Time Of Sperm Penetration Research Articles

Oxygen consumption and ROS production are increased at the time of fertilization and cell cleavage in bovine zygotes

Oxygen consumption is a key indicator of metabolic activity within embryos. Increased oxidative activity and REDOX changes at the time of fertilization have been suggested to signal Ca(2+) oscillations after sperm penetration. The objective of the present study was to determine the oxygen consumption and the REDOX status of zygotes and early embryos at the time of sperm penetration and cell cleavage and to investigate how metabolism relates to key temporal events and developmental competence. Individual oxygen-consumption rates of bovine in vitro matured oocytes and presumptive zygotes (n = 101) were measured using the Nanorespirometer at 0, 7, 12, 17 and 24 h after IVF. Using the Embryoscope, oxygen-consumption profiles of individual oocytes and embryos (n = 75) were recorded repeatedly from 6 h until 30 h after IVF and time-lapse images were acquired, at intervals of ∼36 min. Oocytes and embryos were stained with Hoechst 33342 and visualization of nuclear stage was performed by fluorescence microscopy. To determine the REDOX status, cohorts of oocytes and zygotes (n = 55) were individually stained with REDOX-Sensor Red CC-1 and Hoechst 33342 at 0, 7, 12, 17 and 24 h after IVF and subsequently imaged by confocal microscopy. A peak of oxygen consumption was observed at the time of fertilization and a smaller rise and fall in oxygen consumption could be detected prior to the first cell cleavage. Increased reactive oxygen species production was also observed at 7 h and then at 24 h after IVF, just preceding the first embryonic cleavage. There are specific events during embryo development that appear to be associated with a change in oxygen consumption and REDOX state, indicating that both have a role in sperm-mediated oocyte activation and cell cleavage in bovine embryos.

118 THE PEAK OF OXYGEN CONSUMPTION AT THE TIME OF SPERM PENETRATION IS ASSOCIATED WITH AN INCREASE IN REACTIVE OXYGEN SPECIES PRODUCTION IN BOVINE ZYGOTES

Oxygen is essential for the generation of cellular energy (ATP) via oxidative phosphorylation, and thus oxygen consumption is a key indicator of metabolic activity within cells. Increased oxidative activity at the time of fertilization has been described for marine invertebrate oocytes. The objective of the present study was to determine if changes in oxygen consumption would occur at the time of fertilization and cell cleavage in bovine zygotes. The oxygen consumption of presumptive zygotes (n = 57) was measured individually and continuously from 6 until 30 h after IVF using the Embryoscope™ (Unisense Fertilitech A/S). A control group of in vitro matured oocytes (n = 9) that had not been fertilized was also measured continuously from 6 h after transfer to IVF medium. Time-lapse images were acquired during the measurements at intervals of approximately 36 min and used for evaluation of the developmental progress. Oocytes/zygotes were subsequently stained with Hoechst 33342 for more accurate classification by fluorescence microscopy. Cohorts of oocytes/zygotes (n = 55) were individually stained with REDOX-Sensor Red CC-1 and Hoechst 33342 at 0, 7, 12, 17 and 24 h after IVF and imaged by confocal microscopy to determine the REDOX potential and the distribution pattern as well as nuclear stage. Data was analyzed statistically using Proc Mixed, SAS. The mean oxygen consumption of the developing zygotes peaked markedly at 9 h after fertilization (0.552 nL h–1; value 14% over and above baseline oxygen consumption) and fell abruptly until 20 h after IVF (0.482 nL h–1). Following this, an oxygen peak of lower magnitude (0.487 nL h–1; value 1% over and above baseline oxygen consumption) was observed at the time of the first cleavage (detected between 21 to 32 h). The curve pattern for the control group was statistically different: the mean oxygen consumption was at its greatest from 8 to 12 h after IVF and then fell gradually until 30 h after IVF with no visible small increase in oxygen consumption at the supposed time of first cleavage. Mean respiration rates of the oocytes in the control group were significantly greater than those of the developing zygotes at all time points during the measurements. The mean pixel intensity of the REDOX-Sensor Red CC-1 staining was significantly greater at 7 h and at 24 h after IVF, when compared with the other group. The two distinct peaks of oxygen consumption were coincident with the REDOX pulses occurring at the time of sperm penetration and cell cleavage. However, due to the considerably higher magnitude of the oxygen peak occurring around the time of sperm penetration (signaling fertilization) we were led to speculate that only this peak was associated with an increased reactive oxygen species production. The REDOX pulse observed at the time of cleavage did not seem to be an oxygen-derived REDOX pulse because the oxygen consumption by the bovine zygotes was only changed marginally. ASL is supported by FCT, Portugal. The authors thank Unisense-Fertilitech for their collaboration.

