Orthotopic and heterotopic autografts of frozen-thawed ovarian cortex in sheep.

Freezing ovarian cortex is a new option to preserve the fertility of young patients undergoing cancer treatment or in women facing premature menopause. However, the best way to use this banked tissue remains unclear. The function of heterotopic and orthotopic autografts of frozen-thawed ovarian cortex of sheep was compared in the present study. Fresh and frozen-thawed fragments of ovarian cortex were autografted on the uterine horn of six ewes (orthotopic grafts) and under the skin of the belly in nine ewes (heterotopic grafts). In both orthotopic and heterotopic grafts, the resumption of follicular growth and ovulation was monitored. In orthotopically grafted ewes, fertility was recorded. Oocytes from both types of grafts were collected, matured and fertilized in vitro. In both fresh and frozen-thawed grafts follicular growth resumed normally; preantral and antral follicles were first detectable 4 and 10 weeks respectively following grafting but only 5% of the primordial follicles appeared to have survived. This confirms that grafting procedures are more deleterious for follicle survival than cryopreservation. Although ovulation resumed in most ewes, none of the ewes grafted orthotopically became pregnant at a synchronized mating. Seven months following grafting, oocytes could be collected from heterotopic and orthotopic grafts, matured and some of them fertilized, but none developed to the blastocyst stage. Heterotopic grafting may be an alternative to orthotopic grafting to preserve fertility provided follicle survival in the grafts is markedly improved.     

Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: competence to undergo maturation, embryogenesis, and development to term

Only primordial and primary follicles of frozen-thawed mouse ovaries survive after grafting to the ovarian bursa; large secondary follicles and antral follicles together with the oocytes contained in them degenerate. This study was undertaken to determine whether fully grown oocytes isolated from the antral follicles of frozen-thawed mouse ovaries are viable and can be rescued to undergo maturation, fertilization, and embryo development in vitro. Ovaries were cryopreserved after removal from 22-day-old (C57BL/6J x SJL/J)F(1) mice, with or without prior priming with equine chorionic gonadotrophin, and fresh non-frozen ovaries were used as controls. Only cumulus cell-denuded oocytes were recovered from frozen unprimed ovaries while both cumulus cell-enclosed and denuded oocytes were retrieved from frozen primed ovaries. Oocytes from both groups of frozen-thawed ovaries were able to undergo maturation, fertilization, and development to the blastocyst stage in vitro, though at lower percentages than oocytes from control unfrozen ovaries. Moreover, 19% of 2-cell stage embryos derived from frozen-thawed primed ovaries, compared with 42% of embryos derived from control primed ovaries, developed to term after transfer to pseudopregnant foster mothers (not significantly different). Therefore, fully grown oocytes in antral follicles survive the cryopreservation protocol, as demonstrated by maturation, fertilization and embryo development in vitro, and development to term after embryo transfer.     

Evaluation of the effects of extremely low frequency electromagnetic fields on mammalian follicle development

The aim of this study was to evaluate the effects of pulsed, extremely low-frequency electromagnetic fields (ELF-EMF) on in-vitro mouse pre-antral follicle development. Pre-antral follicles were cultured for 5 days and exposed to ELF-EMF at the frequencies of 33 or 50 Hz. ELF-EMF application did not affect follicular growth over a 3 day culture period, but on day 5 the growth of 33 Hz-exposed follicles was significantly reduced when compared with controls, while the 50 Hz-exposed follicles were not significantly affected. However, ELF-EMF severely impaired antrum formation at both frequencies, as 79 +/- 3% of control follicles developed antral cavities compared with 30 +/- 6% and 51.6 +/- 4% of 33 or 50 Hz-exposed follicles respectively. The follicles with failed antrum formation showed lower oestradiol release and granulosa cell DNA synthesis, but these effects were not related to granulosa cell apoptosis. Furthermore, a high percentage of the in-vitro grown oocytes obtained from exposed follicles had a reduced ability to resume meiotic maturation when compared with controls. These results suggest that ELF-EMF exposure might impair mammalian female reproductive potentiality by reducing the capacity of the follicles to reach a developmental stage that is an essential pre-requisite for reproductive success.     

Pilot study of isolated early human follicles cultured in collagen gels for 24 hours.

