Making a good egg: human oocyte health, aging, and in vitro development.

Mammalian eggs (oocytes) are formed during fetal life and establish associations with somatic cells to form primordial follicles that create a store of germ cells (the primordial pool). The size of this pool is influenced by key events during the formation of germ cells and by factors that influence the subsequent activation of follicle growth. These regulatory pathways must ensure that the reserve of oocytes within primordial follicles in humans lasts for up to 50 years, yet only approximately 0.1% will ever be ovulated with the rest undergoing degeneration. This review outlines the mechanisms and regulatory pathways that govern the processes of oocyte and follicle formation and later growth, within the ovarian stroma, through to ovulation with particular reference to human oocytes/follicles. In addition, the effects of aging on female reproductive capacity through changes in oocyte number and quality are emphasized, with both the cellular mechanisms and clinical implications discussed. Finally, the details of current developments in culture systems that support all stages of follicle growth to generate mature oocytes in vitro and emerging prospects for making new oocytes from stem cells are outlined.

Anti-Mullerian hormone attenuates both cyclophosphamide-induced damage and PI3K signalling activation, while rapamycin attenuates only PI3K signalling activation, in human ovarian cortex in vitro.

STUDY QUESTION: What are the effects of cyclophosphamide exposure on the human ovary and can anti-Mullerian hormone (AMH) and rapamycin protect against these? SUMMARY ANSWER: Exposure to cyclophosphamide compromises the health of primordial and transitional follicles in the human ovarian cortex and upregulates PI3K signalling, indicating both direct damage and increased follicular activation; AMH attenuates both of these chemotherapy-induced effects, while rapamycin attenuates only PI3K signalling upregulation. WHAT IS KNOWN ALREADY: Studies primarily in rodents demonstrate that cyclophosphamide causes direct damage to primordial follicles or that the primordial follicle pool is depleted primarily through excessive initiation of follicle growth. This increased follicular activation is mediated via upregulated PI3K signalling and/or reduced local levels of AMH production due to lost growing follicles. Furthermore, while rodent data show promise regarding the potential benefits of inhibitors/protectants alongside chemotherapy treatment to preserve female fertility, there is no information about the potential for this in humans. STUDY DESIGN, SIZE, DURATION: Fresh ovarian cortical biopsies were obtained from 17 healthy women aged 21-41 years (mean ± SD: 31.8 ± 4.9 years) at elective caesarean section. Biopsies were cut into small fragments and cultured for 24 h with either vehicle alone (DMSO), the active cyclophosphamide metabolite 4-hydroperoxycyclophosphamide (4-HC) alone, 4-HC + rapamycin or 4-HC+AMH. Two doses of 4-HC were investigated, 0.2 and 2 µM in separate experiments, using biopsies from seven women (aged 27-41) and six women (aged 21-34), respectively. Biopsies from four women (aged 28-38) were used to investigate the effect of rapamycin or AMH only. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological analysis of ovarian tissue was undertaken for follicle staging and health assessment. Western blotting and immunostaining were used to assess activation of PI3K signalling by measuring phosphorylation of AKT and phosphorylated FOXO3A staining intensity, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to either dose of 4-HC caused an increase in the proportion of unhealthy primordial (P < 0.0001, both doses) and transitional follicles (P < 0.01 for low dose and P < 0.01 for high dose) compared to vehicle. AMH significantly reduced follicle damage by approximately half in both of the investigated doses of 4-HC (P < 0.0001), while rapamycin had no protective effect on the health of the follicles. Culture with AMH or rapamycin alone had no effect on follicle health. Activation of PI3K signalling following 4-HC exposure was demonstrated by both Western blotting data showing that 4-HC increased in AKT phosphorylation and immunostaining showing increased phosphorylated FOXO3A staining of non-growing oocytes. Treatment with rapamycin reduced the activation of PI3K signalling in experiments with low doses of 4-HC while culture with AMH reduced PI3K activation (both AKT phosphorylation and phosphorylated FOXO3A staining intensity) across both doses investigated. LIMITATIONS, REASONS FOR CAUTION: These in vitro studies may not replicate in vivo exposures. Furthermore, longer experiment durations are needed to determine whether the effects observed translate into irreparable deficits of ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: These data provide a solid foundation on which to explore the efficacy of AMH in protecting non-growing ovarian follicles from gonadotoxic chemotherapies. Future work will require consideration of the sustained effects of chemotherapy treatment and potential protectants to ensure these agents do not impair the developmental competence of oocytes or lead to the survival of oocytes with accumulated DNA damage, which could have adverse consequences for potential offspring. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from TENOVUS Scotland, the Academy of Medical Sciences (to R.R.), the Medical Research Council (G1100357 to R.A.A., MR/N022556/1 to the MRC Centre for Reproductive Health), and Merck Serono UK (to R.A.A.). R.R., H.L.S., N.S., and E.E.T. declare no conflicts of interest. R.A.A. reports grants and personal fees from Roche Diagnostics and Ferring Pharmaceuticals, and personal fees from IBSA and Merck outside the submitted work. TRIAL REGISTRATION NUMBER: N/A.