Artificial Fertilization by Intracytoplasmic Sperm Injection in a Teleost Fish, the Medaka (Oryzias latipes)1

Intracytoplasmic sperm injection (ICSI) is a technique that has been successfully used for assisting reproduction in mammals. However, this method is still not reliable in nonmammalian species, including teleosts. We succeeded in producing medaka individuals by ICSI with a rate of 13.4% (28 hatched embryos out of 209 eggs fertilized by ICSI), the best value reported so far in teleosts, including zebrafish and Nile tilapia. Although the technique was based on that developed for mammalian eggs, some critical modifications were made to adjust it to the medaka egg, which has a thick and hard envelope (the chorion) and a single sperm entry site (the micropyle). Medaka ICSI was performed by injecting a demembranated spermatozoon into an egg cytoplasm through the micropyle 10-15 sec after egg activation induced by a piezo-actuated vibration, the site and timing of sperm penetration being consistent with those in normal fertilization in medaka. To increase the efficiency of ICSI in medaka, we found that the fertilization by ICSI should precisely mimic the fertilization by insemination with intact sperm, both spatially and temporally. The success rate of ICSI was highly variable in batches of eggs (ranging from 0% to 56%), suggesting that the conditions of eggs are important factors in stabilizing the production of individuals by ICSI. The success in medaka ICSI provides a basis for future research to understand the basic mechanisms in gamete biology of teleosts as well as for development of new technology that can yield valuable applications in fisheries science.

Replacement of nuclear protein by histone in pig sperm nuclei during in vitro fertilization

Sperm-specific nuclear protamines are dissociated before decondensation of sperm nuclei during fertilization in pigs. In the present study, replacement of nuclear protein by histone in boar spermatozoa during in vitro fertilization was evaluated by immunohistochemistry using anti-histone antibody. First, the specificity of the antibody used in this study was examined. Immunohistochemistry of the testes and epididymides indicated that somatic nuclei, but not elongated spermatids or maturing spermatozoa, were immunoreactive. Furthermore, immunoreaction was diminished after the antibody had been preincubated with unfractionated histone, indicating that the antibody was specific for the somatic nuclear histone. Immunohistochemistry of serial sections of oocytes, which were matured and co-cultured with boar spermatozoa for 2 to 6 h indicated that, at 2 to 3 h after insemination, penetrating sperm nuclei in the condensed state were not immunoreactive. At 4 to 5 h after insemination, some of the condensed sperm nuclei were immunoreactive in part or over the whole area of the nucleus, and all of the decondensing nuclei and male pronuclei were immunoreactive. At 6 h after insemination, the decondensing sperm nuclei and well-developed male pronuclei were immunoreactive. These results imply that, in pigs, remodelling of sperm nuclear protein from protamine to histone is initiated at the time of sperm penetration, before onset of decondensation and male pronuclear formation.

