Effect of oestrogen on mouse follicle growth and meiotic resumption

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

Reproduction Fertility and Development ◽

10.1071/rd12332 ◽

2014 ◽

Vol 26 (8) ◽

pp. 1084 ◽

Cited By ~ 2

Author(s):

Yu-Ting Shen ◽

Yue-Qiang Song ◽

Xiao-Qin He ◽

Fei Zhang ◽

Xin Huang ◽

...

Keyword(s):

Polar Body ◽

Meiotic Maturation ◽

Dependent Manner ◽

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.

359 ICSI AND ACTIVATION OF OOCYTES RECOVERED FROM TRANSITIONAL AND CYCLING MARES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab359 ◽

2006 ◽

Vol 18 (2) ◽

pp. 286 ◽

Cited By ~ 3

Author(s):

T. Suh ◽

S. Purcell ◽

G. Seidel Jr

Keyword(s):

Growth Factor ◽

Polar Body ◽

Follicular Development ◽

Transitional Period ◽

Developmental Competence ◽

Blastocyst Development ◽

Cleavage Rate ◽

Ovarian follicular development in mares during the transitional period before the breeding season leads to an accumulation of antral follicles of various sizes. The quality of oocytes at this stage may be compromized until the first seasonal ovulation. In this study, we evaluated the developmental competence of oocytes recovered from transitional and cyclic mares, and the effect of zygote activation after intracytoplasmic sperm injection (ICSI). A 2 × 2 × 2 factorial experiment consisting of oocytes from transitional and cyclic mares, two follicle sizes (10 to 20 and 20+ mm), and two treatments (control and activated) was conducted. Follicular oocytes of 14 mares were aspirated in March and April (transitional) and May to July (cyclic) five times per each period at 10-day intervals, without use of hCG. Oocytes aspirated from mares were matured in vitro in a defined medium similar to SOF plus FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF), estradiol (E2), prostaglandin (P4) and 10% FCS, for 30 ± 1 h under 5% CO2 in air at 38.5°C; oocytes with a first polar body were used for ICSI. Motile sperm from frozen-thawed semen were used for sperm injection with a piezo-driven pipet. For activation after ICSI, presumptive zygotes were cultured in G1.3 containing 0.02 µM phorbol 12-myristate 13-acetate (PMA) for 2 h, and then in 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h under 6% CO2 in air at 38.5°C. Zygotes were cultured in 50 µL drops of DMEM/F12 containing 10% FCS for 9 days at 38.5°C in 5% CO2/5% O2/90% N2. Medium was replaced every 3 days. Cleavage and blastocyst rates were calculated based on non-degenerating injected oocytes. Data were analyzed by Fisher's exact test. A total of 115 and 78 oocytes were recovered from cyclic and transitional mares. Average maturation rates to MII in the respective groups were 76.5 and 65.4%, respectively (P < 0.07), and those of 10 to 20 and 20+ mm follicle groups were 70.6 and 80.0%, respectively (P > 0.05). The average cleavage rate in cyclic mares was higher than in transitional mares, and that of the activated group averaged over follicle sizes was higher than that of controls (P < 0.05; Table 1); those of 10 to 20 and 20+ mm follicle groups were not different (P < 0.05; Table 1). Blastocyst rates per oocyte within main effects were not different (P < 0.05; Table 1). Oocytes from transitional mares had lower cleavage rates than those of cyclic mares, but blastocyst development was similar. Activation of zygotes clearly improved cleavage rates of in vivo-derived immature equine oocytes after ICSI. Table 1. Main effect means of responses after ICSI

Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation

Reproduction ◽

10.1530/rep-09-0136 ◽

2009 ◽

Vol 138 (2) ◽

pp. 235-246 ◽

Cited By ~ 25

Author(s):

Svetlana Uzbekova ◽

Mohamad Salhab ◽

Christine Perreau ◽

Pascal Mermillod ◽

Joëlle Dupont

Keyword(s):

