Hypoxia limits mouse follicle growth in vitro

2016 ◽  
Vol 28 (10) ◽  
pp. 1570 ◽  
Author(s):  
J. M. Connolly ◽  
M. T. Kane ◽  
L. R. Quinlan ◽  
P. Dockery ◽  
A. C. Hynes
Keyword(s):  
Ascorbic Acid ◽  
Ovarian Follicle ◽  
Maximum Diameter ◽  
Mouse Serum ◽  
Follicle Growth ◽  
Follicle Culture ◽  
Follicle Diameter ◽  
Serum Type

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin–transferrin–selenium (ITS) and l-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (P < 0.01) inferior to growth in 20% oxygen in terms of follicle diameter. This was likely due to hypoxia, as evidenced by significantly (P < 0.05) increased follicle secretion of vascular endothelial growth factor (VEGF), a marker of cell hypoxia. Follicular growth was not (P > 0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL–1 FSH, 25 μg mL–1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 μm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 μm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

Corrigendum to: Hypoxia limits mouse follicle growth in vitro

2017 ◽  
Vol 29 (2) ◽  
pp. 431 ◽  
Author(s):  
J. M. Connolly ◽  
M. T. Kane ◽  
L. R. Quinlan ◽  
P. Dockery ◽  
A. C. Hynes
Keyword(s):  
Ascorbic Acid ◽  
Ovarian Follicle ◽  
Time Frame ◽  
Maximum Diameter ◽  
Mouse Serum ◽  
Follicle Growth ◽  
Follicle Culture ◽  
Serum Type

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin–transferrin–selenium (ITS) and I-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (PPP>0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL–1 FSH, 25 μgmL–1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 μm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 μm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

The earliest stages of folliculogenesis in vitro

Reproduction ◽  
2002 ◽  
pp. 185-202 ◽  
Author(s):  
JE Smitz ◽  
RG Cortvrindt
Keyword(s):  
Ovarian Tissue ◽  
Ovarian Follicle ◽  
Mammalian Species ◽  
Follicle Development ◽  
Follicle Growth ◽  
Follicle Culture ◽  
In Vitro Techniques ◽  
Suitable Material ◽  
Culture Systems

In recent years several follicle culture systems have been pioneered in different mammalian species for studying ovarian folliculogenesis and culturing immature oocytes. Applications of these in vitro techniques include fertility preservation for humans, conservation of rare animals and development of oocyte banks for research purposes. Immature female gametes in the ovarian cortex can be cryopreserved for later use if culture techniques are available afterwards to promote growth and maturation. This review focuses on biochemical and biophysical factors related to oocyte culture in mice, the only animal in which live offspring have been produced after folliculogenesis in vitro. The advantage of using mice for these studies is that, in parallel to development of follicle culture systems, essential knowledge on folliculogenesis can be obtained from knockout mouse models. Recent experiments in mice stressed the principal role of the oocyte in follicle development and the strict timing of the biological processes underlying oogenesis in vitro. In large domestic animals and humans, study of oocyte culture is confounded by the constitutively prolonged nature of ovarian follicle development. In humans, only some aspects of follicle development have been studied because of the limited availability of suitable material for experimentation, technical difficulties related to manipulation of very small structures and lack of knowledge on physiological regulation of the early stages of follicle growth. Only a few reports describe ovarian follicular growth in vitro. In this review, relevant information on hormonal and growth factor regulation of the earliest stages of follicle growth in mammals is reviewed. Techniques are becoming available for the precise isolation of distinct classes of follicle and powerful molecular biology techniques can be used in studies of ovarian tissue culture.

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.

