Marek Maleszewski - Academia.edu (original) (raw)

Papers by Marek Maleszewski

Acta Biologica Cracoviensia. Series Botanica. Supplement, 2006

Biology of Reproduction, 2019

The full-term development of the xenogeneic embryo in the uterus of the mother of different speci... more The full-term development of the xenogeneic embryo in the uterus of the mother of different species is very restricted and can occur only in certain groups of closely related mammals. In the case of mouse↔rat chimeras, the inter-specific uterine barrier is less hostile to inter-specific chimeric fetuses. In current work, we tested the development of mouse and rat fetuses in uteri of females of the opposite species. We created chimeric mouse↔rat blastocysts by injection of mouse embryonic stem cells (ESCs) into rat 8-cell embryos and rat ESCs into 8-cell mouse embryos. Chimeras were transferred to the foster mothers of the opposite species. Despite a huge number of transferred embryos (>1000 in total for both variants), only one live fetus derived solely from the mouse ESCs was isolated at E13.5 from the rat uterus. All other fetuses and newborns were chimeric or were built only from the cells of the recipient embryo. We examined the possible reason for such an outcome and found t...

Reproduction, Fertility and Development, 2004

Maturation of marsupial oocytes in vitro, an important step in the analysis of early developmenta... more Maturation of marsupial oocytes in vitro, an important step in the analysis of early developmental events, has a low success rate and results from the artificial activation of oocytes, which may not include nuclear maturation. In Sminthopsis macroura, 24-h culture of advanced antral follicles in medium containing 10 μg mL−1 porcine pituitary luteinising hormone (LH) yielded 60% of mature polarised oocytes with the first polar body; follicles cultured in medium without LH yielded only immature oocytes. Parthenogenetic activation of follicular, oviducal or uterine oocytes occurred when a two-step protocol was used. Sixty-one oocytes, exposed to 10 μm calcium ionophore A23187 for 10 min followed by 10 μg mL−1 cycloheximide (protein synthesis inhibitor) for 5 h and then cultured for 20–24 h, were scored for signs of activation, namely extrusion of the second polar body and formation of the pronucleus. In each of 43 oocytes (70%), the extruded second polar body was present. Sixteen oocyt...

The International Journal of Developmental Biology, 2017

PLOS ONE, 2017

The mouse preimplantation embryo generates the precursors of trophectoderm (TE) and inner cell ma... more The mouse preimplantation embryo generates the precursors of trophectoderm (TE) and inner cell mass (ICM) during the 8-to 16-cell stage transition, when the apico-basal polarized blastomeres undergo divisions that give rise to cells with different fate. Asymmetric segregation of polar domain at 8-16 cell division generate two cell types, polar cells which adopt an outer position and develop in TE and apolar cells which are allocated to inner position as the precursors of ICM. It is still not know when the blastomeres of 8-cell stage start to be determined to undergo asymmetric division. Here, we analyze the frequency of symmetric and asymmetric divisions of blastomeres isolated from 8-cell stage embryo before and after compaction. Using p-Ezrin as the polarity marker we found that size of blastomeres in 2/16 pairs cannot be used as a criterion for distinguishing symmetric and asymmetric divisions. Our results showed that at early 8-cell stage, before any visible signs of cortical polarity, a subset of blastomeres had been already predestined to divide asymmetrically. We also showed that almost all of 8-cell stage blastomeres isolated from compacted embryo divide asymmetrically, whereas in intact embryos, the frequency of asymmetric divisions is significantly lower. Therefore we conclude that in intact embryo the frequency of symmetric and asymmetric division is regulated by cell-cell interactions.

