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Histology of the reproductive tract of hybrids between gonochoristic males and parthenogenetic females of Lepidodactylus lugubris in French Polynesia (Reptilia, Gekkonidae).

Parthenogenetic populations of the gecko Lepidodactylus lugubris are widespread throughout Polynesia. They often occur parapatrically, and occasionally syntopically, with the increasingly rare bisexual populations. In these instances, a small number of hybrid individuals occur and include both "female" and "male" external phenotypes, both with greatly reduced gonads. Histological examination demonstrates that these hybrids possess small ovotestes. The differentiation of the cortical tissue is identical in both "male" and "female" hybrids, but the medullary tissue is more developed in "males." The remainder of the genital tract in "females" resembles that of fertile females in the parthenogenetic and bisexual populations. By contrast, the "male" hybrids are markedly intersexual. In one of the two specimens autopsied, the hemipenes are more or less the same size as those of bisexual males, and the sexual segment of the kidney is hypertrophied and serous. In the other hybrid "male," the hemipenes have a structure similar to that seen in females, and the sexual segment of the kidney is poorly differentiated. In both hybrid "males," the ductus deferens is extremely narrow and further reduced in its middle portion; oviducts are present and resemble those of normal or hybrid females. Thus, embryonic-like gonads are associated with complete and normal female reproductive ducts in hybrid "females." Hybrid "males" also have embryonic-like gonads and feminized genital ducts but associated with secondary sexual characters that match those of sexually active or quiescent normal males.

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Interferon-tau in bovine blastocysts following parthenogenetic activation of oocytes: pattern of secretion and polymorphism in expressed mRNA sequences.

A series of experiments were conducted to examine the pattern of production and secretion of interferon-tau (IFN-tau) by blastocysts following parthenogenetic activation of bovine oocytes. In the first experiment, 36.8, 24.1, and 33.2% of IVF-derived and parthenogenetically activated oocytes cultured in the presence or absence of a monolayer of buffalo rat liver cells, respectively, reached the blastocyst stage. Following individual culture of blastocysts, IFN-tau concentration in medium droplets was similar among the three groups, although IVF-derived blastocysts contained significantly more cells. In the second experiment, 156 IVF-derived blastocysts were sexed by PCR with 75 and 81, respectively, being male and female. IFN-tau secretion of these was compared to that of 70 parthenogenetic blastocysts. Female and parthenogenetic blastocysts produced significantly more IFN-tau than their male counterparts. In the third experiment, the ability of hatched blastocysts to form outgrowths and the pattern of their IFN-tau secretion were examined. Of the 48 IVF-derived blastocysts, 44 formed outgrowths compared to 41 of the 42 hatched parthenotes. Parthenogenetic outgrowths were significantly larger after 7 days, but this difference had disappeared after 14 days. IFN-tau secretion did not differ between the two groups. Lastly, sequence analyses of expressed mRNA from individual parthenogenetic blastocyst outgrowths showed four different transcript types which, based on their predicted amino acid sequence, belong to two subgroups, IFN-tau1 and IFN-tau3. In addition, one new transcript sequence was identified, encoding a new protein isoform.

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Porcine oocyte activation: differing roles of calcium and pH.

Intracellular pH has recently been shown to increase during parthenogenetic activation of the porcine oocyte. In the following set of experiments, intracellular pH was monitored during activation and pronuclear development was assessed following activation treatments with calcium, in the absence of calcium, and in oocytes loaded with the calcium chelator BAPTA-AM in calcium-free medium. Intracellular pH increase was not different among groups when treating with 7% ethanol or 50 microM calcium ionophore, or during treatment with thimerosal for 12 or 25 min. Activation with thimerosal (200 microM, 12 min) followed by 8 mM dithiothreitol (DTT, 30 min) resulted in a decreased pronuclear development in calcium-free medium with or without BAPTA-AM loaded oocytes as compared to controls. Activation with 50 microM calcium ionophore resulted in pronuclear development that was different between the calcium-free and BAPTA-AM loaded oocytes in calcium-free medium. Similar incidences of pronuclear formation were observed in all ethanol treatment groups. It was concluded that external calcium as well as large changes in intracellular free calcium are not necessary for the increase in intracellular pH, but normal intracellular calcium signaling is critical for normal levels of pronuclear development. Finally, oocytes were measured for intracellular pH changes for 30 min following subzonal sperm injection. Intracellular pH did not increase, although pronuclear formation was observed 6 hr post SUZI. This suggested that major differences were still present between sperm-induced and parthenogenetic activation of the porcine oocyte.

