scholarly journals Changes in intracellular content of glutathione and thiols associated with γ-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes

Zygote ◽  
1999 ◽  
Vol 7 (4) ◽  
pp. 301-305 ◽  
Author(s):  
Hiroaki Funahashi ◽  
Naoya Bandoh ◽  
Shinobu Nakahira ◽  
She-Hoon Oh ◽  
Seiji Tsuboi
Keyword(s):  
High Performance ◽  
Sperm Nucleus ◽  
Treatment Groups ◽  
Intracellular Content ◽  
Sperm Penetration ◽  
Rat Eggs ◽  
Pronuclear Formation ◽  
Glutamyl Transpeptidase ◽  
Increased Activity ◽  
Sperm Decondensation

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilised oocytes (8.50 ± 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 ± 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 ± 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 ± 74.1 nmol/egg in unfertilised eggs, 146.0 ± 50.0 nmol/egg in DSH eggs and 39.7 ± 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilisation. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 ± 1.5 nmol/egg in unfertilised oocytes and 10.1 ± 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 ± 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilisation but cysteine and γ-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of γ-glutamyl transpeptidase.

229 INFLUENCE OF SPERM TREATMENT ON SPERM HEAD DECONDENSATION FERTILIZING WITH EJACULATED AND EPIDIDYMAL PORCINE SPERM BY ICSI

2009 ◽  
Vol 21 (1) ◽  
pp. 212
Author(s):  
F. A. Garcia-Vazquez ◽  
K. Aviles-Lopez ◽  
C. Matas
Keyword(s):  
Sperm Head ◽  
Oocyte Activation ◽  
Treatment Groups ◽  
Sperm Penetration ◽  
P 16 ◽  
Low Incidence ◽  
High Level ◽  
Pronuclear Formation ◽  
Sperm Decondensation

Intracytoplasmic sperm injection (ICSI) provides a possible remedy to produce high numbers of monospermic zygotes. However, the efficiency of ICSI in pigs is low, mainly due to a failure of oocyte activation and low incidence of sperm head decondensation. The persistence of an intact sperm acrosome, plasma membrane, and perinuclear theca, which are removed during the process of sperm penetration in natural fertilization, are considered major reasons for low male pronuclear formation after ICSI in the oocytes, which are capable of supporting male pronuclear formation after IVF. Failure of male pronucleus formation is the major fertilization defect causing embryo development failure in pig oocytes subjected to ICSI (Lee JW et al. 2003 Theriogenology 59, 305). The main objective of this experiment was to determine the effect of sperm treatment on sperm head decondensation for ejaculated and epididymal spermatozoa used with ICSI. We divided ejaculated (EJ) and epididymal (EP) sperm into 3 treatment groups: 1) sperm without any treatment (control = C), 2) spermatozoa washed through a Percoll® gradient (P), and 3) spermatozoa washed through Percoll® followed by 30 min of incubation in porcine oviductal fluid (POF). Oocytes were matured in vitro 44 h in NCSU-37 and microinjected with sperm from the different treatment. Four hours after injection, the putative zygotes were stained with Hoescht and classificated as: (i) intact, (ii) low level of decondensation, and (iii) high level of decondensation and male pronuclei. A total of 461 oocytes were injected in 6 replicates. Sperm head decondensation (categorical data) was modeled using a binomial model of parameters and analyzed by ANOVA. The EP-C treatment showed a higher level of intact sperm (21.95 ± 3.4) than the other treatments (EP-P: 16 ± 3.6; EP-POF: 6.58 ± 3.6; EJ-C: 12.33 ± 3.7; EJ-P 5 ± 3.5; EJ-POF: 5.3 ± 3.6). Ejaculated sperm showed lower decondensation levels (C: 49.32 ± 5.7; P: 38.75 ± 5.4; POF: 32.00 ± 5.6) compared to epididymal sperm (C: 67.07 ± 5.4; P: 64 ± 5.6; POF: 61.84 ± 5.6). Sperm decondensation and male pronuclear formation were higher in EJ-P (56.25 ± 5.0) and EJ-POF (62.67 ± 5.2) compared to other groups (EP-C: 10.89 ± 4.9; EP-P: 20.00 ± 5.2; EP-POF: 31.58 ± 5.1; EJ-C: 38.36 ± 5.2). In conclusion, the EJ sperm exhibited higher levels of head sperm decondensation and pronuclei formation than EP sperm; the modification of sperm membrane mediated by washing sperm through Percoll® or incubating with POF induced faster decondensation than sperm without any treatment. Supported by MEC (AGL2006-03495).

