Effect of sperm penetration in vitro on completion of first meiosis by bovine oocytes arrested at various stages in culture.

Bovine oocytes cultured for 12-20 h in TC-199 were incubated for 24 h in fertilization medium, Brackett and Oliphant medium with bovine serum albumin (10 mg ml-1), caffeine (5 mmoll-1) and heparin (10 micrograms ml-1), with or without frozen-thawed spermatozoa. High penetration rates (93-96%) and significantly (P < 0.001) higher maturation rates were obtained in oocytes incubated with (93-100%) than without (62-72%) spermatozoa. However, when oocytes at the germinal vesicle stage were cultured for 44 h fertilization medium, maturation of oocytes to metaphase II was reduced. However, all oocytes that were first cultured for 20 h and further for 24 h with spermatozoa were penetrated and 40% of the penetrated oocytes reached metaphase II. All of the remaining oocytes that did not mature arrested at the stages of condensed germinal vesicle (39%) or prometaphase I (22%). These results indicate that oocytes at metaphase I at and after sperm penetration are stimulated by sperm penetration to complete maturation.     

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.     

Sperm penetration in vitro of human oocytes matured in a chemically defined medium

Human oocytes were obtained randomly from the ovaries excised from 28 patients at different stages of the menstrual cycle. When 114 oocytes were cultured for 40-40.5 h in a chemically defined medium 80 showed degeneration. When 16 of the remaining 64 oocytes were examined for their maturation, germinal vesicle breakdown (GVBD) had occurred in 11 oocytes. Another 48 oocytes were inseminated by ejaculated spermatozoa which were washed twice and preincubated for 3 h. When examined 10 h later, 19 (39.6%) were penetrated with an enlarged sperm head or male pronucleus(ei) and its corresponding sperm tail(s).     

Oocyte diameter in relation to meiotic competence and sperm penetration

This study was conducted to determine the diameter of canine oocytes that are able to attain full meiotic competence and sperm penetration. Oocytes were collected from ovaries of bitches at various stages of the estrous cycle. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and were divided into four groups based on diameter: <100, 100 to <110, 110 to <120 and >120 microm. Following in vitro maturation or fertilization, oocytes were stained to assess nuclear maturation and penetration rates. The mean oocyte diameter was 108.5 +/- 0.4 microm. The oocytes displayed size-related ability to undergo meiotic maturation. After culture for 72 h, the rates of oocytes that remained at the germinal vesicle stage in the <110 microm groups were significantly higher (P<0.01) than in the > or = 110 microm groups. None of the oocytes <110 microm reached metaphase II (MU), but 4.9 and 21.5% of the oocytes that were greater than 110 and 120 microm, respectively, progressed to MII. After in vitro fertilization for 20 h, 10 to 25% of oocytes were penetrated by spermatozoa, but there were no clear relationships between oocyte diameter and penetration rates of the oocyte by sperm. In the <120 microm groups, sperm penetration was mostly found in oocytes arrested at the germinal vesicle stage. However, a total of eight oocytes > or = 120 microm in diameter were penetrated by spermatozoa, of which five oocytes reached MII. These results suggest that there is a clear relationship between oocyte diameter and meiotic competence, but no relationship between oocyte diameter and sperm penetration. Canine oocytes may have acquired meiotic competence once they reach at a diameter of 120 microm, but the oocytes may allow the entry of spermatozoa into the ooplasm irrespective of oocyte diameter.     

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.     

