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.     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

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.     

The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content

The relationship between the timing of both sperm nuclear decondensation and male pronucleus formation in the oocyte and the relative level of disulfide bonds within the sperm nucleus was evaluated. Since reduction of sperm nuclear disulfide (S-S) bonds is a prerequisite for sperm nuclear decondensation in vitro and in vivo, we hypothesized that sperm nuclei with relatively few S-S bonds would require less time to decondense in the oocyte than sperm nuclei with higher numbers of S-S bonds, and that male pronucleus formation would occur more rapidly as well. Four types of hamster sperm nuclei, in which the extent of S-S bonding differed, were microinjected into hamster oocytes, and the time course of sperm nuclear decondensation and male pronucleus formation was charted. Cauda epididymal sperm nuclei, which are rich in S-S bonds, required 45-60 min to decondense. In contrast, nuclei containing few S-S bonds (namely sonication-resistant spermatid nuclei and cauda epididymal sperm nuclei treated in vitro with the S-S bond-reducing agent dithiothreitol) decondensed within 5-10 min of microinjection. Caput epididymal sperm nuclei, with intermediate S-S bond content, decondensed in 10-20 min. Regardless of when decondensation occurred, formation of the male pronucleus never preceded that of the female pronucleus, which occurred 1.25-1.5 h after microinjection. However, sperm nuclei with few S-S bonds were more likely than S-S rich nuclei to transform into male pronuclei in synchrony with the formation of the female pronucleus. We conclude that the timing sperm nuclear decondensation and pronucleus formation depends in part upon the S-S bond content of the sperm nucleus.     

Early morphological events of in vitro fertilized bovine oocytes with frozen-thawed spermatozoa

Bovine follicular oocytes were matured and inseminated in vitro with spermatozoa capacitated in vitro. The first evidence of sperm penetration was observed at 3 h after insemination. The penetration rate increased until 5 h, and reached a maximum rate (92%) at 5 h. Decondensation of the sperm head and pronuclear formation were observed 4 h and 7 h after insemination, respectively. Female chromatins of all penetrated oocytes were activated at 3 h, and female pronuclei were formed at 7 h after insemination. Percentages of oocytes with male and female pronuclei at 9 h were 88 and 94%. Polyspermy (4, 7, 19 and 29% at 4, 5, 7 and 9 h after insemination, respectively) and abnormal development of male pronuclei (6 and 7% at 7 and 9 h after insemination, respectively) were also seen.     

The movements and fusion of the pronuclei at fertilization of the sea urchin Lytechinus variegatus: Time-lapse video microscopy

The penetration of the sperm into the egg, and the movements of the male and female pronuclei were followed from sperm attachment through pronuclear fusion, using time-lapse video microscopy of gametes and zygotes of the sea urchin Lytechinus variegatus (23° C). The pronuclei move in four stages: I. Sperm Entry Phase, following sperm-egg fusion and a rapid radiating surface contraction (5.9 ± 1.3 μm/second) when egg microvilli engulf the sperm head, midpiece, and tail to form the fertilization cone and the sperm tail beats in the egg cytoplasm; II. Formation of the Sperm Aster, which pushes the male pronucleus centripetally at a rate of 4.9 ± 1.7 μm/minute starting 4.4 ± 0.5 minutes after sperm-egg fusion, as the male pronucleus undergoes chromatin decondensation; III. Movement of the Female Pronucleus, the greatest and fastest of the pronuclear motions at a rate of 14.6 ± 3.5 μm/minute at 6.8 ± 1.2 minute after sperm-egg fusion, which establishes the contact between the pronuclei; and IV. Centration of the Pronuclei to the egg center at a rate of 2.6 ± 0.9 μm/minute by 14.1 ± 2.6 minutes after sperm-egg fusion. Pronuclear fusion typically occurs after stage IV and proceeds rapidly starting 14.7 ± 3.6 minutes after sperm-egg fusion with the male pronucleus coalescing into the female pronucleus at a rate of 14.2 ± 2.6 μm/minute.     

