In this study, we systematically compared the morphological, functional and molecular characteristics of granulosa cells and oocytes obtained by a three-dimensional in vitro model of ovine ovarian follicular growth with those of follicles recovered in vivo. Preantral follicles of 200 µm diameter were recovered and cultured up to 950 µm over a 20-day period. Compared with in vivo follicles, the in vitro culture conditions maintained follicle survival, with no difference in the rate of atresia. However, the in vitro conditions induced a slight decrease in oocyte growth rate, delayed antrum formation and increased granulosa cell proliferation rate, accompanied by an increase and decrease in CCND2 and CDKN1A mRNA expression respectively. These changes were associated with advanced granulosa cell differentiation in early antral follicles larger than 400 µm diameter, regardless of the presence or absence of FSH, as indicated by an increase in estradiol secretion, together with decreased AMH secretion and expression, as well as increased expression of GJA1, CYP19A1, ESR1, ESR2, FSHR, INHA, INHBA, INHBB and FST. There was a decrease in the expression of oocyte-specific molecular markers GJA4, KIT, ZP3, WEE2 and BMP15 in vitro compared to that in vivo. Moreover, a higher percentage of the oocytes recovered from cultured follicles 550 to 950 µm in diameter was able to reach the metaphase II meiosis stage. Overall, this in vitro model of ovarian follicle development is characterized by accelerated follicular maturation, associated with improved developmental competence of the oocyte, compared to follicles recovered in vivo.
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Véronique Cadoret, Cynthia Frapsauce, Peggy Jarrier, Virginie Maillard, Agnès Bonnet, Yann Locatelli, Dominique Royère, Danielle Monniaux, Fabrice Guérif and Philippe Monget
A Trounson, C Anderiesz and G Jones
Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.
K. M. Henderson, P. Franchimont, Ch. Charlet-Renard and K. P. McNatty
Summary. The ability of bovine granulosa cells to produce inhibin and to synthesize oestradiol-17β increased with increasing follicle size in healthy but not atretic follicles. Granulosa cells from small (≤5 mm diam.) healthy follicles were indistinguishable from cells of atretic follicles in terms of their ability to produce inhibin and to aromatize androgen. However, granulosa cells from healthy and atretic follicles, irrespective of size, differed markedly in their morphological appearance after culture for 24 h. Testosterone (1 μg/ml) stimulated inhibin production by granulosa cells from healthy and atretic follicles while FSH (100 ng/ml) stimulated inhibin production by granulosa cells from healthy follicles only. The relative ability of granulosa cells from different sizes of healthy and atretic follicles to produce inhibin in vitro was reflected in inhibin concentrations in follicular fluid. There was a significant positive correlation between inhibin concentration in follicular fluid and the number of granulosa cells per follicle. There was also a significant positive correlation between follicular diameter and inhibin concentration in follicular fluid, but only in healthy follicles. These findings show that both follicular size and atresia influence follicular inhibin production.
Dianne Moore Smith, C. H. Conaway and W. T. Kerber
Summary. Oocytes obtained from antral follicles of adult and adolescent rhesus monkeys during the annual breeding season extruded polar bodies in vitro at significantly higher rates (50–60%) than oocytes from animals of similar age during the non-breeding season (20–30%) or from infant and prepubertal females at any time of the year (20–30%). The proportion of oocytes degenerating in culture was greatest in groups where maturation was highest.
G. Evans, D. Claire Wathes, G. J. King, D. T. Armstrong and D. G. Porter
Summary. Theca and granulosa layers were isolated from the preovulatory follicles of prepubertal gilts which were untreated (Group A), killed 72 h after 1000 i.u. PMSG (Group B), killed 84 h after PMSG (Group C), or killed 84 h after PMSG + 500 i.u. hCG given at 72 h (Group D). The tissues from individual follicles were cultured for 24 h alone (C), with FSH (F) or with LH (L), and the content of immunoreactive relaxin in the culture media was measured by RIA. Concentrations of relaxin-like material were close to the limit of detection of the assay in all granulosa cell cultures, and in the thecal cultures from the untreated gilts. However, thecal cultures from all 19 treated gilts produced relaxin. The mean ± s.e.m. concentrations (pg/follicle) in Groups AC, BC, CC and DC were 26·5 ± 3·04, 93·1 ± 4·6, 138 ± 16·4 and 285·6 ± 54·1 respectively. Therefore relaxin production was stimulated by PMSG (P < 0·05), with hCG treatment in vivo leading to a further significant increase (P < 0·05). In-vitro treatment with gonadotrophins had no effect in Groups A, C and D, but in Group-B gilts LH produced a significant (P < 0·05) rise in relaxin levels. These studies indicate that the theca is the principal source of relaxin in the porcine preovulatory follicle. The increased production before ovulation suggests that relaxin may be involved in follicular growth or rupture.
