A direct effect of some rifamycin derivatives on the morphology of mammalian mitochondria

Protein synthesis has been investigated in cell-free preparations from mature ovarian oocytes, unfertilized and fertilized eggs, and early embryos of Drosophila melanogaster. Preparations from unfertilized eggs have a specific activity that is 5- to 6-fold higher than the activity of fractions from ovarian oocytes. There is an additional small increase in activity of preparations from fertilized eggs. The specific activity that is rapidly attained in the fertilized egg remains essentially constant for 2 to 2.5 h after fertilization, decreases sharply during blastoderm formation, and again increases during gastrulation. The activities of unfertilized eggs decline slightly during the first 2 h after oviposition, and then decrease more sharply. About 35 % of the ribosomes in preparations from both unfertilized and fertilized eggs sediment in the polyribosome region of sucrose density gradients, whereas no polyribosomes could be detected in preparations from ovarian oocytes. In both ovarian oocytes and fertilized eggs, less than 1 % of the ribosome populations were present as subunits. Additional ribonucleoprotein material of buoyant densities different from those of ribosomal subunits or ribosomes was found throughout the sucrose gradients. About 3.5 % of the ribosomes were found to be membrane-bound in preparations from both unfertilized and fertilized eggs.     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.     

Glucose-6-phosphate and 6-phosphogluconate dehydrogenases from eggs of the sea urchin,Arbacia punctulata

1. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases have been found in homogenates of Arbacia eggs; 95 per cent of the activity toward each substrate is recovered in the supernatant fraction after centrifuging at 20,000 g for 30 minutes. 2. With glucose-6-phosphate as substrate) the rate of TPN reduction by the supernatant fraction from 1 gm. wet weight unfertilized or fertilized eggs was 1.8 to 3.0 micromoles per minute; this rate is sufficient to support a rate of oxygen consumption 24 times that observed for unfertilized, and 6 times that for fertilized, eggs. Pentose was formed from glucose-6-phosphate at a rate 0.3 to 0.5 that of TPN reduction, when both rates were expressed as micromoles per minute. 3. The concentrations of glucose-6-phosphate and 6-phosphogluconate for half maximal activity were each approximately 0.00004 M for the respective enzymes in the supernatant fraction. Maximal activity toward 6-phosphogluconate was 50 to 60 per cent of that toward glucose-6-phosphate. Glucose-6-phosphate dehydrogenase activity was 50 per cent inhibited in presence of 0.00006 M 2,4,5-trichlorophenol. 4. Reduction of DPN by the supernatant fraction in presence of fructose-1,6-diphosphate and ADP was 0.1 to 0.2 micromoles per minute per gm. wet eggs, indicating that the glycolytic pathway can metabolize glucose-6-phosphate at about 5 per cent the rate at which it can be oxidized by the TPN system from unfertilized or fertilized Arbacia eggs. 5. Phosphoglucomutase, hexose isomerase, and a phosphatase for fructose-1,6-diphosphate also appear to be present in Arbacia eggs.     

Bovine oocyte maturation, fertilization and further development in vitro and after transfer into recipients

Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.     

Incidence of chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thawed oocytes fertilized in vitro

To assess the effect of low temperature storage on mouse oocytes we (1) examined the capacity for normal development of embryos derived from frozen oocytes fertilized in vitro after transfer to pseudopregnant foster mothers and (2) analyzed the chromosome complement at the first cleavage division. Fewer frozen than control oocytes were fertilized (36% vs 66%), but after embryo transfer the proportion of fertilized eggs that implanted (67–68%) and formed normal foetuses (50–53%) was similar in the two groups. Freezing did not affect the observed incidence of aneuploidy (1.5–3.3%). The frequency of polyploid embryos derived from frozen oocytes was almost double that of controls (15.8% vs 8.5%), but it is unclear whether this is a real effect of freezing or is an artifact produced by the chromosome preparation technique.     

