scholarly journals Size-specific follicle selection improves mouse oocyte reproductive outcomes

Reproduction ◽  
2015 ◽  
Vol 150 (3) ◽  
pp. 183-192 ◽  
Author(s):  
Shuo Xiao ◽  
Francesca E Duncan ◽  
Lu Bai ◽  
Catherine T Nguyen ◽  
Lonnie D Shea ◽  
...  
Keyword(s):  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Reproductive Outcomes ◽  
Follicle Growth ◽  
Follicle Diameter ◽  
Size Groups ◽  
Cell Embryo ◽  
Embryo Stage ◽  
Follicle Selection

Encapsulated in vitro follicle growth (eIVFG) has great potential to provide an additional fertility preservation option for young women and girls with cancer or other reproductive health threatening diseases. Currently, follicles are cultured for a defined period of time and analyzed as a cohort. However, follicle growth is not synchronous, and culturing follicles for insufficient or excessive times can result in compromised gamete quality. Our objective is to determine whether the selection of follicles based on size, rather than absolute culture time, better predict follicle maturity and oocyte quality. Multilayer secondary mouse follicles were isolated and encapsulated in 0.25% alginate. Follicles were cultured individually either for defined time periods or up to specific follicle diameter ranges, at which point several reproductive endpoints were analyzed. The metaphase II (MII) percentage after oocyte maturation on day 6 was the highest (85%) when follicles were cultured for specific days. However, if follicles were cultured to a terminal diameter of 300–350 μm irrespective of absolute time in culture, 93% of the oocytes reached MII. More than 90% of MII oocytes matured from follicles with diameters of 300–350 μm showed normal spindle morphology and chromosome alignment, 85% of oocytes showed two pronuclei after IVF, 81% developed into the two-cell embryo stage and 38% developed to the blastocyst stage, all significantly higher than the percentages in the other follicle size groups. Our study demonstrates that size-specific follicle selection can be used as a non-invasive marker to identify high-quality oocytes and improve reproductive outcomes during eIVFG.

428. Normal follicle growth and maturation in a three-dimensional in vitro culture system: follicular environment manipulation and assessment of oocyte outcomes

2008 ◽  
Vol 20 (9) ◽  
pp. 108
Author(s):  
K. R. Dunning ◽  
L. K. Akison ◽  
D. L. Russell ◽  
R. J. Norman ◽  
R. L. Robker
Keyword(s):  
Culture System ◽  
Cumulus Cell ◽  
Three Dimensional ◽  
Oocyte Quality ◽  
Metabolic Effects ◽  
Follicle Growth ◽  
Cumulus Expansion ◽  
Follicle Diameter

In vivo, the oocyte matures in a niche environment surrounded by somatic cells, and later in ovarian follicular development, by follicular fluid. Maternal diet influences the environment in which an oocyte matures but the mechanisms by which an altered metabolic profile, such as hyperinsulinemia, affects oocyte quality are not known. We investigated the use of a three dimensional follicle culture system allowing direct manipulation of the follicular environment thus circumventing systemic hormonal and metabolic effects. Secondary follicles (113.4 ± 1.02µm, n = 54) were isolated from mice at d12, encapsulated individually in 2µl of alginate matrix, and cultured in aMEM/5%FCS/10 mIU/mL LH/100 mIU FSH at 37°C/5%CO2, with media sampling and replacement every second day. Following 12 days of culture there was a significant 3-fold increase in follicle diameter (320 ± 10.1µm, n = 51). Histological analysis showed normal follicular morphology and antrum formation. Analysis of oestradiol (15.0ng/mL), androstenedione (7.8ng/mL) and progesterone (23.7ng/mL) in the media at d12 confirmed normal steroidogenesis and differentiation. Treatment of follicles with an ovulatory stimulus (1.5IU/mL hCG/5ng/mL Egf), resulted in cumulus expansion and hyaluronan localising to the cumulus oocyte complex (COC) and follicular basement membrane. These analyses were consistent with follicle growth and induction of ovulation in vivo. Further, COCs isolated from follicles and matured in vitro (IVM) in the presence of Egf and FSH, underwent cumulus expansion (CEI 2.8 ± 0.2) and were capable of fertilisation and blastocyst development. LH did not induce IVM COC expansion (CEI 1.36 ± 0.2), reflecting the normal in vivo differentiation process. However, culturing follicles in high insulin (5ug/mL) led to a significant increase in the degree of IVM cumulus expansion in response to LH (CEI 2.1 ± 0.3) indicating inappropriate cumulus cell differentiation, which may lead to poorer oocyte quality. These results demonstrate that this technique recapitulates normal in vivo folliculogenesis and is useful for manipulation of the follicular environment and assessment of oocyte outcomes.

