Preimplantation Embryogenesis Research Articles

Effects of in vitro oocyte maturation and embryo culture on the expression of glucose transporters, glucose metabolism and insulin signaling genes in rhesus monkey oocytes and preimplantation embryos

Glucose plays a fundamental role during oogenesis and embryogenesis, satisfying the metabolic demands of oocytes and embryos, providing for stored energy reserves in the form of glycogen and supporting nucleotide biosynthesis via the pentose phosphate pathway. Glucose also contributes to the production of amino acids, glycosylated proteins and extracellular components. A detailed understanding of the molecular mechanisms that mediate and regulate glucose uptake and metabolism at different stages of oogenesis and preimplantation embryogenesis could greatly benefit the development of improved methods for in vitro oocyte maturation and in vitro embryo production. Although these processes have been examined in a variety of rodent and agricultural species, detailed information has not yet been described for non-human primates. In this study, we examined the expression of the genes encoding glucose transporters, glucose metabolism enzymes and potential regulators of glucose metabolism in rhesus monkey oocytes and embryos. The data reveal stage-specific regulation of expression of specific types of glucose transporters, stage-specific changes in expression of genes related to different pathways of glucose metabolism and temporal changes in the expression of mRNAs related to insulin signaling. Additionally, the data reveal significant differences in expression of some of these genes in cultured embryos as compared with flushed embryos and between oocytes and embryos obtained following different hormonal stimulation and oocyte maturation protocols.

Role of Phosphocholine Cytidylyltransferase α in Lung Development

Lung development depends upon the differentiation and expansion of a variety of specialized epithelial cell types, including distal type I and type II pneumocytes in the late term. Previous studies have shown a strict dependence on the choline cytidylyltransferase alpha isoform (CCTalpha) to mediate membrane phospholipid formation in cultured cells and during preimplantation embryogenesis. CCTalpha expression is highest in lung, and there has long been speculation about its precise role, due to the dual requirement for phospholipid in proliferating cell membranes and for lung surfactant production from alveolar type II cells. We investigated the function of CCTalpha in lung development, using an inducible, epithelial cell-specific CCTalpha knockout mouse line. Deletion of CCTalpha beginning at embryonic day 7.5 did not restrict lung development but resulted in severe respiratory failure at birth. Alveolar lavage and lung lipid analyses showed significant decreases in the major surfactant phospholipid, dipalmitoyl-phosphatidylcholine. The fatty acids destined for the surfactant phospholipid were redirected to an expanded triglyceride pool. Transcripts encoding type II cell-specific markers were expressed in the knockout mice, indicating the expected progression of differentiation in lung epithelia. However, surfactant protein levels were reduced, with the exception of that for surfactant protein B, which was elevated. Ultrastructural analysis of the type II cells showed Golgi complex abnormalities and aberrant lamellar bodies, which deliver surfactant lipid and protein to the alveolar lumen. Thus, CCTalpha was not required for the proliferation or differentiation of lung epithelia but was essential for the secretory component of phospholipid synthesis and critical for the proper formation of lamellar bodies and surfactant protein homeostasis.

111 CHARACTERIZATION OF BOVINE TELOMERASE REVERSE TRANSCRIPTASE DURING PREIMPLANTATION EMBRYOGENESIS

Telomerase is a specialized reverse transcriptase that extends telomeric DNA of eukaryotic chromosomes and is composed of 2 essential subunits, the telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT). Together, this ribonucleoprotein complex is normally highly active in germ cells and stem/progenitor cells but not in normal somatic cells. In humans, this regulation of telomerase activity is primarily coordinated at the level of transcription of the TERT gene, whereas TERC is virtually ubiquitously expressed. Previous studies from our laboratory have detected telomerase activity in embryos from all stages of early bovine development. However, the regulation of the telomerase subunits remains poorly understood. Therefore, the objective of our study was to characterize the expression of bovine TERT (bTERT) during bovine preimplantation embryogenesis. UsingRT-PCR and immunofluorescence staining procedures (n = 20 embryos at timed stages of development; r = 3), we demonstrate that mRNA transcripts and protein for bTERT were detected in preimplantation bovine embryos from 1-cell to the blastocyst stage. The specificity of bTERT PCR products was confirmed by sequencing and demonstrated 94% sequence homology to the human hTERT cDNA sequence. In the immature oocyte, bTERT protein was localized within the germinal vesicle, and after 18–20 h of in vitro maturation, bTERT was observed as doublet foci co-localized with the condensed metaphase II meiotic stage chromosomes. Post-fertilization, the expression of bTERT was observed within the pronuclei. Through the initial cleavage stages, the expression of bTERT was variable, with some blastomeres showing punctuate staining and others exhibiting a uniform staining pattern. By the 8-16 cell stage, the embryos demonstrated a peri-nuclear presence of bTERT. However, in morula and blastocysts, bTERT was localized to the nuclei as demonstrated by co-localization with 42,6-diamidino-2-phenylindole staining. Based on this pattern of expression, it is tempting to speculate that bTERT could be playing an important role in regulating telomerase activity during early embryo development. The translocation of bTERT protein from the cytoplasm to the nucleus in morulae supports the telomere elongation event that is known to occur in mammalian embryos during the transition from the morula to blastocyst stages.

