Trophoblast Glycoprotein (TPGB/5T4) in Human Placenta: Expression, Regulation, and Presence in Extracellular Microvesicles and Exosomes.

BACKGROUND: Many parallels exist between growth and development of the placenta and that of cancer. One parallel is shared expression of antigens that may have functional importance and may be recognized by the immune system. Here, we characterize expression and regulation of one such antigen, Trophoblast glycoprotein (TPGB; also called 5T4), in the placenta across gestation, in placentas of preeclamptic (PE) pregnancies, and in purified microvesicles and exosomes. METHODS: Trophoblast glycoprotein expression was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. Regulation of 5T4 in cytotrophoblast cells was examined under either differentiating conditions of epidermal growth factor or under varying oxygen conditions. Microvesicles and exosomes were purified from supernatant of cultured and perfused placentas. RESULTS: Trophoblast glycoprotein expression was prominent at the microvillus surface of syncytiotrophoblast and on the extravillous trophoblast cells, with minimal expression in undifferentiated cytotrophoblasts and normal tissues. Trophoblast glycoprotein expression was elevated in malignant tumors. In cytotrophoblasts, 5T4 was induced by in vitro differentiation, and its messenger RNA (mRNA) was increased under conditions of low oxygen. PE placentas expressed higher 5T4 mRNA than matched control placentas. Trophoblast glycoprotein was prominent within shed placental microvesicles and exosomes. CONCLUSION: Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.

The effect of tumor necrosis factor α (TNFα), interleukin 1ß (IL1ß) and interleukin 6 (IL6) on endometrial PGF2α synthesis, metabolism and release in early-pregnant pigs.

Cytokines produced by the porcine uterus and embryos may be involved in the regulation of endometrial prostaglandin synthesis, metabolism, and release. We studied the effect of tumor necrosis factor α (TNFα), interleukin 1ß (IL1ß) and interleukin 6 (IL6) on: 1) endometrial release of prostaglandin F2α (PGF2α), 2) expression of the terminal enzyme of PGF2α synthesis--PGF synthase mRNA (PGFS mRNA), 3) secretion of PGF(2)α metabolite--13,14-dihydro-15-keto PGF2α (PGFM) by the endometrium and 4) presence and activity of endometrial NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The effects of cytokines were determined on days 10-11 and days 12-13, e.g., before and during maternal recognition of pregnancy, and on days 15-16, e.g., during the peri-implantation period and compared with its effect in cyclic gilts on corresponding days of the estrous cycle. TNFα did not affect endometrial release of PGF2α in pregnant and cyclic pigs. IL1ß enhanced endometrial PGF2α release on days 12-13 and 15-16 in pregnant and cyclic pigs, respectively. IL6 increased PGF2α release mainly on days 15-16 of pregnancy. Expression of PGFS mRNA was decreased by IL1ß on days 12-13 of pregnancy (P<0.05) and increased in response to IL1ß, TNFα and IL6 on 12-13 (P<0.05) and 15-16 (P<0.01) of the estrous cycle. IL1ß increased release of PGFM in gravid pigs on days 12-13, 15-16 and in non-gravid pigs 10-11 and 15-16 of the cycle. On days 15-16 of pregnancy TNFα and IL6 increased endometrial secretion of PGFM. We determined that in porcine endometrium NAD-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is present. In gravid pigs, the highest expression of endometrial 15-PGDH occurred during days 12-13 of pregnancy, while in non-gravid pigs during days 10-11 of the estrous cycle. These data provide new evidence that TNFα, IL1ß, IL6 are involved in the regulation of endometrial synthesis, release and metabolism of PGF2α to protect CL during early pregnancy or to facilitate its regression in cyclic females.

In vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian, pituitary, and pineal function in pigs.

