Hypoxia-driven metabolic reprogramming of adipocytes fuels cancer cell proliferation.

Objective: Obesity increases the risk of certain cancers, especially tumours that reside close to adipose tissue (breast and ovarian metastasis in the omentum). The obesogenic and tumour micro-environment share a common pathogenic feature, oxygen deprivation (hypoxia). Here we test how hypoxia changes the metabolome of adipocytes to assist cancer cell growth. Methods: Human and mouse breast and ovarian cancer cell lines were co-cultured with human and mouse adipocytes respectively under normoxia or hypoxia. Proliferation and lipid uptake in cancer cells were measured by commercial assays. Metabolite changes under normoxia or hypoxia were measured in the media of human adipocytes by targeted LC/MS. Results: Hypoxic cancer-conditioned media increased lipolysis in both human and mouse adipocytes. This led to increased transfer of lipids to cancer cells and consequent increased proliferation under hypoxia. These effects were dependent on HIF1α expression in adipocytes, as mouse adipocytes lacking HIF1α showed blunted responses under hypoxic conditions. Targeted metabolomics of the human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes media revealed that culture with hypoxic-conditioned media from non-malignant mammary epithelial cells (MCF10A) can alter the adipocyte metabolome and drive proliferation of the non-malignant cells. Conclusion: Here, we show that hypoxia in the adipose-tumour microenvironment is the driving force of the lipid uptake in both mammary and ovarian cancer cells. Hypoxia can modify the adipocyte metabolome towards accelerated lipolysis, glucose deprivation and reduced ketosis. These metabolic shifts in adipocytes could assist both mammary epithelial and cancer cells to bypass the inhibitory effects of hypoxia on proliferation and thrive.

The histone demethylase KDM4B regulates peritoneal seeding of ovarian cancer.

Epithelial ovarian cancer (EOC) has poor prognosis and rapid recurrence because of widespread dissemination of peritoneal metastases at diagnosis. Multiple pathways contribute to the aggressiveness of ovarian cancer, including hypoxic signaling mechanisms. In this study, we have determined that the hypoxia-inducible histone demethylase KDM4B is expressed in ∼60% of EOC tumors assayed, including primary and matched metastatic tumors. Expression of KDM4B in tumors is positively correlated with expression of the tumor hypoxia marker CA-IX, and is robustly induced in EOC cell lines exposed to hypoxia. KDM4B regulates expression of metastatic genes and pathways, and loss of KDM4B increases H3K9 trimethylation at the promoters of target genes like LOXL2, LCN2 and PDGFB. Suppressing KDM4B inhibits ovarian cancer cell invasion, migration and spheroid formation in vitro. KDM4B also regulates seeding and growth of peritoneal tumors in vivo, where its expression corresponds to hypoxic regions. This is the first demonstration that a Jumonji-domain histone demethylase regulates cellular processes required for peritoneal dissemination of cancer cells, one of the predominant factors affecting prognosis of EOC. The pathways regulated by KDM4B may present novel opportunities to develop combinatorial therapies to improve existing therapies for EOC patients.

Cellular localization and hormonal regulation of inducible nitric oxide synthase in cycling mouse uterus.

Nitric oxide (NO), a potent and versatile free radical, is synthesized in macrophages and mast cells as well as in other types of cells by the inducible form of nitric oxide synthase (iNOS). In this study, cells containing iNOS were identified in the uteri of cycling mice by using a rabbit antibody generated to an iNOS-specific peptide. Macrophages were identified in semiserial sections of the same tissues with the monoclonal antibody, F4/80, and mast cells were identified by toluidine blue staining. In tissue sections of uteri obtained from mice in the four stages of the estrous cycle (8 to 11 mice per stage), iNOS immunoreactivity was strongest in diestrus-I uteri and weakest in diestrus-II uteri. Myometrial mast cells and endometrial epithelial cells were prominent locations of iNOS, and specific protein was also present in myometrial smooth muscle and macrophage-like cells in the endometrial stroma. Because cyclic variations suggested regulation of iNOS expression by ovarian steroid hormones, studies were done using ovariectomized mice. Seven days after ovariectomy, immunoreactive iNOS was low but detectable in mast cells and luminal epithelial cells. In the uteri of ovariectomized, estradiol-17 beta (E2)-treated mice, mast cells were iNOS+ after 24 h whereas epithelial cells were negative; the reverse was observed in progesterone (P4)-treated mice. Both mast cells and epithelial cells were iNOS+ in the uteri of mice that had received a combination of E2 + P4. These results indicate that several types of uterine cells produce iNOS and that expression of this enzyme in specific cell lineages is governed by ovarian steroid hormones. The data are consistent with the postulate that NO derived from uterine leukocytes and other types of cells plays a role in uterine cyclicity and preparation for pregnancy.

