Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1 and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-Tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.
oocyte; ubiquitin; proteasome; UCH
Embryos produced by somatic cell nuclear transfer (SCNT) display low term developmental potential. This is associated with deficiencies in spindle composition prior to activation and at early mitotic divisions, including failure to assemble certain proteins on the spindle. The protein-deficient spindles are accompanied by chromosome congression defects prior to activation and during the first mitotic divisions of the embryo. The molecular basis for these deficiencies and how they might be avoided are unknown. Proteomic analyses of spindles isolated from normal metaphase II (MII) stage oocytes and SCNT constructs, along with a systematic immunofluorescent survey of known spindle-associated proteins were undertaken. This was the first proteomics study of mammalian oocyte spindles. The study revealed four proteins as being deficient in spindles of SCNT embryos in addition to those previously identified; these were clathrin heavy chain (CLTC), aurora B kinase, dynactin 4, and casein kinase 1 alpha. Due to substantial reduction in CLTC abundance after spindle removal, we undertook functional studies to explore the importance of CLTC in oocyte spindle function and in chromosome congression defects of cloned embryos. Using siRNA knockdown we demonstrated an essential role for CLTC in chromosome congression during oocyte maturation. We also demonstrated rescue of chromosome congression defects in SCNT embryos at the first mitosis using CLTC mRNA injection. These studies are the first to employ proteomics analyses coupled to functional interventions to rescue a specific molecular defect in cloned embryos.
somatic cell nuclear transfer; meiosis; oogenesis; gene expression; spindle assembly
To examine whether spontaneous oocyte activation is determined by genetic differences and interacted with culture environment.
Temple University School of Medicine.
C57BL/6, DBA/2, C3H/HeJ, and A/J strains, along with reciprocal F1 hybrid female mice (5–6 weeks).
Immature oocytes from different mouse strains were collected and cultured in different maturation conditions including different serum, serum replacement, bovine serum albumin (BSA) and follicle stimulation hormone (FSH).
Main Outcome Measure(s)
The emission of first polar body, pronucleus formation, meiotic arrest, spontaneous activation, and expression of maturation regulators.
Oocytes from C57BL/6 mice display a high rate of delayed first meiotic division and spontaneous activation after the first meiotic division with in vitro maturation (IVM), and the second meiosis with in vivo maturation (VVM) following superovulation. Spontaneous activation with IVM is sensitive to culture environment. Oocytes spontaneously activated during the first meiotic division with IVM have unusual paired tetrad chromosomes with slight connections at centromeres, whereas oocytes activated in vivo display haploidization from the second meiosis. Spontaneous activation is also seen in F1 hybrid oocytes, indicating a dominant trait from C57BL/6. Delayed meiosis was associated with reduced cylcin B and securin expression.
Both mouse strain and culture environment have a significant effect on the incidence of meiotic defects and spontaneous activation. Reduced expression of meiotic regulators may underlie this effect.
oocyte; meiosis; in vitro maturation; spontaneous activation; assisted reproduction
Mouse embryos display a strain-dependent propensity for blastomere cytofragmentation at the two-cell stage. The maternal pronucleus exerts a predominant, transcription-dependent effect on this phenotype, with lesser effects of the ooplasm and the paternal pronucleus. A parental origin effect has been observed as an inequality in the cytofragmentation rate of embryos produced through genetic crosses of reciprocal F1 hybrid females. To understand the basis for this, we conducted an extensive series of pronuclear transfer studies employing different combinations of inbred and F1 hybrid maternal and paternal genotypes. We find that the parental origin effect is the result of a transgenerational epigenetic modification, whereby the inherited maternal grandpaternal contribution interacts with the fertilizing paternal genome and the ooplasm. This result indicates that some epigenetic information related to grandparental origins of chromosomes (i.e., imprinting of chromosomes in the mother) is retained through oogenesis and transmitted to progeny, where it affects gene expression from the maternal pronucleus and subsequent embryo phenotype. These results reveal for the first time that mammalian embryonic development can be affected by the epigenotype of at least three individuals. Additionally, we observe a significant suppression of fragmentation by F1 hybrid ooplasm when it is separated from the F1 hybrid maternal pronucleus. This latter effect is a striking example of heterosis in the early mammalian embryo, and it provides a new opportunity for examining the molecular mechanisms of heterosis. These results are relevant to our understanding of the mechanisms of epigenetic effects on development and the possible fertility effects of genetic and epigenetic interactions in reproductive medicine.
