Immortalized Rat Granulosa Cells Research Articles

Delta-9-tetrahydrocannabinol increases vascular endothelial growth factor (VEGF) secretion through a cyclooxygenase-dependent mechanism in rat granulosa cells

While the effects of delta-9-tetrahydrocannabinol (THC), the psychoactive component of cannabis, have been studied extensively in the central nervous system, there is limited knowledge about its effects on the female reproductive system. The aim of this study was to assess the effect of THC on the expression and secretion of the angiogenic factor vascular endothelial growth factor (VEGF) in the ovary, and to determine if these effects were mediated by prostaglandins. Spontaneously immortalized rat granulosa cells (SIGCs) were exposed to THC for 24 h. Gene expression, proliferation and TNFα-induced apoptosis were evaluated in the cells and concentrations of VEGF and prostaglandin E2 (PGE2), a known regulator of VEGF production, were determined in the media. To evaluate the role of the prostanoid pathway, cells were pre-treated with cyclooxygenase (COX) inhibitors prior to THC exposure. THC-exposed SIGCs had a significant increase in VEGF and PGE2 secretion, along with an increase in proliferation and cell survival when challenged with an apoptosis-inducing factor. Pre-treatment with COX inhibitors reversed the THC-induced increase in both PGE2 and VEGF secretion. Alterations in granulosa cell function, such as the ones observed after THC exposure, may impact essential ovarian processes including folliculogenesis and ovulation, which could in turn affect female reproductive health and fertility. With the ongoing increase in cannabis use and potency, further study on the impact of cannabis and its constituents on female reproductive health is required.

Inhibition of Sirtuin-1 hyperacetylates p53 and abrogates Sirtuin-1-p53 interaction in Cr(VI)-induced apoptosis in the ovary

Environmental contamination with hexavalent chromium, Cr(VI), has been increasing in the United States as well as in developing countries. Exposure to Cr(VI) predisposes the human population to various diseases, including cancer, infertility, and developmental problems in children. Previous findings from our laboratory reported that prenatal exposure to Cr(VI) caused premature ovarian failure through p53-mediated mechanisms. Sirtuin 1 (SIRT1) is an NAD+ -dependent histone deacetylase class III. SIRT1 deacetylates several histones and non-histone proteins such as p53 and NFkB. The current study determines a role for the SIRT1-p53 network in apoptosis induced by Cr(VI) in the ovary and establishes physical interaction between SIRT1 and p53. Adult pregnant dams were given regular drinking water or Cr(VI) (10 ppm potassium dichromate in drinking water, ad libitum), and treated with SIRT1 inhibitor, EX-527 (50 mg/kg body weight, i.p.,), during 9.5 − 14.5 days post-coitum. On postnatal day-1, ovaries from F1 offspring were collected for various analyses. Results indicated that Cr(VI) increased germ cell and somatic cell apoptosis, upregulated acetyl-p53, activated the apoptotic pathway, and inhibited cell survival pathways. Cr(VI) decreased acetyl-p53-SIRT1 co-localization in the ovary. In an immortalized rat granulosa cell line SIGC, Cr(VI) inhibited the physical interaction between SIRT1 and acetyl-p53 by altering the p53:SIRT1 ratio. EX-527 exacerbated Cr(VI)-induced mechanisms. The current study shows a novel mechanism for Cr(VI)-induced apoptosis in the ovary, mediated through the p53-SIRT1 network, suggesting that targeting the p53 pathway may be an ideal approach to rescue ovaries from Cr(VI)-induced apoptosis.

