The Effects of Floating Granulosa Cells Density on the Developmental Competence of Bovine Oocytes: A Study Using the Well-in-Well Culture System

Group System ◽

Apoptosis Rate ◽

Bovine Oocytes ◽

Hatching Rates

In this study, we succeed to establish the well in the well (WIW) culture system. Using this system we studied the effect of floating granulosa cells density on oocyte developmental competence. Results: Apoptosis rate of the floating cells were high (73.576.3%) and did not differ (P > 0.05) among follicles of different sizes. Hatching rates of blastocysts did not differ (P > 0.05) between oocytes cultured in the WIW system (13.3%) and those cultured in the group system (16.3%). In 4-8mm follicles, oocyte developmental competence is higher (P < 0.05) in follicles with 1030×105/mL floating cells than in follicles with < 10 or >30%×105/mL floating cells.

The Effect of Granulosa Cells Apoptosis on the Cumulus Expansion and the Developmental Competence of Bovine Oocytes

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.997.251 ◽

2014 ◽

Vol 997 ◽

pp. 251-254 ◽

Cited By ~ 1

Author(s):

Wei Guo Feng ◽

Zhi Fang Pan

Keyword(s):

Granulosa Cells ◽

Culture System ◽

Bovine Oocyte ◽

Cumulus Expansion ◽

In this study, we used the well in the well (WIW) culture system to study the effect of granulosa cells apoptosis on the cumulus expansion and the developmental competence of bovine oocytes. Results: The apoptosis of granulosa cells affect the cumulus expansion of bovine oocytes significantly. Especially when the percentage of granulosa cells apoptosis exceed 40%, the cumulus expansion was worse. The cumulus expansion affect the oocyte developmental competence of bovine oocytes significantly. The developmental competence of bovine oocyte increases with the increasing of cumulus expansion.

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 ◽

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.

Presence of LH receptor mRNA in granulosa cells as a potential marker of oocyte developmental competence and characterization of the bovine splicing isoforms

Reproduction ◽

10.1530/reprod/125.3.437 ◽

2003 ◽

Vol 125 (3) ◽

pp. 437-446

Author(s):

C Robert

Keyword(s):

Granulosa Cells ◽

Lh Receptor ◽

Receptor Mrna ◽

Potential Marker ◽

Splicing Isoforms

132 The Developmental Competence of Bovine Oocytes Exposed to Progesterone and Luteotrophic Hormones During the Second Step of Two-Step Maturation

10.1071/rdv30n1ab132 ◽

2018 ◽

Vol 30 (1) ◽

pp. 206

Author(s):

G. Singina ◽

I. Lebedeva ◽

T. Taradajnic ◽

E. Shedova ◽

A. Lopukhov ◽

...

Keyword(s):

Granulosa Cells ◽

Calf Serum ◽

Second Step ◽

Russian Science ◽

Cleavage Rate ◽

System 1 ◽

And Control ◽

Data on effects of progesterone (P4) during in vitro maturation of bovine oocytes on their capacity for embryonic development are contradictory. Our study was aimed at characterising effects of P4 and 2 luteotropic hormones, prolactin (PRL) and LH, on bovine oocyte developmental competence during the second step of two-step maturation (from metaphase (M)I to MII). Slaughterhouse-derived cumulus-enclosed oocytes (CEO) were matured for 12 or 24 h [one-step (OS) Control] in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH at 38.5°C and 5% CO2. The CEO cultured for 12 h were transferred to the following culture systems: (1) TCM-199 containing 10% FCS (Control 1) or (2) a monolayer of granulosa cells (GC) precultured for 12 h in TCM-199 containing 10% FCS (Control 2); then, the oocytes were matured for next 12 h. In both systems, the medium of experimental groups was supplemented with either P4 (50 ng mL−1) or bovine PRL (25 and 50 ng mL−1) or ovine LH (5 μg mL−1). All treatments were repeated 5 to 6 times using 138 to 196 oocytes per group. Following IVM, all oocytes underwent IVF as described previously (Singina et al. 2014 Reprod. Fertil. Dev. 26, 154). Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured to Day 7. Embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst formation. Apoptosis was detected by the TUNEL method using 26 to 47 blastocysts per group (from 4 to 5 separate experiments). For each system, arcsine-transformed data were analysed by one-way ANOVA. In OS Control, the cleavage and blastocyst rates were 68.9 ± 4.4% and 22.0 ± 2.4%, respectively. Regardless of the system or medium of two-step culture, the cleavage rate did not differ from that for OS Control, varying between 57.6 and 68.4%. In the absence of GC (System 1), the blastocyst yield in the P4 group (30.4 ± 0.8%) was greater (P < 0.05) than in OS Control and Control 1 (20.2 ± 2.7%) as well as in the groups treated with LH (19.1 ± 3.0%) and 25 ng mL−1 PRL (20.1 ± 2.7%). In the presence of GC, P4 raised the yield from 16.7 ± 2.3% (Control 2) to 27.7 ± 2.4% (P < 0.05). Furthermore, in System 2, the blastocyst rate in groups treated with P4 and 50 ng mL−1 PRL (25.0 ± 2.8%) was higher (P < 0.05) than in the LH group (13.9 ± 2.6%). Meanwhile, the proportion of apoptotic nuclei (2.3-6.9%) was not associated with the system of oocyte maturation or effects of hormones studied. Our data indicate that P4 (50 ng mL−1) can enhance the developmental competence of bovine oocytes during the second step of two-step maturation regardless of the presence of granulosa cells, whereas the similar effect of PRL (50 ng mL−1) is less pronounced and depends on the granulosa-conditioned environment. This research was supported by the Russian Science Foundation (project 16-16-10069).

