Pronucleus formation, DNA synthesis and metaphase entry in porcine oocytes following intracytoplasmic injection of murine spermatozoa

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilisation. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8-9 h following the injection of porcine sperm, and 6-8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte centre. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. Ultrastructural observation revealed that male pronuclei derived from murine sperm in porcine oocytes are morphologically similar to normal male pronuclei in porcine zygotes. These results suggest that species-specific paternal factors influence the onset of pronucleus formation and DNA synthesis. However, normal nuclear cytoplasmic interactions were observed in porcine S-phase oocytes following murine sperm injection.     

Investigation of the effect of various buffer solutions used for microinjection of gene-engineering constructs on preimplantation development of mouse embryos

We studied the effects of some buffer solutions used for microinjection in mammalian zygotes on preimplantation development of (CBA x C57BL)F1 mouse embryos in vitro. The rate of embryo survival was estimated according to their capacity to develop to the stages of blastocyst and blastocyst hatched from zona pellucida. The results obtained suggested a reduced rate of survival of zygotes to the blastocyst stage after the injection into a pronucleus of the buffers Tris-HCl with EDTA, Tris-HCl with MgCl2 and NaCl, and medium M2 (p < 0.05) and to the stage of blastocyst hatched from zone pellucida after injection of a Dulbecco solution, as compared to the control. No differences were found in the survival rate of zygotes injected with different buffer solutions.     

The Drosophila maternal gene sésame is required for sperm chromatin remodeling at fertilization

The spermatozoon features an extremely condensed and inactive nucleus. The unique sperm chromatin organization is acquired during the late stages of spermatid differentiation by the replacement of somatic histones with sperm-specific chromosomal proteins. At fertilization, the inactive sperm nucleus must be rapidly transformed into a DNA replication competent male pronucleus before the formation of the zygote. The sequential events of this crucial process are well conserved among animals and are controlled by molecules present in the egg. We have previously identified a Drosophila maternal effect mutation called sésame, which specifically arrests male pronucleus formation at a late stage of chromatin decondensation. In this study, we show that sésame affects maternal histone incorporation in the male pronucleus, a situation that is expected to prevent nucleosomal organization of the paternal chromatin. As an apparent consequence, the male pronucleus is arrested before the first S-phase and does not condense mitotic chromosomes. However, centromeric heterochromatin is present on paternal centromeres, which occasionally interact with microtubules. The abnormal chromatin organization of the male pronucleus does not prevent the formation of a male pronuclear envelope, which breaks down and reassembles in synchrony with maternally derived nuclei present in the same cytoplasm.     

The effect of the micromanipulations used for transgenosis on mouse development]

Non-specific effects of micromanipulation techniques used for producing transgenic mice on processes of embryonic development were studied. Zygotes obtained from C57BL and BALBxDD mice were treated as follows: (1) incubated in culture medium; (2) the male pronucleus punctured with a glass microneedle; (3) microinjected with a buffer solution; and (4) DNA (mouse P-35 oncogene with human insulin gene promoter) injected into the male pronucleus. Then zygotes were transferred into oviducts of syngeneic or allogeneic pseudopregnant females. Such treatment resulted in the intrauterine death of embryos, as well as in birth of the dead or non-viable offspring with numerous defects of development. Zygote pronucleus puncturing is the most damaging manipulation, since its effect exceeds that of the zygote incubation and is comparable with the effect of buffer of DNA injections.     

A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes

Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro with in-vitro capacitated spermatozoa. Analysis of 621 penetrated ova fixed at various times after in-vitro insemination led to definition of 6 stages of early development. A time sequence for sperm penetration, sperm head decondensation, male pronucleus formation, the activation of second meiotic division, female chromosome decondensation and pronucleus development was established. First sperm penetration into the ooplasm was recorded 6 h after insemination; 1-2 h was required for the sperm head to decondense and another 4-6 h to develop into the opposing pronucleus stage. Synkaryosis and first cleavage occurred 28 h after fertilization. Examination of the early stages revealed four types of abnormalities, i.e. polyspermy, polygyny, asynchrony between male and female pronucleus development, and preactivation of cytokinesis.     

In vitro development of murine embryos using different types of microinjections

We studied the effects of different types of microinjections, such as the mechanical damage to cytoplasmic and nuclear membranes of the zygote and the injection of various gene-engineering constructs or buffer solutions into the cytoplasm or the pronucleus, on the preimplantation of murine embryos (CBA x x C57BL)F1. The survival rate of the embryos was estimated by their capacity to develop in vitro to the blastocyst or hatched blastocyst stages. Puncture of the cytoplasm using a microneedle and injection of buff or foreign DNA did not affect the zygotes capacity for further in vitro development. But, the puncture of the pronucleus and microinjection of gene-engineering constructs or buffer into it reliably decreased the survival rate of embryos, as compared to the control. The differences were found in the capacity of murine zygotes for in vitro development after injection with gene-engineering constructs.     

