scholarly journals Distribution of cortical granules and meiotic maturation of canine oocytes in bi-phasic systems

2015 ◽  
Vol 27 (7) ◽  
pp. 1082 ◽  
Author(s):  
Maricy Apparicio ◽  
Giuliano Q. Mostachio ◽  
Tathiana F. Motheo ◽  
Aracelle E. Alves ◽  
Luciana Padilha ◽  
...  

The aim of this study was to evaluate the influence of different bi-phasic systems with gonadotrophins and steroids on in vitro maturation rates of oocytes obtained from bitches at different reproductive stages (follicular, luteal, anoestrous). In System A (control) oocytes were matured for 72 h in base medium (BM) with 10 IU mL–1 human chorionic gonadotrophin (hCG), 1 μg mL–1 progesterone (P4) and 1 μg mL–1 oestradiol (E2); in bi-phasic System B oocytes were matured for 48 h in BM with hCG and for 24 h in BM with P4; in bi-phasic System C oocytes were matured for 48 h in BM with hCG, P4 and E2, and for 24 h in BM with P4; in System D, oocytes were cultured in BM without hormonal supplementation. Data were analysed by ANOVA. There was a positive effect of the bi-phasic systems on germinal vesicle breakdown, metaphase I and metaphase II rates, irrespective of reproductive status (P < 0.05). Bi-phasic systems were also beneficial for cortical granule distribution (an indication of cytoplasmic maturation) and its relationship to nuclear status: 74.5% of the oocytes cultured in System B and 85.4% of those cultured in System C presented both nuclear and cytoplasmic maturation (P < 0.001). The stage of the oestrous cycle did not influence maturation rates.

2010 ◽  
Vol 22 (1) ◽  
pp. 324 ◽  
Author(s):  
M. De los Reyes ◽  
D. Luna ◽  
J. Palomino

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 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 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma. Supported by Grant FONDECYT 1080618.


Zygote ◽  
1996 ◽  
Vol 4 (2) ◽  
pp. 145-149 ◽  
Author(s):  
Nam-Hyung Kim ◽  
Billy N. Day ◽  
Hoon Taek Lee ◽  
Kil-Saeng Chung

SummaryIn this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation and in vitro fertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.


Animals ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 163
Author(s):  
Payungsuk Intawicha ◽  
Li-Kuang Tsai ◽  
Shih-Ying Yen ◽  
Neng-Wen Lo ◽  
Jyh-Cherng Ju

The mitogen-activated kinase (MAPK) p38, a member of the MAPK subfamily, is conserved in all mammalian cells and plays important roles in response to various physiologic cues, including mitogens and heat shock. In the present study, MAPK p38 protein expression in porcine oocytes was analyzed during in vitro maturation (IVM) by Western blotting and immunocytochemistry. The levels of p-p38 or activated p38 and p38 expression were at the lowest in the germinal vesicle (GV) stage oocyte, gradually rising at the germinal vesicle breakdown (GVBD) and then reaching a plateau throughout the IVM culture (p < 0.05). Similarly, the expression level of total p38 was also lower in the GV oocyte than in the oocyte of other meiotic stages and uprising after GVBD and remained high until the metaphase III (MII) stage (p < 0.05). In the GV stage, phosphorylated p38 (p-p38) was initially detectable in the ooplasm and subsequently became clear around the nucleus and localized in the ooplasm at GVBD (18 h post-culture). During the metaphase I (MI) and metaphase II (MII) stages, p-p38 was evenly distributed throughout the ooplasm after IVM for 30 or 42 h. We found that the subcellular localization increased in p-p38 expression throughout oocyte maturation (p < 0.05) and that dynamic reorganization of the cytoskeleton, including microfilaments and microtubules, was progressively changed during the course of meiotic maturation which was likely to be associated with the activation or networking of p38 with other proteins in supporting oocyte development. In conclusion, the alteration of p38 activation is essential for the regulation of porcine oocyte maturation, accompanied by the progressive reorganization and redistribution of the cytoskeleton and MAPK p38, respectively, in the ooplasm.


