Surface activity at the egg plasma membrane during sperm incorporation and its cytochalasin B sensitivity: Scanning electron microscopy and time-lapse video microscopy during fertilization of the sea urchin Lytechinus variegatus

Sperm incorporation and the formation of the fertilization cone with its associated microvilli were investigated by scanning electron microscopy of eggs denuded of their vitelline layers with dithiothreitol or stripped of their elevating fertilization coats by physical methods. The activity of the elongating microvilli which appear to engulf the entering spermatozoon was recorded in living untreated eggs with time-lapse video microscopy. Following the acrosome reaction, the elongated acrosomal process connects the sperm head to the egg surface. About 15 microvilli adjacent to the attached sperm elongate at a rate of 2.6 μm/min and appear to engulf the sperm head, midpiece, and sperm tail. These elongate microvilli swell to form the fertilization cone (average height, 6.7 ± 2.0 μm) and are resorbed as the sperm tail enters the egg cytoplasm 10 min after insemination. Cytochalasin B, an inhibitor of microfilament motility, completely inhibits the observed egg plasma membrane surface activity in both control and denuded eggs. These results argue for a role of the microfilaments found in the egg cortex and microvilli as necessary for the engulfment of the sperm during incorporation and indicate that cytochalasin interferes with the fertilization process at this site.     

Surface area change at fertilization: Resorption of the mosaic membrane

Eggs of Strongylocentrotus purpuratus (sea urchin) have a surface area of 41,000 μm² before fertilization as determined by quantitative transmission and scanning electron microscopy. Within a minute after fertilization 18,000 cortical vesicles contribute an additional 57,000 μm² to form a mosaic membrane with the original plasma membrane. However, by 16 min after fertilization the total area of the egg is only 45,000 μm², indicating a rapid resorption of surface. Calculations of surface area depend in large part upon the numbers and dimensions of microvilli, after careful compensations are made for specimen shrinkage. The 134,000 microvilli per egg are 0.35 μm long before fertilization. They elongate to 1.0 μm in the first few minutes and then soon shorten to 0.5 μm. Even at their longest, microvilli do not accommodate all of the surface area of cortical vesicle membrane. The merger of cortical vesicle membranes and the plasma membrane was demonstrated many years ago and is not in doubt; however, this study indicates that the resulting mosaic membrane is not a long-lived, simple arithmetic combination of its components. Rather, the mosaic membrane undergoes a rapid and dynamic shrinkage by a mechanism which is not apparent on the basis of egg topography alone. The absolute values of egg surface area and dynamic changes in the surface are discussed in relation to physiological events accompanying fertilization.     

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

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 NH₄Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.     

Microvillar elongation following parthenogenetic activation of sea urchin eggs

Parthenogenetic activation of unfertilized sea urchin eggs with ammonium chloride at pH 8.0 resulted in a slow, but dramatic, reorganization of surface microvilli in four species of sea urchin eggs. Following NH4Cl treatment, elongation of microvilli on the egg surface was observed concomitant with the formation of microfilament bundles within the microvillar cores. A minimum of 2 h of treatment was required for elongation and microfilament bundle formation to occur. The maintenance of elongated microvilli was pH-sensitive; removal of the activating agent resulted in the retraction of extended microvilli while readdition of NH4Cl caused microvilli to elongate again. Accompanying microvillar elongation in activated eggs, there was an increased calcium uptake as measured by 45Ca uptake. Blocking calcium uptake by incubation in lanthanum chloride or zero-calcium seawater containing 2 mM EGTA prevented microvillar elongation. These results suggested that elongation of microvilli following parthenogenetic activation by NH4Cl is pH- and calcium-dependent and is similar to that observed during normal fertilization.     

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

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 egg's 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.     

Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles

Using improved fixation procedures we have found that extensive endocytotic activity is turned on at fertilization in eggs of three species of sea urchins. Beginning after completion of cortical granule exocytosis and after exocytotic pits have completely smoothed over, the entire activated egg surface engages in a limited period of extensive removal of membrane via uptake into coated vesicles. This “burst phase” lasts about 3–5 min after which the number of invaginating coated vesicles decreases rapidly. At the end of this burst phase all the patches of cortical granule membranes have disappeared, and the egg surface is left uniformly covered by microvilli. For the remainder of the first cell cycle coated pits continue to form at a slower but steady rate. Endocytotic activity continues past the time of first cleavage. There is distinct overlap in onset and duration of the burst phase of endocytosis with the period of medium acidification during normal development. However, activation of eggs in choline sea water, which inhibits acid secretion, results in an endocytic burst whose timing and duration are similar to those in normal eggs. The endocytic burst is, therefore, independent of cytoplasmic alkalinization. These results suggest, in accord with the two-step model of egg activation (D. Epel, R. A. Steinhardt, and R. A. Humphreys, 1974; Dev. Biol.40, 245–255; D. Epel, 1978, Curr. Top. Dev. Biol.12, 185–246) that initiation of endocytosis is most likely a Ca²⁺-dependent event.     