Alterations in follicle development, steroidogenesis, and gonadotropin receptor binding in a model of ovulatory blockade.

Immature female rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) before gonadotropin-induced follicle development and ovulation ovulate significantly fewer ova compared with controls. This study was designed to investigate potential ovarian-specific mechanisms of TCDD-mediated inhibition of ovulation. Immature hypophysectomized rats were treated with TCDD (32 microg/kg) or corn oil vehicle. Follicle development was initiated by injection of 10 IU PMSG 24 h after TCDD, and ovulation was induced 52 h after PMSG by injection of 10 IU hCG. The number of ova flushed from the oviduct was assessed the morning after hCG injection, and ovaries were collected at multiple times throughout the treatment schedule for histological analysis and analysis of FSH and hCG receptor binding and ovarian cAMP levels. In addition, serum levels of estradiol and progesterone were determined. Control rats ovulated 9.3 +/- 1.5 ova, whereas TCDD-treated rats ovulated 0.6 +/- 0.3. Gonadotropin receptor binding was evaluated 52 h after PMSG at the usual time of hCG injection to induce ovulation. Both FSH binding and hCG binding were significantly reduced in animals treated with TCDD. Serum estradiol levels in control animals were increased by 52 h after PMSG administration. In contrast, serum levels of estradiol in TCDD-treated animals remained low. In response to the ovulatory dose of hCG, serum levels of both estradiol and progesterone increased in control animals. Steroid levels also increased in TCDD-treated animals, but did not reach the peak levels observed in controls. TCDD treatment further resulted in lower ovarian cAMP levels at 52 h post-PMSG and at 5 h post-hCG. Together the data indicate that TCDD treatment altered the ability of the ovary to respond to PMSG, resulting in the development of follicles not comparable to controls (lower gonadotropin binding, lower estradiol production, lower levels of cAMP). It appears that critical steps in the development and maturation of follicles are disrupted by TCDD.

Tumor necrosis factor alpha alters follicular steroidogenesis in vitro.

Preovulatory follicles from cyclic proestrous rats were incubated in vitro in M199 for up to 24 hours with various doses of human tumor necrosis factor alpha (TNF). Stepwise increases in progesterone (P) production (20-80 ng/ml/2 follicle/24 hours) were observed with 30-300 pM TNF; 3000 pM TNF reduced (p less than 0.001) P production compared to 300 pM TNF but it was still higher (p less than 0.001) than controls. Follicular androstenedione production was inhibited by low doses of TNF (30-60 pM TNF) and stimulated by 3000 pM TNF. TNF did not alter follicular estradiol (E2) production. The time course studies using 300 pM TNF revealed that follicular P production did not increase significantly until 24 hours in culture unlike LH (160 pM) which increased P by 6 hours. Preincubation of 30 pM TNF with a monoclonal antibody to human recombinant TNF alpha prevented the increase in follicular P production observed at 24 hours in TNF-treated controls. These results indicate that in vitro TNF stimulates follicular P production but only after a lag period and thus provide a basis for future studies elucidating the role of TNF in follicular development.

Mouse endometrial tumor necrosis factor-alpha messenger ribonucleic acid and protein: localization and regulation by estradiol and progesterone.

