Germinal vesicle (GV)-stage oocytes retrieved from antral follicles undergo nuclear maturation in vitro, which typically occurs prior to cytoplasmic maturation. Short-term culture with meiotic inhibitors has been applied to arrest oocytes at the GV stage aiming to synchronize nuclear and ooplasmic maturity. However, the results obtained are still far from the in vivo situation. In order to acquire competence, immature oocytes may require meiotic arrest in vitro for a more extended period. The phosphodiesterase type 3-inhibitor (PDE3-I) is a potent meiotic arrester. The effects of a prolonged culture with PDE3-I on oocyte quality prior to and after reversal from the inhibition are not known. This study tested the impact of long-term in vitro exposure of two PDE3-Is, org9935 and cilostamide, on oocytes using a mouse follicle culture model. The results showed that PDE3-I (maximum of 10 μM) during a 12-day culture of follicle-enclosed oocytes did not alter somatic cell proliferation, differentiation or follicle survival. In addition, the steroid production profile was not significantly modified by a 12-day exposure to PDE3-I. The recombinant human chorionic gonadotrophin/recombinant human epidermal growth factor stimulus induced a characteristic normal progesterone peak of luteinization and normal mucification of the cumulus cells, while the enclosed oocyte remained blocked at the GV stage. In vitro maturation of denuded or cumulus-enclosed oocytes derived from org9935- or cilostamide-exposed follicles progressed through meiosis and formed morphologically normal meiotic spindles with chromosomes properly aligned at the equator. In conclusion, long-term culture with PDE3-I was harmless to somatic cell function, differentiation, oocyte growth and maturation. Our results suggested that PDE3-I can be applied when extended oocyte culture is required to improve ooplasmic maturation.
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D Nogueira, R Cortvrindt, B Everaerdt and J Smitz
R. Braw-Tal and S. Yossefi
Histological sections prepared from cortical parts of 25 bovine ovaries were used to study initiation of follicle growth in vivo. Small follicles were measured and characterized. Initiation of follicle growth consisted of two distinct consecutive phases. The first phase was characterized by transformation of granulosa cells from a flattened to a cuboidal shape and by their proliferation. In the second phase an increase in the number of granulosa cells was accompanied by a rapid increase in the size of the oocyte. Oocytes commenced growth when there were at least 40 granulosa cells in the largest cross-section (fourth generation of follicle cells). The oocyte diameter increased from 29.74 ± 0.30 μm (mean ± sem) in primordial follicles to 92.90 ± 4.50 μm in small antral follicles. The zona pellucida first appeared as an island of periodic acid–Schiff positive material in small preantral follicles, but formed a complete ring around the oocyte when the late preantral stage was reached. Organ culture of ovarian cortical explants was used to study initiation of follicle growth in vitro. Within 2 days of culture most of the primordial follicles entered the growth phase: granulosa cells changed from a flattened to a cuboidal shape and entered S-phase as demonstrated by autoradiography after [3H]thymidine incorporation. On day 2, 48.6% of follicles were labelled compared with 3% on day 0. Follicle growth started in the absence of gonadotrophins, in the serum-free medium, confirming the notion that gonadotrophins are not essential for this process. The culture system used here will be helpful in the study of the involvement of putative factor(s) in the initiation of follicle growth in large domestic animals.
A I Qureshi, S S Nussey, G Bano, P Musonda, S A Whitehead and H D Mason
Histological studies have demonstrated that polycystic ovaries (PCO) contain increased numbers of preantral follicles with a specific increase in primary follicles. Polycystic ovary syndrome is associated with hyperandrogenism and pre- and postnatal androgenisation of primates increases the pool of growing follicles producing changes resembling PCO. In vitro studies could test the hypothesis that androgens alter early folliculogenesis, but conventional culture techniques for small follicles are generally unsuitable in non-rodent species. Our objective was to develop and use a method to investigate the effects of testosterone on early folliculogenesis. We adapted an in ovo technique in which lamb cortical ovarian fragments were grafted onto the chorioallantoic membrane of fertilised chick eggs. Optimal experimental conditions for vascularisation and survival of tissue were determined and the model then used to investigate the effects of testosterone on follicle growth. Eggs were inoculated with testosterone at the time of implantation of the ovarian tissue, which was retrieved 5 days later. Tissue was sectioned and follicles staged and counted. There was no wholesale initiation of primordial follicle growth over the 5-day in ovo culture. Importantly, the proportion of primordial, primary and secondary follicles remained similar to those in unimplanted tissue. Testosterone increased the number of primary follicles by 50% compared with controls, an effect that was largely due to a reduction in atresia. In conclusion, incubation of ovarian cortex with testosterone reproduces the changes in early folliculogenesis reported in histological studies of PCO.
