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Title: GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary}
Article:
Abstract:
Folliculogenesis is a progressive and highly regulated process, which is essential to provide ova for later reproductive life, requires the bidirectional communication between the oocyte and granulosa cells. This physical connection-mediated communication conveys not only the signals from the oocyte to granulosa cells that regulate their proliferation but also metabolites from the granulosa cells to the oocyte for biosynthesis. However, the underlying mechanism of establishing this communication is largely unknown. Here, we report that oocyte geranylgeranyl diphosphate( GGPP), a metabolic intermediate involved in protein geranylgeranylation, is required to establish the oocyte-granulosa cell communication. GGPP and geranylgeranyl diphosphate synthase( Ggpps) levels in oocytes increased during early follicular development. The selective depletion of GGPP in mouse oocytes impaired the proliferation of granulosa cells, primary-secondary follicle transition and female fertility. Mechanistically, GGPP depletion inhibited Rho GTPase geranylgeranylation and its GTPase activity, which was responsible for the accumulation of cell junction proteins in the oocyte cytoplasm and the failure to maintain physical connection between oocyte and granulosa cells. GGPP ablation also blocked Rab27a geranylgeranylation, which might account for the impaired secretion of oocyte materials such as Gdf9. Moreover, GGPP administration restored the defects in oocyte-granulosa cell contact, granulosa cell proliferation and primary-secondary follicle transition in Ggpps depletion mice. Our study provides the evidence that GGPP-mediated protein geranylgeranylation contributes to the establishment of oocyte-granulosa cell communication and then regulates the primary-secondary follicle transition, a key phase of folliculogenesis essential for female reproductive function.
Introduction:
Folliculogenesis is orchestrated by a complex series of cellular and molecular interactions that are evoked by the autocrine, paracrine and endocrine functions of ovarian growth factors, chemokines and steroids[1, 2]. The smaller primordial and primary follicles are abundant in the ovarian cortex, where the hypoxic environment keeps them at a low metabolic rate due to insufficient vascularization and nutrition supplementation. Compared with primordial and primary follicles, the secondary follicles are found in the region closer to the ovarian medulla, where the higher O2 levels facilitate rapid growth and high metabolic rates[3, 4]. The primary-secondary follicle transition, which is independent of the hypothalamic-pituitary-ovarian axis, is characterized by the proliferation of granulosa cells from single monolayer to multiple layers and the rapid expansion in oocyte size[5]. Indeed, this process and the subsequent oocyte development process are dependent on their bidirectional signal and material communication between the oocyte and granulosa cells[6–9]. The bidirectional communication between the oocyte and granulosa cells conveys signals from the oocyte to granulosa cells that regulate granulosa cell proliferation, including growth differentiation factor-9( Gdf9) and bone morphogenetic protein-15( Bmp15)[10, 11]. In addition, this communication involves the transport of metabolites for biosynthesis, such as amino acids and pyruvate, from the granulosa cells to the oocyte[12]. During early follicular development, oocytes begin to express abundant cell-cell communication proteins and receptors as well as G-protein coupled receptors[13, 14]. The junctional proteins expressed during follicular development include connexin 37( gap junction protein alpha 4, Gja4), connexin 43( gap junction protein alpha 1, Gja1), N-cadherin( cadherin 2, Cdh2), E-cadherin( cadherin 1, Cdh1), which are required to establish the bidirectional communication between oocytes and granulosa cells[15, 16]. Connexin 37 localizes to the cell surface of the oocyte and provides the structural basis for the gap junctions between the oocyte and granulosa cells[17]. The loss of connexin 37 blocks oocyte growth and arrests folliculogenesis at the early antral stage[18]. Therefore, establishing communication between oocyte and granulosa cells is critical for early folliculogenesis, but the underlying mechanism remains unclear. We found that the levels of geranylgeranyl diphosphate( GGPP), the substrate of protein geranylgeranylation, and geranylgeranyl diphosphate synthase( Ggpps) increase in oocytes during early follicular development. GGPP, a metabolic intermediate of the mevalonate pathway, is synthesized from farnesyl diphosphate( FPP) by Ggpps[19]. Both FPP and GGPP are used for the prenylation( farnesylation or geranylgeranylation) of proteins, a post-translational modification that is required for the membrane localization and activation of small GTPases such as Ras and Rho[20, 21]. The activation of Rho-family GTPases is critical for the regulation of cell junctions[22]. We recently reported that Ggpps depletion in Sertoli cells enhanced Ras farnesylation and blocked spermatogenesis[23]. Thus, we speculated that GGPP and protein geranylgeranylation in the oocyte might also be essential for events that occur during early folliculogenesis, such as the primary-secondary follicle transition, due to its role in prenylating proteins associated with the regulation of cell junctions. In this study, we generated oocyte-specific Ggpps knockout mice by crossing Ggppsfl/fl mice with Ddx4-Cre transgenic mice and identified a novel function of GGPP-mediated protein geranylgeranylation in the oocyte during the primary-secondary follicle transition. Oocyte GGPP is required for Rho GTPase-regulated physical connection between oocyte and granulosa cells and Rab GTPase-directed secretion of oocyte materials such as Gdf9 and thereby has a profound impact on both the oocyte-granulosa cell communication and ovarian primary-secondary follicle transition.
