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The effects of four rounds of ovarian stimulation spaced 1–6 weeks apart on the normality of metaphase II (MII) spindle formation, chromosomal alignment and cytoplasmic organization were examined in intact ovulated mouse oocytes and at MII for oocytes obtained at the germinal vesicle stage from the same ovaries and matured in vitro. The terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling assay was used to identify DNA strand breaks in chromosomes, and histological studies of ovaries between and at each round of ovarian stimulation were performed. The results demonstrate a progressive and significant increase in the frequency of spindle defects with each round of ovarian stimulation, including those spaced weeks apart. Oocytes with spindle defects were also characterized by the occurrence of detached chromosomes and cytoplasmic asters. In contrast, in-vitro matured oocytes derived from the same ovaries were normal. No evidence of DNA strand breaks with repeated rounds of ovarian stimulation was detected in ovulated or in-vitro matured oocytes. The development and persistence of nodules of hypertrophied granulosa in regions where follicular growth occurs suggest that a progressively increasing proportion of oocytes in the ovulatory pathway may experience an intrafollicular milieu that has negative consequences for competence. The results are discussed with respect to ovarian and oocyte biological ageing and possible adverse implications for human oocyte competence with repeated hyperstimulation. [ABSTRACT FROM PUBLISHER]

The occurrence of a pleiomorphic population of cytoplasmic fragments is a common characteristic of early human embryos fertilized in vitro. Here, temporal, spatial, fine structural, and biochemical aspects of fragmentation were examined in fragmented monospermic and dispermic pronuclear to early cleavage stages human embryos classified as stage-appropriate during the first 3.5 days of culture. The morphodynamics of certain common patterns of fragmentation and the movement and composition of fragments were analysed by time-lapse video, mitochondrial fluorescent probes, and transmission electron microscopy. Plasma membrane and nuclear DNA integrity were assessed by annexin V staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) and single-cell alkaline gel electrophoresis ('comet') assays respectively. Developmental competence for affected embryos was related to outcome after embryo transfer. The results demonstrate that certain common forms of spontaneous fragmentation affecting early human embryos are not lethal, and that clusters of apparent fragments are often transient structures, which disappear by resorption or lysis. The findings suggest that the occurrence and fate of fragments characteristic of these phenotypes may be related to oncosis-like processes associated with transient and focal ATP deficiencies in blastomeres and mitochondrial deficiencies or absence in extracellular fragments. [ABSTRACT FROM AUTHOR]

It has been suggested that mitochondrial DNA defects that effect metabolic capacity may be a proximal cause of failures in oocyte maturation, fertilization, or early embryonic development. Here, the distribution of mitochondria was examined by scanning laser confocal microscopy in living human pronuclear oocytes and cleavage stage embryos, followed either by measurements of the net ATP content of individual blastomeres or anti-tubulin immunofluorescence to determine the relationship between mitochondrial distribution and microtubular organization. The results indicate that specific patterns of perinuclear mitochondrial aggregation and microtubular organization are related, and that asymmetrical mitochondrial distributions at the pronuclear stage can result in some proportion of blastomeres with reduced mitochondrial inheritance and diminished ATP generating capacity. While the inability to divide appears to be a development consequence for an affected blastomere, for the embryo, reduced competence may occur during cleavage if several blastomeres inherit a mitochondrial complement inadequate to support normal cellular functions. The findings provide a possible epigenetic explanation for the variable developmental ability expressed within cohorts of morphologically normal early cleavage stage human embryos obtained by in-vitro fertilization. [ABSTRACT FROM AUTHOR]

Ovarian follicular granulosa cells express temporally and spatially distinct functions throughout the follicle cycle. During the entire cycle, granulosa cells exhibit an unusually broad range of activities including the secretion of steroid hormones, enzymes, growth factors and cytokines. To date, the identity(ies) of these cells (lineage/cell type) remains unknown. We demonstrate expression of the Tie, Tek, cKit, Flt-1, CD-31 and vWF proteins and the ability to rapidly internalize acetylated low density lipoprotein among mural and cumulus subpopulations of human and murine follicular granulosa cells. In addition, we provide evidence that human and murine granulosa cells can engage in tube-forming activity in vitro. To the best of our knowledge, the six phenotypic and two functional markers examined during this study, as a group, are associated only with endothelial or endothelial-like cells. In total, the findings suggest that some granulosa cells may have the potential to actively participate in the vascularization of the corpus luteum, by way of an inherent capacity which is likely to be a characteristic of their unique identity and lineage. This inherent capacity of granulosa cells to behave and respond, at least to some extent, like endothelial cells may be of possible importance in the aetiology of certain follicular pathologies. [ABSTRACT FROM AUTHOR]

