bluuebunny/crossref_metadata_2025 huggingface dataset:
140. THE DEVELOPMENT OF THE PHALLUS IN A MARSUPIAL
Authors: Chew K. , Pask A. , Shaw G. , Renfree M. B.
Published: November 2010

The phallus, the limbs and the tail are all considered appendages in the developing mammal. In mice, several key genes including FGF8, BMP4, SHH, DLX5 and DLX6 are known to control the precise pattering of the limb and phallus in the fetus (reviewed in Yamada et al. 2006). The signalling cascade in both appendages begins with SHH interacting with FGF8. In humans, disruptions to these gene pathways result in malformations of both limbs and phallus because these appendages share conserved elements in patterning and development (Yamada et al. 2003). However, this is a poorly researched area so additional models are needed to provide a greater perspective into mammalian embryonic patterning especially of the external genitalia. In marsupials, since most sexual differentiation occurs after birth, the developing phallus and limbs are accessible at stages that occur in utero in humans and other eutherian mammals. We have used the tammar as an alternative model to understand the differentiation of the phallus and limb. FGF8, SHH and megalin mRNA and protein are all expressedin the limb and phallus from the early embryo until post-natal stages. SHH and megalin were co-localised in the urethral epithelium of the tammar phallus. This is the first study to detect megalin in the developing mammalian phallus. SHH is a critical gene in patterning the appendages, and megalin is a transport protein that ferries steroids into the cell where they can bind to their cognate receptor. It has a high affinity for the potent androgen androstanediol, an androgen critical for virilisation of the tammar phallus (Leihy et al. 2004). Further examination is underway to confirm whether the expression of the genes patterning the phallus in the mouse are conserved in the marsupial and whether megalin plays any role in the patterning of the phallus, possibly mediated by SHH.
141. DEVELOPMENT OF A MODEL SYSTEM TO ASSESS THE REGULATION OF LEYDIG STEM CELL DIFFERENTIATION
Authors: Heng K. , Hafen B. , Kotula-Balak M. , Ivell R. , Anand-Ivell R.
Published: November 2010

The Leydig cells of the testis are responsible for the production of ca. 95% of all circulating androgens in the adult male. There is substantial evidence now that loss of Leydig cell (LC) functionality may account in part for declining androgen levels in the ageing male. Some of this loss of functionality appears to be due to disruption of early LC differentiation processes caused by exposure to environmental endocrine disrupting substances. We have made use of the ability of the alkylating agent ethane dimethane sulfonate (EDS) to destroy specifically all immature and adult LC to develop a model system which allows us to quantitatively assess the differentiation of a new population of LC from resident stem cells in the testis. Following a single bolus injection of EDS into adult Sprague Dawley rats, unless additional testosterone is present to suppress the HPG axis, new LC redifferentiate from the resident stem cells in the testis. These are small spindle-shaped mesenchymal cells which are located in the peritubular region adjacent to the seminiferous tubules. In the absence of mature LC these mesenchymal cells differentiate first into LC progenitor cells, then immature LC, and finally into adult LC, following a precisely defined kinetic which can be mapped using quantitative RT-PCR for LC specific genes in whole testis samples. We are using this model to examine the effect of in vivo application of known environmental endocrine disrupting agents on LC differentiation, and hence their potential to cause long-term loss of LC functionality.
143. THE FIRST EVIDENCE OF HIGH SUSCEPTIBILITY TO COLD SHOCK BY THE SPERMATOZOA OF A MARSUPIAL, THE FAT TAILED DUNNART (SMINTHOPSIS CRASSICAUDATA)
Authors: Czarny N. A. , Rodger J. C.
Published: November 2010

Carnivorous marsupials are native Australian predators including the highly threatened northern quoll (Dasyurus hallucatus) and Tasmanian devil (Sarcophilus harrisii). These species are currently actively managed in captive populations but assisted reproductive techniques such as gamete banking may also contribute to their conservation. Previous studies on a model dasyurid, the fat tailed dunnart (Sminthopsis crassicaudata), have found that spermatozoa do not survive freezing and thawing using a variety of freezing protocols and cryoprotectants. We have re-examined cold shock to investigate problems with sperm cryopreservation in S. crassicaudata. Epididymal spermatozoa were rapidly cooled to 0.5ºC in a pre-cooled tube held in an iced water slurry and upon re-warming the spermatozoa were non-motile (n=6). The addition of up to 20% egg yolk, which is considered protective to the spermatozoa of cold shock sensitive eutherians, did not improve the outcome (n=6). Similarly when S. crassicaudata spermatozoa were rapidly cooled to 4ºC, just 2% remained motile upon re-warming (n=10). However when spermatozoa were combined with at least 10% egg yolk and rapidly cooled to 4ºC only small reductions in motility were observed upon rewarming (n≥8). In order to achieve motile spermatozoa at 0ºC, controlled rate cooling at 0.5ºC/minute was examined. In the absence of egg yolk there was a decline in the percentage of motile spermatozoa below 4ºC (n=6). However if spermatozoa were combined with at least 10% egg yolk there was no significant loss of motility at temperatures as low as 0ºC (n=6). This study has revealed that at least one species of marsupial is highly susceptible to cold shock. These paradigm shifting findings give direction to future experiments aiming to develop a robust technique for sperm preservation in endangered dasyurids.
144. ROLE OF RNA-BINDING PROTEIN, MUSASHI-1 (Msi-1), IN MURINE FOLLICULOGENESIS AND OOCYTE DEVELOPMENT
Authors: Gunter K. M. , Fraser B. A. , Sobinoff A. P. , Siddall N. A. , Hime G. R. , McLaughlin E. A.
Published: November 2010

