Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development.

BACKGROUND: Appropriate gene expression is vital for the regulation of developmental processes. Despite this fact there is a remarkable paucity of information concerning gene activity during preimplantation development. METHODS: We employed reverse transcription and real-time fluorescent PCR to quantify the expression of nine genes (BRCA1, BRCA2, ATM, TP53, RB1, MAD2, BUB1, APC and beta-actin) in oocytes and embryos. A full characterization of all genes was achieved in 42 embryos and four oocytes. The genes analysed have a variety of important cellular functions. RESULTS: Oocytes displayed relatively high levels of mRNA transcripts, while 2-3-cell embryos were seen to contain very little mRNA from any of the genes examined. Recovery of expression levels was not seen until the 4-cell stage or later, with the presumptive activation of the embryonic genome. Some genes displayed sharp increases in expression in embryos composed of 4-8 cells, but, for most, maximum expression was not achieved until the blastocyst stage. CONCLUSIONS: Our data show that it is possible to define characteristic gene expression profiles for each stage of human preimplantation development. The identification of genes active at defined preimplantation phases may provide clues to the cellular pathways utilized at specific stages of development. Expression of genes that function in DNA repair pathways indicate that DNA damage may be common at the cleavage stage. We suggest that specific patterns of gene expression may be indicative of embryo implantation potential.

Mitochondria in human offspring derived from ooplasmic transplantation.

Ooplasmic transfer from fertile donor oocytes into potentially compromised recipient patient oocytes has led to the birth of nearly 30 babies worldwide. Cytoplasmic transplantation has caused apprehension, since the mixing of human ooplasm from two different maternal sources may generate mitochondrial (mt) heteroplasmy (both recipient and donor mtDNA) in offspring. This investigation traced the mitochondrial donor population both during the ooplasmic transfer technique and in the bloods of two 1 year old children using mtDNA fingerprinting. Donor ooplasm stained for active mitochondria was transferred into recipient ooplasm and the mitochondria were visualized by confocal microscopy after the microinjection procedure and fertilization. Heteroplasmy was found in the blood from each of the children. This report is the first case of human germline genetic modification resulting in normal healthy children.

Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR.

Oocytes, in general, are greatly enriched in mitochondria to support higher rates of macromolecular synthesis and critical physiological processes characteristic of early development. An inability of these organelles to amplify and/or to accumulate ATP has been linked to developmental abnormality or arrest. The number of mitochondrial genomes present in mature mouse and human metaphase II oocytes was estimated by fluorescent rapid cycle DNA amplification, which is a highly sensitive technique ideally suited to quantitative mitochondrial DNA (mtDNA) analysis in individual cells. A considerable degree of variability was observed between individual samples. An overall average of 1.59 x 10(5) and 3.14 x 10(5) mtDNA molecules were detected per mouse and human oocyte, respectively. Furthermore, the mtDNA copy number was examined in polar bodies and contrasted with the concentration in their corresponding oocytes. In addition, the density of mtDNA in a cytoplasmic sample was estimated in an attempt to determine the approximate number of mitochondria transferred during clinical cytoplasmic donation procedures as well as to develop a clinical tool for the assessment and selection of oocytes during in vitro fertilisation procedures. However, no correlation was identified between the mtDNA concentration in either polar bodies or cytoplasmic samples and their corresponding oocyte.

Characterization of the full fragile X syndrome mutation in fetal gametes.

Fragile X syndrome results from the expansion of the CGG repeat in the FMR1 gene. Expansion has been suggested to be a postzygotic event with the germline protected. From an analysis of intact ovaries of full mutation fetuses, we now show that only full expansion alleles can be detected in oocytes (but in the unmethylated state). Similarly, the testes of a 13-week full mutation fetus show no evidence of premutations while a 17-week full mutation fetus exhibits some germ cells with attributes of premutations. These data discount the hypothesis that the germline is protected from full expansion and suggest full mutation contraction in the immature testis. Thus, full expansion may already exist in the maternal oocyte, or postzygotic expansion, if it occurs, arises quite early in development prior to germline segregation.

