Frank J. Longo

Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus.

Experiments were carried out to determine the origin of cortical polarity in mouse eggs and its possible relation to the meiotic apparatus. Cortices of mature eggs overlying the meiotic apparatus (microvillus-free area) were distinguished by an absence of microvilli and a thickened layer of actin. In contrast, the surfaces of immature oocytes were covered entirely with a dense population of microvilli and were subtended by a uniform layer of actin. When induced to undergo maturation, meiotic spindles formed in the center of immature oocytes and then moved peripherally. Coincident with the cortical localization of the meiotic spindle was the formation of a microvillus-free area, i.e., a loss of microvilli and a thickening of the actin layer associated with this region of the egg cortex. If immature oocytes were incubated in cytochalasin B, meiotic spindles formed; however, they failed to move peripherally and microvillus-free areas did not develop. Oocytes incubated in colchicine did not form meiotic spindles, although the chromosomes condensed and became localized to cortices where microvillus-free areas developed. Cytochalasin B-treated mature eggs maintained intact meiotic spindles and exhibited a disappearance of microvillus-free areas and a reduction in cortical actin. The chromosomes of mature eggs treated with colchicine remained associated with microvillus-free areas despite the disappearance of meiotic spindles. Occasionally, colchicine-treated eggs possessed more than one cortically located mass of chromosomes, each of which was associated with a microvillus-free area. These observations indicate that mechanisms involving the movement of the meiotic spindle to the oocyte cortex and development and maintenance of cortical polarity are cytochalasin B sensitive. Commensurate with the localization of meiotic chromosomes to the egg cortex is the reorganization of cortical actin and the formation of a microvillus-free area.

Effects of cytochalasin B on sperm-egg interactions

Abstract The effects of cytochalasin B (CB) on the interaction of sea urchin ( Arbacia ), molluscan ( Spisula ), and mammalian ( Mus ) gametes have been examined. Despite the absence of sperm incorporation and gamete membrane fusion, CB-treated Arbacia and Spisula eggs (1–10 μg/ml for 1–10 min) mixed with sperm activated. Unlike the situation observed in Arbacia and Spisula , mouse eggs treated with CB (1–50 μg/ml for 1 hr) are capable of sperm incorporation. These data are discussed with reference to possible mechanisms by which sperm may induce eggs to activate.

Correlative ultrastructural and electrophysiological studies of sperm-egg interactions of the sea urchin, Lytechinus variegatus☆

The sequence of ultrastructural events following the onset of the sperm-induced conductance increase in eggs of the sea urchin, Lytechinus variegatus, was investigated. Eggs voltage clamped at -20 mV were fixed 1 to 20 sec after onset of the conductance increase caused by single sperm. Continuity between the plasma membranes of the sperm and egg was first detected 5 sec after onset of the conductance increase. The earliest stages of formation of the fertilization cone coincided with the establishment of continuity of the gamete plasma membranes. At 6 to 8 sec after the initial conductance increase cortical granule dehiscence was first observed in the immediate vicinity where continuity of the gamete plasma membranes had occurred. These observations are consistent with the conclusion that opening of ion channels at fertilization precedes fusion of the sperm and egg plasma membranes, while exocytosis of cortical granules is initiated following fusion of the sperm and egg plasma membranes.

Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg

Abstract Experiments have been carried out to test the proposal that the pH increase at fertilization in sea urchin eggs promotes microvillar elongation. Results presented herein show that microvillar elongation and microfilament formation occurred when sea urchin eggs were incubated in sodium-free seawater containing the calcium ionophore A23187, a treatment which initiates activation, i.e., induces a transient increase in intracellular free calcium, but prevents subsequent cytoplasmic alkalinization. Within elongated microvilli and cortices of these eggs, microfilaments were arranged in a loose meshwork. However, if the pH of the egg cytoplasm was increased experimentally, microfilament bundles appeared within individual microvilli. These findings suggest that: (1) microvillar elongation and microfilament formation in the sea urchin egg at fertilization may occur when cytoplasmic alkalinization is inhibited, and (2) formation of the microvillus bundle of microfilaments at egg activation is pH sensitive. Additionally, if the cytoplasmic pH of unfertilized eggs was experimentally elevated by NH 4 Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.

Insemination of immature sea urchin (Arbacia punctulata) eggs

Abstract Sea urchin previtellogenic, vitellogenic, and meiotically dividing oocytes, and eggs having completed meiosis (pronuclear ova) were examined with light and electron microscopy at increasing periods after mixing with sperm. Electron microscopic observations demonstrated that spermatozoa were able to fuse with ova at all of the stages of development that were studied. Transformations of incorporated sperm nuclei into male pronuclei, i.e., disappearance of the sperm nuclear envelope, chromatin dispersion, and formation of a pronuclear envelope were only observed in inseminated pronuclear eggs. Sperm nuclei that enter meiotically dividing oocytes decondense to a limited extent but not to the same degree as observed in inseminated pronuclear ova. Cisternae do not become associated with the dispersed chromatin and a nuclear envelope is not formed in these specimens. Sperm nuclei incorporated into pre-and vitellogenic oocytes become surrounded by membranous elements of undetermined origin and do not undergo chromatin dispersion. These results suggest that factors or conditions required for development of male pronuclei are active (or appear) at progressive stages of nuclear maturation.