The aims of the study were: (1) to examine 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and/or prolactin (PRL) effects on in vitro secretion of progesterone (P(4)) and estradiol (E(2)) by luteinized granulosa and theca cells from porcine preovulatory follicles; and (2) to determine the effects of TCDD on PRL, luteinizing hormone (LH), and melatonin luteal phase in pigs. We found that TCDD itself did not affect progesterone secretion, but it abolished the stimulatory effect of PRL in the follicular cells. TCDD stimulated PRL secretion during the luteal phase and inhibited during the follicular phase. Moreover, TCDD increased luteinizing hormone secretion by pituitary cells during the follicular phase. In contrast to protein and steroid hormones, melatonin secretion in vitro was not affected by TCDD. In conclusion, it was found that the pituitary-ovarian axis in pigs is sensitive to TCDD, and the dioxin exhibited a profound ability to disrupt the ovarian actions of prolactin.

Temporal and anatomical sensitivities to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin leading to premature acyclicity with age in rats.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates diverse dioxin toxicities. While the acute effects of activation of the AhR pathway by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been a focus of past study, the role of this pathway in normal physiology and ageing is unclear. The purpose of this study was to identify the portion of the reproductive axis [ovary vs. hypothalamus and pituitary gland (H-H axis)] and the stages of the reproductive lifespan (foetal and early post-natal life vs. adolescence and adulthood) that are particularly sensitive to the effects of TCDD during female reproductive ageing. Adult pregnant Lewis rat dams were dosed with corn oil vehicle or TCDD (50 ng/kg-week by gavage) on days 14 and 21 of gestation and post-natal days 7 and 14 to provide in utero and lactational (IUL) exposure to pups. Female pups (n = 96) were weaned on post-natal day 21 and dosed with TCDD or vehicle weekly. Half of the pups were used as donors for ovary transplantation while the remainder were recipients. Following ovary transplantation, rats (n = 6-8 per group) received weekly TCDD or vehicle again until sacrifice at 8 months of age. Beginning at vaginal opening, reproductive cycles were monitored by vaginal cytology for 10 days each month. Blood samples were collected at 22.00 h on proestrus to measure concentration of 17beta-oestradiol in serum. Real-time PCR was used to determine differences in Cyp1a1, Cyp19a1, Cyp17a1, LH receptor (LHR), FoxA2 and FoxJ1 genes expression between control and remaining groups. IUL exposure of the H-H axis plus adult exposure of the whole body to TCDD significantly delayed puberty in females rats. Data analysis revealed an accelerated onset of acyclicity by 5 months in all groups involving IUL exposure of the developing ovary to TCDD. 17beta-oestradiol was significantly decreased in animals receiving TCDD during IUL exposure of the H-H axis. CYP1a1 expression was markedly greater in the liver than in ovarian tissue and correlated with ongoing TCDD exposure. Aromatase, 17alpha-hydroxylase and LHR gene expressions were largely unchanged (or occasionally elevated) by TCDD. FoxA2 and FoxJ1 mRNAs were similarly of limited value mechanistically, although FoxJ1 was much higher in TTT females (receiving TCDD as donor, recipient and adult). This study reveals a particular sensitivity of the developing ovary to TCDD leading to early loss of reproductive function with age.

Effects of phytoestrogen daidzein and estradiol on steroidogenesis and expression of estrogen receptors in porcine luteinized granulosa cells from large follicles.

The aims of the study were to compare the in vitro effects of daidzein or 17beta-estradiol (E(2)) on: 1) progesterone (P(4)) secretion by luteinized granulosa cells harvested from large porcine follicles, as well as 2) estrogen receptor alpha and beta (ERalpha and ERbeta) mRNA and protein expression in the cells. In addition, the effect of daidzein on E(2) secretion and viability of the granulosa cells was examined. We found that basal and gonadotropin-stimulated P(4) secretion were inhibited in granulosa cells cultured in the presence of daidzein either for 24 or 48 hours. In contrast to daidzein, E(2) reduced P(4) secretion only during 24-hour cell cultures increasing it during longer cultures. Daidzein did not affect E(2) secretion by granulosa cells. The expression of ERalpha and ERbeta mRNA, as well as ERbeta protein, was up-regulated by daidzein but unaffected by E(2). To conclude, the soy estrogen daidzein acts directly on the porcine ovary to decrease progesterone production and to increase expression of ERbeta mRNA and protein. Daidzein actions in porcine luteinized granulosa cells differ from those of estradiol and it may suggest disadvantageous effects of the phytoestrogen on reproductive processes in females.

