Expression of secreted frizzled-related protein 4 in the primate placenta.

Secreted frizzled-related protein 4 (sFRP4) blocks the Wnt signalling pathway by competitively binding Wnt ligands (frizzled receptors). This pathway is important during development and oncogenesis. It is, however, complex with a large number of interacting proteins, isoforms and receptors. The Wnt signalling pathway has a role in human placental development and implantation, particularly in the trophoblast. Humans and macaque monkeys exhibit a similar remodelling of the decidual spiral arteries. The expression of sFRP4 in human and macaque placentas at different gestational ages have been examined with immunohistochemistry, in-situ hybridization, real-time polymerase chain reaction, and western blotting. This study demonstrates that sFRP4 is expressed predominantly in the villous syncytiotrophoblast and the invasive intermediate cytotrophoblast, and in the amnion. These observational studies suggest that sFRP4 has a role in placental development and implantation, and may be an important factor in the development of the decidual fibrinoid zone, and in trophoblast apoptosis and a band of apoptosis in the underlying decidua deep into the trophoblast.

Expression of secreted frizzled-related protein 4 (SFRP4) in primary serous ovarian tumours.

OBJECTIVE: Serous ovarian cancer is the most prevalent type of ovarian cancer. The majority of women present at an advanced stage and patient survival is poor. Resistance to chemotherapy is thought to relate to failure of tumours to undergo apoptosis. Secreted frizzled-related protein 4 (SFRP4) has been demonstrated to be involved in apoptosis in the ovary but not in ovarian tumours as yet. This study examined SFRP4 expression in ovarian cancers and correlated this with expression of beta-catenin, a main component of the wNT-signalling pathway it inhibits. METHODS: We examined 153 primary serous ovarian carcinomas for SFRP4 and B-catenin expression using immunohistochemistry on tissue microarrays and correlated this with clinical information. RESULTS: SFRP4 expression was inversely associated with beta-catenin expression in 84% of samples. However, high-level SFRP4 expression was not significantly associated with patient survival (p = 0.08). CONCLUSION: Elevated SFRP4 expression in serous ovarian tumours appears to correlate with reduced beta-catenin expression but long-term survival appears unaffected by this.

Secreted Frizzled-related protein 4 (sFRP4) chemo-sensitizes cancer stem cells derived from human breast, prostate, and ovary tumor cell lines.

This study investigated molecular signals essential to sustain cancer stem cells (CSCs) and assessed their activity in the presence of secreted frizzled-related protein 4 (sFRP4) alone or in combination with chemotherapeutic drugs. SFRP4 is a known Wnt antagonist, and is also pro-apoptotic and anti-angiogenic. Additionally, sFRP4 has been demonstrated to confer chemo-sensitization and improve chemotherapeutic efficacy. CSCs were isolated from breast, prostate, and ovary tumor cell lines, and characterized using tumor-specific markers such as CD44+/CD24-/CD133+. The post-transcription data from CSCs that have undergone combinatorial treatment with sFRP4 and chemotherapeutic drugs suggest downregulation of stemness genes and upregulation of pro-apoptotic markers. The post-translational modification of CSCs demonstrated a chemo-sensitization effect of sFRP4 when used in combination with tumor-specific drugs. SFRP4 in combination with doxorubicin/cisplatin reduced the proliferative capacity of the CSC population in vitro. Wnt/ß-catenin signaling is important for proliferation and self-renewal of CSCs in association with human tumorigenesis. The silencing of this signaling pathway by the application of sFRP4 suggests potential for improved in vivo chemo-responses.

Wnt signaling in triple-negative breast cancer.

Wnt signaling regulates a variety of cellular processes, including cell fate, differentiation, proliferation and stem cell pluripotency. Aberrant Wnt signaling is a hallmark of many cancers. An aggressive subtype of breast cancer, known as triple-negative breast cancer (TNBC), demonstrates dysregulation in canonical and non-canonical Wnt signaling. In this review, we summarize regulators of canonical and non-canonical Wnt signaling, as well as Wnt signaling dysfunction that mediates the progression of TNBC. We review the complex molecular nature of TNBC and the emerging therapies that are currently under investigation for the treatment of this disease.

