Decondensed Sperm Nuclei Research Articles

Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization.

Oocyte-produced glutathione (the tripeptide gamma-glutamyl-cysteinyl-glycine; GSH) has been implicated in the reduction of disulfide bonds in the sperm nucleus during fertilization and thus in the development of the male pronucleus (PN). In this study, we show that the depletion of endogenous glutathione by 10 mM buthionine sulfoximine (BSO; specific inhibitor of GSH synthesis) during bovine oocyte maturation (24 h in vitro; represents prophase I to metaphase II transition in this species) blocks the formation of a male PN in > 85% of treated oocytes (vs. 6.8% in controls) and prevents the assembly of the sperm aster microtubules in approximately 35%. Consequently, the pronuclear migration and apposition do not occur. Ultrastructural observations suggest that the effect of BSO on pronuclear apposition might be due to incomplete disassembly of the sperm tail connecting piece, which normally leads to the release of the sperm centriole and to the reconstitution of the zygotic centrosome during fertilization. The sperm nucleus decondensation and migration blocks were reversed by the treatment of the GSH-depleted oocytes with 1-10 mM dithiothreitol (a disulfide bond-reducing agent) applied 8 h after insemination: 82% of these oocytes exhibited a normal male PN and pronuclear apposition 20 h after insemination. The pool of glutathione seems to be generated during oocyte maturation since > 80% of oocytes that were matured in the absence of BSO displayed a normal male PN, as apposed to a female PN, when inseminated and cultured in the presence of 10 mM BSO. These data suggest that the reduction of disulfide bonds in the sperm after incorporation is important for the formation of the male PN, as well as for the disassembly of the sperm tail connecting piece and pronuclear apposition. The lack of disulfide-reducing power in the GSH-depleted oocytes can be reversed by treatment with disulfide bond-reducing agents.

Conformation of nucleoplasmin and its interaction with DNA-protamine complex as a simple model of fish sperm nuclei

Nucleoplasmin was isolated from Xenopus laevis eggs and purified by an improved method using an open column. Its conformation was investigated spectrophotometrically by UV, CD and fluorescence. It was shown that α-helix content of nucleoplasmin was 30–40%, and one of the two tryptophan residues in nucleoplasmin located in the hydrophobic surroundings and the other in the relatively hydrophilic surroundings. The isolated nucleoplasmin was found to decondense sperm nuclei of salmon also, suggesting a possibility of the existence of nucleoplasmin-like protein in fish as well. Collapse of the protamine (salmine)-DNA complex as a simple model for fish sperm nuclei by nucleoplasmin was directly observed by measuring OD 320 of aqueous protamine-DNA mixtures. This is a molecular level observation for the removal of protamine from DNA-protamine complex.

Transgenic Xenopus embryos from sperm nuclear transplantations reveal FGF signaling requirements during gastrulation.

We have developed a simple approach for large-scale transgenesis in Xenopus laevis embryos and have used this method to identify in vivo requirements for FGF signaling during gastrulation. Plasmids are introduced into decondensed sperm nuclei in vitro using restriction enzyme-mediated integration (REMI). Transplantation of these nuclei into unfertilized eggs yields hundreds of normal, diploid embryos per day which develop to advanced stages and express integrated plasmids nonmosaically. Transgenic expression of a dominant negative mutant of the FGF receptor (XFD) after the mid-blastula stage uncouples mesoderm induction, which is normal, from maintenance of mesodermal markers, which is lost during gastrulation. By contrast, embryos expressing XFD contain well-patterned nervous systems despite a putative role for FGF in neural induction.

Aneuploidy detection in human sperm nuclei using PRINS technique

Rapid and specific identification of chromosomes can be attained in situ using the PRimed IN Situ (PRINS) labelling technique. We have adapted this technique to mature human sperm in combination with a protocol for simultaneous decondensation and denaturation of sperm nuclei. This strategy allowed us to obtain double labelling of human spermatozoa in a <2-hr reaction. In the present study, we report the estimates of disomy for chromosomes 3, 7, 10, 11, and 17 on 64,642 spermatozoa from 2 normal males. The incidences of disomy ranged from 0.28–0.34%. There were no significant interindividual or interchromosomal differences in disomy rates. © 1996 Wiley-Liss, Inc.

