Clinical pregnancy in Turner syndrome following re-implantation of cryopreserved ovarian cortex.

Turner syndrome (TS) leads to a characteristic phenotype, including premature ovarian insufficiency and infertility. Ovarian tissue cryopreservation (OTC) is becoming an established fertility preservation strategy for both pre- and post-pubertal females and may offer the chance of having a biological family to selected patients with TS. To date, women with TS have had ovarian tissue cryopreserved but there are few reports of autologous re-implantation and none of pregnancy. We herein report, to our knowledge, the first clinical pregnancy in a patient with TS, conceived naturally following re-implantation of cryopreserved ovarian tissue which had been removed soon after spontaneous puberty. This provides proof of concept for OTC as a means of fertility preservation in TS.

The existence and potential of germline stem cells in the adult mammalian ovary.

It has long been accepted that the complement of follicles within the ovary is formed before birth in humans, or shortly after birth in rodents, and that no follicles are formed thereafter. This follows entry of all oogonia into meiosis in fetal life, with no remaining germ stem cells in the ovary, in contrast to the presence of spermatogonia in the testis. This has been brought back into debate in recent years, following the demonstration of isolation of cells expressing both germline and stem markers from the postnatal ovary in several species, including humans. We describe these cells as putative ovarian stem cells. Isolation of these cells is challenging, adding to the debate as to their existence, and the validity of DDX4 as the main marker used for their isolation has also to be questioned. While different groups have used varying techniques and indeed terminology to describe these cells, the body of evidence regarding their initial characterization after isolation is growing. There remain very limited data regarding their developmental potential, but the demonstration of the production of functional oocytes from induced pluripotent stem cells and the advances in ovarian follicle culture techniques provide a basis for such studies.

Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system.

STUDY QUESTION: Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle? SUMMARY ANSWER: Development of human oocytes from primordial/unilaminar stages to resumption of meiosis (Metaphase II) and emission of a polar body was achieved within a serum free multi-step culture system. WHAT IS KNOWN ALREADY: Complete development of oocytes in vitro has been achieved in mouse, where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. Human oocytes have been grown in vitro from the secondary/multi-laminar stage to obtain fully grown oocytes capable of meiotic maturation. However, there are no reports of a culture system supporting complete growth from the earliest stages of human follicle development through to Metaphase II. STUDY DESIGN, SIZE, DURATION: Ovarian cortical biopsies were obtained with informed consent from women undergoing elective caesarean section (mean age: 30.7 ± 1.7; range: 25-39 years, n = 10). PARTICIPANTS/MATERIALS, SETTING, METHODS: Laboratory setting. Ovarian biopsies were dissected into thin strips, and after removal of growing follicles were cultured in serum free medium for 8 days (Step 1). At the end of this period secondary/multi-laminar follicles were dissected from the strips and intact follicles 100-150 µm in diameter were selected for further culture. Isolated follicles were cultured individually in serum free medium in the presence of 100 ng/ml of human recombinant Activin A (Step 2). Individual follicles were monitored and after 8 days, cumulus oocyte complexes (COCs) were retrieved by gentle pressure on the cultured follicles. Complexes with complete cumulus and adherent mural granulosa cells were selected and cultured in the presence of Activin A and FSH on membranes for a further 4 days (Step 3). At the end of Step 3, complexes containing oocytes >100 µm diameter were selected for IVM in SAGE medium (Step 4) then fixed for analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Pieces of human ovarian cortex cultured in serum free medium for 8 days (Step 1) supported early follicle growth and 87 secondary follicles of diameter 120 ± 6 µm (mean ± SEM) could be dissected for further culture. After a further 8 days, 54 of the 87 follicles had reached the antral stage of development. COCs were retrieved by gentle pressure from the cultured follicles and those with adherent mural granulosa cells (n = 48) were selected and cultured for a further 4 days (Step 3). At the end of Step 3, 32 complexes contained oocytes >100 µm diameter were selected for IVM (Step 4). Nine of these complexes contained polar bodies within 24 h and all polar bodies were abnormally large. Confocal immuno-histochemical analysis showed the presence of a Metaphase II spindle confirming that these IVG oocytes had resumed meiosis but their developmental potential is unknown. LIMITATIONS, REASONS FOR CAUTION: This is a small number of samples but provides proof of concept that complete development of human oocytes can occur in vitro. Further optimization with morphological evaluation and fertilization potential of IVG oocytes is required to determine whether they are normal. WIDER IMPLICATIONS OF THE FINDINGS: The ability to develop human oocytes from the earliest follicular stages in vitro through to maturation and fertilization would benefit fertility preservation practice. STUDY FUNDING/COMPETING INTEREST(S): Funded by MRC Grants (G0901839 and MR/L00299X/1). No competing interests.

