Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro

Effects of gonadotropins on the maturation of isolated oocytes and production of progesterone by porcine ovarian follicles from gonadotropin treated gilts have been studied in vitro. The addition of gonadotropins (2 I. U./ml, PMSG, HGC or 2 mg/ml FSH) to the culture medium resulted in increasing the number (84 - 90 %) of isolated oocytes which reached metaphase II. Expansion of the whole cumulus mass was observed only in media containing PMSG, whereas FSH or HCG alone did not cause these marked changes in the cumulus cells. Denudation of the eggs prior to culture gave no significant differences in the maturation rates between oocytes cultured in media with or without gonadotropins. In vitro maturation of follicle-enclosed oocytes took place only in HCG treated animals. Removing the ovary at 15 or 60 minutes after intravenous HCG administration induced oocyte maturation only in 22% and 17% respectively. A sharp increase in the number of oocytes which resume meiosis during follicle culture was observed 4 hours after HCG injection (84 %) and all of the oocytes of the gilts ovariectomized at 8 hours after HCG injection matured during the culture period. The progesterone production of isolated follicles from control gilts (only PMSG injected) increased slowly during a 96-hour culture period (from 48 to 240 ng progesterone/follicle), whereas the secretion of progesterone was drastically increased after a 15 minute interval between HCG injection and ovariectomy (from 42 to 950 ng progesterone/follicle). Follicles removed 24 hours after HCG injection showed a further increase in steroid production (2000 ng progesterone/follicle) and consistently secreted large amounts of progesterone during the culture period.     

Effect of aging of recipient oocytes on the development of bovine nuclear transfer embryos in vitro

The present study was undertaken to examine the effect of the length of in vitro maturation of oocytes on the efficiency of enucleation, parthenogenetic activation and blastomere fusion by electric stimulus. In vitro development of reconstituted oocytes receiving a blastomere from 8 to 16-cell bovine embryos fertilized in vitro was investigated to assess the effect of aging of the oocytes. The proportion of oocytes with a first polar body at 22 to 24 hours after maturation was high (80%) compared with those obtained at 16 to 18, 28 to 30 or 42 to 44 hours (50 to 75%). The success rate of enucleation significantly decreased with aging (88, 85, 74 and 55%). The activation rate significantly increased with the length of maturation in vitro (P<0.01) (1 to 4, 24 to 41, 57 to 70 and 80 to 87%). The proportion of oocytes fused with a blastomere from 8- to 16-cell embryos was not dependent on the age of the oocytes (54 approximately 59%). The ability of the reconstituted oocytes to develop to the 2-cell and the 8- to 16-cell stage increased with the length of maturation of recipient oocytes. When oocytes enucleated and a blastomere at 22 to 24 hours were incubated further for 22 to 23 hours until electrofusion. The proportions of oocytes which developed to the 2-cell and the 8- to 16-cell stages (74 and 17%) were similar to those obtained at 42 to 44 hours after maturation. However, only 1 to 6% of reconstituted eggs receiving a blastomere from 8- to 16-cell embryos fertilized in vitro developed into a blastocyst in vitro.     

Comparison of bulk enucleation methods for porcine oocytes

Cloning of mammalian oocytes requires that the recipient oocyte is enucleated to remove all genetic material associated with the chromosomes. The procedure currently used in most species requires careful micromanipulation of oocytes treated with cytochalasin B to prevent structural damage. Although functional, this procedure requires time and limits the number of oocytes available for cloning, and our ability to understand the mechanisms of nuclear reprogramming. Therefore, this study aimed at evaluating different procedures to enucleate large pools of oocytes in a time-efficient manner. Two different approaches were tested. The first approach involved centrifugation of zona-free oocytes through a percoll gradient to separate the portion containing the chromatin from the cytoplasmic portion. The second used etoposide to prevent chromatin segregation at first metaphase and resulting in the expulsion of all chromosomes in the polar body. Using the chemical approach an average enucleation rate of 39.4 +/- 7.5% was obtained, while the centrifugation approach resulted in an average enucleation rate of 66.9 +/- 6. In terms of time efficiency, the control manipulation method takes 0.11 min and the centrifugation took an average of 0.52 min per oocyte. The MPF activity at the end of procedure was estimated through the measurement of H1 activity and as expected, the etoposide-cycloheximide treated oocytes had lower H1 activity which was restored by further incubation in the maturation medium for 5 hr while the centrifugation gave a nonsignificant intermediary result. In conclusion, the results presented suggest that both the chemical and the mechanical methods are usable alternatives to micromanipulation of oocytes to generate a large number of chromosome free cytoplasm for biochemical analysis. Mol. Reprod. Dev. 67: 70-76, 2004.     