The human ovarian cortex contains mainly primordial and primary follicles. The ability to mature these follicles in vitro could be of great importance for infertility treatments. Fresh and frozen-thawed ovarian tissue was incubated with collagenase and DNase. Follicles with one layer or an incomplete second layer of granulosa cells were then dissected. The follicles were embedded in collagen gels and cultured with Earle's balanced salt solution, 10% fetal calf serum and 0.5 IU/ml follicle stimulating hormone. Increases in the number of granulosa cell layers and in oocyte size were observed in 40 and 38.7% of the follicles from fresh and frozen-thawed tissue respectively, during a 24 h culture period. All the growing follicles were surrounded by cellular outgrowths. Attempts to culture the follicles longer resulted in deterioration of the follicles and oocyte release. Since our study was purely morphological, further growth parameters, e.g. DNA synthesis, should be examined in the future.     

Ultrastructure and viability of isolated bovine preantral follicles

Techniques for the isolation of ovarian follicles and maturation of oocytes in vitro have enormous reproductive potential. Preservation of normal tissue function is vital. This study emphasizes the ultrastructure and viability of mechanically isolated bovine small (diameter 40-100 microm) preantral and large (140-450 microm) preantral/early antral follicles. Viability studies were performed for small preantral follicles. The presence of esterase activity, active mitochondria and dead cells served as parameters of oocyte and granulosa cell viability. After 1 day of culture, all follicles had a viable granulosa, displaying active mitochondria and/or esterase activity in all their cells, although a few (generally <5) dead granulosa cells were present in 17% of the follicles. Of the oocytes, 35 and 80% had esterase activity and active mitochondria respectively, whereas 8% appeared dead. The percentages of oocytes showing esterase activity and active mitochondria decreased during culture, whereas the percentage of follicles with dead oocytes or dead granulosa cells strongly increased. More than 90% of the isolated small follicles showed a poor ultrastructure, especially of their oocyte, which points to a negative selective isolation of poor follicles in the present study and/or an isolation procedure-induced damage of follicles. With respect to large preantral follicles, 42% of those distributed in the cortex and 64% of those isolated and cultured for 1 day had a poor ultrastructure. In contrast with the small ones, the percentage of ultrastructurally poor large preantral follicles had decreased to 27% after 5 days of culture, possibly due to better isolation and culture conditions. It is recommended to use ultrastructural and/or viability cell markers for in-vitro grown follicles to evaluate their quality, and particularly that of their oocytes.     

Development of antral follicles in human cryopreserved ovarian tissue following xenografting

This study reports the first gross morphological and histological evidence of antral follicle development in human ovarian tissue following cryopreservation. Human ovarian tissue was cryopreserved using propanediol and sucrose and grafted under the renal capsule of bilaterally oophorectomized severe combined immunodeficient (SCID) mice. Follicles at all stages of development were observed in the grafted tissue whereas, prior to grafting, only primary and primordial follicles were present. Antral follicles were rarely observed on grafts examined <20 weeks after grafting either non-frozen tissue (fresh, 1/7) or cryopreserved tissue (0/11). In contrast, development of at least one antral follicle was evident on the majority of sites > or = 20 weeks after grafting (fresh 7/9, cryopreserved 18/24). Antral follicle diameters ranged from 0.1 to 5.0 mm. Histological examination of these antral follicles indicated normal follicular morphology, i.e. antral cavities encapsulated by concentric layers of theca and granulosa cells. Pedicles containing germinal vesicle oocytes were observed protruding from the granulosa cell layers. The development and morphology of the cryopreserved and fresh tissue following grafting was similar.     

Ovary and ovulation: Follicular development in cryopreserved marmoset ovarian tissue after transplantation

Pieces of marmoset ovary were frozen by slow cooling in 1.5M dimethylsulphoxide. The follicles in fresh and frozen tissuewere counted and examined for morphological appearance in stainedserial sections. The proportion of normal follicles was similarin fresh tissue and frozen tissue examined immediately afterthawing. Follicles at all stages of folliculogenesis up to thesmall antral stage survived freezing and thawing. Fresh andfrozen tissue was transplanted underneath the kidney capsulesof ovariectomized immunodeficient mice. The establishment ofgrafts was similar, and oestrogenic activity (cornificationof the vaginal epithelium) was observed in the recipients 20and 16 days after transplantation of fresh and frozen graftsrespectively. The total number of follicles and the proportionof normal follicles were similar in fresh and frozen grafts.Grafts of frozen tissue recovered between 7 and 15 days aftertransfer contained follicles up to the small antral stage ofdevelopment. Grafts recovered between 21 and 32 days containedfollicles at all stages of folliculogenesis, including largeantral follicles (1–2 mm diameter). Our results suggestthat freezing and thawing do not substantially damage marmosetovarian tissue, and the cryopreserved tissue retains its abilityto support the development of large antral follicles.     