Tumor necrosis factor-alpha (TNF) messenger RNA (mRNA) and protein have been identified in the uteri of cycling rats, mice, and women. In this study, a mouse model was used to investigate the cell-specific expression and regulation of the TNF gene in the endometrium. Uteri from cycling and ovariectomized hormone-reconstituted mice were tested by in situ and Northern blot hybridizations as well as by immunohistochemistry. Cyclic variations were observed in TNF mRNA. Although weak to undetectable during proestrus, estrus, and diestrus-I, TNF mRNA was present in both epithelial and stromal cells on diestrus-II. TNF protein was observed in the endometrium on each day of the estrous cycle, primarily in the epithelial cells. Stromal TNF immunoreactivity was observed only during diestrus-I. Seven days after ovariectomy, TNF mRNA and protein were undetectable in the endometrium. Specific message and protein were restored in both epithelial and stromal cells after administration of 17 beta-estradiol (E2), progesterone (P4), and E2 plus P4. E2 treatment resulted in a biphasic pattern of TNF mRNA expression. mRNA was present in epithelial cells 1 and 6 h posttreatment, was not detectable after 24 h, and then was present in both the epithelium and stroma after 72 h. In contrast, TNF mRNA was detectable at all time points after P4 administration. TNF mRNA was localized to both the epithelium and stroma until 72 h of P4 treatment, when stromal TNF mRNA was no longer detectable. TNF mRNA was also present at all time points examined after the combination E2 and P4 treatment. TNF mRNA was invariably localized to the epithelium; however, stromal mRNA fluctuated over the course of treatment. TNF mRNA was in the stroma 1 and 24 h post-E2 plus P4 treatment, but was not present at the 6 and 72 h points. In general, the pattern of TNF protein matched that of the TNF mRNA, with a few exceptions. Twenty-four hours after E2 treatment, TNF mRNA was not detectable; however, TNF protein was present in the epithelium. The opposite was observed 24 h after E2 plus P4 administration when the epithelium and stroma contained TNF mRNA; however, no protein was observed. In addition, with the exception of 72 h of E2 treatment, stromal TNF protein was not abundant, although TNF mRNA was often detected. In summary, the results of this study indicate that both epithelial and stromal cells contain TNF mRNA and protein and that E2 and P4 exert individual influences on the cell-specific expression of TNF transcripts and protein.

Effects of tumor necrosis factor-alpha in vitro on steroidogenesis of healthy and atretic follicles of the rat: theca as a target.

Tumor necrosis factor-alpha (TNF), a pleiotropic cytokine localized within the ovary, alters follicular steroidogenesis. Preovulatory follicles dissected from ovaries of normal cyclic adult rats on the morning of proestrus exhibit steroidogenic and histological signs of atresia after 24 h of culture under the conditions of 5% CO2 and air. Follicles cultured for 24 h in 5% CO2 and 95% O2 appeared histologically and steroidogenically healthy. Under both culture conditions, human recombinant TNF (5 ng/ml) significantly increased the production of pregnenolone, progesterone, 20 alpha-dihydroprogesterone, and 17 alpha-hydroxyprogesterone by the follicles. Follicles cultured in 5% CO2 and air exhibited no change in androstenedione or estradiol production compared to control follicles incubated without TNF. In contrast, follicles cultured in 5% CO2 and 95% O2 responded to TNF with increased androstenedione and estradiol production. Separation of the thecal and granulosa compartments indicated that the increased progestin production observed in the whole follicle in response to TNF originated from the theca. TNF significantly inhibited basal and FSH-stimulated progesterone production from the granulosa of preovulatory follicles. Exogenous substrate added to whole follicles cultured in the presence or absence of TNF indicated that TNF enhanced the conversion of 25-hydroxycholesterol to pregnenolone. These studies reveal that TNF enhanced steroidogenesis in both healthy and atretic follicles and that this action of TNF is on the theca, where TNF increases the conversion of cholesterol to pregnenolone. The data imply that TNF has differential effects on thecal and granulosa steroidogenesis.

Recapitulation of the pathway for trophoblast giant cell differentiation in vitro: stage-specific expression of members of the prolactin gene family.

