bluuebunny/crossref_metadata_2025 huggingface dataset:
419. Rational design of bifunctional siRNAs that silence genes and recruit the innate immune system to treat ectopic pregnancies
Authors: Tong S. , Gantier M. P. , Belhke M. , Williams B. R. G.
Published: November 2010

RNA interference (RNAi) is a new therapeutic approach, silencing genes to disrupt diseases. However, short interfering siRNAs (molecule used in RNAi) can have off-target effects, activating the immune system through RNA sensing toll-like receptors (TLR) 3, 7 and 8. We have previously proposed that in some diseases (cancers, ectopic pregnancies) it may be useful to enhance the immune response. A novel class of immunostimulatory siRNAs could be developed, silencing genes important to disease and recruiting the immune system to further aid disease clearance. We set out to develop a rational design strategy that enhances immunostimulatory properties to any siRNA sequence but maintains effective gene silencing. We screened a set of siRNAs targeting lamin. All were of the same sequence, except for different immunostimulatory motifs on the 3′ end of the sense strand. We also investigated a different design where we added a small micro-RNA like poly-uridine bulge (potentially immunostimulatory) on the sense strand. We used human peripheral blood mononuclear cells (PBMCs) to test for immunostimulation, and HEK 293-T-cells to test for lamin gene knockdown.Of all strategies tested, the poly-uridine bulge was best. It silenced the lamin gene as effectively as control, but caused a 2–3 fold increase of IFN-α and TNF-α. We verified this approach by adding the poly-uridine bulge onto an siRNA of low immunostimulatory potential targeting GFP. The bulge markedly enhanced immunostimulation in a dose response manner, and did not compromise gene knockdown. The addition of a poly-uridine bulge to siRNAs can increase immunostimulation without affecting gene silencing efficacy. Immunostimulatory siRNAs might be particularly efficacious to treat ectopic pregnancies where there are abundant immune cells, and functional TLR 7/8 in the trophoblast (unpublished observations). We now plan to test this immunostimulatory siRNA approach in an in vivo ectopic pregnancy model using JEG-3 cells xenographted in NOD-SCID mice.
420. The effect of maternal dietary protein on the expression of genes involved in TGF-β superfamily bioavailability in the bovine ovarian cortex of female offspring
Authors: Rodgers R. , Prodoehl M. , Irving-Rodgers H. , Sullivan T. , Micke G. , Bonner W. , Gibson M. , Perry V.
Published: November 2010

Although numerous familial studies have indicated a genetic component to polycystic ovary syndrome (PCOS), a consistent and repeatable association with any particular gene has not been definitively established. However, one genetic marker, the microsatellite D19S884, has shown repeatable and consistent association with PCOS. D19S884 maps to intron 55 of the fibrillin-3 gene. The family of fibrillin extracellular matrix proteins includes the family of latent TGF-β binding proteins which are known to have roles in ovarian function. These provide structural support to tissues and regulate TGF-β bioavailability and are candidates for involvement in clinical manifestation of PCOS. Many of the risk factors associated with PCOS including type II diabetes, heart disease, and obesity are also known to be affected by in utero experience. In addition, although diet has not been examined in the PCOS outcome of offspring, some fetal experiences including androgen exposure have been linked to PCOS outcome. We therefore investigated the effect of protein content of maternal diet on expression of fibrillins-1, –2, and –3 and LTBPs-1 and –2 in the tunica albuginea of ovaries from female offspring by QRT–PCR. Diets were either high (13% protein per total dry weight) or a low protein (5%) during the first two trimesters. All three fibrillins were expressed in the tunica albuginea with the highest level of expression observed for fibrillin-1 followed by fibrillin-2, with low levels of expression of fibrillin-3. Both LTBP-1 and LTBP-2 were expressed, with the highest expression observed for LTBP-2. No significant (P > 0.05) effects of diet upon gene expression were observed. Expression levels of all three fibrillins were positively correlated with each other, and fibrillins-1 and 2 were positively correlated with LTBP-2. This is the first demonstration of the expression of fibrillins and LTBPs in the ovary, however these were unaffected by the maternal diets examined.
421. Seminal fluid TGFβ regulates follistatin mRNA expression human Ect1 cervical epithelial cells
Authors: Sharkey D. J. , Robertson S. A.
Published: November 2010

Introduction of seminal fluid into the female reproductive tract following coitus stimulates a local inflammatory response. Inflammatory leukocyte recruitment is regulated by induction of cytokine and chemokine synthesis in female tract epithelial cells by seminal fluid signalling agents. Affymetrix microarray analysis in immortalised ectocervical epithelial (Ect1) cells identified the potent anti-inflammatory cytokine follistatin (FST) as the most strongly differentially expressed gene, with a ~12-fold increase in mRNA expression induced by seminal fluid. Follistatin has recently been implicated as a key cytokine in early pregnancy by studies in female follistatin null mice, which exhibit infertility as a consequence of failure to resolve the uterine post-mating inflammatory response. The aim of this study was to investigate seminal plasma regulation of follistatin in human Ect1 cervical cells, and to examine the role of the major active seminal fluid constituent, TGFβ, in controlling Ect1 cells follistatin mRNA expression. To confirm Affymetrix findings, qRT–PCR experiments were undertaken in Ect1 cells incubated with 10% pooled human seminal plasma (SP). Primers specific for the tissue bound isoform of follistatin (FST288) as well as both FST288 and the circulating 315 isoforms (FSTall) were used. Ect1 cell incubation with 10%SP elicited 3.8-fold and 4-fold increases in FST288 and FSTall respectively. Incubation of Ect1 cells with TGFβ1, TGFβ2 and TGFβ3 showed differential effects of the three isoforms, with rTGFβ2 inducing FST288 and FSTall, while rTGFβ1 and TGFβ3 exerted little effect.. These results suggest that seminal plasma induces follistatin synthesis after coitus and that TGFβ2 is at least partly responsible for this effect. Follistatin induced by seminal fluid may act to limit the course of inflammation after intercourse, and thereby prevent uncontrolled inflammatory damage. Follistatin induced in the female tissues would be augmented by follistatin delivered from the male, since human seminal plasma also contains a high concentration of this cytokine.
422. Data mining: relationship of sperm kinetics and DNA integrity
Authors: Osman K. , Ibrahim S. , Ismail M. , Das S. , Rahman M. Abd , Othman A.
Published: November 2010