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

scholarly journals In vitro growth and steroidogenesis of dog follicles are influenced by the physical and hormonal microenvironment

Reproduction ◽  
2011 ◽  
Vol 142 (1) ◽  
pp. 113-122 ◽  
Author(s):  
N Songsasen ◽  
T K Woodruff ◽  
D E Wildt
Keyword(s):  
Structural Integrity ◽  
Cell Function ◽  
Separate Experiment ◽  
In Vitro Growth ◽  
Follicle Growth ◽  
Steroid Production ◽  
Follicle Diameter ◽  
Equine Chorionic Gonadotropin ◽  
Over Time

The present study examined the influences of the physical and hormonal microenvironment on in vitro growth and steroidogenesis of dog follicles. Follicles were enzymatically isolated and individually encapsulated in 0.5% (w/v; n=17) or 1.5% (n=10) alginate and cultured with 0.5 IU/ml equine chorionic gonadotropin for 192 h. In a separate experiment, follicles were encapsulated in 0.5% alginate and cultured with 0 (n=22), 1 (n=23), 10 (n=20) or 100 (n=21) μg/ml FSH for 240 h. Follicle diameter and steroid production were assessed every 48 h in both studies. Follicles encapsulated in the 0.5% alginate grew faster (P<0.05) than those cultured in the 1.5% concentration. Oestradiol (E2) and progesterone (P4) increased consistently (P<0.05) over time, and follicles in the 1.5% alginate produced more (P<0.05) P4 than those in the 0.5% solution. Follicles cultured in the highest FSH concentration (100 μg/ml) increased 100% in size after 240 h compared with 50 to 70% in lower dosages. E2 concentration remained unchanged over time (P>0.05) across FSH dosages. However, P4 increased (P<0.05) as culture progressed and with increasing FSH concentration. Results demonstrate that dog follicles cultured in alginate retain structural integrity, grow in size and are hormonally active. Lower alginate and increasing FSH concentrations promote in vitro follicle growth. However, the absence of an E2 rise in follicles cultured in FSH alone suggests the need for LH supplementation to support theca cell differentiation and granulosa cell function.

scholarly journals DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Alma López ◽  
Miguel Betancourt ◽  
Yvonne Ducolomb ◽  
Juan José Rodríguez ◽  
Eduardo Casas ◽  
...  
Keyword(s):  
Dna Damage ◽  
Embryo Development ◽  
Tail Length ◽  
Cumulus Cells ◽  
Cell Types ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Development Rates

Abstract Background The evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. Results The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length (CTL) and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69%, Toxicity; 66.7 ± 4.57%, Vitrification) and cumulus cells viability (32.7 ± 5.85%, Toxicity; 7.7 ± 2.21%, Vitrification) compared to control (95.5 ± 4.04%, oocytes; 89 ± 4.24%, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65%, 22.6 ± 3.05%, respectively) and vitrification (32.3 ± 9.29%, 20 ± 1%, respectively). It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in embryo development to the blastocyst stage was observed after CPAs exposure (1.66 ± 0.57%) or vitrification (2 ± 1%) compared to control (22.3 ± 2.51%). This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells. Conclusion This study demonstrates that oocyte exposure to CPAs or vitrification reduced viability in oocytes and cumulus cells, and generated DNA damage in the cumulus cells, affecting fertilization and embryo development rates. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification that compromise their developmental capacity, as well as the search for new vitrification strategies to increase fertilization and embryo development rates by preserving the integrity of the cumulus cells.

