Jonathan Van Blerkom

Mitochondrial function in the human oocyte and embryo and their role in developmental competence

The role of mitochondria as a nexus of developmental regulation in mammalian oogenesis and early embryogenesis is emerging from basic research in model species and from clinical studies in infertility treatments that require in vitro fertilization and embryo culture. Here, mitochondrial bioenergetic activities and roles in calcium homeostasis, regulation of cytoplasmic redox state, and signal transduction are discussed with respect to outcome in general, and as possible etiologies of chromosomal defects, maturation and fertilization failure in human oocytes, and as causative factors in early human embryo demise. At present, the ability of mitochondria to balance ATP supply and demand is considered the most critical factor with respect to fertilization competence for the oocyte and developmental competence for the embryo. mtDNA copy number, the timing of mtDNA replication during oocyte maturation, and the numerical size of the mitochondrial complement in the oocyte are evaluated with respect to their relative contribution to the establishment of developmental competence. Rather than net cytoplasmic bioenergetic capacity, the notion of functional compartmentalization of mitochondria is presented as a means by which ATP may be differentially supplied and localized within the cytoplasm by virtue of stage-specific changes in mitochondrial density and potential (ΔΨm). Abnormal patterns of calcium release and sequestration detected at fertilization in the human appear to have coincident effects on levels of mitochondrial ATP generation. These aberrations are not uncommon in oocytes obtained after ovarian hyperstimulation for in vitro fertilization. The possibility that defects in mitochondrial calcium regulation or bioenergetic homeostasis could have negative downstream development consequences, including imprinting disorders, is discussed in the context of signaling pathways and cytoplasmic redox state.

The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting

This paper reports the proceedings of an international consensus meeting on oocyte and embryo morphology assessment. Following background presentations about current practice, the expert panel developed a set of consensus points to define the minimum criteria for oocyte and embryo morphology assessment. It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes. This document is intended to be referenced as a global consensus to allow standardized reporting of the minimum dataset required for the accurate description of embryo development. This paper reports the proceedings and outcomes of an international consensus meeting on human oocyte and embryo morphology assessment. An expert panel developed a series of consensus points to define the minimum criteria for such assessments. The definition of common terminology, and standardization of laboratory practices related to these morphological assessments, will permit more effective comparisons of treatment outcomes around the world. This report is intended to be referenced as a global consensus to allow standardized reporting of the minimum descriptive criteria required for routine clinical evaluations of human embryo development in vitro.

Mitochondria in early mammalian development.

The role of mitochondria as central determinants of development competence of oocytes and preimplantation stage embryos is considered in the context of the diverse activities these organelles have in normal cell function. Stage- and cell-cycle-specific mitochondrial translocations and redistributions are described with respect to mechanisms of cytoplasmic remodeling that may establish domains of autonomous regulation of mitochondrial function and activity during early development. The functions of mitochondria as intracellular signaling elements, as regulators of signaling pathways, and oxygen sensors in differentiated cells are suggested to have similar capacities during mammalian oogenesis and early embryogenesis. Questions concerning the numerical size of the oocyte mitochondrial complement, the energy required to support normal preovulatory oogenesis and preimplantation embryogenesis, and the regulation of mitochondrial activity by extrinsic and intrinsic factors are addressed with respect the potential they may have for new investigational approaches to study the origin of the differential developmental competence of human oocytes and preimplantation stage embryos.

Qualitative patterns of protein synthesis in the preimplantation mouse embryo: I. Normal pregnancy☆

Abstract Qualitative patterns of protein synthesis in preimplantation mouse embryos were examined by SDS-polyacrylamide-gel electrophoresis followed by autoradiography. The results demonstrate that the qualitative pattern of protein synthesis in newly fertilized eggs (day 1) is very similar to the protein pattern obtained from ovulated, unfertilized eggs. By late day 1 or early day 2, most of these “maternal” proteins are no longer being synthesized by the embryo, and many new autoradiographic bands are apparent. The most intriguing aspect of this study is the observation that all major changes in the qualitative pattern of protein synthesis take place between fertilization and the four- to eight-cell stage (day 3). From early day 3 onward, the qualitative pattern of protein synthesis remains essentially unchanged. Many of the major autoradiographic bands observed in mouse embryos from the four- to eight-cell stage and onward are also observed in protein patterns obtained from blastocyst-stage rabbit embryos. The changing patterns of protein synthesis revealed in this study occur before any gross differentiation of the embryos is evident (delineation of the inner cell mass and trophoblast) and before a marked increase in the relative rate of incorporation of l -[35S]methionine takes place. However, the qualitative changes in the pattern of protein synthesis do coincide with a period of extensive fine structural differentiation.

Mitochondria as regulatory forces in oocytes, preimplantation embryos and stem cells.

