Morphological events during in vitro fertilization of prepubertal goat oocytes matured in vitro

Experiments were carried out to study morphological changes temporally associated with in vitro fertilization (IVF) of prepubertal goat oocytes and to elucidate some of the abnormalities occurring during this process. The effects of different intervals of insemination on subsequent embryonic development were also studied. Prepubertal goat oocytes collected at slaughter were matured in TCM199 supplemented with estrous goat serum (20%), FSH (10 microg/ml), LH (10 microg/ml) and estradiol-17 beta (1 microg/ml) for 27 h at 38.5 degrees C. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation as described by Younis et al. (37) but with 100 microg/ml heparin. Representative oocytes were fixed every 2 to 4 h from 2 to 28 h after insemination for a study of sperm penetration, sperm head decondensation, meiotic activation, female chromosome decondensation, and male and female pronuclear formation. At the same intervals after insemination, some of the ova were co-cultured on granulosa cell monolayers for up to 9 d. Sperm penetration into the ooplasm was first observed at 4 h post insemination; decondensation of male and female chromatin and formation of male and female pronuclei occurred at 6 to 8 and 10 to 16 h after insemination, respectively. Highest proportions of oocytes were penetrated after exposure to spermatozoa for 8 h. There were no significant differences in ovum penetration after longer insemination intervals. Cleavage was first observed 24 h after insemination. Three types of abnormalities were observed. These were polyspermy, polygyny and asynchrony in the development of the female and male pronuclei, apparently due to a delay in the decondensation of the male pronucleus. Significantly higher proportions of oocytes cleaved (31.2 to 45.5%) after 20, 24 or 28 h insemination intervals than following shorter intervals of exposure to spermatozoa. However, the sperm exposure interval did not significantly affect subsequent embryonic development to the blastocyst stage. Embryos resulting from oocytes exposed to sperm cells for at least 12 h developed further than the 8-cell stage.     

Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes

The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matured in vitro was assessed. For this purpose, protamine was purified from boar sperm nuclei and an antiserum against protamine was developed. After affinity purification, the antiserum reacted exclusively with boar protamine during western blotting, showing no crossreactivity with core histones. Immunohistochemical evaluation revealed that only fully developed spermatid nuclei in boar testes stained strongly with the antiserum. When pig oocytes matured in vitro were fertilized in vitro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermic penetration. Paraffin wax sections of the inseminated oocytes were examined by immunohistochemical analysis with the antiserum. The proportion of condensed sperm nuclei that reacted with the antiserum was 87% of the sperm nuclei that penetrated by 2 h after insemination, and this decreased to 20 and 13% at 3 and 5 h after insemination, respectively. However, none of the decondensing sperm nuclei or male pronuclei reacted with the antiserum during the entire insemination period. These results indicate that a specific antiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei before decondensation during in vitro fertilization.     

In vitro fertilization rate of horse oocytes with partially removed zonae

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 mu M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2 57 ), 12 (7 58 ), 52 (31 60 ), and 86% (44 51 ) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2 2 ), 57 (4 7 ), 58 (18 31 ), and 34% (15 44 ), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11 49 ), and increased to 38% (21 55 ) at 5 h, to 46% (23 50 ) at 10 h, and to 56% (27 48 ) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.     

Maturation, fertilization, and development of dog oocytes in vitro.

Preovulatory oocytes were collected from ovaries of beagle bitches that had received superovulatory treatment. They were cultured in a modified Krebs-Ringer bicarbonate solution containing 10% fetal calf serum and 30 mg/L gentamicin sulfate for up to 72 h. About 32% of oocytes reached metaphase II by 72 h of culture. When these in vitro-matured oocytes were inseminated with ejaculated beagle spermatozoa that had been preincubated for 4 h, sperm penetration of the zona pellucida started about 1 h after insemination, and both male and female pronuclei were seen in the ooplasm at 8 h after insemination. At 18-20 h after insemination, oocytes were transferred to Whitten's medium and cultured for 76-78 h. The first cleavage was observed at 48 h after insemination, and 15 of 45 oocytes developed to the 2-8-cell stage. These results demonstrate that in vitro-matured canine oocytes can be fertilized and develop to the 8-cell stage in vitro.     

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm^?1 AC followed by a 30 μsec pulse at 120 V mm^?1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88–100%) and polyspermic rates (79–100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 ± 0.5 vs. 4.6 ± 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. © 1993 Wiley-Liss, Inc.     

