Restraint Stress on Female Mice Diminishes the Developmental Potential of Oocytes: Roles of Chromatin Configuration and Histone Modification in Germinal Vesicle Stage Oocytes1

In the pig, the efficiency of in vitro embryo production procedures is still limited. It has been suggested that prematuration treatments could improve the developmental capability of oocytes. In particular, recent studies conducted in the bovine (Luciano, 2011, BOR, in press) indicate that the prolongation of a patent bidirectional crosstalk between the oocyte and the surrounding cumulus cells, together with the maintenance of a proper level of cAMP during the prematuration culture, could be beneficial to oocytes that have not yet acquired full meiotic and developmental capability. The aim of the present study was to assess the effect of treatment with cilostamide, an inhibitor of the phosphodiesterase 3 (PDE3), which degrades cAMP, on the functional status of gap junction-mediated communication (GJC) in pig cumulus–oocyte complexes (COC). Moreover, since chromatin configuration represents a marker of oocyte differentiation and competence, the effect of cilostamide on the process of chromatin remodeling was also evaluated during the culture period. To this aim, COC were collected from 3- to 6-mm antral follicles and cultured for up to 24 h in defined culture medium supplemented with 0.1 IU mL–1 of FSH in the presence or absence of 1 μM cilostamide. The GJC functionality was assessed by Lucifer Yellow fluorescent dye microinjection at the time of collection (0 h) and after 12, 18, or 24 h of culture. Chromatin configuration was evaluated by fluorescence microscopy after removal of cumulus cells and DNA staining with Hoechst and oocytes were classified according to Bui et al. (2004 BOR 70, 1843–1851) as SC, (with stringy chromatin within the germinal vesicle), GVI (with chromatin condensed in a rim around the nucleolus), GVII-IV (where the beginning of formation of chromatin strands is typical), ProMI (prometaphase I) and MI (metaphase I). The administration of cilostamide sustained functional coupling for up to 24 h of culture as the percentage of COC with open GJC was significantly higher when compared with the control group (62.2% vs 30%; P < 0.05) and not significantly different from the time 0 h (80%). The maintenance of the coupling during the culture period was accompanied by a delay of the meiotic resumption as only 26.3% of cilostamide-treated oocytes underwent germinal-vesicle breakdown and reached ProMI stage compared to the control group (62.1%; P < 0.05). Moreover the transition towards advanced stages of differentiation, as judged by the chromatin configuration, was slowed down in the presence of cilostamide. In conclusion, our study indicates that the maintenance of elevated cAMP levels through the inhibition of PDE3 sustains a functional bidirectional communication between the oocyte and cumulus cells and delays meiotic resumption in the pig oocyte. This could be a useful approach for the development of prematuration treatments aimed at improving the embryonic developmental potential of pig oocytes. Experiments are in progress in our laboratories to confirm this hypothesis. This study has been supported by EU FP6 grant n LSHB-CT-2006-037377 (Xenome) EU FP7- n°223485 (Plurisys).

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Restraint Stress on Female Mice Diminishes the Developmental Potential of Oocytes

Obstetrical & Gynecological Survey ◽

10.1097/01.ogx.0000467231.03943.2d ◽

2015 ◽

Vol 70 (6) ◽

pp. 395-396

Author(s):

Xiu-Fen Wu ◽

Hong-Jie Yuan ◽

Hong Li ◽

Shuai Gong ◽

Juan Lin ◽

...

Keyword(s):

Restraint Stress ◽

Developmental Potential ◽

Female Mice

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RASGRF1 in CRF cells controls the early adolescent female response to repeated stress

Journal of Endocrinology ◽

10.1530/joe-19-0375 ◽

2020 ◽

Vol 245 (3) ◽

pp. 397-410

Author(s):

Shan-xue Jin ◽

David A Dickson ◽

Jamie Maguire ◽

Larry A Feig

Keyword(s):

