Temporary work and depressive symptoms in South Korean workers.

BACKGROUND: In many countries, including South Korea, labour market changes have led to an increase in unstable, temporary jobs. There is evidence that workers in such jobs may experience poorer mental health than those in more stable employment. AIMS: To investigate the association between temporary employment and depressive symptoms in South Korean workers. METHODS: We analysed data from the 2010-2014 Korean Welfare Panel Study (KOWEPS). Employment type was categorized into workers paid per day of labour (day labourers), those on short-term contracts (fixed-term workers) and permanent workers. The association between employment type and depressive symptoms, measured using the Center for Epidemiological Studies Depression scale (CES-D 11), was examined using the generalized estimating equation model. RESULTS: A total of 3756 workers aged 20-59 were included in the 2010 baseline population. Day labourers had the highest mean CES-D 11 score, followed by fixed-term workers and permanent workers. With the day labourer group as reference, fixed-term workers (ß: -1.5027, P < 0.001) and permanent workers (ß: -2.1848, P < 0.001) showed statistically significant decreases in depression scores. CONCLUSIONS: Compared with day labourers, fixed-term workers and permanent workers had progressively lower depression scores. The findings of this study suggest that mental health inequalities based on employment type exist in South Korea.

Nonrigid PET motion compensation in the lower abdomen using simultaneous tagged-MRI and PET imaging.

PURPOSE: We propose a novel approach for PET respiratory motion correction using tagged-MRI and simultaneous PET-MRI acquisitions. METHODS: We use a tagged-MRI acquisition followed by motion tracking in the phase domain to estimate the nonrigid deformation of biological tissues during breathing. In order to accurately estimate motion even in the presence of noise and susceptibility artifacts, we regularize the traditional HARP tracking strategy using a quadratic roughness penalty on neighboring displacement vectors (R-HARP). We then incorporate the motion fields estimated with R-HARP in the system matrix of an MLEM PET reconstruction algorithm formulated both for sinogram and list-mode data representations. This approach allows reconstruction of all detected coincidences in a single image while modeling the effect of motion both in the emission and the attenuation maps. At present, tagged-MRI does not allow estimation of motion in the lungs and our approach is therefore limited to motion correction in soft tissues. Since it is difficult to assess the accuracy of motion correction approaches in vivo, we evaluated the proposed approach in numerical simulations of simultaneous PET-MRI acquisitions using the NCAT phantom. We also assessed its practical feasibility in PET-MRI acquisitions of a small deformable phantom that mimics the complex deformation pattern of a lung that we imaged on a combined PET-MRI brain scanner. RESULTS: Simulations showed that the R-HARP tracking strategy accurately estimated realistic respiratory motion fields for different levels of noise in the tagged-MRI simulation. In simulations of tumors exhibiting increased uptake, contrast estimation was 20% more accurate with motion correction than without. Signal-to-noise ratio (SNR) was more than 100% greater when performing motion-corrected reconstruction which included all counts, compared to when reconstructing only coincidences detected in the first of eight gated frames. These results were confirmed in our proof-of-principle PET-MRI acquisitions, indicating that our motion correction strategy is accurate, practically feasible, and is therefore ready to be tested in vivo. CONCLUSIONS: This work shows that PET motion correction using motion fields measured with tagged-MRI in simultaneous PET-MRI acquisitions can be made practical for clinical application and that doing so has the potential to remove motion blur in whole-body PET studies of the torso.

Bacterial motility: membrane topology of the Escherichia coli MotB protein.

The MotB protein of Escherichia coli is an essential component of the force generators that couple proton movement across the cytoplasmic membrane to rotation of the flagellar motors. The membrane topology of MotB was examined to explore the possibility that it might form a proton channel. MotB--alkaline phosphatase fusion proteins were constructed to identify likely periplasmic domains of the MotB molecule. Fusions distal to a putative membrane-spanning segment near the amino terminus of MotB exhibited alkaline phosphatase activity, indicating that an extensive carboxyl-terminal portion of MotB may be located on the periplasmic side of the membrane. Protease treatment of MotB in spheroplasts confirmed this view. The simple transmembrane organization of MotB is difficult to reconcile with a role as a proton conductor.

CDX2 promotes anchorage-independent growth by transcriptional repression of IGFBP-3.

