Kimihisa Tajima

Cell-specific regulation of apoptosis by glucocorticoids: implication to their anti-inflammatory action.

Glucocorticoids play a major role in attenuation of the inflammatory response. These steroid hormones are able to induce apoptosis in cells of the hematopoietic system such as monocytes, macrophages, and T lymphocytes that are involved in the inflammation reaction. In contrast, it was discovered recently that in glandular cells such as the mammary gland epithelia, hepatocytes, ovarian follicular cells, and in fibroblasts glucocorticoids protect against apoptotic signals evoked by cytokines, cAMP, tumor suppressors, and death genes. The anti-apoptotic effect of glucocorticoids is exerted by modulation of several survival genes such as Bcl-2, Bcl-x(L), and NFkB, in a cell-specific manner. Moreover, upregulation or downregulation of the same gene product can occur in a cell-dependent manner following stimulation by glucocorticoids. This phenomenon is probably due to composite regulatory cross-talk among multiple nuclear coactivators or corepressors, which mediate the transcription regulation of the genes, by their interaction with the glucocorticoid receptor. These observations suggest that the anti-inflammatory action of glucocorticoids is exerted by two complementary mechanisms: on one hand, they induce death of the cells that provoke the inflammation, and on the other hand they protect the resident cells of the inflamed tissue by arresting apoptotic signals. Moreover, the complementary action of glucocorticoids provides a new insight to the therapeutic potential of these hormones.

Leptin attenuates follicular apoptosis and accelerates the onset of puberty in immature rats.

Human and rat granulosa cells express receptors to leptin which synergies with glucocorticoid hormones in stimulation of ovarian steroidogenesis. To examine whether leptin affects follicular development and maturation, we injected recombinant ovine leptin (300 ng-10 microg/animal) daily to immature 21 day-old female rats. Non-treated rats reached puberty at 44.5+/-1.6 (n=9) days. In contrast, in leptin treated animals, puberty was reached at 34.5+/-1.6 (n=9) days. Ovarian sections revealed hypertrophy of granulosa cells in leptin treated animals. Moreover, the number of ovulations was 2-fold higher in the treated animals compared to controls (3-4 ovulations versus 7-8 on the first three estrous cycles, P<0.001). Leptin dramatically reduced incidence of follicular apoptosis measured by TUNEL, and was already evident after 7 days of leptin injection (12% of apoptosis in leptin treated group compared to 52% in controls, P<0.001). Maximal protection against apoptosis was achieved at 1-3 microg leptin/animal. The levels of FSH, LH, progesterone and the steroidogenic factors ADX and STAR were elevated earlier in development in the leptin treated animals compared to control animals which is in line with the achievement of early puberty in the leptin treated animals compared to non treated ones. To reveal whether modulation of death and survival genes is involved in leptin attenuation of follicular apoptosis, we examined the expression of the survival gene Bcl-2 and the death gene Bax in Western blots of ovarian homogenates. There was a pronounced elevation in Bcl-2 expression during 7-14 days of leptin injections up to 16.3-fold (P<0.001) compared to Bcl-2 expression in controls. Bax expression was elevated only 3.4 fold (P<0.001), leading to an increase in the Bcl-2/Bax ratio of 4.7 fold (P<0.001). Expression of the tumor suppressor gene p 53 and the oncogene Mdm2 did not change significantly. Our data suggests that leptin may be involved in accelerating follicular maturation by attenuating follicular atresia and increasing the ratio of Bcl-2/Bax.