Different roles of cAMP/PKA and PKC signaling in regulating progesterone and PGE2 levels in immortalized rat granulosa cell cultures

Follicular cells from various species secrete steroids and prostaglandins, which are crucial for reproduction, in response to gonadotropins. Here, we examined prostaglandin E2 (PGE2) secretion from immortalized rat granulosa cells derived from preovulaotry follicles expressing the rat follicle stimulating hormone receptor (denoted as FSHR cells) that produce progesterone in response to gonadotropins. The cells were stimulated with a) pregnant mare’s serum gonadotropin (PMSG; a rat FSH receptor agonist), b) activators of the protein kinase A (PKA) pathway (forskolin and a cell permeable cAMP analog Dibutyryl-cAMP (DB-cAMP)) and c) protein kinase C (PKC) (12-O-tetradecanoylphorbol 13-acetate; TPA), alone and in combination for 24 h. Thereafter, PGE2 and progesterone levels in the culture media were determined. In accordance with previous studies, while PMSG and the PKA pathway activators induced progesterone accumulation in the media, TPA did not. In contrast, our data indicate that TPA, but neither PMSG, forskolin and DB-cAMP evoked PGE2 accumulation in the media. Western Blot analysis of cell lysate showed a drastic TPA induced increase of COX-2 levels, which was not seen with neither PMSG nor forskolin treatment. This association between the COX-2 and PGE2 levels suggests that the enzyme activity is the likely factor that determines the synthesis and levels of the prostaglandin in the culture media of the granulosa-derived cells. The addition of the PKA inhibitor H-89 to the FSHR cultures suppressed the gonadotropin and forskolin induction of progesterone secretion. Incubation in the presence of GF109203X (a PKC inhibitor) attenuated the TPA induced PGE2 accumulation in the culture media of the cells (a dose dependent reduction of 40–70%). In addition, while TPA inhibited the PMSG and forskolin induced-accumulation of progesterone in the media, the gonadotropin and forskolin inhibited the elevation of PGE2 levels evoked by TPA (a dose dependent decrease of 35–55%). These data suggest that cAMP/PKA and PKC signaling have opposite effects on PGE2 and progesterone synthesis in FSHR cells. We propose that this PKA and PKC interplay on progesterone and PGE2 may be advantageous for the coordination of these key mediators for successful ovulation and luteinization.

Progesterone receptor membrane component-1 (PGRMC1) and PGRMC-2 interact to suppress entry into the cell cycle in spontaneously immortalized rat granulosa cells.

Progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 are expressed in rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs) but their biological roles are not well defined. The present studies demonstrate that depleting either Pgrmc1 or Pgrmc2 in SIGCs increases entry into the cell cycle but does not increase cell proliferation. Rather, PGRMC1 and/or PGRMC2-deplete cells accumulate in metaphase and undergo apoptosis. Because both PGRMC1 and PGRMC2 localize to the mitotic spindle, their absence likely accounts for cells arresting in metaphase. Moreover, pull-down assays, colocalization studies and in situ proximity ligation assays (PLA) indicate that PGRMC1 binds PGRMC2. Disrupting the PGRMC1:PGRMC2 complex through the use of siRNA or the cytoplasmic delivery of a PGRMC2 antibody increases entry into the cell cycle. Conversely, overexpressing either PGRMC1-GFP or GFP-PGRMC2 fusion protein inhibits entry into the cell cycle. Subsequent studies reveal that depleting PGRMC1 and/or PGRMC2 reduces the percentage of cells in G0 and increases the percentage of cells in G1. These observations indicate that in addition to their role at metaphase, PGRMC1 and PGRMC2 are involved in regulating entry into the G1 stage of the cell cycle. Interestingly, both PGRMC1 and PGRMC2 bind GTPase-activating protein-binding protein 2 (G3BP2) as demonstrated by pull-down assays, colocalization assays, and PLAs. G3bp2 siRNA treatment also promotes entry into the G1 stage. This implies that dynamic changes in the interaction among PGRMC1, PGRMC2, and G3BP2 play an important protein regulating the rate at which SIGCs enter into the cell cycle.

