R.S. Robinson

Inhibition of nuclear factor kappa B (NFκB) activity induces nerve growth factor-resistant apoptosis in PC12 cells

The mechanism(s) underlying nerve growth factor (NGF)‐mediated rescue of neurons from apoptosis is poorly understood, although it is well established that the high‐affinity NGF receptor (TrkA) plays a pivotal role in mediating NGF effects. The report that the low‐affinity NGF receptor (p75NGFR) can induce apoptosis prompted us to analyze the role played by a putative p75NGFR‐associated signal‐transduction element, the transcription factor nuclear factor kappa B (NFκB), in the modulation of apoptosis in PC12 cells. Here, we report that inhibition of NFκB function results in apoptosis of rat PC12 cells, a neuroblast‐like cell line model of NGF‐responsive neural tissues. Furthermore, NGF did not protect PC12 cells from cell death induced by the inhibition of NFκB. These results indicate that NFκB function is essential to maintain PC12 cell survival and to permit NGF‐mediated rescue, consistent with the idea that signaling elements potentially associated with both TrkA‐ and p75NGFR are involved in the regulation of apoptosis. J. Neurosci. Res. 47:155–162, 1997.

Angiogenesis and vascular function in the ovary

Ovarian function is dependent on the establishment and continual remodelling of a complex vascular system. This enables the follicle and/or corpus luteum (CL) to receive the required supply of nutrients, oxygen and hormonal support as well as facilitating the release of steroids. Moreover, the inhibition of angiogenesis results in the attenuation of follicular growth, disruption of ovulation and drastic effects on the development and function of the CL. It appears that the production and action of vascular endothelial growth factor A (VEGFA) is necessary at all these stages of development. However, the expression of fibroblast growth factor 2 (FGF2) in the cow is more dynamic than that of VEGFA with a dramatic upregulation during the follicular-luteal transition. This upregulation is then likely to initiate intense angiogenesis in the presence of high VEGFA levels. Recently, we have developed a novel ovarian physiological angiogenesis culture system in which highly organised and intricate endothelial cell networks are formed. This system will enable us to elucidate the complex inter-play between FGF2 and VEGFA as well as other angiogenic factors in the regulation of luteal angiogenesis. Furthermore, recent evidence indicates that pericytes might play an active role in driving angiogenesis and highlights the importance of pericyte-endothelial interactions in this process. Finally, the targeted promotion of angiogenesis may lead to the development of novel strategies to alleviate luteal inadequacy and infertility.

Corpus Luteum–Endometrium–Embryo Interactions in the Dairy Cow: Underlying Mechanisms and Clinical Relevance

Conception rates of dairy cows are currently declining at an estimated 1% every year. Approximately, 35% of embryos fail to prevent luteolysis during the first three weeks of gestation. Interactions between the corpus luteum, endometrium and embryo are critical to the successful establishment of pregnancy and inadequacies will result in the mortality of the embryo. For example, as little as a one day delay in the post-ovulatory rise of progesterone has serious consequences for embryo development and survival. Recently, we found that LH support, degree of vascularization and luteal cell steroidogenic capacity were not the major factors responsible for this luteal inadequacy, but are nevertheless essential for luteal development and function. Progesterone acting on its receptor in the endometrium stimulates the production of endometrial secretions on which the free-living embryo is dependent. However, their exact composition and effects of inadequate progesterone remains to be determined. The embryo is recognized through its secretion of interferon tau (IFNT), which suppresses luteolytic pulses of prostaglandin F(2 alpha). In the cow, it is most likely that IFNT inhibits oxytocin receptor up-regulation directly and does not require the prior inhibition of oestrogen receptor alpha (ESR1). Unravelling the precise luteal-endometrium and embryo interactions is essential for us to understand pregnancy establishment and development of strategies to reverse the declining fertility of dairy cows.