Non-growing follicle density is increased following adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) chemotherapy in the adult human ovary.

STUDY QUESTION: Do the chemotherapeutic regimens of ABVD (adriamycin, bleomycin, vinblastine and dacarbazine) or OEPA-COPDAC (combined vincristine, etoposide, prednisone, doxorubicin (OEPA) and cyclophosphamide, vincristine, prednisone, dacarbazine (COPDAC)) used to treat Hodgkin lymphoma (HL), affect the density, morphology and in vitro developmental potential of human ovarian follicles? SUMMARY ANSWER: Ovarian tissue from women treated with ABVD contained a higher density of non-growing follicles (NGFs) per cubic millimetre and increased numbers of multiovular follicles but showed reduced in vitro growth compared with patients with lymphoma who had not received chemotherapy, patients treated with OEPA-COPDAC, age-matched healthy women and age-related model-predicted values. WHAT IS KNOWN ALREADY: Chemotherapy regimens can cause a loss of follicles within the ovary, which depends on the drugs given. Early stage HL is commonly treated by ABVD, a non-alkylating regimen that apparently has ovarian sparing qualities; thus it is important to investigate the histological appearance and distribution of follicles within ABVD-treated ovarian tissue. STUDY DESIGN, SIZE, DURATION: Thirteen ovarian biopsies were obtained from HL patients (six adolescents and seven adults) and one biopsy from a non-HL patient. Two HL patients and the non-HL patient had received no treatment prior to biopsy collection. The remaining 11 HL patients received one of two regimens: ABVD or OEPA-COPDAC. Tissue was analysed histologically and compared to biopsies from healthy women, and in a subgroup of patients, tissue was cultured for 6 days in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian biopsies were obtained from patients undergoing ovarian cryopreservation for fertility preservation and from healthy women at the time of Caesarian section ('obstetric tissue'). Follicle number and maturity were evaluated in sections of ovarian cortical tissue, and compared to an age-related model of mean follicle density and to age-matched contemporaneous biopsies. The developmental potential of follicles was investigated after 6 days of tissue culture. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 6877 follicles were analysed. ABVD-treated tissue contained a higher density of NGFs per cubic millimetre (230 ± 17) (mean ± SEM) than untreated (110 ± 54), OEPA-COPDAC-treated (50 ± 27) and obstetric (20 ± 4) tissue (P < 0.01), with follicle density 9-21 SD higher than predicted by an age-related model. Biovular and binucleated NGFs occurred frequently in ABVD-treated and in adolescent-untreated tissue but were not observed in OEPA-COPDAC-treated or obstetric tissue, although OEPA-COPDAC-treated tissue contained a high proportion of morphologically abnormal oocytes (52% versus 23% in untreated, 22% in ABVD-treated and 25% in obstetric tissue; P < 0.001). Activation of follicle growth in vitro occurred in all groups, but in ABVD-treated samples there was very limited development to the secondary stage, whereas in untreated samples from lymphoma patients growth was similar to that observed in obstetric tissue (untreated; P < 0.01 versus ABVD-treated, NS versus obstetric). LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: Although a large number of follicles were analysed, these data were derived from a small number of biopsies. The mechanisms underpinning these observations have yet to be determined and it is unclear how they relate to future fertility. WIDER IMPLICATIONS OF THE FINDINGS: This study confirms that the number of NGFs is not depleted following ABVD treatment, consistent with clinical data that female fertility is preserved. Our findings demonstrate that immature follicle density can increase as well as decrease following at least one chemotherapy treatment. This is the first report of morphological and follicle developmental similarities between ABVD-treated tissue and the immature human ovary. Further experiments will investigate the basis for the marked increase in follicle density in ABVD-treated tissue. STUDY FUNDING/COMPETING INTERESTS: Funded by UK Medical Research Council Grants G0901839 and MR/L00299X/1. The authors have no competing interests.