Granulosa Cells ◽

Map Kinases ◽

Glycogen Synthase ◽

Polar Body ◽

Germinal Vesicle ◽

Glycogen Synthase Kinase ◽

Aurora A Kinase ◽

Glycogen synthase kinase 3 (GSK3) regulates cellular metabolism and cell cycle via different signalling pathways. In response to insulin and growth factors GSK3 is serine-phosphorylated and inactivated. We analysed GSK3B expression and activation in bovine cumulus cells (CC) and oocytes at different meiotic stagesin vitroin parallel with MAP kinases ERK (MAPK3/MAPK1) and p38 (MAPK14). GSK3B localised to cytoplasm in granulosa cells and in oocytes throughout folliculogenesis. In mature metaphase-II (MII) oocytes, GSK3B was concentrated to the region of midzone between the oocyte and the first polar body, as well as active phospho-Thr Aurora A kinase (AURKA). Duringin vitromaturation (IVM), in oocytes, phospho-Ser9-GSK3B level increased as well as phospho-MAPK3/MAPK1, while phospho-MAPK14 decreased. In CC, phospho-MAPK14 increased upon germinal vesicle breakdown (GVBD)/metaphase-I (MI) and then decreased during transition to MII. Administration of inhibitors of GSK3 activity (lithium chloride or 2′Z,3′E -6-bromoindirubin-3′-oxime) rapidly increased phospho-Ser9-GSK3B, and led to transient decrease of phospho-MAPK3/MAPK1 and to durable enhancing of phospho-MAPK14 in granulosa primary cell culture. GSK3 inhibitors during IVM diminished cumulus expansion and delayed meiotic progression. In cumulus, phospho-MAPK14 level was significantly higher in the presence of inhibitors, comparing with control, through the time of MI/MII transition. In oocytes, phospho-GSK3B was increased and phospho-MAPK3/MAPK1 was decreased before GVBD and oocytes were mainly arrested at MI. Therefore, GSK3B might regulate oocyte meiosis, notably MI/MII transition being the part of MAPK3/1 and MAPK14 pathways in oocytes and CC. GSK3B might be also involved in the local activation of AURKA that controls this transition.

Thioglycolic acid inhibits mouse oocyte maturation and affects chromosomal arrangement and spindle configuration

Toxicology and Industrial Health ◽

10.1177/0748233708095862 ◽

2008 ◽

Vol 24 (4) ◽

pp. 227-234 ◽

Cited By ~ 8

Author(s):

SY Hou ◽

L Zhang ◽

K Wu ◽

L Xia

Keyword(s):

Thioglycolic Acid ◽

Polar Body ◽

Germinal Vesicle ◽

Chromosomal Arrangement ◽

First Polar Body ◽

Inverted Microscope ◽

Polar Body Formation ◽

Spindle Elongation

Previous studies have shown that thioglycolic acid (TGA) leads to potential reproductive toxicology. To clarify the exact effects of this compound on reproduction, mice oocytes were treated with different TGA doses. At the end of the culture period, the nuclear status of mice oocytes was assessed under an inverted microscope. After immunofluorescence staining, the chromosomal arrangement and spindle configuration of oocytes were evaluated. The results indicated that TGA decreases the percentage of first polar body formation but does not influence that of germinal vesicle breakdown. TGA induces abnormal chromosomal arrangement and spindle elongation. In conclusion, TGA inhibits in-vitro maturation of mice oocytes and affects chromosomal arrangement and spindle configuration. Furthermore, it probably interferes with biochemical changes that occur during meiosis, resulting in aberrant development.

NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.704341 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yuting Xiang ◽

Chuanchuan Zhou ◽

Yanyan Zeng ◽

Qi Guo ◽

Jiana Huang ◽

...

Keyword(s):

Oocyte Maturation ◽

High Throughput Sequencing ◽

Epigenetic Modification ◽

Binding Protein ◽

Polar Body ◽

Germinal Vesicle ◽

Translation Efficiency ◽

N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.

Ultrastructure of Baboon Zygotes Produced By Microinjection of Spermatozoa

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100119946 ◽

1985 ◽

Vol 43 ◽

pp. 650-651

Author(s):

T. Caceci ◽

A.A. Shaikh ◽

D.C. Kraemer

Keyword(s):

Polar Body ◽

Follicular Development ◽

Male And Female ◽

Menstrual Cycles ◽

Follicular Aspiration ◽

First Polar Body ◽

Preovulatory Follicles ◽

Lh Release

Five baboons were treated during seven menstrual cycles with 5.0 mg of FSH-P for five days, starting on either day 3 or day 5 of the cycle. On day 5 of the treatment, the ovaries were examined by laparoscopy to evaluate follicular development. All animals exhibited multiple preovulatory follicles and at that time 100 mg GnRH was administered intramuscularly to induce LH release. Between 24 and 30 hours after injection of GnRH, laparoscopic follicular aspiration was used to collect oocytes. These were matured in vitro (determined by extrusion of the first polar body) and fertilized by microinjection with frozen-thawed baboon spermatozoa. Male and female pronuclei were observed in 32% of the resulting zygotes within 24 hours. These zygotes were compared to a zygote in the same stage that had been fertilized in vivo and obtained by laparoscopy and flushing of the oviduct.

Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression

Journal of Cell Science ◽

10.1242/jcs.22.3.531 ◽

1976 ◽

Vol 22 (3) ◽

pp. 531-545

Author(s):

P.M. Wassarman ◽

W.J. Josefowicz ◽

G.E. Letourneau

Keyword(s):

Cyclic Amp ◽

Cytochalasin B ◽

Polar Body ◽

Germinal Vesicle ◽

Meiotic Maturation ◽

Dibutyryl Cyclic Amp ◽

Mouse Oocytes ◽

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.

