scholarly journals Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse.

1986 ◽  
Vol 102 (3) ◽  
pp. 1039-1046 ◽  
Author(s):  
H J Clarke ◽  
Y Masui
Keyword(s):  
Morphological Changes ◽  
Small Mass ◽  
Sperm Nucleus ◽  
Metaphase Chromosomes ◽  
Oocyte Meiosis ◽  
Maturation In Vitro ◽  
Sperm Penetration ◽  
Sperm Nuclei ◽  
Metaphase I

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.

Transformation of sperm nuclei into metaphase chromosomes in maturing pig oocytes penetrated in vitro

Zygote ◽  
1997 ◽  
Vol 5 (2) ◽  
pp. 183-191 ◽  
Author(s):  
Wei-Hua Wang ◽  
Koji Niwa
Keyword(s):  
Culture Medium ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Mitogen Activated Protein Kinase ◽  
Sperm Chromatin ◽  
Metaphase Chromosomes ◽  
Mitogen Activated Protein ◽  
Sperm Penetration ◽  
Sperm Nuclei

SummaryCumulus-free pig oocytes at the germinal vesicle (GV) stage were incubated in modified Brackett & Oliphant's medium with 5% fetal calf serum and 5mM caffeine with or without cryopreserved, ejaculated spermatozoa. When oocytes were transferred into modified tissue culture medium (TCM- 199B at pH 7.4) supplemented with 1OIU/ml eCG, 1OIU/ml hCG and 1 μg/ml oestradiol-17p after 8h of incubation with spermatozoa and cultured for 0–48 h, 86–99% of oocytes were penetrated. Most (95–100%) oocytes penetrated 0–16 h after transfer had decondensed sperm chromatin. However, 24 h after transfer 47% and 33% of penetrated oocytes contained recondensed sperm chromatin and sperm metaphase chromosomes, respectively. The proportion of penetrated oocytes containing sperm metaphase chromosomes increased after 36–48 h of transfer (51–65%). The transformation of sperm nuclei to metaphase chromosomes was obtained in 75% and 79% of anaphase I (AI) to telophase I (TI) and metaphase II (Mil) oocytes, respectively, but only in 38% of metaphase I (MI) oocytes. Moreover, such transformation was observed only in 1 of 30 oocytes at the stages of GV breakdown to prometaphase I and none of 69 oocytes at the GV stage. The transformation of sperm nuclei into metaphase chromosomes was completely inhibited in oocytes penetrated by eight or more spermatozoa. Well-developed male and female pronuclei were observed in only 3 (4%) of 77 oocytes penetrated 48 h after transfer. The proportion of oocytes reaching Mil was greatly inhibited by sperm penetration; only 18% of penetrated oocytes, but 87% of non-inseminated oocytes, reached Mil by 48 h after transfer. None of the oocytes penetrated by seven or more spermatozoa reached MIL Most (75%) oocytes were inhibited from the transition from MI to Mil even though they were cultured for 48 h. The present results indicate that: (1) the cytoplasm of maturing oocytes possesses an activity for transforming sperm nuclei into metaphase chromosomes, (2) immature pig oocytes penetrated by spermatozoa can undergo meiotic maturation to MI, and (3) the transition of such oocytes from MI to Mil is inhibited, suggesting that an activity of mitogen-activated protein kinase may be retarded.

Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes

Reproduction ◽  
2000 ◽  
pp. 247-256 ◽  
Author(s):  
A Shimada ◽  
K Kikuchi ◽  
J Noguchi ◽  
K Akama ◽  
M Nakano ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Morphological Changes ◽  
Affinity Purification ◽  
Immunohistochemical Analysis ◽  
Sperm Chromatin ◽  
Vitro Fertilization ◽  
Boar Sperm ◽  
Sperm Penetration ◽  
Sperm Nuclei

The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matured in vitro was assessed. For this purpose, protamine was purified from boar sperm nuclei and an antiserum against protamine was developed. After affinity purification, the antiserum reacted exclusively with boar protamine during western blotting, showing no crossreactivity with core histones. Immunohistochemical evaluation revealed that only fully developed spermatid nuclei in boar testes stained strongly with the antiserum. When pig oocytes matured in vitro were fertilized in vitro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermic penetration. Paraffin wax sections of the inseminated oocytes were examined by immunohistochemical analysis with the antiserum. The proportion of condensed sperm nuclei that reacted with the antiserum was 87% of the sperm nuclei that penetrated by 2 h after insemination, and this decreased to 20 and 13% at 3 and 5 h after insemination, respectively. However, none of the decondensing sperm nuclei or male pronuclei reacted with the antiserum during the entire insemination period. These results indicate that a specific antiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei before decondensation during in vitro fertilization.

