Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development

The ovary contains a pool of primordial follicles containing oocytes arrested in meiosis that are the source of developing follicles for the female. Growth and differentiation factor-9 (GDF-9) is a member of the transforming growth factor beta superfamily of growth factors, and follicles of GDF-9 knockout mice arrest in the primary stage of development. The effect of GDF-9 treatment on the primordial to primary follicle transition and on subsequent follicle progression was examined using a rat ovary organ culture system. Ovaries from 4-day-old rats were cultured under serum-free conditions in the absence or presence of growth factors. GDF-9 treatment caused a decrease in the proportion of stage 1 early primary follicles and a concomitant increase in the proportion of stage 2 mature primary follicles. GDF-9 did not effect primordial follicles or stage 0 to stage 1 follicle transition. GDF-9 also did not influence stage 3 or 4 secondary follicle numbers. Isolated antral follicle granulosa and theca cell cultures were used to analyze the actions of GDF-9. GDF-9 treatment did not directly influence either granulosa or theca cell proliferation. The ability of GDF-9 to influence the expression of another growth factor was examined. GDF-9 treatment increased kit ligand (KL) mRNA expression in bovine granulosa cells after 2 days of culture. Ovaries from 4-day-old rats were also cultured with or without GDF-9 treatment, and total ovary expression of KL mRNA was increased by GDF-9. In summary, GDF-9 was found to promote the progression of early primary follicle development but did not influence primordial follicle development. The actions of GDF-9 on specific stages of follicle development may in part be mediated through altering the expression of KL.     

Regulation of Pcsk6 expression during the preantral to antral follicle transition in mice: opposing roles of FSH and oocytes

Several secreted products of the TGFbeta superfamily have important roles during follicular development and are produced by both oocytes and somatic cells (granulosa and theca) in the follicle. The proprotein convertases are a family of seven known proteins that process TGFbeta ligands and other secreted products to their mature active form. The present study examined the regulation of steady-state levels of Pcsk6 mRNA, which encodes a convertase protein known to process members of the TGFbeta superfamily, during mouse follicular development. Pcsk6 mRNA and protein were expressed in preantral but not cumulus or mural granulosa cells. Pcsk6 mRNA levels in preantral granulosa cells were not regulated by growing oocytes of preantral follicles, but were elevated by FSH. Furthermore, Pcsk6 mRNA in preantral granulosa cells was potently suppressed by factor(s) secreted by fully grown oocytes from antral follicles, in part through SMAD2/3-mediated pathways. Oocytes acquired the ability to suppress the steady-state levels of Pcsk6 mRNA in granulosa cells during the preantral to antral follicle transition. Suppression of Pcsk6 mRNA by oocytes could reflect a change in the mechanism(s) regulating the activity of members of the TGFbeta superfamily.     

Growth differentiation factor 9 is antiapoptotic during follicular development from preantral to early antral stage

Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia.     

Mouse oocytes regulate metabolic cooperativity between granulosa cells and oocytes: amino acid transport

A search for genes expressed more highly in mouse cumulus cells than mural granulosa cells by subtraction hybridization yielded Slc38a3. SLC38A3 is a sodium-coupled neutral amino acid transporter having substrate preference for l-glutamate, l-histidine, and l-alanine. Detectable levels of Slc38a3 mRNA were found by in situ hybridization in granulosa cells of large preantral follicles, but levels were higher in all granulosa cells of small antral follicles; expression became limited to cumulus cells of large antral follicles. Expression of Slc38a3 mRNA in granulosa cells was promoted by fully grown oocytes from antral follicles but not by growing oocytes from preantral follicles. Fully grown oocytes were dependent on cumulus cells for uptake of l-alanine and l-histidine but not l-leucine. Fully grown but not growing oocytes secreted one or more paracrine factors that promoted cumulus cell uptake of all three amino acids but of l-alanine and l-histidine to a much greater extent than l-leucine. Uptake of l-leucine appeared dependent primarily on contact-mediated signals from fully grown oocytes. Fully grown oocytes also promoted elevated levels of Slc38a3 mRNA and l-alanine transport by preantral granulosa cells, but growing oocytes did not. Therefore, fully grown oocytes secrete one or more paracrine factors that promote cumulus cell uptake of amino acids that oocytes themselves transport poorly. These amino acids are likely transferred to oocytes via gap junctions. Thus, oocytes use paracrine signals to promote their own development via metabolic cooperativity with cumulus cells. The ability of oocytes to mediate this cooperativity is developmentally regulated and acquired only in later stages of oocyte development.     

Mouse oocytes suppress cAMP-induced expression of LH receptor mRNA by granulosa cells in vitro

Mouse oocytes suppress follicle-stimulating hormone (FSH)–induced luteinizing hormone receptor (LHR) messenger ribonucleic acid (mRNA) expression in cultured granulosa cells. The objective of this study was to assess the mechanism by which oocytes suppress FSH-induced LHR expression. The effect of cumulus cell–denuded, germinal-vesicle-stage oocytes, isolated from antral follicles, on FSH-induced cyclic adenosine monophosphate (cAMP) production by cultured granulosa cells was determined by radioimmunoassays. In addition, the effect of oocytes on 8Br-cAMP–induced LHR mRNA steady-state expression by granulosa cells was assessed by RNase protection assays. Oocytes had no detectable effect on FSH-induced cAMP production. However, oocytes dramatically suppressed 8Br-cAMP–induced LHR mRNA steady-state expression by granulosa cells. It was concluded that the mechanism by which oocytes suppress FSH-induced steady-state expression of LHR mRNA is not by inactivating FSH, preventing functional interactions of FSH with its granulosa cell receptors, or by interfering with the signal-transduction mechanisms required for FSH-dependent cAMP production. In addition, since oocytes suppressed the 8Br-cAMP–induced increase in steady-state expression of mRNA for LHR, oocyte-derived factors probably suppress expression by acting downstream of FSH-induced elevation of granulosa cell cAMP. Mol. Reprod. Dev. 49:327–332, 1998. © 1998 Wiley-Liss, Inc.     