In addition to their role in generating energy (ATP), other possible regulatory functions for mitochondria in the mature oocyte, preimplantation stage embryo and differentiating embryonic stem cell are discussed. The question of the numerical size of a normal mitochondrial complement in the mature oocyte, which has been suggested to be a critical factor in the determination of oocyte and embryo developmental competence, is addressed in the context of mitochondrial DNA copy numbers and the apparent number of organelles detected in living mouse and human blastocysts with mitochondria-specific fluorescent probes. The existence of a spatially stable subplasmalemmal domain of high-polarized mitochondria in the mature oocyte and early embryo is proposed to form a microzone of differential activity that may locally influence the normality of fertilization. High-polarized mitochondria may also influence developmentally significant activities in the peri-implantation blastocyst and differentiating embryonic stem cell. The work discussed indicates that mitochondria may have multiple functions during early development, and specific areas of investigation required to confirm these possibilities are suggested.

Epigenetic influences on oocyte developmental competence: perifollicular vascularity and intrafollicular oxygen.

Purpose:Studies indicating that the developmental competence of the human oocyte is influenced by the level of intrafollicular oxygen are described.Methods:Perifollicular vascularity and dissolved oxygen content were determined by color Doppler ultrasonography and analysis of follicular fluid at ovum retrieval, respectively, in stimulated cycles for in vitro fertilization.Results:Differences in the degree of perifollicular vascularity correlate with differences in the dissolved oxygen content of the corresponding follicular fluid. Oocytes with cytoplasmic and chromosomal disorders and embryos with multinucleated blastomeres and limited developmental ability were derived predominantly from underoxygenated follicles. Findings from several studies indicate that embryos with the highest implantation potential originate from follicles that are well-vascularized and oxygenated.Conclusions:Follicular vascularity and oxygen content appear to be important determinants of oocyte competence. Possible causes of differences in follicle-specific vascularity and the potential effects of severe hypoxia on the normality of molecular and cellular processes during follicle growth and preovulatory development are discussed.Purpose: Studies indicating that the developmental competence of the human oocyte is influenced by the level of intrafollicular oxygen are described. Methods: Perifollicular vascularity and dissolved oxygen content were determined by color Doppler ultrasonography and analysis of follicular fluid at ovum retrieval, respectively, in stimulated cycles for in vitro fertilization. Results: Differences in the degree of perifollicular vascularity correlate with differences in the dissolved oxygen content of the corresponding follicular fluid. Oocytes with cytoplasmic and chromosomal disorders and embryos with multinucleated blastomeres and limited developmental ability were derived predominantly from underoxygenated follicles. Findings from several studies indicate that embryos with the highest implantation potential originate from follicles that are well-vascularized and oxygenated. Conclusions: Follicular vascularity and oxygen content appear to be important determinants of oocyte competence. Possible causes of differences in follicle-specific vascularity and the potential effects of severe hypoxia on the normality of molecular and cellular processes during follicle growth and preovulatory development are discussed.

Molecular differentiation of the rabbit ovum: I. During oocyte maturation in vivo and in vitro

The normality of nuclear and cytoplasmic maturation of rabbit oocytes, matured in vivo and in vitro, has been assessed by cytogenetic and electrophoretic criteria. The findings indicate not only that nuclear maturation in vivo and in vitro are directly comparable, but also, as observed by high-resolution, two-dimensional polyacrylamide gel electrophoresis, (1) that both qualitative and quantitative changes in the pattern of polypeptide synthesis occur during maturation, (2) that these patterns are directly comparable in oocytes that had been matured either in vivo or in vitro, and (3) that each stage of maturation is associated with the appearance of specific polypeptides in the autoradiographic patterns. The major differences observed between oocytes matured under these two conditions are (1) that several polypeptides fail to appear in in vitro matured oocytes at the time they are detected in vivo and (2) that the synthesis of some polypeptides is prolonged in vitro compared to in vivo matured oocytes.

Patterns of polypeptide synthesis of porcine oocytes during maturation in vitro

Abstract Changes in the pattern of polypeptide synthesis during the in vitro maturation of the porcine oocyte have been studied by high-resolution, two-dimensional, polyacrylamide gel electrophoresis. The findings reveal not only that pig oocytes mature in vitro , but also that they are engaged in the synthesis of a complex pattern of polypeptides throughout maturation. The results also demonstrate the presence of molecular markers, the appearance, disappearance, and significant change in intensity of which are temporally related with the sequential stages of nuclear meiotic maturation. Collectively, the evidence indicates the presence of a developmental program during oocyte maturation.

Qualitative patterns of protein synthesis in the preimplantation mouse embryo: II. During release from facultative delayed implantation

Qualitative patterns of newly synthesized protein were examined in mouse embryos released from facultative delayed implantation either by removal from the uterus with subsequent culture in vitro, or by the administration of progesterone and estradiol 17β (in vivo) to the ovariectomized mother. It was found that the qualitative pattern of protein synthesis during the first 25 hr following release from delay is the same as the pattern obtained from normal blastocysts immediately prior to implantation. These results are discussed in relation to possible mechanisms of activation of delayed blastocysts.

The Influence of Intrinsic and Extrinsic Factors on the Developmental Potential and Chromosomal Normality of the Human Oocyte

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 cytoplasmic 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.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.