Maturation, fertilization and development of bovine oocytes in vitro using TCM199 and a simple defined medium with co-culture

Bovine oocytes obtained from ovarian follicles (2 to 5 mm in diameter) from slaughtered cattle were cultured in TCM199 with 10% heat-inactivated estrous cow serum (ECS) for 24 to 25 h at 39 degrees C under 5% CO(2) in air. The 10% ECS was selected on the basis of preliminary studies in which in vitro fertilization rates of oocytes with 10, 15 and 20% ECS in the medium were 46, 30 and 31%, respectively (P<0.05). Of 120 oocytes cultured for 24 to 25 h, 63% were classified as being in Metaphase II. The rate of oocytes matured in vitro was 55% (69 125 ), the proportion of penetrated oocytes which contained male and female pronuclei was 94% (65 69 ), and the incidence of polyspermy was very low (0 to 9%). Of 122 oocytes fertilized in vitro and cultured in TCM199 medium with 10% fetal bovine serum for 7 d, 53% were cleaved, but only 2% developed beyond the 16-cell block. However, in simple semi-defined Chatot-Ziomek-Bavister medium co-cultured with bovine oviduct epithelial cells (BOEC), 75% of 138 oocytes cleaved, and 38% of those which cleaved developed into morulae or blastocysts. The results of this study indicate that co-culture with BOEC exerted a pronounced beneficial effect on development of in vitro fertilized bovine oocytes through the 16-cell block. The medium required in the co-culture system was simple and semi-defined.     

RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.     

The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation

In a previous study we have shown that the addition of growth hormone (GH) during in vitro maturation accelerates nuclear maturation, induces cumulus expansion, and promotes subsequent cleavage and embryonic development. The aim of this study was to investigate whether the promotory effect of GH on subsequent cleavage and blastocyst formation is due to an improved fertilization and whether this effect is caused by an improved cytoplasmic maturation of the oocyte. Therefore, bovine cumulus oocyte complexes (COCs) were cultured for 22 hours in M199 supplemented with 100 ng/ml bovine GH (NIH-GH-B18). Subsequently the COCs were fertilized in vitro. Cultures without GH served as controls. To verify whether the promoted fertilization is caused by the effect of GH on cumulus expansion or oocyte maturation, cumulus cells were removed from the oocytes after in vitro maturation (IVM) and denuded MII oocytes were selected and fertilized in vitro. Both IVM and in vitro fertilization (IVF) were performed at 39°C in a humidified atmosphere with 5% CO₂ in air. At 18 hours after the onset of fertilization, the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenylindole (DAPI) staining. Oocytes with either an metaphase I (MI) or MII nuclear stage and without penetrated sperm head were considered unfertilized; oocytes with two pronuclei, zygotes, and cleaved embryos were considered normally fertilized; and oocytes with more than two pronuclei were considered polyspermic. To evaluate cytoplasmic maturation, the distribution of cortical granules 22 hours after the onset of IVM, and sperm aster formation 8 hours after the onset of fertilization were assessed. In addition, to assess the sperm-binding capacity, COCs were fertilized in vitro, and 1 hour after the onset of fertilization the number of spermatozoa bound to the oocytes was counted. The addition of GH during IVM significantly (P < 0.001) enhanced the proportion of normal fertilized oocytes. Removal of the cumulus cells prior to fertilization and selection of the MII oocytes did not eliminate the positive effect of GH on fertilization. No effect of GH on the sperm-binding capacity of the oocyte was observed. In addition, GH supplementation during IVM significantly (P < 0.001) enhanced the migration of cortical granules and sperm aster formation. It can be concluded that the promotory effect of GH on the developmental competence of the oocyte is due to a higher fertilization rate as a consequence of an improved cytoplasmic maturation. Mol. Reprod. Dev. 49:444–453, 1998. © 1998 Wiley-Liss, Inc.     

Chromosome studies in human in vitro fertilization

Summary The chromosome constitution of 22 human preimplantation embryos from donor oocytes fertilized in vitro by donor sperm was studied to assess the contribution of lethal chromosome anomalies to the high failure rate of implantation of in vitro fertilized embryos after embryo transfer in infertile women. Evidence was found of nondisjunction, resulting in trisomy, monosomy, and nullosomy; structural abnormalities; haploidy; and triploidy. Despite the lethality of their chromosome complements, these embryos could not be distinguished morphologically from those with normal chromosomes.     