MPF (M-phase promoting factor) is an essential regulator of meiotic division in oocytes. It consi... more MPF (M-phase promoting factor) is an essential regulator of meiotic division in oocytes. It consists of catalytic subunit CDK1 / p34 cdc2 (cyclin-dependent ki-nase1) and regulatory subunit-cyclin B. During meiotic maturation of the mouse oocyte MPF activity increases, as oocytes progress towards metaphase of the first meiotic division (metaphase I). Next MPF activity is reduced at metaphase I/anaphaseI transition and rises again in the metaphase of the second meiotic division (metaphase II) (Verlhac et al., 1994). After fertilization of metaphase II oocyte, MPF activity is diminished in process involving calcium-dependent proteolysis of cyclin B (Nixon et al., 2002; Verlhac et al., 1994). Spermatozoa introduced into maturing oocytes trigger transient calcium increases in ooplasm, which are similar to these observed after fertilization of mature, metaphase II oocytes, but less numerous (Carroll et al., 1994). We showed that polispermic (about 4-5 spermatozoa per an oocyte) fertilization of maturing oocytes 2h after resumption of meiosis is accompanied by downregulation of MPF activity. One and 2 hours after this precocious fertilization we observed 1,5-fold decrease in MPF activity. However it is not caused by proteolysis of cyclin B, since fertilization of maturing oocytes did not affected the cyclin B level. Moreover, the decrease in MPF activity is not dependent on increase of the concentration of Ca 2+. In experiments in which we preincubated maturing oocytes in Ca 2+-chelator, BAPTA prior to fertilization, MPF activity was also diminished. There are two main conclusions from our data. Firstly, a signal transduction pathway, which acts between the activating factor delivered by spermatozoon, and cyclin B proteolysis does not function in maturing oocytes. Secondly, there must be other mechanism that enables a decrease in MPF activity in fertilized maturing oocytes. It might involve changes in the phosphorylation of CDK1, a catalytic subunit of MPF. Further experiments are planned in order to elucidate this problem.

The International Journal of Developmental Biology, 2008

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in co... more Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperminduced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/ Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca 2+ transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (α α α α α, δ δ δ δ δ and ζ ζ ζ ζ ζ), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP 3 receptors which participate in calcium release during oocyte fertilization.

Zygote, 2002

Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 mi... more Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 min or 3 h after egg activation and DNA replication and RNA synthesis were analysed in remodelled thymocyte nuclei and female pronuclei. We show that thymocyte nuclei which transform into pronucleus-like nuclei (thymocytes injected not later than 60 min after activation) enter S-phase 1 h earlier than the female pronuclei. At the beginning of the first cell cycle they remain transcriptionally silent, but in G2 undertake transcription earlier than the female pronuclei. Partly remodelled thymocyte nuclei (injected 3 h after activation) start to replicate DNA at the same time as the female pronuclei. They reinitiate RNA synthesis within 2 h after transfer and continue to transcribe irrespective of the transcriptional activity of the female pronucleus. We show that the observed transcription is only nuclear, i.e. RNA polymerase II-dependent.

Zygote, 2006

SummaryCD9 is a member of the tetraspanin superfamily proteins and is the only protein on the mou... more SummaryCD9 is a member of the tetraspanin superfamily proteins and is the only protein on the mouse oocyte which is known to be indispensable in sperm–egg fusion. Here, using indirect immunofluorescence we show that CD9 appears on the oolemma during the early stages of the growth of the oocyte, when it measures 13–22 μm in diameter. When the oocyte reaches a diameter of 17–22 μm, the density of CD9 in its oolemma is similar to the density of this protein in the cell membrane of the fully grown secondary oocyte. The appearance of CD9 in growing oocytes correlates with the previously reported time of the acquisition of fusibility between the spermatozoon and the egg. Accordingly we propose that during oogenesis the development of the ability of the oolemma to fuse with sperm may be regulated by synthesis of CD9 by the oocyte.