Spatio-temporal remodelling of the composition and architecture of the human ovarian cortical extracellular matrix during in vitro culture.

STUDY QUESTION: How does in vitro culture alter the human ovarian cortical extracellular matrix (ECM) network structure? SUMMARY ANSWER: The ECM composition and architecture vary in the different layers of the ovarian cortex and are remodelled during in vitro culture. WHAT IS KNOWN ALREADY: The ovarian ECM is the scaffold within which follicles and stromal cells are organized. Its composition and structural properties constantly evolve to accommodate follicle development and expansion. Tissue preparation for culture of primordial follicles within the native ECM involves mechanical loosening; this induces undefined modifications in the ECM network and alters cell-cell contact, leading to spontaneous follicle activation. STUDY DESIGN, SIZE, DURATION: Fresh ovarian cortical biopsies were obtained from six women aged 28-38 years (mean ± SD: 32.7 ± 4.1 years) at elective caesarean section. Biopsies were cut into fragments of ∼4 × 1 × 1 mm and cultured for 0, 2, 4, or 6 days (D). PARTICIPANTS/MATERIALS, SETTING, METHODS: Primordial follicle activation, stromal cell density, and ECM-related protein (collagen, elastin, fibronectin, laminin) positive area in the entire cortex were quantified at each time point using histological and immunohistological analysis. Collagen and elastin content, collagen fibre characteristics, and follicle distribution within the tissue were further quantified within each layer of the human ovarian cortex, namely the outer cortex, the mid-cortex, and the cortex-medulla junction regions. MAIN RESULTS AND THE ROLE OF CHANCE: Primordial follicle activation occurred concomitantly with a loosening of the ovarian cortex during culture, characterized by an early decrease in stromal cell density from 3.6 ± 0.2 × 106 at day 0 (D0) to 2.8 ± 0.1 × 106 cells/mm3 at D2 (P = 0.033) and a dynamic remodelling of the ECM. Notably, collagen content gradually fell from 55.5 ± 1.7% positive area at D0 to 42.3 ± 1.1% at D6 (P = 0.001), while elastin increased from 1.1 ± 0.2% at D0 to 1.9 ± 0.1% at D6 (P = 0.001). Fibronectin and laminin content remained stable. Moreover, collagen and elastin distribution were uneven throughout the cortex and during culture. Analysis at the sub-region level showed that collagen deposition was maximal in the outer cortex and the lowest in the mid-cortex (69.4 ± 1.2% versus 53.8 ± 0.8% positive area, respectively, P < 0.0001), and cortical collagen staining overall decreased from D0 to D2 (65.2 ± 2.4% versus 60.6 ± 1.8%, P = 0.033) then stabilized. Elastin showed the converse distribution, being most concentrated at the cortex-medulla junction (3.7 ± 0.6% versus 0.9 ± 0.2% in the outer cortex, P < 0.0001), and cortical elastin peaked at D6 compared to D0 (3.1 ± 0.5% versus 1.3 ± 0.2%, P < 0.0001). This was corroborated by a specific signature of the collagen fibre type across the cortex, indicating a distinct phenotype of the ovarian cortical ECM depending on region and culture period that might be responsible for the spatio-temporal and developmental pattern of follicular distribution observed within the cortex. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Ovarian cortical biopsies were obtained from women undergoing caesarean sections. As such, the data obtained may not accurately reflect the ECM distribution and structure of non-pregnant women. WIDER IMPLICATIONS OF THE FINDINGS: Clarifying the composition and architecture signature of the human ovarian cortical ECM provides a foundation for further exploration of ovarian microenvironments. It is also critical for understanding the ECM-follicle interactions regulating follicle quiescence and awakening, leading to improvements in both in vitro activation and in vitro growth techniques. STUDY FUNDING/COMPETING INTEREST(S): Medical Research Council grant MR/R003246/1 and Wellcome Trust Collaborative Award in Science: 215625/Z/19/Z. The authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: N/A.