278 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION AND SUPERSTIMULATION ON THE NORMALITY OF BOVINE EMBRYOS PRODUCED IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 296 ◽  
Author(s):  
K. Imai ◽  
T. Somfai ◽  
M. Ohtake ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
...  
Keyword(s):  
Cell Division ◽  
Development Program ◽  
Time Lapse ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Cell Stage ◽  
Follicular Wave ◽  
Significant Difference

We previously reported that follicular wave synchronization by dominant follicle removal on Day 5 and the start of a superstimulatory treatment on Day 7 after ovum pick-up (OPU) was effective to increase oocyte quality (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). The present study was designed to examine the effect of superstimulatory treatment-induced follicular wave synchronization on quality of embryos obtained by OPU and in vitro production. Japanese Black cows were reared under the same feeding and environmental conditions and 2 OPU sessions were conducted in each cow. The first OPU session was performed in 7 cows at arbitrary days of estrous cycle using a 7.5-MHz linear transducer with needle connected to an ultrasound scanner. Then, follicles larger than 8 mm in diameter were aspirated and CIDR was inserted on Day 5 (the day of first OPU session = Day 0). The cows then received 30 mg of FSH twice a day from Days 7 to 10 in decreasing doses (4, 4, 3, 3, 2, 2, 1, 1 mg per shot) by i.m. injections. Cloprostenol (PGF; 0.75 mg) was administered in the morning of Day 9. The second OPU session was performed 48 h after PGF administration (Day 11) and only follicles larger than 5 mm in diameter were aspirated. The CIDR was removed from the cows just before OPU. Grade 1 and 2 cumulus oocyte complexes were in vitro matured, fertilized (IVF), and cultured as described by Imai et al. (2006 J. Reprod. Dev. 52, Suppl. S19-29). Some zygotes were fixed and stained to check their sperm penetration. Embryo development was monitored by time-lapse cinematography for 168 h after IVF. Cleavage pattern of embryos was classified morphologically into normal and abnormal (including those with multiple fragments, protrusions, 3 to 4 blastomeres, and uneven cell division) groups at their first cleavage. Normal penetration rate of second OPU session was significantly (P < 0.05) higher than that of the first OPU session. There were no differences in the mean percentage of total blastocyst and grade 1 blastocyst rates between the first (45.2 and 46.9%, respectively) and second (47.5 and 41.8%, respectively) OPU sessions. However, the rates of blastocysts developing from embryos that were beyond the 4-cell stage at 48 h after IVF was significantly (P < 0.05) higher after the second OPU session (81.2%) than after the first OPU session (67.4%). Furthermore, a significant difference (P < 0.05) was found in the rates of normal cleavage at the first cell division in embryos that developed to the blastocyst stage between the first and second OPU sessions (53.3% and 73.9%, respectively). These results indicate that superstimulatory treatment-induced follicular wave synchronization improved the normality of fertilization and development of cattle oocytes obtained by OPU. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

MiR-290 family maintains developmental potential by targeting p21 in mouse pre-implantation embryos

2021 ◽  
Author(s):  
Xiangnan Li ◽  
Yueshi Liu ◽  
Qier Mu ◽  
Junliang Tian ◽  
Haiquan Yu
Keyword(s):  
Embryonic Development ◽  
Untranslated Region ◽  
Target Genes ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Cell Embryo ◽  
Embryo Stage ◽  
Genome Activation ◽  
Zygotic Genome

Abstract The miR-290 family is a mouse-specific microRNA cluster, which maintains mouse embryonic stem cells (ESCs) pluripotency by increasing OCT3/4 and C-MYC expression. However, its functions in mouse pre-implantation embryos remain unclear, especially during zygotic genome activation (ZGA). In this study, miR-290 family expression increased from the two-cell embryo stage through the blastocyst stage. Inhibition of miR-294-3p/5p did not affect ZGA initiation or embryo development, whereas pri-miR-290 knockdown decreased ZGA gene expression and slowed embryonic development. In addition, pluripotency decreased in ESCs derived from pri-miR-290 knockdown blastocysts. To clarify the mechanism of action, 33 candidate miR-294-3p target genes were screened from three databases, and miR-294-3p directly targeted the 3′-untranslated region of Cdkn1a (p21) mRNA. Similar to pri-miR-290 knockdown, P21 overexpression impeded embryonic development, whereas simultaneous overexpression of P21 and pri-miR-290 partially rescued embryonic development. The results indicate that the miR-290 family participates in promoting ZGA process and maintaining developmental potency in embryos by targeting p21.