Summary: Cytofragmentation in two-cell mouse embryos is controlled by strain-specific factors, epigenetic information from the maternal grandfather, and a striking hybrid vigor effect mediated by the ooplasm
apoptosis; cytofragmentation; embryo; gene regulation; genomic imprinting; heterosis; mitochondria; nuclear transfer; oocyte development; parental origin effects; superovulation; transgenerational effect
Over the last decade, we have witnessed an explosion of information on genetic factors underlying common human diseases and disorders. This ‘human genomics’ information revolution has occurred as a backdrop to a rapid increase in the rates of many human disorders and diseases. For example, obesity, Type 2 diabetes, asthma, autism spectrum disorder and attention deficit hyperactivity disorder have increased at rates that cannot be due to changes in the genetic structure of the population, and are difficult to ascribe to changes in diagnostic criteria or ascertainment. A likely cause of the increased incidence of these disorders is increased exposure to environmental factors that modify gene function. Many environmental factors that have epidemiological association with common human disorders are likely to exert their effects through epigenetic alterations. This general mechanism of gene–environment interaction poses special challenges for individuals, educators, scientists and public policy makers in defining, monitoring and mitigating exposures.
aging; behavior; DNA methylation; endocrine disruptors; maternal diet; windows of sensitivity
To determine if preimplantation embryos are targets for relaxin secreted from the corpus luteum of the menstrual cycle.
Rhesus monkey oocytes obtained from females undergoing controlled ovarian hyperstimulation were inseminated and the resulting embryos were cultured in medium with or without recombinant human relaxin (20 ng/ml) for 8 days.
Controlled ovarian stimulation to obtain oocytes for in vitro produced embryos that were cultured with or without human recombinant relaxin.
MAIN OUTCOME MEASURES
The rate of blastocyst development and the percentage of blastocysts and ICM/TE ratio were measured on Day 8 of culture. The presence of relaxin receptor (RXFP1) mRNA in 8 cell embryos was observed by array hybridization.
RXFP1 receptor expression was localized to the inner cell mass of blastocysts as shown by immunohistochemistry. The percentage of embryos that developed to blastocyst and the inner cell mass/ trophectoderm cell ratio was unchanged with relaxin supplementation, however the relaxin-treated embryos developed into blastocysts significantly sooner than untreated embryos.
These results are the first evidence that the preimplantation primate embryo is a target for relaxin and that the addition of relaxin to in vitro culture medium enhances rhesus monkey embryo development.
gene expression; granulosa cells; blastocyst; rhesus macaque
Two essential aspects of mammalian development are the progressive specialization of cells toward different lineages, and the maintenance of progenitor cells that will give rise to the differentiated components of each tissue and also contribute new cells as older cells die or become injured. The transition from totipotentiality to pluripotentiality, to multipotentiality, to monopotentiality, and then to differentiation is a continuous process during development. The ontological relationship between these different stages is not well understood. We report for the first time an ontological survey of expression of 45 putative “stemness” and “pluripotency” genes in rhesus monkey oocytes and preimplantation stage embryos, and comparison to the expression in the inner cell mass, trophoblast stem cells, and a rhesus monkey (ORMES6) embryonic stem cell line. Our results reveal that some of these genes are not highly expressed in all totipotent or pluripotent cell types. Some are predominantly maternal mRNAs present in oocytes and embryos before transcriptional activation, and diminishing before the blastocyst stage. Others are well expressed in morulae or early blastocysts, but are poorly expressed in later blastocysts or ICMs. Also, some of the genes employed to induce pluripotent stem cells from somatic cells (iPS genes) appear unlikely to play major roles as stemness or pluripotency genes in normal embryos.
stem cell; cell lineage; embryo; trophoblast
Growth hormone (GH) in rhesus macaque in vitro oocyte maturation (IVM) has been shown to increase cumulus expansion and development of embryos to the 9–16 cell stage in response to 100 ng/ml recombinant human GH (r-hGH) supplementation during IVM. Although developmental endpoints for metaphase II (MII) oocytes and embryos are limited in the macaque, gene expression analysis can provide a mechanism to explore GH action on IVM. In addition, gene expression analysis may allow molecular events associated with improved cytoplasmic maturation to be detected. In this study, gene expression of specific mRNAs in MII oocytes and cumulus cells that have or have not been exposed to r-hGH during IVM was compared. In addition, mRNA expression was compared between in vitro and in vivo-matured MII oocytes and germinal vesicle (GV)- stage oocytes. Only two of 17 genes, insulin-like growth factor 2 (IGF2) and steroidogenic acute regulator (STAR), showed increased mRNA expression in MII oocytes from the 100 ng/ml r-hGH treatment group compared with other IVM treatment groups, implicating insulin-like growth factor (IGF) and steroidogenesis pathways in the oocyte response to GH. The importance of IGF2 is notable, as expression of IGF1 was not detected in macaque GV-stage or MII oocytes or cumulus cells.
