Sperm Chromatin Decondensation Research Articles

Two Steps Required for Male Pronucleus Formation in the Sea Urchin Egg

We have developed a system for microinjection of sea urchin sperm nuclei into live eggs and oocytes in order to study conditions which are required for pronuclear development. The system bypasses sperm-initiated surface events that result in egg activation. By comparison to male nuclei of normally fertilized eggs or immature oocytes, the injected nuclei faithfully reflect the ability of the egg cytoplasm to promote male pronuclear development. Isolated nuclei injected into fertilized eggs swell and transform into male pronuclei like the endogenous male pronuclei. Nuclei injected into immature oocytes remain conical like fertilizing sperm nuclei. If sperm nuclei are injected into unfertilized, unactivated eggs, they undergo incomplete decondensation and fail to form pronuclei. This partial decondensation can be inhibited with 6-dimethylaminopurine (6-DMAP). If unfertilized eggs are first incompletely activated by raising internal pH with ammonia, injected sperm nuclei will complete pronuclear development. Increasing pH or Ca2+ does not cause sperm nuclear swelling or chromatin decondensation in immature oocytes. These experiments reveal two conditions necessary for pronuclear formation. The first condition, resulting in partial decondensation, develops during meiotic maturation and can be blocked by 6-DMAP. The second requires cytoplasmic alkalinization, but not the fertilization-induced internal Ca2+ increase or inositol triphosphate pathway activation.

Dynamics of activation of in vitro matured bovine follicular oocytes following combined ethanol and cycloheximide treatment

Among sperm morphology abnormalities, macrocephalic and large-headed spermatozoa are commonly associated with a low chance of pregnancy, mainly in relation to meiotic abnormalities during spermatogenesis. Here is reported the case of a patient with 98% of spermatozoa showing abnormal morphology, many having enlarged heads (47%). Sperm-head measurement, sperm fluorescent in situ hybridization analysis, sperm chromatin decondensation and molecular biology were performed. Fifty-six percent of the sperm displayed a large head (length >4.7 μm and width >3.2 μm), and the mean sperm-head area was 15.8 ± 3.8 μm2 (9.7 ± 1.5 μm2 and 9.3 ± 1.4 μm2 for two controls). Normal chromosomal content was found in 97% of the cells and no aurea kinase C-gene mutation was found. Mean sperm chromatin decondensation rate was 46%, 64% for large-head forms and 10% for other forms. This is, as far as is known, one of the first cases of semen with enlarged-head spermatozoa linked to sperm chromatin condensation dysfunction with no major meiotic dysfunction. The study centre advised the couple to undergo intracytoplasmic sperm injection with the patient’s spermatozoa.Among sperm morphology abnormalities, macrocephalic and large-headed spermatozoa are commonly associated with a low chance of pregnancy, mainly in relation to meiotic abnormalities during spermatogenesis. We report the case of a 51-year-old referred to our centre for a 3-year history of secondary infertility who had 98% of spermatozoa with abnormal morphology and many with enlarged sperm heads (47%). Sperm head measurements, sperm fluorescent in situ hybridization analysis, and sperm chromatin decondensation were performed to propose the best treatment for this couple. In contrast with previous studies indicating a high abnormal chromosome content correlated with the percentage of macrocephalic spermatozoa, 97% of the spermatozoa in this case had a normal content for chromosomes analysed. Aniline blue staining showed a high rate of decondensed spermatozoa (46%), compared with controls (19% and 20%). We showed that the mean sperm head area for decondensed sperm heads was greater (P < 0.0001) than that for condensed sperm heads, and an increased rate (P < 0.0001) of decondensed sperm heads was found for macrocephalic spermatozoa (>14 μm2), compared with normal sperm heads; respectively 64% and 10%. Because during the ICSI procedure the operator select apparently normal sperm form (mainly with normal condensed head), we advised, in this particular case, the couple to undergo ICSI with the patient’s spermatozoa as they had a good prognosis compared with previous data published for macrocephalic spermatozoa.