Enhancement of Cell Surface Expression and Receptor Functions of Membrane Progestin Receptor α (mPRα) by Progesterone Receptor Membrane Component 1 (PGRMC1): Evidence for a Role of PGRMC1 as an Adaptor Protein for Steroid Receptors

A variety of functions have been proposed for progesterone receptor membrane component 1 (PGRMC1), including acting as a component of a membrane progestin receptor and as an adaptor protein. Here we show that stable overexpression of human PGRMC1 in nuclear progesterone receptor (PR)-negative breast cancer cell lines causes increased expression of PGRMC1 and membrane progesterone receptor α (mPRα) on cell membranes that is associated with increased specific [(3)H]progesterone binding. The membrane progestin binding affinity and specificity were characteristic of mPRα, with a Kd of 4.7 nM and high affinity for the mPR-specific agonist, Org OD 02-0, and low affinity for corticosteroids. Progestin treatment caused activation of G proteins, further evidence for increased expression of functional mPRs on PGRMC1-transfected cell membranes. Immunocytochemical and coimmunoprecipitation studies showed a close association of PGRMC1 with mPRα in cell membranes. Transfection of PGRMC1 into spontaneously immortalized rat granulosa cells was associated with membrane expression of PGRMC1 and mPRα as well as antiapoptotic effects of progestins that were abolished after cotransfection with small interfering RNA for mPRα. These data demonstrate that PGRMC1 can act as an adaptor protein, transporting mPRα to the cell surface, and that the progestin binding and apoptotic functions previously ascribed to PGRMC1 are dependent on cell surface expression of mPRα. Collectively, the results suggest PGRMC1 and mPRα are components of a membrane progesterone receptor protein complex. Increased expression of estrogen receptor β was also observed in the membranes of PGRMC1-transfected cells, suggesting that PGRMC1 can act as an adaptor protein for multiple classes of steroid receptors.

Cross Talk Between Estradiol and mTOR Kinase in the Regulation of Ovarian Granulosa Proliferation

Treatment of ovarian granulosa cells and follicles with the mammalian target of rapamycin (mTOR) kinase inhibitor results in biphasic effects where nanomolar rapamycin (RAP) results in reduced proliferation, mitotic anomalies, and attenuated follicle growth, while the picomolar RAP results in accelerated follicle growth. Here, we tested whether such effects are specific to RAP or could be mimicked by 2 alternative mTOR inhibitors, everolimus (EV) and temsirolimus (TEM), and whether these effects were dependent on the presence of estradiol (E2). Spontaneously immortalized rat granulosa cells (SIGCs) were cultured in dose curves of RAP, EV, TEM, or vehicle with or without E2. Proliferation and phosphorylation of mTOR targets p70S6 kinase and 4E-binding protein (BP) were determined. Cell cycle gene array analysis and confirmatory quantitative reverse transcriptase polymerase chain reaction were performed upon cells treated with picomolar RAP versus controls. Nanomolar RAP, EV, and TEM reduced SIGC proliferation and decreased phospho-p70 and 4E-BP. Picomolar concentrations accelerated proliferation without affecting mTOR substrate phosphorylation. Acceleration of growth by picomolar inhibitor required E2. Picomolar drug treatment altered the transcription of cell cycle regulators, increasing Integrin beta 1 and calcineurin expression, and decreasing inhibin alpha, Chek1, p16ARF, p27/Kip1, and Sestrin2 expression. At nanomolar concentrations, mTOR inhibitors attenuated granulosa proliferation. Accelerated growth and alterations in cell cycle gene transcription found with picomolar concentrations required E2 within the intrafollicular concentration range. The low concentrations of inhibitors required to increase granulosa proliferation suggest a novel use to support the growth of ovarian follicles.

MTOR Controls Ovarian Follicle Growth by Regulating Granulosa Cell Proliferation

We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed.

