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Biomedical subjects

E E Telfer

Publications and source records attributed to E E Telfer.

14 recordsLinked to original sources
PubMed2003

Analysis of atresia in equine follicles using histology, fresh granulosa cell morphology and detection of DNA fragmentation.

Follicular atresia has been examined previously by various biochemical and histological methods. The aim of this study was to compare, for the first time, detection of granulosa cell apoptosis by biochemical DNA analysis and microscopic examination of fresh granulosa cell morphology with the established method of detecting atresia by histology in equine follicles. DNA extracted from granulosa cells was examined by staining with ethidium bromide and end-labelling with [(32)P]dideoxy-ATP, which labels the free 3'-end of DNA fragments. In 25 of 26 follicles (96%) there was agreement between end-labelling and staining of DNA with ethidium bromide (P < 0.001). Granulosa cell apoptosis was distinguished more easily in the end-labelled samples than by staining with ethidium bromide. Histological atresia and apoptosis as detected by biochemical DNA analysis were significantly correlated (P < 0.02) with 20 of 22 follicles (91%) receiving corresponding classifications with the two methods. No follicles with granulosa cell apoptosis as detected by biochemical DNA analysis were histologically viable, but some of the histologically early atretic follicles did not display DNA laddering. Stereomicroscopic evaluation of morphology of the fresh granulosa cells was significantly correlated (P < 0.001) with the histological findings, with 29 of 33 follicles (88%) receiving corresponding classifications. There was a potential error in determining follicle health by biochemical DNA analysis only, as both histologically early and late atretic follicles in some cases did not show DNA laddering. Thus, if relying solely on biochemical detection of apoptosis, severely atretic follicles could wrongly be classified as healthy follicles.

Animals↗
PubMed2003

Domestic ruminants as models for the elucidation of the mechanisms controlling ovarian follicle development in humans.

It is necessary to understand the basic physiology underlying the complex process of folliculogenesis to address common causes of infertility and to devise innovative strategies to increase the efficiency of assisted reproduction technologies. Availability of suitable ovarian tissue is a major constraint to research in this area in humans, and monovulatory domestic ruminants represent a physiologically relevant model to elucidate basic mechanisms before more focused clinical investigations. This paper reviews the development of several whole animal and cell culture models in ruminants that have allowed basic investigations into the endocrine and local mechanisms regulating preantral and antral follicle development in monovulatory species. Studies on preantral follicle development using the ovarian autograft model have shown, contrary to accepted dogma, that FSH may mediate the rate at which preantral follicles grow and have provided evidence to support the existence of local regulatory feedback mechanisms that influence the rate of primordial follicle initiation and preantral follicle development. Studies on the endocrine control of antral follicle development using the GnRH-antagonist model have shown that a pulsatile mode of LH delivery is not a requirement for normal patterns of follicle development and ovarian hormone secretion. Studies on the local control of somatic cell differentiation using physiological cell culture models have highlighted the essential relationship between somatic cell communication and expression of differentiative markers. We conclude that the domestic ruminant represents a valuable model system for the elucidation of the endocrine and local mechanisms controlling both early and terminal stages of follicle development in monovulatory species. The results of these investigations have direct strategic relevance within clinical medicine.

Animals↗
PubMed2001

Effect of ascorbic acid on health and morphology of bovine preantral follicles during long-term culture.

During ovarian folliculogenesis, ascorbic acid may be involved in collagen biosynthesis, steroidogenesis and apoptosis. The aims of this study were to determine the effects of ascorbic acid on bovine follicle development in vitro. Preantral follicles were cultured for 12 days in serum-free medium containing ascorbic acid (50 microg ml(-1)). Half of the medium was replaced every 2 days, and conditioned medium was analysed for oestradiol and matrix metalloproteinase 2 (MMP-2) and MMP-9 secretion. On day 12, cell death was assessed by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In the absence of serum, there was significant (P < 0.05) follicle growth and oestradiol secretion over the 12 day culture period. Ascorbic acid had no effect on these parameters. The addition of serum from day 0 stimulated follicle growth (P < 0.05), but compromised follicle integrity. By day 12 of culture, a higher proportion of follicles remained intact in the presence of ascorbic acid in serum-free conditions (P < 0.05), and significantly (P < 0.01) less granulosa and theca cell death was observed in these follicles than in control follicles. Moreover, ascorbic acid significantly (P < 0.05) increased production of MMP-9, an enzyme involved in basement membrane remodelling. In conclusion, this culture system was capable of supporting follicle differentiation over the 12 day culture period. Furthermore, ascorbic acid maintains bovine follicle health and basement membrane remodelling in vitro.

Animals↗
PubMed2001

In vitro development of pig preantral follicles.