O-146: The clinical impact associated with the use of PICSI TM - derived embryos

Oligozoospermic men requiring intracytoplasmic sperm injection (ICSI) often carry seminal populations demonstrating increased levels of chromosomal aberrations and compromised DNA integrity. The in vitro selection of sperm for ICSI is critical and directly influences the paternal contribution to preimplantation embryogenesis. Hyaluronan (H) is a major constituent of the cumulus oophorous matrix and may play a critical role in the selection of functionally competent sperm during in vivo fertilization. Research has demonstrated that H-bound (HB) sperm carry enhanced levels of developmental maturity, sperm chromatin integrity and normal morphology. HB-sperm may contain increased levels of functional competence. The PICSI ™ plate provides microdrops of H for sperm selection and was used in the study. The pilot study examined the use of HB-sperm in the treatment of ICSI patients. 26 ICSI patients were consented for the IRB-approved study and randomized as to their treatment group. ICSI patients in Group A (ICSI or control) received embryos created using standard sperm selection criteria. Group B (PICSI) patients received PICSI embryos created using HB-sperm and Group C (PICSI + ICSI) patients received control and PICSI embryos. 147 and 126 oocytes received sperm selected using the conventional criteria and HB-sperm, respectively. Resulting embryos (n=177) were scored by two independent embryologists. Oocytes of Group A patients received ICSI using sperm selected via the evaluation of motility and morphology. HB-sperm meeting the same criteria were used in the ICSI of Group B oocytes. In Group C, one-half of the oocytes received sperm selected via standard criteria and the other half received HB-sperm. Embryos were assessed on Days 1, 3, 5 and 6 for morphology. Selection for embryo transfer (ET) was based upon morphological criteria. Statistical significance was tested by the Pearson Chi-Squared Statistic using SPSS for Windows. The fertilization rate carried by the PICSI oocytes (61.1%, n=126) was not statistically different (p = 0.071) from that of the control ICSI oocytes (66.6%, n=147). There were no statistically significant differences observed in zygote, embryo and blastocyst morphologies. Differences in the blastocyst conversion rate were not statistically significant (Group A: 53.2%, B: 52.4%, C: 51.9%, p=0.081). The clinical pregnancy rate (CPR) for Group A patients (25.0%, n=8) was statistically lower than that in B (57.1%, n=7) and in C (57.1%, n=7). The CPR of Groups B and C were statistically greater than the non-study ICSI patients during the same time period (33.3%, n=24). Miscarriage rates (MR) were statistically greater in Group A (p=0.013). The current study suggests that the use of H in sperm selection may enhance our ability to isolate optimal sperm from a compromised seminal population. HB-sperm carry a reduced frequency of chromosomal aneuploidy and may exert a positive paternal influence on preimplantation embryogenesis. Patients receiving PICSI-derived or a combination of PICSI and ICSI embryos demonstrated statistically greater CPR and lower MR over those patients receiving only ICSI embryos. Although the study is in its early stages, the increased CPR and reduced MR associated with the use of HB-sperm in ICSI is promising. The study is being continued to further explore the enhanced embryonic potential demonstrated by PICSI-derived embryos.