Replacement of nuclear protein by histone in pig sperm nuclei during in vitro fertilization

Sperm-specific nuclear protamines are dissociated before decondensation of sperm nuclei during fertilization in pigs. In the present study, replacement of nuclear protein by histone in boar spermatozoa during in vitro fertilization was evaluated by immunohistochemistry using anti-histone antibody. First, the specificity of the antibody used in this study was examined. Immunohistochemistry of the testes and epididymides indicated that somatic nuclei, but not elongated spermatids or maturing spermatozoa, were immunoreactive. Furthermore, immunoreaction was diminished after the antibody had been preincubated with unfractionated histone, indicating that the antibody was specific for the somatic nuclear histone. Immunohistochemistry of serial sections of oocytes, which were matured and co-cultured with boar spermatozoa for 2 to 6 h indicated that, at 2 to 3 h after insemination, penetrating sperm nuclei in the condensed state were not immunoreactive. At 4 to 5 h after insemination, some of the condensed sperm nuclei were immunoreactive in part or over the whole area of the nucleus, and all of the decondensing nuclei and male pronuclei were immunoreactive. At 6 h after insemination, the decondensing sperm nuclei and well-developed male pronuclei were immunoreactive. These results imply that, in pigs, remodelling of sperm nuclear protein from protamine to histone is initiated at the time of sperm penetration, before onset of decondensation and male pronuclear formation.

Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro.

The effects of oviductal fluid on sperm penetration and cortical granule exocytosis in pigs were examined. Cortical granule exocytosis in oocytes matured in vivo and in vitro was observed by staining with fluorescent-labelled lectin and laser-scanning confocal microscopy. Exocytosis of matured oocytes was classified into three categories after in vitro fertilization: complete cortical granule exocytosis and even distribution of exudate in the entire perivitelline space (type I); complete exocytosis and partial distribution of exudate (type II) and incomplete cortical granule exocytosis (type III). The incidence of oocytes with type I exocytosis was higher in oocytes matured in vivo than in those matured in vitro. The addition of oviductal fluid at a concentration of 1% or 10% to the fertilization medium decreased sperm penetration and the mean number of spermatozoa present in penetrated eggs. The distribution of cortical granule exudate was not different in the presence of 1% oviductal fluid after sperm penetration from that of control groups. When oocytes were cultured for 1.5 h in medium containing 10% or 30% oviductal fluid before insemination, the incidence of monospermy increased without a decrease in sperm penetration. Preculture of oocytes in medium containing 30% oviductal fluid increased type I cortical granule reaction and increased resistance of the zona pellucida to dissolution by 0.1% (w/v) pronase at the time of sperm penetration. These results suggest that a factor(s) from the oviductal secretion is required for the complete cortical granule reaction and in the modification of the zona pellucida.

Dose-dependent effect of heparin on fertilizing ability of goat spermatozoa

Intact bovine oocytes were used to study the effect of heparin on goat IVF. Oocytes were matured in Medium 199 plus estrous sheep serum. Fresh semen was incubated for 4 h at room temperature, and spermatozoa were then resuspended in medium Talp plus serum and incubated further for 1 h at 39 °C in 5% CO 2 in air. Later, spermatozoa were resuspended in Talp plus serum and heparin and were then incubated in microdrops until the oocytes were matured. In Experiment 1, the effect of heparin on spermatozoa from individual males was studied by a dose-response curve. In Experiment 2, the timing of sperm penetration in matured oocytes was studied to assess the stage at which the action of heparin could be expressed in the fertilization process. In Experiment 3, heparin from the same source but at different grades of bioactivity was adjusted for bioactivity and its effect on spermatozoa was compared in terms of penetration rates in order to identify heparin-dependent variations on goat IVF. In Experiment 4, the influence of calcium on the effect of heparin at different levels of bioactivity on the fertilizing ability spermatozoa was assessed as in Experiment 3. In Experiment 5, different batches of heparin from the same source and grade of bioactivity were compared as above. The results suggest that 1) heparin stimulates fertilization rates following a comparable pattern between males; 2) the most probable site of action is at the stage of sperm capacitation; and 3) provided that the source and grade of bioactivity is preserved, heparin maintains the efficiency of sperm penetration into matured oocytes.

