Morphometric and gene expression analyses of stromal expansion during development of the bovine fetal ovary

During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n = 27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P  0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P < 0.05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.M.D. Hartanti, A K. Hummitzsch, H.F. Irving-Rodgers, W.M. Bonner, K.J. Copping, R.A. Anderson, I.C. McMillen, V.E.A. Perry and R.J. Rodger

Morphometric and gene expression analyses of stromal expansion during development of the bovine fetal ovary

During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n = 27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P  0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P < 0.05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.M.D. Hartanti, A K. Hummitzsch, H.F. Irving-Rodgers, W.M. Bonner, K.J. Copping, R.A. Anderson, I.C. McMillen, V.E.A. Perry and R.J. Rodger

Endothelial dysfunction of bypass graft: Direct comparison of In Vitro and In Vivo models of ischemia-reperfusion injury

BACKGROUND: Although, ischemia/reperfusion induced vascular dysfunction has been widely described, no comparative study of in vivo- and in vitro-models exist. In this study, we provide a direct comparison between models (A) ischemic storage and in-vitro reoxygenation (B) ischemic storage and in vitro reperfusion (C) ischemic storage and in-vivo reperfusion. METHODS AND RESULTS: Aortic arches from rats were stored for 2 hours in saline. Arches were then (A) in vitro reoxygenated (B) in vitro incubated in hypochlorite for 30 minutes (C) in vivo reperfused after heterotransplantation (2, 24 hours and 7 days reperfusion). Endothelium-dependent and independent vasorelaxations were assessed in organ bath. DNA strand breaks were assessed by TUNEL-method, mRNA expressions (caspase-3, bax, bcl-2, eNOS) by quantitative real-time PCR, proteins by Western blot analysis and the expression of CD-31 by immunochemistry. Endothelium-dependent maximal relaxation was drastically reduced in the in-vivo models compared to ischemic storage and in-vitro reperfusion group, and no difference showed between ischemic storage and control group. CD31-staining showed significantly lower endothelium surface ratio in-vivo, which correlated with TUNEL-positive ratio. Increased mRNA and protein levels of pro- and anti-apoptotic gens indicated a significantly higher damage in the in-vivo models. CONCLUSION: Even short-period of ischemia induces severe endothelial damage (in-vivo reperfusion model). In-vitro models of ischemia-reperfusion injury can be limitedly suited for reliable investigations. Time course of endothelial stunning is also described

Localization of the trace elements iron, zinc and selenium in relation to anatomical structures in bovine ovaries by X-ray fluorescence imaging

X-ray fluorescence (XRF) was used to image 40 histological cross-sections of bovine ovaries (n=19), focusing on structures including: antral follicles at different stages of growth or atresia, corpora lutea at three stages of development (II-IV), and capillaries, arterioles, and other blood vessels. This method identified three key trace elements [iron (Fe), zinc (Zn), and selenium (Se)] within the ovarian tissue which appeared to be localized to specific structures. Owing to minimal preprocessing of the ovaries, important high-resolution information regarding the spatial distribution of these elements was obtained with elemental trends and colocalizations of Fe and Zn apparent, as well as the infrequent appearance of Se surrounding the antrum of large follicles, as previously reported. The ability to use synchrotron radiation to measure trace element distributions in bovine ovaries at such high resolution and over such large areas could have a significant impact on understanding the mechanisms of ovarian development. This research is intended to form a baseline study of healthy ovaries which can later be extended to disease states, thereby improving our current understanding of infertility and endocrine diseases involving the ovary.Melanie J. Ceko, Katja Hummitzsch, Wendy M. Bonner, Jade B. Aitken, Kathryn M. Spiers, Raymond J. Rodgers and Hugh H. Harri

X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function

Corrected by: Erratum: X-Ray fluorescence imaging and other analyses identify selenium and GPX1 as important in female reproductive function, in Vol. 7, Issue 1, 188. The author H. H. Harris is listed with affiliation ‘c’, however, he should be listed with affiliation ‘a’; i.e. ‘The University of Adelaide’. The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.Studies of selenium (Se) status indicate that Se is necessary for fertility but how precisely is not known. We aimed to show that Se was important in bovine female reproductive function. The elemental distribution in the bovine ovary (n = 45 sections) was identified by X-ray fluorescence (XRF) imaging. Se was consistently localized to the granulosa cell layer of large (>10 mm) healthy follicles. Inductively Coupled Plasma - Mass Spectrometry revealed tenfold higher Se in the bovine follicle wall compared to corpora lutea. Gene expression analysis of selenoprotein genes GPX1, GPX3, VIMP and SELM in bovine granulosa cells revealed that only GPX1 was significantly up-regulated in large healthy follicles compared to the small healthy or atretic follicles (P < 0.05). Western immunoblotting identified GPX1 protein in bovine granulosa cells of large healthy follicles, but not of small healthy follicles. To assess if GPX1 was important in human follicles, cumulus cells from women undergoing IVF/ICSI with single embryo transfer were collected. Oocytes and embryos were cultured and transferred independently in 30 patients undergoing elective single embryo transfer. Gene expression of GPX1 was significantly higher in human cumulus cells from cumulus-oocyte complexes yielding a pregnancy (P < 0.05). We present the first XRF imaging of mammalian ovaries showing that Se is consistently localized to the granulosa cells of large healthy follicles. We conclude that Se and selenoproteins are elevated in large healthy follicles and may play a critical role as an antioxidant during late follicular development.M. J. Ceko, K. Hummitzsch, N. Hatzirodos, W. M. Bonner, J. B. Aitken, D. L. Russell, M. Lane, R. J. Rodgers and H. H. Harri