The human pronuclear ovum: Fine strcture of monospermic and polyspermic fertilization in vitro

The fine structure of pronuclear ova (monospermy and polyspermy) and one-cell embryos has been investigated in our IVF programme. Sixteen oocytes were collected at laparoscopy after appropriate hormonal stimulation and were matured and fertilized in vitro by methods that have given rise to normal pregnancies. Pronuclear ova showing monospermic fertilization had two vesicular pronuclei surrounded by aggregations of cellular organelles. The male pronucleus was closely associated with a sperm axoneme, while the female pronucleus was dismantling its envelope and condensing its chromatin ahead of its counterpart in late pronuclear ova. Each pronucleus had dispersed chromatin, dense compact nucleoli, and intranuclear annulate lamellae. Smooth endoplasmic reticulum, annulate lamellae, Golgi complexes, and mitochondria formed a conspicuous part of the perinuclear ooplasm. The one-cell embryos were either in syngamy or in the process of undergoing first cleavage. Positive evidence of cortical granule release and second polar bodies were detected in the perivitelline space. A block to polyspermy seemed to operate at the level of the inner zona. Dispermic and polyspermic ova had 3–16 pronuclei resembling those of monospermic ova and had sperm tails in the ooplasm. Sperm were also seen penetrating the inner zona and were occasionally found in the perivitelline space. Incomplete cortical granule release and early signs of cytoplasmic fragmentation were noted in polyspermic ova. Both normal and abnormal features of these ova are reported and compared with pronuclear structure in vivo and in vitro.     

Remodeling the sperm nucleus into a male pronucleus at fertilization

After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.     

Nascent protein requirement for completion of meiotic maturation and pronuclear development: examination of fertilized and A-23187-activated surf clam (Spisula solidissima) eggs

The involvement of newly synthesized proteins and calcium in meiotic processes, sperm nuclear transformations, and pronuclear development was examined in emetine-treated, fertilized, and A-23187-activated Spisula eggs by observing changes in the morphogenesis of the maternal and paternal chromatin. Emetine treatment (50 micrograms/ml) initiated 30 min before fertilization or A-23187 activation inhibited incorporation of [3H]leucine into TCA-precipitable material and blocked second polar body formation. Sperm incorporation and the initial enlargement of the sperm nucleus were unaffected; however, the dramatic enlargement and transformation of the sperm nucleus into a male pronucleus, which normally follow polar body formation, were delayed 10 to 20 min. Unlike the situation in untreated, control eggs, male pronuclear development took place while the maternally derived chromosomes remained condensed. It was not until approximately 20 min after the normal period of pronuclear development that the maternal chromosomes dispersed and formed a female pronucleus in emetine-treated, fertilized eggs. Formation of pronuclei, however, was unaffected in both emetine-treated, A-23187-activated eggs and fertilized eggs incubated with A-23187. These observations indicate that germinal vesicle breakdown, first polar body formation, and initial transformations of the sperm nucleus are independent of newly synthesized proteins. Inhibition of second polar body formation and the delay in pronuclear development brought about by emetine, as well as the appearance of silver grains over pronuclei in autoradiographs of control eggs incubated with [3H]leucine demonstrate that nascent proteins are involved with the completion of meiotic maturation and the development of male and female pronuclei. The ability of A-23187 to override the inhibitory effects of emetine on pronuclear development suggests that both nascent protein and calcium signals are involved in regulating the status of the maternal and paternal chromatin during pronuclear development.     

Chromatin transitions in the fertilizing sperm nucleus of the sea urchin,strongylocentrotus purpuratus

Electron microscopic analysis of fertilization in the sea urchin, Strongylocentrotus purpuratus, has been carried out in an effort to establish the sequence of events involving dispersion of the paternal chromatin. Subsequent to loss of the nuclear envelope the condensed sperm chromatin begins to disperse under the influence of egg cytoplasmic factors. However, this process does not proceed at a uniform rate as is observed in other species examined to date. Portions of the paternal genome rapidly transform into dispersed chromatin while other adjacent regions disperse at a reduced rate. This variation in the time sequence of dissociation of the paternally derived chromosomes results in a reticulum of electron lucent and electron dense chromatin within the developing male pronucleus. As the paternally derived chromatin is dispersing and migrating centrad, membranous vesicles of maternal origin become aligned along the peripheral aspect of the chromatin. Deposition of a continuous bilaminar nuclear envelope around the dispersing sperm chromatin results in the formation of the definitive male pronucleus. At the time the male pronucleus is formed the paternally derived chromosomes have not completely dispersed and are visualized as a reticulum of condensed and dispersed chromatin. These results indicate that not all the paternally derived chromatin is modified in the same manner during pronuclear development.     

