RESUMEN
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.
Asunto(s)
Envejecimiento/fisiología , Ciclo Estral/efectos de los fármacos , Ovario/efectos de los fármacos , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/agonistas , Reproducción/efectos de los fármacos , Animales , Femenino , Factores de Transcripción Forkhead/genética , Factor Nuclear 3-beta del Hepatocito/genética , Lactancia , Ovario/fisiopatología , Ovario/trasplante , Dibenzodioxinas Policloradas/farmacología , Embarazo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Ratas , Ratas Endogámicas LewRESUMEN
The first wave of follicular development following ovulation in cattle is characterized by selection and growth of a large, estrogenic dominant follicle. After the follicle becomes morphologically dominant, concentrations of estradiol in its follicular fluid decrease abruptly. The purpose of this study was to determine whether this decrease in estrogen production is caused by an insufficient supply of androgen from theca interna or decreased aromatization of androgen precursor by granulosa cells. Dominant follicles were collected from Holstein heifers on d 4, 6, or 8 of the first follicular wave (n = 5/d). Amounts of 17alpha-hydroxylase mRNA in theca interna were sevenfold higher (P < 0.01) on d 4 than on d 8. After 3 h in culture, secretion of androstenedione by theca interna collected on d 4 (236 +/- 44 pg/microg of protein) tended to be lower (P = 0.055) compared with d 6 (517 +/- 162 pg/microg protein) and was lower (P < 0.05) compared with d 8 (387 +/- 51 pg/microg of protein). In granulosa cells, amounts of aromatase mRNA decreased (P < 0.05) on d 8 compared with d 6 but not d 4. In vitro secretion of estradiol was higher in granulosa cells collected on d 4 (3.5 +/- 0.8 ng/[10(5) cells x 3 h]) compared with d 6 (1.8 +/- 0.6 ng/[10(5) cells x 3 h]; P < 0.05) and tended to be higher on d 4 than on d 8 (2.2 +/- 0.2 ng/[10(5) cells x 3 h]; P = 0.058). We conclude that the decrease in estradiol production observed during atresia of the dominant follicle is not due to lack of androgen substrate for aromatization or downregulated expression of the aromatase gene, but may be the direct result of decreased activity of the aromatase enzyme within granulosa cells.
Asunto(s)
Bovinos/fisiología , Estradiol/biosíntesis , Folículo Ovárico/fisiología , Células Tecales/fisiología , Androstenodiona/metabolismo , Animales , Estradiol/análisis , Estradiol/genética , Estradiol/metabolismo , Femenino , Líquido Folicular/química , Regulación de la Expresión Génica/fisiología , Células de la Granulosa/química , Células de la Granulosa/metabolismo , Células de la Granulosa/fisiología , Folículo Ovárico/química , ARN Mensajero/metabolismo , Reproducción/fisiología , Células Tecales/metabolismo , Factores de TiempoRESUMEN
Pregnancy-associated glycoprotein (PAG)-1 (PAG1) and pregnancy-specific protein B are either identical or closely related antigens released by trophoblast binucleate cells of placentas of cattle. Sheep and other ruminants produce similar products. There is evidence, however, that these antigens, which are related structurally to the pepsinogens and other aspartic proteinases, are not single gene products but members of an extensive family. Here, the sequential use of ammonium sulfate precipitation and Sepharose blue, anion-exchange, and cation-exchange chromatographies, as well as isoelectric elution from a Mono P column, has allowed several PAG1-related molecules to be purified from the medium after culture of explants from Day 100 sheep placentas. Each of these PAGs cross-reacted to a varying extent with a panel of three different anti-PAG1 antisera. Four of them, all of which were major secretory products of the placenta, were subjected to amino-terminal microsequencing. Although each was related to ovine (ov) PAG1, none was identical. Reverse transcription-polymerase chain reaction was then used to amplify PAG1-related cDNA from Day 100 placental RNA. Seven novel full-length cDNA, all distinct from ovPAG1, were identified from 25 cDNA selected for sequencing. Only two of these (ovPAG3 and ovPAG7) encoded polypeptides identical in sequence at their inferred amino termini to one of the PAGs (ovPAG65) purified from explant cultures. Even so, they were only 84% identical in overall sequence. The remaining five cDNA were unique. In situ hybridization analysis revealed that expression of ovPAG3 and ovPAG7, like that of ovPAG1, is confined to trophoblast binucleate cells. The data confirm that at Day 100 of pregnancy the ovine placenta produces many different PAGs, which differ considerably in sequence and immunological cross-reactivity.