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Nucleolar proteins and ultrastructure in bovine in vivo developed, in vitro produced, and parthenogenetic cleavage-stage embryos.

In the present study, ribosomal RNA (rRNA) gene activation, monitored through nucleolus development, was studied by autoradiography following (3)H-uridine incubation, transmission electron microscopy, and immunofluorescence confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (topoisomerase I, upstream binding factor, and RNA polymerase I) and processing (fibrillarin, nucleolin, and nucleophosmin) in in vivo developed, in vitro produced, and parthenogenetic bovine embryos. In general, in vivo developed embryos displayed formation of fibrillo-granular nucleoli during the 4th post-fertilization cell cycle. During the previous stages of development, nucleolus precursor bodies (NPBs) were observed. However, on some occasions the initial steps of nucleolus formation were observed already at the 2- and 4-cell stage in cases where such embryos were collected from superovulated animals together with later embryonic stages presenting nucleolar development and autoradiographic labeling. The in vitro produced embryos displayed very synchronous formation of fibrillo-granular nucleoli and autoradiographic labeling during the 4th cell cycle. In vivo developed and in vitro produced embryos displayed allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the 4th cell cycle. The parthenogenetic embryos typically displayed formation of fibrillo- granular nucleoli during the 5th cell cycle and autoradiographic labeling was not observed until the morula stage. Moreover, the 1-, 2-, and 4-cell parthenogenetic embryos practically lacked NPBs. On the other hand, parthenogenetic embryos displayed allocation of nucleoar proteins to nuclear entities during the 4th cell cycle. In conclusion, both in vivo developed and in vitro produced bovine embryos displayed activation of transcription and nucleolar development during the 4th cell cycle. However, in vivo developed embryos flushed together with later developmental stages displayed premature activation of these processes. Parthenogenetic bovine embryos, on the other hand, displayed a delayed activation.

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Epidermal growth factor induces Bcl-xL gene expression and reduces apoptosis in porcine parthenotes developing in vitro.

The aim of this study was to determine the interactive effects of bovine serum albumin (BSA) and epidermal growth factor (EGF) on the viability and development of porcine presumptive diploid parthenotes developing in vitro. The addition of 0.1 and 0.4% BSA to the culture medium enhanced the development of 4-cell parthenotes to the blastocyst stage but EGF had no effect. However, while BSA also increased cell numbers, it did so only when EGF was also present. Either agent on its own had no effect. Similarly, apoptosis in the blastocysts was not influenced by either agent on its own but was reduced when both BSA and EGF were present. Furthermore, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that EGF enhanced the mRNA expression of Bcl-xL in the presence of 0.4% BSA but BSA and EGF alone had no effect. EGF and/or BSA did not influence Bak gene expression in the blastocyst stage parthenotes. These results suggest that EGF and BSA synergistically enhance Bcl-xL gene expression, which may result in a net increase in cell number in porcine presumptive diploid parthenotes developing in vitro.

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Activation of cumulus-free mouse oocytes.

Attempts were made to activate parthenogenetically oocytes from random-bred mice under conditions required for nuclear transfer, viz. in the absence of cumulus cells. Of the large number of stimulants previously described and presumed to be generally effective, only exposure to strontium ion (as a replacement for calcium ion in the medium), or to calcium ionophore, activated a substantial proportion of cumulus-free oocytes from TRf mice, a synthetic, random-bred strain. These stimuli were less effective in oocytes from random-bred ICR mice. The relevance of the findings to nuclear-transfer cloning attempts in mice is discussed.

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Spontaneous and induced activation of rat oocytes.

