Plasticity of the inner cell mass in mouse blastocyst is restricted by the activity of FGF/MAPK pathway.

In order to ensure successful development, cells of the early mammalian embryo must differentiate to either trophectoderm (TE) or inner cell mass (ICM), followed by epiblast (EPI) or primitive endoderm (PE) specification within the ICM. Here, we deciphered the mechanism that assures the correct order of these sequential cell fate decisions. We revealed that TE-deprived ICMs derived from 32-cell blastocysts are still able to reconstruct TE during in vitro culture, confirming totipotency of ICM cells at this stage. ICMs isolated from more advanced blastocysts no longer retain totipotency, failing to form TE and generating PE on their surface. We demonstrated that the transition from full potency to lineage priming is prevented by inhibition of the FGF/MAPK signalling pathway. Moreover, we found that after this first restriction step, ICM cells still retain fate flexibility, manifested by ability to convert their fate into an alternative lineage (PE towards EPI and vice versa), until peri-implantation stage.

Curcumin disrupts meiotic and mitotic divisions via spindle impairment and inhibition of CDK1 activity.

OBJECTIVES: Curcumin, a natural compound, is a potent anti-cancer agent, which inhibits cell division and/or induces cell death. It is believed that normal cells are less sensitive to curcumin than malignant cells; however, the mechanism(s) responsible for curcumin's effect on normal cells are poorly understood. The aim of this study was to verify the hypothesis that curcumin affects normal cell division by influencing microtubule stability, using mouse oocyte and early embryo model systems. MATERIALS AND METHODS: Maturating mouse oocytes and two-cell embryos were treated with different concentrations of curcumin (10-50 microm), and meiotic resumption and mitotic cleavage were analysed. Spindle and chromatin structure were visualized using confocal microscopy. In addition, acetylation and in vitro polymerization of tubulin, in the presence of curcumin, were investigated and the damage to double-stranded DNA was studied using gammaH2A.X. CDK1 activity was measured. RESULTS AND CONCLUSIONS: We have shown for the first time, that curcumin, in a dose-dependent manner, delays and partially inhibits meiotic resumption of oocytes and inhibits meiotic and mitotic divisions by causing disruption of spindle structure and does not induce DNA damage. Our analysis indicated that curcumin affects CDK1 kinase activity but does not directly affect microtubule polymerization and tubulin acetylation. As our study showed that curcumin impairs generative and somatic cell division, its future clinical use or of its derivatives with improved bioavailability after oral administration, should take into consideration the possibility of extensive side-effects on normal cells.

Delayed sperm incorporation into parthenogenetic mouse eggs: sperm nucleus transformation and development of resulting embryos.

In this study we examined the effect of experimentally induced asynchrony between male and female pronuclei on male pronucleus formation and developmental potential of the resulting mouse embryos. We demonstrate that when the interval between oocyte activation and sperm incorporation is up to 1.5-2 hr, the spermatozoa transform into normal pronuclei. These male pronuclei can replicate their chromosomes during the first embryonic cell cycle and are transcriptionally competent. During the first cleavage these "delayed" male pronuclei condense into discrete mitotic chromosomes and when resulting embryos are transplanted into oviducts of pregnant females at least some of them can develop to term. In contrast, when sperm nuclei are introduced into parthenogenetic eggs 3 hr or more after activation, their transformation into pronuclei is significantly impaired, and they neither replicate nor transcribe. During the first mitosis they form a group of condensed chromatin, which is displaced into one of the resulting blastomeres leading to formation of haploid/diploid mosaic embryos. These mosaic embryos have poor developmental potential: only a few can reach blastocyst stage in vitro and no full-term development of such embryos was observed after transfer into pregnant females. We conclude that the cytoplasmic factors that make possible the transformation of a sperm nucleus into a functional male pronucleus exhaust within 1.5-2 hr after fertilization and that the male genome which had skipped the first cell cycle cannot become a functional partner in the embryonic genome.

Behavior of transgenic mouse spermatozoa with galline protamine.

