Marie McLaughlin

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

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

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

Quiescent follicles of large mammals initiate growth within cultured pieces of ovarian cortex. Systems capable of sustaining in vitro development from this early stage until oocyte maturation would allow investigation of mechanisms regulating oocyte development in its entirety. The aims of this study were 1) to determine whether bovine follicles initiated to grow in vitro could be isolated from the cortical environment, and could undergo further development and 2) to evaluate the effect of activin and FSH on the development of secondary follicles derived from primordial follicles. Fragments of bovine ovarian cortex were cultured in serum-free medium for 6 days; thereafter, secondary follicles were isolated for further culture. After a maximum total of 21 days in vitro, follicles were either processed for histological assessment or opened to release the oocyte-cumulus complexes for inspection by light microscopy. Compared with control, significant follicle and oocyte growth were observed in activin-exposed follicles, with or without FSH, with some oocyte diameters measuring over 100 microns following a total in vitro period of 15 days. Significant oestradiol secretion was observed in follicles cultured in activin alone after a total of 9 days in vitro compared with other treatment groups; however, this effect was not sustained. In summary, this study demonstrates the promotion of primordial bovine follicle development within a two-step serum-free culture system with oocyte diameters >100 mum achieved over 15 days in vitro. Further development of this system is needed to support complete oocyte growth and thereafter in vitro maturation.

In vitro development of ovarian follicles.

Tissue banking of ovarian material is being increasingly offered to a variety of patients as a means of fertility preservation. This tissue comprises thin cortical surface biopsies that contain predominantly primordial follicles, and currently the only option to restore fertility is by transplantation. However, this is not a viable option for all patients. The potential of this tissue could be realized by the development of in vitro systems to support complete growth from the early primordial stages through to maturity. This technology would have many therapeutic applications including the production of competent oocytes for assisted reproduction technologies, determination of toxicological effects on germ cell development, assessment of cryopreserved ovarian tissue before transplantation for fertility preservation as well as providing an experimental model to address basic scientific questions concerning human oocyte development. Complete oocyte development in vitro from the primordial stage has been achieved in mice, but the larger size and longer growth period of human follicles has made the interspecies translation of these techniques difficult. Recently progress has been made in defining conditions that support different stages of follicle development in vitro that make a complete in vitro system from primordial to maturation a possible reality. This article deals with our current understanding of in vitro development.

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.

Inhibition of phosphatase and tensin homologue (PTEN) in human ovary in vitro results in increased activation of primordial follicles but compromises development of growing follicles

In the mammalian ovary a small number of follicles are steadily recruited from the quiescent pool to undergo development. Follicle loss, maintenance and growth are strictly controlled by complex molecular interactions including the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signalling pathway. Stimulation of PI3K promotes phosphorylation of Akt resulting in follicle survival and activation of growth whereas this pathway is suppressed by the actions of the phosphatase and tensin homologue (PTEN). The aim of this study was to determine the effect of dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate (bpV), a reversible inhibitor of PTEN, on the activation, survival and development of human ovarian follicles in vitro. Biopsied ovarian tissue fragments were obtained from 17 women aged 23–46 years and exposed to 1 µM bpV(HOpic) (n = 146) or control medium (n = 128) for 24 h. Media were then replaced with control medium and all tissue incubated for a further 5 days. Ovarian tissue from each treatment group was fixed after the initial 24 h culture period and phosphorylated Akt was quantified by western blotting. After 6 days incubation all tissue fragments were inspected under light microscopy and any secondary follicles ≥100 µm isolated. Isolated follicles were cultured individually in control medium supplemented with 100 ng/ml recombinant human activin A. Tissue fragments without follicles suitable for isolation were fixed and processed for histological and immunohistochemical analysis. During 6 days culture, follicle activation occurred in tissue samples from both treatment groups but with significantly more follicles progressing to the secondary stage of development in the presence of 1 µM bpV(HOpic) compared with control (31 versus 16%; P < 0.05). Increased activation was associated with increased Akt phosphorylation and increased nuclear export of FOXO3. However isolated and cultured follicles that had been exposed to bpV(HOpic) showed limited growth and reduced survival compared with follicles from control fragments (P < 0.05). This study demonstrates that inhibition of PTEN with bpV(HOpic) affects human ovarian follicle development by promoting the initiation of follicle growth and development to the secondary stage, as in rodent species, but severely compromises the survival of isolated secondary follicles.

