Acquisition of 2C-like totipotency through defined maternal-effect factors.

Fully grown oocytes have the natural ability to transform 2 terminally differentiated gametes into a totipotent zygote representing the acquisition of totipotency. This process wholly depends on maternal-effect factors (MFs). MFs stored in the eggs are therefore likely to be able to induce cellular reprogramming to a totipotency state. Here we report the generation of totipotent-like stem cells from mESCs using 4MFs Hsf1, Zar1, Padi6, and Npm2, designated as MFiTLSCs. MFiTLSCs exhibited a unique and inherent capability to differentiate into embryonic and extraembryonic derivatives. Transcriptomic analysis revealed that MFiTLSCs are enriched with 2-cell-specific genes that appear to synergistically induce a transcriptional repressive state, in that parental genomes are remodeled to a poised transcriptional repression state while totipotency is established following fertilization. This method to derive MFiTLSCs could help advance the understanding of fate determinations of totipotent stem cells in a physiological context and establish a foundation for the development of oocyte biology-based reprogramming technology.

A highly active mineral-based ice nucleating agent supports in situ cell cryopreservation in a high throughput format.

Cryopreservation of biological matter in microlitre scale volumes of liquid would be useful for a range of applications. At present, it is challenging because small volumes of water tend to supercool, and deep supercooling is known to lead to poor post-thaw cell viability. Here, we show that a mineral ice nucleator can almost eliminate supercooling in 100 µl liquid volumes during cryopreservation. This strategy of eliminating supercooling greatly enhances cell viability relative to cryopreservation protocols with uncontrolled ice nucleation. Using infrared thermography, we demonstrate a direct relationship between the extent of supercooling and post-thaw cell viability. Using a mineral nucleator delivery system, we open the door to the routine cryopreservation of mammalian cells in multiwell plates for applications such as high throughput toxicology testing of pharmaceutical products and regenerative medicine.

Trophectoderm non-coding RNAs reflect the higher metabolic and more invasive properties of young maternal age blastocysts.

Increasing female age is accompanied by a corresponding fall in her fertility. This decline is influenced by a variety of factors over an individual's life course including background genetics, local environment and diet. Studying both coding and non-coding RNAs of the embryo could aid our understanding of the causes and/or effects of the physiological processes accompanying the decline including the differential expression of sub-cellular biomarkers indicative of various diseases. The current study is a post-hoc analysis of the expression of trophectoderm RNA data derived from a previous high throughput study. Its main aim is to determine the characteristics and potential functionalities that characterize long non-coding RNAs. As reported previously, a maternal age-related component is potentially implicated in implantation success. Trophectoderm samples representing the full range of maternal reproductive ages were considered in relation to embryonic implantation potential, trophectoderm transcriptome dynamics and reproductive maternal age. The long non-coding RNA (lncRNA) biomarkers identified here are consistent with the activities of embryo-endometrial crosstalk, developmental competency and implantation and share common characteristics with markers of neoplasia/cancer invasion. Corresponding genes for expressed lncRNAs were more active in the blastocysts of younger women are associated with metabolic pathways including cholesterol biosynthesis and steroidogenesis.

Inconsistencies in fertility preservation for young people with cancer in the UK.

OBJECTIVE: To assess the utilisation of and funding structure for fertility preservation for children diagnosed with cancer in the UK. DESIGN: Survey of paediatric oncologists/haematologists. Questionnaires were sent electronically with reminder notifications to non-responders. SETTING: UK Paediatric Oncology Principal Treatment Centres (PTCs). PARTICIPANTS: Paediatric oncologists/haematologists with an interest in the effects of treatment on fertility representing the 20 PTCs across the UK. MAIN OUTCOME MEASURES: Referral practices, sources and length of funding for storage of gametes or gonadal tissue for children diagnosed with cancer in the preceding 12 months. RESULTS: Responses were received from 18 PTCs (90%) with responses to 98.3% of questions. All centres had referred patients for fertility preservation: ovarian tissue collection/storage 100% (n=18 centres), sperm banking 100% (n=17; one centre was excluded due to the age range of their patients), testicular tissue storage 83% (n=15), mature oocyte collection 35% (n=6; one centre was excluded due to the age range of their patients). All centres with knowledge of their funding source reported sperm cryopreservation was NHS funded. Only 60% (n=9) centres reported the same for mature oocyte storage. Of the centres aware of their funding source, half reported that ovarian and testicular tissue storage was funded by charitable sources; this increased in England compared with the rest of the UK. CONCLUSIONS: Inequality exists in provision of fertility preservation for children with cancer across the UK. There is lack of formalised government funding to support international guidelines, with resultant geographical variation in care. Centralised funding of fertility preservation for children and young adults is needed alongside establishment of a national advisory panel to support all PTCs.

