Retrieval of mouse oocytes

To date, only a few studies have reported successful manipulations of Peromyscus embryogenesis or reproductive biology. Together with the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu), we are characterizing the salient differences needed to develop this system. A primary goal has been to optimize oocyte/early embryo retrieval.     

Enucleolation of porcine oocytes

Germinal vesicles (GVs) in immature mammalian oocytes contain prominent nucleoli whose role in the process of oocyte maturation is not fully understood. Here we report that the microsurgical removal of nucleoli from immature fully grown porcine oocytes permits germinal vesicle breakdown and chromosome condensation and the enucleolated oocytes mature up to the second metaphase. Interestingly, the enucleolation of growing oocytes which, although unable to mature, resulted in germinal vesicle breakdown and the formation of a cluster of condensed chromatin. These results indicate that the nucleolus in fully grown oocytes is dispensable at least for nuclear maturation. On the other hand, the results obtained in growing oocytes suggest the role of the nucleolus in the cell cycle regulation.     

Cryopreservation of starfish oocytes

Research from many laboratories over the past several decades indicates that invertebrate oocytes and eggs are extraordinarily difficult to freeze. Since starfish oocytes, eggs, and embryos are an important cell and developmental biology model system, there is great interest to cryopreserve these cells. Previous starfish oocyte cryopreservation studies using slow cooling protocols revealed that these cells are highly sensitive to osmotic stress and form intracellular ice at very high sub-zero temperatures, suggesting that common freezing methodologies may not prove useful. We report here that a short exposure to 1.5 M Me2SO/1 M trehalose in hypotonic salt solution followed by ultra-rapid cooling to cryogenic temperatures allows starfish oocytes to be cryopreserved with the average survival rate of 34% when normalized to control oocytes that were exposed to CPA, but not frozen. On average, 51% of the oocytes in 77% of the batches of frozen oocytes underwent meiotic maturation in response to the starfish maturation hormone, 1-methyladenine. In one experiment, eggs developing from thawed oocytes were capable of being fertilized and two developed into embryos. These data suggests that successful cryopreservation of starfish oocytes is possible, but will need further refinement to increase the numbers of fully competent embryos.     

Oogenesis and oocytes

The morphological features of polychaete ovarian morphology and oogenesis are reviewed. Some basic information on ovarian structure and/or oogenesis is known for slightly more than half of recognized polychaete families although comprehensive studies of oogenesis have been conducted on 0.1 of described species. Relative to other major metazoan groups, ovarian morphology is highly variable in the Polychaeta. While some species appear to lack a defined ovary, most have paired organs that are segmentally repeated to varying degrees depending on the family. Ovaries vary widely in their location but are most frequently associated with the coelomic peritoneum, parapodial connective tissue, or elements of the circulatory system. The structural complexity of the ovary is correlated with the type of oogenesis expressed by the species. In some polychaetes, extraovarian oogenesis occurs in which previtellogenic oocytes are released into the coelom from a simple ovary where differentiation occurs in a solitary fashion or in association with nurse cells or follicle cells. In other species, intraovarian oogenesis occurs in which oocytes undergo vitellogenesis within the ovary, often in association with follicle cells that may provide nutrition. Vitellogenesis probably includes both autosynthetic and heterosynthetic processes; autosynthesis involves the manufacture of yolk bodies via the proteosynthetic organelles of the oocyte whereas heterosynthesis involves the extraovarian production of female-specific yolk proteins that are incorporated into the oocyte through a receptor-mediated process of endocytosis. Variation in the speed of egg production varies widely and appears to be correlated with the vitellogenic mechanism employed. Mature ova display a wide range of egg envelope morphologies that often show some intrafamilial similarities.     

