169 CHANGES OF PROTEIN PROFILES DURING FOLLICLE DEVELOPMENT AND IN VITRO OOCYTE MATURATION IN THE PIG

2011 ◽  
Vol 23 (1) ◽  
pp. 187 ◽  
Author(s):  
M. R. Ji ◽  
D. M. Jang ◽  
Y. S. Lee ◽  
H. T. Cheong ◽  
B. K. Yang ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Protein Extraction ◽  
Protein Profile ◽  
Follicular Development ◽  
Germinal Vesicle ◽  
Maldi Mass Spectrometry ◽  
Protein Patterns ◽  
Total Proteins ◽  
Follicular Fluids

When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro conditions is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development. In the first study, to investigate protein patterns during oocyte maturation in vitro, immature oocytes (germinal vesicle stage; GV stage) as control and oocytes matured (M-II phase) in vitro were analysed. The porcine oocytes in the GV stage were put in culture with TCM-199 for 44 h for M-II-stage oocytes. Total proteins were extracted from 1200 oocytes for GV and M-II stages, separated on 2-D gels, and stained with silver. In general, the overall protein staining pattern between the 2 gels was remarkably similar for most protein spots. Analysis of the gels identified proteins that were up- or down-regulated between GV and M-II stages. Up-regulated proteins were identified as PDE4D, GPKOW, PGM5, HSP70, ZPG4, galK1, GST-β, PDX1, PDX2, and PDX3. In contrast, down-regulated proteins were identified as PRKAB1, GRP78, TD-pozl, ERP57, MPP1, DTNA, ZP3B, HSP90, HSP86, and HSP27. This study has identified a novel protein, named myomegalin, that interacts with cyclic nucleotide phosphodiesterase (PDE4D). The second study analysed changes in proteins in follicular fluids during porcine follicular development. Follicular fluids were collected from follicles of 1- to 2-, 2- to 6-, and 6- to 10-mm diameter from ovaries of slaughtered pigs. Total proteins were extracted from follicular fluids by M-PER Mammalian Protein Extraction Reagent. Differentially expressed proteins were analysed by MALDI mass spectrometry and searched on NCBInr. As a result, Spot No. 28 from the 2- to 6-mm follicle was Ig lambda chain C region, and Spot No. 32 and 33 from 6- to 10-mm were Apolipoprotein A-IV (APOA4). Increases of those proteins were correlated with follicular development. These results indicate that in vitro maturation changes the protein profile of porcine oocytes, which play important roles in the sequence of molecular events in porcine oocyte maturation and follicular development. This work was supported by Korean Research Foundation Grant funded by the Korean Government (2009-0071610).

Levels of MMP2, TIMP1 and TIMP2 in Follicular Fluids in Women Undergoing in Vitro Fertilization and Their Relationship to Oocyte Quality

2019 ◽  
Vol 12 (2) ◽  
pp. 100-107
Author(s):  
Nina P. Ayvazova ◽  
Lyubomira O. Ilieva ◽  
Emiliana I. Konova ◽  
Milena A. Atanasova
Keyword(s):  
Matrix Metalloproteinases ◽  
Ovarian Tissue ◽  
Follicular Development ◽  
Germinal Vesicle ◽  
Enzyme Linked Immunosorbent Assay ◽  
Oocyte Maturity ◽  
Vitro Fertilization ◽  
Follicular Fluids

Summary Recently, the important role of matrix metalloproteinases (MMPs) has been identified in follicular development and subsequent ovulation. Although the role of MMP in ovarian tissue remodeling during folliculogenesis has been well studied, the relationship between matrix protease activity and their inhibitors - Tisue inhibitors of matrix metalloproteinases (TIMP) and aging of the oocytes is still unclear. The present study aimed to establish the probable relationship between the expression levels of MMP-2 and TMP-1 and TIMP-2 in follicular fluid with the degree of oocyte maturity and quality. Follicular fluids from 20 women collected on the day of follicular puncture were tested for the presence of MMP-2, TIMP-1, and TIMP-2 using enzyme-linked immunosorbent assay (ELISA). The oocytes obtained were described in terms of maturity, morphology, and fertilization, as well as the embryo’s quality and rate of development. MMP-2 was significantly higher in follicular aspirates in the first prophase of meiosis - germinal vesicle (GV), compared to aspirates with first metaphase (MI) (p=0.011) and second metaphase (MII) of mature oocytes (p=0.010). The MMP-2/TIMP-1 ratio was significantly higher for GV compared to M1 (p=0.011), M2 (p=0.006) and atretic oocytes (p=0.032); (F(3, 71)=2.909, p=0.040). Based on our results, we can conclude that MMP-2 concentration in follicular fluids during the IVF / ICSI procedure had a significant relationship to oocyte maturation levels. It was significantly higher in the case of immature oocytes. On the other hand, oocytes with normal morphology were associated with a significantly higher MMP-2 concentration in follicular fluids.

