Melatonin improves developmental competence of oocyte-granulosa cell complexes from porcine preantral follicles

SummaryIn vitro culture of the oocyte granulosa cell complexes (OGCs) from early antral follicles (EAFs) shows granulosa cell (GC) proliferation, but to a lesser extent than that observed in vivo during follicle development. As the number of GCs closely relates to energy sufficiency of the oocytes, enhancement of GC proliferation influences oocyte development. GC proliferation depends on glycolysis and insulin-mediated AKT/mTOR signaling pathway; therefore, addition of culture medium containing insulin and glucose may potentially promote GC proliferation and hence improve oocyte development. In the present study, we assessed the effect of exogenous insulin and glucose concentration on GC proliferation and oocyte energy status as well as developmental abilities of porcine oocytes grown in vitro. In the presence of 5.5 mM of glucose (Low), a comparison of 10 versus 20 μg/ml insulin showed that high insulin enhanced GC proliferation but exhausted glucose from the medium, which resulted in low energy status including lipid and adenosine triphosphate of the oocyte. Whereas, in the presence of 20 μg/ml insulin, medium with 11 mM glucose (High) enhanced GC proliferation and oocyte energy status as well as developmental ability up to the blastocyst stage. Considering that there was no difference in OGCs development observed with medium (10 μg/ml insulin) containing 5.5 versus 11 mM glucose, we concluded that the combination of high insulin and glucose enhanced GC proliferation and energy status of oocytes as well as the developmental ability of the oocytes grown in vitro.

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Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles

Reproduction ◽

10.1530/rep.0.1230593 ◽

2002 ◽

pp. 593-600 ◽

Cited By ~ 57

Author(s):

EC dela Pena ◽

Y Takahashi ◽

S Katagiri ◽

EC Atabay ◽

M Nagano

Keyword(s):

Ethylene Glycol ◽

Survival Rates ◽

Subsequent Development ◽

Developmental Competence ◽

Preantral Follicles ◽

Cell Complexes ◽

Vitrification Solution ◽

First Time

Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups.

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Meiotic Competence of Porcine Oocyte-Granulosa Cell Complexes Isolated From Preantral Follicles Was Maintained During in Vitro Culture

Fertility and Sterility ◽

10.1016/j.fertnstert.2005.07.1031 ◽

2005 ◽

Vol 84 ◽

pp. S394-S395

Author(s):

Y. Miyata ◽

N. Harauma ◽

K. Ohsumi ◽

Y. Tsuji ◽

A. Fukuda ◽

...

Keyword(s):

In Vitro Culture ◽

Granulosa Cell ◽

Preantral Follicles ◽

Cell Complexes ◽

Porcine Oocyte ◽

Meiotic Competence

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Supplementation of c-type natriuretic peptide during in vitro growth period benefits the development of murine preantral follicles

Zygote ◽

10.1017/s096719942000060x ◽

2020 ◽

pp. 1-5

Author(s):

Li Ang ◽

Cao Haixia ◽

Li Hongxia ◽

Li Ruijiao ◽

Guo Xingping ◽

...

Keyword(s):

Natriuretic Peptide ◽

Granulosa Cells ◽

Culture System ◽

Early Stage ◽

Developmental Competence ◽

Control Group ◽

Follicle Development ◽

Control Groups ◽

Preantral Follicles

Summary The present study investigated the effects of c-type natriuretic peptide (CNP) on the development of murine preantral follicles during in vitro growth (IVG). Preantral follicles isolated from ovaries of Kunming mice were cultured in vitro. In the culture system, CNP was supplemented in the experimental groups and omitted in the control groups. In Experiment 1, CNP was only supplemented at the early stage and follicle development was evaluated. In Experiments 2 and 3, CNP was supplemented during the whole period of in vitro culture. In Experiment 2, follicle development and oocyte maturity were evaluated. In Experiment 3, follicle development and embryo cleavage after in vitro fertilization (IVF) were assessed. The results showed that in the control groups in all three experiments, granulosa cells migrated from within the follicle and the follicles could not reach the antral stage. In the experimental groups in all three experiments, no migration of granulosa cells was observed and follicle development was assessed as attaining the antral stage, which was significantly superior to that of the control group (P < 0.0001). Oocyte meiotic arrest was effectively maintained, hence giving good developmental competence. In conclusion, CNP supplementation in the culture system during IVG benefited the development of murine preantral follicles.

