Immunocytochemical localization of cyclin/proliferating cell nuclear antigen (PCNA) in fertilized eggs of mice.

Immunolocalization of cyclin/PCNA (proliferating cell nuclear antigen) was performed with monoclonal antibody using immunogold methods on ultrathin cryosections of fertilized mouse eggs. Immunolabeling in pronuclei was checked 20, 22, 24 and 26 h after HCG injection. A relation between onset of pronuclei migration (early S-phase) and appearance of colloidal particle clusters was found. Afterwards, (mid S-phase) the increase of labelling and the localization of cyclin/PCNA were found throughout the pronuclei, except in the nucleolar bodies. Lower labelling appeared at the time of close reciprocal pronuclei contact (late S-phase). It is concluded that bulk and distribution of cyclin/PCNA in pronuclei is closely related to the progression of first interphase after fertilization.     

The first cycle of DNA replication in parthenogenetic mouse embryos

Dynamics of the first cell cycle in parthenogenetic mouse embryos derived from ethanol-activated eggs was studied using 3H-thymidine. DNA synthesis starts within 5 h and is terminated within 10 h after activation: it lasts ca. 6 h. Changes in the intensity of 3H-thymidine incorporation and in the distribution of radioactive label between haploid and diploid parthenogens were observed. 3H-thymidine was shown to incorporate into pronucleoli of diploid parthenogens and late-labeled heterochromatin blocks were bound in both diploid and haploid pronuclei. The structure of the first cell cycle in parthenogenetic and normal embryos is discussed.     

Analysis of the first cell cycle in the cross between hamster eggs and human sperm

Golden hamster eggs fused with human sperm were pulsed with bromodeoxyuridine to determine the timing of S-phase and the length of the first ceil cycie in this hybrid cross. Fused eggs were fixed and pronuclei scored for incorporation of the thymidine analogue detected by indirect immunofiuorescence. Although S-phase started synchronously 3–3.5 hr after coincuba-tion of sperm and eggs, its duration was variable such that two-cell stages appeared at 16 hr while a proportion of pronuclei was still engaged in DNA synthesis. Unlike rodent sperm chromatin, human sperrn chromatin was able to participate in DNA synthesis well before its maturation into a fully developed pronucleus. Human sperm chromatin appears able to function under conditions different in several respects from those in human eggs.     

Effect of near ultraviolet and visible light on amoeba.

The near ultraviolet and visible light (VL) impinging at an intensity of 2-5 x 10(2) J s-1 m-2 for 2-5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.     

Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation

The exposure of mouse zygotes pre-stained with Hoechst 33342 to u.v. irradiation for 20-30 sec significantly or completely inhibited development to blastocysts in vitro. However, development to the blastocyst stage of enucleated eggs receiving pronuclei from untreated eggs was as good as that of control reconstituted eggs when the cytoplasm originated from eggs exposed to u.v. irradiation for 20-30 sec, but was significantly lower when the cytoplasm was from eggs exposed for 40 sec. The chromosomes at the second metaphase stage could be removed with 15 sec of exposure to u.v. irradiation under a fluorescence microscope. Most eggs enucleated at the second metaphase that received a single inner cell mass nucleus (75%) showed pronuclear formation 6 h after activation; 23% of them developed to morphologically normal 2-cell eggs and 5% developed to blastocysts. These results demonstrate that the cytoplasm of mouse zygotes is more resistant to u.v. irradiation after Hoechst staining. Eggs at the second metaphase, from which chromosomes have been removed under a fluorescence microscope, can therefore be used as cytoplasm recipients for nuclear transplantation of inner cell mass nuclei.     

Early events of in-vitro fertilization of cat eggs by epididymal spermatozoa

Newly ovulated eggs from mature queens treated with PMSG and hCG were inseminated in modified KRB solution with spermatozoa recovered from the cauda epididymidis of male cats. When 5 eggs were examined 15 min after insemination, no signs of sperm penetration into the vitellus were observed. However, in an egg examined before fixation 20 min after insemination, a spermatozoon whose head had passed through the zona pellucida was observed. Very high proportions (90-100%) of the eggs were penetrated when they were examined 0.5-5 h after insemination. Male and female pronuclei were first observed in eggs examined 4 h after insemination.     

