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This paper describes an approach for accurately simulating the reset noise of CTIA-based pixels. Using a circuit simulator to find the reset noise of a CTIA based pixel is not straightforward, due to the noise sampling and charge redistribution after the reset switch opens. This often leads to an equation-based analysis of the pixel noise, which is cumbersome for actual design work and incompatible with a mixed-signal design flow for advanced ROIC designs. In a CTIA-based ROIC, the start of pixel integration is defined by the opening of the CTIA reset switch. The opening of this switch down-converts the wideband noise of the circuit to DC, and the charge is then redistributed by the CTIA to create an output reset noise. This reset noise can be removed by correlated double sampling (CDS). However, it is important to understand the magnitude of the reset noise in order to evaluate the effectiveness of the CDS scheme. CDS can be performed either in the pixel, or externally in the analog or digital domains. The specifications of the signal chain depend on the amount of reset noise and the degree of cancellation required. Simulation of the reset noise in SPICE is not straightforward, since the charge is redistributed after the switch opens, and the noise on the two capacitors is correlated and cannot be treated independently. We describe a simulation technique that gives accurate estimates of the pixel reset noise, and verify the results using Spectre-RF.

The magnitude of the inner mitochondrial membrane potential (deltapsim) appears to influence the level of certain mitochondrial activities including regulation of ionic fluxes and ATP liberation, activities that are often compartmentalized or location dependent in cells. Recent evidence suggests that within cells, mitochondria can be heterogeneous with respect to deltapsim, and that high-polarized mitochondria (high deltapsim) may occur in the subplasmalemmal cytoplasm where intercellular contact is absent. Here, we investigated whether deltapsim in oocytes and preimplantation embryos was heterogeneous and cell contact-associated.

BACKGROUND: The magnitude of the inner mitochondrial membrane potential (ΔΨm) appears to influence the level of certain mitochondrial activities including regulation of ionic fluxes and ATP liberation, activities that are often compartmentalized or location dependent in cells. Recent evidence suggests that within cells, mitochondria can be heterogeneous with respect to ΔΨm, and that high-polarized mitochondria (high ΔΨm) may occur in the subplasmalemmal cytoplasm where intercellular contact is absent. Here, we investigated whether ΔΨm in oocytes and preimplantation embryos was heterogeneous and cell contact-associated. METHODS: Mouse and human oocytes and preimplantation stage embryos stained with mitochondria-specific probes rhodamine 123, MitoTracker Orange, and the ΔΨm-sensitive probe JC-1, (5,5′,6,6'-tetrachloro-1,1,3,3′-tetraethylbenzimidazoylcarbocyanine iodide), were examined by epifluorescence, scanning laser confocal, and transmission electron microscopy. The possibility that intercellular contact and ΔΨm are associated was examined for oocytes, where transzonal coronal cell contacts were terminated naturally or experimentally, and for intact, disaggregated, and reconstructed cleavage stage mouse embryos. RESULTS: For both oocytes and embryos, clusters of apparently high-polarized mitochondria occur in the pericortical cytoplasm in regions free from intercellular contact. CONCLUSIONS: The findings suggest that mitochondria in oocytes and preimplantation embryos may be heterogeneous with respect to ΔΨm. We propose that high-polarized pericortical mitochondria may have a role in the acquisition of oocyte competence and the regulation of early developmental processes that may be associated with elevated metabolism or intracellular signalling through calcium-induced calcium release pathways.

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.

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.

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.

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.

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.

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.

The relationship between factors external to the human oocyte and the developmental potential of the female gamete after meiotic maturation in vivo is discussed. The findings indicate very different intrafollicular oxygen contents exist among follicles from the same and different patients, and marginally hypoxic conditions in some follicles are associated with a comparatively low adenosine triphosphate content and a reduction in intracellular pH for the corresponding metaphase II-stage oocyte. For such oocytes, the finding of an increased frequency of cytoplsmic pathology and chromosomal scattering (aneuploidy) suggests a compromised developmental potential. Three distinctly different patterns of cumulus cell attachment and proliferation during the first 24 hours of culture after insemination are described. As reported earlier, different cumulus cell phenotypes observed in vitro are independent of the presence of the oocyte or fertilized egg and unrelated to the degree of expansion of the cumulus at retrieval. However, developmental viability and implantation potential after in vitro fertilization-embryo transfer appear to be related to an intrinsic pattern of cumulus cell behavior in vitro. The results are discussed with respect to the role of factors external to the human oocyte that may influence or determine the ability of the oocyte to develop progressively after fertilization.