Oocyte-granulosa-theca cell interactions during preantral follicular development

The preantral-early antral follicle transition is the penultimate stage of follicular development in terms of gonadotropin dependence and follicle destiny (growth versus atresia). Follicular growth during this period is tightly regulated by oocyte-granulosa-theca cell interactions. Formation of the theca cell layer is a key event that occurs during this transitional stage. Granulosal factor(s) stimulates the recruitment of theca cells from cortical stromal cells, while oocyte-derived growth differentiation factor-9 (GDF-9) is involved in the differentiation of theca cells during this early stage of follicular development. The preantral to early antral transition is most susceptible to follicular atresia. GDF-9 promotes follicular survival and growth during transition from preantral stage to early antral stage by suppressing granulosa cell apoptosis and follicular atresia. GDF-9 also enhances preantral follicle growth by up-regulating theca cell androgen production. Thecal factor(s) promotes granulosa cell proliferation and suppress granulosa cell apoptosis. Understanding the intraovarian mechanisms in the regulation of follicular growth and atresia during this stage may be of clinical significance in the selection of the best quality germ cells for assisted reproduction. In addition, since certain ovarian dysfunctions, such as polycystic ovarian syndrome and gonadotropin poor-responsiveness, are consequences of dysregulated follicle growth at this transitional stage, understanding the molecular and cellular mechanisms in the control of follicular development during the preantral-early antral transition may provide important insight into the pathophysiology and rational treatment of these conditions.

Novel genes modulated by FSH in normal and immortalized FSH-responsive cells: new insights into the mechanism of FSH action

Follicle‐stimulating hormone (FSH) controls the development of follicle‐enclosed oocytes in the mammalian ovary by interacting with specific receptors located exclusively on granulosa cells. Its biological activity involves stimulation of intercellular communication, intracellular signaling, and up‐regulation of steroidogenesis; the entire spectrum of genes regulated by FSH is not yet fully characterized. We have established monoclonal rat FSH‐responsive granulosa cell lines that express FSH receptors at 20‐fold higher rates than with primary cells, and thus increased the probability of yielding a distinct spectrum of genes modulated by FSH. Using Affymetrix DNA microarrays, we discovered 11 genes not reported earlier to be up‐regulated by FSH and 9 genes not reported earlier to be down‐regulated by FSH. Modulation of signal transduction associated with G‐protein signaling, phosphorylation of proteins, and intracellular‐extracellular ion balance was suggested by up‐regulation of decay accelerating factor GPI‐form precursor (DAF), membrane interacting protein RGS16, protein tyrosine phosphatase (PTPase), oxidative stress‐inducible protein tyrosine phosphatase (OSIPTPase), and down‐regulation of rat prostatic acid phosphatase (rPAP), Na+, K+‐ATPase, and protein phosphatase 1β. Elevation in granzyme‐like proteins 1 and 3, and natural killer (NK) cell protease 1 (NKP‐1) along with reduction in carboxypeptidase E indicates possible FSH‐mediated preparation of the cells for apoptosis. Up‐regulation of vascular endothelial growth factors indicates the ability of FSH to produce angiogenic factors upon their maturation; whereas, reduction in insulin‐like growth factor binding protein (IGFBP3) indicates its increased potential to promote p53‐induced apoptosis. Striking similarities in FSH modulation of gene expression were found in primary cultures of human granulosa cells obtained from IVF patients although these cells expressed only 1% of FSH receptor compared with immortalized rat cells, as indicated by microarray technique, which probably is in the normal range of expression of this receptor in nontransformed cells. These findings should increase our understanding of the mechanism of FSH action in stimulating development of the ovarian follicular cells, of intracellular and intercellular communication, and of increasing the potential of ovarian follicular cells to undergo apoptosis during the process of selection of the dominant follicle.—OSasson, R., Dantes, A., Tajima, K., Amsterdam, A. Novel genes modulated by FSH in normal and immortalized FSH‐responsive cells: new insights into the mechanism of FSH action. FASEB J. 17, 1256–1266 (2003)

Glucocorticoids protect against apoptosis induced by serum deprivation, cyclic adenosine 3',5'-monophosphate and p53 activation in immortalized human granulosa cells: involvement of Bcl-2.

