Behavior of sperm nuclei incorporated into parthenogenetic mouse eggs prior to the first cleavage division.

When artificially activated mouse eggs are inseminated in the middle of the first cell cycle, sperm nuclei remain condensed until the first mitosis. During mitosis of the first cleavage division sperm nuclei decondense, subsequently recondense and are passively displaced to the daughter blastomeres. In the 2-cell embryos sperm nuclei form interphase nuclei which are able to replicate DNA and to condense into discrete chromosomes during the following mitotic division. These observations suggest that the mitotic cytoplasm of 1-cell embryos creates similar conditions for the transformation of sperm nuclei into male pronuclei as the cytoplasm of metaphase II oocytes.     

体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

为研究微管在体外受精与孤雌活化过程中的动态变化,本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况,采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化,以分析微管在减数分裂过程中的作用及其对早期发育的影响.结果显示,体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率(P<0.05),而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著.在体外受精中,精子入卵,激活卵母细胞,减数分裂恢复,纺锤丝牵拉赤道板卜致密排列的母源染色体向纺锤体两侧迁移;后期将染色体拉向两极;末期时,微管分布于两组已去凝集的母源染色体之间,卵母细胞排出第二极体(the second polarbody,Pb2),解聚的母源染色体形成雌原核.同时,在受精后5～8 h精子染色质发生去浓缩与再浓缩,形成雄原核.在原核形成的同时,胞质星体在雌、雄原核的周围重组形成长的微管,负责雌、雄原核的迁移靠近.孤雌活化过程中,卵母细胞恢复减数分裂,姐妹染色单体分离,被拉向两极,经细胞松弛素B处理后,活化4～6 h,卵周隙中未见Pb2,而在胞质中出现两个混合的单倍体原核,之间由微管相连接,负责两个单倍体原核的迁移靠近.与体外受精相比较,孤雌活化时卵母细胞更容易被激活,减数分裂期间微管的发育早且更完善.     

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.     

Completion of mouse embryogenesis requires both the maternal and paternal genomes

Transplantation of pronuclei between one-cell-stage embryos was used to construct diploid mouse embryos with two female pronuclei ( biparental gynogenones ) or two male pronuclei ( biparental androgenones ). The ability of these embryos to develop to term was compared with control nuclear-transplant embryos in which the male or the female pronucleus was replaced with an isoparental pronucleus from another embryo. The results show that diploid biparental gynogenetic and androgenetic embryos do not complete normal embryogenesis, whereas control nuclear transplant embryos do. We conclude that the maternal and paternal contributions to the embryonic genome in mammals are not equivalent and that a diploid genome derived from only one of the two parental sexes is incapable of supporting complete embryogenesis.     

Transformations of sperm nuclei incorporated into sea urchin (Arbacia punctulata) embryos at different stages of the cell cycle

In order to test the hypothesis that regulators of male pronuclear development may have a more general role, sharing some relation to factors involved with the cell cycle, Arbacia zygotes and 2- to 8-cell stage embryos were inseminated during different phases of the cell cycle and examined by light and electron microscopy. Differences in the development and morphology of fertilization cones and sperm asters were observed in embryos inseminated during different stages of the cell cycle. Extremely large fertilization cones, approximately four times the length of those found in fertilized eggs, formed in embryos inseminated during metaphase to telophase. Sperm asters developed only in embryos inseminated during prophase to anaphase. These variations are believed to reflect changes in the status of the cortex and cytoskeletal system of the embryo. Although sperm nuclei underwent morphological changes subsequent to incorporation, in general, they failed to develop into male pronuclei. There was a consistent correlation in sperm nuclear transformations and the cell cycle which was expressed in two patterns of morphogenesis: (1) sperm nuclei incorporated into embryos just prior to prophase and at telophase failed, for the most part, to disperse and transformed into aggregations of chromatin granules approximately 40 nm in diameter; and (2) sperm nuclei incorporated into prometaphase-anaphase embryos dispersed and then condensed into chromatin masses, morphologically similar to chromosomes of the embryo. Evidence is discussed which indicates that following the normal period of fertilization changes occur in the zygote, rendering it unable to fully support the transformation of sperm nuclei into male pronuclei.     

Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro

Virtually all mammalian species including mouse, rat, pig, cow, and human, but not sheep and rabbit, undergo genome-wide epigenetic reprogramming by demethylation of the male pronucleus in early preimplantation development. In this study, we have investigated and compared the dynamics of DNA demethylation in preimplantation mouse and rat embryos by immunofluorescence staining with an antibody against 5-methylcytosine. We performed for the first time a detailed analysis of demethylation kinetics of early rat preimplantation embryos and have shown that active demethylation of the male pronucleus in rat zygotes proceeds with a slower kinetic than that in mouse embryos. Using dated mating we found that equally methylated male and female pronuclei were observed at 3 hr after copulation for mouse and 6 hr for rat embryos. However, a difference in methylation levels between male and female pronuclei could be observed already at 8 hr after copulation in mouse and 10 hr in rat. At 10 hr after copulation, mouse male pronuclei were completely demethylated, whereas rat zygotes at 16 hr after copulation still exhibited detectable methylation of the male pronucleus. In addition in both species, a higher DNA methylation level was found in embryos developed in vitro compared to in vivo, which may be one of the possible reasons for the described aberrations in embryonic gene expression after in vitro embryo manipulation and culture.     