This report describes spatial and temporal aspects of sperm penetration and intracytoplasmic migration, pronuclear evolution and the specificity of presyngamic opposition, stage-specific changes in cytoskeletal organization and the relative contribution of maternal and paternal components to mitotic spindle formation. These studies involved observations of living human oocytes during conventional insemination in vitro and after intracytoplasmic deposition of spermatozoa, analysis of chromatin organization and distribution during pronuclear evolution, and detection of actin and alpha-, beta- and gamma-tubulin by confocal immunofluorescence microscopy. Immature and mature oocytes, penetrated but unfertilized oocytes, fertilized but arrested eggs, and cleavage-stage embryos from normal and dispermic fertilizations were examined. The results demonstrate that sperm nuclear migration to the maternal perinuclear region is rapid and linear, occurs in the absence of a detectable cytoskeletal system and appears to be assisted by an unusual configuration of the sperm tail principal piece which results from either retained intracytoplasmic motility or the process by which the sperm tail is progressively incorporated into the oocyte. Our findings also show a specificity of pronuclear alignment that is associated with a polarized distribution of both maternal and paternal chromatin, and with the position of the sperm centrosome and the presence of microtubules nucleated from this structure. The results also indicate that a maternal microtubule nucleating capacity is present in the immature oocyte but is apparently inactive until spindle formation. The poles of the first mitotic spindle appear to be derived from the sperm centrosome, although some maternal contribution cannot be excluded. The sperm tail and centrosome persist in a single cell through the cleavage stages, and the latter serves as a prominent site of cytoplasmic microtubule nucleation. The results provide a detailed understanding of the cellular and nuclear morphodynamics of the human fertilization process and indicate subtle defects that may be responsible for early developmental failure. [ABSTRACT FROM AUTHOR]

This study examined the feasibility of isolating and transferring the centrosome-containing region of spermatozoa to mature human oocytes. The findings demonstrate that individual sperm centrosomes can be transferred and are capable of nucleating maternal tubulin to form a well-developed sperm aster in the recipient oocyte. The results are discussed with respect to centrosome function in early human development and applications in clinical in-vitro fertilization in the treatment of certain forms of male factor infertility. [ABSTRACT FROM AUTHOR]

The relationship between the ATP content of mature human oocytes and developmental potential after uterine transfer of sibling embryos was examined in 20 non-male factor in-vitro fertilization (IVF) patients matched for age, fertility history, ovarian stimulation protocol, oocyte quality and number, stage and morphology of embryos at uterine transfer. ATP content was determined for uninseminated and unfertilized oocytes for each cohort, and for embryos that developed after dispermic fertilization or which showed significant fragmentation. The effect of reduced ATP content on meiotic maturation, fertilization and preimplantation development was examined in cultured mouse oocytes treated with uncouplers of mitochondrial oxidative phosphorylation. The results demonstrated that meiotic maturation occurs in both mouse and human oocytes over a wide range of ATP contents, and that the ATP content of normal-appearing, metaphase II human oocytes can differ significantly between cohorts; however, a higher potential for continued embryogenesis and implantation in the human is associated with embryos that develop from cohorts of oocytes with ATP contents > or = 2 pmol/oocyte. The findings are discussed with respect to possible aetiologies and developmental consequences for embryonic development of different oocyte ATP contents, and the extent to which mitochondrial function may determine or influence the continued developmental capacity of embryos which appear normal and developmentally viable at the early cleavage stages. [ABSTRACT FROM AUTHOR]

This study demonstrates that sperm penetration into the ooplasm occurs at high frequency in germinal vesicle (GV)stage human oocytes which failed to resume meiosis after ovulation induction in cycles of ovarian hyperstimulation for in-vitro fertilization. The capacity of the immature human oocyte to prevent polyspermic penetration at the cell surface level was suggested by the finding that despite the presence of numerous spermatozoa within the zona pellucida and on the oocyte surface within 3 h after insemination, all normal-appearing GV stage oocytes examined in this study were penetrated by a single spermatozoon. This notion was also supported by scanning confocal microscopic analysis of oocytes double-stained for DNA and cortical granules– which showed highly localized regions of cortical granule-free cytoplasm in proximity to the penetrated spermatozoon. The developmental ability of these oocytes was assessed by culture in vitro. The results show that oocytes penetrated by a spermatozoon at the GV stage resume meiosis, develop the capacity to decondense sperm DNA, abstrict both first and second polar bodies, and form a male pronucleus from the spermatozoon which enters the oocyte prior to the resumption of meiotic maturation. After penetration, sperm nuclei rapidly migrate to the centre of the oocyte and become juxtaposed with the germinal vesicle, suggesting the presence of a cellular mechanism which permits directed movement within the cytoplasm. The developmental ability of these oocytes and the normality of the resulting embryos are discussed [ABSTRACT FROM PUBLISHER]

