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Evelyn E Telfer

Ovarian cryopreservation rapidly developed from basic science to clinical application and can now be used to preserve the fertility of girls and young women at high risk of sterility. Primordial follicles can be cryopreserved in ovarian cortex for long-term storage and subsequently autografted back at an orthotopic or heterotopic site to restore fertility. However, autografting carries a risk of re-introducing cancer cells in patients with blood-born leukaemias or cancers with a high risk of ovarian metastasis. For these women fertility restoration could only be safely achieved in the laboratory by the complete in vitro growth (IVG) and maturation (IVM) of cryopreserved primordial follicles to fertile metaphase II (MII) oocytes. Culture systems to support the development of human oocytes have provided greater insight into the process of human oocyte development as well as having potential applications within the field of fertility preservation. The technology required to culture human follicles is extremely challenging, but significant advances have been made using animal models and translation to human. This review will detail the progress that has been made in developing human in vitro growth systems and consider the steps required to progress this technology towards clinical application.

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Marie McLaughlin and Evelyn E Telfer

Quiescent follicles of large mammals initiate growth within cultured pieces of ovarian cortex. Systems capable of sustaining in vitro development from this early stage until oocyte maturation would allow investigation of mechanisms regulating oocyte development in its entirety. The aims of this study were 1) to determine whether bovine follicles initiated to grow in vitro could be isolated from the cortical environment, and could undergo further development and 2) to evaluate the effect of activin and FSH on the development of secondary follicles derived from primordial follicles. Fragments of bovine ovarian cortex were cultured in serum-free medium for 6 days; thereafter, secondary follicles were isolated for further culture. After a maximum total of 21 days in vitro, follicles were either processed for histological assessment or opened to release the oocyte–cumulus complexes for inspection by light microscopy. Compared with control, significant follicle and oocyte growth were observed in activin-exposed follicles, with or without FSH, with some oocyte diameters measuring over 100 microns following a total in vitro period of 15 days. Significant oestradiol secretion was observed in follicles cultured in activin alone after a total of 9 days in vitro compared with other treatment groups; however, this effect was not sustained. In summary, this study demonstrates the promotion of primordial bovine follicle development within a two-step serum-free culture system with oocyte diameters >100 μm achieved over 15 days in vitro. Further development of this system is needed to support complete oocyte growth and thereafter in vitro maturation.

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Evelyn Telfer and R. G. Gosden

Summary. The incidence of polyovular types in the growing follicle population was estimated using quantitative cytology. Of 15 species studied, polyovular follicles were recorded in the following species and in ascending order of abundance: rabbits, rhesus monkeys, humans, cats, dogs. The incidence in bitches was 14% in animals aged 1–2 years but only 5% at 7–11 years old. The frequency of the various types of polyovular preantral follicle varied inversely with the numbers of oocytes per follicle and the probability of finding a follicle with more than 5 oocytes was remote. In young ovaries the frequency was constant in the early stages of growth but decreased in the largest preantral stage. The pattern in ageing ovaries was, by contrast, one of declining frequency such that few if any polyovular types completed development. The ovary of the ageing bitch was also characterized by a higher incidence of degenerating follicles and a much smaller pool of primordial stages. Polyovular follicles were larger than uniovular types at comparable stages which were defined by the number of granulosa cell layers. Their oocytes were smaller but the overall ooplasmic mass was increased with a corresponding increase in the mass of granulosa cells.

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C. Torrance, Evelyn Telfer, and R. G. Gosden

Summary. Follicles were isolated from the ovaries of 10-day-old C57BL6/CBA F1 hybrids by mechanical and enzymic treatment, embedded in a collagen-gel matrix to maintain the 3 dimensional integrity of the follicle and cultured for up to 14 days. Gels were removed at various times during the culture period and prepared for histology. Follicles grew from unilaminar to multilaminar stages within 6 days of the culture period. A more detailed assessment of growth by counting follicles at different stages and measuring oocyte and follicle diameters showed that follicle growth was maintained for up to 14 days in culture. Initially the proportion of unhealthy follicles was high but this declined after 6 days in culture.

Keywords: pre-antral follicles; collagen gel; culture; mouse

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Evelyn Telfer, C. Torrance, and R. G. Gosden

Summary. Isolated ovarian follicles taken from 10-day-old mice and cultured in collagen gel for 5 days, in the presence or absence of serum, were transplanted under the kidney capsule of ovariectomized mice. Hosts showed vaginal opening within 5 days and cornified vaginal smears by 9 days. Follicles proceeded to Graafian stages and luteinization occurred. Ovulation was not observed and oocytes degenerated within the luteinized follicle. Theca formation was preceded by the appearance of blood vessels within the graft. In-vitro fertilisation of harvested oocytes resulted in embryos.

Keywords: preantral follicles; collagen gel; culture; in vivo; mouse

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Evelyn Telfer, J. D. Ansell, Helen Taylor, and R. G. Gosden

Summary. The number of clonal precursors of granulosa cells in mouse ovarian follicles has been estimated using a technique based on the phenomenon of random X-chromosome inactivation of somatic cells and the use of an X-linked alloenzyme variant of the glycolytic enzyme PGK-1. The granulosa cells of follicles were oligoclonal in origin and founded by a small number of cells (about 5) which was consistent with histological observations. When the analysis was extended to two subcompartments of the follicle, the mural and cumulus granulosa cells, the results indicated that the cumulus and mural granulosa cells had a common origin.