The International Journal of Developmental Biology, 2003

Fertilization of a mouse egg results in modification of the cytoplasmatic membrane (oolemma) whic... more Fertilization of a mouse egg results in modification of the cytoplasmatic membrane (oolemma) which makes fusion with additional sperm impossible. CD9 is a transmembrane protein reported to be responsible for gamete fusion. Since the molecular mechanism of zygote membrane modification after fertilization remains unknown, we were interested to check whether lack of CD9 is the reason for non-penetrability of zona-free zygotes. We wanted also to determine the effect of different methods of zona pellucida removal on the presence of CD9 on the surface of unfertilized eggs and their ability to be fertilized afterwards. We demonstrated that CD9 is present on the surface of both zygotes and parthenogenotes. We showed also that the treatment of eggs with pronase completely removes CD9 from the membrane of eggs making them infertile. Eggs treated with chymotrypsin and acid Tyrode still posses CD9 on their surface and remain fertile. The results of our experiments indicate that modification of the zygote oolemma does not involve a lack of CD9. We cannot exclude however, that the amount of CD9 decreases after fertilization. In addition, our studies indicate that the previously reported infertility of eggs treated with different proteases may result from the decrease or removal of CD9 and probably other proteins responsible for gamete fusion from the surface of eggs.

The International Journal of Developmental Biology, 2008

How did you, in mid 50', come up with the idea to study the regulative potential of mammalian emb... more How did you, in mid 50', come up with the idea to study the regulative potential of mammalian embryo? As far as I know at that time nobody at Warsaw University was studying this particular subject. Did you want to extrapolate experimental approach of Driesch, Horstadius and Spe-Early mammalian embryo: my love

The International Journal of Developmental Biology, 2008

In this article, we describe the history (between the XIX century and World War II) of embryologi... more In this article, we describe the history (between the XIX century and World War II) of embryological research conducted at Warsaw University, together with current research activities being carried out at the Department of Embryology. During the partition of Poland, the Imperial (Russian) Warsaw University conducted research on avian embryology (and to a smaller extent, on reptilian embryology). When Poland regained independence in 1918, these studies were continued under the Chair of Comparative Anatomy headed by Professor Jan Tur. A new Department of Embryology created in 1954 was first headed by Professor Stanislaw Bilewicz and since 1964 by Professor Andrzej Tarkowski, who in 2003 was succeeded by Dr. Marek Maleszewski D.Sc. During the last 45 years, embryological research at Warsaw University has concentrated mainly on mammalian development with special emphasis on the regulative capabilities of early embryos and also on experimental chimaeras, nucleo-cytoplasmic interactions in oogenesis and early embryogenesis (including regulation of DNA replication and transcription), experimental parthenogenesis and fertilization.

The International Journal of Developmental Biology, 2008

Reproductive Biology, 2011

Oct-4, the marker of pluripotent cells, is crucial for murine preimplantation development. During... more Oct-4, the marker of pluripotent cells, is crucial for murine preimplantation development. During the formation of the blastocyst Oct-4 is downregulated in the trophectoderm (TE) and its expression becomes restricted to the inner cell mass (ICM). In order to determine the exact timing of the disappearance of Oct-4 protein from TE we analyzed the localization and level of Oct-4 at different stages of blastocyst development. The presence of Oct-4 protein was determined by immunohistochemistry using confocal microscopy. We found that the downregulation of Oct-4 protein in TE of mouse blastocysts progresses gradually during development, and Oct-4 protein persists in some of the TE cells at least until the expanded blastocyst (120-140 cells) stage. Our findings indicate that the switching-off of the Oct-4 expression is not necessary for the trophectoderm formation. The complete elimination of Oct-4 protein from TE occurs at the period of blastocyst implantation, when lack of Oct-4 is required for the proper functioning of the trophectoderm.

Reproductive Biology, 2008

Oocytes of most mammalian species, including mouse and human, are fertilized in metaphase of the ... more Oocytes of most mammalian species, including mouse and human, are fertilized in metaphase of the second meiotic division. A fertilizing spermatozoon introduces an oocyte-activating factor, phospholipase C zeta, triggering oscillations of the cytoplasmic concentration of free calcium ions ([Ca 2+ ] i) in the oocyte. [Ca 2+ ] i oscillations are essential for the activation of the embryonic development. They trigger processes such as resumption and completion of meiosis, establishment of the block to polyspermy and recruitment of maternal mRNAs necessary for the activation of the embryo genome. Moreover, it has been recently shown that [Ca 2+ ] i oscillations may also influence the development of the embryo. The ability to generate [Ca 2+ ] i oscillations develops in mammalian oocytes during meiotic maturation and requires several cytoplasmic changes, including: 1/ reorganization of endoplasmic reticulum, the main stockpile of calcium in the oocyte, 2/ increase in the number of 1,4,5-inositol triphosphate (IP) receptors, Sperm-induced Ca 2+ oscillations in mammalian oocytes / changes in their biochemical properties (e.g.: sensitivity to IP), and possibly both 4/ an increase in the concentration of Ca 2+ ions stored in endoplasmic reticulum (ER) and 5/ redistribution of Ca 2+-binding ER proteins. The aim of this review is to present the state of current knowledge about these processes.