Development of rabbit parthenogenetic oocytes and nuclear-transferred oocytes receiving cultured cumulus cells

The present study determined a suitable parthenogenetic activation procedure for rabbit oocytes and examined the developmental potential of enucleated oocytes receiving cultured cumulus cells. Unfertilized oocytes recovered from superovulated rabbits were activated with one or two sets of electrical pulses, with or without subsequent administration of 6-dimethylaminopurine (6-DMAP). The proportion of oocytes treated with one or two sets of electrical pulses and 6-DMAP that cleaved (87% and 98%, respectively) and developed into blastocysts (77% and 85%, respectively) was significantly higher (P < 0.05) than those activated with electrical pulses alone (30% and 42% for cleavage, 7% and 17% for blastocysts). Cumulus cells separated from ovulated oocytes obtained from mature rabbits were cultured for three to five passages and then induced to quiescence by serum starvation before nuclear transfer. The enucleated oocytes receiving cumulus cells were activated with electrical pulses followed by the addition of 6-DMAP, and cultured in vitro for 5 to 6 d or transferred to pseudopregnant recipient females 1 d after activation. Of 186 nuclear-transferred oocytes, 123 (66%) cleaved and 42 (23%) developed into blastocysts. After transfer of 174 nuclear-transferred oocytes to 8 recipient females, a total of 3 implantation sites were observed in 3 recipient females but no fetuses were obtained.     

In vitro parthenogenetic development of mouse oocytes following reciprocal transfer of the chromosome spindle between in vivo-matured oocytes and in vitro-matured oocytes

Mouse follicles grown in vitro from preantral to mature stages yield oocytes that can be fertilized in vitro, but embryonic development is poor. To investigate whether this poor development is due to a nuclear or a cytoplasmatic factor, we designed an experiment in which the MII chromosome spindle was exchanged between in vitro-matured oocytes and in vivo-matured oocytes by electrofusion. Subsequent embryo development was evaluated by blastocyst formation rate and blastocyst cell number after parthenogenetic activation. Electrofusion was successful in 62-78% of the oocytes. Transfer of the spindle apparatus from in vitro-matured oocytes to the in vivo MII cytoplasmic environment resulted in a high rate of blastocyst development, whereas in the reverse situation (transfer of the nucleus from in vivo-matured oocytes into in vitro-matured MII cytoplasm) poor quality embryos and a low rate of blastocyst formation was observed. These results indicate that the low developmental competence of in vitro-matured oocytes from mouse preantral follicles after activation is caused by the cytoplasmic component rather than the nuclear component.     

Centrifugal bisection of starfish oocytes

Immature oocytes or mature eggs of starfish were centrifuged in a sucrose density gradient. They were then separated into two fractions of fragments, nucleate light fragments and anucleate heavy fragments. Vital-staining experiments showed that the oocytes were elongated along the animal-vegetal (AV) axis during the centrifugation in a contrast to centrifuged eggs whose centrifugal axis was not related to the AV axis. The light and heavy oocyte fragments were comprised of animal and vegetal halves of oocytes, respectively. When matured and fertilized, most of the light oocyte fragment-derived embryos failed gastrulation and developed into Dauerblastulae. Two-dimensional gel electrophoretic analysis of fragments revealed that three basic proteins were predominantly enriched in the heavy oocyte fragments but scarcely detected in the light oocyte fragments. One of these proteins, App20, was identified as a homologue of cyclophilin (peptidyl-prolyl cis-trans isomerase). The present study provides a simple means of separating a population of starfish oocytes into animal and vegetal halves, thereby enabling us to analyze any difference of components between animal and vegetal cytoplasm of the oocytes.     

卵母细胞的生发泡移植

生发泡(germinal vesicle,GV)移植到去核的GV期卵母细胞后,获得重构卵,重构卵在体外能成熟,受精和进行胚胎发育。GV移植到去核的第二次减数分裂中期(metaphase Ⅱ,MII)卵母细胞后,重构卵能发生GV破裂,但难以排出第一极体。GV移植后,通过连续核移植,重构合子具有发育到终期的能力。GV移植为研究卵母细胞的发育提供了一种重要工具。     

Electrical activation of mouse oocytes

Activation of the oocyte is the least efficient step in nuclear transplantation in the rabbit. We report the influence of age of oocytes, field strength, pulse duration and number, and shape of field on the rate of activation of mouse oocytes by electrical pulses. Regardless of oocyte age, activation rates were similar over a wide range of field strengths and pulse durations. Aged oocytes activated at a higher rate than recently ovulated oocytes (32 vs 3%), which lysed more frequently (13 vs 2%). Fragmentation rate was also higher among aged oocytes (42 vs 6%). The rate of activation increased with the number of pulses, from 9% with a single pulse, to 61% with six pulses. It also increased with the interval between pulses. Comparison of activation chamber geometries showed that the rate of activation was higher in a nonuniform field than in a uniform field and, for a particular field strength, varied from one electrode gap to another. These observations indicate that the rate of activation can be greatly increased by multiple electrical pulses. The activation rate also varied with consistent field strength in chambers with different electrode configurations.     