Paternal and maternal factors in preimplantation embryogenesis: interaction with the biochemical environment

Paternal effect on embryonic development occurs as early as fertilization. Incorrect formation of the spermatozoon due to centrosome defects and abnormal concentrations of any components involved in the activation process lead to failure immediately or in the subsequent cell cycles. Sperm chromosomal abnormalities result in early embryo developmental arrests. Generally poor spermatozoa lead to poor blastocyst formation. Sperm DNA fragmentation may impair even late post-implantation development. The DNA repair capacity of the oocytes is of major importance. Early preimplantation development, i.e. until maternal to zygotic transition, is maternally driven. Maternal mRNAs and proteins are of major importance, as there is an unavoidable turnover of these reserves. Polyadenylation of these mRNAs is precisely controlled, in order to avoid too early or too late transcription and translation of the housekeeping genes. An important set of maternal regulations, such as DNA stability, transcriptional regulation and protection against oxidative stress, are impaired by age. The embryo biochemical endogenous pool is very important and may depend upon the environment, i.e. the culture medium. Paternal, maternal and environmental factors are unavoidable parameters; they become evident when age impairs oocyte quality.

Expression Pattern of UCH-L1 and UCH-L3 during Mouse Oogenesis and Preimplantation Embryogenesis

Expression Pattern of UCH-L1 and UCH-L3 during Mouse Oogenesis and Preimplantation Embryogenesis

Mitogen-activated protein kinase (MAPK) blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase (ERK) pathways

Blastocyst formation, as a critical period during development, is an effective indicator of embryonic health and reproductive efficiency. Out of a number of mechanisms underlying blastocyst formation, highly conserved mitogen-activated protein kinase (MAPK) signaling has emerged as a major mechanism involved in regulating murine preimplantation embryo development. The objective of our study was to ascertain the role of MAPK signaling in regulating bovine development to the blastocyst stage. Using reverse transcriptase PCR and immunohistochemical staining procedures we have demonstrated that mRNA transcripts and polypeptides encoding p38 MAPK pathway constituents are detectable in preimplantation bovine embryos from the one-cell to the blastocyst stage. Further, the effects on bovine embryo development following inhibition of p38 alpha/beta and extracellular signal-regulated kinase (ERK) signaling by treatment with SB220025 and U0126, respectively, were investigated. Eight-cell bovine embryos (50 per group; three replicates) were placed into treatments consisting of synthetic oviductal fluid (SOF) medium: SOF + SB202474 (inactive analogue), SOF + SB220025, SOF + U0124 (inactive analogue), SOF + U0126, and SOF + SB220025 + U0126. Inhibition of p38 MAPK or ERK signaling individually did not affect development to the blastocyst stage. However, when both pathways were blocked simultaneously there was a significant reduction (P < 0.05) in blastocyst formation, cell number and immunofluorescence of phosphorylated downstream pathway constituents. We have determined that, in variance to what was observed during murine preimplantation development, bovine early embryos progress at normal frequencies to the blastocyst stage in the presence of p38 MAPK inhibitors.

Chromosomal disorders and nuclear and cell destruction in cleaving human embryos

Three types of defects of preimplantation embryogenesis contribute to the developmental arrest of cleaving human embryos: blastomere fragmentation, abnormal nuclear status and chromosomal disorders. Data concerning the relation and succession of these abnormalities during first mitotic cycles of the human zygote are controversial and mainly empirical at present. In this study we have performed simultaneous evaluation of blastomere fragmentation, nuclear apoptotic changes and the ploidy of four chromosomes (1, 5, 19 and X or 18, 21, X and Y) in 193 human embryos. Another group of 28 embryos was subjected to TUNEL for confirmation of apoptosis in blastomere nuclei. Nuclei with apoptotic chromatin were seen in nearly 1/10 of blastomeres of embryos with good morphology and in more than 1/5 of blastomeres of embryos with more than 20% fragmentation. The correct number of investigated chromosomes was registered in 85.2% of successfully tested embryos. Chromatin apoptotic changes are the only limiting factor for the success of chromosomal FISH tests. Nearly 1/2 of embryos with at least one apoptotic nucleus were chromosomally abnormal. For the embryos that contain only normal nuclei, the rate of ploid normality was more than 89%. The rate of euploidy was higher (66%) in embryos with a significant degree of cell fragmentation. Moderate cell fragmentation was not related to significant increase of chromatin and chromosomal disorders. In a substantial portion of abnormal blastomeres, chromatin damage preceded cell fragmentation. Nuclear destruction in human blastomeres was illustrated by fluorograms of different stages of chromatin lesions.