CDX2 is a Drosophila caudal-related homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. We have reported that CDX2 promotes tumorigenicity in a subset of human colorectal cancer cell lines. Here, we present evidence that CDX2 negatively regulates the well-documented growth inhibitor insulin-like growth factor binding protein-3 (IGFBP-3). Specifically, CDX2 binds to the IGFBP-3 gene promoter and can repress IGFBP-3 transcription, protein expression and secretion. Furthermore, inhibition of IGFBP-3 partially rescues the decreased anchorage-independent growth phenotype observed in CDX2 knockout cells. These data demonstrate for the first time that (1) CDX2 can function as a transcriptional repressor, and (2) one mechanism by which CDX2 promotes anchorage-independent growth is by transcriptional repression of IGFBP-3.

CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48.

CDX2 is a Drosophila caudal-related homeobox transcription factor that is important for the establishment and maintenance of intestinal epithelial cells. CDX2 is a marker of colon cancer, with strong staining in up to 90% of colonic adenocarcinomas. CDX2 heterozygous-null mice develop colonic neoplasms, which have suggested that CDX2 is a tumor suppressor. However, CDX2 has not been reported to affect xenograft growth. Furthermore, CDX2 is rarely mutated in colon cancer, which has led to suggestions that it may play only a minor role as a tumor suppressor in colon cancer. To understand the functional contributions of CDX2 to colon cancer, we disrupted CDX2 in LOVO and SW48 human colon cancer cell lines by targeted homologous recombination. Consistent with the literature, disruption of CDX2 enhanced anchorage-dependent cell proliferation. However, homozygous loss of CDX2 led to significant inhibition of anchorage-independent growth in LOVO cells, and cell lethality in SW48 cells. Further analyses revealed that disruption of CDX2 led to anchorage-independent G1 to S growth arrest and anoikis. In vivo xenograft studies confirmed that disruption of CDX2 inhibited LOVO tumor growth. These data demonstrate that CDX2 mediates anchorage-independent growth and survival. Thus, CDX2 has tumorigenic potential in the human colon cancer cell lines LOVO and SW48.

Identification and characterization of a 19q12 amplicon in esophageal adenocarcinomas reveals cyclin E as the best candidate gene for this amplicon.

Genomic DNA amplification in tumors is frequently associated with an increased gene copy number of oncogenes or other cancer-related genes. We have used a two-dimensional whole-genome scanning technique to identify gene amplification events in esophageal adenocarcinomas. A multicopy genomic fragment from a tumor two-dimensional gel was cloned, and genomic amplification encompassing this fragment was confirmed by Southern blot analysis. The corresponding DNA sequence was matched by BLAST to a BAC contig, which allowed the use of electronic-PCR to localize this amplicon to 19q12. Sequence tagged site-amplification mapping, an approach recently implemented in our laboratory (Lin, L. et al., Cancer Res., 60: 1341-1347,2000), was used to characterize the amplicon. Genomic DNA from 65 esophageal and 11 gastric cardia adenocarcinomas were investigated for 19q12 amplification using quantitative PCR at 11 sequence tagged site markers neighboring the cloned fragment. The amplicon was narrowed from >8 cM to a minimal critical region spanning <0.8 cM, between D19S919 and D19S882. This region includes the cyclin E gene. Fourteen expressed sequence tags (ESTs) covering the minimal region were then assayed for potential gene overexpression using quantitative reverse transcription-PCR. Seven of the selected ESTs were found to be both amplified and overexpressed. Among these seven ESTs, cyclin E showed the highest frequency of gene amplification and overexpression in the tumors examined, which allowed us to finalize the core-amplified region to <300 kb. These results indicate that cyclin E is the likely target gene selected by the amplification event at 19q12. The fact that cyclin E overexpression was found only in the amplified tumors examined indicates that gene amplification underlies the cyclin E gene overexpression. Our study represents the first extensive analysis of the 19q12 amplicon, and is the first to physically map the core-amplified domain to a region of <300 kb that includes cyclin E. Amplification of 19q12 was found neither in the 28 esophageal squamous cancers nor in the 39 lung adenocarcinomas examined but was observed in 13.8% of esophageal and 9.1% of gastric cardia adenocarcinomas.

Blocking BRE expression in Leydig cells inhibits steroidogenesis by down-regulating 3beta-hydroxysteroid dehydrogenase.

