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J C Sadeu, T Adriaenssens, and J Smitz

Growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and anti-Müllerian hormone (AMH) play an important role in the primary to secondary follicle transition and follicle activation in vivo. In organ culture of neonatal mouse ovaries, it was observed that significantly fewer primary follicles develop to the secondary stage. The objectives of this study were: (1) to compare ovarian follicular populations between organ-cultured neonatal mouse ovaries and freshly isolated age-matched control ovaries; (2) to quantify RNA levels of Gdf9, Bmp15, and Amh in cultured primary follicles; and (3) to immunolocalize GDF9 and AMH in cultured ovaries. Ovaries from 3-day-old (PND 3) mice were cultured for 7 or 10 days in the absence or presence of FSH. Follicular populations were counted in freshly isolated 13-day-old (PND 13) ovaries and organ-cultured ovaries. Transcripts were quantified in isolated primary follicles using real-time RT-PCR, and protein expressions were localized using immunohistochemistry. The number of secondary follicles in organ-cultured ovaries was significantly lower than in vivo controls. Gdf9 and Bmp15 mRNA expression levels were similar as in controls. Amh mRNA levels were significantly (P<0.05) lower after day 10 of culture in the absence of FSH. GDF9 and AMH proteins were respectively detected in the oocytes and the granulosa cells (GC) beginning at the primary and primordial stages onward. GDF9 and BMP15 production in cultured primary follicles are not different from in vivo controls; hence abnormal early follicular growth was not related to a deficient transcription of these factors.

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MA Driancourt, K Reynaud, and J Smitz

After in vitro maturation, fertilization and development, the percentage of fertilized eggs developing to the blastocyst stage is usually lower in calves compared with cows. It is unknown whether this low ability to develop in vitro is inherent to calf oocytes or is caused by altered follicular maturation. The latter possibility was explored in the present study using two markers of follicle function: in vitro steroidogenesis by intact follicles and aromatase activity of follicular walls. Calf follicles > 9 mm in diameter had a low ability to produce oestradiol (ten times reduction compared with cows) despite a testosterone output by theca cells which was similar to that observed in cows. This finding is in agreement with the low aromatase activity of granulosa cells of calf follicles measured by tritiated water release assay. Qualitative and quantitative differences between calf and cow follicular fluids were assessed using western blotting (inhibin and activin, heat shock protein 90, Mullerian inhibiting substance) and assays (inhibin and activin) to determine whether this defective aromatase could be produced by alterations in the amounts of follicular proteins modulating aromatase (inhibin and activin, heat shock protein 90, Mullerian inhibiting substance). Western blotting of follicular fluid proteins demonstrated three main bands (59, 57 and < 30 kDa) and one minor band (34 kDa) with the anti-alpha inhibin antibody, whereas a single 18 kDa band was detected when an anti-beta inhibin antibody was used. Calf follicular fluid contained similar amounts of all main inhibin forms (alpha and beta) but a 34 kDa alpha inhibin form was missing. The amounts of dimeric inhibin were similar between cows and calves but small follicles from calves contained more activin. Single bands at 70 kDa (Mullerian inhibiting substance) and 90 kDa (heat shock protein 90) were detected by western blotting. Mullerian inhibiting substance was missing from calf follicular fluid and heat shock protein 90 was present in smaller amounts in calf versus cow follicular fluid. None of the above differences could explain the defective aromatase of calf follicles. Two-dimensional separation of the [35S]-labelled proteins secreted by follicular walls originating from calf or cow follicles matched for size and follicle health was performed and 151 spots were observed on the master gel, which summarized all the spots present at least once. Fifteen spots were present in calves and not in cows. Quantitative differences were also detected with three spots containing more proteins in cows than in calves. Whether some of these proteins can alter maturation of follicles or oocytes requires further investigation.