Quercetin induces p53-independent apoptosis in human prostate cancer cells by modulating Bcl-2-related proteins: a possible mediation by IGFBP-3.

Quercetin, a flavonoid found in onion, grapes, green vegetables, etc., has been shown to possess potent antiproliferative effects against various malignant cells. We report insulin-like growth factor-binding protein-3 (IGFBP-3) as an effector of quercetin-induced apoptosis in human prostate cancer cell lines in a p53-independent manner. We evaluated the production of IGFBP-3 in quercetin-treated cells. Apoptosis was studied in quercetin-treated cells to study the IGFBP-3-mediated role with flow cytometry and DNA fragmentation. Protein expressions of Bcl-2, Bcl-x(L), and Bax were studied by Western blot. Increased production of IGFBP-3 was associated with the increased ratio of proapoptotic to antiapoptotic members of the Bcl-2 family. In quercetin-treated PC-3 cells, an increase in Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein were observed. As PC-3 is a p53-negative cell line, these modulations of proapoptotic proteins and induction of apoptosis were independent of p53. The level of IGFBP-3 on the response of PC-3 cells to quercetin was examined. There was a twofold increase in IGFBP-3 level in conditioned media of 100 microM quercetin-treated cells. Quercetin also brought a peak at sub-G1 in PC-3 cells. Thus, increased level of IGFBP-3 was associated with increased proapoptotic proteins and apoptosis in response to quercetin, suggesting it may be a p53-independent effector of apoptosis in prostate cancer cells via its modulation of the Bax/Bcl-2 protein ratio.

Physiological apoptosis in hormone-dependent tissues: involvement of caspases.

Physiological apoptosis in mammals is a type of programmed cell death, an important element in the developmental repertoire ensuring tissue homeostasis and proper disposal of cells that are no longer needed, such as milk-producing epithelial cells in the mammary gland after lactation, luteal cells in the post partum Corpus luteum or secretory cells in the prostate after castration. Although incompletely described, apoptosis in hormone-dependent tissues is apparently initiated and executed using common biochemical strategies. These include survival pathways governed by local and systemic factors and hormones, diverse regulatory pathways and caspase-dependent execution pathways. Using an antibody that recognizes processed effector caspases or a fluorogenic caspase substrate, we present for the first time evidence that caspases are activated in the mammary gland, in the prostate and in the ovary at the time when apoptosis occurs. Most likely phagocytosis of apoptotic cells by neighboring cells may represent an important step, since only a modest involvement of professional phagocytes is apparent. Here, we will summarize and discuss recent data and will attempt to draw a generalized picture of how physiological apoptosis may occur in these organs.

Role of DDC-4/sFRP-4, a secreted frizzled-related protein, at the onset of apoptosis in mammary involution.

Using differential display, we isolated DDC-4, a secreted frizzled-related protein (sFRP), which is induced in the physiological apoptosis of hormonally regulated, reproductive tissues such as mammary gland, prostate, corpus luteum and uterus. The role of this gene in apoptosis was studied in animals overexpressing ectopic DDC-4/sFRP-4. Transgenic mice bearing the DDC-4/sFRP-4 cDNA under the control of the MMTV-LTR promoter showed lactational insufficiency and many apoptotic cells in the alveoli between day 19 of pregnancy and day 4 of lactation as demonstrated by TUNEL reaction and the presence of activated caspase-3. We performed a PKB/Akt kinase assay and studied several of its substrates using phosphorylation-specific antibodies to show reduced phosphorylation in PKB/Akt itself, as well as in glycogen synthetase kinase-3beta (GSK-3beta), BAD, and Forkhead. Taken together, our results show a role for DDC-4/sFRP-4 in abrogating an epithelial cell survival pathway at the onset of mammary gland involution.

Effect of interleukin-1 beta on ovulation in the in vitro perfused rabbit ovary.

