Oocyte bone morphogenetic protein 15, but not growth differentiation factor 9, is increased during gonadotropin-induced follicular development in the immature mouse and is associated with cumulus oophorus expansion

Bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9 are oocyte-secreted growth factors that are critical local regulators of ovarian function and may be involved in preovulatory cumulus expansion. As cumulus expansion occurs in response to the ovulatory surge, the present study was designed: 1) to investigate whether GDF9 and BMP15 are regulated by gonadotropins in the mouse ovary; and 2) to visualize changes in both GDF9 and BMP15 immunostaining in response to gonadotropins. Immature 21-day-old mice were sequentially treated with recombinant human FSH (r-hFSH), 5 IU daily, at Days 21, 22, and 23 of life, then injected with 5 IU hCG at Day 24 of life. In response to r-hFSH, steady-state Bmp15 mRNA expression levels increased in both total ovaries and cumulus-oocyte complexes, whereas Gdf 9 mRNA levels did not. In addition, BMP15 protein levels increased in total ovaries. The GDF9 immunostaining was exclusively seen in growing oocytes in both control and gonadotropin-treated mice, whereas that of BMP15, which was also primarily seen in growing oocytes, exhibited important changes in response to gonadotropins. Strong BMP15 immunostaining was observed in the follicular fluid of atretic antral follicles after FSH treatment and in expanded, but not in compact, cumulus cells after hCG. The present results show for the first time that BMP15 levels increase during gonadotropin-induced follicular development, in parallel with oocyte maturation, and that this local factor is likely involved in cumulus expansion as previously suggested by studies in Bmp15-null mice.     

Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.     

Oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells

Mammalian oocytes are deficient in their ability to carry out glycolysis. Therefore, the products of glycolysis that are necessary for oocyte development are provided to oocytes by companion cumulus cells. Mouse oocytes secrete paracrine factors that promote glycolysis in cumulus cells. The objective of this study was to identify paracrine factors secreted by oocytes that promote glycolysis and expression of mRNA encoding the glycolytic enzymes PFKP and LDHA. Candidates included growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and fibroblast growth factors (FGFs). Bmp15-/- and Gdf9+/- Bmp15-/- (double mutant, DM) cumulus cells exhibited reduced levels of both glycolysis and Pfkp and Ldha mRNA, and mutant oocytes were deficient in promoting glycolysis and expression of Pfkp and Ldha mRNA in cumulus cells of wild-type (WT) mice. Alone, neither recombinant BMP15, GDF9 nor FGF8 promoted glycolysis and expression of Pfkp and Ldha mRNA in WT cumulus cells. Co-treatment with BMP15 and FGF8 promoted glycolysis and increased expression of Pfkp and Ldha mRNA in WT cumulus cells to the same levels as WT oocytes; however, the combinations of BMP15/GDF9 or GDF9/FGF8 did not. Furthermore, SU5402, an FGF receptor-dependent protein kinase inhibitor, inhibited Pfkp and Ldha expression in cumulus cells promoted by paracrine oocyte factors. Therefore, oocyte-derived BMP15 and FGFs cooperate to promote glycolysis in cumulus cells.     

Differential gene expression of bone morphogenetic protein 15 and growth differentiation factor 9 during in vitro maturation of porcine oocytes and early embryos

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) belong to the TGF-beta superfamily and are involved in the regulation of folliculogenesis. Though there are many reports concerning the expression and regulation of GDF9 in the process of oocyte maturation, expression of BMP15 during oocyte maturation is still not clearly understood. It has been reported that BMP15 and GDF9 expression is important in folliculogeneiss and that the regulation of these two proteins is complex and species-specific. In this report, we investigated the expression of BMP15 and GDF9 genes during in vitro maturation (IVM) at 0, 6, 12, 18, 24, 30, 36, 42 and 48 h for porcine oocytes. Porcine GDF9 gene was found to be highly expressed in immature oocytes and declined slowly during the oocyte maturation process. BMP15mRNA and its encoded protein were expressed at low levels in immature oocytes and increased to the highest level at 18 h of IVM, which coincides with the time of cumulus cell expansion. Thus, these two genes were differentially expressed during the oocyte maturation process and BMP15 is specifically expressed during cumulus cell expansion in porcine oocytes.     

Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9+/-Bmp15-/- [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.     

Regulation of oocyte and cumulus cell interactions by intermedin/adrenomedullin 2

Ovarian folliculogenesis has been studied as a model of hormonal regulation of development and differentiation, cell death, and cell-cell communication. In addition to gonadotropins from the pituitary and follicular paracrine factors, oocyte secreted factors have been shown to play critical roles in the regulation of follicular cell functions. Except for the well characterized BMP family proteins, including GDF9 and BMP15, oocytes are known to secrete oocyte secreted factors that are important for the regulation of cumulus cell survival and the maintenance of tertiary structure of cumulus cell-enclosed oocyte complexes (COCs). Based on genomic screening and studies of COCs cultured in vitro, we showed that intermedin (IMD)/adrenomedullin 2 (ADM2) is a novel oocyte-derived ligand important for the regulation of cell interactions in COCs that functions, in part, by suppressing cumulus cell apoptosis. Consistently, we showed that suppression of IMD/ADM2 signaling in growing rat ovaries in vivo leads to oocyte atresia and aberrant cell cycle progression in follicular cells. Together, our studies indicated that mammalian oocytes deploy a G protein-coupled receptor ligand to coordinate normal interactions of oocytes and cumulus cells and provided a better understanding of how the tertiary structure of a COC is maintained as follicles undergo exponential growth during the late stages of folliculogenesis.     

