Co-culture of buffalo (Bubalus bubalis) preantral follicles with antral follicles: a comparative study of developmental competence of oocytes derived from in vivo developed and in vitro cultured antral follicles

Zygote ◽  
2012 ◽  
Vol 21 (3) ◽  
pp. 286-294 ◽  
Author(s):  
G. Taru Sharma ◽  
Pawan K. Dubey ◽  
Amar Nath ◽  
G. Saikumar
Keyword(s):  
Growth Factor ◽  
Bubalus Bubalis ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Term Survival ◽  
Preantral Follicles ◽  
Antral Follicles ◽  
Epidermal Growth

SummaryThe present study was undertaken to examine whether the presence of antral follicles (AFs) affects the survival, growth and steroidogenesis of preantral follicles (PFs) and compare the maturation and developmental competence of buffalo oocytes derived from in vivo developed and in vitro cultured AFs. Two experiments were carried out. In experiment I, PFs (200–250 μm) were isolated and cultured with or without AFs (3–5 mm) in TCM-199 medium that contained 10% fetal bovine serum (FBS), 1% insulin transferin selenium (ITS), 20 ng/ml epidermal growth factor (EGF), 0.5 μg/ml follicle-stimulating hormone (FSH) and 100 ng/ml insulin-like growth factor (IGF)-I. In experiment II, in vitro developmental competence was compared for the cumulus–oocyte complexes (COCs) recovered from in vivo developed and in vitro cultured AFs. Survival, growth, development of antrum, accumulation of estradiol and progesterone was (P < 0.05) higher when PFs were co-cultured with AFs. Developmental competence of both types of follicular oocytes did not differ significantly in terms of maturation and cleavage rate, but morula and blastocyst production rate were (P < 0.05) higher with in vivo developed AFs as compared with the in vitro cultured antral follicular oocytes. In conclusion, co-culture of PFs with AFs supports long-term survival and growth of buffalo PFs and this co-culture system plays a dual role for in vitro production of embryos as well as understanding the relationship between developing PFs and AFs.

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

2006 ◽  
Vol 18 (2) ◽  
pp. 286 ◽  
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 ◽  
First Polar Body

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

scholarly journals Immunolocalization of the hepatocyte growth factor (HGF) system in the rat ovary and the anti-apoptotic effect of HGF in rat ovarian granulosa cells in vitro

Reproduction ◽  
2006 ◽  
Vol 132 (2) ◽  
pp. 291-299 ◽  
Author(s):  
Mehmet Uzumcu ◽  
Zui Pan ◽  
Yi Chu ◽  
Peter E Kuhn ◽  
Rob Zachow
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Interstitial Cells ◽  
Preantral Follicles ◽  
Antral Follicles ◽  
Apoptotic Effect ◽  
Equine Chorionic Gonadotropin ◽  
Hepatocyte Growth

Hepatocyte growth factor (HGF) regulates granulosa cell (GC) steroidogenesis and suppresses apoptosis in non-ovarian cells. The hypothesis was thus developed that intraovarian HGF supports folliculogenesis by mediating steroidogenesis and suppressing apoptosis. To investigate the latter, the anti-apoptotic actions of HGF were tested in GCs and follicles isolated from immature rats. Results showed that HGF suppressed apoptosis in GC and follicle cultures as visualized using apoptosis indicator dye, YO-PRO-1. Immunohistochemistry was used to investigate the distribution of HGF, c-met, and HGF activator (HGFA) protein during folliculogenesis in equine chorionic gonadotropin (eCG)-primed rats. Immunoreactive HGF content was the greatest in GCs within preantral follicles. Following eCG, large antral follicles showed elevated HGF staining in theca and interstitial cells when compared with GCs. Intense c-met staining was observed in GCs within non-primed small preantral follicles; following eCG, the level of c-met was diminished in GCs, but increased within theca and interstitial cells. Theca, interstitium, and GCs in non-primed and primed ovaries contained HGFA. Following eCG, HGFA was more apparent in theca cells and the interstitium when compared to that in GCs within large antral follicles. The presence of HGF, c-met, and HGFA in preantral follicles would potentially enable the anti-apoptotic effects of HGF that were observed in vitro to occur in vivo. Advanced folliculogenesis led to a change in the cellular distribution of the HGF, c-met, and HGFA, suggesting that the ovarian HGF system is hormonally regulated in vivo.

