scholarly journals Expression of Transcripts in Marmoset Oocytes Retrieved during Follicle Isolation Without Gonadotropin Induction

2019 ◽  
Vol 20 (5) ◽  
pp. 1133 ◽  
Author(s):  
Yoon Kim ◽  
Byeong-Cheol Kang ◽  
Jun Yun ◽  
Jae Ahn ◽  
Yong Kim ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
World Monkey ◽  
Strong Association ◽  
Assisted Reproductive Technique ◽  
Early Maturation ◽  
Sperm Binding ◽  
Maturation Rate ◽  
Pre Treatment

The in vitro maturation of oocytes is frequently used as an assisted reproductive technique (ART), and has been successfully established in humans and rodents. To overcome the limitations of ART, novel procedures for the in vitro maturation of early follicles are emerging. During the follicle isolation procedure, the unintended rupture of each follicle leads to a release of extra oocytes. Such oocytes, which are obtained during follicle isolation from marmosets, can be used for early maturation studies. Marmoset (Callithrix jacchus), which is classified as a new-world monkey, is a novel model that has been employed in reproductive biomedical research, as its reproductive physiology is similar to that of humans in several aspects. The ovaries of female marmosets were collected, and the excess oocytes present during follicle isolation were retrieved without pre-gonadotropin induction. Each oocyte was matured in vitro for 48 h in the presence of various concentrations of human chorionic gonadotropin (hCG) and epidermal growth factor (EGF), and the maturity of oocytes and optimal maturation conditions were evaluated. Each oocyte was individually reverse-transcribed, and the expression of mRNAs and microRNAs (miRs) were analyzed. Concentrations of hCG significantly affected the maturation rate of oocytes [the number of metaphase II (MII) oocytes]. The expression of BMP15 and ZP1 was highest when the oocytes were matured using 100 IU/L of hCG without pre-treatment with gonadotropins, and that of Cja-mir-27a was highest when cultured with follicle stimulating hormone. These results suggest that these up-regulated miRs affect the maturation of oocytes. Interactions with other protein networks were analyzed, and a strong association of BMP15 and ZP1 with sperm binding receptor (ACR), anti-Müllerian hormone (AMH), and AMH receptor was demonstrated, which is related to the proliferation of granulosa cells. Collectively, on the basis of these results, the authors propose optimal maturation conditions of excess oocytes of marmoset without in vivo gonadotropin treatment, and demonstrated the roles of miRs in early oocyte maturation at the single-cell level in marmosets.

314 MELATONIN ON IN VIVO AND IN VITRO MATURATION OF MOUSE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 246 ◽  
Author(s):  
H. Fernandes ◽  
L. Schefer ◽  
F. C. Castro ◽  
C. L. V. Leal
Keyword(s):  
Ovarian Stimulation ◽  
In Vitro Maturation ◽  
F1 Hybrids ◽  
Saline Control ◽  
Control Group ◽  
Mouse Oocytes ◽  
Maturation Rate ◽  
Different Doses

Melatonin is a pineal hormone related to the control of the circadian cycle, besides the reproductive seasonality of some animal species, and has shown positive effects on oocyte maturation and embryo development. The aim of this study was to assess the effects of melatonin on in vivo and in vitro maturation of mouse oocytes. Female F1 hybrids (C57BL/6 × CBA; n = 8 per group/treatment) were used in 3 different treatments (trt) groups: (I) in vivo trt: mice received 2 different doses of melatonin injections, 10 and 20 mg kg–1 per IP including a saline control dose (0 mg kg–1 per IP) for 4 days along with ovarian stimulation trt of 5 IU of eCG IP, followed by 5 IU of hCG IP 48 h later, and cumulus-oocyte complexes (COC) were collected 16 h after hCG; (II) mice received a similar in vivo melatonin trt, but ovarian stimulation trt was only 5 IU of eCG, no hCG, and COC were collected after 48 h and subsequently matured in vitro with 0.5 µg mL–1 of FSH for 16 h; (III) in vitro maturation of oocytes: COC were collected 48 h after 5 IU of eCG and maturated in the presence of 3 different doses of melatonin (10–9, 10–6, and 10–3 M) or 0.5 µg mL–1 of FSH (control) for 16 h. In vitro-maturing oocytes were in incubated at 37°C, 5% CO2, and 95% humidity. Maturation rates were evaluated according to the presence of the first polar body under an inverted microscope. Statistical analyses were performed by ANOVA followed by Tukey's test (4 replicates). In the first treatment, 20 mg kg–1 of melatonin showed the highest in vivo maturation rate, 80.3% (61/76), while 10 mg kg–1 of melatonin was 62.4% (53/85) and the saline control group was 69.4% (77/111), but differences were not significant (P > 0.05). For in vitro maturation of oocytes from animals previously treated with melatonin, the 10 mg kg–1 of melatonin group had the highest maturation rate, 53.2% (99/186), in comparison with the saline and 20 mg kg–1 of melatonin groups, which showed 46.6 (88/189) and 39.0% (85/218), respectively; again, no differences were detected (P > 0.05). In the last treatment, the maturation rates increased from 48.9 (43/88) to 53.7 (51/95) and 56.0% (56/100) as the melatonin concentrations decreased from 10–3, 10–6, and 10–9 M, respectively. The control group had the highest rate of 57.3% (55/96), but no statistical differences were observed (P = 0.706). In conclusion, under the conditions studied, melatonin was unable to improve the maturation rate neither after in vivo nor in vitro treatment. However, during in vitro maturation, melatonin alone was as efficient as FSH in promoting maturation in murine oocytes, indicating its potential effect on stimulating meiosis. Therefore, the role of melatonin in stimulating meiosis needs further investigation.Acknowledgments to FAPESP for fellowship (HF) and funding (CLVL).