Cytogenetics of unfertilized human oocytes

During an in-vitro fertilization programme 150 oocytes from 62 women with a mean age of 31 years (range 24-39) remained unfertilized. Successful chromosome analysis was carried out on 96 oocytes by Q-banding: 59 (61.5%) oocytes bore a normal haploid complement, 8 (8.3%) were diploid and 3 (3.1%) tetraploid. In 26 (27.1%) oocytes aneuploidy was observed; these included 9 (9.4%) nullisomic, 5 (5.2%) double nullisomic, 4 (4.2%) triple nullisomic and 2 (2.1%) disomic oocytes. The remaining 54 (36.0%) oocytes could not be evaluated. A nearly uniform rate of aneuploidy was found for unfertilized oocytes among different donor age groups.     

Morphology of immature bovine oocytes

Bovine cumulus oocyte complexes (COCs) as used for in vitro maturation and fertilization can be classified into different categories by light microscopical inspection. We have distinguished four categories based on compactness and transparency of the cumulus investment and homogeneity and transparency of the ooplasm. The four categories were studied for their morphological characteristics at the ultrastructural level and for their developing capacity in an in vitro maturation system. In categories 1 and 2 oocytes, organelles were evenly distributed. In categories 3 and 4, oocytes organelles were clustered and the distribution of the organelles mimicked the characteristics of oocytes during final maturation. Cumulus cell process endings penetrated the cortex of the oocyte or were located superficial to the cortex of the oocyte. In category 1 oocytes, most of the process endings penetrated the cortex. In category 4 oocytes, most of the process endings did not penetrate. In categories 2 and 3 oocytes, both forms of process endings did occur. After in vitro maturation, only category 4 oocytes showed a decreased developing capacity. Categories 1–3 oocytes showed equal developing capacity in an in vitro maturation system.     

Actin in Xenopus oocytes

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.     

RNA-binding proteins of Rana temporaria oocytes: incorporation into informosomes of oocytes in vivo]

The participation of RNA-binding protein in the formation of informosomes in vivo was studied using an intracellular microinjection technique. The RNA-binding protein of the frog Rana temporaria oocytes was isolated by affinity chromatography and was labelled in vitro without any loss of its activity. It was shown that during cultivation of the oocytes the specific incorporation of the injected RNA -- binding [3H]-protein into the ribonucleoprotein particles occurred. These particles were further described as informosomes, characteristic ribonucleoprotein particles of animal cells.     

In vivo aging of oocytes influences the behavior of nuclei transferred to enucleated rabbit oocytes

The present study examined nuclear remodeling in rabbit nuclear transfer (NT) embryos formed from metaphase II (MII) oocytes aged in vivo until 19 hr postcoitum (hpc), enucleated, and fused at 22–26 hpc with 32-cell morula blastomeres by means of electric fields, which also induced recipient oocyte activation. Post-activation events observed during the first hour following the fusion/activation pulse were studied in terms of chromatin, lamins, and micro-tubules, and revealed that transferred nuclei underwent premature chromosomes condensation (PCC) in only one-third of NT embryos and remained in interphase in others. Recipient oocytes were mostly not activated by manipulations performed before the fusion/activation pulse. The persistance of transferred nuclei in interphase resulted from the rapid progression of recipient oocytes to interphase after activation, suggesting that the cytoplasmic state of MII oocytes aged in vivo was poised for the approach to interphase. Studying micro-tubular organization in MII oocytes before nuclear transfer manipulations, we found that 19 hpc MII oocytes aged in vivo differed from 14 hpc MII oocytes (freshly ovulated) and from 19-hpc MII oocytes aged in vitro (collected at 14 hpc and cultured for 5 hr), notably by the presence of microtubule asters and tubulin foci or only tubulin foci dispersed throughout the cytoplasm. When PCC was avoided, remodeling of the transferred nucleus was well advanced 1 hr after nuclear transfer, and NT embryos developed better to the blastocyst stage. Mol. Reprod. Dev. 46:325–336, 1997. © 1997 Wiley-Liss, Inc.