Strategies to support human oocyte development in vitro

Many young cancer patients are now being given the option to store ovarian cortical biopsies before undergoing potentially damaging chemo- or radiotherapy. This tissue mainly contains large numbers of immature primordial follicles. Currently the only option to restore fertility using this tissue is by transplantation which may not be a viable option for all patients. Greater options to realise the potential of this tissue to restore fertility could be achieved by the development of in vitro systems that support oocyte development. The ability to develop human oocytes from the most immature stages of follicles (primordial) through to maturation and fertilisation in vitro would revolutionise fertility preservation practice. This has been achieved in mouse where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. However, developing IVG systems to support complete development of human oocytes has been more difficult because of differences in scale of timing and size. Our lab has been working on a multi-step culture system to support growth and development of bo-vine and human oocytes from primordial through to fully grown, using fresh and cryopreserved ovarian cortical tissue. This review outlines the approaches being taken to obtain complete in vitro development of human oocytes and strategies for assessing the health and viability of IVG oocytes.

Natural history of the mammalian oocyte

Combining cryopreservation of immature oocytes with in-vitro growth/maturation techniques is the ambition of many IVF clinics. Whilst these techniques have been demonstrated in rodents their application to humans and domestic species has been slow. There are many technical reasons for the lack of progress in these species, but the major problem is that we have very little knowledge of how the oocyte acquires developmental competence during its growth within the follicle. The life history of the mammalian oocyte involves a complex series of co-ordinated developmental processes that in the human take place over several months. This review will consider: (i) growth and development of the oocyte; (ii) the newly regenerated debate on the existence of germ-line stem cells in the mammalian ovary; and (iii) strategies for producing oocytes in vitro.

The controversial existence and functional potential of oogonial stem cells

The regenerative potential of the mammalian ovary has been a controversial area over the last decade. Isolation of cells, termed oogonial stem cells (OSCs), from adult rodent and human ovaries has been reported, with these cells exhibiting both germ and stem cell markers in culture. When re-introduced into an ovarian somatic environment these cells have generated follicles capable of producing healthy offspring in rodents, and there is some evidence of human OSCs being able to form oocyte-like structures in a xenotransplant model. Importantly, there are no data on their potential physiological role within the ovary, and specifically no evidence that they contribute to the primordial follicle pool and thus to later stages of follicle development. The cues required for oocyte differentiation from these cells are not well understood either in vivo or in vitro, and these will need to be further elucidated to maximise their potential for therapeutic intervention. OSCs may also be of value as a model to investigate normal human germ cell differentiation. It is likely that their interactions with ovarian somatic cells and/or extracellular signals will be important in these processes. This review summarises our current knowledge on the isolation and characterisation of mammalian oogonial stem cells.

Human oocytes express ATP-sensitive K+ channels

BACKGROUND ATP-sensitive K(+) (K(ATP)) channels link intracellular metabolism with membrane excitability and play crucial roles in cellular physiology and protection. The K(ATP) channel protein complex is composed of pore forming, Kir6.x (Kir6.1 or Kir6.2) and regulatory, SURx (SUR2A, SUR2B or SUR1), subunits that associate in different combinations. The objective of this study was to determine whether mammalian oocytes (human, bovine, porcine) express K(ATP) channels. METHODS Supernumerary human oocytes at different stages of maturation were obtained from patients undergoing assisted conception treatments. Bovine and porcine oocytes in the germinal vesicle (GV) stage were obtained by aspirating antral follicles from abattoir-derived ovaries. The presence of mRNA for K(ATP) channel subunits was determined using real-time RT-PCR with primers specific for Kir6.2, Kir6.1, SUR1, SUR2A and SUR2B. To assess whether functional K(ATP) channels are present in human oocytes, traditional and perforated patch whole cell electrophysiology and immunoprecipitation/western blotting were used. RESULTS Real-time PCR revealed that mRNA for Kir6.1, Kir6.2, SUR2A and SUR2B, but not SUR1, were present in human oocytes of different stages. Only SUR2B and Kir6.2 mRNAs were detected in GV stage bovine and porcine oocytes. Immunoprecipitation with SUR2 antibody and western blotting with Kir6.1 antibody identified bands corresponding to these subunits in human oocytes. In human oocytes, 2,4-dinitrophenol (400 µM), a metabolic inhibitor known to decrease intracellular ATP and activate K(ATP) channels, increased whole cell K(+) current. On the other hand, K(+) current induced by low intracellular ATP was inhibited by extracellular glibenclamide (30 µM), an oral antidiabetic known to block the opening of K(ATP) channels. CONCLUSIONS In conclusion, mammalian oocytes express K(ATP) channels. This opens a new avenue of research into the complex relationship between metabolism and membrane excitability in oocytes under different conditions, including conception.