The induction of reversible and irreversible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes

We investigated the effects of puromycin on mouse oocyte chromosomes during meiotic maturation in vitro. Puromycin treatment for 6 hr at 100 μg/ml almost completely, but reversibly, suppressed [³⁵S]methionine incorporation into oocyte protein at all stages of maturation tested. Nevertheless, oocytes treated at the germinal vesicle stage underwent germinal vesicle breakdown (GVBD) and chromosome condensation. These oocytes completed nuclear maturation to metaphase II (MII) if the inhibitor was withdrawn. Prolonged (24-hr) treatment, however, caused the chromsomes to degenerate. The chromosomes of oocytes treated shortly after GVBD for 6 hr remained condensed, but the oocytes failed to form a polar body. However, 24-hr treatment caused the chromosomes to decondense to form an interphase nucleus. Oocytes treated near MI for 6 hr gave off a polar body during the treatment, and their chromosomes decondensed to form a nucleus, which remained as long as the treatment was continued. However, if the puromycin was withdrawn, the chromosomes recondensed to a state morphologically similar to that at MII. Thus, the chromosome decondensation induced by protein synthesis inhibition at MI was reversible. Oocytes treated at MII, several hours after first polar body formation, also underwent chromosome decondensation to form a nucleus. In the continuous presence of puromycin, the chromosomes remained decondensed, but neither DNA synthesis nor mitosis occurred. However, following puromycin withdrawal, these occytes synthesised DNA and underwent mitosis. Thus, protein synthesis inhibition at MII, by parthenogenetically activating the oocytes, caused irreversible chromosome decondensation. Based on these observations, we discussed the roles of protein synthesis in the regulation of oocyte chromosome behaviour during meiotic maturation.     

Maturation of pig oocytes: observations on membrane potential

The membrane-potential changes of pig oocytes during maturation are described. Cumulus-enclosed oocytes have a resting potential of -41.81 +/- 0.60 mV; the removal of cumulus cells caused this potential to drop to -30.95 +/- 0.43 mV. Adding LH to the culture medium did not influence the potential of denuded oocytes but depolarized the potential of cumulus-enclosed oocytes to -32.90 +/- 0.43 mV. FSH did not affect the membrane potential of denuded or cumulus-enclosed oocytes, but significantly reduced the amplitude of the depolarization induced by LH. The effect of gonadotropins on cultured granulosa cells was also investigated. Plated granulosa cells have a resting potential of -45.21 +/- 0.72 mV, similar to that of cumulus-enclosed oocytes. As recorded in cumulus-enclosed oocytes, LH depolarized granulosa cell membrane potential (-30.33 +/- 0.69 mV) and FSH reduced this effect. To evaluate if oocyte maturation in vivo is accompanied by membrane-potential depolarization, follicular growth and oocyte maturation were induced in 6 prepubertal gilts by using an eCG-hCG treatment. Twenty hours after the beginning of oocyte maturation in vivo (induced by hCG), the membrane potential of the oocyte was depolarized to -28.84 +/- 1.01 mV, a value similar to that observed in vitro. These data indicate that both LH and FSH can influence the membrane potential of follicular somatic cells and, consequently, that of the oocyte. The electrical coupling between somatic cell and oocyte may represent a means by which the gonadotropin message is passed to the germinal cell by the somatic compartment.     