雌核发育和两性融合发育鱼卵调控精核受精发育的生化特性研究

两性融合生殖的鱼卵受精后,精核能疏松、解凝,形成雄性原核：雌核发育银鲫卵子受精后,精核发育受到抑制,无法形成原核。采用显微注射去膜精核以及细胞学和电镜观察的方法,本文对两类鱼卵受精后精核早期发育的生化性质进行了初步探讨,并着重研究了雌核发育银鲫卵子控制精核发育的生化特征。实验结果显示,两性融合生殖鱼类卵质中,一定量的Ｃａ２＋的存在,二硫键的还原作用对于精核的发育显然是必要的;而在雌核发育银鲫卵中,Ｃａ２＋的功能和二硫键的还原作用与精核发育受到抑制之间并无直接联系。银鲫卵质中似乎显示出异常的磷酸酶脂解活性,导致磷酸化过程无法进行,使精核解凝受到阻碍。另外,两性融合生殖的鱼卵重质层中具有大量诱导精核原核化的有关因子,而银鲫卵质中则缺少该因子（或活性极低）。银鲫卵质中还可能缺乏某些与雄性原核的核膜重组装有关的大分子物质。     

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.     

Fertilization Reaction without Changes in Intracellular Ca2+ in Medaka Eggs—An Experiment with Acetone-Treated Eggs

To investigate whether or not causal relationship exists between the increase in intracellular Ca²⁺ and other cortical reactions at fertilization in the medaka, Oryzias latipes , intracellular Ca²⁺ was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca²⁺. Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e. completion of meiosis, fusion of pronuclei, and accumulation of cortical cytoplasm with intact cortical alveoli in the animal pole region were observed in normal time sequence in these eggs. When denuded eggs were treated with acetone, contraction of the egg and slow resumption of meiosis (extrusion of polar body) were observed without insemination. When denuded eggs were inseminated immediately after acetone-treatment, the number of spermatozoa that penetrated into the egg was greater in the animal hemisphere than in the vegetal hemisphere. These results may indicate that acetone inactivates the egg plasma membrane or its adjacent cortical cytoplasm so that it cannot participate in a propagative increase in intracellular Ca²⁺ and exocytosis, while it also induces cytoplasmic activation leading to egg contraction, resumption of meiosis and formation of pronuclei. The present results suggest that sperm penetration, resumption of meiosis and ooplasmic segregation are regulated separately from the release of intracellular Ca²⁺ and exocytosis.     

First cleavage of enucleated rat eggs following transplantation of karyoplast removed from pronuclear eggs stored at 2 to 6 degrees C for various durations

Pronuclear rat eggs were cultured for 24 to 48 hours at 37 degrees C after storage at 2 to 6 degrees C for 0 to 216 hours in medium. Very high proportions (89 to 97%) of eggs cleaved to the two-cell stage after storage for 0 to 48 hours. The proportion, however, decreased rapidly in eggs stored for 72 hours (60%), and eggs stored for more than 120 hours cleaved poorly. However, when male and female pronuclei from stored eggs were transplanted into enucleated fresh eggs, 92 to 100% of the fused eggs with karyoplast stored for 0 to 144 hours cleaved. Although the cleavage rate was reduced to 50% when karyoplast from eggs stored for 168 hours was transplanted, this reduction was not significant. Complete loss of cleaving ability was observed in fused eggs with the karyoplast stored for 216 hours. These results clearly indicate that the pronuclei of rat eggs can be stored for a longer period than the cytoplasm at low temperatures (2 to 6 degrees C).     

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.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

The early embryonic mitosis in normal and cooled eggs of the silkworm,Bombyx mori

Early embryonic mitosis of the silkworm, Bombyx mori, was morphologically studied in the normal eggs and in the eggs treated by low temperature (?10°C). The first embryonic mitosis is observed in the eggs at 120 to 150 minutes after deposition at 26°C. After egg and sperm pronuclei unite, a spindle is formed in each of the pronuclei independently. At metaphase and anaphase paternal and maternal chromosomes are in separate groups on a spindle (gonomeric) and karyogamy takes place at telophase when they reach the poles. The second embryonic mitosis is shown in the eggs at 180 to 210 minutes after deposition. The division of two nuclei is not synchronous in the silkworm, and the mitosis is not gonomeric. In the eggs treated by low temperature, spindle fibers are not observed at all at ?10°C, and chromosomes, which form two deeply stained masses of irregular shape, are seen in the less stained area of spindle shape. When the eggs are returned to 26°C, some eggs go into normal gonomeric division, while some form two small and compact spindles, which seem to be derived from each of the pronuclei. It was observed that these compact spindles are able to continue mitosis.     

Pronuclear synthesis of DNA in fertilized and parthenogenetically activated mouse eggs : A cytophotometric study