Structure and formation of the unusual sperm of Patelloida latistrigata (Mollusca : Patellogastropoda): implications for fertilization biology

The structure of the spermatozoa and spermatogenesis of the lottiid limpet Patelloida latistrigata is described by transmission electron microscopy. Although the lengths of the spermatozoa (about 60 μm) and their head region (about 12 μm) are similar to those of other patellogastropods, the structure of the sperm head and midpiece are very different. The head consists of an unusually large acrosome (about 11-μm long) with a broad posterior invagination that houses the relatively small nucleus. The midpiece mitochondria, which are rather elongate with large folded tubular cristae, are housed in a cytoplasmic sheath posterior to the nucleus. The proximal centriole is unusually elongate (about 2-μm long). The axoneme that emerges from the distal centriole is surrounded anteriorly by the cytoplasmic sheath in which the cytoplasmic side of the plasma membrane has electron-dense material. The flagellum is enlarged at its terminal end. Spermatogenesis is similar to that described for other patellogastropods. Patelloida latistrigata, therefore, has spermatozoa that seem to meet the morphological criteria of ent-aquasperm, which raises the question of whether fertilization is truly external in this limpet. However, it is also possible that the modifications to the sperm are linked to unknown specializations of the egg or egg envelope.     

ELECTRON MICROSCOPIC OBSERVATIONS ON THE EXTRA-GRANULAR ZONE OF THE EMBRYO OF THE SEA URCHIN,HEMICENTROTUS PULCHERRIMUS*

“Extra-granular zone” proposed by Dan and Dan (3) was studied with the electron microscope. Two stages are noticed during the process of its formation; at first, a relatively yolk granule-free zone constitutes the egg periphery which should correspond to that observed by Dan and Dan , and later, this zone is occupied by a layer of the vacuoles (about 1.3 μm in mean diameter) with highly electron-transparent inclusions which migrated to the egg surface from the inner cytoplasm after fertilization. These vacuoles are scarcely found in the micromeres of 16-cell stage embryo.     

Microfilaments during sea urchin fertilization: Fluorescence detection with rhodaminyl phalloidin

Rhodaminyl-labeled phalloidin is used to demonstrate the distribution of microfilaments during fertilization and early development in eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The surface of unfertilized eggs have numerous punctate fluorescence sites at which rhodaminyl phalloidin binds, indicating the presence of actin oligomers or polymers. During fertilization this punctate pattern of fluorescence begins to change. Within thirty seconds of insemination, the fertilization cone is first detectable with this technique as an erect structure on the surface of the egg. The fertilization cone grows to a maximum size by 8–9 minutes, and is resorbed by 16 minutes after insemination. The surface of the fertilized egg displays numerous fluorescent fibers by 10 minutes after insemination rather than the punctate fluorescence observed in unfertilized eggs, indicative of the burst of microfilament assembly resulting in microvillar elongation. The elongated microfilaments persist through cytokinesis. Staining is also detected throughout the cortices of unfertilized, fertilized, and cleaving eggs. Cytochalasin E (10 μM, 30 min) prevents microfilament elongation and cytokinesis and reduces the cortical staining intensity after fertilization. At cleavage, contractile rings, appearing as narrow equatorial bundles of fibers, have been detected in Lytechinus variegatus as transient structures.     

Cytoskeletal and contractile proteins in coelomic oocytes,unfertilized and fertilized eggs of discoglossus pictus (Anura)

The distribution of actin, myosin, and tubulin has been investigated in coelomic oocytes, unfertilized and fertilized eggs of Discoglossus pictus utilizing: (1) immunofluorescence; (2) electron microscopy; (3) incubation with heavy meromyosin (HMM), and (4) SDS-polyacrylamide gel electrophoresis (PAGE). In coelomic oocytes, the germinative area (GA) has long, irregular microvilli containing microfilaments. In the rest of the oocyte, the microvilli are shallow. During the transit of the oocyte in the oviduct, a dimple forms by the invagination of the GA. A palisade of microfialment bundles is present in the finger-shaped microvilli of the dimple and extends for about 10 μm in the cytoplasm. In the rest of the egg, microvilli are absent and only random filaments appear in the cortex. Following HMM incubation, the dimple microfilaments are decorated with arrowheads pointing toward the bulk of the cytoplasm. SDS-PAGE of egg extracts shows bands co-migrating with actin (43K), pyruvate kinase (57K), and phosphorylase (94K). As result fertilization, the pattern of microfilament bundles in the dimple disappers in parallel with the dimple invergination itself. Generally, the entire oocyte cortex is positive to immunofluorescent staining with anti-actin, antimyosin, and antitubulin antibodies. However, the pattern of distribution and intensity of immunofluorescent staining changes for each antiserum, during different stages. It is concluded that a contractile system is present in Discoglossus eggs, and it is particularly developed in the dimple. The dimple is probably a major compartment for the storage of unpolymerized tubulin.     