Organization of microfilaments in sea urchin (Arbacia punctulata) eggs at fertilization: effects of cytochalasin B.

Abstract Investigations were carried out to examine more closely the aggregations of microfilaments associated with the elongation of microvilli and formation of fertilization cones and the effects of cytochalasin B (CB) on these processes in Arbacia eggs following insemination. At 1 to 5 min postinsemination fertilized eggs were treated with 1–10 μg/ml CB and then prepared for electron microscopy at periodic intervals. Examination of CB-treated and untreated specimens demonstrated that: (1) Reorganization of the eggs microvilli took place soon after insemination; this process, as well as formation of fertilization cones, was correlated with the appearance of fascicles of microfilaments. (2) CB inhibited the formation of fertilization cones and the elongation of microvilli. Bundles of microfilaments were not observed in CB-treated zygotes. (3) CB prevented the normal movements (rotation) of the incorporating spermatozoon into the egg cortex but did not inhibit the migration or fusion of the male and female pronuclei.

Chapter 6 Transformations of Sperm Nuclei Upon Insemination

Publisher Summary This chapter describes the transformations of the sperm nuclei upon insemination. Normal interactions between the nucleus and cytoplasm are of fundamental importance at all stages of embryonic development. However, during fertilization, a unique situation exists that demonstrates rather dramatically the presence of the conditions within the eggs that bring about major changes in nuclear morphology and function. The relationship and possible functional equivalence of the transformations, the maternally and paternally derived chromatin, undergoes at fertilization and the changes in size and behavior of the transplanted nuclei has been discussed in the chapter. At fertilization, the sperm nucleus is reorganized to form the male pronucleus. Transformation of the sperm nucleus into a male pronucleus involves morphological changes similar to those that have been described for the transplanted nuclei; they include: chromatin decondensation and nuclear enlargement. The activity of the genes has been shown to depend on the association of regulatory proteins with particular regions of chromosomes. The transformations of the sperm nucleus at fertilization into a male pronucleus may be grouped into three processes that are described ultrastructurally in a number of animals and include: (1) breakdown of the sperm nuclear envelope, (2) chromatin dispersion, and (3) formation of a “new” nuclear envelope.

Calreticulin Modulates Capacitative Ca2+ Influx by Controlling the Extent of Inositol 1,4,5-Trisphosphate-induced Ca2+ Store Depletion

Calreticulin (CRT) is a highly conserved Ca2+-binding protein that resides in the lumen of the endoplasmic reticulum (ER). We overexpressed CRT in Xenopusoocytes to determine how it could modulate inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ influx. Under conditions where it did not affect the spatially complex elevations in free cytosolic Ca2+ concentration ([Ca2+] i ) due to InsP3-induced Ca2+ release, overexpressed CRT decreased by 46% the Ca2+-gated Cl− current due to Ca2+influx. Deletion mutants revealed that CRT requires its high capacity Ca2+-binding domain to reduce the elevations of [Ca2+] i due to Ca2+ influx. This functional domain was also required for CRT to attenuate the InsP3-induced decline in the free Ca2+concentration within the ER lumen ([Ca2+]ER), as monitored with a “chameleon” indicator. Our data suggest that by buffering [Ca2+]ER near resting levels, CRT may prevent InsP3 from depleting the intracellular stores sufficiently to activate Ca2+ influx.

Expression and localization of DNA topoisomerase II during rat spermatogenesis

The potential role(s) of DNA topoiosmerase II (topo II) during chromatin changes that characterize different stages of spermatogenesis was investigated in the rat by an analysis of the expression and localization of topo II mRNA and protein in individual spermatogenic cells. Expression of topo II was restricted to spermatogonia, spermatocytes, and round and early‐elongating spermatids. Two protein bands of 177 and 170 kDa were detected in immunoblots of spermatocytes and round spermatids, while bands of 148 and 142 kDa were prominent in preparations of elongating spermatids. Topo II levels and distribution patterns, as observed by immunofluorescent microscopy, exhibited cell type‐specific variations. Differences in topo II staining patterns were also apparent when nuclear matrices of spermatogenic cells were prepared with different extraction conditions. In addition to its possible function as a structural component, topo II, associated with nuclear matrix preparations from spermatogenic cells, possessed catalytic activity. These observations indicate that both the 177 and 170 kDa and the 148 and 142 kDa forms of topo II share similar structural and functional properties. Topo IIβ mRNA was transcribed in rat spermatogenic cells at 6.2 kb. Relative levels of topo IIβ mRNA were high in spermatogonia and spermatocytes, and decreased in both round and early‐elongating spermatids. Changes in topo II expression levels and localization patterns represent distinct stage‐specific markers for the maturation of spermatogenic cells, and are consistent with the involvement of topo II in mediating DNA modifications and chromatin changes during spermatogenesis.

Relation of intracellular pH and pronuclear development in the sea urchin, Arbacia punctulata. A fine structural analysis.

Abstract Zygotes, treated with sodium-free artificial seawater (Na-free ASW) in order to inhibit the elevation of intracellular pH at fertilization, were examined by light and electron microscopy. Although such specimens elevated fertilization membranes, male pronuclear development and sperm aster formation were suppressed. Inhibition of these events was reversed when zygotes were resuspended in seawater or in Na-free ASW containing ammonia. These results indicate that alkalinization following insemination or processes accompanying this alteration induce a pervasive change within the zygote which supports events of fertilization.