A limiting factor to realizing the full potential of many of the new reproductive techniques is the lack of abundant numbers of fertilizable oocytes. This problem could be addressed by using the large source of oocytes available from preantral and primordial follicles by developing systems for in vitro growth. In vitro systems that use early growing follicles as a source of oocytes have been developed for laboratory species and these have been successful in producing live young. If successful, in vitro growth in association with in vitro maturation (IVM) and cryopreservation would optimize in vitro production systems. In vitro growth systems that support the growth of pig preantral follicles have been developed and have been successful in producing meiotically competent oocytes but, to date, no live young have been produced. However, these systems remain to be characterized and their main application is as experimental models to study the processes of early oocyte and follicle development. This review provides an overview of culture systems that have been developed for domestic species and discusses how these are furthering our basic knowledge of early follicular development, as well as considering the benefits and potential problems associated with in vitro growth systems.

Animals↗
PubMed2000

In vitro development of oocytes from porcine and bovine primary follicles.

A limiting factor to realising the full potential of many of the new reproductive techniques is the lack of availability of fertile oocytes. Methods for maturing oocytes in vitro (IVM) have been developed to address this problem but the success rate and quality of embryos produced by IVM is variable. The variation in success may be due to the poor quality of oocytes that are being selected for maturation, since these would be taken from developed antral follicles. To attempt to eliminate this variation and increase the numbers produced, it may be better to use the large source of oocytes from preantral and primordial follicles by developing systems for in vitro growth (IVG). In vitro systems that utilise early growing follicles as a source of oocytes have been developed for laboratory species and these have been successful in producing live young. If successful, IVG in association with IVM would supercede existing technology for assisted reproduction in both humans and animals by making it possible to develop the desired number of high quality oocytes from small amounts of ovarian tissue. However, developing IVG systems for species with follicles that develop over several months presents enormous technical challenges. We have developed systems that permit the growth of individual porcine and bovine preantral follicles for periods of up to 20 days. Porcine follicles grown in micro-wells show a higher rate of survival if grown in the presence of serum than follicles grown under serum free conditions. Oocytes recovered from in vitro grown porcine follicles are capable of reaching metaphase II after in vitro maturation. A similar system has been developed for bovine follicles and survival rate is high under serum free conditions but as yet no oocytes from in vitro grown oocytes have been capable of completing meiotic maturation.

Animals↗
PubMed2000

Ovarian autografts in sheep as a model for studying folliculogenesis.

The studies outlined in this review indicate that the cortical autograft, in addition to its clinical application as a means to restore fertility, represents a valuable experimental model that can be exploited to examine aspects of both early and terminal follicle development. The autograft procedure is a means to experimentally deplete the follicle population in an individual and this procedure results in similar endocrine changes and reproductive cycle perturbances as those observed in aged sheep and women with incipient ovarian failure. This methodology therefore represents a non-primate large animal model to study the consequences of, and possible interventions to overcome, reproductive problems associated with depleted ovarian follicular reserves. Without the necessity of keeping animals for large periods of time so that this depletion can occur naturally. In terms of early follicle development, the fact that the ischaemia that occurs during revascularisation of the autograft effectively synchronises follicle development at the primordial stages of development means that the autograft can be used as a model to study the control of early follicle development. This model has been used to examine the role of FSH (follicle stimulating hormone) in the control of early follicle development and the preliminary data presented provides strong evidence that FSH does indeed modulate early folliculogenesis, confirming the value of this model as a means of performing experimental investigations in this area. Further work using this model will concentrate on the role of other endocrine and local factors in the control of early folliculogenesis and the identification of the key developmental checkpoints during this process, with a view to designing physiological culture systems to support early follicle and oocyte development.

Animals↗
PubMed2000

Growth and antrum formation of bovine preantral follicles in long-term culture in vitro.

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 micrometer), 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.

Animals↗
PubMed2000

Culture of bovine preantral follicles in a serum-free system: markers for assessment of growth and development.

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.

Animals↗
PubMed1998

In vitro models for oocyte development.

The mammalian ovary has a large store of primordial follicles, which are a potential source of oocytes for in vitro production of embryos. Several culture systems have been developed to support the growth and development of oocytes from rodent primordial and preantral follicles and progress is slowly being made in modifying these techniques to support the in vitro growth of porcine and bovine follicles. Oocytes from porcine preantral follicles can acquire competence to resume meiosis and proceed to Metaphase II after in vitro growth (IVG) but fertilisation has yet to be demonstrated. This paper presents the current status of technology for the in vitro growth and development of immature mammalian oocytes. Culture systems used successfully to grow immature rodent oocytes are compared and adaptations of these methods to support porcine and bovine oocyte growth discussed.

Animals↗
PubMed1995

Bovine cumulus cell expansion does not depend on the presence of an oocyte secreted factor.