Ultraviolet irradiation of tilapia spermatozoa and the Hertwig effect: electron microscopic analysis

Electron microscopic analysis of U V‐irradiated tilapia sperm showed that with irradiation dose of 1800 J m−2 min−1, an irradiation duration of 0.5 min caused decondensation of sperm chromatin. This phenomenon of chromatin decondensation reached a peak after l.5min of irradiation, where ∼ 15% of the sperm showed total decondensation, and was less apparent after 3 min of irradiation or more. Damage to the cytoplasmic membrane and nuclear envelope could be seen in cells that underwent total decondensation. As the duration of irradiation increased, cytoplasmic membrane and nuclear envelope defects appeared more severe, the mitochondria were affected and appeared as empty capsules, and sperm cells tended to lose their tails. Based on these results and others reported in the literature, we propose an explanation for the ‘Hertwig curve’ obtained in tilapia using UV irradiation. Sperm cells with decondensed chromatin and damaged cytoplasmic membrane and nuclear envelope, activate the ‘developmental switch’ when they penetrate the egg, but their pronuclei are subjected to cytoplasmic nuclease digestion. Consequently, the maternal pronucleus is the only functional pronucleus in the zygote, and therefore, only haploid embryos with the exclusive maternal genome are formed. If the paternal pronucleus is not digested, these embryos will die due to improper expression of the paternal genes.

Marsupial fertilization: Some further ultrastructural observations on the dasyurid Sminthopsis Crassicaudata

Various morphological aspects of in vivo egg maturation and sperm-egg interaction were investigated in the Australian marsupial Sminthopsis crassicaudata with the transmission and scanning electron microscopes. Cortical granules invariably occurred in primary oocytes, with the number increasing after resumption of the first meiotic division. They generally occurred close to the oolemma, including the region near the oocyte nucleus. After mating, spermatozoa with intact acrosomes, which had a homogeneous electron-dense matrix, were found on the outer zona surface, but loss of acrosomal contents had occurred by the time of zona penetration. Sperm incorporation into the egg took place at the metaphase II stage of meiosis, and, at this time, cortical granules disappeared from the egg cortex. Sperm heads with condensed chromatin in the egg cytoplasm had an electron-dense layer of subacrosomal material over part of the dorsal nuclear surface, but no membranes were present around these incorporated spermatozoa. Sperm chromatin decondensation resulted in an elevation of egg cytoplasm, and the cell membrane over this area lacked microvilli. The pronuclear envelope was not laid down until after chromatin decondensation had occurred. By this time the fertilized egg had reached the uterus, and a smooth, electron-dense, shell membrane had been deposited. These observations, together with our previous findings, indicate that some of the processes of sperm-egg interaction are similar to those in eutherian mammals, whereas others appear highly divergent.

Parallel pathways of cell cycle control during Xenopus egg activation.

Transit from M phase into interphase in many eukaryotic cells is preceded by an increase in intracellular free calcium ([Ca2+]i), which may act via calcium-dependent enzymes to trigger the M-phase/interphase transition. To test the role of the calcium- and phospholipid-dependent enzyme protein kinase C (PKC) in the M-phase/interphase transition, PKC was activated in M-phase-arrested Xenopus eggs by treatment with the phorbol ester phorbol 12-myristate 13-acetate under conditions that prevent a rise in [Ca2+]i and activation of other calcium-dependent enzymes. Under these conditions, several cellular events characteristic of transit into interphase occur: sperm chromatin decondenses, the Golgi and the nuclear envelope reassemble, and endocytosis resumes. These events are also triggered by treatment of eggs with the diacylglycerol 1,2-dioctanoyl-sn-glycerol. Surprisingly, the activity of M-phase-promoting factor (MPF), a universal regulator of M phase, remains high under these conditions. If [Ca2+]i is subsequently raised, MPF activity is rapidly destroyed. Similarly, lysates made from eggs treated with phorbol 12-myristate 13-acetate support sperm chromatin decondensation in vitro and yet retain high MPF activity, measured either as the ability to induce meiotic resumption in oocytes or as histone H1 kinase activity. These effects are not triggered by the 4 alpha-phorbol ester isomer, which does not activate PKC, and are sensitive to the PKC "pseudosubstrate" peptide. The results suggest that two, parallel signals are generated by the rise in [Ca2+]i both of which contribute to cell cycle regulation. One pathway inactivates MPF; the other pathway activates PKC.

