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J. W. OVERSTREET

Summary.

When does were inseminated with spermatozoa from the lower part of the corpus epididymidis of individual bucks, fertilization rates varied greatly and in almost every case, cleavage occurred later than has been reported following the insemination of ejaculated spermatozoa. The retardation occurred both when spermatozoa were deposited in the uterus and in the Fallopian tube, with the exception of those animals showing the greatest delay, where tubal insemination resulted in fewer eggs being fertilized and in cleavage being more retarded.

It appears that the phenomenon of delayed fertilization resulting from the use of epididymal spermatozoa is not due to an abnormal rate of sperm migration, but to the need for an extended period of capacitation. The epididymal spermatozoa of some bucks required more than twice as much time for capacitation as that reported to be necessary for ejaculated spermatozoa. It is suggested that the process of sperm capacitation is primarily a continuation of the maturation process begun in the epididymis of the male.

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J. W. OVERSTREET

Summary.

Rabbit ova can be labelled with fluorescein isothiocyanate (FITC) or rhodamine blue isothiocyanate (RBITC) for use in mixed egg-transfer experiments. Both FITC and RBITC bind to the zona pellucida of the treated egg, allowing recognition of experimental and control groups in a mixed population. Labelling ova with one of these dyes is not detrimental to their penetrability or fertilizability and can be of significant value in a fertilization assay in which experimental and control ova are transferred to the same tubal environment.

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J. W. OVERSTREET

Summary.

Groups of does were tubally inseminated with concentrations of 500 to 10,000 motile capacitated spermatozoa in 10 μ1 of serum acidic saline about the time of ovulation, or with 1000 to 10,000 motile untreated spermatozoa 10 hr before the expected time of ovulation. The minimum number of spermatozoa required for fertilization was in the 500 to 1500 range. Fertilization levels >90% were not reached until 10,000 motile capacitated spermatozoa were inseminated at the time of ovulation, while 10,000 untreated spermatozoa deposited 10 hr earlier, fertilized less than 70% of the eggs.

A higher level of fertilization was consistently observed when the spermatozoa were inseminated in 10-μ1 volumes than when the same number of spermatozoa was deposited in 100 μ1. There was little difference in the level of fertilization irrespective of whether tubal insemination of capacitated spermatozoa took place about the time of, or 4 hr before, ovulation.

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W. F. Blazak and J. W. Overstreet

Summary. In the present study of 15 fertile men, a mean of 89 ± 3% of ejaculated cells had nuclei which were stable after treatment with 1% sodium dodecyl sulphate (SDS). Examination of repeated ejaculates demonstrated the constancy of the response of ejaculated spermatozoa from different men to SDS treatment. After 5 cycles of washing, the percentages of cells with stable nuclei after SDS treatment declined from 85 ± 3 to 46 ± 6% and ranged from 12 to 66% for different men. The supernatant derived from the first wash inhibited the nuclear chromatin decondensation of washed spermatozoa. Treatment of washed spermatozoa with 1 mm-zinc (which binds to –SH groups) or 1 mm-copper (which promotes oxidation of –SH groups to –S—S–) resulted in a significant increase in the proportion of cells with stable nuclear chromatin. However, the stabilizing effects of zinc could be reversed with continued washing of the cells, whereas the nuclei of copper-treated cells remained stable to SDS after additional washing. We conclude that (1) the number of spermatozoa with nuclei that are insufficiently stabilized by disulphide bonds is much higher in the semen of fertile men than was previously thought, (2) significant differences exist among fertile men in the proportions of ejaculated spermatozoa extensively stabilized by disulphide bonds, and (3) removal of seminal plasma and/or cellular contaminants (zinc) is necessary to unveil the instability of the nuclear chromatin in the ejaculated spermatozoa of fertile men.

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J. W. Overstreet and G. W. Cooper

Summary. Few spermatozoa were present in the ampullae of females 12 h after intravaginal artificial insemination (AI) when there was no ovulation-inducing stimulus. When ovulation was induced, sperm distributions in the female tract 12 h after AI did not differ from those observed 12 h after natural mating. The number of spermatozoa in the oviductal isthmus was similar in all 3 groups as was the percentage of isthmic spermatozoa exhibiting 'activated' motility. When fertile mating was delayed for 8 or 12 h after coitus with a vasectomized male (i.e. 2 h before or after ovulation), spermatozoa were not present in the ampulla 4 h later. The numbers of spermatozoa recovered from the cranial isthmus after delayed matings and 12 h after natural matings did not differ, but after delayed matings the motility of isthmic spermatozoa was non-progressive or poorly progressive and none exhibited 'activated' motility. Flagellar activity of isthmic spermatozoa recovered 4 h after delayed matings and after natural matings was similarly depressed. These observations indicate that sperm ascent to the tubal ampulla in the sustained phase of transport, though enhanced by ovulation, must also depend on changes in flagellar activity and a specific pattern of motility, both of which appear only after spermatozoa have resided for more than 4 h in the female tract.

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J. W. OVERSTREET and J. M. BEDFORD

Summary.