scholarly journals Follicular development and preovulatory oocyte maturation in Hungarian Mangalica and Landrace gilts

2001 ◽  
Vol 44 (4) ◽  
pp. 413-420 ◽  
Author(s):  
I. Egerszegi ◽  
H. Torner ◽  
J. Rátky ◽  
K.-P. Brüssow
Keyword(s):  
Oocyte Maturation ◽  
Critical Level ◽  
Follicular Development ◽  
Germinal Vesicle ◽  
Follicular Growth ◽  
Physiological Basis ◽  
Meiotic Configuration ◽  
Preovulatory Follicles ◽  
Chromatin Configuration ◽  
Follicular Fluids

Abstract. Preservation of native pig breeds of different values has got increasing public interest. Hungarian Mangalica, likewise other native races, became uninteresting because of economic reasons or other characteristics, and were replaced by modern breeds. Its population decreased rapidly and reached a critical level. However, the exceptional taste of the meat, and the robustness and motherliness do support the propagation of this breed. Nevertheless, low prolificacy and marked seasonality remains a problem. The aim ofthe present study was to find possible implications of the physiological basis with regard to the low fecundity. Therefore, preovulatory follicular development and intrafollicular oocyte maturation of Mangalica and of Landrace gilts were compared. A total of 18 pubertal Blond and Swallow Belly Mangalica and 19 Landrace gilts (8.5 to 9 month of age, body weight of 120 to 125 kg) were used. Oestrus of gilts was synchronized by feeding Regumate®, follicular growth was stimulated by administering 1,000 IU PMSG 24 h after the last Regumate® feeding and the LH peak simulated with 750 IU hCG 80 h after PMSG. Cumulus-oocyte-complexes (COCs) were recovered 34 h after hCG by endoscopic Ovum Pick Up. Follicular fluids from follicles per ovary were pooled and the morphology of COCs was determined. COCs were classified as compact, expanded or denuded. Thereafter, COCs were prepared for evaluation of nuclear configuration. Based on their nuclear status the oocytes were classified as 1) immature – germinal vesicle (GV), with diplotene chromatin; 2) meiosis resumed – G V breakdown, diakinesis, M-I to A-I; or 3) mature – T-I and M-II. The average number (+SD) of preovulatory follicles was 6.8 + 1.4 in Mangalica and 19.6 ± 6.6 in Landrace gilts (p<0.05). Differences were obtained conceming the morphology of recovered COCs between breeds. The per cent of oocytes with compact cumulus was higher in Mangalica compared to Landrace gilts (31 vs. 16 %) but less oocytes possess expanded cumulus (62 vs. 78 %, p<0.05). The meiotic configuration of oocytes was unlike between Mangalica and Landrace gilts. The rate of oocytes with mature chromatin configuration (Telophase I /Metaphase II) was higher (27 vs. 62 %, p<0.05) in Landrace sows. It is assumed that both diminished follicular development and protracted intrafollicular oocyte maturation may be involved in low fecundity in Mangalica.

scholarly journals Feasibility of Secondary Follicle Isolation, Culture and Achievement of In-Vitro Oocyte Maturation from Superovulated Ovaries: An Experimental Proof-of-Concept Study Using Mice

2021 ◽  
Vol 10 (13) ◽  
pp. 2757
Author(s):  
Xia Hao ◽  
Amandine Anastácio ◽  
Kenny A. Rodriguez-Wallberg
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Follicular Development ◽  
Clinical Situation ◽  
Experimental Proof ◽  
Secondary Follicle ◽  
17Β Estradiol ◽  
Follicle Size ◽  
And Control