Mechanisms of displacement of sperm basic nuclear proteins in mammals. An in vitro simulation of post-fertilization results

1982 ◽  
Vol 53 (1) ◽  
pp. 227-244
Author(s):  
T.C. Rodman ◽  
F.H. Pruslin ◽  
V.G. Allfrey
Keyword(s):  
Sperm Nucleus ◽  
Nuclear Proteins ◽  
Mouse Sperm ◽  
Basic Proteins ◽  
Molecular Events ◽  
In Vitro Simulation ◽  
Sperm Nuclei ◽  
Two Parameters

A standardized cytological preparation of mature mouse sperm has been devised to serve as an in vitro system for probing the intra-ooplasmic molecular events of transformation of the fertilizing sperm. Two parameters of the early phase of transformation in vivo are defined at the resolution of the light microscope: deletion of sperm-unique nuclear proteins, detectable by immunofluorescence, and retention of homogeneity of the residual DNA complex, with intact chromatin boundaries detectable by ethidium bromide staining. These studies show that both parameters are conserved when in vitro sperm preparations are treated with NaCl under reducing conditions. The deletion of 2 different classes of the unique basic proteins of mouse sperm nuclei is specified by the NaCl concentration: 0.7 M-NaCl displaces the non-protamine class but not the protamines, while 1 M-NaCl displaces both. On the other hand the effects of treatment with trypsin at various concentrations and intervals are less consistent with the in vivo parameters, indicating fragmentation and displacement, not only of the sperm-unique basic proteins, but also of structural proteins believed to maintain the fundamental cohesive organization of the DNA matrix. These observations suggest that mechanisms other than proteolysis, e.g. localized changes in ionic concentrations, may participate in the post-fertilization displacement of the sperm-unique nuclear proteins in vivo. This study also supports the validity of the in vitro simulation as a model with which to probe the progression of transformation of the sperm nucleus to the zygote pronucleus.

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 < 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

255 SPERM NUCLEAR DECONDENSATION ABILITY OF IN VITRO MATURED CANINE OOCYTES

2011 ◽  
Vol 23 (1) ◽  
pp. 225
Author(s):  
M. De los Reyes ◽  
J. Vergara ◽  
J. Palomino
Keyword(s):  
Cumulus Cells ◽  
Sperm Nucleus ◽  
Culture Conditions ◽  
Sperm Chromatin ◽  
Chromatin Decondensation ◽  
Chi Square ◽  
Cytoplasmic Maturation ◽  
Sperm Nuclei ◽  
Nuclear Decondensation

