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.     

The first cycle of DNA replication in mouse embryogenesis studied by microinjections of 3H-thymidine into the cytoplasm of fertilized ova

H-thymidine was injected into cytoplasm of the eggs taken at different intervals after fertilization and the eggs were fixed immediately thereafter. DNA synthesis was shown to begin in pronuclei when they are still in the marginal zones of cytoplasm, immediately after their formation. S-phase lasts 5-6 h in every pronucleus and is terminated at 1-2 h before the first cleavage division when the pronuclei are closely approached and located in the center of cytoplasm. At the end of S-phase late replicating heterochromatic regions are distinctly localized near the nuclear envelope and in pronuclei. Male and female pronuclei display asynchrony in the course of S-phase and differences in 3H-thymidine incorporation into chromatin. Structural features of the first cell cycle in mouse embryogenesis are discussed.     

DNA synthesis in the fertilizing hamster sperm nucleus: sperm template availability and egg cytoplasmic control

To assess the role of the availability of sperm nuclear templates in the regulation of DNA synthesis, we correlated the morphological status of the fertilizing hamster sperm nucleus with its ability to synthesize DNA after in vivo and in vitro fertilization. Fertilized hamster eggs were incubated in 3H-thymidine for varying periods before autoradiography. None of the decondensed sperm nuclei nor early (Stage I) male pronuclei present after in vivo or in vitro fertilization showed incorporation of label, even in polyspermic eggs in which more advanced pronuclei were labeled. In contrast, medium-to-large pronuclei (mature Stage II pronuclei) consistently incorporated 3H-thymidine. To investigate the contribution of egg cytoplasmic factors to the regulation of DNA synthesis, we examined the timing of DNA synthesis by microinjected sperm nuclei in eggs in which sperm nuclear decondensation and male pronucleus formation were accelerated experimentally by manipulation of sperm nuclear disulfide bond content. Although sperm nuclei with few or no disulfide bonds decondense and form male pronuclei faster than nuclei rich in disulfide bonds, the onset of DNA synthesis was not advanced. We conclude the the fertilizing sperm nucleus does not become available to serve as a template for DNA synthesis until it has developed into a mature Stage II pronucleus, and that, as with decondensation and pronucleus formation, DNA synthesis also depends upon egg cytoplasmic factors.     

Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs

A cell-free cytoplasmic preparation from activated Rana pipiens eggs could induce in demembranated Xenopus laevis sperm nuclei morphological changes similar to those seen during pronuclear formation in intact eggs. The condensed sperm chromatin underwent an initial rapid, but limited, dispersion. A nuclear envelope formed around the dispersed chromatin and the nuclei enlarged. The subcellular distribution of the components required for these changes was examined by separating the preparations into soluble (cytosol) and particulate fractions by centrifugation at 150,000 g for 2 h. Sperm chromatin was incubated with the cytosol or with the particulate material after it had been resuspended in either the cytosol, heat-treated (60 or 100 degrees C) cytosol or buffer. We found that the limited dispersion of chromatin occurred in each of these ooplasmic fractions, but not in the buffer alone. Nuclear envelope assembly required the presence of both untreated cytosol and particulate material. Ultrastructural examination of the sperm chromatin during incubation in the preparations showed that membrane vesicles of approximately 200 nm in diameter, found in the particulate fraction, flattened and fused together to contribute the membranous components of the nuclear envelope. The enlargement of the sperm nuclei occurred only after the nuclear envelope formed. The pronuclei formed in the cell-free preparations were able to incorporate [3H]dTTP into DNA. This incorporation was inhibited by aphidicolin, suggesting that the DNA synthesis by the pronuclei was dependent on DNA polymerase-alpha. When sperm chromatin was incubated greater than 3 h, the chromatin of the pronuclei often recondensed to form structures resembling mitotic chromosomes within the nuclear envelope. Therefore, it appeared that these ooplasmic preparations could induce, in vitro, nuclear changes resembling those seen during the first cell cycle in the zygote.     