Oocyte in-vitro maturation and follicle culture: current clinical achievements and future directions

This paper reviews two recent developments in the field of assisted reproduction: in-vitro culture (IVC) of follicles and in-vitro maturation (IVM) of oocytes. Both in-vitro procedures for oocytes and immature follicles were developed so as to enable storage of ovarian cortical tissue and oocyte-cumulus complexes (OCC) from small follicles. Until now, complete in-vitro development from a primordial follicle up to ovulation has been achieved only in mice. Culture of ovarian cortex from large mammals and humans has revealed that the proliferation and differentiation of pre-granulosa cells is easily switched on, but that within a few days oocyte growth is often compromised. Culture of OCC from preantral follicles from sheep yielded antral follicles after 1 month in a serum-free system and the enclosed oocytes had almost doubled their diameter. Similar work in humans is ongoing, but no consistent successes have been reported as yet. In-vitro maturation of OCC from antral follicles has already been used clinically for more than 10 years. Inspired by the work on bovines, several in-vitro fertilization (IVF) clinics have applied IVM on OCC from different sizes of follicles. The culture conditions needed to provide a reasonable yield of developmentally competent oocytes have not been clearly defined, although some IVF groups have reported live births. To enable the development of consistent techniques of IVC and IVM, a more systematic and scientific approach is needed: use of defined culture media in combination with follicle stage-dependent supplements might improve our understanding of the minimal requirements for oocyte growth and maturation.     

Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.

The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C.     

Expression of receptors for insulin-like growth factor-I and transforming growth factor-beta in human follicles

The in-vitro growth of immature oocytes in early follicles from cryopreserved human ovarian tissues is a new concept in in-vitro fertilization programmes for the treatment of infertile and cancer patients. To better understand the regulatory mechanism of follicular development, immunohistochemistry was used to study the expression of insulin-like growth factor (IGF) type I receptor (IGF-IR) and transforming growth factor-beta (TGFbeta) type I (TbetaR-I) and type II (TbetaR-II) receptors in fresh and frozen ovarian tissues from 14 women. Immunoreactivities for IGF-IR and TbetaR-I were present simultaneously in the oocytes of primordial, pre-antral and antral follicles. Staining for both IGF-IR and TbetaR-I was also observed in granulosa cells of primordial, pre-antral and antral follicles. IGF-IR and TbetaR-I also stained in thecal cells of pre-antral and antral follicles. Stromal cells in surrounding ovarian tissue expressed IGF-IR and TbetaR-I at various follicular stages. Unlike TbetaR-I, TbetaR-II was expressed only in the oocytes of primordial and primary follicles, and with weak staining intensity in thecal cells. No significant staining for TbetaR-II was found in oocytes and granulosa cells of antral follicles. There was no difference in staining patterns for IGF-IR, TbetaR-I and TbetaR-II between fresh and frozen ovarian tissues, indicating that cryopreservation might not significantly alter the immunoreactivities of these receptors in frozen ovarian tissue. The results suggest that IGF-I and TGFbeta may participate in the regulation of follicular growth by binding to their receptors through an autocrine or paracrine mechanism. IGF-I and TGFbeta may be useful in regulating the in-vitro or in-vivo maturation of oocytes not only in later follicles but also very early follicles, from cryopreserved ovarian tissues for clinical use in the future.     