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Conditional Deletion of the Retinoblastoma (Rb) Gene in Ovarian Granulosa Cells Leads to Premature Ovarian Failure

Molecular Endocrinology ◽

10.1210/me.2008-0033 ◽

2008 ◽

Vol 22 (9) ◽

pp. 2141-2161 ◽

Cited By ~ 22

Author(s):

Claudia Andreu-Vieyra ◽

Ruihong Chen ◽

Martin M. Matzuk

Keyword(s):

Granulosa Cell ◽

Target Genes ◽

Ovarian Function ◽

Specific Pattern ◽

Tumor Formation ◽

Conditional Knockout ◽

Mrna Levels ◽

Preantral Follicles ◽

Recombination System ◽

Ovarian Tumorigenesis

Abstract The retinoblastoma protein (RB) regulates cell proliferation and survival by binding to the E2F family of transcription factors. Recent studies suggest that RB also regulates differentiation in a variety of cell types, including myocytes, neurons, adipocytes, and chondrocytes. Rb mutations have been found in ovarian cancer; however, the role of RB in normal and abnormal ovarian function remains unclear. To test the hypothesis that loss of Rb induces ovarian tumorigenesis, we generated an ovarian granulosa cell conditional knockout of Rb (Rb cKO) using the Cre/lox recombination system. Rb cKO females showed 100% survival and no ovarian tumor formation through 9 months of age, but they developed progressive infertility. Prepubertal Rb cKO females showed increased ovulation rates compared with controls, correlating with increased follicle recruitment, higher Fshr and Kitl mRNA levels, and lower anti-Müllerian hormone levels. In contrast, the ovulation rate of 6-wk-old females was similar to that of controls. Morphometric analysis of Rb cKO ovaries from 6-wk-old and older females showed increased follicular atresia and apoptosis. Rb cKO ovaries and preantral follicles had abnormal levels of known direct and indirect target genes of RB, including Rbl2/p130, E2f1, Ccne2, Myc, Fos, and Tgfb2. In addition, preantral follicles showed increased expression of the granulosa cell differentiation marker Inha, decreased levels of Foxl2 and Cyp19a1 aromatase, and abnormal expression of the nuclear receptors Nr5a1, Nr5a2, and Nr0b1. Taken together, our results suggest that RB is required for the temporal-specific pattern of expression of key genes involved in follicular development.

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Cumulus and granulosa cell biomarkers: a good predictor for successful oocyte and embryo developmental competence in human in vitro fertilization

Journal of Genetic Medicine ◽

10.5734/jgm.2021.18.1.1 ◽

2021 ◽

Vol 18 (1) ◽

pp. 1-7

Author(s):

Eun Jeong Yu ◽

Sang Woo Lyu

Keyword(s):

In Vitro Fertilization ◽

Granulosa Cell ◽

Good Predictor ◽

Developmental Competence ◽

Vitro Fertilization ◽

Human In Vitro Fertilization

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340 IN VITRO MATURATION OF PREPUBERTAL BOVINE OOCYTES ON A GRANULOSA CELL MONOLAYER FROM ADULT ANIMALS IMPROVES THEIR DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab340 ◽

2006 ◽

Vol 18 (2) ◽

pp. 277

Author(s):

S. Ponebsek ◽

C. Wrenzycki ◽

K.-G. Hadeler ◽

D. Herrmann ◽

K. Korsawe ◽

...

Keyword(s):

Granulosa Cell ◽

Relative Abundance ◽

Glucose Transporter ◽

Cell Monolayer ◽

Mrna Abundance ◽

Developmental Competence ◽

Hsp 70 ◽

Developmental Capacity ◽

Calf Oocytes

Oocytes from prepubertal calves have a decreased developmental competence compared with oocytes from adult animals. The goal of this study was to improve the developmental competence of juvenile oocytes by maturation on granulosa cell (GC) monolayers from adult animals. Oocytes were recovered by ovum pickup (OPU) from 48 Holstein Friesian calves at 7-8 months of age and 18 adult cows. Animals received intramuscular injections of 60 mg FSH 48 h prior to each OPU session. Follicles were punctured twice per week in six consecutive OPU sessions. Cumulus oocyte complexes (COCs) recovered from calves were divided into three quality groups (classes I-III) and were then randomly distributed into three maturation groups: COCs were matured for 24 h on either GC or fibroblasts or without co-culture. Cow oocytes were matured without co-culture. TCM-199 supplemented with BSA (0.1%), hCG (5 IU/mL), and eCG (10 IU/mL) served as the medium in all groups. After maturation, all COCs were fertilized in vitro; after 18 h, presumptive zygotes were cultured in SOF+BSA for 8 days (37�C, 5% CO2). On Day 3, cleavage rates and, on Day 8, blastocyst rates were determined. The relative mRNA abundance of the following transcripts, critically involved in early embryonic development was determined: growth differentiation factor-9 (GDF-9), heat shock protein 70 (Hsp-70), and glucose transporter-3 (Glut-3). Single immature and matured oocytes (for GDF-9 and Hsp-70) and 8-16-cell embryos and expanded blastocysts (for Hsp-70 and Glut-3) from calves and cows were examined by semiquantitative RT-PCR. Cleavage and blastocyst rates were similar in oocytes derived from cows and calves matured on GC (74.3% vs. 70.0% and 22.3% vs. 22.3%, respectively), but were significantly higher (P < 0.05; one way ANOVA, Student-Newman-Keuls Method) than in the group without co-culture on fibroblasts (55.2% vs. 53.6% and 11.7% vs. 5.5%, respectively). GDF-9 expression was similar in immature calf and cow oocytes. After maturation, a significant decrease in GDF-9 expression was observed in calf oocytes. Matured cow oocytes showed a significantly higher mRNA abundance of GDF-9 than matured calf oocytes. The relative abundance of Hsp-70 was decreased in matured oocytes of all groups. Expanded blastocysts derived from adult oocytes expressed Hsp-70 significantly higher than blastocysts derived from oocytes of the control calves. The relative abundance of Glut-3 mRNA was similar in 8-16 cell embryos and expanded blastocysts in all groups. Overall, mRNA expression pattern for Hsp-70 and Glut-3 in blastocysts from GC matured oocytes were similar to that of cow blastocysts. Results indicate that maturation of juvenile calf oocytes on granulosa cells from adult animals improves their developmental competence. These findings provide clues toward identification of factors critically involved in acquiring full developmental capacity at puberty.