The effects of granulosa cell apoptosis on the developmental competence of bovine oocytes: A study using the well-in-well culture system

2013 ICME International Conference on Complex Medical Engineering ◽

10.1109/iccme.2013.6548301 ◽

2013 ◽

Cited By ~ 2

Author(s):

Weiguo Feng ◽

Guohui Wang ◽

Zhifang Pan ◽

Chune Ren

Keyword(s):

Granulosa Cell ◽

Cell Apoptosis ◽

Culture System ◽

325RETINOID-DEPENDENT POLY(A) MRNA CONTENTS IN BOVINE OOCYTES PREMATURED AND/OR MATURED IN VITRO

10.1071/rdv16n1ab325 ◽

2004 ◽

Vol 16 (2) ◽

pp. 282

Author(s):

L.J. Royo ◽

A. Rodriguez ◽

A. Gutierrez-Adan ◽

C. Diez ◽

E. Moran ◽

...

Keyword(s):

Cumulus Cells ◽

Defined Medium ◽

Mrna Levels ◽

Atp Production ◽

Beneficial Effects ◽

Grant Support ◽

Retinoic acid (RA) can induce cell differentiation and plays a role in controlling events within the cell cycle, but little is known of RA post-transcriptional modifications in the oocyte. Bovine oocyte and cumulus cells express most of RA receptors, and the presence of 9-cis-RA during in vitro prematuration and maturation (IVM) improves oocyte developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003 Reproduction 125, 409–416). This work analyzes the mRNA stability in bovine oocytes during in vitro prematuration and/or maturation. Cumulus-oocyte complexes (COCs) were cultured in defined medium with polyvinyl alcohol (DM). Those COCs undergoing prematuration were cultured for 24h in DM with 25μM roscovitine. For IVM, COCs were cultured in DM containing pFSH, LH and E2 for 24h, and some prematured COCs were then allowed to mature. Incubations were made at 39°C in 5% CO2 in air and high humidity. Within experiments, COCs were cultured with 5nM 9-cis-RA, in 1% ethanol (both as a vehicle and as an inhibitor of endogenous RA synthesis), 3% ethanol, 5% ethanol and untreated. Groups of 10 COCs per treatment were cultured, and oocytes detached from cumulus cells were analyzed. Poly(A) mRNA quantification was based on the pyrophosphorylation property of the DNA polymerase (Klenow). ATP production was measured by luminometric assay as a function of numbers of poly(A) tails. Data (4 replicates) were analyzed by ANOVA and Duncan’s test (v,x,y,zP<0.01; a,bP<0.05), and poly(A) mRNA (pg oocyte−1) was expressed as LSM±SE. After prematuration, poly(A) mRNA contents differed between 9-cis-RA (125.7±4.8x) and untreated (95.5±4.8y) oocytes, as compared to 1% ethanol (72.2±4.8z) and immature (71.5±4.8z) oocytes. After IVM, untreated oocytes (23.0±2.2v) showed the lowest poly(A) mRNA amount, and poly(A) mRNA in 9-cis-RA (36.2±2.2y) basically equalled that in 1% ethanol (35.2±2.2y), while 3% (44.5±2.2yz) and 5% ethanol (52.0±2.2z) increased poly(A) mRNA levels. All groups of matured oocytes showed poly(A) mRNA contents lower than in immature (71.5±4.8x). After prematuration+maturation, poly(A) mRNA values were 34.2±2.2v (untreated+untreated), 36.5±2.2v (9-cis-RA+untreated), 49.5±2.2xa (untreated+9-cis-RA), 41.0±2.2vxb (9-cis-RA+9-cis-RA) and 59.0±2.2y (untreated+1% ethanol). Levels of poly(A) mRNA from prematured+matured oocytes were again lower than in immature (71.5±4.8x). Our study shows that beneficial effects of RA on the oocyte developmental competence can be represented in part as a gain in the quality of mRNAs stored. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

