It has long been realized that follicular somatic cells support oocyte development. The close physical association of these two types of cell indicated this to early biologists, but physiological evidence was first presented by Pincus and Enzmann (1935), who found that fully grown oocytes removed from antral follicles underwent a spontaneous, gonadotrophin-independent resumption of meiosis in culture, and concluded that follicular somatic cells maintain oocytes in meiotic arrest. Subsequent studies demonstrated that follicular somatic cells promote the reinitiation of meiosis and its progression to metaphase II (nuclear maturation). Follicular somatic cells also promote oocyte competence to undergo fertilization and preimplantation embryogenesis (cytoplasmic maturation) (Buccione et al., 1990a) and granulosa cells participate in the global suppression of transcription in oocytes that occurs before nuclear maturation (Fig. 1) (De la Fuente and Eppig, 2001). In contrast to granulosa cell-to-oocyte communication, knowledge of oocyte-to-granulosa cell communication is relatively recent. The studies of Nalbandov and colleagues pioneered this field, observing a precocious luteinization of rabbit follicles in vivo after removal of the oocyte-cumulus complex (El-Fouly et al., 1970). Similarly, granulosa cells from antral follicles cultured in the absence of oocytes were observed to resemble luteinized granulosa cells, whereas those cultured in the proximity of oocytes appeared to maintain a granulosa cell-like appearance (Nekola and Nalbandov,

The objective of these studies was to achieve complete oocyte development in vitro beginning with the oocytes in the primordial follicles of newborn mouse ovaries. A two-step strategy was developed: first the ovaries of newborn mice were grown in organ culture for 8 days, and then the developing oocyte-granulosa cell complexes were isolated from the organ-cultured ovaries and cultured for an additional 14 days. The oocytes of primordial follicles are approximately 4190 microns3 in volume (20 microns in diameter), and this volume increased by approximately 53,810 microns3 to a final size of 58,000 microns3--a 13.8-fold increase--during the 8 days of organ culture. In the first experiment the oocyte-granulosa cell complexes were grown in control medium or in medium supplemented with FSH (0.5 ng/ml), epidermal growth factor (EGF; 1.0 ng/ml), or EGF plus FSH. Only 50-60% of the complexes cultured in control medium or in medium supplemented with FSH were recovered at the end of the 14-day culture period. In contrast, more than 90% of the complexes cultured in medium supplemented with EGF were recovered. The median size of the oocytes grown in control medium was 176,800 microns3 (69-microns diameter), while the median size of those grown in medium supplemented with EGF was slightly smaller (136,400-microns3 volume; 63-microns diameter), due to the survival of more smaller-size oocytes in EGF-containing medium. Thirty percent of the oocytes recovered after development in FSH-containing medium were competent to undergo germinal vesicle breakdown (GVB). In the second set of experiments, oocyte-granulosa cell complexes isolated from organ-cultured ovaries were cultured in medium supplemented with either 0.5 or 5.0 ng/ml FSH or with these same concentrations of FSH plus 1.0 ng/ml EGF. Again, increased oocyte recovery was observed in the groups cultured with EGF. There was no difference among the groups in the percentage of the oocytes that acquired competence to undergo GVB (32%) or in the percentage of GVB oocytes that produced a polar body, thus indicating progression of meiosis to metaphase II (22%). When the mature oocytes were inseminated, 21% underwent fertilization and cleavage to the 2-cell stage in the groups without EGF during oocyte development, while 42% underwent fertilization and cleavage to the 2-cell stage in the groups cultured with EGF. Less than 2% of the 2-cell-stage embryos developed to the blastocyst stage in any of the groups. One hundred and ninety 2-cell-stage embryos were transferred to the oviducts of pseudopregnant females; two females produced one pup each; one was living and the other had apparently died recently. The results reported here clearly show that complete development of oocytes in vitro from the primordial follicle stage is possible and establish the framework for further studies using oocytes from laboratory animals as model systems for the development of oocytes from humans as well as from animals of agricultural and zoological importance.

Granulosa cells of mammalian Graafian follicles maintain oocytes in meiotic arrest, which prevents the precocious maturation. We show that mouse mural granulosa cells, which line the follicle wall, express natriuretic peptide precursor type C, Nppc, mRNA while cumulus cells surrounding oocytes express mRNA of the NPPC receptor NPR2, a guanylyl cyclase. NPPC elevated cGMP levels in cumulus cells and oocytes and inhibited meiotic resumption in vitro. Meiotic arrest was not sustained in most Graafian follicles of Nppc or Npr2 mutant mice, and meiosis resumed precociously. Oocyte-derived paracrine factors promoted cumulus cell expression of Npr2 mRNA. Therefore, the granulosa cell ligand NPPC and its receptor NPR2 in cumulus cells prevent precocious meiotic maturation, which is critical for maturation and ovulation synchrony and for normal female fertility.Meiosis is a germ cell-specific process that reduces the number of chromosomes from the diploid to the haploid number. It begins in human and mouse ovaries during fetal life but meiotic progression becomes arrested for prolonged periods at the diplotene stage of meiotic prophase. Fully-grown mammalian oocytes in Graafian follicles are maintained in meiotic prophase arrest until the preovulatory surge of luteinizing hormone (LH) triggers the resumption of meiosis and ovulation. The mature oocytes (eggs) are then available for fertilization within the oviduct. The somatic cell compartment of Graafian follicles, consisting of mural granulosa cells lining the inside of the follicle wall and cumulus cells surrounding the oocyte, plays a crucial role in maintaining oocyte meiotic arrest in mammals since removal of the oocyte-cumulus cell complex from these follicles results in gonadotropin-independent meiotic resumption in culture (1,2). Cyclic nucleotides cAMP and cGMP are crucial to the maintenance of meiotic arrest. Cyclic AMP is generated within oocytes downstream of GPR3 and GPR12, regulators of Gs proteins controlling adenylyl cyclase (3,4). Inability to sustain oocyte cAMP levels leads to precocious gonadotropinindependent resumption of meiosis, which interrupts the synchrony between oocyte maturation and ovulation and compromises female fertility (3-5). PDE3A, an oocytespecific phosphodiesterase, becomes activated after the LH-surge to decrease cAMP levels in oocytes and thereby initiates pathways governing meiotic resumption (6). Before the LHsurge, cGMP, originating in granulosa cells of the follicular somatic compartment and transferred to the oocyte via gap junctions, inhibits activity of PDE3A in the oocyte (7,8). Therefore, control of cGMP production by granulosa cells is crucial for maintaining meiotic arrest in fully-grown oocytes. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptExploration of the mouse cumulus cell transcriptome for mRNAs encoding guanylyl cyclases using microarray analysis (9) revealed abundant expression of natriuretic peptide receptor 2 (Npr2, also called GC-B) mRNA. The presence of this guanylyl cyc...