Conversion of cholesterol to biologically active steroids is a multi-step enzymatic process. Along with some important enzymes, like cholesterol side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD), several proteins play key role in steroidogenesis. The role of steroidogenic acute regulatory (StAR) protein is well established. A novel protein, BRE, found mainly in brain, adrenals and gonads, was highly expressed in hyperplastic rat adrenals with impaired steroidogenesis, suggesting its regulation by pituitary hormones. To further elucidate its role in steroidogenic tissues, mouse Leydig tumor cells (mLTC-1) were transfected with BRE antisense probes. Morphologically the BRE antisense cells exhibited large cytoplasmic lipid droplets and failed to shrink in response to human chorionic gonadotropin. Although cAMP production, along with StAR and P450scc mRNA expression, was unaffected in BRE antisense clones, progesterone and testosterone yields were significantly decreased, while pregnenolone was increased in response to human chorionic gonadotropin stimulation or in the presence of 22(R)OH-cholesterol. Furthermore, whereas exogenous progesterone was readily converted to testosterone, pregnenolone was not, suggesting impairment of pregnenolone-to-progesterone conversion, a step metabolized by 3beta-HSD. That steroidogenesis was compromised at the 3beta-HSD step was further confirmed by the reduced expression of 3beta-HSD type I (3ss-HSDI) mRNA in BRE antisense cells compared with controls. Our results suggest that BRE influences steroidogenesis through its effects on 3beta-HSD action, probably affecting its transcription.

Wilms' tumor protein WT1 as an ovarian transcription factor: decreases in expression during follicle development and repression of inhibin-alpha gene promoter.

WT1, a gene deleted in some Wilms' tumors, encodes a transcription factor with zinc fingers and shares homology with proteins in the early growth response gene family. Although defects in the WT1 gene are associated with nephroblastoma and genitourinary malformation, the specific function of WT1 in the gonads remains unclear. We investigated the expression of WT1 transcripts in rat ovary during follicle development by Northern blotting, RNase protection assay, and in situ hybridization. Abundant WT1 transcripts were found in the ovary, testis, uterus, and kidney, with lower levels in the heart and pancreas. Treatment with estrogen or gonadotropins did not affect the concentration of ovarian WT1 mRNA. In situ hybridization analysis indicated that ovarian WT1 mRNA is expressed exclusively in the surface epithelium and granulosa cells of primordial, primary, and secondary follicles, and its levels decrease during follicle growth. Although RNase protection assay suggested the presence of four alternatively spliced forms of WT1 mRNA, the ratio of these transcripts remains constant during ovarian growth. Developmental changes in the expression of two granulosa cell differentiation marker genes, inhibin-alpha and FSH receptor, were found to be inversely correlated with WT1 levels. Because potential WT1-binding sites were found in the promoter of inhibin-alpha gene, we further tested whether WT1 might regulate the expression of this gene. Cotransfection of a WT1 expression vector with a promoter reporter plasmid of inhibin-alpha resulted in the repression of promoter activities in CHO cells in a dose-dependent manner. These results suggest that WT1 is expressed in high levels in granulosa cells of primordial, primary, and secondary follicles but decreases with follicle development. This transcription factor might be a repressor of ovarian differentiation genes in the granulosa cells and play a role in arresting the differentiation of immature follicles.

Interaction of SecB with intermediates along the folding pathway of maltose-binding protein.

SecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative. In order to better understand the nature of the binding, we have examined the interaction of SecB with intermediates along the folding pathway of maltose-binding protein. Taking advantage of forms of maltose-binding protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state.

Interleukin-1 beta suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production.

A growing body of evidence suggests that intraovarian interleukin-1 beta (IL-1 beta) may play an intermediary role in the ovulatory process. Furthermore, induction of nitric oxide (NO) by IL-1 beta has been reported in a wide variety of tissues. As the majority of ovarian follicles undergo an atretic degeneration process involving apoptotic cell death, we set out to determine whether IL-1 beta rescues follicles from apoptosis and the possible involvement of NO. Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 h in serum-free medium with or without hormone treatments. After culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends with [32P]dideoxy-ATP. Follicular NO production was also determined by a colorimetric method. Treatment with IL-1 beta dose-dependently suppressed the spontaneous onset of apoptosis in cultured follicles, but stimulated NO production. In contrast, the addition of IL-1 receptor antagonist eliminated both effects of IL-1 beta, confirming receptor mediation. Follicles treated with sodium nitroprusside, a NO generator or an analog of cGMP, the second messenger for NO, also showed decreased follicle apoptosis. Moreover, the addition of NG-monomethyl-L-arginine, a NO synthase inhibitor, reversed both IL-1 beta stimulation of NO production and suppression of apoptosis, suggesting a mediatory role of NO in these IL-1 beta effects. Gonadotropins also prevent follicle apoptosis. Of interest, treatment with hCG stimulated NO production, and the hCG suppression of follicle apoptosis and stimulation of NO production were partially blocked by cotreatment with IL-1 receptor antagonist, indicating the mediation of endogenous IL-1 beta. Treatment with IL-1 beta also stimulated a small increase in the production of cAMP, estrogen, and progesterone. Taken together, these findings suggest that IL-1 beta is a survival factor for ovarian follicles, and its action is partially mediated via NO and cGMP generation. Moreover, part of the suppressive action of gonadotropins on follicle apoptosis is mediated by endogenously produced IL-1 beta.

Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles.

In the mammalian ovary, only a small fraction of follicles fully mature and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian mediators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell types, and the apoptotic effect of TNF alpha is, partially, coupled to the sphingomyelin signaling pathway with ceramide as a second messenger. Because TNF alpha has been localized in the rat ovary, and TNF alpha treatment increases granulosa cell ceramide production, we studied the effect of treatment with TNF alpha and ceramide on follicle apoptosis. Immature rats were implanted with diethylstilbestrol to stimulate the development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treatments. During culture, spontaneous follicle apoptosis occurred (10-fold increase in DNA fragmentation), which was partially blocked by 100 ng/ml FSH (60% suppression). The effect of FSH was counteracted by TNF alpha in a dose-dependent manner, with the maximal effect at 100 ng/ml TNF alpha (90% reversal of FSH action). In situ analysis indicated that the granulosa cell is the follicle cell type undergoing DNA fragmentation. A membrane-permeable ceramide analog, C2-ceramide N-acetyl sphingosine, mimicked the effect of TNF alpha and was able to completely abolish the action of FSH at 50 microM. In contrast, another ceramide analog, C2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To study the mechanism of TNF alpha and ceramide action, the effect of sodium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-converting enzyme/ced-3-related cystine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with ATM (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating follicle apoptosis. Treatment with either TNF alpha or ceramide increased both basal and FSH-stimulated progesterone production by cultured follicles. Concomitant treatment by ATM did not alter the stimulatory effect of TNF alpha or ceramide on progesterone production, ruling out nonspecific toxic effect of the inhibitor and indicating that the apoptotic and steroidogenic pathways are independent. In summary, treatment with TNF alpha or its second messenger, ceramide, stimulates apoptosis of early antral follicles in culture, suggesting a potential role for TNF alpha as an intraovarian regulator of follicle atresia by acting through the ceramide signaling pathway.

Hormonal regulation of apoptosis in early antral follicles: follicle-stimulating hormone as a major survival factor.

Hormonal regulation of apoptosis has been studied in cultured preovulatory follicles. Because early antral follicles are most vulnerable to undergo atretic degeneration under physiological conditions in vivo, the present studies were designed to investigate the hormonal regulation of apoptosis using in vitro culture of early antral follicles. Rats were implanted with diethylstilbestrol at 24 days of age to stimulate the development of early antral follicles, and ovaries were collected at day 27 of age. Early antral follicles were dissected and cultured (four per vial) for 24 h with or without hormonal treatments. After culture, DNA was extracted from follicles, and the degree of apoptotic DNA fragmentation was determined using 3'-end labeling and gel electrophoresis. In situ analysis of apoptotic DNA fragmentation revealed that granulosa cells in these follicles are the main cell type undergoing apoptosis. Follicles cultured in the absence of hormones showed a 12-fold increase in the level of apoptotic DNA fragmentation which was prevented by treatment with FSH in a dose-dependent manner (60% maximal suppression and apparent ED50 of 30 ng/ml). Similarly, treatment with (Bu)2cAMP also suppressed follicle apoptosis. Treatment with LH or human CG, however, minimally suppressed apoptotic DNA fragmentation (35% maximal suppression). Insulin-like growth factor-I (IGF-I) also suppressed apoptosis by 45%. Moreover, the suppressive effect of FSH on apoptosis was partially reversed by coincubation with IGF-binding protein-3, suggesting a potential mediatory role of endogenous IGF-I. However, recombinant bovine GH had no effect on follicle apoptosis despite its ability to stimulate IGF-I messenger RNA (mRNA) levels. Incubation of follicles with epidermal growth factor (EGF) and basic fibroblast growth factor maximally suppressed follicle apoptosis by only 32% and 42%, respectively. Ligand binding analysis indicated the minimal effectiveness of EGF on apoptosis in early antral follicles, as compared with its potent action in preovulatory follicles reported earlier, may be due to a 3.5 fold increase in EGF receptor concentration in the mature follicles. High doses (150 or 500 ng/ml) of interleukin-1beta also suppressed apoptosis by 48% whereas treatment with an NO generator, sodium nitroprusside, or a cyclic GMP analog suppressed apoptosis as effectively as that of FSH. Furthermore, treatment with activin resulted in a dose-related suppression of follicle apoptosis, reaching a maximal 40% suppression. In contrast, cotreatment of activin with its binding protein, follistatin, abolished this effect. Collectively, these data demonstrated a stage-dependent difference in the hormonal regulation of follicle apoptosis. Although FSH, LH/human CG, GH, IGF-I, EGF, basic fibroblast growth factor, and interleukin-1beta are all effective survival factors for preovulatory follicles, FSH is a major survival factor for early antral follicles, the stage during which a majority of follicle undergo atresia under physiological conditions.

Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor I.

Although the majority of ovarian follicles undergo atresia through a mechanism involving apoptotic cell death, in vivo studies concerning the hormonal regulation of atresia have been difficult due to the presence of heterogeneous population of follicles in the ovary. In the present study, the regulation of follicle apoptosis by gonadotropins, insulin-like growth factor I (IGF-I), and IGF-binding protein 3 (IGFBP-3) was examined using a serum-free culture of preovulatory follicles. Immature rats at 26 days of age received a single dose of PMSG. Two days later, the largest preovulatory follicles were collected for in vitro culture with or without hormones. After 24 h of culture, follicular apoptotic DNA fragmentation was analyzed by autoradiography of size-fractionated DNA labeled at 3'-ends by [32P]dideoxy-ATP. A spontaneous increase in apoptotic DNA fragmentation occurred after 24 h of culture in the absence of hormones, whereas treatment with human CG (hCG) or FSH suppressed follicular apoptosis in a dose-dependent manner, with 0.1 microgram/ml causing maximal suppression by 60-62%. Cotreatment with hCG and FSH had no additional effect. Like gonadotropins, treatment with IGF-I and insulin also suppressed the spontaneous onset of apoptosis, with IGF-I being more effective than insulin. Cotreatment with IGFBP-3 and hCG dose-dependently reversed the suppressive effect of hCG on apoptosis by 42%, suggesting a mediatory role of endogenously produced IGF-I. The addition of IGFBP-3 also blocked the suppressive action of IGF-I by 49%, whereas it did not affect the suppressive action of an IGF-I agonist or insulin. Treatment with IGFBP-3 alone had no effect on apoptotic DNA fragmentation. Estrogen and progesterone production by the cultured follicles were also analyzed by RIA. Gonadotropin treatment resulted in a marked stimulation of the production of both steroid productions. In contrast, treatment with IGF-I caused a small increase in estrogen but decreased progesterone production. Although treatment with IGFBP-3 alone decreased both estrogen and progesterone production, cotreatment with IGFBP-3 and hCG resulted in a slight decrease in estrogen production but an increase in progesterone production. Furthermore, IGFBP-3 did not affect IGF-I action on steroid production. To further substantiate the hypothesis that IGFBP-3 blocks the suppressive effect of hCG on apoptosis by neutralizing endogenously produced IGF-I, solution hybridization analysis was performed, and hCG treatment was shown to increase IGF-I messenger RNA levels in cultured follicles by 1.9-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Type 4 cyclic adenosine monophosphate-specific phosphodiesterases are expressed in discrete subcellular compartments during rat spermiogenesis.