Pronucleus formation, DNA synthesis and metaphase entry in porcine oocytes following intracytoplasmic injection of murine spermatozoa

The onset of pronucleus formation and DNA synthesis in porcine oocytes following the injection of porcine or murine sperm was determined in order to obtain insights into species-specific paternal factors that contribute to fertilisation. Similar frequencies of oocytes with female pronuclei were observed after injection with porcine sperm or with murine sperm. In contrast, male pronuclei formed 8-9 h following the injection of porcine sperm, and 6-8 h following the injection of murine sperm. After pronucleus formation maternally derived microtubules were assembled and appeared to move both male and female pronuclei to the oocyte centre. A few porcine oocytes entered metaphase 22 h after the injection of murine sperm, but normal cell division was not observed. The mean time of onset of S-phase in male pronuclei was 9.7 h following porcine sperm injection and 7.4 h following mouse sperm injection. Ultrastructural observation revealed that male pronuclei derived from murine sperm in porcine oocytes are morphologically similar to normal male pronuclei in porcine zygotes. These results suggest that species-specific paternal factors influence the onset of pronucleus formation and DNA synthesis. However, normal nuclear cytoplasmic interactions were observed in porcine S-phase oocytes following murine sperm injection.     

DNA Synthesis and pronucleus development in pig zygotes obtained in vivo: An autoradiographic and ultrastructural study

Porcine zygotes flushed from oviducts 48,52,56,60, or 64 hr after hCG were incubated 30 min in ³H-thymidine, transferred to nonradioactive medium for 2 hr, and incubated for 30 min with ¹⁴C-thymidine. After this procedure, ova were prepared (i.e., at 51,55,59,63, or 67 hr after hCG) for autoradiography and ultrastructural observations, respectively. The first autoradiographic labelling, i.e., DNA synthesis, was observed at 56–56.5 hr after hCG, while the latest labelling was seen at 60–60.5 hr. At 51 hr after hCG, formation of the pronuclear envelope was observed, while no nucieolus precursor bodies or prestages to these structures were found. At 55 hr a few clusters of small electron-dense granules were observed, together with condensed chromatin in the pronuclei. At 59 hr the apposed regions of both pronuclei contained nucleolus precursor bodies and condensed chromatin, in close contact with both clusters of small granules and clusters of an additional category of large granules and the nuclear envelope. Additionally, large accumulations of the small granules were found in the vicinity of similarly sized accumulations of the large granules without chromatin association. At 63 hr the spherical accumulations large granules on some occasions presented a central vacuole, and condensed chromatin and clusters of small granules were attached to its periphery. Within the vacuole, electrondense material was found. It is concluded that (1) the S-phase in porcine zygotes is initiated around 56 hr post-hCG injection and is of a duration of 4.5–7.5 hr and (2) the progress of the S-phase is paralleled by the appearance of and complex interaction between different granules in the nucleoplasm. © 1995 Wiley-Liss, Inc.     

Spatial distribution of sperm-derived chromatin in zygotes determined by fluorescence in situ hybridization.

Fluorescence in situ hybridization was used to determine the spatial distribution of chromatin in zygote pronuclei. A hybrid system involving golden hamster eggs and individual human sperm permitted use of DNA probes specific for the entire human chromosome 4, for the heterochromatic region on the long arm of the human Y chromosome and for unique DNA sequences on human chromosome 19. Chromosome 4 occupied a circumscribed domain in the pronuclei, similar to findings in somatic interphases. Unlike the situation in somatic interphases, the Y heterochromatin was extended throughout the first cell cycle. Pronuclear chromatin was extended 3- to 4-fold compared to somatic interphase chromatin. The extended pronuclear chromatin conformation is likely to affect a zygote's susceptibility to environmental hazards.     