The trophoblast giant cell lineage is characterized by endoreduplication and expression of members of the PRL gene family. This report describes the functional consequences following in vitro manipulation of a rat trophoblast cell line, termed Rcho-1. Rcho-1 cells can be cultured under conditions that promote proliferation or differentiation. Proliferation is maintained by culturing the cells in the presence of fetal bovine serum under subconfluent conditions. Differentiation is induced by growing the cells to confluence and removing the mitogenic source. Differentiation is characterized by continued synthesis of DNA in the absence of proliferation (endoreduplication) and the sequential expression of members of the PRL gene family. Western and Northern blot analyses demonstrated that placental lactogen-I (PL-I) was first expressed, followed sequentially by PL-II, PRL-like protein-A, and PRL-like protein-C. The ontogeny of expression of members of the PRL gene family by the Rcho-1 cells recapitulated the pattern of in situ expression by trophoblast giant cells of the junctional zone of the chorioallantoic placenta. A notable difference between in vivo trophoblast giant cell differentiation and in vitro Rcho-1 cell differentiation is the termination of PL-I expression in normal trophoblast giant cells developing in vivo and the continued expression of PL-I in differentiated Rcho-1 cell cultures. The Rcho-1 cell line provides a unique in vitro model for investigating the initiation and maintenance of the trophoblast giant cell differentiation pathway.

Identification and immunochemical characterization of a major placental secretory protein related to the prolactin-growth hormone family, prolactin-like protein-C.

Trophoblast cells of the rat chorioallantoic placenta synthesize and secrete a number of proteins structurally related to pituitary PRL. During the purification of one member of the placental PRL family, PRL-like protein-A (PLP-A), we identified a major contaminating protein with a similar mol wt but possessing a more acidic pI (5.9-6.1) and different immunoreactivities. After isolation by two-dimensional gel electrophoresis, the more acidic contaminating protein was electroeluted, and its N-terminal amino acid sequence was determined by gas phase sequencing. The N-terminal sequence showed considerable homology with members of the PRL family, including characteristic positioning of cysteine residues at amino acids 4 and 11. The newly identified protein species have been termed PLP-C based on their structural similarity with pituitary PRL. The protein was further characterized by the generation of specific immunological probes. Antibodies were generated to electrophoretically purified protein and to a chemically synthesized peptide representing amino acids 11-32 of its N-terminal sequence. Each antiserum specifically recognized two major species migrating at approximately 25 and 29 kDa, respectively. The 29-kDa species specifically bound to Concanavalin-A, while the 25-kDa species failed to bind to the lectin. Furthermore, the 29-kDa species could be converted to the 25-kDa species by enzymatic deglycosylation. The antisera have also been used to examine the cell- and temporal-specific patterns of expression. The immunoreactive protein species (25 and 29 kDa) were localized primarily to spongiotrophoblast cells present in the junctional zone of the chorioallantoic placenta. Expression was initiated after midgestation and increased during the remaining part of gestation. In summary, PLP-C is a major secretory protein produced by spongiotrophoblast cells during the second half of gestation.

Immunological evidence for a human ovarian tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF alpha) was localized to follicular and luteal compartments of the human ovary using a biotin strept-avidin immunocytochemical technique with polyclonal antibodies to human recombinant TNF alpha. Immunoreactive TNF alpha (I-TNF) was observed in granulosa cells of healthy antral and atretic follicles and appeared to be secreted by the granulosa cells, since it was present in the follicular fluid surrounding antral and degenerating granulosa. Preantral follicles did not exhibit I-TNF. I-TNF was in the cytoplasm of large granulosa-lutein cells and small paraluteal cells. Granulosa cells cultured on an Immobilon membrane exhibited I-TNF in the cytoplasm and I-TNF released onto the membrane, which appeared as a dark-red stain after immunolocalization. Granulosa cells cultured on plastic slides exhibited I-TNF throughout the cytoplasm. To determine if I-TNF was secreted by granulosa cells, granulosa cell-conditioned medium was examined for the presence of TNF by a double sandwich enzyme-linked immunosorbant assay using both monoclonal and polyclonal antibodies to human recombinant TNF alpha. Conditioned medium contained up to 800 pg TNF/500,000 cells after 24 h of culture. I-TNF was also found (100-170 pg/mL) in human follicular fluid. Immunoblot assay of I-TNF in cultured granulosa revealed approximately 240 pg TNF/250,000 cells. The results indicate that the human ovary contains I-TNF, and thus, a paracrine and/or autocrine role of TNF in human ovarian follicular function seems likely.

Trophoblast cell differentiation and organization: role of fetal and ovarian signals.