The effect of progesterone on oxytocin-stimulated intracellular mobilization of Ca2+ and prostaglandin E2 and F2alpha secretion from porcine myometrial cells.

UNLABELLED: Our past studies have shown that porcine myometrium produce prostaglandins (PG) during luteolysis and early pregnancy and that oxytocin (OT) and its receptor (OTr) support myometrial secretion of prostaglandins E2 and F2alpha (PGE2 and PGF2alpha) during luteolysis. This study investigates the role of intracellular Ca2+ [Ca2+]i as a mediator of OT effects on PG secretion from isolated myometrial cells in the presence or absence of progesterone (P4). Basal [Ca2+]i was similar in myometrial cells from cyclic and pregnant pigs (days 14-16). OT (10(-7)M) increased [Ca2+]i in myometrial cells of cyclic and pregnant pigs, although this effect was delayed in myometrium from pregnant females. After pre-incubation of the myocytes with P4 (10(-5)M) the influence of OT on [Ca2+]i)was delayed during luteolysis and inhibited during pregnancy. Myometrial cells in culture produce more PGE2 than PGF2alpha regardless of reproductive state of the female. OT (10(-7)M) increased PGE2 secretion after 6 and 12 h incubation for the tissue harvested during luteolysis and after 12 h incubation when myometrium from gravid females was used. In the presence of P4 (10(-5)M), the stimulatory effect of OT on PG secretion was diminished. IN CONCLUSION: (1) porcine myometrial cells in culture secrete PG preferentially during early pregnancy and produce more PGE2 than PGF2alpha, (2) OT controls myometrial PGF2alpha secretion during luteolysis, (3) release of [Ca2+]i is associated with the influence of OT on PG secretion, and (4) the effects of OT on PG secretion and Ca2+ accumulation are delayed by P4 during luteolysis and completely inhibited by P4 during pregnancy.

Expression of enzymes of cyclooxygenase pathway and secretion of prostaglandin E2 and F2alpha by porcine myometrium during luteolysis and early pregnancy.

UNLABELLED: Past studies of uterine prostaglandin (PGs) and pig reproduction have focused on endometrial rather than myometrial PGs. This study documents the synthesis and secretion of myometrial prostaglandins (PGs) in pigs and the involvement of oxytocin (OT) in these processes. Cyclooxygenase-2 (COX-2) expression was similar in myometrial explants from cyclic and pregnant pigs (days 14-16) and OT (10(-7) M) in vitro significantly increased COX-2 protein regardless of reproductive state. Basal expression of prostaglandin E2 synthase (PGES) was higher during pregnancy than during luteolysis. Conversely, prostaglandin F synthase (PGFS) was highest during luteolysis and lower in myometrium from gravid animals. OT had no influence on the expression of PGES and PGFS. In another tissue culture experiment, myometrial slices produced more PGE2 than PGF2alpha regardless of reproductive state of the female. OT stimulated PGE2 production in myometrium harvested during luteolysis and increased PGF2alpha production in all tissues examined. Progesterone (P4; 10(-5) M) blocked stimulatory effect of OT on myometrial PG release. Myometrial OTr mRNA was higher (P=0.03) during luteolysis than during pregnancy. IN CONCLUSION: (1) oxytocin increases myometrial COX-2 expression, but does not influence the expression of terminal enzymes of PGs synthesis (PGES and PGFS); (2) porcine myometrium preferentially produces PGs during early pregnancy and secretes more PGE2 than PGF2alpha; (3) myometrial OT and OTr support secretion of PGs from myometrium during luteolysis.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on weight gain and hepatic ethoxyresorufin-o-deethylase (EROD) induction vary with ovarian hormonal status in the immature gonadotropin-primed rat model.

