Robert W. McGaughey

Activation of protein kinase C after fertilization is required for remodeling the mouse egg into the zygote

Fertilization of the mammalian egg initiates numerous biochemical and structural changes which remodel the egg into a single‐celled zygote. To date, the most extensively studied phenomenon of fertilization in virtually all species has been the relationship between sperm penetration and the induction of the initial rise in intracellular‐free calcium ([Ca2+]i) concentration within the egg. In contrast, relatively few studies have focused on the biochemical events following this rise in calcium, and even fewer studies have directly linked the biochemical events to the structural changes which must ensue for proper development of the embryo. In this study, we exploited recently developed technologies to investigate the action of protein kinase C (PKC), a presumed downstream transducer of the initial rise in [Ca2+]i, during fertilization and artificial activation with calcium ionophore or phorbol 12‐myristate 13‐acetate (PMA). The newly developed myristoylated PKC pseudosubstrate (myrPKCΨ) was used to specifically inhibit PKC, thereby averting the trauma of injecting the egg with nonmyristoylated PKCΨ. Following fertilization, eggs which were pretreated with myrPKCΨ were not capable of forming a second polar body and pronuclear formation was significantly inhibited. Spatial and temporal localization of PKC using confocal microscopy to visualize the PKC reporter dye, Rim‐1, demonstrated localization of PKC to the lateral aspects of the forming second polar body after fertilization, or after artificial activation with calcium ionophore or PMA. In vivo biochemical analysis of eggs which were fertilized or artificially activated demonstrated that PKC activity rose at the same time (40 min) as the second polar body formed and then subsided over the next 5 hr post activation. From these data, we conclude that PKC plays an integral role in directing the transformation from egg to embryo. Mol. Reprod. Dev. 46:587–601, 1997.

Notch pathway genes are expressed in mammalian ovarian follicles

Folliculogenesis is the process of development of ovarian follicles that ultimately results in the release of fertilizable oocytes at ovulation. This is a complex program that involves the proliferation and differentiation of granulosa cells. Granulosa cells are necessary for follicle growth and support the oocyte during folliculogenesis. Genes that regulate the proliferation and differentiation of granulosa cells are beginning to be elucidated. In this study, the expression patterns of Notch receptor genes and their ligands, which have been shown to regulate cell-fate decisions in many systems during development, were examined in the mammalian ovary. In situ hybridization data showed that Notch2, Notch3, and Jagged2 were expressed in an overlapping pattern in the granulosa cells of developing follicles. Jagged1 was expressed in oocytes exclusively. Downstream target genes of Notch also were expressed in granulosa cells. These data implicate the Notch signaling pathway in the regulation of mammalian folliculogenesis.

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.

Molecular differentiation of the rabbit ovum: II. During the preimplantation development of in vivo and in vitro matured oocytes

Abstract The developmental capacity of in vitro matured rabbit oocytes was assessed after transfer to inseminated, ovariectomized recipients such that fertilization and preimplantation development occurred in vivo . The results demonstrate that of the total number of transferred oocytes (1) 75% were fertilized, (2) 50% underwent cleavage, and (3) 13% developed into expanded blastocysts. By light microscopic criteria, embryos recovered at representative stages of preimplantation development were morphologically indistinguishable from embryos recovered at comparable stages from normally mated animals. Autoradiographs produced by high resolution, two-dimensional polyacrylamide gel electrophoresis demonstrated that changes in the pattern of polypeptide synthesis during the preimplantation stages were directly and entirely comparable for embryos derived either from normally mated animals or from in vivo or in vitro matured and transferred oocytes. Up to approximately the eight-cell stage, the translational patterns indicate the progressive disappearance of numerous oocyte-characteristic polypeptides from the autoradiographs as well as the appearance of some new species of polypeptides. Between the eight-cell and early blastocyst period, extensive and complex changes (qualitative and quantitative) occur in the patterns, whereas, in contrast, the phase of blastocyst growth and expansion that occurs during the latter portion of the preimplantation period is characterized by a fairly uniform and constant translational pattern.

