Short Communication Microsatellite loci in the tetraploid spined loach, Cobitis biwae, and cross-species amplification in four related species.

Fifteen microsatellite loci were identified in the tetraploid spined loach, Cobitis biwae (Teleostei: Cobitidae). Among these, 14 were polymorphic (5-31 alleles) and showed moderate to high cross-species amplification transferability in four related species, Cobitis matsubarai, Cobitis taenia, Misgurnus anguillicaudatus, and Misgurnus fossilis. The loci, described herein, will be useful for population genetics, phylogeny, parentage analysis, and detection of hybridization among Cobitis species.

The expression of aryl hydrocarbon receptor in porcine ovarian cells.

The aryl hydrocarbon receptor (AHR) is an intracellular transcription factor best known for mediating the toxicity of dioxins. The AHR is phylogenetically highly conserved among invertebrates and vertebrates and may play a significant role in the regulation of physiological processes including female reproduction. This study was performed to determine the partial nucleotide sequence of porcine AHR and to evaluate the AHR mRNA (real-time PCR) and AHR protein (Western blot) expression in granulosa and theca interna cells harvested from medium (3-6 mm) and pre-ovulatory (≥8 mm) follicles as well as in luteal cells obtained from corpora lutea collected during the mid-luteal phase (days: 8-10) of the porcine oestrous cycle. In the study, the partial nucleotide sequence of porcine AHR containing 1021 bp (GenBank accession no: HM488957.1) was determined. The AHR transcript and protein were found in all ovarian cells obtained during both phases of the porcine oestrous cycle. The highest AHR transcript level was detected in theca interna cells isolated from pre-ovulatory follicles as well as in luteal cells. Higher AHR protein expression was found in granulosa cells isolated from pre-ovulatory follicles in comparison with all remaining cell types. The presence of AHR in the examined ovarian cells may account for their sensitivity to some environmental pollutants. Moreover, the differences found in AHR mRNA expression between granulosa and theca cells as well as between cells originating from follicles of different size suggest the involvement of AHR in the modulation of reproductive processes in the porcine ovary.

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.

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.