Development ◽  
1997 ◽  
Vol 124 (9) ◽  
pp. 1845-1850
Author(s):  
L.K. Berg ◽  
G.M. Wessel

Cortical granules are secretory vesicles poised at the cortex of an egg that, upon stimulation by sperm contact at fertilization, secrete their contents. These contents modify the extracellular environment and block additional sperm from reaching the egg. The role of cortical granules in blocking polyspermy is conserved throughout much of phylogeny. In the sea urchin, cortical granules accumulate throughout the cytoplasm during oogenesis, but in mature eggs the cortical granules are attached to the plasma membrane, having translocated to the cortex at some earlier time. To study the process of cortical granule translocation to the cell surface we have devised a procedure for maturation of sea urchin oocytes in vitro. Using this procedure, we examined the rate of oocyte maturation by observing the movement and breakdown of the germinal vesicle, the formation of polar bodies and the formation of the egg pronucleus. We find that oocyte maturation takes approximately 9 hours in the species used here (Lytechinus variegatus), from the earliest indication of maturation (germinal vesicle movement) to formation of a distinct pronucleus. We then observed the translocation of cortical granules in these cells by immunolocalization using a monoclonal antibody to hyalin, a protein packaged specifically in cortical granules. We found that the translocation of cortical granules in in vitro-matured oocytes begins with the movement of the germinal vesicle to the oocyte cell surface, and is 50% complete 1 hour after germinal vesicle breakdown. In the in vitro-matured egg, 99% of the cortical granules are at the cortex, indistinguishable from translocation in oocytes that mature in vivo. We have also found that eggs that mature in vitro are functionally identical to eggs that mature in vivo by four criteria. (1) The matured cells undergo a selective turnover of mRNA encoding cortical granule contents. (2) The newly formed pronucleus begins transcription of histone messages. (3) Cortical granules that translocate in vitro are capable of exocytosis upon activation by the calcium ionophore, A23187. (4) The mature egg is fertilizable and undergoes normal cleavage and development. In vitro oocyte maturation enables us to examine the mechanism of cortical granule translocation and other processes that had previously only been observed in static sections of fixed ovaries.


Development ◽  
2002 ◽  
Vol 129 (18) ◽  
pp. 4315-4325
Author(s):  
Gary M. Wessel ◽  
Sean D. Conner ◽  
Linnea Berg

Cortical granules exocytose after the fusion of egg and sperm in most animals, and their contents function in the block to polyspermy by creating an impenetrable extracellular matrix. Cortical granules are synthesized throughout oogenesis and translocate en masse to the cell surface during meiosis where they remain until fertilization. As the mature oocyte is approximately 125 μm in diameter (Lytechinus variegatus), many of the cortical granules translocate upwards of 60 μm to reach the cortex within a 4 hour time window. We have investigated the mechanism of this coordinated vesicular translocation event. Although the stimulus to reinitiate meiosis in sea urchin oocytes is not known, we found many different ways to reversibly inhibit germinal vesicle breakdown, and used these findings to discover that meiotic maturation and cortical granule translocation are inseparable. We also learned that cortical granule translocation requires association with microfilaments but not microtubules. It is clear from endocytosis assays that microfilament motors are functional prior to meiosis, even though cortical granules do not use them. However, just after GVBD, cortical granules attach to microfilaments and translocate to the cell surface. This latter conclusion is based on organelle stratification within the oocyte followed by positional quantitation of the cortical granules. We conclude from these studies that maturation promoting factor (MPF) activation stimulates vesicle association with microfilaments, and is a key regulatory step in the coordinated translocation of cortical granules to the egg cortex.


2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal


Reproduction ◽  
2002 ◽  
pp. 557-564 ◽  
Author(s):  
M Shimada ◽  
N Kawano ◽  
T Terada

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.


2018 ◽  
Vol 26 (11) ◽  
pp. 1519-1537
Author(s):  
Maxim Filatov ◽  
Yulia Khramova ◽  
Maria Semenova

Mechanisms of meiotic prophase I arrest maintenance (germinal vesicle [GV] stage) and meiotic resumption (germinal vesicle breakdown [GVBD] stage) in mammalian oocytes seem to be very complicated. These processes are regulated via multiple molecular cascades at transcriptional, translational, and posttranslational levels, and many of them are interrelated. There are many molecular cascades of meiosis maintaining and meiotic resumption in oocyte which are orchestrated by multiple molecules produced by pituitary gland and follicular cells. Furthermore, many of these molecular cascades are duplicated, thus ensuring the stability of the entire system. Understanding mechanisms of oocyte maturation is essential to assess the oocyte status, develop effective protocols of oocyte in vitro maturation, and design novel contraceptive drugs. Mechanisms of meiotic arrest maintenance at prophase I and meiotic resumption in mammalian oocytes are covered in the present article.