Immature female rats received 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during an induced proestrus or diestrus. The inhibitory effect of TCDD on acute weight gain and the induction of hepatic ethoxyresorufin-o-deethylase (EROD) activity by TCDD were greatest during proestrus. In a second experiment, ovariectomized rats received estradiol cypionate (ECP) or progesterone followed by TCDD. TCDD and estradiol each alone significantly inhibited weight gain. Progesterone potentiated the effects of TCDD on weight gain. The highest dose of ECP was associated with greater induction of hepatic EROD activity by TCDD than seen with TCDD alone. Estradiol modulates the induction of hepatic EROD activity by TCDD. Differential effects of TCDD on acute weight gain during proestrus vs. diestrus in this model do not mimic changes induced by estrogen alone. Hepatic responses to TCDD may vary according to phase of the female reproductive cycle.

Mechanisms of cytokine-induced death of cultured bovine luteal cells.

Tumour necrosis factor alpha (TNF-alpha) and gamma-interferon (IFN-gamma) are cytotoxic to bovine luteal cells in vitro and may contribute to cell death during luteolysis in vivo. In this study, the mechanism by which luteal cells are killed by TNF-alpha and IFN-gamma was investigated. Luteal cells were cultured for 7 days in the presence or absence of TNF-alpha and IFN-gamma. Inhibitors of arachidonate metabolism or scavengers of free radicals were included in the culture media. In addition, the effect of IFN-beta on the viability of cytokine-treated luteal cells was tested. Lastly, untreated and cytokine-treated cells were subjected to single cell gel electrophoresis for quantification of DNA fragmentation. Neither indomethacin nor nordihydroguaiaretic acid, which are inhibitors of cyclooxygenase and lipoxygenase, respectively, were able to prevent cytokine-induced cell death. Similarly, both the phospholipase A(2) inhibitor arachidonyltrifluoromethyl ketone and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine, were largely without effect. In contrast, while vitamin C did not significantly affect viability, superoxide dismutase plus catalase increased viability of cytokine-treated cells (P < 0.05), and IFN-beta prevented cell death (P < 0.05). Finally, while control cells remained free of DNA damage, TNF-alpha plus IFN-gamma induced significant amounts of DNA damage by 48 h after initiation of treatment (P < 0.05). In conclusion, reactive oxygen species, but not arachidonate metabolism or nitric oxide, contribute to cytokine-induced luteal cell death in vitro, and the process of cell death may be via apoptosis. Furthermore, IFN-beta may confer protective effects against cytokine-induced cell death in bovine luteal cells.

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.

Gonadotropin-releasing hormone (GnRH) partially reverses the inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on ovulation in the immature gonadotropin-treated rat.

Several studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has inhibitory effects on ovulation. This action may be the result of either direct effect(s) of TCDD on ovarian function or via altered secretion of pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) which regulate ovarian follicular development and ovulation. To further evaluate the effects of TCDD on pituitary gonadotropins and their regulation, the potential role of gonadotropin-releasing hormone (GnRH) was investigated in the current study. Immature (23-day-old) female Sprague-Dawley rats were dosed with TCDD (32 microg/kg) in corn oil or vehicle alone. Equine chorionic gonadotropin (eCG) was injected subcutaneously (5 IU, sc) 24 h later to induce follicular development. Immediately prior to the expected time of the LH/FSH surges, 54 h after eCG injection, half of TCDD- or corn oil-treated rats were injected with GnRH (2 microg/rat, sc). Blood and ovaries were collected at 54, 56, 58, 60 and 72 h after eCG. Serum concentrations of 17beta-estradiol (E(2)), progesterone (P(4)), LH, and FSH were determined by radioimmunoassay. An indication of ovulation rate was assessed at 72 h after injection of eCG by irrigating the ova from oviducts. TCDD reduced the number of ova in the oviducts by 70-80% (2-3 ova/rat) and this was confirmed by the number of corpora lutea. GnRH partially restored ovulation (6-7 ova/rat) in TCDD-treated rats without reversing its effect on ovarian weight reduction. In controls, the LH and FSH surges at 58 h after eCG were significantly reduced at that time in TCDD-treated rats. However, in rats treated with TCDD and GnRH, a huge LH/FSH surges occurred at 56 h after eCG injection. GnRH alone enhanced E(2) and P(4) serum levels at 56-58 h after eCG injection. In rats treated with both TCDD and GnRH, E(2) secretion was significantly lower at 58, 60, and 72 h when compared with GnRH alone, whereas serum P(4) was only decreased at 72 h after eCG injection. The results indicate that exogenous GnRH induces LH and FSH surges in TCDD-treated rats, but only partially restores the inhibitory effects of TCDD on ovulation.

