Lyndsey Boswell

Bone Morphogenetic Proteins Are Mediators of Luteolysis in the Human Corpus Luteum

Bone morphogenetic proteins (BMPs), members of the transforming growth factor β (TGFβ) superfamily, play important roles in folliculogenesis in various species; however, little is known about their role in luteal function. In this study, we investigated the expression, regulation, and effects of BMP2, BMP4, and BMP6 in carefully dated human corpora lutea and cultured human luteinized granulosa cells. The mRNA abundance of BMPs was increased in the regressing corpus luteum in vivo (P<.01-.001). Human chorionic gonadotropin (hCG) down-regulated BMP2, BMP4, and BMP6 transcripts both in vivo (P=.05-.001) and in vitro (P<.001), and decreased the mRNA abundance of BMP receptors (BMPR1A, BMPR1B, BMPR2; P<.05-.01) in vitro. Three BMPs were regulated by differential signaling pathways. H89, a protein kinase A inhibitor, increased the expression of both BMP2 (P<.05) and BMP4 (P<.05) while decreasing BMP6 (P<.01). PMA, a protein kinase C activator, decreased both BMP4 and BMP6 expression (P<.0001) while enhancing the mRNA abundance of BMP2 (P<.01). BMPs significantly down-regulated transcripts for LH/choriogonadotropin receptor (LHCGR; P<.001) and steroidogenic acute regulatory protein (STAR; P<.001), whereas up-regulating those of follicular stimulating hormone receptor (FSHR; P<.01) and aromatase (CYP19A1; P<.05-.01) in vitro, possessing an effect opposite to hCG but similar to Activin A. Like Activin A, BMP4 and BMP6 stimulated the expression of Inhibin/Activin subunits with a marked effect on INHBB expression (P<.05-.01). These data confirm that BMPs are increased during luteal regression and negatively regulated by hCG via differential mechanisms, suggesting that BMPs are one of the mediators of luteolysis in women.

The Association between Smoking and Ectopic Pregnancy: Why Nicotine Is BAD for Your Fallopian Tube

Epidemiological studies have shown that cigarette smoking is a major risk factor for tubal ectopic pregnancy but the reason for this remains unclear. Here, we set out to determine the effect of smoking on Fallopian tube gene expression. An oviductal epithelial cell line (OE-E6/E7) and explants of human Fallopian tubes from non-pregnant women (n = 6) were exposed to physiologically relevant concentrations of cotinine, the principle metabolite of nicotine, and changes in gene expression analyzed using the Illumina Human HT-12 array. Cotinine sensitive genes identified through this process were then localized and quantified in Fallopian tube biopsies from non-pregnant smokers (n = 10) and non-smokers (n = 11) using immunohistochemistry and TaqMan RT-PCR. The principle cotinine induced change in gene expression detected by the array analysis in both explants and the cell line was significant down regulation (P<0.05) of the pro-apoptotic gene BAD. We therefore assessed the effect of smoking on cell turnover in retrospectively collected human samples. Consistent with the array data, smoking was associated with decreased levels of BAD transcript (P<0.01) and increased levels of BCL2 transcript (P<0.05) in Fallopian tube biopsies. BAD and BCL2 specific immunolabelling was localized to Fallopian tube epithelium. Although no other significant differences in levels of apoptosis or cell cycle associated proteins were observed, smoking was associated with significant changes in the morphology of the Fallopian tube epithelium (P<0.05). These results suggest that smoking may alter tubal epithelial cell turnover and is associated with structural, as well as functional, changes that may contribute to the development of ectopic pregnancy.

The Loss of Luteal Progesterone Production in Women Is Associated With a Galectin Switch via α2,6-Sialylation of Glycoconjugates

