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Regular Article |
a Program for In Vitro Fertilization, Reproductive Surgery and Infertility, New York University School of Medicine, New York, New York 10016
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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fertilization, gametogenesis, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Numerous studies of GV maturation have utilized recipient ooplasts created from enucleated, cumulus-denuded oocytes [6–8]. However, embryos generated from reconstructed oocytes that had matured in vitro in the absence of cumulus cells from the GV stage onward displayed poor progression to morula- or blastocyst-stage embryo development and poor embryo morphology [9]. Recently, Kono et al. [10] reported that a GV from a fully grown, immature oocyte supports embryonic and fetal development to term when, following completion of the first meiotic division in the absence of cumulus-enclosed cells, it is transferred to and activated in the cytoplasm of an enucleated oocyte that had matured in vivo. The GV from a growing oocyte (diameter, 
60 µm) has been reported to be similarly competent [11]. These observations suggest that cytoplasmic factors may account for a decline in embryonic developmental potential when oocytes are denuded of their cumuli and are matured in vitro. In this study, we assessed individually the role of the nucleus and cytoplasm of mouse oocytes denuded of cumulus cells and matured in vitro to determine the potential for normal embryonic development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recovery of In Vitro- and In Vivo-Matured Oocytes and Fertilized Zygotes
Female mice (age, 6–8 wk) were injected i.p. with 5 IU of eCG (Sigma, St. Louis, MO). One group was killed by cervical dislocation 48 h after eCG to collect denuded GV-stage oocytes, which were then cultured in vitro to M-II (in vitro-matured oocytes). A second group was injected with 5 IU of hCG (Sigma) at 48 h after eCG and then killed 16–18 h later to collect M-II oocytes (in vivo-matured oocytes). A final group of mice was mated immediately after hCG injection and killed 22 h later to collect fertilized zygotes.
Cumulus-denuded, GV-stage oocytes were collected from each ovary by puncturing the follicles with a needle in Hepes-HTF. The oocytes were stripped of cumulus cells by repeated gentle pipetting. After washing in Hepes-HTF, the cumulus-denuded oocytes were then transferred to HTF containing 50 µg/ml of 3-isobutyl-1-methylxanthine (Sigma) and cultured for 4 h in an incubator (5% CO2 in air, 37°C). Oocytes with a diameter of approximately 80 µm and a visible perivitelline space were selected for a final 16-h maturation culture in HTF.
The M-II oocytes and zygotes were harvested from ampullae of excised fallopian tubes. Cumulus cells were removed by pipetting during a brief exposure to serum-free Hepes-HTF containing 300 IU/ml of hyaluronidase (Sigma).
Artificial Activation of In Vivo- and In Vitro-Matured Oocytes
In vivo- and in vitro-matured, M-II oocytes were activated as previously described [9, 12]. After incubation in Dulbecco PBS (Irvine Scientific) containing 3 µM Ca2+ ionophore A23187 (Sigma) for 5 min at room temperature, the oocytes were rinsed and cultured for 4–5 h in HTF containing 5 µg/ml of cycloheximide (ICN Biomedicals, Inc., Aurora, OH). The oocytes were then monitored under a dissection microscope for polar body extrusion and pronucleus (PN) formation 1 and 4 h later, respectively. Oocytes that displayed a second polar body and a single PN at these times were used.
Reconstructing Zygotes
Activated oocytes and in vivo-fertilized zygotes were placed in a micromanipulation droplet overlaid with oil. After preincubation in HEPES-HTF containing 7.5 µg/ml of cytochalasin B (Sigma) for 15 min at 30°C, the cells were subjected to micromanipulation. When reconstructing a zygote, the crucial step is to distinguish the female from the male PN in the fertilized zygotes. In each independent experiment, we pooled, in one dish, numerous zygotes from more than three superovulated and mated mice. We micromanipulated only those zygotes in which the female PN was in close proximity to the second polar body but also at a distinct distance from the male PN [12–14]. To remove the female PN, the zona pellucida was pierced with a sharp glass needle near the second polar body. An injection pipette was then passed through the slit to aspirate the female PN; in many instances, female PN manipulation triggered similar movements of the second polar body. The male PN was identified and then removed by aspiration. The PN for transfer were then inserted into the perivitelline space of the recipient cell. The procedure for electrofusion of the recipient cytoplasts and the transferred karyoplast has been described elsewhere [6, 9]. A similar micromanipulation procedure was used to transfer the M-II spindle between oocytes; in mice, the spindle is easily visualized using Hoffman optics.