Embryonic development of in vitro matured and in vitro fertilized dog oocytes

In several studies, early cleavage stage canine embryos have been derived from in vitro fertilized oocytes cultured under various conditions. Despite these results, IVF protocols for canine oocytes have yielded low fertilization rates. In this study, Experiment I compared the effects of tissue culture medium (TCM)-199 supplemented with either (A) 1 microg/ml estradiol or (B) 20 microg/ml estradiol + 1 microg/ml human somatotropin (hST) on the in vitro nuclear maturation rate of canine oocytes. Meiotic progression to the metaphase I and II (MI/MII) stages at 72 hr of in vitro culture (IVC) was 10.2% (11/108) in medium A versus 14.1% (30/142) in medium B (P = 0.802). In Experiment II, cleavage rate was determined among oocytes recovered from ovaries of bitches at different reproductive stages. Oocytes (n = 888) were retrieved from bitches at the follicular, anestrous, and luteal stages and selected for high morphological quality. Oocytes were matured for 48 hr in TCM-199 supplemented with 1 microg/ml hST + 20 microg/ml estradiol. Oocytes were in vitro fertilized with fresh canine spermatozoa that had been isolated on a Percoll gradient, and were cultured in synthetic oviduct fluid (SOF) medium with bovine serum albumin (BSA; 4 mg/ml) up to 5 days in 5% CO(2) in air at 37 degrees C. A proportion of oocytes (30.6%) with identifiable nuclear material had cytoplasm penetrated or fertilized by sperm. The percentage of oocytes developing into early stage embryos was 10.1% (27/267). Although pronuclear development was observed to be higher for oocytes recovered at the follicular phase, the cleavage rate was similar among oocytes recovered from bitches at the follicular, anestrus, and luteal stages. There was no correlation between the proportion of capacitated or acrosome reacted spermatozoa and pronuclei formation and/or percent cleavage. It was concluded that TCM-199 supplemented with 1 microg/ml hST and estradiol (20 microg/ml) supports nuclear and cytoplasmic maturation of canine oocytes. In this study, meiotic competence was verified by the in vitro production (IVP) and development of embryos up to the 8 cell-stage. Furthermore, the results indicate that, under the described conditions and despite the influence of reproductive status of the bitch on the developmental competence of in vitro fertilized oocytes to the pronuclei stage, cleavage was independent of donor's reproductive estrous cycle stage.     

Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4-cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x ) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41). In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt-stored zonae, zonae-free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt-stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona-free hamster ova that were penetrated by sperm cells from the same sample. In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt-stored zonae, zona-free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy-six of 80 salt-stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different. The salt-stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt-stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.     

Evaluation of boar spermatozoa penetrating capacity using pig oocytes at the germinal vesicle stage

We evaluated the ability of immature pig oocytes (at germinal vesicle stage) to detect differences in the in vitro penetration rates of boar spermatozoa. In Experiment 1, immature and ovulated oocytes (n=303) were exposed to capacitated boar spermatozoa to determine if the penetrability of immature pig oocytes was comparable to that of ovulated oocytes. The percentages of penetrated oocytes and the mean number of spermatozoa per oocyte were similar for immature (88.82 and 7.42+/-0.41) and ovulated oocytes (90.97 and 7.95+/-0.34, respectively). In Experiment 2, immature oocytes (n=1230) were inseminated with semen from 2 boars (A and B) with satisfactory semen characteristics to establish the variability of in vitro penetrating capacity between the boars. Semen was examined for motility, movement quality, acrosome integrity and plasma membrane integrity at various stages of the in vitro procedure. Although the sperm evaluation results were similar between boars, Boar A exhibited a significantly higher (P<0.001) penetration rate (91.49%) and number of spermatozoa penetrated per oocyte (5.90+/-0.25) than Boar B (52.87% and 2.03+/-0.12, respectively). Increasing the sperm concentration at insemination from 1x10(6) to 10x10(6) cells/ml resulted in an increased penetrating capacity for both boars, and the differences in the number of spermatozoa per oocyte between boars also increased. These results indicate that immature pig oocytes can be used in a homologous in vitro fertilization assay, and that despite similarities in semen characteristics a significant boar effect is evident for parameters of in vitro penetration of oocytes.     

Ability of in vitro maturing bovine oocytes to transform sperm nuclei to metaphase chromosomes.