The rat chorioallantoic placenta is organized into two distinct zones (junctional and labyrinth) and expresses six members of the placental prolactin (PRL) family: placental lactogen-I (PL-I), PL-I variant (PL-Iv), PL-II, PRL-like protein-A (PLP-A), PLP-B, and PLP-C. These placental hormones are expressed in distinct cell- and temporal-specific patterns and can be used to monitor the state of differentiation of rat trophoblast cells. This study was initiated to examine the role of the fetus and maternal ovaries in the regulation of trophoblast cell differentiation and organization. Expression of the placental PRL family was monitored by Northern and Western blotting and immunocytochemical analysis. The roles of the fetus and maternal ovaries were examined by chemically induced fetal death (DFX, induced by intraamniotic injection of digoxin) and surgical removal of the ovaries (OVX), respectively. The endocrine differentiation of the placenta was assessed on day 19 of gestation (sperm positive = day 0, parturition = day 21). Day 10 of gestation was the earliest day that DFX could be reliably performed. Day 10 is a time point during pregnancy preceding the onset of expression of all members of the placental PRL family except PL-I. DFX on day 10 of gestation did not affect the endocrine differentiation of the trophoblast cells but did alter the organization of the chorioallantoic placenta. PL-II, PL-Iv, and the PLPs were all expressed in their appropriate molecular forms and cell types in placentas developing in the absence of fetal influence. The maternal ovaries, in the absence of the fetus, had two distinct actions on the placenta: ovarian signals were essential during a period at midgestation for the maintenance of the placenta and exposure of the developing placenta to ovarian signals during the second half of gestation, in the absence of the fetus, arrested labyrinth zone development. The active ovarian signals were progesterone and estrogen. We conclude that trophoblast cell differentiation occurs independent of the fetus and maternal ovaries. However, signals from both the fetus and maternal ovaries are required for normal organization of the chorioallantoic placenta.

Expression of the transporter for antigen processing-1 (Tap-1) Gene in subpopulations of human trophoblast cells.

Heterodimers of transporter for antigen processing proteins, Tap-1 and Tap-2, are essential components of the pathway that leads to expression of conventional HLA-A, -B class I transplantation antigens on cell surfaces. In this study, expression of the Tap-1 gene in trophoblast cells, some of which display novel and unconventional HLA class I molecules that include HLA-G and an HLA-C-like antigen, was investigated by using in situ hybridization to identify Tap-1 mRNA and immunohistochemistry to detect Tap-1 protein. The experiments were done on semiserial sections of paraformaldehyde-fixed tissues. In first trimester placentas, expression of the Tap-1 gene correlated with expression of HLA class I antigens in trophoblast cells. HLA-G/C positive extravillous cytotrophoblast cells exhibited high intensity in situ hybridization signals for Tap-1 mRNA and strong staining with anti-Tap-1 whereas Tap-1 gene products were rarely detected in HLA class I antigen negative syncytiotrophoblast and villous cytotrophoblast cells. Relationships were less definitive in term tissues. Although Tap-1 protein was detectable in extravillous cytotrophoblast cells (chorionic cytotrophoblast cells) as expected, HLA class I negative syncytiotrophoblast contained low intensity hybridization and immunostaining signals. Collectively, the data suggest that (1) as with conventional HLA class I antigens in other types of cells, the pathway leading to expression of novel HLA class I antigens in trophoblast cells includes transport of peptides by Tap-1, and that (2) deficiencies in Tap-1 might account in (whole or in) part for the failure of some trophoblast cells to express HLA class I antigens.

Differential responses of phenotypically distinct rat trophoblast cell lines to MHC class I antigen-inducing cytokines.