Therapeutic Potential of In Vitro-Derived Oocytes for the Restoration and Treatment of Female Fertility.

Considerable progress has been made with the development of culture systems for the in vitro growth and maturation (IVGM) of oocytes from the earliest-staged primordial follicles and from the more advanced secondary follicles in rodents, ruminants, nonhuman primates, and humans. Successful oocyte production in vitro depends on the development of a dynamic culture strategy that replicates the follicular microenvironment required for oocyte activation and to support oocyte growth and maturation in vivo while enabling the coordinated and timely acquisition of oocyte developmental competence. Significant heterogeneity exists between the culture protocols used for different stages of follicle development and for different species. To date, the fertile potential of IVGM oocytes derived from primordial follicles has been realized only in mice. Although many technical challenges remain, significant advances have been made, and there is an increasing consensus that complete IVGM will require a dynamic, multiphase culture approach. The production of healthy offspring from in vitro-produced oocytes in a secondary large animal species is a vital next step before IVGM can be tested for therapeutic use in humans.

Intracellular oxygen metabolism during bovine oocyte and preimplantation embryo development.

We report a novel method to profile intrcellular oxygen concentration (icO2) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO2 and decreasing oxygen consumption to increase icO2. Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9-4 × 106 copies and did not correlate with icO2. These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.

The effects of aging on molecular modulators of human embryo implantation.

Advancing age has a negative impact on female fertility. As implantation rates decline during the normal maternal life course, age-related, embryonic factors are altered and our inability to monitor these factors in an unbiased genome-wide manner in vivo has severely limited our understanding of early human embryo development and implantation. Our high-throughput methodology uses trophectoderm samples representing the full spectrum of maternal reproductive ages with embryo implantation potential examined in relation to trophectoderm transcriptome dynamics and reproductive maternal age. Potential embryo-endometrial interactions were tested using trophectoderm sampled from young women, with the receptive uterine environment representing the most 'fertile' environment for successful embryo implantation. Potential roles for extracellular exosomes, embryonic metabolism and regulation of apoptosis were revealed. These biomarkers are consistent with embryo-endometrial crosstalk/developmental competency, serving as a mediator for successful implantation. Our data opens the door to developing a diagnostic test for predicting implantation success in women undergoing fertility treatment.

Probing morphological, genetic and metabolomic changes of in vitro embryo development in a microfluidic device.

Assisted reproduction technologies for clinical and research purposes rely on a brief in vitro embryo culture which, despite decades of progress, remain suboptimal in comparison to the physiological environment. One promising tool to improve this technique is the development of bespoke microfluidic chambers. Here we present and validate a new microfluidic device in polydimethylsiloxane (PDMS) for the culture of early mouse embryos. Device material and design resulted embryo compatible and elicit minimal stress. Blastocyst formation, hatching, attachment and outgrowth formation on fibronectin-coated devices were similar to traditional microdrop methods. Total blastocyst cell number and allocation to the trophectoderm and inner cell mass lineages were unaffected. The devices were designed for culture of 10-12 embryos. Development rates, mitochondrial polarization and metabolic turnover of key energy substrates glucose, pyruvate and lactate were consistent with groups of 10 embryos in microdrop controls. Increasing group size to 40 embryos per device was associated with increased variation in development rates and altered metabolism. Device culture did not perturb blastocyst gene expression but did elicit changes in embryo metabolome, which can be ascribed to substrate leaching from PDMS and warrant further investigation.

Metabolomic Analysis Evidences That Uterine Epithelial Cells Enhance Blastocyst Development in a Microfluidic Device.

Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1 × B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher (p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrimidine metabolism) in the CM group, suggesting a beneficial effect of CM on embryo development. The metabolomic study carried out in a microfluidic environment confirms our hypothesis on the potential of uterine epithelial cells to enhance blastocyst development. Further investigations are required to highlight specific pathways involved in embryo development and implantation.

Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation.

Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below -20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.

Survival from cancer in young people: An overview of late effects focusing on reproductive health.