Investigation of the Effect of Various Buffer Solutions Used for Microinjection of Gene-Engineering Constructs on Preimplantation Development of Mouse Embryos

We studied the effects of some buffer solutions used for microinjection in mammalian zygotes on preimplantation development of (CBA × C57BL) F₁ mouse embryos in vitro. The rate of embryo survival was estimated according to their capacity to develop to the stages of blastocyst and blastocyst hatched from zona pellucida. The results obtained suggested a reduced rate of survival of zygotes to the blastocyst stage after the injection into a pronucleus of the buffers Tris-HCl with EDTA, Tris-HCl with MgCl₂ and NaCl, and medium M2 (p < 0.05) and to the stage of blastocyst hatched from zone pellucida after injection of a Dulbecco solution, as compared to the control. No differences were found in the survival rate of zygotes injected with different buffer solutions.     

In vitro Development of Murine Embryos Using Different Types of Microinjections

We studied the effects of different types of microinjections, such as the mechanical damage to cytoplasmic and nuclear membranes of the zygote and the injection of various gene-engineering constructs or buffer solutions into the cytoplasm or the pronucleus, on the preimplantation of murine embryos (CBA × C57BL)F₁. The survival rate of the embryos was estimated by their capacity to develop in vitro to the blastocyst or hatched blastocyst stages. Puncture of the cytoplasm using a microneedle and injection of buff or foreign DNA did not affect the zygotes capacity for further in vitro development. But, the puncture of the pronucleus and microinjection of gene-engineering constructs or buffer into it reliably decreased the survival rate of embryos, as compared to the control. The differences were found in the capacity of murine zygotes for in vitro development after injection with gene-engineering constructs.     

Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii

The gynogenetic fish, Carassius auratus langsdorfii (the ginbuna, a crucian carp), provides an interesting model for the study of the mechanisms controlling male pronucleus formation. When the sperm nucleus of a different subspecies (C. a. cuvieri) is incorporated into the gynogenetic egg, the nuclear envelope of the spermatozoon is not broken down, and the pronucleus fails to develop, although dispersion of the sperm chromatin occurs to some extent within the space limited by the nuclear envelope. When spermatozoa without plasma membranes and nuclear envelopes were microinjected into mature activated eggs, the sperm nuclei underwent chromatin dispersion, nuclear envelope formation, DNA synthesis, and transformation into male pronuclei. These results indicate that the failure of the male pronucleus to form in ginbuna is primarily due to the failure of sperm nuclear envelope breakdown. We conclude that sperm nuclear envelope breakdown is an indispensable step for the development of the male pronucleus.     

Nuclear envelope breakdown is under nuclear not cytoplasmic control in sea urchin zygotes

Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.     

Sperm penetration in vitro of pig follicular oocytes matured in culture.

Boar ejaculated and epididymal spermatozoa were preincubated in modified KRB or the isolated oviduct and uterine horn of an oestrous sow for 4.5-5 h at 37 degrees C before introduction into medium containing ovarian oocytes previously cultured for 24 h. At examination 17-20 h after insemination 60.6% of the total oocytes had reached at least the 2nd metaphase. The proportions of oocytes penetrated (i.e. enlarged sperm head or male pronucleus and corresponding sperm tail) were 0, 10.0 and 16.7% with ejaculated spermatozoa, and 3.3, 19.6 and 26.4% with epididymal spermatozoa preincubated in modified KRB, oviduct and uterus, respectively. Although the proportion of oocytes with morphologically normal male and female pronuclei was low (10/36 = 27.8%), the results suggest that boar spermatozoa can be capacitated in the isolated genital tract of an oestrous sow and that capacitation of epididymal is better than that of ejaculated spermatozoa.     

Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos

Cloning by somatic cell nuclear transfer is critically dependent upon early events that occur immediately after nuclear transfer, and possibly additional events that occur in the cleaving embryo. Embryo culture conditions have not been optimized for cloned embryos, and the effects of culture conditions on these early events and the successful initiation of clonal development have not been examined. To evaluate the possible effect of culture conditions on early cloned embryo development, we have compared a number of different culture media, either singly or in sequential combinations, for their ability to support preimplantation development of clones produced using cumulus cell nuclei. We find that glucose is beneficial during the 1-cell stage when CZB medium is employed. We also find that potassium simplex optimized medium (KSOM), which is optimized to support efficient early cleavage divisions in mouse embryos, does not support development during the 1-cell or 2-cell stages in the cloned embryos as well as other media. Glucose-supplemented CZB medium (CZB-G) supports initial development to the 2-cell stage very well, but does not support later cleavage stages as well as Whittten medium or KSOM. Culturing cloned embryos either entirely in Whitten medium or initially in Whittens medium and then changing to KSOM at the late 4-cell/early 8-cell stage produces consistent production of blastocysts at a greater frequency than using CZB-G medium alone. The combination of Whitten medium followed by KSOM resulted in an increased number of cells per blastocyst. Because normal embryos do not require glucose during the early cleavage stages and develop efficiently in all of the media employed, these results reveal unusual culture medium requirements that are indicative of altered physiology and metabolism in the cloned embryos. The relevance of this to understanding the kinetics and mechanisms of nuclear reprogramming and to the eventual improvement of the overall success in cloning is discussed.     

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.     

DNA synthesis in the fertilizing hamster sperm nucleus: sperm template availability and egg cytoplasmic control

To assess the role of the availability of sperm nuclear templates in the regulation of DNA synthesis, we correlated the morphological status of the fertilizing hamster sperm nucleus with its ability to synthesize DNA after in vivo and in vitro fertilization. Fertilized hamster eggs were incubated in 3H-thymidine for varying periods before autoradiography. None of the decondensed sperm nuclei nor early (Stage I) male pronuclei present after in vivo or in vitro fertilization showed incorporation of label, even in polyspermic eggs in which more advanced pronuclei were labeled. In contrast, medium-to-large pronuclei (mature Stage II pronuclei) consistently incorporated 3H-thymidine. To investigate the contribution of egg cytoplasmic factors to the regulation of DNA synthesis, we examined the timing of DNA synthesis by microinjected sperm nuclei in eggs in which sperm nuclear decondensation and male pronucleus formation were accelerated experimentally by manipulation of sperm nuclear disulfide bond content. Although sperm nuclei with few or no disulfide bonds decondense and form male pronuclei faster than nuclei rich in disulfide bonds, the onset of DNA synthesis was not advanced. We conclude the the fertilizing sperm nucleus does not become available to serve as a template for DNA synthesis until it has developed into a mature Stage II pronucleus, and that, as with decondensation and pronucleus formation, DNA synthesis also depends upon egg cytoplasmic factors.     

DNA methylation state is preserved in the sperm-derived pronucleus of the pig zygote

DNA methylation reprogramming (DMR) during preimplantation development erases differentiation-associated, unessential epigenetic information accumulated during gametogenesis, and ultimately brings pluripotency to the resulting embryo. Two patterns of DMR of sperm-derived pronucleus have been reported in mammals. In the first, the male pronucleus is actively demethylated whereas in the second, the methylation state seems to be maintained. The maintenance-type DMR has been seen only through immunocytochemical observations, and waits to be proven by additional molecular-level evidence. We demonstrate that, in pig, paternally derived DNA methylation is preserved during pronucleus development, based on the following observations. First, immunostaining of pig zygotes at different time points showed the DNA methylation state to be balanced between parental pronuclei throughout pronucleus development. Second, bisulfite analysis of PRE-1 repetitive sequences found mono- and polyspermic eggs to have similar methylation states. Third, the methylation state of a human erythropoietin gene delivered by transgenic pig spermatozoa was maintained in the male pronucleus. Finally, 5-aza-2'-deoxycytidine treatment, which blocks re-methylation, did not show the male pronucleus to be stalled in a demethylated state. In pig zygotes, paternally derived cytosine methylation was preserved throughout pronucleus development. These findings from multilateral DMR analyses provide further support to the view that DMR occurs in a non-conserved manner during early mammalian development.     

Fertilization of rat eggs in vitro at various times before and after ovulation with special reference to fertilization of ovarian oocytes matured in culture.

Oocytes recovered at various times from immature rats treated with PMSG and HCG were incubated with capacitated epididymal spermatozoa of mature rats. In the presence of follicular cells, sperm penetration was not observed 4 hr after incubation in the oocytes at stages from the intact germinal vesicle to the chromatin mass, but 7 to 55% of oocytes were penetrated at stages from the condensed germainal vesicle to metaphase II. After the removal of follicular cells, 15 to 72% of the oocytes at any stage were penetrated. After further incubation for 15 hr, the proportion of penetrated oocytes increased from 8 to 98% from early to late stages and that of penetrated oocytes with a male and female pronucleus increased from 9 to 100% as maturation progressed. Although the average number of spermatozoa/oocyte was not correlated with its maturation, transformation of the sperm head into a male pronucleus was retarded or failed, especially in the younger oocytes. Following incubation in a defined medium for 13 hr, 85% of oocytes at the intact germinal vesicle stage matured to the stage of the first polar body formation, but only 18 to 22% of these mature oocytes were penetrated by spermatozoa and only a few of the penetrated oocytes cleaved into normal two-cell eggs. When eggs recovered from oviducts 14 to 20 hr after ovulation were exposed to capacitated spermatozoa, the proportion of penetrated eggs (86 to 98%) and that of polyspermic eggs (11 to 27%) were not related to the ages of the eggs, but failure of transformation of the sperm head and the proportion of abnormal eggs increased 14 to 20 hr after ovulation.     

Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro.

The present study was carried out to examine the effect of maturation media on male pronucleus formation of pig oocyte matured and fertilized in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughter house were cultured (36 h) in three different media (mTCM-199, Waymouth MB 752/l, and mTLP-PVA), fertilized in vitro, and assessed for nuclear maturation and male pronucleus formation. The addition of 10% (v/v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (P less than 0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 752/l with or without pFF than in oocytes matured in the other two media (P less than 0.01). In experiment 2, the addition of cysteine (the same concentration as in Waymouth medium, 0.57 mM), to mTLP-PVA significantly increased the rate of male pronucleus formation of pig oocytes compared with the control (P less than 0.01). The results indicate that the composition of maturation medium affects the ability of pig oocytes to form male pronuclei following sperm penetration; media containing a high concentration of cysteine (possibly as a substrate of glutathione), such as Waymouth MB 752/l, can remarkably promote this ability.     

克服昆明小鼠体外受精卵发育阻滞方法的研究

本研究应用CZB和WM培养液进行昆明小鼠体外受精胚胎的发育培养,建立了一个可行的胚胎体外培养的新方法,并通过改变培养液的成分及其含量,对胚胎发育的阻滞机理和突破方法进行了初步的探索。培养于WM中的受精卵发生阻滞,有48％停留于2细胞阶段;而CZB中的胚胎有81％发育为桑椹胚和囊胚。在WM中添加EDTA和谷氨酰胺得到了66％的囊胚;加大WM中乳酸钠和丙酮酸钠的比值未能克服发育的阻滞现象。实验结果表明,EDTA和谷氨酰胺在克服阻滞时具有协同作用。     

人二倍体化合子的DNA 甲基化变化模式

目的:探讨人类三原核合子及二倍体化合子中DNA甲基化模式的变化情况。方法:我们采用显微操作技术去除三原核合子中两个雄原核中的一个,观察恢复了二倍体状态的胚胎的发育情况,并检测了三原核和二倍体化的合子及早期胚胎中DNA甲基化模式的动态变化。结果:二倍体化的合子的囊胚形成率与三原核合子的囊胚形成率无显著性差异;在人三原核合子中两个雄原核发生主动地DNA去甲基化而雌原核在受精后的20h后仍保持甲基化。三原核与二倍体化合子中,DNA甲基化模式没有差别。结论:去除一个雄原核不会影响合子和胚胎的DNA甲基化模式。去除多余雄原核并不能改善胚胎的发育。     

The formation of the pronuclei and their associated perinuclear cytoplasm; the determination of the phases of the first cleavage cycles in Crepidula fornicata (Mollusca,Gastropoda)

Summary

The behaviour of the male and the female pronuclei in Crepidula fornicata is studied, beginning at the formation of the second polar body. Shortly after the extrusion of the second polar body the female pronucleus is formed, and then the male pronucleus enters the yolk-free cytoplasm near the animal pole. Both pronuclei are enveloped by a typical nuclear membrane, and increase in size until the prophase; a zygote nucleus is not formed (“Ascaris type” of fertilization). In the meantime, the chromatin of both pronuclei is arranged in a meshwork in the centre of the pronuclei.

Shortly after the formation of the second polar body a special cytoplasm, the “perinuclear cytoplasm”, is formed in the vicinity of each of the pronuclei. During the early stages of the first cleavage cycle this cytoplasm is composed of numerous Golgi complexes, small dense Golgi vesicles, smooth endoplasmic reticulum vesicles, mitochondria and rosettes of glycogen-like granules. At later stages, when the pronuclei have met and their plasms coalesced, the number of Golgi elements decreases; at the same time, the small dense Golgi vesicles increase in number and aggregate in clusters.

The phases of the first three cleavage cycles are determined by cytophotometry. The nuclear DNA of the male pronucleus and that in the nuclei of the blastomeres of the 2- and the 4-cell stage is reduplicated between 7 and 33% of the normalized cleavage cycles; the G₂-phase is between 33 and 57%, while the mitotic phase occupies the last part of each cleavage cycle and the first 7% of the next cleavage cycle. There is no G j-phase. Since the female pronucleus lies just beneath the polar bodies, its DNA content could not be measured separately.