Effects of male accessory sex glands on fertilization, polyspermy and morphometry of early embryos in the golden hamster

Preimplantation embryos sired by hamsters without accessory sex glands (ASG) were found to have a higher mortality rate and a slower cleavage rate than those sired by sham-operated males at 72 h post coitum (p.c.). A time-course study of fertilization in vivo was conducted to determine whether this effect was due to delayed fertilization. Ultrastructural morphometry of 48 h embryos was also undertaken to establish the earliest manifestation of developmental anomalies. Compared to sham-operated controls (SH), ablation of all the ASG (TX), or just the ventral prostate (VPX) or ampullary gland (AGX) had no effect on the timing of sperm penetration, extrusion of the second polar body and pronuclear formation. Females mated with AGX males tended to have more polyspermic embryos (9.7%; p < 0.05). The volumes, volume fractions (VV) of the blastomere nuclei, mitochondria and yolk material of the four-cell embryos sired by these same groups of males were assessed using point counting techniques. No difference in the VV of yolk and mitochondria could be observed between groups. However, the SH group did have a significantly larger proportion of the cell occupied by the nucleus (p < 0.05), and the TX group had a higher proportion of the nucleus occupied by nucleoli when compared with the SH group (p < 0.01). Smaller nuclei and larger nucleoli in the TX group was interpreted as an early manifestation of a slower division rate of the blastomeres.

Effect of the donor oocyte breed on in vitro fertilization results in cattle

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO2 in air and heparin.In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO 2 in air and heparin. In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.

Effect of bovine serum albumin concentration and source on sperm capacitation in the golden hamster.

Since virtually all experiments on sperm capacitation routinely include serum albumin in the culture media, the relative effect of BSA concentration on sperm capacitation was investigated. The fertile life of hamster sperm, as measured by the ability to penetrate the zona pellucida, decreased from 5 to 3 h as the concentration of BSA was increased from 3 to 18 mg/ml. To determine the extent of sperm capacitation in sperm populations that resulted in 100% penetration, the rate and timing of sperm penetration through the zona pellucida was determined. For each insemination, the rate of sperm penetration (%/min), the time when 50% of the eggs were zona-penetrated (T50), and the sperm capacitation index (T50/rate) were calculated. The extent of sperm capacitation as measured by all three parameters increased as the duration of preincubation or the BSA concentration was increased. The data on the duration of preincubation and the BSA concentration matrix indicated two properties of the effect of BSA on sperm capacitation: 1) the extent of sperm capacitation after the first hour of preincubation was independent of BSA concentration; and 2) when sperm were preincubated for two or more hours, the capacitating effectiveness of BSA was saturable; the saturating concentration of BSA decreased as the duration of preincubation increased. To determine whether the first hour of preincubation is independent of BSA, sperm were preincubated for 1 h without BSA and then 2 h with BSA. The extent of sperm capacitation of these sperm was equal to that of the 3-h control sperm; thus the first hour of capacitation is independent of BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The influence of postovulatory age on the rate of cleavage in in‐vitro fertilized rat oocytes

AbstractThe purpose of this study was to analyze the effect of postovulatory ‘aging’ in the oviduct on the rate of zygotic development. Two ovulatory ages were tested: oocytes collected from the oviducal ampullae 1) soon after ovulation (denoted freshly ovulated) or 2) 7‐hour postovulation. All the oocytes were from superovulated immature rats. By manipulation of the timing of the ovulatory hormone treatment, it was possible to place both types of oocytes into sperm suspension from the same pool and at the same time. The oocytes and spermatozoa were coincubated overnight. Cleavage was established by interference contrast microscopy. The time of the first cleavage of ova from the 7‐hour postovulation group was clearly advanced. Because the cleavage time curves were not parallel, no reliable estimate of the time difference could be made, but it was clearly in the range of 2 hr. This shift could not be related to any difference in the time of sperm penetration. Both groups of oocytes underwent penetration by spermatozoa at the same time. The time interval between maximal sperm penetration (94% of oocytes in both groups) and maximal cleavage (50% in both groups) was 23 hr in the freshly ovulated and 21 hr in the 7‐hour postovulatory eggs. Nor was the difference related to polyspermy, which was approximately 14% in both groups. These results support the hypothesis that developmental processes are under way in the oocyte before fertilization, but at a much slower rate than after fertilization.