REPRODUCTION, 2008

Fertilization affects levels of cyclin B1 and M-phase promoting factor (MPF) activity in maturing... more Fertilization affects levels of cyclin B1 and M-phase promoting factor (MPF) activity in maturing and metaphase II mouse oocytes in two distinct ways. In metaphase II oocytes, it leads to a Ca2+-dependent, continuous degradation of cyclin B1 and inactivation of cyclin dependent kinase (CDC2A)–cyclin B1 complex (MPF). In this paper, we show that neither mono- nor polyspermic fertilization of prometaphase I and metaphase I oocytes triggered degradation of cyclin B1. However, polyspermic fertilization of prometaphase I oocytes led to a transient decrease in MPF activity that lasted for 2 h. The inactivation of MPF in polyspermic prometaphase I oocytes did not depend on the fertilization-induced increase in the cytoplasmic concentration of free Ca2+ions, but was caused, at least in part, by dephosphorylation of CDC2A at threonine 161 (Thr161). We found that polyspermic fertilization did not affect glutathione levels in prometaphase I oocytes, and concluded that the decrease in MPF activ...

Molecular Reproduction and Development, 1992

When artificially activated mouse eggs are inseminated in the middle of the first cell cycle, spe... more 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.

Molecular Reproduction and Development, 2004

To track the lineage of both blastomeres of 2-cell embryos during mouse preimplantation developme... more To track the lineage of both blastomeres of 2-cell embryos during mouse preimplantation development, each cell was injected with dextran solutions conjugated with different fluorochromes. The fate of the progeny of the first two blastomeres was followed with confocal microscopy during cleavage and during the formation of the blastocyst. We observed that in most of cleaving embryos the cells derived from the two first blastomeres intermingled in both the trophectoderm and the inner cell mass (ICM) and did not form two discrete groups. We conclude that embryonic parts of blastocysts contain descendants of both blastomeres of 2-cell embryo.

Molecular Reproduction and Development, 1990

A micromethod is presented which makes possible the analysis of mouse sperm nucleus decondensatio... more 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.

Molecular Reproduction and Development, 2004

Activity of the sperm-derived oocyte-activating factor persists in zygotes and can be detected by... more Activity of the sperm-derived oocyte-activating factor persists in zygotes and can be detected by a fusion with metaphase II (MII) oocytes leading to the activation of the hybrids. We have shown, that in the great majority of oocytes inseminated 1-2 hr after germinal vesicle breakdown (GVBD) the sperm-derived activating ability was eliminated. Only few hybrids produced by fusion of MII oocytes with oocytes inseminated during in vitro maturation (M x IVM-P + sperm hybrids) underwent activation, whereas almost all of MII oocyte x zygote hybrids entered interphase. However, frequency of activation of M x IVM-P + sperm hybrids was higher than that of control hybrids, which were obtained by fusion of MII oocytes with oocytes uninseminated during in vitro maturation. Although the difference was not statistically significant, it suggested that in a certain number of oocytes inseminated after GVBD the sperm-derived oocyte-activating factor remained partially active. This was confirmed by our observation that several oocytes, which were inseminated during in vitro maturation and managed to accomplish MII, underwent activation and formed pronuclei when examined 25-26 hr after the beginning of maturation. We have also demonstrated that parthenogenotes, could acquire the sperm-derived activity, as a consequence of sperm injection. MII oocytes were fused with parthenogenotes inseminated by ICSI and all hybrids underwent activation. This result indicated that the ability to induce activation in hybrid, was sperm-derived.

