Anita Franczak

Ovarian Endocrine Disruption Underlies Premature Reproductive Senescence Following Environmentally Relevant Chronic Exposure to the Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

Abstract The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. While previous work has focused on mechanisms of short-term endocrine disruption and ovotoxicity in response to AHR ligands, we have shown recently that chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces premature reproductive senescence in female rats without depletion of ovarian follicular reserves. In the current study, premature reproductive senescence was induced using a range of low-dose exposure to TCDD (0, 1, 5, 50, and 200 ng kg−1 wk−1) beginning in utero and continuing until the transition to reproductive senescence. Doses of 50 and 200 ng TCDD kg−1 wk−1 delayed the age at vaginal opening and accelerated the loss of normal reproductive cyclicity with age without depletion of follicular reserves. Serum estradiol concentrations were decreased in a dose-dependent fashion (≥5 ng kg−1 wk−1) across the estrous cycle in perisenescent rats still displaying normal cyclic vaginal cytology. Serum FSH, LH, and progesterone profiles were unchanged by TCDD. The loss of reproductive cyclicity following chronic exposure to TCDD was not accompanied by decreased responsiveness to GnRH. Ovarian endocrine disruption is the predominant functional change preceding the premature reproductive senescence induced by chronic exposure to low doses of the AHR-specific ligand TCDD.

Effects of Acute and Chronic Exposure to the Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the Transition to Reproductive Senescence in Female Sprague-Dawley Rats

Abstract Activation of the aryl hydrocarbon receptor (AHR) can occur in polluted environments, either from smoking-related toxicants or from endogenous ligands. We tested whether acute or chronic exposure to the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the transition to reproductive senescence in female Sprague-Dawley rats. In experiment 1, rats (n = 6 per experimental group) received a single dose of 0 or 10 μg/kg of TCDD orally (p.o.) on Postnatal Day 29. Vaginal cytology was monitored for 1 wk each month until rats were killed at 1 yr of age. The single prepubertal exposure to TCDD hastened the transition to reproductive senescence in female rats and was associated with delayed puberty, abnormal cyclicity, and premature reproductive senescence. In a second experiment, rats were exposed to TCDD chronically through weekly dosing (0, 50, or 200 ng kg−1 wk−1 p.o., n = 7 each dose) beginning in utero. Lifelong exposure to these lower doses of TCDD induced a dose- and time-dependent loss of normal cyclicity and significantly hastened the onset of the transition to reproductive senescence (P < 0.05). This premature transition to reproductive senescence was associated with prolonged estrous cycles and, at the highest dose of TCDD, persistent estrus or diestrus. The number and size of ovarian follicles were not altered by TCDD. Diestrous concentrations of LH in rats exposed chronically to TCDD were similar to those in controls, whereas progesterone tended to be elevated at both doses of the dioxin (P < 0.08). Serum FSH was elevated in the group exposed to 50 ng/kg of TCDD (P < 0.02), whereas estradiol was decreased at both doses of dioxin (P < 0.01). Data thus far support endocrine disruption rather than depletion of follicular reserves as a primary mechanism of the premature transition to reproductive senescence following activation of the AHR pathway by TCDD in female rats.

The concentrations of catecholamines and oxytocin receptors in the oviduct and its contractile activity in cows during the estrous cycle