Decondensation of mouse sperm chromatin in cell‐free extracts: A micromethod

A micromethod is presented which makes possible the analysis of mouse sperm nucleus decondensation in vitro using very small volumes of cytoplasmic preparations, even smaller than 1 microliters. We show that cell-free extracts obtained from interphase HeLa cells as well as lysates from mouse eggs and embryos can sustain early stages of mouse sperm nucleus transformation, provided the sperm nuclear envelope is damaged or removed.

Subzonal transfer of multiple sperm (MIST) into early human embryos

Microinsemination sperm transfer (MIST) is a technique whereby sperm are transferred into the perivitelline space (PVS) with the aid of a micromanipulator. MIST is now used to investigate whether blastomere membranes of early human embryos are capable of fusing with the sperm as in the metaphase II oocyte. Between 10 and 30 sperm were transferred into 11 donated human embryos between pronuclear and 16 cell stage. After culture for 6-24 hr in vitro, the embryos were fixed for transmission electron microscopy (TEM). Both acrosome-intact and acrosome-reacted sperm were located in the PVS and between blastomeres. Sperm in the PVS were sometimes penetrating the inner regions of the zona. Sperm-blastomere membrane fusion was not observed, but sperm tail incorporation by phagocytosis was occasionally evident. Sperm heads incorporated into blastomeres were often located in membrane-bound vesicles. Both acrosome-intact and acrosome-reacted sperm heads were found in vacuoles. Acrosome-reacted sperm heads were lying passively in vacuoles or were undergoing degenerative changes at their surfaces. Sperm chromatin decondensation was not observed in any of the sperm heads that were detected in the blastomeres. The evidence presented clearly shows that sperm heads are incapable of expanding their chromatin to form typical male pronuclei following MIST into early human embryos.

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 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 nucleolar 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.

Human sperm‐egg interaction in vitro

AbstractThe early events of gamete membrane fusion and sperm incorporation are portrayed, which are possibly the earliest pictures of human conception on record.Oocytes recovered at laparoscopy from an in vitro fertilization programme using clomiphene and human menopausal and chorionic gonadotrophin stimulation were zona‐punctured and examined for polyspermic penetration 3–6 hours after insemination. Three eggs were penetrated, and one donor became pregnant in the same cycle.Fusion occurred between the oolemma and the midsegment of the sperm cell membrane extending from the equatorial vestige of the acrosome to the anterior region of the postacrosomal segment. Only acrosome‐reacted sperm were capable of fusing with the egg. Fusion was followed by incorporation of the spermhead into the ooplasm by a process akin to phagocytosis. Sperm chromatin decondensation occurred by progressive inflation and disorganization of the original nuclear envelope.The mechanism of gamete fusion and sperm incorporation resembles that observed during human monospermic fertilization and generally conforms to that reported for eutherian mammals.

Sperm‐oocyte membrane fusion in the human during monospermic fertilization

AbstractSperm‐oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro.Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH‐surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy.Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome‐reacted and had been partly incorporated into the ooplasm, while the anterior two‐thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized.The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.

Effect of in vivo oocyte aging on sperm chromatin decondensation in the golden hamster

AbstractAged spontaneously activated hamster oocytes recovered from adult females 18 and 24 hours after ovulation were at the pronuclear stage. These oocytes and fresh controls were inseminated in vitro with capacitated hamster spermatozoa and observed with the phase‐contrast microscope. The percentage of fertilization in fresh control oocytes was 98%, as compared to 36% and 18% when the oocytes were recovered 18 and 24 hours after ovulation, respectively. The mean number of sperm decondensations per egg in control oocytes was 10, and in the aged ones it was 0.69 and 0.12 when the oocytes were recovered 18 and 24 hours after ovulation, respectively. When similarly treated oocytes were studied with scanning and transmission electron microscopy, it was found that the degree of gamete membrane fusion was greater than that observed with the phase‐contrast microscope, but that most of the spermatozoa failed to decondense the chromatin. We suggest that parthenogenetic oocytes at the pronuclear stage are in a similar stage of the cell cycle as in fertilized eggs, in which the cytoplasm does not have the ability to decondense the sperm chromatin.

The human sperm nucleus takes up zinc at ejaculation.

Ejaculated and vasal sperm were obtained from men referred for vasectomy, and sperm nuclear elements were determined by X-ray microanalysis. Sperm head zinc concentrations, expressed as the ratio Zinc to Sulphur, were significantly higher in ejaculated than in vasal sperm. A physiological sperm nuclear zinc uptake is discussed in relation to sperm chromatin decondensation.