Interleukin-1 (IL-1), a prominent 17-kilodalton member of a group of immune mediators referred to as cytokines, is secreted by a variety of immuno- and nonimmunocompetent cells. As IL-1 is an established mediator of inflammation, and ovulation may constitute an inflammatory-like reaction, consideration may be given to the possibility that IL-1 may play an intermediary role in the ovulatory process. Such a hypothesis is supported by the recent demonstration of the gonadotropin-dependent preovulatory induction of IL-1 transcripts at the level of the murine and human ovary. To date, however, the direct effect of IL-1 beta on the ovulatory process has not been examined. The objective of this study was to investigate the potential role of IL-1 beta in ovulation, oocyte maturation (nuclear and cytoplasmic), and subsequent fertilizability of in vitro ovulated oocytes. Rabbit ovaries perfused in vitro were used for these experiments. Ovarian arteries were cannulated in situ, and the ovaries were excised and perfused in vitro with or without IL-1 beta (18 ng/ml). The ovulatory efficiency of 18 ng/ml IL-1 beta-treated ovaries was 73.1%, similar to that of hCG (71.2%). Recovered oocytes were examined for their maturation and were inseminated in vitro to investigate fertilization, cleavage, and embryonic development. The fertilization rates of the 18 ng/ml IL-1 beta-treated and hCG-treated groups were 65.8% and 95.8% (P < 0.01), respectively. Cleavage rates of the IL-1 beta-treated and hCG-treated groups were 50% and 83.3% (P < 0.01), respectively. Most of the cleaved embryos from the IL-1 beta-treated group arrested at the four-cell stage, and only 2.6% of the fertilized embryos developed into the morula stage, whereas 54.2% of the hCG-treated group developed to the morula stage (P < 0.01). A cytotoxic effect of IL-1 beta is unlikely in this model. A more likely explanation is the induction of other factors by IL-1 beta, which may inhibit cytoplasmic maturation. Taken together, our findings demonstrate that in the absence of an ovulatory gonadotropic trigger, IL-1 beta can induce ovulation and oocyte maturation, facilitate fertilization, and influence subsequent embryonic development. Although fertilization and embryonic development occurred after IL-1 beta treatment, these rates were lower than those after hCG treatment. These observations give credence to the possibility that IL-1 may play an intermediary role in the ovulatory process.

Effects of a gonadotropin-releasing hormone analog on rabbit ovarian function.

This study was undertaken to elucidate the effects of a GnRH analog (GnRH-a) on rabbit ovulation, oocyte maturation, and steroidogenesis, and to verify whether treatment with a GnRH-a interferes with ovarian response to exogenous gonadotropin (hCG), both in vivo and in vitro. Three approaches were used. In the first, adult New Zealand White (NZW) rabbits were divided into two groups. Both received PMSG and hCG administered 72 h after PMSG. In the test group a GnRH-a, leuprolide acetate (LA; 20 microg/kg) was administered s.c. every 24 h. Treated rabbits showed a significant decrease in ovulatory efficiency (control = 88%; treated = 72%), and an increase in degeneration rate of preimplantation embryos (control = 30% vs. treated = 40%). For the second approach, in vitro perfusion experiments were designed to compare the direct effects of LA (10.000 ng/ml) and hCG (50 IU) on ovarian function and to verify whether the presence of a GnRH-a in the perfusate modifies the actions of hCG. LA reduced the ovulatory efficiency of hCG-treated ovaries perfused in vitro (hCG-treated = 87%; hCG-treated LA-perfused = 70%), reduced the potential for preimplantation development (morula stage: hCG-treated = 53%; hCG-treated LA-perfused = 31%; LA-perfused = 12%), and increased the degeneration rate of early embryos (21%, 48%, and 56% respectively). In the third approach, the direct effect of LA (Group I: control, Group II:1.000 ng/ml, and Group III:10.000 ng/ml) on the in vitro maturation of denuded rabbit oocytes was evaluated. LA induced meiotic maturation, but increased oocyte degeneration rate. The potential for preimplantation development was reduced (Morula stage: control = 16%, Group II = 8%, and Group III = 6%), and degeneration rate was increased (38%, 65%, 63% respectively). This study suggests that pharmacological doses of LA may exert a negative effect on oocyte function by direct action on the oocyte, indirectly via alteration of the intrafollicular environment and/or through interference with gonadotropin-induced biological effects within the ovary.

