Evelyn E. Telfer

Growth and Antrum Formation of Bovine Preantral Follicles in Long-Term Culture In Vitro

Abstract Culture of preantral follicles has important biotechnological implications through its potential to produce large quantities of oocytes for embryo production and transfer. A long-term culture system for bovine preantral follicles is described. Bovine preantral follicles (166 ± 2.15 μm), surrounded by theca cells, were isolated from ovarian cortical slices. Follicles were cultured under conditions known to maintain granulosa cell viability in vitro. The effects of epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, FSH, and coculture with bovine granulosa cells on preantral follicle growth were analyzed. Follicle and oocyte diameter increased significantly (P < 0.05) with time in culture. FSH, IGF-I, and EGF stimulated (P < 0.05) follicle growth rate but had no effect on oocyte growth. Coculture with granulosa cells inhibited FSH/IGF-I-stimulated growth. Most follicles maintained their morphology throughout culture, with the presence of a thecal layer and basement membrane surrounding the granulosa cells. Antrum formation, confirmed by confocal microscopy, occurred between Days 10 and 28 of culture. The probability of follicles reaching antrum development was 0.19 for control follicles. The addition of growth factors or FSH increased (P < 0.05) the probability of antrum development to 0.55. Follicular growth appeared to be halted by slower growth of the basement membrane, as growing follicles occasionally burst the basement membrane, extruding their granulosa cells. In conclusion, a preantral follicle culture system in which follicle morphology can be maintained for up to 28 days has been developed. In this system, FSH, EGF, and IGF-I stimulated follicle growth and enhanced antrum formation. This culture system may provide a valuable approach for studying the regulation of early follicular development and for production of oocytes for nuclear/embryo transfer, but further work is required.

A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin

BACKGROUND The objective of this study was to determine whether follicles grown within human ovarian cortical strip culture for 6 days in serum-free medium could be isolated at the secondary stage of pre-antral development and grown in vitro to the late pre-antral/early antral stage during a 4 day culture period. METHODS Ovarian cortical biopsies were obtained from six women aged 26-40 years, with informed consent, during elective Caesarean section. Small tissue slices of ovarian cortex, with underlying stromal tissue removed, were cultured in serum-free medium for 6 days and at the end of this period pre-antral (secondary) follicles were dissected from the strips. Seventy-four intact pre-antral follicles ranging in size (66-132 microm) (mean size 100 microm +/- 3.4) were selected for further culture. Follicles were placed individually within V-shaped microwell culture plates in serum-free medium in the presence (n = 38) or absence (n = 36) of 100 ng/ml of human recombinant activin A. RESULTS Pre-antral follicles grown for 4 days in the presence of activin A grew to a larger size (mean diameter 143 microm +/- 7.4) than those grown in control medium (mean diameter 111 microm +/- 8) (P < 0.005). Ninety percent of follicles cultured in the presence of activin A increased in size during the first 2 days of culture compared with only 36% of follicles in control medium (P > 0.005). Of the follicles surviving the entire culture period, 30% of those cultured in the presence of activin A showed normal morphology with intact oocytes and antral formation. None of the follicles grown in control medium developed antral cavities and >90% of those follicles collected at the end of the culture period showed signs of oocyte degeneration. CONCLUSIONS The results reported here demonstrate that under certain conditions, it is possible to achieve accelerated oocyte/follicle development from human primordial/primary follicles. This provides the first encouraging step towards achieving full in vitro growth of human oocytes.

On Regenerating the Ovary and Generating Controversy

For more than a half a century, biologists have upheld the theory that in most mammalian species, oocytes are formed before or shortly after birth, but never in adulthood. This foundation of reproductive science has survived the rapid growth of new technology and knowledge and has remained virtually unchallenged until two recent papers were published by the group headed by Jonathan Tilly. The first paper claims that mouse germline stem cells (GSCs) replace ovarian follicles that have been rapidly lost through follicle death (Johnson et al., 2004).