The type 4 cAMP-specific phosphodiesterases (PDE4) are a family of closely related enzymes with similar catalytic domains and divergent amino- and carboxyl-terminus domains. Multiple PDE proteins with heterogeneous amino termini are derived from each gene. To understand the significance of this heterogeneity, the expression and localization of variants derived from PDE4A and PDE4D genes was investigated during spermatogenesis in the rat. RNase protection analysis with mRNA for testes at different ages of development showed that two transcripts (PDE4D1 and PDE4D2) are expressed at day 10 and 15 of age and become undetectable thereafter. An additional PDE4D transcript appears at day 30 and increased during testid maturation. This latter transcript codes for a long variant of the PDE4D gene and is expressed in germ cells as demonstrated by RNase protection with RNA from isolated pachytene spermatocytes and round spermatids. The presence of a corresponding PDE4D protein with a molecular mass of 98 kDa was established by immunoprecipitation and Western blot analysis with antibodies specific for PDE4D and by immunoaffinity chromatography purification of the 98 kDa variant from isolated germ cells. PDE4A transcripts were also expressed in pachytene spermatocytes and round spermatids. Two polypeptides encoded by these PDE4A transcripts were expressed in pachytene spermatocytes, reached a maximum in round spermatids, and declined thereafter. Immunofluorescence analysis demonstrated a localization of the PDE4D protein in the manchette and in a periacrosomal region of the developing spermatid, a localization confirmed by immunogold electron microscopy. Conversely, the PDE4A was mostly soluble in the cytoplasm of round spermatids. These data demonstrate that PDE4D and PDE4A variants are expressed at different stages and localized in distinct subcellular structures of developing spermatids. Different properties of the mRNAs derived from the two genes and localization signals are responsible for the temporal and spatial expression of the different PDE4 isoenzymes.

Characterization of the antiapoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) and the stimulation of its message by gonadotropins in the rat ovary.

The majority of ovarian follicles undergo atresia mediated by apoptosis. Bcl-2-related proteins act as regulators of apoptosis via the formation of dimers with proteins inside and outside the Bcl-2 family. Previous studies have identified BAD as a proapoptotic Bcl-2 family member expressed in the ovary. It is known that BAD phosphorylation induced by survival factors leads to its preferential binding to 14-3-3 and suppression of the death-inducing function of BAD. To identify ovarian binding partners for hypophosphorylated BAD, we performed a yeast two-hybrid screening of a rat ovary complementary DNA library using as bait a mutant BAD incapable of binding to 14-3-3. Screening of yeast transformants yielded positive clones encoding the rat ortholog of Mcl-1 (myeloid cell leukemia-1), an antiapoptotic Bcl-2 protein. Amino acid sequence analysis revealed that rat and human Mcl-1 showed a complete conservation of the Bcl-2 homology domains BH1, BH2, and BH3. In the yeast two-hybrid system, Mcl-1 binds to the hypophosphorylated mutant of BAD and interacts preferentially with different proapoptotic (Bax, Bak, Bok, Bik, and BOD) compared with antiapoptotic Bcl-2 family members (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, CED-9, and BHRF-1). Northern blot hybridization demonstrated expression of Mcl-1 transcripts of 2.3 and 3.7 kb in the ovary and diverse other rat tissues. In immature rats, PMSG treatment led to a transient increase in the 2.3-kb Mcl-1 transcript, peaking at 6 h after injection and returning to baseline levels after 24 h. Moreover, the same transcript was induced in the PMSG-primed preovulatory rat ovary 6 h after the administration of ovulatory doses of either hCG or FSH. In situ hybridization studies revealed that the gonadotropin stimulation of ovarian Mcl-1 message occurs in both granulosa and thecal cells. In conclusion, rat Mcl-1 was identified as an ovarian BAD-interacting protein and the message for the antiapoptotic Mcl-1 protein was induced after treatment with gonadotropins in granulosa and thecal cells of growing follicles.

Preantral ovarian follicles in serum-free culture: suppression of apoptosis after activation of the cyclic guanosine 3',5'-monophosphate pathway and stimulation of growth and differentiation by follicle-stimulating hormone.

