Hyaluronan Synthesis by Mouse Cumulus Cells Is Regulated by Interactions between Follicle-stimulating Hormone (or Epidermal Growth Factor) and a Soluble Oocyte Factor (or Transforming Growth Factor β1)

Abstract

Expansion of the cumulus cell-oocyte complex (COC) in the preovulatory mammalian follicle requires a transient induction of hyaluronan (HA) synthesis by the cumulus cells. We studied the interactions of known factors that regulate this process by isolating compact COCs from mice and inducing their expansion in vitro. Maximum HA synthesis requires either follicle-stimulating hormone (FSH) or epidermal growth factor (EGF) in combination with either a soluble factor(s) produced by the oocyte or transforming growth factor ␤ 1 . FSH (or EGF) exerts its effects during the first 2 h of incubation, before HA synthesis actually begins. The oocyte factor(s) (or transforming growth factor ␤ 1 ) exerts its effects from 2 h onwards and must be continuously present throughout the subsequent ϳ10 h to achieve a maximum level of HA synthesis. FSH stimulates intracellular cAMP synthesis, which correlates with net HA production up to ϳ14 fmol/COC at 5 ng/ml FSH; however, higher concentrations of FSH increase cAMP levels ϳ10-fold higher with no additional effect on HA synthesis. EGF at saturating concentrations for HA synthesis does not stimulate cAMP above basal levels. Tyrosine kinase inhibitors genistein and tyrphostin AG18 nearly abolish the HA synthesis response to EGF and inhibit the response to FSH by ϳ60%, suggesting that a tyrosine kinase activity is involved for both factors, whereas FSH also operates partially through another signaling pathway. Actinomycin D abolishes HA synthesis if added at the beginning of culture and reduces HA synthesis by ϳ50% if added between 6 -12 h when HA synthesis is normally maximal. The results suggest that regulation of HA synthesis is primarily controlled at the transcriptional level.In the mammalian preovulatory follicle, the cumulus oophorus expands dramatically when the cumulus cells (CCs), 1 consisting of a few layers of granulosa cells closely surrounding the oocyte, are induced to synthesize large amounts of hyaluronan (HA) (1). The secreted HA interacts with specific matrix components, thereby forming a highly hydrated and viscoelastic matrix in the intercellular spaces, which results in an ϳ20-fold increase in volume of the cumulus cell-oocyte complex (COC) (2-4). Recent findings show that CCs synthesize a dermatan sulfate (DS) proteoglycan with high molecular weight that also accumulates in the extracellular matrix, most likely through specific binding to HA (5). The expansion of the COC may facilitate the extrusion of the oocyte through the ruptured follicle wall during ovulation and assist its capture by the oviductal fimbria and entry into the oviduct (6, 7). Moreover, HA and DS proteoglycans may contribute to the success of oocyte fertilization by stabilizing the structure of the egg zona pellucida (8) and by stimulating sperm activation (9) and motility (10, 11). Presently, it is not known if COC expansion in vivo is induced directly by gonadotropins or is mediated indirectly by the mural granulosa cells (12). However, the time course of mouse COC expansion and HA synthesis i...

Genistein-mediated inhibition of glycosaminoglycan synthesis as a basis for gene expression-targeted isoflavone therapy for mucopolysaccharidoses

“…16,17 The EGF influences expression of certain genes by binding to its transmembrane receptor, which upon interaction with EGF becomes an active protein kinase, initiating a specific kinase cascade that finally results in regulation of activity of particular transcription factors. An independent research line led to discovery that tyrosine-specific protein kinase activity of the EGF receptor is inhibited by genistein (4 0 , 5, 7-trihydroxyisoflavone or 5, 7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one).…”

The type I BMP receptor BmprIB is essential for female reproductive function

“…The molecular basis for the defective cumulus expansion in BmrpIB mutants is currently unknown. Cumulus expansion requires the activity of FSH, prostaglandins, and soluble oocyte factor(s); TGF-␤ can substitute for the oocyte-derived factor(s) (23,24). BmprIB mutants do not exhibit decreased Fshr mRNA expression, suggesting that decreased activation of FSH receptor does not contribute to defective expansion.…”

“…Cumulus cell expansion is thought to require the combined activity of FSH and a soluble factor(s) produced by the oocyte (24). We therefore examined levels of Fshr mRNA in ovaries from WT and mutant hormone-primed immature mice (before the onset of estrous cycles).…”

Cyclic AMP-elevating Agents Promote Cumulus Cell Survival and Hyaluronan Matrix Stability, Thereby Prolonging the Time of Mouse Oocyte Fertilizability

“…Compact COCs were mechanically dissociated, and oocyte was discarded. Isolated cumulus cells were then stimulated with FSH in the presence of TGF␤, a growth factor able to substitute for the oocyte in promoting expansion (33). Apoptotic rate as well as the temporal pattern of HA release from the expanded matrix were similar in cumulus cells cultured with (intact COC) and without oocytes (CC) (Fig.…”