Restraint Stress ◽

Elevated Serum ◽

Early Adolescent ◽

Nucleotide Exchange ◽

Female Response ◽

Female Mice ◽

Repeated Stress ◽

Guanine Nucleotide Exchange ◽

Rac Gtpases ◽

Nucleotide Exchange Factor

RASGRF1 (GRF1) is a calcium-stimulated guanine-nucleotide exchange factor that activates RAS and RAC GTPases. In hippocampus neurons, it mediates the action of NMDA and calcium-permeable AMPA glutamate receptors on specific forms of synaptic plasticity, learning, and memory in both male and female mice. Recently, we showed GRF1 also regulates the HPA axis response to restraint stress, but only in female mice before puberty. In particular, we found that after 7 days of restraint stress (7DRS) (30 min/day) both elevated serum CORT levels and induction of an anxiolytic phenotype normally observed in early adolescent (EA) female mice are blocked in GRF1-knockout mice. In contrast, no effects were observed in EA male or adult females. Here, we show this phenotype is due, at least in part, to GRF1 loss in CRF cells of the paraventricular nucleus of the hypothalamus, as GRF1 knockout specifically in these cells suppressed 7DRS-induced elevation of serum CORT levels specifically in EA females, but only down to levels found in comparably stressed EA males. Nevertheless, it still completely blocked the 7DRS-induced anxiolytic phenotype observed in EA females. Interestingly, loss of GRF1 in CRF cells had no effect after only three restraint stress exposures, implying a role for GRF1 in 7DRS stress-induced plasticity of CRF cells that appears to be specific to EA female mice. Overall, these findings indicate that GRF1 in CRF cells makes a key contribution to the distinct response EA females display to repeated stress.

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Meiotic Spindle Characteristics of Oocytes After Vitrification at Germinal Vesicle Stage, Warming and Maturation In Vitro and Developmental Potential After ICSI

Journal of Equine Veterinary Science ◽

10.1016/j.jevs.2018.05.091 ◽

2018 ◽

Vol 66 ◽

pp. 202 ◽

Cited By ~ 2

Author(s):

L.J. Maclellan ◽

J.N. Hatzel ◽

F. Amoroso ◽

J.E. Stokes ◽

E.M. Carnevale

Keyword(s):

Germinal Vesicle ◽

Meiotic Spindle ◽

Developmental Potential ◽

Maturation In Vitro

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Mice homozygous for an insertional mutation in the Zp3 gene lack a zona pellucida and are infertile

Development ◽

10.1242/dev.122.9.2903 ◽

1996 ◽

Vol 122 (9) ◽

pp. 2903-2910 ◽

Cited By ~ 15

Author(s):

T. Rankin ◽

M. Familari ◽

E. Lee ◽

A. Ginsberg ◽

N. Dwyer ◽

...

Keyword(s):

Zona Pellucida ◽

Genetic Defect ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Early Embryo ◽

Preimplantation Embryos ◽

Germinal Vesicle Breakdown ◽

Female Mice ◽

Insertional Mutation ◽

Homozygous Mutant

Mammalian oocytes synthesize and secrete a zona pellucida that surrounds the growing oocytes, ovulated eggs and preimplantation embryos. The extracellular zona matrix is composed of three glycoproteins (ZP1, ZP2, ZP3) that are involved in folliculogenesis, species-specific fertilization, and passage of the early embryo down the oviduct. We have established a mouse line in which Zp3 has been inactivated by homologous recombination with an insertional mutation. Neither Zp3 transcripts nor ZP3 protein was detected in female mice homozygous for the mutation (Zp3−/−), whereas both ZP1 and ZP2 were present in mutant oocytes. Homozygous mutant Zp3−/− mice had follicles with germinal-vesicle-intact oocytes but that lacked a zona pellucida matrix and had a disorganized corona radiata. Although mutant oocytes underwent germinal vesicle breakdown (GVBD) prior to ovulation, the cumulus-oocyte complex was markedly disrupted and the oocytes were often separate from the cumulus cells. After hormone-induced ovulation, cumulus masses were present in the oviducts of homozygous mutant mice, but zona-free eggs were observed in only half of the females and, in these, less than 10% of the normal number [correction of mumber] of eggs were detected. No zona-free 2-cell embryos were recovered from homozygous mutant Zp3−/− female mice after mating with males proven to be fertile, and none became visibly pregnant or produced offspring. These results demonstrate that a genetic defect in a zona pellucida gene causes infertility and, given the conserved nature of the zona pellucida, a similar phenotype is expected in other mammals.