Preantral Follicle Growth is Regulated by c-Jun-N-Terminal Kinase (JNK) Pathway

c-Jun N-terminal kinase (JNK) pathway has been shown to be essential for cell cycle progression and mitosis. We previously showed that this pathway is activated in mitotic granulosa cells of follicles from transitional to antral stages. In this study, we, therefore, aimed to investigate whether this signaling pathway has any effect on in-vitro growth of murine preantral follicles and granulosa cell cycle control. Two structurally different pharmacologic JNK inhibitors, SP600125 and AS601245, were used in the experiments. First their inhibitory concentrations were determined in granulosa cells by Western blot analysis. Then preantral follicles isolated from immature and adult C57BL/6 mice were cultured in matrigel and standard culture plates for 6 days with these inhibitors. Spontaneously immortalized rat granulosa cells (SIGCs) were first synchronized at G1/S and G2/M stages of cell cycle and then treated with JNK inhibitors. Cell cycle progression was analyzed with Bromodeoxyuridine (BrdU) assay and flow cytometry analysis. Both inhibitors significantly inhibited phosphorylation of c-Jun in granulosa cells at 25, 50, and 100 μmol/L concentrations. Isolated preantral follicles cultured with these inhibitors exhibited arrested growth in culture in a dose-dependent manner. Cell cycle analyses showed that both inhibitors impair the progression of cell cycle at S phase and G2/M transition of granulosa cells. These results suggest that JNK pathway is essential for in vitro growth of preantral follicle growth and regulates both S phase and G2/M stages of cell cycle in granulosa cells.

C-Jun N-terminal kinase (JNK) pathway controls granulosa cell mitosis: a novel mechanism in regulation of preantral follicle growth

OBJECTIVE: Molecular regulation of preantral follicle growth is not well elucidated. c-Jun N-terminal kinase (JNK) is a member of the MAP Kinase (MAPK) family which plays a role in cell growth and survival. We previously showed that JNK is essential for cell cycle progression, and that cJun, a downstream target of JNK, is activated in preantral follicles. Here we aimed to determine the role of JNK in preantral follicle development in vitro. To achieve this, we used two structurally different reversible inhibitors of JNK, SP600125 and AS601245. DESIGN: In vitro study. MATERIALS AND METHODS: Preantral follicles isolated from immature C57BL/6 mice were cultured in 3-D extracellular matrix (matrigel) for 6 days with/without SP600125 and AS601245 at 3 different doses. To specify the role of JNK in granulosa cell growth, spontaneously immortalized rat granulosa cells (SIGC) were synchronized and treated with SP600125 and AS601245. SIGC have similar characteristics to preantral follicle granulosa cells. Cell cycle progression was assessed by BrdU assay and FACS analysis. RESULTS: Both SP600125 and AS601245 caused a dose dependent arrest in follicle growth compared to controls after 6d (Table-1).Table 1Growth characteristics of preantral follicles culturedGROUPSNFollicle Diamater Mean ± [SEM] Day 0Follicle Diamater Mean ± [SEM] Day 6% Growth Mean ± [SEM]Control (DMSO)13120.6 ±1.4208.1 ± 7.472.1 ± 5.4ap<0.01bp<0.01cp<0.0001dp<0.01ep<0.01fp<0.0001SP600125 (25mM)10130.9 ± 11161.1±15.122.8 ± 1.4ap<0.01SP600125 (50mM)11125.8 ± 12.6141.1 ± 20.79.75 ± 6.0bp<0.01SP600125 (100mM)12113.6 ± 9.9103.1± 5.3-8.02 ± 4.1cp<0.0001AS601245 (25mM)9145.5 ± 9.6169.1 ± 13.216.2 ± 12.9dp<0.01AS601245 (50mM)10107.2 ± 19.4117.2 ± 23.89.23 ± 6.4ep<0.01AS601245 (100mM)10126.9 ± 10.7113.2 ± 9.910.5 ± 1.8fp<0.0001a,b,d,e p<0.01c,f p<0.0001 Open table in a new tab 17 and 21 % of cells treated with 50 μM SP600125 and AS601245 at S phase, respectively, were BrdU positive compared to 55 % of controls (p<0.001). Both inhibitors impaired cell cycle progression at G2/M in a dose dependent manner. Activation of the other members of MAPK (ERK and P38) was not blocked, indicating that the inhibition was specific to JNK. CONCLUSIONS: JNK controls preantral follicle growth by regulating S and G2/M phases of granulosa cell cycle. This may revolutionize our understanding of preantral folliculogenesis and lead to targeted approaches to ovulatory dysfunction and contraception.