Transformation of sperm nuclei into male pronuclei in nucleate and anucleate fragments of parthenogenetic mouse eggs

Our objective was to examine the ability of nucleate and anucleate fragments of artificially activated mouse eggs to transform sperm nucleus into male pronucleus. To this end, zona-free oocytes in metaphase II were activated by ethanol and bisected into halves (one with the spindle, the other anucleate) either within 10 to 20 min (series A) or 3 or 5 hr later (series B). In series A, the fragments were inseminated 3,5, and 8 h after activation, and in series B. 3 and 5 h after activation. Both nucleate and anucleate fragments lose the capability of transforming sperm nucleus into fully formed pronucleus sometime between 3 and 5 h after activation. In 8 h old parthenogenetic fragments, the majority of sperm nuclei remain unchanged or begin decondensation but never reach the stage of an early pronucleus. In over 1/3 of anucleate fragments of this age group, sperm nuclei develop defectively: chromatin decondenses inside the persisting nuclear envelope. In other experimental groups, the incidence of these abnormal sperm nuclei varies between 0 and 10%. In general, the anuclcate fragments retain the capability to transform sperm nuclei (fully or partially) longer than their nuclear counterparts. This difference may be accounted for by a different level of substances required for pronuclcar growth (extrachromosomal constituents of the germinal vesicle and nuclear lamins): high and constant in the cytoplasm of anucleate egg halves and low and progressively decreasing in the nucleate halves because of their putative uptake by the female pronucleus. However, the cytoplasmic factors responsible for the initial stages of transformation (nuclear envelope breakdown, chromatin decondensation) become eventually inactivated both in the presence and in the absence of a female pronucleus.     

Fertilization of parthenogenetically activated mouse eggs : I. Behaviour of sperm nuclei in the cytoplasm of parthenogenetically activated eggs

Oocytes from Swiss albino females were activated by heat-shock (44.5 °C) as described previously [8] and fertilized in vitro [10]. Time of insemination varied from 10 min to 3 h after activation. It has been found that spermatozoa may penetrate the zona pellucida and into the cytoplasm of the activated eggs. Sperm penetration may still occur as late as in the 3rd h after activation. The results indicate that the decondensation factor remains present in the activated eggs for at least 1.5 h after activation. Dispersion and transformation of the sperm chromatin into the early male pronucleus takes place at that time. In the pronucleus formed, no growth was registered. This may be caused by the fact that the processes of artificial activation precede those which accompany fertilization. The cytoplasm therefore loses the properties displayed in the course of the normal process of fertilization, when activation is the result of the penetrating spermatozoon.     

Failure of male pronucleus formation is the major cause of lack of fertilization and embryo development in pig oocytes subjected to intracytoplasmic sperm injection

The objectives of this study were 1) to compare the efficiency of intracytoplasmic sperm injection (ICSI) with and without additional artificial stimulation using frozen-thawed sperm and in vitro-matured porcine oocytes and 2) to determine the nuclear anomalies of ICSI oocytes that failed to fertilize or develop. In experiments 1 and 2, we evaluated the effects of additional activation treatments, e.g., electrical stimulus, Ca ionophore (A23187), and/or cycloheximide, on fertilization and development of ICSI porcine oocytes. Significantly higher fertilization, cleavage, and blastocyst rates were obtained for oocytes treated with a combination of ICSI and electrical activation (EA) (P < 0.05) than for those treated with ICSI alone. However, different combinations of electrical and chemical activation treatments did not further improve the rates of fertilization, cleavage, and blastocyst development for ICSI embryos. To elucidate the association between sperm head decondensation and oocyte activation and to investigate the cause of embryonic development failure, in experiment 3 we evaluated the nuclear morphology of oocytes 16-20 h after ICSI. Nearly 100% of oocytes showed female pronucleus formation after ICSI regardless of activation treatment. However, failure of male pronucleus formation with intact or swelling sperm heads was observed in some ICSI embryos, suggesting that these embryos underwent cell division with the female pronucleus only. Artificial activation (EA and A23187) had a beneficial effect on embryonic development, sperm decondensation was independent of the resumption of meiosis, and the failure of formation of a male pronucleus was the major cause for fertilization failure in porcine ICSI embryos.     

Chromatin and microtubule organisation in maturing and pre-activated porcine oocytes following intracytoplasmic sperm injection

Chromatin and microtubule organisation was determined in maturing and activated porcine oocytes following intracytoplasmic sperm injection in order to obtain insights into the nature of sperm chromatin decondensation and microtubule nucleation activity. Sperm chromatin was slightly decondensed at 8 h following injection into germinal vesicle stage oocytes. Sperm-derived microtubules were not seen in these oocytes. Following injection into metaphase I (MI)-stage oocytes, sperm chromatin went to metaphase in most cases. A meiotic-like spindle was seen in the sperm metaphase chromatin. In a few MI-stage oocytes, sperm chromatin decondensed at 8 h after injection, and a small sperm aster was seen. Sperm injection into oocytes at 5 h following activation failed to yield pronuclear formation. Maternally derived microtubules were organised near the female chromatin in these oocytes, and seemed to move condensed male chromatin closer to the female pronucleus. At 18 h after sperm injection into pre-activated oocytes, a condensed sperm nucleus was located in close proximity to the female pronucleus. These results suggest that the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent. In the absence of a functional male centrosome, microtubules of female origin take over the role of microtubule nucleation for nuclear movement.     

Role of glutathione in the maturation and fertilization of pig oocytes in vitro

The present study examined, by treatment of buthionine sulfoximine (BSO), which is a specific inhibitor of glutathione (GSH) synthesis, the role of GSH in the maturation and fertilization of pig oocytes in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughterhouse were cultured for 36 h in Waymouth MB 752/1 with or without BSO (1 mM), fertilized in vitro, and assessed for GSH concentration (before insemination), maturation, and fertilization. The addition of BSO to maturation medium immediately after culture (Group I), 12 h after culture (Group II), or 24 h after culture (Group III) significantly decreased the GSH concentration in pig oocytes compared with the control (P < 0.01), whereas the rate of cumulus mass expansion at 36 h of culture and the rates of nuclear maturation and sperm penetration following in vitro insemination did not differ. However, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the control and Group III than in oocytes matured in Groups I and II (P < 0.01). In experiment 2, when BSO was added to maturation media 15, 18, 21, or 24 h after culture, the rate of pig oocytes having condensed sperm heads was significantly lower and the rate of male pronucleus formation of pig oocytes was significantly higher in oocytes matured in the medium supplemented with BSO at 21 or 24 h of culture than in oocytes matured in other groups (P < 0.05 or P < 0.01). The results indicate that GSH synthesis occurs throughout in vitro maturation of pig oocytes and GSH is an important cytoplasmic factor for regulating sperm nuclear decondensation and male pronucleus formation following sperm penetration in pig oocytes. © 1993 Wiley-Liss, Inc.     

Improvement of male pronuclear formation during cross‐fertilization between Chinese hamster spermatozoa and Syrian hamster oocytes by nocodazole,and chromosome analysis of hybrid zygotes

During cross‐fertilization between Chinese hamster spermatozoa and Syrian hamster oocytes, incorporated sperm heads frequently fail to develop into male pronuclei, whereas the group of oocyte chromosomes develop into female pronuclei. The present study applies this cross‐fertilization system to the cytogenetic investigation of mammalian hybrid embryos. Immediately after insemination, oocytes were exposed to 0.1 μg/ml nocodazole for 1 hr (1 hr group) or 2 hr (2 hr group), then further cultured. Although the rates of sperm penetration in the 1 hr (48.0%) and 2 hr (75.8%) groups were significantly lower than that in the control group (89.8%), the ratios of male pronuclear formation were higher in both exposed groups (79.4% and 74.2%, respectively) than in the control group (10.6%). These results were apparently due to sperm head decondensation induced during the meiotic arrest of oocytes at metaphase II by nocodazole. Chromosomes of hybrid zygotes obtained after nocodazole exposure were analyzed at the first cleavage metaphase. The incidence of structural chromosome aberrations in the Chinese hamster genome of hybrid zygotes was high in the control (42.1%) and 1 hr (48.8%) groups. This incidence was reduced to 14.4% in the 2 hr group. Because the lag of sperm head decondensation behind the second meiotic division of oocytes was greater in the control and 1 hr groups than in the 2 hr group, untimely sperm head decondensation may be implicated in occurrence of structural chromosome aberrations in the male genomes of hybrid zygotes. Mol. Reprod. Dev. 52:117–124, 1999. © 1999 Wiley‐Liss, Inc.     

A Fine Structural Study of the Fertilization Process of the Jellyfish Cladonema uchidai

This study described the fertilization process of the jellyfish Cladonema uchidai by means of transmission electron microscopy. Female pronucleus was situated in close vicinity to the animal pole of the spawned egg, where the surface of the egg was flat or slightly depressed. Microvilli were observed except on the surface at the animal pole. The egg was entirely covered with a coat composed of fibrous materials. The spermatozoon was of the primitive type, and the proacrosomal vesicles were found immediately beneath the plasma membrane of the antero-lateral region of the sperm head. Within 15 sec after insemination, spermatozoa were incorporated in the egg cytoplasm only at the microvilli-free surface at the animal pole. Neither opening of the proacosomal vesicles nor formation of the acrosomal process was observed. No appreciable changes of cortical cytoplasm could be detected, although the egg became sticky after fertilization. Decondensation of the incorporated sperm nucleus occurred without breakdown of the original nuclear envelope. Within 10 min after insemination, the sperm nucleus still under the process of its decondensation fused with the female pronucleus. These findings were discussed in comparison with the fertilization process of higher metazoans as well as of other cnidarians.     

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.     