Workflow Optimization for Identification of Female Germline or Oogonial Stem Cells in Human Ovarian Cortex Using Single-Cell RNA Sequence Analysis.

In 2004, the identification of female germline or oogonial stem cells (OSCs) that can support post-natal oogenesis in ovaries of adult mice sparked a major paradigm shift in reproductive biology. Although these findings have been independently verified, and further extended to include identification of OSCs in adult ovaries of many species ranging from pigs and cows to non-human primates and humans, a recent study rooted in single-cell RNA sequence analysis (scRNA-seq) of adult human ovarian cortical tissue claimed that OSCs do not exist, and that other groups working with OSCs following isolation by magnetic-assisted or fluorescence-activated cell sorting have mistaken perivascular cells (PVCs) for germ cells. Here we report that rare germ lineage cells with a gene expression profile matched to OSCs but distinct from that of other cells, including oocytes and PVCs, can be identified in adult human ovarian cortical tissue by scRNA-seq after optimization of analytical workflow parameters. Deeper cell-by-cell expression profiling also uncovered evidence of germ cells undergoing meiosis-I in adult human ovaries. Lastly, we show that, if not properly controlled for, PVCs can be inadvertently isolated during flow cytometry protocols designed to sort OSCs because of inherently high cellular autofluorescence. However, human PVCs and human germ cells segregate into distinct clusters following scRNA-seq due to non-overlapping gene expression profiles, which would preclude the mistaken identification and use of PVCs as OSCs during functional characterization studies.

Protecting life in a time of war.

Despite centuries of lessons from history, war endures. Across Earth, during nearly every year from the beginning of the twentieth century to present day, over 30 wars have been fought resulting in 187 million casualties, excluding the most recent conflict, which is the impetus for this essay (Timeline of 20th and 21st century wars). We are, sadly, a war-mongering people. The word "war" word infiltrates our vernacular, e.g., the war on poverty, on drugs, on cancer, on COVID, and, apropos, on terror. How did rational approaches to disagreement and conflict evade the world's progress? Reproductive physicians and scientists are dedicated to safeguard lives and build families. Violence is antithetical to our mission as professionals, and moral integrity as humans. We are deeply concerned for, and stand in unity with, our Ukrainian colleagues-the embryologists, scientists, OBGYN and REI physicians, infertility patients, and all people under siege. Reproductive health services for Ukrainians (as with many other war-torn regions) have collapsed. Deeply disturbing reports have emerged that cite civilian hospitals (including maternity centers) being targeted. Liquid nitrogen supplies are scarce. Pregnant mothers and gestational carriers are at emergent risk of delivering in extremely harsh conditions, cold underground bunkers and refugee queues.

Inhibition of PTEN activates bovine non-growing follicles in vitro but increases DNA damage and reduces DNA repair response.