Induction of myometrial 11beta-hydroxysteroid dehydrogenase type 1 messenger ribonucleic acid and protein expression late in rat pregnancy.

Myometrial function in pregnancy is regulated by a range of hormonal stimuli, including glucocorticoids, particularly in the period leading up to parturition. Glucocorticoid hormone action is dependent not only on expression of glucocorticoid receptor (GR) within target cells, but also on local expression of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD). Therefore, this study examined changes in myometrial 11betaHSD bioactivity and expression (messenger RNA and protein) of the 11betaHSD-1 isoform and whether 11betaHSD-1 and GR are colocalized to myometrial cells. Myometrial 11-oxoreductase activity (conversion of [3H]11-dehydrocorticosterone to [3H]corticosterone) was only just detectable (<6%) at the postestrus stage of the cycle and on days 5 and 10 of pregnancy, but then increased markedly by day 16 (45 +/- 2%). This activity increased further to maximal levels on day 22 of pregnancy (55 +/- 3%) and remained high on day 23 (term; 34 +/- 3%) before decreasing by 24 h postpartum (9 +/- 2%). High 11beta-dehydrogenase activity was evident before (87 +/- 1%) and during the first half (day 5,91 +/- 1%; day 10, 88 +/- 2%) of pregnancy, was lower on days 16 (55 +/- 2%), 22 (39 +/- 3%), and 23 (58 +/- 1%), then returned to prepregnancy levels 24 h postpartum (86 +/- 1%). The marked induction of 11-oxoreductase activity late in pregnancy was strongly and positively correlated with both 11betaHSD-1 messenger RNA expression (by Northern analysis) and protein (by Western analysis; r = 0.96 and 0.98, respectively; P < 0.001). Moreover, 11betaHSD-1 and GR immunoreactivity were colocalized to the smooth muscle cells of the myometrium and the uterine epithelium late in pregnancy. Collectively, these data demonstrate that a marked induction of 11betaHSD-1 expression occurs in the rat myometrium near term, and this is associated with increased 11-oxoreductase bioactivity. As GR is coexpressed in the myometrium, we suggest that the induction 11betaHSD-1 serves to enhance local glucocorticoid actions and thus facilitate parturition.

Antioxidants mimic the ability of chorionic gonadotropin to suppress apoptosis in the rabbit corpus luteum in vitro: a novel role for superoxide dismutase in regulating bax expression.

We have recently reported that members of the bcl-2 gene family are expressed and estradiol regulated in rabbit luteal cells during corpus luteum (CL) regression, and that estradiol and hCG are effective inhibitors of apoptosis in the rabbit CL in vivo and in vitro. As Bcl-2 and related proteins are known to regulate levels of reactive oxygen species or their intermediates in cells as one possible mechanism to control apoptosis, the present studies were designed to examine if oxidative stress plays a role in luteal cell apoptosis during CL regression in the rabbit. In the first set of experiments, healthy CL obtained from day 11 pseudopregnant rabbits were incubated in serum-free medium for 2 h in the absence or presence of superoxide dismutase (SOD; 1.5-150 U/ml), ascorbic acid (1-100 mM), N-acetyl-L-cysteine (25 and 50 mM), or catalase (10-1000 U/ml). Cells within CL incubated in medium alone exhibited extensive apoptosis (examined by analysis of extracted DNA using 3'-end labeling), and this onset of apoptosis was blocked in a dose-dependent fashion by treatment with SOD, ascorbic acid, N-acetyl-L-cysteine, or catalase. In the second set of experiments, expression of bax and bcl-x in CL after in vitro treatment without and with 100 U/ml SOD was examined. Although SOD treatment did not alter the levels of bcl-x messenger RNA (mRNA) over the 2-h incubation period, this antioxidant enzyme significantly reduced the levels of bax mRNA in incubated CL. In the final set of experiments, we observed that expression of mitochondrial- or manganese-containing SOD was significantly increased by treatment of isolated CL with 1 microg/ml hCG in vitro, whereas bax mRNA levels were significantly reduced under the same culture conditions. Collectively, these data indicate that the gonadotropin-mediated inhibition of apoptosis in rabbit luteal cells involves enhanced expression of the oxidative stress response gene, manganese-containing SOD, whose protein product may then function to protect luteal cells directly from the damaging effect of reactive oxygen species and/or indirectly by acutely down-regulating expression of Bax, a prooxidant member of the Bcl-2 protein family.