non-human primate; growth hormone; oocyte; gene expression
There is a vital need to identify factors that enhance human and nonhuman primate in vitro embryo culture and outcome, and to identify the factors that facilitate that objective. Granulosa and cumulus cells were obtained from rhesus monkeys that had either been FSH-primed (in vitro maturation [IVM]) or FSH and hCG-primed (in vivo maturation [VVM]) and compared for the expression of mRNAs encoding follistatin (FST), inhibin, and activin receptors. The FST mRNA displayed marginally decreased expression (P = 0.05) in association with IVM in the granulosa cells. The ACVR1B mRNA was more highly expressed in cumulus cells with IVM compared with VVM. Cumulus-oocyte complexes from FSH-primed monkeys exposed to exogenous FST during the 24-h IVM period exhibited no differences in the percentage of oocytes maturing to the metaphase II stage of meiosis compared to controls. However, embryos from these oocytes had significantly decreased development to the blastocyst stage. The effect of FST on early embryo culture was determined by exposing fertilized VVM oocytes to exogenous FST from 12 to 60 h postinsemination. FST significantly improved time to first cleavage and embryo development to the blastocyst stage compared with controls. The differential effects of exogenous FST on embryo development, when administered before and after oocyte maturation, may depend on the endogenous concentration in cumulus cells and oocytes. These results reveal evolutionary conservation of a positive effect of FST on embryogenesis that may be broadly applicable to enhance in vitro embryogenesis, with potential application to human clinical outcome and livestock and conservation biology.
Follistatin supplementation of culture medium after fertilization improves primate embryo development, while addition during in vitro maturation decreases oocyte developmental potential.
embryo; gene expression; gene regulation; granulosa cells; oocyte development; ovary; rhesus macaque
The ooplasm plays a central role in forming the paternal pronucleus, and subsequently in regulating the expression of paternally inherited chromosomes. Previous studies in mice have revealed genetic differences in paternal genome processing by ooplasm of different genotypes. Ooplasm donation coupled to intracytoplasmic sperm injection (ICSI) has been used in human assisted reproductive technology (ART). This procedure exposes the developing paternal pronucleus to ‘foreign’ ooplasm, which may direct aberrant epigenetic processing. The potential effects of the foreign ooplasm on epigenetic information in the paternal pronucleus are unknown; however, some human progeny from ooplasm donation procedures display abnormalities.
In this study, we employed inter-genotype ooplasm transfer followed by ICSI using two mouse strains, C57BL/6 and DBA/2, to explore the influence of foreign ooplasm on paternal pronucleus function. In order to assay for effects on the paternal genome without masking effects of the maternal genome, we examined ooplasm effects in diploid androgenones, which are produced by pronuclear transfer to contain exclusively two paternal sets of chromosomes, in combination with ICSI.
There was no significant effect of intra-strain ooplasm transfer among androgenones made with either C57BL/6 or DBA/2 oocytes. There was a significant negative effect on androgenone blastocyst development with inter-genotype transfer (10% volume) of DBA/2 ooplasm to C57BL/6 oocytes (P < 0.05). The reciprocal inter-genotype ooplasm transfer had no significant effect.
