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K Yang

During mammalian pregnancy, the circulating concentration of cortisol (in rodents, corticosterone) in the mother is much higher than that in the fetus. Since the placenta is the only barrier, apart from the uterus, between the mother and her fetus, this gradient in cortisol concentrations suggests that there is a placental barrier preventing maternal cortisol from crossing into the fetus. The intracellular enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) is an ideal candidate for this barrier because it interconverts cortisol and corticosterone to their inactive metabolites cortisone and 11-dehydrocorticosterone. Indeed, 11 beta-HSD enzyme is expressed in the placenta of humans and a range of other animal species. Moreover, it is well positioned to serve as the barrier since it is localized to the syncytiotrophoblast, the site of maternal-fetal exchange. Given that fetal exposure to excessive amounts of glucocorticoids leads to intrauterine growth retardation, it has been hypothesized that the physiological significance of this placental 11 beta-HSD barrier is to protect the fetus from adverse effects of maternal glucocorticoids.

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D J Kwon, C K Park, B K Yang and H T Cheong

We attempted to control the nuclear remodelling of somatic cell nuclear transfer embryos (NTs) and examined their subsequent development and DNA methylation patterns in pigs. Porcine foetal fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h. After activation, NTs were cultured in vitro for 6 days to examine their development. The nuclear remodelling type of the reconstituted embryos was evaluated 1 h after fusion. Methylated DNA of in vitro-fertilised (IVF) embryos and NTs at various developmental stages and of donor cells was detected using a 5-methylcytosine (5-MeC) antibody. Caffeine-treated NTs induced premature chromosome condensation at a high rate (P<0.05), whereas most vanadate-treated NTs formed a pronucleus-like structure. Although cleavage rates to the two-cell stage did not differ among groups, delayed cleavage was observed in the vanadate-treated group. The blastocyst formation rate was significantly reduced by vanadate treatment compared with caffeine-treated and non-treated (control) NT groups (P<0.05). The apoptotic cell index of NT blastocysts was lower in the caffeine-treated group than in other groups (P<0.05). The methylation patterns were similar among NTs, but more hypermethylated DNA was observed at the four-cell stage of control and vanadate-treated NTs when compared with that in IVF embryos (P<0.05). Thus, the nuclear remodelling type controlled by caffeine or vanadate treatment can affect in vitro development and the methylation status of NTs in relation to nuclear reprogramming.

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Z-M. Yang, S-P. Le, D-B. Chen, K. Yasukawa and M. J. K. Harper

The presence of leukaemia inhibitory factor (LIF) binding and expression of the gp130 component of the LIF receptor were studied in the rabbit uterus during early pregnancy. LIF binding to myometrium was moderate in oestrous and non-oestrous animals and on day 1 of pregnancy, declined on days 2 and 3, and increased to a peak value on days 5 and 6 of pregnancy. Binding to stromal cells was not observed. Binding of LIF to luminal and glandular epithelium was low in unmated animals and on days 1 and 2 of pregnancy. Binding to luminal epithelium increased from day 3, and to glandular epithelium from day 5 of pregnancy. Highest binding was seen on days 5 and 6, with a slight decline observed on day 7, and with little difference between the mesometrial and antimesometrial regions of the implantation site. In all cases, binding of LIF was similar in the uteri of day 6 pseudopregnant and pregnant animals. At all stages, gpl30 was absent from stroma and almost absent from myometrium and glandular epithelium. It was expressed in luminal epithelium, reaching maximal expression on day 6 of pregnancy and pseudopregnancy, but diminished on day 7 of pregnancy, particularly in the antimesometrial area of the implantation site. The coexpression of LIF binding and gpl30 may indicate the presence of high-affinity LIF receptor, which matches the pattern of LIF protein expression and, as in mice, suggests its importance for implantation.