Following fusion, reconstructed zygotes were cultured in S1 at 37°C with 5% CO2 overnight and then transplanted to the oviducts of foster mother mice. Some embryos were subjected to cytogenetic analysis.
Experimental Design
Several types of reconstructed zygotes were made using pronuclear transfer. In vivo-type zygotes served as controls to assess the efficiency of the oviduct transfer procedure.
Type 1 zygotes Oocytes are matured in vivo or in vitro (Fig. 1A) and then artificially activated. The activated egg is then the recipient for the transfer of a male PN from a fertilized zygote.
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Type 2 zygotes Oocytes are matured in vivo or in vitro (Fig. 1B) and then artificially activated. The resultant PN is then exchanged with the female PN of a fertilized zygote. Thus, type 2 zygotes compare the developmental capacities between artificially activated PN from eggs that matured in vivo or in vitro.
Type 3 zygotes Oocytes are matured in vivo or in vitro (Fig. 1C) and then artificially activated. The resultant PN is then removed and exchanged with male and female PN from a fertilized zygote. Thus, type 3 zygotes compare the functional competencies between activated cytoplasts from eggs that matured in vivo or in vitro.
Type 4 zygotes Oocytes are matured in vitro (Fig. 1D), and the resultant M-II spindles are removed and transferred to enucleated M-II cytoplasts from eggs that matured in vivo. The reconstructed oocytes are then artificially activated, and the resultant PN is then exchanged with the female PN of a fertilized zygote.
Type 5 zygotes Oocytes are matured in vitro (Fig. 1E), and the resultant M-II spindles are removed and transferred to enucleated M-II cytoplasts from eggs that matured in vivo. The reconstructed oocytes are then artificially activated and outfitted with a male PN transferred from a fertilized zygote.
Cytogenetic Analysis
Artificially activated oocytes with a single PN and reconstructed zygotes were cultured in S1 containing 1 µg/ml of nocodazole overnight at 37°C to produce metaphase arrest. Cells were fixed for cytogenetic analysis. Each oocyte was transferred into a 1% hypotonic trisodium citrate solution for 10 min before fixation with methanol:acetic acid (3:1 [w/v]) on a clean glass slide [15]. The chromosome spreads were air-dried, stained with a microdroplet of phosphate buffer containing 3 ng/ml of 4',6'-diamidino-2-phenylindole (Vysis Inc., Downers Grove, IL), and then covered with a thin glass slide. The number and the structure of the chromosomes were visualized immediately using fluorescence microscopy; digital images were saved for computer analysis.
Embryo Transfer
Type 2 embryos were surgically transferred into oviducts of CD-1 foster mothers mated with vasectomized CD-1 males [16]. Types 3–5 embryos were transferred into each oviduct on Day 1 of pseudopregnancy (i.e., the day on which the copulation plug was found). Foster mothers carrying only one fetus were delivered by cesarean section at 20 days postcoitus.
Data Analysis
Data were analyzed using 
2 or Fisher exact probability tests when appropriate. Significance was determined at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The majority of denuded GV-stage oocytes extruded a first polar body (69%, 286 of 415). When allowed to mature in culture for 16–18 h following exposure to A23187 and cycloheximide, 257 of these 286 oocytes (90%) extruded a second polar body and formed a PN. Oocytes that matured to M-II in vivo responded to this treatment with polar body extrusion and PN formation at a similar rate (94%, 169 of 179).
Cytogenetic Analysis of Activated Oocytes and Reconstructed Zygotes
A uniform haploid complement of 20 chromosomes was noted in 33 of 37 oocytes that extruded a second polar body after in vitro maturation and activation (Fig. 2A). Oocytes that failed to extrude a second polar body displayed 40 chromosomes and were discarded (Fig. 2B). Although four activated oocytes had less than 20 chromosomes on analysis, each had overlapping chromosomes in the spread (Fig. 2C). In vitro type 2 zygotes (n = 10) were also subjected to cytogenetic analysis, and eight presented two haploid complements of 40 chromosomes (Fig. 2D).
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Development of Type 1 Zygotes Derived from Oocytes Matured In Vivo or In Vitro
Birth rates were 17% (n = 30) (Table 1) for in vivo type 1 zygotes constructed by simply transferring male PN into oocytes matured in vivo after activation. In contrast, no pregnancies were observed in the nine foster mothers that received in vitro type 1 zygotes, even though the electrofusion and embryo cleavage rates before embryo transfer were comparable to those of the in vivo type 1 zygotes (Table 1).