An Update on Embryo Culture for Human Assisted Reproductive Technology: Media, Performance, and Safety

Several culture medium formulations are now available for the successful production and propagation of viable human embryos. In the most popular format, nutrients are provided in a temporal sequence that matches metabolic and amino acid composition with the requirements of specific developmental stage. An alternative philosophy, that all nutritional requirements for preimplantation embryogenesis can be met with a single medium formulation, is represented in commercially available formulations as well. Regardless of format employed, it is not widely appreciated in assisted reproductive technology laboratories that medium performance is strongly influenced by other components of the culture system, such as transitional conditions employed during the retrieval, pH, gas phase, and patient-specific characteristics. There is now further concern that in vitro culture, in general, modifies normal embryonic epigenetic processes and gene expression, genetic changes that may relate to specific ingredients of culture media.

Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte–cumulus cell complex in mice: genetic evidence for an oocyte–granulosa cell regulatory loop

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9 +/− Bmp15 −/− [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 ( Has2) mRNA was impaired in DM oocyte–cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte–granulosa cell regulatory loop.

Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence.

Mitochondria are the most abundant organelles in the mammalian oocyte and early embryo. While their role in ATP production has long been known, only recently has their contribution to oocyte and embryo competence been investigated in the human. This review considers whether such factors as mitochondrial complement size, mitochondrial DNA copy numbers and defects, levels of respiration, and stage-specific spatial distribution, influence the developmental normality and viability of human oocytes and preimplantation-stage embryos. The finding that mitochondrial polarity can differ within and between oocytes and embryos and that these organelles may participate in the regulation of intracellular Ca(2+)homeostasis are discussed in the context of how focal domains of differential respiration and intracellular-free Ca(2+)regulation may arise in early development and what functional implications this may have for preimplantation embryogenesis and developmental competence after implantation.

Role of endonuclease G in neuronal excitotoxicity in mice

Excitotoxicity is a process by which excitatory amino acids induce neuronal cell death. To what extent excitotoxicity is regulated by apoptotic molecules is currently unclear. We previously found that endonuclease G (EndoG) plays an important role in both normal apoptosis in vivo and in pre-implantation embryogenesis. To investigate whether EndoG participates in neuronal cell death, we compared EndoG expression and kainic acid (KA)-induced seizure behavior and excitotoxicity in EndoG +/− and wild-type mice. We found that EndoG expression in the hippocampus of EndoG +/− mice is reduced compared to that in the wild-type mice. The reduction of EndoG expression levels in the hippocampus did not result in altered KA-induced seizure severity in EndoG +/− mice compared to that in wild-type mice. However, both CA3 and CA1 pyramidal neurons in EndoG +/− mice are more resistant to KA-induced cell death than that in wild-type mice. These results indicate that reduced expression of EndoG in the hippocampi of EndoG +/− mice leads to resistance to excitotoxicity.

Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy

Nitric oxide (NO) has been demonstrated to mediate events during ovulation, pregnancy, blastocyst invasion and preimplantation embryogenesis. However, less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this structure is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development, an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO (L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species (ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor that modulates normal yolk sac vascular development, and decreased NO bioavailability and NO-mediated sequela may underlie high glucose induced vasculopathy.

Expression of Smac/DIABLO in mouse preimplantation embryos and its correlation to apoptosis and fragmentation

Regulation of early embryonal development during fertilization and implantation is crucial for mammalian reproduction. Several studies have described cell death during preimplantation embryogenesis in a range of mammalian species, both in vivo and in vitro. Therefore, apoptosis may be involved in early embryonic arrest and the characteristic cytoplasmic fragments are the equivalents of apoptotic bodies, the end-product of apoptosis. Although apoptosis is expected to associate with fragmentation in early preimplantation embryos, the mechanism through which this fragmentation occurs has not been elucidated. Recently, second mitochondria-derived activator of caspase/Direct IAP Binding Protein with Low pI (Smac/DIABLO) was identified as a mitochondrial protein that is released into the cytosol during apoptosis. Once released, the Smac/DIABLO blocks the anti-apoptotic activity of inhibitor of apoptosis proteins (IAPs). We hypothesized that the Smac/DIABLO may be involved in the fragmentation of mouse preimplantation embryos. Therefore, we investigated the expression of Smac/DIABLO mRNA and protein and its localization in mouse oocytes and preimplantation embryos. Smac/DIABLO mRNA was detected by RT-PCR in the oocytes and the preimplantation embryos. Immunohistochemistry studies showed that the Smac/DIABLO protein localized in mitochondria and was released into the cytosol in both fragmented embryos and embryos in which apoptosis was induced by staurosporine. These observations indicate that the Smac/DIABLO is involved in the fragmentation and apoptosis of preimplantation embryos.