Cytoskeletal reorganization during early mammalian development: Analysis using embedment-free sections

We have examined cytoskeletal reorganization during early embryonic development in the hamster by employing detergent extraction to remove soluble components of the embryos and reveal the underlying structural network. This procedure allows examination of both the cortical cytoskeleton and the cytoskeleton of the egg interior. Sections of eggs and embryos were prepared for transmission electron microscopy with the removable embedding medium, diethylene glycol disterate which allows thicker sections than conventional embedment procedures thereby providing more spatial cues for studying organization. The cytoskeleton reorganizes after fertilization, at the time of compaction and again at the blastocyst stage. These cytoskeletal reorganizations are considered in terms of the blastomere polarity hypothesis and the involvement of the cytoskeleton with early embryonic development.

The cortical cytoskeleton and its role in sperm penetration of the mammalian egg.

In this study isolated cortical regions of both penetrated and nonpenetrated Syrian hamster eggs were examined in whole mounts and platinum replicas of detergent-extracted cortical patches. Two types of cytoskeletal organization were observed in the egg cortex: Loose networks (LN regions) with integrated localized dense networks (LDN regions). Decoration with heavy meromyosin and labeling with antiactin/protein G gold both indicate that the cortical cytoskeleton consists mainly of a LN of actin microfilaments and several types of nonactin filaments, whereas LDN regions dispersed within the LN were comprised of nonactin filaments. Cortical patches and replicas of eggs incubated with sperm for 10-15 min provide evidence that cortical microfilaments may be intimately associated with penetrating spermatozoa. The results of this investigation provide the first high resolution view of the cortical cytoskeletal domain of a mammalian egg and suggest that actin microfilaments might play a role in sperm penetration of the egg cortex.

Patterns of polypeptide synthesis in mouse oocytes during germinal vesicle breakdown and during maintenance of the germinal vesicle stage by dibutyryl cAMP

Mouse oocytes undergo spontaneous meiotic maturation when cultured in vitro in the absence, but not in the presence, of dibutyryl cyclic AMP (cAMP). Furthermore, the inhibition by dibutyryl cAMP is reversible. Oocytes synthesize stage-specific polypeptides (i.e., developmental polypeptides) during meiotic maturation. Dibutyryl cAMP affects the synthesis of four of these developmental polypeptides by reversibly inhibiting the synthesis of one polypeptide, and irreversibly inhibiting the synthesis of three other polypeptides. Dibutyryl cAMP also reversibly inhibits the synthesis of at least two specific nondevelopmental polypeptides.

An alternative to in vitro fertilization-embryo transfer: the successful transfer of human oocytes and spermatozoa to the distal oviduct

In vitro fertilization-embryo transfer (IVF-ET) for treatment of irreparable tubal damage provides a means for otherwise hopelessly infertile couples to obtain offspring. Recently a simpler and less expensive procedure was applied as an alternative to IVF-ET to patients with at least one patent fallopian tube. This procedure employs stimulated ovarian cycles, washed and cultured sperm, and laparoscopic aspiration of oocytes. Recovered oocytes and sperm are deposited in the ampulla of the oviduct by means of a catheter. From February 1 to September 28, 1985, 137 cycles yielded 60 laparoscopies (51 patients). Elevated human chorionic gonadotropin observed in 18 cases was confirmed in 12 cases by ultrasound. Nine uterine pregnancies continued with no tubal pregnancy. Success is comparable for this new procedure and IVF-ET. The results suggest that many IVF-ET patients should be offered the simpler and less expensive option of tubal transfer (TT).

Correlation of estrogen levels with oocytes aspirated and with pregnancy in a program of clinical tubal transfer.

Transfer of human spermatozoa and mature oocytes to patent fallopian tubes (tubal transfer, TT) in cases of infertility provides a successful, simple alternative to in vitro fertilization-embryo transfer. The authors report their second TT series performed between October 1, 1985 and February 1, 1986. Of 59 transfers, 18 clinical pregnancies were obtained, of which 2 were ectopic and 1 spontaneously aborted. A triplet pregnancy, 3 sets of twins, and 11 singleton pregnancies delivered normally. Pregnancy was highly correlated with number of mature oocytes aspirated and with number of oocytes transferred. The study demonstrates that, in combination, serum estrogen levels and number of developing follicles observed by ultrasound are predictive of the number of mature oocytes obtained at laparoscopy. These predictive values provide excellent criteria for selecting stimulation cycles for TT and for informing individual patients of the probability for success before surgery.