The relationship between cumulus cell-oocyte coupling,oocyte meiotic maturation,and cumulus expansion

The timing of the reduction of cumulus cell-oocyte coupling was correlated with oocyte meiotic maturation and the expansion (mucification) of the cumulus oophorus using immature mice treated with gonadotropins. Three hours after the injection of an ovulatory dose of human chorionic gonadotropin (hCG), more than 90% of the oocytes isolated from large Graafian follicles had undergone germinal vesicle breakdown, indicating that oocyte meiotic maturation had been initiated. However, no cumulus expansion or reduction of intercellular coupling was detected at this time. By 6 hr after hCG injection, the index of oocyte-cumulus cell coupling was still not less than that found in oocyte-cumulus cell complexes isolated from control mice not receiving hCG. Cumulus expansion at 6 hr post-hCG was limited to the outer cumulus cells while those adjacent to the oocyte were still tightly packed. Cumulus expansion appeared complete by 9 hr after hCG injection and the cumulus cell-oocyte coupling index was greatly reduced. These results show that oocyte meiotic maturation in the mouse is not initiated by a reduction in cumulus cell-oocyte coupling or by cumulus expansion. However, the results suggest that the reduction of intercellular coupling in vivo may be a result of cumulus expansion.     

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.     

The mechanism of cumulus cell-oocyte uncoupling: Evidence for the participation of both cumulus cells and oocytes

Cumulus cells are metabolically coupled to oocytes via heterologous gap junctions. This coupling terminates near the time of ovulation, and the termination appears to be correlated with the mucification of the cumulus cells lying immediately adjacent to the oocytes. The first objective of this project was to determine whether follicle stimulating hormone (FSH) induction of cumulus cell-oocyte uncoupling could occur independently of FSH-stimulated cumulus mucification (expansion). Intercellular coupling was measured as a percentage of radiolabeled choline (or its metabolites) that was incorporated into the oocyte relative to the total amount of radiolabel incorporated into the entire cumulus cell-oocyte complex. It was found that the complete suppression of FSH-stimulated cumulus expansion with chondroitin sulfate B had no suppressive effect on FSH-stimulated cumulus cell-oocyte uncoupling. This finding showed that FSH-stimulated cumulus expansion was not required for cumulus cell-oocyte uncoupling. Since 17β-estradiol, testosterone, or progesterone could not induce maximal cumulus cell uncoupling, it was concluded that the uncoupling-promoting action of FSH was probably not mediated by steroid hormones. A partial uncoupling of cumulus cells and oocytes was found when spontaneous oocyte maturation had occurred in the absence of FSH. This partial uncoupling was prevented by incubation of cumulus cell-oocyte complexes in concentrations of dibutyryl cyclic adenosine monophosphate (dbcAMP) or 3-isobutyl-1-methyl xanthine (IBMX) (0.25 and 0.10 mM respectively) that suppressed spontaneous oocyte maturation without inducing cumulus expansion. These inhibitors also prevented the maximal induction of uncoupling that would have been provoked by biological grade preparations of either FSH or luteinizing hormone (LH). It was concluded that two factors were required to bring about maximal cumulus cell-oocyte uncoupling: one factor was dependent upon the action of gonadotropins on cumulus cell function, the other factor appeared to be a function of the oocytes, since maximal uncoupling could occur only after the germinal vesicles had broken down.     

The effects of plasma-derived extracellular vesicles on cumulus expansion and oocyte maturation in mice

Cumulus cell expansion is required for the ovulation of a fertilizable oocyte. Extracellular vesicles (EVs) are bilayer-lipid membrane vesicles that may be found in a variety of bodily fluids and play an important role in biological processes. This study aimed to examine the effects of plasma-derived EVs on cumulus expansion and in vitro maturation (IVM) of the oocyte. EVswere isolated using ultracentrifugation from the plasma of female mice. The morphology and size of EVs were analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Western blotting allowed us to identify CD63, CD81, CD9, and HSP70 protein markers of EVs; the expression of the genes related to cumulus cell expansion, including hyaluronan synthase 2 (Has2) and prostaglandinendoperoxide synthase 2 (Ptgs2), were assessed using real-time polymerase chain reaction. Plasma-derived EVs labeled with Dil dye were successfully incorporated with cumulus cells during IVM. Plasma-derived EVs significantly induced cumulus expansion and maturation of oocytes. The percentage of oocytes that reached the MII stage was significantly greater in the EVs treatment group compared with other groups. Although treatment with epidermal growth factor (EGF) significantly increased cumulus expansion in cumulus-oocyte complexes (COCs), the impact was less than that seen with plasma-derived EVs. Furthermore, EVs generated from plasma substantially enhanced Has2 and Ptgs2 mRNA expression in the cumulus-oocyte complex. This research indicates that EVs derived from plasma are capable of promoting cumulus expansion and oocyte maturation.     

Influence of hyaluronan accumulation during cumulus expansion on in vitro porcine oocyte maturation

During oocyte maturation, the cumulus-oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process.     

Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass

The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down-regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down-regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.