This study was designed to examine the relationship between lateralization and field dependence-independence. Through prior screening, 48 right-handed college students (24 men and 24 women) were identified as either field dependent or field independent on the Embedded Figures Test. Using a unilateral haptic task, field-independent men were more lateralized than field-dependent men. No such differences were found for women. These results are discussed in relation to Witkin's theory of psychological differentiation. Possible differences between men and women are also discussed.

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.

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.

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.

Two studies on developmental trends in dichhaptic lateralization for nonlinguistic stimuli were conducted on school-aged children. Study 1 included 60 students in Grades 1, 5, and 9. Study 2 included 200 students from Grades 2, 4, 6, 8, and 10. Both studies showed significant age-related increases in over-all dichhaptic accuracy. In Study 2 a significant left-hand advantage for all subjects as well as an over-all advantage for boys were noted. Significant interactions indicated different developmental trends for boys and girls but did not show clear patterns in the development of lateralization.

A total of 518 normal-appearing, meiotically mature human oocytes that were judged unfertilized after insemination in vitro were examined for sperm penetration by conventional fluorescence and laser scanning confocal microscopy with DNA-specific probes. A similar analysis was performed on 29 single pronuclear oocytes that were presumed to originate by spontaneous (parthenogenetic) activation. The results demonstrate that 22% of the unfertilized oocytes and 52% of the presumed parthenogenetic oocytes were actually penetrated. Sperm penetration occurred in both normozoospermic and male factor cases. The findings indicate the importance of penetration analysis in determining the causes of fertilization failure that may reside with the male or female gamete, especially when assessing the utility of and necessity for assisted fertilization in subsequent attempts. The results also suggest that the cytoplasmic capacity to decondense sperm DNA may decline more rapidly than the ability of the oocyte to be penetrated and to mount an effective block to polyspermy.

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.

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.

This study determined the occurrence of two molecular markers of apoptosis, chromosomal DNA strand breaks and oolemma phosphatidylserine redistribution, in >200 uninseminated and unfertilized human oocytes, and >800 newly ovulated and cultured mouse oocytes. DNA breaks were analysed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and phosphatidylserine by annexin V staining, with imaging by conventional epifluorescence and scanning laser confocal fluorescence microscopy. More than 300 intact and 500 fragmented mouse oocytes were examined at 24 h intervals during 6 days of culture in three different types of medium. For the human, 205 oocytes were examined at retrieval or at 24 h intervals during 7.5 days of culture in two types of medium. The perifollicular vascularity and the dissolved oxygen content of follicular fluid were determined for most of the follicles from which human oocytes were derived. The results demonstrate that TUNEL fluorescence of metaphase II (MII) chromosomes and annexin V staining of the oolemma in newly ovulated and cultured mouse and human oocytes are rare, and, when detected, are not spatially or temporally related. This finding also applied to mouse oocytes that fragmented during culture and exhibited morphological features that grossly resembled apoptotic body formation. In contrast, TUNEL but not annexin V staining occurred in the first polar body of a relatively high proportion of newly ovulated mouse oocytes, but was rarely detected in newly aspirated human oocytes. For the human, the occurrence of MII chromosomal TUNEL fluorescence was patient-specific and unrelated to perifollicular vascularity or dissolved oxygen content of the corresponding follicular fluid. The pattern of chromosomal TUNEL fluorescence observed in the first polar body and in the MII chromosomes of a very small number of mouse and human oocytes, especially after many days of culture, suggests that DNA strand breaks may not arise by apoptosis-associated endonuclease digestion. The results with these two markers suggest that it is premature to conclude that apoptosis occurs in ovulated oocytes or that such a mechanism is involved in the elimination or prevention of fertilization of oocytes with cytoplasmic or chromosomal defects.

We investigate the performance of free-space optical interconnection systems at the technology level. Specifically, three optical transmitter technologies, lead-lanthanum-zirconate-titanate and multiple-quantum-well modulators and vertical-cavity surface-emitting lasers, are evaluated. System performance is measured in terms of the achievable areal data throughput and the energy required per transmitted bit. It is shown that lead-lanthanum-zirconate-titanate modulator and vertical-cavity surface-emitting laser technologies are well suited for applications in which a large fan-out per transmitter is required but the total number of transmitters is relatively small. Multiple-quantum-well modulators, however, are good candidates for applications in which many transmitters with a limited fan-out are needed.