Follicular development and oocyte maturation in mammals requires the temporal and spatial control of protein production. Consequently, it is hypothesised that the preovulatory follicle represses mRNA translation until specific proteins are required during oocyte maturation. Increasingly RNA-binding proteins are being recognised as important contributors to germ cell development, particularly during oocyte transcriptional quiescence. We have identified the presence of RNA-binding protein musashi-1 (Msi-1) mRNA within the mouse ovary and mature mouse oocyte, where the protein is believed to act as a translational repressor by binding to specific sequences within the 3' UTR of target mRNA molecules. Recent studies in various mammalian systems have identified p21 WAF1, cdkn2a, notch and m-numb as potential targets of Msi-1. We have also identified morf4l1 as a potential target through preliminary pulldown and microarray analysis using a GST tagged Msi-1 recombinant protein. To further study these potential targets, a transgenic Msi-1 mouse was produced to overexpress the RNA-binding protein in the developing oocyte. Real time PCR, performed on intact ovaries of WT and Tg mice, has so far demonstrated a 1.5-fold increase in Msi-1 expression in tgMsi-1/+ ovaries, above WT ovary expression. Real time PCR analysis of Msi-1 target mRNA expression has also shown an overall increase in expression in the tgMsi-1/+ ovaries of p21 WAF1 (~2.5-fold), cdkn2a (~2-fold), and notch (~3-fold). However m-numb and morf4l1 do not appear to be targets of Msi-1 in the oocyte, with no significant difference in expression between the WT and tgMsi-1/+ ovaries analysed. Functional quantification of oocyte development reveals a significantly less oocytes produced from superovulated juvenile mice compared with wild type litter mates. Therefore, preliminary analysis suggests that Msi-1 may play a role in binding the transcripts of genes necessary for cell cycle regulation and chromatin remodelling, characteristic of meiotic progression and oocyte development.
146. MACROPHAGES ARE ESSENTIAL FOR MAINTENANCE OF EARLY PREGNANCY THROUGH REGULATION OF CORPUS LUTEUM FUNCTION
Authors: Care A. S. , Jasper M. J. , Ingman W. V. , Robertson S. A.
Published: November 2010

Macrophages are abundant within the ovary, and have been identified in the corpus luteum (CL) of most species studied, including in rodents and human. Through their secretory products, macrophages are thought to be involved in ovarian tissue remodelling, including luteinization, and in regulating steroidogenesis [1, 2]. Macrophages co-cultured with granulosa or luteal cells act to stimulate progesterone secretion [3]. To determine the impact of macrophage ablation during early pregnancy, we utilised the macrophage specific CD11b-DTR diphtheria toxin receptor (DTR) transgenic mouse to cause transient systemic ablation of macrophages via the administration of DT (25 ng/g). The effects of macrophage ablation during the pre-implantation phase of pregnancy were evaluated and implantation sites were counted. Ablation of macrophages on day 1 pc (n=13 wild-type; n=9 CD11b-DTR) or day 4 pc (n=6 CD11b-DTR; n=9 wild-type) caused complete pregnancy loss in all DT-treated CD11b-DTR mice, while wild-type mice maintained viable pregnancies [mean ± SEM implantation sites = 5.0 ± 1.4 (day 1 treated), and 6.0 ± 1.9 (day 4 treated)]. Serum progesterone was analysed 24 h following macrophage ablation. A single DT injection on day 3 pc significantly reduced serum progesterone (P4) levels [n=7 wild-type P4 (ng/ml)= 29.6 ± 3.3; n=8 CD11b-DTR = 11.1 ± 2.1]. The administration of exogenous P4 (2 mg) on each of day 4-7 pc prevented fetal loss in DT-treated CD11b-DTR mice (n=6; implantation sites = 7.8 ± 1.6), while no pregnancies remained viable in DT-treated mice administered vehicle only (n=9). In conclusion, luteal insufficiency appears to be the cause of pregnancy failure following macrophage ablation. These data indicate a critical role for macrophages in corpus luteum function in early pregnancy.
147. PATERNAL OBESITY IMPAIRS SPERM FUNCTION AND SUBSEQUENT EMBRYO AND PREGNANCY OUTCOMES
Authors: Bakos H. W. , Mitchell M. , Setchell B. P. , Lane M.
Published: November 2010

Despite the increased prevalence of obesity in males of reproductive age, the effects of male obesity on conception and pregnancy have been largely ignored. Hence, the aim of this study was to elucidate the effects of paternal Diet Induced Obesity (DIO) on sperm function, embryo development and pregnancy. Six week old C57BL/6 male mice (n=36) were allocated to either standard chow or a high fat diet (HFD). After eight weeks, mice were either sacrificed and spermatozoa assessed, for motility, reactive oxygen species (ROS) and DNA damage or mated and zygotes collected and cultured to the blastocyst stage. Blastocyst development, cell number and apoptosis were assessed, and fetal outcomes analyzed following embryo transfer. Differences between treatments were assessed using GLM. The percentage of motile spermatozoa was decreased (36% vs. 44%, p<0.05) in the HFD group compared to controls. Intracellular ROS were elevated (692units vs. 409units, p<0.01) in the HFD group compared to controls. Overall levels of sperm DNA damage were also increased (1.64% vs. 0.17%, p<0.05) in the HFD group. Blastocyst development was reduced when males were fed a HFD (64% vs. 84%, p<0.05). Similarly, blastocyst cell number (37.9±2.8 vs. 46.6±2.5, p<0.05), inner cell mass number (11.4±0.9 vs. 15.3±0.9, p<0.05) were reduced and apoptosis (12.8±1.9 vs. 6.6±0.6, p<0.05) increased in embryos sired by a male fed a HFD. Implantation (86.7% vs. 72.5%, p<0.05) and fetal development (38.7% vs. 22.5% p<0.05) were also significantly reduced when blastocysts came from a male fed a HFD. This is the first report providing comprehensive evidence that paternal DIO significantly impairs embryo quality and pregnancy rates. These effects may be related to the observed increase in oxidative stress and sperm DNA damage. These data provide compelling evidence that male obesity impacts on male fertility, embryos as well as pregnancy and therefore studies in human are warranted.
148. HORMONAL REGULATION OF miRNA IN THE TESTIS
Authors: Nicholls P. K. , Harrison C. A. , O'Donnell L. , Stanton P. G.
Published: November 2010