224. Platelet derived growth factors and receptors in the rat ovary contribute towards preantral follicle growth

2005 ◽  
Vol 17 (9) ◽  
pp. 87
Author(s):  
L. S. Sleer ◽  
C. C. Taylor
Keyword(s):  
Growth Factors ◽  
Preantral Follicle ◽  
Follicle Growth ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Follicle Diameter ◽  
The Family ◽  
Rat Ovary ◽  
In Cells

In this study, the family of platelet derived growth factors (PDGF) and receptors were identified and characterized in the rat ovary and a role in contributing towards growth of preantral follicles was revealed. Real-time polymerase chain reaction revealed the presence of mRNA for all platelet derived growth factors (PDGF-A, PDGF-B, PDGF-C and PDGF-D) and receptors (PDGF-Rα and PDGF-Rβ). In situ hybridization identified oocytes of primordial/primary follicles and cells of the theca layer as a source of PDGF-B, PDGF-C and PDGF-D mRNA. Protein expression was explored through immunohistochemistry. In rats aged days 0 and 4, PDGF-Rα, PDGF-A and PDGF-C immunoreactivity was observed within oocyte clusters, and PDGF-Rβ and PDGF-B immunoreactivity in cells surrounding oocyte clusters. In primordial follicles, PDGF-Rβ and PDGF-C was observed in the oocyte, and PDGF-Rα and A in the either the oocyte or pregranulosa cells. In primary follicles, PDGF-A, PDGF-C, PDGF-Rα and PDGF-Rβ are expressed in the oocyte. PDGF-Rβ is also expressed in cells surrounding primordial and primary follicles, possibly the precursors to theca cells. In secondary and antral follicles, all four PDGF isoforms and both receptors are expressed in either theca or vascular cells of the theca layer, and PDGF-Rα and A are also expressed in some granulosa cells in rats aged day 20 and older. A role in preantral follicle growth was identified by in vitro culture of preantral follicles. Preantral follicles cultured in serum free medium increased in diameter by 11.0 ± 1.57% over 5 days. Addition of PDGF-AA, PDGF-AB or PDGF-BB to the medium resulted in increases in follicle diameter after 5 days of 18.32 ± 2.18%, 17.72 ± 2.3% and 17.6 ± 1.81%, respectively, representing a significant increase over control diameters. In summary, this study has identified and characterized the presence and localization of all members of the family of platelet derived growth factors and receptors in the rat ovary and revealed a role for these growth factors in positively influencing early follicle growth.

Expression and localization of Nlrp4g in mouse preimplantation embryo

Zygote ◽  
2014 ◽  
Vol 23 (6) ◽  
pp. 846-851 ◽  
Author(s):  
Hui Peng ◽  
Xiujiao Lin ◽  
Wenhao Li ◽  
Wenchang Zhang
Keyword(s):  
Preimplantation Embryo ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Preimplantation Embryo Development ◽  
Gene Copies ◽  
Mouse Preimplantation Embryo ◽  
Cell Embryo ◽  
Embryo Stage ◽  
Genome Activation ◽  
Zygotic Genome

SummaryThe Nlrp gene family contains 20 members and plays a pivotal role in the innate immune and reproductive systems in the mouse. During evolution, seven Nlrp4 gene copies (named from Nlrp4a to Nlrp4g). Nlrp4a–Nlrp4g have arisen that display specific or preferential ovarian expression patterns. However, the expression pattern and localization of Nlrp4g in mouse preimplantation embryo development are unknown. Here we report that Nlrp4g was highly expressed in mature oocytes and zygotes, then downregulated and not detected after the 2-cell embryo stage. NLRP4G protein remained present through the blastocyst stage and was mainly localized in the cytoplasm. Furthermore, overexpression of Nlrp4g in zygotes did not affect normal development in terms of the rate of blastocyst formation. These results provide the first evidence that NLRP4G is a maternal factor that may play essential role during zygotic genome activation in the mouse.

scholarly journals Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation

2017 ◽  
Vol 114 (29) ◽  
pp. E5796-E5804 ◽  
Author(s):  
Ye Yuan ◽  
Lee D. Spate ◽  
Bethany K. Redel ◽  
Yuchen Tian ◽  
Jie Zhou ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Fourfold Increase

Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called “FLI medium,” improves nuclear maturation of oocytes in cumulus–oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus–oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.

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