Acta Biologica Cracoviensia. Series Botanica. Supplement, 2006

Biology of Reproduction, 2019

The full-term development of the xenogeneic embryo in the uterus of the mother of different speci... more The full-term development of the xenogeneic embryo in the uterus of the mother of different species is very restricted and can occur only in certain groups of closely related mammals. In the case of mouse↔rat chimeras, the inter-specific uterine barrier is less hostile to inter-specific chimeric fetuses. In current work, we tested the development of mouse and rat fetuses in uteri of females of the opposite species. We created chimeric mouse↔rat blastocysts by injection of mouse embryonic stem cells (ESCs) into rat 8-cell embryos and rat ESCs into 8-cell mouse embryos. Chimeras were transferred to the foster mothers of the opposite species. Despite a huge number of transferred embryos (>1000 in total for both variants), only one live fetus derived solely from the mouse ESCs was isolated at E13.5 from the rat uterus. All other fetuses and newborns were chimeric or were built only from the cells of the recipient embryo. We examined the possible reason for such an outcome and found t...

Reproduction, Fertility and Development, 2004

Maturation of marsupial oocytes in vitro, an important step in the analysis of early developmenta... more Maturation of marsupial oocytes in vitro, an important step in the analysis of early developmental events, has a low success rate and results from the artificial activation of oocytes, which may not include nuclear maturation. In Sminthopsis macroura, 24-h culture of advanced antral follicles in medium containing 10 μg mL−1 porcine pituitary luteinising hormone (LH) yielded 60% of mature polarised oocytes with the first polar body; follicles cultured in medium without LH yielded only immature oocytes. Parthenogenetic activation of follicular, oviducal or uterine oocytes occurred when a two-step protocol was used. Sixty-one oocytes, exposed to 10 μm calcium ionophore A23187 for 10 min followed by 10 μg mL−1 cycloheximide (protein synthesis inhibitor) for 5 h and then cultured for 20–24 h, were scored for signs of activation, namely extrusion of the second polar body and formation of the pronucleus. In each of 43 oocytes (70%), the extruded second polar body was present. Sixteen oocyt...

The International Journal of Developmental Biology, 2017

PLOS ONE, 2017

The mouse preimplantation embryo generates the precursors of trophectoderm (TE) and inner cell ma... more The mouse preimplantation embryo generates the precursors of trophectoderm (TE) and inner cell mass (ICM) during the 8-to 16-cell stage transition, when the apico-basal polarized blastomeres undergo divisions that give rise to cells with different fate. Asymmetric segregation of polar domain at 8-16 cell division generate two cell types, polar cells which adopt an outer position and develop in TE and apolar cells which are allocated to inner position as the precursors of ICM. It is still not know when the blastomeres of 8-cell stage start to be determined to undergo asymmetric division. Here, we analyze the frequency of symmetric and asymmetric divisions of blastomeres isolated from 8-cell stage embryo before and after compaction. Using p-Ezrin as the polarity marker we found that size of blastomeres in 2/16 pairs cannot be used as a criterion for distinguishing symmetric and asymmetric divisions. Our results showed that at early 8-cell stage, before any visible signs of cortical polarity, a subset of blastomeres had been already predestined to divide asymmetrically. We also showed that almost all of 8-cell stage blastomeres isolated from compacted embryo divide asymmetrically, whereas in intact embryos, the frequency of asymmetric divisions is significantly lower. Therefore we conclude that in intact embryo the frequency of symmetric and asymmetric division is regulated by cell-cell interactions.

