Prophase Oocytes Research Articles

A dynamic population of prophase CENP-C is required for meiotic chromosome segregation.

The centromere is an epigenetic mark that is a loading site for the kinetochore during meiosis and mitosis. This mark is characterized by the H3 variant CENP-A, known as CID in Drosophila. In Drosophila, CENP-C is critical for maintaining CID at the centromeres and directly recruits outer kinetochore proteins after nuclear envelope break down. These two functions, however, happen at different times in the cell cycle. Furthermore, in Drosophila and many other metazoan oocytes, centromere maintenance and kinetochore assembly are separated by an extended prophase. We have investigated the dynamics of function of CENP-C during the extended meiotic prophase of Drosophila oocytes and found that maintaining high levels of CENP-C for metaphase I requires expression during prophase. In contrast, CID is relatively stable and does not need to be expressed during prophase to remain at high levels in metaphase I of meiosis. Expression of CID during prophase can even be deleterious, causing ectopic localization to non-centromeric chromatin, abnormal meiosis and sterility. CENP-C prophase loading is required for multiple meiotic functions. In early meiotic prophase, CENP-C loading is required for sister centromere cohesion and centromere clustering. In late meiotic prophase, CENP-C loading is required to recruit kinetochore proteins. CENP-C is one of the few proteins identified in which expression during prophase is required for meiotic chromosome segregation. An implication of these results is that the failure to maintain recruitment of CENP-C during the extended prophase in oocytes would result in chromosome segregation errors in oocytes.

2022 Winners: Kathleen Scheffler, Federica Giannini and Tom Lemonnier

2022 Winners: Kathleen Scheffler, Federica Giannini and Tom Lemonnier

First person – Federica Giannini

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Federica Giannini is co-first author on ‘ The prophase oocyte nucleus is a homeostatic G-actin buffer’, published in JCS. Federica is a PhD student in the lab of Binyam Mogessie at the School of Biochemistry, University of Bristol, UK, investigating the molecular mechanisms of how healthy mammalian eggs are formed.

The prophase oocyte nucleus is a homeostatic G-actin buffer.

ABSTRACTFormation of healthy mammalian eggs from oocytes requires specialised F-actin structures. F-actin disruption produces aneuploid eggs, which are a leading cause of human embryo deaths, genetic disorders and infertility. We found that oocytes contain prominent nuclear F-actin structures that are correlated with meiotic developmental capacity. We demonstrate that nuclear F-actin is a conserved feature of healthy mammalian oocytes and declines significantly with female reproductive ageing. Actin monomers used for nuclear F-actin assembly are sourced from an excess pool in the oocyte cytoplasm. Increasing monomeric G-actin transfer from the cytoplasm to the nucleus or directly enriching the nucleus with monomers led to assembly of stable nuclear F-actin bundles that significantly restrict chromatin mobility. By contrast, reducing G-actin monomer transfer by blocking nuclear import triggered assembly of a dense cytoplasmic F-actin network that is incompatible with healthy oocyte development. Overall, our data suggest that the large oocyte nucleus helps to maintain cytoplasmic F-actin organisation and that defects in this function are linked with reproductive age-related female infertility.This article has an associated First Person interview with Federica Giannini, joint first author of the paper.

Development of WEE2 kinase inhibitors as novel non-hormonal female contraceptives that target meiosis†.

WEE2 oocyte meiosis inhibiting kinase is a well-conserved oocyte specific kinase with a dual regulatory role during meiosis. Active WEE2 maintains immature, germinal vesicle stage oocytes in prophase I arrest prior to the luteinizing hormone surge and facilitates exit from metaphase II arrest at fertilization. Spontaneous mutations at the WEE2 gene locus in women have been linked to total fertilization failure indicating that selective inhibitors to this kinase could function as non-hormonal contraceptives. Employing co-crystallization with WEE1 G2 checkpoint kinase inhibitors, we revealed the structural basis of action across WEE kinases and determined type I inhibitors were not selective to WEE2 over WEE1. In response, we performed in silico screening by FTMap/FTSite and Schrodinger SiteMap analysis to identify potential allosteric sites, then used an allosterically biased activity assay to conduct high-throughput screening of a 26 000 compound library containing scaffolds of known allosteric inhibitors. Resulting hits were validated and a selective inhibitor that binds full-length WEE2 was identified, designated GPHR-00336382, along with a fragment-like inhibitor that binds the kinase domain, GPHR-00355672. Additionally, we present an in vitro testing workflow to evaluate biological activity of candidate WEE2 inhibitors including; (1) enzyme-linked immunosorbent assays measuring WEE2 phosphorylation activity of cyclin dependent kinase 1 (CDK1; also known as cell division cycle 2 kinase, CDC2), (2) in vitro fertilization of bovine ova to determine inhibition of metaphase II exit, and (3) cell-proliferation assays to look for off-target effects against WEE1 in somatic (mitotic) cells.

