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.

scholarly journals Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes

Zygote ◽  
2002 ◽  
Vol 10 (2) ◽  
pp. 95-104 ◽  
Author(s):  
Mike Katayama ◽  
Takashi Miyano ◽  
Masashi Miyake ◽  
Seishiro Kato
Keyword(s):  
Plasma Membrane ◽  
Intracytoplasmic Sperm Injection ◽  
Acrosome Reaction ◽  
Sperm Chromatin ◽  
Oocyte Activation ◽  
Freezing And Thawing ◽  
Chromatin Decondensation ◽  
Progesterone Treatment ◽  
Pronuclear Formation ◽  
Boar Spermatozoa

Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.

Sperm nuclear envelope: breakdown of intrinsic envelope and de novo formation in hamster oocytes or eggs

Zygote ◽  
1997 ◽  
Vol 5 (1) ◽  
pp. 35-46 ◽  
Author(s):  
Noriko Usui ◽  
Atsuo Ogura ◽  
Yasuyuki Kimura ◽  
Ryuzo Yanagimachi
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
De Novo ◽  
Germinal Vesicle ◽  
Mature Oocyte ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Early Prophase ◽  
Nuclear Envelope Breakdown ◽  
M Phase

SummaryDuring fertilisation of a fully mature oocyte, the sperm intrinsic nuclear envelope (SINE) disappears soon after sperm-oocyte fusion. A new nuclear envelope appears around the decondensed sperm chromatin when the oocyte reaches telophase II. Whether the SINE persists or rapidly disappears after sperm entery into immature oocytes or fertilised eggs has been controversial. Nuclear envelopes have been demonstrated around the sperm chromatin, which cannot be decondensed within the ooplasm of these oocytes or eggs, but whether these envelopes are persisting SINEs or newly formed envelopes has been apoint of dispute. To resolve this issue, the fate of the germinal vesicle stage(GV oocytes) or fertilised eggs at the pronuclear stage(PN eggs). The SINEs disappeared quikly within these oocytes or eggs, like those within maturing or mature oocytes, suggesting that the envelops around the sperm chromatin must be newly formed after SINE breakdown. To obtain further evidence, a detergent-treated, SINE-free sperm nucleus was injected into a PN egg. A new envelope appeared around the still-condensed or partially decondensed sperm chromatin within 3h after injection. Thus, disassembly of the SINE within ooplasm, unlike that of nuclear envelopes of other cells at prophase, is independent of the cell cycle stage of the oocyte or egg, whereas the ability of the ooplasm to assemble the new envelope is restricted to certain periods of the cycle. i.e. early prophase and telophase during meiosis and interphase, periods when active M-phase Promoting factor (MPF) is absent from the ooplasm.

scholarly journals 282 EFFECT OF THE MATURATION TIME ON THE OUTPUT OF THE INTRACYTOPLASMIC SPERM INJECTION (ICSI) OF PIG OOCYTES PRECULTURED WITH ROSCOVITINE

2005 ◽  
Vol 17 (2) ◽  
pp. 291
Author(s):  
E. García-Roselló ◽  
C. Matás ◽  
F. García-Vázquez ◽  
S. Cánovas ◽  
P. Coy
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Kinase Activity ◽  
Specific Inhibitor ◽  
Maturation Time ◽  
Cell Stage ◽  
Female Pronucleus ◽  
Pre Treatment ◽  
Activation Rates ◽  
Pronuclear Formation ◽  
Further Development