Ultrastructure of pig tubal ova

Summary The unfertilized ova of the pig are characterized by the first polar body situated in the perivitelline space. The metaphase chromosomes of the ova are found free in a cortical area, predominantly inhabited by the spindle fibers. Mitochondria show morphological changes in the form of swelling of their matrices. Frequently, the membranes of the individual cristae mitochondriales meet each other, forming meeting points, at regular intervals. The endoplasmic reticulum increases in quantity when compared with that of the pig follicular oocytes (Norberg, 1972b). The Golgi complexes are sparse and scattered. Occasionally, remnants of the end bulbs of the corona radiata cell processes occur below the surface membrane of the ova.Usually, the sperm-penetrated ova contain the first and the second polar body within the perivitelline space. Intranuclear annulate lamellae are observed within the male and female pronucleoplasm, and of particular interest are extended linear structures in one of the pronuclei. These structures may be considered as precursor stage in the formation of the intranuclear annulate lamellae. The parapronuclear cytoplasm is rich in organelles, especially the cytoplasmic annulate lamellae. In contrast to the scarcity of Golgi complexes in the unfertilized ova, many newly formed Golgi vesicles and lamellae reappear in the pronuclear stage. The zona pellucida displays ultrastructural changes following sperm penetration of the ova.This work was supported by the Agricultural Research Council of Norway.     

Microtubule distribution during fertilization in the rabbit.

The distribution of microtubules was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti-alpha-tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronuclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.     

THE EFFECTS OF NICOTINE ON FERTILIZATION IN THE SEA URCHIN, ARBACIA PUNCTULATA

The number of sperm incorporated into eggs made polyspermic with varying concentrations of nicotine (0.025–0.25%, v/v) appears to be directly related to the concentrations employed. The cortical response is morphologically equivalent to that observed in control preparations. Shortly after their incorporation all of the spermatozoa undergo structural events normally associated with the development of the male pronucleus in monospermic eggs. During the reorganization of the spermatozoa, sperm asters are formed. The number of male pronuclei that initially migrate to and encounter the female pronucleus is usually one to three. When pronuclei come into proximity to one another the surface of the female pronucleus proximal to the advancing male pronuclei flattens and becomes highly convoluted. Subsequently, the pronuclei contact each other and the outer and inner membranes of the pronuclear envelopes fuse, thereby producing the zygote nucleus. The male pronuclei remaining in the zygote after this initial series of pronuclear fusions continue to differentiate, i.e. they enlarge, form nucleolus-like bodies, and undergo further chromatin dispersion. In approximately 90% of the zygotes, all of the remaining male pronuclei progressively migrate to the zygote nucleus and fuse to form one large nucleus by 80 min postinsemination. Mitosis and cleavage of the polyspermic zygote occurs later than in monospermic eggs.     

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.     

Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii

The gynogenetic fish, Carassius auratus langsdorfii (the ginbuna, a crucian carp), provides an interesting model for the study of the mechanisms controlling male pronucleus formation. When the sperm nucleus of a different subspecies (C. a. cuvieri) is incorporated into the gynogenetic egg, the nuclear envelope of the spermatozoon is not broken down, and the pronucleus fails to develop, although dispersion of the sperm chromatin occurs to some extent within the space limited by the nuclear envelope. When spermatozoa without plasma membranes and nuclear envelopes were microinjected into mature activated eggs, the sperm nuclei underwent chromatin dispersion, nuclear envelope formation, DNA synthesis, and transformation into male pronuclei. These results indicate that the failure of the male pronucleus to form in ginbuna is primarily due to the failure of sperm nuclear envelope breakdown. We conclude that sperm nuclear envelope breakdown is an indispensable step for the development of the male pronucleus.     