Fertility preservation through ovarian stimulation, aiming at cryopreserving mature oocytes or embryos, is sometimes unsuccessful. This clinical situation deserves novel approaches to overcome infertility following cancer treatment in patients facing highly gonadotoxic treatment. In this controlled experimental study, we investigated the feasibility of in-vitro culturing secondary follicles isolated from superovulated ovaries of mice recently treated with gonadotropins. The follicle yields of superovulated ovaries were 45.9% less than in unstimulated controls. Follicles from superovulated ovaries showed faster growth pace during the initial 7 days of culture and secreted more 17β-estradiol by the end of culture vs controls. Parameters reflecting the outcome of follicular development and oocyte maturation competence in vitro were similar between superovulated and control groups, with a similar follicle size at the end of culture and around 70% survival. Nearly half of cultured follicles met the criteria for in-vitro maturation in both groups and approximately 60% of those achieved a mature MII oocyte, similarly in both groups. Over 60% of obtained MII oocytes displayed normal-looking spindle and chromosome configurations, without significant differences between the groups. Using a validated follicle culture system, we demonstrated the feasibility of secondary follicle isolation, in-vitro culture and oocyte maturation with normal spindle and chromosome configurations obtained from superovulated mice ovaries.

scholarly journals Requirement of mosXe protein kinase for meiotic maturation of Xenopus oocytes induced by a cdc2 mutant lacking regulatory phosphorylation sites.

1992 ◽  
Vol 12 (7) ◽  
pp. 3192-3203 ◽  
Author(s):  
K M Pickham ◽  
A N Meyer ◽  
J Li ◽  
D J Donoghue
Keyword(s):  
Protein Kinase ◽  
Oocyte Maturation ◽  
Xenopus Oocytes ◽  
Germinal Vesicle ◽  
Cyclin B1 ◽  
Phosphorylation Sites ◽  
Germinal Vesicle Breakdown ◽  
Regulatory Phosphorylation

The p34cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14----Ala and Tyr-15----Phe did not induce germinal vesicle breakdown (BVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15p34cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mosXe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34cdc2 single mutants Ala-161 and Glu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin B1 in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mosXe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin B1 and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mosXe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.

scholarly journals The Role of Androgens in Mammals Folliculogenesis

2018 ◽  
Vol 44 (1) ◽  
pp. 15
Author(s):  
Livia Brunetti Apolloni ◽  
Jamily Bezerra Bruno ◽  
Benner Geraldo Alves ◽  
José Ricardo de Figueiredo
Keyword(s):  
Androgen Receptor ◽  
In Vitro Culture ◽  
Steroid Hormones ◽  
Oocyte Maturation ◽  
Follicular Development ◽  
Follicular Growth ◽  
Preantral Follicles ◽  
Ovarian Cells

Introduction: Steroid hormones production is a physiological process termed steroidogenesis. An important stage of this process is the conversion of androgens into estrogens through aromatase enzyme. Furthermore, androgens are important in the process of folliculogenesis, promoting follicular growth in different species. Thus, the aim of this review was to present the process of synthesis, mechanism of action, and importance of androgens in folliculogenesis. Additionally, the main results of in vitro culture of ovarian cells in the presence of these hormones were emphasized.Review: Folliculogenesis begins in prenatal life in most of species and can be defined as the process of formation, follicular growth, and oocyte maturation. Preantral follicles represent 95% of the follicular population and assisted reproductive technologies have been developed (e.g., Manipulation of Oocytes Enclosed in Preantral Follicles - MOEPF) in order to avoid the great follicle loss that occurs naturally in vivo by atresia. The MOEPF aim to obtain a large number of competent oocytes from preantral follicles and then subject to in vitro maturation, fertilization, and culture for embryo production. However, the development of an efficient medium to ensure the follicular survival and oocyte maturation is the major challenge of this biotechnology. To achieve the success on in vitro culture, the effects of substances as androgens on follicular development have been evaluated. Androgens are steroid hormones produced in theca cells (TC) that are fundamental for follicular growth. These cells provide all the androgens required by the developing follicles for conversion into estrogens by the granulosa cells (GC). Androgens receptors (AR) are localized in cell cytoplasm of all follicular categories, being more expressed in preantral follicles. The androgen pathway initiates through its connection to its receptor, making a complex androgen-AR, that in the nucleus helps on the process of gene transcription related with follicular survival. This mechanism is androgen receptor genomic activity. In addition to genomic action, there is an androgen receptor non-genomic activity. This occurs through activation of AR and its interaction with different signaling molecules located on the cell membrane, triggering events that aid in the follicular development. Regardless of the androgens actions, ovarian cells of several species subjected to in vitro culture have shown the importance of these hormones on the follicle development. Recent studies demonstrated that androgens addition on the culture medium stimulated the activation of preantral follicles (bovine and caprine), antrum formation (swine), survival (non-primate), and oocyte maturation (antral follicles; bovine). Also, some studies suggest that the addition of these hormones on in vitro culture is dose-dependent and species-specific.Conclusion: This review shows the role of androgens in different stages of follicular development and its action as a substrate for steroidogenesis and transcription of genes related to follicular survival and oocyte maturation. However, when these hormones should be added during in vitro follicular culture and which concentration is required remains unclear, being necessary more studies to elucidate these aspects.