Effect of hormone pre-treatment of prepubertal sheep on the production and developmental capacity of oocytes in vitro and in vivo

1997 ◽  
Vol 9 (6) ◽  
pp. 625 ◽  
Author(s):  
J. K. O'Brien ◽  
N. F. G. Beck ◽  
W. M. C. Maxwell ◽  
G. Evans
Keyword(s):  
In Vitro Maturation ◽  
Single Treatment ◽  
Oocyte Collection ◽  
Oestradiol Benzoate ◽  
Developmental Capacity ◽  
Pre Treatment ◽  
Pregnant Mare Serum Gonadotrophin

Twenty 36-week-old Merino lambs were given either 3, 1 or 0 treatments of 50 µg oestradiol benzoate and (48 h later) a 1·5 mg Norgestamet implant left in situ for 9 days (3-, 1- and 0CYCLE+G). On Day 7 after the last implant insertion, and on the same day for 0CYCLE+G, each lamb received 400 I.U. pregnant mare serum gonadotrophin and 6 mg follicle-stimulating hormone (FSH). The reproductive tracts were removed for oocyte collection 24 h after FSH. Reproductive tracts were also collected from 16-24-week-old lambs (n = 31) (0CYCLEG). The number of antral follicles per ovary was similar for the 3-, 1- and 0CYCLE+G treatments. Similar rates of in vitro maturation and monospermic fertilization were obtained for all groups. The proportion of blastocysts per cleaved oocyte was higher for 1CYCLE+G (50·5%) than for 3CYCLE+G (32·9%), 0CYCLE+G (24·3%), and 0CYCLEG (11·8%) (P < 0·05). Viable fetuses were obtained at Day 93 of pregnancy after transfer of embryos from all treatments. These results indicate that a single treatment with oestrogen and progesterone, prior to gonadotrophin stimulation, will increase the yield and developmental capacity of oocytes from prepubertal sheep.

Investigations of oocyte in vitro maturation within a mouse model

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Boon Chin Alexis Heng ◽  
Ng Soon Chye
Keyword(s):  
In Vitro Maturation ◽  
Culture Medium ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Standard Duration ◽  
Maturation Rate ◽  
Meiotic Competence

This study attempted to develop a ‘less meiotically competent’ murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze–thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.

157 EFFECT OF FSH STARVATION (COASTING) FOLLOWING SUPEROVULATION ON OOCYTE COMPETENCE AND CLONING EFFICIENCY IN GOATS

2017 ◽  
Vol 29 (1) ◽  
pp. 187 ◽  
Author(s):  
C. E. Méndez-Calderón ◽  
C. R. Lazzarotto ◽  
L. H. Aguiar ◽  
F. L. Ongaratto ◽  
K. C. S. Tavares ◽  
...  
Keyword(s):  
Pregnancy Outcome ◽  
In Vitro Maturation ◽  
Animal Health ◽  
Oocyte Quality ◽  
Control Group ◽  
In Vitro Production ◽  
Oocyte Competence ◽  
Maturation Rate