Separation and Loss of Centrioles From Primordidal Germ Cells To Mature Oocytes In The Mouse

Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Synapsis and recombination of autosomes and sex chromosomes in two terns (Sternidae, Charadriiformes, Aves)

The frequency of recombination and the patterns of crossover site distribution along the chromosomes vary considerably among animal species, including closely related species. Several hypotheses concerning the adaptive value and evolution of these variations were proposed. It was supposed that the recombination patterns of the species’ genomes are influenced by their phylogenetic history and ecology. However, most original data were obtained from mammals. The mammals show high karyological variability, which strongly influences the recombination patterns. Therefore it is important to study recombination rate and distribution in more karyologically stable taxa, such as reptiles and birds. We used immunolocalization of SYCP3, the protein of the lateral element of the synaptonemal complex (SC), centromere proteins and the mismatch-repair protein MLH1, which is associated with the recombination nodules, at the synaptonemal complex spreads of prophase oocytes of two tern species, black tern (Chlidonias niger) and common tern (Sterna hirundo). We first described the karyotype of Ch. niger (2n = 74, FN= 94) and identified suggestive rearrangements by which its karyotype differs from that of S. hirundo (2n = 68, FN = 90). We found that these species significantly differed by the numbers of the MLH1 foci per cell (Ch. niger: 53.0±4.2; S. hirundo: 44.1±5.0). We showed that the difference in the crossover numbers per cell was determined by the difference in the SC length (total and of individual bivalents) and by chromosomal rearrangements, which also influenced the distributions of crossover sites along the chromosomes. The difference in recombination patterns was higher between the rearranged homeologues than between the non-rearranged ones. We investigated the synaptic patterns of the heteromor phic Z and W chromosomes, localized the pseudoautosomal regions and estimated their lengths. In spite of several autosomal rearrangements, which differentiate these species, the structure and synaptic patterns of the sex chromosomes have not changed over 9 MY, which have passed since the moment of divergence between the genera Sterna and Chlidonias.

Cellular development of the germinal epithelium during the gametogenic cycle of the golden mussel Limnoperna fortunei (Bivalvia: Mytilidae).

The golden mussel Limnoperna fortunei is an invasive species that has quickly dispersed and colonized several potential different habitats distributed all over the world, causing environmental and economic impacts. Thus, in order to contribute to a better understanding of the reproductive aspects of L. fortunei, we described the cellular dynamic of the male and female germinal epithelium during the annual reproductive life history of this species, with the use of high resolution histology. An approximate of 1 200 specimens of L. fortunei were sampled periodically on the upper Paraná River floodplain (Brazil), from March 2010 to December 2012. Based on the activity of the germinal epithelium and consequent germ cell development, this study has resulted in the recognition of the following reproductive phases: Developing, Spawning Capable, Regressing and Regenerating. In the characterization of these phases, the following germ cells were described for males: spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa. Cell nests, oogonia, early prophase oocytes, previtellogenic oocytes and vitellogenic oocytes (early vitellogenic oocytes, middle vitellogenic oocytes and full-grown oocytes) were described for females. The morphological data and reproductive parameters obtained, showed the value of the cellular dynamics of the germinal epithelium, for the understanding of the cyclic gonadal events during the adult reproductive life of the mollusk in general. These results on the gametogenesis of this invasive species may be a fundamental tool for the development of control strategies and programs implementation, to reduce their proliferation and impacts in natural local environments.

Effects of dam size and nutrition during pregnancy on fetal ovarian development of their offspring in sheep

This study examined the effects of maternal size at conception and nutrition during pregnancy on ovarian development in their offspring. Romney ewes were selected at mating for heavy (H) or light (L) live weights and body condition, and then allocated to either ad libitum (A) or pregnancy maintenance (M) nutrition from Days 21–140 of pregnancy, resulting in four treatment groups; HA, HM, LA and LM. Ewes were euthanised on Days 65 (P65), 100 (P100) and 140 (P140) of pregnancy to collect fetal ovarian tissue, which was processed for morphological evaluation and immunohistochemical study of AMH and GDF9 (P140 only). Fetuses from H-dams had more oocytes in the first meiotic prophase on P65 (P=0.05) than those from L-dams. On P100, LA-fetuses had more primordial and fewer primary follicles than did LM-fetuses (P<0.05). On P140, fetuses from M dams had more primordial and fewer primary follicles compared with fetuses from A dams. Also on P140, H–M fetuses had greater (P=0.05) AMH expression than L-M fetuses. These results indicate that maternal size at conception and nutrition during pregnancy exert minor effects on fetal ovarian cell number.