Lactational exposure to hexavalent chromium delays puberty by impairing ovarian development, steroidogenesis and pituitary hormone synthesis in developing Wistar rats

Hexavalent chromium (Cr-VI) is used in a wide range of industries. Cr-VI from chromate industries and atmospheric emissions contribute to the Cr contamination in the environment. Cr is a reproductive metal toxicant that can traverse the placental barrier and cause a wide range of fetal effects including ovotoxicity. Therefore, the goal of this study was to investigate the basic mechanisms involved in Cr(VI)-induced ovotoxicity, and the protective role of vitamin C on ovarian follicular development and function in Cr(VI)-induced reproductive toxicity using both in vivo and in vitro approaches. Lactating rats received potassium dichromate (200 mg/L) with or without vitamin C (500 mg/L), through drinking water from postpartum days 1–21. During postnatal days (PND) 1–21 the pups received Cr(VI) via the mother's milk. Pups from both control and treatment groups were continued on regular diet and water from PND-21 onwards, and euthanized on PND-21, -45 and -65. Cr(VI) decreased steroidogenesis, GH and PRL, increased FSH and did not alter LH. Cr(VI) delayed puberty, decreased follicle number, and extended estrous cycle. Spontaneously immortalized rat granulosa cells were treated with 12.5 μM (IC 50) potassium dichromate for 12 and 24 h, with or without vitamin C pre-treatment. Cr(VI) decreased the mRNA expressions of StAR, SF-1, 17β-HSD-1, 17β-HSD-2, FSHR, LHR, ERα and ERβ. Vitamin C pre-treatment protected ovary and granulosa cells from the deleterious effects of Cr(VI) toxicity, both in vivo and in vitro. Therefore, Cr(VI) toxicity could be a potential risk to the reproductive system in developing females, and vitamin C plays a protective role against Cr(VI)-induced ovotoxicity.

The configuration of the alpha and beta subunit domains in single-chain bovine LH analogs influences the secretion and steroidogenic response

The gonadotrophins LH, FSH and human (h) CG are non-covalent heterodimers composed of a common alpha and the hormone-unique beta subunit. LH regulates the production of androgens and progestins in the follicle, and the levels of these steroids are critical for the ovarian function. Structural features of the gonadotrophins involved in the steroidogenic response of the ovary are not completely understood. As an approach to address how the topology of the ligand affects steroidogenesis we exploited the single-chain (SC) gonadotrophin methodology because manipulating the relative position of the tethered subunit domains in SC hCG analogs enabled to change in the conformation, secretion, receptor binding and adenylyl cyclase activity. We genetically engineered a SC bovine LH analog with a linker derived from the CTP domain of the hCGbeta subunit, NH2-alpha-CTP-LHbeta-COOH (denoted as alphaCTPLHbeta; AB configuration) and evaluated the secretion form transfected CHO cells and steroidogenesis in follicular derived cells in comparison to the variant NH2-LHbeta-CTP-alpha-COOH (LHbetaCTPalpha; BA configuration). The secretion of the analogs from CHO cells was quantitative, and that of alphaCTPLHbeta was more efficient than that of LHbetaCTPalpha The experiments suggested that both variants were N- and O- glycosylated, though the posttranslational modifications are likely to be non-identical in the AB and BA analogs. The analogs stimulated progesterone secretion by immortalized rat granulosa cells that express the rat LH receptor but the EC50 of alphaCTPLHbeta (AB orientation) was higher by 20 fold, as compared to LHbetaCTPalpha (BA). In primary cultures of bovine theca cells, alphaCTPLHbeta stimulated progesterone release with a reduced sensitivity (by at least 50 folds) and smaller magnitude over the basal levels (about 3 folds) relative to LHbetaCTPalpha. In contrast, the accumulation of androstenedione in the media of the same primary cultures appeared to be nearly identical. As a result, the androstenedione/progesterone ratio for the alphaCTPLHbeta analog was significantly increased relative to LHbetaCTPalpha (2-3 folds). This unequal response suggests a distinct regulation of progesterone and androstenedione biosynthesis. Our data demonstrate major differences in steroid balance following stimulation of the receptor with structural LH analogs and provide further insight into gonadotrophin regulation of ovarian steroid production.