胞浆内精子注射技术生产小鼠

以piezo操作系统为技术支撑 ,在掌握小鼠卵母细胞胞浆内精子注射技术 (ICSI)的基础上 ,进行了ICSI技术生产试管小鼠的尝试。来自成年昆明 (KM)小鼠附睾尾的新鲜精子 ,剪切去尾后 ,直接将精子头注射到B6D2F1小鼠卵母细胞质中 ,注射后 1h ,83.3%的卵母细胞存活。6h时 ,84.0 %的成活卵子成功受精 ,形成原核 ,排出PB2 体外培养的ICSI胚胎 ,卵裂率 (98%vs 94.7% )和 4-细胞期胚胎比率 (89.5%vs 92.1% )均与培养的体内受精卵没有差异 (P >0.05 ) ;但是 ,桑椹胚(63.8%vs84.2% )和囊胚发育率 (25.7%vs68.4% )极显著地 (P <0.01)低于对照组。120枚原核期胚胎移植给 7只假孕受体后 ,4只受孕小鼠共产出 28只ICSI小鼠 (23.3% )。健康成年的 25只ICSI小鼠都没有明显的生理和行为异常。随机选择其中的 20只小鼠 ,分别进行ICSI小鼠间、ICSI与KM小鼠间共 12组的交配 ,结果所有雌鼠妊娠产仔。在成功建立小鼠ICSI技术的基础上,成功获得了我国的首例ICSI小鼠,并且证明这些ICSI小鼠都具有正常的繁殖后代的能力。     

In vitro fertilization of bovine follicular oocytes recovered by laparoscopy

Thirty-three laparoscopies were performed on 16 follicle stimulating hormone (FSH)-superovulated cows (one to four laparoscopies per cow). Two hundred and four follicles were aspirated (6.18/cow) and 157 oocytes (77% recovery rate) were isolated. Sixty percent of the oocytes found were considered to be of good to excellent quality. In cows with ongoing ovulations at laparoscopy only 32% of the oocytes were suitable for fertilization. Twenty-four percent of the oocytes from cows with no ovulation and 16% from cows with ovulations were fertilized. Higher proportions of oocytes were fertilized in cows showing estrous behavior vs cows with no signs of estrus (25 vs 15%). Using lysolecithin-treated spermatozoa, 28% of the oocytes with good to excellent quality were fertilized, and 35% of the fertilized eggs cleaved in vitro to two- to four-cell stages.     

Superovulation of immature hypothyroid rdw rats by thyroxine therapy and the development of eggs after in vitro fertilization.

The aim of this study was to examine the effect of thyroxine on ovulation in immature rdw rats and the fertilization and development of the eggs. Serum thyroxine concentrations at 30 days of age were significantly lower in rdw rats than in normal rats (P < 0.001), and greatly increased after thyroxine replacement therapy (P < 0.001). Although few eggs (1-5 +/- 1-2) were obtained from immature rdw rats treated with gonadotrophins alone, females treated with gonadotrophins and thyroxine ovulated significantly more eggs (85 +/- 5). As a control, normal littermates ovulated 21-45 eggs when treated with gonadotrophins alone, and 68 eggs when administered with gonadotrophins and thyroxine. Of the eggs collected from rdw rats treated with gonadotrophins and thyroxine, and inseminated with spermatozoa from mature F1 males, 98% were penetrated and in almost all (99%) of these eggs, male and female pronuclei formed. Forty-seven per cent of the pronuclear eggs developed to the blastocyst stage in vitro. After transfer to recipients, 21% (14/66) of one-cell and 22% (8/37) of two-cell embryos developed to offspring, and 62% (8/13) of pups were of rdw/rdw genotype. The average body weight (6.9 versus 7.8 g) of offspring derived from one-cell embryos was lower than that for two-cell embryos. The morulae and blastocysts did not develop to term, although 41% implanted in the uterine horns of recipients. In conclusion, in immature rdw rats, superovulation was induced by gonadotrophins combined with thyroxine therapy and the superovulated oocytes were fertilized and developed in vitro and developed to term after embryo transfer.     

