A functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. Here we report a microarray analysis showing that treatment of cultured bovine theca cells (TC) with BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. RNAi-mediated knockdown of INSL3 reduced INSL3 mRNA and secreted protein level (75 and 94%, respectively) and elicited a 77% reduction in CYP17A1 mRNA level and 83% reduction in androstenedione secretion. Knockdown of RXFP2 also reduced CYP17A1 mRNA level (81%) and androstenedione secretion (88%). Conversely, treatment with exogenous (human) INSL3 increased androstenedione secretion ~2-fold. The CYP17 inhibitor abiraterone abolished androgen secretion and reduced expression of both INSL3 and RXFP2. Collectively, these findings indicate a positive autoregulatory role for INSL3 signaling in maintaining thecal androgen production, and visa versa. Moreover, BMP6-induced suppression of thecal androgen synthesis may be mediated, at least in part, by reduced INSL3-RXFP2 signaling

Leaf diseases of wheat

Trial 90PE10 Strategic variety mixtures to reduce septoria diseases of wheat. Evaluation of wheat lines for suitability to mixing for septoria control. Location: Mt. Barker Research Station To determine components of partial resistance to both Septoria species to ensure that lines with components that are complimentary are chosen for evaluation as mixtures for Septoria control. Trial 90MT12 and 90ME10 Assessing effects of a paired variety mixture to reduce Septoria. Location: Borden and Mt Barker Research Station. To assess the effects of a variety mixture on Septoria diseases. Trial 90MT11 and 90JE9 Evaluating variety mixtures to reduce septoria, using a range of crossbreds. Location: Mt. Barker Research Station To identify varietal combinations from a range of genotypes for using in mixtures to reduce Septoria diseases. Trial 90BA50 and 90MT60 Mixing varieties with a range of maturities for possible disease and agronomic benefits. Location: Badgingarra Research Station and Mt Barker Research Station. To evaluate disease control and other agronomic effects from growing varieties with different maturities as mixtures. Glasshouse screening for resistance to S. nodorum in wheat (with R.E. Wilson) During the year 16 sets of material were screened. Material comprised stage 1.2 and stage 2 lines as well as experimental Triticum tauschii material. Tests were conducted on Triticum tauschii lines and synthetic hexaploid derivatives. Derivatives are called synthetic because their production imitates the natural cross of durum with the primitive grass species %. tauschii which in theory resulted in the first bread wheat. Trial 90MT15 and 90E12 Time of planting and variety effects on septoria diseases of wheat. (with W. Smith, Esperance) Location: Mt. Barker Research Station and Esperance Downs Research Station. To establish earliest practical planting times for differing varieties to minimize the impact of Septoria diseases and maximize yield. Trial 90BA15 (90ES20 was discontinued because of very severe Rhizoctonia and absence of leaf disease) Location: Badgingarra Research Station. To compare a range of new products for control of Septoria diseases with the current standard chemical - TILT. Trial 90A4 Is seed infection a significant source of early inoculum of septoria nodorum? Location: Avondale Research Station. To evaluate the effect of seed borne infection of S. nodorum as early season inoculum. Are ascospores a significant source of infection of septoria nodorum? Location: Badgingarra and Mt Barker Research Station. To determine the extent of ascospore dispersal of Leptosphaeria nodorwn from wheat stubble. Unusual disease occurrence. Leaf rust (Puccinia recondita) of wheat Location: Esperance and Mt. Barker, Jerramungup Leaf rust has occurred in Western Australia, perhaps for the first time in over 10 years. A late wheat leaf rust epidemic developed along the south coast between Esperance and Mt Barker. It was first apparent at trace levels on early-sown crops in September around Esperance. Because rainfall was below average in many areas until October, the rust did not increase until late in the season. It reached yield-damaging levels in some later sown crops with severe epidemics observed in the Esperance and Jerramungup districts. The rust strain is new to Western Australia. Its mode of entry is unknown but is likely to have been by wind from South Australia and its appearance relatively early in 1990 suggests it reached Western Australia in 1989. It apparently remained undetected, surviving through a summer with favourable rains

Cell-free protein synthesis as a tool to study RXFP3- Relaxin-3 protein interactions

With the discovery of the relaxin family peptide receptors there is interest in obtaining a clearer understanding of the structure of these proteins and the molecular mechanism of receptor-ligand interaction. As G-protein coupled receptors, obtaining milligram quantities for structural investigations is hampered by the inherent instability of these integral membrane proteins. In the current context, understanding of GPCR structural biology has increased dramatically with crystal structures of several inactive and now active forms solved. In addition, the first nuclear magnetic resonance structure of a GPCR was obtained which is of crucial importance to studying these receptors in a more “biologically relevant” setting. However despite this expansion in the field, most structures have been solved on modified systems so as to increase stability and are not necessarily representative of the native receptors. In relation to the relaxin family peptide receptors, we chose to investigate relaxin-family peptide receptor-3 expressed by cellfree protein synthesis. In contrast to in-vivo expression, cell-free was capable of producing large amounts of native receptor which makes it amenable to demanding structural studies