Deciphering early folliculogenesis: Jun Amino Terminal Kinase (JNK) plays a key role in preantral follicle growth

Objective: The factors regulating the preantral follicle growth are largely unknown. Our preliminary data indicated that extracellular matrix (ECM) and integrin signaling pathways might play a role in preantral follicle growth (1). JNK is one of the key signaling molecules involved in integrin signaling. In this study, we sought to determine whether ECM and integrins regulate preantral ovarian follicle growth by signaling via JNK.Design: Controlled laboratory study involving rodent ovarian tissue, granulosa cells, and isolated preantral follicles.Materials and Methods: Ovarian sections from 8 cycling rats (4 months old) as well as spontaneously immortalized rat granulosa cells (SIGC) were stained with anti-phospho-c-Jun antibodies to determine the activation of c-Jun by JNK. Synchronized SIGC were treated with a JNK inhibitor, SP600125 (JNKi), and a FACS analysis was performed to determine progression through mitosis. To determine the role of JNK on preantral follicle development, preantral follicles were isolated by an enzymatic and mechanical dissection method from 3-week-old mice. They were then cultured in DMEM-F12 supplemented with 10% FBS, FSH, and growth factors for 9 days with or without 100uM JNKi. Follicle measurements were performed using an image analyzer every other day.Results: Phospho-c-jun was specifically expressed in mitotic granulosa cells of growing follicles (Fig. 1), and mitotic SIGC as shown by immunohistochemistry and immunofluorescence, respectively. JNKi arrested granulosa cell proliferation at G2/M phase in a dose-dependent manner as shown by FACS analysis (Fig. 2). Likewise, JNKi treatment resulted in the arrest and reversal of preantral follicle growth in vitro (Day 0: 125.2±6.5μm vs. day 9: 114.56±5.8μm, p < 0.001). Whereas in controls, follicles continued to grow (144.88±17.5μm vs. 164.34±19.1μm, p = 0.001). View Large Image Figure ViewerDownload (PPT)Conclusion: These data strongly suggest a key role for JNK in preantral ovarian follicle growth in a rodent model. Considered together with previous data (1), ECM and integrins may regulate preantral follicle growth via JNK. Furthermore, pharmacological manipulation of preantral follicle growth via JNK inhibitors or stimulators may bode well for future therapeutic applications and in vitro growth.1. Oktay et al. Biol Reprod 2000(63):457.Supported by: NIH HDO43339-01 (to K.O). Objective: The factors regulating the preantral follicle growth are largely unknown. Our preliminary data indicated that extracellular matrix (ECM) and integrin signaling pathways might play a role in preantral follicle growth (1). JNK is one of the key signaling molecules involved in integrin signaling. In this study, we sought to determine whether ECM and integrins regulate preantral ovarian follicle growth by signaling via JNK. Design: Controlled laboratory study involving rodent ovarian tissue, granulosa cells, and isolated preantral follicles. Materials and Methods: Ovarian sections from 8 cycling rats (4 months old) as well as spontaneously immortalized rat granulosa cells (SIGC) were stained with anti-phospho-c-Jun antibodies to determine the activation of c-Jun by JNK. Synchronized SIGC were treated with a JNK inhibitor, SP600125 (JNKi), and a FACS analysis was performed to determine progression through mitosis. To determine the role of JNK on preantral follicle development, preantral follicles were isolated by an enzymatic and mechanical dissection method from 3-week-old mice. They were then cultured in DMEM-F12 supplemented with 10% FBS, FSH, and growth factors for 9 days with or without 100uM JNKi. Follicle measurements were performed using an image analyzer every other day. Results: Phospho-c-jun was specifically expressed in mitotic granulosa cells of growing follicles (Fig. 1), and mitotic SIGC as shown by immunohistochemistry and immunofluorescence, respectively. JNKi arrested granulosa cell proliferation at G2/M phase in a dose-dependent manner as shown by FACS analysis (Fig. 2). Likewise, JNKi treatment resulted in the arrest and reversal of preantral follicle growth in vitro (Day 0: 125.2±6.5μm vs. day 9: 114.56±5.8μm, p < 0.001). Whereas in controls, follicles continued to grow (144.88±17.5μm vs. 164.34±19.1μm, p = 0.001). Conclusion: These data strongly suggest a key role for JNK in preantral ovarian follicle growth in a rodent model. Considered together with previous data (1), ECM and integrins may regulate preantral follicle growth via JNK. Furthermore, pharmacological manipulation of preantral follicle growth via JNK inhibitors or stimulators may bode well for future therapeutic applications and in vitro growth. 1. Oktay et al. Biol Reprod 2000(63):457. Supported by: NIH HDO43339-01 (to K.O).