2003 ◽  
Vol 19 (3-4) ◽  
pp. 1-8 ◽  
Author(s):  
Tatjana Smiljakovic ◽  
Melo Sterza ◽  
M. Kubelka ◽  
Z. Vohnikova ◽  
W. Tomek

Bovine oocytes are arrested in the germinal vesicle stage (GV stage)and mature spontaneously when they are removed from their follicles and transferred to a suitable culture medium. This process, known as meiotic maturation is characterized among others, by germinal vesicle breakdown followed by metaphase I (MI) stage and further development to metaphase II (MII), where they become arrested again. During GVBD to MI transition, the overall protein synthesis reaches the highest level and it rapidly declines in MII. We have previously shown that transcription completely declines during meiotic maturation. Therefore we suppose that gene expression is exclusively regulated on translational level at this stage of development. This means that mRNAs, which were stored in repressed form during oocyte growth, were actively translated during meiotic maturation. Therefore we have investigated specific regulators of translation, namely the eukaryotic initiation factor of translation eIF4E (cap binding protein) and a specific repressor of eIF4E function, the 4E-binding protein 4E-BP1. Furthermore, we have elucidated pathways, which lead to eIF4E and 4E-BP1 phosphorylation by using specific M-phase kinase inhibitors, and we compare these results with transcription and cytoplasmic polyadenylation events during the course of meiotic maturation. The detailed knowledge of such regulatory processes can help to improve in vitro bio-techniques and to estimate the risk of these techniques.


2011 ◽  
Vol 23 (1) ◽  
pp. 224 ◽  
Author(s):  
E. C. Curnow ◽  
J. P. Ryan ◽  
D. M. Saunders ◽  
E. S. Hayes

During oocyte growth chromatin configuration of the germinal vesicle (GV) oocyte undergoes modification in relation to changes in transcriptional activity crucial for conferring meiotic as well as developmental competence on the oocyte. In the macaque oocyte, there are 3 distinct GV states: GV1, noncondensed chromatin; GV2, an intermediate state; and GV3, condensed chromatin. The aim of this study was to test the effects of a prematuration culture (PMC) system, using the phosphodiesterase type 3 inhibitor milrinone (MIL), on the synchronization of GV chromatin to the GV3 stage and assess metaphase II (MII) oocyte reduced glutathione (GSH) content as a measure of cytoplasmic maturation. Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. To assess the effect of PMC on GV chromatin status, immature oocytes retrieved from unstimulated ovaries were either fixed (2% paraformaldehyde+0.1% Triton-X100) immediately after follicular aspiration (t = 0) or after culture in a humidified atmosphere of 6% CO2 in air at 37°C for 24 h in modified Connaught Medical Research Laboratories medium (mCMRL) supplemented with 10% FCS (Hyclone, Logan, UT, USA) and 12.5 μM MIL in the absence (MILNil) or presence of 1.0 IU of FSH (MILFSH). For chromatin assessment, fixed GV oocytes were stained with 5 μg mL–1 of 4′,6-diamidino-2-phenylindole (Molecular Probes, Leiden, the Netherlands) and imaged using confocal microscopy. Following PMC, MILFSH oocytes were transferred to fresh mCMRL+FCS supplemented with 1.0 IU of recombinant human FSH and 1.0 IU of hLH and cultured for a further 30 h. Control and MILFSH oocytes were denuded of cumulus cells and assessed for maturation. The MII oocytes were prepared for GSH analysis, and total GSH content was determined using a commercial 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay kit (North-West Life Science). The MII rates were compared using chi-square. Differences in oocyte GSH content were compared using t-test. Significant differences were determined at P < 0.05. There was no significant difference in the proportion of oocytes remaining at the GV stage following 24 h of PMC in MILNil or MILFSH (42/44, 96% v. 32/35, 91%, respectively). However, there was a significant reduction in GV1 chromatin (15/49, 31% v. 28/54, 52% and 22/58, 38%) and a significant increase in GV3 chromatin (23/49, 47% v. 14/54, 26% and 16/58, 28%) observed in MILFSH oocytes compared with both MILNil and t = 0 oocytes, respectively. The MII rate of MILFSH oocytes following in vitro maturation was significantly higher compared with the MII rate of control in vitro matured oocytes (91/167, 55% v. 83/243, 34%). There was no significant difference in the GSH content of GV oocytes from the time of oocyte collection (t = 0) or GV oocytes following PMC in MILFSH (3.69 ± 0.16 and 4.14 ± 0.28 pmol/oocyte, n = 39–49 oocytes). The GSH content of control in vitro matured MII oocytes was significantly greater than that of MILFSH-treated MII oocytes (3.13 ± 0.16 v. 2.02 ± 0.04 pmol/oocyte, n =53–54 oocytes). The PMC supported high rates of nuclear maturation, but cytoplasmic maturation, assessed by GSH content, was negatively affected. Further assessment following fertilization and development is required to determine the practical utility of PMC in a primate in vitro maturation setting.


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