scholarly journals Inactivation of glucocorticoids by 11β-hydroxysteroid dehydrogenase enzymes increases during the meiotic maturation of porcine oocytes

Reproduction ◽  
2008 ◽  
Vol 136 (6) ◽  
pp. 725-732 ◽  
Author(s):  
Rachel J Webb ◽  
Neera Sunak ◽  
Lisa Wren ◽  
Anthony E Michael
Keyword(s):  
Oocyte Maturation ◽  
Enzyme Activities ◽  
Germinal Vesicle ◽  
Hydroxysteroid Dehydrogenase ◽  
Target Cells ◽  
Cortisol Metabolism ◽  
Enzymatic Inactivation ◽  
Glucocorticoid Metabolism ◽  
Ovarian Cyst Fluid

Recent reports have shown that glucocorticoids can modulate oocyte maturation in both teleost fish and mammals. Within potential target cells, the actions of physiological glucocorticoids are modulated by 11β-hydroxysteroid dehydrogenase (HSD11B) isoenzymes that catalyse the interconversion of cortisol and cortisone. Hence, the objective of this study was to establish whether HSD11B enzymes mediate cortisol–cortisone metabolism in porcine oocytes and, if so, whether the rate of glucocorticoid metabolism changes during oocyte maturation. Enzyme activities were measured in cumulus–oocyte complexes (COCs) and denuded oocytes (DOs) using radiometric conversion assays. While COCs and DOs oxidised cortisol to inert cortisone, there was no detectable regeneration of cortisol from cortisone. The rate of cortisol oxidation was higher in expanded COCs than in compact COCs containing germinal vesicle (GV) stage oocytes (111±6 vs 2041±115 fmol cortisone/oocyte.24 h; P<0.001). Likewise, HSD11B activities were 17±1 fold higher in DOs from expanded COCs than in those from compact COCs (P<0.001). When GV stage oocytes were subject to a 48 h in vitro maturation protocol, the enzyme activities were significantly increased from 146±18 to 1857±276 fmol cortisone/oocyte.24 h in GV versus MII stage oocytes respectively (P<0.001). Cortisol metabolism was inhibited by established pharmacological inhibitors of HSD11B (glycyrrhetinic acid and carbenoxolone), and by porcine follicular and ovarian cyst fluid. We conclude that an HSD11B enzyme (or enzymes) functions within porcine oocytes to oxidise cortisol, and that this enzymatic inactivation of cortisol increases during oocyte maturation.

scholarly journals In vitro development of mouse fetal germ cells into mature oocytes

Reproduction ◽  
2007 ◽  
Vol 134 (2) ◽  
pp. 223-231 ◽  
Author(s):  
Wei Shen ◽  
Lan Li ◽  
Zhaodai Bai ◽  
Qingjie Pan ◽  
Mingxiao Ding ◽  
...  
Keyword(s):  
Genomic Imprinting ◽  
Germ Cells ◽  
Follicular Development ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown ◽  
Primordial Follicles ◽  
Ovarian Cells ◽  
Fetal Mice ◽  
Vitro Development