Intracytoplasmic sperm injection (ICSI) is a popular technique in treating infertile male that bypasses sperm natural selection. Due to this, the occasional and unintentional use of spermatozoa in ICSI with high amount of DNA fragmentation seems to be unpreventable. The objective of this study was to develop single sperm selection technique and in the process to determine the relationship between sperm kinetic parameters and sperm DNA damage. Semen from sexually matured male Boer buck cross species were collected and cryo-preserved. After taking into consideration semen sperm count, sperm were isolated individually in an ELISA plate by diluting semen in extender. Then every sperm’s kinetics was assessed by computer-assisted sperm analyzer (CASA) while neutral comet assay was used to quantitate and categorise its DNA damage condition. DNA damage was categorised from minimal damage (category 0) to extensive damage (category 4). Relationship between CASA parameters and DNA damage category of 490 sperms was determined using a Classification and Regression modelling (C&R). A total of 208 sperm data was used to generate a suitable C&R model. A further 250 sperm data was then used to determine accuracy of the model. Results obtained indicated that VSL, WOB and VCL were important factors in determining the overall condition of a particular sperm. A low value of VSL would indicate minimal DNA damage. Identification of higher category of DNA damage would require combination assessment of VSL, WOB and VCL. Accuracy of the developed C&R model was at 83.6%. Based on the above procedure it has been shown that sperm kinetics and DNA integrity can be considered together in selecting potential sperm for ICSI procedure.
423. Effects of Eurycoma longifolia Jack on spermatogenesis and sperm counts of male rats treated with oestradiol
Authors: Norhazlina A. , Heriza A. Wan Nurul , Norfilza M. , Moratv P.
Published: November 2010

Eurycoma longifolia has been known for its aphrodisiac effects in male. Our previous study showed that with the dosage of 8 mg/kg bodyweight (BW) Eurycoma longifolia root extract increased plasma total testosterone levels in male rats when given for 14 days. Oestrogen is a potent inhibitor for testosterone production and spermatogenesis. The aim of the present study is to determine the effects of Eurycoma longifolia (8 mg/kg BW) on the spermatogenic cell count and sperm count of testosterone-suppressed male rats. Adult male Sprague-Dawley healthy male rats weighed 200–250 g were treated with, either control vehicle (no active ingredients given), oestradiol (500 mg/kg BW), Eurycoma longifolia (8 mg/kg BW) or combination of Eurycoma longifolia and oestradiol for fourteen consecutive days. Results showed that sperm count and spermatogenic cell count were increased in Eurycoma longifolia treated group compared with control group (P < 0.05) and to oestradiol treated group (P < 0.05). While in oestradiol treated group, sperm count and spermatogenic cell count were reduced significantly (P < 0.05) compared with control group. Combination of Eurycoma longifolia and oestradiol did not affect sperm count and spermatogenic cell count when compared with control group but they were significantly increased compared with oestradiol treated group (P < 0.05). Thus, the study has shown that Eurycoma longifolia is potentially capable to suppress harmful effects of oestradiol on spermatogenesis and sperm counts in healthy male rats when given for fourteen consecutive days.
424. Polyunsaturated fatty acids and mitochondrial reactive oxygen species production in human spermatozoa and male infertility
Authors: Koppers A. J. , Aitken R. J.
Published: November 2010

Reactive oxygen species (ROS) are traditionally considered detrimental by-products of cellular metabolism. However, ROS have conflicting roles in human spermatozoa, either as a functional mediator of sperm capacitation or generating a state of oxidative stress that is associated with male infertility. Using the probe MitoSOX Red, we have shown that defective human spermatozoa generate mitochondrial ROS in manner that was negatively correlated with motility (R2 = 0.8048). Previous research has shown higher levels of polyunsaturated fatty acids (PUFAs) in defective spermatozoa. However, the addition of PUFA to normal human spermatozoa results in increased mitochondrial ROS production (P < 0.001) and lipid peroxidation (P < 0.001) determined by MitoSOX Red and BODIPY C11 assays, as a consequence human spermatozoa also exhibited decreased sperm motility (P < 0.001). Ongoing research is currently evaluating the relationship between cellular levels of PUFAs in human spermatozoa and mitochondrial ROS generation and decreased sperm motility. This research demonstrates that mitochondrial ROS generation in human spermatozoa may have significant consequences for their function and we propose that elevated PUFA content may be a primary cause of increased oxidative stress and therefore male infertility.
427. MDM2 maintains the latency of expression of TRP53 in the mouse preimplantation embryo
Authors: Jin X. , Chandrakanthan V. , O.'Neill C.
Published: November 2010

