatozoa irradiated at 10,000 rad were inseminated together with comparable aliquots of normal spermatozoa from the same ejaculate, the irradiated spermatozoa fertilized 50% of the ova.…”

“…In one series of experiments designed to study the effect of incubation at a slightly elevated temperature on the fertility of spermatozoa (Ulberg & Burfening, 1967), an ejaculate sample was incubated at 40°C for 3 hr, a control aliquot of the sample being incubated likewise at 38°C. The control or the experimental sample was then subjected to X-irradiation at a rate of 500 rad/ min, as described by Bedford & Hunter (1968), a total dose of 6000 to 7000 rad being given. The two equal volumes containing motile irradiated or nonirradiated spermatozoa in equal numbers were mixed thoroughly, and the mixture was inseminated intravaginally into an oestrous rabbit which also received an ovulating injection of 100 i.u.…”

“…The ova were assessed by phase microscopy for the stage of cleavage, for perivitelline spermatozoa, and for the stage of the nuclei or mitotic figures in the blastomeres, as described elsewhere (Bedford & Hunter, 1968). In the eggs which had only reached the two-to four-cell stage with or without fragmentation, the presence of one or more perivitelline spermatozoa was taken as evidence of fertilization when the fertilizing sperm tail could not be found.…”