The developmental competence of mouse and human early embryos appears to be directly related to the metabolic capacity of a finite complement of maternally inherited mitochondria that appear to begin to replicate after implantation. Mitochondrial dysfunctions resulting from a variety of intrinsic and extrinsic influences, including genetic abnormalities, hypoxia and oxidative stress, can profoundly influence the level of ATP generation in oocytes and early embryos, which in turn may result in aberrant chromosomal segregation or developmental arrest. Deletions and mutations in oocyte mitochondrial DNA may subtend metabolic deficiencies or replication disorders in some infertile women and in women of increased reproductive age. Here, we describe methods for (i) the compartmentalization of mouse and human oocyte mitochondria into unique cytoplasts enriched for these organelles, and (ii) their transfer by microinjection into intact recipient oocytes. Metabolically active mitochondria in donor and recipient metaphase II stage oocytes were labelled with mitochondria-specific fluorescent probes, and the fate and location of donated mitochondria in recipient oocytes were followed by conventional epifluorescence and scanning laser confocal fluorescence microscopy. The net ATP content of undisturbed and recipient oocytes from the same cohort(s) was measured quantitatively at timed intervals after mitochondrial injection. The results demonstrate the feasibility of isolating and transferring mitochondria between oocytes, an apparent increase in net ATP production in the recipients, and the persistence of activity in the transferred mitochondria. The findings are discussed with respect to mitochondrial function and dysfunction in mammalian oocytes and embryos, and to the potential clinical applications of mitochondrial donation as they relate to the creation of heteroplasmic embryos.Keywords:heteroplasmy/mitochondria/mitochondrial donation/mitochondrial function in pre-implantation embryos

This study examined the relationship between blastomere fragmentation in cultured human embryos obtained by in-vitro fertilization and the effect of fragmentation on the distribution of the following eight regulatory proteins found to be: (i) localized in the mature oocyte in subplasmalemmal, polarized domains; and (ii) unequally inherited by the blastomeres during cleavage: leptin, signal transducer and activator of transcription 3 (STAT3), Bax, Bcl-x, transforming growth factor beta 2 (TGF beta 2), vascular endothelial growth factor (VEGF), c-kit and epidermal growth factor R (EGF-R). Four basic patterns of fragmentation were observed. The severity of the impact of each type of fragmentation on the affected blastomere(s) and the developmental competence of the embryo appeared to be a function of the unique temporal and spatial features associated with the particular fragmentation pattern(s) involved in each instance. The findings demonstrate that certain patterns of fragmentation can result in the partial or near total loss of the eight regulatory proteins from specific blastomeres and that the developmental potential of the affected embryo can be particularly compromised if it occurs during the 1- or 2-cell stages. In contrast, fragmentation from portions of a fertilized egg or a blastomere(s) in a 2-cell embryo that do not contain the protein domains, or the complete loss by fragmentation of a regulatory protein domain-containing blastomere after the 4-cell stage does not necessarily preclude continued development to the blastocyst, although the normality and developmental potential of the embryo may be compromised. The possible association between fragmentation and apoptosis was examined by annexin V staining of plasma membrane phosphatidylserine and TUNEL analysis of blastomere DNA. No direct correlation between fragmentation and apoptosis was found following the analyses of fragmented embryos with these two markers. However, while we suggest that changes in cell physiology unrelated to apoptosis are the more likely causes of fragmentation, we cannot exclude the possibility that fragmentation itself may be an initiator of apoptosis if critical ratios or levels of developmentally important proteins are altered by partial or complete elimination of their polarized domains. The findings are discussed with respect to the possible developmental significance of regulatory protein polarization in human oocytes and preimplantation stage embryos.

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, multinucleation 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.