Re-implantation of cryopreserved ovarian cortex resulting in restoration of ovarian function, natural conception and successful pregnancy after haematopoietic stem cell transplantation for Wilms tumour.

With the improvement of long-term cancer survival rates, growing numbers of female survivors are suffering from treatment-related premature ovarian insufficiency (POI). Although pre-treatment embryo and oocyte storage are effective fertility preservation strategies, they are not possible for pre-pubertal girls or women who cannot delay treatment. In these cases, the only available treatment option is ovarian cortex cryopreservation and subsequent re-implantation. A 32-year-old woman had ovarian cortex cryopreserved 10 years previously before commencing high-dose chemotherapy and undergoing a haematopoietic stem cell transplant for recurrent adult Wilms tumour, which resulted in POI. She underwent laparoscopic orthotopic transplantation of cryopreserved ovarian cortex to the original site of biopsy on the left ovary. She ovulated at 15 and 29 weeks post-re-implantation with AMH detectable, then rising, from 21 weeks, and conceived naturally following the second ovulation. The pregnancy was uncomplicated and a healthy male infant was born by elective Caesarean section at 36+4 weeks gestation. This is the first report of ovarian cortex re-implantation in the UK. Despite the patient receiving low-risk chemotherapy prior to cryopreservation and the prolonged tissue storage duration, the re-implantation resulted in rapid restoration of ovarian function and natural conception with successful pregnancy.

An externally validated age-related model of mean follicle density in the cortex of the human ovary.

PURPOSE: The ability to accurately estimate a woman's ovarian reserve by non-invasive means is the goal of ovarian reserve prediction. It is not known whether a correlation exists between model-predicted estimates of ovarian reserve and data generated by direct histological analysis of ovarian tissue. The aim of this study was to compare mean non-growing follicle density values obtained from analysis of ovarian cortical tissue samples against ovarian volume models. METHODS: Non-growing follicle density values were obtained from 13 ovarian cortical biopsies (16-37 years). A mean non-growing follicle density was calculated for each patient by counting all follicles in a given volume of biopsied ovarian cortex. These values were compared to age-matched model generated densities (adjusted to take into consideration the proportion of ovary that is cortex) and the correlation between data sets tested. RESULTS: Non-growing density values obtained from fresh biopsied ovarian cortical samples closely matched model generated data with low mean difference, tight agreement limits and no proportional error between the observed and predicted results. CONCLUSION: These findings validate the use of the adjusted population and ovarian volume models, to accurately predict mean follicle density in the ovarian cortex of healthy adult women.

The immature human ovary shows loss of abnormal follicles and increasing follicle developmental competence through childhood and adolescence.