Bovine oocytes at the germinal vesicle stage were inseminated in Brackett & Oliphant's medium with bovine serum albumin, caffeine and heparin. Eight hours after insemination, oocytes were transferred into tissue culture medium-199 containing 10% fetal calf serum and cultured for 5-40 h at 39 degrees C in 5% CO2 in air. The proportions of unpenetrated and penetrated oocytes reaching metaphase II increased as the time of examination increased, reaching 70 and 65% 40 h after transfer, respectively. When oocytes were penetrated by more than four spermatozoa, meiotic maturation was greatly retarded. Sperm nuclei were decondensed in most (81%) penetrated oocytes 5 h after transfer. The decondensed sperm nuclei were recondensed and then transformed to metaphase chromosomes which were morphologically compacted at first but became slightly dispersed later. The formation of the metaphase chromosomes was observed in 86% of penetrated oocytes examined 40 h after transfer, and occurred in all metaphase II oocytes at that time. In oocytes penetrated by more than nine spermatozoa, no such transformation of sperm nuclei was observed. Well-developed male and female pro-nuclei were observed in only three (6%) of 51 oocytes penetrated 40 h after transfer.     

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.     

Comparison of the penetrability of the egg vestments in follicular oocytes, unfertilized and fertilized ova of the rabbit

Mixed populations of rabbit ovulated eggs and follicular oocytes, one labelled with a fluorescent marker, were transferred to the same tubal ampulla of an inseminated recipient female and were then recovered 3 hr later. There was no significant difference in the number spermatozoa penetrating to the perivitelline space or within the substance of the zona pellucida of follicular oocytes (immature or atretic) and mature ovulated ova. In contrast to mature ovulated ova, however, none of the spermatozoa reaching the perivitelline space of vesicular (dictyate) oocytes had attached to or penetrated the vitelline surface to enter the ooplasm.The same approach involving transfer of nonpenetrated eggs together with eggs penetrated previously in a donor female, demonstrated that prior entry of spermatozoa does not reduce the penetrability or receptivity of the rabbit zona pellucida to subsequent spermatozoa.These experiments indicate: (a) that the penetrability of the granulosa cell investment and/or zona pellucida of the rabbit follicular oocyte does not change from the time of antrum formation until the point at which follicular atresia ensures; (b) that between the time of initial LH stimulation and ovulation important changes mediating the onset of the fertizability of the dictyate oocyte of the rabbit probably occur at the vitelline surface; and (c) that in neither a qualitative nor quantitative sense has the demonstrably greater resistance of the rabbit zona pellucida to proteolysis following fertilization any physiological significance for sperm penetration.     

Effect of in vivo oocyte aging on sperm chromatin decondensation in the golden hamster

Aged spontaneously activated hamster oocytes recovered from adult females 18 and 24 hours after ovulation were at the pronuclear stage. These oocytes and fresh controls were inseminated in vitro with capacitated hamster spermatozoa and observed with the phase-contrast microscope. The percentage of fertilization in fresh control oocytes was 98%, as compared to 36% and 18% when the oocytes were recovered 18 and 24 hours after ovulation, respectively. The mean number of sperm decondensations per egg in control oocytes was 10, and in the aged ones it was 0.69 and 0.12 when the oocytes were recovered 18 and 24 hours after ovulation, respectively. When similarly treated oocytes were studied with scanning and transmission electron microscopy, it was found that the degree of gamete membrane fusion was greater than that observed with the phase-contrast microscope, but that most of the spermatozoa failed to decondense the chromatin. We suggest that parthenogenetic oocytes at the pronuclear stage are in a similar stage of the cell cycle as in fertilized eggs, in which the cytoplasm does not have the ability to decondense the sperm chromatin.     

Effects of spermatozoa during in vitro meiosis progression in the porcine germinal vesicle oocytes