A genetic test of the mechanism of Wolbachia-induced cytoplasmic incompatibility in Drosophila

Cytoplasmic bacteria of the genus Wolbachia are best known as the cause of cytoplasmic incompatibility (CI): many uninfected eggs fertilized by Wolbachia-modified sperm from infected males die as embryos. In contrast, eggs of infected females rescue modified sperm and develop normally. Although Wolbachia cause CI in at least five insect orders, the mechanism of CI remains poorly understood. Here I test whether the target of Wolbachia-induced sperm modification is the male pronucleus (e.g., DNA or pronuclear proteins) or some extranuclear factor from the sperm required for embryonic development (e.g., the paternal centrosome). I distinguish between these hypotheses by crossing gynogenetic Drosophila melanogaster females to infected males. Gynogenetic females produce diploid eggs whose normal development requires no male pronucleus but still depends on extranuclear paternal factors. I show that when gynogenetic females are crossed to infected males, uniparental progeny with maternally derived chromosomes result. This finding shows that Wolbachia impair the male pronucleus but no extranuclear component of the sperm.     

Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid

Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona-free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.     

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.     

The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content

The relationship between the timing of both sperm nuclear decondensation and male pronucleus formation in the oocyte and the relative level of disulfide bonds within the sperm nucleus was evaluated. Since reduction of sperm nuclear disulfide (S-S) bonds is a prerequisite for sperm nuclear decondensation in vitro and in vivo, we hypothesized that sperm nuclei with relatively few S-S bonds would require less time to decondense in the oocyte than sperm nuclei with higher numbers of S-S bonds, and that male pronucleus formation would occur more rapidly as well. Four types of hamster sperm nuclei, in which the extent of S-S bonding differed, were microinjected into hamster oocytes, and the time course of sperm nuclear decondensation and male pronucleus formation was charted. Cauda epididymal sperm nuclei, which are rich in S-S bonds, required 45-60 min to decondense. In contrast, nuclei containing few S-S bonds (namely sonication-resistant spermatid nuclei and cauda epididymal sperm nuclei treated in vitro with the S-S bond-reducing agent dithiothreitol) decondensed within 5-10 min of microinjection. Caput epididymal sperm nuclei, with intermediate S-S bond content, decondensed in 10-20 min. Regardless of when decondensation occurred, formation of the male pronucleus never preceded that of the female pronucleus, which occurred 1.25-1.5 h after microinjection. However, sperm nuclei with few S-S bonds were more likely than S-S rich nuclei to transform into male pronuclei in synchrony with the formation of the female pronucleus. We conclude that the timing sperm nuclear decondensation and pronucleus formation depends in part upon the S-S bond content of the sperm nucleus.     

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).     

Kinetochore appearance during meiosis,fertilization and mitosis in mouse oocytes and zygotes

The events of mammalian fertilization overlap with the completion of meiosis and first mitosis; the pronuclei never fuse, instead the parental genomes first intermix at the mitotic spindle equator at metaphase. Since kinetochores are essential for the attachment of chromosomes to spindle microtubules, this study explores their appearance and behavior in mouse oocytes, zygotes and embryos undergoing the completion of meiosis, fertilization and mitoses. Kinetochores are traced with immunofluorescence microscopy using autoimmune sera from patients with CREST (CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) scleroderma. These sera cross-react with the 17 kDa centromere protein (CENP-A) and the 80 kDa centromere protein (CENP-B) found at the kinetochores in human cell cultures. The unfertilized oocyte is ovulated arrested at second meiotic metaphase and kinetochores are detectable as paired structures aligned at the spindle equator. At meiotic anaphase, the kinetochores separate and remain aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis is lost. The female pronucleus and the second polar body nucleus each receive a detectable complement of kinetochores. Mature sperm have neither detectable centrosomes nor detectable kinetochores, and shortly after sperm incorporation kinetochores become detectable in the decondensing male pronucleus. In pronuclei, the kinetochores are initially distributed randomly and later found in apposition with nucleoli. At mitosis, the kinetochores behave in a pattern similar to that observed at meiosis or mitosis in somatic cells: irregular distribution at prophase, alignment at metaphase, separation at anaphase and redistribution at telophase. They are also detectable in later stage embryos. Colcemid treatment disrupts the meiotic spindle and results in the dispersion of the meiotic chromosomes along the oocyte cortex; the chromosomes remain condensed with detectable kinetochores. Fertilization of Colcemid-treated oocytes results in the incorporation of a sperm which is unable to decondense into a male pronucleus. Remarkably kinetochores become detectable at 5 h post-insemination, suggesting that the emergence of the paternal kinetochores is not strictly dependent on male pronuclear decondensation.     