STUDY QUESTION: Does ovarian follicle activation by phosphatase homologue of chromosome-10 (PTEN) inhibition affect DNA damage and repair in bovine oocytes and granulosa cells? SUMMARY ANSWER: PTEN inhibition promotes bovine non-growing follicle activation but results in increased DNA damage and impaired DNA repair capacity in ovarian follicles in vitro. WHAT IS KNOWN ALREADY: Inhibition of PTEN is known to activate primordial follicles but may compromise further developmental potential. In breast cancer cells, PTEN inhibition represses nuclear translocation of breast cancer susceptibility 1 (BRCA1) and Rad51; this impairs DNA repair resulting in an accumulation of damaged DNA, which contributes to cell senescence. STUDY DESIGN, SIZE, DURATION: Bovine ovarian tissue fragments were exposed to control medium alone or containing either 1 or 10 µM bpv(HOpic), a pharmacological inhibitor of PTEN, in vitro for 24 h. A sub-group of tissue fragments were collected for Western blot analysis after bpv(HOpic) exposure. The remainder were incubated in control medium for a further 5 days and then analysed histologically and by immunohistochemistry to detect DNA damage and repair pathways. PARTICIPANTS/MATERIALS, SETTING, METHODS: Bovine ovaries were obtained from abattoir-slaughtered heifers. Tissue fragments were exposed to either control medium alone or medium containing either 1 µM or 10 µM bpv(HOpic) for 24 h. Tissue fragments collected after 24 h were subjected to Akt quantification by Western blotting (six to nine fragments per group per experiment). Follicle stage and morphology were classified in remaining fragments. Immunohistochemical analysis included nuclear exclusion of FOXO3 as a marker of follicle activation, γH2AX as a marker of DNA damage, meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM), Rad51, breast cancer susceptibility 1 (BRCA1) and breast cancer susceptibility 2 (BRCA2) as DNA repair factors. A total of 29 550 follicles from three independent experiments were analysed. MAIN RESULTS AND THE ROLE OF CHANCE: Tissue fragments exposed to bpv(HOpic) had increased Akt phosphorylation at serine 473 (pAkt/Akt ratio, 2.25- and 6.23-fold higher in 1 and 10 µM bpv(HOpic) respectively compared to control, P < 0.05). These tissue fragments contained a significantly higher proportion of growing follicles compared to control (78.6% in 1 µM and 88.7% in 10 µM versus 70.5% in control; P < 0.001). The proportion of morphologically healthy follicles did not differ significantly between 1 µM bpv(HOpic) and control (P < 0.001) but follicle health was lower in 10 µM compared to 1 µM and control in all follicle types (P < 0.05). DNA damage in oocytes, indicated by expression of γH2AX, increased following exposure to 1 µM bpv(HOpic) (non-growing, 83%; primary follicles, 76%) and 10 µM (non-growing, 77%; primary, 84%) compared to control (non-growing, 30% and primary, 59%) (P < 0.05 for all groups). A significant reduction in expression of DNA repair proteins MRE11, ATM and Rad51 was observed in oocytes of non-growing and primary follicles of treatment groups (primary follicles in controls versus 10 µM bpv(HOpic): MRE, 68% versus 47%; ATM, 47% versus 18%; Rad51, 48% versus 24%), P < 0.05 for all groups. Higher dose bpv(HOpic) also resulted in lower expression of BRCA1 compared to control and 1 µM bpv(HOpic) (P < 0.001) in non-growing and primary follicles. BRCA2 expression was increased in oocytes of primary follicles in 1 µM bpv(HOpic) (36%) compared to control (20%, P = 0.010) with a marked decrease in 10 µM (1%, P ≤ 0.001). Granulosa cells of primary and secondary follicles in bpv(HOpic) groups showed more DNA damage compared to control (P < 0.05). However, bpv(HOpic) did not impact granulosa cell DNA repair capacity in secondary follicles, but BRCA1 declined significantly in higher dose bpv(HOpic). LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study focuses on non-growing follicle activation after 6 days culture and may not reflect DNA damage and repair capacity in later stages of oocyte and follicle growth. WIDER IMPLICATIONS OF THE FINDINGS: In vitro activation of follicle growth may compromise the bidirectional signalling between oocyte and granulosa cells necessary for optimal oocyte and follicle health. This large animal model may be useful in optimising follicle activation protocols with a view to transfer for clinical application. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Indonesia endowment fund for education. No competing interest. TRIAL REGISTRATION NUMBER: Not applicable.

FERTILITY PRESERVATION: Progress and prospects for developing human immature oocytes in vitro.

Ovarian cryopreservation rapidly developed from basic science to clinical application and can now be used to preserve the fertility of girls and young women at high risk of sterility. Primordial follicles can be cryopreserved in ovarian cortex for long-term storage and subsequently autografted back at an orthotopic or heterotopic site to restore fertility. However, autografting carries a risk of re-introducing cancer cells in patients with blood-born leukaemias or cancers with a high risk of ovarian metastasis. For these women fertility restoration could only be safely achieved in the laboratory by the complete in vitro growth (IVG) and maturation (IVM) of cryopreserved primordial follicles to fertile metaphase II (MII) oocytes. Culture systems to support the development of human oocytes have provided greater insight into the process of human oocyte development as well as having potential applications within the field of fertility preservation. The technology required to culture human follicles is extremely challenging, but significant advances have been made using animal models and translation to human. This review will detail the progress that has been made in developing human in vitro growth systems and consider the steps required to progress this technology towards clinical application.

In vitro growth of immature bovine follicles and oocytes.