Interaction of estradiol and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an ovulation model: evidence for systemic potentiation and local ovarian effects.

Immature rats were treated with estradiol cypionate, (ECP, 0, 0.1, 1, or 2 mg/kg s.c.) followed 24 h later by TCDD (0 or 10 microg/kg orally). Follicular development was induced with eCG [5 or 10 IU subcutaneously (s.c.)] followed by an ovulatory dose of hCG (10 IU s. c.). Inhibition of ovulation by TCDD was potentiated by ECP in hypophysectomized but not intact rats. Only hypophysectomized rats exposed systemically to TCDD and ECP exhibited weight loss. Pair feeding mimicked the combined effects of TCDD and ECP in hypophysectomized rats. In another experiment, intact rats received ECP s.c. (0 or 2 mg/kg) and TCDD into the ovarian bursa (0 or 250 ng). Another group of intact rats received TCDD orally (10 microg/kg) and ECP into the ovarian bursa (0 or 1.5 microg). Blockade of ovulation by systemic or local TCDD was alleviated by ECP pretreatment. Estrogen increased the systemic toxicity of TCDD in rats whereas antagonizing its direct ovarian effects.

Expression of cytokine messenger ribonucleic acids in the bovine corpus luteum.

There is considerable evidence that luteolysis in the cow and other species involves components of the immune system. In this study, we examined the expression of the mRNAs for TNF-alpha, IFN-gamma, IL-1beta, IL-2, and IL-2 receptor (IL-2R) by reverse transcription-polymerase chain reaction (RT-PCR) using bovine-specific primers. Expression was examined in corpora lutea (CL) of the early (day 5), mid (days 11-12), and late (day 18) luteal phase, and at 1, 4, and 24 hours following a luteolytic dose of prostaglandin (PG) F2alpha. Tumor necrosis factor-alpha, IFN-gamma, and IL-1beta mRNAs were detectable by RT-PCR at all stages of the cycle examined. Densitometric intensities of the electrophoresed IFN-gamma PCR products revealed a drop in RNA expression during late diestrus and at one hour of prostaglandin-induced luteolysis (P < 0.05). The mRNA for TNF-alpha seemed to remain constant during the cycle, and rose slightly during luteolysis. Interleukin-1beta mRNA also did not vary during the cycle or during luteolysis. Finally, expression of mRNAs for IL-2 and IL-2 receptor was not evident in CL by the methods employed in this study. These results are the first to describe mRNA expression for TNF-alpha, IFN-gamma, and IL-1beta in the bovine corpus luteum, and support a role for these cytokines in luteal function and regression.

Assessment of the mechanism by which prolactin stimulates progesterone production by early corpora lutea of pigs.

Previously, we reported that administration of prolactin (PRL) during the early luteal phase in sows increases plasma progesterone concentrations. In the current study, we searched for the mechanisms by which PRL exerts this luteotrophic effect. The objectives of the study were (1) to examine the effect of PRL and/or low-density lipoproteins (LDL) on progesterone production by porcine luteal cells derived from early corpora lutea, and (2) to assess the ability of PRL to activate phosphoinositide-specific phospholipase C (PI-PLC) and protein kinase C (PKC) in these luteal cells. Ovaries with early corpora lutea (day 1-2 of the oestrous cycle) were obtained from the slaughterhouse. Progesterone production by dispersed luteal cells was measured after treatment with PRL, phorbol 12-myristate 13-acetate or inhibitors of PKC in the presence or absence of LDL. LDL increased progesterone concentration in the incubation medium (304.5 vs 178.6 ng/ml in control, P<0.05). PRL augmented LDL-stimulated progesterone secretion by luteal cells (to 416 ng/ml, P<0.05), but PRL alone did not affect progesterone production (209.6 ng/ml, P>0.05). Staurosporine, a PKC inhibitor, inhibited progesterone secretion stimulated by the combined action of LDL and PRL; however, such inhibition was not demonstrated when cells were treated with the PKC inhibitor, H-7. PKC activation was assessed by measuring the specific association of [H]phorbol dibutyrate (H-PDBu) with luteal cells after treatment with PRL or ionomycin (a positive control). PRL and ionomycin increased H-PDBu-specific binding in early luteal cells by 28+/-5.5% (within 5 min) and 70.2+/-19.3% (within 2 min) over control binding respectively (P<0.05). In addition, PRL did not augment the LDL-stimulated progesterone production in PKC-deficient cells. In contrast with PKC, total inositol phosphate accumulation, as well as intracellular free calcium concentrations, were not affected by PRL in the current study. We conclude that PRL, in the presence of LDL, stimulates progesterone production by early corpora lutea in vitro. Moreover, PRL appears to activate PKC, but not PI-PLC, in these cells. Thus intracellular transduction of the PRL signal may involve activation of PKC that is not dependent on PI-PLC.