Characterization and expression analysis of a cyclin B gene from black tiger shrimp (Penaeus monodon).

The open reading frame of black tiger shrimp (Penaeus monodon) cyclin B (Pmcyclin B) was identified, based on cDNA sequence registered in GenBank (accession No. EF015590). The target sequence was 1206 bp, corresponding to 401 amino acids. Two conserved signature sequences of the cyclin B gene family were found in the Pmcyclin B deduced aa sequence. Temporal expression of Pmcyclin B in different tissues, including ovary, lymphoid organ, brain, blood, muscle, heart, gill, hepatopancreas, and intestine, were quantified by quantitative real time PCR. Messenger RNA expression levels of Pmcyclin B were greatest in the ovary, compared to other tissues (P < 0.05). Temporal expression of Pmcyclin B in the ovary at six different developmental stages was investigated by real-time PCR; no significant difference was observed (P < 0.05). Recombinant Pmcyclin B protein and its polyclonal antibody were successfully produced. Western blot analysis revealed differential expression of Pmcyclin B in ovaries in developmental stages II to IV; a positive signal (45 kDa) was observed in all ovarian stages assessed, but was most intense at stage III. Pmcyclin B protein was assessed by immunohistochemistry and was localized to the cytoplasm of prophase oocytes at stage II and enriched in the nuclei of pro-metaphase oocytes at stages III and IV. Results from this study indicate that Pmcyclin B is constitutively expressed and plays an important role in ovarian maturation in P. monodon.

Molecular cloning, expression profiles and subcellular localization of cyclin B in ovary of the mud crab, Scylla paramamosain

A full-length cDNA of cyclin B was isolated from ovary of the mud crab (Scylla paramamosain) in this study. This transcript encodes a polypeptide of 401 amino acids, which is highly homologous to cyclin B protein family. Reverse transcription-PCR (RT-PCR) showed that cyclin B mRNA was expressed at highest levels in ovary of the mud crab. During the ovarian maturation process, real-time RT-PCR revealed that the abundance of cyclin B mRNA increased from the second stage (early-developing stage) to the fourth stage (nearly-ripe stage) and reached the peak level at the fifth stage (ripe stage). This result indicates the identified cyclin B gene might be related to the cell proliferation in ovary, both mitotically and meiotically. Immunohistochemistry showed that cyclin B protein was localized in the cytoplasm of prophase oocytes at the second stage while enriched in the nuclei of pro-metaphase oocytes at the fourth stage. It suggests the tested cyclin B protein might play different roles in ovary at the two stages.

Female germ cell renewal during the annual reproductive cycle in Ostariophysians fish

The objective was to characterize female germ cell renewal during the annual reproductive cycle in two species of ostariophysian fish with distinct reproductive strategies: a siluriform, Pimelodus maculatus, in which oocyte development is group synchronous and the annual reproductive period is short; and a characiform, Serrasalmus maculatus, with asynchronous oocyte development and a prolonged reproductive period. These reproductive strategies result in fish determinate and indeterminate fecundity, respectively. Annual reproductive phases were determined by biometric and histologic analysis of gonads and interpreted according to new proposals for phase classification and stages of oocyte development (with special attention to germinal epithelium activity). Histologically, there were two types of oogonia in the germinal epithelium: single oogonia and those in mitotic proliferation. Oogonial proliferation and their entry into meiosis resulted in formation of cell nests (clusters of cells in the ovarian lamellae). Morphometric analysis was used to estimate germ cell renewal. Based on numbers of single oogonia in the lamellar epithelium, and nests with proliferating oogonia or early prophase oocytes throughout the annual reproductive cycle, oogonial proliferation and entrance into meiosis were more intense during the regenerating phase and developing phase, but decreased sharply (P < 0.05) during the spawning-capable phase. Oogonial proliferation gradually recovered during the regressing phase. We concluded that, independent of species or features of the reproductive cycle, germ cell renewal occurred during the regenerating phase, ensuring availability of eggs for the spawning event.

