Marilyn J. O'Brien

Bio: Marilyn J. O'Brien is an academic researcher from National Institutes of Health. The author has contributed to research in topics: Oocyte & Ovarian follicle. The author has an hindex of 29, co-authored 35 publications receiving 5700 citations.

Development in vitro of mouse oocytes from primordial follicles.

Abstract: 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.

A Revised Protocol for In Vitro Development of Mouse Oocytes from Primordial Follicles Dramatically Improves Their Developmental Competence

Abstract: 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.

Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells

Abstract: Oocyte-derived bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are key regulators of follicular development. Here we show that these factors control cumulus cell metabolism, particularly glycolysis and cholesterol biosynthesis before the preovulatory surge of luteinizing hormone. Transcripts encoding enzymes for cholesterol biosynthesis were downregulated in both Bmp15(-/-) and Bmp15(-/-) Gdf9(+/-) double mutant cumulus cells, and in wild-type cumulus cells after removal of oocytes from cumulus-cell-oocyte complexes. Similarly, cholesterol synthesized de novo was reduced in these cumulus cells. This indicates that oocytes regulate cumulus cell cholesterol biosynthesis by promoting the expression of relevant transcripts. Furthermore, in wild-type mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol and Ebp, which encode enzymes required for cholesterol synthesis, were highly expressed in cumulus cells compared with oocytes; and oocytes, in the absence of the surrounding cumulus cells, synthesized barely detectable levels of cholesterol. Furthermore, coincident with reduced cholesterol synthesis in double mutant cumulus cells, lower levels were also detected in cumulus-cell-enclosed double mutant oocytes compared with wild-type oocytes. Levels of cholesterol synthesis in double mutant cumulus cells and oocytes were partially restored by co-culturing with wild-type oocytes. Together, these results indicate that mouse oocytes are deficient in synthesizing cholesterol and require cumulus cells to provide products of the cholesterol biosynthetic pathway. Therefore, oocyte-derived paracrine factors, particularly, BMP15 and GDF9, promote cholesterol biosynthesis in cumulus cells, probably as compensation for oocyte deficiencies in cholesterol production.

Initial and cyclic recruitment of ovarian follicles.

Abstract: Mammalian ovaries consist of follicles as basic functional units. The total number of ovarian follicles is determined early in life, and the depletion of this pool leads to reproductive senescence. Each follicle develops to either ovulate or, more likely, to undergo degeneration. The dynamics of ovarian follicle development have interested endocrinologists and developmental biologists for many years. With the advent of assisted reproductive techniques in humans, the possibility of regulating follicle development in vivo and in vitro has gained clinical relevance. In this review, we focus upon key branching points during the development of ovarian follicles as well as factors involved in determining the eventual destiny of individual follicles. We discuss inconsistencies in the literature regarding the definitions of follicle recruitment and selection and propose to name the two major steps of follicle development as initial and cyclic recruitment, respectively. Because some of these disparities have arisen due to differences in the animal systems studied, we also compare the development of the ovarian follicles of both humans and rats. We also review the status of knowledge of several puzzling clinical issues that may provide important clues toward unlocking the mechanisms of follicle development.

Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions

Abstract: Saez, Juan C., Viviana M. Berthoud, Maria C. Branes, Agustin D. Martinez, and Eric C. Beyer. Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions. Physiol Rev 83: 1359-1400,...

Derivation of oocytes from mouse embryonic stem cells

Abstract: Continuation of mammalian species requires the formation and development of the sexually dimorphic germ cells. Cultured embryonic stem cells are generally considered pluripotent rather than totipotent because of the failure to detect germline cells under differentiating conditions. Here we show that mouse embryonic stem cells in culture can develop into oogonia that enter meiosis, recruit adjacent cells to form follicle-like structures, and later develop into blastocysts. Oogenesis in culture should contribute to various areas, including nuclear transfer and manipulation of the germ line, and advance studies on fertility treatment and germ and somatic cell interaction and differentiation. In the early mammalian embryo, the germ line and soma are indistinguishable from each other. In the mouse, germ cell competence is induced at embryonic day 6.5 in proximal epiblast cells by signals emanating from the

Oocyte control of ovarian follicular development and function in mammals.

Abstract: A new perspective on ovarian follicular development has emerged over the last decade. Whereas the oocyte was previously considered only a passive recipient of developmental signals from oocyte-associated granulosa cells, it is now clear that communication between oocytes and granulosa cells is bidirectional. A complex interplay of regulatory factors governs the development of both types of cell. This interplay is essential not only for oocyte development but also for follicular development, beginning with the initial assembly of the primordial follicle and continuing throughout ovulation. The existence of an oocyte-granulosa cell regulatory loop, essential for normal follicular differentiation as well as for the production of an oocyte competent to undergo fertilization and embryogenesis, is proposed. Although gonadotrophins are essential for driving the differentiation of granulosa cell phenotypes, within its sphere of influence, the oocyte is probably the dominant factor determining the direction of differentiation and the function of the granulosa cells associated with it.

Development in vitro of mouse oocytes from primordial follicles.

Abstract: 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.