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A prematuration approach to equine IVM: considering cumulus morphology, seasonality, follicle of origin, gap junction coupling and large-scale chromatin configuration in the germinal vesicle

Reproduction Fertility and Development ◽

10.1071/rd19230 ◽

2019 ◽

Vol 31 (12) ◽

pp. 1793 ◽

Cited By ~ 1

Author(s):

Valentina Lodde ◽

Silvia Colleoni ◽

Irene Tessaro ◽

Davide Corbani ◽

Giovanna Lazzari ◽

...

Keyword(s):

Gap Junction ◽

Embryo Development ◽

Large Scale ◽

Germinal Vesicle ◽

Culture Conditions ◽

Developmental Competence ◽

Morphological Criteria ◽

Chromatin Configuration ◽

Follicle Size ◽

Meiotic Competence

Several studies report that a two-step culture where mammalian oocytes are first kept under meiosis-arresting conditions (prematuration) followed by IVM is beneficial to embryo development. The most promising results were obtained by stratifying the oocyte population using morphological criteria and allocating them to different culture conditions to best meet their metabolic needs. In this study, horse oocytes were characterised to identify subpopulations that may benefit from prematuration. We investigated gap-junction (GJ) coupling, large-scale chromatin configuration and meiotic competence in compact and expanded cumulus–oocyte complexes (COCs) according to follicle size (<1, 1–2, >2cm) and season. Then we tested the effect of cilostamide-based prematuration in compact COCs collected from follicles <1 and 1–2cm in diameter on embryo development. Meiotic competence was not affected by prematuration, whereas COCs from follicles 1–2cm in diameter yielded embryos with a higher number of cells per blastocyst than oocytes that underwent direct IVM (P<0.01, unpaired Mann–Whitney test), suggesting improved developmental competence. Oocytes collected from follicles <1cm in diameter were not affected by prematuration. This study represents an extensive characterisation of the functional properties of immature horse oocytes and is the first report of the effects of cilostamide-based prematuration in horse oocyte IVM on embryo development.

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Production of good-quality blastocyst embryos following IVF of ovine oocytes vitrified at the germinal vesicle stage using a cryoloop

Reproduction Fertility and Development ◽

10.1071/rd12215 ◽

2013 ◽

Vol 25 (8) ◽

pp. 1204 ◽

Cited By ~ 18

Author(s):

Adel R. Moawad ◽

Jie Zhu ◽

Inchul Choi ◽

Dasari Amarnath ◽

Wenchao Chen ◽

...

Keyword(s):

Germinal Vesicle ◽

Blastocyst Stage ◽

Control Groups ◽

Blastocyst Development ◽

Chromosome Configuration ◽

Nuclear Maturation ◽

Chromatin Configuration ◽

And Control ◽

First Time

The cryopreservation of immature oocytes at the germinal vesicle (GV) stage would create an easily accessible, non-seasonal source of female gametes for research and reproduction. The present study investigated the ability of ovine oocytes vitrified at the GV stage using a cryoloop to be subsequently matured, fertilised and cultured in vitro to blastocyst-stage embryos. Selected cumulus–oocyte complexes obtained from mature ewes at the time of death were randomly divided into vitrified, toxicity and control groups. Following vitrification and warming, viable oocytes were matured in vitro for 24 h. Matured oocytes were either evaluated for nuclear maturation, spindle and chromosome configuration or fertilised and cultured in vitro for 7 days. No significant differences were observed in the frequencies of IVM (oocytes at the MII stage), oocytes with normal spindle and chromatin configuration and fertilised oocytes among the three groups. Cleavage at 24 and 48 h post insemination was significantly decreased (P < 0.01) in vitrified oocytes. No significant differences were observed in the proportion of blastocyst development between vitrified and control groups (29.4% v. 45.1%, respectively). No significant differences were observed in total cell numbers, the number of apoptotic nuclei or the proportion of diploid embryos among the three groups. In conclusion, we report for the first time that ovine oocytes vitrified at the GV stage using a cryoloop have the ability to be matured, fertilised and subsequently developed in vitro to produce good-quality blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

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Effects of roscovitine on maintenance of the germinal vesicle in horse oocytes, subsequent nuclear maturation, and cleavage rates after intracytoplasmic sperm injection

Reproduction ◽

10.1530/rep.0.1250693 ◽

2003 ◽

pp. 693-700 ◽

Cited By ~ 21

Author(s):