Acute suppression of circulating reproductive hormones (FSH and testosterone) inhibits sperm release (spermiation) (1), although the molecular mechanisms of spermiation failure are poorly understood. Micro-RNAs (miRNAs) are small non-coding RNAs that regulate protein expression, and are essential for normal spermatogenesis. Recent studies suggest that miRNAs are exquisitely sensitive to hormonal control by FSH, LH and testosterone (2–4). This suggests that hormonal regulation of miRNAs in the testis following acute hormonal suppression may contribute to spermiation failure. Therefore, we hypothesised that gonadotrophin regulated miRNAs control spermiation outcome. We used array analysis to show that miRNA expression is hormonally regulated by FSH and testosterone in our rat in vivo model of spermiation failure and also in primary rat Sertoli cells by. qPCR validation revealed that miR-7b, -23a, -30c, -125b, -148b, -197, -483, -592, and -690 are all hormonally sensitive testicular miRNAs. Bioinformatic analyses of potential gene targets of these miRNAs predicted numerous protein components localised in the testicular tubulobulbar complex (TBC). The TBC is a podosome-like structure found between Sertoli cells and adjacent germ cells in the testis, and is thought to internalise intact inter-cellular structures and regulate spermatid head shape prior to spermiation. WASP, a TBC protein that regulates actin filament dynamics, contained a conserved binding site for miR-690 within its 3'UTR. Increased miR-690 expression following hormone suppression corresponded to a decrease in WASP protein expression in vivo and in vitro. In addition, transfection of miR-690 into HEK293T cells down-regulated WASP protein. Our results suggest that following hormone suppression, miR-690 is stimulated in the Sertoli cell, thereby inhibiting WASP protein expression. We conclude that miRNA-mediated disruption of TBC integrity potentially regulates spermatid disengagement. This study describes new molecular mechanisms in the testis that may control spermiation outcome of potential significance in male hormonal contraception.
149. Smad3 DOSAGE INFLUENCES TESTICULAR MATURATION
Authors: Itman C. , Wong C. , Jans D. A. , Ernst M. , Loveland K. L.
Published: November 2010

Activin A, a TGF-beta superfamily ligand which signals via Smad2 and Smad3, is critical for normal mouse testis development and quantitatively normal sperm production. Whereas activin enhances immature Sertoli cell proliferation (1), excessive activin production causes Sertoli cell tumours (2); this is alleviated when mice lack Smad3 (3). Sertoli cells exhibit developmentally regulated Smad utilization in activin signalling. Immature Sertoli cells signal via Smad3 while the onset of Smad2-mediated signal transduction correlates with Sertoli cell maturation (4). This change coincides with decreased testicular Smad3 production at puberty and a shift in follicle stimulating hormone (FSH)-induced Smad transcription, from Smad3 in 6 dpp (days post partum) Sertoli cells to Smad2 in 15 dpp cells. These findings suggest that Smad3 is more important for testis development than adult spermatogenesis. To test this hypothesis, we examined testis development in Smad3+/– and Smad3–/– mice. At 7 dpp, testis weight and cord diameter were reduced in Smad3–/–mice, indicating impaired Sertoli cell proliferation. Levels of FSH, a potent Sertoli cell mitogen, were unaltered. Histological analysis revealed advanced spermatogenesis in heterozygous mice, with round spermatids already present at 16 dpp. Quantitative PCR also identified advanced Sertoli and germ cell maturation in Smad3+/– mice, while Leydig cell maturation appeared unaltered. Adult Smad3+/– and Smad3–/– mice were fertile, but had smaller testes. This is the first study relating Smad3 levels to puberty onset and identifies the Smad3+/– mouse as a model of peripheral precocious puberty with otherwise normal physiological status, i.e. no gonadal tumours and normal FSH levels. These results demonstrate that FSH influences testis growth and maturation by regulating Smad3 expression and highlights the importance of testing whether environmental factors, toxicants and endocrine disruptors affect Smad3 expression, thereby leading to altered testis development.
150. THE CAPACITATION INDUCED FORMATION OF A MULTIMERIC SPERM - ZONA PELLUCIDA RECEPTOR COMPLEX
Authors: Dun M. D. , Nixon B. , Aitken R. J.
Published: November 2010

The ability of mammalian spermatozoa to fertilize the oocyte is dependent on a complex cascade of biophysical and biochemical changes collectively known as capacitation. This final phase of sperm maturation is characterised by a dramatic remodelling of the sperm surface architecture to render the cell competent to recognise and bind to the zona pellucida. Although the current paradigm suggests this interaction is mediated by a single receptor-ligand interaction, recent evidence emerging from our laboratory suggests that this event is more complex and involves the capacitation-dependent formation of a multimeric zona pellucida receptor complex. Through the novel application of Blue Native PAGE and Far Western blotting, we have recently identified the first such complex that displays the anticipated affinity for the zona pellucida. LCMS analysis revealed that this high molecular weight complex is comprised of a family of 8 chaperonin subunits that form an active t-complex polypeptide-1 (TCP-1) complex. In addition, this complex was also shown to contain an N-acetylglucosaminyltransferase, GCnT2, an enzyme with affinity for N-acetylglucosamine. Importantly, this sugar forms part of the zona pellucida glyco-matrix and has previously been implicated as a ligand for sperm interaction. Consistent with this notion, incubation of sperm with anti-GCnT2 antibodies or N-acetylglucosamine competitively inhibited sperm-zona pellucida interactions in a dose dependent manner. Collectively, this data raises the intriguing possibility that chaperonin proteins participate in the assembly and/or presentation of key zona adhesion molecules during capacitation. Future work is aimed at identifying additional zona receptors that may reside within this complex.
151. LIPOTOXICITY MEDIATED ENDOPLASMIC RETICULUM STRESS, MITOCHONDRIAL DYSFUNCTION AND APOPTOSIS CONTRIBUTE IMPAIRED OOCYTE QUALITY IN RESPONSE TO OBESITY
Authors: Wu L. L. Y. , Yang X. , Dunning K. R. , Norman R. J. , Robker R. L.
Published: November 2010