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

Journal of Animal Science ◽

10.2527/1990.6841182x ◽

1990 ◽

Vol 68 (4) ◽

pp. 1182-1187 ◽

Cited By ~ 25

Author(s):

E. Sato ◽

M. Matsuo ◽

H. Miyamoto

Keyword(s):

Polar Body ◽

Calf Serum ◽

Germinal Vesicle ◽

Razor Blade ◽

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.

Heparin effect on in vitro nuclear maturation of bovine oocytes

Zygote ◽

10.1017/s0967199407004418 ◽

2008 ◽

Vol 16 (1) ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Juan Carlos Flores-Alonso ◽

Leticia Lezama-Monfil ◽

María Luisa Sánchez-Vázquez ◽

Rosalina Reyes ◽

Néstor M. Delgado

Keyword(s):

Oocyte Maturation ◽

Morphological Changes ◽

Polar Body ◽

Germinal Vesicle ◽

Salt Medium ◽

Maturation Factor ◽

First Polar Body ◽

Bovine Oocytes

SummaryOocytes undergo numerous biochemical and morphological changes during their development from preantral to preovulatory phases. In vitro studies have suggested several compounds that might induce oocyte maturation. Heparin is a natural component of ooplasm, follicular fluid and uterine fluid and previous studies indicated that it might act as a chromatin maturation factor in bovine oocytes. We tested this hypothesis in vitro by timing germinal vesicle breakdown (GVBD) and first polar body (PB) formation without any other natural or introduced factors that might influence the rate of oocyte maturation. We also determined if these oocytes could be fertilized.Bovine oocytes were incubated in a salt medium and TCM 199 supplemented with different concentrations of heparin for 24 h at 37.5 °C in a humidified atmosphere of 5% CO2. With 1.0 and 6.5 mg/ml heparin, the time of GVBD was reduced from 4.7 ± 1.1 h to about 1.5 h and the time of first PB formation was reduced from 22.0 ± 1.1 h to 9.0–11.0 h in salt medium. In TCM 199, only 6.5 mg/ml heparin significantly reduced the time of PB formation. In both incubation media, 1.0 and 6.5 mg/ml heparin induced GVBD, extrusion of the first PB and formation of the metaphase II nucleus. Moreover, heparin did not interfere with the fertilization of oocytes matured in TCM 199. Based on the results, we propose that heparin plays an important role in the rearrangement of the oocyte chromatin and acts as an oocyte maturation factor.

Melatonin Alleviates the Suppressive Effect of Hypoxanthine on Oocyte Nuclear Maturation and Restores Meiosis via the Melatonin Receptor 1 (MT1)-Mediated Pathway

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.648148 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jing Wang ◽

Zhiyong Zhuo ◽

Xiao Ma ◽

Yunjie Liu ◽

Jing Xu ◽

...

Keyword(s):

Cholesterol Biosynthesis ◽

Polar Body ◽

Membrane Receptor ◽

Intracellular Camp ◽

Mrna Levels ◽

Nuclear Maturation ◽

Oocyte Meiosis ◽

It is well known that hypoxanthine (HX) inhibits nuclear maturation of oocytes by elevating the intracellular cAMP level, while melatonin (MT) is a molecule that reduces cAMP production, which may physiologically antagonize this inhibition and restore the meiosis process. We conducted in vitro and in vivo studies to examine this hypothesis. The results showed that 10–3 M MT potentiated the inhibitory effect of HX on mouse oocyte meiosis by lowering the rate of germinal vesicle breakdown (GVBD) and the first polar body (PB1). However, 10–5 M and 10–7 M MT significantly alleviated the nuclear suppression induced by HX and restored meiosis in 3- and 6-week-old mouse oocytes, respectively. We identified that the rate-limiting melatonin synthetic enzyme AANAT and melatonin membrane receptor MT1 were both expressed in oocytes and cumulus cells at the GV and MII stages. Luzindole, a non-selective melatonin membrane receptor antagonist, blocked the activity of MT on oocyte meiotic recovery (P < 0.05). This observation indicated that the activity of melatonin was mediated by the MT1 receptor. To understand the molecular mechanism further, MT1 knockout (KO) mice were constructed. In this MT1 KO animal model, the PB1 rate was significantly reduced with the excessive expression of cAPM synthases (Adcy2, Adcy6, Adcy7, and Adcy9) in the ovaries of these animals. The mRNA levels of Nppc and Npr2 were upregulated while the genes related to progesterone synthesis (Cyp11a11), cholesterol biosynthesis (Insig1), and feedback (Lhcgr, Prlr, and Atg7) were downregulated in the granulosa cells of MT1 KO mice (P < 0.05). The altered gene expression may be attributed to the suppression of oocyte maturation. In summary, melatonin protects against nuclear inhibition caused by HX and restores oocyte meiosis via MT1 by reducing the intracellular concentration of cAMP.