J M Young, S Henderson, C Souza, H Ludlow, N Groome and A S McNeilly
Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1–3 to >6 mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3–4 mm, and TCs more inhibin α mRNA at >4 mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1–3 mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).
Davina A Cossigny, Jock K Findlay and Ann E Drummond
Numerous studies have reported on the roles of activins in gonadal regulation; however, little is known about their specific roles in early folliculogenesis. Ovarian follicular growth was investigated in 10-day cultures of day 4 postnatal whole ovaries treated with activin A (ActA; 50 ng/ml), with or without FSH (100 ng/ml) in vitro. We hypothesized that treatment with ActA±FSH would affect rates of growth and atresia in follicles. None of the treatments affected primordial follicle activation, and antral follicles were not observed after 10 days in culture. Primordial follicle numbers from all treatment groups were ∼20% of those in day 4 fresh ovaries, indicating that activation had occurred. In the presence of ActA, preantral follicle numbers increased significantly (P<0.0001). ActA alone decreased the proportion of atretic follicles in the primary and preantral classes, whereas the combined treatment of ActA+FSH increased the proportion of atretic preantral oocytes. Real-time PCR analysis revealed that follistatin, FSH receptor, and activin βA and βB subunits were all expressed at significantly higher levels in the ActA-only treated group but not in the ActA+FSH group. Here, we report novel findings supporting the role of FSH in primordial follicle survival through an action on apoptosis and a stimulatory role of ActA in the primordial to primary and preantral stages of follicle development, suggesting an inhibitory action of activin on oocyte apoptosis.
Y. Hirao, T. Nagai, M. Kubo, T. Miyano, M. Miyake and S. Kato
Preantral follicles containing oocytes of 70–89.5 μm in diameter were isolated from pig ovaries and cultured in collagen gel for up to 16 days, in the presence of serum, FSH and oestradiol. Formation of follicular antra occurred as the culture proceeded. The oocytes had been enclosed by granulosa cells and contacts between the oocytes and processes of the enclosing cumulus cells were maintained over the culture period. After 16 days of culture, 30–40% of the oocytes were of normal appearance, and the diameters of about half of these oocytes were larger than 100 μm. When the oocytes grown in vitro were liberated from the follicles and cultured for a further 48 h in modified Krebs–Ringer bicarbonate solution, 6, 30 and 60% of the oocytes larger than 90, 100 and 110 μm underwent germinal vesicle breakdown, respectively. Progression to metaphase II was observed in 40% of oocytes that were over 110 μm in diameter, whereas no oocyte less than 90 μm in diameter resumed meiosis. The relationship between the size and meiotic competence of oocytes was similar for oocytes grown in vitro or in vivo. Oocytes grown and matured in vitro were penetrated by spermatozoa and formed a female pronucleus, but decondensation of the sperm head was incomplete. The results demonstrate for the first time that pig oocytes from preantral follicles can grow up to their final size, acquire meiotic competence, and be penetrated by spermatozoa in vitro.
Miwa Segino, Mario Ikeda, Fumiki Hirahara and Kahei Sato
In a previous report, we showed that follicles isolated from frozen/thawed mouse ovarian tissues reached the mature follicle stage on the 12th day of culture. However, the developmental ability was lower than that of fresh ovarian tissue. The purpose of this study was to define a culture system with some technical modification for preantral follicles isolated from frozen/thawed ovarian tissue and to confirm cell injury. Ovaries obtained from three-week-old female mice were cryopreserved by the rapid freezing method. Preantral follicles isolated from frozen/thawed ovarian tissues were cultured for 12–16 days. The follicles were then stimulated with human chorionic gonadotropin. In vitro fertilization was performed on the released cumulus–oocyte complexes (COCs). Preantral follicle viability was assessed by supravital staining using Hoechst 33258. Using this stain cell death was found in part of the granulosa cells of a follicle obtained from frozen/thawed ovarian tissue. On the 14th and 16th days of culture, the diameters of follicles isolated from frozen/thawed ovaries were larger than on the 12th day of culture. The released COCs were fertilized and developed to the blastocyst stage in 15.8% (12/76) of the oocytes taken from the fresh group, and in 0% (0/73), 2.9% (2/69) and 19.1% (22/115) of the oocytes taken from the frozen/thawed group that had been cultured for 12, 14 and 16 days respectively. The preantral follicles isolated from frozen/thawed mouse ovarian tissues developed slowly compared with the freshly prepared preantral follicles. During prolonged culture from 12 to 16 days, these follicles obtained the potential to fertilize and develop to the blastocyst stage.