Peri-ovulatory putrescine to reduce aneuploid conceptions

Women experience diminished fertility, increased risk of miscarriages and congenital birth defects in their late 30s and early 40s, 10-15 years before reaching menopause. Egg aneuploidy (having an incorrect number of chromosomes) is the most important etiology for these reproductive problems in older women. Science correspondent Jon Cohen wrote a News piece in 2002 [1] on the quest to understand the mechanisms of maternal aging-related aneuploidies in humans, which is still applicable today. Although we have made impressive progress in understanding how aneuploidies arise, we know little about providing medical interventions to reduce them. “My Holy Grail is to deliver some sort of aneuploidy-reduction pill, …”, Dr. Terry Hassold, a leading geneticist in the field, is quoted as saying. Our recent study [2] may be a step towards developing such a pill. Aneuploid eggs result in aneuploid conceptions, most of which are lost either prior to clinical recognition of pregnancies or by miscarriages. The small proportion that survive inevitably carry major birth defects. Accurate egg aneuploidy rates in the general population are not known, but numerous analyses of IVF samples have revealed staggeringly high rates. Notably, a recent study of more than 20,000 IVF eggs revealed significant age-related aneuploidies, reaching >40% in the 40 year old age group [3]. Furthermore, this study [3] employs fluorescence in situ hybridization of polar bodies and is therefore limited in the number of chromosomes (five) that can be analyzed simultaneously. The true aneuploidy rates in older women, considering all 23 chromosomes, would be expected to be much higher. This unusually high egg aneuploidy rate in humans is likely due to a combination of our longevity and the peculiar oogenesis (the process of generating eggs) in all vertebrates including humans. Unlike vertebrate males that continuously generate mature sperm from germ line stem cells through adulthood, in vertebrates oogenesis begins during the embryonic stage when germ line stem cells initiate meiosis and develop into primary oocytes. By birth, the females have developed a finite number of primary oocytes arrested in meiotic prophase that comprises their lifetime egg supply. The prophase oocytes contain chromosome bivalents each having four chromatids linked together by a combination of sister chromotid cohesion and non-sister cross-over (Figure ​(Figure1),1), with this linkage persisting for decades in women. During ovulation the two sister chromatids are segregated to the same cell (egg or 1st polar body) in a process called oocyte maturation; in the subsequent fertilization, the sisters are segregated between the 2nd polar body and the haploid egg. As females age, chromosome cohesion becomes progressively weakened [4], predisposing the oocytes to greater risk of premature separation of sister chromatids (PSSC) during oocyte maturation [2-5] (Figure ​(Figure11). Figure 1 Premature separation of sister chromatids (PSSC) during oocyte maturation Given the great time lapse, it seems daunting that any measure can be found to keep these primary oocytes “fresh” while women age. Our study however suggests that a metabolic deficiency during ovulation in older females, which can be easily corrected, is an important compounding factor in age-related egg aneuploidies. It has been known for more than four decades [6] that during mammalian ovulation, the ovaries exhibit a transitory (i.e. a few hours) rise in the enzyme ornithine decarboxylase (ODC) and its product putrescine and yet its physiological function remains enigmatic. We found that inhibition of ODC during ovulation results in significant increase of incidence of PSSC in eggs of young mice. Interestingly, older mice exhibit a diminished ODC rise in the ovaries, and a corresponding increase of PSSC in eggs. Most remarkably, a combination of putrescine supplementation in mouse drinking water leading up to oocyte retrieval and in oocyte maturation medium reduced the incidence of PSSC in eggs by more than 50% [2]. Putrescine is a naturally occurring, small metabolite easily absorbed and distributed throughout the organism. A high dose of putrescine supplementation similar to that used in our study has been given safely to neonatal calves and pigs as an ingredient of soybean-based milk replacement [7]. However, post-conception putrescine supplementation in mouse drinking water compromises fetal development, likely due to excess reactive oxygen species produced by placental diamine oxidase oxidizing the exogenous putrescine [8]. In contrast, periovulatory putrescine supplementation is safe for gestation, because exogenous putrescine administered around ovulation is cleared long before placental development (Tao and Liu, in preparation). Nonetheless, further studies, including that employing primates, are required to formulate effective and safe putrescine supplementation protocols to reduce egg aneuploidies, both in IVF clinics and in natural conception.

Bim Mediates Germ Cell Death During Ovarian Development.