Pronuclear formation and intracellular glutathione content of in vitro-matured porcine oocytes following in vitro fertilisation and/or electrical activation

Zygote ◽  
1995 ◽  
Vol 3 (3) ◽  
pp. 273-281 ◽  
Author(s):  
Hiroaki Funahashi ◽  
Todd T. Stumpf ◽  
Thomas C. Cantley ◽  
Nam-Hyung Kim ◽  
Billy N. Day
Keyword(s):  
Electrical Stimulation ◽  
Electrical Activation ◽  
In Vitro Fertilisation ◽  
Oocyte Activation ◽  
Intracellular Content ◽  
Intracellular Glutathione ◽  
Sperm Penetration ◽  
Sperm Factor ◽  
Pronuclear Formation

SummaryPronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes matured in vitro were determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisation in vitro differ from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.

In vitro fertilization in inbred BALB/c mice I: isotonic osmolarity and increased calcium-enhanced sperm penetration through the zona pellucida and male pronuclear formation

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 249-257 ◽  
Author(s):  
Seiji Kito ◽  
Yuki Ohta
Keyword(s):  
Calcium Concentration ◽  
Detrimental Effect ◽  
Choline Chloride ◽  
Positive Control ◽  
Basic Medium ◽  
Vitro Fertilization ◽  
Medium Osmolarity ◽  
Sperm Penetration ◽  
Pronuclear Formation

SummaryTo optimize IVF conditions for BALB/c mice, which are known to have poor in vitro fertilizability, the requirements for sperm–ova interaction were studied by use of modified simplex optimization medium (mKSOM) as a basic medium. Modified human tubal fluid (mHTF) was used for sperm preincubation and acted as a positive control. When the two media were compared, neither capacitation nor fertilization was supported in mKSOM. Increasing the calcium concentration in mKSOM to 5 mM or more during sperm: ova coincubation improved zona penetration but not male pronuclear (MPN) formation to the same level as those cells incubated in mHTF. When medium osmolarity was varied from 230–305 mOsmol by NaCl at 5 mM CaCl2, MPN formation improved at 280 mOsmol or higher osmolarity to the same level as that found when using mHTF. When NaCl equivalent to 25–75 mOsmol was substituted with trehalose, no significant reduction in fertilization was observed. Substitution of NaCl equivalent to 75 mOsmol with other osmotic reagents (sucrose, choline chloride and sorbitol) resulted in similar levels of fertilization as found with mHTF, except for sorbitol, which reduced fertilization significantly caused by its detrimental effect on sperm viability. At isotonic osmolarity (305 mOsmol), maximum fertilization was observed at 5 mM CaCl2; lower or higher concentrations of CaCl2 resulted in reduced fertilization. Calcium and osmolarity, therefore, are important for sperm : ova interaction in BALB/c mice and the increases in calcium to 5 mM and osmolarity to 305 mOsmol are optimal for BALB/c sperm to penetrate through the zona and to form MPN.

Evaluation of pteridine metabolism in battery workers chronically exposed to lead

2006 ◽  
Vol 25 (7) ◽  
pp. 353-359 ◽  
Author(s):  
A B Engin ◽  
D Tuzun ◽  
G Sahin
Keyword(s):  
Neurological Disorders ◽  
High Performance ◽  
Aminolevulinic Acid ◽  
Control Group ◽  
Occupationally Exposed ◽  
Lead Levels ◽  
Urinary Lead ◽  
Urinary Neopterin ◽  
Battery Workers ◽  
Increased Activity

Occupationally-exposed lead affects the neuromuscular junction and might cause disturbances in the locomotor activity. This study was undertaken to evaluate pteridine metabolism, in which neurotransmitters are synthesized in battery workers. Urinary neopterin, biopterin and creatinine were measured using high performance liquid chromatography. Serum neopterin concentrations were detected by enzyme-linked immunoassay. Blood dihydropteridine reductase (DHPR) activities and delta-aminolevulinic acid (delta-ALA) were measured spectrophotometrically. Blood and urinary lead were detected by atomic absorption spectroscopy. Significantly increased blood and urinary lead levels, urinary neopterin, biopterin and delta-ALA were found in workers, while DHPR activities were indifferent compared to control group. Urinary creatinine decreased. This is the first study to demonstrate that increased activity of the pteridine pathway results in the accumulation of the neurotransmitters that may be responsible for the neurological disorders.