Aneuploidy detection in human sperm nuclei using PRINS technique.

Rapid and specific identification of chromosomes can be attained in situ using the PRimed IN Situ (PRINS) labelling technique. We have adapted this technique to mature human sperm in combination with a protocol for simultaneous decondensation and denaturation of sperm nuclei. This strategy allowed us to obtain double labelling of human spermatozoa in a < 2-hr reaction. In the present study, we report the estimates of disomy for chromosomes 3, 7, 10, 11, and 17 on 64,642 spermatozoa from 2 normal males. The incidences of disomy ranged from 0.28-0.34%. There were no significant interindividual or interchromosomal differences in disomy rates.

Decondensation of Bull and Human Sperm Nuclei by Dithiothreitol and/or Heparin

Various concentrations of dithiothreitol (DTT) and/or heparin were examined for their ability to induce in vitro decondensation of bovine and human sperm nuclei. Bull sperm nuclei decondensed when DTT (10 mM) and heparin (10 μg /ml) were added simultaneously or when heparin (100 μg/ml) was added after the pretreatment with DTT (100 mM). Neither DTT (500 mM) nor heparin (10 mg/ml) alone, even at high concentration, could induce decondensation of bull sperm nuclei. In contrast, human sperm nuclei decondensed when DTT alone (10 mM) or heparin alone (1 mg/ml) was added. Pretreatment of human sperm with DTT (0.1 mM) decreased the concentration of heparin required for decondensation to 10 μg/ml, a concentration incapable of inducing decondensation by itself. Overall, higher concentrations of DTT and heparin were required for bovine than for human sperm for nuclear decondensation to occur. The difference in nuclear decondensation between bull and human sperm in response to DTT and heparin is likely related to the differences between the two species in the DNA-protamine complex of sperm nuclei.

Use of fresh or aged bovine oocytes for intracytoplasmic sperm injection (ICSI)

Following their incorporation into oocytes, sperm nuclei (SN) of the surf clam, Spisula solidissima, undergo an initial expansion, followed by condensation and then a dramatic enlargement during their development into male pronuclei. These changes are temporally correlated with alterations in the maternal chromatin: germinal vesicle breakdown (GVBD), meiotic maturation, and female pronuclear development, respectively. To analyze possible changes occurring in SN at fertilization, surf clam oocyte extracts; prepared before and after parthenogenetic activation, were examined for their ability to affect SN in vitro. Sperm heads were incubated in extracts for variable periods up to 5 hr. Extracts prepared from oocytes following GVBD (15 min postactivation) induced an expansion in ∼90% of SN by 60 min incubation. However, when SN were incubated in extracts from unactivated or 4-min-activated oocytes only ∼30% underwent expansion. Ultrastructural examination of specimens taken at increasing periods of incubation in oocyte extracts revealed that SN expansion in vitro resembled chromatin decondensation in vivo. SN incubated 1 to 5 hr in extracts prepared from oocytes following GVBD consisted of decondensed chromatin surrounded to varying degrees by membranous cisternae. Staining with anti-lamin antibody was variable: some specimens (60-70%) were positive while others (30-40%) were weak to negative. In contrast, all decondensed SN incubated in extracts from postmeiotic oocytes (65 min postactivation) were delimited by an intact nuclear envelope possessing nuclear pores and reactive to anti-lamin antibody. Decondensation of SN in 15- or 65-min extracts was blocked by EDTA, 2,6-dimethylami-nopurine, histone, and protamine. The presence (65-min extract) and absence (unactivated, 4- and 15-min extracts) of sperm nuclear envelope assembly in vitro is consistent with events in vivo, where such a structure forms after meiotic maturation in concert with the development of the female pronucleus.

Double and triple in situ chromosomal labeling of human spermatozoa by PRINS.