Normal ovarian development in mice is characterized by the initial expansion of germ cells followed by the death of more than two thirds of their maximum number. The final result of germ cell mitosis and apoptosis during embryogenesis is a fixed number of oocytes stored within the ovary as primordial follicles just after birth, from which all ovulated oocytes are derived. Thus, apoptosis plays a critical role in determining the number of oocytes endowed within the ovary and the fertile lifespan of the female. In this study, we investigated the role of Bim (Bcl-2 interacting mediator of cell death), a pro-apoptotic protein belonging to the BH3-only subgroup of the Bcl-2 family of apoptotic regulators, in mediating cell death during the establishment and maintenance of the primordial follicle pool. Immunohistochemical (IHC) staining revealed that Bim protein is expressed within a small subset of mouse oocytes in meiotic prophase at embryonic day (E) 15.5 to E17.5, with minimal expression detected in oocytes once primordial follicle assembly is complete at post natal (PN) day 5. Analysis of follicle numbers at PN10 showed that bim-/- mice had significantly more primordial follicles than WT (2.76 ± 0.25 vs 1.51 ± 0.23 follicles/104μm2, P<0.05), supporting an important role for Bim in determining the initial size of the primordial follicle pool. Furthermore, while quantitative real-time polymerase chain reaction (QRT-PCR), in situ hybridisation (ISH) and IHC analysis revealed the induction of Bim expression within primordial follicle oocytes following ?-irradiation-induced DNA damage, Bim deficiency could not rescue oocytes from ?-irradiation induced apoptosis, probably due to the presence of other BH3-only family members, most likely Puma and Noxa. In summary, this study supports a non-redundant and essential role for pro-apoptotic Bim in the elimination of germ cells during ovarian development and in the establishment of the primordial follicle reserve. This work was supported by the NHMRC of Australia (Program Grants #494802 and #257502, Fellowships JKF (#441101), KJH (#494836), CLS (#406675), AS (#461299); Victorian Government's Operational Infrastructure Support Program.

DNA Methylation, Histone Modifications and Behaviour of AKAP95 during Mouse Oocyte Growth and Upon Nuclear Transfer of Foreign Chromatin into Fully Grown Prophase Oocytes

The poor efficiency of mammalian cloning is due to inappropriate/incomplete epigenetic reprogramming of the donor chromatin. As the success in reprogramming of the donor nucleus may require activity of similar mechanisms which reprogram the chromatin in the course of gametogenesis, we decided to follow the status of some epigenetic markers in the late phase of oogenesis in mice, i.e. in prophase oocytes during their growth and after completion of the growth phase. Our analysis reveals an increase in the level of global DNA methylation starting in oocytes with diameters around 60 microm which was further elevated until completion of oocyte growth. A similar increase was observed in respect to the acetylation of histone H4. On the other hand, the methylation of histone H4 Arg3 was constantly high until the end of oocyte growth, although it differed between fully grown oocytes depending on the type of spatial chromatin organization. We have also studied the AKAP95 protein which was abundant at earlier stages but decreased in fully grown oocytes according to changes in their chromatin organization. The nuclear transfer of different types of donor nuclei with hypomethylated DNA into fully grown prophase oocytes did not increase the global level of methylation of transferred foreign chromatin, regardless if the recipient oocyte was devoid of its own nucleus or its nucleus was left intact. This suggests a major problem in the ability of recipient oocytes to modify donor DNA methylation.

Changes in the activity of defense mechanisms against induced acidosis during meiotic maturation in mouse oocytes

Previous work has indicated that although activity of the HCO3-/Cl- exchanger (AE), which regulates intracellular alkalosis, is high in the germinal vesicle (GV) stage, the oocyte is inhibited as it progresses through meiotic maturation. In this study, we aimed to investigate the defense mechanisms against acidosis during the meiotic maturation stages. Intracellular pH (pHi) was recorded using a microspectrofluorometric technique, and Na+/H+ (NHE) and Na+-dependent HCO3-/Cl- exchanger (NDCBE) activity were determined by measuring the recovery rate from induced acidosis. Additionally, SLC9A1 (for NHE) and SLC4A8 (for NDCBE) gene transcription levels were determined by real-time PCR. The recovery rate of first meiotic prophase (GV) oocytes was high, but it decreased during the meiotic metaphase II (MII) stage in HCO3--free medium; it became high again at the pronuclear zygote (PN) stage. Recovery rate was significantly inhibited by 5-(N-ethyl-N-isopropyl) amiloride and cariporide or in the absence of extracellular Na+, implicating NHE, specifically NHE1 activity. Moreover, the level of SLC9A1 transcription correlated with the observed changes in NHE activity. The changes in NHE activity during meiotic maturation displayed a similar pattern to that of AE. The recovery rate from acidosis was significantly higher in MII stage oocytes and PN zygotes in HCO3--containing medium; however, the increase was significantly inhibited in Na+-free medium or 4,4'-diisocyanatostilbene-2,2'-disulfonic acid. Furthermore, changes in the transcription of SLC4A8 during meiotic maturation were concordant with the level of exchanger activity. These results indicate that NDCBE activity is present in mouse oocytes and zygotes, and that this activity exhibits a different pattern than that of AE and NHE during meiotic maturation.