This paper provides a summary of the areas of survival from childhood, teenage and young adult cancers and the significant late effects that can arise from treatment; with particular focus on the area of reproductive health and the impact on both fertility and pregnancy. To complete this review, Web of Science and MEDLINE were used. Search terms included: ""survival AND childhood OR teenage OR young adult cancer", "late effects", "childhood cancer", "teenage AND/OR young adult cancer", AND "fertility after cancer" OR "pregnancy AND cancer" OR "fertility preservation". Additionally, the clinical expertise of the authors was drawn upon. Childhood cancer is a thankfully rare occurrence; however, the incidence is increasing. Survival rates remain high and this means that a growing population of childhood and young adult cancer survivors are reaching adulthood. For some of these adults, although cured of their cancer, they are now facing a future with lasting effects on their health from their treatments. These effects, commonly referred to as late effects, are defined as health problems related either directly to the underlying cancer or to its treatment and which occur months or years after treatment has finished. Reproductive health is an important consideration for these patients, and although many will be able to conceive naturally, some will exhibit impaired fertility after their treatments. This can include difficulties at all points along the path from conception to delivery of a live, healthy offspring. High-quality, large-population evidence is sparse in many areas relating to fertility risk from treatment and the maternal and fetal health of childhood cancer survivors. Yet given the potential for complications, the authors advocate consideration of fertility at the time of diagnosis and before potentially gonadotoxic treatment.

Fertility preservation options in prepubertal females: feasibility, safety and outcomes.

Fertility preservation is a developing area of reproductive biology and successes in adult populations has led to an emerging interest in prepubertal populations. Advances in treatment strategies for many disease processes including childhood cancer means that many patients now survive their initial diagnosis. Ovarian insufficiency is a recognized side-effect of chemotherapy and as such advances in reproductive technologies are now possible to attempt to overcome the so called late effects. Questions regarding the outcomes and safety of these treatments still exist. A review of the available literature with regard to fertility preservation options in prepubertal females was undertaken with consideration given to the need for options, availability of options, safety of available techniques and current outcomes. The new discipline of onco-fertility is rapidly advancing. Work to facilitate accurate and robust identification of those in need of fertility preservation and the optimization of strategies for fertility restoration are ongoing. With time it is hoped that the techniques used to freeze-store ovarian tissue and to conduct auto-transplantation will undergo refinement and so advance from the research arena into mainstream clinical practice. Further research is likely to be fruitful and will offer significant hope to those at risk of premature ovarian insufficiency whether from underlying genetic causes or as a result of ablative therapies. It is important that research advancements are disseminated widely as this will help to inform clinical practitioners, nurses and counsellors of strategies and to ensure that appropriate treatments are offered to patients who are at risk of fertility loss.

Isolation and expression of the human gametocyte-specific factor 1 gene (GTSF1) in fetal ovary, oocytes, and preimplantation embryos.

PURPOSE: Gametocyte-specific factor 1 has been shown in other species to be required for the silencing of retrotransposons via the Piwi-interacting RNA (piRNA) pathway. In this study, we aimed to isolate and assess expression of transcripts of the gametocyte-specific factor 1 (GTSF1) gene in the human female germline and in preimplantation embryos. METHODS: Complementary DNA (cDNA) libraries from human fetal ovaries and testes, human oocytes and preimplantation embryos and ovarian follicles isolated from an adult ovarian cortex biopsy were used to as templates for PCR, cloning and sequencing, and real time PCR experiments of GTSF1 expression. RESULTS: GTSF1 cDNA clones that covered the entire coding region were isolated from human oocytes and preimplantation embryos. GTSF1 mRNA expression was detected in archived cDNAs from staged human ovarian follicles, germinal vesicle (GV) stage oocytes, metaphase II oocytes, and morula and blastocyst stage preimplantation embryos. Within the adult female germline, expression was highest in GV oocytes. GTSF1 mRNA expression was also assessed in human fetal ovary and was observed to increase during gestation, from 8 to 21 weeks, during which time oogonia enter meiosis and primordial follicle formation first occurs. In human fetal testis, GTSF1 expression also increased from 8 to 19 weeks. CONCLUSIONS: To our knowledge, this report is the first to describe the expression of the human GTSF1 gene in human gametes and preimplantation embryos.

A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys.

