Electrical maturation of the murine oocyte: an increase in calcium current coincides withacquisition of meiotic competence

Zygote ◽  
1993 ◽  
Vol 1 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Joan M. Murnane ◽  
Louis J. DeFelice
Keyword(s):  
Polar Body ◽  
Reversal Potential ◽  
Germinal Vesicle ◽  
Outward Current ◽  
Ca Channels ◽  
Oocyte Growth ◽  
Inward Current ◽  
First Polar Body ◽  
Meiotic Competence

SummaryWe have used the whole-cell recording technique to compare three stages of primary and secondary oocytes from F1 hybrid mice (C57BL/6J x SJL/J):neonatal germinal vesicle (NGV) stage primary oocytes from 10- to 20-day-old, prepubescent mice; mature germinal vesicle (MGV) stage primary oocytes from 12-week-old, post-pubescent, superovulated mice; first polar body (FPB) stage secondary oocytes from 12-week-old, post-pubescent mice during the normal oestrus cycle or following superovulation. NGV, MGV and FPB oocytes all exhibit two voltage-dependent currents: an inward, rapidly activating/inactivating current, and an outward, slowly activating/non-inactivating current. In 1.5 mmol/1 external Ca the average peak inward current is − 2.9, − 12.4 and − 13.8 μA/cm2 in NGV, MGV and FPB oocytes, respectively. In 20 mmol/1 Ca these currents increase and the reversal potential shifts to the right. The outward current decreases slightly with growth and development: at 40 mV test potentials, NGV oocytes have average outward currents of 8.9 μA/cm2, and MGV and FPB oocytes have currents of 5.0 and 5.5 μA/cm2, respectively. Thus, MGV oocytes express FPB current patterns. The reversal potentials, kinetics and pharmacology of the currents indicate that Ca channels carry the inward current and K channels carry the outward current. During growth in vivo a gradual depolarisation accompanies maturation. Resting potentialsranged from − 45 to − 30 mV in NGV oocytes to − 35 to − 17 mV in MGV oocytes to − 20 mV to − 3 mV in FPB oocytes. These data suggest that a selective increase occurs in the number of Ca channels during oocyte growth. This increase precedes nuclear maturation and coincides with the acquisition of meiotic competence.

Transcriptional activity associated with meiotic competence in fully grown mouse GV oocytes

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 327-332 ◽  
Author(s):  
Honglin Liu ◽  
Fugaku Aoki
Keyword(s):  
Transcriptional Activity ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Meiotic Maturation ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Active Transcription ◽  
Subsequent Activation ◽  
Meiotic Competence

The involvement of cumulus cells and chromatin organisation in transcriptional activity was investigated. In addition, the relationship between transcriptional activity and meiotic competence in fully grown mouse oocytes was surveyed. Transcriptional activity was detected in fully grown oocytes in which chromatin did not surround the nucleolus in the germinal vesicle (NSN-type oocytes), but not in oocytes in which chromatin surrounded the nucleolus (SN-type oocytes). Cumulus cells seemed to downregulate transcriptional activity in NSN-type oocytes, since transcriptional activity was 3 times greater in the denuded NSN-type oocytes free of cumulus cells (DO oocytes) than in NSN-type oocytes enclosed in cumulus cells (COC oocytes). Higher transcriptional activity corresponded to lower germinal vesicle breakdown (GVB) competence of fully grown oocytes in culture. Although GVB occurred in nearly all (99%) the SN-type oocytes, it occurred in 88% of COC/NSN-type oocytes (cumulus-oocyte complex with SN-type configuration) and in 61% of DO/NSN-type oocytes (denuded oocytes with NSN-type configuration). There was a negative correlation between transcriptional activity and the capacity of a cell to complete the progression to the second metaphase (MII). In GVB oocytes, the percentage of first polar body (PBI) extrusion differed among COC/NSN-type (81%), DO/SN-type (66%), COC/NSN-type (47%) and DO/NSN-type (29%) oocytes. After activation with 10 mM Sr2+, the frequency of parthenogenetic activation was greater in SN-type oocytes (46.9%) than in transcriptionally active NSN-type oocytes (27.5%). These results suggest that transcriptional activity has a detrimental effect on the competence of meiotic maturation and subsequent activation in fully grown GV oocytes. Alternatively, active transcription in the fully grown oocytes suggests that they are still in the process of synthesising substances required for meiotic maturation and are not yet competent for these processes.