Glucocorticoid hormones are known to enhance gonadotropin/cAMP-induced steroidogenesis in rat and human granulosa cells. As glucocorticoids induce apoptosis in numerous cell types, we investigated the role of glucocorticoids in the control of apoptosis in immortalized human granulosa cells (HO-23) transfected with a temperature-sensitive mutant of p53 (Val135). When HO-23 were incubated with forskolin in the presence or absence of dexamethasone (Dex) at 32 or 37 C, progesterone production was higher by 4- and 8-fold in the presence of Dex at 37 or 32 C, respectively (P < 0. 01). The expression of adrenodoxin (ADX), which is an intrinsic part of the cytochrome P450 side-chain cleavage enzyme system, remained the same in the presence or absence of Dex in forskolin-stimulated cells. Dex reduced apoptosis (to 33% of control) in cultures after activation of p53 by shifting the temperature from 37 to 32 C. Moreover, Dex suppressed apoptosis induced by serum deprivation (to 40% of control) or forskolin stimulati...

Mechanisms of gonadotropin desensitization.

The gonadotropic hormones, FSH and LH exert a major effect on ovarian and testicular function through interaction with specific seven-transmembrane domain glycoprotein receptors. Desensitization to the hormones, which can occur both in vivo and in vitro, is essential for prevention of overstimulation of the gonadal cells. The long-term process of desensitization to the gonadotropic hormones is probably mediated, in part, by extensive clustering and internalization of the hormone-receptor complex. Short-term desensitization may occur as a result of phosphorylation of serine or threonine residues on the receptor molecules, although a specific receptor kinase has not yet been identified. Recently, we have discovered a novel mechanism of gonadotropin desensitization, which is exerted by down-regulation of StAR expression and steroidogenesis mediated by MAPK activation as a result of hormone-receptor interaction, cAMP accumulation and PKA activation. Thus, PKA not only mediates gonadotropin-induced steroidogenesis, it also activates the down-regulation mechanism that can silence steroidogenesis under certain conditions. Moreover, our findings raise the possibility that activation or inhibition of ERK by other pathways could be an important mechanism for diminution or amplification of gonadotropin-stimulated steroidogenesis. This could contribute to functional luteolysis, a process in which luteinized granulosa cells show reduced sensitivity to LH despite maintenance of LH receptors, or to up-regulation of the steroidogenic machinery during luteinization of granulosa cells.

Analysis of signal transduction stimulated by gonadotropins in granulosa cells

Gonadotropins exert their effect on ovarian follicular cells through the activation of the hormone sensitive adenylate cyclase and consequent elevation of intracellular cyclic AMP (cAMP). Desensitization to the hormone in cultured primary granulosa cells can occur within a short period and internalization of the hormone-receptor complex has been observed both in vivo and in vitro. It was recently documented that the gonadotropins as well as cAMP activate MAP kinase (MAPK) in granulosa cells. Moreover we discovered that specific inhibitors of extracellular signal-regulated kinase phosphorylation, 1 and 2, augment steroidogenesis in granulosa cells up-regulating steroidogenic acute regulatory (StAR) protein expression, and that this modulation is blocked by specific inhibitors of protein kinase A. It is therefore suggested that gonadotropins may activate both stimulatory and inhibitory pathways which regulate steroidogenesis. Moreover the ratio between the activity of these two pathways may determine the rate of steroidogenesis, and rapid activation of MAPK may account as part of the mechanism of desensitization to the hormonal action. Steroidogenic factor-1 and DAX-1 may be involved in the regulation of the MAPK-dependent attenuation of steroidogenesis, since they exhibit sites that could be potentially phosphorylated by the MAPK cascade.