THE FINE STRUCTURE OF PRONUCLEAR DEVELOPMENT AND FUSION IN THE SEA URCHIN, ARBACIA PUNCTULATA

Fertilization events following coalescence of the gamete plasma membranes and culminating in the formation of the zygote nucleus were investigated by light and electron microscopy in the sea urchin, Arbacia punctulata. Shortly after the spermatozoon passes through the fertilization cone, it rotates approximately 180° and comes to rest lateral to its point of entrance. Concomitantly, the nonperforated nuclear envelope of the sperm nucleus undergoes degeneration followed by dispersal of the sperm chromatin and development of the pronuclear envelope. During this reorganization of the sperm nucleus, the sperm aster is formed. The latter is composed of ooplasmic lamellar structures and fasciles of microtubules. The male pronucleus, sperm mitochondrion, and flagellum accompany the sperm aster during its migration. As the pronuclei encounter one another, the surface of the female pronucleus proximal to the advancing male pronucleus becomes highly convoluted. Subsequently, the formation of the zygote nucleus commences with the fusion of the outer and the inner membranes of the pronuclear envelopes, thereby producing a small internuclear bridge and one continuous, perforated zygote nuclear envelope.     

Nuclear envelope dynamics during male pronuclear development

Upon fertilization, the sperm nucleus undergoes reactivation. The poreless sperm nuclear envelope is replaced by a functional male pronuclear envelope and the highly compact male chromatin decondenses. Here some recent evidence is examined: that disassembly of the sperm lamina is required for chromatin decondensation, that remnant portions of the sperm nuclear envelope target the binding of egg membrane vesicles that form the male pronuclear envelope, that functional male pronuclear envelopes containing lamin B receptor assemble prior to lamin import and lamina formation, and that lamina assembly drives male pronuclear swelling. Several unresolved issues are discussed.     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

The maternal effect mutation sésame affects the formation of the male pronucleus in Drosophila melanogaster

After entering the oocyte and before the formation of the diploid zygote, the sperm nucleus is transformed into a male pronucleus, a process that involves a series of conserved steps in sexually reproducing animals. Notably, a major modification of the male gamete lies in the decondensation of the highly compact sperm chromatin. We present here the phenotype of sésame (ssm), a maternal effect mutation which affects the formation of the male pronucleus in Drosophila melanogaster. Homozygous ssm(185b) females produce haploid embryos which develop with only the maternally derived chromosomes. These haploid embryos die at the end of embryogenesis. Cytological analyses of the fertilization in eggs laid by ssm(185b) mutant females showed that both pronuclear migration and pronuclear apposition occurred normally. However, a dramatic alteration of the male pronucleus by which its chromatin failed to fully decondense was systematically observed. Consequently, the affected male pronucleus does not enter the first mitotic spindle, which is organized around only the maternally derived chromosomes. Immunodetection of lamina antigens indicates that a male pronuclear envelope is able to form around the partially decondensed paternal chromatin. This suggests that the maternally provided sésame(+) function is required for a late stage of sperm chromatin remodeling.     

Selective association of some hamster-egg-synthesized proteins with decondensing human sperm chromatin

Zona-free hamster eggs were fertilized in vitro with human spermatozoa in a culture medium enriched with either 3H-arginine or 3H-tryptophan. Autoradiography was used to investigate decondensing sperm heads and all pronuclei for the presence of newly synthesized, 3H-labelled proteins. In the case of 3H-arginine-labelled proteins, an intense accumulation of radioactivity was detected in all autoradiograms of chromatin structures. On the other hand, no comparable accumulation was seen for 3H-tryptophan-labelled proteins up to the progressed-pronucleus stage. It is concluded that, as a part of changes of the nucleoproteins in decondensing sperm chromatin, there is an accumulation in the male (as well as in the female) pronucleus of basic nuclear proteins synthesized by the egg during fertilization. Since non-histone, 3H-tryptophan-labelled proteins were not incorporated in the same way, these 3H-arginine-labelled proteins accumulating in pronuclear chromatin during the earliest phase of pronucleus formation are probably histones.     

Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin.

Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin LIII did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.     