This study was conducted to evaluate the effect of different maturation times on the output of intracytoplasmic sperm injection (ICSI) with pig oocytes precultured with roscovitine, a specific inhibitor of MPF kinase activity (Meijer and Raymond 2003 Acc. Chem. Res. 36, 417–425). Cumulus-oocyte complexes (COCs) were collected from nonatretic follicles from slaughtered gilts. Just after recovery, oocytes (n = 456) were cultured in the presence of 50 μ­M roscovitine for 28–30 h, which maintains them at the GV stage until use without affecting further development (Coy et al. 2004 Human Reprod. 19, 41–42). Then, oocytes were matured in a conventional maturation system (Coy et al. 2002 Reproduction 124, 279–288) for 36, 40, or 44 h. Matured oocytes were fertilized by ICSI. Injection was performed as described by Martin (2000 Biol. Reprod. 63, 109–112) but in D-PBS medium with 10% FCS. Injected oocytes were transferred to TALP medium (Rath et al. 1999 J. Anim. Sci. 77, 3346–3352) for 24 h, after which they were fixed and Hoechst-stained to check for fertilization. Results (data were analyzed by ANOVA and Tukey test) showed similar activation rates (oocytes with female pronucleus) for 36, 40, and 44 h (83.6%, 81.7%, and 90.7%, respectively). Differences were observed in male pronuclear formation (80.2%, 67.6%, and 57.8%) and in putative embryos (zygotes showing two pronuclei or two cell stage 24 hpi; 74.3%, 62.9%, and 55.1%) for 36, 40, and 44 h, respectively (P < 0.05), where the 36-h group showed the best result. Data show that pre-treatment of pig oocytes with roscovitine and further maturation for 36 h can improve the output of ICSI. This work was supported by Ministerio de Ciencia y Tecnología (AGL2003-03144).

scholarly journals The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation

Reproduction ◽  
2005 ◽  
Vol 130 (1) ◽  
pp. 29-39 ◽  
Author(s):  
Luisa Gioia ◽  
Barbara Barboni ◽  
Maura Turriani ◽  
Giulia Capacchietti ◽  
Maria Gabriella Pistilli ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone Acetylation ◽  
Hdac Inhibitors ◽  
Germinal Vesicle ◽  
Fertilization Rate ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Germinal Vesicle Breakdown

The present experiments compared the ability of pig oocytes matured eitherin vivoorin vitroto structurally reorganize the penetrated sperm chromatin into male pronucleus (PN) and to carry out, in parallel, the epigenetic processes of global chromatin methylation and acetylation, 12–14 h afterin vitrofertilization (IVF). In addition, PN distribution of histone deacetylase (HDAC), a major enzyme interfacing DNA methylation and histone acetylation, was investigated. The ability of the oocyte to operate an efficient block to polyspermy was markedly affected by maturation. The monospermic fertilization rate was significantly higher forin vivothan forin vitromatured (IVM) oocytes(P< 0.01) which, furthermore, showed a reduced ability to transform the chromatin of penetrated sperm into male PN(P< 0.01). Indirect immunofluorescence analysis of global DNA methylation, histone acetylation and HDAC distribution (HDAC-1, -2 and -3), carried out in monospermic zygotes that reached the late PN stage, showed that IVM oocytes also had a reduced epigenetic competence. In fact, while in about 80% ofin vivomatured and IVF oocytes the male PN underwent a process of active demethylation and showed a condition of histone H4 hyperacetylation, only 40% of IVM/IVF zygotes displayed a similar PN remodelling asymmetry. Oocytes that carried out the first part of maturationin vivo(up to germinal vesicle breakdown; GVBD) and then completed the processin vitro, displayed the same PN asymmetry as oocytes matured entirelyin vivo. A crucial role of HDAC in the establishment of PN acetylation asymmetry seems to be confirmed by the use of HDAC inhibitors as well as by the abnormal distribution of the enzyme between the two PN in IVM zygotes. Collectively, these data demonstrated that some pig IVM oocytes fail to acquire full remodelling competence which is independent from their ooplasmic ability to morphologically reorganize the sperm nucleus into PN.