Growth differentiation factor-9 stimulates rat theca-interstitial cell androgen biosynthesis

Growth differentiation factor-9 (GDF-9) was shown recently to be essential for early follicular development, including the appearance of the theca layer. Theca cells provide the androgen substrate for aromatization and estrogen production by granulosa cells. Using biologically active recombinant GDF-9 (rGDF-9) and an androgen-producing immortalized theca-interstitial cell (TIC) line or primary TIC, we have examined the action of this paracrine hormone on theca cell steroidogenesis. The effect of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells, GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived growth factor enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product, on TIC androgen production suggests a regulatory role of the oocyte on theca cell function and hence on follicle development and differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions on thecal cell recruitment and differentiation.     

Paracrine actions of growth differentiation factor-9 in the mammalian ovary.

Although the transforming growth factor-beta (TGF-beta) superfamily is the largest family of secreted growth factors, surprisingly few downstream target genes in their signaling pathways have been identified. Likewise, the identities of oocyte-derived secreted factors, which regulate important oocyte-somatic cell interactions, remain largely unknown. For example, oocytes are known to secrete paracrine growth factor(s) which are necessary for cumulus expansion, induction of hyaluronic acid synthesis, and suppression of LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated that absence of the TGF-beta family member, growth differentiation factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary follicle stage leading to infertility. In the present study, we demonstrate that mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles. To explore the biological functions of GDF-9 in the later stages of folliculogenesis and cumulus expansion, we produced mature, glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary cell expression system. A granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semiquantitative RT-PCR. We find that recombinant GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppresses urokinase plasminogen activator (uPA) and LHR mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increases granulosa cell progesterone synthesis in the absence of FSH. Since induction of HAS2 and suppression of the protease uPA in cumulus cells are key events in the production of the hyaluronic acid-rich extracellular matrix which is produced during cumulus expansion, we determined whether GDF-9 could mimic this process. Using oocytectomized cumulus cell-oocyte complexes, we show that recombinant GDF-9 induces cumulus expansion in vitro. These studies demonstrate that GDF-9 can bind to receptors on granulosa cells to regulate the expression of a number of gene products. Thus, in addition to playing a critical function as a growth and differentiation factor during early folliculogenesis, GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte microenvironment, processes which are essential for normal ovulation, fertilization, and female reproduction.     

Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism

Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell-oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development.     

Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles

In addition to pituitary gonadotropins and paracrine factors, ovarian follicle development is also modulated by oocyte factors capable of stimulating granulosa cell proliferation but suppressing their differentiation. The nature of these oocyte factors is unclear. Because growth differentiation factor-9 (GDF-9) enhanced preantral follicle growth and was detected in the oocytes of early antral and preovulatory follicles, we hypothesized that this oocyte hormone could regulate the proliferation and differentiation of granulosa cells from these advanced follicles. Treatment with recombinant GDF-9, but not FSH, stimulated thymidine incorporation into cultured granulosa cells from both early antral and preovulatory follicles, accompanied by increases in granulosa cell number. Although GDF-9 treatment alone stimulated basal steroidogenesis in granulosa cells, cotreatment with GDF-9 suppressed FSH-stimulated progesterone and estradiol production. In addition, GDF-9 cotreatment attentuated FSH-induced LH receptor formation. The inhibitory effects of GDF-9 on FSH-induced granulosa cell differentiation were accompanied by decreases in the FSH-induced cAMP production. These data suggested that GDF-9 is a proliferation factor for granulosa cells from early antral and preovulatory follicles but suppresses FSH-induced differentiation of the same cells. Thus, oocyte-derived GDF-9 could account, at least partially, for the oocyte factor(s) previously reported to control cumulus and granulosa cell differentiation.     

小鼠腔前卵泡体外培养中BMP-6和GDF-9的表达

目的观察BMP-6及GDF-9蛋白在小鼠体外培养卵泡中的定位和定量表达,探讨二者与卵泡发育的关系。方法采用免疫荧光和western blot技术观察BMP-6及GDF-9在体外培养的第6、10天卵泡的定位和定量表达情况。结果在体外培养中,腔前卵泡和有腔卵泡的卵母细胞和颗粒细胞中均检测到BMP-6和GDF-9蛋白的表达;western blot定量显示,在卵泡体外发育的不同阶段,BMP-6和GDF-9蛋白的表达水平不同。结论 BMP-6及GDF-9蛋白存在于体外培养卵泡的卵母细胞和颗粒细胞中;二者的表达水平随卵泡的发育成熟而发生变化。     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.