Site‐specific sperm agglutination and the timed release of a sperm chemo‐attractant by the egg of the leptomedusan, Orthopyxis caliculata

AbstractIn Orthopyxis sperm entry apparently occurs at the site of emission of the polar bodies. If sperm are present from the time of spawning of the egg, they agglutinate head to head shortly after emission of the second polar body but only at a point on the egg surface under the second polar body. Since sperm agglutination does not occur elsewhere on the egg, it appears that this part of the surface of the egg of Orthopyxis and probably other hydromedusae is a special membrane patch which causes sperm to bind reversibly both to it, and to each other. The patch develops at a specific time during egg maturation and ceases function at or just after fertilization. Concomitant with the appearance of the patch is the production of a species‐specific sperm attractant by the egg. These results imply that the egg has strict control not only of the site and timing of sperm penetration but also of the time during which sperm are attracted to the egg.

Penetration of human eggs by human spermatozoa in vitro.

The timing of sperm penetration through the zona peliucida, and fertilization of human eggs had received little attention in the literature. Edwards et al. (1969) inseminated eggs matured in vitro with ejaculated spermatozoa which had been washed and found that spermatozoa did not penetrate the anna pellucida earlier than 7 h after insemination. A similar time interval between insemination and anna penetration by spermatozoa has been reported by others (Bavister et al., 1969; Overstreet and Hembree, 1976). This time interval has been considered to represent the time required for sperm capacitation, acrosome reaction and passage of the reacted spermatozoa through the zona pellucida (Austin, 1969; Austin et al., 1973). We report here that under our experimental conditions, sperm penetration into human eggs can occur much faster than previously reported. MATERIALS AND METHODS Immature oocytes were collected at laparoscopy by flushing ovarian follicles with Dulbecco’s phosphate buffered saline supplemented with 556 mM glucose and 0.1% bovine serum albumin. The recovery rate and criteria used for selection of viable oocytes were as reported by Lopata and coworkers (1974). The selected oocytes were thoroughly rinsed with the same buffer and transferred into 0.2 ml of a culture medium under liquid paraffin in a plastic petri dish. The culture medium was Ham’s F-b supplemented with 1 mM sodium pyruvate, 10 mM sodium lactate, penicillin G (100 units/mi) and 15% heat-inactivated human serum. The oocytes were incubated for 2 days at 37#{176}C under 5% CO2 in air. When examined at the end of incubation, the follicle cells that were originally surrounding each oocyte were found as a compact cell mass attached to an almost naked oocyte. The oocytes were mechanically freed from the follicle cell mass and then washed twice with BWW medium (Biggers et al., 1971), supplemented with 15% heat-inactivated human serum and placed in 0.2 ml of fresh, BWW medium (serum-supplemented) under paraffin oil in a petri dish. Freshly ejaculated spermatozoa were washed 3 times with BWW medium by centrifugation (1,000 rpm for 10 mm each) and added to the droplet containing the oocytes. The final concentration of spermatozoa in the droplet was about 5 X io spermatozoa/mI. The dish was incubated at 37#{176}C Accepted January 20, 1978. Received November 10, 1977. under 5% CO2 in air. Two oocytes, selected at random, were removed from the dish at 2, 4 or 7 h after insemination (total number of oocytes studied = 6) and were fixed in 5% glutaraldehyde, postfixed with 2% osmium tetroxide, dehydrated in graded ethanol and embedded in an epon-araldite mixture. The oocytes were studied using both light and transmission electron microscopy for signs of zona penetration and fertilization.