General morphology, physical and chemical stability of nuclei, and postfertilization behavior of spermatozoa from transgenic mice [TgN (Prml Gal) 223 Bri] containing nuclear avian protamine (galline) were compared to those in the spermatozoa of wild-type (Wild) mice. Galline to protamine I ratios in spermatozoal nuclei of transgenic mice, strains 3175 (T75) and 3177 (T77), were 1.94 and 5.62, respectively. Live T75 and T77 spermatozoa were indistinguishable in their gross morphology from Wild spermatozoa. However, unlike Wild and T75 spermatozoa, T77 spermatozoa were vulnerable to mechanical handling, as about 40% of heads and tails were separated after gentle pipetting in suspension. Motility of T77 spermatozoa was markedly inferior to that of T75 and Wild. Chromatin heterogeneity and instability of transgenic spermatozoal nuclei were evident by transmission electron microscopy, staining reaction to Giemsa, and, as apparent by both light microscopy and flow cytometry, reaction to SDS detergent. Wild and T75 spermatozoa fertilized 90% and 60% of zona-intact oocytes in vitro, respectively. T77 spermatozoa completely failed to fertilize and bound to zona surfaces very weakly, and none of them inserted their heads into the zona. Although inefficiently, T77 spermatozoa could fertilize zona-free oocytes in vitro, indicating some ability to undergo capacitation and spontaneous acrosome reaction in vitro. After microsurgical injection into oocytes, the rate of nuclear decondensation was the greatest in rooster spermatozoa, followed by T77, T75, and Wild spermatozoa.

Mouse oocytes and parthenogenetic eggs lose the ability to be penetrated by spermatozoa after fusion with zygotes.

Fertilised mouse eggs develop the oolemma block to sperm penetration within 1 h. This block makes zona-free eggs at the pronuclear stage (zygotes) fully resistant to sperm penetration. In this study we investigated whether this block can spread--as a result of cell fusion--to the oolemma of eggs that are competent to be penetrated by spermatozoa. Preovulatory (GV) oocytes, ovulated oocytes in metaphase II (MII) and 1-cell parthenotes were fused with zygotes and the hybrid cells inseminated at various intervals after fusion. Sperm penetration was assessed on the basis of the presence of Giemsa-positive sperm heads in the air-dried preparations. The oolemma block to sperm penetration develops in all types of hybrids although at different speeds: it develops fast (2-3 h) in oolemma derived from MII oocytes and artificially activated eggs, and slowly in oolemma derived from GV oocytes. In the GV oocyte-zygote hybrids the time of formation of the block varied: while 50% of cells lost the ability to fuse with sperm by 2 h after fusion, in the remaining cells the block must have developed some time between 5 and 18 h after fusion. How these sperm-induced modifications of the oolemma of fertilised egg spread in the hybrid cell and render the 'virgin' part of oolemma resistant to sperm penetration remains unknown.

Inadequate function of sterile tw5/tw32 spermatozoa overcome by intracytoplasmic sperm injection.

Mice carrying two t complementary haplotypes (tw5/tw32) are totally sterile. Their spermatozoa have poor motility and fertilize neither zona-intact nor zona-free oocytes, even though they are structurally indistinguishable from control (wild-type) spermatozoa. However, when injected directly into oocytes, these infertile spermatozoa are able to participate in normal development. This suggests that infertility of tw5/tw32 male (spermatozoa) is more likely to be due to poor sperm-oocyte interaction than to genetic incompetence of sperm nuclei.

Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: evidence based on intracytoplasmic sperm injection experiments in the mouse.

Mouse oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and reinseminated after the removal of zonae pellucidae at pronuclear stage or at the 2-cell stage. Although these oocytes were activated normally by ICSI, as evidenced by resumption of meiosis and cortical granule exocytosis, they did not develop oolemma block to sperm penetration. They could be penetrated by spermatozoa at pronuclear stage and even at the 2-cell stage. This supports the notion that incorporation of sperm plasma membrane into oolemma contributes to the changes in oolemma that block sperm penetration.

Mouse oocytes penetrated by sperm at GV or GVBD stage lose the ability to fuse with additional spermatozoa.