Fibroblast Growth Factor 2 Is More Dynamic than Vascular Endothelial Growth Factor A During the Follicle-Luteal Transition in the Cow

Abstract Luteal inadequacy is a major cause of infertility in a number of species. During the early luteal phase, progesterone production requires the rapid growth of the corpus luteum (CL), which is in turn dependent on angiogenesis. In the present study, we examined the temporal changes in vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) during the follicular-luteal transition and CL development in the cow. Luteal VEGFA concentrations increased as the CL developed but were lower in the regressing CL. Conversely, luteal FGF2 concentrations were highest immediately postovulation in the collapsed follicle and declined as the CL developed. Furthermore, three FGF2 isoforms were present in the collapsed follicle, but only one isoform was detected in older CL. Interestingly, FGF2 concentrations increased in the regressing CL. Western blot analysis for SPARC showed the presence of two isoforms, which were constitutively expressed throughout CL development. Further studies investigated the regulation of FGF2 by LH, which showed that FGF2 concentrations in preovulatory follicular fluid were higher in those animals that had experienced an LH surge. Moreover, LH stimulated FGF2 production in dispersed luteal cells. Conversely, the LH surge had no effect on follicular fluid VEGFA concentrations. In conclusion, FGF2 was more dynamic than VEGFA and SPARC during the follicular-luteal transition, which suggests that FGF2 plays a key role in the initiation of angiogenesis at this time. Furthermore, it is likely that this is stimulated by the LH surge. The results also suggest that VEGFA and SPARC have a more constitutive, but essential, role in the development of the CL vasculature.

FGF2 is crucial for the development of bovine luteal endothelial networks in vitro.

The development of the corpus luteum requires angiogenesis, and involves the complex interplay between factors such as vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). However, the relative role of these factors remains to be elucidated. This study used a new physiologically relevant mixed luteal cell culture system to test the hypotheses that: a) FGF2 and VEGFA are critical for bovine luteal angiogenesis; and b) local luteal PDGF signalling stimulates the formation of endothelial networks. Cells were treated with receptor tyrosine kinase inhibitors against VEGFA (SU1498), FGF2 (SU5402) or PDGF (AG1295) activity. After 9 days in culture, endothelial cells were immunostained for von Willebrand factor (VWF) and quantified by image analysis. Highly organised intricate endothelial networks were formed in the presence of exogenous VEGFA and FGF2. The inhibition of FGF2 activity reduced the total area of VWF staining versus controls (>95%; P<0.001). Inhibition of VEGF and PDGF activity reduced the endothelial network formation by more than 60 and 75% respectively (P<0.05). Progesterone production increased in all treatments from day 1 to 7 (P<0.001), and was unaffected by FGF2 or PDGF receptor kinase inhibition (P>0.05), but was reduced by the VEGF receptor inhibitor on days 5 and 7 (P<0.001). In conclusion, this study confirmed that VEGF signalling regulates both bovine luteal angiogenesis and progesterone production. However, FGF2 was crucial for luteal endothelial network formation. Also, for the first time, this study showed that local luteal PDGF activity regulates bovine luteal endothelial network formation in vitro.

Quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels

This study describes a digital technique for uterine morphometry and its application to endometrial structure during the bovine oestrous cycle. Neither the number nor the size of uterine gland ducts changed during the cycle but a reduction in total endometrial area from days 0 to 8 after oestrus led to an increase in the proportion of the endometrium occupied by gland ducts (gland duct density). This effect on day 8 was maintained to day 16. When endometrial morphology was related to circulating progesterone concentrations on days 5 and 8 of the luteal phase, no relationships were found on day 5, but on day 8, a high progesterone concentration was associated with an increased number of gland ducts. Furthermore, in animals slaughtered on day 8, a high progesterone concentration on day 5 was associated with decreased gland duct size, though a simultaneous decrease in endometrial area led to an increase in gland duct density. The results suggest that contrary to expectation, endometrial glands do not grow and regress during the oestrous cycle, although cyclic changes in endometrial area controlled by progesterone lead to changes in gland duct density.

A novel physiological culture system that mimics luteal angiogenesis

Luteal inadequacy is a major cause of poor embryo development and infertility. Angiogenesis, the formation of new blood vessels, is an essential process underpinning corpus luteum (CL) development and progesterone production. Thus, understanding the factors that regulate angiogenesis during this critical time is essential for the development of novel strategies to alleviate luteal inadequacy and infertility. This study demonstrates the development of a physiologically relevant primary culture system that mimics luteal angiogenesis. This system incorporates all luteal cell types (e.g. endothelial, steroidogenic cells, fibroblasts and pericytes). Using this approach, endothelial cells, identified by the specific marker von Willebrand factor (VWF), start to form clusters on day 2, which then proliferate and develop thread-like structures. After 9 days in culture, these tubule-like structures lengthen, thicken and form highly organized intricate networks resembling a capillary bed. Development of the vasculature was promoted by coating wells with fibronectin, as determined by image analysis (P<0.001). Progesterone production increased with time and was stimulated by LH re-enforcing the physiological relevance of the model in mimicking in vivo luteal function. LH also increased the area stained positively for VWF by twofold (P<0.05). Development of this endothelial cell network was stimulated by fibroblast growth factor 2 and vascular endothelial growth factor A, which increased total area of VWF positive staining on day 9, both independently (three- to fourfold; P<0.01) and in combination (tenfold; P<0.001). In conclusion, the successful development of endothelial cell networks in vitro provides a new opportunity to elucidate the physiological control of the angiogenic process in the developing CL.