CONTEXT Luteal progesterone is fundamental for reproduction, but the molecular regulation of the corpus luteum (CL) in women remains unclear. Galectin-1 and galectin-3 bind to the sugar chains on cells to control key biological processes including cell function and fate. METHODS The expression and localization of LGALS1 and LGALS3 were analyzed by quantitative PCR and histochemical analysis, with special reference to α2,6-sialylation of glycoconjugates in carefully dated human CL collected across the menstrual cycle and after exposure to human chorionic gonadotrophin (hCG) in vivo. The effects of hCG and prostaglandin E2 on the expression of galectins and an α2,6-sialyltransferase 1 (ST6GAL1) in granulosa lutein cells were analyzed in vitro. RESULTS Galectin-1 was predominantly localized to healthy granulosa lutein cells and galectin-3 was localized to macrophages and regressing granulosa lutein cells. Acute exposure to luteotrophic hormones (hCG and prostaglandin E2) up-regulated LGALS1 expression (P < .001). ST6GAL1, which catalyzes α2,6-sialylation to block galectin-1 binding, increased during luteolysis (P < .05) as did LGALS3 (P < .05). Luteotrophic hormones reduced ST6GAL1 and LGALS3 in vivo (P < .05) and in vitro (P < .001). There was an inverse correlation between the expression of ST6GAL1 and HSD3B1 (P < .01) and a distinct cellular relationship among α2,6-sialylation, 3β-hydroxysteroid dehydrogenase, and galectin expression. CONCLUSIONS Galectin-1 is a luteotrophic factor whose binding is inhibited by α2,6-sialylation in the human CL during luteolysis. ST6GAL1 and galectin-3 expression is increased during luteolysis and associated with a loss of progesterone synthesis. Luteotrophic hormones differentially regulate galectin-1 and galectin-3/α2,6-sialylation in granulosa lutein cells, suggesting a novel galectin switch regulated by luteotrophic stimuli during luteolysis and luteal rescue.

In an Ovine Model of Polycystic Ovary Syndrome (PCOS) Prenatal Androgens Suppress Female Fetal Renal Gluconeogenesis

Increased maternal androgen exposure during pregnancy programmes a polycystic ovary syndrome (PCOS)-like condition, with metabolic dysfunction, in adult female offspring. Other in utero exposures associated with the development of insulin resistance, such as intrauterine growth restriction and exposure to prenatal glucocorticoids, are associated with altered fetal gluconeogenesis. We therefore aimed to assess the effect of maternal androgenisation on the expression of PEPCK and G6PC in the ovine fetus. Pregnant Scottish Greyface sheep were treated with twice weekly testosterone propionate (TP; 100mg) or vehicle control from day 62 to day102 of gestation. At day 90 and day 112 fetal plasma and liver and kidney tissue was collected for analysis. PEPCK and G6PC expression were analysed by quantitative RT-PCR, immunohistochemistry and western blotting. PEPCK and G6PC were localised to fetal hepatocytes but maternal androgens had no effect on female or male fetuses. PEPCK and G6PC were also localised to the renal tubules and renal PEPCK (P<0.01) and G6PC (P = 0.057) were lower in females after prenatal androgenisation with no change in male fetuses. These tissue and sex specific observations could not be explained by alterations in fetal insulin or cortisol. The sexual dimorphism may be related to the increase in circulating estrogen (P<0.01) and testosterone (P<0.001) in females but not males. The tissue specific effects may be related to the increased expression of ESR1 (P<0.01) and AR (P<0.05) in the kidney when compared to the fetal liver. After discontinuation of maternal androgenisation female fetal kidney PEPCK expression normalised. These data further highlight the fetal and sexual dimorphic effects of maternal androgenisation, an antecedent to adult disease and the plasticity of fetal development.

Expression and localization of inhibitor of differentiation (ID) proteins during tissue and vascular remodelling in the human corpus luteum

Members of the transforming growth factor-β (TGF-β) superfamily are likely to have major roles in the regulation of tissue and vascular remodelling in the corpus luteum (CL). There are four inhibitor-of-differentiation (ID1-4) genes that are regulated by members of the TGF-β superfamily and are involved in the transcriptional regulation of cell growth and differentiation. We studied their expression, localization and regulation in dated human corpora lutea from across the luteal phase (n = 22) and after human chorionic gonadotrophin (hCG) administration in vivo (n = 5), and in luteinized granulosa cells (LGCs), using immunohistochemistry and quantitative RT-PCR. ID1-4 can be localized to multiple cell types in the CL across the luteal phase. Endothelial cell ID3 (P < 0.05) and ID4 (P < 0.05) immunostaining intensities peak at the time of angiogenesis but overall ID1 (P < 0.05) and ID3 (P < 0.05) expression peaks at the time of luteolysis, and luteal ID3 expression is inhibited by hCG in vivo (P < 0.01). In LGC cultures in vitro, hCG had no effect on ID1, down-regulated ID3 (P < 0.001), and up-regulated ID2 (P < 0.001) and ID4 (P < 0.01). Bone morphogenic proteins (BMPs) had no effect on ID4 expression but up-regulated ID1 (P < 0.01 to P < 0.005). BMP up-regulation of ID2 (P < 0.05) was additive to the hCG up-regulation of ID2 expression (P < 0.001), while BMP cancelled out the down regulative effect of hCG on ID3 regulation. As well as documenting regulation patterns specific for ID1, ID2, ID3 and ID4, we have shown that IDs are located and differentially regulated in the human CL, suggesting a role in the transcriptional regulation of luteal cells during tissue and vascular remodelling.