Chronic low-dose antiprogestin impairs preimplantation embryogenesis, but not oocyte nuclear maturation or fertilization in rhesus monkeys

Continual administration of low doses of the antiprogestin ZK 137 316 permits ovarian/menstrual cyclicity, but prevents pregnancy in female rhesus monkeys. The sites of contraceptive action remain unknown. This study determined whether chronic, low-dose antiprogestin exposure during follicular development impairs oocyte maturation in vivo, as well as fertilization and preimplantation embryogenesis in vitro. Adult, female rhesus monkeys exhibiting normal menstrual cycles received vehicle ( n=9) or 0.03 mg ZK 137 316 ( n=8)/kg body weight i.m. daily for 3 months. Controlled ovarian stimulation with recombinant gonadotropins was initiated in the 3rd month. Oocytes collected from preovulatory follicles were evaluated for nuclear maturity and inseminated in vitro. Preimplantation embryonic development was monitored in vitro. The total number of oocytes and percentage collected at each nuclear stage were similar in both groups. More ( P<0.05) atretic oocytes were recovered following antiprogestin relative to vehicle treatment. Fertilization rates and percentages of embryos that progressed to the morula stage were similar between groups, but antiprogestin-treated females exhibited less ( P<0.05) normal cleavage. Embryonic development was accelerated by 1 day ( P<0.05) from the 16-cell to the morula stage in the antiprogestin group relative to vehicle. Despite this, the majority of embryos became blastocysts within 6 days in vitro in the antiprogestin group, but fewer expanded ( P=0.09) and hatched ( P<0.05) compared to vehicle. During in vivo treatment with chronic, low-dose antiprogestin, oocytes retained their ability to resume and complete meiosis as well as fertilize following insemination in vitro. However, preimplantation embryogenesis in vitro was impaired, particularly during the later stages of blastocyst development. Thus, antiprogestin exposure during follicular development altered oocyte functions that are critical for normal preimplantation embryogenesis; this may contribute to pregnancy prevention.

A retrospective analysis: seasonal decline in implantation rates (IR) and its correlation with increased levels of volatile organic compounds (VOC)

A retrospective analysis: seasonal decline in implantation rates (IR) and its correlation with increased levels of volatile organic compounds (VOC)

Identification of Gasz, an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats, a sterile-alpha motif, and a basic leucine zipper.

To discover causes of infertility and potential contraceptive targets, we used in silico subtraction and genomic database mining to identify conserved genes with germ cell-specific expression. In silico subtraction identified an expressed sequence tag (EST) present exclusively in a newborn mouse ovary library. The full-length cDNA sequence corresponding to this EST encodes a novel protein containing four ankyrin (ANK) repeats, a sterile-alpha motif (SAM), and a putative basic leucine zipper (bZIP) domain. Northern blot and semiquantitative RT-PCR analyses demonstrated that the mRNA is exclusively expressed in the mouse testis and ovary. The expression sites were localized by in situ hybridization to pachytene spermatocytes in the testis and oocytes in the ovary. Immunohistochemistry showed that the novel protein is localized to the cytoplasm in pachytene spermatocytes and early spermatids, oocytes at all stages of oogenesis, and in early preimplantation embryos. Based on its germ cell-specific expression and the presence of ANK, SAM, and basic leucine zipper domains, we have termed this novel protein GASZ. The mouse Gasz gene, which consists of 13 exons and spans 60 kb, is located on chromosome 6 between the Wnt2 and cystic fibrosis transmembrane conductance regulator (Cftr) genes. Using genomic database mining, orthologous genes encoding GASZ were identified in the rat, cow, baboon, chimpanzee, and human. Phylogenetic analyses reveal that the GASZ proteins are highly conserved among these species. Human and mouse GASZ proteins share 85.3% amino acid identity, and human and chimpanzee GASZ proteins differ by only 3 out of 475 amino acids. In humans, the GASZ gene resides on chromosome 7 and is similarly composed of 13 exons. Because both ANK repeats and the SAM domain function as protein-protein interaction modules that mediate signal transduction cascades in some systems, GASZ may represent an important cytoplasmic signal transducer that mediates protein-protein interactions during germ cell maturation in both males and females and during preimplantation embryogenesis.