Evidence for alternative pathways of granulosa cell death in healthy and slightly atretic bovine antral follicles.

Granulosa cell death is an early feature of atresia; however, there are many apparent contradictions in the literature concerning the mode of granulosa cell death. We have therefore examined this process in bovine healthy and atretic antral follicles, using a variety of established techniques. Light and electron microscopic observations indicated the presence of pyknotic or shrunken nuclei in both the membrana granulosa and the antrum. In the membrana granulosa, these nuclei were frequently crescent shaped and uniformly electron dense and were approximately the same size as healthy nuclei, all of which are typical of early apoptosis. However, these nuclei were within the membranes of a healthy granulosa cell, suggesting that phagocytosis by a neighboring granulosa cell is an unusually early event in the apoptotic pathway of granulosa cells. In the membrana granulosa, pyknotic nuclei stained intensely with hematoxylin but weakly with the DNA-intercalating stain propidium iodide. A percentage of these pyknotic nuclei stained by TUNEL (terminal deoxy-UTP nick end-labeling). However, in the antrum, the pyknotic nuclei and larger globules of DNA stained intensely with both hematoxylin and propidium iodide, but were not TUNEL positive. The comet assay of cell death produced a streak tail of randomly nicked DNA, rather than the plume of low mol wt apoptotic DNA. Globules collected from fresh follicular fluid stained intensely with propidium iodide and were shown by PAGE to contain DNA, the majority of which was high mol wt. In conclusion, granulosa cells within the membrana granulosa die by apoptosis, with phagocytosis by a neighboring cell preceding any potential budding of the nucleus or cell itself. Granulosa cells near the antrum are sloughed off into the antrum, and their death has features more consistent with that of other cell types that undergo death as a result of terminal differentiation.

Interleukin-1 is both morphogenic and cytotoxic to cultured rat ovarian cells: obligatory role for heterologous, contact-independent cell-cell interaction.

An increasing body of information now suggests that intraovarian interleukin-1 (IL-1) may play an intermediary role in the ovulatory process. Given that follicular rupture inevitably requires marked tissue remodeling and possibly cell death, we set out to examine the morphogenic potential of IL-1 under in vitro circumstances. Treatment of freshly plated whole ovarian dispersates from immature rats with any one of several batches of IL-1 (10 ng/ml) for up to 96 h produced marked time-dependent morphological alterations, including cellular retraction, rounding, clumping, aggregation, blebbing, swelling, and, ultimately, irreversible detachment. Evidence of (asynchronous) cell death consisted of reduced total cell number, diminished cellular protein content, enhanced cellular release of lactic dehydrogenase, failure to exclude trypan blue, and attenuated reduction of the tetrazolium dye 3-[4,5-dimethylthiazol-2-y]2,5-diphenyltetrazolium bromide to spectrophotometrically detectable formazan. Comparable results were obtained when using established day 4 cultures, arguing against a possible critical action of IL-1 at the time of plating. Dose-response curves revealed IL-1 beta (EC50, 0.2-0.4 ng/ml) to be substantially more potent than IL-1 alpha (EC50, 2.7-2.8 ng/ml). Importantly, the concurrent provision of an IL-1 beta-directed polyclonal antibody yielded complete immunoneutralization of the IL-1 beta effect, arguing against the possible involvement of a non-IL-1 contaminant. An unrelated polyclonal antiserum raised against insulin-like growth factor-I was without effect. IL-1 action proved relatively specific, in that tumor necrosis factor-alpha (10 ng/ml), a putative cytotoxic principle, as well as IL-1-inducible ILs (IL-2 and -6; 100 U/ml) were without effect. Although minimally effective at the level of the isolated granulosa or theca-interstitial cell, IL-1 proved highly potent in heterologous (but not homologous), contact-dependent and independent cocultures of these somatic cell types, strongly suggesting obligatory cell-cell cooperation. These observations further indicate that IL-1 action is indirect and may require the induction of an intermediary soluble principle to serve as the final effector. Taken together, these findings indicate that relatively low concentrations of IL-1 (beta >> alpha), possible of somatic ovarian cell or resident ovarian macrophage origin, are capable of exerting specific dose- and time-dependent (immunoneutralizable) morphogenic as well as cytotoxic effects at the level of ovarian cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of protein kinase-C in gonadotropin-induced ovulation in the in vitro perfused rabbit ovary.