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Development of Type 2 and 3 Zygotes Derived from Nucleus or Cytoplasm of Oocytes Matured In Vivo
We used in vivo type 2 and 3 zygotes to assess, respectively, the developmental capacities of the nuclei and cytoplasm derived from oocytes that had matured in vivo before activation. Construction of in vivo type 2 zygotes occurred without incident, and each zygote divided during overnight culture. All the cleaving embryos were transferred to the oviducts of seven pseudopregnant foster mothers. Six mice became pregnant and delivered a total of 11 normal, live offspring (Table 1). Construction of in vivo type 3 zygotes also proceeded routinely, and all fused zygotes cleaved during overnight culture. When transferred to oviducts of pseudopregnant foster mice, five normal, live offspring were generated by in vivo type 3 zygotes (Table 1). Significantly, no differences were found between the live birth rates generated by in vivo type 2 and 3 zygotes (21% vs. 18%). Thus, following chemical activation, the nuclei and cytoplasm of in vivo-matured oocytes are functionally competent to support embryonic development to term.
Development of Type 2 and 3 Zygotes Derived from Nucleus or Cytoplasm of In Vitro-Matured Oocytes
When in vitro type 2 zygotes were transferred, only 1 of 10 foster mothers became pregnant, bearing only a single pup. This live birth rate (1%) was significantly less than that generated by transfer of in vivo type 2 zygotes (21%) (Table 1). These results suggest that PN of oocytes matured and activated in vitro cannot support embryonic development to term and, thus, are functionally different from PN of oocytes matured in vivo. However, different results were obtained when we tested the cytoplasm of oocytes that matured in vitro. When 49 embryos derived from in vitro type 3 zygotes were transferred to the oviducts of six foster mothers, five became pregnant, bearing nine live pups (18%) (Table 1). This pregnancy rate was no different than that of in vivo type 3 zygotes.
Development of Type 4 and 5 Zygotes Constructed with Nuclei of In Vitro-Matured Oocytes Activated in the Cytoplasm of In Vivo-Matured, Enucleated Oocytes
Spindles (n = 98) of in vitro-matured oocytes were transferred to enucleated, in vivo-matured oocytes. Eighty-two percent fused after electrical stimulation, and 77 of these 80 fused oocytes transformed into PN gametes that extruded a second polar body. When a female PN of these activated oocytes (n = 42) was exchanged with the female PN of in vivo zygotes, most (98%) of the reconstructed in vitro type 4 zygotes divided during overnight culture in S1. After transfer of type 4 zygotes to the oviduct of three pseudopregnant mice, two became pregnant, bearing a total of seven normal, live pups at 20 days after transfer (Table 1). Similarly, when the remainder of these activated oocytes (n = 42) were outfitted with a male PN, all the resultant in vitro type 5 zygotes divided during overnight culture. After transfer of in vitro type 5 zygotes to the oviducts of four pseudopregnant mice, two became pregnant, bearing a total of eight normal, live pups (Table 1). The live birth rates of type 4 and 5 zygotes were 17% and 23%, respectively, which are not significantly different from those observed with in vivo type 2 or both groups of type 3 zygotes (Table 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, remarkably similar birth rates were observed following the transfer of several groups of experimentally constructed zygotes to the uteri of foster mother mice. However, two groups of constructed zygotes—in vitro type 1 and in vitro type 2—displayed birth rates that were significantly lower than those of all the other groups; in fact, only 1 of 152 transferred zygotes survived to term. In vitro type 1 zygotes were constructed by denuding and maturing immature eggs to M-II in vitro, activating them with A23187 + cycloheximide, and then "fertilizing" them with insertion of a male PN. In vitro type 2 zygotes were similarly constructed through artificial activation, but the resultant PN was then transferred into a fertilized zygote enucleated of its female PN. Although the failure of term development for in vitro type 1 and 2 zygotes may arise from any one of the experimental procedures used in their construction, the significantly higher rates seen with the in vivo type 1 and 2 and the in vitro type 4 and 5 zygotes allow us to rule out the cumulus stripping/maturation conditions, the activation treatments, and the PN transfer procedures as causal factors. In addition, the similar birth rates observed for the in vivo type 1, 2, and 3 zygotes and the in vitro type 3, 4, and 5 zygotes also rule out the oviductal transfer procedure as a cause for the poor pregnancy outcome with in vitro type 1 and 2 zygotes.