Sucrose pretreatment for enucleation: an efficient and non-damage method for removing the spindle of the mouse MII oocyte

Oocytes enucleated at metaphase II stage can support reprogramming of transferred nucleus and further developing to term. However, the first polar body in mice sometimes migrates away from the original place of expulsion, so the chromosomes of the oocyte will displace from the first polar body. Thus, it is not always possible to successfully enucleate according to the position of the first polar body. Here we use sucrose treatment to visualize metaphase spindle fibers and chromosomes with standard light microscopy. In the manipulation medium containing 3% sucrose, oocytes of poor quality become shrunken, deformed or fragmented, while oocytes of good quality in the same medium would show a swelling around the metaphase chromosomes and a transparent spindle area, shaped like "infinity" and "0". So it is easy to remove the well-distinguished spindle and chromosomes in oocytes of good quality. Re-examined by Hoechst 33342 stain under the UV light, the enucleation rate was 100%. There was no significant difference in IVF and cleavage rates between the sucrose treatment and the control group. In conclusion, this study demonstrated that 3% sucrose pretreatment can give a method for evaluating embryo quality and more importantly, it can, under a common microscope, allow the visualization of the spindle and chromosomes in oocytes of good quality and hence efficiently improve enucleation rate without any harm.     

Analyses of mitogen-activated protein kinase function in the maturation of porcine oocytes

The function of mitogen-activated protein kinase (MAPK) during porcine oocyte maturation was examined by injecting oocytes with either mRNA or antisense RNA of porcine c-mos protein, an upstream kinase of MAPK. The RNAs were injected into the cytoplasm of porcine immature oocytes immediately after collection from ovaries, then the oocytes were cultured for maturation up to 48 h. The phosphorylation and activation of MAPK were observed at 6 h after injection of the c-mos mRNA injected-oocytes, whereas in control oocytes, MAPK activation was detected at 24 h of culture. The germinal vesicle breakdown (GVBD) rate at 24 h of culture was significantly higher in c-mos mRNA-injected oocytes than in control oocytes. In contrast, although injection of c-mos antisense RNA completely inhibited phosphorylation and activation of MAPK throughout the maturation period, the GVBD rate and its time course were the same in noninjected oocytes. The degree of maturation-promoting factor (MPF) activation was, however, very low in oocytes in the absence of MAPK activation. Most of those oocytes had both abnormal morphology and decondensed chromosomes at 48 h of culture. These results suggest that MAPK activation is not required for GVBD induction in porcine oocytes and that the major roles of MAPK during porcine oocyte maturation are to promote GVBD by increasing MPF activity and to arrest oocytes at the second metaphase.     

The effect of hormones and growth factor on the course of meiosis in murine oocytes in culture]

The role of some intraovarian regulators of the final stages of gametogenesis is analysed. It is shown that the epidermal growth factor (EGF) in concentration of 1 and 10 ng/ml is able to induce reinitiation of meiosis from dictyotene stage during cultivation of the ovarian follicles of prepuberal mice in the serum-free medium after gonadotrophic stimulation. The pattern of maturation was analogous to that of maturation after HCG (LH) administration. Also, the EGF is able to stimulate meiosis reinitiation in the culture of cumulus-free oocytes blocked with cAMP at the stage of dictyotene. At the same time fibroblast growth factors and insulin do not demonstrate such an activity. Taking into consideration a high sensibility of oocytes to the EGF action, and also the fact that the character of changes of steroid hormones secreted by the ovary in culture under the action of EGF is the same as that under the influence of LH it is suggested that, the EGF and EGF-like proteins secreted by somatic follicle cells are the paracrinic regulators of the mammalian oocyte maturation which modulate neuroendocrine factors of the oogenesis control.     

Response of large oocytes of Xenopus laevis to progesterone in vitro in relation to oocyte size and time after previous HCG-induced ovulation.