Tumor-promoting phorbol esters are believed to affect ovarian granulosa cell progesterone and prostaglandin (PG) production and possibly ovulation by activating protein kinase-C (PKC). The effects of phorbol esters and PKC inhibitors on ovulation, progesterone, and PG production were examined in an in vitro perfused rabbit ovary. The effect of tranexamic acid, an inhibitor of the conversion of plasminogen activator to plasmin, on phorbol ester-induced ovulation was also examined. Phorbol 12,13-dibutyrate (PdBU), a PKC stimulator, induced ovulation in a dose-related manner in the absence of gonadotropins (56%, 200 nM PdBU; 0%, 0 nM PdBU; P < 0.05). Perfusate progesterone levels were increased only after 600 nM PdBU treatment, and perfusate PGF2 alpha, PGE2, and 6-keto-PGF1 alpha were increased in a dose-dependent fashion (P < 0.05). Staurosporine, a potent inhibitor of the catalytic domain of PKC, and calphostin-C, a specific inhibitor of the diacylglycerol-binding region, inhibited hCG-induced ovulation in a dose-related manner. Gonadotropin-induced ovulation decreased from 73% without staurosporine to 19% with 1.0 microM staurosporine (P < 0.01). Calphostin-C reduced ovulatory efficiency from 60% to 24% (P < 0.01). However, neither inhibitor decreased progesterone or PGF2 alpha production by ovaries exposed to hCG. hCG-induced oocyte maturation was also unaffected by exposure to either staurosporine or calphostin-C. Tranexamic acid reduced phorbol ester-induced ovulatory efficiency from 67% to 37% (P < 0.05). These findings demonstrate that the calcium-dependent PKC pathway is instrumental in gonadotropin-mediated follicular rupture in the rabbit. Although PGs may play an important role in ovulation, they do not appear to be directly responsible for PKC-mediated follicular rupture.

Estradiol regulation of the rabbit corpus luteum: in vivo and in vitro studies.

The objective of this study was to determine whether estradiol has a direct effect on progesterone secretion by the rabbit corpus luteum. Empty or estradiol-filled Silastic capsules were implanted sc into pseudopregnant rabbits (day 0). Ten days later (day 10), peripheral blood was obtained via the marginal ear vein, and Silastic capsules were removed. Twenty-four hours after capsule removal (day 11), blood samples were obtained and ovaries removed for in vitro perfusion. The artery and vein of each ovary were individually cannulated, and ovaries were perfused in vitro for 6 h. Mean progesterone secretion rates were determined from perfusate samples taken every 30 min. On day 10, serum progesterone concentrations were similar in control and estradiol-treated animals. On day 11, 24 h after withdrawal of Silastic capsules, serum progesterone concentration in the estradiol-treated rabbits decreased significantly compared to controls. The withdrawal of estradiol also significantly reduced the secretion of progesterone by in vitro perfused ovaries in estradiol-withdrawn rabbits compared to empty capsule controls. Addition of estradiol or 25-hydroxycholesterol (25-OH) to the perfusion medium significantly increased progesterone secretion by ovaries from estradiol-withdrawn rabbits but not to control values. In contrast, a combination of estradiol plus 25-OH restored progesterone secretion to control levels. Although estradiol together with 25-OH stimulated progesterone secretion 24 h after estradiol withdrawal, progesterone secretion in vitro was unaffected 48 h after capsule removal, whereas pregnenolone stimulated secretion 5-fold. These results demonstrate that estradiol has a direct and acute stimulatory effect on progesterone secretion by the rabbit corpus luteum.