Penetration in vitro of bovine oocytes during maturation by frozen-thawed spermatozoa

Bovine immature oocytes cultured for various times in TC-199 medium were inseminated with frozen-thawed spermatozoa in Medium BO with caffeine (5 mM) and heparin (10 micrograms/ml). Very high penetration rates (95-100%) were obtained in all oocytes which had been cultured for 0-20 h. When oocytes cultured for 0 and 4 h were inseminated, 100% of them were penetrated and had a decondensing sperm head and most of the oocytes remained at the stage of condensed germinal vesicle (GV) to telophase-I 20-22 h after insemination. The formation of male and female pronuclei was first observed in oocytes inseminated 8 h after culture. The proportions of polyspermy and average number of spermatozoa in penetrated oocytes gradually decreased as oocyte maturation proceeded. Penetration of at least one spermatozoon with a decondensing head into oocytes at the GV stage (without culture) was almost completed up to 8 h after insemination and at that time most of the penetrated oocytes were still at the stage of GV or condensed GV. These results indicate that maturation of bovine oocytes is not required for sperm penetration into the vitellus or for sperm nuclear decondensation under the in-vitro conditions used.     

Histone H1 kinase activity during in vitro fertilization of pig follicular oocytes matured in vitro

The present study was conducted to clarify the relationship between histone H1 kinase (H1K) activity and events associated with in vitro fertilization of pig follicular oocytes matured in vitro. Histone H1 kinase has been shown to be homologous with a maturation promoting factor (MPF). Cumulus-oocyte complexes obtained from prepubertal gilts were cultured for 46 h in a modified Waymouth's MB752/1 medium and were then inseminated in vitro with frozen-thawed and preincubated epididymal boar spermatozoa. At 4, 6, 8 and 10 h post insemination, the oocytes were stained with 10 microg/ml Hoechst-33342 and examined under a fluorescent microscope for the stage of fertilization, according to morphological changes of oocyte nuclear chromatin and the extent of sperm penetration. Sperm penetration was observed to occur within 4 h post insemination (20.5%), and the percentage of fertilized oocytes increased (P < 0.01) to 72.9% at 8 h post insemination. Pronuclear formation was observed from 6 h post insemination (3.3%) and the percentage increased (P < 0.01) to 46.8% at 10 h post insemination. In each examination period, H1K activities in unfertilized oocytes at metaphase-II remained unchanged (112.0 fmol/h/oocyte) and were higher (P < 0.01) than those in fertilized oocytes (30.1 fmol/h/oocyte). The H1K activity in fertilized oocytes such as oocytes emitting a second polar body, oocytes with an enlarging sperm head(s) and oocytes with multiple pronuclei did not differ significantly. These results suggest that MPF in pig oocytes is inactivated shortly after sperm penetration and is maintained at the basal level throughout pronuclear formation.     

Protein phosphorylation is essential for formation of male pronucleus in bovine oocytes

This study examined the association between the morphological and protein phosphorylation events following sperm penetration of in vitro matured and in vitro fertilized bovine oocytes. Oocytes were labeled with [³²P]‐orthophosphate at 3 hr intervals from 3 to 18 hr of following insemination. The phosphorylation of protein complexes of 23 kDa and 18 kDa specifically increased with the formation of male and female pronuclei. In addition, oocytes were treated with 6‐Dimethylaminopurine (6‐DMAP) or Okadaic acid (OA) at 0, 3, 6, and 9 hr respectively following insemination. Although the formation of female pronucleus was not affected by 6‐DMAP, the male pronuclear formation was completely inhibited by the presence of 6‐DMAP at 0 and 3 hr of post insemination. The formation of both pronuclei was inhibited by the presence of OA at any time following insemination. These results suggest that the male pronuclear formation is associated with protein phosphorylation and that the formation of the male and the female pronuclei may involve different factors in bovine zygotes since they respond differently to the kinase modulations. Mol. Reprod. Dev. 52:43–49, 1999. © 1999 Wiley‐Liss, Inc.     

Early events of in-vitro fertilization of cat eggs by epididymal spermatozoa

Newly ovulated eggs from mature queens treated with PMSG and hCG were inseminated in modified KRB solution with spermatozoa recovered from the cauda epididymidis of male cats. When 5 eggs were examined 15 min after insemination, no signs of sperm penetration into the vitellus were observed. However, in an egg examined before fixation 20 min after insemination, a spermatozoon whose head had passed through the zona pellucida was observed. Very high proportions (90-100%) of the eggs were penetrated when they were examined 0.5-5 h after insemination. Male and female pronuclei were first observed in eggs examined 4 h after insemination.     

Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection.

Development of bovine oocytes after intracytoplasmic sperm injection (ICSI) was investigated. Oocytes were matured for 24-26 h in vitro and injected with isolated sperm heads. When treated with 7% ethanol (v/v) for 5 min, 71.7% of ICSI oocytes were activated as shown by the resumption of meiosis and the formation of female pronuclei. However, 41.5% of injected sperm heads remained condensed at 18-20 h after injection into the ooplasm. The incidence of decondensing sperm and that of male pronuclei at this stage were 15.1% and 26.4%, respectively. A total of 55.5% of oocytes reached the 2-cell stage following sperm head injection and 54.7% after sham-ICSI; these percentages were not significantly different from those following in vitro fertilisation (IVF) (73.1%). The percentage of 2-cell embryos reaching the 8-cell stage following ICSI was 37.5%, and 27.6% after sham-ICSI, which were significantly lower (p < 0.01) than the equivalent percentage following IVF (62.4%). The percentages of parthenogenetic embryos reaching the 2-cell, 4-cell and 8-cell stages following ICSI were 56.4%, 48.9% and 30.0%, respectively. These results indicate that the low rate of normal embryonic development of bovine oocytes following ICSI is largely due to the parthenogenetic activation of the oocytes.     

Effect of duration of in vitro maturation on nuclear maturation and fertilizability of feline oocytes

This study was conducted to improve in vitro production of embryos from domestic cats using TCM-199 as an IVM medium. The time sequence of nuclear maturation and the optimal timing of in vitro insemination were examined. Most oocytes were at the germinal vesicle stage immediately after collection; however, 8.3% had already resumed meiosis before IVM culture. After 30 h of IVM culture, the percentage of oocytes at metaphase II (MII) reached a peak (75.5%) and did not change (P>0.05) from 30 to 48 h after IVM culture. The percentage of oocytes with two pronuclei was higher (P<0.05) for oocytes matured for 30 and 36 h (38.2 and 33.0%, respectively) than for those after IVM culture for only 24 h (18.5%). Total sperm penetration rate was highest (P<0.05) for oocytes that had been matured for 30 h (46.1%). After 30 h of IVM and 18 h of IVF culture, 66.3 and 24.8% of inseminated oocytes had cleaved and developed to the blastocyst stage, respectively. We concluded that IVM of feline oocytes for 30 h in TCM-199 resulted in optimal nuclear maturation and sperm penetration.     

Effect of semen preparation on IVF of prepubertal goat oocytes

The aim of these experiments was to study the effects of different methods of washing and selection of spermatozoa on the IVF of IVM oocytes from prepubertal goats. Fresh ejaculates from 3 males of proven fertility were processed according to the following treatments: 1) centrifugation in TALP, 2) centrifugation in sucrose-based Ficoll medium, 3) centrifugation in Percoll gradients at 40 and 80%, 4) by swim-up and 5) by dilution of spermatozoa (1:40) in (1:1) TALP. In all 5 treatments spermatozoa were incubated for 45 min with 100 microg/mL of heparin and then added to Fert-TALP. Oocytes were matured for 27 h in TCM-199 supplemented with 20% estrous goat serum (EGS), FSH, LH and estradiol-17beta. Spermatozoa (4x10(6) cells/mL) were coincubated with oocytes in 100 microL of Fert-TALP with hypotaurine for 24 h, after which the oocytes were transferred to a granulosa cells monolayer in TCM-199 plus 10% of EGS for 24 h (48 h post insemination). At 17 h post insemination a sample of sperm-exposed oocytes was taken and stained in lacmoid to observe sperm penetration and the formation of pronuclei. At 48 h post insemination the cleavage rate of oocytes was evaluated. Motility, viability and acrosome status of the spermatozoa were evaluated immediately after the mixing of the ejaculates, after washing and selection treatments, and after incubation with heparin and at 17 h post insemination. The different ejaculate treatments did not affect the penetration and cleavage rates of oocytes. At 48 h post insemination the cleavage rate was 46.9, 36.6 and 29.0% for dilution, Ficoll and swim-up preparations, respectively. Only the swim-up protocol improved sperm motility and viability compared with that of the initial semen sample and with the other sample treatments. At 17 h post insemination the semen parameters were the same for all sperm sample treatments.     