TRP53 is a tumour suppressor that causes cell-cycle arrest or cell death in response to a range of stressors. Normal preimplantation embryo development requires that TRP53 is maintained in a labile state1. Culture of mouse C57BL6 preimplantation embryos causes this latency to be breached and this is a cause of the low embryo viability following culture. MDM2 is an ubiquitin ligase that targets TRP53 for degradation by the proteosome. MDM2 is activated by Serine 166 phosphorylation (pMDM2). This is commonly catalysed by the phosphatidylinositol-3 kinase (PI3K) and AKT signalling pathway. A range of embryotrophins activate the PI3K and AKT pathway. This study tested the hypothesis that TRP53 is maintained in a latent state in the normal embryo by the activation of MDM2 by the actions of embryotrophins via a PI3K and AKT signalling pathway. Genetic deletion of Mdm2 prevents normal preimplantation development in a Trp53 dependent manner2. Addition of an MDM2 inhibitor (Nutlin-3) to culture medium caused a dose-dependent inhibition of zygote development (P < 0.001) that did not occur in Trp53−/−embryos. Immunofluorescence and western blot analysis detected pMDM2 throughout mouse preimplantation development. Zygote culture reduced the levels of pMDM2 formation. Furthermore, blocking the actions of Paf, PI3K or AKT in vitro reduced in the expression of pMDM2, and also resulted in higher levels of TRP53 expression in embryos. The embryopathy resulting from increased TRP53 could be partially ameliorated by the addition of the TRP53 antagonist α-pifithrin to media (P < 0.05). The results show MDM2 was activated by an embryotrophin (Paf), PI3K and AKT signalling pathway and was required for the latency of TRP53 expression in the preimplantation embryos.
(1) Li A, Chandrakanthan V, Chami O, O’Neill C. (2007) Biology of Reproduction 76: 362–367.
(2) Montes de Oca Luna R, Wagner DS, Lozano G. (1995) Nature 378: 203–205.
428. Normal follicle growth and maturation in a three-dimensional in vitro culture system: follicular environment manipulation and assessment of oocyte outcomes
Authors: Dunning K. R. , Akison L. K. , Russell D. L. , Norman R. J. , Robker R. L.
Published: November 2010

In vivo, the oocyte matures in a niche environment surrounded by somatic cells, and later in ovarian follicular development, by follicular fluid. Maternal diet influences the environment in which an oocyte matures but the mechanisms by which an altered metabolic profile, such as hyperinsulinemia, affects oocyte quality are not known. We investigated the use of a three dimensional follicle culture system allowing direct manipulation of the follicular environment thus circumventing systemic hormonal and metabolic effects. Secondary follicles (113.4 ± 1.02µm, n = 54) were isolated from mice at d12, encapsulated individually in 2µl of alginate matrix, and cultured in aMEM/5%FCS/10 mIU/mL LH/100 mIU FSH at 37°C/5%CO2, with media sampling and replacement every second day. Following 12 days of culture there was a significant 3-fold increase in follicle diameter (320 ± 10.1µm, n = 51). Histological analysis showed normal follicular morphology and antrum formation. Analysis of oestradiol (15.0ng/mL), androstenedione (7.8ng/mL) and progesterone (23.7ng/mL) in the media at d12 confirmed normal steroidogenesis and differentiation. Treatment of follicles with an ovulatory stimulus (1.5IU/mL hCG/5ng/mL Egf), resulted in cumulus expansion and hyaluronan localising to the cumulus oocyte complex (COC) and follicular basement membrane. These analyses were consistent with follicle growth and induction of ovulation in vivo. Further, COCs isolated from follicles and matured in vitro (IVM) in the presence of Egf and FSH, underwent cumulus expansion (CEI 2.8 ± 0.2) and were capable of fertilisation and blastocyst development. LH did not induce IVM COC expansion (CEI 1.36 ± 0.2), reflecting the normal in vivo differentiation process. However, culturing follicles in high insulin (5ug/mL) led to a significant increase in the degree of IVM cumulus expansion in response to LH (CEI 2.1 ± 0.3) indicating inappropriate cumulus cell differentiation, which may lead to poorer oocyte quality. These results demonstrate that this technique recapitulates normal in vivo folliculogenesis and is useful for manipulation of the follicular environment and assessment of oocyte outcomes.
429. Culture of mouse zygotes altered epigenetic states of the agouti viable yellow allele in the resulting progeny
Authors: Li A. , Morgan H. , Jin X. , Whitelaw E. , O.'Neill C.
Published: November 2010