Epidermal growth factor inhibits follicular response to human chorionic gonadotropin: possible role of cell to cell communication in the response to gonadotropin.

Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol and progesterone secretion and ovulation were examined in the in vitro perfused rabbit ovary. We also examined the effects of EGF on hCG-induced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P less than 0.05) reduced hCG-induced secretion of estradiol (control, 117 +/- 12 pg/min.follicle; 10 ng/ml EGF, 55 +/- 10) and progesterone (control, 18.2 +/- 1.2 ng/min.follicle; 10 ng/ml EGF, 11.9 +/- 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency (number of ovulated ova per number of mature follicles x 100) when EGF was given 30 min before hCG was reduced dose-dependently from 58.2% with no EGF to 8.3% with 10 ng/ml EGF (P less than 0.001). Ovulation was not inhibited by EGF when it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication.

Prostaglandins and progesterone secretion in the in vitro perfused pseudopregnant rabbit ovary.

The relationship between progesterone and prostaglandin (PG) secretion in the pseudopregnant rabbit corpus luteum was investigated using isolated in vitro perfused ovaries. Progesterone and PG secretion were measured on days 1, 11, and 18 of hCG-induced pseudopregnancy. The mean progesterone secretion increased significantly from days 1 to 11, and then decreased significantly by day 18. PG secretion was inversely correlated with progesterone secretion, suggesting that PG might inhibit progesterone secretion. To test this hypothesis, indomethacin, an inhibitor of PG secretion, was administered to intact rabbits from days 11-18 of pseudopregnancy and/or on day 18 ovaries were perfused in vitro with indomethacin. Indomethacin administered in vivo, in vitro, or both in vivo and in vitro significantly reduced PG secretion compared to that in controls, but did not affect progesterone secretion. In addition, perfusion of ovaries in vitro with PGF2 alpha did not alter progesterone secretion on either day 11 or day 18. Thus, although there is an inverse relationship between progesterone and PG secretion during pseudopregnancy, PGF2 alpha alone had no effect on progesterone secretion. These results question the hypothesis that PGF2 alpha alone is the luteolytic factor.

Transforming growth factor-beta 1 inhibits ovarian androgen production: gene expression, cellular localization, mechanisms(s), and site(s) of action.