In a prospective randomized study, excess embryos from 100 women undergoing in-vitro fertilization were cultured from the 2-cell to the hatched-blastocyst stage in the presence or absence of a confluent monolayer of Vero cells. The frequencies of fragmentation, developmental arrest, multi-nucleation and blastocyst formation were observed for 254 embryos over 7 days in culture. The number of nucleated cells, and fine structure of trophectoderm and inner cell mass were analysed at the expanded blastocyst stage on day 5.5 post-insemination. The frequency of hatching from the zona pellucida was determined between days 6 and 7 post-insemination. With respect to these developmental parameters, the findings indicate that no overt or statistically significant improvement in early human embryogenesis occurs in the co-culture system. [ABSTRACT FROM PUBLISHER]

The frequency of aneuploidy in 583 newly aspirated, uninseminated metaphase II-stage human oocytes which exhibited seven distinct forms of cytoplasmic dysmorphism [Van Blerkom (1990) J. Electron Microsc. Tech., 16,324] after ovarian stimulation and ovulation induction was determined in the living state by DNA fluorescence followed by fixation and air-drying for karyotyping. The findings demonstrate that as many as half of the oocytes with dysmorphic phenotypes which arise early in meiotic maturation are aneuploid, with hypohaplidy predominant. In contrast, cytoplasmic defects which occur at or after metaphase I are associated with a relatively low frequency of aneuploidy (less than 15%), which is comparable to that previously reported for human oocytes with a normal cytoplasmic appearance [Van Blerkom and Henry (1988) Hum. Reprod., 3, 777]. The aetiologies of aneuploidy in dysmorphic oocytes, as well as the clinical implications for oocyte selection in laboratory-assisted conception are discussed. [ABSTRACT FROM AUTHOR]

After thawing and culture in vitro, greater than 90% of germinal-vesicle (GV)-stage mouse oocytes cryopreserved by vitrification, were capable of resuming meiosis and undergoing normal chromosomal and cytoplasmic maturation to metaphase II. This high frequency of development occurred against a background of profound alterations in the structure and organization of the cytoplasm, nucleus, nucleolus and chromatin during the dehydration stage of vitrification. Most, but not all, cytoplasmic and nuclear perturbations returned to a normal state during post-thaw culture. However, the results clearly demonstrate that vitrification is associated with chromosomal and cellular disorders that could adversely affect development after fertilization. Irreversible changes of potential developmental significance observed after vitrification at the GV stage include (i) premature chromosomal condensation, (ii) mixing of nucleoplasmic and cytoplasmic components prior to GV breakdown, and (iii) externalization of chromatin fragments into the cytoplasm after reformation of the oocyte nucleus, which shows the potential for the generation of fertilizable oocytes containing deleted segments of DNA. [ABSTRACT FROM AUTHOR]

Chromosome complement and location were examined by fluorescence microscopy for 225 meiotically mature (metaphase II) human oocytes after staining with DNA-specific probes. Both preovulatory oocytes and oocytes that failed to fertilize in vitro were analysed. After inspection in the living state, oocytes were selected for karyotyping or transmission electron microscopy. The findings demonstrate a high correlation between assessments of chromosome complement in living oocytes and the results from subsequent karyotypes. In addition to numerical aberrations (aneuploidy), the results also demonstrate the ability to detect abnormalities in chromosome structure and distribution. Specifically, this approach identified living oocytes that (1) contained no apparent chromosomes in the ooplasm, (2) contained chromosome not associated with the MII spindle and (3) had weak or no detectable chromosomal fluorescence in the first polar body. The findings demonstrate that approximately 8% of the oocytes were aneuploid (hypohaploid or hyperhaploid). Another 6.5% displayed anomalies in chromosome structure or distribution that could lead to aneuploid situations. The results are discussed with respect to the origin, occurrence and developmental consequences for such oocytes. [ABSTRACT FROM AUTHOR]

The presence of three or more presumed pronuclei in 27 of 884 (3%) in-vitro fertilized human eggs suggested the occurrence of polyspermic fertilization. Detailed examination by differential interference contrast microscopy indicated that approximately 40% of presumed multipronuclear eggs were normally fertilized but contained a cytoplasmic vacuole(s) (pseudo-pronucleus) of pronuclear dimension and gross morphology. Closely timed observations of presumed multipronuclear eggs revealed that pseudo-pronuclei (PPN) can co-migrate and become juxtaposed with true pronuclei, thus presenting the impression of an aberrant fertilization. Fluorescent probe and electron microscopic analyses demonstrated that PPN are enclosed by a plasma membrane and contain neither DNA nor nucleoli. PPN can develop either in the mature oocyte or the newly fertilized egg. Pseudo-multipronuclear eggs progress through the preimplantation stages in an apparently normal fashion and, as demonstrated by one birth and two ongoing pregnancies, are developmentally viable. Criteria for the unambiguous designation of pseudo-multipronuclear eggs are based on: the absence of normal morphodynamic changes associated with pronuclear development; and the failure of nucleoli to appear during the perisyngamic stage. The findings strongly suggest that pseudo-multipronuclear human eggs may represent a class of morphological variants of normally fertilized and developmentally competent eggs. [ABSTRACT FROM AUTHOR]