UNLABELLED In vitro experiments on oviducts of cyclic cows were undertaken to study: (1) the content of dopamine (DA), noradrenaline (NA) and adrenaline (A) in infundibulum, ampulla and isthmus, (2) the concentration of oxytocin receptors (OTR) in oviductal tissues and (3) the motility of ampulla and isthmus. Changes of DA content were observed in the infundibulum and the ampulla with maximal values occurring on Days 6-10 of the estrous cycle. The mean NA content was greatest in infundibulum<ampulla<isthmus. NA concentrations were the highest in the isthmus on Days 1-5 and 16-21, whereas in infundibulum NA levels were low during the whole estrous cycle. Mean A content was the highest (P<0.001) in the isthmus and in the studied regions of the oviduct A content decreased from Days 1-5 to Days 16-21 (P<0.01). Oxytocin receptors densities were measured in oviducts collected from cows on Days 16-21 (277.1+/-151.4 fmol/mg protein; K(d) 20.5+/-10.9 nM). Oxytocin (10(-7) M) increased the area under the contractile curve (AUC) of the ampulla and the isthmus on Days 16-21 and of the isthmus on Days 1-5 (P<0.01). Acetylcholine (Ach) (10(-7) M) stimulated the ampulla and the isthmus contractions on Days 1-5 (P<0.05) and ampulla contractions on Days 6-10 and 16-21 (P<0.01). NA (10(-5) M) relaxed the ampulla and the isthmus during most of the studied days but was most effective on Days 1-5 and 16-21 (P<0.01). IN CONCLUSION (a) catecholamine content in the bovine oviduct varies by region and phase of the estrous cycle, (b) the presence of OTR and the stimulatory effect of oxytocin on oviduct motility are evident in the follicular-phase cows, (c) Ach and NA modify contractile activity in the oviduct of cows during follicular and early-luteal phases and (d) the studied oviductal parameters did not differ by ipsilateral and contralateral relationships to the active ovary in cow.

Transcriptomic analysis of the porcine endometrium during early pregnancy and the estrous cycle

The goal of this study was to describe the alterations in the transcriptome of the endometrium in pigs during the beginning of implantation (days 15-16 of pregnancy) compared to cyclic pigs during the onset of luteolysis (days 15-16 of the estrous cycle). The global expression of genes in porcine gravid and non-gravid endometria was investigated using the Porcine (V2) Two-color gene expression microarray, 4 × 44 (Agilent, USA). Analysis of the microarray data showed that, of 589 accurately annotated genes, the expression of 266 genes was up-regulated and expression of 323 was down-regulated in the endometrium harvested during early pregnancy compared with the endometrium during the estrous cycle. In pregnant pigs, genes with the most significantly altered expression were involved in the following biological processes: the metabolic process, cellular process, cell communication, immune system process, developmental process, cell adhesion, antigen processing and presentation, antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class II, immune response, and the polysaccharide metabolic process. In the pregnant endometrium, cell adhesion molecules and steroid hormone biosynthesis pathways were the most significantly enriched biological pathways. Analysis of the interaction network among selected genes showed that androgen receptor (AR) encoding genes interact with genes involved in important processes occurring during early pregnancy. The bioinformatic analysis revealed information about the meaning of differentially expressed genes. The data provided new insight into the dynamic changes of the endometrial gene expression profile during days 15-16 of pregnancy.

Effect of an oxytocin antagonist on prostaglandin F2α secretion and the course of luteolysis in sows