Analysis of sea urchin egg cortical transformation in the absence of cortical granule exocytosis

A burst of endocytosis accompanying microvillar elongation follows cortical granule exocytosis in normal sea urchin development. By 5 min postfertilization the burst is over and a lower level of endocytosis ensues (constitutive phase). To determine whether microvillar elongation and initiation of endocytosis are necessary concommitants of cortical granule exocytosis we utilized Chase's (1967, Ph.D. thesis, University of Washington, Seattle) high-hydrostatic pressure technique to block the latter and then examined developing eggs for endocytosis and microvillar elongation. To accomplish this, eggs were fertilized, after which hydrostatic pressure was quickly raised to 6000-7000 psi at the start of cortical granule exocytosis and maintained for 5 min. Only the cortical granules immediately surrounding the sperm penetration site were secreted (about 3% or less of the egg's total number of cortical granules). Blockage of major cortical granule exocytosis had the following consequences on surface events during first division: (1) The endocytosis burst normally associated with cortical granule exocytosis was effectively eliminated as was early microvillar elongation and elevation. Both occurred to a limited extent around the sperm penetration site which resulted in a highly localized surface transformation. (2) By 20 min after fertilization endocytosis began over the rest of the egg surface in the absence of any further cortical granule exocytosis. (3) Subsequently, during a 30-min period starting midway between fertilization and first cleavage microvilli more than doubled in length and endocytosis levels increased severalfold. These events brought about a complete surface transformation similar to that which normally occurs in early development but in the absence of cortical granule exocytosis. By first cleavage surfaces and cortices of high-pressure-treated and control eggs were nearly indistinguishable except for the presence of cortical granules in cortices of the former. Pressure-treated eggs cleaved normally and developed to larval forms overnight. The period of late surface transformation in high-pressure-treated Strongylocentrotus purpuratus eggs corresponds in timing and some of its characteristics to second phase microvillar elongation observed in normal development in this species and also in S. droebachiensis development. These observations suggest, therefore, that microvillar elongation and endocytosis are necessary membrane remodelling events which must occur for normal development even in the absence of membrane addition from the cortical granules.     

Movement of a non-flagellate spermatozoon: A study of the male gamete of Nematospiroides dubius (Nematoda)

The spermatozoa of Nematospiroides dubius were studied using the scanning electron microscope and time-lapse cinematography. Spermatozoa undergo a profound change in morphology after insemination: they change from an elongate structure, 16–18 μm long, to a more rounded form about 5–10 μm in diameter. Spermatozoa from female worms stuck to, and migrated across a glass surface by the production of pseudopodia, but they adhered more readily to a glass surface coated with egg albumin. The average speed of a sample of six differentiated spermatozoa was 7·3 μm/min. Their locomotion is not considered to be amoeboid but resembles the movement of monopodial neutrophils. A hypothesis for the mechanism of movement is presented, and other possible functions of the pseudopodial region are discussed.     

Novel surface specialization on a sea anemone egg: “Spires” of actin-filled microvilli

The ultrastructure of the “spiny” surface of Tealia crassicornis eggs is examined in detail by scanning and transmission electron microscopy in order to understand its function. Long microvilli are clustered together in spiral aggregates of 50–75 microvilli called “spires.” There are about 15,000 spires per egg. Dense bundles of microfilaments making up the cores of these microvilli are shown to be composed of actin by staining with the fluorescent dye nitrobenzoxadiazole (NBD)-phallacidin. It is postulated that the bundles of actin and the spires of microvilli are stiff and provide reinforcement to the egg surface. Such postulated properties would provide physical protection for these large eggs which, unlike the eggs of most invertebrates, appear to lack all extracellular investing coats.     

