Mammalian oocytes go through a long and complex developmental process while acquiring the competencies that are required for fertilization and embryogenesis. Recent advances in molecular genetics and quantitative live imaging reveal new insights into the molecular basis of the communication between the oocyte and ovarian somatic cells as well as the dynamic cytoskeleton-based events that drive each step along the pathway to maturity. Whereas self-organization of microtubules and motor proteins direct meiotic spindle assembly for achieving genome reduction, actin filaments are instrumental for spindle positioning and the establishment of oocyte polarity needed for extrusion of polar bodies. Meiotic chromatin provides key instructive signals while being 'chauffeured' by both cytoskeletal systems.

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

The physical basis for communication between animal cells is thought largely to depend on the presence of gap junctions which are wide-spread among various vertebrate and invertebrate tissues (14,20). Studies on cultured cell lines have indicated that, in addition to mediating electrical impulse propagation between neighboring cells (electrotonic coupling), gap junctions mediate certain metabolic aspects of cellular function by permitting the transcellular passage of "informational" cytoplasmic molecules between physically conjoined cells (metabolic cooperation) (10,15,23). Moreover, the morphological demonstration that gap junctions conjoin different cell types within tissues such as the vertebrate retina (19), an arachnid midgut (16), and the mammalian kidney (18) establishes a precedent for gap junction-mediated cooperation between distinct cell types in other tissues.Recent physiological findings (3, 13) suggest that metabolic cooperativity between folliculargranulosa cells and oocytes within developing mammalian ovarian follicles may importantly regulate both the meiotic maturation of the egg and the transformation of the follicular epithelium into the corpus luteum. As an extension of our studies into the nature of coordinative cellular interactions in the ovarian follicle (2), we report here that gap junctions conjoin oocytes to companion follicular cells. MATERIALS AND METHODS Thin Section and Tracer AnalysisOvaries from adult cycling female mice, rats, and rhesus monkeys (Macaca mulatta), from 1-and 5-6-day old rats and from estrous rabbits were fixed for freezefracture studies by immersion for 20 rain at room temperature in 0.1 M cacodylate buffer (pH 7.4) containing 2% paraformaldehyde, 3% glutaraldehyde, and 5% sucrose. Thin-section and tracer samples were fixed for a total of 60 rain. Final fixation in all cases was carried out on individual follicles which had been dissected free from surrounding stromal tissue (with the exception of the 5-6-day-old rat ovaries) and cut into l-ram cubes. Better fixation of rabbit follicles from animals in estrous was achieved by omitting the paraformaldehyde from the primary fixative,. Ionic lanthanum was used as an extracellular tracer as previously described (2). After several washes in buffer, the tissues were postfixed in 1% OsO4 in 0.1 M cacodylate buffer at room temperature for 1 h, washed, dehydrated in a graded series of ethanol, and embedded in a mixture of Epon-Araldite (5). Thin sections were cut on a diamond knife, collected on 300-mesh copper grids, and examined in either a Philips 200 or 300 electron microscope, both unstained and after staining with uranyl acetate (25) and lead (21). Freeze-Fracture AnalysisAfter primary fixation for 20 min, cubes of tissue were washed thoroughly, equilibrated for 90 rain in 20% glycerol in 0.1 M cacodylate buffer, frozen on paper disks in liquid Freon 22, and stored in liquid nitrogen. A

Although the physiological, molecular and genetic determinants that initiate and sustain follicular growth and maturation have received much attention (see reviews by McNatty et al., 1999;McGee and Hsueh, 2000), comparatively little information has been forthcoming with regard to the changes in cellular behaviour that attend the process of folliculogenesis. When viewed in the context of other developmental systems, in which paracrine control of cell differentiation is considered implicitly relative to cell signalling, cell-cell adhesion and cell-extracellular matrix (ECM) interactions, our knowledge of cellular behaviours in the ovarian follicle is lacking, despite the wealth of information available on the role of growth factors and hormones in ovarian follicle development (for a review see Elvin and Matzuk, 1998). Recently, Rodgers et al. (1999 have drawn attention to the role of the ECM during bovine follicle growth as related to changes in the follicular basal lamina that may influence the growth and differentiation of both granulosa and theca cells. Given the

Homologous gap junctions are generally recognized as a means of coordinating cellular behavior under developmental and homeostatic conditions. In the mammalian ovary, heterologous gap junctions between the oocyte and the granulosa cells have been widely implicated in the regulation of meiotic maturation late in oogenesis. However, the role of oocyte-granulosa cell gap junctions at earlier stages of oogenesis is poorly understood. Stage-specific defects in both oocyte and follicle development have been identified in juvenile mice deficient in heterologous oocyte-granulosa cell gap junctions due to targeted deletion of Gja4, the gene encoding connexin-37. Follicle development arrests at the type 4 preantral stage and although oocytes commence growth, oocyte growth ceases at a diameter of 52 microm (74.3% of control size). Analysis of cell cycle and cytoskeletal markers indicates that oocytes arrest in a G(2) state based on uniform decondensed GV chromatin, interphase microtubule arrays, and nonphosphorylated cytoplasmic centrosomes. Functional assays of meiotic competence confirm that oocytes from connexin-37-deficient mice are unable to enter M phase (initiate meiotic maturation) unless treated with the phosphatase inhibitor okadaic acid (OA). Unlike growing oocytes from heterozygous control animals, OA-treated oocytes from connexin-37-deficient mice respond acutely and progress rapidly to the circular bivalent stage of meiosis I and upon removal from OA rapidly revert to an interphase state. In contrast, OA-treated control incompetent oocytes are slow to respond, exhibit a lower proportion of chromosomal bivalent stage oocytes, but remain in and progress into meiotic M phase upon removal from OA. This study demonstrates that heterologous gap-junctional communication is required for the completion of oocyte growth and the acquisition of cytoplasmic meiotic competence.