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285 RELATIONSHIP BETWEEN FOLLICLE FLUID STEROID CONCENTRATIONS, GRANULOSA CELL GENE EXPRESSION, AND BOVINE OOCYTE DEVELOPMENTAL COMPETENCE

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab285 ◽

2010 ◽

Vol 22 (1) ◽

pp. 299

Author(s):

S. Matoba ◽

S. Mamo ◽

E. Gallagher ◽

A. G. Fahey ◽

T. Fair ◽

...

Keyword(s):

Gene Expression ◽

Granulosa Cell ◽

Granulosa Cells ◽

Target Genes ◽

Transcript Abundance ◽

Blastocyst Stage ◽

Developmental Competence ◽

Oocyte Competence ◽

Cell Gene Expression ◽

The Relationship

The ability to culture oocytes and embryos in an individually identifiable manner facilitates the study of the relationship between follicle param- eters and oocyte development, in order to identify markers of competent oocytes. The aim of this study was to examine the predictive value of intrafollicular steroid concentrations and granulosa cell transcript abundance on the ability of immature bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles (n = 214, 11 replicates, 49 animals) were dissected from the ovaries of slaughtered animals. Following measure- ment of diameter, follicles were carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through maturation, fertilization, and culture on the cell adhesive Cell-Tak (20 oocytes/100 μL; Matoba and Lonergan 2009 Reprod. Fertil. Dev. 21, 160). Cleavage and blastocyst rates were assessed on Days 2 and 9, respectively. Follicular fluid was recovered and stored at -80°C until analysis for concentrations of the steroids estradiol, progesterone, and testosterone by RIA. Granulosa cells were collected from each follicle for analysis of gene expression by quantitative RT-PCR. Primers were designed for 7 target genes (AMH, CYP19A, ESR1, ESR2, FSHR, HSD3B1 and LHCGR) and 2 reference genes (PPIA and H2AZ). Transcript abundance of target genes in granulosa cells associated with embryos that cleaved and developed to the blastocyst stage (competent) and those that cleaved but failed to develop (incompetent) was examined. Mean steroid concentrations were compared by ANOVA and Spearman correlations, and logistical regression were used to test the relationship between follicle size and steroid con- centration and the ability of steroid concentration to predict developmental competence. Gene expression data were analyzed using the delta-delta CT (cycle threshold) method. Values were normalized to the average values of the reference genes and means were compared by the Student’s t-test In total, 79.1% of oocytes cleaved after IVF and 28.3% developed to the blastocyst stage. The mean (±SEM) follicular concentrations of testosterone (62.8 ± 4.8 ng mL-1), progesterone (616.8 ± 31.9 ng mL-1), or estradiol (14.4 ± 2.4 ng mL-1 were not different (P ≥ 0.05) between competent and incompetent oocytes. Follicular diameter was negatively correlated with testosterone, progesterone, testosterone:estradiol, and pro- gesterone:estradiol (P ≤ 0.01) and positively correlated with estradiol (P ≤ 0.01). Logistical regression analysis showed that steroid concentrations or the ratio of steroids were not satisfactory predictors of oocyte competence. Transcript abundance of AMH, ESR1, ESR2, FSHR, and HSD3B1 was significantly higher (P ≤ 0.05) in granulosa cells associated with competent compared with incompetent oocytes. In conclusion, follicular steroid concentrations were not associated with oocyte development. In contrast, granulosa cell gene expression may be a useful predictor of oocyte competence. Supported by Science Foundation Ireland (07/SRC/B1156).

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