The PRimed IN Situ (PRINS) labeling method allows rapid, specific detection of human chromosomes in situ. We have adapted the PRINS protocol to mature human sperm in combination with a 3 M NaOH protocol for simultaneous in situ decondensation and denaturation of sperm nuclei. Using fluorochrome-labeled dNTPs in a sequential PRINS reaction, the direct detection of two or three distinct chromosomes must be performed within a timespan of 3 h. The method was tested with primers specific for chromosomes 8, 9, 12, 13, 16, 18, and 21 and the X. The frequencies of disomy ranged from 0.11% to 0.34%. Chromosome-specific primers have been defined for most of the human chromosomes, including some that are indistinguishable by fluorescence in situ hybridization (FISH) with centromeric probes. Consequently, this new strategy constitutes a rapid and efficient alternative to FISH for detecting nondisjunction in human sperm.

Structure of the rRNA genes in the hamster sperm nucleus.

We have examined the structure of the major ribosomal RNA (rRNA) genes in the hamster sperm nucleus, using fluorescent in situ hybridization (FISH). The rRNA genes are present as tandemly repeated clusters located at the telomeric ends of the short arms of five pairs of acrocentric chromosomes in the Syrian golden hamster (as they are in humans). In somatic cells, these five chromosome pairs come together to form the nucleolus, the site of rRNA synthesis. The nucleolus remains intact through S phase of the cell cycle, breaking apart only during late G2 and mitosis when the chromosomes condense. Mammalian sperm nuclei are the final products of meiotic division and morphological differentiation that includes a dramatic chromatin condensation. Consequently, it was not immediately obvious whether the rRNA genes would be condensed into a nucleolus-like structure in the mature spermatozoa, or separated, as they are in mitotic chromosomes. We found that of 117 sperm nuclei examined, 91.5% contained between two and five FISH signals for the rRNA gene clusters, and 64.0% contained four (29%) or five (35%) signals. In decondensed hamster sperm nuclei, the rRNA hybridized signals were separated into independent strands. These data collectively indicate that the chromosomes containing the rRNA genes are not bound together into a pre-nucleolar structure in fully condensed mammalian sperm nuclei.

Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation.

In the present study we investigated the relevance of sperm immobilization prior to intracytoplasmic sperm injection (ICSI) in the fertilization process. Using supravital staining of the spermatozoa with eosin and studying sperm decondensation with 2 mM dithiothreitol (DTT) in conditions imitating sperm handling during ICSI, we demonstrated that immobilization of the spermatozoon by squeezing its tail between the glass pipette and the bottom of the dish damages the sperm plasma membrane. Polyvinylpyrrolidone (PVP), which is usually present in the drop with the spermatozoon to facilitate its handling, was found to impede the access of both eosin and DTT to the sperm nucleus. We conclude that (i) sperm immobilization prior to ICSI damages the sperm plasma membrane, that (ii) this damage is sufficient for thiol-reducing agents to gain access to the sperm nucleus, and finally that (iii) PVP possibly interferes with sperm nucleus decondensation.

Molecular Localization of Free Thiols in Human Sperm Chromatin1

The presence in human sperm nuclear proteins of a limited amount of unoxidized thiol groups, stabilized by reversible binding to zinc ions, has been presumed to play a role in the decondensation of sperm within the oocyte. In the present study, the number and molecular localization of free sulfhydryls in the major proteins of human sperm chromatin, protamines P1 and P2, were determined by alkylation of reactive thiols with 14C-iodoacetamide, isolation of protamines, and peptide mapping. Less than 1.5% of the cysteines of protamines were found as reactive thiols, a proportion strikingly lower than that reported previously for whole human sperm proteins. The amount of sulfhydryls was unaffected by the zinc chelating agent EDTA. Labeling was evenly distributed on every cysteine of protamines P1 and P2. The results confirm the extensive stabilization of sperm chromatin by disulfide bridges and show that the unoxidized cysteines remaining at the end of epididymal transit in some protamine molecules may be one of the six (protamine P1) or five (protamine P2) cysteines present in the sequence of each class of protamines. This even distribution of the reactive cysteines could facilitate decondensation of sperm nuclei initiated by a thiol-disulfide exchange.

