Several fibroblast growth factors (FGFs), including FGF1, FGF4 and FGF10, alter ovarian granulosa cell function. These ligands exhibit different patterns of receptor activation, and their mechanisms of action on granulosa cells remain unknown. The objective of this study was to identify the major pathways and target genes activated by FGF1, FGF4 and FGF10 in primary oestrogenic granulosa cells cultured under serum-free conditions. FGF1 and FGF4 increased levels of mRNA encoding Sprouty family members, SPRY2 and SPRY4, and the orphan nuclear receptors NR4A1 and NR4A3. Both FGF1 and FGF4 decreased levels of mRNA encoding SPRY3 and the pro-apoptotic factor BAX. FGF1 but not FGF4 stimulated expression of the cell cycle regulator, GADD45B. In contrast, FGF10 altered the expression of none of these genes. Western blot demonstrated that FGF4 activated ERK1/2 and Akt signalling rapidly and transiently, whereas FGF10 elicited a modest and delayed activation of ERK1/2. These data show that FGF1 and FGF4 activate typical FGF signalling pathways in granulosa cells, whereas FGF10 activates atypical pathways.
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Zhongliang Jiang and Christopher A Price
Anthony Estienne and Christopher A Price
Several growth factor families have been shown to be involved in the function of the female reproductive tract. One subfamily of the fibroblast growth factor (FGF) superfamily, namely the FGF8 subfamily (including FGF17 and FGF18), has become important as Fgf8 has been described as an oocyte-derived factor essential for glycolysis in mouse cumulus cells and aberrant expression of FGF18 has been described in ovarian and endometrial cancers. In this review, we describe the pattern of expression of these factors in normal ovaries and uteri in rodents, ruminants and humans, as well as the expression of their receptors and intracellular negative feedback regulators. Expression of these molecules in gynaecological cancers is also reviewed. The role of FGF8 and FGF18 in ovarian and uterine function is described, and potential differences between rodents and ruminants have been highlighted especially with respect to FGF18 signalling within the ovarian follicle. Finally, we identify major questions about the reproductive biology of FGFs that remain to be answered, including (1) the physiological concentrations within the ovary and uterus, (2) which cell types within the endometrial stroma and theca layer express FGFs and (3) which receptors are activated by FGF8 subfamily members in reproductive tissues.
Lauriane Relav and Christopher A Price
Controling the duration and amplitude of mitogen-activated protein kinase (MAPK) signaling is an important element in deciding cell fate. One group of intracellular negative regulators of MAPK activity is a subfamily of the dual specificity phosphatase (DUSP) superfamily, of which up to 16 members have been described in the ovarian granulosa cells. Growth factors stimulate proliferation of granulosa cells through MAPK, protein kinase C (PKC), and AKT pathways, although it is not known which pathways control DUSP expression in these cells. The aim of the present study was to identify which pathways were involved in the regulation of DUSP expression using a well-established serum-free culture system for bovine granulosa cells. Stimulation of cells with FGF2 increased DUSP1, DUSP5, and DUSP6 mRNA abundance in a time- and dose-dependent manner, and increased DUSP5 and DUSP6 protein accumulation. None of the other eleven DUSP measured were regulated by FGF2. Pharmacological inhibition of MAPK3/1 signaling decreased FGF2-stimulated DUSP1, DUSP5, and DUSP6 mRNA levels (P < 0.05), whereas inhibition of PKC did not affect the expression of these three DUSPs. Abundance of FGF2-dependent DUSP6 mRNA was reduced by inhibition of phospholipase C (PLC) or by chelating calcium, but DUSP5 mRNA abundance was not affected. Abundance of basal DUSP1 and DUSP6, but not DUSP5 mRNA was increased by the addition of the calcium ionophore A23187. We conclude that FGF2 stimulation of DUSP5 abundance requires MAPK3/1 whereas DUSP6 mRNA accumulation is dependent on calcium signaling as well as MAPK3/1 activation, suggesting complex regulation of physiologically important DUSPs in the follicle.