The injection of Xenopus laevis females with human chorionic gonadotropin (HCG) leads to ovulation (and maturation) of oocytes whose diameters are 1.2 mm or larger. However, when Xenopus oocytes are removed from their follicular investments by manual dissection and exposed to the steroid, progesterone, in vitro, they exhibit maturation down to about 0.90 mm in diameter with the majority larger than 1.0 mm showing a positive response. Within each female the larger of the oocytes undergo maturation earlier than smaller ones.The response of oocytes also was shown to depend on the length of time since females were last stimulated to ovulate. Similar-sized oocytes from recently ovulated (stimulated) females matured much faster than those of untreated, unstimulated females. Indeed, even the smaller oocytes from stimulated females often matured before the largest oocytes of females without previous HCG injection.The experiments demonstrate that the physiological state of an oocyte cannot be accurately deduced solely from its size nor response to gonadotropins; unresponsiveness presumably being due to inability of follicular elements to respond to the trophic hormones or transfer the stimulus to the oocyte via the appropriate steroid.     

Efficient production of nuclear transferred rat embryos by modified methods of reconstruction

In this study we investigated spontaneous oocyte activation and developmental ability of rat embryos of the SD-OFA substrain. We also tried to improve the somatic cell nuclear transfer (SCNT) technique in the rat by optimizing methods for the production of reconstructed embryos. About 20% of oocytes extruded the second polar body after culture for 3 hr in vitro and 84% of oocytes were at the MII stage. MG132 blocked spontaneous activation but decreased efficiency of parthenogenetic activation. Pronuclear formation was more efficient in strontium-activated oocytes (66.1-80.9%) compared to roscovitine activation (24.1-54.5%). Survival rate after enucleation was significantly higher (89.4%) after slitting the zona pellucida and then pressing the oocyte with a holding pipette in medium without cytochalasin B (CB) compared to the conventional protocol using aspiration of the chromosomes after CB treatment (67.7%). Exposure of rat ova to UV light for 30 sec did not decrease their in vitro developmental capacity. Intracytoplasmic cumulus cell injection dramatically decreased survival rate of oocytes (42%). In contrast, 75.9% of oocytes could be successfully electrofused. Development to the 2-cell stage was reduced after SCNT (24.6% compared 94.6% in controls) and none from 244 reconstructed embryos developed in vitro beyond this stage. After overnight in vitro culture, 74.4% of the SCNT embryos survived and 56.1% formed pronuclei. The pregnancy rate of 33 recipients after the transfer of 695 of these cloned embryos was, however, very low (18.2%) and only six implantation sites could be detected (0.9%) without any live fetuses and offspring.     

Acceleration of onset of oocyte maturation in vitro by luteinizing hormone

The purpose of this study was to analyze the effect of luteinizing hormone (LH) on the earliest stage of oocyte maturation - the stage of breakdown of the dictyate nucleus. Oocytes were isolated from the preovulatory follicles of adult, cyclic rats. They were incubated in culture medium with or without 10 μg/ml LH. The cultures were observed continuously for up to 3 hours. Analysis of the rate of disappearance of the germinal vesicle nucleolus revealed that LH accelerated the breakdown process. The median times of disappearance were 91.3 minutes without LH and 62.3 minutes with LH. This is in accord with earlier reports on enhancement of fertilizability of oocytes matured in vitro with LH. Thus, although oocytes mature spontaneously in culture, the maturation remains LH sensitive.     