Fluorescent in situ hybridization evaluation of human Y-bearing spermatozoa separated by albumin density gradients

Fluorescent in situ hybridization of decondensed sperm nuclei was employed to evaluate the effectiveness of the Ericsson albumin gradient enrichment technique of Y-bearing sperm. Mature human spermatozoa were separated from donor- and randomly selected patient semen using three different albumin density layers. Sperm nuclei, in the albumin layer with highest density, were decondensed and the percentage Y-bearing sperm was determined by fluorescent in situ hybridization. A slight, although statistically significant, increase in percentage Y-bearing sperm was observed. Although statistically significant, the clinical relevance of the small increase in Y-bearing sperm remains uncertain. A randomized controlled clinical study should help to clarify the above laboratory results.

Calicin in human sperm fertilizing zona-free hamster eggs in vitro.

Calicin, a basic cytoskeletal protein has been proposed to be involved in the formation and maintenance of the highly regular organization of the postacrosomal perinuclear theca, the calyx of mammalian spermatozoa. The fate of human sperm calicin in the zona-free hamster egg fertilized in vitro has been analysed at the light and electron microscope level using polyclonal mouse anti-calicin antibodies. Calicin was localized in the postacrosomal dense lamina of the capacitated acrosome-reacted spermatozoa attached on the surface of the egg as well as in the spermatozoa at an early stage of ooplasmic incorporation. As determined by the aid of the DNA-specific fluorescent dye 4,6-diamidino-2-phenylindole (DAPI), the pattern of staining with anti-calicin changed from a funnel-shape into a ring soon after the onset of the nuclear decondensation. Later, no anti-calicin labelling could be detected around the more decondensed sperm nuclei. Because the residual, ring-like accumulation of calicin was associated with the least decondensed chromatin, it appears that the degradation of calicin-containing perinuclear theca is intimately involved with the posteriorly advancing nuclear disintegration. The ring formation also suggests that the calyx of human spermatozoa is not structurally homogeneous at least in terms of sensitivity to ooplasmic degradation. Calicin appears to be unaffected by lytic enzymes of the acrosome. The present study further shows that DAPI can be effectively used to analyse sperm nuclear decondensation in vitro.

Effects of chronic low-dose cyclophosphamide exposure on the nuclei of rat spermatozoa.

Previous studies from our laboratory have shown that chronic exposure of the father to low doses of cyclophosphamide results in a significant increase in early embryonic death with little effect on the male reproductive system in rats. Such effects on progeny outcome are hypothesized to be mediated by an action of the drug on the nucleus of spermatozoa. The purpose of the present studies was to investigate the effects of cyclophosphamide treatment for 1 or 6 wk on the pattern of decondensation of sperm nuclei and on the sulfhydryl content of sperm nuclear proteins. Adult male rats were treated with cyclophosphamide (6.1 mg/kg/day) daily for 1 or 6 wk. Cauda epididymal spermatozoa were collected, demembranated, and then incubated with dithiothreitol (DTT) and proteinase K. The in vitro decondensation pattern of the nuclei of spermatozoa was divided into two phases: nuclear swelling and nuclear elongation. Spermatozoa from animals treated for 1 wk with cyclophosphamide showed the same decondensation pattern as those treated with vehicle (saline) alone. However, spermatozoa from animals treated for 6 wk with cyclophosphamide showed normal initial nuclear swelling but had a markedly affected nuclear elongation pattern. The changes with time in the decondensation pattern of these spermatozoa were quantitated by morphometric analysis of the head region of the spermatozoa. The nuclear area, curvature, and length of spermatozoa obtained from chronically drug-treated males were all significantly smaller than for those obtained from controls, while cell diameter was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies to decondensed sperm nuclear deoxyribonucleic acid in patients with antisperm antibodies and systemic lupus erythematosus detected by immunofluorescence flow cytometry