Ovulated rat oocytes undergo spontaneous activation during in vitro culture. After extrusion of the second polar body, they do not enter interphase but are arrested again in next metaphase-like stage (M III arrest). The present study demonstrates that puromycin and chloral hydrate can trigger transition to interphase of metaphase II and spontaneously (incompletely) activated rat oocytes. The response of oocytes to these activators depends on their stage at the time of application of a stimulus. Metaphase II oocytes enter interphase at 86.8% when treated with puromycin and in 28.7% after chloral hydrate activation. Oocytes activated with chloral hydrate at the time of spontaneously induced anaphase II enter interphase at 64.8%, but after reaching the stage of telophase II their capability to shift to interphase is again low (28.8%). Finally, M III oocytes cannot be forced to enter interphase by either chloral hydrate or puromycin treatment. This study shows that resumption of the second meiotic division and transition to interphase--the two processes that normally occur in succession as a response to oocyte activatin--can be experimentally separated.

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Zona pellucida modifications in the mouse in the absence of oocyte activation.

Mouse oocytes arrested in metaphase II exhibit zona hardening and a reduced fertilization rate after exposure to the cryoprotectant dimethylsulfoxide (Johnson J, In Vitro Fertil Embryo Transfer 6:168-175, 1989) but do not undergo parthenogenetic activation (Johnson and Pickering, Development 100:313-324, 1987). This paper shows that dimethylsulfoxide causes proteolytic modification of the zona pellucida glycoprotein ZP2 and inhibition of sperm binding. These effects of dimethylsulfoxide are caused by premature exocytosis of the cortical granules, a process that is initiated usually on fertilization. A model for the mechanism of action of dimethylsulfoxide is proposed based on the combined effects of cytoskeletal modification and osmotic shock. The presence of serum before and during the exposure to dimethylsulfoxide was found to reduce significantly these deleterious effects on the mouse zona pellucida without inhibiting the cortical granule release. These results highlight the suitability of dimethylsulfoxide as a tool to study the mechanisms leading to cortical granule release. Use of dimethylsulfoxide allows the separation of oocyte parthenogenetic activation from cortical granule release, and addition of serum allows separation of cortical granule release from the action of the cortical granule contents. Their use allows a dissection of the mechanisms underlying each of these three related events.

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Anucleate fragments of parthenogenetic eggs and of maturing oocytes contain complementary factors required for development of a male pronucleus.

Eight-h-old anucleate fragments of artificially activated mouse oocytes produced within 30 min after activation were fused with anucleate fragments of ovarian oocytes bisected at the GV stage and matured in vitro for 4-5 h. The resulting cybrids were inseminated soon after fusion and fixed 3-4 h or 14 h later. After 3-4 h of culture most of the sperm nuclei underwent decondensation. Early male pronuclei were observed sporadically. After overnight culture small or fully formed male pronuclei were present in monospermic and dispermic cybrids. The abortive nuclei, designated as stage IIa (earlier described by Borsuk and Tarkowski, 1989) were also observed and their occurrence was strongly correlated with the rate of polyspermy. In unfused (control) anucleate fragments of maturing and activated oocytes inseminated at the same time as the cybrids, male pronuclei were never observed. These results show that the cytoplasms of activated eggs and of maturing oocytes contain complementary factors required for the transformation of the sperm nucleus into the male pronucleus. The former has lost the capability to break down the sperm nucleus envelope but contains factors required for pronuclear growth. The latter is able only to initiate the process of transformation, i.e., to break down the nuclear envelope and decondense the denuded sperm chromatin. However, the activity of the factor responsible for nuclear envelope breakdown is limited because in cybrids penetrated by more than two spermatozoa, transformation of sperm nuclei was usually abnormal.

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Vitrification of mouse oocytes: improved rates of survival, fertilization, and development to blastocysts.

Rall and Fahy's (1985) vitrification procedure for the cryopreservation of 8-cell embryos was applied to unfertilized mouse oocytes. Unchanged, this method resulted in a mean of 24.1% of vitrified oocytes fertilizing and developing to blastocysts in vitro. Exposure of oocytes to the cryoprotectant media, but without the vitrification, resulted in 30.8% developing to blastocysts. Modifications to the durations of and media used in the dilution and equilibration steps of the procedure produced a final protocol giving a mean of 55.4% of vitrified oocytes and 72.4% of nonvitrified VS1-exposed oocytes developing to blastocysts; 85.7% of control oocytes develop to blastocysts. Osmotically induced damage was found to be the most important cause of loss of viability in these methods. Cooling of oocytes to 5-8 degrees C during the procedure had no significant effect on their viability. No parthenogenetic activation of oocytes occurred as a result of exposure to the procedure.