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F. Du, J. R. Giles, R. H. Foote, K. H. Graves, X. Yang and R. W. Moreadith

Rabbit embryonic stem-like cells, characterized by embryoid body formation and differentiation into cell types representative of all three germ layers, were studied for their ability to promote early embryonic development after nuclear transfer. After culture of the reconstructed embryos, 23% (n = 35) developed successfully into morulae or blastocysts, compared with 34% (n = 62) for cloned embryos derived from nuclear transfer with embryonic blastomeres. The cloned embryos from the embryonic stem-like cells appeared normal, with an average of 26% inner cell mass cells, similar to that of control non-manipulated embryos (25%) or cloned embryos from blastomeres (25%). Thus, nuclear transfer of rabbit embryonic stem-like cells leads to early embryonic development that is indistinguishable from blastomere fusion. These results have implications for the development of gene targeting in a species (rabbit) that may be a more suitable model for studying certain human diseases. In addition, this technique may be applicable to other species from which putative embryonic stem cells have been derived, particularly agriculturally important animals.

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A. N. Brooks, N. B. Haynes, K. Yang and G. E. Lamming

Summary. In June, 16 mature ewes were ovariectomized and allocated to four groups: 1, saline; 2, naloxone; 3, progesterone implant plus naloxone; 4, oestrogen implant plus naloxone. Steroids were implanted at the time of ovariectomy. At 5 days after ovariectomy, the animals were intravenously infused with saline for 8 h and naloxone (50 mg/h) in saline for 8 h the following day. Three intact ewes were given naloxone in a similar way. During infusions and for 8 h on the day after naloxone, jugular venous blood samples were taken every 15 min and assayed for LH.

Naloxone resulted in significant increases in mean LH concentration (P < 0·01), LH episode frequency and episode height (P < 0·05) in Group 3 ewes, but was without effect in any other group. These results provide evidence that the progesterone status of the ewe affects its response to naloxone, that progesterone negative feedback on LH release may be mediated by an opioid system, and that increased oestradiol negative feedback during seasonal anoestrus is unlikely to work via increased opioid inhibition of LH.

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K. Yang, N. B. Haynes, G. E. Lamming and A. N. Brooks

Summary. The opioid antagonist WIN-44441-3 (WIN-3, Sterling-Winthrop) caused significant increases in LH secretion in ovariectomized ewes treated with progesterone but not in ovariectomized animals treated with oestradiol-17β. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17β or oestradiol-17β and progesterone together were not affected by treatment with WIN-3 on Day 6 after ovariectomy (there was a significant increase in LH as a result of WIN-3 treatment 13 days after ovariectomy in sheep given no steroid therapy). However, WIN-3 treatment of ovariectomized sheep given progesterone resulted in a significant increase in plasma LH. WIN-3 was ineffective when given to intact ewes treated with progesterone during the non-breeding season. With ovariectomized sheep during the breeding season there was again no response to WIN-3 at 6 days after ovariectomy in sheep given oestradiol-17β, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17β. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17β did not result from a reduced pituitary response to GnRH since such animals responded normally to exogenous GnRH treatment. Overall, these results are consistent with the idea that, irrespective of the time of year, progesterone exerts negative feedback upon LH release at least in part through an opioidergic mechanism, whereas oestradiol-17β exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17β during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.

Keywords: opioids; LH; oestradiol-17β; progesterone; ewes

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K-P. Yang, G. E. Lamming, N. B. Haynes and A. N. Brooks

Summary. In May mature seasonally anoestrous ewes were implanted with melatonin which advanced the onset of cycles by about 1 month. The LH response to an opioid antagonist, WIN-3, was determined 5, 15, 25 and 60 days after melatonin implantation, by intravenous administration of WIN-3 (12·5 mg/dose) 4 times at 15-min intervals during both the 1st and the 5th hour of an 8-h treatment period. There was no effect of WIN-3 at 5, 15 and 25 days after melatonin implantation. At 60 days LH concentration and pulse frequency were significantly increased (P < 0·05 and <0·01 respectively) in response to WIN-3 treatment, but only in those animals which had begun reproductive cycles, an effect known to be mediated by the presence of progesterone. We were therefore unable to find evidence to support the hypothesis that the influence of melatonin in advancing the breeding season may be via an opioidergic pathway.