Thus, inter-genotype ooplasm transfer in conjunction with ICSI can alter the function of the paternal genome. However, the effect of foreign ooplasm is restricted to a negative effect, with no evidence of a positive effect. This study provides important new information about the possible consequences of ooplasm donation in human ART.
ooplasm donation; androgenone; parthenogenone; IVF; ICSI
DDK syndrome is the polar-lethal embryonic death that occurs at the morula-blastocyst transition when female mice of the DDK strain are mated with males from many other inbred strains (so-called “alien” males). Embryonic death is caused by incompatibility between a DDK oocyte factor and an alien male gene, both of which map to the Om locus on mouse chromosome 11. We have compared global transcription patterns of DDK X DDK embryos (high viability) and DDK X C57BL/6 embryos (low viability) at the morula stage, approximately 24 h before any morphological manifestations of DDK syndrome are observed. Of the transcripts that are differentially more abundant in the DDK X C57BL/6 embryos, we noted that many are the products of genes induced by the “unfolded protein response”. We confirmed that a number of genes in this pathway are up-regulated in the DDK X C57BL/6 embryos by quantitative RT-PCR. Immunostaining of the endoplasmic reticulum (ER) marker BIP/GRP78 (immunoglobin-binding protein/glucose-regulated protein of 78 kDa), official symbol HSPA5, heat shock protein 5. revealed an accompanying abnormal HSPA5 accumulation and ER structure in the DDK X C57BL/6 embryos. Immunostaining for HERPUD1 (homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1) and ATF4 (activating transcription factor 4) also revealed accumulation of these stress-response products. Our results indicate that the unfolded protein response is induced in embryos destined to die from DDK syndrome and that the embryonic death observed is associated with inability to resolve the associated ER stress.
DDK syndrome is the polar-lethal embryonic death that occurs at the morula-blastocyst transition when female mice of the DDK strain are mated with males from many other inbred strains (so-called alien males). Embryonic death is caused by incompatibility between a DDK oocyte factor and an alien male gene, both of which map to the Om locus on mouse chromosome 11. We compared global transcription patterns of DDK × DDK embryos (high viability) and DDK × C57BL/6 embryos (low viability) at the morula stage, approximately 24 h before any morphological manifestations of DDK syndrome are observed. Of the transcripts that are differentially more abundant in the DDK × C57BL/6 embryos, many are the products of genes induced by the “unfolded protein response.” We confirmed by quantitative RT-PCR that a number of genes in this pathway are upregulated in the DDK × C57BL/6 embryos. Immunostaining of the endoplasmic reticulum (ER) marker BIP/GRP78 (immunoglobin-binding protein/glucose-regulated protein of 78 kDa), official symbol HSPA5, heat shock protein 5 revealed an accompanying abnormal HSPA5 accumulation and ER structure in the DDK × C57BL/6 embryos. Immunostaining for HERPUD1 (homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1) and ATF4 (activating transcription factor 4) also revealed accumulation of these stress-response products. Our results indicate that the unfolded protein response is induced in embryos destined to die of DDK syndrome and that the embryonic death observed is associated with inability to resolve the associated ER stress.
The unfolded protein response contributes to embryonic death in the DDK syndrome.
DDK syndrome; early development; embryo; epigenetics; unfolded protein response
MicroRNAs (miRNAs) are a class of small RNAs that silence gene expression. In animal cells, miRNAs bind to the 3′ untranslated regions of specific mRNAs and inhibit their translation. The correct regulation of mRNA expression by miRNAs is believed to be important for oocyte maturation, early development and implantation. We examined the expression of 25 mRNAs involved in the microRNA processing pathway in a non human primate oocyte and embryo model. We observed that mRNAs related to miRNA splicing are downregulated during oocyte maturation while those related to miRNA processing are upregulated, indicating that there may exist a temporal difference in their activities related to transcriptional activity in germinal vesicle stage oocytes. We also observed that the vast majority of mRNAs examined were insensitive to α-amanitin at the 8-16 cell stage. The expression data did not reveal a major impact of embryo culture, and hormonal stimulation protocol affected only a small number of mRNAs, suggesting that the components of the pathway may be accumulated in the oocyte during oogenesis and resistant to exogenous insults. In comparison to published mouse array data, we observed species differences and similarities in the temporal expression patterns of some genes, suggesting that miRNA processing may be regulated differently. These data extend our understanding of the potential roles of miRNA during primate embryogenesis.