We have investigated the effect of co-culture with porcine spermatozoa on in vitro maturation of porcine germinal vesicle (GV) oocytes before fertilization. Most oocytes were arrested at the first prophase of meiosis when oocytes were cultured in TCM 199 alone, but the proportion of oocytes that reached metaphase II was significantly elevated by co-incubation with spermatozoa in vitro. The oocyte maturation effect was observed with intact and parts of spermatozoa (head and tail) collected from adult swine (regardless of source). However, gonocytes from the newborn porcine testis were not able to enhance in vitro maturation of porcine germinal vesicle oocytes. Interestingly, the oocyte maturation effect by spermatozoa was not decreased with heat treatment, but the maturation effect of oocyte treatment disappeared with exposure to detergent in sperm suspension. Porcine spermatozoa were also observed to stimulate meiosis of oocytes, which was maintained at meiotic arrest using dibutyryl cyclic AMP or forskolin. The study suggests that (i) membrane of porcine spermatozoa contains a substance(s) that can enhance in vitro maturation of oocytes prior to fertilization, (ii) the putative meiosis-enhancing substance(s) of spermatozoa from adult testes retains the oocyte maturation effect during transportation of spermatozoa through epididymis, and (iii) the putative meiosis-enhancing substance(s) is able to overcome the inhibitory effect of dibutyryl cyclic AMP or forskolin by inducing germinal vesicle breakdown of porcine cumulus-oocyte complexes maintained in meiotic arrest.     

Intracellular localization of argyrophilic proteins in the maturing oocyte,fertilized egg,and spermatozoa of the starfish,Asterina pectinifera

Changes in intracellular localization of argyrophilic proteins visualized as silver-stained particles by nuclear organizer region (NOR)-silver staining were investigated in starfish oocyte maturation. The silver-stained particles were localized in the germinal vesicle and nucleolus of immature oocytes and dispersed into the cytoplasm at the time of germinal vesicle breakdown (GVBD). In the mature egg cytoplasm, silver-stained particles were distributed on yolk-like granules with diameters of 0.3–1.0 μm. In spermatozoa, silver-stained particles were detected heavily in the acrosome and centrosomes but few were detected in the nucleus, whereas they were present in the male pronucleus of fertilized eggs. The silver-stained particles were removed by pretreatment of eggs with protease but not with nuclease. These results indicate that argyrophilic proteins disperse to the egg cytoplasm during GVBD and might be incorporated to the male pronucleus from the egg cytoplasm in fertilization. The morphological changes from chromosomes through chromosome vesicles to female pronucleus were also observed with light microscopy after NOR-silver staining.     

Effects of different serum supplements in maturation medium on meiotic and cytoplasmic maturation of pig oocytes

The temporal progression of meiotic and cytoplasmic maturation of pig oocytes cultured in a medium supplemented with 0.4% polyvinylalcohol (PVA), 10% fetal calf serum (FCS), 10% newborn piglet serum (NPS), 10% porcine follicular fluid (PFF) or 10% porcine seminal fluid (PSF) was examined after 20, 30, 40 and 50 hours of culture. There were no differences in germinal vesicle breakdown (GVBD) among FCS and NPS supplements. After 20 hours of culture, the frequency for GVBD was higher (P < 0.05) in FCS and NPS (54% and 52%, respectively) than in PVA (32%) and PFF (33%) culture media but were not different at 40 and 50 hours of culture. Supplementation with PSF resulted in a rapid chromosome condensation of pig oocytes after 20 hours culture, but all GVBD oocytes stopped developing at the condensed germinal vesicle stage. Oocytes were not penetrated by spermatozoa when inseminated following 20 hours of culture, while high penetration (87 to 100%) and polyspermy rates (86 to 100%) were consistently obtained in all the supplement groups when inseminated after 30, 40 or 50 hours of culture. Male pronuclear formation rates at 10 to 12 hours after insemination, following a 50-hour culture period in FCS and NPS, were 28 and 28%, respectively, in comparison with 54% in PVA and 59% in PFF. The results indicate that supplementing maturation media with serum such as FCS and NPS reduced the ability of pig oocytes to form a male pronucleus, and further suggest that the detrimental effects may be due to accelerated progression of maturation events.     