As noted above, cytogenetic analyses indicated that female PN which developed in the in vitro type 1 zygotes were consistently haploid. Thus, it is also unlikely that an anomalous segregation of chromosomes is the cause of the poor birth rate noted following transfer of these zygotes. The presence of a haploid genome also suggests that the activation process was properly completed. Such a conclusion is supported by our observations that diploid eggs were occasionally observed at 4 h following activation but failed to extrude a second polar body. Each in vitro type 2 zygote received a PN from an egg that had extruded a second polar body.
Previously, Schroeder and Eppig [24] reported that the pregnancy and birth rates of in vitro-matured mouse eggs are identical to those of eggs that matured in vivo. However, those authors also commented that such results were specific to their experimental conditions and could be influenced by a variety of factors, including the media employed and the experimental procedures under investigation. Thus, it is not surprising that other investigators have reported pregnancy and live birth rates following fertilization of in vitro-matured eggs that are appreciably lower than those of in vivo-matured eggs [25–27]. Our results suggest an explanation for this latter observation—despite being morphologically and cytogenetically normal, the ability of female PN to support normal embryonic development may, nonetheless, be functionally compromised when they are activated in mouse eggs that have matured in vitro. The normal live birth rate observed following oviduct transfer of type 4 and 5 zygotes indicates that this anomaly in pronuclear function actually has its origins in the ooplasm at the time of activation. Thus, the M-II spindle of an in vitro-matured egg must be transferred into the cytoplasm of an in vivo-matured egg before activation for the resultant PN to be capable of supporting a normal birth rate. Similar results have been reported previously, even for less immature oocytes from growing follicles [10, 11]. Clearly, whether the maturation from GV to M-II occurs in vivo or in vitro has little influence on the functional potential of that nucleus to support later embryonic development.
Taken together, our results suggest the hypothesis that, during the transition from M-II to interphase following artificial activation, the ooplasmic milieu plays an important role in determining the functional competence of the resultant PN to support embryonic development to term. Previous studies from this laboratory suggest that functional competence is compromised during the preimplantation period [9]. A similar situation may also apply in the eggs of mice and other species when activation and PN formation is normally achieved by fertilization with a sperm.
During the final stages of maturation from GV to M-II, mouse oocytes undergo pronounced morphologic and biochemical changes that include the synthesis of new cytoplasmic proteins [28], protein phosphorylation [29], and intracellular Ca2+ oscillations [30]; similar changes have also been reported for other mammalian species, including humans [31–33]. The newly synthesized proteins appear to be necessary for the oocyte to complete normal meiosis to M-II, and they may be involved in development of the PN after activation (mouse [34] and other mammals [35–37]). Comparative studies of protein synthesis between in vitro- and in vivo-matured oocytes have revealed that several proteins with distinct molecular weights are present only in the cytoplasm of oocytes that matured in vivo (mouse [28] and other mammals [38, 39]). Studies in the rabbit and cow suggest that some of these proteins may depend on cumulus cell—oocyte interactions since more proteins are present in the cytoplasm when oocytes are cultured within an intact cumulus mass [33, 40, 41]. However, because mouse oocytes can complete meiosis and activation even when denuded of cumulus cells at the GV stage [6, 12, 24], the proteins important for these processes would appear to occur independently of cumulus cell contact. A similar argument can be raised for LH-stimulated protein synthesis, because these oocytes come from animals not treated with exogenous LH before collection or during culture.
In summary, we hypothesize that, during the oocyte activation process, its genome undergoes a functional transition, or switch, during the M-II to interphase interval that is important for normal embryonic development to continue until birth. The role of the ooplasm during this transition is to ensure that it is completed properly. The ooplasm of oocytes denuded of cumulus cells and matured in vitro appears to be incapable of switching the M-II nucleus to the interphase PN with full function. We speculate that gonadotropins and/or cumulus cell-dependent proteins normally deposited in the ooplasm in vivo may play a crucial role in this transition of nuclear function.
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FOOTNOTES |
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1 Correspondence: Lewis C. Krey, Program for In Vitro Fertilization, New York University School of Medicine, 660 First Avenue, 5th Floor, New York, NY 10016. FAX: 212 263 8827; kreyivf{at}yahoo.com 
Accepted: August 2, 2001.
Received: September 19, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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