Fibroblast growth factor 2 is a key determinant of vascular sprouting during bovine luteal angiogenesis

Fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF) A are thought to be key controllers of luteal angiogenesis; however, their precise roles in the regulation and coordination of this complex process remain unknown. Thus, the temporal and spatial patterns of endothelial network formation were determined by culturing mixed cell types from early bovine corpora lutea on fibronectin in the presence of FGF2 and VEGFA (6 h to 9 days). Endothelial cells, as determined by von Willebrand factor immunohistochemistry, initially grew in cell islands (days 0-3), before undergoing a period of vascular sprouting to display a more tubule-like appearance (days 3-6), and after 9 days in culture had formed extensive intricate networks. Mixed populations of luteal cells were treated with SU1498 (VEGF receptor 2 inhibitor) or SU5402 (FGF receptor 1 inhibitor) or control on days 0-3, 3-6 or 6-9 to determine the role of FGF2 and VEGFA during these specific windows. The total area of endothelial cells was unaffected by SU1498 treatment during any window. In contrast, SU5402 treatment caused maximal reduction in the total area of endothelial cell networks on days 3-6 vs controls (mean reduction 81%; P<0.001) during the period of tubule initiation. Moreover, SU5402 treatment on days 3-6 dramatically reduced the total number of branch points (P<0.001) and degree of branching per endothelial cell island (P<0.05) in the absence of changes in mean island area. This suggests that FGF2 is a key determinant of vascular sprouting and hence critical to luteal development.

Nerve Growth Factor, Central Nervous System Apoptosis, and Adrenocortical Activity in Aged Fischer-344/Brown Norway F1 Hybrid Rats

During aging there is a progressive loss of neuronal function in the basal forebrain that results in cognitive impairment and cholinergic deficits. While altered neurotrophin (NT)-mediated signal transduction may account for some age-associated deficits, there are differences in the extent of NT responsiveness among different laboratory rat strains. Here we measured nerve growth factor (NGF) protein levels and fragmented DNA in the CNS, and basal and NGF-stimulated activity levels of the hypothalamus-pituitary-adrenocortical axis (HPAA) in 3-, 18-, and 30-month-old Fischer-344/Brown Norway rats. Our results show that while there is no age-associated differences in NGF protein levels, in aged Fischer-344/Brown Norway rats, there are increases in levels of immunoreactive fragmented DNA in the CNS and in adrenocortical responses to the peripheral administration of NGF. These data contribute to the characterization of the Fischer-344/Brown Norway F1 hybrid rat and provide baseline values useful for future studies on aged CNS.

Assessing the influence of gender, learning style, and pre-entry experience on student response to delivery of a novel veterinary curriculum.

We investigated whether a novel veterinary curriculum was biased toward a particular gender, learning style, or pre-university experience (entry following undergraduate degree or direct entry from secondary school). We found no significant difference (p>0.05) in overall performance of first-year male, female, graduate-entry, or school-entry students. Students rated live-animal practical classes and facilitated problem-based learning as the most favored method of teaching, and this response was not biased by gender or pre-vet school experience. Men rated multiple-choice question (MCQ) assessment more highly than women, but there was no significant difference (p>0.05) in male or female performance on MCQ examinations. Men and women also performed comparably well in essays (both knowledge based and critical), suggesting that the retention of knowledge and depth of understanding was not gender biased. However, men performed significantly (p<0.05) better on critical essays compared with knowledge-based essays, and this trend was shown for both graduate-entry and school-entry students alike. We found no significant difference (p>0.05) in performance between groups of students with multimodal, kinesthetic, or reading-writing learning styles. Students with an auditory preference consistently performed less well in all types of assessment (p<0.05), but the number of students in this group was very small. Students whose learning style could not be specifically determined by Visual, Auditory, Read/write, Kinesthetic (VARK) tests consistently performed better than other groups, but this finding was not significant. Our results indicate that the Nottingham veterinary course does not bias for or against any of the variables we investigated.