The effect in vivo of alterations in gonadotropins and cyclic nucleotides on oocyte maturation in the hamster

The temporal relationship of changes in cAMP and cGMP to oocyte maturation was examined in proestrous hamsters (day 4). The first series of experiments showed, in normal cycling hamsters, an increase in cAMP and a decrease in cGMP at 1400 h shortly after the rise in LH with oocyte maturation beginning at 1800 h. When a second group of animals was injected with phenobarbital at 1200 h to block the LH surge, no significant change occurred in either cyclic nucleotide and oocyte maturation was prevented. In the second series of experiments single injections of either saline, hCG (30 IU), LH (10 micrograms) or FSH (10 micrograms) were given each to a group of animals at 0900 h on day 4. Animals were killed at five time intervals between 15 min and 3 h following the injection. LH and hCG stimulated a simultaneous increase in cAMP and decline in cGMP. The injection of FSH, however, did not cause an increase in cAMP but still produced a sharp decline in cGMP. Oocyte maturation occurred at 3 h in those animals injected with gonadotropins. Animals injected with saline showed neither cyclic nucleotide changes nor oocyte maturation. When cAMP and cGMP levels were expressed as a ratio (cAMP/cGMP) a significant increase occurred in the normal cycling animals and in those injected at 0900 h with gonadotropins. Phenobarbital and saline injected control animals showed no significant increase in the cAMP/cGMP ratio and no oocyte maturation. The results of these experiments and previous studies by this investigator indicate that cGMP may play an important role in oocyte maturation in the hamster prior to the LH surge. Since, in the presence of gonadotropins, the cAMP/cGMP ratio increases prior to oocyte maturation, it may be that the cyclic nucleotide ratio is also of importance in this process. Previous work by Hubbard and Terranova (1) has shown that guanosine 3':5' cyclic monophosphate (cGMP), can inhibit spontaneous maturation of hamster oocytes in vitro. This inhibitory action was dose dependent and overcome by LH. The cGMP-mediated inhibition occurred only in cumulus-enclosed oocytes, while adenosine 3':5' cyclic monophosphate (cAMP) inhibited spontaneous maturation in both cumulus-enclosed and denuded oocytes. The results of this study suggested that cGMP may play a role in inhibiting oocyte maturation prior to the LH surge. LH, the initiator of oocyte maturation, has also been shown in the intact proestrous rat and hamster to cause a decrease in cGMP at the same time that cAMP is rising (2,3).(ABSTRACT TRUNCATED AT 400 WORDS)     

LH与hCG对昆明小鼠卵母细胞体外成熟的影响

目的研究促黄体素（LH）、人绒毛膜促性腺激素（hCG）对昆明小鼠卵母细胞体外成熟的影响。方法小鼠经注射孕马血清促性腺激素（PMSG）48h后,摘取卵巢获得未成熟卵母细胞,分别在含不同浓度的LH和hCG的成熟液中,或将LH和hCG以不同的浓度组合加入到成熟液,进行体外成熟。结果经15.16h的成熟培养,5个浓度LH组中的极体率均高于对照组,其中200IU/mL组显著高于50IU/mL、400IU/mL、300IU/mL组和对照组（P〈0.05）;5个浓度hCG组的极体率与对照组极体率无显著差异（P〉0.05）;协同组中15IU/mL hCG＋200IU/mL LH组的极体率显著高于对照组和其它各处理组。结论LH对小鼠卵母细胞的体外成熟有一定的促进作用。     

一种改进的牛体细胞核移植去核方法

为了提高传统的盲吸法牛体细胞核移植去核效率,将0.5mL离心管底部截掉,在截口处蒙上一层400目的细胞筛网,再与1 mL的离心管套在一起。实验1,将成熟的卵母细胞置于改造过的离心管内膜上,分别以1,000r/min、2,000r/min及3,000r/min离心10min,Hoechst 33342染色后,在荧光显微镜下计算第一极体与染色体的位置关系及去核效率;实验2,将卵母细胞以2,000r/min离心后去核做受体,颗粒细胞做供体进行核移植,检查重构胚胎的早期发育情况。结果表明:以2,000 r/min将卵母细胞离心10min后,有86.6%卵母细胞的极体与染色体之间夹角在20°以内,此时去核效率最高(87.4%);将卵母细胞离心后,对随后的重构胚发育无影响。因此,采用离心辅助去核的方法可以显著提高牛卵母细胞的去核效率。     

GnRH improves the recovery rate and the in vitro developmental competence of oocytes obtained by transvaginal follicular aspiration from superstimulated heifers