MPF (M-phase promoting factor) is an essential regulator of meiotic division in oocytes. It consi... more MPF (M-phase promoting factor) is an essential regulator of meiotic division in oocytes. It consists of catalytic subunit CDK1 / p34 cdc2 (cyclin-dependent ki-nase1) and regulatory subunit-cyclin B. During meiotic maturation of the mouse oocyte MPF activity increases, as oocytes progress towards metaphase of the first meiotic division (metaphase I). Next MPF activity is reduced at metaphase I/anaphaseI transition and rises again in the metaphase of the second meiotic division (metaphase II) (Verlhac et al., 1994). After fertilization of metaphase II oocyte, MPF activity is diminished in process involving calcium-dependent proteolysis of cyclin B (Nixon et al., 2002; Verlhac et al., 1994). Spermatozoa introduced into maturing oocytes trigger transient calcium increases in ooplasm, which are similar to these observed after fertilization of mature, metaphase II oocytes, but less numerous (Carroll et al., 1994). We showed that polispermic (about 4-5 spermatozoa per an oocyte) fertilization of maturing oocytes 2h after resumption of meiosis is accompanied by downregulation of MPF activity. One and 2 hours after this precocious fertilization we observed 1,5-fold decrease in MPF activity. However it is not caused by proteolysis of cyclin B, since fertilization of maturing oocytes did not affected the cyclin B level. Moreover, the decrease in MPF activity is not dependent on increase of the concentration of Ca 2+. In experiments in which we preincubated maturing oocytes in Ca 2+-chelator, BAPTA prior to fertilization, MPF activity was also diminished. There are two main conclusions from our data. Firstly, a signal transduction pathway, which acts between the activating factor delivered by spermatozoon, and cyclin B proteolysis does not function in maturing oocytes. Secondly, there must be other mechanism that enables a decrease in MPF activity in fertilized maturing oocytes. It might involve changes in the phosphorylation of CDK1, a catalytic subunit of MPF. Further experiments are planned in order to elucidate this problem.

The International Journal of Developmental Biology, 2008

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in co... more Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperminduced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/ Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca 2+ transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (α α α α α, δ δ δ δ δ and ζ ζ ζ ζ ζ), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP 3 receptors which participate in calcium release during oocyte fertilization.

Zygote, 2002

Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 mi... more Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 min or 3 h after egg activation and DNA replication and RNA synthesis were analysed in remodelled thymocyte nuclei and female pronuclei. We show that thymocyte nuclei which transform into pronucleus-like nuclei (thymocytes injected not later than 60 min after activation) enter S-phase 1 h earlier than the female pronuclei. At the beginning of the first cell cycle they remain transcriptionally silent, but in G2 undertake transcription earlier than the female pronuclei. Partly remodelled thymocyte nuclei (injected 3 h after activation) start to replicate DNA at the same time as the female pronuclei. They reinitiate RNA synthesis within 2 h after transfer and continue to transcribe irrespective of the transcriptional activity of the female pronucleus. We show that the observed transcription is only nuclear, i.e. RNA polymerase II-dependent.

Zygote, 2006

SummaryCD9 is a member of the tetraspanin superfamily proteins and is the only protein on the mou... more SummaryCD9 is a member of the tetraspanin superfamily proteins and is the only protein on the mouse oocyte which is known to be indispensable in sperm–egg fusion. Here, using indirect immunofluorescence we show that CD9 appears on the oolemma during the early stages of the growth of the oocyte, when it measures 13–22 μm in diameter. When the oocyte reaches a diameter of 17–22 μm, the density of CD9 in its oolemma is similar to the density of this protein in the cell membrane of the fully grown secondary oocyte. The appearance of CD9 in growing oocytes correlates with the previously reported time of the acquisition of fusibility between the spermatozoon and the egg. Accordingly we propose that during oogenesis the development of the ability of the oolemma to fuse with sperm may be regulated by synthesis of CD9 by the oocyte.