Optimization of Novel Follicle Culture System to Generate Developmentally Competent Oocytes

2020 ◽  
Author(s):  
Jongwon Kim ◽  
Jung Kyu Choi
Keyword(s):  
Culture System ◽  
Ovarian Follicle ◽  
Formation Rate ◽  
Germinal Vesicle ◽  
Ovarian Follicles ◽  
Germinal Vesicle Breakdown ◽  
Follicle Culture ◽  
Nuclear Maturation ◽  
Follicle Diameter

This study aimed to develop a novel culture system for porcine ovarian follicles that yields developmentally competent oocytes. We mechanically isolated ovarian follicles of various sizes 325–500 mm and treated them with ovine follicle stimulating hormone OFSH at different concentrations 0–400 mIU. Follicle diameter, antrum formation and cumulus oocyte complex COC recovery rate were significantly higher p andlt; 0.05 under the 0 and 50 mIU OFSH treatments compared with the remaining concentrations 100, 200 and 400 mIU. Additionally, follicles cultured for 3 and 4 d differed significantly p andlt; 0.05 in follicle diameter, antrum formation rate and COC recovery from those cultured for 5 and 6 d. Follicle characteristics did not differ across diameter: those at 250–300, 301–400 and 401–500 mm in vitro had antrum formation rates of 90%, 92% and 90%, along with COC recovery of 78%, 82% and 85%, respectively. Furthermore, nuclear maturation percentages for oocytes that experienced germinal vesicle breakdown (GVBD) were 10%, 13% and 14%, depending on the size of the originating follicle (250–300, 301–400 and 401–500 mm). Nuclear maturation for metaphase II (MII) oocytes derived from follicles of those three sizes were 1%, 2% and 1%, respectively. After 3 d of culture, the 250–300 mm group differed significantly from other size groups in follicle diameter and COC recovery. This study provides insight into establishing effective protocols of ovarian follicle culture, thus improving efforts to preserve large-mammal fertility.

scholarly journals Melatonin as an endogenous hormone to slow down the exhaustion of ovarian follicle reserve in mice–a novel insight into its roles in early folliculogenesis and ovarian aging

2020 ◽  
Author(s):  
Chan Yang ◽  
Qinghua Liu ◽  
Yingjun Chen ◽  
Xiaodong Wang ◽  
Zaohong Ran ◽  
...  
Keyword(s):  
Ovarian Follicle ◽  
Primordial Follicle ◽  
High Dose ◽  
Follicle Growth ◽  
Ovarian Aging ◽  
Neonatal Mice ◽  
Primordial Follicle Activation ◽  
Follicle Activation

Abstract Previous studies have shown that long-term intake of exogenous melatonin can effectively delay ovarian aging, but the mechanism has not been fully elucidated. We observed that SNAT, the rate-limiting enzyme in the melatonin synthetic pathway, is localized in primordial and early follicle, and that granulosa cells isolated from follicle can synthesize melatonin. In vitro cultured neonatal mice ovaries with melatonin inhibited primordial follicle activation and early follicle growth. In vivo experiments further indicated that daily injections of melatonin to neonatal mice during the primordial follicle activation phase can reduce the number of activated follicles by inhibiting the PI3K-AKT-FOXO3 pathway; during the early follicle growth phase, injections of melatonin significantly suppressed early follicle growth and atresia, and transcriptome data showed that multiple pathways involved in folliculogenesis, including PI3K-AKT, were suppressed. Further, SNAT knockout in mice resulted in a significant increase in follicle activation and atresia, and eventually accelerated ovarian aging. We also demonstrated that prolonged high-dose melatonin intake had no obvious adverse effect on the health condition of mice. This study confirms that endogenous melatonin is involved in the regulation of ovarian aging, and reveals that melatonin delays ovarian aging by inhibiting primordial follicle activation, early follicle growth and atresia.

scholarly journals Anti-Müllerian Hormone Attenuates the Effects of FSH on Follicle Development in the Mouse Ovary