Oocyte competence plays a key role in the overall efficiency of reproductive biotechnologies. In cattle, FSH starvation following superovulation (coasting) improves oocyte competence, blastocyst yield and pregnancy outcome when used in ovum pickup-in vitro production programs. The aim of this study was to compare the effect of coasting after exogenous FSH stimulation on goat oocyte quality and competence to support in vitro maturation and in vivo embryo development following cloning procedures in goats. Donor and recipient preparation, cumulus-oocyte complex (COC) retrieval and selection, IVM, cloning by somatic cell nuclear transfer, embryo transfer, and pregnancy diagnosis (Days 23–26) were performed according to our established procedures [Martins et al. 2016 doi: 10.1089/cell.2015.0082]. Cumulus-oocyte complexes were obtained in vivo from 71 cycling FSH-stimulated mature Nubian-crossed goats, combined or not with FSH starvation (coasting period). Donor females were oestrous synchronized with a progesterone intravaginal insert (Day 0). On Day 10, a 0.75-mg D-cloprostenol dose was given IM, with the onset of the superovulation treatment, composed of five 20-mg FSH doses (Folltropin®, Bioniche Animal Health, Pullman, WA, USA), via IM at 12-h intervals. Donors were subjected to laparoscopic ovum pickup either 9 h (control group, n = 36) or 21 h (coasting group, n = 35) after the last FSH dose, respectively. Skin fibroblast cell cultures from a male neonate were co-transfected with a mammary gland expression vector with the human lactoferrin (hLF) coding sequence and with CRISPR/Cas9 system either for the PRNP prion gene or the Rosa26 locus. A bi-allelic hLF-PRNP and a mono-allelic hLF-Rosa26 cell colony were used for cloning. Data were compared by ANOVA or the χ2 test (P < 0.05). No differences were observed between control and coasting for number of follicles (18.7 ± 1.4 v. 21.2 ± 1.7), and retrieved (17.3 ± 1.2 v. 20.7 ± 1.9), viable (15.9 ± 1.1 v. 19.6 ± 1.8), Grade I (1.5 ± 0.3 v. 2.5 ± 0.5), and Grades III+IV (6.0 ± 0.6 v. 5.7 ± 0.7) COC, as well as for COC retrieval (92.4%, 574/621 v. 94.5%, 685/725) and fusion (62.8%, 273/435 v. 61.3%, 311/507) rates, respectively, irrespective of the cell lines. However, the coasting group rendered higher number of Grade II COC (11.3 ± 1.2 v. 8.4 ± 0.7), number and proportion of Grades I+II COC (13.9 ± 1.5 v. 9.9 ± 0.9, 70.8% v. 62.4%), and maturation rate (70.9% v. 65.4%) than the control group, respectively, for a lower proportion of Grades III+IV (29.2% v. 37.6%, respectively). A total of 213 and 233 Day-1 cloned embryos from the control and the coasting groups were transferred to 18 (96/9 hLF-PRNP and 117/9 hLF-Rosa26 cells) and 19 (128/11 hLF-PRNP and 105/8 hLF-Rosa26 cells) female recipients, respectively, resulting in 1/9 (11.1%) and 4/11 (36.4%) pregnancies from the hLF-PRNP cells, and 3/9 (33.3%) and 3/8 (37.5%) from the hLF-Rosa26 cells, for the control (4/18, 22.2%) and coasting (7/19, 36.8%) groups, respectively, for an overall pregnancy rate of 29.7% (11/37). In conclusion, the use of coasting improved oocyte quality and in vitro maturation rate, also appearing to increase pregnancy outcome after goat cloning.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

scholarly journals Protein oligomerization is the biochemical process highly up-regulated in porcine oocytes before in vitro maturation (IVM)

2018 ◽  
Vol 6 (4) ◽  
pp. 155-162 ◽  
Author(s):  
Sylwia Borys-Wójcik ◽  
Ievgenia Kocherova ◽  
Piotr Celichowski ◽  
Małgorzata Popis ◽  
Michal Jeseta ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Protein Oligomerization ◽  
Dynamic Nature ◽  
Affymetrix Microarray ◽  
Expression Of Genes ◽  
Before And After ◽  
Porcine Oocyte ◽  
Lower Expression

AbstractA wide variety of mechanisms controlling oligomerization are observed. The dynamic nature of protein oligomerization is important for bioactivity control. The oocyte must undergo a series of changes to become a mature form before it can fully participate in the processes associated with its function as a female gamete. The growth of oocytes in the follicular environment is accompanied by surrounding somatic cumulus (CCs) and granulosa cells (GCs). It has been shown that oocytes tested before and after in vitro maturation (IVM) differ significantly in the transcriptomic and proteomic profiles. The aim of this study was to determine new proteomic markers for the oligomerization of porcine oocyte proteins that are associated with cell maturation competence. The Affymetrix microarray assay was performed to examine the gene expression profile associated with protein oligomerization in oocytes before and after IVM. In total, 12258 different transcriptomes were analyzed, of which 419 genes with lower expression in oocytes after IVM. We found 9 genes: GJA1, VCP, JUP, MIF, MAP3K1, INSR, ANGPTL4, EIF2AK3, DECR1, which were significantly down-regulated in oocytes after IVM (in vitro group) compared to oocytes analyzed before IVM (in vivo group). The higher expression of genes involved in the oligomerization of the protein before IVM indicates that they can be recognized as important markers of biological activation of proteins necessary for the further growth and development of pig embryos.

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