Effect of medium milieu on sperm penetration and pronuclear formation of bovine oocytes matured in vitro

2002 ◽  
Vol 57 (8) ◽  
pp. 2093-2104 ◽  
Author(s):  
Byung-Ki Kim ◽  
Sang-Chan Lee ◽  
Kwang-Sun Lee ◽  
Bok-Kyu Lee ◽  
Chang-Hee Han ◽  
...  
Keyword(s):  
Sperm Penetration ◽  
Pronuclear Formation ◽  
Bovine Oocytes

348 CO-CULTURE OF PORCINE OOCYTES - CUMULUS COMPLEXES DERIVED FROM SMALL FOLLICLE WITH CUMULUS-CELL MASSES FROM MIDDLE FOLLICLE IMPROVES MEIOTIC MATURATION OF THE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 330
Author(s):  
R. Matsunaga ◽  
H. Funahashi
Keyword(s):  
Early Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Protein Profiles ◽  
Blastocyst Formation ◽  
Maturation Rate ◽  
Sperm Penetration ◽  
Functional Peptide ◽  
Pronuclear Formation

It is known that maturation rate of oocytes derived from small follicles (SF) is lower than that of oocytes from middle follicles (MF). Since it has been reported that cumulus cells have important role during oocytes maturation, the ability of SF oocytes to complete the meiotic maturation may be affected by additional cumulus-cell mass. The present study was undertaken to examine the effects of co-culture of oocyte-cumulus complexes (OCCs) derived from SF with additional cumulus-cell masses on in vitro maturation and developmental competence of the oocytes. OCCs were aspirated from small (SF; 1-2 mm in diameter) or middle follicles (MF; 3-6 mm in diameter) of prepuberal ovaries. OCCs were cultured in porcine oocyte medium (POM; Research Institute for the Functional Peptide, Yamagata, Japan) supplemented with gonadotropins and dbcAMP for a first 20-h period and then in gonadotropin-free and dbcAMP-free POM for another 24 h. Culture medium was collected after the first 20-h culture and the end of IVM, and analyzed for the protein profiles. Following IVM, some oocytes were co-incubated with spermatozoa in a drop of modified Medium199 containing 0.4% BSA and 5 mM caffeine for 8 h and then incubated in PZM5 (Research Institute for the Functional Peptide, Yamagata, Japan) for 6 days. Sperm penetration, cleavage, and the early development of the oocytes were examined before culture in PZM5 or Day 2 and Day 6 of culture, respectively. OCCs derived from SF were co-cultured with cumulus-cell masses derived from SF or MF during IVM (SFO-SFC and SFO-MFC groups, respectively). Some OCCs derived from SF or MF were cultured for IVM without additional cumulus-cell masses (SFO and MFO, respectively). After culture, meiotic maturation of the oocytes was examined. To analyze the developmental competence of oocytes of SF, MF, and SFO-MFC groups, sperm penetration, pronuclear formation, cleavage, and blastocyst formation were examined. Protein profiles in the IVM media were examined by 10% SDS-PAGE. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P ≤ 0.05). After culture for IVM, the diameters of SFO and SFO-MFC were not different from that of MFO (113.3-114.5 μm). The maturation rate of SFO-MFC oocytes (75.5 ± 6.2%) was higher than SFO (52.2 ± 2.8%) and comparable with the rate of MFO oocytes (83.2 ± 6.3%), while there was not significant difference between the mature rate of SFO+SFC oocytes (63.6 ± 4.0%) and SFO oocytes. There were no significant differences between groups in sperm penetration, pronuclear formation, and cleavage. Blastocyst formation of SF oocytes was not improved by co-culture with MF cumulus-cell masses. Certain band was detected only in MF medium of collected at 20 h (24.5 kD). From these results, we conclude that secretions from cumulus-cell masses derived from MF well improve the meiotic progress of oocytes derived from SF, but not the early development following IVF.

119 HETEROLOGOUS BOVINE IN VITRO FERTILIZATION USING CRYOPRESERVED BOTTLENOSE DOLPHIN SPERMATOZOA

2012 ◽  
Vol 24 (1) ◽  
pp. 172 ◽  
Author(s):  
M. J. Sanchez-Calabuig ◽  
P. Beltran-Brena ◽  
E. Martinez-Nevado ◽  
D. Rizos ◽  
J. F. Perez-Gutierrez ◽  
...  
Keyword(s):  
Bottlenose Dolphin ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Sperm Chromatin ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Sperm Penetration ◽  
Pronuclear Formation ◽  
Bovine Oocytes