Unique protein domains, concentration gradients, and asymmetric protein distributions or polarities are principle forces establishing the identity and fate of individual cells during early development in lower vertebrates and invertebrates. Here, we present evidence that these same forces exist during mammalian development in the form of two representative regulatory proteins, leptin and STAT3. Leptin, the 16 kDa cytokine product of the obese gene (ob) is involved in the activation of STAT3, a member of the signal transducer and activation of transcription family of proteins. We examined the temporal and spatial aspects of leptin and STAT3 immunofluorescence in mouse and human oocytes and preimplantation stage embryos. The findings demonstrate that both leptin and STAT3 are polarized in the oocyte and, as a consequence of their location and the position of the cleavage planes with respect to these protein domains: (i) differences in allocation of these proteins between blastomeres occur at the first cell division such that by the 8-cell stage; (ii) unique cellular domains consisting of leptin/STAT3 rich and leptin/STAT3 poor populations of cells are generated. By the morula stage, a cell-borne concentration gradient of these proteins extending along the surface of the embryo is observed. A potential role of these proteins in early development is indicated at the morula stage where the 'inner' cells consist of blastomeres that contain little, if any, leptin/STAT3 while 'outer' cells contain both leptin/STAT3 rich and poor cells. This pattern persists through the hatched blastocyst stage with little, if any, leptin/STAT3 detected in the inner cell mass and populations of leptin/STAT3 rich and poor cells forming the trophoblast. We have examined oocytes from mutant C57BL/6J ob/ob mice which are both obese and infertile (although fertility can be restored by the exogenous provision of leptin) and have found STAT3 and the mutant (truncated) leptin protein to be present and polarized, suggesting the possibility that the truncated leptin protein may still contain operational domains which are functional during oocyte development and early embryogenesis. Furthermore, analysis of leptin and STAT3 in intact ovarian follicles suggests that these proteins may be maternally derived and in particular, that a subpopulation of follicle cells may be partly responsible for the establishment of their polarized distribution in the oocyte. The results are discussed with respect to the proposition that leptin and STAT3 have critical roles in early mammalian development, and may be involved in the determination of the animal pole of the oocyte and in the establishment of the inner cell mass and trophoblast in the preimplantation stage embryo.

The synthesis of stage-related proteins characteristic of meiotic maturation and early preimplantation development occurs in the absence of significant transcription. Previous work indicated that some of the stage-related proteins typical of the early postfertilization period are synthesized in unfertilized oocytes at the same time that they are detected in fertilized eggs. This observation has led to the suggestion that protein synthesis in newly fertilized eggs is regulated by an intrinsic developmental program initiated during the resumption of meiosis (meiotic maturation) and supported in part by previously untranslated mRNA. It also has been proposed that the rapid and complex changes in protein synthesis that characterize this period may involve differential gene expression or selective protein degradation, or both. To date, cell-free translation of oocyte RNA has not demonstrated the existence of a sizeable population of preformed mRNA that could support the observed changes in protein synthesis. I have tested the notion that the apparent changes in protein synthesis during early development in the mouse may be derived from families of proteins related both in amino acid sequence and posttranslational modification. The findings show that many changes in protein synthetic patterns related to early development after fertilization are independent of fertilization and involve the posttranslational modification of proteins with identical or very similar primary structures. The results are discussed with respect to current interpretations of quantitative and qualitative changes in protein synthesis during early mammalian development as they relate to differential gene expression and presumed activation of preformed mRNA.

The extent to which differential gene expressions can be correlated with organ development was examined at the level of protein synthesis during pre- and postnatal development in the mouse. High resolution, equilibrium, two-dimensional polyacrylamide gel electrophoresis detected, for each of five to ten successive stages for each of seven organ systems, between 850 and 1000 separate newly synthesized proteins. The possibility that the 1000 detectable proteins synthesized at any one time during organ development represent a sampling bias was contra-indicated (a) because a different and larger population of [14C]- amino acid-incorporating protein syntheses gave similar results and (b) because nonequilibrium isoelectricfocusing, electrophoresis, isoelectric points between pH 5·5 and 8·7, confirmed the results from yet a different population of protein syntheses. Within limits of the sampling of protein syntheses, the entire period of organ development examined proceeds with altered expression of small proportion of the total proteins being synthesized. While all protein changes were stage specific, approximately three organ-specific protein syntheses were detected per organ system. One family of five protein syntheses seen in 16-day foetuses had homologous primary structures and presumably are keratins derived from a single genomic expression. These selected stage-specific protein syntheses examined by electrophoresis of partial proteolytic digests disclosed a programme for post-translational changes in protein syntheses. The current observations indicate that the examined pre- and postnatal organ development of the seven organs occurs in the presence of greater than 99% similarity among proteins synthesized in the same and different organ systems. Functional differentiation during organogenesis, therefore, occurs in the presence of less than 1 % change in qualitative or quantitative switch in protein syntheses. Evidence is presented to indicate that even this remarkably small number of changes in protein syntheses during functional organ differentiation may be derived from an even smaller subset of gene expressions. Collectively, the data suggest that explanatory mechanisms for molecular organogenesis must encompass both selective gene expressions along with post-translational programmed events.

The occurrence of grossly normal-appearing morula stage human embryos (8- to 12-cell) that developed from eggs fertilized in vitro containing three pronuclei is described. Serial section analysis by transmission electron microscopy demonstrated that 25% of the blastomeres were multinucleate, with as many as five deoxyribonucleic acid-containing nuclei (determined by fluorescence microscopy) in a single cell. Light and electron microscopy indicated that normal cortical and zona reactions had taken place. Fine-structural development of the cytoplasm was characteristic of morula stage human embryos. The results suggest the need to determine pronuclear number prior to syngamy and are discussed with respect to the notion that human embryos may have the capacity to develop normally in spite of the presence of abnormal cells.