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MA Driancourt, K Reynaud, R Cortvrindt, and J Smitz

Evidence from mouse mutants indicates that the Kit gene encoding KIT, a receptor present on the oocyte and theca cells, and the Mgf gene encoding KIT LIGAND, the ligand of KIT, are important regulators of oogenesis and folliculogenesis. Recently, in vitro cultures of fetal gonads, of follicles and of oocytes have identified specific targets for the KIT-KIT LIGAND interaction. In fetal gonads, an anti-apoptotic effect of KIT-KIT LIGAND interactions on primordial germ cells, oogonia and oocytes has been demonstrated. In postnatal ovaries, the initiation of follicular growth from the primordial pool and progression beyond the primary follicle stage appear to involve KIT-KIT LIGAND interactions. During early folliculogenesis, KIT together with KIT LIGAND controls oocyte growth and theca cell differentiation, and protects preantral follicles from apoptosis. Formation of an antral cavity requires a functional KIT-KIT LIGAND system. In large antral follicles, the KIT-KIT LIGAND interaction modulates the ability of the oocyte to undergo cytoplasmic maturation and helps to maximize thecal androgen output. Hence, many steps of oogenesis and folliculogenesis appear to be, at least in part, controlled by paracrine interactions between these two proteins.

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Anamaria C Herta, Francesca Lolicato, and Johan E J Smitz

The currently available assisted reproduction techniques for fertility preservation (i.e. in vitro maturation (IVM) and in vitro fertilization) are insufficient as stand-alone procedures as only few reproductive cells can be conserved with these techniques. Oocytes in primordial follicles are well suited to survive the cryopreservation procedure and of use as valuable starting material for fertilization, on the condition that these could be grown up to fully matured oocytes. Our understanding of the biological mechanisms directing primordial follicle activation has increased over the last years and this knowledge has paved the way toward clinical applications. New multistep in vitro systems are making use of purified precursor cells and extracellular matrix components and by applying bio-printing technologies, an adequate follicular niche can be built. IVM of human oocytes is clinically applied in patients with polycystic ovary/polycystic ovary syndrome; related knowhow could become useful for fertility preservation and for patients with maturation failure and follicle-stimulating hormone resistance. The expectations from the research on human ovarian tissue and immature oocytes cultures, in combination with the improved vitrification methods, are high as these technologies can offer realistic potential for fertility preservation.

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D Nogueira, R Cortvrindt, B Everaerdt, and J Smitz

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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K Reynaud, R Cortvrindt, J Smitz, F Bernex, JJ Panthier, and MA Driancourt

The KIT receptor, present on oocyte and theca cells in ovarian follicles, and its ligand, KIT LIGAND, produced by granulosa cells, are encoded at the Kit gene and the Mgf gene, respectively. Both Kit and Mgf mutations affect oogenesis and folliculogenesis. In this study, the ovarian function of heterozygous mice with a mutation Kit(W-lacZ) was examined. Firstly, the amounts of KIT and KIT LIGAND proteins in the ovaries of mice at different ages were determined. Secondly, in vivo and in vitro folliculogenesis of wild type and heterozygous mice were compared. Western blotting showed that the amounts of both KIT and KIT LIGAND proteins were decreased in mutant mice. Ovarian follicle populations were counted and more type 5a follicles and fewer type 5b (preantral follicles) were present in ovaries from Kit(W-lacZ/+) ovaries. Furthermore, the relationships between oocyte size and follicle size differed between wild type and heterozygous mice. This finding may be a consequence of altered proliferation of granulosa cells or of altered oocyte growth in mutant mice. Other features of folliculogenesis, such as initiation of follicular growth, total follicle population and follicular atresia, were not affected by the mutation. Analysis of in vitro folliculogenesis did not reveal other differences between wild type and mutant mice. It is concluded that the Kit(W-lacZ) mutation affects the expression of KIT and KIT LIGAND proteins, resulting in alterations in granulosa cell proliferation and/or oocyte growth in preantral follicles.

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R B Gilchrist, A M Luciano, D Richani, H T Zeng, X Wang, M De Vos, S Sugimura, J Smitz, F J Richard, and J G Thompson

The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP – inhibiting the hydrolysis of intra-oocyte cAMP – and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect – the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.