Hilda M Guerrero-Netro, Younès Chorfi, and Christopher A Price
Mycotoxins can reduce fertility and development in livestock, notably in pigs and poultry, although the effect of most mycotoxins on reproductive function in cattle has not been established. One major mycotoxin, deoxynivalenol (DON), not only targets immune cells and activates the ribotoxic stress response (RSR) involving MAPK activation, but also inhibits oocyte maturation in pigs. In this study, we determined the effect of DON on bovine granulosa cell function using a serum-free culture system. Addition of DON inhibited estradiol and progesterone secretion, and reduced levels of mRNA encoding estrogenic (CYP19A1) but not progestogenic (CYP11A1 and STAR) proteins. Cell apoptosis was increased by DON, which also increased FASLG mRNA levels. The mechanism of action of DON was assessed by western blotting and PCR experiments. Addition of DON rapidly and transiently increased phosphorylation of MAPK3/1, and resulted in a more prolonged phosphorylation of MAPK14 (p38) and MAPK8 (JNK). Activation of these pathways by DON resulted in time- and dose-dependent increases in abundance of mRNA encoding the transcription factors FOS, FOSL1, EGR1, and EGR3. We conclude that DON is deleterious to granulosa cell function and acts through a RSR pathway.
Ela Lutwak, Christopher A Price, Sagit-Sela Abramovich, Shiri Rabinovitz, Irit Granot, Nava Dekel, and Dina Ron
Similar expression to FGF (SEF or IL17RD), is a tumor suppressor and an inhibitor of growth factors as well as of pro-inflammatory cytokine signaling. In this study, we examined the regulation of Sef expression by gonadotropins during ovarian folliculogenesis. In sexually immature mice, in situ hybridization (ISH) localized Sef gene expression to early developing oocytes and granulosa cells (GC) but not to theca cells. Sef was also expressed in mouse ovarian endothelial cells, in the fallopian tube epithelium as well as in adipose tissue venules. SEF protein expression, determined by immunohistochemistry (IHC), correlated well with Sef mRNA expression in GC, while differential expression was noticed in oocytes. High Sef mRNA but undetectable SEF protein levels were observed in the oocytes of primary/secondary follicles, while an inverse correlation was found in the oocytes of preantral and small antral follicles. Sef mRNA expression dropped after pregnant mare's serum gonadotropin (PMSG) administration, peaked at 6–8 h after human chorionic gonadotropin (hCG) treatment, and declined by 12 h after this treatment. ISH and IHC localized the changes to oocytes and mural GC following PMSG treatment, whereas Sef expression increased in mural GC and declined in granulosa–lutein cells upon hCG treatment. The ovarian expression of SEF was confirmed using human samples. ISH localized SEF transcripts to human GC of antral follicles but not to corpora lutea. Furthermore, SEF mRNA was detected in human GC recovered from preovulatory follicles. These results are the first to demonstrate Sef expression in a healthy ovary during folliculogenesis. Hormonal regulation of its expression suggests that Sef may be an important factor involved in intra-ovarian control mechanisms.
Peng Han, Hilda Guerrero-Netro, Anthony Estienne, Binyun Cao, and Christopher A Price
Fibroblast growth factors (FGF) modify cell proliferation and differentiation through receptor tyrosine kinases, which stimulate the expression of transcription factors including members of the early growth response (EGR) family. In ovarian granulosa cells, most FGFs activate typical response genes, although the role of EGR proteins has not been described. In the present study, we determined the regulation of EGR mRNA by FGFs and explored the role of EGR1 in the regulation of FGF-response genes. Addition of FGF1, FGF2, FGF4 or FGF8b increased EGR1 and EGR3 mRNA levels, whereas FGF18 increased only EGR1 mRNA abundance. No mRNA encoding EGR2 or EGR4 was detected. Overexpression of EGR1 increased EGR3 mRNA levels as well as the FGF-response genes SPRY2, NR4A1 and FOSL1 and also increased the phosphorylation of MAPK3/1. Knockdown of EGR3 did not alter the ability of FGF8b to stimulate SPRY2 mRNA levels. These data demonstrate the regulation of EGR1 and EGR3 mRNA abundance by FGFs in granulosa cells and suggest that EGR1 is likely an upstream component of FGF signaling in granulosa cells.