Regardless of whether fertilization occurs in vivo or in vitro, a large proportion of human embryos do not develop progressively through the pre-implantation stages or arrest development after implantation. This study examined the association between the chromosomal/spindle normality of the mature human oocyte and the dissolved oxygen content, vascular endothelial growth factor concentration (VEGF) and perifollicular blood flow characteristics of the corresponding ovarian follicles. Findings from >1000 samples of follicular fluid show that developmentally significant differences in dissolved oxygen content occur in follicular fluids aspirated from follicles of equivalent size and ultrasonographic appearance. Oocytes from severely hypoxic follicles were associated with high frequencies of abnormalities in the organization of the chromosomes on the metaphase spindle that could lead to segregation disorders and catastrophic mosaicisms in the early embryo. Oocytes with cytoplasmic defects and cleavage stage embryos with multinucleated blastomeres are derived predominantly from severely hypoxic follicles. VEGF measurements of follicular fluid and colour pulsed Doppler ultrasonographic analysis of follicle-specific blood flow characteristics indicated a potentially important role for this factor both in perifollicular angiogenesis and in the regulation of intrafollicular oxygen levels. The results are discussed with respect to how severe intrafollicular hypoxia may influence the normality of chromosomal organization and segregation in the oocyte, and whether detailed pulsed Doppler analysis of individual pre-ovulatory follicles may provide an indirect indication of the 'health' of the follicle and possibly the developmental competence of the corresponding oocyte.

This study describes the occurrence of a highly specialized subpopulation of granulosa and cumulus oophorus cells that accumulate and sequester specific growth factors by a novel mechanism. These cells are characterized by multiple balloon-like processes tethered to the cell by means of a slender stalk of plasma membrane. Time-lapse analyses demonstrate that these tethered structures (TS) form in minutes and frequently detach from the cell with the bulbous portion remaining motile on the cell surface. Serial section reconstruction of transmission electron microscopic images shows a specific and stable intracellular organization in which an apparent secretory compartment composed of densely packed vacuoles, vesicles, and cisternae is separated by a thick filamentous network from a nuclear compartment containing mitochondria, polyribosomes, lipid inclusions, and rough-surfaced endoplasmic reticulum. Immunofluorescent analysis performed during the formation of these structures showed a progressive accumulation of vascular endothelial growth factor, leptin, and transforming growth factor-beta2 in the bulbous region. TS were identified in newly aspirated masses of granulosa and cumulus oophorus, and their production persists for months in culture. Observations of TS-forming cells made over several days of culture indicates that their production is episodic and factor release from these cells may be pulsatile. The findings suggest that a novel method of growth factor storage and release by an apparent apocrine-like mechanism occurs in the human ovarian follicle. The results are discussed with respect to possible roles in pre- and post-ovulatory follicular development.

Treatment of epithelial BSC-1 cells with low concentrations of the detergent Brij 58 results in partial or complete removal of the plasmalemma and partial extraction of internal membrane-bound organelles without causing massive release of "cytosolic" proteins from the cytoplasmic ground substance. Stereoscopic high-voltage electron microscopy of such extracted and fixed cells demonstrates a system of slender (4-20 nm) strands in a three-dimensional "microtrabecular" arrangement similar to that observed in unextracted whole-mount preparations. Extraction of Brij-extracted cells with Triton X-100 dissolves many of the microtrabecular strands, leaving, as a more stable structure, a characteristic cytoskeletal network composed of various filaments and microtubules. Two-dimensional polyacrylamide gel electrophoresis of 35S-labeled polypeptides performed concurrently with the morphological studies demonstrates that Triton extraction of Brij-extracted cells releases a large number of polypeptides. This release parallels the loss of structural components observed by electron microscopy. Labeling of Brij-extracted cells with heavy meromyosin subfragment 1 decorates actin filaments with characteristic arrowhead complexes which are readily visualized only after subsequent Triton extraction. These observations support the concept that many cytoplasmic proteins are structure-bound and, in addition to the components comprising the cytoskeleton, are structure-forming. We conclude that a metastable association of various proteins of the cytoplasmic ground substance exists whose morphological integrity is maintained, at lest temporarily, after removal of the plasmalemma in solutions containing Brij 58.

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.

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament "branches" are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the "microtrabecular network" (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).