LC Franz ◽

YH Choi ◽

EL Squires ◽

K Hinrichs ◽

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Germinal Vesicle ◽

Meiotic Maturation ◽

Developmental Potential ◽

Nuclear Maturation ◽

Maturation Medium

This study was conducted to evaluate the effects of roscovitine on suppression of meiosis, subsequent meiotic maturation, and cleavage rates after intracytoplasmic sperm injection of horse oocytes. Oocytes were classified as having compact or expanded cumuli (Com or Exp oocytes) and were divided into three culture groups: 30 h culture in maturation medium (30 h Mat); 54 h culture in maturation medium (54 h Mat), or 24 h culture in medium containing 66 micro mol roscovitine l(-1) and then 30 h culture in maturation medium (Ros+M). After maturation, oocytes were subjected to intracytoplasmic sperm injection and cultured in G1.2 medium for 96 h. Among oocytes fixed immediately after roscovitine culture, 26 of 31 (84%) Com oocytes and 16 of 28 (57%) Exp oocytes were at the germinal vesicle stage (P<0.05). After maturation culture, there were no differences in maturation rates or morphological cleavage rates among treatments. Among Com oocytes, significantly more embryos in the Ros+M treatment than in the 54 h Mat treatment had cleaved with > or = two normal nuclei (63 versus 36%; P<0.05); whereas among Exp oocytes, significantly more embryos in the 30 h Mat treatment than in the Ros+M treatment (63 versus 42%; P<0.05) had cleaved with > or = two normal nuclei. The average number of nuclei in embryos at 96 h was significantly higher (P<0.05) in Ros+M Com oocytes (13.5) than in any other Com or Exp group. These results demonstrate that roscovitine can reversibly maintain equine oocytes in the germinal vesicle stage for up to 24 h, and that such suppression may increase the developmental potential of Com, but not Exp, oocytes.

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The position of the germinal vesicle and the chromatin organization together provide a marker of the developmental competence of mouse antral oocytes

Reproduction ◽

10.1530/rep-09-0230 ◽

2009 ◽

Vol 138 (4) ◽

pp. 639-643 ◽

Cited By ~ 26

Author(s):

Michele Bellone ◽

Maurizio Zuccotti ◽

Carlo Alberto Redi ◽

Silvia Garagna

Keyword(s):

Germinal Vesicle ◽

Chromatin Organization ◽

Developmental Competence ◽

Cell Stage ◽

Morphological Marker ◽

Good Marker ◽

Chromatin Configuration ◽

Meiotic Competence

Based on their chromatin organization, antral oocytes can be classified into two classes, namely surrounded nucleolus (SN, chromatin forms a ring around the nucleolus), and not surrounded nucleolus (NSN, chromatin has a diffuse pattern). Oocytes of both classes are capable of meiotic resumption, but while SN oocytes, following fertilization, develop to term, NSN oocytes never develop beyond the two-cell stage. A recent study has shown that the position of the germinal vesicle (GV) can be used as a morphological marker predictive of oocyte meiotic competence, i.e. oocytes with a central GV have a higher meiotic competence than oocytes with an eccentric GV. In the present study, we have associated both markers with the aim of identifying, with more accuracy, the oocytes' developmental competence. Following their isolation, antral oocytes were classified on the basis of both SN and NSN chromatin configuration and their GV position, matured to metaphase II and fertilized in vitro. We demonstrated that the position of the GV is a good marker to predict the oocytes' developmental competence, but only when associated with the observation of the chromatin organization.

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Graft site and gonadotrophin stimulation influences the number and quality of oocytes from murine ovarian tissue grafts

Reproduction ◽

10.1530/rep.1.00916 ◽

2006 ◽

Vol 131 (5) ◽

pp. 851-859 ◽

Cited By ~ 40

Author(s):

Hsiao Yun Yang ◽

Shae-Lee Cox ◽

Graham Jenkin ◽

Jock Findlay ◽

Alan Trounson ◽

...

Keyword(s):

Ovarian Tissue ◽

Germinal Vesicle ◽

Fertilization Rate ◽

Ovarian Tissue Cryopreservation ◽

Control Group ◽

Developmental Potential ◽

Oocyte Yield ◽

Tissue Grafts

Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured beforein vitrofertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.

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