The role of oxytocin (OT) in the regulation of prostaglandin F2 alpha (PGF2 alpha) secretion during luteolysis in gilts was studied using a highly specific OT antagonist (CAP-581). In Experiment 1 gilts on Days 14 to 19 of the oestrous cycle in Latin square design were used, to determine the dose and time of application of OT and CAP. In Group I (n = 6) gilts were treated intravenously with saline or with 10, 20 and 30 IU of OT. Concentrations of the main PGF2 alpha metabolite i.e. 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) were measured in blood samples as uterine response to the treatment. Twenty IU of OT was the most effective to stimulate PGFM release and this dose was used after CAP treatment in gilts of Groups II, III and IV. Gilts of Group II (n = 3) were injected into the uterine horns (UH) with saline (5 ml/horn) or CAP (2 mg, 3 mg and 4 mg; half dose/horn) and OT was injected (i.v.) 30 min thereafter. Any of the CAP doses given into the UH affected PGFM plasma concentrations stimulated by OT. In Group III (n = 4) gilts were infused (i.v.) for 30 min with CAP (9 mg, 14 mg and 18 mg/gilt) followed by 20 IU of OT. All doses of CAP effectively inhibited OT-stimulated PGF2 alpha release, therefore 9 mg was selected for the further studies. Gilts of Group IV (n = 4) received OT 4, 6 and 8 h after CAP to define how long CAP blocks the OT receptors. Concentrations of PGFM increased after any of this period of time. Thus, we concluded that 9 mg of CAP infused every 4 h will effectively block OT receptors. In Experiment 2, gilts (n = 4) received CAP as a 30-min infusion every 4 h on Days 12-20 of the oestrous cycle. Control gilts (n = 3) were infused with saline. CAP infusions diminished the height of PGFM peaks (P < 0.05). Frequency of the PGFM (P < 0.057) and OT (P < 0.082) peaks only tended to be lower in the CAP-treated gilts. Peripheral plasma concentrations of progesterone (P4) and oestradiol-17 beta (E2) and the time of luteolysis initiation as measured by the decrease of P4 concentration were the same in CAP- and saline-treated gilts. The macroscopic studies of the ovaries in gilts revealed lack of differences between groups. We conclude that OT is involved in the secretion of luteolytic PGF2 alpha peaks but its role is limited to controlling their height and frequency. Blocking of OT receptors did not prevent luteolysis in sows.

The effect of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) 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 1β (IL1β) 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. IL1β 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 IL1β on days 12-13 of pregnancy (P<0.05) and increased in response to IL1β, TNFα and IL6 on 12-13 (P<0.05) and 15-16 (P<0.01) of the estrous cycle. IL1β 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, IL1β, 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.

Endometrial and myometrial secretion of androgens and estrone during early pregnancy and luteolysis in pigs

UNLABELLED Previously, we found that in addition to embryos, the uterine tissues may be a source of estradiol-17beta (E(2)) during early pregnancy in the pig. The aim of the present study was to determine whether porcine endometrium and myometrium secrete androgens - androstenedione (A(4)), testosterone (T) and estrone (E(1)) during early pregnancy and luteolysis (Days 14-16) in pigs. Individual endometrial and myometrial slices (200 mg) were first pre-incubated (24 h) and then incubated (6 h, 37 degrees C in an atmosphere of 95% O(2) and 5% CO(2)) in the presence or absence of progesterone (P(4); 10(-5) M), oxytocin (OT; 10(-7) M) or OT plus P(4). Basal endometrial and myometrial secretion of A(4) and T did not differ between pregnant and cyclic gilts. Endometrial secretion of E(1) was higher in pregnant than cyclic gilts (p<0.05) while myometrial secretion of E(1) did not differ between the two groups of the examined pigs (p>0.05). Progesterone significantly increased A(4) and T secretion (p<0.001) by uterine tissues regardless of the reproductive status. In the presence of P(4), endometrial and myometrial secretion of E(1) was increased only during luteolysis (p<0.001). In both tissues, OT did not affect the examined steroid secretion and did not change the effect of P(4). IN CONCLUSION 1) porcine endometrium and myometrium was found to produce A(4), T and E(1) in vitro; 2) basal endometrial and myometrial production of A(4) and T did not differ between the examined reproductive periods; 3) the endometrium released more E(1) during early pregnancy than luteolysis; 4) in the presence of substrate (P(4)), uterine tissues increased secretion of A(4) and T during early pregnancy and luteolysis; and 5) P(4) increased uterine production of E(1) only during luteolysis. These data demonstrated the presence of the active steroid pathway in porcine endometrium and myometrium which may serve as an alternative source of androgens and estrogens in pigs.

Androgens and estradiol-17β production by porcine uterine cells: in vitro study.