Guangyin Xi, Wenjing Wang, Sarfaraz A Fazlani, Fusheng Yao, Mingyao Yang, Jing Hao, Lei An and Jianhui Tian
Compared to ovarian antral follicle development, the mechanism underlying preantral follicle growth has not been well documented. Although C-type natriuretic peptide (CNP) involvement in preantral folliculogenesis has been explored, its detailed role has not been fully defined. Here, we used mouse preantral follicles and granulosa cells (GCs) as a model for investigating the dynamic expression of CNP and natriuretic peptide receptor 2 (NPR2) during preantral folliculogenesis, the regulatory role of oocyte-derived growth factors (ODGFs) in natriuretic peptide type C (Nppc) and Npr2 expression, and the effect of CNP on preantral GC viability. Both mRNA and protein levels of Nppc and Npr2 were gradually activated during preantral folliculogenesis. CNP supplementation in culture medium significantly promoted the growth of in vitro-cultured preantral follicles and enhanced the viability of cultured GCs in a follicle-stimulating hormone (FSH)-independent manner. Using adult and prepubertal mice as an in vivo model, CNP pre-treatment via intraperitoneal injection before conventional superovulation also had a beneficial effect on promoting the ovulation rate. Furthermore, ODGFs enhanced Nppc and Npr2 expression in the in vitro-cultured preantral follicles and GCs. Mechanistic study demonstrated that the regulation of WNT signaling and estrogen synthesis may be implicated in the promoting role of CNP in preantral folliculogenesis. This study not only proves that CNP is a critical regulator of preantral follicle growth, but also provides new insight in understanding the crosstalk between oocytes and somatic cells during early folliculogenesis.
Oocyte quality in pigs is defined as the potential of that oocyte to develop into a viable offspring. There is increasing evidence that the programming of the oocyte must be completed before leaving the ovarian follicle, including both nuclear and cytoplasmic maturation. Pig oocytes matured in vitro under basic conditions are deficient in some, as yet unidentified, cytoplasmic factors and thus developmentally incompetent. This developmental incompetence can be overcome to some extent by follicular supplementation (with follicular fluid or granulosa cells) of the culture medium, emphasizing the importance of somatic signals during oocyte maturation. Furthermore, evidence is accumulating that the status of the follicle has a critical impact on the competence of the oocyte in vitro and in vivo and this has been demonstrated by co-culture with follicles at different maturational stages or from breeds with enhanced embryo survival. It now also appears that manipulation of maternal nutrition before mating or oocyte collection can enhance embryo survival in vivo and oocyte developmental competence in vitro, presumably by altering follicular secretions and hence the environment in which the oocyte is nurtured. Identification of both the key follicular factors influencing pig oocyte quality and reliable markers of oocyte quality will undoubtedly yield major improvements in embryo survival in vivo and embryo production in vitro.
Cecilia L. Schmidt, June Z. Kendall, Pramila V. Dandekar, M. M. Quigley and Karmen L. Schmidt
Summary. To determine the effects of prolonged hCG treatment in vitro upon granulosa cells from follicles of various sizes previously exposed to clomiphene citrate and hCG in vivo, progesterone and relaxin concentrations of spent media were correlated with light microscopic and ultrastructural characteristics. Intact, freshly dispersed cells were characterized by numerous lipid droplets, elliptical mitochondria with tubular or lamellar cristae, moderate rough-surfaced endoplasmic reticulum (RER), sparse smooth-surfaced endoplasmic reticulum (SER), and few Golgi. After 10–24 days in culture, 2 morphologically distinct cell types, 'granulosa-type' and 'luteal-type', were noted at the light microscopic level. Ultrastructurally, lipid droplets decreased in number, mitochondria became pleomorphic, RER became more prominent and dilated, and Golgi became more widely dispersed. Tubular SER became abundant and annular nexuses became more numerous after hCG treatment in vitro. Granulosa cells generated from all follicles responded to hCG treatment with significantly increased progesterone secretion after 4 days in culture. Relaxin was not detectable in any sample of medium. This study shows that human granulosa cells from 15–25-mm follicles retain their differentiated function of progesterone secretion in long-term culture and recover responsiveness to hCG in vitro, as demonstrated by enhanced progesterone secretion and development of prominent SER and increased annular nexuses.
N Songsasen, T K Woodruff and D E Wildt
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.
N Crozet, M Dahirel and L Gall
The objective of the present study was to grow meiotically incompetent goat oocytes from early antral follicles in vitro and to render them competent to undergo germinal vesicle breakdown. Cumulus-oocyte complexes with pieces of parietal granulosa cells were isolated from follicles 0.35-0.45 mm in diameter using both mechanical and enzymatic methods. The cumulus-oocyte complexes were divided into two groups according to oocyte diameter (group A: < 95 microm; group B: > 95 microm) and cultured for 8 or 9 days on granulosa cell monolayers. Within 8 days of culture, the mean oocyte diameter increased from 86 +/- 0.4 microm to 95 +/- 0.7 microm in group Aand from 106 +/- 0.2 microm to 109 +/- 0.5 microm in group B. After 9 days of culture, the mean diameter of oocytes from groups A and B were 99 +/- 0.5 microm and 112 +/- 0.4 microm, respectively. The meiotic competence of oocytes grown in vitro was evaluated by in vitro maturation. Within 8 days of culture, only 3% of oocytes from group A and 6% of oocytes from group B acquired the ability to undergo germinal vesicle breakdown. After 9 days of culture, 7% of group A oocytes and 42% of group B oocytes were competent to resume meiosis. The expression of p34(cdc2) in oocytes grown in vitro was analysed by the western blot technique. During 9 days of culture, p34(cdc2) accumulated in both groups of growing oocytes, but its concentration was lower than in fully grown oocytes used as controls. The results showed for the first time that goat oocytes from early antral follicles can grow, accumulate p34(cdc2) and acquire the ability to resume meiosis, when cultured for 9 days on granulosa cell monolayers.