122 A New Maturation Medium Improves Porcine Embryo Production In Vitro

2018 ◽  
Vol 30 (1) ◽  
pp. 200 ◽  
Author(s):  
A. Lucas-Hahn ◽  
B. Petersen ◽  
M. Nowak-Imialek ◽  
U. Baulain ◽  
R. Becker ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Developmental Competence ◽  
Gestation Length ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Maturation Medium

Recently (Spate et al. 2017 Reprod. Fertil. Dev. 29, 150), a new medium [TCM-199 supplemented with hCG 10 IU, pregnant mare serum gonadotropin (PMSG) 10 IU mL−1, fibroblast growth factor (FGF) 40 ng mL−1, leukemia inhibitory factor (LIF) 2000 U mL−1, IGF-1 20 ng mL−1, epidermal growth factor (EGF) 10 ng mL−1], termed FLI medium, was demonstrated to improve porcine oocyte maturation in vitro. The effects on embryo development and quality have not yet been investigated. The purpose of the present study was to compare the FLI medium in porcine in vitro embryo production (IVP) with our standard maturation medium (DMEM supplemented with 10 IU mL−1 PMSG and hCG, 50 ng mL−1 EGF, 100 ng mL−1 IGF1, and 5 ng mL−1 FGF). Briefly, gilt oocytes were collected via aspiration of follicles from abattoir ovaries and matured for 44 h in either FLI or standard DMEM medium at 39°C, 5% CO2 in humidified air. In vitro fertilization was performed with freshly ejaculated sperm (250,000 mL−1) of a multi-transgenic boar (GGTA1-KO/hCD46/hCD55/hCD59/hHO-1/hA20) by co-incubation with the matured oocytes in PGMTac4 medium for 4 h. Zygotes were washed twice and then cultured for 6 days in PZM3 medium. Development to the blastocyst stage was recorded at Day 6 of culture. Blastocysts were fixed and Hoechst33342 stained for counting the nuclei. Each of the experiments was repeated 3 times. In a second step, Day 5 blastocysts derived from the FLI medium were transferred to synchronized pubertal gilts to test the in vivo developmental competence of the IVF embryos. Maturation of oocytes in FLI medium resulted in a significantly higher blastocyst rate (49.3 vs. 13.5; P ≤ 0.001, Chi-squared test) and nuclei number (41.3 ± 12.2 vs. 35.3 ± 10.8; P ≤ 0.001, one-way ANOVA) compared with the standard medium, whereas the cleavage rate was not affected. Transfer of Day 5 blastocysts (average 35 embryos/recipient) derived from the FLI system using 8 recipients resulted in 7 pregnancies (87.5%) as determined by ultrasound scanning on Day 25 of gestation. At the time of writing, one recipient had delivered 5 healthy piglets after a gestation length of 114 days. Results indicate that the FLI medium significantly improves blastocyst rates and the cell number of the resulting blastocysts (Table 1) and yields pig IVF embryos with a high developmental capacity in vivo. By producing high-quality porcine embryos, this FLI-based IVF system provides an efficient method to modify the porcine genome by cytoplasmic microinjection of CRISPR/Cas molecules into IVF-derived zygotes. Table 1.Results of maturation of oocytes in FLI medium compared with DMEM

Role of follicle stimulating hormone and epidermal growth factor in the development of porcine preantral follicle in vitro

Zygote ◽  
2007 ◽  
Vol 15 (3) ◽  
pp. 233-240 ◽  
Author(s):  
Ji Wu ◽  
Qi Tian
Keyword(s):  
Growth Factor ◽  
Embryonic Development ◽  
Follicle Stimulating Hormone ◽  
Preantral Follicle ◽  
Preantral Follicles ◽  
Antral Follicles ◽  
Estradiol Secretion ◽  
Epidermal Growth

SummaryThe aim of the present study was to assess the role of follicle stimulating hormone (FSH), epidermal growth factor (EGF) or a combination of EGF and FSH on the in vitro growth of porcine preantral follicles, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. Porcine preantral follicles were cultured for 3 days in the absence or in the presence of FSH or EGF. Oocytes from these follicles were then matured, fertilized in vitro and embryos were cultured. Estradiol secretion and histological analysis of cultured follicles were also carried out. The results showed that when FSH, or a combination of EGF and FSH, was added to the culture medium, most of preantral follicles grew to antral follicles with high estradiol secretion and the oocytes from these antral follicles could mature, fertilize and develop to the blastocyst stage. Without FSH, or a combination of EGF and FSH, preantral follicles were unable to develop to the antral stage. Histology demonstrated that the resulting follicles were nonantral, estradiol production was reduced and none of their oocytes matured after in vitro maturation. The results indicate the essential role of FSH in promoting in vitro growth of porcine preantral follicle, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. EGF with FSH treatment of porcine preantral follicles improves the quality of oocytes, shown by a higher frequency of embryonic development.