Electron microscopic observations on sperm entry and pronuclear formation in naked eggs of the rose bitterling in polyspermic fertilization

Unfertilized eggs of the rose bitterling (Rhodeus ocellatus ocellatus) were squeezed out of females that had an elongated ovipositor and were dechorionated mechanically with fine forceps in physiological saline. The dechorionated eggs were transferred into fresh water then inseminated at once by spermatozoa of the same species. A large number of spermatozoa was found on the surface of eggs that had not yet had cortical reaction following insemination. The surface of the naked eggs responded by formation of many small cytoplasmic protrusions (viz., fertilization cones) at sperm attachment sites. The formed fertilization cones were rosettelike structures formed by the aggregation of some bleblike swellings devoid of microvilli and microplicae. About 10 min after insemination, the fertilization cones retracted, but marks of their presence characterized by less microvilli and microplicae remained in the eggs 15 min after insemination. Many spermatozoa penetrated into the cytoplasm of each naked egg. The sperm nuclear envelope disappeared by means of vesiculation resulting from fusion of the inner and outer membranes. The sperm nucleus decondensed and developed into a larger male pronucleus. Smooth-surfaced vesicles surrounded the decondensing sperm nucleus and formed the new male pronuclear envelope. Sperm mitochondria and flagella were found in the egg 15 min after insemination. The response of the egg surface to sperm entry and pronucleus formation are discussed.     

The changes in sperm nuclei after penetrating fish oocytes matured without germinal vesicle material in their cytoplasm

The processes occurring from sperm penetration to chromosome formation in the cytoplasm of Oocytes matured in vitro, after removal of the germinal vesicle (GV) and before hormonal stimulation, were observed with electron microscope. The dechorionated oocytes, matured without the participation of the GV material, responded to sperm penetration by initiating a cortical reaction within 20 seconds after insemination. The pentrating sperm nuclei transformed to male pronuclei with vesiculation of the nuclear membrane, chromatin decondensation, and formation of a pronuclear membrane. Before cleavage, however, no chromosome formation was observed in these oocytes. Instead, the fully grown pronuclei change to a picnotic chromatin mass without or with an only fragmented nuclear membrane, then disappeared. On the contrary, sperm nuclei that penetrated into the cytoplasm of naked eggs containing GV material during maturation underwent pronuclear and chromosomal formation. Judging from these observation in Oryzias oocytes, the GV material seems to be unnecessary for the formation of pronucleus from the compact sperm nucleus, but is essential for the process of chromosomal formation.     

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.     

Premature sperm incorporation into the primary oocyte of the polychaete Pectinaria: Male pronuclear formation and oocyte maturation

Immature oocytes of the annelid Pectinaria were prematurely fertilized while in the germinal vesicle stage. Fertilization was morphologically normal except for the formation of an enlarged fertilization cone which persisted even after sperm incorporation. However, at 30 min postinsemination, no signs of male pronuclear morphogenesis were detected. Ultrastructural data show that in the cytoplasm of a GV-stage oocyte the sperm nuclear envelope remains intact and the enclosed chromatin remains condensed. Prematurely fertilized eggs were then induced to undergo germinal vesicle breakdown (GVBD). Subsequently male pronuclear development occurred. Thus, the factors in the Pectinaria oocyte which are necessary for sperm transformation develop in the maturing cytoplasm and are dependent upon GVBD. Such prematurely fertilized oocytes fail to display the normal arrest of meiosis at Metaphase I, but instead progress directly to formation of the female pronucleus. Occurrences of normal first cleavage were observed suggesting that prematurely incorporated sperm can be recruited for participation in development.     

Development of human male pronucleus: Ultrastructure and timing

The process of human male pronuclear formation was studied using an experimental model based on in vitro inseminated human zona-free eggs prepared from oocytes that failed to fertilize in a clinical in vitro fertilization program. The main ultrastructural changes in penetrated sperm nuclei transforming into pronuclei were used to define four stages of pronuclear development. The first two stages, representing partial (Stage 1) and total (Stage 2) sperm chromatin decondensation, appeared as early as 1 hr after mixing of gametes. This rapid initial phase was followed by a more lengthy array of events leading to transformation of decondensed sperm nuclei into fully developed male pronuclei (Stages 3 and 4). Stage 3 was characterized by reformation of the nuclear envelope, reorganization of chromatin, and the assembly of nuclcolar precursors. It was not completed until 12 hr after in vitro insemination when fully developed male pronuclei (Stage 4) were first observed. In some eggs pronuclei did not reach Stage 4 at all. The results of this study provide a morphological background for further research into molecular aspects of human male pronuclear development and its regulation.