Expression of genes for alcohol and aldehyde metabolizing enzymes in mouse oocytes and preimplantation embryos

Alcohols and aldehydes are metabolized primarily by alcohol (ADH) and aldehyde (ALDH) dehydrogenase isozymes. Although significant progress has been made towards understanding the involvement of these isozymes in the oxidation of alcohol and aldehydes in the body, it is not known how these compounds are handled during fertilization and preimplantation embryogenesis. In this study, reverse transcription and the polymerase chain reaction (RT-PCR) was used to determine which ADH and ALDH isozymes are expressed at the oocyte, zygote, morula, and blastocyst stages of preimplantation development in the mouse. Transcripts of β-actin and vimentin, assayed as controls, were detected at all stages, as well as Class III ADH ( Adh-2) and Class 3 ALDH ( Ahd-4), involved in the detoxification of formaldehyde and aromatic aldehydes, respectively. In contrast, transcripts for the major ethanol oxidizing isozyme, Class I ADH ( Adh-1) was not detected during preimplantation development. Cytosolic retinol dehydrogenase ( Adh-3) transcripts were marginally detected in oocytes and zygotes. The mRNA for cytosolic retinal dehydrogenase ( Ahd-2), microsomal short-chain retinol dehydrogenases (RoDH Type I), and the mitochondrial low-Km acetaldehyde dehydrogenase ( Ahd-5) only appeared as maternal transcripts. Microsomal ALDH ( Ahd-3), which is induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), was not expressed until the blastocyst stage. ADH and ALDH enzyme systems may guard mouse preimplantation embryos against the toxic effects of industrial pollutants, such as formaldehyde and TCDD, as well as peroxidatic aldehydes generated during lipid peroxidation. The absence of enzymes to convert ethanol to acetaldehyde, coupled with oocyte expression of the acetaldehyde-degrading enzyme, Ahd-5, may be protective for the early embryo.

A retrospective analysis: the examination of a potential relationship between particulate (P) and volatile organic compound (VOC) levels in a class 100 IVF laboratory cleanroom (CR) and specific parameters of embryogenesis and rates of implantation (IR).

Objective: Successful preimplantation embryogenesis is critically dependent upon the culture environment and changing organic chemistry of the ambient air provided by the IVF laboratory and clinical procedure rooms. Constituents of ambient air are influenced by the dynamic nature of the environment both external and internal to the IVF laboratory suite. To date, the role that ambient air constituents exert on preimplantation toxicology is poorly understood. CR protocols dictate aggressive monitoring of ambient air within the IVF CR. Our retrospective study sought to examine the potential relationship between critical parameters of ambient air, specifically, P and VOC levels, and preimplantation embryogenesis and IR.

Estrogenic upregulation of DNA polymerase beta in oocytes of preovulatory ovine follicles.

The basic premise of this investigation was that local hormonal control of stockpiling of the base excision repair polymerase (poly) beta within oocytes of preovulatory follicles occurs as a function of cytoplasmic maturation. There was an increase in immunoreactive poly beta in sectioned oocytes of preovulatory ovine follicles during a 12-36-hour interval following the onset of prostaglandin (PG) F2alpha-induced (Day 14 of the estrous cycle) luteal regression; this response was not observed in subordinate (nonovulatory) follicles. Accumulation of poly beta in oocytes at 36 hr after PGF2alpha was negated by treatment of ewes at 12 hr with the aromatase inhibitor Arimidex or an ovulatory dose of GnRH (which, via surge gonadotropin stimulation, acutely downregulates the proestrous rise in follicular estrogen biosynthesis). Estradiol-17beta stimulated poly beta expression (transcriptional control) in oocytes of explanted (12 hr after PGF2alpha) follicles (24-hour incubation). We suggest that a critical period of estrogen amplification in the preovulatory follicle underscores the capacity of its oocyte to efficiently repair DNA and therefore reconcile spontaneous infidelities in genomic integrity that inevitably occur during preimplantation embryogenesis.