207 DEVELOPMENT OF A ROUTINE IN VITRO EMBRYO PRODUCTION SYSTEM USING SINGLY IN VITRO-MATURED, FERTILIZED, AND CULTURED BOVINE OOCYTES

10.1071/rdv21n1ab207 ◽

2009 ◽

Vol 21 (1) ◽

pp. 201

Author(s):

I. G. F. Goovaerts ◽

J. L. M. R. Leroy ◽

J. B. P. De Clercq ◽

S. Andries ◽

P. E. J. Bols

Keyword(s):

Production System ◽

Cumulus Cell ◽

Control Individual ◽

Embryo Production ◽

Group Culture ◽

In Vitro Embryo Production ◽

Bovine Oocytes ◽

Hatching Rates

An in vitro embryo production system (IVP), in which a single oocyte can be tracked from the moment of retrieval up to the blastocyst stage, would be a valuable tool for studies linking developmental competence and embryo metabolism to oocyte quality and follicular environment. Unfortunately, to date, data on individual IVP are inconsistent, and in most cases show unsatisfactory blastocyst rates. Earlier studies revealed that individual culture on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos as obtained after regular group culture (Goovaerts et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 190). Because, in the latter study, single culture was performed after group maturation and fertilization, the aim of this study was to develop and test an individual IVP system using bovine oocytes or zygotes obtained after single maturation and single fertilization. Therefore, 532 grade I COC from slaughterhouse ovaries (3 replicates) were randomly assigned to 1 of 2 treatments: a complete individual IVP protocol, or a routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2) on a 6-day-old monolayer of matured CC (5% CO2 in air). Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were assessed 2, 8, and 10 days postfertilization, respectively. Possible effects of individual and group culture were evaluated with binary logistic regression (SPSS 15.0). No interactions between replicate and treatment could be found (P > 0.05). Cleavage and blastocyst rates were significantly lower after individual IVP, compared with group IVP, whereas the blastocyst rates on cleaved zygotes and the hatching rates did not differ significantly (Table 1). In conclusion, acceptable blastocyst rates (25.1%) could be obtained after individual IVP. The lower blastocyst rates were associated with the lower cleavage rates, and no effect of the individual embryo culture system on embryo development could be found. Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro embryo production (IVP)

Maturation, fertilisation and culture of bovine oocytes and embryos in an individually identifiable manner: a tool for studying oocyte developmental competence

10.1071/rd09277 ◽

2010 ◽

Vol 22 (5) ◽

pp. 839 ◽

Cited By ~ 27

Author(s):

Satoko Matoba ◽

Trudee Fair ◽

Patrick Lonergan

Keyword(s):

Blastocyst Stage ◽

Standard Group ◽

Success Rates ◽

Group Culture ◽

Polyester Mesh ◽

Development In Vitro ◽

The ability to successfully culture oocytes and embryos individually would facilitate the study of the relationship between follicle parameters and oocyte developmental competence, in order to identify markers of competent oocytes, as well as the ability to use small numbers of oocytes from an individual donor such as when ovum pick-up is carried out. Using a total of 3118 oocytes, the aim of the present study was to develop a system capable of supporting the development of immature bovine oocytes to the blastocyst stage in an individually identifiable manner. Initially, post-fertilisation embryo culture in the Well-of-the-Well (WOW) system, on the cell adhesive Cell-Tak or in polyester mesh was tested and shown to result in similar development to embryos cultured in standard group culture. The results demonstrate that it is possible to culture bovine oocytes to the blastocyst stage in an individually identifiable manner in all three culture systems with comparable success rates. This permits the localisation and identification of individual embryos throughout preimplantation development in vitro while retaining the developmental benefits of group culture. In terms of ease of preparation and use, culture in isolation within the strands of a polyester mesh is preferable.