Progression of preantral follicle development is essential to further follicle maturation and ovulation, but there are few models for studying the regulation of preantral follicle survival and growth. We have evaluated preantral follicle survival in vivo and in vitro, and have developed a serum-free rat follicle culture system that can be used to characterize the regulation of preantral follicle growth and differentiation. Analysis of ovarian cell DNA fragmentation during the first wave of follicle growth in the infantile rat indicated negligible apoptosis up to day 16 of age. However, a major increase in apoptosis was found by day 18, a time point associated with the appearance of large antral follicles. In situ analysis confirmed that apoptotic DNA fragments were limited to antral follicles. Culture of individual preantral follicles mechanically dissected from ovaries of 12- or 14-day-old rats in serum-free conditions led to major increases in follicle cell apoptosis, similar to that seen in cultures of antral and preovulatory follicles. In contrast to antral and preovulatory follicles, treatment of preantral follicles with gonadotropins or cAMP analogs did not prevent apoptosis. However, treatment with 8-bromo-cGMP or 10% serum suppressed apoptosis by 75% in cultured preantral follicles. In situ analysis identified granulosa cells as the cell type susceptible to apoptosis regulation. Taking advantage of the ability of the cGMP analog to suppress apoptosis, we evaluated the potential of FSH as a growth factor. In the absence of serum, FSH treatment for 48 h did not affect follicle size compared to controls; however, treatment with the cGMP analog together with FSH increased follicle diameter (13%; P < 0.01) and viable cells (2.4-fold; P < 0.01) compared to control values. Immunoblot analysis further indicated that the inhibin-alpha content of the cultured follicles was increased by treatment with the combination of FSH and 8-bromo-cGMP, demonstrating the induction of follicle cell differentiation during culture. Therefore, we demonstrated that activation of the cGMP pathway promotes the survival of cultured preantral follicles and that in the presence of alpha cGMP analog, FSH is a growth and differentiation factor for preantral follicles. The present serum-free follicle culture model system will be useful in further evaluation of the regulation of growth and differentiation of preantral follicles.

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide type I receptor messenger ribonucleic acid during ovarian follicle development in the rat.

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with considerable homology to vasoactive intestinal peptide, has been shown to be stimulated by gonadotropins in the ovary. The present studies further evaluated the cell-type specific expression and gonadotropin regulation of PACAP type I receptor (PACAPR) messenger RNA in immature rat ovaries and in cultured preovulatory follicles. Northern blot analysis of ovaries obtained from prepubertal rats revealed the increased expression of PACAPR during prepubertal development. The major cell types expressing PACAPR messenger RNA were granulosa cells of large preantral follicles. Treatment of immature rats with PMSG caused a decrease in ovarian PACAPR expression. In contrast, treatment with human (h) CG at 2 days after PMSG treatment stimulated ovarian PACAPR messenger RNA within 3-6 h in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and dose-dependent stimulation of PACAPR by gonadotropins in granulosa cells of preovulatory follicles. Moreover, RNase protection assay revealed that the short variant of ovarian PACAPR was the predominant form stimulated during prepubertal development and by gonadotropins. These results demonstrate the expression of PACAPR messenger RNA in granulosa cells of growing follicles and of preovulatory follicles stimulated by gonadotropins, and suggest that PACAP may play a role in the growth of developing follicles and in ovulation as an autocrine/paracrine factor.

Gonadotropin stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) messenger ribonucleic acid in the rat ovary and the role of PACAP as a follicle survival factor.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel neuropeptide with considerable homology to vasoactive intestinal peptide and GH-releasing hormone, exists in two biologically active forms, PACAP-38 and -27. The presence of PACAP in the ovary has been demonstrated, where it stimulates steroidogenesis and cAMP accumulation in cultured granulosa cells. In the present study, gonadotropin regulation of PACAP gene expression was examined in PMSG/human (h)CG-treated immature rat ovaries and cultured preovulatory follicles. Northern blot analysis of ovaries obtained from PMSG/hCG-treated immature animals revealed the transient induction of PACAP transcripts by hCG, reaching a maximum at 6 h. The major cell types expressing PACAP messenger RNA were granulosa cells of preovulatory follicles and some theca/interstitial cells. In preovulatory follicles cultured in serum-free medium, PACAP transcripts were transiently induced by LH and FSH, reaching a maximum 6-9 h after stimulation in granulosa cells but not in theca cells. Treatment with cycloheximide or alpha-amanitin abolished LH-induced PACAP transcripts, indicating that new protein synthesis and transcription are necessary. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-induced PACAP messenger RNA, and forskolin mimicked the LH action, implying the role of adenylate cyclase activation. In contrast, treatment with chelerythrine, an inhibitor of protein kinase C, and 2-O-tetradecanol-phorbol-13-acetate had no effect. We further tested the role of PACAP in follicle apoptosis using apoptotic DNA fragmentation analysis. Treatment with PACAP-38 suppressed follicle apoptosis in a dose-dependent manner. Moreover, the LH suppression of follicle apoptosis was partially blocked by cotreatment with PACAP-38 antagonist, indicating mediation by endogenous PACAP-38. These results suggest that PACAP, transiently induced by the gonadotropin surge, could be a local regulator of a number of events and may act as a follicle survival factor during the periovulatory period.