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Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes.

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.

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Early development and X-chromosome inactivation in mouse parthenogenetic embryos.

Early development and X-chromosome inactivation were studied in ethanol-induced mouse parthenogenones. About 24% of oocytes transferred to 0.5-day pseudopregnant recipients successfully implanted. However, only 49%, 20%, and 16% of implanted parthenogenones survived 5, 6, and 7 days later, respectively. Abnormal development was evident in every parthenogenone as early as 5 days after activation with the degenerating polar trophectoderm. These embryos were destined to become either small disorganized embryos or embryonic ectoderm vesicles bounded by the visceral endoderm. Only 2 of 51 representative 6- to 8-day parthenogenones sectioned had morphology of the normal egg cylinder, although growth retardation was evident. Spontaneous LT/Sv parthenogenones shared similar morphological features. In late blastocysts, the frequency of cells with an apparently inactivated X chromosome was lower in parthenogenones than in fertilized embryos. The failure of X-inactivation in the trophectoderm seems to contribute to the defective development of parthenogenones.

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Cleavage rate of haploid and diploid parthenogenetic mouse embryos during the preimplantation period.

The lack of a paternal genome in parthenogenetic embryos clearly limits their postimplantation development, but apparently not their preimplantation development, since morphologically normal blastocysts can be formed. The cleavage rate of these embryos during the preimplantation period gives a better indication of the influence of their genetic constitution than blastocyst formation. Conflicting results from previous studies prompted us to use a more suitable method of following the development of haploid and diploid parthenogenetic embryos during this period. Two classes of parthenogenetic embryos were analysed following the activation of oocytes in vitro with 7% ethanol: 1) single pronuclear (haploid) embryos and 2) two pronuclear (diploid) embryos. Each group was then transferred separately during the afternoon to the oviducts of recipients on the 1st day of pseudopregnancy. Control (diploid) 1-cell fertilised embryos were isolated in the morning of finding a vaginal plug, and transferred to pseudopregnant recipients at approximately the same time of the day as the parthenogenones. Embryos were isolated at various times after the HCG injection to induce ovulation, from each of the three groups studied. Total cell counts were made of each embryo, and the log mean values were plotted against time. The gradient of the lines indicated that 1) the cell doubling time of the diploid parthenogenones was 12.25 +/- 0.34 h, and was not significantly different from the value obtained for the control group (12.74 +/- 1.17 h), and that 2) the cell doubling time of the haploid parthenogenones (15.25 +/- 0.99 h) was slower than that of the diploid parthenogenones and the control diploid group.(ABSTRACT TRUNCATED AT 250 WORDS)

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The kinetics of oocyte activation and polar body formation in bovine embryo clones.

The kinetics of polar body formation were examined in parthenogenetically activated, in vitro matured and aged bovine oocytes. Subsequently, the presence or absence of polar body formation was determined in bovine embryo clones. Polar body formation, defined as telophase II, occurred by 1 (13/40, 43%) and 2 h (15/21, 71%) postparthenogenetic activation of metaphase II stage oocytes. Parthenogenetically activated oocytes readily formed pronuclei by 4 h. Some oocytes had chromatin in a highly condensed state at 1, 2, and 4 h postactivation (13/72, 18%). These oocytes often (10/13, 77%) appeared to be "self-enucleated," as the condensed chromatin was found in a membrane-bound extrusion. The phenomenon was most prevalent when oocytes were handled at room temperature (25-27 degrees C). Nuclear transfer procedures were established to bring about synchronous blastomere fusion and oocyte activation conditions. Synchronous conditions were achieved only when oocytes were handled and manipulated at 37-39 degrees C. Embryo clones examined 2 h postfusion did not form a polar body. Conversely, nucleate demi-oocyte controls were at the late telophase II stage of meiosis. The results are discussed in relation to cell cycle effects on bovine nuclear transfer.

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Behavior of sperm nuclei incorporated into parthenogenetic mouse eggs prior to the first cleavage division.

When artificially activated mouse eggs are inseminated in the middle of the first cell cycle, sperm nuclei remain condensed until the first mitosis. During mitosis of the first cleavage division sperm nuclei decondense, subsequently recondense and are passively displaced to the daughter blastomeres. In the 2-cell embryos sperm nuclei form interphase nuclei which are able to replicate DNA and to condense into discrete chromosomes during the following mitotic division. These observations suggest that the mitotic cytoplasm of 1-cell embryos creates similar conditions for the transformation of sperm nuclei into male pronuclei as the cytoplasm of metaphase II oocytes.