Oocyte maturation: aberrant post-fusion responses of the rabbit primary oocyte to penetrating spermatozoa

1979 ◽  
Vol 39 (1) ◽  
pp. 1-12
Author(s):  
M. Berrios ◽  
J.M. Bedford
Keyword(s):  
Anterior Part ◽  
Light And Electron Microscopy ◽  
Germinal Vesicle ◽  
Sperm Chromatin ◽  
Fallopian Tubes ◽  
Cortical Granules ◽  
Germinal Vesicle Breakdown ◽  
Acrosomal Membrane ◽  
Primary Oocyte ◽  
Inner Acrosomal Membrane

Primary oocytes cannot be fertilized normally; they begin to develop this capacity as meiosis resumes. To elucidate the changes involved in acquisition of their fertilizability, rabbit primary oocytes displaying a germinal vesicle (GV oocytes) were placed in Fallopian tubes inseminated previously with spermatozoa, recovered 2–5 h later and examined by light and electron microscopy. At least 4 aspects of GV oocyte/sperm interaction were abnormal. Although the vestments and oolemma seem normally receptive to spermatozoa, fusion with the oolemma of the primary oocyte did not elicit exocytosis of cortical granules, and consequently multiple entry of spermatozoa into the ooplasm was common. Secondly, the GV oocyte cortex failed to achieve a normal englufment of the anterior part of the sperm head. It sank into the ooplasm capped by only a small rostral vesicle or left the stable inner acrosomal membrane as a patch in the oolemma. Only rarely then was there significant dispersion of the sperm chromatin, and this remained surrounded by nuclear envelope. The persistence of this envelope constitutes a further aberrant feature, for it disappears immediately in secondary oocytes and was absent in primary oocytes in which germinal vesicle breakdown had occurred. The results are discussed with particular reference to current ideas about male pronucleus formation.

scholarly journals Mitogen-Activated Protein Kinase and Cell Cycle Progression During Mouse Egg Activation Induced by Various Stimuli

1999 ◽  
Vol 54 (3-4) ◽  
pp. 285-294 ◽  
Author(s):  
Q. Y. Sun ◽  
Y. Lax ◽  
S. Rubinstein ◽  
D. Y. Chen ◽  
H. Breitbart
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Protein Phosphatase ◽  
Kinase Activity ◽  
Polar Body ◽  
Sperm Chromatin ◽  
Mitogen Activated Protein ◽  
Second Polar Body ◽  
Pronuclear Formation ◽  
Mouse Egg

Abstract A very sensitive method was established for detecting the activity of mitogen-activated protein (MAP) kinase in mouse eggs, and used to follow temporal changes of this kinase during fertilization and sponatenous or chemically-induced parthenogenic activation. MAP kinase activity increased between 1 and 2.5 h post-insemination, at which time the second polar body was emitted and sperm chromatin was dispersed; its activity decreased sharply at 8 h, when pronuclei were formed. Both calcium ionophore A23187 and ethanol simulta­ neously induced pronuclear formation and MAP kinase inactivation in aged eggs 8 h after incubation but less effectively in fresh eggs. The protein kinase inhibitor staurosporine in­duced pronuclear formation and MAP kinase inactivation more quickly than other treat­ ments, with MAP kinase inactivation occurring slightly proceeding pronuclear formation. Okadaic acid, a specific inhibitor of protein phosphatase 1 and 2A , induced increase in MAP kinase activity, and overcame pronuclear formation induced by various stimuli. MAP kinase inactivation preceded pronuclear formation in eggs spontaneously activated by aging in vitro, perhaps due to cytoplasmic degeneration and thus delayed response of nuclear envelope precursors to MAP kinase inactivation. These data suggest that MAP kinase is a key protein kinase regulating the events of mouse egg activation. Increased MAP kinase activity is temporally correlated with the second polar body emission and sperm chromatin decondensation. Although different stimuli (including sperm) may initially act through different mechanisms, they finally inactivate MAP kinase, probably by allowing the action of protein phosphatase, and thus induces the transition to interphase.

Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes

1993 ◽  
Vol 104 (3) ◽  
pp. 861-872 ◽  
Author(s):  
M.S. Szollosi ◽  
J.Z. Kubiak ◽  
P. Debey ◽  
H. de Pennart ◽  
D. Szollosi ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Kinase Inhibitor ◽  
Polar Body ◽  
Histone H1 ◽  
Sperm Chromatin ◽  
Oocyte Activation ◽  
Mouse Oocytes ◽  
Spindle Rotation ◽  
Polar Body Extrusion ◽  
Pronuclear Formation

Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.

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