The acrosome reaction of rabbit spermatozoa, an essential prerequisite for penetration of the zona, occurs usually in the vicinity of the egg, suggesting that the rabbit egg may produce a factor akin to the `fertilizin' of some invertebrates. Specific inactivation of such a factor should render eggs impenetrable and possibly point to the nature of a `fertilizin' in mammals.

Rabbit eggs with granulosa cells removed were treated for different periods with trypsin, chymotrypsin, neuraminidase or anti-progesterone antiserum, and then transferred alone, or together with control eggs (one group labelled with fluorescein isothiocyanate), to the oviducts of inseminated recipients. Three hours later the eggs were recovered and the experimental and control groups were compared for penetration of the vitellus and for numbers of spermatozoa within the perivitelline space or in the zona pellucida. None of these treatments affected the penetrability of the zona pellucida significantly since the number of spermatozoa within treated eggs in any one experiment was always comparable to that of untreated eggs exposed to the same fertilization environment.

If there is a specific substance emanating from or present on the surface of the rabbit egg which induces the acrosome reaction, its activity seems unaffected by trypsin or chymotrypsin; the charged radicals of N-acetyl neuraminic acid or local concentrations of progesterone do not appear to be involved.

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J. M. BEDFORD and J. W. OVERSTREET

Summary.

X-irradiation with 6000 to 10,000 rad can be used as a method of `marking' a population of rabbit spermatozoa without affecting their fertilizing ability. Following a mixed insemination, irradiated spermatozoa compete equally well with non-irradiated spermatozoa from the same population, as judged by differential egg development 50 hr after insemination, at which time eggs fertilized by irradiated spermatozoa display obviously retarded cleavage.

In this paper, the technique has been used to demonstrate: (a) a subtle decrease in the fertility of spermatozoa after incubation in vitro at 40° C, which was not detectable by insemination of the treated sample alone, (b) the ability of spermatozoa from the proximal cauda epididymidis to initiate contact with and fertilize ova as readily as ejaculated spermatozoa following mixed insemination, there being no delay in the time of egg penetration by the epididymal spermatozoa, (c) the existence of selective fertilization between individuals of the same breed, as well as those differing genetically.

The irradiation marking technique could have many applications in comparative assessment of the fertility of semen from different individuals, irrespective of their genetic make-up, and in testing the effect of various treatments or environments on the fertilizing capacity of spermatozoa.

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J. W. OVERSTREET and J. M. BEDFORD

The capacitation process has been suggested as a prerequisite for every stage of gamete interaction, including penetration of the granulosa cell investment (Austin, 1960), contact with the zona pellucida (Bedford, 1967; Hartmann & Gwatkin, 1971), penetration of the zona substance (Bedford, 1970) and, for the hamster at least, incorporation into the vitellus (Yanagimachi & Noda, 1970).

Morphological studies have demonstrated a characteristic fusion and vesiculation of the plasma membrane and outer acrosomal membrane of the spermatozoon during its passage through the granulosa cell mass (Barros, Bedford, Franklin & Austin, 1967). Capacitation is considered to be a prerequisite for this acrosome reaction and it may be inferred that membrane changes are related to capacitation as well (Bedford, 1969, 1970). It is uncertain,however, whether the

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J. W. Overstreet and R. A. Tom

Summary. When non-motile spermatozoa were used in seminal plasma for artificial insemination, rapid sperm transport to the oviducts was observed in every animal examined 15 min after insemination. Rapid transport never occurred when non-motile spermatozoa were suspended in artificial media, but when motile spermatozoa were suspended in artificial media they were frequently recovered from the oviducts within 15 min. Two successive artificial inseminations 15 min apart were each followed by rapid transport when both inseminates contained seminal plasma; when the second inseminate consisted of spermatozoa suspended in saline, only the first sperm population reached the oviducts. Rapid sperm transport was effectively blocked with the α-adrenergic antagonist phenoxybenzamine. We therefore conclude that: (1) sperm motility is not required for rapid sperm transport in rabbits, (2) constituents of the seminal plasma may initiate rapid transport by stimulation of vaginal contractions, (3) independent contractions of the uterus under control of the sympathetic nervous system may continue the transport process once the spermatozoa reach the uterus.

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J. W. OVERSTREET and C. E. ADAMS

Summary.

The incidence of selective fertilization in rabbits was evaluated on the basis of the proportion of degenerating, fertilized eggs recovered 40 to 50 hr after X-irradiation of one part of a mixed inseminate. The results so obtained accord with our earlier observations on the proportion of colour-marked offspring.

Labelling of living spermatozoa with fluorescein isothiocyanate was used to establish the identity of spermatozoa recovered from the female tract and/or associated with eggs following mixed inseminations. Little difference in the proportion of the two sperm populations present in the female tract was apparent 6 hr after insemination but, by 13 hr, the spermatozoa of the superior buck predominated in all segments of the tract. Moreover, a higher proportion of the spermatozoa found in association with the eggs was contributed by the superior buck following either intravaginal or intratubal mixed insemination 10 hr before ovulation.

Although one factor in selective fertilization is a differential survival of the two sperm populations, compensation for this factor by intratubal deposition of equivalent numbers of active uterine spermatozoa did not raise the level of fertilization achieved by the inferior buck. It seems likely, therefore, that differences may exist between spermatozoa with regard to their speed of penetration of the egg membranes.