Olga Jablonska

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.

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.

The expression of the aryl hydrocarbon receptor in reproductive and neuroendocrine tissues during the estrous cycle in the pig.

The aryl hydrocarbon receptor (AhR) has been recognized as a mediator of xenobiotic-induced toxicity. In addition, it was demonstrated that the AhR is able to influence the regulation of reproductive processes in females. The aim of this study was to examine AhR mRNA (real-time PCR) and protein (Western-blot) expression in ovarian follicles and stroma, corpora lutea (CL), oviducts, endometrium, myometrium as well as in medial basal hypothalami (MBH), and anterior (AP) and posterior (PP) pituitaries harvested during the follicular (days 17-19) and luteal (days 8-10) phase of the porcine estrous cycle. The AhR transcript and protein were found in all structures collected during both phases. AhR mRNA expression tended (p=0.06) to be higher in the CL than in follicles. The AhR protein expression in ovarian stroma was higher (p≤0.01) during the follicular than in the luteal phase. Endometrial expression of AhR mRNA was higher (p≤0.01), while AhR protein was lower (p≤0.01) during the follicular phase in comparison to the luteal phase. Within neuroendocrine tissues, AhR mRNA and protein content in hypothalamus were relatively low and did not differ (p>0.05) between phases. In contrast, higher AhR mRNA expression in AP (p≤0.001) and protein expression in PP (p≤0.01) were found during the luteal phase compared to the follicular phase. Differences in AhR expression observed in reproductive and neuroendocrine tissues during the follicular and luteal phase of the estrous cycle indicate the involvement of AhR in the regulation of reproductive function in pigs.

DAIDZEIN AFFECTS STEROIDOGENESIS AND OESTROGEN RECEPTOR EXPRESSION IN MEDIUM OVARIAN FOLLICLES OF PIGS

Daidzein, a phytoestrogen present in soybean products used in swine feed, has been demonstrated to affect both reproductive and endocrine functions. The aims of this study were to examine the in vitro effects of daidzein on (1) progesterone (P4) and oestradiol (E2) secretion by porcine luteinised granulosa cells harvested from medium follicles, and (2) the mRNA and protein expression of oestrogen receptors α and β (ERα and ERβ) in these cells. The influence of E2 on P4 secretion and ERα and ERβ expression in the granulosa cells of pigs was also investigated. It was found that daidzein inhibited progesterone secretion by luteinised granulosa cells isolated from medium follicles. In contrast, E2 did not affect progesterone production by these cells. Moreover, daidzein did not alter the granulosal secretion of E2. Both daidzein and E2 decreased mRNA expression of ERα in the cells examined. The expression of ERβ mRNA was not affected by daidzein but was inhibited by E2. ERα protein was not detected while ERβ protein was found in the nuclei of the cells. Daidzein and E2 upregulated the expression of ERβ protein in the cells. In summary, the phytoestrogen daidzein directly affected the porcine ovary by inhibiting progesterone production and increasing ERβ protein expression. Daidzein-induced changes in follicular steroidogenesis and granulosal sensitivity to oestrogens may disturb reproductive processes in pigs.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters steroid secretion but does not affect cell viability and the incidence of apoptosis in porcine luteinised granulosa cells.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a by-product of human industrial activity, was found to affect ovarian steroidogenesis in animals, but the mechanism of its action is still unclear. The aims of the study were to examine the effect of TCDD on (1) progesterone (P4) and oestradiol (E2) production by granulosa cells isolated from medium (3-6 mm) and preovulatory (≥ 8 mm) porcine follicles, (2) the viability of the cells, and (3) the incidence of apoptosis. Porcine granulosa cells were cultured (48 h) with or without TCDD (100 pM, 100 nM). Steroid hormone concentrations in the medium were determined by radioimmunoassay. The viability of granulosa cells was tested spectrophotometrically (alamarBlue™ assay). Apoptosis was evaluated by flow cytometry using Annexin V and by TUNEL assay. The higher dose of TCDD (100 nM) significantly inhibited P4 and stimulated E2 production by luteinised granulosa cells isolated from medium follicles. The lower dose of TCDD (100 pM) significantly stimulated P4 and inhibited E2 secretion by the cells isolated from preovulatory follicles. None of the two TCDD doses affected cell viability or induced apoptosis in granulosa cells. In conclusion, TCDD directly affected steroid production by granulosa cells obtained from mature pigs, but the effect of TCDD was not due to its cytotoxicity.

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces, Cobitidae)

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n = 48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n = 98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2 % triploids and 44.8 % tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2 days after hatching, approximately 65.1 % of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2 % on average during 2–5 and 10–15 months of life, respectively. The karyotype of tetraploid progeny (4n = 98) included 3n = 74 chromosomes of the parental female and n = 24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66 % of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.

Evidence of the sterility of allotetraploid Cobitis loaches (Teleostei, Cobitidae) using testes ultrastructure

The diploid-polyploid populations of Cobitis distributed in Poland are usually composed of the spined loach Cobitis taenia or, less often, the Danubian loach C. elongatoides and their triploid (females) and tetraploid hybrids (females and males). The aim of this study was to determine whether tetraploid males participate in the reproduction process by analyzing their testis ultrastructure and the process of spermatogenesis in comparison with diploid males of both parental species. Tetraploid loaches were obtained from three different diploid-polyploid populations distributed in Poland. The structure of Cobitis testes are typical for most Teleostei fish with cystic-type spermatogenesis. The successive stages of developing germ cells are enclosed within cysts formed by the Sertoli cells. This paper morphologically describes the different germ cell stages of spermatogenesis (spermatogonia, spermatocytes, spermatids, and spermatozoa) of C. taenia and C. elongatoides and provides a pioneering ultrastructural analysis of tetraploid Cobitis testes which reveals their unusual structure for the first time. Thus, cysts with normal spermatogonia and spermatocytes (pachyten or leptoten stages) containing synaptonemal complexes were present and no spermatids or spermatozoa were observed. Moreover, in contrast to previously analyzed diploid species, single cells or all of the cells within the cysts displayed chromatin condensation and/or chromatin fragmentation. The obtained results clearly demonstrated that tetraploid males are sterile and diploids are fertile and are the only sperm donors in the reproduction processes of diploid-polyploid Cobitis populations.

The combined effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phytoestrogen genistein on steroid hormone secretion, AhR and ERβ expression and the incidence of apoptosis in granulosa cells of medium porcine follicles

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor β (ERβ) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERβ and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.