FMRP associates with polyribosomes as an mRNP, and the I304N mutation of severe fragile X syndrome abolishes this association.

Fragile X mental retardation is caused by the lack of FMRP, a selective RNA-binding protein associated with ribosomes. A missense mutation, I304N, has been found to result in an unusually severe phenotype. We show here that normal FMRP associates with elongating polyribosomes via large mRNP particles. Despite normal expression and cytoplasmic mRNA association, the I304N FMRP is incorporated into abnormal mRNP particles that are not associated with polyribosomes. These data indicate that association of FMRP with polyribosomes must be functionally important and imply that the mechanism of the severe phenotype in the I304N patient lies in the sequestration of bound mRNAs in nontranslatable mRNP particles. In the absence of FMRP, these same mRNAs may be partially translated via alternative mRNPs, although perhaps abnormally localized or regulated, resulting in typical fragile X syndrome.

The fragile X mental retardation protein is a ribonucleoprotein containing both nuclear localization and nuclear export signals.

Fragile X syndrome is a frequent cause of mental retardation resulting from the absence of FMRP, the protein encoded by the FMR1 gene. FMRP is an RNA-binding protein of unknown function which is associated with ribosomes. To gain insight into FMRP function, we performed immunolocalization analysis of FMRP truncation and fusion constructs which revealed a nuclear localization signal (NLS) in the amino terminus of FMRP as well as a nuclear export signal (NES) encoded by exon 14. A 17 amino acid peptide containing the FMRP NES, which closely resembles the NES motifs recently described for HIV-1 Rev and PKI, is sufficient to direct nuclear export of a microinjected protein conjugate. Sucrose gradient analysis shows that FMRP ribosome association is RNA-dependent and FMRP is found in ribonucleoprotein (RNP) particles following EDTA treatment. These data are consistent with nascent FMRP entering the nucleus to assemble into mRNP particles prior to export back into the cytoplasm and suggests that fragile X syndrome may result from altered translation of transcripts which normally bind to FMRP.

Non-disjunction in human sperm: results of fluorescence in situ hybridization studies using two and three probes.

Fluorescence in situ hybridization using two or three probes was utilized to estimate the incidence of diploidy, the incidence of disomy for the sex chromosomes and chromosomes 16 and 18, and the proportion of Y- and X-chromosome bearing sperm, in a series of normal males. Our results demonstrate the importance of using an approach capable of distinguishing disomy from diploidy, as most donors had levels of diploidy higher than the disomy levels of individual chromosomes. Our analyses suggest the existence of chromosome-specific mechanisms of paternal non-disjunction, as sex chromosome disomy was approximately 1.5 times as common as disomy 16, and over two times as common as disomy 18. In studies of gametic sex ratio, we found little evidence for marked deviation from an expected 1:1 ratio.

Repeated sperm injection under the zona following initial fertilization failure.

When fertilization fails following micromanipulative under-zona insemination, it is possible to repeat the procedure adding more spermatozoa to achieve fertilization, embryonic development and pregnancy. We report on 18 human in-vitro fertilization cycles where this approach was used. In nine cycles only late-fertilized embryos were available for transfer, and these gave rise to two viable pregnancies (22.2% per transfer). In six cycles, where a mixture of late- and timely fertilized embryos were available for transfer, two viable pregnancies arose (33.3% per transfer). In three cycles no fertilization was achieved even after reinsemination by repeated under-zona insemination.

Pure FSH alone induces ovulation and subsequent pregnancy in the mouse resulting in fetal development.