Mouse one-cell embryos were taken 1, 1.5, 2, 3, 4, 6, 8, 10, 13 and 18 h after insemination. One-cell parthenogenones were induced by treatment of mouse eggs obtained 20 h after HCG injection with hyaluronidase and cultured for 0.5, 1, 3, 4.5, 6, 8, 10, 12 and 24 h. Some parthenogenones were pulse-labelled with tritiated thymidine, cut and autoradiographed. Both the embryos and parthenogenones were Feulgen-stained, and integrated relative optical absorption of either pronuclei or nuclei of polar bodies was measured with a cytophotometer. In some fertilized eggs and parthenogenones the DNA synthesis sets in 4–6 h after either insemination or parthenogenetic stimulus. Between the 8th and 13th hour after insemination the fraction of DNA synthesizing embryonic pronuclei remained at the level 30–40%. Most parthenogenones duplicated their DNA content between the 8th and 12th hour after hyaluronidase treatment. The DNA synthesis time in pronuclei of embryos was determined to be 3.5–4.0 h and that of pronuclei of parthenogenones approx. 4 h. The minimal time of the G2 phase was estimated to be 3–5 h. The first labelled pronuclei of parthenogenones were detected 6 h after stimulus. Male pronuclei started and ended DNA synthesis earlier than female pronuclei. Differences in the DNA content between pronuclei of the parthenogenones (when there are two in one parthenogenone) were observed beginning with the 10th h after hyaluronidase treatment.The DNA content in the nuclei of the second polar bodies (PB) of embryos increased slowly between the 8th and 22nd hour after insemination, up to an overall value of 1.4 C. That of the nuclei of the polar bodies of parthenogenones accompanied the synthesis of DNA in pronuclei to the 10th hour after hyaluronidase treatment, up to an overall value of 1.4 C.     

Asynchronous DNA replication and origin licensing in the mouse one‐cell embryo

To prevent duplicate DNA synthesis, metazoan replication origins are licensed during G1. Only licensed origins can initiate replication, and the cytoplasm interacts with the nucleus to inhibit new licensing during S phase. DNA replication in the mammalian one‐cell embryo is unique because it occurs in two separate pronuclei within the same cytoplasm. Here, we first tested how long after activation the oocyte can continue to support licensing. Because sperm chromatin is licensed de novo after fertilization, the timing of sperm injection can be used to assay licensing initiation. To experimentally skip some of the steps of sperm decondensation, we injected mouse sperm halos into parthenogenetically activated oocytes. We found that de novo licensing was possible for up to 3 h after oocyte activation, and as early as 4 h before DNA replication began. We also found that the oocyte cytoplasm could support asynchronous initiation of DNA synthesis in the two pronuclei with a difference of at least 2 h. We next tested how tightly the oocyte cytoplasm regulates DNA synthesis by transferring paternal pronuclei from zygotes generated by intracytoplasmic sperm injection (ICSI) into parthenogenetically activated oocytes. The pronuclei from G1 phase zygotes transferred into S phase ooplasm were not induced to prematurely replicate and paternal pronuclei from S phase zygotes transferred into G phase ooplasm continued replication. These data suggest that the one‐cell embryo can be an important model for understanding the regulation of DNA synthesis. J. Cell. Biochem. 107: 214–223, 2009. © 2009 Wiley‐Liss, Inc.     

Abortive second meiosis detected in cytochalasin-treated eggs in androgenetic diploid Corbicula fluminea

The hermaphroditic diploid clam Corbicula fluminea reproduces by androgenesis. In the control (androgenetic development), all maternal chromosomes and maternal centrosomes at the meiotic poles were extruded as two first polar bodies and subsequently second meiosis did not occur. In eggs treated with cytochalasin D (CD) to inhibit the polar body extrusion, the second meiosis was abortive. After the first meiosis, two centrosomes at the spindle poles remained in the cytoplasm because of the effect of CD. The chromosomes divided into two groups at anaphase-I as observed in the control eggs. Two centrosomes divided into four just after the first meiosis but did not separate completely. The microtubules from the centrosomes were rather short. So at the second meiosis, two monoasters or tetrapolar spindles were formed. The fluorescence signal from microtubules of the monoaster or tetrapolar spindle was weak compared with the spindle at the first meiosis. The maternal chromosomes on the monoaster or tetrapolar spindle did not move, and became large female pronuclei. The pronuclei became the metaphase chromosomes on the spindle for the first cleavage. The present study suggests that second meiosis regulating factors may be abortive in androgenetic diploid C. fluminea.     

DNA synthesis and distribution in parthenogenetic bovine embryos

Parthenogenetically activated, in vitro-matured bovine oocytes and parthenogenotes obtained at 2 to 4 days post activation were analyzed by 3H-thymidine autoradiography for the timing of the S-phase and for distribution of newly replicated DNA, respectively. Spread pronuclear parthenogenotes revealed that the DNA synthesis in electrically stimulated oocytes commenced at 14 h post activation. At 20 to 24 h, a maximum number of labeled pronuclei was reached (25 to 38%), and DNA synthesis persisted in some parthenogenotes up to 30 h post activation. The DNA labeling detected on semi-thin sections showed that the distribution of newly synthesized DNA in the nuclei of 3- to 16-cell parthenogenotes was mostly irregular or abnormal, documenting that the apparent morphological normalcy of parthenogenotes was in contrast to the data concerning the DNA synthesis and distribution.     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).