Ultrastructural aspects of in vivo fertilization in the cow

In vivo fertilization of cow eggs has been studied by electron microscopy. Eggs were recovered from intracervically inseminated heifers 30 to 42 hr after the onset of oestrus. The corona cells remained attached to 4 out of the 15 eggs studied, but no sign of sperm phagocytosis was noted. Spermatozoa close to the zona pellucida, but not in contact with it, were not acrosome reacted. In contrast, all sperm penetrating the zona pellucida had completed the acrosome reaction. Vesiculated products of the reaction were present at the zona surface of every penetrated egg, indicating that in this species, the acrosome reaction occurs at the surface of the zona pellucida. During sperm passage through the zona pellucida, the equatorial segment overlaid by its plasma membrane remained intact. Soon after penetration into the ooplasm, the sperm nucleus decondensed; at the same time, the female chromosomes resulting from the second meiotic division aggregated in a few masses of condensed chromatin. A nuclear envelope started to form around the condensed female chromatin, while it was not yet present around the decondensing male nucleus. After swelling, the two pronuclei presented similar ultrastructural morphology; they contained small, compact, agranular nucleoli with a large fibrillar center and unevenly distributed chromatin. The pronuclear envelope contained pores and presented characteristic blebbing. The endoplasmic reticulum was closely apposed to the nuclear envelope and large Golgi structures were proximal to the pronuclei.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

Mouse Zygotes Respond to Severe Sperm DNA Damage by Delaying Paternal DNA Replication and Embryonic Development

Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.     

Development of pronuclei from human spermatozoa injected microsurgically into frog (Xenopus) eggs

Human spermatozoa were demembranated with Triton X-100 (TX) and injected into the mature eggs of Xenopus laevis. The nuclei of these spermatozoa decondensed and developed into pronuclei. Chromosomes did not appear in the eggs until the end of a 5-hr incubation period. When the demembranated human spermatozoa were further treated with dithiothreitol (DTT) before they were injected into the eggs, the sperm nuclear decondensation and pronuclear development took place considerably faster than in spermatozoa treated with the detergent alone. By the end of the 5-hr incubation period, decondensed chromatin threads or chromosome-like structures appeared, but none of the eggs cleaved. When human spermatozoa were injected into full-grown ovarian oocytes with intact germinal vesicle (GV) or oocytes which had matured without GV, the nuclei of a proportion of TX-treated and all TX-DTT-treated sperm decondensed but showed no sign of developing into pronuclei. Sperm nuclei injected into maturing oocytes formed condensed chromatin fragments as long as the oocytes were not activated, but they transformed into pronuclei when the oocytes were stimulated with electric shock. These results indicate that the cytoplasmic factors responsible for the decondensation of human sperm nuclei are present in egg cytoplasm independent of GV-materials. We also suggest that the factors controlling development of decondensed sperm nuclei into pronuclei are dependent on GV materials.     

Experimental hybridization among Oryzias species. II. Karyogamy and abnormality of chromosome separation in the cleavage of interspecific hybrids between Oryzias latipes and O. javanicus

In interspecific hybridization between Oryzias latipes and O. javanicus, all hybrid embryos failed to develop and died before hatching. Cytological examination of fertilization and early development was performed to discover the cause of lethal development. When O. latipes eggs were inseminated by sperm of O. javanicus, the cortical reaction was induced normally. Chromosomal material in the fertilized eggs was visualized using the DNA-specific fluorochrome Hoechst. The spermatozoon was capable of penetrating into the egg cytoplasm through the micropyle, and the sperm nucleus transformed to the male pronucleus. The female pronucleus that formed after extrusion of the second polar body migrated towards the male pronucleus. The female and the male pronuclei underwent DNA synthesis and encountered each other in the center of the blastodisc, fused with one another and formed a zygote nucleus before breakdown of the nuclear envelope. Metaphase chromosomes with electron dense chromatin regions were abnormally divided into each blastomere in cleavage. The abnormally separating chromatin masses were also labeled by BrdU. The abnormal separation resulting in partial loss of fragmented chromatin might be a cause of abortive development in the interspecific hybrids between O. latipes and O. javanicus.     