The role of FSH in inducing folliculogenesis is well established. Recently, the availability of pure FSH has led to a reevaluation of its role in the process of ovulation. Previously, these functions have been examined separately, usually with pregnant mares' serum gonadotropin (PMSG) followed by FSH for ovulation or FSH for folliculogenesis followed by hCG for ovulation. To determine if FSH alone can induce both folliculogenesis, ovulation and establish a functioning corpus luteum without exogenous LH, we injected sexually mature intact mice (CD-1) with either ovine FSH (oFSH, 5 micrograms; < 0.2% LH contamination) or recombinant FSH (RCFSH, 1 IU; devoid of any LH activity) to stimulate folliculogenesis, followed forty-eight hours later by a second injection of the same preparation (oFSH, 15 micrograms; RCFSH, 1 IU, respectively) to induce ovulation. Injected female mice were mated individually with a fertile male. On days 15-17, pregnancy rates and fetal development were obtained for each animal and were compared with controls, mice injected with PMSG (1 IU) followed by hCG (1 IU). oFSH/oFSH and RCFSH/RCFSH results were combined since no statistical significant differences were detected between these groups. The pregnancy rate for the group receiving FSH/FSH (78.3%, n = 23) was higher than that of the PMSG/hCG group (48.3%, n = 27; p = .02). The number of fetuses produced per mouse in animals receiving FSH alone (8.5 +/- 1.1; mean +/- S.E.) also was greater than the controls (4.5 +/- 99; p = .01). We conclude that the ability of these animals to proceed beyond ovulation to implantation with fetal development demonstrates FSH's ability to cause not only follicular maturation and rupture, but also granulosa cell luteinization, further identifying the potentially important role of FSH in the ovulatory process.

Microsurgical fertilization procedures: the absence of stringent criteria for patient selection.

Subzonal sperm insertion and partial zona dissection were applied in 250 in vitro fertilization cycles in couples (n = 200) with abnormal semen analyses; 61 clinical pregnancies were established (24% per egg retrieval). Patients were selected without using minimal cutoff criteria. The study included patients with 0% normal sperm forms (strict criteria), no motile sperm (but some live cells), and sperm counts which could be assessed only after centrifugation. Patients were categorized into three subsets. Group A (n = 116 cycles) failed to fertilize in a previous cycle. Group B (n = 40) was excluded from IVF due to the severity of sperm profiles, such as a maximum of 2% normal forms. Group C (n = 94) constitutes those patients for whom a standard cycle could possibly result in failure. Monospermic fertilization rates were 18% (A), 19% (B), and 24% (C). The incidences of embryo replacement were 63% (A), 53% (B), and 69% (C). Rates of clinical pregnancy were 22% (A), 23% (B), and 28% (C). The presence of one, two, or three semen abnormalities did not correlate with the outcome of microsurgical fertilization. Twenty-two percent of patients with combined oligoasthenoteratozoospermia became pregnant. Moreover, ongoing pregnancies were established in instances with 0% normal sperm forms and no progressively motile spermatozoa. It is concluded that stringent cutoff criteria may not be necessary when both partial zona dissection and subzonal sperm insertion are performed efficiently.

Subzonal sperm insertion and the frequency of gamete fusion.

The subzonal sperm insertion technique was applied to assess the potential of motile human spermatozoa to form pronuclei. In 184 mature human oocytes, subzonal sperm insertion was used as the primary mode of insemination in cases with abnormal semen analyses. Oocytes (n = 131) that failed to fertilize in vitro in cases with normal semen profiles were also micromanipulated for secondary insemination. The frequency of sperm fusion, expressed as a percentage, was defined as the total number of male pronuclei formed divided by the total number of spermatozoa inserted subzonally. Our results indicate that 37% of spermatozoa from men with normal semen are able to fuse with the oolemma and decondense within the ooplasm, when placed in the perivitelline space of the oocyte. Excluding the oocytes that appeared morphologically abnormal (presence of cytoplasmic inclusions such as refractile bodies within the ooplasm), the frequency of sperm fusion increased to nearly 60%. Moreover, 14% of subzonally inserted spermatozoa from men with abnormal semen analyses demonstrated an ability to form a pronucleus. The incidence of polyspermy was high, ranging from 30 to 80% in the different groups studied. It is therefore concluded that the human oolemma provides little protection against multiple sperm fusion and that the frequency of gamete fusion is unexpectedly high, even when the spermatozoa are derived from infertile men.

Evaluation of Vero cell co-culture system for mouse embryos in various media.