Effects of Ovarian Theca Cells on Apoptosis and Proliferation of Granulosa Cells: Changes During Bovine Follicular Maturation

Abstract We have investigated the role of theca cells in the control of apoptosis and proliferation of granulosa cells during bovine ovarian follicular development using a coculture system in which granulosa and theca cells were grown on opposite sides of a collagen membrane. A DNA fluorescence flow cytometry was used to determine the extent of apoptosis and proliferation in populations of granulosa cells. When granulosa cells were isolated from small follicles (3–5 mm), the percentage of apoptotic cells gradually increased by 1.8-fold during the 3 days of culture. This change was reduced (3.1-fold) by the presence of theca cells. When the cells were isolated from large follicles (15–18 mm), the percentage of apoptotic granulosa cells was gradually reduced (3.4-fold) during the 3 days of culture in single-cultured groups. The percentage of apoptosis on Day 1 was reduced (1.6-fold) by the presence of theca cells. However, such an effect was not detected on Days 2 and 3 of the culture. Theca cells did not affect the proliferation of granulosa cells obtained from either small or large follicles. The present study suggests that theca cells regulate the fate of granulosa cells throughout the follicular maturation process by secreting factors that suppress apoptosis.

Granulosa Cells Promote Differentiation of Cortical Stromal Cells into Theca Cells in the Bovine Ovary

Abstract Formation of a theca cell (TC) layer is an important physiologic event that occurs during early follicular development. Nevertheless, little is known concerning the nature and regulation of the formation of the TC layer during follicular growth. Using an established coculture system in this study, we examined the hypothesis that stromal cells differentiate into TCs during early follicular development and that this process involves interaction with granulosa cells (GCs). Ovarian stromal cells from the bovine ovarian cortex (SC) and medulla (SM) were cultured with or without GCs from small antral follicles. The presence of GCs increased the number of lipid droplets and mitochondria, and it stimulated androstenedione production in SC and SM. However, luteinizing hormone/choriogonadotropin receptor (LHCGR) mRNA abundance and hCG-induced cAMP and androstenedione production were increased in SC but not in SM by the presence of GCs. The present results indicate that GCs are involved in the functional differentiation and the acquisition of LH responsiveness in stromal cells of the ovarian cortex. We suggest that GC-SC interaction is important in the formation of the TC layer during early follicular development, although the nature of this interaction remains to be determined.

Luteinizing hormone-induced Akt phosphorylation and androgen production are modulated by MAP Kinase in bovine theca cells

BackgroundTheca cells play an important role in controlling ovarian steroidogenesis by providing aromatizable androgens for granulosa cell estrogen biosynthesis. Although it is well established that the steroidogenic activity of theca cells is mainly regulated by LH, the intracellular signal transduction mechanisms that regulate thecal proliferation and/or steroidogenesis remain obscure. In this study, we examined whether and how LH controls the PI3K/Akt signaling pathway and androgen production in bovine theca cells. We also explored whether this LH-induced PI3K/Akt activation is modulated with other signaling pathways (i.e. PKA and MAPK).MethodsOvarian theca cells were isolated from bovine small antral follicles and were incubated with LH for various durations. Phospho-Akt and total-Akt content in the cultured theca cells were examined using Western blotting. Androstenedione levels in the spent media were determined using EIA. Semi-quantitative RT-PCR analyses were conducted to analyze the mRNA levels of CYP17A1 and StAR in the theca cells. To examine whether Akt activity is involved in theca cell androgen production, the PI3K inhibitors wortmannin and LY294002 were also added to the cells.ResultsAkt is constitutively expressed, but is gradually phosphorylated in cultured bovine theca cells through exposure to LH. LH significantly increased androstenedione production in bovine theca cells, whereas addition of the wortmannin and LY294002 significantly decreased LH-induced androstenedione production. LH significantly increased CYP17A1 mRNA level in theca cells, whereas addition of LY294002 significantly decreased LH-induced CYP17A1 expression. Neither LH nor PI3K inhibitors alter the mRNA levels of StAR in theca cells. Although H89 (a selective inhibitor of PKA) does not affect LH-mediated changes in Akt, U0126 (a potent MEK inhibitor) suppressed LH-induced Akt phosphorylation, CYP17A1 expression, and androgen production in theca cells.ConclusionThese results indicate that LH stimulates CYP17 mRNA expression and androgen production in theca cells via activation of the PI3K/Akt pathway. The LH-induced Akt phosphorylation and androgen production are modulated by the MAPK signaling in bovine theca cells.