Xiaofeng Zheng, Christopher A Price, Yves Tremblay, Jacques G Lussier, and Paul D Carrière
Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-β1 (TGFB1) in the regulation of estradiol-17β (E2) and progesterone (P4) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E2 secretion and mRNA expression of E2-related enzymes cytochrome P450 aromatase (CYP19A1) and 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E2 secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P4 secretion and mRNA expression of 3β-hydroxysteroid dehydrogenase (HSD3B) and α-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P4 and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P4 secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E2, but did not decrease the conversion of estrone (E1) to E2 and pregnenolone to P4. Overall, these results indicate that TGFB1 counteracts stimulation of E2 and P4 synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E2 and cholesterol to P4 without shutting down HSD17B reducing activity and HSD3B activity.
Mingju Cao, José Buratini Jr, Jacques G Lussier, Paul D Carrière, and Christopher A Price
Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.
Mathilde Daudon, Yves Bigot, Joëlle Dupont, and Christopher A Price
Hormones secreted by muscle cells (myokines) are involved in the adaptive response to nutritional and metabolic changes. In this review, we discuss how one family of myokines may alter fertility in response to sudden changes in energy balance.
Dietary stress such as obesity and short-term changes in energy balance can disrupt ovarian function leading to infertility. Adipose tissue secretes hormones (adipokines), such as leptin and adiponectin, that are known to alter ovarian function. Muscles can also secrete endocrine factors, and one such family of myokines, the eleven Fibronectin type III domain-containing (FNDC) proteins, is emerging as important for energy sensing and homeostasis. In this review, we summarize the known roles the FNDC proteins play in energy homeostasis and explore potential impacts on fertility in females. The most well-known member, FNDC5, is the precursor of the ‘exercise hormone’, irisin, secreted by both muscle and adipose tissue. The receptors for irisin are integrins, and it has recently been shown to alter steroidogenesis in ovarian granulosa cells although the effects appear to be species or context specific, and irisin may improve uterine and placental function in women and rodent models. Another member, FNDC4, is also cleaved to release a bioactive protein that modulates insulin sensitivity in peripheral tissues and whose receptor, ADGRF5, is expressed in the ovary. As obese women and farm animals in negative energy balance (NEB) both have altered insulin sensitivity, secreted FNDC4 may impact ovarian function. We propose a model in which NEB or dietary imbalance alters plasma irisin and secreted FNDC4 concentrations, which then act on the ovary through their cognate receptors to reduce granulosa cell proliferation and follicle health. Research into these molecules will increase our understanding of energy sensing and fertility and may lead to new approaches to alleviate post-partum infertility.
Paula F Lima, Cinthia M Ormond, Ester S Caixeta, Rodrigo G Barros, Christopher A Price, and José Buratini
In vitro maturation (IVM) of oocytes in cattle is inefficient, and there is great interest in the development of approaches to improve maturation and fertilization rates. Intraovarian signalling molecules are being explored as potential additives to IVM media. One such factor is kit ligand (KITL), which stimulates the growth of oocytes. We determined if KITL enhances oocyte maturation in cattle. The two main isoforms of KITL (KITL1 and KITL2) were expressed in bovine cumulus–oocyte complexes (COC), and levels of mRNA increased during FSH-stimulated IVM. The addition of KITL to the culture medium increased the percentage of oocytes that reached meiosis II but did not affect cumulus expansion after 22 h of IVM. Addition of KITL reduced the levels of mRNA encoding natriuretic peptide precursor C (NPPC), a protein that holds oocytes in meiotic arrest, and increased the levels of mRNA encoding YBX2, an oocyte-specific factor involved in meiosis. Removal of the oocyte from the COC resulted in increased KITL mRNA levels and decreased NPPC mRNA levels in cumulus cells, and addition of denuded oocytes reversed these effects. Taken together, our results suggest that KITL enhances bovine oocyte nuclear maturation through a mechanism that involves NPPC, and that the oocyte regulates cumulus expression of KITL mRNA.