A. A. Murray, R. G. Gosden, V. Allison and N. Spears
The effects of androgens on ovarian follicular development have been investigated using a whole follicle culture system. Follicles obtained from mouse ovaries and cultured in the presence of anti-androgen serum grew more slowly than control follicles. This effect was reversed by the addition of androstenedione to the medium. A similar effect was obtained when receptor-mediated effects of androgens were blocked using an androgen receptor antagonist. When follicles were grown in concentrations of FSH that are marginal for follicle development, they developed faster in the presence of a non-aromatizable androgen, dihydroxytestosterone. The results indicate that androgens exert a direct, stimulatory role on the growth and development of mouse antral follicles, in vitro.
J. Motlik, Nicole Crozet and J. Fulka
Summary. Pig oocytes were isolated from early antral follicles of different sizes and their abilities to resume and complete meiotic maturation in vitro were compared. After 24 h of culture, more than 80% of the oocytes from follicles 0·3–0·7 mm in diameter remained at the germinal vesicle stage, while 66, 94·3 and 100% oocytes from follicles 0·8–1·6, 1·7–2·2 and 3–5 mm in diameter, respectively, completed germinal vesicle breakdown. After 48 h of culture, 35% of the oocytes in the smallest follicle class progressed to prometaphase and only 4% to metaphase I. Of the oocytes from follicles 0·8–1·6 mm in diameter, 23% reached metaphase I and 17·3% metaphase II. About 50 and 76% of the oocytes from follicles 1·8–2·2 mm and 3–5 mm in diameter, respectively, extruded the first polar body.
The ability to resume meiosis (i.e. to undergo germinal vesicle breakdown) is reached by porcine oocytes when they approach their full size in antral follicles >0·8 mm in diameter and before they are capable of completing it (i.e. reaching metaphase II). The ability to complete meiotic maturation acquired in antral follicles of about 2 mm in diameter coincided with a significant decrease in the nucleolar transcriptional activity of the oocytes.
Jing Xu, Marcelo P Bernuci, Maralee S Lawson, Richard R Yeoman, Thomas E Fisher, Mary B Zelinski and Richard L Stouffer
A three-dimensional culture system supports the development of primate preantral follicles to the antral stage with appreciable steroid production. This study assessed i) whether in vitro developmental competence of follicles is age dependent, ii) the role of gonadotropins and insulin in supporting folliculogenesis, and iii) anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) production by growing follicles. Ovaries were obtained from prepubertal, young, and older adult rhesus macaques. Secondary follicles were encapsulated into alginate beads and cultured individually for 40 days in media containing 0.05 or 5 μg/ml insulin, with or without recombinant human (rh) FSH (500 mIU/ml). No follicles survived in the culture without rhFSH. In the presence of rhFSH, survival was lower for follicles from older animals, whereas growth, i.e. follicle diameter, was less by day 40 for follicles from prepubertal animals. The surviving follicles were categorized as no-grow (NG; ≤250 μm), slow-grow (SG; 250–500 μm), and fast-grow (FG; ≥500 μm) according to their diameters. SG follicles cultured with 5 μg/ml insulin produced more ovarian steroids than those cultured with 0.05 μg/ml insulin by week 5. SG and FG follicles produced more AMH and VEGF than the NG, and levels peaked at weeks 2 and 5 respectively. After 100 ng/ml rh chorionic gonadotropin treatment for 34 h, more healthy oocytes were retrieved from young adults whose follicles were cultured with 5 μg/ml insulin. This culture system offers an opportunity to characterize the endocrine and paracrine function of primate follicles that influence follicle growth and oocyte maturation.