Our objective was to evaluate and compare the efficacy and mechanisms of co-culturing mouse embryos with Vero cells in both Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) and human tubal fluid (HTF) culture medium. Two-cell CB6F1 mouse embryos were cultured either in the presence of Vero cells (group A) or in culture medium alone (group B). In DMEM/F12 significantly more morulae developed in group A than in group B on day 3 (91 versus 23%; P less than 0.01). In contrast, the mouse embryos grew rapidly in HTF and significant differences were noted only in later embryonic stages (on day 5; 86% and 50%, P less than 0.01 of group A and B respectively, hatching or hatched). Similar experiments using DF1 and ICR mouse strains also revealed enhanced embryo development in the presence of Vero cells. To determine whether the embryo-enhancing effects of Vero cells were due to the removal of toxins or to the secretion of embryotrophic factors, ICR mouse embryos were cultured in fresh media with cells (group A), without cells (group B) and in cell-free conditions using cell-conditioned media which were obtained in the presence (group C) or absence (group D) of embryos. These results demonstrated that completion of hatching was highest (52%; P less than 0.01) in group A after 6 days in culture. There were no significant differences between groups B, C and D (rates of total hatching 18, 17 and 17%, respectively). It is concluded that Vero cells improve the development of mouse embryos and this is likely to be due to removal of substances inhibitory to development.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology.

OBJECTIVE: To establish guidelines for application of partial zona dissection, subzonal sperm insertion, and regular in vitro fertilization (IVF) in severe male factor patients. DESIGN: Two studies were performed: partial zona dissection and IVF was applied in 57 couples during the first period, and subzonal sperm insertion was also applied in a second group of 47 couples. SETTING: Procedures were performed in an academic research environment. PATIENTS, PARTICIPANTS: Couples who failed fertilization previously, others not acceptable for IVF, and a third group in whom IVF was expected to fail. INTERVENTIONS: Oocytes were micromanipulated with either partial zona dissection or subzonal sperm insertion, or the zona pellucida was left intact. Embryos were replaced in patients prophylactically treated with methylprednisolone and antibiotics. MAIN OUTCOME MEASURES: Because several microsurgical fertilization techniques are now available, this study was performed to compare sperm parameters, embryo morphology, fertilization, and implantation rates after application of two successful micromanipulation procedures. RESULTS: Twenty-one pregnancies were established in 104 patients, 5 definitely from subzonal sperm insertion and 4 from partial zona dissection. Patients who failed IVF before had a similar chance of pregnancy after the use of micromanipulation, as first time patients (9/53 versus 12/51). In a subgroup of 15 patients who failed IVF with insufficient numbers of motile sperm, fertilization was significantly higher after subzonal sperm insertion. Partially zona-dissected embryos from couples with severe teratozoospermia (less than or equal to 5% normal forms; strict criteria) had significantly more morphological abnormalities than those from patients with moderate teratozoospermia (6% to 10% normal forms). In severely teratozoospermic patients, significantly fewer partially zona-dissected than subzonally inserted embryos implanted. CONCLUSIONS: The decision of which micromanipulation method to perform can possibly be based on careful analysis of sperm morphology.

A preferential site for sperm-egg fusion in mammals.

The tendency of mammalian sperm-egg fusion to occur at a site away from the first polar body was investigated in a homologous (mouse oocytes and mouse spermatozoa) and in a heterologous model (hamster oocytes and mouse spermatozoa). Following micromanipulation of the zona pellucida either in proximity to or opposite the first polar body, in vitro fertilization was performed and subsequent differences in sperm-egg interaction were evaluated. Since spermatozoa from random-bred mice do not readily penetrate intact zonae pellucidae in vitro, it is likely that zona penetration occurred through the artificial holes in both models. The creation of a gap in the zona pellucida opposite the first polar body was associated with levels of sperm fusion that were significantly higher than those resulting from manipulation near the first polar body. Spermatozoa were rarely found to penetrate the hole completely, and in general few spermatozoa were observed in the pervitelline space. The proximity between pronuclei following sperm penetration was correlated with the position of the incision with respect to the polar body. The findings suggest that breaching the zona pellucida for microsurgical fertilization should be performed away from the microvillus-free area of the oocyte.