miRNA; embryo; oocyte; gene regulation; oocyte quality; in vitro maturation; cleavage; preimplantation embryo
New techniques to boost male and female fertility are being pioneered at a rapid pace in fertility clinics to increase the efficiency of assisted reproduction methods in couples in which natural conception has not been achieved. This study investigates the possible epigenetic effects of ooplasm manipulation methods on postnatal growth and development using a mouse genetic model, with particular emphasis on the possible effects of intergenotype manipulations. We performed interstrain and control intrastrain maternal pronuclear transfers, metaphase-II spindle transfers, and ooplasm transfer between C57BL/6 and DBA/2 mice, and found no major, long-term growth defects or epigenetic abnormalities, in either males or females, associated with intergenotype transfers. Ooplasm transfer itself was associated with reduced viability, and additional subtle effects of ooplasm strain of origin were observed. Both inter- and intrastrain ooplasm transfer were associated with subtle, transient effects on growth early in life. We also performed inter- and intrastrain germinal vesicle transfers (GVTs). Interstrain GVT females, but not males, had significantly lower body weights at birth and thereafter compared with the intrastrain GVT and non-GVT controls. No GVT-associated changes were observed in DNA methylation of the Mup1, Rasgrf1, H19, Snrpn, or Peg3 genes, nor any difference in expression of the imprinted Rasgrf1, Igf2r, or Mest genes. These results indicate that some ooplasm manipulation procedures may exert subtle effects on growth early in life, while intergenotype GVT can result in significant growth deficiencies after birth.
Ooplasm manipulation studies in mice reveal effects of cytoplasm transfer and germinal vesicle transfer on growth.
assisted reproduction; assisted reproductive technology; DNA methylation; embryo; germinal vesicle transfer; nuclear transfer; oocyte; ovum
CTCF is a multifunctional nuclear factor involved in epigenetic regulation. Despite recent advances that include the systematic discovery of CTCF-binding sites throughout the mammalian genome, the in vivo roles of CTCF in adult tissues and during embryonic development are largely unknown. Using transgenic RNAi, we depleted maternal stores of CTCF from growing mouse oocytes, and identified hundreds of misregulated genes. Moreover, our analysis suggests that CTCF predominantly activates or derepresses transcription in oocytes. CTCF depletion causes meiotic defects in the egg, and mitotic defects in the embryo that are accompanied by defects in zygotic gene expression, and culminate in apoptosis. Maternal pronuclear transfer and CTCF mRNA microinjection experiments indicate that CTCF is a mammalian maternal effect gene, and that persistent transcriptional defects rather than persistent chromosomal defects perturb early embryonic development. This is the first study detailing a global and essential role for CTCF in mouse oocytes and preimplantation embryos.
CTCF; Mouse; Oocyte; Preimplantation embryo; Meiosis
Genomic imprinting has dramatic effects on placental development, as has been clearly observed in interspecific hybrid, somatic cell nuclear transfer, and uniparental embryos. In fact, the earliest defects in uniparental embryos are evident first in the extraembryonic trophoblast. We performed a microarray comparison of gynogenetic and androgenetic mouse blastocysts, which are predisposed to placental pathologies, to identify imprinted genes. In addition to identifying a large number of known imprinted genes, we discovered that the Polycomb group (PcG) gene Sfmbt2 is imprinted. Sfmbt2 is expressed preferentially from the paternal allele in early embryos, and in later stage extraembryonic tissues. A CpG island spanning the transcriptional start site is differentially methylated on the maternal allele in e14.5 placenta. Sfmbt2 is located on proximal chromosome 2, in a region known to be imprinted, but for which no genes had been identified until now. This possibly identifies a new imprinted domain within the murine genome. We further demonstrate that murine SFMBT2 protein interacts with the transcription factor YY1, similar to the Drosophila PHO-RC.
Genomic Imprinting; Sfmbt2; PcG gene; Extraembryonic tissues; placenta; MMU2; YY1; microarrays; uniparental embryos
Cloned embryos produced by somatic cell nuclear transfer (SCNT) display a plethora of phenotypic characteristics that make them different from fertilized embryos, indicating defects in the process of nuclear reprogramming by the recipient ooplasm. To elucidate the extent and timing of nuclear reprogramming, we used microarrays to analyze the transcriptome of mouse SCNT embryos during the first two cell cycles. We identified a large number of genes mis-expressed in SCNT embryos. We found that genes involved in transcription and regulation of transcription are prominent among affected genes, and thus may be particularly difficult to reprogram, and these likely cause a ripple effect that alters the transcriptome of many other functions, including oxidative phosphorylation, transport across membrane, and mRNA transport and processing. Interestingly, we also uncovered widespread alterations in the maternal (i.e. non transcribed) mRNA population of SCNT embryos. We conclude that gene expression in early SCNT embryos is grossly abnormal, and that this is at least in part the result of incomplete reprogramming of transcription factor genes.
embryo; somatic cell nuclear transfer; reprogramming; microarray; transcription; transcriptome