To evaluate a flow cytometric method to detect and quantitate serum anti-DNA antibodies using unfixed, swollen and decondensed human sperm nuclei and to examine the relationship between antibodies against sperm surface antigens to the presence of antibodies against nuclear antigens. Serum IgG and IgG subclass antibodies to decondensed sperm nuclei were detected by indirect immunofluorescence (IIF) flow cytometry. Sera were screened by IIF for anti-double-stranded DNA antibodies using the protozoan Crithidia luciliae as the substrate and for antinuclear antibodies using human epithelial (HEp 2) cells, respectively. All sera were assessed for antibodies against the sperm plasma membrane by an indirect immunobead test. Infertility laboratory at the University of Oklahoma Health Sciences Center and rheumatology laboratory at the Oklahoma Medical Research Foundation. Sera from 33 antisperm antibody-positive patients (5 subgroups), 33 patients with systemic lupus erythematosus (SLE; 6 subgroups), and 20 normal controls were selected. IgG antibodies against decondensed sperm nuclear DNA were detected in 11 (33.3%) of 33 antisperm antibody-positive patients versus 14 (42.4%) of 33 patients with SLE. Anti-DNA antibodies were most prevalent in vasectomized men and in antisperm antibody positive women with SLE. In the sera from patients with SLE, the presence of the anti-nuclear ribonucleoprotein antibody was associated with the presence of sperm head-directed antisperm antibodies. Anti-double-stranded DNA antibodies were found in 6 (18.1%) of 33 sera from patients with antisperm antibody and 17 (51.5%) of 33 sera from patients with SLE. Antinuclear antibodies were found in only 9 (27.2%) of 33 sera from patients with antisperm antibody and 30 (90.9%) of 33 sera from patients with SLE. All 20 of the control sera gave negative results in the three tests. Serum IgG reactivity to sperm nuclei was predominantly of the IgG1 and IgG3 subclasses. Anti-DNA is frequently found in either patients with antisperm antibodies or patients with SLE. Our results indicated that decondensed sperm nuclei can provide a specific substrate for screening serum anti-DNA antibodies.

Epidermal growth factor enhances oocyte maturation in pigs.

Epidermal Growth Factor (EGF) has been reported to stimulate nuclear maturation in porcine oocytes (Sommer et al., 1992). The objective of this experiment was to test the effect of EGF alone or in combination with gonadotropins and follicular shell coculture, on cytoplasmic maturation of oocytes in vitro. A preliminary experiment tested the effective dose of EGF for stimulation of oocyte nuclear maturation in our culture system. Nuclear maturation rates (MII%) of 67.2 +/- 4.6, 81.1 +/- 8.6, and 80.7 +/- 5.1 for oocyte complexes cultured in the presence of 0.1, 1, and 10 ng/ml EGF, respectively, were significantly higher (P < 0.05) than for oocytes cultured in the absence of EGF (18.1 +/- 9.4%). In the main experiment a 2 x 2 x 2 factorial random complete block design was used to examine the effect of EGF (1.0 ng/ml) alone or in combination with gonadotropins and follicular shell coculture on cytoplasmic maturation. Cytoplasmic maturation was evaluated by the ability of oocytes to decondense sperm nuclei after sperm penetration. EGF alone did not stimulate cytoplasmic maturation in oocytes in vitro (P > 0.05). However, EGF showed a positive interaction (P < 0.05) with gonadotropin treatment on male pronuclear formation. We conclude that EGF alone can stimulate nuclear maturation in pig oocytes, and EGF can interact with gonadotropins to enhance cytoplasmic maturation. A high level of cytoplasmic maturation of in vitro matured pig oocytes could be achieved using a combination of EGF, gonadotropin, and follicular shell supplementation to the culture environment.