Preovulatory changes in ovarian expression of collagenases and tissue metalloproteinase inhibitor messenger ribonucleic acid: role of eicosanoids.

The preovulatory surge of gonadotropins activates a cascade of proteolytic enzymes resulting in the rupture of the follicular wall and the release of a fertilizable ovum during ovulation. In the rat the process is initiated by a rise in follicular tissue-type plasminogen activator, produced predominantly in granulosa cells. Recent studies revealed a preovulatory increase in ovarian collagenolytic activity in vivo and an increase in activatable collagenase in vitro. In view of the complicated control of mammalian collagenase synthesis and activity by local inhibitors and activators, we examined the expression of ovarian interstitial and type IV collagenases and tissue inhibitor of metalloproteinase (TIMP) mRNA after an ovulatory stimulus. Ovarian mRNA was isolated from immature PMSG-treated rats 3, 6, and 9 h after hCG stimulation. Northern blot analyses revealed a mRNA of 1.7 kilobases (kb) hybridizing with the human interstitial collagenase cDNA probe. The levels of this mRNA showed a 25-fold increase between 3-6 h after hCG stimulation. The human cDNA probe of collagenase IV hybridized with a mRNA of 3.1 kb, which showed only a 4-fold increase 9 h after hCG treatment. The interstitial collagenase mRNA was expressed in both granulosa cells of preovulatory follicles and the residual ovarian tissue, whereas the expression of collagenase IV mRNA was limited to the residual tissue. Inhibitors of eicosanoid synthesis, previously shown to block ovulation and the LH/hCG-induced rise in ovarian collagenolysis, suppressed the gonadotropic stimulation of interstitial collagenase mRNA, but slightly stimulated that of collagenase IV. The mouse cDNA probe of TIMP hybridized with a 0.9-kb mRNA, which was stimulated by hCG to reach a maximum (7- to 8-fold increase) between 6-9 h after stimulation. TIMP was expressed and stimulated in both the granulosa cells and the residual tissue. Inhibitors of eicosanoid synthesis did not affect the gonadotropic stimulation of TIMP mRNA. These data support the suggested role of interstitial collagenase in follicle rupture and the essential role of eicosanoids in the mediation of gonadotropic stimulation of interstitial collagenase production and action. The observed stimulation of TIMP mRNA expression by the gonadotropin and the lack of any effect of eicosanoid synthesis inhibitors on this action of LH/hCG offer an additional mechanism by which these inhibitors may block ovulation. Thus, the suppression of ovulation by inhibitors of eicosanoid synthesis may result from selective inhibition of interstitial collagenase expression and undisturbed gonadotropin-stimulated TIMP expression.

Expression of messenger ribonucleic acid for the antiapoptosis gene P11 in the rat ovary: gonadotropin stimulation in granulosa cells of preovulatory follicles.

P11, a member of the S100 family of calcium-binding proteins, has been shown to interact with BAD (Bcl-xL/Bcl-2-associated death promoter) in the yeast two-hybrid protein-protein interaction assay. Because overexpression of P11 dampens the proapoptotic activity of BAD in transfected cells, we tested the possibility that the expression of this antiapoptotic protein may be regulated by gonadotropins and other survival factors in the ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of P11 messenger RNA (mRNA) during prepubertal development in the theca cells of preantral and early antral follicles. Treatment of immature rats with PMSG did not affect P11 expression, whereas treatment of PMSG-primed rats with an ovulatory dose of human (h)CG stimulated ovarian P11 mRNA within 6-9 h in the granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time-dependent stimulation of P11 by gonadotropins. In addition, treatment of cultured preovulatory follicles with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-stimulated P11 mRNA, whereas treatment with forskolin, an adenylate cyclase activator, but not the protein kinase C activator, 2-O-tetradecanol-phorbal-13-acetate, mimicked the LH action, suggesting the role of adenylate cyclase activation in P11 expression. Treatment with other follicle survival factors, including the epidermal growth factor, the basic fibroblast growth factor, and interleukin-1beta, could also stimulate P11 expression in cultured preovulatory follicles. These results demonstrate the expression of P11 mRNA in theca cells of different-sized follicles and in granulosa cells of preovulatory follicles following gonadotropin stimulation, and suggest that P11 may mediate, at least partially, the survival action of gonadotropins during the ovulatory process.