Timing of sperm transport, sperm penetration and cleavage in the rat.

Times of sperm entry into the oviduct from the uterus, into the ampulla from the isthmus; of sperm penetration into oocytes, and of cleavage, were determined using three mating regions. Time intervals and their errors of estimation were calculated. Spermatozoa were first found in the isthmus of the oviduct no earlier than 15 minutes after coitus, but required four hours to ascend the oviduct to the ampulla. The rate of sperm arrival was equal to the rate of sperm penetration, i.e., about 3 sperms/hour. Time of cleavage in vivo was 20.6 hours after sperm penetration in ad libitum mated animals. In culture, oocytes cleaved at exactly the same time as in vivo. Delaying sperm arrival to the site of fertilization (by delaying mating) shortened the time interval between median time of sperm penetration and median time of cleavage. It was concluded that the time of cleavage of the oocyte reflects primarily the time of sperm penetration, but is also influenced by the postovulatory age of the oocyte.

Timing of sperm penetration, pronuclear formation, pronuclear DNA synthesis, and first cleavage in naturally ovulated mouse eggs.

Timing of development of naturally ovulated mouse eggs from sperm penetration to first cleavage, including that of DNA synthesis, was established. In an attempt to limit variability, partial synchronization of ovulation was accomplished by shortening the length of the dark period to five hours, and partial synchronization of sperm entry was attempted by mating the females soon after the ovulated eggs reached the ampulla and by limiting the period at which mating could occur to only 20 minutes. Evidence of sperm penetration (presence of one or more sperm in perivitelline space or inside the vitellus) was found beginning 1.75 hours after the end of the mating period. Pronuclei were formed three to four hours after sperm entry. Pronuclear DNA, synthesis began about eight hours postmating, 3.25 to 4.5 hours after pronuclear formation, or about 6.25 to 8.5 hours after sperm entry; it was completed in almost all zygotes by 16 hours postmating. The first completed cleavage division was found 17 to 18 hours postmating, and almost all eggs had cleaved by 20 hours.

In vitro fertilization of hamster eggs by ejaculated or epididymal spermatozoa in the presence of male accessory secretions.

In vitro fertilization of hamster eggs by ejaculated or epididymal spermatozoa in the presence of seminal plasma or male accessory gland secretions was examined. There was no difference in the penetration rates, the time of sperm penetration and the optimal sperm concentration between ejaculated and epididymal spermatozoa. The swelling of the zona pellucida, a high incidence of polyspermy, distortion of the vitellus and the degeneration of eggs were observed after incubation of ejaculated or epididymal spermatozoa in the presence of seminal plasma or accessory gland secretions. Using ejaculated spermatozoa, the fertilization rates of eggs with or without follicular cells were similar but fertilization by epididymal spermatozoa was inhibited by secretion of the seminal vesicle or ventral prostrate gland but not by that of the coagulating gland or the dorsal prostrate.

The sperm acrosome reaction and fertilization in the guinea-pig: a study in vivo

Female guinea-pigs were naturally or artificially inseminated before or after ovulation and the distribution of spermatozoa in the oviducts and the time of sperm penetration into the eggs were determined. When animals were inseminated before ovulation, the spermatozoa stayed in the distal half of the oviduct until about the time of ovulation. Only a very few spermatozoa were present in the proximal half of the oviduct at the time of ovulation, but these were sufficient to effect fertilization. When animals were inseminated after ovulation, the spermatozoa ascended the oviduct faster than when animals were inseminated before ovulation, and fertilization commenced in 4 hr. Regardless of the time of insemination, the spermatozoa participating in fertilization appeared to undergo the acrosome reaction after they reached the proximal part of the oviduct or when they were very near the eggs.

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.