Endocrinology ◽  
2001 ◽  
Vol 142 (11) ◽  
pp. 4891-4899 ◽  
Author(s):  
Alexandra L. L. Durlinger ◽  
Maria J. G. Gruijters ◽  
Piet Kramer ◽  
Bas Karels ◽  
T. Rajendra Kumar ◽  
...  
Keyword(s):  
Ovarian Follicle ◽  
Inhibitory Effect ◽  
Ovarian Follicles ◽  
Follicle Development ◽  
Wild Type ◽  
Follicle Growth ◽  
Primordial Follicles ◽  
Ovarian Follicle Development

Abstract Although ovarian follicle growth is under the influence of many growth factors and hormones of which FSH remains one of the most prominent regulators. Therefore, factors affecting the sensitivity of ovarian follicles to FSH are also important for follicle growth. The aim of the present study was to investigate whether anti-Müllerian hormone (AMH) has an inhibitory effect on follicle growth by decreasing the sensitivity of ovarian follicles to FSH. Furthermore, the combined action of AMH and FSH on ovarian follicle development was examined. Three different experiments were performed. Using an in vitro follicle culture system it was shown that FSH-stimulated preantral follicle growth is attenuated in the presence of AMH. This observation was confirmed by an in vivo experiment showing that in immature AMH-deficient females, more follicles start to grow under the influence of exogenous FSH than in their wild-type littermates. In a third experiment, examination of the follicle population of 4-month-old wild-type, FSHβ-, AMH-, and AMH-/FSHβ-deficient females revealed that loss of FSH expression has no impact on the number of primordial and preantral follicles, but the loss of inhibitory action of AMH on the recruitment of primordial follicles in AMH-deficient mice is increased in the absence of FSH. In conclusion, these studies show that AMH inhibits FSH-stimulated follicle growth in the mouse, suggesting that AMH is one of the factors determining the sensitivity of ovarian follicles for FSH and that AMH is a dominant regulator of early follicle growth.

scholarly journals Multiple follicle culture supports primary follicle growth through paracrine-acting signals

Reproduction ◽  
2013 ◽  
Vol 145 (1) ◽  
pp. 19-32 ◽  
Author(s):  
J E Hornick ◽  
F E Duncan ◽  
L D Shea ◽  
T K Woodruff
Keyword(s):  
Feedback Mechanism ◽  
Follicle Growth ◽  
Primary Follicle ◽  
Growth And Survival ◽  
Follicle Culture ◽  
Secreted Factors ◽  
Secondary Stage ◽  
Survival And Growth

In vitro follicle growth in alginate hydrogels is a unique and versatile method for studying ovarian and follicle biology that may also have implications for fertility preservation. Current culture systems support the development of isolated mouse follicles from the secondary stage onward. However, it has been a challenge to grow smaller follicles in vitro due to the dissociation of the oocyte from companion somatic cells. Recent work has demonstrated that coculturing primary follicles with mouse embryonic fibroblasts or ovarian stromal cells supports follicle survival and growth. In this study, we demonstrate that follicles themselves can exert a beneficial coculture effect. When primary follicles were cultured in groups of five or ten (multiple follicle culture), there was increased growth and survival. The multiple follicle culture approach maintained follicle integrity and resulted in the formation of antral stage follicles containing meiotically competent gametes. The growth and survival of primary follicles were highly number dependent, with the most significant enhancement observed when the largest number of follicles was grown together. Our data suggest that the follicle unit is necessary to produce the secreted factors responsible for the supportive effects of multiple follicle culture, as neither denuded oocytes, oocyte-secreted factors, nor granulosa cells alone were sufficient to support early follicle growth in vitro. Therefore, there may be signaling from both the oocyte and the follicle that enhances growth but requires both components in a feedback mechanism. This work is consistent with current in vivo models for follicle growth and thus advances the movement to recapitulate the ovarian environment in vitro.

scholarly journals C-Type Natriuretic Peptide Stimulates Ovarian Follicle Development