Graafian follicles are cooler than neighbouring ovarian tissues and deep rectal temperatures

In order to establish appropriate culture temperatures for in-vitro maturation of pig ovarian oocytes, large Graafian follicles (7-10 mm diameter) were sensed by infra-red technology during the latter part of a spontaneous oestrous cycle. Temperatures were measured under systemic anaesthesia almost instantaneously upon revealing the ovaries at mid- ventral laparotomy. Temperature differentials were observed within all 16 ovaries sensed in 14 animals. Ovaries were always cooler than deep rectal temperatures (mean rectal temperature was 38.0 +/- 0.4 degrees C; range 37.5-38.6 degrees C) and mature follicles always cooler than ovarian stroma (35.6 +/- 0.3 degrees C versus 37.3 +/- 0.2 degrees C respectively; P < 0.01). Such follicles were frequently 1.5-1.8 degrees C cooler than the adjacent stroma, the mean being 1.7 +/- 0.4 degrees C. Small Graafian follicles (< 5-6 mm diameter) and recent ovulations did not show this differential. The control experiment of excising an ovary, deep freezing it in liquid nitrogen, and then restoring it to the body cavity before further sensing indicated that intra-ovarian temperature gradients depended on the activity of living tissues and/or a functional blood supply. Furthermore, calculation of anticipated rates of cooling for exposed Graafian follicles strongly suggested that artefacts could not have been solely responsible for the observed temperatures. Endothermic reactions within mature follicles were thus brought into focus. It is concluded that follicular temperatures may influence the meiotic progression and cytoplasmic maturation of oocytes and act to regulate enzymatic activity in the biosynthetic pathways for steroid and/or peptide hormones.      

Ovary and ovulation: In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system

A simplified culture system was developed for the in-vitro maturationof early preantral mouse ovarian follicles. The follicles werecultured singly in 20 (µ droplets under oil in mediumsupplemented with recombinant follicle stimulating hormone (r-FSH)at 37°C and 5% CO² in air. The follicles grew and becameattached to the bottom of the dish, progressively lost theirspherical structure by outgrowth of the granulosa cells throughthe basal membrane and developed follicles with antral-likecavities. The normal three-dimensional follicular structurewas lost but all components, i.e. theca, granulosa and oocyte,remained functional, as was proven by the oestradiol, inhibinand progesterone secretion patterns. Follicle survival exceeded80% and histological analysis proved the absence of atresiaand cell death in granulosa cells up to day 16. Oocytes of 55(± 4) µm diameter on the day of isolation reached74 (± 3) µm by day 16 of culture. The optimal momentfor inducing the final meiotic maturation with human chorionicgonadotrophin was investigated: the highest absolute numbersof metaphase II oocytes were obtained on days 12 and 14 (39and 41%). The fertilizing potential of the in-vitro maturedoocytes was comparable to in-vivo matured controls. A 50% hatched-blastocystdevelopment rate was observed.     

A new source of human oocytes: preliminary report on the identification and maturation of human preantral follicles from follicular aspirates

This study aimed to investigate the development of human preantral follicles and oocyte maturation in vitro. Preantral follicles were obtained from follicular aspirates during egg retrieval carried out during an in-vitro fertilization (IVF) programme. They were first incubated in Ham's F10 medium with 15% fetal cord serum (FCS). After 28 days, the medium was supplemented with different doses of human menopausal gonadotrophin (HMG), human follicular fluid (hFF) and epidermal growth factor (EGF) by orthogonal design. Promotion of final maturation was completed in the presence of HMG and hFF. Development from preantral to antral follicles was found within 6-12 days of culture. With time, the proportion of follicles with diameters of >300 microm increased at 21-28 days of culture (P < 0.005). The maximum number of oocytes extruded, and first polar body formation, occurred in the presence of 0.15 IU/ml HMG 40% (v/v) hFF and 6 ng/ml EGF. We conclude that follicular aspirates obtained during egg retrieval in an IVF programme contain many preantral follicles which could develop into antral follicles with extrusion of oocytes in culture, and that the oocytes can mature in vitro. Hence, a new source of human oocytes is available.      