Reduced polyspermic penetration in porcine oocytes inseminated in a new in vitro fertilization (IVF) system: straw IVF

High incidence of polyspermy is still a major problem in the in vitro fertilization (IVF) of porcine oocytes matured in vitro. This study was designed to examine whether embryo cryopreservation straws can be used to conduct IVF in porcine oocytes. The efficiency of this system was further compared with traditional microdrop IVF. Immature oocytes were aspirated from antral follicles and matured in vitro. After maturation, oocytes were inseminated either in straws or in microdrops with frozen-thawed boar spermatozoa. For straw IVF, sperm concentration and the presence of air columns between insemination segment and oil column were examined. Sperm-oocyte binding and cortical granules (CGs) before and after sperm penetration were examined by confocal microscopy. When various sperm concentrations were used for IVF in the straws with air columns, it was found that 5 x 106 cells/ml of sperm concentration was the optimal concentration; a high penetration rate (94.0%) and normal fertilization (oocytes with both male and female pronuclei) rate (38.2%) were obtained. Increasing sperm concentration to 10 x 106 cells/ml increased polyspermic penetration (61.9%) without affecting sperm penetration (86.9%). Reducing sperm concentration to 1 x 106 cells/ml reduced polyspermic penetration (25.6%), but sperm penetration rate (69.9%) was also reduced. When IVF was conducted in the straws with or without air columns, and in the microdrops, it was found that sperm penetration in the straws with air columns (96.5%) was significantly (p < 0.05) higher than that in the straws without air columns (81.7%) and in the microdrop (72.9%). However, the incidence of polyspermic penetration in the straws with air columns (34.2%) and without air columns (36.6%) was significantly (p < 0.05) lower than that (52.4%) in the microdrops. The number of spermatozoa bound to the oocytes was increased gradually in the straws but not in the microdrops in which more spermatozoa bound to the oocytes soon after insemination. CG exocytosis was more complete and faster in the oocytes inseminated in the straws than in the microdrops. These findings indicate that IVF of porcine oocytes in the straws provides a better condition in which more oocytes are fertilized normally than that in the microdrop IVF.     

Pronucleus formation, DNA synthesis and metaphase entry in porcine oocytes following intracytoplasmic injection of murine spermatozoa

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilisation. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8-9 h following the injection of porcine sperm, and 6-8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte centre. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. Ultrastructural observation revealed that male pronuclei derived from murine sperm in porcine oocytes are morphologically similar to normal male pronuclei in porcine zygotes. These results suggest that species-specific paternal factors influence the onset of pronucleus formation and DNA synthesis. However, normal nuclear cytoplasmic interactions were observed in porcine S-phase oocytes following murine sperm injection.     

Fertilisability of ovine, bovine or minke whale (Balaenoptera acutorostrata) spermatozoa intracytoplasmically injected into bovine oocytes

This study was conducted to investigate the possibility of using bovine oocytes for a heterologous fertility test by intracytoplasmic sperm injection (ICSI) and to compare the pronuclear formation of ram, bull and minke whale spermatozoa after injection into bovine oocytes. Bovine oocytes were cultured in vitro for 24 h and those with a polar body were selected for ICSI. Frozen-thawed semen from the three species were treated with 5 mM dithiothreitol for 1 h and spermatozoa were killed by storing them in a -20 degrees C refrigerator before use. ICSI was performed using a Piezo system. Three experiments were designed. In experiment 1, a higher (p < 0.05) male pronuclear formation rate was found in the oocytes injected with ram (52.6%) or bull (53.4%) spermatozoa than with minke whale spermatozoa (39.1%). In experiment 2, sperm head decondensation was detected at 2 h after ICSI in the oocytes injected with a spermatozoon of each species. Male pronuclei were first observed at 4 h in the oocytes injected with ram or bull spermatozoa and at 6 h in oocytes injected with minke whale spermatozoa. The mean diameters of male pronuclei derived from both whale and bull spermatozoa were larger than those from ram spermatozoa (30.4 microm and 28.3 microm vs 22.4 microm, p < 0.005). The mean diameter of female pronuclei in the oocytes injected with whale spermatozoa was also larger than with ram spermatozoa (29.3 microm vs 24.7 microm, p < 0.05). The development of male and female pronuclei was synchronous. In experiment 3, ethanol-activated oocytes injected with a spermatozoon from any of the three species achieved significantly higher (p < 0.05-0.001) cleavage rates than control oocytes. Blastocyst formation was only observed when bull spermatozoa were used. The results of this study indicate that dead foreign spermatozoa can participate in fertilisation activities in bovine oocytes after ICSI.     