Synergetic effects of epidermal growth factor and estradiol on cytoplasmic maturation of porcine oocytes

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 349-354 ◽  
Author(s):  
Yong-Hai Li ◽  
Rui-Hua Liu ◽  
Li-Hong Jiao ◽  
Wei-Hua Wang
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Early Embryo ◽  
Antral Follicles ◽  
Nuclear Maturation ◽  
17Β Estradiol ◽  
Epidermal Growth ◽  
Cytoplasmic Maturation ◽  
Pronuclear Formation

This study was conducted to examine the effect of epidermal growth factor (EGF) and 17β-estradiol (E2) on nuclear and cytoplasmic (male pronuclear formation and early embryo development) maturation of porcine oocytes. Oocytes were aspirated from antral follicles and cultured in modified TCM-199 medium supplemented with 0.57 mM cysteine, 10 IU/ml eCG, 10 IU/ml hCG, with or without EGF and/or E2. In vitro fertilisation of matured oocytes was performed in a modified Tris-buffered medium (mTBM) with frozen-thawed ejaculated spermatozoa. Oocytes were transferred to NCSU-23 supplemented with 0.4% bovine serum albumin at 6 h after in vitro fertilisation. Significantly higher (p < 0.05) rates of nuclear maturation, pronuclear formation and cleavage (91.7%, 65.2% and 37.3%, respectively) were observed when oocytes were cultured in the medium containing both EGF (10 ng/ml) and E2 (1 μg/ml) than in the medium supplemented with either EGF or E2 or without both. Intracellular glutathione concentration in the oocytes cultured in the medium containing both E2 and EGF was also significantly higher (12.1 pmol per oocyte) than that of oocytes cultured in the medium with E2 or EGF alone or without both. These findings suggested that EGF and E2 have a synergestic effect on both nuclear and cytoplasmic maturation of porcine oocytes.

scholarly journals Disruption of TP63-miR-27a* Feedback Loop by Mutant TP53 in Head and Neck Cancer

2019 ◽  
Vol 112 (3) ◽  
pp. 266-277 ◽  
Author(s):  
Nikhil S Chari ◽  
Cristina Ivan ◽  
Xiandong Le ◽  
Jinzhong Li ◽  
Ainiwaer Mijiti ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Oral Cavity ◽  
Head And Neck ◽  
Feedback Loop ◽  
Growth Factor Receptor ◽  
Epidermal Growth

Abstract Background Alterations in the epidermal growth factor receptor and PI3K pathways in head and neck squamous cell carcinomas (HNSCCs) are frequent events that promote tumor progression. Ectopic expression of the epidermal growth factor receptor–targeting microRNA (miR), miR-27a* (miR-27a-5p), inhibits tumor growth. We sought to identify mechanisms mediating repression of miR-27a* in HNSCC, which have not been previously identified. Methods We quantified miR-27a* in 47 oral cavity squamous cell carcinoma patient samples along with analysis of miR-27a* in 73 oropharyngeal and 66 human papillomavirus–positive (HPV+) samples from The Cancer Genome Atlas. In vivo and in vitro TP53 models engineered to express mutant TP53, along with promoter analysis using chromatin immunoprecipitation and luciferase assays, were used to identify the role of TP53 and TP63 in miR-27a* transcription. An HNSCC cell line engineered to conditionally express miR-27a* was used in vitro to determine effects of miR-27a* on target genes and tumor cells. Results miR-27a* expression was repressed in 47 oral cavity tumor samples vs matched normal tissue (mean log2 difference = −0.023, 95% confidence interval = −0.044 to −0.002; two-sided paired t test, P = .03), and low miR-27a* levels were associated with poor survival in HPV+ and oropharyngeal HNSCC samples. Binding of ΔNp63α to the promoter led to an upregulation of miR-27a*. In vitro and in vivo findings showed that mutant TP53 represses the miR-27a* promoter, downregulating miR-27a* levels. ΔNp63α and nucleoporin 62, a protein involved in ΔNP63α transport, were validated as novel targets of miR-27a*. Conclusion Our results characterize a negative feedback loop between TP63 and miR-27a*. Genetic alterations in TP53, a frequent event in HNSCC, disrupt this regulatory loop by repressing miR-27a* expression, promoting tumor survival.

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