Epigenetic modification of the genome is one potential mechanism for the persistence of altered gene expression. Recent evidence indicates that the growth environment of the preimplantation embryo may have long-term consequences in the gene expression of progeny (Mahsoudi et al. 2007; Watkins et al. 2008). This study assessed whether culture of zygotes induced persistent epigenetic changes in gene expression. The agouti viable yellow (Avy) allele is epigenetically sensitive, and is a widely used model for studying epigenetic phenomena. It has active (hypomethyalted) and inactive (hypermethylated) epialleles. Progeny with an active epiallele have yellow fur; those with an inactive epiallele have an agouti coat. Some progeny are mosaics for the two epigenetic states and have a mottled coat color. Males (Avy/a C57BL/6) were mated to wildtype females (A/A FVB). Embryo treatments were: (1) zygotes collected and cultured for 96 h in Sydney IVF Media suite and transferred to day 3 pseudopregnant Swiss females; (2) blastocysts collected and transferred to recipients without being subjected to culture; (3) naturally mated females left to give birth without interventioin. The number, sex, genotype, and epi-phenotype of the resulting progeny were recorded. The culture of zygotes caused a significant shift in the expression of the epialleles compared with embryos that were transferred without culture (P = 0.014), or those born following natural matings (P < 0.001). Offspring that resulted from culture are more likely to have active epialleles, resulting in a higher proportion of progeny with yellow fur. The results of this study show that culture of mouse zygotes changed the expected expression of the Avy allele. This study provides evidence for the epigenetic basis for the long-term ‘memory’ of preimplantation embryo’s growth environment, and for robust and readily accessible methodology for addressing the underlying molecular bases for this phenomenon.
(1) Mahsoudi B, Li A, O’Neill C. Assessment of the Long-Term and Transgenerational Consequences of Perturbing Preimplantation Embryo Development in Mice. Biol Reprod 2007; 77: 889–896.
(2) Watkins AJ, Ursell E, Panton R, Papenbrock T, Hollis L, Cunningham C, Wilkins A, Perry VH, Sheth B, Kwong WY, Eckert JJ, Wild AE, Hanson MA, Osmond C, Fleming TP. Adaptive responses by mouse early emb.
430. Investigation of pluripotency in derived embryonic stem cell (ESC) lines
Authors: Asl M. Pashai , Khodadadi K. , Holland M. K. , Richings N. M. , Verma P. J.
Published: November 2010

To produce autologous ESCs for a bovine model of cell therapy, we activated oocytes by calcium ionophore (CI) and 6 dimethylaminopurine (6 DMAP) and isolated ESCs from the resulting parthenotes. Parthenote ESC lines (pbESC) would also provide a valuable tool for epigenetic studies on ESCs. Five pbESC like-cell lines were expanded for 12 passages over 120 days and differentiated to form embryoid bodies by suspension culture. The pbESC lines demonstrated typical ESC morphology and expressed ESC markers including alkaline phosphate and stage-specific embryonic antigen, SSEA1 and SSAE4 asssessed by histochemical and immuno-fluorescence staining, respectively. In addition, gene expression of Oct4, Rex1, SSEA1 and ALP was confirmed using RT–PCR. These cells had a normal karyotype. The cells formed EBs and showed expression of the markers of three embryonic germ layers. In summary, we show that ESCs can be derived from bovine parthenogenetic blastocysts and that these cells express pluripotent markers and have ability to form EBs and differentiate into cells indicative of the three embryonic germ layers. Additional work will focus on imprinted gene expression and will provide further evidence of the parthenogenetic origin of the pbESC lines.
431. Importin proteins and their role in maintenance of the stem cell state
Authors: Young J. C. , Miyamoto Y. , Major A. , Dias V. L. , Jans D. A. , Loveland K. L.
Published: November 2010

The importin (IMP) family of proteins mediates transport into the nucleus for many proteins larger than 40 kD. Through differential cargo recognition, IMPs regulate cellular events by controlling nuclear access of transcription factors and chromatin remodelling agents. During spermatogenesis, many IMPs change expression and localisation in a manner concordant with specific stages of spermatogenic development. To assess the potential role of IMPs in the transition between the stem cell and subsequent differentiation, we undertook analysis of the expression and subcellular localisation of several key murine IMPs in both pluripotent embryonic stem cells (mESCs) and embryoid bodies (EBs). All of the IMPs analysed (IMPα2, 3, 4, IMPβ1 and IMP5) were detected in undifferentiated mESCs by immunofluorescence, and each exhibited distinctive nucleocytoplasmic distribution patterns. Subcellular localisation of most IMPs altered after 10 days of mESC differentiation as EBs. This was paralleled by changes in the mRNA levels of IMPα1–4, IMPα6, IMPβ1 and IMP5, concomitant with alterations in the expression level of the pluripotency marker, Oct3/4. Reducing IMP-dependent nuclear import through overexpression of specific dominant-negative IMP constructs led to alterations in import or production of Oct3/4 protein, depending on the specific IMP. These findings indicate that IMPs may play very specific but distinct roles in cell fate choice between maintenance of pluri/multipotency and commitment to differentiation in ESCs and potentially in spermatogenesis or other organs that contain stem cells.
432. How do antiphospholipid antibodies contribute to preeclampsia?
Authors: Chen Q. , Viall C. , Stone P. R. , Chamley L. W.
Published: November 2010