Meiotic maturation of bovine oocytes in vitro: improvement of meiotic competence by dibutyryl cyclic adenosine 3′,5′-monophosphate.

1990 ◽  
Vol 68 (4) ◽  
pp. 1182-1187 ◽  
Author(s):  
E. Sato ◽  
M. Matsuo ◽  
H. Miyamoto
Keyword(s):  
Polar Body ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Razor Blade ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Polar Body Formation ◽  
Bovine Oocytes ◽  
Meiotic Competence

Abstract The present study was undertaken to determine the precise stage of growth at which the ability to resume meiosis is acquired in bovine oocytes. Oocytes of various sizes were isolated from ovaries by mechanical dissection using an 18-gauge needle followed by a razor blade. This method yielded an average of 26.2 ± 7.4 growing and fully grown oocytes from an ovary. Cumulus-enclosed oocytes were cultured in vitro in tissue culture medium 199 containing 10% fetal calf serum. Oocytes ≤ 90 µm in diameter did not resume meiosis. However, germinal vesicle breakdown was observed in oocytes whose diameters exceeded 91 µm. Polar body formation was observed in oocytes with diameters exceeding 101 µm. About 80% of the oocytes with diameters ≥ 121 µm were able to extrude the polar body. The percentage of large oocytes (101 to 120 µm) with first polar body increased when incubated in medium containing dibutyryl cyclic adenosine 3′,5′-monophosphate; however, oocytes 90 to 101 µm did not extrude the first polar body even when cultured in a medium containing dibutyryl cyclic adenosine 3′,5′-monophosphate. These observations indicate that the capability to resume meiosis is acquired gradually during development of oocytes and that dibutyryl cyclic adenosine 3′,5′-monophosphate can improve the meiotic competence of bovine oocytes in culture.

Effect of oestrogen on mouse follicle growth and meiotic resumption

Zygote ◽  
2021 ◽  
pp. 1-8
Author(s):  
Heng Chi ◽  
Zuowu Cao
Keyword(s):  
Polar Body ◽  
Follicular Development ◽  
Germinal Vesicle ◽  
Membrane Receptor ◽  
Follicle Growth ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Two Phases

Summary Many studies have shown that oestrogen affects late follicular development, but whether oestrogen is involved in other aspects of folliculogenesis remains unclear. In this study, two antagonists of oestrogen, tamoxifen and G15, were used to determine the effects of oestrogen on folliculogenesis. Mouse preantral follicles and cumulus–oocyte complexes (COCs) were cultured in vitro. The results showed that follicle growth stimulated using pregnant mare serum gonadotrophin (PMSG) was inhibited using tamoxifen, whether in vivo or in vitro. The average diameters, the maximum diameters of follicles and the numbers of follicles with a diameter of more than 300 μm decreased significantly following a 4-day culture with tamoxifen. G15, the antagonist of oestrogen via the membrane receptor, did not change follicular growth stimulated by PMSG in vitro. Results of in vitro maturation of COCs showed that germinal vesicle breakdown (GVBD) occurred spontaneously (95.1%) after 2 h in culture, and the GVBD ratio changed little with the addition of either oestrogen or 10 μM G15. However, first polar body (PBI) extrusion was driven by oestrogen markedly and supplementation with 10 μM G15 inhibited PBI extrusion (82.4% vs 55.0%) significantly. These results demonstrated that oestrogen promotes follicle growth through the nuclear receptor during follicle growth and then triggers the transition of metaphase to anaphase through the membrane receptor during meiotic resumption. So oestrogen plays a progressive role in the two phases of follicle growth and oocyte meiotic resumption.

Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

2014 ◽  
Vol 26 (8) ◽  
pp. 1084 ◽  
Author(s):  
Yu-Ting Shen ◽  
Yue-Qiang Song ◽  
Xiao-Qin He ◽  
Fei Zhang ◽  
Xin Huang ◽  
...  
Keyword(s):  
Polar Body ◽  
Meiotic Maturation ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes ◽  
First Polar Body ◽  
Triphenyltin Chloride ◽  
Dose Dependent

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

Whole cell current analyses of pancreatic acinar AR42J cells. I. Voltage- and Ca(2+)-activated currents

1991 ◽  
Vol 260 (5) ◽  
pp. C934-C948 ◽  
Author(s):  
K. Kusano ◽  
H. Gainer
Keyword(s):  
Reversal Potential ◽  
Outward Current ◽  
Pancreatic Acinar Cells ◽  
Equilibrium Potential ◽  
Channel Blockers ◽  
Whole Cell ◽  
Inward Current ◽  
Tail Current ◽  
Ar42j Cells ◽  
Conductance Increase

Voltage- and Ca(2+)-activated whole cell currents were studied in AR42J cells, a clonal cell line derived from rat pancreatic acinar cells, using a patch electrode voltage-clamp technique. Four kinds of ionic currents were identified by their ionic dependencies, pharmacological properties, and kinetic parameters: 1) an outward current flow due mainly to a voltage-dependent K(+)-conductance increase, 2) an initial transient inward current due to an Na(+)-conductance increase, 3) transient and long-duration inward current due to a Ca(2+)-conductance increase, and 4) a slowly activating inward current that persists over the duration of the depolarizing pulse and deactivates slowly upon repolarization, producing a slow inward tail current. The slow inward tail current was particularly robust and was interpreted as due to a Ca(2+)-activated Cl(-)-conductance increase, since 1) the generation of this current was blocked by removing the extracellular Ca2+, applying Ca(2+)-channel blockers (Cd2+, nifedipine), or by lowering the intracellular Ca2+ concentration [( Ca2+]i) with EGTA; and 2) the reversal potential (Erev) of the slow inward tail current was close to 0 mV in the control condition (152 mM [Cl-]o/154 mM [Cl-]i), and changes of the [Cl-]o/[Cl )i ratio shifted the Erev toward the predicted Cl- equilibrium potential.

Effects of norepinephrine upon superficial layer neurons in the superior colliculus of the hamster: In vitro studies

1999 ◽  
Vol 16 (3) ◽  
pp. 557-570 ◽  
Author(s):  
HONGJING TAN ◽  
RICHARD D. MOONEY ◽  
ROBERT W. RHOADES
Keyword(s):  
Superior Colliculus ◽  
Optic Tract ◽  
Reversal Potential ◽  
Outward Current ◽  
Input Resistance ◽  
Superficial Layer ◽  
Membrane Properties ◽  
Induced Response

Intracellular recording techniques were used to evaluate the effects of norepinephrine (NE) on the membrane properties of superficial layer (stratum griseum superficiale and stratum opticum) superior colliculus (SC) cells. Of the 207 cells tested, 44.4% (N = 92) were hyperpolarized by ≥3 mV and 8.7% (N = 18) were depolarized by ≥3 mV by application of NE. Hyperpolarization induced by NE was dose dependent (EC50 = 8.1 μM) and was associated with decreased input resistance and outward current which had a reversal potential of −94.0 mV. Depolarization was associated with a very slight rise in input resistance and had a reversal potential of −93.1 mV for the single cell tested. Pharmacologic experiments demonstrated that isoproterenol, dobutamine, and p-aminoclonidine all hyperpolarized SC cells. These results are consistent with the conclusion that NE-induced hyperpolarization of SC cells is mediated by both α2 and β1 adrenoceptors. The α1 adrenoceptor agonists, methoxamine and phenylephrine, depolarized 35% (6 of 17) of the SC cells tested by ≥3 mV. Most of the SC cells tested exhibited responses indicative of expression of more than one adrenoceptor. Application of p-aminoclonidine or dobutamine inhibited transsynaptic responses in SC cells evoked by electrical stimulation of optic tract axons. Inhibition of evoked responses by these agents was usually, but not invariably, associated with a hyperpolarization of the cell membrane and a reduction in depolarizing potentials evoked by application of glutamate. The present in vitro results are consistent with those of the companion in vivo study which suggested that NE-induced response suppression in superficial layer SC neurons was primarily postsynaptic and chiefly mediated by both α2 and β1 adrenoceptors.