STUDY QUESTION: What clinical practices, patient management strategies and experimental methods are currently being used to preserve and restore the fertility of prepubertal boys and adolescent males? SUMMARY ANSWER: Based on a review of the clinical literature and research evidence for sperm freezing and testicular tissue cryopreservation, and after consideration of the relevant ethical and legal challenges, an algorithm for the cryopreservation of sperm and testicular tissue is proposed for prepubertal boys and adolescent males at high risk of fertility loss. WHAT IS KNOWN ALREADY: A known late effect of the chemotherapy agents and radiation exposure regimes used to treat childhood cancers and other non-malignant conditions in males is the damage and/or loss of the proliferating spermatogonial stem cells in the testis. Cryopreservation of spermatozoa is the first line treatment for fertility preservation in adolescent males. Where sperm retrieval is impossible, such as in prepubertal boys, or it is unfeasible in adolescents prior to the onset of ablative therapies, alternative experimental treatments such as testicular tissue cryopreservation and the harvesting and banking of isolated spermatogonial stem cells can now be proposed as viable means of preserving fertility. STUDY DESIGN, SIZE, DURATION: Advances in clinical treatments, patient management strategies and the research methods used to preserve sperm and testicular tissue for prepubertal boys and adolescents were reviewed. A snapshot of the up-take of testis cryopreservation as a means to preserve the fertility of young males prior to December 2012 was provided using a questionnaire. PARTICIPANTS/MATERIALS, SETTING, METHODS: A comprehensive literature review was conducted. In addition, survey results of testis freezing practices in young patients were collated from 24 European centres and Israeli University Hospitals. MAIN RESULTS AND THE ROLE OF CHANCE: There is increasing evidence of the use of testicular tissue cryopreservation as a means to preserve the fertility of pre- and peri-pubertal boys of up to 16 year-old. The survey results indicate that of the 14 respondents, half of the centres were actively offering testis tissue cryobanking as a means of safeguarding the future fertility of boys and adolescents as more than 260 young patients (age range less than 1 year old to 16 years of age), had already undergone testicular tissue retrieval and storage for fertility preservation. The remaining centres were considering the implementation of a tissue-based fertility preservation programme for boys undergoing oncological treatments. LIMITATIONS, REASONS FOR CAUTION: The data collected were limited by the scope of the questionnaire, the geographical range of the survey area, and the small number of respondents. WIDER IMPLICATIONS OF THE FINDINGS: The clinical and research questions identified and the ethical and legal issues raised are highly relevant to the multi-disciplinary teams developing treatment strategies to preserve the fertility of prepubertal and adolescent boys who have a high risk of fertility loss due to ablative interventions, trauma or genetic pre-disposition.

Analysis of DNA Methylation Patterns in Single Blastocysts by Pyrosequencing®.

Extensive epigenetic reprogramming occurs during mammalian gametogenesis and preimplantation development. DNA methylation patterns that are laid down during these stages are essential for subsequent normal foetal development. The requirement for more precise assessment of the epigenetic programming of in vitro-derived human preimplantation embryo has become of paramount importance following the identification of epigenetic diseases that are associated with assisted reproduction and/or infertility. Such techniques are also useful and applicable to experimental reproductive biology. In order to expand our knowledge of epigenetic marks, including DNA methylation, during mammalian reproduction and early development, it is necessary to test new and sufficiently sensitive protocols. There are, however, unique challenges to obtain DNA methylation data from the small cell numbers that are present in the preimplantation embryo. In this protocol, we describe the successful application of Pyrosequencing(®) to yield quantitative DNA methylation data over several CpG sites at differentially methylated regions (DMRs) at imprinted loci in single blastocysts, in this case, human blastocysts. Future developments of the protocol will allow DNA methylation analysis of a more extensive panel of genes for each embryo and at the same time, since the protocol allows for the extraction of mRNA from the embryo, the comparison between DNA methylation and gene expression.

Deoxyribonucleic acid methylation profiling of single human blastocysts by methylated CpG-island amplification coupled with CpG-island microarray.

OBJECTIVE: To study whether methylated CpG-island (CGI) amplification coupled with microarray (MCAM) can be used to generate DNA (deoxyribonucleic acid) methylation profiles from single human blastocysts. DESIGN: A pilot microarray study with methylated CpG-island amplification applied to human blastocyst genomic DNA and hybridized on CpG-island microarrays. SETTING: University research laboratory. PATIENT(S): Five cryopreserved sibling 2-pronuclear zygotes that were surplus to requirements for clinical treatment by in vitro fertilization were donated with informed consent from a patient attending Bourn Hall Clinic, Cambridge, United Kingdom. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Successful generation of genome-wide DNA methylation profiles at CpG islands from individual human blastocysts, with common genomic regions of DNA methylation identified between embryos. RESULT(S): Between 472 and 734 CpG islands were methylated in each blastocyst, with 121 CpG islands being commonly methylated in all 5 blastocysts. A further 159 CGIs were commonly methylated in 4 of the 5 tested blastocysts. Methylation was observed at a number of CGIs within imprinted-gene, differentially methylated regions (DMRs), including placental and preimplantation-specific DMRs. CONCLUSION(S): The MCAM method is capable of providing comprehensive DNA methylation data in individual human blastocysts.

The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro.

Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37°C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 ± 205, n = 8) to mature MII oocytes (744 633 ± 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge.

Variable imprinting of the MEST gene in human preimplantation embryos.

There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos.

Characterisation of the cellular and molecular responses of ovine oocytes and their supporting somatic cells to pre-ovulatory levels of LH and FSH during in vitro maturation.

The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100  ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30  min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.