The International Journal of Developmental Biology, 2003

Fertilization of a mouse egg results in modification of the cytoplasmatic membrane (oolemma) whic... more Fertilization of a mouse egg results in modification of the cytoplasmatic membrane (oolemma) which makes fusion with additional sperm impossible. CD9 is a transmembrane protein reported to be responsible for gamete fusion. Since the molecular mechanism of zygote membrane modification after fertilization remains unknown, we were interested to check whether lack of CD9 is the reason for non-penetrability of zona-free zygotes. We wanted also to determine the effect of different methods of zona pellucida removal on the presence of CD9 on the surface of unfertilized eggs and their ability to be fertilized afterwards. We demonstrated that CD9 is present on the surface of both zygotes and parthenogenotes. We showed also that the treatment of eggs with pronase completely removes CD9 from the membrane of eggs making them infertile. Eggs treated with chymotrypsin and acid Tyrode still posses CD9 on their surface and remain fertile. The results of our experiments indicate that modification of the zygote oolemma does not involve a lack of CD9. We cannot exclude however, that the amount of CD9 decreases after fertilization. In addition, our studies indicate that the previously reported infertility of eggs treated with different proteases may result from the decrease or removal of CD9 and probably other proteins responsible for gamete fusion from the surface of eggs.

The International Journal of Developmental Biology, 2008

How did you, in mid 50', come up with the idea to study the regulative potential of mammalian emb... more How did you, in mid 50', come up with the idea to study the regulative potential of mammalian embryo? As far as I know at that time nobody at Warsaw University was studying this particular subject. Did you want to extrapolate experimental approach of Driesch, Horstadius and Spe-Early mammalian embryo: my love

The International Journal of Developmental Biology, 2008

In this article, we describe the history (between the XIX century and World War II) of embryologi... more In this article, we describe the history (between the XIX century and World War II) of embryological research conducted at Warsaw University, together with current research activities being carried out at the Department of Embryology. During the partition of Poland, the Imperial (Russian) Warsaw University conducted research on avian embryology (and to a smaller extent, on reptilian embryology). When Poland regained independence in 1918, these studies were continued under the Chair of Comparative Anatomy headed by Professor Jan Tur. A new Department of Embryology created in 1954 was first headed by Professor Stanislaw Bilewicz and since 1964 by Professor Andrzej Tarkowski, who in 2003 was succeeded by Dr. Marek Maleszewski D.Sc. During the last 45 years, embryological research at Warsaw University has concentrated mainly on mammalian development with special emphasis on the regulative capabilities of early embryos and also on experimental chimaeras, nucleo-cytoplasmic interactions in oogenesis and early embryogenesis (including regulation of DNA replication and transcription), experimental parthenogenesis and fertilization.

The International Journal of Developmental Biology, 2008

Reproductive Biology, 2011

Oct-4, the marker of pluripotent cells, is crucial for murine preimplantation development. During... more Oct-4, the marker of pluripotent cells, is crucial for murine preimplantation development. During the formation of the blastocyst Oct-4 is downregulated in the trophectoderm (TE) and its expression becomes restricted to the inner cell mass (ICM). In order to determine the exact timing of the disappearance of Oct-4 protein from TE we analyzed the localization and level of Oct-4 at different stages of blastocyst development. The presence of Oct-4 protein was determined by immunohistochemistry using confocal microscopy. We found that the downregulation of Oct-4 protein in TE of mouse blastocysts progresses gradually during development, and Oct-4 protein persists in some of the TE cells at least until the expanded blastocyst (120-140 cells) stage. Our findings indicate that the switching-off of the Oct-4 expression is not necessary for the trophectoderm formation. The complete elimination of Oct-4 protein from TE occurs at the period of blastocyst implantation, when lack of Oct-4 is required for the proper functioning of the trophectoderm.

Reproductive Biology, 2008

Oocytes of most mammalian species, including mouse and human, are fertilized in metaphase of the ... more Oocytes of most mammalian species, including mouse and human, are fertilized in metaphase of the second meiotic division. A fertilizing spermatozoon introduces an oocyte-activating factor, phospholipase C zeta, triggering oscillations of the cytoplasmic concentration of free calcium ions ([Ca 2+ ] i) in the oocyte. [Ca 2+ ] i oscillations are essential for the activation of the embryonic development. They trigger processes such as resumption and completion of meiosis, establishment of the block to polyspermy and recruitment of maternal mRNAs necessary for the activation of the embryo genome. Moreover, it has been recently shown that [Ca 2+ ] i oscillations may also influence the development of the embryo. The ability to generate [Ca 2+ ] i oscillations develops in mammalian oocytes during meiotic maturation and requires several cytoplasmic changes, including: 1/ reorganization of endoplasmic reticulum, the main stockpile of calcium in the oocyte, 2/ increase in the number of 1,4,5-inositol triphosphate (IP) receptors, Sperm-induced Ca 2+ oscillations in mammalian oocytes / changes in their biochemical properties (e.g.: sensitivity to IP), and possibly both 4/ an increase in the concentration of Ca 2+ ions stored in endoplasmic reticulum (ER) and 5/ redistribution of Ca 2+-binding ER proteins. The aim of this review is to present the state of current knowledge about these processes.