The production of functional female gametes is essential for the propagation of all vertebrate species. The growth of oocytes within ovarian follicles and their development to mature eggs have fascinated biologists for centuries, and scientists have long realized the importance of the ovarian follicle's somatic cells in nurturing oogenesis and delivering the oocyte to the oviduct by ovulation. Recent studies have revealed key roles of the oocyte in folliculogenesis and established that bidirectional communication between the oocyte and companion somatic cells is essential for development of an egg competent to undergo fertilization and embryogenesis. The challenge for the future is to identify the factors that participate in this communication and their mechanisms of action.

THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.

A system is described here by which live mice can be produced from oocytes isolated from 12-day-old mice, be grown, matured, and fertilized in vitro, and then be transferred to pseudopregnant females. These oocytes were, at the time of isolation from preantral follicles, in about mid-growth phase and incompetent of undergoing germinal vesicle breakdown (GVB) without further development. The developmental competence of mouse oocytes that grew and underwent maturation in vitro was compared to oocytes that grew in vivo and underwent maturation in vitro. After isolation from mice 16 through 28 days old, oocytes were found to increase in size and to sequentially acquire the ability to undergo GVB, produce a polar body, cleave to the 2-cell stage after insemination, and develop to the blastocyst stage. Moreover, the number of cells per blastocyst increased with the age of the mice from which the immature oocytes were isolated. Oocyte-granulosa cell complexes isolated from 12-day-old mice were cultured for 10 days. At the end of the culture period, the oocytes had grown to a size equivalent to oocytes isolated from 16-day-old mice, and 87% of the in-vitro-grown (IVG) oocytes underwent GVB; 79% of these produced a clearly visible polar body when maturation occurred in the presence of follicle-stimulating hormone (FSH). The IVG oocytes cleaved to the 2-cell stage after insemination in vitro with a frequency equivalent to superovulated ova and ova that matured in vitro after isolation from 22-day-old mice.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to improve the conditions for oocyte development in vitro beginning with the primordial follicles of newborn mice. Previous studies showed that oocytes competent of meiotic maturation, fertilization, and preimplantation could develop in vitro from primordial follicles. However, the success rates were low and only one live offspring was produced (0.5% of embryos transferred). A revised protocol was compared with the original protocol using oocyte maturation and preimplantation development as end points. The percentage of oocytes maturing to metaphase II and developing to the blastocyst stage was significantly improved using the revised protocol. In addition, we compared the production of offspring from two-cell stage embryos derived from in vitro-grown and in vivo-grown oocytes. Of 1160 transferred two-cell stage embryos derived from in vitro-grown oocytes, 66 (5.7%) developed to term and 7 pups (10.6%) died at birth. The remaining 59 pups (27 females, 32 males) survived to adulthood. By comparison, of 437 transferred two-cell stage embryos derived from in vivo-grown oocytes, 76 (17.4%) developed to term and 4 (5.3%) died at birth. The remaining 72 pups (35 females, 37 males) survived to adulthood. These studies provide proof of the principle that fully competent mammalian oocytes can develop in vitro from primordial follicles and present a significant advance in oocyte culture technology.

The female gamete (the oocyte) serves the distinct purpose of transmitting the maternal genome and other maternal factors that are critical for post-ovulation events. Through the identification and characterization of oocyte-specific factors, we are beginning to appreciate the diverse functions of oocytes in ovarian folliculogenesis, fertilization and embryogenesis. To understand these processes further, we identified genes called zygote arrest 1 (Zar1 and ZAR1 in mouse and human, respectively) as novel oocyte-specific genes. These encode proteins of 361 amino acids and 424 amino acids, respectively, which share 59% amino-acid identity and an atypical plant homeo-domain (PHD) motif. Although Zar1-null (Zar1(-/-)) mice are viable and grossly normal, Zar1(-/-) females are infertile. Ovarian development and oogenesis through the early stages of fertilization are evidently unimpaired, but most embryos from Zar1(-/-) females arrest at the one-cell stage. Distinct pronuclei form and DNA replication initiates, but the maternal and paternal genomes remain separate in arrested zygotes. Fewer than 20% of the embryos derived from Zar1(-/-) females progress to the two-cell stage and show marked reduction in the synthesis of the transcription-requiring complex, and no embryos develop to the four-cell stage. Thus, Zar1 is the first identified oocyte-specific maternal-effect gene that functions at the oocyte-to-embryo transition and, as such, offers new insights into the initiation of embryonic development and fertility control in mammals.