2012 ◽  
Vol 26 (7) ◽  
pp. 1158-1166 ◽  
Author(s):  
Yorino Sato ◽  
Yuan Cheng ◽  
Kazuhiro Kawamura ◽  
Seido Takae ◽  
Aaron J.W. Hsueh
Keyword(s):  
Natriuretic Peptide ◽  
Granulosa Cells ◽  
Ovulation Induction ◽  
Ovarian Follicle ◽  
Antral Follicle ◽  
In Vivo Studies ◽  
Follicle Growth ◽  
Secondary Stage

Abstract C-type natriuretic peptide (CNP) encoded by the NPPC (Natriuretic Peptide Precursor C) gene expressed in ovarian granulosa cells inhibits oocyte maturation by activating the natriuretic peptide receptor (NPR)B (NPRB) in cumulus cells. RT-PCR analyses indicated increased NPPC and NPRB expression during ovarian development and follicle growth, associated with increases in ovarian CNP peptides in mice. In cultured somatic cells from infantile ovaries and granulosa cells from prepubertal animals, treatment with CNP stimulated cGMP production. Also, treatment of cultured preantral follicles with CNP stimulated follicle growth whereas treatment of cultured ovarian explants from infantile mice with CNP, similar to FSH, increased ovarian weight gain that was associated with the development of primary and early secondary follicles to the late secondary stage. Of interest, treatment with FSH increased levels of NPPC, but not NPRB, transcripts in ovarian explants. In vivo studies further indicated that daily injections of infantile mice with CNP for 4 d promoted ovarian growth, allowing successful ovulation induction by gonadotropins. In prepubertal mice, CNP treatment alone also promoted early antral follicle growth to the preovulatory stage, leading to efficient ovulation induction by LH/human chorionic gonadotropin. Mature oocytes retrieved after CNP treatment could be fertilized in vitro and developed into blastocysts, allowing the delivery of viable offspring. Thus, CNP secreted by growing follicles is capable of stimulating preantral and antral follicle growth. In place of FSH, CNP treatment could provide an alternative therapy for female infertility.

In vitro and in vivo reduction of erythrocyte sorbitol by ascorbic acid

Diabetes ◽  
1989 ◽  
Vol 38 (8) ◽  
pp. 1036-1041 ◽  
Author(s):  
J. A. Vinson ◽  
M. E. Staretz ◽  
P. Bose ◽  
H. M. Kassm ◽  
B. S. Basalyga
Keyword(s):  
Ascorbic Acid

Maturation, iron deficiency, and ligands in enteric radioiron transport in vitro

1963 ◽  
Vol 204 (1) ◽  
pp. 171-175 ◽  
Author(s):  
W. S. Ruliffson ◽  
J. M. Hopping
Keyword(s):  
Ascorbic Acid ◽  
Iron Deficiency ◽  
Iron Absorption ◽  
Deficiency Anemia ◽  
Anaerobic Incubation ◽  
Reverse Effect ◽  
Factors Affecting ◽  
Everted Gut Sac

The effects in rats, of age, iron-deficiency anemia, and ascorbic acid, citrate, fluoride, and ethylenediaminetetraacetate (EDTA) on enteric radioiron transport were studied in vitro by an everted gut-sac technique. Sacs from young animals transported more than those from older ones. Proximal jejunal sacs from anemic animals transported more than similar sacs from nonanemic rats, but the reverse effect appeared in sacs formed from proximal duodenum. When added to media containing ascorbic acid or citrate, fluoride depressed transport as did anaerobic incubation in the presence of ascorbic acid. Anaerobic incubation in the presence of EDTA appeared to permit elevated transport. Ascorbic acid, citrate, and EDTA all enhanced the level of Fe59 appearing in serosal media. These results appear to agree with previously established in vivo phenomena and tend to validate the in vitro method as one of promise for further studies of factors affecting iron absorption and of the mechanism of iron absorption.

Sign in / Sign up

Export Citation Format

Share Document