Dendritic arbor of locus coeruleus neurons contributes to opioid inhibition

1996 ◽  
Vol 75 (5) ◽  
pp. 2029-2035 ◽  
Author(s):  
R. A. Travagli ◽  
M. Wessendorf ◽  
J. T. Williams
Keyword(s):  
Locus Coeruleus ◽  
Horizontal Plane ◽  
Reversal Potential ◽  
Outward Current ◽  
Membrane Properties ◽  
Equilibrium Potential ◽  
Intrinsic Membrane Properties ◽  
In Cells

1. The nucleus locus coeruleus (LC) is made up of noradrenergic cells all of which are hyperpolarized by opioids. Recent work has shown that the reversal potential of the opioid-induced current is more negative than the potassium equilibrium potential. The aim of the present study was to determine whether the extent of the dendritic field could contribute to the very negative opioid reversal potential. 2. Individual LC cells were labeled in the brain slice preparation. The number of dendrites found on cells in slices sectioned in the horizontal plane was greater than cells in coronal slices. However, the dimensions of the cell body slices from each plane were not significantly different. 3. The resting conductance of neurons from slices cut in the horizontal plane was significantly larger than in cells from coronal plane. 4. The amplitude of the outward current induced by [Met5]-enkephalin (ME) was larger in cells from horizontal slices and the reversal potential was more negative than that of cells in coronal slices. 5. The results show that the plane of section influences the membrane properties and opioid actions of LC neurons in vitro and suggest that these differences correlate with the numbers of dendrites. The results suggest that in vivo, in addition to intrinsic membrane properties and synaptic inputs, the structural makeup of the nucleus is an important factor in determining the activity.

JNK2 Participates in Spindle Assembly during Mouse Oocyte Meiotic Maturation

2011 ◽  
Vol 17 (2) ◽  
pp. 197-205 ◽  
Author(s):  
Xin Huang ◽  
Jing-Shan Tong ◽  
Zhen-Bo Wang ◽  
Cai-Rong Yang ◽  
Shu-Tao Qi ◽  
...  
Keyword(s):  
Polar Body ◽  
Specific Inhibitor ◽  
Germinal Vesicle ◽  
Spindle Assembly ◽  
Mouse Oocyte ◽  
Meiotic Maturation ◽  
Cytoplasmic Microtubule ◽  
Spindle Poles ◽  
First Polar Body ◽  
Oocyte Meiotic Maturation

AbstractIt is well known that c-Jun N-terminal kinase (JNK) plays pivotal roles in various mitotic events, but its function in mammalian oocyte meiosis remains unknown. In this study, we found that no specific JNK2 signal was detected in germinal vesicle stage. JNK2 was associated with the spindles especially the spindle poles and cytoplasmic microtubule organizing centers at prometaphase I, metaphase I, and metaphase II stages. JNK2 became diffusely distributed and associated with the midbody at telophase I stage. Injection of myc-tagged JNK2α1 mRNA into oocytes also revealed its localization on spindle poles. The association of JNK2 with spindle poles was further confirmed by colocalization with the centrosomal proteins, γ-tubulin and Plk1. Nocodazole treatment showed that JNK2 may interact with Plk1 to regulate the spindle assembly. Then we investigated the possible function of JNK2 by JNK2 antibody microinjection and JNK specific inhibitor SP600125 treatment. These two manipulations caused abnormal spindle formation and decreased the rate of first polar body (PB1) extrusion. In addition, inhibition of JNK2 resulted in impaired localization of Plk1. Taken together, our results suggest that JNK2 plays an important role in spindle assembly and PB1 extrusion during mouse oocyte meiotic maturation.

Involvement of GABAA receptor in Bufo arenarum oocyte maturation

Zygote ◽  
2008 ◽  
Vol 16 (2) ◽  
pp. 135-144
Author(s):  
G. Sánchez Toranzo ◽  
L. Zelarayán ◽  
F. Bonilla ◽  
J. Oterino ◽  
M.I. Bühler
Keyword(s):  
Receptor Agonist ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Dependent Manner ◽  
Germinal Vesicle Breakdown ◽  
First Polar Body ◽  
Bufo Arenarum ◽  
Turn Over ◽  
Pkc Activation