Preeclampsia is characterised by elevated maternal blood pressure which is preceded by endothelial activation. The cause of this endothelial cell dysfunction is unclear but it appears to be triggered by a placental factor. One of the risk factors for developing preeclampsia is the presence of antiphospholipid antibodies (aPL) in the maternal blood but exactly how aPL predispose women to developing preeclampsia is unclear. A second feature known to be associated with preeclampsia is excessive shedding and deportation of dead trophoblasts. We have previously shown that shed trophoblasts are phagocytosed by endothelial cells and that phagocytosis of necrotic trophoblasts leads to endothelial cell activation1. In this study we examined the hypothesis that aPL alter the number or nature of trophoblasts shed from the placenta resulting in endothelial cell activation. Using our published model of trophoblast shedding 2 human first trimester placental explants were treated with monoclonal aPL, IIC5 or ID2, or control antibody CD45 for 72 h. Shed trophoblasts then were harvested and counted using a Cellometer AutoT4 automated cell counter. The activity of caspases 3&7 was analysed in all treated shed trophoblasts using a FLICA™ kit. The treated shed trophoblasts also were exposed to the endothelial cell line HMEC-1 for 24 h. The level of ICAM-1 by HMEC-1 was determined by cell-based ELISA. The number of trophoblasts shed from placental explants was increased 2 fold following aPL treatment whereas, treatment with CD45 resulted in only a 1.3 fold increase in shedding. Trophoblasts shed from aPL-treated explants contained less active caspases 3 & 7 compared with control shed trophoblasts. Moreover, phagocytosis of trophoblasts shed from aPL-treated explants induced significantly increased expression of ICAM-1 compared with controls. aPL treatment affected the number and nature of trophoblasts shed from placentae in such a way that phagocytosing endothelium become activated. These findings suggest that aPL treatment may have shifted the type of cell death that shed trophoblasts are undergoing from apoptosis to a more necrotic or aponecrotic mechanism. This type of shedding of trophoblasts in vivo might contribute to the endothelial cell activation which is a hallmark feature of preeclampsia.
(1) Chen Q, Stone PR, McCowan LM et al. Phagocytosis of necrotic but not apoptotic trophoblasts induces endothelial cell activation. Hypertension. 2006;47:116–121.
(2) Abumaree MH, Stone PR, Chamley LW. An in vitro model of human placental trophoblast deportation/shedding. Mol Hum Reprod. 2006;12:687–694.
434. Identification of differentially expressed proteins in ovine chorion rupture sites at preterm and term using proteomics
Authors: Fazlic M. , Fairclough R. , Fraser S. , Young R. , Jenkin G. , Knight M. , McDonagh M.
Published: November 2010

Introduction: A significant number of babies are delivered preterm and many of these deliveries are due to spontaneous rupture of fetal membranes. However, the mechanisms underlying fetal membrane rupture are not well defined. The sheep has proved to be a valuable model for elucidating the physiology of birth, with many of the findings clearly relevant to human parturition. In the current study we have used the sheep model to investigate changes in protein expression in the chorion in relation to the onset of spontaneous full term labour. Methods: Proteomic analysis was used to compare the protein profiles at the chorion rupture site between mid/late gestation (136 days, n = 6) and term delivery (145days, n = 6). Proteins were solubilised and separated into soluble and insoluble fractions which were separated by 2D-electrophoresis. Relative protein expression was quantified using the PROGENESIS software and protein spots were picked and identified using Matrix Assisted Laser Desorption Ionisation-Time of Flight (MALDI-TOF) mass spectrometry. Data-mining used gene ontology (GO terms) for each protein and these were clustered into networks using DAVID bioinformatics resource (http://david.abcc.ncifcrf.gov/home.jsp). Results: A total of 150 protein spots were identified of which 60 proteins were found to differ by more than 2-fold between preterm and term samples. This group was significantly enriched for proteins from the several functional GO categories including; Oxidoreductase activity (7 proteins); negative regulation of apoptosis (4); structural molecule activity (7); protease inhibitor activity (7); Carbohydrate metabolism (10); Glucose metabolism (5); Glycolysis (5); response to stress (8) and heat shock (3). Of the differentially regulated proteins, 10 were found to be significantly upregulated at term (ANOVA P < 0.05) including isocitrate dehydrogenase, glutamate dehydrogenase 1, malate dehydrogenase 2. A further 10 proteins, including serpin peptidase inhibitor SERPINA12, serpin peptidase inhibitor SERPINH1 precursor and heat shock protein 90 kDa β were found to be downregulated.
435. Follicle differentiation and luteinisation in the mouse is associated with hypoxia inducible factor activity
Authors: Tam K. , Russell D. , Kind K. , Thompson J.
Published: November 2010

Hypoxia inducible factors (HIF) are transcription factors that mediate the response to hypoxic stress. Under hypoxic conditions, HIF is stabilised, translocates to the nucleus, and binds to the Hypoxia Response Elements (HRE) upstream of numerous target genes involved in angiogenesis and glycolysis, including Vegf, Glut-1 and Ldha. Little is known about the role of HIFs in regulating ovarian function. In rat granulosa cells, FSH stimulates HIF 1α via the PI3K/Akt pathway, demonstrating a role for HIFs during follicular development. In contrast, there is limited information regarding the role of HIFs during corpus luteum formation. In this study we investigated whether HIFs play a role in follicle differentiation and luteinisation. Prepubertal C57Bl6 females were stimulated with eCG (5 IU) followed 46 h later by hCG (5 IU). Mice were sacrificed at 0, 4, 8, 12, 16 and 24 h post hCG and granulosa cells were collected for Western analysis of HIF-1a protein. To investigate HIF activation in the ovary, a transgenic reporter mouse line was developed by lenti-viral incorporation of an HRE (4)-SV40-eGFP construct. Ovaries were collected from mice plugged day 1, 4 and 8 for CL analysis in vivo.A time- dependent increase of HIF 1α protein levels in granulosa cells, maximal around time of ovulation, was observed. Ovaries from cycling HRE-eGFP transgenic mice exhibited no eGFP in primordial, primary or preantral follicles. Upon antrum formation, eGFP was evident in occasional sections in antral follicles but HIF signalling was restricted within the theca. In contrast, corpora lutea on pregnancy day 1, 4 and 8 readily expressed eGFP and eGFP expression increases as luteinisation progresses.These results demonstrate that in vivo HIFs may play a role in folliculogenesis, but this is restricted to theca cells of antral follicles before hCG stimulation. Following hCG, maximal HIF activity is associated with the time of ovulation. In addition, HIF activity is maintained during luteinisation.
436. Investigation of the role of SRC in capacitation associated tyrosine phosphorylation of human spermatozoa
Authors: Mitchell L. A. , Nixon B. , Baker M. A. , Aitken R. J.
Published: November 2010