In obesity, accumulation of lipid in non-adipose tissues, a process termed lipotoxicity, is associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction and ultimately apoptosis . We have previously shown that diet-induced obesity in mice causes impaired oocyte developmental competence, but whether this is due to activation of lipotoxicity pathways in the ovary is not known. The present study examined the hypothesis that diet-induced lipid accumulation in the cumulus oocyte complex (COC) disrupts ER homeostasis and mitochondrial membrane potential which leads to apoptosis. COCs and mural granulosa cells were collected from ovaries of adult mice fed a high fat (HFD) or control diet for 4 weeks. ER homeostasis was assessed by measuring expression of known ER stress marker genes, GRP78, ATF4 and CHOP. COCs from mice fed HFD showed significantly increased expression of GRP78 and ATF4. There was a similar trend towards increased expression in granulosa cells. Mitochondrial function was assessed by measuring membrane potential using the dual emission probe JC-1. In COCs from mice fed HFD there were reduced numbers of active mitochondria but instead large aggregated clusters of inactive mitochondria. Apoptosis in granulosa cells was determined by DNA laddering assay which showed significantly increased DNA fragmentation in cells from mice fed HFD. Apoptosis was also assessed by TUNEL staining of paraffin embedded ovaries from identical treatment groups. Ovaries from HFD mice appeared to have increased TUNEL positivity in both granulosa and cumulus cells. Our results demonstrate that the ER stress, mitochondrial dysfunction and apoptosis are markedly increased in granulosa cells and COCs from mice fed HFD, suggesting that lipotoxicity contributes to the impaired oocyte quality and reduced fertility observed in response to obesity.
152. INVOLVEMENT OF TRP53 IN SIRT1 FUNCTION DURING EMBRYO DEVELOPMENT
Authors: Mitchell M. , O'Neill C. , Filby A. N. , Cashman K. , Lane M.
Published: November 2010

Sirtuin 1 (SIRT1) is an NAD+-dependant deacetylase with significant functions in cell survival and metabolism, including glucose homeostasis and mitochondrial physiology. TRP53 is a universal effector of cellular stress responses and is an important target of SIRT1. Transcriptional activity of TRP53 in the pre-implantation embryo is associated with retarded development, however examination of SIRT1 function and how it relates to TRP53 activity remains to be elucidated. We therefore assessed whether SIRT1 is involved in pre-implantation embryo development and determined whether TRP53 interacts with SIRT1 function. Zygotes were collected from superovulated female mice and cultured to the blastocyst stage in optimised conditions (F1 mouse strain, G1/G2 series sequential media, 5%CO2, 5%O2, 90%N2, group culture) or compromised conditions (C57Bl/6 strain, mHTF static media, 5%CO2 in air, individual culture). Embryo development and blastocyst cell number was assessed following exposure to a SIRT1 inhibitor (0, 1, 10, 100 or 1000µM sirtinol). In subsequent experiments, embryos were cultured in a 2x2 factorial design (±1µM sirtinol and ±30µM pifithrin-α (TRP53 inhibitor)) and embryo development and cell number determined. Sirtinol caused a dose-dependent reduction in total cell number in blastocysts during culture in both optimised and compromised conditions (p<0.05), while the rate of development of zygotes was reduced for embryos in compromised but not optimised conditions (p<0.05). When SIRT1 was inhibited, in the presence or absence of TRP53 inhibition, blastocyst development and cell number for embryos in optimal conditions was unchanged. However, blastocyst development (83% vs 55%, p<0.05), and cell number (39 vs 54 cells, p<0.05) was reduced when SIRT1 was inhibited in compromised embryos, and in the absence of TRP53, development was resolved to control levels. These results show that SIRT1 is important for embryo development, particularly under compromised conditions, and that TRP53 is a likely target for SIRT1 deacetylase activity in the mammalian embryo.
153. XENOBIOTICS; INFLUENCE ON OVARIAN FOLLICULAR DEVELOPMENT
Authors: Sobinoff A. P. , Pye V. , Nixon B. , Roman S. D. , McLaughlin E. A.
Published: November 2010