A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes

Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro with in-vitro capacitated spermatozoa. Analysis of 621 penetrated ova fixed at various times after in-vitro insemination led to definition of 6 stages of early development. A time sequence for sperm penetration, sperm head decondensation, male pronucleus formation, the activation of second meiotic division, female chromosome decondensation and pronucleus development was established. First sperm penetration into the ooplasm was recorded 6 h after insemination; 1-2 h was required for the sperm head to decondense and another 4-6 h to develop into the opposing pronucleus stage. Synkaryosis and first cleavage occurred 28 h after fertilization. Examination of the early stages revealed four types of abnormalities, i.e. polyspermy, polygyny, asynchrony between male and female pronucleus development, and preactivation of cytokinesis.     

Development potential of bovine oocytes matured in vitro or in vivo

Bovine oocytes matured in vivo or in vitro were evaluated after sperm-oocyte incubation for frequency of sperm penetration, frequency of male pronuclei formation, and embryonic development. The frequency of sperm penetration was not different for in vitro matured oocytes (216/295, 73%) vs. in vivo matured oocytes (119/176, 70%). However, formation of male pronuclei was reduced (p less than 0.05) for oocytes matured in vitro (149/216, 69%) vs. in vivo (104/119, 88%). Early embryonic development was evaluated 48 h after the onset of sperm-egg incubations. In vitro matured and fertilized oocytes failed to develop to the 2-cell stage (3/88, 3%), whereas oocytes matured in vivo showed normal development (23/56, 40%) to the 2- and 4-cell stage. Development to the blastocyst stage was evaluated after 5 days in ovine oviducts (in vivo). Morulae and blastocysts were obtained only after in vitro fertilization from oocytes that were in vivo-matured (recovered from oviduct, 14/56, 25%; recovered from follicle, 36/80, 45%). Oocytes that were matured in vitro and fertilized in vitro failed to develop to morulae (0/33) in vivo.     

Sperm penetration in vitro of pig follicular oocytes matured in culture.

Boar ejaculated and epididymal spermatozoa were preincubated in modified KRB or the isolated oviduct and uterine horn of an oestrous sow for 4.5-5 h at 37 degrees C before introduction into medium containing ovarian oocytes previously cultured for 24 h. At examination 17-20 h after insemination 60.6% of the total oocytes had reached at least the 2nd metaphase. The proportions of oocytes penetrated (i.e. enlarged sperm head or male pronucleus and corresponding sperm tail) were 0, 10.0 and 16.7% with ejaculated spermatozoa, and 3.3, 19.6 and 26.4% with epididymal spermatozoa preincubated in modified KRB, oviduct and uterus, respectively. Although the proportion of oocytes with morphologically normal male and female pronuclei was low (10/36 = 27.8%), the results suggest that boar spermatozoa can be capacitated in the isolated genital tract of an oestrous sow and that capacitation of epididymal is better than that of ejaculated spermatozoa.     

The effects of spermatozoal irradiation with X-rays on chromosome abnormalities and on development of mouse zygotes after delayed fertilization

The chromosome complements of zygotes derived from oocytes aged post ovulation and fertilized in vivo with X-ray-irradiated sperm were studied. Ovulation was induced by an injection of luteinizing hormone-releasing hormone (LHRH) at pro-estrus and fertilization was achieved by artificial insemination at 13 h and 24 h after LHRH in order to obtain embryos from unaged and aged (12 h post-ovulation) oocytes respectively. Post-ovulatory aging prior to fertilization did not significantly affect the percentage of zygotes with irradiation-induced chromosome abnormalities. However, post-ovulatory aging had a negative effect on the morphology of male as well as female pronuclear chromosomes of the first cleavage metaphase. When fertilized with control spermatozoa this effect was apparent in both the male and the female pronucleus. When unaged oocytes were fertilized with X-irradiated spermatozoa chromosome morphology was also adversely affected in both pronuclei. In zygotes from aged oocytes, there was an extra negative effect of X-rays on the male pronuclear chromosomes only. After fertilization with X-irradiated sperm 27% of zygotes from aged oocytes were arrested at interphase compared to 7% from unaged oocytes. We suggest that post-ovulatory aging and X-rays affect the male and female pronuclear chromatin structure after fertilization. These chromatin alterations could interact with DNA lesions induced in the spermatozoa prior to fertilization, such that development to first cleavage can be blocked.