Loss of an intrinsic capacity for human sperm chromatin decondensation.

Chromatin decondensation of human ejaculated spermatozoa was studied in vitro, at various points of time after ejaculation, by sperm exposure to the detergent sodium dodecyl sulphate (SDS) containing zinc chelating EDTA. Within 5 min after ejaculation EDTA revealed a capacity for decondensation in 90% of the spermatozoa. This sperm capacity decreased rapidly upon storage. The results support the concept that the capacity to decondense is a normal property of freshly ejaculated spermatozoa and that this property may be rapidly lost. The loss is most probably due to an inability of thiol groups to take part in a thiol-disulphide exchange in the sperm chromatin. A loss of functional thiols may hinder a capacity for chromatin decondensation inherent to the spermatozoon. A loss of thiols due to oxidation, that is, surplus S-S bridge formation, may also delay hypothetical extrinsic S-S cleaving factors in the ooplasm. In either case, the complete and non-delayed sperm chromatin decondensation in the ovum may be hindered. This may result in the abnormal embryonic development observed in ova fertilized with aged spermatozoa.

Zinc preserves an inherent capacity for human sperm chromatin decondensation.

The effect of zinc on the nuclear chromatin decondensation ability of human, ejaculated spermatozoa was studied by exposing washed spermatozoa to the detergent sodium dodecyl sulphate (SDS) before and after sperm storage. Treatment with EDTA increased the proportion of decondensing spermatozoa before storage. Zinc supplementation before storage reversibly inhibited spontaneous decondensation as well as EDTA-enhanced decondensation. Treatment with EDTA before storage decreased the proportion of spermatozoa decondensing after storage. Zinc supplementation during storage reduced the decrease in the proportion of spermatozoa decondensing after storage. Two effects of zinc were observed: one immediate effect (reversible inhibition of the decondensation) and one long-term effect (protection of the intrinsic capacity for decondensation during storage). Both effects may be explained by a zinc-thiol interaction in the chromatin. Spermatozoal zinc is suggested to protect an inherent capacity for decondensation, thereby helping to extend the functional life-span of the ejaculated spermatozoon.

Phosphorylation of sea urchin sperm H1 and H2B histones precedes chromatin decondensation and H1 exchange during pronuclear formation

Immediately following fertilization in the sea urchin, sperm-specific histones Sp H1 and Sp H2B are phosphorylated. Then, in parallel with chromatin decondensation, nearly all phosphorylated Sp H1 is lost from the pronuclear chromatin, with the concurrent assimilation of the egg phosphoprotein CS H1. Chemical cleavage of in vivo labeled Sp H1 and Sp H2B shows that serine phosphorylation occurs in the unusually long N-terminal region of these proteins. These regions contain tandemly repeated tetra- and pentapeptide units each containing serine, proline, and two basic amino acids. It is proposed that sperm chromatin decondensation may require prior phosphorylation of these unusual N-terminal regions, whose function in the mature sperm may be to condense or stabilize its highly compact chromatin.

Effects of Ca 2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs

Nuclei transplanted into unactivated amphibian eggs are known to condense into metaphase chromosomes whereas those transplanted into activated eggs decondense and enlarge. We have made cell-free cytoplasmic preparations from Rana pipiens eggs which can induce demembranated Xenopus laevis sperm to undergo changes similar to those seen in intact eggs. Sperm chromatin which is incubated for 3 hr in unactivated egg preparations made using a buffer containing 3 m M EGTA is induced to form metaphase chromosomes. However, decondensed interphase nuclei are formed when chromatin is incubated in unactivated egg preparations made without EGTA as well as in activated egg preparations. When Ca 2+ ions are added to unactivated egg preparations made with EGTA, the preparations lose the ability to induce metaphase chromosome formation and become capable of decondensing sperm chromatin. Once the ability to decondense chromatin has developed, either in unactivated or activated egg preparations, it cannot be suppressed by the addition of EGTA. However, decondensation of sperm chromatin in activated egg preparations can be suppressed by the addition of unactivated egg preparations made with EGTA. In this case, the incubated sperm chromatin is induced to form metaphase chromosomes. These results may indicate that the chromosome condensation activity of unactivated egg cytoplasm can be sustained in cell-free preparations when Ca 2+ ion levels are kept low, but when Ca 2+ ion levels increase this activity is lost and replaced by a new activity which can decondense chromatin. Since this change in cytoplasmic activities is comparable to that occurring in the intact egg following fertilization, these results suggest that Ca 2+ ions play a crucial role during activation in altering the cytoplasmic activities which control nuclear behavior.