Observation on the incorporation cone in the rat

At the time of in vivo sperm–egg fusion in the rat, a small region of the oolemma under the head of the fertilizing sperm is observed to be free of microvilli. The microvilli-free region increases in area, and by one hour after sperm–egg contact extends over an area 20–30 μ in circumference and bulges out to form an “incorporation cone” visible by light microscopy. The microvilli-free incorporation cone reaches its maximum size at about two hours after sperm–egg interaction. It soon becomes smaller and has disappeared three to four hours after sperm–oocyte fusion. The cone cytoplasm is characterized by a 0.1 μ zone of thin filaments below the plasma membrane. Cytochalasin-B, 2.5 μg/ml, prevents formation of the cone or destroys the intact cone. It is suggested that micro filaments may be involved in the formation of the incorporation cone.     

Ultrastructure of the spermatozoon of Lepidogalaxias salamandroides and its phylogenetic significance

The spermatozoon of Lepidogalaxias salamandroides possesses an acrosome (putative), one or two perforatoria (putative) but no nine-triplet centrioles. Two elongated mitochondria (12 μm long) are situated in parallel between the nucleus (20 μm long) and the axoneme (53 μm long). The above features are unique among other teleosts with internal fertilization. The presence of an “acrosome” in this primitive teleost supports the hypothesis that this structure has been secondarily lost in teleosts during evolution. The uncertainty of phylogenetic placement of this fish is reflected by its unique sperm ultrastructure.     

Formation and structure of the fertilization envelope in Xenopus laevis

This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro.     

Fertilization in Pinus Bungeana

Pinus bungeana is a species endemic to China and as yet its embryology has not been reported. The present paper deals with its process of fertilization in some details. 1. The development of the male gamete and the structure of the archegonium. The spermatogenous cell has already divided into two uniqual male gametes in the middle of May (in 1978, at Peking), about ten days before fertilization. Both sperms are spheroidal to ellipsoidal. The larger sperm is about 94 × 65 μm and the smaller one, about 72 × 58 μm in size. As the pollen tube approaches the archegonium the two sperms move toward the apex of the tube together with the remaining contents. Generally the larger sperm precedes the smaller one. The cytoplasmic contents also contain a sterile cell, 3—43×2—29 μm in size and a tube nuleus, 15—30 μm in diamter, besides the sperms. A mass of starch grains of more or less similar to sperm in size is also included in the contents of the pollen tube. Generally 3—4, even up to 7–8 pollen grains germinate normally within an ovule. Therefore, many sperms (up to 14—16) may be present on the same nucellus. The archegonium is elongato-ellipsoidal, about 870 ×500 μm in size. Arehegonia are single, 2—(3—5) in number, with 2 neck cells and a layer of jacket cells. The central cell divided in the middle of May and gave rise to the ventral canal cell and the egg. As the archegonium matures the cytoplasm becomes radiate fibrillae around the egg nucleus. The egg nucleus is large, 150—226 μm in diameter. One large nucleolus, 22—25 μm in diameter and sometimes up to 50; small nueleoli are present within the nucleus. 2. Fertilization Pollination takes place in the first week of May and fertilization will be effected from the end of May to the first week of June of next year. The interval between pollinatin and fertilization in P. bungeana is about thirteen months and the lapse of time is almost similar to most of the Pinus so far recorded. When the pollen tube contacts the archegonium through the neck cells all its contents are discharged into the egg cell. Usually the larger sperm fuses with the egg nucleus and the rest of the contents stays in the upper part of the egg cell. It is interesting to note that the nonfunctional second sperm also moves toward the egg nucleus and often divides by mitosis; and this phenomenon is not reported elsewhere. At the earlier stage of the fusion between male and female nuclei the male nucleoplasm is dense and finely granular while the female nucleoplasm is thin and coarsely granular, hence the boundary between them is very clear. The nuclear membranes of both nuclei persist for a long time. After the male nucleus sinks into the female nucleus completely, both nuclei begin to divide and enter into the prophase and then the metaphase simultaneously. By this time the paternal and maternal chromosome sets with their spindles still remain at certain distance from each other. Then the paternal chromosomes with their spindle move gradually toward the maternal ones. At first a multipolar common spindle appears as the maternal and paternal spindles with their chromosomes merge together. Finally a regular bipolar spindle is formed and both the maternal and paternal chromosomes become arranged on the equatorial plate. In the meantime, the process of fusion is complete and the zygote is at the stage of metaphase. At the moment the spindle looks greater in width than in length, being about 80×65—70 μm in size. 3. Supernumerary nuclei and sperms. The ventral canal cell degenerates soon after its formation. While the supernumerary sperms divide usually after their entrance into the egg cell. Therefore, the supernumerary nuclei probably derive directly from the smaller sperms or indirectly from mitoses of the larger ones Generally the nucleoplasm of the supernumerary nuclei is rather thin while the nucleoplasm of the undivided sperms is rather dense. This shows that the former is in the state of degeneration. The supernumerary nuclei of P. bungeana are as many as 7, their usual size being 43—58×32—43 μm. In the upper part of some egg cells there are still secondary smaller sperms about the size of 36 × 29 μm, Their volume is just about half of the usual smaller sperm. Probably they are derived from the division of the smaller sperms.     