Keywords: opiods; LH; melatonin; ewes

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LJ Xiao, HL Diao, XH Ma, NZ Ding, K Kadomatsu, T Muramatsu and ZM Yang

Basigin is essential for fertilization and implantation. The aim of this study was to determine the expression and hormonal regulation of the basigin gene in the rat uterus during the peri-implantation period. Basigin mRNA was localized strongly in the luminal epithelium on day 1 of pregnancy and gradually decreased to a basal concentration from day 3 to day 5 of pregnancy. Basigin mRNA and protein were expressed strongly in the implanting blastocyst and primary decidua on day 6 of pregnancy. A similar expression pattern was also induced in the uterus after delayed implantation was terminated by oestrogen treatment and the embryo implanted, whereas expression was not detected during delayed implantation. Basigin expression was not detected on day 6 of pseudopregnancy. Basigin mRNA was expressed strongly in the decidua on days 7 and 8 of pregnancy. Furthermore, both basigin mRNA and protein were induced in the decidua during artificial decidualization. In addition, oestrogen stimulated strong expression of basigin mRNA in the uterine epithelium of ovariectomized rats. These findings indicate that basigin may play a role during implantation and decidualization in rats.

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Ying Fang, Hsun-Ming Chang, Jung-Chien Cheng, Christian Klausen, Peter C K Leung and Xiaokui Yang

Lysyl oxidase (LOX), a key enzyme in the formation and stabilization of the extracellular matrix, is expressed in granulosa cells and plays a critical role in the regulation of granulosa cell differentiation, oocyte maturation and ovulation. To date, the regulation of LOX expression in human granulosa cells remains largely unknown. In this study, using primary and immortalized human granulosa lutein cells, we demonstrated that transforming growth factor (TGF)-β1 (TGFB1) upregulated LOX expression and downregulated microRNA-29a (MIR29A) expression via a TGF-β type I receptor-mediated signaling pathway. Additionally, we showed that MIR29A downregulated the expression of LOX in both types of cells. Furthermore, the downregulation of MIR29A contributed to the TGFB1-induced increase in LOX expression because the inhibition of MIR29A with a MIR29A inhibitor not only reversed the MIR29A-induced downregulation of LOX but also enhanced the TGFB1-induced upregulation of LOX. Our findings suggest that TGFB1 and MIR29A may play essential roles in the regulation of extracellular matrix remodeling during the periovulatory phase.

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G. B. Kudolo, Y-Q. Yang, D-B. Chen, M. A. Jones and M. J. K. Harper

Significant changes in platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) concentration have been observed in rabbit endometrium during the preimplantation period, but, under in vitro conditions, constitutive PAF biosynthesis by isolated endometrial tissues was not easily demonstrable. Relative changes in enzymes involved in the synthesis and metabolism of PAF in the tissues may account for this disparity. In addition, during this period of preimplantation, marked changes in PAF receptor concentration have been noted. The present study examines the factors that may modulate the metabolism of exogenous [3H]PAF in the endometrium of rabbits on day 6 of pregnancy. Since preferential [3H]PAF binding in situ by the glandular epithelial, but not by the stromal, cells was demonstrated, their cell-specific metabolism of exogenous [3H]PAF was also examined. After entry into the endometrial cell, [3H]PAF was rapidly metabolized by the sequential action of cytosolic Ca2+-independent acetylhydrolase to [3H]lyso-PAF and this was in turn acylated by membrane-associated transacylase to [3H]alkylacylglycerylphosphorylcholine. PAF resynthesis was not observed and, in stromal cells, there was a significant build-up of [3H]lyso-PAF, suggesting that lyso-PAF:acetyl-CoA acetyltransferase may be a limiting factor. In the glandular epithelial cells, however, there was a significant accumulation of a neutral lipid without a significant build-up of [3H]lyso-PAF or [3H]PAF. The neutral lipid co-migrated with the product of phospholipase C-catalysed metabolism of PAF and authentic 1-O-hexadecyl-2-acetyl-glycerol. In addition, the elution times of phospholipase C digestion of C18 PAF and the neutral lipid produced by cellular metabolism of [3H]PAF, determined by gas chromatography/flame ionization detection, were similar. It seems that it is the synthesis of the neutral lipid from reacetylated [3H]lyso-PAF that prevented [3H]PAF accumulation under in vitro conditions. This is the first documentation of the synthesis of this lipid in the mammalian uterus. The lipid may serve as the precursor for de novo PAF synthesis in the glandular epithelial cells during endometrial proliferation.