The mammalian female reproductive lifespan is largely defined by a finite pool of ovarian follicles established around the time of birth. It is now understood that certain synthetic chemical compounds, known as xenobiotics, can cause premature ovarian senescence through the destruction of small ovarian follicles. Although the ovotoxic effects of these chemicals are well documented, the exact molecular mechanisms behind their action are only just becoming understood. Recent evidence suggests that bioactivation of xenobiotics by Phase I detoxifying enzymes may lead to the generation of free oxygen radicals (ROS), which we suspect may perturb intracellular signalling pathways in primordial follicles. In this study we attempted to identify ovarian follicle signalling pathways activated by xenobiotic exposure using ovotoxic agents which target immature follicles. Neonatal ovaries obtained from 3/4-day old Swiss mice were exposed to either 4-Vinylcyclohexene (25µM), Methoxychlor (25µM) or Menadione (5µM) for 96hrs using our in vitro culture system. Total RNA was then collected and analysed using Affymetrix Mouse Genome 430 2.0 Arrays. Bioinformatic analysis identified between ~500–1000 genes with a two-fold significant difference in gene expression (p<0.05) for each xenobiotic compared to the control. Differentially expressed genes were analysed for pathways and molecular functions using Ingenuity Pathways Analysis (Ingenuity Systems). In agreement with the current literature, many of the genes belonged to toxic response pathways, such as; Xenobiotic metabolism (10); p53 (15) and Apoptosis (11) signalling. However, the vast majority of the differentially expressed genes belonged to canonical pathways implicated in follicular development, such as PI3K/AKT (18), Wnt/ b -catenin (21), and JAK/Stat (8) signalling. Further qPCR analysis has confirmed a substantial increase in the transcription factor Sox4 and cell cycle inhibitor Cdkn2a in 4-Vinylcyclohexene and Menadione treated ovaries respectively. These results suggest that xenobiotics which target primordial follicles may exert part of their ovotoxic effects by perturbing signalling pathways involved in follicular activation and development.
154. EFFECT OF KOREAN RED GINSENG EXTRACT IN STEROID-INDUCED POLYCYSTIC OVARY MURINE MODEL
Authors: Pak S. , Kim S. , Bae C. , Lim S.
Published: November 2010

Experimental induction of polycystic ovary (PCO) resembling some aspects of human PCO syndrome was produced using the long-acting compound estradiol valerate (EV). Our previous study on the role of Korean red ginseng total saponins in a steroid-induced PCO rat model demonstrated that electro-acupuncture modulates nerve growth factor (NGF) concentration in the ovaries. In fact, the involvement of a neurogenic component in the pathology of PCO-related ovarian dysfunction is preceded by an increase in sympathetic outflow to the ovaries. In the present study, we tested the hypothesis that Korean red ginseng extract (KRGE) administration modulates sympathetic nerve activity in rats with PCO. This was done by analysing NGF protein and NGF mRNA expression involved in the pathophysiological process underlying steroid-induced PCO. EV injection resulted in significantly higher ovarian NGF protein and NGF mRNA expression in PCO rats compared to control rats, and PCO ovaries were counteracted by KRGE administration with significantly lower expression of NGF protein and NGF mRNA compared to EV treated ovaries. These results indicate that EV modulates the neurotrophic state of the ovaries, which may be a component of the pathological process by which EV induces cyst formation and anovulation in rodents.
155. CD44 SIGNALLING IN THE CUMULUS OOCYTE COMPLEX DURING OVULATION
Authors: Alvino E. R. , Robker R. L. , Russell D. L.
Published: November 2010

Oocytes develop within ovarian follicles that nurture and regulate oocyte maturation. The LH surge induces a cascade of gene expression leading to formation of the hyaluronan (HA) rich cumulus matrix around the oocyte. This cumulus oocyte complex (COC) is composed of high concentrations of HA cross-linked by several HA-binding proteins. Null mutation of several COC matrix genes results in ovulation defects demonstrating the importance of the composition and structure of the COC; but the mechanisms by which the matrix promotes ovulation are unknown. We hypothesised that HA, via activation of its receptor CD44 on cumulus cells, regulates cytoskeletal function, cell adhesion and migration, and that acquired cumulus cell motility facilitates ovulation. We investigated cellular signaling and cellular phenotypes occurring in response to the formation of the HA-rich COC matrix. Expression of CD44 was upregulated 5 to 6-fold in cumulus cells following 6 or 12h hCG (LH analog) stimulation. Signal transducers of CD44 action; Tiam1, a guanine exchange factor, and Rac1, an actin cytoskeleton remodelling Rho-family GTPase, were present in cumulus cells but not regulated by hCG. Induction of migratory and invasive capacity of cumulus cells by hCG was demonstrated using transwell migration and ECM invasion assays. Cumulus cell migration increased 8-fold 10h after hCG compared with cumulus cells from untreated mice. These cumulus cells also showed the capacity to invade through a matrigel barrier. Inhibitors of the CD44-assembled cell migration complex demonstrated the importance of this pathway in the migratory and invasive phenotype of cumulus cells. These results demonstrate that CD44 is a key factor in the assembly of a macromolecular complex facillitating cell motility in cumulus cells at the time of ovulation, and suggest that cumulus cells in the expanded COC undergo epithelial-mesenchymal transition to become invasive motile cells which may play a key role mediating ovulation.
156. EFFECTS OF ALBENDAZOLE ON OVARIAN OESTROGEN SYNTHESIS IN THE RAT
Authors: Zulkafli I. S. , Mark P. J. , Martin G. B. , Waddell B. J.
Published: November 2010

Albendazole is a drug commonly used for treatment of helminth infestation in human and livestock populations. Recent studies show that albendazole reduces ovarian follicular fluid oestrogen levels in sheep1, but the mechanism involved is unknown. The aims of this study were to determine whether albendazole exerts similar effects on ovarian oestrogen levels in the rat, and to assess the effects of albendazole on expression of key steroidogenic genes in the rat ovary. Oestrus cycles were continuously monitored in Wistar rats by vaginal smears. Commencing at proestrus, albendazole was administered for 12 days in drinking water (approximate dose 15 mg/kg/day). Plasma and whole ovaries were collected on the fourth proestrus (1500–1600h). A second group of rats were treated similarly except that pseudopregnancy (PSP) was induced by mating with a vasectomised male at the second proestrus. Plasma and the non-luteal ovary were collected on day 8 of PSP. Oestradiol was extracted from plasma and ovaries with ethyl acetate and concentrations measured by a chemiluminescent assay. Expression of steroidogenic acute regulatory protein (StAR), P450 side chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), aromatase and 20α-hydroxysteroid dehydrogenase (20α-HSD) mRNAs were measured by RT-PCR. Oestrus cyclicity, ovarian weight and mating behaviour were all unaffected by albendazole in cycling and PSP rats, although as expected levels of oestradiol were lower in PSP. In ovaries of cycling rats albendazole did not affect oestradiol concentrations but reduced ovarian P450scc mRNA expression (by 65%; P=0.024) and there was a trend for an increase in 3β-HSD (P=0.09) and aromatase expression (P=0.12). Expression of the other steroidogenic genes was unaffected and no changes in gene expression were observed in PSP rats. In conclusion, albendazole treatment reduced ovarian P450scc in cycling rats but did not inhibit ovarian oestradiol synthesis or reproductive function.
157. EXPRESSION PATTERNS OF EXTRACELLULAR MATRIX IN MICE OVARIES
Authors: Hummitzsch K. , Irving-Rodgers H. F. , Murdiyarso L. S. , Bonner W. M. , Sado Y. , Ninomiya Y. , Couchman J. R. , Sorokin L. M. , Rodgers R. J.
Published: November 2010