SummaryAmphibian oocytes meiotic arrest is released under the stimulus of progesterone; this hormone interacts with the oocyte surface and starts a cascade of events leading to the activation of a cytoplasmic maturation promoting factor (MPF) that induces germinal vesicle breakdown (GVBD), chromosome condensation and extrusion of the first polar body.The aim of this work was to determine whether the activation of a GABAA receptor is able to induce GVBD in fully grown denuded oocytes of Bufo arenarum and to analyse its possible participation in progesterone-induced maturation. We also evaluated the role of purines and phospholipids in the maturation process induced by a GABAA receptor agonist such as muscimol.Our results indicated that the activation of the GABAA receptor by muscimol induces maturation in a dose- and time-dependent manner and that this activation is a genuine maturation that enables oocytes to form pronuclei. Assays with a receptor antagonist, picrotoxine, showed that the maturation induced by muscimol was inhibited. Treatment with picrotoxine, however, shows that the participation of GABAA receptor in progesterone-induced maturation is not significant.In addition, our results indicate that high intracellular levels of purines obtained by the use of db-AMPc and theophylline or the inhibition of the phosphatidylinositol 4,5-bisphosphate (PIP2 hydrolysis by neomycin and PIP2 turn over by LiCl, respectively, inhibited the maturation induced by muscimol. Treatment with H-7 indicated, however, that PKC activation is not necessary for GVBD induced by the GABAA receptor agonist. Results suggest that the transduction pathway used by the GABAA receptor to induce maturation is different from those used by progesterone.

74 IN VIVO SURVIVAL OF DOMESTIC CAT OOCYTES AFTER VITRIFICATION, INTRACYTOPLASMIC SPERM INJECTION, AND TRANSFER TO RECIPIENTS

2008 ◽  
Vol 20 (1) ◽  
pp. 118 ◽  
Author(s):  
M. C. Gómez ◽  
N. Kagawa ◽  
C. E. Pope ◽  
M. Kuwayama ◽  
S. P. Leibo ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Oocyte Retrieval ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
Minimal Amount ◽  
Vitro Fertilization ◽  
First Polar Body

The ability to cryopreserve female gametes efficiently holds immense economic and genetic implications. The purpose of the present project was to determine if domestic cat oocytes could be cryopreserved successfully by use of the Cryotop method. We evaluated (a) cleavage frequency after in vitro fertilization (IVF) v. intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification, and (b) fetal development after transfer of resultant embryos into recipients. In vivo-matured cumulus–oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment, denuded of cumulus cells, and examined for the presence of the first polar body (PB). In vitro-matured COCs were obtained from ovaries donated by local clinics and placed into maturation medium for 24 h before cumulus cells were removed and PB status was determined. Oocytes were cryopreserved by the Cryotop method (Kuwayama et al. 2005 Reprod. Biomed. Online 11, 608–614) in a vitrification solution consisting of 15% DMSO, 15% ethylene glycol, and 18% sucrose. For IVF, oocytes were co-incubated with 1 � 106 motile spermatozoa mL–1 in droplets of modified Tyrode's medium in 5% CO2/air at 38�C (Pope et al. 2006 Theriogenology 66, 59–71). For ICSI, an immobilized spermatozoon was loaded into the injection pipette, which was then pushed through the zona pellucida into the ooplasm. After a minimal amount of ooplasm was aspirated into the pipette, the spermatozoon was carefully expelled, along with the aspirated ooplasm. After ICSI, or at 5 or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006). As assessed by normal morphological appearance after liquefaction, the survival rate of both in vivo- and in vitro-matured oocytes was >90% (93–97%). For in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 73% (16/22) and 53% (30/57), respectively, as compared to 68% (19/28) after ICSI of vitrified oocytes (P > 0.05). For in vivo-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes were 55% (18/33) and 35% (6/17), respectively, compared to 50% (10/20) after ICSI of vitrified oocytes (P > 0.05). At 18–20 h after ICSI, 18 presumptive zygotes and four 2-cell embryos derived from vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo-matured and 12 in vitro-matured vitrified oocytes were transferred by laparoscopy into the oviducts of two recipients at 24–26 h after oocyte retrieval. The two recipients were 9-month-old IVF/ET-derived females produced with X-sperm sorted by flow cytometry. At ultrasonography on Day 22, both recipients were pregnant, with three live fetuses observed in one recipient and one live fetus seen in the second recipient. On Day 63 and Day 66 of gestation, four live kittens were born, without assistance, to the two recipients. The one male and three female kittens weighed an average of 131 g. In summary, in vivo viability of zygotes/embryos produced by ICSI of cat oocytes vitrified by the Cryotop method was demonstrated by the birth of live kittens following transfer to recipients.

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