The site of the acrosome reaction during in vivo penetration of the sheep oocyte

In vivo fertilization of sheep eggs has been studied by electron microscopy. Remnants of the acrosome reaction were present at the zona surface of every penetrated egg, indicating that the acrosome reaction in sheep occurs at the surface of the zona pellucida. To determine whether follicular oocytes could specifically bind spermatozoa, oocytes isolated from different size classes of antral follicles were transferred into the oviducts of mated ewes, recovered 4 hr 30 min later, and analyzed by electron microscopy. Oocytes from follicles up to 1 mm in diameter failed to bind spermatozoa and were not penetrated. In contrast, the zona of oocytes from follicles ? 2 mm in diameter induced the acrosome reaction. These oocytes were penetrated but failed to achieve cortical granule exocytosis and so to mount a block to polyspermy. Moreover, sperm nuclei incorporated into the ooplasm did not decondense although the sperm nuclear envelope was dispersed.     

Maturation-associated changes in the rat zona pellucida

Rat follicular oocytes, arrested at prophase I, cannot be fertilized in vitro. This capacity is acquired following resumption of meiosis and a series of changes involving both the oocyte and the cumulus cells surrounding it. Oocytes exposed to sperm at different hours before ovulation show a gradual increase in the permeability of their zona pellucida (ZP). Our study examined whether the ZP, in response to the physiological stimulus for maturation and concomitant with the other oocyte--cumulus components, undergoes maturational changes. Two ZP characteristics were assessed, sensitivity to proteolysis and sperm binding. ZP surrounding oocytes and eggs were collected from five sources: 1) germinal vesicle (GV)-intact oocytes, 2) preovulatory eggs, 3) ovulated eggs isolated from oviducts of immature females, 4) fertilized eggs, 5) ovulated eggs isolated from oviducts of mature females. All ZP surrounding oocytes/eggs from groups 1-5 were dissolved by trypsin. When solubility by pronase and alpha-chymotrypsin was examined, a large variation between groups was found. All ZP from group 2 were dissolved by 0.001% pronase, compared to 0% solubility in group 4. Only 10% of the ZP surrounding GV-intact oocytes (group 1) were dissolved by this enzyme, compared to 82% in group 3. Solubility in 0.01% alpha-chymotrypsin showed a similar pattern. Capacitated sperm were incubated with eggs from groups 1 and 3. The number of sperm binding to ZP in group 3 was repeatedly higher than that in group 1. In both tests it was found that the ZP surrounding the mature eggs differ in their characteristics from ZP of GV-intact oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The changes in sperm nuclei after penetrating fish oocytes matured without germinal vesicle material in their cytoplasm

The processes occurring from sperm penetration to chromosome formation in the cytoplasm of Oocytes matured in vitro, after removal of the germinal vesicle (GV) and before hormonal stimulation, were observed with electron microscope. The dechorionated oocytes, matured without the participation of the GV material, responded to sperm penetration by initiating a cortical reaction within 20 seconds after insemination. The pentrating sperm nuclei transformed to male pronuclei with vesiculation of the nuclear membrane, chromatin decondensation, and formation of a pronuclear membrane. Before cleavage, however, no chromosome formation was observed in these oocytes. Instead, the fully grown pronuclei change to a picnotic chromatin mass without or with an only fragmented nuclear membrane, then disappeared. On the contrary, sperm nuclei that penetrated into the cytoplasm of naked eggs containing GV material during maturation underwent pronuclear and chromosomal formation. Judging from these observation in Oryzias oocytes, the GV material seems to be unnecessary for the formation of pronucleus from the compact sperm nucleus, but is essential for the process of chromosomal formation.     

Influence of follicular components on oocyte meiosis in vitro

Oocytes and follicular components obtained from ovaries recovered from mature Hereford cows at slaughter were used to determine follicular influence on oocyte maturation. Some oocytes were fixed immediately to determine the stage of maturation. The remaining oocytes were cultured for 32 to 34 hr in various environments to determine the influences of the granulosum and follicular fluids on meiotic changes. All noncultured oocytes had dictyate nuclei except one in premetaphase. Oocytes cultured in 50 or 100% follicular fluid or in contact with stratum granulosum cells showed some meiotic inhibition both before and after germinal vesicle breakdown (GVB). The least resumption of meiosis occurred in oocytes cultured in their intact follicles.     

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.