Extracellular matrix (ECM) has been shown to have distinct expression patterns during bovine follicle and corpus luteum (CL) formation and regression. To date little is known about ECM patterns during follicle and CL formation in the mouse ovary. Twenty nine day old mice were treated with PMSG on experimental day 0 and 1 to induce follicle development, and subsequently with hCG on day 2 to induce ovulation. Ovaries were collected for immunohistochemistry on days 0, 2 and 5 (n = 10 per group). Another group was similarly treated but was additionally mated, and ovaries examined in the pregnant mice on experimental day 11 (n = 7). The follicular basal lamina (BM) of all developmental stages contained collagen IV α1 and α2, laminin α1, β1 and γ1 chains, nidogen 1 and 2, and perlecan. Collagen XVIII was only found in BMs of primordial, primary and some preantral follicles, whereas laminin α2 was only present in some preantral and antral follicles. BMs of atretic follicles showed similar composition to healthy follicles. A specialized matrix of the membrana granulosa (focimatrix) was detected in both healthy and atretic follicles. The focimatrix contained collagen IV α1 and α2, laminin α1, β1 and γ1 chains, nidogen 1 and 2, perlecan and collagen XVIII, but not laminin α2. The immunostaining in CL was restricted to capillary sub-endothelial basal laminas and contained collagen IV α1 and α2, laminin α1, β1 and γ1 chains, nidogen 1 and 2, perlecan and collagen XVIII. No parenchymal matrix, as observed in cow and human, was detected. In addition, laminin α4 and α5 were not immunolocalised to any structure in the mouse ovary. The composition of the BM of follicles in the mouse ovary is similar to cow and rat, but the appearance and composition of the focimatrix differs from bovine ovaries.
158. EFFECT OF A59V LOCUS IN THE LEPTIN GENE ON LENGTH OF PREGNANCY IN IRANIAN HOLSTEIN COWS
Authors: Yazdani H. , Rahmani H. , Edris M. , Dirandeh E.
Published: November 2010

We investigated effect of A59V polymorphism in the leptin gene on length of pregnancy. Blood was collected from 255 Holstein cattles belonging to four different herd managements in Isfahan province. Genomic DNA extracted from whole blood. Genotypes of A59V locus were identified PCR-RFLP technique. Amplified region is located in exon three of leptin gene. The genomic bovine leptin sequences, which consist of three exons, were obtained from Gene Bank (Accession number U50365). The polymerase chain reaction was used to amplify the 331 bp DNA fragments from genomic DNA. The PCR reaction contained 100 ng of genomic DNA, 0.3 µM of each primer, 1.5 mM MgCl2, 200 µM dNTP, 10mM Tris HCl, 50 mM KCl and 1 U Taq-polymerase in total volume of 20 µL. Sequences of primers that were used in PCR were reported previously by Haegeman et al. (2000). Conditions for PCR were 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 30 s. Followed by final extension for at for 15 min 72°C. Digestion of PCR product of 331 bp with 5 U of HphI (Fermentas) in 20 µL of reaction volume at 37°c for 8 h and analyzed on 8% no denature polyacrylamyde gel. Allele A in the A59V locus was the allele not digested by restriction enzyme, allele B was the restriction enzyme-digested PCR product. Digestion revealed 3 genotypes, AA (331 bp), AB (331, 311, and 20 bp), and BB (311 and 20 bp). Significances of the genotype effects were estimated by GLM procedure of SAS. This study showed that genotype effect on length of pregnancy were significant (P<0.01). Animals homozygous for allele A had higher length of pregnancy ((P<0.01, AA=279.17±0.47, AB=276.96±0.57, BB=274.8±2.2).
159. MATERNAL NUTRITION AND GESTATIONAL AGE AFFECT PLACENTAL microRNA EXPRESSION IN THE GUINEA PIG
Authors: Grant P. A. , Kind K. L. , Sohlstrom A. , Roberts C. T. , Owens J. A.
Published: November 2010

Maternal undernutrition restricts placental growth and nutrient supply to the fetus, but induces compensatory alterations in structure and function of the placenta. Maternal undernutrition in guinea pigs also restricts placental growth and alters structure, and changes expression of Igf1, Igf2, Slc2a1, Slc38a2 mRNA in mid and late gestation, consistent with nutritionally induced changes in nutrient transport across the placenta. MicroRNAs are non-coding RNAs that regulate expression of target genes by translational inhibition and mRNA degradation and are present in the mammalian placenta. Effects of maternal undernutrition on their expression are unknown. We hypothesised that altered expression of key functional genes in the placenta in maternal undernutrition are in part due to altered expression of regulatory microRNAs. The effect of maternal food restriction on the expression of microRNAs in the guinea pig placenta was examined at D30 and D60 of gestation (term = D70). Guinea pigs were fed either ad libitum (AL) or restricted (R). MicroRNA expression was determined by Exiqon microarray v.8.1. In AL placentas, 119 microRNAs were upregulated (p<0.05), whilst 40 were down-regulated (p<0.05) at late compared to early gestation. In R placentas, 163 microRNAs were upregulated (p<0.05), whilst 123 were down-regulated (p<0.05) at late compared to early gestation. Of the 20 most abundant up-regulated microRNAs miR-Plus (ID 17871) and hsa-miR-411 were altered only in AL and hsa-miR-376a and -376b were altered only in R placenta. Of the 20 most abundant down-regulated microRNAs, 13 were altered only in AL and 14 only in R placentas. Placental expression of microRNAs changed with gestation, and maternal undernutrition modified this pattern and altered expression of many additional microRNAs in the guinea pig placenta. This suggests that miRNAs and factors that influence their expression may play a role in the structural and/or functional development of the placenta and hence fetal growth.
160. THE POTENTIAL ROLE OF microRNAs IN THE DEVELOPMENT OF THE HUMAN PLACENTA IN EARLY PREGNANCY
Authors: Kong W. , Nowak R. , Roberts C. T. , Owens J. A.
Published: November 2010