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Genome exclusion and gametic DAPI-DNA content in the hybridogenetic Bacillus rossius-grandii benazzii complex (Insecta Phasmatodea).

Among Sicilian stick insects, two hybridogenetic complexes have been discovered: Bacillus rossius-grandii benazzii and B. rossius-grandii grandii, which also produce androgenetic offspring. The egg maturation of the former is analyzed here through DAPI fluorometry, which, besides the assessment of the meiotic stages, also allows their DNA measurements and the analysis of sperm-head evolution into male pronuclei in these polyspermic eggs. Hybridogenetic eggs undergo an extrasynthesis of chromosomes, because two groups of n autobivalents (4C each) are segregated at metaphase 1st; the two groups must correspond to the pure parental species haplosets. Then the grandii chromosomes degenerate (1st polar body), while the rossius chromosomes divide further to produce two groups of n autodiads (2C each); one of them degenerates (2nd polar body), and the other is ready to perform syngamy (female pronucleus). Meanwhile, several B. grandii sperm evolve into male pronuclei by doubling their DNA (from 1C to 2C content) and assuming an interphase nucleus appearance. If regular mixis occurs, the F1 hybrid constitution is restored but, if it fails, a fusion between two sperms may occur, originating fully paternal descendants (natural androgenesis). The genome exclusion mechanism of stick-insect hybridogens appears to be more primitive than those observed in the already known hybridogenetic complexes of Poeciliopsis and Rana esculenta. Unfertilized eggs of hybridogens are capable of self-activation, but the cytology of the related clonally reproducing B. whitei indicates that its parthenogenetic mechanism stems from the hybridization event (hybrid theory) rather than from tychoparthenogenetic potentialities (spontaneous theory).

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Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin B after in vitro maturation.

The present study was conducted to investigate the effects of different culture durations (24-36 hr) on bovine oocyte maturation in vitro and the effect of the presence or absence of cumulus cells at the time of treatment to induce parthenogenetic activation (exposure to ethanol and cytochalasin B; CB) (experiment I). The effects of dosage (2.5 or 5.0 micrograms/ml) and incubation time (2.5, 5, or 10 hr) in CB (experiment II) on the subsequent development to the blastocyst stage in vitro was also investigated. In experiment I, cleavage and development to the blastocyst stage were not affected by the presence or absence of cumulus cells at the time of parthenogenetic activation. However, the 24-hr culture duration for in vitro maturation had a significantly lower rate of development to the blastocyst stage than the longer culture durations (27-36 hr). In experiment II, treatment with 5 micrograms/ml CB for 5 hr showed the highest percentage of development to blastocyst in the oocytes matured for both 27 and 30 hr. To determine the viability of the parthenogenetic embryos (morulae and blastocysts), four recipient heifers received two embryos each, and one heifer was found to be pregnant on day 35 following transfer. Although fetal heartbeat was not observed, the subsequent estrus was prolonged in all heifers. The present results demonstrate development of in vitro-matured, parthenogenetically activated bovine embryos up to the preimplantation stage.

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Chromatin and microtubule organization in the first cell cycle in rabbit parthenotes and nuclear transplant embryos.

Artificial activation and nuclear transfer in rabbit oocytes have been used in past years in an attempt to develop viable techniques for cloning in cattle. The procedures established in our laboratory, using the rabbit as a model, consistently lead to high rates of development to the blastocyst stage. However, the rate of embryos developing to term is considerably lower. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of electrical pulse-activated oocytes and of nuclear transfer embryos. Our goal was to investigate the responses of the cell to the different stimuli applied and to establish the sequence of events leading to first cleavage in the absence of normal fertilization. Our results show that, in both electrically activated oocytes and nuclear transfer embryos, although the initial development patterns are rather unusual, embryos become synchronized at the time of the formation of a pronuclear-like structure, and then organize metaphase spindles and cleave. These spindles consistently present small defects, suggesting that problems in the formation of the mitotic apparatus during the first cell cycle may have a long-term effect leading to embryo mortality.

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