In this study we assessed the effect of GnRH on the recovery rate, meiotic synchronization and in vitro developmental competence of oocytes recovered close to the expected time of ovulation. Twenty-three heifers were superstimulated with FSH, and luteolysis was induced by PGF(2alpha) injection 48 h after the start of treatment Twelve heifers received 200 microg GnRH at 34 h after PGF(2alpha) treatment, Blood samples were collected between 35 to 47 h after PGF(2alpha) administration to determine the time of the LH surge. Transvaginal follicular aspiration was performed at 60 h after PGF(2alpha), and the recovered oocytes were fertilized or fixed either immediately or after 24 h of maturation in vitro. GnRH-treated heifers showed an LH surge within 3 h after treatment, while only 4 of the 10 heifers in the control group exhibited an LH surge by 47 h after treatment with PGF(2alpha). The average number of large follicles (> 10 mm) was 21.3 +/- 2.3 and 19.3 +/- 2.4 for GnRH-treated and control heifers, respectively. The oocyte recovery rate was 87.7 and 63.1% (P < 0.05), respectively, and most of the cumulus-oocyte-complexes (COC) recovered from the 2 groups had an expanded cumulus (80.4 and 80.5%, respectively). Oocytes with an expanded cumulus from the GnRH group had completed meiotic maturation at higher rate than the controls (97 vs 20%;P < 0.05). In vitro development to the blastocyst stage of cumulus-expanded oocytes fertilized immediately after recovery was higher in GnRH-treated than in control heifers (60.3 vs 40.0%; P < 0.05). No difference was observed when oocytes with compact or expanded cumulus were matured in vitro for 24 h before fertilization. These results indicate that GnRH injections improve the oocyte recovery rate and that oocytes have a higher development competence than those obtained from non-GnRH-treated animals. We propose that this higher in vitro developmental competence may result from a more synchronous or further advanced meiotic maturation. However, due to the small number of oocytes in our study, we must emphasize that our findings on meiotic resumption are of preliminary nature.     

Reconstruction of enucleated mouse germinal vesicle oocytes with blastomere nuclei

We have investigated the possibility that mitotic nuclei originating from preimplantation stage embryos and placed in the oocyte cytoplasm can undergo remodelling that allows them to undergo meiosis in the mouse. To address this question, we have used enucleated germinal vesicle (GV) ooplasts as recipients and blastomeres from the 2-, 4- or 8-cell stage as nuclear donors. We employed two methods to obtain ooplasts from GV oocytes: cutting and enucleation. Although efficiency of the reconstruction process was higher after enucleation than after cutting (90% and 70% respectively), the developmental potential of the oocytes was independent of how they had been produced. Nuclei from the 2-, 4-, or 8-cell stage embryos supported maturation in about 35%, 55% and 60% of cases, respectively. The time between nuclear envelope breakdown and the first meiotic division was shortened by up to 5 h in reconstructed oocytes, a period equivalent to the mitotic division of control blastomeres. About one-third of oocytes reconstituted with blastomere nuclei divided symmetrically instead of extruding a polar body; however, in the majority of them metaphase plates were found, suggesting that reconstructed oocytes (cybrids) underwent a meiotic rather than mitotic division. The highest percentage of asymmetric divisions accompanied by metaphase plates was found in cybrids with 8-cell-stage blastomere nuclei, suggesting that the nuclei from this stage appear to conform best to the cytoplasmic environment of GV ooplasts. Our results indicate that the oocyte cytoplasm is capable of remodelling blastomere nuclei, allowing them to follow the path of the meiotic cell cycle.     