Assisted reproductive technologies are of great importance for increasing genetic diversity in captive animals without displacing them. The development and improvement of these techniques require accurate methods to assess sperm function. The ability of the sperm to bind the zona pellucida and the formation of a male pronucleus have been shown to have a high predictive value for fertilization outcome. The use of zona-intact bovine in vitro–matured oocytes in heterologous fertilization with dolphin spermatozoa could provide valuable information on its fertilizing ability. The aim of the present study was to evaluate male pronuclear formation in zona-intact bovine oocytes after coincubation with frozen-thawed bottlenose dolphin spermatozoa. A total of 1546 immature cumulus oocytes complexes (COC) were obtained from bovine ovaries collected at slaughter. The COC were matured for 24 h in TCM-199 supplemented with 10 ng mL–1 of epidermal growth factor and 10% FCS. Matured COC were inseminated with frozen-thawed Bovi-pure (Nidacon International, Mölndal, Sweden) separated bovine (control) or dolphin spermatozoa. At 18, 20, 22, 24, 26 and 28 h post-insemination (hpi), half of the presumptive zygotes from each group were fixed and stained with Hoechst 33342 to examine sperm penetration, polyspermy and pronuclear formation and the remainder were cultured in synthetic oviduct fluid supplemented with 5% FCS for evaluating fertilization rates by cleavage on Days 2 and 4 (Day 0 = day of IVF). As expected, in the control a higher percentage of 2 pronuclear formation was observed at 18 hpi (74.5%), with a decrease at 20 and 22 hpi (57.4 and 43.2%, respectively) and was significantly lower (P ≤ 0.001) at 24 hpi (13.3%), reaching the lowest values at 26 and 28 hpi. However, in the heterologous group significantly less oocytes with both pronuclear formed (P ≤ 0.001) were observed at 18, 20 and 22 hpi (1.2, 3.4 and 3.0%, respectively) compared with 24, 26 and 28 hpi (22.5, 11.4 and 8.9%, respectively). No polyspermy was detected in oocytes coincubated with dolphin spermatozoa. Moreover, the cleavage rate at Day 2 and 4 in heterologous fertilization was 13.0 and 34.8%, respectively, whereas for the control it was 90.0%. In conclusion, these results indicate that dolphin spermatozoa can penetrate bovine oocytes and induce the block to polyspermy and the differences found regarding pronuclear formation times between the 2 species could be due to distinct sperm chromatin organisation or condensation. In conclusion, our preliminary results show that heterologous fertilization using bovine oocytes is useful for characterising the viability of dolphin thawed spermatozoa, which also could be helpful in performing a more complete sperm evaluation. Further studies are necessary to provide more consistent evidence of the efficiency of this test. The authors thank the staff at Zoo Aquarium Madrid for their dedicated work toward dolphin semen collection.

376 EFFECT OF SPERM TREATMENT ON THE ABILITY TO ACTIVATE OOCYTES AFTER ICSI IN PIGS

2007 ◽  
Vol 19 (1) ◽  
pp. 303
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
N. Maedomari ◽  
M. Ozawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Oocyte Activation ◽  
Sham Injection ◽  
Oocyte Meiosis ◽  
Freeze Dried ◽  
Sperm Penetration ◽  
Sperm Factor ◽  
Pre Treatment ◽  
Pronuclear Formation

During fertilization, sperm penetration (gamete membrane fusion and exposure of sperm cytoplasm) allows oocyte activation (resumption of oocyte meiosis, pronuclear formation, etc.) by inducing an elevation of the intracellular free Ca2+ concentration. So a spermatozoon ought to be able to fully activate an oocyte. However, in pig ICSI oocytes, although a spermatozoon is injected successfully into ooplasm, complete activation is deficient in some of the oocytes. A variety of sperm pre-treatments before ICSI have been reported; however, there is a possibility that the treatment affects the ability to activate oocytes after the injection. We examined the effect of sperm treatments (freezing, freeze-drying, and sonication) on the ability to activate oocytes. Ejaculated boar semen was centrifuged (10 min, 600g) and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze 1991 Reprod. Domest. Anim. (Suppl. 1), 231–253). The sperm were then treated with or without sonication for 10 s (fresh whole and sonicated sperm, respectively). The freezing of sperm was carried out as was described (Kikuchi et al. 1998 Theriogenology 50, 615–623). Frozen–thawed spermatozoa were then treated with or without sonication (frozen–thawed sonicated and whole sperm, respectively). The fresh whole and sonicated sperm were subjected to a freeze-drying system and the sperm were then re-hydrated (freeze-dried whole and sonicated sperm, respectively). A whole sperm or 1 or 3 sonicated sperm heads were then injected into in vitro-matured oocytes, as described previously (Nakai et al. 2003 Biol. Reprod. 68, 1003–1008; 2006 Reproduction 131, 603–611). Sham injection was also performed. No artificial stimulation was added to the injected oocytes. The oocytes with more than one pronucleus(i) at 10 h after the injection were defined as being activated. As shown in Table 1, the rates of activated oocytes after injection of one sonicated head or sham injection were significantly lower than those of the oocytes injected with whole sperm or 3 sonicated sperm heads in each sperm source (P &lt; 0.05 by ANOVA and Duncan's multiple range test). Furthermore, the rates of activated oocytes for each injection category were not different among the 3 sperm sources. These results suggest that sonication before ICSI may reduce the quantity of activation-inducing sperm factor. It is also suggested that sperm pre-treatment such as freezing or freeze-drying does not affect the ability for oocyte activation. Table 1. Effect of sperm treatment on oocyte activation after ICSI