Role of the cytoskeleton in sperm entry during fertilization in the freshwater bivalve Dreissena polymorpha

The present study examined the role of the cytoskeleton in sperm entry and migration through the egg cytoplasm during fertilization in the zebra mussel, Dreissena polymorpha (Bivalvia: Veneroida: Dreissenidae). Fertilization in this freshwater bivalve occurs outside the mantle cavity, permitting detailed observations of fertilization. After its initial binding to the egg surface, the sperm is incorporated in two stages: (1) a gradual incorporation of the sperm nucleus into the egg cortex, followed by (2) a more rapid incorporation of the sperm axoneme, and translocation of the sperm head through the egg cytoplasm. Initial incorporation into the egg cortex was shown to be microfilament dependent. Microfilaments were found in the sperm's preformed acrosomal filament, the microvilli on the egg surface, and in an actin-filled insemination cone surrounding the incorporating sperm. Treatment of eggs with cytochalasin B inhibited sperm entry in a dose- and time-dependent manner. Microtubule polymerization was not necessary for initial sperm entry. Following incorporation of the sperm head, the flagellar axoneme entered the egg cytoplasm and remained active for several minutes. Associated with the incorporated axoneme was a flow of cytoplasmic particles originating near the proximal end of the flagella. Inhibition of microtubule polymerization prevented entry of the sperm axoneme, and the subsequent cytoplasmic current was not observed. After sperm incorporation into the egg cortex, no appreciable microfilaments were associated with the sperm nucleus. A diminutive sperm aster was associated with the sperm nucleus during its decondensation, but no obvious extension toward the female pronucleus was observed. The sperm aster was significantly smaller than the spindle associated with the female pronucleus, suggesting a reduced role for the sperm aster in amphimixis.     

Fertilization of rat eggs in vitro at various times before and after ovulation with special reference to fertilization of ovarian oocytes matured in culture.

Oocytes recovered at various times from immature rats treated with PMSG and HCG were incubated with capacitated epididymal spermatozoa of mature rats. In the presence of follicular cells, sperm penetration was not observed 4 hr after incubation in the oocytes at stages from the intact germinal vesicle to the chromatin mass, but 7 to 55% of oocytes were penetrated at stages from the condensed germainal vesicle to metaphase II. After the removal of follicular cells, 15 to 72% of the oocytes at any stage were penetrated. After further incubation for 15 hr, the proportion of penetrated oocytes increased from 8 to 98% from early to late stages and that of penetrated oocytes with a male and female pronucleus increased from 9 to 100% as maturation progressed. Although the average number of spermatozoa/oocyte was not correlated with its maturation, transformation of the sperm head into a male pronucleus was retarded or failed, especially in the younger oocytes. Following incubation in a defined medium for 13 hr, 85% of oocytes at the intact germinal vesicle stage matured to the stage of the first polar body formation, but only 18 to 22% of these mature oocytes were penetrated by spermatozoa and only a few of the penetrated oocytes cleaved into normal two-cell eggs. When eggs recovered from oviducts 14 to 20 hr after ovulation were exposed to capacitated spermatozoa, the proportion of penetrated eggs (86 to 98%) and that of polyspermic eggs (11 to 27%) were not related to the ages of the eggs, but failure of transformation of the sperm head and the proportion of abnormal eggs increased 14 to 20 hr after ovulation.     

Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid

Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona-free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.     

Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm

Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.     

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.     

Nuclear and Cytoplasmic Maturation of Bovine Oocytes Cultured with dbc AMP,FSH AND hCG

The present investigation was undertaken to study the effect of addition of dbc AMP on bovine oocyte maturation and fertilization in vitro. The bovine oocytes isolated from 2–8 mm follicles were cultured for 26 h in TCM-199. The maturation rate (71.4 %) did not significantly increase after supplementation of the culture medium with dbc AMP (86.3 %.) or FSH + hCG (86.3 %). The in vitro fertilization rate of oocytes based on sperm penetration and presence of sperm tail in the ooplasm increased significantly in the dbc AMP (34.7 %) and the dbc AMP + FSH + hCG (33.9 %) treated groups when compared with untreated controls (17.9 %). However, dbc AMP treated oocytes were not able to secure the formation of male pronucleus 20 h after in vitro fertilization, while in oocytes matured in dbc AMP free medium both pronuclei were present in approximately 15 % of the penetrated oocytes. Also, the sperm head decondensation was blocked or slowed down by the dbc AMP treatment. It is concluded (1) that dbc AMP may improve the condition for the interaction of oocytes with spermatozoa, and (2) that the ooplasm of such dbc AMP treated oocytes apparently is not able to decandense the sperm head and transform it to the male pronucleus.