STUDY QUESTION: Do the ovarian follicles of children and adolescents differ in their morphology and in vitro growth potential from those of adults? SUMMARY ANSWER: Pre-pubertal ovaries contained a high proportion of morphologically abnormal non-growing follicles, and follicles showed reduced capacity for in vitro growth. WHAT IS KNOWN ALREADY: The pre-pubertal ovary is known to contain follicles at the early growing stages. How this changes over childhood and through puberty is unknown, and there are no previous data on the in vitro growth potential of follicles from pre-pubertal and pubertal girls. STUDY DESIGN, SIZE, DURATION: Ovarian biopsies from five pre-pubertal and seven pubertal girls and 19 adult women were analysed histologically, cultured in vitro for 6 days, with growing follicles then isolated and cultured for a further 6 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian biopsies were obtained from girls undergoing ovarian tissue cryopreservation for fertility preservation, and compared with biopsies from adult women. Follicle stage and morphology were classified. After 6 days in culture, follicle growth initiation was assessed. The growth of isolated secondary follicles was assessed over a further 6 days, including analysis of oocyte growth. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-pubertal ovaries contained a high proportion of abnormal non-growing follicles (19.4 versus 4.85% in pubertal ovaries; 4004 follicles analysed; P = 0.02) characterized by indistinct germinal vesicle membrane and absent nucleolus. Follicles with this abnormal morphology were not seen in the adult ovary. During 6 days culture, follicle growth initiation was observed at all ages; in pre-pubertal samples there was very little development to secondary stages, while pubertal samples showed similar growth activation to that seen in adult tissue (pubertal group: P = 0.02 versus pre-pubertal, ns versus adult). Isolated secondary follicles were cultured for a further 6 days. Those from pre-pubertal ovary showed limited growth (P < 0.05 versus both pubertal and adult follicles) and no change in oocyte diameter over that period. Follicles from pubertal ovaries showed increased growth; this was still reduced compared with follicles from adult women (P < 0.05) but oocyte growth was proportionate to follicle size. LIMITATIONS, REASONS FOR CAUTION: These data derive from only a small number of ovarian biopsies, although large numbers of follicles were analysed. It is unclear whether the differences between groups are related to puberty, or just age. WIDER IMPLICATIONS OF THE FINDINGS: These findings show that follicles from girls of all ages can be induced to grow in vitro, which has important implications for some patients who are at high risk of malignant contamination of their ovarian tissue. The reduced growth of isolated follicles indicates that there are true intrafollicular differences in addition to potential differences in their local environment, and that there are maturational processes occurring in the ovary through childhood and adolescence, which involve the loss of abnormal follicles, and increasing follicle developmental competence. STUDY FUNDING/COMPETING INTEREST(S): Funded by MRC grants G0901839 and G1100357. No competing interests.

Activin promotes follicular integrity and oogenesis in cultured pre-antral bovine follicles.

The aim of this study was to determine the individual and combined effect of activin and follicle stimulating hormone (FSH) on somatic and germ cell development in cultured pre-antral follicles. Pre-antral bovine follicles (mean diameter 157 +/- 3, range 132-199 microm) were cultured for 8 days in serum-free medium in the presence of either 100 ng/ml of recombinant human activin A (rhAct A), 100 ng/ml rhAct A combined with a high (100 ng/ml) or low (50 ng/ml) concentration of recombinant FSH (rFSH) or 50 ng/ml rFSH alone. Intrafollicular connexin 43 expression and actin-based cell adhesion were assessed on Day 2 and 4 of culture. Steroidogenesis was evaluated after Day 4 and 8. Follicles exposed to 100 ng/ml activin maintained expression of connexin 43 at the follicular periphery. In the presence of activin, with or without 100 ng/ml or 50 ng/ml FSH, follicles were steroidogenic undergoing significant growth (P < 0.01), granulosa cell proliferation (P < 0.01) and antral cavity formation (P < 0.05) compared with cultured controls. Maximum oocyte growth occurred in the presence of 100 ng/ml activin alone with a significant percentage of these oocytes maintaining normal morphology over controls (P < 0.05). These results are consistent with a role for activin in maintaining oocyte granulosa cell interactions due to increased peripheral granulosa cell adhesion to the basement membrane and retention of adhesion at the surface of the zona pellucida. Thus, the polarized expression of cell contact interactions promoted by activin supports ongoing folliculogenesis.

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.

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.

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.

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.

Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.

BACKGROUND Female cancer patients are offered 'banking' of gametes before starting fertility-threatening cancer therapy. Transplants of fresh and frozen ovarian tissue between healthy fertile and infertile women have demonstrated the utility of the tissue banked for restoration of endocrine and fertility function. Additional methods, like follicle culture and isolated follicle transplantation, are in development. METHODS Specialist reproductive medicine scientists and clinicians with complementary expertise in ovarian tissue culture and transplantation presented relevant published literature in their field of expertise and also unpublished promising data for discussion. As the major aims were to identify the current gaps prohibiting advancement, to share technical experience and to orient new research, contributors were allowed to provide their opinioned expert views on future research. RESULTS Normal healthy children have been born in cancer survivors after orthotopic transplantation of their cryopreserved ovarian tissue. Longevity of the graft might be optimized by using new vitrification techniques and by promoting rapid revascularization of the graft. For the in vitro culture of follicles, a successive battery of culture methods including the use of defined media, growth factors and three-dimensional extracellular matrix support might overcome growth arrest of the follicles. Molecular methods and immunoassay can evaluate stage of maturation and guide adequate differentiation. Large animals, including non-human primates, are essential working models. CONCLUSIONS Experiments on ovarian tissue from non-human primate models and from consenting fertile and infertile patients benefit from a multidisciplinary approach. The new discipline of oncofertility requires professionalization, multidisciplinarity and mobilization of funding for basic and translational research.