Sperm aster in rabbit zygotes: its structure and function

Microscope observations of rabbit zygotes demonstrate that a sperm aster forms in association with the male pronucleus approximately 1 h postinsemination and consists of two regions. One, the centrosphere, contains a dense aggregation of cisternae of smooth endoplasmic reticulum and microtubules. The second consists of fascicles of microtubules which emanate from the centrosphere. Fertilized rabbit eggs were cultured in medium containing colcemid in order to determine its effects on various events of fertilization, such as movements of the male and female pronuclei and DNA synthesis. No evidence was obtained to indicate that a sperm aster is formed in colcemid-treated zygotes. In addition, migration and close apposition of the pronuclei do not take place. Breakdown of the pronuclear envelopes and condensation of the maternally and paternally derived chromosomes occur even though the pronuclei fail to migrate centrad. Autoradiographic analysis of the synthesis of DNA by both pronuclei demonstrates that their migration into close apposition to one another is not required for the incorporation of tritiated thymidine.     

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).     

Asynchronous DNA replication and origin licensing in the mouse one‐cell embryo

To prevent duplicate DNA synthesis, metazoan replication origins are licensed during G1. Only licensed origins can initiate replication, and the cytoplasm interacts with the nucleus to inhibit new licensing during S phase. DNA replication in the mammalian one‐cell embryo is unique because it occurs in two separate pronuclei within the same cytoplasm. Here, we first tested how long after activation the oocyte can continue to support licensing. Because sperm chromatin is licensed de novo after fertilization, the timing of sperm injection can be used to assay licensing initiation. To experimentally skip some of the steps of sperm decondensation, we injected mouse sperm halos into parthenogenetically activated oocytes. We found that de novo licensing was possible for up to 3 h after oocyte activation, and as early as 4 h before DNA replication began. We also found that the oocyte cytoplasm could support asynchronous initiation of DNA synthesis in the two pronuclei with a difference of at least 2 h. We next tested how tightly the oocyte cytoplasm regulates DNA synthesis by transferring paternal pronuclei from zygotes generated by intracytoplasmic sperm injection (ICSI) into parthenogenetically activated oocytes. The pronuclei from G1 phase zygotes transferred into S phase ooplasm were not induced to prematurely replicate and paternal pronuclei from S phase zygotes transferred into G phase ooplasm continued replication. These data suggest that the one‐cell embryo can be an important model for understanding the regulation of DNA synthesis. J. Cell. Biochem. 107: 214–223, 2009. © 2009 Wiley‐Liss, Inc.     

The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro

Cell-free extracts prepared from Xenopus eggs support chromosome decondensation and pronuclear formation on demembranated sperm heads. 32P-dCTP pulse-labelling studies demonstrate that DNA synthesis occurs in multiple bursts of 30-40 min in extracts containing pronuclei, each burst being followed by a period of 20-50 min during which no synthesis occurs. Density substitution with bromodeoxyuridine indicates that the synthesis in each burst is semiconservative and results from new initiations, and that, following multiple bursts of synthesis, reinitiation events can occur. Changes in nuclear morphology have been characterized in the extract by phase-contrast microscopy and by fluorescence microscopy following pulse labelling with biotin-11-dUTP and staining with anti-lamin antibodies. Lamin accumulation occurs as DNA decondenses and parallels the acquisition of membrane structures. Biotin-11-dUTP incorporation is first observed in small nuclei having decondensed DNA and an extensive lamina. While DNA synthesis is occurring nuclei remain relatively small, but rapid swelling accompanied by chromosome condensation occurs when biotin incorporation ceases. Nuclear swelling and chromatin condensation is followed by nuclear membrane breakdown, lamin dispersal and chromosome formation. Mitosis lasts for approximately 20 min. Nuclear reassembly is recognized by the appearance of membrane vesicles around small pieces of decondensed DNA, which parallels the appearance of lamin islands within a chromatin mass. These 'islands' incorporate biotin, indicating that DNA synthesis is occurring, and apparently fuse as larger S-phase nuclei are formed. Extensive protein synthesis occurs for at least 4 h in most extracts. This synthesis is required for the initiation of mitotic events and the reinitiation of DNA synthesis.     