Decondensation of human spermatozoal chromatin by nuclear actin polymerization.

A critical relationship exists between nuclear actin polymerization and decondensation of sperm chromatin. Characteristic decondensation phenomena were brought about by the protein S-1 of heavy meromyosin of rabbit skeletal muscle in sperm that had undergone the acrosome reaction. Sperm treated with only calcium or only ionophore were not affected by the S-1 trigger, and the nucleus remained in the condensed state. Since S-1 specifically binds to actin, it was possible to demonstrate this phenomenon at the ultrastructural level. Maybe there are switches that operate at different steps for events leading to syngamy and fertilization. No switch can be operative until the preceding event has prepared the sperm for entering the next phase. The operations are performed in a perfect sequential order. This investigation leads to the conclusion that decondensation of sperm chromatin is brought about by nuclear actin polymerization.

Effect of Cryopreservation on the Nuclear Chromatin Decondensation Ability of Human Spermatozoa

The possible effect of cryopreservation on human sperm chromatin decondensation ability has been investigated. Comparisons of the actions of the decondensation-inducing agent 1% (w/v) sodium dodecyl sulphate + 6 mM EDTA were made on 30 ejaculates between spermatozoa in seminal plasma, spermatozoa in semen diluted with cryoprotective medium (CPM) and spermatozoa frozen and thawed in the semen-CPM mixture. The results, analyzed as paired series, showed no significant differences between the spermatozoa under the three treatment conditions. Thus, spermatozoa cryopreserved by a method routinely used for semen storage for subsequent artificial insemination showed a nuclear stability equivalent to that of fresh semen. The CPM by itself had no effect upon chromatin instability. No correlation was found between the percentage recovery of post-thaw motility (an usual index for judging semen cryopreservation) and the tests of chromatin decondensation.

Decondensation of mouse sperm chromatin and reassembly into nucleosomes mediated by polyglutamic acid in vitro

The organization of the mammalian sperm nucleus was probed with staphylococcal nuclease. Although isolated nuclei are resistant to cleavage, following reduction and alkylation, 30% of the sperm DNA could be digested and the remaining DNA had a heterodisperse size distribution. By morphological criteria, a model acidic protein, polyglutamic acid was capable of decondensing purified sperm nuclei that had been reduced and alkylated. The maximal extent of nuclease digestion increased to 85–90%. The subsequent addition of purified, exogenous core histones in 0.1 M NaCl partially reversed this vulnerability to nuclease cleavage such that only 55% of the DNA was digested. Furthermore, analysis of the remaining DNA revealed a nucleosome ladder pattern with unit length repeat of 150 bp. These results strongly suggest that polyglutamic acid can mediate not only decondensation of sperm nuclei but also the assembly of sperm chromatin into nucleosomes.

Changes in the stainability and sulfhydryl level in the sperm nucleus during sperm‐oocyte interaction in mice

AbstractDuring maturation in the epididymis, mouse sperm nuclei become difficult to stain with Giemsa and its component basic dyes. Mature sperm from the cauda epididymis can be stained only after DTT treatment. Stainable sperm such as those from the testis accumulate 3H NEM when examined by autoradiography, while unstainable sperm do not, indicating a close correlation between the basic dye binding capacity and SH levels in the sperm nuclei. During insemination of zonafree ovarian oocytes with a germinal vesicle (GV), mature sperm nuclei become stainable and capable of binding with 3H NEM. At the same time, sperm have established pronase‐resistant contact with the oocyte. Similarly, sperm nuclei become stainable during fertilization when the sperm attachment to the egg becomes pronase resistant. However, these changes occur before sperm chromatin decondensation begins. Therefore, it is suggested that S‐S bonds in sperm nucleoproteins are reduced when the sperm establish a stable contact with the egg plasma membrane, thus reversing sperm maturational changes. The reduction of S‐S bonds may be a prerequisite for sperm chromatin decondensation.