Porcine (Sus scrofa domestica) uterine slices harvested during both early pregnancy and luteolysis produce steroid hormones. The aim of the present study was to determine (1) which porcine separated uterine cells secrete androgens: androstenedione (A(4)) and testosterone (T), and estradiol-17beta (E(2)) in culture; (2) if the production of A(4), T and E(2) in the uterine cells is regulated by P4 and OT; (3) if uterine tissues expressed cytochrome P450arom gene (CYP19). Uteri were collected on Days 14 to 16 of early pregnancy and the estrous cycle. Enzymatically separated epithelial cells, stromal cells, and myocytes were cultured in vitro for 2, 6, and 12h with control medium, progesterone (P(4); 10(-5) M), oxytocin (OT; 10(-7) M), and both hormones (P(4)+OT). The studied cells secreted A(4), T, and E(2) in vitro. Progesterone served as a substrate for steroid synthesis in the uterine cells. Isolated uterine cells, cultured separately, contributed in equal portion to the basal production of androgens (A(4) and T) during both early pregnancy and luteolysis. In pregnant pigs, the epithelial and stromal cells were rich sources of E(2) compared with myocytes. Myocytes produced E(2) mainly during luteolysis. Pregnant porcine endometrium and myometrium expressed the gene CYP19, which encodes for P450 aromatase, a steroidogenic enzyme. The results indicate an active steroidogenic pathway in porcine uterine cells. The epithelial cells, stromal cells, and myocytes participate in steroid production as an alternative source for their action in pigs.

The activity and localization of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase and release of androstenedione and progesterone by uterine tissues during early pregnancy and the estrous cycle in pigs.

Abstract Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3β-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3β-HSD mRNA, 2) uterine 3β-HSD protein activity and 3) in vitro production of A4 and P4 by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3β-HSD and the presence and activity of 3β-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A4 by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P4 did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P4 in pregnant pigs on days 12 to 13. The release of P4 by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs.

The effect of interleukin 1β and interleukin 6 on estradiol-17β secretion in the endometrium of pig during early pregnancy and the estrous cycle

Estrogens are produced by porcine embryos during early pregnancy. It was found that the uterus of pigs might be a source of steroid hormones, including estrogens. However, the factors involved in the regulation of endometrial steroidogenesis remain unknown. We hypothesize that interleukin (IL) 1β and IL6, which are cytokines produced by porcine embryos and uterine cells, might be involved in the regulation of endometrial estrogen synthesis. Porcine endometrial explants were harvested from gravid (N = 15) and cyclic (N = 15) pigs on days 10 to 11, 12 to 13, and 15 to 16. Samples were analyzed to determine: (1) the expression of CYP19 mRNA and the presence of aromatase cytochrome P450 protein in the tissue; and (2) the release of endometrial estradiol-17β (E2) in response to IL1β and IL6 after 6 and 12 hours of in vitro incubation. The effects observed in pregnant gilts were compared with the effects in nonpregnant gilts on corresponding days of the estrous cycle. On days 15 to 16 of pregnancy the expression of CYP19 in the endometrium was markedly decreased when compared with other periods, and the quantity of endometrial P450 aromatase protein was higher on these days than on days 12 to 13. In nongravid pigs, the expression of CYP19 was lower on days 15 to 16 when compared with days 12 to 13 and the quantity of P450 aromatase protein did not differ during the studied days of the estrous cycle. Basal endometrial E2 release was higher in pregnant gilts when compared with cyclic gilts only on days 15 to 16. In gravid pigs IL1β and IL6 did not affect endometrial E2 release on days 10 to 11 and 12 to 13 of pregnancy (P > 0.05); however, increased E2 release was observed on days 15 to 16 (P < 0.05). In cyclic pigs neither IL1β, nor IL6 affected endometrial E2 release (P > 0.05). These results provide evidence that: (1) the endometrium possesses a potential for steroidogenesis and produces E2in vitro in gravid and nongravid pigs between days 10 to 16; and (2) IL1β and IL6 increase in vitro endometrial synthesis of E2 in pregnant pigs on days 15 to 16. Therefore, IL1β and IL6 might act as stimulators of endometrial E2 secretion in vitro during the time of decreased production of embryonic estrogens. This correlates with a rapid remodeling of the endometrial tissue and the beginning of hemotrophic nutrition of the embryos occurring on days 15 to 16 of pregnancy.