Ovulation and fertilization of primary and secondary oocytes in LT/Sv strain mice

In this study, the chromosome constitution of both unfertilized oocytes and fertilized eggs isolated from the oviducts of LT/Sv strain mice were analyzed. Air-dried chromosome preparations from unfertilized oocytes revealed that about one-third of those examined were ovulated as primary oocytes. These were arrested at metaphase of the first meiotic division and exhibited the characteristic “tetrad” chromosome configuration. The remaining two-thirds of the unfertilized oocytes were ovulated at metaphase of the second meiotic division. The fertilized eggs were isolated from the oviducts of LT/Sv females previously mated to (C57BL × CBA) F1 hybrid males. Analysis of the fertilized eggs at metaphase of their first cleavage mitosis revealed that about one-third of the eggs examined were digynic triploids, whereas the remaining two-thirds had the normal diploid chromsome constitution. In the triploids, the 40 female chromosomes present (mouse, n = 20) were derived from a single diploid pronucleus formed after the extrusion of a first polar body, and following the monospermic fertilization of primary oocytes. The female pronuclear-derived chromosomes invariably exhibited “homologous pairing,” and these were associated at their centromeres. The ovulation, penetration, and subsequent fertilization of primary oocytes is an extremely unusual phenomenon in mammals and only appears to occur on a regular basis in LT/Sv mice. The premature “cytoplasmic maturation” of these oocytes is of interest, as they clearly have the same developmental capacity as secondary oocytes. The significance of these observations in relation to folliculogenesis and litter size in LT/Sv mice is discussed.     

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.     

Birth of lambs after in vitro maturation, fertilization, and coculture with oviductal cells.

Control ovine oocytes matured and fertilized in vitro were transferred to intermediate recipient ewes. After 5 days, 59% of eggs were recovered. Thirty-one (38%) reached morula/blastocyst stage. Twenty-one embryos at the morula or blastocyst stage were transferred to six recipient ewes, resulting in five pregnancies, of which four were maintained. Nine lambs were born (43%). In the experiment, 72 ooctyes matured and fertilized in vitro were cocultured for 5 days with sheep oviductal epithelial cells. Thirty-one eggs (43%) developed to the noncompacted morula stage. Transfer of 26 embryos to 11 recipient ewes resulted in two pregnancies (18%). Two male lambs were born. The result indicates that the coculture of in vitro matured and fertilized ovine eggs with sheep oviductal epithelial cells throughout the preimplantation period is compatible with further development to term.     

The dependence of annulate lamellae formation on the nucleus in parthenogenetic rabbit eggs

Previously it has been shown that, in the rabbit, although annulate lamellae (AL) are absent in the follicular oocytes, they appear in the fertilized eggs after the formation of the pronuclei. Furthermore, neither pronuclei nor AL appear when unfertilized eggs are aged in vivo or in vitro. This study was undertaken to determine whether AL formation requires presence of an intact nucleus, or whether the sperm alone contains the stimulatory factors essential to AL synthesis. Rabbit eggs were exposed to 10 degrees C, then incubated for 24 hours. Control eggs were incubated without cold-treatment. Electron microscopic observations indicated that two-thirds of the eggs formed one to two 'pronuclei', or subnuclei. The remainder one-third of the cold-treated eggs and the control eggs failed to form 'pronuclei'. AL were present in large amounts only in those activated eggs (parthenogenones) which formed 'pronuceli.' AL were absent in the control and the non-activated experimental eggs, both of which failed to form a 'pronucleus.' A few small AL were observed in eggs with subnuclei. Condensed fine textured nucleoli appeared precociously during cold-treatment in some eggs and they were present in the 'pronuclei' of activated eggs. It was concluded that the sperm is not necessary for AL formation, but the presence of an intact nucleus is mandatory.     