Timing of meiotic stages in oocytes of the Syrian hamster (Mesocricetus auratus) and analysis of induced chromosome aberrations

Summary The stages of maturation of oocytes (meiotic stages) in adult Syrian hamsters (Mesocricetus auratus) have been timed following superovulation treatment. Timing and superovulation were induced by an injection of pregnant mares' serum on the first morning of the estrus cycle, followed by injection of human chorionic gonadotropin (HCG) in the evening of the fourth day. The oocytes completed the prophase of the first maturation division 3 h after the injection of HCG. Ovulation began 91/2h after the injection. A method of analysis of metaphase II chromosomes of Syrian hamster oocytes 16h after injection of HCG is described. This method seems to be appropriate for the examination of the induction of genetic defects during oogenesis. Female hamsters were treated with 2,3,5-triethyleneimono-benzoquione-1,4. The induced chromosome aberrations were analysed. Dosis-dependent response could be demonstrated.     

Centriole behavior during meiosis in oocytes of the sea urchin Hemicentrotus pulcherrimus

Ultrastructural changes in the maturing oocyte of the sea urchin Hemicentrotus pulcherrimus were observed, with special reference to the behavior of centrioles and chromosomes, using oocytes that had spontaneously started the maturation division process in vitro after dissection from ovaries. The proportion of oocytes entering the maturation process differed from batch to batch. In those eggs that accomplished the maturation division, it took ~4.5-5 h from the beginning of germinal vesicle breakdown to the formation of a second polar body. Serial sections revealed that a young oocyte before germinal vesicle breakdown had a pair of centrioles with procentrioles, located between the presumed animal pole and the germinal vesicle and accompanied by amorphous aggregates of moderately dense material and dense granules (granular aggregate). Just before germinal vesicle breakdown, a pair of fully grown centrioles located in the granular aggregate, which is present until this stage and then disappears, had already separated from another pair of centrioles. In meiosis I, each division pole had two centrioles, whereas in meiosis II each had only one. The two centrioles in the secondary oocyte separated into single units and formed the mitotic figure of meiosis II. The first polar body had two centrioles and the second had only one. The two centrioles in the first polar body did not form the mitotic figure nor did they separate at the time of meiosis II. These results indicate that, in sea urchins, duplication of the centrioles does not occur during the two successive meiotic divisions and the egg inherits only one centriole from the primary oocyte, confirming the results previously reported for starfish oocytes.     

The incidence of aneuploidy after single pulse electroactivation of mouse oocytes

A brief electric pulse often produces a high rate of activation of recently ovulated oocytes. Some other efficient parthenogenetic stimuli, such as alcohol, however, disrupt the spindle apparatus and increase the incidence of aneuploidy. In this paper, we have determined whether electroactivation per se increases the incidence of chromosomal segregation errors in haploid parthenogenones as evidenced at first cleavage mitosis. Superovulated F1 hybrid female mice were killed at 15.5, 18.5, 22.5, and 25 h after the HCG injection. Batches of 10–12 cumulus-denuded oocytes were transferred to an electroactivation chamber containing mannitol which was connected to a high voltage pulse stimulator and the pulse was triggered once. A high proportion of oocytes activated following this treatment, but only the single-pronuclear haploid parthenogenones were incubated overnight in medium containing colcemid, to determine the incidence of aneuploidy as evidenced at first cleavage mitosis. “Sham” electroactivation groups were also examined for evidence of activation and aneuploidy as described above. In these cases, cumulus-denuded oocytes were put through the electroactivation chamber but the pulse was not triggered. A further group of oocytes was studied to determine the effect of handling and exposure to hyaluronidase on activation frequency and parthenogenetic pathways. Finally, the spontaneous rate of aneuploidy was examined in fertilised embryos of F1 hybrid female mice × Rb(1.3)1Bnr male mice at first cleavage mitosis. The results show that single pulse electroactivation does not increase the level of aneuploidy in single-pronuclear parthenogenones compared to the “sham” group or the spontaneous rate observed in 1-cell fertilised embryos, nor does aneuploidy appear to increase with postovulatory age. The developmental pathways observed in the electroactivation group are significantly different to those observed in the “sham” group, and the level of activation observed in both groups is increased through handling of oocytes and their exposure to hyaluronidase. © 1993 Wiley-Liss, Inc.     

A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72-78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.