The sperm chromatin decondensation occurs when a spermatozoon enters into an oocyte during fertilization, and the effectiveness of this process is connected to the grade of oocyte cytoplasmic maturation. In this study chromatin sperm decondensation was evaluated after IVF of canine oocytes matured in vitro (IVM), comparing different durations of maturation. Cumulus–oocytes complexes (COC) for IVM were obtained from bitch ovaries after ovariohysterectomy, selecting those COC with compact cumulus cells and a homogeneous dark cytoplasm. The COC were matured in vitro for 0, 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM HEPES, 10% FCS, 0.25 mM pyruvate, 10 IU mL–1 of hCG, 300 IU mL–1 of penicillin, and 20 mg mL–1 of streptomycin at 38.5°C and 5% CO2. Fresh ejaculates from 3 adult dogs were centrifuged, and the sperm pellet was resuspended in fert-TALP medium. In each replicate, 100-μL fert-TALP drops containing 10 to 12 IVM oocytes after each culture time were co-culture with 2.5 × 106 spermatozoa mL–1 for 24 h under culture conditions. Soon after co-culture, all oocytes were denuded from cumulus cells and fixed in 3% paraformaldehyde. The nuclear stage of the oocytes and the appearance of the sperm nucleus were determined by 4′,6-diamidino-2-phenylindole staining under a fluorescence inverted microscope. For each treatment, at least 4 replicates were performed, and the data were compared statistically by chi-square test, using InfoStat Professional Program. A total of 800 oocytes were evaluated, and the percentages of oocytes with sperm penetration were 58% (138/238), 61% (108/177), 72% (118/165), and 70% (153/220) at 0, 48, 72, and 96 h of IVM, respectively. The percentage of sperm nuclear decondensation at each time point significantly increased up to 72 h of culture, showing 12, 34, 81, and 85% of sperm nuclei deconsated, respectively. The percentages of nuclear maturation also increased (P ≤ 0.05) with time, showing 0, 8, 20, and 27% of oocytes at second metaphase (MII) stage at 0, 48, 72, and 96 h of culture. The percentage of MII stage was much lower than that of chromatin decondensation in all maturing groups. These results suggest that canine oocytes matured in vitro are able to decondense the sperm chromatin during IVF, and this ability increases up to 72 h of culture. Nevertheless, cytoplasmic maturation, as evaluated by sperm chromatin decondensation, in canine oocytes matured in vitro may not be completely connected with nuclear development. This work was supported by Grant FONDECYT 1080618.

Immunofluorescent anti-tubulin staining of spindles during meiotic maturation of mouse oocytes in vitro

1978 ◽  
Vol 29 (1) ◽  
pp. 171-188
Author(s):  
P.M. Wassarman ◽  
K. Fujiwara
Keyword(s):  
Polar Body ◽  
Immunofluorescent Staining ◽  
Meiotic Maturation ◽  
Cytoplasmic Bridge ◽  
Specific Staining ◽  
Mouse Oocytes ◽  
Maturation In Vitro ◽  
Reproducible Manner ◽  
Metaphase I

Immunofluorescent anti-tubulin staining has been used to follow nuclear progression from dictyate to metaphase II during meiotic maturation of mouse oocytes in vitro. Antibody directed against tubulin isolated from sea-urchin eggs decorates the metaphase I and metaphase II spindles, as well as the cytoplasmic bridge, midbody, and polar body of the maturing mouse oocytes. Changes in the tubulin-specific staining pattern during meiotic maturation in vitro take place in a highly reproducible manner. Oocytes exposed continuously to cytochalasin B arrest at metaphase I and display a spindle which by immunofluorescent staining is virtually indistinguishable from the spindle of untreated oocytes.

scholarly journals FGF2/FGFR signaling promotes cumulus-oocyte complex maturation in vitro

Reproduction ◽  
2020 ◽  
Author(s):  
Chao Du ◽  
John S Davis ◽  
Chao Chen ◽  
Zan Li ◽  
Ye Cao ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Mapk Pathway ◽  
Expression Patterns ◽  
Cumulus Cells ◽  
Cumulus Expansion ◽  
Meiotic Progression ◽  
Oocyte Meiosis ◽  
Maturation In Vitro ◽  
Fgfr Signaling

Fibroblast growth factor 2 (FGF2), a member of FGF family, binds with FGF receptors (FGFR) to initiate biological functions in various somatic cells. However, little is known regarding the role of FGF2/FGFR on oocyte meiosis. In this study, we investigated expression patterns and functions of FGF2/FGFR during in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs). Among four FGFRs, Ffgr1 was the most abundant in COCs. The transcripts for Fgf2 and Ffgr1 in COCs increased during IVM. Ffgr1 was present in oocytes and cumulus cells, while Fgf2 was present in only cumulus cells. Treatment of COCs with the selective FGFR inhibitor SU5402 blocked oocyte meiotic progression and downregulated expression of Bmp15 and Gdf9. In contrast, supplement of FGF2 promoted oocyte meiotic progression and upregulated Bmp15 and Gdf9 expression. Inhibition of FGFR with SU5402 reduced cumulus expansion and expressions of Ptx3, Has2 and Tnfaip6. Treatment with FGF2 increased Ptx3 and Has2 expression. Inhibition of FGFR had no effect on meiotic progression of denuded oocytes (DOs). However, co-culture of DOs with COCs or supplementation with FGF2 promoted meiotic progression of DOs. Inhibition of FGF2/FGFR signaling also downregulated Ffgr1 expression, while supplemental FGF2 upregulated Fgfr1 expression. Furthermore, inhibition of FGFR in COCs interrupted the c-Mos/MAPK pathway and maturation-promoting factor (MPF), as indicated by downregulation of oocyte c-mos and Ccnb1 transcripts, respectively. Overall, this study suggests that FGF2 produced by cumulus cells, activates a FGF2/FGFR autocrine/paracrine loop within COCs to regulate cumulus expansion and oocyte meiosis. These findings reveal a novel role for FGF2/FGFR signaling during in vitro maturation of COCs.

