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ST Leung, K Derecka, GE Mann, AP Flint, and DC Wathes

Both the production of cytokines and the distribution of immune cells within the uterus change during early pregnancy. Evidence obtained mainly from mice indicates that these changes are important for implantation and in preventing a maternal immune response to the conceptus. The ruminant embryo also produces interferon tau at this time, the signal for the maternal recognition of pregnancy. The relationship between these events in cows was studied using uteri from three groups of animals on day 16 after observed oestrus: (i) cyclic controls, (ii) pregnant and (iii) inseminated but with no embryo present. Embryo size and the antiviral activity in uterine flushings (indicative of the interferon tau concentration) were measured. Sections of intact uterus were frozen for the localization and quantitation of CD4(+) (T lymphocytes), CD14(+) (macrophages) and CD21(+) (B lymphocytes) uterine cells by immunohistochemistry. The expression of interleukin (IL)-1alpha, IL-2, IL-6 and IL-10 mRNAs in uterine extracts was measured by RT-PCR. Neither embryo size, interferon tau concentration nor pregnancy status influenced the distribution of CD4(+), CD14(+) or CD21(+) cells in the day 16 uterus. Endometrial IL-1alpha mRNA was detected in most cows across the groups, whereas IL-2 mRNA was only present in the non-pregnant uterus. IL-6 and IL-10 mRNAs were not detectable in any uteri. In conclusion, IL-2 mRNA expression is detectable in the non-pregnant but not the pregnant uterus on day 16 and interferon t is unlikely to play a role in the redistribution of immune cells in the uterus during early bovine pregnancy.

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Hsun-Ming Chang, Jung-Chien Cheng, Yingtao Liu, Christian Klausen, Congjian Xu, and Peter C K Leung

Lysyl oxidase (LOX) is the key enzyme involved in the crosslinking of collagen and elastin that is essential for the formation of extracellular matrix (ECM). LOX-mediated ECM remodeling plays a critical role in follicle development, oocyte maturation and corpus luteum formation. To date, the regulation of LOX in human ovary has never been elucidated. Activin A and its functional receptors are highly expressed in ovarian follicles from an early developmental stage. They locally regulate follicle progression. The aim of this study was to investigate the effects of activin A on the expression of LOX and its extracellular enzyme activity in primary and immortalized human granulosa–lutein cells obtained from patients undergoing an in vitro fertilization procedure. We demonstrated that activin A significantly upregulated the expression of connective tissue growth factor (CTGF) and LOX via an activin/TGF-β type I receptor mediated-signaling pathway. Using a target depletion small interfering RNA knockdown approach, we further confirmed that the upregulation of CTGF expression resulted in an activin-A-induced increases in LOX expression and activity. These findings may provide insight into the mechanisms by which intrafollicular growth factors regulate the expression of LOX for ECM formation and tissue remodeling in the human ovary.

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K. Leung, V. Padmanabhan, L. J. Spicer, H. A. Tucker, R. E. Short, and E. M. Convey

Summary. Thirty primiparous suckling beef cows were slaughtered on Day 7, 14, 28,42 or 56 after parturition. Some had resumed oestrous cyclicity by the time they were slaughtered on Days 42 and 56. Amongst acyclic cows between Days 7 and 42, pituitary LH concentrations and basal and GnRH-induced release of LH from pituitary explants doubled. Pituitary FSH concentration and basal release in FSH increased only by 15–20%, while GnRH-induced release of FSH in vitro was unchanged. During post-partum anoestrus, overall mean concentrations of serum FSH did not change, whereas overall mean concentrations and pulse amplitudes of serum LH increased. Numbers and affinity constants of GnRH-binding sites in pituitary glands remained constant during the post-partum period studied. We conclude that, under these experimental conditions, numbers and affinity constants of GnRH-binding sites in the pituitary gland of post-partum beef cows do not limit the ability of the anterior pituitary gland to release gonadotrophins.