Phenotypically distinct rat trophoblast cell lines, the Rcho-1 and R8RP.3 cells, were compared for their responses to cytokines known to induce major histocompatibility (MHC) class I antigens, tumour necrosis factor (TNF), transforming growth factor-beta (TGF-beta), and interferon-gamma (IFN-gamma). Cell enzyme immunosorbent assays and flow cytometry experiments showed that only IFN-gamma could induce RT1 class I antigens on the Rcho-1 cells. Non-adherent cells were slightly less responsive than adherent, giant cell-like Rcho-1 cells. By contrast, RT1 class I antigens on the R8RP.3 cells were induced by both TGF-beta 1 and IFN-gamma. The cytokines also had different effects on mitochondrial enzyme activity in the two lines. TNF and TGF-beta 1 mRNAs were demonstrated in both lines by using Northern blot hybridization. Rcho-1 but not R8RP.3 cells contained two TNF messages (approximately 2.2, 1.9 kb). Steady state levels of transcripts from the TNF gene, and, to a lesser extent, the TGF-beta 1 gene, were increased in cultures of Rcho-1 cells that contained high proportions of giant cells. Thus, phenotypically distinct rat trophoblast cell lines do not respond identically to TNF, TGF-beta 1 or IFN-gamma, transcription of cytokine genes does not prevent the cells from responding to paracrine cytokine signals, and the cells contain novel TNF transcripts that might be important in cell maturation or differentiation.

Generation and characterization of antipeptide antibodies to rat cytochrome P-450 side-chain cleavage enzyme.

In this report, we describe the generation of immunologic probes to rat P-450scc. Two regions of the P-450scc amino acid sequence were identified (internal domain: amino acids 421-441; carboxy terminal domain: amino acids 509-526), chemically synthesized and used as immunogens in rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA) and Western blot analyses. Antisera were successfully generated to each of the P-450scc regions that recognized the entire 49 kDa rat P-450scc protein. Antiserum directed to the internal domain of P-450scc showed broad species crossreactivity, whereas antiserum directed to the carboxy terminal domain of P-450scc crossreacted with only rat and mouse. Both antisera were useful for Western blot and immunocytochemical analyses of rat P-450scc expression. In addition to recognizing the major 49 kDa P-450scc protein, each antiserum also recognized lower molecular weight species. Antiserum directed to the internal domain of P-450scc specifically recognized a 42 kDa species, whereas antiserum directed to the carboxy terminal domain specifically recognized an 8 kDa species. We hypothesize that the two lower molecular weight immunoreactive species are generated by proteolytic cleavage of rat P-450scc between the internal and carboxy terminal epitopes.

Female steroid hormones regulate production of pro-inflammatory molecules in uterine leukocytes.

Estrogens and progesterone could be among the environmental signals that govern uterine immune cell synthesis of pro-inflammatory substances. In order to investigate this possibility, we first mapped expression of the inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) genes in the leukocytes of cycling and pregnant mouse uteri, then tested the ability of estradiol-17 beta (E2) and progesterone to influence gene expression. Immunohistochemistry, in situ hybridization, and other experimental approaches, revealed that the iNOS and TNF-alpha genes are expressed in mouse uterine mast cells, macrophages and natural killer cells (uNK). Gene expression in each cell type was noted to be dependent upon stage of the cycle or stage of gestation, implying potential relationships with levels of female hormones and state of cell differentiation or activation. Further in vivo and in vitro experiments showed that individual hormones have cell type-specific effects on synthesis of iNOS and TNF-alpha that are exerted at the level of transcription. In uterine mast cells, iNOS and TNF-alpha are promoted by E2 whereas preliminary studies in macrophages suggest that transcription and translation of the two genes are unaffected by E2 but are inhibited by progesterone. Uterine NK cell production of iNOS and TNF-alpha is strongly related to cell differentiation, which is initiated and sustained by progesterone. Collectively, the results indicate that regulation of synthesis of pro-inflammatory molecules by hematopoietic cells in cycling and pregnant uterus comprises a new and potentially critical role for female steroid hormones.

A review of mechanisms controlling ovulation with implications for the anovulatory effects of polychlorinated dibenzo-p-dioxins in rodents.

Polychlorinated dibenzo-p-dioxins (PCDDs) can impinge on female fertility by preventing ovulation. In this review, the aspects of normal ovulatory physiology most relevant to our current understanding of PCDD action on the ovary are briefly reviewed. This is followed by a comprehensive assessment of data relevant to the effects of PCDDs during ovulation in the rat. PCDDs interrupt ovulation through direct effects on the ovary in combination with dysfunction of the hypothalamo-hypophyseal axis.

Recombinant porcine prorelaxin produced in Chinese hamster ovary cells is biologically active.

Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studied so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay.

Impaired ovulation by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) in immature rats treated with equine chorionic gonadotropin.