Discussion:
After primordial follicles are activated, dormant oocytes are awakened into a phase of rapid growth, and their metabolic demands are dramatically increased[37]. During the primary-secondary follicle transition, the granulosa cells actively proliferate to form multiple layers and thereby support rapid oocyte growth[1]. Thus, the follicle must adjust to the dramatic alterations of metabolic demand that follow primordial follicle activation. In addition, gene expression profiling during early folliculogenesis showed that oocytes begun to express abundant cell-cell communication proteins( including connexin 37 and N- and E-cadherin) and receptors as well as G-protein coupled receptors[13, 14]. Consistent with this, we also found that Ggpps protein and the GGPP level were associated with early folliculogenesis. The depletion of GGPP from oocytes inhibited Rho-mediated oocyte-granulosa cell contact and impaired Rab-directed cell secretion, which might be responsible for the arrest of granulosa cells proliferation and the failure of the primary-secondary follicle transition. The bidirectional communication between oocytes and the surrounding granulosa cells relies on the localization of junction proteins to the cell membrane[15, 38]. E- and N-cadherin localize to the oocyte membrane and establish oocyte-granulosa cell contacts[16, 39]. Under calcium-free conditions that block cadherin-mediated cell adhesion, the contacts between oocytes and granulosa cells are maintained in primordial follicles but are lost in primary follicles[16]. Consistent with this, we also found that oocyte-granulosa cell contact was maintained in the primordial follicles of Ggppsfl/fl, Ddx4-Cre mice but was lost in primary follicles due to the accumulation of N- and E-cadherin in the oocyte cytoplasm. In connexin 37-deficient mice, folliculogenesis arrested at the early antral stage. Mouse ovarian follicles lacking connexin 43 did not develop beyond the primary follicle stage, and oocyte growth was disrupted[18, 40, 41]. This phenotype is similar to that of GGPP-deficient mice, and the expression of both connexin 37 and connexin 43 decreased in GGPP-deficient mice. The enzymatic activity of Rho GTPases relies on geranylgeranylation via the attachment of GGPP to the C-terminus, which is required for their membrane anchoring and activation. We found that GGPP depletion inhibited the geranylgeranylation and activation of Rho GTPase families, which is responsible for the accumulation of N- and E-cadherin in the oocyte cytoplasm and the failure to maintain oocyte-granulosa cell contact. Here, we establish a novel link between metabolic intermediate GGPP and junction integrity through Rho GTPases. In addition, as a metabolic intermediate of the mevalonate pathway, other groups and we have reported that GGPP-mediated protein geranylgeranylation is essential for maintaining cellular homeostasis[42–45]. Therefore, identifying protein geranylgeranylation-regulated small GTPases or signaling pathways will shed a new light on the molecular mechanisms underlying oocyte development. Intracellular vesicle formation and transport play an important role in secretion and protein turnover at the cell membrane[28, 46]. It has been reported that the abnormal aggregation of membrane structures including secretory vesicles in the oocyte resulted in the failure of primary-secondary follicle transition[47]. Rab GTPases mediate the trafficking of specific cargo molecules to the cell membrane. The activity of those Rab proteins depends on GGPP-mediated geranylgeranylation and membrane anchoring[29]. It was reported that Rab27a was highly expressed in the oocyte and Rab27a knockout mice exhibited a reduced average litter size compared with the control[32]. In this study, we found the geranylgeranylation and the hydrophobic properties of Rab27a were significantly reduced in Ggppsfl/fl, Ddx4-Cre mice. In addition, we recently reported loss of GGPP in β-cells disrupted insulin secretion due to its role in Rab27a geranylgeranylation[31]. Thus, we suggested that GGPP-mediated geranylgeranylation might be essential for the material secretion and protein membrane localization of by Rab-mediated endocytic trafficking pathways. Statins are used to lower cellular cholesterol levels in patients with hypercholesterolemia by inhibiting the rate-limiting enzyme HMG-CoA reductase of the mevalonate pathway[48, 49]. Statins are also used to treat polycystic ovarian syndrome( PCOS), which is a common endocrine-metabolic disorder associated with dyslipidemia[50]. In addition to their cholesterol-lowering benefits, statins also reduce the synthesis of metabolic intermediates of the mevalonate pathway, including FPP and GGPP. As a result, prolonged statin treatment is associated with significant side effects, including myopathy and liver injury[51–53]. In this study, we found that oocyte GGPP depletion induced ovarian dysfunction and female subfertility by inhibiting protein geranylgeranylation. It was also reported that statins could inhibit blastocyst formation by preventing geranylgeranylation[54]. Therefore, the reduction of metabolic intermediates of the mevalonate pathway by statin therapy should be considered with caution. In summary, we report that GGPP-mediated protein geranylgeranylation is essential for the membrane dynamics and the remodeling of cell communication between oocytes and granulosa cells. These findings provide the evidence of protein geranylgeranylation involving in regulating primary-secondary follicle transition and establish a novel connection between intermediate metabolites, ovarian follicular development and female fertility.
Folliculogenesis is a progressive and highly regulated process that requires the tight coordination of metabolism and bidirectional communication between the oocyte and granulosa cells . How this communication is established remains unclear . Here , we find that GGPP-mediated protein geranylgeranylation , a post-translational modification , is essential for the oocyte-granulosa cell communication . GGPP depletion in oocytes inhibits Rho GTPase geranylgeranylation-regulated cell adhesion and impairs Rab GTPase geranylgeranylation-directed cell secretion , which are responsible for the failure to maintain oocyte-granulosa cell communication . This communication defect is probably not able to support the proliferation of granulosa cells from one layer to multiple layers and ultimately results in the failure of the primary-secondary follicle transition and female subfertility . Our findings provide the evidence of GGPP-mediated protein geranylgeranylation involving in regulating primary-secondary follicle transition and establish a novel link between folliculogenesis and GGPP-regulated membrane dynamics .