It is the aim of this study to establish ovarian transforming growth factor-beta 1 (TGF beta 1) gene expression, to reevaluate its cellular localization, and to explore potential interactions of this regulatory peptide on ovarian androgen biosynthesis. Northern analysis of whole ovarian polyadenylated RNA revealed a single 2.5-kilobase transcript corresponding to the TGF beta 1 precursor. Immunohistochemical staining localized the protein to the thecal-interstitial (interfollicular) compartment. To explore potential autocrine effects of TGF beta 1, use was made of whole ovarian dispersates from immature rats the differentiation of which was monitored by the acquisition of androgen biosynthetic capacity. The accumulation of androsterone, the major androgenic steroid detectable in this culture system, increased 5.4-fold over baseline in response to treatment with hCG (1 ng/ml). This effect was further optimized (2- to 4-fold) by supplementation with insulin (1 microgram/ml) and insulin-like growth factor-I (50 ng/ml). In the absence of these optimizing supplements, TGF beta 1 (10 ng/ml) was without effect on basal androsterone accumulation, producing distinct, albeit relatively limited (25%), inhibition of hCG hormonal action. In contrast, supplement-mediated optimization of ovarian androgen biosynthesis revealed TGF beta 1 to be a highly potent inhibitor (greater than 80%) of hCG hormonal action. This reversible TGF beta 1 action proved time and dose dependent, with a minimal time requirement of 72 h and a median inhibitory dose of 2.6 ng/ml. TGF beta 1 action was not due to diminution in the viable cell mass or altered cAMP generation and, therefore, most likely involved a site(s) of action distal to or independent of cAMP generation. Cellular radiolabeling studies of TGF beta 1-treated ovarian cells disclosed the accumulation of steroid intermediates proximal to the 17 alpha-hydroxylation step, suggesting TGF beta 1-mediated blockade at the level of the steroidogenic enzyme 17 alpha-hydroxylase/17-20-lyase. Taken together, these observations are in keeping with the view that TGF beta 1, possibly of thecal-interstitial origin, may not only play a positive paracrine role at the level of the adjacent granulosa cell (as previously reported), but may also constitute one of several autocrine signals concerned with the regulation of ovarian androgen economy. As such, these findings reaffirm the polyfunctional nature of TGF beta 1 action, as manifested by its diametrically opposed effects in different ovarian compartments.

Postischemic cerebral blood flow recovery in the female: effect of 17 beta-estradiol.

Female reproductive hormones are considered to be protective agents in atherosclerotic vascular disease and stroke. The present study determined if there are unique cerebrovascular responses in female animals to global cerebral ischemia and if 17 beta-estradiol is important to postischemic outcome in brain. Three groups of anesthetized, sexually mature rabbits were treated with normotensive four-vessel occlusion (6 min) and 3 h of reperfusion: females chronically instrumented with 17 beta-estradiol implants (EFEM; n = 8, plasma estradiol level = 365 +/- 48 pg/ml), untreated females (FEM; n = 8, estradiol = 13 +/- 3 pg/ml), and untreated males (M; n = 8, estradiol < limit of radioimmunoassay). CBF (microspheres) and somatosensory evoked potential (SEP) amplitude were measured during ischemia/reperfusion. Baseline hemispheric blood flow and regional flow distribution were not altered by chronic estradiol treatment. Hemispheric blood flow was equivalently reduced during ischemia in FEM and M (6 +/- 1 and 9 +/- 2 ml min-1 100 g-1, respectively); however postischemic hyperemia was greater in FEM than M (CBF = 257 +/- 27 and 183 +/- 27 ml min-1 100 g-1. However, EFEM experienced higher CBF during ischemia (e.g., 13 +/- 2 ml min-1 100 g-1) and less hyperemia (134 +/- 4 ml min-1 100 g-1 in hemispheres) in numerous brain regions than FEM. CBF at 3 h reperfusion was not different among the groups. Recovery of SEPs was incomplete and similar in all groups. We conclude that chronic exogenous 17 beta-estradiol treatment increases CBF during global incomplete ischemia and ameliorates postischemic hyperemia in the female animal.

DDC-4, an apoptosis-associated gene, is a secreted frizzled relative.

The differential display method has been used in our laboratory as a coincidence analysis to isolate genes expressed in common in each of three different rat tissues undergoing physiological apoptosis: mammary gland, ovarian corpus luteum and ventral prostate. The most interesting of these isolates, DDC-4, shows a clear association with apoptosis, its expression being confined to these three organs, and only during their involution. Using DDC-4 as probe, we screened a rat ovarian cDNA library to obtain full-length isolates. One isolate, Y81 clone 40, gives rise to a protein of approximately 40 kDa with coupled in vitro transcription/translation. Sequencing of this clone indicates an open reading frame of 1044 nucleotides encoding a protein of 39.7 kDa with a putative signal sequence. This clone exhibits a high homology with the cysteine-rich domain, i.e. the ligand-binding domain, of the fizzled gene family originally defined as tissue polarity genes in Drosophila. The homology of Y81 clone 40 is most extensive with the newly described secreted frizzled relatives, the frzb subfamily.