Capacitation is a pre-requisite for mammalian spermatozoa allowing them to gain the ability to fertilise an oocyte. A fundamental part of this mechanism is a dramatic increase in tyrosine phosphorylation. Implicated in this process in the mouse is a unique cAMP/PKA-mediated pathway involving a PKA-activated tyrosine kinase suggested to be pp60c-src (SRC). The Src kinases examined were predominantly expressed in the human sperm tail, a site compatible with a role in mediating the capacitation-associated tyrosine phosphorylation cascade. Co-immunoprecipitation revealed that PKA-c could be isolated from sperm and this interaction was restricted to capacitated cells, suggesting PKA-mediated activation of SRC forms an integral part of the signalling cascade assembled during capacitation. Upon activation, SRC undergoes autophosphorylation of Y416 and thus phosphorylation of this residue indicates the presence of active SRC kinase. The phosphorylation status of SRC was compared using both 2D-immunoblotting and immunocytochemical studies, both revealing a significant increase in SRC activation during capacitation. Furthermore, suppression of PKA and SRC through application of SU6656, or H89, a PKA inhibitor, led to a dramatic decrease in tyrosine phosphorylation and SRC activity. In conclusion, this study has provided evidence for the involvement of non-receptor tyrosine kinase, SRC, in regulating tyrosine phosphorylation associated with capacitation. Inhibition of SRC did not completely suppress tyrosine phosphorylation suggesting this complex signal transduction pathway exhibits a degree of functional redundancy.
437. Whole body heat stress induces selective germ cell apoptosis in mice
Authors: Wechalekar H. , Setchell B. P. , Breed W. G. , Ricci M. , Leigh C. , Peirce E.
Published: November 2010

Introduction: In scrotal mammals, heat stress (43°C/ 20 min) to the scrotum results in germ cell death in the testes1, abnormal spermatozoa, and infertility2 whereas two days of whole body heating (36°C, 12 h/ day) reduces testes weight, sperm numbers and fertility3. The aim of the present study was to determine the intratesticular effects of whole body heating on germ cell maturation and apoptosis. Methods: C57BL/6 mice (n = 16) were housed at 37–38°C for 8 h/ day for 3 days while controls (n = 4) were kept at 23–24°C. Animals from heat treated (n = 4), and control groups (n = 1) were sacrificed at 16 h, 7, 14 and 21 days post exposure to heat. Testes were weighed and analysed by t-test. In testes from each animal, two sections 70µm apart were end labelled for TdT-mediated-dUTP nick (TUNEL). Apoptosis was determined in 200 seminiferous tubules by a colour threshold set in the particle analysis program (Olympus).The tubules were staged as I-VI (early), VII-VIII, IX-X and XI-XII (late) and results analysed using Wilcoxon test. Results: The weights of testes were significantly reduced in heat-treated animals (P < 0.05) at 16 h, 7 and 14 days with no significant difference at 21 days. Apoptosis was significantly higher in the heat-treated group in stages I-VI and XI-XII at 16 h, 7 and 14 days (P < 0.05). In addition, in stages VII-VIII and IX-X apoptosis was significantly higher at 16 h (P < 0.05) with no statistical difference between other time intervals. By day 21, the levels of apoptosis did not differ significantly from the controls in any of the stages (P > 0.05).Conclusion: Whole body heat stress can induce stage and cell specific degeneration of the germ cells in the seminiferous epithelium. The germ cells undergoing apoptosis are spermatogonia, primary spermatocytes and early spermatids. In addition, heat stress produces significant apoptosis of germ cells in the hormone dependent stages VII-VIII immediately after heat stress.
(1) Rockett, J.C. et al. (2001) Biol. Reprod. 65:229–239.
(2) Banks, S. et al. (2005) Reproduction 129:505–514.
(3) Yaeram, J. et al. (2006) Reprod. Fert. Dev. 18:647–653.
438. PLZF is a spermatogonia stem cell-specific marker in the sheep testis: application to enrichment of ovine spermatogonial stem cell
Authors: Borjigin U. , Davey R. , Hutton K. , Herrid M.
Published: November 2010

Identification and isolation of spermatogonial stem cells (SSCs) are prerequisite for long-term culture, genetic manipulation, and transplantation research. The promyelocytic leukemia zinc-finger (PLZF) has been identified as a spermatogonia stem cell marker in rodent and other species, however its expression in sheep testis has not been reported yet. In this study, we validated an antibody that specifically binds to spermatogonia stem cell in sheep testis, thus demonstrated that PLZF is a spermatogonia stem cell marker and can be used for its identification. Testes from 12 Merino rams were selected to represent four stages of testis development at testis weights of 3–5 g (neonatal), 30 g (peripubertal), 50 g (prepubertal) and 100 g (mature). Three testes sections from 4 different developmental stage were stained with PLZF antibody and 25 individual tubules in each section were counted. In the sections, the percentage of PLZF positive cells/per tubule was increased nearly 2-fold from neonatal (6. 4 ± 0. 4%) to peripubertal (1 2.2 ± 2.8%), and then the percentage begin to decline in prepubertal (4.6 ± 0. 7%) and mature testes (3.1 ± 0.6%). A single cell suspension of testicular cells was generated by a two step enzymic digestion (n = 4) and spermatogonia stem cells were enriched by overnight differential plating with 0.2% gelatine coated flask. The percentages of spermatogonia stem cells in the single cell suspensions were assessed by PLZF antibody staining of smears. Compared with the initial isolation (3.1 ± 0.6%), spermatogonia were enriched 11-fold in overnight differential plating (34.0 ± 5.7,%) (P < 0.05). These data provide the basis for future studies aimed at refining conditions of spermatogonial stem cell culture and manipulation before male germ stem cell transplantation in sheep.
439. GDF9 and BMP15 are germ-free regulators of Sertoli cell function
Authors: Nicholls P. K. , Harrison C. A. , Gilchrist R. B. , Farnworth P. G. , Stanton P. G.
Published: November 2010