scholarly journals Double fertilization in Helianthus

2015 ◽  
Vol 42 (2) ◽  
pp. 323-343 ◽  
Author(s):  
J. Telżyńska ◽  
H. Telżyński
Keyword(s):  
Cell Cycle ◽  
Pollen Tube ◽  
Helianthus Annuus ◽  
Morphological Changes ◽  
Sperm Nucleus ◽  
Double Fertilization ◽  
Average Cell ◽  
Controlled Pollination ◽  
Sperm Nuclei ◽  
Ribosome Formation

After controlled pollination of <i>Helianthus annuus</i> L. florets, the whole course of fertilization is described and documented on 24 microphotos. The timing of events is evaluated. The average cell cycle in the proembryo is 2 hours and the nuclear cycle in endosperm - 60 minutes.Plasmoptysis is suggested as the mechanism of pollen tube opening in the synergid. The structure of the thread-like sperm nucleus is interpreted as an end to end union of chromosomes, and the morphological changes of the sperm nuclei are explained as folding and coiling, based on a spiralization mechanism of chromosomes. Cytochemical observations indicating ribosome formation in the course of the nuclear cycles in the endosperm are described. The mechanisms accelerating nuclear cycles in the endosperm are discussed.

Activation of bovine oocytes penetrated after germinal vesicle breakdown

Zygote ◽  
1994 ◽  
Vol 2 (4) ◽  
pp. 273-279 ◽  
Author(s):  
Lalantha R. Abeydeera ◽  
Kiyoshi Okuda ◽  
Koji Niwa
Keyword(s):  
Culture Medium ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Nuclear Transformation ◽  
Metaphase Chromosomes ◽  
Germinal Vesicle Breakdown ◽  
Maturation Medium ◽  
Sperm Penetration ◽  
Sperm Nuclei ◽  
Bovine Oocytes

SummaryThe present study was designed to examine the ability of bovine oocytes, after germinal vesicle breakdown (GVBD), to be activated by sperm penetration and the sequence of sperm nuclear transformation. Bovine oovytes cultured for 8 h in maturation medium (tissue culture medium TCM-199 containing 10% fetal calf serum) were inseminated in Brackett and Oilphant's medium supplemented with bovine serum albumin (10 mg/ml), caffeine (5mM) and heparin (10 μg/ml). When oocytes were transferrred to the maturation medium 8 h after insemination and additionally cultured for 5−40 h at 39°C in 5% CO2 in air, 71−76% of oocytes were penetrated and polyspermy (67–75%) was common. The proportions of penetrated oocytes that were activated significantly increased with the lapse of the additional culture time, reaching 88% and 87% by 25 and 40 h after additional culture, respectively. When compared with unpenetrated oocytes, signifcantly higher proportions of penetrated oocytes, reached metaphase II or beyond 15 and 25 h after additional culture. After penetration, sperm nuclei were transformed into metaphase chromosomes and then to telophase chromomes before the formation of male pronuclei. These results provide evidence that bovine oocytes acquire the ability to respond to sperm-mediated activation soon after GVBD.

scholarly journals CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dalileh Nabi ◽  
Hauke Drechsler ◽  
Johannes Pschirer ◽  
Franz Korn ◽  
Nadine Schuler ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Polar Body ◽  
Spindle Assembly ◽  
V Protein ◽  
Oocyte Meiosis ◽  
Spindle Microtubules ◽  
Polar Body Extrusion ◽  
Metaphase I

AbstractProper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V−/− oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V−/− oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V−/− oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

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