Comparison of reproductive development in triploid and diploid female rainbow trout Oncorhynchus mykiss

The diploid rainbow trout Oncorhynchus mykiss reached sexual maturity 3 years after hatching and its oogenesis underwent four stages, which were oogonia, primary oocyte, secondary oocyte and egg. Reproductive development and hormone changes of 4 to 35 month-old female O. mykiss were investigated using histological and radioimmunoassay methods in order to provide a theoretical and practical basis for the use of triploid female O. mykiss. The oogonium of the triploid female could develop into the oocytes of the prophase with abortion occurring later; the oogonium was surrounded by stroma cells to form the oogonium cluster and the gonads showed a virilescent tendency when the oogonium clusters were gradually replaced by spermatogenic-like cytocysts. After 13 months, amounts of gonadotropic hormone (GtH-I, GtH-II) and oestradiol (17beta-E(2)) in triploid females were lower than in diploid fish at corresponding time periods, but the amounts of testosterone (T) increased consistently after 21 months and were more than in diploid fish in the corresponding time periods (P > 0.05). The infertility of triploid females resulted from meiosis failure, which caused developmental abortion of oocytes and oogonium formed cytocysts before the prophase oocytes. The cytocyst formation was due to the lack of the normal interaction of ovum and follicular cells, the development of follicular cells producing steroids were inhibited, the arylate path from T to 17beta-E(2) was interrupted, concentration of 17beta-E(2) decreased and concentration of T increased in the blood, the content of vitellogenin (Vg) decreased in the liver with a low 17beta-E(2) and high T caused to ovaries to show a tendency to be virilescent.

Histone H3 lysine 4 methylation is associated with the transcriptional reprogramming efficiency of somatic nuclei by oocytes

BackgroundWhen the nuclei of mammalian somatic cells are transplanted to amphibian oocytes in the first meiotic prophase, they are rapidly induced to begin transcribing several pluripotency genes, including Sox2 and Oct4. The more differentiated the donor cells of the nuclei, the longer it takes for the pluripotency genes to be activated after the nuclear transfer to oocytes. We have used this effect in order to investigate the role of histone modifications in this example of nuclear reprogramming.ResultsReverse transcription polymerase chain reaction analysis shows that the transcriptional reprogramming of pluripotency genes, such as Sox2 and Oct4, takes place in transplanted nuclei from C3H10T1/2 cells and from newly differentiated mouse embryonic stem cells. We find that the reprogramming of 10T1/2 nuclei is accompanied by an increased phosphorylation, an increased methylation and a rapidly reduced acetylation of several amino acids in H3 and other histones. These results are obtained by the immunofluorescent staining of transplanted nuclei and by Western blot analysis. We have also used chromatin immunoprecipitation analysis to define histone modifications associated with the regulatory or coding regions of pluripotency genes in transplanted nuclei. Histone phosphorylation is increased and histone acetylation is decreased in several regulatory and gene coding regions. An increase of histone H3 lysine 4 dimethylation (H3K4 me2) is seen in the regulatory regions and gene coding region of pluripotency genes in reprogrammed nuclei. Furthermore, histone H3 lysine 4 trimethylation (H3K4 me3) is observed more strongly in the regulatory regions of pluripotency genes in transplanted nuclei that are rapidly reprogrammed than in nuclei that are reprogrammed slowly and are not seen in β-globin, a gene that is not reprogrammed. When 10T1/2 nuclei are incubated in Xenopus oocyte extracts, histone H3 serine 10 (H3S10) is strongly phosphorylated within a few hours. Immunodepletion of Aurora B prevents this phosphorylation.ConclusionWe conclude that H3K4 me2 and me3 are likely to be important for the efficient reprogramming of pluripotency genes in somatic nuclei by amphibian oocytes and that Aurora B kinase is required for H3S10 phosphorylation which is induced in transplanted somatic cell nuclei.

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes – A method for quantitative transcriptional reprogramming

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis.Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein.