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Biomedical subjects

R G Gosden

Publications and source records attributed to R G Gosden.

At least 19 recordsLinked to original sources
PubMed1997

Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue.

OBJECTIVE: To develop an efficient isolation technique for human primordial follicles. DESIGN: Prospective, experimental study of ovarian biopsies collected from healthy women undergoing elective cesarean section. Ovarian blocks either were fixed for histology and follicle counting or partially disaggregated with type 1A collagenase before or after cryopreservation. After partial disaggregation, follicles were isolated by microdissection. SETTING: Leeds General Infirmary. MAIN OUTCOME MEASURE(S): Follicle viability was assessed with live-dead stains using 5-(and 6-) carboxyfluorescein diacetate, succinimidyl ester and propidium iodide, respectively, and using electron microscopy. The numbers recovered were expressed as a percentage of the numbers of primordial follicles in comparable blocks of tissue and the viability of the whole follicle and oocyte were scored separately. RESULT(S): On average, 18.0 +/- 3.8 and 15.9 +/- 2.2 (mean +/- SEM) follicles per block were recovered from fresh and cryopreserved ovarian tissue, respectively, corresponding to recovery rates of 57.9% +/- 8.8% and 56.2% +/- 16.7%. In the fresh group, the percent viability of whole follicles and oocytes were 71.6% +/- 2.4% and 91.3% +/- 2% compared with 71.5% +/- 4.7% and 95% +/- 4.3% in the frozen-thawed group. Electron microscopy confirmed that the majority of the cells lacked ultrastructural signs of damage after isolation and cryopreservation. CONCLUSION(S): Primordial follicles can be isolated from fresh and cryopreserved human ovarian tissue with similar high efficiency and viability rates.

Adult↗
PubMed1996

A model conforming the decline in follicle numbers to the age of menopause in women.

The store of primordial follicles in the ovary is fixed before birth and dwindles with age until it is unable to provide enough Graafian stages to sustain menstrual cyclicity. According to a simple bi-exponential model of ageing, the rate of follicle disappearance increases at age 37.5 years (or when 25 000 follicles remain) so that the numbers fall to approximately 1000 at 51 years, the median age of menopause in the population. This study attempts to produce a biologically more realistic model of follicle disappearance and harmonizes follicle dynamics with the distribution of menopausal ages from an American survey. The step-change in the rate of follicle attrition was replaced by a model which assumed that this rate changes more gradually with the size of the follicle store. This produced a distribution of predicted menopausal ages (based on an assumed threshold of 1000 follicles) which was closer to observed data. The fit further improved when the model was modified by having a threshold that varied across the population. Using such a stochastic threshold model for menopause, the number of fertile years remaining could be forecast with an acceptable margin of uncertainty if it ever becomes possible to estimate the size of the follicle store in vivo.

Adult↗
PubMed1996

The establishment of follicular dominance in co-cultured mouse ovarian follicles.

An in vitro model of dominant and subordinate ovarian follicles was developed to allow a closer investigation of the phenomenon of follicular dominance. Preantral mouse ovarian follicles were cultured either alone or in pairs. Pairs of follicles were either in direct contact or in shared medium, but without physical contact. The experiments showed that where contact was allowed to develop between follicles one follicle invariably became dominant, while the other would grow and develop little during the culture period. In contrast, there was no effect of co-culture on follicle development in the absence of contact between the follicles. There was, therefore, no evidence of secretion of a diffusible factor by a dominant follicle that could affect the development of neighbouring follicles. After 6 days of co-culture with contact, histological examination of the subordinate follicle showed that it was healthy, in spite of remarkably little growth during culture. In a further experiment, the subordinate follicle was separated from the dominant one after 2 days of co-culture (when a significant difference in size had already developed), and cultured alone. These 'released' follicles exhibited a spurt of growth during the remaining culture period, attaining a size and appearance indistinguishable from those of controls by the end of culture. This confirms that the dominant follicle, while depressing the growth of its neighbour, is not inducing irreversible atresia in the subordinate follicle in this model. The in vitro model will allow a more detailed study of direct influences of dominant-subordinate follicle interactions, and should increase our knowledge of a poorly understood phenomenon.

Animals↗
PubMed1995

A mathematical model of follicle dynamics in the human ovary.

A mathematical model has been developed to describe the rates of growth and death of follicles in human ovaries between 19 and 50 years of age. It was based on the numbers of follicles at three successive stages of development, which were obtained by counting follicles in histological sections of ovaries from 52 normal women. The model indicated that follicle dynamics were age dependent, with a transition at 38 years of age when the rate of follicle disappearance increased. The rates of follicle growth increased at successive stages but did not change with age. The annual egress from stage III (consisting of follicles with two or more granulosa cell layers) was affected by the declining numbers of small follicles, and corresponded to 31, nine and one follicles per day at 29-30, 39-40 and 49-50 years of age respectively. The rate of death at stage I (representing small, resting follicles) was the only parameter which varied significantly with age: no evidence of significant atresia was found for this stage in ovaries < or = 38 years old, but there was significant death above this age. As a consequence, only 40% of follicles leaving stage I reached stage III in older ovaries and just 1500 follicles in toto remained at 50 years of age from the 300,000 present at 19 years. This high death rate of small follicles appears to be responsible for advancing the timing of ovarian failure, and therefore of menopause, to midlife in our species.

Adult↗
PubMed1994

Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile.

A model culture system has been developed whereby individual, primary ovarian mouse follicles can be grown in vitro to the Graafian stage in the normal physiological time course, and then ovulated in response to luteinizing hormone. We report here on the successful fertilization and subsequent embryo development of the oocytes from such follicles. This is the first time that oocytes from in-vitro matured whole follicles have been fertilized and shown to produce viable offspring in host animals. The study demonstrates that the culture system mimics physiological conditions for normal follicle development.

Animals↗
PubMed1994

Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196 degrees C.

Cortical slices were prepared from the right ovaries of six lambs and either grafted directly to the ovarian pedicles of origin or cooled slowly to liquid nitrogen temperatures in medium containing dimethylsulphoxide. Three weeks later, the contra-lateral ovary was removed and replaced with frozen-thawed slices from the same animal. Two of the animals mated during their second oestrous cycle 3-4 months later and the remainder had at least one ovulatory cycle. The pregnancies reached full-term development, one lamb being derived from an ovulation in a fresh graft and the other from a frozen-thawed graft. None of the sheep had peripheral plasma concentrations of follicle stimulating hormone or luteinizing hormone consistently in the castrate range, and only one graft was devoid of follicles when the animals were slaughtered 9 months after the operations. Grafts with primordial follicles always contained developing follicles, which occasionally attained pre-ovulatory sizes of 7 mm in diameter. A corpus albicans was present in five grafts. Since all developing follicles had degenerated 1 week after grafting in an additional ewe, the large follicles in long-term grafts had presumably commenced growing after the operation. There were no obvious differences between fresh and frozen-thawed grafts in either appearance or weight, and all had apparently grown since implantation. Despite substantial depletion of primordial follicle numbers, the results indicated that frozen storage and replacement of a patient's own ovarian tissue might be practicable when fertility potential is threatened by chemotherapy/radiotherapy.

Animals↗
PubMed1994

Characterization of follicular energy metabolism.

It has been shown that mouse ovarian follicles have a large glycolytic capacity, and this study was undertaken to determine whether follicles can develop normally using glycolysis alone. Pre-antral mouse follicles were grown using an in-vitro system which supports development to the preovulatory stage within 5 days. Cultures were maintained in either aerobic conditions in the presence of the inhibitor of oxidative phosphorylation, sodium malonate, or under anaerobic conditions. Samples of media were removed every 24 h and analysed for oestradiol using an enzyme-linked immunosorbent assay (ELISA) technique and for lactate and glucose using a fluorometric assay. Follicle size, oestradiol production and glycolytic rate were not significantly different between control and sodium malonate-treated follicles. Follicles cultured under anaerobic conditions showed significantly slower rates of growth and oestradiol production compared with controls. However, the rate of glycolysis was significantly higher during anoxia. Results indicated that anaerobic glycolysis may sustain limited periods of growth during the pre-antral phase, but that the presence of oxygen is vital to ensure normal development. It is concluded that pre-antral follicles can undergo development to the preovulatory stage using glycolysis alone, a feature which may allow them to conserve their limited supply of oxygen for other vital biosynthetic processes.

Animals↗
PubMed1994

The effect of glucose metabolism on murine follicle development and steroidogenesis in vitro.

Previous studies have shown that mouse ovarian follicles produce large amounts of lactate during growth and maturation in vitro, suggesting a metabolic preference for glycolysis. Further investigations were therefore undertaken to determine firstly the effect of gonadotrophins on glucose uptake and secondly the role of glucose in follicle lactate production, development and steroidogenesis. Pre-antral mouse follicles were cultured individually in media containing different concentrations of glucose (1-5.5 mM) using a system which supported development to the pre-ovulatory stage within 5 days. Samples of media were removed every 24 h and analysed for oestradiol using an enzyme-linked immunosorbent assay technique and for glucose uptake and lactate production using a fluorimetric assay. Results showed that all the glucose consumed was converted to lactate, irrespective of the glucose concentration. Growth and steroidogenesis were also dependent on glucose concentration, and at concentrations below 2 mM, follicle development was significantly retarded. Results confirm that follicles adopt a predominantly glycolytic mode of energy production to sustain growth and steroidogenesis, and that the glycolytic rate is stimulated by gonadotrophins.

Animals↗
PubMed1994

Clonal analysis of chimaeric mouse ovaries using DNA in situ hybridization.

Analysis of chimaeric mouse ovaries using DNA in situ hybridization was undertaken to (i) investigate the morphogenesis of follicular cell clonal expansion, (ii) evaluate whether the different cell populations within the ovary are derived from the same or unrelated progenitor cells and (iii) estimate the number of progenitor cells giving rise to the different types of ovarian cell. Chimaeras were produced by aggregation of eight-cell morulae from normal mice and those transgenic for the beta-globin gene. Chimaeric blastocysts were transferred to pseudopregnant hosts and the ovaries of resultant adult offspring were prepared for in situ hybridization using a digoxigenin-labelled cDNA probe to the beta-globin gene. Results showed that follicles are constructed by the non-random, radial proliferation of granulosa cell clones, which form long, thin, unbranched columns across the follicle wall. Qualitative and quantitative studies revealed that both peripheral and central granulosa cells are derived from the same progenitor cells. Phenotypic differences may therefore be due to positional cues within the follicle rather than being cell lineage dependent. It is suggested that granulosa cells and germinal epithelium may be partly derived from the same progenitor cells and that theca externa is probably derived from interstitial tissue. However, results from this study did not support the contention that theca interna and theca externa-interstitial tissue have the same origin, and it is suggested that the former cell type may exist in an undifferentiated state from early stages of follicle development. Furthermore, granulosa and germinal epithelium appear to be derived from different progenitor cells from either theca interna or theca externa-interstitial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effects of epidermal growth factor on the growth and differentiation of cultured mouse ovarian follicles.

The aim of this investigation was to determine the influence of epidermal growth factor (EGF) on follicular growth and steroidogenesis in mice. Follicles were cultured in medium containing human recombinant EGF at concentrations of 1-20 ng ml-1. Oestradiol production was assayed immunoenzymatically, and growth was measured by recording follicle diameter daily and by analysing the total DNA content of follicles. The effect of EGF on cumulus-oocyte complexes isolated from cultured follicles was also assessed. Results showed that EGF inhibited oestradiol production in a dose-dependent manner, but had no mitogenic effect. Despite almost complete inhibition of oestradiol production at concentrations of EGF > or = 10 ng ml-1, follicles were still able to achieve preovulatory size and morphology, although the incidence of atresia was increased over controls. Conversely, at a concentration of only 1 ng EGF ml-1, a significantly greater number of follicles reached the Graafian stage compared with control follicles. Cumulus expansion and meiotic maturation by isolated cumulus-oocyte complexes from cultured follicles was dramatically stimulated in the presence of EGF and FSH, but not by FSH alone. These findings suggest that EGF may have a modulatory effect on oestradiol production in vivo, and that follicular growth and differentiation may be uncoupled from steroidogenesis. Finally, ovulatory changes in the cumulus-oocyte complex may require the presence of this factor.

Animals↗
PubMed1994

Follicular development from ovarian xenografts in SCID mice.

Cortical slices of either cat or sheep ovaries were grafted under the renal capsules of ovariectomized SCID mice. The grafts became vascularized and were still surviving with large follicles present at autopsy up to nine months later. As developing follicles undergo atresia during the period of ischaemia after ovarian grafting, those found in long-term grafts at autopsy had presumably started to grow from the primordial stage after transplantation. Some follicles had reached a diameter of 3 mm with a normal antrum and appeared to be cytologically normal, but the latent period for the emergence of antral follicles was shorter in cat compared with sheep grafts. Oestradiol production from grafts, as indicated by vaginal cornification and plasma measurements collected at autopsy, was not constant and circulating concentrations varied among animals, and were sometimes far in excess of the normal physiological range of the host. The vaginal smears never presented cytological patterns like those of the normal mouse oestrous cycle, and ovulation had not occurred in any of the grafts. These results demonstrate that ovarian xenografts in SCID mice can serve as experimental models for investigating follicle development in species in which follicle growth in vitro and studies of the parent animal are impracticable.

Animals↗
PubMed1994

Effect of inhibin immunoneutralization on steroidogenesis in rat ovarian follicles in vitro.

Inhibin is a putative gonadal paracrine factor produced by FSH-stimulated granulosa cells. To assess the paracrine function of inhibin further, preantral follicles (approx. 240 microns) with attached thecal cells were isolated mechanically from immature rat ovaries and cultured individually in vitro for 5 days in medium containing homologous serum and FSH. After 5 days, follicles had grown to preovulatory size (approx. 470 microns) with a commensurate increase in oestradiol secretion but not progesterone. Immunoneutralization of endogenous inhibin resulted in a significant decrease in oestradiol secretion and an increase in progesterone accumulation. When antiserum-treated follicles were supplemented with exogenous inhibin, oestradiol secretion was restored and progesterone accumulation was reduced. Aromatase substrate (androstenedione) levels were too low to measure, regardless of antiserum treatment. However, follicles treated with inhibin antiserum in the presence of exogenous androstenedione also exhibited oestradiol levels similar to untreated controls while progesterone accumulation remained elevated. We interpret these data as evidence that inhibin secreted by FSH-stimulated granulosa cells exerts a physiologically significant paracrine function in the follicle wall. Based on previous observations that inhibin stimulates androgen synthesis by isolated thecal/interstitial cells, it is proposed that granulosa-derived inhibin promotes thecal androgen synthesis and hence oestrogen synthesis during preovulatory follicle development. The antiserum-induced increase in progesterone accumulation is most probably explained by reduced metabolism of C21 steroid substrate to androgen in thecal/interstitial cells deprived of inhibin. It is concluded that inhibin is a physiological modulator of follicular steroidogenesis which exerts its effect at the level of thecal cell androgen synthesis.

Androstenedione↗
PubMed1993

Transplantation of frozen-thawed mouse primordial follicles.

Primordial follicles were isolated from juvenile mouse ovaries and cryopreserved by slow freezing with dimethylsulphoxide as the cryoprotectant. After thawing, approximately 80% of the oocytes and 65% of the somatic cells excluded Trypan Blue dye, indicating that cell membranes were still intact. Frozen-thawed cells were suspended in plasma clots and transplanted to the ovarian bursas of host animals that had been sterilized by oophorectomy. The grafts of frozen-thawed cells reorganized into morphologically distinguishable ovaries which produced signs of oestrogenic activity. After natural mating, host females produced normal offspring that were demonstrated by genetic markers to be derived from the transplanted frozen-thawed primordial follicles.

Animals↗
PubMed1993

Pattern of lactate production and steroidogenesis during growth and maturation of mouse ovarian follicles in vitro.

A model culture system for the study of follicular metabolism has been developed from existing methods of whole-follicle culture. The modified system mimics the in vivo growth and maturation of mouse ovarian follicles from primary to preovulatory stages and the modulating influences of the LH and FSH. This is the first study to demonstrate ovulation in vitro from individual ovarian follicles. The pattern of follicular lactate production relative to steroidogenesis was studied throughout in vitro follicular development over a period of 6 days. Twenty-four-hour samples of medium from individual follicles were analyzed for lactate and pyruvate by an automated analytical technique, and for estradiol and progesterone by an immunoenzymatic method. Follicles produced remarkably large quantities of lactate and estradiol during FSH-stimulated development in vitro. LH further stimulated lactate production but resulted in a significant decrease in estradiol secretion and an ovulation rate of 30%. Progesterone production was not detectable throughout the culture period, and follicles showed no evidence of pyruvate uptake. These findings demonstrate the validity of using this model culture system for the study of follicular metabolism and provide new information on the pattern of carbohydrate metabolism relative to steroidogenesis during follicular growth and maturation.

Animals↗
PubMed1993

Quantitative and cytochemical studies of mast cell proteases in rat ovaries and uteri in various reproductive states.

A role for mast cell proteases (RMCP I and II) in the cyclical remodelling of ovarian and uterine tissues of rats was investigated in the oestrous and pregnancy cycles using immunocytochemistry and enzyme-linked immunosorbent assays. The concentrations of RMCP I exceeded that of RMCP II by 100-fold in both tissues, but were always much higher in uteri than ovaries. Most of the protease activity in the uterus was located in the myometrium, whereas it was more focally distributed in the hilus and medulla of the ovary. Protease activity was confined to mast cells identified by metachromatic staining and no single cell contained both proteases. The concentrations of RMCP I and II in the two organs did not fluctuate throughout the 4-day oestrous cycle. Neither were RMCP I concentrations in the uterus altered by administration of diethylstilboestrol to ovariectomized animals, although total amounts per uterus were substantially greater than in the controls. Concentrations of RMCP I were substantially reduced in the uterus after day 6 of pregnancy and rose during the puerperium. The reduction was greater in pregnant than in pseudopregnant horns and tended to be lower in the vicinity of conceptuses than between them. The physiological significance of the lower mast cell protease concentrations is unclear, although their absence may contribute to the decreased protein catabolism during pregnancy.

Animals↗
PubMed1992

Transplantation of fetal germ cells.

Oogonial stem cells are short-lived and endow the ovary with its lifetime store of follicles during fetal life. No compensatory mechanisms exist to replace germ cells that are lost for whatever reason after birth. Fetal germ cells and the abundant primordial follicles of immature animals can be successfully stored at low temperatures and transplanted to hosts to generate normal ovulatory cycles. Sterilized hosts are restored to fertility. Such results suggest that the abundant reserves of germ cells in the ovaries of human abortuses offer opportunities for treating patients whose sterility is due to afollicular ovaries uncomplicated by autoimmune disease. The prospects for this treatment depend largely on the vigilance of the recipient's immune system and public attitudes to a radical treatment, though one that promises to overcome sterility and hypoestrogenism in women with either premature menopause or gonadal dysgenesis.

Adult↗
PubMed1992

Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause.

Menopause is triggered by the number of ovarian follicles falling below a threshold number and is irreversible because oogonial stem cells disappear after birth. Since it is the result of programmed disappearance of a limited store of follicles, menopause can be predicted using mathematical models based on total follicle counts at different ages. Our model shows follicle numbers decline bi-exponentially rather than as a simple exponential function of age, as had been assumed, with a first exponential rate parameter of -0.097 and a second of -0.237. The change occurred when numbers had fallen to the critical figure of 25,000 at age 37.5 years. The unexpectedly faster rate of ovarian ageing afterwards lowers the follicle population to 1000 at approximately 51 years, and was adopted as the menopausal threshold because it corresponds to the median age of menopause in the general population. Had the earlier rate persisted menopause would not be expected until 71 years. The impact of step reductions of follicle numbers on the prospective span of menstrual life was predicted by the model. A reduction by 50% before age 30 years resulted in the threshold being reached at 44 years and 0.6 year later for every subsequent year until age 37.5 years after which it is reached at 48 years. A reduction of 90% in childhood before age 14 years could result in menopause as early as 27 years, with increments of 0.6 year per year afterwards until after 37.5 years when it is expected at age 41 years.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