11β-Hydroxysteroid dehydrogenase (11βHSD) enzymes modulate the target cell actions of corticosteroids by catalysing metabolism of the physiological glucocorticoid (GC), cortisol, to inert cortisone. Recent studies have implicated GCs in boar sperm apoptosis. Hence, the objective of this study was to characterise 11βHSD enzyme expression and activities in the boar testis and reproductive tract. Although 11βHSD1 and 11βHSD2 mRNA transcripts and proteins were co-expressed in all tissues, cortisol–cortisone interconversion was undetectable in the corpus and cauda epididymides, vas deferens, vesicular and prostate glands, irrespective of nucleotide cofactors. In contrast, homogenates of boar testis, caput epididymidis and bulbourethral gland all displayed pronounced 11βHSD activities in the presence of NADPH/NADP+ and NAD+, and the penile urethra exhibited NAD+-dependent 11β-dehydrogenase activity. In kinetic studies, homogenates of boar testis, caput epididymidis and bulbourethral gland oxidised cortisol with K m values of 237–443 and 154–226 nmol/l in the presence of NADP+ and NAD+ respectively. Maximal rates of NADP+-dependent cortisol oxidation were 7.4- to 28.5-fold greater than the V max for NADPH- dependent reduction of cortisone, but were comparable with the rates of NAD+-dependent cortisol metabolism. The relatively low K m estimates for NADP+ -dependent cortisol oxidation suggest that either the affinity of 11βHSD1 has been increased or the cortisol inactivation is catalysed by a novel NADP+-dependent 11βHSD enzyme in these tissues. We conclude that in the boar testis, caput epididymidis and bulbourethral gland, NADP+- and NAD+-dependent 11βHSD enzymes catalyse net inactivation of cortisol, consistent with a physiological role in limiting any local actions of GCs within these reproductive tissues.
Victoria Sharp, Lisa M Thurston, Robert C Fowkes and Anthony E Michael
Jordan E Read, Victoria Cabrera-Sharp, Victoria Offord, Samantha M Mirczuk, Steve P Allen, Robert C Fowkes and Amanda M de Mestre
Equine chorionic girdle trophoblast cells play important endocrine and immune functions critical in supporting pregnancy. Very little is known about the genes and pathways that regulate chorionic girdle trophoblast development. Our aim was to identify genes and signalling pathways active in vivo in equine chorionic girdle trophoblast within a critical 7-days window. We exploited the late implantation of the equine conceptus to obtain trophoblast tissue. An Agilent equine 44K microarray was performed using RNA extracted from chorionic girdle and chorion (control) from equine pregnancy days 27, 30, 31 and 34 (n = 5), corresponding to the initiation of chorionic girdle trophoblast proliferation, differentiation and migration. Data were analysed using R packages limma and maSigPro, Ingenuity Pathway Analysis and DAVID and verified using qRT-PCR, promoter analysis, western blotting and migration assays. Microarray analysis showed gene expression (absolute log FC >2, FDR-adjusted P < 0.05) was rapidly and specifically induced in the chorionic girdle between days 27 and 34 (compared to day 27, day 30 = 116, day 31 = 317, day 34 = 781 genes). Pathway analysis identified 35 pathways modulated during chorionic girdle development (e.g. FGF, integrin, Rho GTPases, MAPK) including pathways that have limited description in mammalian trophoblast (e.g. IL-9, CD40 and CD28 signalling). Rho A and ERK/MAPK activity was confirmed as was a role for transcription factor ELF5 in regulation of the CGB promoter. The purity and accessibility of chorionic girdle trophoblast proved to be a powerful resource to identify candidate genes and pathways involved in early equine placental development.