Microsurgical fertilization and teratozoospermia.

Semen parameters were correlated with the outcome of partial zona dissection (PZD) in 42 couples with male factor infertility. Although fertilization rates were reduced, 12% of the embryos implanted following replacement. Spermatozoa from teratozoospermic sperm populations were able to fuse with oocytes following zona penetration through the artificial gaps. PZD followed by insemination with less than 5% normal spermatozoa led to 20 embryos which, upon replacement, did not implant. Motility and sperm count were not clearly correlated with the outcome of PZD and are therefore less useful indicators for patient selection. Teratozoospermic patients who previously failed to fertilize were compared to a group of similar patients who had not attempted IVF before. Although fertilization was significantly improved in first-time patients, 41% of the patients whose spermatozoa were initially unable to fertilize had at least one embryo when PZD was performed. Several pregnancies were established in this group. Subzonal sperm insertion (SZI) and PZD were compared in 19 patients using sibling oocytes. A significant fraction of spermatozoa from infertile men were able to fuse with the oolemma when directly inserted into the perivitelline area. Using a sucrose solution to shrink the ooplasm, only 1% of the oocytes were damaged during SZI. Monospermic fertilization rates following PZD and SZI were 15 and 16%, respectively. Both micromanipulation methods were successful in most patients. However, in two small groups of patients, only one technique resulted in fertilization.

Immunosuppression supports implantation of zona pellucida dissected human embryos.

The effect of low dose immunosuppression with methylprednisolone during the first 4 days after oocyte retrieval on potential immune cell invasion of partially zona dissected embryos in utero was investigated in alternate in vitro fertilization patients (n = 32). The incidence of pregnancy was significantly higher in patients receiving methylprednisolone (7 of 18, 39%) than in control patients (1 of 14, 7%). Twenty-eight percent (11 of 39) of the embryos replaced in the corticosteroid treated patients implanted, whereas only 7% (2 of 31) of embryos in control patients had a fetal heart beat. There were no side effects reported in any of the patients receiving corticosteroids. It can be concluded that methylprednisolone supports implantation of embryos with small holes in their zonae. However, the actual mechanisms of corticosteroid support on the interaction between immune cells and micromanipulated embryos are not well understood.

Ion channels in the nuclear envelope.

Cell nuclei are capable of partitioning a wide variety of molecules from the cytosol, including macromolecules such as proteins and RNA, and smaller peptides, amino acids, sugars and Na+ and K+ ions, all of which can be accumulated in or excluded from the nuclear domain. There are two mechanisms behind this compartmentalization: selective retention of freely diffusible molecules, and selective entry through the nuclear envelope. It is generally accepted that the nuclear envelope restricts only the larger molecules. Here we apply the patch-clamp technique to isolated murine pronuclei and show that the nuclear envelope contains K(+)-selective channels which have multiple conductance states, the maximal conductance being 200 pS. These channels, which contribute to the nuclear membrane potential, may be important in balancing the charge carried by the movement of macromolecules in and out of the nucleus.

Recognition of paternal pronuclei in human zygotes.

The possibility of pronuclear gender determination using morphological criteria only was investigated in 140 two-pronucleate and 39 tripronucleate zygotes. Zygotes were videotaped on different focal points and the positions of the polar bodies, pronuclear diameters, number and distribution of nucleoli, and presence of sperm tail remnants were indicated on diagrams. The three known criteria used for recognition of the paternal pronucleus in rodent zygotes were investigated. These criteria are (a) the presence of sperm tail remnants, (b) an increased pronuclear diameter, and (c) the farthest distance from the second polar body. Sperm tail remnants were observed in only 3/342 (1%) of the pronuclei. Pronuclear diameters and positions of the largest pronuclei did not reveal any trends. Pronuclei of tripronucleate zygotes were frequently smaller than those of two-pronucleate ones. The parental origin of human pronuclei cannot be determined morphologically using standard light optics. Microsurgical removal of paternal pronuclei from polyspermic zygotes should therefore be implemented with caution.