The role of structural integrity of the fertilising spermatozoon in early human embryogenesis.

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a 'sum of parts' is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.     

Analysis of the first cell cycle in the cross between hamster eggs and human sperm

Golden hamster eggs fused with human sperm were pulsed with bromodeoxyuridine to determine the timing of S-phase and the length of the first ceil cycie in this hybrid cross. Fused eggs were fixed and pronuclei scored for incorporation of the thymidine analogue detected by indirect immunofiuorescence. Although S-phase started synchronously 3–3.5 hr after coincuba-tion of sperm and eggs, its duration was variable such that two-cell stages appeared at 16 hr while a proportion of pronuclei was still engaged in DNA synthesis. Unlike rodent sperm chromatin, human sperrn chromatin was able to participate in DNA synthesis well before its maturation into a fully developed pronucleus. Human sperm chromatin appears able to function under conditions different in several respects from those in human eggs.     

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 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.     

DDK egg-foreign sperm incompatibility in mice is not between the pronuclei

A high rate of normal postimplantation development was achieved when the pronuclei of embryos from matings of DDK females with (CBA X C57BL/6J)F1 males were transplanted into enucleated embryos of non-DDK origin. This shows that the DDK egg cytoplasm, not the maternal pronucleus, is involved in the late preimplantation-lethal incompatibility.     

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.     

Improvement of male pronuclear formation during cross‐fertilization between Chinese hamster spermatozoa and Syrian hamster oocytes by nocodazole,and chromosome analysis of hybrid zygotes

During cross‐fertilization between Chinese hamster spermatozoa and Syrian hamster oocytes, incorporated sperm heads frequently fail to develop into male pronuclei, whereas the group of oocyte chromosomes develop into female pronuclei. The present study applies this cross‐fertilization system to the cytogenetic investigation of mammalian hybrid embryos. Immediately after insemination, oocytes were exposed to 0.1 μg/ml nocodazole for 1 hr (1 hr group) or 2 hr (2 hr group), then further cultured. Although the rates of sperm penetration in the 1 hr (48.0%) and 2 hr (75.8%) groups were significantly lower than that in the control group (89.8%), the ratios of male pronuclear formation were higher in both exposed groups (79.4% and 74.2%, respectively) than in the control group (10.6%). These results were apparently due to sperm head decondensation induced during the meiotic arrest of oocytes at metaphase II by nocodazole. Chromosomes of hybrid zygotes obtained after nocodazole exposure were analyzed at the first cleavage metaphase. The incidence of structural chromosome aberrations in the Chinese hamster genome of hybrid zygotes was high in the control (42.1%) and 1 hr (48.8%) groups. This incidence was reduced to 14.4% in the 2 hr group. Because the lag of sperm head decondensation behind the second meiotic division of oocytes was greater in the control and 1 hr groups than in the 2 hr group, untimely sperm head decondensation may be implicated in occurrence of structural chromosome aberrations in the male genomes of hybrid zygotes. Mol. Reprod. Dev. 52:117–124, 1999. © 1999 Wiley‐Liss, Inc.     

Protamine withdrawal from human sperm nuclei following heterologous ICSI into hamster oocytes

During late stages of spermatogenesis in mammals, most histones bound to DNA are replaced by protamines (PRM), which results in formation of supercondensed and genetically inert sperm chromatin. At fertilization, mature spermatozoon penetrates oocyte and chromatin is remodeled "back" from nucleoprotamine to nucleohistone state. While being crucial for activation of male genome and ultimately for initiation of embryonic development, this process is poorly studied, especially in humans. Data on model animals concerning PRM to histones exchange post fertilization are few and contradictory. As direct experimentation with human embryos is impossible due to ethical, legal and technical reasons, we evaluate the timing and mode of PRM removal in a heterologous ICSI system using hamster ova injected with human sperm. Localization of human PRM 1 and 2 in hybrid zygotes was established using immunofluorescence. We observed a marked zygote to zygote variability in male pronuclei size for any time point post ICSI and demonstrated that PRM removal correlates with the developing pronuclei area rather than time after injection. Overall, the disappearance of protamines from sperm is rather rapid and most likely completed within 1 hr. We propose that the critical characteristic influencing PRM removal after heterologous fertilization is the intrinsic heterogeneity of the human sperm population. The same yet unexplored variance may be one of the reasons for canceled, delayed or aberrant early embryonic development during natural or artificial fertilization in humans.