Ovarian follicle culture: advances and challenges for human and nonhuman primates

The removal and cryostorage of ovarian cortical biopsies is now offered as a fertility preservation option for young women. The only available option to restore fertility using this tissue is by transplantation, which may not be possible for all patients. The full potential of this tissue to restore fertility could be achieved by the development of in vitro systems that support oocyte development from the most immature stages to maturation. The techniques of in vitro growth (IVG) combined with in vitro maturation (IVM) are being developed with human tissue, but comparing different systems has been difficult because of the scarcity of tissue so nonhuman primates are being used as model systems. There are many challenges to developing a complete culture system that would support human oocyte development, and this review outlines the approaches being taken by several groups using tissue from women and nonhuman primate models to support each of the stages of oocyte development.

The role of neurotrophin receptors in female germ-cell survival in mouse and human

During mammalian ovary formation, the production of ovarian follicles is accompanied by an enormous loss of germ cells. It is not known how this loss is regulated. We have investigated the role of the Trk tyrosine kinase receptors, primarily TrkB, in this process. The ovaries of TrkB–/– and TrkC–/– mice with a mixed (129Sv × C57BL/6) genetic background were examined shortly after birth. Around 50% of TrkB–/– mice had grossly abnormal ovaries that contained greatly reduced numbers of follicles. No defects were found in the ovaries of TrkC–/– mice. Congenic TrkB–/– mice were generated on 129Sv and C57BL/6 backgrounds: whereas the former had a mixed ovarian phenotype similar to that of the original colony of mice, the ovaries of all offspring of the C57BL/6 congenic line contained reduced numbers of follicles. RT-PCR showed that mRNA encoding TrkB and its two ligands, neurotrophin 4 (NT4) and brain-derived neurotrophic factor (BDNF), were present throughout the period of follicle formation in the mouse. In situ hybridisation showed that TrkB was expressed primarily in the germ cells before and after follicle formation. Mouse neonatal and fetal ovaries and human fetal ovaries were cultured in the presence of K252a, a potent inhibitor of all Trk receptors. In mice, K252a inhibited the survival of germ cells in newly formed (primordial) follicles. This effect was rescued by the addition of basic fibroblast growth factor (bFGF) to the culture medium. Combined addition of both BDNF and NT4 blocking antibodies lowered germ-cell survival, indicating that these TrkB ligands are required in this process. The results indicate that signalling through TrkB is an important component of the mechanism that regulates the early survival of female germ cells.

The ageing ovary and uterus: new biological insights

BACKGROUND Advanced maternal age is associated with reduced fertility and adverse pregnancy outcomes. This review details recent developments in our understanding of the biology and mechanisms underlying reproductive ageing in women and the implications for fertility and pregnancy. METHODS Sociological online libraries (IBSS, SocINDEX), PubMed and Google Scholar were searched for relevant demographic, epidemiological, clinical and biological studies, using key words and hierarchical MeSH terms. From this, we identified and focused on key topics where it was judged that there had been clinically relevant advances in the understanding of ovarian and uterine ageing with implications for improved diagnostics and novel interventions. RESULTS Mapping of the ovarian reserve, follicular dynamics and associated biomarkers, across the reproductive lifespan has recently been performed. This now allows an assessment of the effects of environmental, lifestyle and prenatal exposures on follicular dynamics and the identification of their impact during periods of germ cell vulnerability and may also facilitate early identification of individuals with shorter reproductive lifespans. If women choose to time their family based on their ovarian reserve this would redefine the meaning of family planning. Despite recent reports of the potential existence of stem cells which may be used to restore the primordial follicle and thereby the oocyte pool, therapeutic interventions in female reproductive ageing at present remain limited. Maternal ageing has detrimental effects on decidual and placental development, which may be related to repeated exposure to sex steroids and underlie the association of ageing with adverse perinatal outcomes. CONCLUSIONS Ageing has incontrovertible detrimental effects on the ovary and the uterus. Our enhanced understanding of ovarian ageing will facilitate early identification of individuals at greatest risk, and novel therapeutic interventions. Changes in both ovary and uterus are in addition to age-related co-morbidities, which together have synergistic effects on reducing the probability of a successful pregnancy outcome.