DNA methylation pattern during the encystment of Physarum flavicomum

1990 ◽  
Vol 68 (6) ◽  
pp. 944-948 ◽  
Author(s):  
Chengming Zhu ◽  
Henry R. Henney Jr.
Keyword(s):  
Dna Methylation ◽  
High Performance ◽  
Restriction Enzymes ◽  
Methylation Pattern ◽  
Dna Base Composition ◽  
Metabolic Role ◽  
Growing Cell ◽  
Dna Base ◽  
Intracellular Content ◽  
Methylation Patterns

In Physarum flavicomum Berk., haploid myxamoebae convert to dormant microcysts under conditions of nutrient imbalance. Exogenous adenine increases the intracellular content of S-adenosylmethionine (SAM) and inhibits this process. However, treatments that reduce the intracellular SAM levels relieve the inhibition of encystment induced by adenine. SAM plays a major metabolic role in cellular transmethylation reactions. In this study, we compared the DNA methylation patterns of growing cells, encysting cells, adenine-inhibited cells, and cysts using three different approaches: incubation of the cells with [14C]methylmethionine and detection of the labeled methyl group in purified DNA samples; analyses of DNA base composition by high performance liquid chromatography; and restriction endonuclease analyses of DNA. We found that DNA from the adenine-treated cells was labelled 1.3 times more with [14C]methylmethionine than was the DNA of untreated encysting cells. The DNA G + C content of this species was about 41%. The DNA of growing cells had the highest 5-methylcytosine (5MC) content, while DNA from the cysts had the lowest (about 27% that of growing cells). Adenine-inhibited cells had about 1.2 times more DNA-5MC than did encysting cells. Using the restriction enzymes SmaI, PvuI, and XhoI (which are inhibited by C residue methylation), we found that cyst DNA had more cutting sites than did amoebal DNA. By using the restriction enzyme DpnI which cuts DNA at GmATC sites, we found that cyst DNA, but not growing cell DNA, contained N6-methyladenine.Key words: amoebae, cysts, methylation, 5-methylcytosine, N6-methyladenine, DNA, encystment, Physarum flavicomum, development, inhibition.

Chromatin and microtubule organisation in maturing and pre-activated porcine oocytes following intracytoplasmic sperm injection

Zygote ◽  
2002 ◽  
Vol 10 (2) ◽  
pp. 123-129 ◽  
Author(s):  
Bong-Ki Kim ◽  
Youn-Jeong Lee ◽  
Xiang-Shun Cui ◽  
Nam-Hyung Kim
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Germinal Vesicle ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Nuclear Movement ◽  
Microtubule Nucleation ◽  
Close Proximity ◽  
Female Pronucleus ◽  
Cycle Dependent ◽  
Pronuclear Formation

Chromatin and microtubule organisation was determined in maturing and activated porcine oocytes following intracytoplasmic sperm injection in order to obtain insights into the nature of sperm chromatin decondensation and microtubule nucleation activity. Sperm chromatin was slightly decondensed at 8 h following injection into germinal vesicle stage oocytes. Sperm-derived microtubules were not seen in these oocytes. Following injection into metaphase I (MI)-stage oocytes, sperm chromatin went to metaphase in most cases. A meiotic-like spindle was seen in the sperm metaphase chromatin. In a few MI-stage oocytes, sperm chromatin decondensed at 8 h after injection, and a small sperm aster was seen. Sperm injection into oocytes at 5 h following activation failed to yield pronuclear formation. Maternally derived microtubules were organised near the female chromatin in these oocytes, and seemed to move condensed male chromatin closer to the female pronucleus. At 18 h after sperm injection into pre-activated oocytes, a condensed sperm nucleus was located in close proximity to the female pronucleus. These results suggest that the sperm nuclear decondensing activity and microtubule nucleation abilities of the male centrosome are cell cycle dependent. In the absence of a functional male centrosome, microtubules of female origin take over the role of microtubule nucleation for nuclear movement.

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