Fertilization and early development in Beroe ovata

Fertilization in the clear egg (1 mm in diameter) of the ctenophore Beroe ovata and, in particular, the positioning and movements of pronuclei, and their relationship to the larval oral-aboral axis have been observed. Fertilization can take place anywhere on the egg surface. The sperm pronucleus remains at its entry site and becomes surrounded by a specialized zone (30–50 μm in diameter) beneath the surface referred to as the sperm pronuclear zone or SPZ and devoid of large cortical granules. Polyspermy has been observed to be frequent; each pronucleus is surrounded by its own SPZ. Only the egg pronucleus migrates with a continuous velocity (averaging 18 μm/min) and moves beneath the surface directly toward the immobile sperm pronucleus. In polyspermic eggs, the egg pronucleus can probe several SPZ, each containing a single sperm nucleus, before it finally enters one SPZ and fuses with the chosen sperm pronucleus. These migrations of the egg pronucleus occur over several millimeters and take hours, but the mechanism underlying the motion or how the egg pronucleus decides which SPZ to enter is not yet known. Under our experimental conditions the mitotic apparatus and the first cleavage plane which defines the oral-aboral axis of the larva (see Reverberi (1971). “Experimental Embryology of Marine and Fresh-Water Invertebrates.” North-Holland, Amsterdam. for review) pass through the point of sperm entry. During fertilization and cleavage, movements of a cortical autofluorescent material are clearly seen. This material is segregated into micromeres as cleavage progresses.     

Egg structures of four Nabis species (Rhynchota : Nabidae)

Despite the numerous works on insect egg structure, detailed studies on Nabis genus (Rhynchota : Nabidae) have not been carried out previously. The external morphology and internal chorionic structure of the eggs of Nabis pseudoferus pseudoferus Remane, Nabis occidentalis Rieger, Nabis punctatus Costa and Nabis rugosus L. were investigated, using scanning electron microscopy, to improve our knowledge of their organization. To assess their role in taxonomy, a comparison between the eggs of the 4 species under consideration was carried out. The eggs are jar-shaped with the front end narrowed in a “collar” and closed by an operculum. The chorion, except in the regions of the collar and the operculum, is organized in an outer layer of about 4–5 μm, separated from the inner surface by a “pillars” layer of about 0.5–1 μm. In the region of the collar, the chorion has small internal channels which represent the aeropyles; their number varies very considerably even within the same species. The operculum is made up of closed spaces filled with air in communication with the aeropyles and the “pillars” layer, thus forming a single space that represents the respiratory system. The general shape and characteristics of the eggs of N. pseudoferus, N. occidentalis and N. rugosus are very similar; only N. punctatus can be identified with certainty at the egg stage.     

The movements and fusion of the pronuclei at fertilization of the sea urchin Lytechinus variegatus: Time-lapse video microscopy

The penetration of the sperm into the egg, and the movements of the male and female pronuclei were followed from sperm attachment through pronuclear fusion, using time-lapse video microscopy of gametes and zygotes of the sea urchin Lytechinus variegatus (23° C). The pronuclei move in four stages: I. Sperm Entry Phase, following sperm-egg fusion and a rapid radiating surface contraction (5.9 ± 1.3 μm/second) when egg microvilli engulf the sperm head, midpiece, and tail to form the fertilization cone and the sperm tail beats in the egg cytoplasm; II. Formation of the Sperm Aster, which pushes the male pronucleus centripetally at a rate of 4.9 ± 1.7 μm/minute starting 4.4 ± 0.5 minutes after sperm-egg fusion, as the male pronucleus undergoes chromatin decondensation; III. Movement of the Female Pronucleus, the greatest and fastest of the pronuclear motions at a rate of 14.6 ± 3.5 μm/minute at 6.8 ± 1.2 minute after sperm-egg fusion, which establishes the contact between the pronuclei; and IV. Centration of the Pronuclei to the egg center at a rate of 2.6 ± 0.9 μm/minute by 14.1 ± 2.6 minutes after sperm-egg fusion. Pronuclear fusion typically occurs after stage IV and proceeds rapidly starting 14.7 ± 3.6 minutes after sperm-egg fusion with the male pronucleus coalescing into the female pronucleus at a rate of 14.2 ± 2.6 μm/minute.