Little is known about the mechanisms underlying primordial follicular formation and the acquisition of competence to resume meiosis by growing oocytes. It is therefore important to establish anin vitroexperimental model that allows one to study such mechanisms. Mouse follicular development has been studiedin vitroover the past several years; however, no evidence has been presented showing that mature oocytes can be obtained from mouse fetal germ cells prior to the formation of primordial follicles. In this study, a method has been established to obtain mature oocytes from the mouse fetal germ cells at 16.5 days postcoitum (dpc). From the initiation of primordial follicular formation to the growth of early secondary follicles, ovarian tissues from 16.5 dpc fetal mice were culturedin vitrofor 14 days. Subsequently, 678 intact secondary follicles were isolated from 182 mouse fetal ovaries and cultured for 12 days. A total of 141 oocytes inside antral follicles were maturedin vitro, and 102 oocytes underwent germinal vesicle breakdown. We found that 97 oocytes were fertilized and 15 embryos were able to form morula–blastocysts. We also analyzed various genomic imprinting markers and showed that the erasure of genomic imprinting markers in the parental generation was also imposed on the oocytes that developed from fetal germ cells. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with ovarian cells, and that oocytes within the growing follicles are able to mature normallyin vitro.

Oocyte maturation in the mummichog (Fundulus heteroclitus): Effects of steroids on germinal vesicle breakdown of intact follicles in vitro

1986 ◽  
Vol 62 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Mark S. Greeley ◽  
Dan R. Calder ◽  
Malcolm H. Taylor ◽  
Hans Hols ◽  
Robin A. Wallace
Keyword(s):  
Oocyte Maturation ◽  
Fundulus Heteroclitus ◽  
Germinal Vesicle ◽  
Germinal Vesicle Breakdown

scholarly journals Effects of gonadotrophin in vivo and 2-hydroxyoestradiol-17β in vitro on follicular steroid hormone profile associated with oocyte maturation in the catfish Heteropneustes fossilis

2006 ◽  
Vol 189 (2) ◽  
pp. 341-353 ◽  
Author(s):  
A Mishra ◽  
K P Joy
Keyword(s):  
Oocyte Maturation ◽  
Germinal Vesicle ◽  
Hplc Method ◽  
Heteropneustes Fossilis ◽  
Germinal Vesicle Breakdown ◽  
Steroid Profile ◽  
17 Hydroxyprogesterone ◽  
Envelope Layer

An HPLC method was used to tentatively identify progesterone (P4) and its metabolites (17-hydroxyprogesterone (17-P4) and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)), corticosteroids (cortisol and corticosterone) and testosterone in ovary/follicular preparations of the catfish Heteropneustes fossilis associated with in vivo or in vitro oocyte maturation/ovulation. A single i.p. injection of human chorionic gonadotrophin (100 IU/fish, sampled at 0, 8 and 16 h) induced oocyte maturation and ovulation, which coincided with significant and progressive increases in 17,20β-P, and P4 and 17-P4, the precursors of the former. Both cortisol and corticosterone also increased significantly. Conversely, testosterone decreased significantly and progressively over time. Under in vitro conditions, incubation of post-vitellogenic (intact) follicles or follicular envelope (layer) with 2-hydroxyoestradiol (2-OHE2, 5 μM for 0, 6 and 24 h) elicited a sharp significant increase in 17,20β-P, the increase being higher in the follicular envelope incubate. P4 and 17-P4 also registered significant increases over the time with the peak values at 24 h. Cortisol and corticosterone increased significantly in the intact follicle, but not in the follicular envelope incubate. Testosterone decreased significantly in the intact follicle, but increased significantly (24 h) in the follicular envelope incubate. Coincident with these changes, the percentage of germinal vesicle breakdown (GVBD) increased over the time in the intact follicle incubate (48.9% at 6 h and 79.8% at 24 h). Denuded oocytes on incubation with 2-OHE2 (5 μM) did not produce any significant change in the percentage of GVBD or in the steroid profile. While corticosterone and 17,20β-P were undetected, P4, 17-P4, cortisol and testosterone were detected in low amounts. The results show that the 2-OHE2-induced GVBD response seems to be mediated through the production of 17,20β-P and corticosteroids. It is suggested that hydroxyoestrogens seem to be a component in the gonadotrophin cascade of regulation of oocyte maturation/ovulation in the catfish.

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