Follicular oocyte maturation and sperm penetration in vitro in the silver fox ( Vulpes vulpes)

Of the 950 follicular oocytes recovered, a high percentage (12%) were found to be degenerate and 7% had undergone germinal vesicle breakdown. Incubation of the remaining cumulus oocyte complexes in TCM 199 medium supplemented with 10% fetal calf serum, with or without human chorionic gonadotropin (HCG), in a 5% CO 2 in air atmosphere at 38.5°C resulted in expansion of the cumulus in 45% of the HCG supplemented group and in 22% of the non-supplemented group. Cumulus expansion was accompanied by germinal vesicle breakdown in almost all instances. Nineteen per cent of oocytes reached Metaphase I or II by 48 h of culture in the HCG supplemented group. Following insemination in vitro, decondensing sperm nuclei were found in the vitellus of 11% of the oocytes that had not been cultured and in the vitellus of 18% of those that had been cultured for 48 h. The results presented indicate that: (1) silver fox follicular oocytes can be matured in vitro, (2) HCG can promote development of the cumulus oocyte complexes, (3) maturation of the oocytes is not required for sperm passage through the zona pellucida, (4) the spermatozoa required capacitation which took approximately 5–8 h in vitro.

Sperm Nuclear Transformations in Cytoplasmic Extracts from Surf Clam (Spisula solidissima) Oocytes

Following their incorporation into oocytes, sperm nuclei (SN) of the surf clam, Spisula solidissima, undergo an initial expansion, followed by condensation and then a dramatic enlargement during their development into male pronuclei. These changes are temporally correlated with alterations in the maternal chromatin: germinal vesicle breakdown (GVBD), meiotic maturation, and female pronuclear development, respectively. To analyze possible changes occurring in SN at fertilization, surf clam oocyte extracts; prepared before and after parthenogenetic activation, were examined for their ability to affect SN in vitro. Sperm heads were incubated in extracts for variable periods up to 5 hr. Extracts prepared from oocytes following GVBD (15 min postactivation) induced an expansion in ∼90% of SN by 60 min incubation. However, when SN were incubated in extracts from unactivated or 4-min-activated oocytes only ∼30% underwent expansion. Ultrastructural examination of specimens taken at increasing periods of incubation in oocyte extracts revealed that SN expansion in vitro resembled chromatin decondensation in vivo. SN incubated 1 to 5 hr in extracts prepared from oocytes following GVBD consisted of decondensed chromatin surrounded to varying degrees by membranous cisternae. Staining with anti-lamin antibody was variable: some specimens (60-70%) were positive while others (30-40%) were weak to negative. In contrast, all decondensed SN incubated in extracts from postmeiotic oocytes (65 min postactivation) were delimited by an intact nuclear envelope possessing nuclear pores and reactive to anti-lamin antibody. Decondensation of SN in 15- or 65-min extracts was blocked by EDTA, 2,6-dimethylami-nopurine, histone, and protamine. The presence (65-min extract) and absence (unactivated, 4- and 15-min extracts) of sperm nuclear envelope assembly in vitro is consistent with events in vivo, where such a structure forms after meiotic maturation in concert with the development of the female pronucleus.

Preferential binding of cross-reactive group A streptococcal anti-DNA monoclonal antibodies to decondensed human sperm DNA.

Four murine anti-streptococcal monoclonal antibodies (mAbs) that cross-react with human DNA were evaluated by immunofluorescence flow cytometry for their reactivity with condensed and decondensed human sperm nuclei. All 4 anti-DNA mAbs reacted with condensed sperm nuclei. The reactivity of 3 of these mAbs with decondensed sperm nuclei was 13 to 177 times higher than that found with condensed nuclei. Under identical conditions, mAbs to cytoskeletal/cytocontractile proteins lacked reactivity with decondensed sperm nuclei. Binding of monoclonal anti-DNA antibodies to decondensed sperm nuclei was abolished by preincubation with double-stranded DNA. The preferential binding of anti-DNA antibodies to decondensed sperm DNA suggests the utility of decondensed sperm nuclei as the antigenic substrate for screening anti-DNA antibodies by flow cytometry.

Effect of acrosome defects and sperm chromatin decondensation on fertility and litter size in the rabbit. Preliminary electron-microscopic study.

Sections of ejaculated spermatozoa from rabbits with known fertility were observed by transmission electron microscopy. Acrosome defects, odd head shapes, the presence of spermatids and the decondensation of sperm nuclei were recorded. The fertility negatively correlated with the number of acrosomal defects, while litter size was negatively related to nuclear decompaction. Sperm nuclear decondensation was present in all ejaculates. Its frequency was correlated with that of spermatids in ejaculates.