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.     

Sperm chromatin remodeling after intracytoplasmic sperm injection differs from that of in vitro fertilization

Intracytoplasmic sperm injection (ICSI) is a popular method used in assisted conception, and live offspring have been born from a variety of species, including humans. In ICSI, sperm chromatin is introduced into the oocyte together with the acrosome, a structure that does not enter the oocyte during normal fertilization. We compared sperm chromatin remodeling, the potential of embryos to develop in vitro, and DNA synthesis in mouse embryos obtained from in vitro fertilization (IVF) and ICSI. We also tested whether sperm pretreatment prior to ICSI (i.e., capacitation, acrosome reaction, membrane removal, and reduction of disulfide bonds in protamines) facilitates chromatin remodeling and affects embryo development. Sperm chromatin was examined on air-dried, Giemsa-stained preparations at 30-min intervals for up to 4.5 h postfertilization. In all experimental groups, the oocytes underwent activation and formed pronuclei with similar rates. However, the dynamics of sperm chromatin remodeling in ICSI and IVF embryos varied. In ICSI, chromatin remodeling was more asynchronous than in IVF. Sperm capacitation prior to injection enhanced remodeling asynchrony and resulted in delayed pronuclei formation and DNA synthesis. The removal of the acrosome prior to injection with calcium ionophore A23187 but not with detergent Triton X-100 allowed more synchronous chromatin remodeling, timely DNA synthesis, and good embryo development. Our data have significance for the refinement of the molecular and biologic mechanisms associated with ICSI for current and future applications.     

An ultrastructural study of cross-fertilization (Arbacia female x Mytilus male)

Insemination of sea urchin (Arbacia) ova with mussel (Mytilus) sperm has been accomplished by treating eggs with trypsin and suspending the gametes in seawater made alkaline with NaOH. Not all inseminated eggs undergo a cortical granule reaction. Some eggs either elevate what remains of their vitelline layer or demonstrate no cortical modification whatsoever. After its incorporation into the egg, the nucleus of Mytilus sperm undergoes changes which eventually give rise to the formation of a male pronucleus. Concomitant with these transformations, a sperm aster may develop in association with the centrioles brought into the egg with the spermatozoon. Both the male pronucleus and the sperm aster may then migrate centrad to the female pronucleus. Evidence is presented which suggests that fusion of the male pronuclei from Mytilus sperm with female pronuclei from Arbacia eggs may occur, although this was not directly observed. These results demonstrate that Mytilus sperm nuclei are able to react to conditions within Arbacia eggs and differentiate into male pronuclei.     

Technical and physiological aspects associated with the lower fertilization following intra cytoplasmic sperm injection (ICSI) in human

The fertilization rates with ICSI range from 30% to 70% and suggest that, despite injecting sperm into mature oocytes, significant fertilization failure still occurs in humans. The objective of this study was to determine technical and physiological factors which may contribute to lower fertilization following ICSI. Eggs that failed to show two pronuclei (PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). The eggs were analyzed by staining with DNA fluorochromes, Hoescht 33258 and DAPI. The extent of sperm head as well as maternal chromatin decondensation in unfertilized ICSI eggs was determined by high resolution fluorescence microscopy. The average fertilization rate (FR) from all ICSI cycles in these two groups was 45%. The FR in Groups A and B were 35% and 59%, respectively (P < 0.05). In Group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In Group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin while 45% were partially decondensed. Sperm chromatin was not detected in 24% of all unfertilized eggs studied. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause for failure of fertilization rates in ICSI cycles. The increased percentage of eggs undergoing sperm head decondensation may be related to subtle changes in technique as experience is gained over time. The failure of sperm head decondensation in some of the ICSI eggs may be associated with cytoplasmic immaturity but not nuclear maturity.