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D. C. Wathes, H. Flick Smith, S. T. Leung, K. R. Stevenson, S. Meier, and G. Jenkin

The development of uterine oxytocin receptors is an important regulatory step in the initiation of labour. Paracrine production of oxytocin by uterine and placental tissues may also be involved in some species. Placentome, intercotyledonary endometrium, myometrium and fetal membranes were collected from 3–5 ewes each, at regular intervals throughout pregnancy and from eight ewes during labour. Localization of mRNA encoding oxytocin and its receptor was by in situ hybridization; oxytocin peptide concentrations were measured by radioimmunoassay and oxytocin receptor concentrations were measured by autoradiography and radioreceptor assay. In the intercotyledonary endometrium, mRNA encoding the oxytocin receptor was located in the luminal epithelium only. Both the epithelial and myometrial receptors were detected at low concentrations from the fourth week of gestation onwards, with a major increase associated with the onset of labour. In the placentomes, oxytocin receptors were localized to a stromal capsule surrounding the placental villi. Expression in this region was maximal in mid-gestation, declining in the second half of pregnancy and remaining low during labour. Cervical oxytocin receptors were detected at low concentrations in the epithelium and the muscular/connective tissue layers from day 22 of pregnancy onwards. There was no evidence for the local uterine production of oxytocin in the ewe; mRNA encoding oxytocin was undetectable and oxytocin concentrations were always <23 pg g−1 wet mass of tissue. These results suggest that regulation of the timing of oxytocin receptor development varies between the different tissue types, despite a similar steroidal background. The receptors in the luminal epithelium are probably associated with the ability of exogenous oxytocin to induce the release of PGF throughout most of pregnancy. The increase in receptors in both the intercotyledonary endometrium and myometrium at term suggest an involvement in labour, whereas their role in caruncular stroma in mid-pregnancy is unknown.

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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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Pang-Pin Liu, Hsun-Ming Chang, Jung-Chien Cheng, and Peter C K Leung

Activin A is one of the members of transforming growth factor-β superfamily that is expressed in human large luteal cells, and may act in an autocrine/paracrine manner to regulate luteal function. Prostaglandin-endoperoxide synthase 2 (PTGS2) enzyme and its derivative, prostaglandin E2 (PGE2), play significant roles in the regulation of corpus luteum formation and maintenance. To date, whether activin A can induce the expression of PTGS2 and the production of PGE2 in human granulosa-lutein cells is largely unknown. The aim of this study was to examine the effects of activin A on the regulation of PTGS2 expression and PGE2 production in human granulosa-lutein cells, and to investigate the underlying signal transduction mechanisms. In this study, the immortalized (SVOG cells) and primary human granulosa-lutein cells were used as the cell models. A TGF-β/activin type I receptor inhibitor, SB431542 and small interfering RNAs were used to investigate the activin A-induced downstream signaling pathway. We have demonstrated that activin A upregulated the expression of PTGS2 and increased the production of PGE2 via an ACVR1B-mediated SMAD2/3–SMAD4 signaling pathway. Our results suggest that activin A may be involved in the modulation of human corpus luteum formation via the induction of PTGS2 expression and PGE2 production.

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Hsien-Ming Wu, Hsin-Shih Wang, Hong-Yuan Huang, Yung-Kuei Soong, Colin D MacCalman, and Peter C K Leung

Type I GnRH (GnRH-I, GNRH1) and type II GnRH (GnRH-II, GNRH2), each encoded by separate genes, have been identified in humans. The tissue distribution and functional regulation of GnRH-I and GnRH-II clearly differ despite their comparable cDNA and genomic structures. These hormones exert their effects by binding to cell surface transmembrane G protein coupled receptors and stimulating the Gq/11 subfamily of G proteins. The hypothalamus and pituitary are the main origin and target sites of GnRH, but numerous studies have demonstrated that extra-hypothalamic GnRH and extra-pituitary GnRH receptors exist in different reproductive tissues such as the ovary, endometrium, placenta, and endometrial cancer cells. In addition to endocrine regulation, GnRH is also known to act in an autocrine and paracrine manner to suppress cell proliferation and activate apoptosis in the endometrium and endometrial cancer cells through several mechanisms. Both GnRH-I and GnRH-II exhibit regulatory roles in tissue remodelling during embryo implantation and placentation, which suggests that these hormones may have important roles in embryo implantation and early pregnancy. The presence of varied GnRH and GnRH receptor systems demonstrate their different roles in distinct tissues using dissimilar mechanisms. These may result in the generation of new GnRH analogues used for several hormone-related diseases.