Development of human cells with RXFP1 knockdown using retroviral delivery of microRNA against human RXFP1

To study the specifi c actions of relaxin through RXFP1 in human cells, it would be advantageous to develop cell populations with permanent RXFP1 knockdown (KD). We have developed and assessed four microRNA against human RXFP1. One of the four designed microRNA displayed signifi cant RXFP1 KD as assessed by reduced relaxin binding when co-transfected with human RXFP1 into HEK-293T cells. The selected microRNA sequence was subsequently retrovirally delivered into the human dermal fi broblast cell line BJ3 which natively expresses RXFP1. The RXFP1 KD BJ3 cells displayed diminished RXFP1 mRNA expression and complete loss of ability of relaxin treatment to reduce collagen deposition after TGF-β1 stimulation. The retroviral expression of miRNA to successfully silence RXFP1 expression is an invaluable tool to investigate receptor specifi city, signalling and possible off -target eff ects of newly developed relaxin analogs

Effect of diluent type, cryoprotectant concentration, storage method and freeze/thaw rates on the post-thaw quality and fertility of cryopreserved alpaca spermatozoa

This study compared protocols for cryopreservation of ejaculated, papain-treated alpaca spermatozoa. This included different concentrations of egg yolk (EY; 5, 10 or 15%) and glycerol (2, 5 or 10%), diluent types (SHOTOR, lactose, skim milk or INRA-96™), freeze rates (2, 4 or 8 cm above liquid nitrogen; LN), thaw rates (37 °C for 1 min or 42 °C for 20 sec) and storage vessels (pellets, 0.25 mL straws or 0.5 mL straws). Spermatozoa were assessed pre-freeze and 0, 30, 60 and 90 min post-thaw. Forty-one hembras were inseminated with either fresh, papain-treated or frozen-thawed spermatozoa. Motility was affected by EY concentration (P < 0.001), diluent type (P < 0.001), freeze rate (P = 0.003) and storage vessel (P = 0.001). Viability was affected by EY concentration (P < 0.001), diluent type (P < 0.001), storage vessel (P = 0.002) and thaw rate (P = 0.03). For artificial insemination (AI), semen was diluted 1:3 in a lactose-based diluent, with 5% EY and glycerol. Freezing was in 0.5 mL straws, 2 cm above LN for 4 min then thawing at 37 °C for 1 min. Pregnancy rates of those ovulated (n = 26) were not different (1/5 fresh, 1/4 papain-treated, 0/17 frozen-thawed; P = 0.10). Pregnancy can be achieved after AI with papain-treated spermatozoa. Further work is needed to determine the optimal dose, timing and location for insemination

Development of a scaffold displaying exoloops of RXFP1

Relaxin family peptide receptor 1 (RXFP1), the cognate receptor for relaxin, is a G-protein coupled receptor (GPCR) possessing a unique extracellular region consisting of a domain of 10 leucine rich repeats (LRRs) linked to an N-terminal low density lipoprotein Class A module. Relaxin binds to its receptor primarily by a high affinity interaction with the LRRs. An additional low-affinity interaction has been proposed to occur between relaxin and the the exoloops (ELs) of the transmembrane domain, however the molecular detail of this interaction remains undefined. While site directed mutagenesis and subsequent functional characterisation of these mutants traditionally allows identification of residues contributing to receptor function, in this case results are complicated by the presence of the high affinity binding site in the LRRs. To create a tool to investigate the low-affinity interaction, a protein scaffold system displaying exoloops 1 and 2 from RXFP1 was designed. This was achieved by inserting RXFP1 exoloops 1 and 2 into the native loops of a thermostabilised 6 kDa GB1 protein creating EL1/EL2-GB1. This protein has been expressed and purified in milligram quantities and used in conjunction with biophysical techniques such as NMR to explore relaxin binding to the exoloops of RXFP1

Benefits of a College STEM Faculty Development Initiative: Instructors Report Increased and Sustained Implementation of Research-Based Instructional Strategies

The Summer Institutes on Scientific Teaching (SI) is a faculty development workshop in which science, technology, engineering, and mathematics (STEM) instructors, particularly from biology, are trained in the Scientific Teaching (ST) pedagogy. While participants have generally reported positive experiences, we aimed to assess how the SI affected participants’ teaching practices. Building on a previously developed taxonomy of ST practices, we surveyed SI participants from the 2004–2014 SI classes regarding specific ST practices. Participants’ self-reported use and implementation of ST practices increased immediately after SI attendance as well as over a longer time frame, suggesting that implementation persisted and even increased with time. However, instructors reported implementation gains for some practices more than others. The practices with the highest gains were engaging students in their own learning, using learning goals in course design, employing formative assessment, developing overarching course learning goals, representing science as a process, and facilitating group discussion activities. We propose that the ST practices showing the greatest gains may serve as beneficial focal points for professional development programs, while practices with smaller gains may require modified dissemination approaches or support structures