Culture of Bovine Preantral Follicles in a Serum-Free System: Markers for Assessment of Growth and Development

Abstract Satisfactory development of bovine follicles in vitro remains elusive. This study used a serum-free system to evaluate the effects of insulin-like growth factor-1 (IGF-1) on bovine preantral follicles in culture and to identify the activity of gelatinase matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) in vitro to assess their potential as markers of development. Preantral follicles were cultured for 6 days in serum-free medium containing insulin and IGF-1 (10 ng/ml). No difference was observed in follicular growth, health, or antrum formation between IGF-1-treated follicles and controls. However, IGF-1 had a negative effect (P < 0.01) on oocyte size and granulosa cell proliferation. When MMP-9 was secreted, the probability of follicles having healthy granulosa or theca cells at the end of the culture period was 0.85 and 0.60, respectively. If TIMP-1 was released, the probability of follicles having healthy somatic cells was 0.79. When TIMP-2 was detected, the probability of granulosa and theca cell health was 0.78 and 0.67, respectively. These results demonstrate no positive effects of IGF-1 on bovine follicles in this system. Furthermore, MMP-9 and TIMPs are related to follicular health and, therefore, can be used as markers of follicular development.

Oocyte development in bovine primordial follicles is promoted by activin and FSH within a two-step serum-free culture system

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 mum achieved over 15 days in vitro. Further development of this system is needed to support complete oocyte growth and thereafter in vitro maturation.

Cancer treatment and gonadal function: experimental and established strategies for fertility preservation in children and young adults

Preservation of gonadal function is an important priority for the long-term health of cancer survivors of both sexes and all ages at treatment. Loss of opportunity for fertility is a prime concern in both male and female cancer survivors, but endocrine effects of gonadal damage are likewise central to long-term health and wellbeing. Some fertility preservation techniques, such as semen and embryo cryopreservation, are established and successful in adults, and development of oocyte vitrification has greatly improved the potential to cryopreserve unfertilised oocytes. Despite being recommended for all pubertal male patients, sperm banking is not universally practised in paediatric oncology centres, and very few adolescent-friendly facilities exist. All approaches to fertility preservation have specific challenges in children and teenagers, including ethical, practical, and scientific issues. For young women, cryopreservation of ovarian cortical tissue with later replacement has resulted in at least 40 livebirths, but is still regarded as experimental in most countries. For prepubertal boys, testicular biopsy cryopreservation is offered in some centres, but how that tissue might be used in the future is unclear, and so far no evidence suggests that fertility can be restored. For both sexes, these approaches involve an invasive procedure and have an uncertain risk of tissue contamination in haematological and other malignancies. Decision making for all these approaches needs assessment of the individuals risk of fertility loss, and is made at a time of emotional distress. Development of this specialty needs better provision of information for patients and their medical teams, and improvements in service provision, to match technical and scientific advances.

The development of methods for isolation and culture of preantral follicles from bovine and porcine ovaries

Abstract The recent advances in reproductive technologies such as, in vitro fertilisation, embryo transfer, transgenesis, sexing and cloning cannot be widely applied to domestic species because of the shortage of suitable oocytes to manipulate in vitro. Current methods of superovulation and In vitro maturation of oocytes from antral follicles has not provided sufficient material to allow rapid progress. The ovaries of fetal and prepubertal animals contain a large number of preantral follicles which could be a potential supply of mature oocytes for in vitro manipulation. Culture systems that support the growth and differentiation of rodent preantral follicles have been successful at producing large numbers of meiotically competent oocytes. Methods of isolating and culturing preantral follicles from bovine and porcine ovaries are now being developed. This paper highlights the latest developments in these techniques and discusses how close we are to achieving ‘oocyte farming’ for domestic species.