Fertilization following intracytoplasmic sperm injection of in vivo and in vitro matured oocytes from an Australian marsupial, the tammar wallaby (Macropus eugenii)

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm-oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.e. development to the two-pronuclei stage) of in vivo and in vitro matured oocytes following ICSI and sham injection was assessed at 17-19 h after injection. Fertilization occurred in 48% (45/93) of in vivo matured oocytes that were injected with spermatozoa. Significantly fewer sham-injected oocytes (6/82, P < 0.005) and uninjected control oocytes (5/84, P < 0.005) formed two pronuclei. In a direct comparison, the numbers of in vivo and in vitro matured oocytes that formed two pronuclei after ICSI were 22/28 (78.6%) and 23/40 (57.6%), respectively, which are not significantly different. There was also no significant difference in the nuclear response of in vivo and in vitro matured oocytes to sham injection. The numbers of oocytes forming a single pronucleus after sham injection were 10/24 (41.7%) and 24/37 (64.9) for in vivo and in vitro matured oocytes, respectively. Immature germinal-vesicle-stage oocytes were unable to decondense sperm injected during ICSI or to form pronuclei. These results demonstrate that both in vitro and in vivo matured tammar wallaby oocytes can be fertilized by ICSI. The success of ICSI not only offers the opportunity for fundamental analysis of marsupial fertilization but could, in conjunction with development of appropriate culture conditions and embryo transfer technologies, contribute to increased production of offspring from rare or valuable marsupials.     

Dependence of Ca2+ release from intracellular stores on NADH and FAD levels in fertilized and unfertilized bovine oocytes

Relation between NADH and FAD concentrations and the quantity of calcium released from intracellular stores in fertilized and unfertilized bovine oocytes was investigated using luminescent analysis. Inhibition of Ca2+ exit from intracellular stores was detected in degenerative oocytes at metaphase II and 2-cell embryos. The intensity of both NADH and FAD fluorescence increased in 2-cell degenerated embryos, whereas the increase in only NADH fluorescence intensity occurred in degenerated oocytes at metaphase II stage. Degeneration exerted no influence on NADH fluorescence intensity or Ca2+ exit from intracellular stores, whereas a decreased FAD fluorescence intensity was noted in degenerated pronuclei. The obtained data testify that in degenerated zygotes and early embryos Ca2+ release may occur from different intracellular stores.     

Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization

The objective of this study was to determine an optimum maturation period of canine oocytes for the development in vitro after in vitro fertilization (IVF). Canine oocytes larger than 110 micrometers in diameter, which were collected from ovaries at the follicular phase of the reproductive cycle, were cultured for each time (48, 72 and 96 h) in TCM 199 medium supplemented with 10% canine serum, fertilized, and then cultured in vitro for 8 days. Significantly more oocytes reached metaphase II (MII) in the 72-h culture group than in the 48-h culture group (25.6% vs. 41.0%). The percentages of oocytes that reached MII or beyond after maturation culture did not differ significantly between the 72- and 96-h culture groups, but the percentage of parthenogenetically activated oocytes in the 96-h culture group was significantly higher than that in the 72-h culture group. The percentages of cleaved embryos after IVF were significantly higher in the 48- and 72-h culture groups than in the 96-h culture group. In the 48-h culture group, 3.9% of fertilized oocytes developed to the 16-cell stage or beyond, but none of the cleaved embryos in the 72- and 96-h culture groups developed to the same stage. These results indicate that full nuclear maturation of oocytes collected from ovaries at the follicular phase occurs after 72 h of in vitro culture. However, an optimum maturation period (48 h) for the in vitro development of canine oocytes after IVF may be different from the period necessary to reach the maximal oocyte maturation rate, when based on the developmental stage of the cleaved embryos.     

Fertilization initiates the transition from anaphase I to metaphase II during female meiosis in C. elegans

Oocytes from most animals arrest twice during the meiotic cell cycle. The universally conserved prophase I arrest is released by a maturation hormone that allows progression to a second arrest point, typically metaphase I or II. This second arrest allows for short-term storage of fertilization-competent eggs and is released by signaling that occurs during fertilization. Nematodes are unique in that the maturation hormone is secreted by sperm rather than by the mother's somatic tissues. We have investigated the nature of the second arrest in matured but unfertilized Caenorhabditis elegans embryos using time-lapse imaging of GFP-tubulin or GFP-histone. Unfertilized embryos completed anaphase I but did not form polar bodies or assemble meiosis II spindles. Nevertheless, unfertilized embryos assembled female pronuclei at the same time as fertilized embryos. Analysis of embryos fertilized by sperm lacking the SPE-11 protein indicated that fertilization promotes meiotic cytokinesis through the SPE-11 protein but assembly of the meiosis II spindle is initiated through an SPE-11-independent pathway.