Depletion of vitamin C from pig corpora lutea by prostaglandin F2 alpha-induced secretion of the vitamin.

The luteolytic effects of prostaglandin F2 alpha (PGF2 alpha) are thought to be mediated in part by the promotion of an increasingly oxidative cellular environment. Loss of antioxidants is one mechanism by which PGF2 alpha might induce or exacerbate oxidative damage within the corpus luteum. This study was performed to establish whether depletion of vitamin C is an acute effect of PGF2 alpha on the pig corpus luteum and to gain insight into the mechanism of luteal vitamin C loss at luteolysis. Gilts (n = 4) were anaesthetized and both utero-ovarian veins and an ear vein were catheterized. Each corpus luteum on the treated ovary received an intraluteal injection of PGF2 alpha (1 microgram) followed by a sustained release implant containing 100 micrograms of the prostaglandin. The other ovary served as the control and each corpus luteum received corresponding volumes of injection vehicle and blank implant. Blood was collected from the ear vein and both utero-ovarian veins every 15 min beginning 15 min before the onset of treatment. Collection of blood stopped when animals were ovariectomized and corpora lutea were collected at 2 h after treatment. Progesterone and vitamin C (ascorbate) concentrations were measured in tissue and plasma samples. PGF2 alpha-treated luteal tissue had similar progesterone, but significantly lower ascorbate, concentrations when compared with control corpora lutea. PGF2 alpha treatment resulted in a rapid and sustained increase in plasma ascorbate within the treatment-side utero-ovarian vein, while the control utero-ovarian vein and ear vein showed little change in plasma ascorbate during the experimental period. No effect of PGF2 alpha on plasma progesterone was evident. This finding suggests that PGF2 alpha depletes the pig corpus luteum of vitamin C by inducing secretion of the vitamin into the bloodstream. Further studies are necessary to determine whether the depletion of vitamin C that is induced by PGF2 alpha contributes to the demise of the pig corpus luteum.

Total ascorbate and dehydroascorbate concentrations in porcine ovarian stroma, follicles and corpora lutea throughout the estrous cycle and pregnancy.

Ovarian tissues are thought to require ascorbate as an antioxidant and enzymatic cofactor for the processes of steroid and collagen synthesis. We measured the concentrations of total ascorbate and oxidized ascorbate (dehydroascorbate, DHA) in ovarian stroma, follicles and corpora lutea (CL) throughout the estrous cycle and pregnancy of the sow. Both total ascorbate and DHA concentrations were greatest in luteal tissue and lowest in ovarian stroma across all stages examined. Within the CL, total ascorbate levels were lowest during the early, early-mid, and late luteal phase and were elevated during the mid-luteal phase. Luteal total ascorbate concentrations were further elevated during early pregnancy and were comparable to mid-luteal phase concentrations during the remainder of gestation. Luteal DHA concentrations decreased from mid to late luteal phase, and were elevated throughout pregnancy. As the CL aged during the cycle, the DHA/total ascorbate ratio decreased and remained low throughout pregnancy. Total ascorbate concentrations in follicular tissue increased during the follicular phase and were lowest during the early luteal phase. The DHA concentrations and DHA/total ascorbate ratios in follicular tissue did not differ with stage. Total ascorbate and DHA concentrations in ovarian stroma were low and did not vary with stage. We conclude that periods of maximal luteal and follicular function are associated with increased concentrations of total ascorbate within the tissue. Furthermore, luteolysis appears to be associated with depletion of luteal ascorbate species.