Partial sequencing of the rat steroidogenic acute regulatory protein message from immortalized granulosa cells: regulation by gonadotropins and isoproterenol

Steroidogenic acute regulatory protein (StAR), a 30-kDa protein involved in the transport of cholesterol to inner mitochondrial membrane during stimulation of steroid hormone biosynthesis, has recently been cloned from human adrenals and MA-10 mouse Leydig tumor cells. We examined the regulation of StAR mRNA accumulation upon induction of steroidogenesis in immortalized rat granulosa cells. Granulosa cells were transfected with SV40 DNA alone (POGS5); with SV40 DNA and Ha- ras oncogene (POGRS1); with SV40 DNA, Ha- ras oncogene and LH CG receptor (GLHR15) or with FSH receptor (GFSHR17) or with the β 2-adrenergic receptor (G β 2AR13) expression plasmids. Cells were cultured to confluency and then stimulated for 24 h with oFSH (4 nM), hCG (2.4 nM), isoproterenol (10 μM) or forskolin (50 μM). By quantitative RT-PCR, StAR mRNA was undetectable in non-steroidogenic cells (transfected with SV40 DNA alone, POGS5) either in the presence or in the absence of forskolin. In contrast, variable amount of the message was detected in all steroidogenic cell lines cotransfected with SV40 DNA and Ha- ras. Moreover, an increase in the StAR mRNA expression was evident in all steroidogenic cells upon stimulation with their respective agonists, concomitantly with enhanced progesterone production. The RT-PCR product was sequenced and the 379 base pairs of rat StAR were found to be 93% and 86% identical to mouse and human cDNA, respectively. The deduced 126 amino acid sequence was 95%, 88% and 88% identical to the mouse, human and bovine deduced protein sequences. We conclude that StAR message is expressed only in the steroidogenic rat granulosa cells and can be upregulated by FSH, hCG, isoproterenol and forskolin in the appropriate cell lines. In addition, we find that the rat StAR cDNA exhibit a high degree of homology with the mouse and human sequences.

Gap junctions in myometrial cell cultures: evidence for modulation by cyclic adenosine 3':5'-monophosphate.