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.

Method for isolating preantral follicles from mare ovaries.

The aims of this study were to evaluate the use of collagenase treatment to isolate preantral follicles from mare ovaries and to assess the effect of this treatment on follicular morphology. Intact mare ovaries were chopped into pieces, incubated individually with 1, 3 or 5 mg collagenase (type 1A) ml(-1) in a shaking waterbath at 37 degrees C for up to 2 h and passed through a series of stainless steel filters with pore size 50-300 microm to remove large clumps and stromal cells. The samples were prepared for histological analysis and sections were examined by light microscopy. Isolated follicles and oocytes were measured and the quality of the follicles was assessed by microscopic examination. Very few intact preantral follicles were isolated after 30 min incubation with 1, 3 or 5 mg collagenase ml(-l). After 60 and 90 min incubations, between eight and 71 intact preantral follicles were isolated with 3 or 5 mg collagenase ml(-1), whereas very few were isolated after incubation with 1 mg collagenase ml(-1). The number of intact follicles isolated after incubation with either 3 or 5 mg collagenase ml(-1) was not significantly different. The quality of the isolated follicles decreased with increasing incubation time and no intact follicles were observed after 2 h of incubation. Preantral follicles 60-300 microm in diameter were isolated from ovaries after treatment with either 3 or 5 mg collagenase ml(-1). Most of the follicles isolated were 90-150 microm in diameter. This study indicates that equine preantral follicles can be isolated from equine ovaries using collagenase and that collagenase does not have a deleterious effect on follicle morphology when used at the appropriate concentration for a minimum period. However, oocyte quality and follicle viability remain to be determined.

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)

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.

Apoptosis in equine granulosa cells and its relationship to cumulus expansion and oocyte chromatin configuration in ovarian follicles.

During the oestrous cycle follicles grow and either ovulate or regress. Regressing follicles undergo atresia and in many species apoptosis has been identified as the underlying mechanism in this process. The aims of this study were to establish whether equine granulosa cells degenerate via an apoptotic mechanism and whether the presence of apoptotic cell death in granulosa cells is correlated with oocyte quality. Ovaries from mares at unknown stages of the oestrous cycle were obtained from an abattoir. In Expt 1, follicles (n=352) from 37 mares were processed. DNA was extracted from granulosa cells, fractionated by agarose gel electrophoresis, stained with ethidium bromide and visualized with ultraviolet light or end-labelled with [32P]dideoxy-ATP, and autoradiography was performed after electrophoresis. In Expt 2, follicles (n=34) from four mares with at least one follicle >35 mm in diameter were processed. DNA was extracted from the granulosa cells; the cumulus oophorus was classified and the oocyte chromatin was stained with Hoechst 33,258 fluorescent stain. In Expt 1, apoptosis, as determined by the characteristic laddering of internucleosomal DNA fragments, was present in 45% of all follicles. Apoptosis was apparent primarily in follicles <20 mm in diameter and was present with greatest frequency in follicles 6-10 mm in diameter. Apoptosis was not detected in follicles >27 mm in diameter. The presence of sheared DNA of a wide range of different molecular masses, possibly indicative of necrotic cell death, was positively correlated with follicle size. In Expt 2, apoptosis was detected in 50% of follicles <20 mm in diameter but not in follicles > 30 mm in diameter. The oocytes had an expanded cumulus oophorus in 58% of follicles <20 mm in diameter, whereas 80% of follicles > 30 mm in diameter had a compact cumulus oophorus. In these mares, follicles <20 mm in diameter appeared to undergo apoptotic changes as well as cumulus expansion. These findings indicate that degeneration occurs in many follicles that are not destined for ovulation and that detection of apoptosis can be used as an indicator of follicular degeneration in mares. Apoptosis, as a marker of cell death, can be used to study the growth and selection of follicles, and to correlate the health of granulosa cells with oocyte quality.