Keywords: granulosa cells; primordial follicles; phosphoglycerate kinase; ovary

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Fiona H Thomas, Bruce K Campbell, David G Armstrong, and Evelyn E Telfer

The aim of this study was to determine the effect of regulation of IGF-I bioavailability on preantral follicle development in vitro. Bovine preantral follicles were cultured for 6 days in serum-free medium with increasing doses of Long R3 (LR3) IGF-I (an analog with low affinity for IGF-binding proteins (IGFBPs)), or human recombinant IGF-I (hrIGF-I). Follicle diameter and estradiol production were measured every second day. On day 6, ratios of oocyte/follicle diameter and oocyte morphology were assessed by histological examination, and IGFBP-2 and -3 were detected by immunocytochemistry and in situ hybridization respectively. Both types of IGF-I increased follicle diameter in a dose-dependent manner (P < 0.05) and increased estradiol production over control levels (P < 0.05). However, follicles treated with LR3 IGF-I and the highest concentration of hrIGF-I (1000 ng/ml) had smaller oocyte/follicle ratios, and increased oocyte degeneration, compared with controls or follicles treated with physiological concentrations of hrIGF-I (P < 0.05). IGFBPs were detected in cultured preantral follicles, indicating a requirement for regulation of IGF bioavailability during the early stages of follicular development. Specifically, IGFBP-3 mRNA was found to be expressed in oocytes, and IGFBP-2 immunoreactivity was detected in oocytes and granulosa cells of cultured follicles. In summary, the regulation of IGF-I bioavailability by IGFBPs is necessary for the co-ordination of oocyte and follicle development in vitro.

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R. G. Gosden, Evelyn Telfer, M. J. Faddy, and D. J. Brook

Summary. Halving the numbers of follicles in young adult mice by unilateral ovariectomy resulted in compensatory Graafian follicle growth with a reduction by about 25% of the expected number of oestrous cycles. The impact of the operation on the numbers and dynamics of preantral follicles during the first 2 months after ovariectomy was studied using a compartmental mathematical model to analyse differential follicle counts. There were changes in growth and/or death rates at all stages of follicle development, and the patterns emerging were time-dependent. The rate of follicle survival from the pool of unilaminar stages was paradoxically reduced, but those forming two granulosa cell layers continued to develop towards Graafian size. As the frequency of follicle death declined, the numbers of healthy large preantral and antral stages in unpaired ovaries rose to approach those in pairs of age-matched control ovaries, suggesting that follicles otherwise undergoing atresia were being rescued. In the long-term, follicle dynamics after unilateral ovariectomy at young ages did not appear to compromise fecundity seriously.

Keywords: follicles; ovary; unilateral ovariectomy; oestrous cycle; mouse

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R. G. Gosden, R. H. F. Hunter, Evelyn Telfer, C. Torrance, and N. Brown

In common with all other cells, the oocyte and granulosa cells are bathed in extracellular fluid. It has, however, become conventional to reserve the term ‘follicular fluid’ for that fraction of the extracellular fluid that accumulates in the antrum of larger follicles. This pool of fluid is of considerable biological significance since its composition indicates the environment in which the oocyte and granulosa cells are growing and maturing. Furthermore, it buffers the internal environment of the follicle against the influence of external conditions presented by the blood stream.

The chemical composition of follicular fluid has been studied extensively and found to consist of substances derived from blood as well as from local secretion and metabolism. Particular attention has been paid to the proteins and hormonal steroids. Rather than attempt a comprehensive review, this paper will focus on general physical characteristics of the fluid and the physiological factors that influence its formation. These properties determine the rate at which extracellular fluid is accumulating and, hence, the size and morphogenesis of the Graafian follicle. It is important to reveal the mechanism and dynamics of follicular fluid formation if the composition of the fluid is to be fully understood.

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Kirsty A Walters, John P Binnie, Bruce K Campbell, David G Armstrong, and Evelyn E Telfer

This study aimed to determine the effect of insulin-like growth factor-I (IGF-I) on early antral bovine follicular development, and the expression of insulin-like growth factor-binding protein-2 (IGFBP-2). Antral follicles separated into three different size groups were cultured for 6 days in medium supplemented with either a low (10 ng/ml) or high (1 μg/ml) dose of human recombinant IGF-I. Oestradiol production by follicles in all size ranges, cultured in the presence of the high concentration of IGF-I, significantly increased by day 6 (P < 0.05). Follicles in the smallest size range, 165–215 μm, cultured in a high dose of IGF-I, were found to be significantly increased in size (P < 0.01). Oocyte health of the largest follicles (281–380 μm) was significantly improved by the addition of IGF-I to the culture medium. mRNA expression of IGFBP-2 was decreased in the granulosa cells of follicles, size range 216–280 μm, cultured with a high dose of IGF-I (P < 0.05). Granulosa cells (P < 0.05) and oocytes (P < 0.01) of the largest follicles (281–380 μm) showed a decrease in IGFBP-2 expression (protein) when cultured in the control and low-IGF-I treatment groups. Therefore, the response of a bovine follicle to IGF-I is both dose and stage dependent. This work supports a role for IGF-I in modulating somatic and germ-cell maturation and development in early antral follicles. Furthermore, the inverse relationship between the level of IGF-I stimulation and IGFBP-2 expression suggests a local regulatory system modulating IGF-I availability.