REPRODUCTION, 2008

Fertilization affects levels of cyclin B1 and M-phase promoting factor (MPF) activity in maturing... more Fertilization affects levels of cyclin B1 and M-phase promoting factor (MPF) activity in maturing and metaphase II mouse oocytes in two distinct ways. In metaphase II oocytes, it leads to a Ca2+-dependent, continuous degradation of cyclin B1 and inactivation of cyclin dependent kinase (CDC2A)–cyclin B1 complex (MPF). In this paper, we show that neither mono- nor polyspermic fertilization of prometaphase I and metaphase I oocytes triggered degradation of cyclin B1. However, polyspermic fertilization of prometaphase I oocytes led to a transient decrease in MPF activity that lasted for 2 h. The inactivation of MPF in polyspermic prometaphase I oocytes did not depend on the fertilization-induced increase in the cytoplasmic concentration of free Ca2+ions, but was caused, at least in part, by dephosphorylation of CDC2A at threonine 161 (Thr161). We found that polyspermic fertilization did not affect glutathione levels in prometaphase I oocytes, and concluded that the decrease in MPF activ...

Molecular Reproduction and Development, 1992

When artificially activated mouse eggs are inseminated in the middle of the first cell cycle, spe... more 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.

Molecular Reproduction and Development, 2004

To track the lineage of both blastomeres of 2-cell embryos during mouse preimplantation developme... more To track the lineage of both blastomeres of 2-cell embryos during mouse preimplantation development, each cell was injected with dextran solutions conjugated with different fluorochromes. The fate of the progeny of the first two blastomeres was followed with confocal microscopy during cleavage and during the formation of the blastocyst. We observed that in most of cleaving embryos the cells derived from the two first blastomeres intermingled in both the trophectoderm and the inner cell mass (ICM) and did not form two discrete groups. We conclude that embryonic parts of blastocysts contain descendants of both blastomeres of 2-cell embryo.

Molecular Reproduction and Development, 1990

A micromethod is presented which makes possible the analysis of mouse sperm nucleus decondensatio... more 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.

Molecular Reproduction and Development, 2004

Activity of the sperm-derived oocyte-activating factor persists in zygotes and can be detected by... more Activity of the sperm-derived oocyte-activating factor persists in zygotes and can be detected by a fusion with metaphase II (MII) oocytes leading to the activation of the hybrids. We have shown, that in the great majority of oocytes inseminated 1-2 hr after germinal vesicle breakdown (GVBD) the sperm-derived activating ability was eliminated. Only few hybrids produced by fusion of MII oocytes with oocytes inseminated during in vitro maturation (M x IVM-P + sperm hybrids) underwent activation, whereas almost all of MII oocyte x zygote hybrids entered interphase. However, frequency of activation of M x IVM-P + sperm hybrids was higher than that of control hybrids, which were obtained by fusion of MII oocytes with oocytes uninseminated during in vitro maturation. Although the difference was not statistically significant, it suggested that in a certain number of oocytes inseminated after GVBD the sperm-derived oocyte-activating factor remained partially active. This was confirmed by our observation that several oocytes, which were inseminated during in vitro maturation and managed to accomplish MII, underwent activation and formed pronuclei when examined 25-26 hr after the beginning of maturation. We have also demonstrated that parthenogenotes, could acquire the sperm-derived activity, as a consequence of sperm injection. MII oocytes were fused with parthenogenotes inseminated by ICSI and all hybrids underwent activation. This result indicated that the ability to induce activation in hybrid, was sperm-derived.