Current perspective on primordial follicle cryopreservation and culture for reproductive medicine

Mature oocytes are rare and precious cells. A technology which generates larger numbers would be very welcome in clinical practice, animal production technology and research. Since de-novo formation of female germ cells has ceased by the time of birth, the most attractive strategy, in theory, is to harvest and culture primordial follicles, the most abundant stage in the ovary at all ages. So far, there has been more success with cryopreservation of primordial follicles than with culture, and frozen-thawed ovarian tissue grafts have restored fertility to a number of species after oophorectomy. However, in-vitro development of isolated follicles is not sustained beyond the primary follicle stage. To meet their requirements for growth, metabolism and differentiation, a multistage protocol will probably be required for the prolonged period of development to maturity. The mouse is the only model, to date, in which a live offspring has ever been produced after growing follicles completely in vitro. A triple-stage process was required, involving culture of ovarian explants followed by isolation of granulosa-oocyte complexes and, finally, suitable conditions for completing meiotic maturation. Achievement of this goal for the larger and more slowly developing follicles from human and farm animal ovaries is still a remote possibility.     

Microfluidic Encapsulation of Ovarian Follicles for 3D Culture

The ovarian follicle that contains one single oocyte is the fundamental functional tissue unit of mammalian ovary. Therefore, isolation and in vitro culture of ovarian follicles to obtain fertilizable oocytes are regarded as a promising strategy for women to combat infertility. In this communication, we performed a brief survey of studies on microfluidic encapsulation of ovarian follicles in core–shell hydrogel microcapsules for biomimetic 3D culture. These studies highlighted that recapitulation of the mechanical heterogeneity of the extracellular matrix in ovary is crucial for in vitro culture to develop early pre-antral follicles to the antral stage, and for the release of cumulus–oocyte complex (COC) from antral follicles in vitro. The hydrogel encapsulation-based biomimetic culture system and the microfluidic technology may be invaluable to facilitate follicle culture as a viable option for restoring women’s fertility in the clinic.     

Fertilization and early embryology: Cryopreservation of immature mouse oocytes

Mouse oocytes enclosed in cumulus cells were isolated from antralfollicles at the germinal vesicle (GV) stage. They were storedin straws at – 196°C by a conventional mouse embryofreezing method using dimethylsulphoxide (1.5 M) as the cryoprotectant.Overall survival assessed after removal of the cumulus cellswas 93% (299/320). A significantly greater proportion of freshoocytes remained arrested at the GV stage during culture (11versus 1%), but the rate of maturation to metaphase II was notsignificantly different between frozen and fresh oocytes (83versus 74%). The rate of fertilization in vitro was similarfor frozen and fresh oocytes matured in vitro (70 versus 81%)but significantly less than with mature ovulated oocytes (96%).Fertilization of frozen and fresh oocytes arrested after germinalvesicle breakdown was similar (77 versus 95%. No evidence ofparthenogenetic activation was found in the different groupsafter overnight incubation of metaphase II oocytes. Implantationwas similar for embryos derived from fresh and frozen GV-stageoocytes matured in vitro and mature ovulated oocytes, but theloss of embryos after implantation was significantly higherin the in-vitro matured groups (frozen, 40% and fresh, 46% versus24%). The overall survival of oocytes frozen at the GV stagewas 27%. This compares favourably with the estimated overallsurvival of mature oocytes cryopreserved by a similar procedure.We conclude that the increased post-implantation loss is dueto suboptimal conditions for maturation in vitro rather thanfreezing injury.     

Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation.

Ovarian cortical tissue, donated by 20 women aged 25-43 years during gynaecological laparoscopies or laparotomies, was first cultured for 7-9 days as tissue slices, 0.1-0.3 mm in thickness, in extracellular matrix, to initiate the growth of the primordial and primary follicles. It was then divided into two parts, one of which was cultured further as slices, and the other one used for enzymatic (collagenase at 1, 0.5 or 0.25 mg/ml; 17 patients) or mechanical (four patients) partial isolation of the follicles. The tissue slices and the partially isolated follicles were cultured for a further 1-3 weeks in the matrix. After approximately 2 weeks in culture, some oocytes began to extrude from the follicles, which were usually at the secondary stage. They were small, 20-80 micrometer in diameter, and had a thin or absent zona. Polar bodies and meiotic chromosomes could be seen in these naked oocytes. This premature extrusion probably resulted from sub-optimal culture conditions. It occurred sooner in follicles that had been partially isolated using collagenase. Histologically, larger numbers of oocytes were observed in non-isolated slice cultures than in the partially isolated cultures. Initiation of growth of the follicles occurred during the first 7-9 days in culture within slices. In non-isolated slices and following mechanical partial isolation there were significantly more secondary follicles after 11-18 days in culture than following isolation with collagenase. The proportion of atretic follicles increased during all cultures, and it was significantly higher after partial isolation. Because partial isolation did not improve the survival or development of the follicles the optimal method for human ovarian follicles could be to culture them non-isolated within small tissue slices.     

Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles.

In-vitro maturation (IVM) of human ovarian follicles and oocytes could benefit infertile women, and allow the development of in-vitro systems for the study of human follicular development. Little is known about the initiation of growth of primordial follicles and the regulation of early folliculogenesis. An ovarian tissue-slice culture system was used to examine the effects of media composition, follicle stimulating hormone (FSH) and serum substitution on the development of small human follicles in vitro. Human ovarian cortex biopsies were cut into small pieces and cultured for 5, 10 or 15 days. Control (non-cultured) and cultured tissue was fixed, serially sectioned, and stained. The follicles contained within the tissue pieces were counted, measured, and assessed for stage of development and viability. Comparison of the ability of alpha-minimum essential medium (alpha-MEM), Waymouth's, or Earle's balanced salt solution (EBSS) culture media (all with 10% human serum) to support follicle growth demonstrated significantly increased initiation and growth of follicles in alpha-MEM during the first 10 days of culture. The supplementation of alpha-MEM with 300 mIU/ml FSH significantly reduced levels of atresia and increased the mean diameter of healthy follicles. Follicles in tissue cultured for 10 days with human serum albumin and ITS (insulin/transferrin/selenium mix) were significantly larger, more developed and showed significantly less atresia than those cultured with serum alone. Primordial to small preantral follicles can be grown under serum-substituted conditions in tissue-slice culture, and are responsive to FSH, which is thought to be acting mainly as a survival factor at these early stages.     

Assessment of the need for follicle stimulating hormone in early preantral mouse follicle culture in vitro

In two consecutive controlled experiments 160 early preantral follicles were cultured in order to evaluate effects of recombinant follicle stimulating hormone (r-FSH) on survival, differentiation, oestradiol and inhibin secretion, cumulus mucification and cumulus-corona-oocyte detachment by human chorionic gonadotrophin (HCG) stimulation. Nuclear maturation in oocytes was also assessed following addition of HCG. A histological analysis of cultured follicles was carried out on semi- thin sections at various culture stages. Addition of r-FSH was essential for follicle survival for 16 days: without r-FSH only 11% of the follicles survived for 12 days (with r-FSH: 79%) and none of these mucified after the HCG stimulus. r-FSH promoted granulosa cell proliferation and antral-like cavity formation. Without r-FSH, histology of the cultures demonstrated degeneration and reduced granulosa cell proliferation; oestradiol and inhibin production were reduced. This study illustrates the essential role of FSH in promoting the in-vitro growth of early preantral mouse ovarian follicles and in maintaining the oocyte under meiotic arrest.      

Immunocytogenetic detection of normal and abnormal oocytes in human fetal ovarian tissue in culture.

This study aimed to: (i) determine whether oocytes are present in cultures of human fetal ovary; (ii) identify whether meiotic anomalies are evident; and (iii) assess whether preparation or culture conditions influence oocyte survival and meiotic progression. Ovaries were collected from fetuses after termination at 13-16 weeks. Oocyte assessment utilized antibodies specific for synaptonemal complex proteins (associated with chromosomes only during meiosis), and antibodies to centromeric proteins. Fragments of tissue were cultured in minimal essential medium + 10% serum +/- follicle stimulating hormone (100 mIU/ml). The sera were fetal calf serum (FCS), FCS for embryonic stem cells (ES-FCS) and human female serum. The numbers and stages of oocytes were assessed after 7-40 days, and particular arrangements of chromosome synapsis identified. Results in fresh tissue included oocytes at leptotene, zygotene, pachytene and diplotene in three of five samples. Four specimens remained viable in vitro, and three had detectable oocytes after culture. The numbers of oocytes and the proportions of zygotene and pachytene cells increased with time in culture. The proportion of degenerate cells in culture was initially higher than in fresh samples, but declined subsequently. More oocytes were detected in ES-FCS and human serum than in FCS. We conclude that human oocytes survive in culture and that progression through prophase I continues.