Mouse oocytes penetrated by spermatozoa during germinal vesicle (GV) breakdown undergo maturation and are arrested at metaphase of the second meiotic division despite the presence of sperm nuclei within the ooplasm. When these oocytes were re-inseminated, none was penetrated by newly added spermatozoa. When GV oocytes were inseminated and cultured in the presence of dibutyryl cAMP, the oocytes remained at GV stage, yet they did not permit entry of additional spermatozoa. These observations suggest that the plasma membrane of maturing oocytes is modified by precociously penetrating spermatozoa independently from cortical granule exocytosis. Sperm components incorporated into the oocytes seem to be responsible for the modification of the oocyte's plasma membrane.

Sperm nuclei entering parthenogenetically activated mouse oocytes before the first mitosis transform into pronuclei. An ultrastructural study.

BACKGROUND: This report is an extension of previous observations (Maleszewski 1992. Mol. Reprod. Dev., 33:215-221) on the behavior of mouse sperm nuclei incorporated into parthenogenetically activated mouse oocytes prior to the first cleavage division and undergoing transformation during mitosis. METHOD: Artificially activated mouse oocytes were inseminated in vitro and an ultrastructural analysis was performed of sperm-derived nuclei present in two parthenogenetic two-cell embryos. RESULTS: Both chromatin and nuclear envelope of sperm derived-nuclei are structurally identical with those of oocyte-derived nuclei and of the nuclei of blastomeres of normal two-cell embryos. CONCLUSIONS: Cytoplasm of the parthenogenote during the first mitotic division has the ability to transform sperm nucleus into a male pronucleus just like the cytoplasm of a metaphase II oocyte.

Activation of hamster zona-free oocytes by homologous and heterologous spermatozoa.

Spermatozoa of a wide variety of species can fuse with zona-free hamster oocytes. Zona-free hamster oocytes were inseminated with spermatozoa of homologous (hamster) and other (mouse, guinea-pig and human) species, and their responses were closely examined to determine whether such interspecific sperm-oocyte fusion always induces normal oocyte activation. While guinea-pig and human spermatozoa could activate hamster oocytes as efficiently as hamster spermatozoa, mouse spermatozoa could not. Mouse spermatozoa fused readily with hamster oocytes, yet most oocytes remained inactivated at least during the first 1.5-2 h. The amount of M-phase (metaphase) promoting factor was reduced in hamster oocytes fused with one or several mouse spermatozoa; however, repetitive Ca2+ transients failed to occur unless oocytes were inseminated with a concentrated sperm suspension and penetrated by very many spermatozoa. These observations suggest that sperm-oocyte membrane fusion per se is not sufficient to trigger oocyte activation, and that putative sperm-derived oocyte activating factors show some degree of species specificity.

Sulphydryl reagent iodoacetamide inhibits progression of meiosis and sperm transformation in mouse oocytes fertilised in vitro.

The effect of iodoacetamide (IA), a sulphydryl blocking agent, on fertilisation in the mouse was examined by transferring zona-free oocytes into IA (500 microM)-containing medium at various times after insemination. When inseminated oocytes were transferred into IA medium 10 min after insemination, the oocyte chromosomes remained aggregated in one or two masses and the sperm nucleus failed to decondense. When oocytes were transferred during the second meiosis, oocyte meiosis was more or less arrested. The sperm nucleus decondensed but remained blocked at an early stage of decondensation. These observations suggest that thiol groups in the oocyte's cytoplasm and perhaps microtubules of the meiotic spindle play crucial roles in the completion of meiosis and the transformation of sperm nucleus into pronucleus.

Sperm penetration in parthenogenetic mouse embryos triggers a plasma membrane block to polyspermy.

Mouse oocytes activated parthenogenetically do not generate a plasma membrane block against spermatozoa over the first three cell cycles. We show that they lose this fusibility spontaneously at the 8-cell stage. Insemination of 1-cell parthenogenetic embryos induces loss of fusibility earlier, at the 2-cell stage. This observation suggests that incorporation of the sperm cell membrane components into the oolemma may be responsible for the development of the membrane block.

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.

Decondensation of mouse sperm chromatin in cell-free extracts: a micromethod.

A micromethod is presented which makes possible the analysis of mouse sperm nucleus decondensation in vitro using very small volumes of cytoplasmic preparations, even smaller than 1 microliters. We show that cell-free extracts obtained from interphase HeLa cells as well as lysates from mouse eggs and embryos can sustain early stages of mouse sperm nucleus transformation, provided the sperm nuclear envelope is damaged or removed.