Several studies have established that 2,3,7,8 tetrachloro-p-dioxin (TCDD) blocks ovulation. The main purpose of this study was to determine if induced ovulation was delayed temporarily by TCDD. The ovulation model used was that of the gonadotropin-primed intact or hypophysectomized rat. Immature intact female Sprague-Dawley rats (IIR) were given 32 microg TCDD/kg by gavage on day 24 of age. The next day equine chorionic gonadotropin (eCG) (5 IU) was injected sc to stimulate follicular development. The number of ova in the oviducts, the ovulation rate, and steroid concentrations were determined at 72, 96, 120, and 144 h after eCG. Immature female Sprague-Dawley rats (IHR) were hypophysectomized on day 23 of age. On day 26, the IHR were given 20 microg TCDD/kg by gavage. The next day eCG (10 IU) was injected sc to stimulate follicle development and at 52 h after eCG, 10 IU human chorionic gonadotropin (hCG) was given to induce ovulation. The same parameters as in IIR were determined in IHR at 72, 96, and 120 h after eCG. TCDD decreased body and ovarian weight gains in both IIR and IHR. In IIR, TCDD delayed ovulation by 24 to 48 h reducing the number of ova shed as well as the number of animals ovulating at 72 and 96 h after eCG. In IHR, however, TCDD reduced only the number of ova shed but caused no delay in ovulation. The IIR treated with TCDD had low levels of progesterone (P4) at 72 and 96 h after eCG but high levels of estradiol (E2) at the same time points. This sustained high level of E2 production coincided with a transient decrease in serum concentrations of androstenedione (A4). The alteration of steroid hormones by TCDD was restored to normal by 48 h after ovulation in IIR. Serum P4 concentration was not altered by TCDD in IHR at 72 h after eCG but was decreased thereafter. The delay in ovulation induced by TCDD in IIR indicates the disruption of the hypothalamus-pituitary-ovary axis during proestrus. The decrease in number of ova shed in IHR induced by exogenous gonadotropins indicates an additional direct ovarian effect of TCDD in blocking ovulation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro.

The main purpose of this study was to investigate the direct effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian function including ovulation and steroidogenesis. In vivo effects of TCDD were investigated on ovulation and alteration of circulating and ovarian steroid hormones in immature hypophysectomized rats (IHR) primed with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In addition, in vitro effects of TCDD on the steroidogenesis of granulosa cells (GC), theca-interstitial cells (TIC), and whole ovarian dispersates derived from the ovary of IHR were investigated. In the ovulation model, rats were hypophysectomized on Day 23 of age. On Day 26, the IHR were given 20 microg TCDD/kg by gavage. The next day eCG (10 IU) was injected sc to stimulate follicular development. Fifty-two hours after eCG, 10 IU hCG was given to induce ovulation. TCDD (20 microg/kg) blocked ovulation and reduced ovarian weight in IHR. Concentrations of progesterone (P4), androstenedione (A4), and estradiol (E2) in sera and ovaries were not altered by TCDD at 12, 24, 48, and 72 h after eCG. except for a two-fold increase in ovarian concentration of A4 at 48 h after TCDD. However, this higher concentration of A4 at 48 h after TCDD did not reflect that of A4 in sera and did not correlate with E2 in either sera or ovaries. In isolated GC from untreated IHR, TCDD (0.1 to 100 nM) had no significant effect on P4 and E2 after stimulation by LH or FSH. In TIC and whole ovarian dispersates containing GC, TIC, and other ovarian cells, TCDD (0.1 to 800 nM) had no effect on A4 and P4 secretion stimulated by LH. Using RT-PCR, AhR mRNA was shown to be expressed constitutively in the whole ovary of IHR with maximum down-regulation at 6 h after TCDD (20 microg/kg). Ovarian CYP1A1 was induced maximally at 6 h after TCDD, whereas CYP1B1 could not be detected. The induction of AhR related genes by TCDD in the ovary implies the existence of AhR-mediated signal transduction pathways. In summary, these results indicate that TCDD does not affect ovulation in IHR by altering ovarian steroidogenesis. It seems that inhibition of ovulation by TCDD is due to processes related to follicular rupture.