Oocyte-secreted Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are critical regulatory factors in female reproduction. Together they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study, we show that the expression and stage-specific localisation of both factors are limited to the germ cells of the rat seminiferous epithelium; with GDF9 being principally localised in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of the GDF9 and BMP15 signalling receptors (ALK5, ALK6, and BMPRII). Individual receptor types were expressed throughout the seminiferous epithelium, but co-expression of type I and type II receptors was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFβ ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction formation and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localisation of the junctional proteins claudin-11, occludin and ZO-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 upregulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together these results demonstrate that GDF9 and BMP15 are germ cell specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.
440. A-kinase anchoring protein 4 in the marsupial
Authors: Hu Y. , Yu H. , Pask A. J. , O.'Brien D. A. , Shaw G. , Renfree M. B.
Published: November 2010

A-Kinase Anchor Protein 4 (AKAP4) is an X-linked member of the AKAP family of scaffold proteins that anchor cAMP-dependent protein kinases and play an essential role in fibrous sheath assembly during spermatogenesis and flagellar function in spermatozoa. Marsupial spermatozoa differ in structural organisation from those of eutherian mammals but data on the molecular control of their structure and function are limited. We therefore cloned and characterised the AKAP4 gene in a marsupial, the tammar wallaby (Macropus eugenii). The gene structure, sequence and predicted protein of AKAP4 were highly conserved with that of eutherian orthologueues and it mapped to the marsupial X-chromosome. There was no AKAP4 expression detected in the developing young and in the adult, expression was limited to the testis with a major transcript of 2.9kb identified by northern blotting. AKAP4 mRNA was detected by in situ hybridisation in the cytoplasm of round and elongated spermatids in the adult testis while its protein was found in the sperm tail from principal piece of the flagellum. This is consistent with its expression in other mammals. Thus this gene appears to have a conserved role in spermatogenesis for at least the last 166 million years of mammalian evolution.
441. Renin angiotensin system polymorphisms are associated with pregnancy complications
Authors: Zhou A. , Thompson S. , Nowak R. , Heinemann G. , Zhang J. , Dekker G. A. , Roberts C. T.
Published: November 2010

Preeclampsia (PE), small for gestational age (SGA) and preterm birth (PTB) together affect 20% of first pregnancies. Currently there is no reliable way to identify women at risk. Polymorphims in genes in the renin angiotensin system (RAS) may be associated with impaired placentation and poor maternal response to pregnancy and hence predict risk for pregnancy complications. We aimed to determine if three functional polymorphisms in RAS genes, namely, AGT M235T, ACE (I/D) and AT1R A1166C are associated with pregnancy complications. Pregnancy trios were prospectively recruited from two public hospitals in Adelaide. Pregnancies were classified into normal (n = 110), PE (n = 26), SGA (n = 47), PE+SGA (n = 12), gestational hypertension (GH, n = 17), PTB (n = 20). PE, PE +SGA and GH were also grouped together as hypertension. Parental blood and maternal blood pressure was sampled or measured at 15 weeks gestation. Cord blood was sampled after delivery. DNA was extracted from buffy coats and genotyped using high resolution melt analyses. Maternal plasma [ACE] was measured by ELISA. Data were analysed by ANOVA and Fisher's exact test. Likelihood ratios (LR) were calculated where appropriate. Maternal ACE I/D was associated with hypertension (P = 0.001, LR = 14.8) and SGA (P = 0.019, LR = 10). Paternal AT1R A1166C was associated with PTB (P = 0.01, LR = 7.4). For ACE I/D, plasma [ACE] in women with DD was 33% and 62% higher than ID and II, respectively (both P < 0.001). Systolic blood pressure in women with DD was 6% higher than II (P = 0.04). Our data suggest that RAS polymorphisms are associated with pregnancy complications. Furthermore, maternal ACE genotype determines plasma [ACE] and affects blood pressure at 15 weeks gestation, well before symptoms manifest. Our data also suggest that paternal genotype may be important in determining risk for pregnancy complications, consistent with the role of paternity in their aetiology.
442. Functional perspectives of caspase-14 in the human placenta
Authors: White L. J. , Charles A. K. , Dharmarajan A. M.
Published: November 2010