Communication between the oocyte and its somatic cells has been shown to be important in oocyte development. Here we examined how the oocyte may be involved in bovine cumulus cell expansion. Intact bovine cumulus oocyte complexes (COC) were obtained by puncturing antral follicles. From the intact COC, oocytectomised complexes (OOX) were produced by micro surgical removal of the oocyte. Clumps of cumulus cells (CC) were obtained by micro-dissection. Intact or OOX complexes or CC were matured in the presence of fetal calf serum and hFSH (6 mlU/ml) for 24 hr and the degree of expansion measured. The presence of the oocyte is not essential to allow bovine cumulus expansion to occur as expansion occurred in all groups. Murine OOX complexes from eCG primed 35-40-day-old C57BL6/CBA F1 hybrids (known to require the presence of an oocyte secreted factor for cumulus expansion) were cultured with or without denuded bovine oocytes (1 oocyte/microliter). Murine OOX complexes expanded only in the presence of denuded bovine oocytes. Thus some factor produced by bovine oocytes enabled expansion of murine OOX complexes. To determine whether the factor is secreted by bovine oocytes, murine OOX were cultured with or without media conditioned by bovine oocytes (1 oocyte/microliter for 4 hr). Significant expansion of murine OOX occurred in media conditioned by bovine oocytes. This shows that the cumulus expansion enabling effect of bovine oocytes is released into the surrounding media. Media conditioned by bovine oocytes and then frozen for up to 1 month showed that the activity by the factor can withstand freezing.

Animals↗
PubMed1993

Production of cumulus expansion enabling factor by mouse oocytes grown in vitro: preliminary characterization of the factor.

The objective of this study was to determine whether fully grown oocytes, obtained after isolation from preantral follicles and growth in vitro, secrete paracrine factors affecting granulosa cell development and function. If so, the relative ease in producing oocytes in this way could facilitate the identification and characterization of the factors. As a test of this idea, the ability of in vitro grown oocytes to produce a paracrine factor that is known to enable the isolated cumulus oophorus to undergo expansion in response to follicle stimulating hormone (FSH) was determined. Initial experiments compared culture systems, which differed in the orientation of the oocyte-granulosa cell complexes from preantral follicles to an extracellular matrix, for their ability to support oocyte growth and the acquisition of competence to resume meiosis. The systems for culture on the surface of the matrix produced larger oocytes and the highest percentage of oocytes having competence to resume meiosis. Oocytes grown using this system secreted active cumulus expansion enabling factor, albeit at levels about half that of oocytes grown in vivo. A preliminary characterization of the cumulus expansion enabling factor secreted by the oocytes grown in vitro showed that activity was lost upon treatment with either heat (65 degrees C for 15 min) or proteinase K. Activity did not pass through a membrane having a nominal molecular weight limit (NMWL) of 100 kd but did pass through a membrane having a NMWL of 300 kd. It is concluded that cumulus expansion enabling factor is secreted by oocytes grown in vitro. This factor is probably a protein or depends upon a protein for its activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Mouse oocytes promote proliferation of granulosa cells from preantral and antral follicles in vitro.

Evidence is now emerging that the oocyte plays a role in the development and function of granulosa cells. This study focuses on the role of the oocyte in the proliferation of (1) undifferentiated granulosa cells from preantral follicles and (2) more differentiated mural granulosa cells and cumulus granulosa cells from antral follicles. Preantral follicles were isolated from 12-day-old mice, and mural granulosa cells and oocyte-cumulus complexes were obtained from gonadotropin-primed 22-day-old mice. Cell proliferation was quantified by autoradiographic determination of the 3H-thymidine labeling index. To determine the role of the oocyte in granulosa cell proliferation, oocyte-cumulus cell complexes and preantral follicles were oocytectomized (OOX), oocytectomy being a microsurgical procedure that removes the oocyte while retaining the three-dimensional structure of the complex or follicle. Mural granulosa cells as well as intact and OOX complexes and follicles were cultured with or without FSH in unconditioned medium or oocyte-conditioned medium (1 oocyte/microliter of medium). Preantral follicles were cultured for 4 days, after which 3H-thymidine was added to each group for a further 24 h. Mural granulosa cells were cultured as monolayers for an equilibration period of 24 h and then treated for a 48-h period, with 3H-thymidine added for the last 24 h. Oocyte-cumulus cell complexes were incubated for 4 h and then 3H-thymidine was added to each group for an additional 3-h period. FSH and/or oocyte-conditioned medium caused an increase in the labeling index of mural granulosa cells in monolayer culture; however, no differences were found among treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Primate and bovine immature oocytes and follicles as sources of fertilizable oocytes.

This review discusses the mechanisms underlying ovarian follicle development and the potential of immature follicles and oocytes from non-rodent mammalian species particularly human and bovine to serve as sources of oocytes for the in-vitro production of embryos. Factors that regulate growth and differentiation of unilaminar (primordial and primary) and multilaminar (secondary) follicles and the maturation of oocytes are highlighted. We conclude that many obstacles must still be overcome before fertilizable oocytes can be obtained from human and bovine ovaries, and more research on the quality of and culture conditions for immature oocytes and follicles is required before these can be considered as a source for in-vitro production.

Abortion, Induced↗