Arachidonic acid inhibits hCG-stimulated progesterone production by corpora lutea of primates: potential mechanism of action.

Arachidonic acid (AA) is a precursor of metabolites known to affect the corpus luteum (CL) in many species, including primates. We have shown that some of these products (prostaglandins F2 alpha and E2) inhibit pro-gesterone (P4) production and activate the phosphatidylinositol (PI) pathway in CL of rhesus monkeys. A direct role of AA in luteal function has also been suggested. The current experiments were designed to investigate the effect of AA on P4 synthesis and to examine the ability of AA to activate the PI pathway in CL of rhesus monkeys. Basal and hCG-stimulated P4 production by luteal cells collected during the midluteal phase was measured after treatment with AA (1, 5, and 10 microM) or linoleic acid (1, 5, and 10 microM). Dispersed cells (50,000/tube) were incubated at 37 degrees C for 2 h. AA elicited a dose-dependent decrease in hCG-stimulated, but not in basal, P4 production. hCG-stimulated P4 production was reduced (P < 0.01) at AA doses of 5 microM (12.1 +/- 1.5 ng/mL) and 10 microM (8.6 +/- 1.8 mg/mL) to hCG alone (18 +/- 1.6 ng/mL). There was no significant effect of 1 microM AA (15.2 +/- 1.6). Response to linoleic acid was dissimilar and was not dose-dependent. Viability of cells was not affected by any treatment. Indomethacin, a prostaglandin synthesis inhibitor, and nordihydroguaiaretic acid, an inhibitor of lipoxygenase, did not interfere with the inhibitory effect of AA. Activation of the PI pathway was assessed by monitoring the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol phosphates and by monitoring increases in intracellular free calcium concentrations ([Ca2+]i) in individual cells. Moreover, the ability of AA to activate protein kinase C (PKC) in luteal cells was measured using a [3H]phorbol dibutyrate (PDBu) binding assay. AA did not alter PIP2 hydrolysis or [Ca2+]i, however, AA (10 microM) increased specific binding of [3H]PDBu to luteal cells (P < 0.05). We conclude that AA inhibits hCG-stimulated P4 production by primate luteal cells. AA exerts this action without being converted to prostaglandins or leukotrienes. This inhibition may be mediated through the activation of PKC. These results suggest a possible role for AA in the regulation of luteal function in primates, and that PKC-activation by AA may promote its effects.

CaBP9K levels during the luteal and follicular phases of the estrous cycle in the bovine uterus.

The expression of calbindin-D9K (CaBP9K) and calbindin-D28K (CaBP28K) genes in the reproductive system is well established for rodent and avian species, but not for domestic livestock. This investigation expanded the study of these proteins to include the bovine uterus and examined the levels of CaBP9K and CaBP9K mRNA in the nonpregnant bovine uterus during the estrous cycle. Immunohistochemical studies revealed that CaBP9K was present in all uterine glandular and luminal epithelial cells. In contrast, the closely related calcium binding protein CaBP28K was present in only one to two glandular cells in the samples examined. Neither protein was localized in the myometrium or in the stromal cells of the endometrium. RIA and dot blot hybridization were used to quantify the amount of CaBP9K and CaBP9K mRNA. The levels of both the protein and its mRNA were threefold higher during the luteal phase than during the follicular phase. RIA was also used to determine bovine uterine levels of 17 beta-estradiol and progesterone. Progesterone levels were higher during the luteal phase than during the follicular phase, while 17 beta-estradiol levels were higher during the follicular phase. This investigation represents the first characterization of CaBP9K gene expression in the bovine uterus. It demonstrated that the expression of CaBP9K and CaBP9K mRNA was greatest during the progesterone-dominated luteal phase of the bovine estrous cycle. These results indicated that CaBP9K may be involved in uterine glandular function during the luteal phase.