The functional barrier for exchange between the mother and fetus in the placenta is created by the fusion of cytotrophoblasts with one another to form a continuous, multinuclear syncytiotrophoblast, which is maintained by the incorporation of underlying proliferative cytotrophoblasts. Disruption to this process has been hypothesised to be involved in the aetiology of preeclampsia. Recently we investigated caspase-14 in the context of trophoblast differentiation as it is crucial to epidermal differentiation and keratin stabilisation, revealing disparate expression of caspase-14 in the differentiating BeWo cell line. Consequently, further examination as to its functional role in trophoblast differentiation was conducted using RNA Interference (RNAi), with the hypothesis that differentiation would be suppressed following caspase-14 silencing. 100nM siRNA were delivered into the BeWo cell line for 16 h before the addition of 20µM Forskolin. Cultures were incubated for a further 24, 48 or 72 h before the extraction of RNA and protein. Transcription of the trophoblast hormone β-hCG, the endothelial mediator of eNOS, and cytokeratin 18 were found to be increased after both 24 and 48 h of differentiation following silencing, implicating caspase-14 in the regulation of these pathways. As both β-hCG and eNOS are significantly increased with trophoblast differentiation, this indicates that caspase-14 functionally suppresses BeWo differentiation. Furthermore, the differential expression of cytokeratin 18 indicates a conserved role for caspase-14 in keratin homeostasis in barrier formation. In conclusion, the suppression of caspase-14 in the BeWo cell line resulted in augmented differentiation, a trait often observed in preeclampsia. Further investigation of caspase-14 activity would provide important insight into mechanisms of trophoblast differentiation, particularly in relation to preeclampsia.
445. Focal adhesions disassemble during early pregnancy in rat uterine epithelial cells
Authors: Kaneko Y. , Lindsay L. A. , Murphy C. R.
Published: November 2010

Successful blastocyst implantation require uterine epithelial cells (UECs) to undergo the 'plasma membrane transformation' followed by the removal of these cells around the implantation sites. The present study investigated the distribution and expression of two principal focal adhesion proteins talin and paxillin in rat UECs during early pregnancy and their role in the loss of these cells at the time of implantation. Results from immunofluorescence microscopy have demonstrated a major distributional change of talin and paxillin in UECs where an intense basal staining of these proteins on day 1 of pregnancy was lost at time of implantation. This was consistent with the significant decrease in paxillin seen through western blotting analysis. Interestingly the amount of talin was not significantly different between day 1 of pregnancy and at the time of implantation with a calpain 2 mediated proteolytic fragment of talin seen on both of these days of pregnancy. Calpain 2 activity was further investigated through western blotting analysis and increases in the active form of calpain 2 at the time of implantation. These observations suggest that talin and paxillin have disassembled from the site of focal adhesions where talin is undergoing cleavage by active calpain 2 at the time of implantation. This allows UECs to become less adherent to the underlying basal lamina facilitating their removal during blastocyst invasion. Hence, disassembly of focal adhesions at the time of implantation is a critical event for successful implantation and placentation.
001. ISOLATION AND CHARACTERISATION OF PORCINE ES CELLS
Authors: Nottle M. B. , Vassiliev I. M. , Vassilieva S. , Beebe L. F. S. , Harrison S. J. , McIlfatrick S. M.
Published: November 2010

Embryonic stem (ES) cellshave the capacity for self renewal, can remain undifferentiated in long term culture and can contribute to all the cells in the body including the germ cells. EScells have been isolated in mice and have also been described for humans. However despite considerable effort for more than two decades ES cellswhich can contribute to the germline are yet to be isolated for the pig or any domestic species for that matter. We have developed a new method for isolating porcine ES cells which uses whole embryos cultured in alpha MEM with 10% serum replacement plus additives under 5% O2. Unlike methods employed previously this method results in homogenous outgrowths whose cells resemble ES cells and which express Oct 4 and Nanog and SSEA-1 [1]. These cells can be passaged and cryopreserved repeatedly resulting in the establishment of cell lines at similar efficiencies to that reported previously for 129Sv mice [2]. These cells can form embryoid bodies and can be differentiated to various cell types representative of all three germ layers [3]. Following their injection into blastocysts these cells localise /become incorporated in the inner cell mass and can be used to produce chimaeras when these embryos are transferred to recipient animals [2]. To date we have produced chimaeric pigs from one male ES cell line [2]. These are currently being mated to demonstrate germline transmission. Future studies will examine the applicability of our method to other species commencing with mice and cattle before extending these to humans.
002. REGULATION OF PLURIPOTENCY AND CELL CYCLE IN FETAL GERM CELLS
Authors: Western P. , Van Den Bergen J. , Miles D. , Ralli R. , Sinclair A.
Published: November 2010

The germ cell lineage is unique in that it must ensure that the genome retains the complete developmental potential (totipotency) that supports development in the following generation. This is achieved through a number of mechanisms that prevent the early germ cell lineage from somatic differentiation and promote the capactity for functional totipotency. Part of this process involves the retained germ line expression of key genes that regulate pluripotency in embryonic stem cells, embryonic germ cells and some embryonal carcinoma cells, the stem cells of testicular tumours. Despite this, germ cells are not intrinsically pluripotent and must differentiate along the male or female pathways, a process which requires commitment of the bi-potential primordial germ cells to the spermatogenic (male) pathway and their entry into mitotic arrest, or to the oogenic pathway (females) and entry into meiosis. This involves robust regulation of regulatory networks controlling pluripotency, cell cycle and sex specific differentiation. Our work aims to further understand the mechanisms controlling differentiation, pluripotency and cell cycle in early male and female germ cells. Our data shows that mitotic arrest of male germ cells involves strict regulation of the G1-S phase check-point through the retinoblastoma protein. In addition, suppression of pluripotency in differentiating male germ cells involves post-transcriptional regulation of OCT4, transcriptional regulation of Sox2 and Nanog and methylation of the Sox2 and Nanog promoters. Further understanding of these processes promises to lead to a greater understanding of the molecular mechanisms underlying control of pluripotency, cell cycle and differentiation in the germ line and the initiation of germ cell derived testis tumours.