The limitation in the supply of mature, fertilisable oocytes constitutes a major impediment to increasing the success of assisted reproduction, stem cell derivation and cloning in domestic species. Techniques are being developed to grow immature oocytes invitro that have the potential to increase the supply of oocytes. Mouse oocytes can be cultured from initial stages of development to maturity, and live young have been produced, but for domestic species, such as cows, with long growth periods, invitro systems that allow complete growth of oocytes contained within primordial follicles to maturity is technically challenging and has not yet been achieved. For cows, several culture systems have been developed that support specific developmental stages, but a multistep culture system will be required for complete growth invitro. This review highlights the steps that will be required to achieve the goal of growing oocytes invitro.

Future developments: In vitro growth (IVG) of human ovarian follicles.

Removal and storage of ovarian cortical tissue is currently offered to young female cancer patients undergoing potentially sterilizing chemotherapy and/or radiotherapy. For patients at high risk of reintroduction of malignancy through auto-transplantation, the ultimate aim is to achieve complete oocyte development from this tissue in vitro. The ability to develop human oocytes from the earliest follicular stages through to maturation and fertilization in vitro would revolutionize fertility preservation practice. This has been achieved in mice where in vitro grown oocytes from primordial follicles have resulted in the production of live offspring. Systems that support growth and development of oocytes from human ovarian cortex are being developed by several groups. This review focuses on the steps required to recapitulate in vitro the process of human oocyte development from the primordial stage and the systems currently available to support this.

Being a good egg in the 21st century.

Introduction: Women are increasingly having children at a later age, but this can conflict with declining fertility in the later 30's and thereafter. Areas of agreement: Declining egg quality and quantity with age are well-established, although egg quality can only be surmised from reproductive success or failure. Areas of controversy: Whether increasing the number of eggs that can be obtained from ovarian stimulation is of value, and whether there are precursor cells within the adult ovary that could become mature eggs. Growing points: There is increasing use of donated eggs by older women to enhance their chances of conception. The storage of frozen eggs for potential use later in life is also becoming more common. Areas timely for developing research: Understanding of growth initiation of follicles and development of an artificial ovary may lead to the ability to affect fertility and reproductive lifespan.

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.

Age-dependent loss of cohesion protection in human oocytes.

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

Relationship between Amino Acid Metabolism and Bovine In Vitro Follicle Activation and Growth.

The amino acid metabolism of bovine follicles during in vitro growth (IVG) was evaluated to identify potential indicators of health during culture. The bovine ovarian cortex was sliced, prepared as strips, and cultured for 6 days. Tissue samples were examined histologically before and after 6 days of culture, and the degree of follicle activation was classified as either high or low based on the number of growing secondary follicles present (high: 7~11; low: 0~1). In a separate experiment, secondary follicles (diameter range: 100~200 µm) were manually isolated and cultured, and their growth was monitored for 6 days. Cultured follicles were classified as growth or degenerate based on diameter change during culture (growth: +60.5~74.1 µm; degenerate: -28~15.2 µm). Free amino acids and their metabolites were measured in the spent culture medium from each group. In cultured ovarian cortical strips, the concentration of α-aminoadipic acid was significantly higher in the low activation group than in the high group (p < 0.05), while those of methionine, lysine, and arginine were higher in the high activation group. In cultured isolated secondary follicles, concentrations of methionine, tyrosine, histidine, and hydroxyproline were higher in the degenerate group (p ≤ 0.05). In conclusion, amino acid metabolism has the potential to serve as an indicator of primordial follicle activation and subsequent growth rate during bovine IVG.

In vitro growth and maturation of primordial follicles and immature oocytes.

Cryopreservation of ovarian tissue to preserve the fertility of girls and young women at high risk of sterility is now widely practiced. Pieces of cryopreserved ovarian cortex can be thawed and autografted to restore fertility, but because of the risks of reintroduction of the cancer, transplantation may not be possible for girls and women with blood-borne leukemias or cancers with a high risk of ovarian metastasis. Cryopreserved ovarian tissue contains mainly primordial follicles but also provides access to immature oocytes from small antral follicles, which may be matured in vitro to provide an additional source of mature oocytes. So in cases in which transplantation is contraindicated, fertility restoration could be safely achieved in the laboratory either by in vitro maturation (IVM) of oocytes aspirated from growing follicles or by the complete in vitro growth (IVG) and maturation (IVM) of primordial follicles to produce fertile metaphase II (MII) oocytes. The development of IVM and IVG methods to support all stages of oocytes available within ovarian tissue will maximize the potential for all patients undergoing fertility preservation.

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.

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.