Primary cultures of myometrial cells from juvenile rats, continuous cultures maintained by serial passage, and a pSV3neotransfected myometrial cell line were established and utilized for the study of development and modulation of gap junctional intercellular communication (GJIC) in vitro. The smooth muscle origin and homogeneity of the cultures were verified by immunofluorescence staining of alpha-smooth muscle actin and cellular desmin. Although gap junctions were not detected in thin sections of juvenile and adult myometrial tissues by transmission electron microscopy, they were detected in cultured myometrial cells derived from juvenile and adult animals. The presence of GJIC in cultured cells was confirmed using a fluorescence recovery after photo-bleaching assay. Administration of exogenous estradiol-17 beta (10(-7) M) resulted in an increase in GJIC in primary and passage 9 myometrial cultures, whereas pSV3neo-transfected myometrial cells were not significantly different from untreated controls. The lack of estrogen responsiveness in pSV3neo-transfected cultures correlated with lower levels of estrogen receptors than in primary cultures. Addition of 1 mM 8-bromo-cAMP resulted in rapid (within 2 min) increases in dye transfer in both control and estradiol-17 beta-primed primary cultures. Uncoupling of cells by treatment with 1 mM 1-octanol, followed by addition of 1 mM 8-bromo-cAMP, resulted in increased GJIC in control and estradiol-17 beta-primed cultures, although up-regulation of GJIC in estradiol-17 beta-primed cultures was much greater than in control cultures. Comparative experiments carried out on a spontaneously immortalized rat granulosa cell line (SIGC), which expresses the same connexin43 species as myometrial cells, exhibited similar responses to exogenous 8-bromo-cAMP following uncoupling of gap junctions with octanol. While the results of these investigations may not be extrapolated to myometrium in vivo, they suggest that myometrial cell culture may offer additional opportunities to explore the temporal expression and modulation of GJIC in myometrium.

Patulin-induced cellular toxicity: A vital fluorescence study

The mechanisms of patulin-induced cellular toxicity in an immortalized rat granulosa cell line were examined using several vital fluorescence bioassays. Monochlorobimane and 5-chloromethylfluorescein diacetate were used to monitor cellular glutathione (GSH) levels and revealed dose- and time-dependent depletion of GSH by patulin. A significant reduction in the fluorescence of the monochlorobimane-GSH conjugate by 0.1 μ m patulin was observed between 1 and 2 hr. Similar GSH depletion by the mycotoxin was also observed in parallel studies on a liver (Clone 9) and a renal (LLC-PK 1) cell line, although reduction of fluorescence occurred within 1 hr at the same dosage. Analysis of the electrical potential-dependent partitioning of rhodamine 123 into mitochondria also revealed significant effects of patulin within 1 hr at 0.1 μ m. An initial dose-dependent reduction in mitochondrial fluorescence was followed by loss of selective partitioning of the fluorophore into mitochondria at higher doses and/or a longer exposure of cells to patulin. The reduction in mitochondrial fluorescence was paralleled by a dose-dependent decrease in intracellular pH detected with 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Analysis of [Ca 2+] i with indo-1 and fluo-3 revealed a significant dose-dependent influx of Ca 2+ at 10 μ m and an alteration of the pattern of ionomycin-induced Ca 2+ influx at 1.0 μ m following patulin treatment. A carboxy-fluorescein fluorescence photobleaching assay was used to examine the effects of patulin on gap junction-mediated intercellular communication. Dose-dependent reduction in intercellular communication was observed within 2 hr with 1.0 μ m patulin. These observations indicate that the fluorescence assays used in this study provide a sensitive index of toxicity caused by exposure to patulin. Further, the toxic effects of patulin may involve direct effects on cellular glutathione levels and mitochondrial function in addition to direct effects on the plasma membrane.

Reduced cell-cell communication between mitotic and nonmitotic coupled cells

The effects of mitosis on gap junctional intercellular communication (GJIC) were quantified in a clonal cell line of spontaneously immortalized rat granulosa cells (SIGC) using a fluorescence recovery after photobleaching assay. Reduction of GJIC was associated with the process of mitosis and was first apparent at the onset of prophase. Resumption of GJIC between newly divided cells and surrounding cells occurred slowly, requiring several hours following cytokinesis before reestablishment of maximal rates. Mitotic rates of GJIC in SIGC were comparable to values obtained in interphase cells partially uncoupled by 0.5 m M octanol. Limited studies of other cell lines generalized the mitotic-associated reduction of GJIC observed in SIGC. The data suggest that mitosis is one process which alters GJIC. This could be of significance when there is a change in the rate of proliferation, such as in the acquisition of immortalization, an early stage of transformation.