Placental functional development is characterised by dynamic and co-ordinated changes in expression of genes that drive invasion, differentiation and growth. These changes may arise in part from altered expression of microRNAs (miRNAs) via their regulatory networks. MiRNAs are short, single-stranded, non-coding RNAs involved in the post-transcriptional repression of gene expression. MiRNAs bind to complementary sites in the 3'UTR of target mRNAs to repress or silence translation. MiRNAs have been detected in the mammalian placenta, but their patterns of expression throughout pregnancy have not been systematically characterized. Using microarrays, miRNA gene expression was compared at two stages (6–8 weeks, 10–12 weeks) in early gestation, in chorionic villi of human placentas (term ~40 weeks). Putative and validated targets of differentially expressed miRNAs were extracted from freely accessible databases, miRBase [1], PicTar [2], TargetScan [3] and miRecords [4]. 15 miRNAs were differentially expressed between these gestational ages (p<0.05). 11 of these miRNAs were upregulated in 10–12 week villi and 4 were downregulated. Many of the differentially expressed miRNAs are members of the same polycistronic clusters, suggesting that these miRNAs may be co-expressed. Shared targets of differentially expressed miRNAs from the same clusters were assessed using Ingenuity Pathways Analysis, to search for significantly represented molecular networks. All downregulated miRNAs at 10–12 weeks shared 35 putative targets and fell into 1 of 2 clusters, on chromosome 13 or X. Previously validated targets include PTEN [5], Notch1 [6], VEGFA [7], CDKN2A [8] and DHFR [9] . Six of the upregulated miRNAs at 10–12 weeks are members of 3 clusters on chromosome 19, 9 and X. Networks targeted by these cluster members include PTEN, HIF1α and IL-12 signalling. Together all of these processes are active and important in early placentation and their predicted targeting by differentially expressed miRNAs is consistent with an important role in placental development.
161. PATERNAL AND FETAL SINGLE NUCLEOTIDE POLYMORPHISMS IN KDR GENE ASSOCIATE WITH PREECLAMPSIA AND INTRAUTERINE GROWTH RESTRICTION
Authors: Andraweera P. H. , Thompson S. D. , Nowak R. C. , Zhang V. J. , Dekker G. A. , Roberts C. T.
Published: November 2010

Introduction: Preeclampsia (PE) and intrauterine growth restriction (IUGR) together contribute to maternal and neonatal morbidity and mortality. Abnormal placental angiogenesis is implicated in these pregnancy complications. KDR is the main receptor for vascular endothelial growth factor, a potent angiogenic factor which regulates placental angiogenesis. Derangements in KDR expression are known to result in abnormal angiogenesis. We aimed to determine whether polymorphisms in KDR gene (KDR T604C and KDR C1192C) are associated with PE and IUGR. Methods: 1169 nulliparous pregnant women and their partners were recruited prospectively at the Lyell McEwin Hospital and women monitored throughout pregnancy. PE and IUGR were classified using strict guidelines. Uncomplicated pregnancies were deemed controls. Peripheral blood was collected from couples and cord blood collected at delivery. DNA extraction from buffy coats and genotyping were performed at the Australian Genome Research Facility using the Sequenom MassARRAY system. Genotypes for PE (n=63) and IUGR (n=94) were compared with controls (n=373) and analysed using ANOVA and Chi Square. Odds Ratios (OR) were calculated. Results: Paternal and neonatal KDR T604C were associated with PE (p=0.028, OR=1.9, 95%CI=1.08–3.34 and p=0.008, OR=2.5, 95%CI=1.3–4.78). Paternal and neonatal KDR T604C were associated with IUGR (p=0.005, OR=2.01, 95%CI=1.24–3.25 and p=0.01, OR=1.15, 95% CI=1.22–3.79). Neonates with KDR T604C CC genotype were 144.5g lighter than those with the TT genotype (p=0.05). Mean customised birth weight centile was 8.7 lower for fathers with KDR T604C CC genotype compared to CT (p=0.041). KDR C1192T SNP was not associated with outcome. Conclusion: Our results suggest that KDR T604C polymorphism is associated with both PE and IUGR. We are the first to demonstrate an association between paternal KDR polymorphisms and pregnancy complications. Paternal genes acting via the placenta appear to contribute to the risk of PE and IUGR. Ongoing research will determine the role of these polymorphisms in placental angiogenesis.
162. THE EFFECT OF MATERNAL FOLIC ACID SUPPLEMENTATION THROUGHOUT PREGNANCY ON NEURODEVELOPMENT, MOTOR FUNCTION AND BEHAVIOUR OF PROGENY IN THE RAT
Authors: Pearce B. L. , Owens J. A. , Dziadek M. , Grant P. A. , Wlodek M. , Robinson J. S. , Pitcher J. B.
Published: November 2010

Background: Maternal folic acid supplementation (mFAS) during early pregnancy is recommended to reduce the incidence of neural tube defects and has recently been associated with improved neurodevelopment in children. However, the effect on neurodevelopment of mFAS from before conception and throughout pregnancy is unknown. We examined the effect of mFAS throughout the gestational period on postnatal growth, neurodevelopment and early adult motor function and behaviour in rat offspring. Methods: Female Wistar Rats were fed either a control (folic acid 2mg/kg, n=6) or moderate mFAS diet (folic acid 6mg/kg, n=6) from two weeks before mating with Lewis males, until birth of progeny. Male and female progeny (Control=36, mFAS=36) were weighed on postnatal day (PD) 3, 7, 14, 21, 40 and 90, and underwent various tests between PD4 and 14: righting reflex, palm-grasp reflex, negative geotaxis, forelimb hanging, ascent test and eye opening. Locomotor/ exploratory behaviour, motor coordination and anxiety were assessed using an open field test (PD52), rotarod (PD55) and elevated plus maze (PD58) (Control=24, mFAS=24). Results: mFAS did not alter maternal weight gain, litter-size at birth or progeny growth between PD3-90. mFAS tended to increase righting reflex time (p=0.057) and impair ascent ability (p=0.085). Negative geotaxis time was reduced at PD7 but not later (Diet x Age p=0.051). mFAS increased the proportion of progeny with eyes open at PD14 (p=0.008) and tended to increase forelimb hanging time(p=0.097). mFAS did not alter motor learning/function (rotarod), but increased ambulatory and exploratory behaviour (open field test; p=0.027). Conclusions: mFAS delays some early aspects of neurodevelopment including neonatal postural reflex maturation and proprioceptive/vestibular function, but accelerates others such as eye opening. However, the open field test indicated that mFAS improved the offspring's locomotion and exploratory behaviours in adulthood. Further studies will differentiate the neurodevelopmental effects of mFAS around conception from gestation-long mFAS.
163. MATERNAL FOLIC ACID SUPPLEMENTATION INDUCED ALTERATIONS IN METABOLIC HEALTH OF PROGENY: ROLE OF microRNA REGULATORY NETWORKS
Authors: Chu W. H. , Harland L. , Grant P. , De Blasio M. , Kong W. , Moretta S. , Robinson J. S. , Dziadek M. E. , Owens J. A.
Published: November 2010

Background: Nutrition in early life can influence metabolic functionality in later life, in part via heritable epigenetic changes, which modify gene expression without altering DNA sequence. Folate supplies methyl groups for the methylation of DNA and histones, both major epigenetic marks that change dynamically in utero. We have recently shown that maternal folic acid supplementation (MFAS) in the pregnant rat increases insulin sensitivity in adult male progeny, while decreasing that of females. The molecular basis of this is unknown but microRNAs may play a role. MicroRNAs are epigenetically regulated non-coding RNAs that downregulate post-transcriptional expression of their targets. MFAS may modulate epigenetics and expression of microRNAs and their targets in adult progeny to alter insulin sensitivity. Aims/Hypotheses: The effect of MFAS before and throughout pregnancy on microRNA expression in liver and skeletal muscle of adult progeny was determined. Methods: Female Wistar rats were fed Control (n=11) or Folic Acid Supplemented (n=9) diets containing either 2 or 6 mg folic acid/kg respectively, from two weeks before mating and throughout pregnancy. One male and female progeny per litter were sacrificed on postnatal day 90 and microRNA expression was determined by Exiqon microRNA microarray v.8.1. Results: MFAS altered hepatic microRNA expression in adult male progeny, but did not alter that in females. Sixteen hepatic microRNAs were differentially expressed, with five predicted in silico (rno-miR: 23a, 23b, 212, 298 and 325-5p) to target several key insulin signalling molecules (p85α, p110β, Akt2, and Prkcz). miR-122a, which promotes cholesterol and lipid synthesis in vivo, was also downregulated. MFAS did not alter microRNA expression in skeletal muscle of adult male or female progeny. Conclusions: MFAS alters hepatic microRNA expression in adult male progeny. Changes in their expression together with their targets in insulin signalling pathway may initiate increased insulin sensitivity in adult male progeny.
164. THE IMPRINT STATUS AND EXPRESSION OF INS IN THE TAMMAR WALLABY, MACROPUS EUGENII
Authors: Stringer J. M. , Shaw G. , Pask A. , Renfree M. B.
Published: November 2010

Genomic imprinting is an epigenetic mechanism that differentially regulates the expression of certain genes, resulting in expression from only one parental allele. It is presumed to have first evolved after the divergence of therian mammals from the monotremes. One imprinted gene, INS is maternally imprinted (paternally expressed) in the eutherian and marsupial yolk sac1,2. INS encodes the precursor to the hormone insulin, which regulates carbohydrate metabolism and has a role in cell growth and, by regulating amino acid and fatty acid transporters, protein synthesis. In rats, mice and several other mammals insulin, in addition to cortisol and prolactin, is an absolute requirement for the onset of lactation and the synthesis of milk3. As imprinting plays an important role in regulating nutrition and growth the role of imprinted genes in the placenta has been the focus for imprinting research. Since the mammary gland provides a critical source of nutrition for the neonate in all mammals it is possible that genomic imprinting may have developed and been maintained in this organ. Given that marsupials deliver tiny, altricial young, it is in the relatively long and complex lactation phase where the mother has most control of the young's growth. Therefore, there may be greater selection for genomic imprinting in the marsupial mammary gland than in the eutherian mammary gland. This study examined the expression and the imprint status of INS in the mammary gland and neonatal tissues of the tammar wallaby, Macropus eugenii. INS expression was detected using PCR and direct sequencing provides evidence of INS imprinting in the mammary gland. This is the first study to identify imprinting in the mammary gland of a marsupial and the first to identify INS imprinting outside of the yolk sac.