bluuebunny/crossref_metadata_2025 huggingface dataset:
020. HORMONAL REGULATION OF SPERMATOGENESIS
Authors: Handelsman D.
Published: November 2010

Spermatogenesis is a spatially and temporally co-ordinated proliferation of the germinal epithelium in the semeniferous tubules. The germ cells are embedded in a scafolding formed by adjacent Sertoli cells linked tightly by intercellular junctions and with each germ cell enshrouded by elongations of Sertoli cell cytoplasm. Spermatogenesis comprises serial stages from the mitotic replication of the stem and early germ cells, followed by meiosis, the reductive division producing haploid, amorphous gametes which subsequently undergo spermiogenesis, the metamorphosis into terminally differentiated and functional spermatozoa. Although long known that all but the earliest stages are hormonally regulated by pituitary secretion of LH and FSH, it has remained difficult to separate gonadotrophin effects by classical endocrine ablate-replace methods as these two heterodimeric hormones have identical α and homologous β subunits, are secreted from the same pituitary gonadotrophs to target cognate receptors expressed on adjacent testicular cells as equally homologous, heptahelical G-coupled protein receptors. Over two decades our laboratory has developed a variety of complementary genetic and pharmacological approaches to dissect the individual and co-operative effects of LH, its main effector testosterone and FSH on spermatogenesis. Using the gonadotrophin and testosterone deficient hpg mouse, double transgenic human FSH secreting mouse and the androgen receptor knockout mouse lines together with steroidal depot homone delivery, we have explored systematically annd defined the individual primary actions of FSH and testosterone and their interactions in the regulation of testis growth, spermatogenesis and ultimately male fertility.
031. THE ORIGIN OF ANEUPLOIDY IN HUMANS: WHERE WE'VE BEEN, WHERE WE'RE GOING
Authors: Hassold T.
Published: November 2010

With the advent of the human genome project in the 1990s, DNA markers became available, allowing us to determine the parent and meiotic stage of origin of human aneuploid conditions. This approach has been extensively used to study the origin of trisomies, with one over-arching conclusion: the vast majority of trisomies derive from errors in the development of the egg and, in particular, from nondisjunction occurring in the first maternal meiotic division (MI). However, against this general background, it has also become apparent that there is considerable chromosome-to-chromosome variation. For example, certain aneuploidies (e.g., the XXY condition, or Klinefelter syndrome) are commonly paternal in origin, while others (e.g., trisomy 16, the most common human trisomy) almost always originate from maternal MI errors. Thus, we now know that individual chromosomes behave differently with respect to mechanisms of meiotic nondisjunction. In this presentation, we will summarize results of these parental origin studies of aneuploidy and discuss the involvement of the first identified molecular correlate of nondisjunction – aberrant meiotic recombination – in the genesis of these abnormalities. We will also look forward, discussing the development of mouse models of human aneuploidy, and recent advances in molecular cytogenetics that make it possible to directly analyze human meiosis ‘as it happens’ in fetal oocytes and in spermatocytes.
032. Cdh1: A CELL CYCLE PROTEIN INVOLVED IN FEMALE MEIOSIS AND PREVENTION OF ANEUPLOIDY
Authors: Jones K. T.
Published: November 2010

Mammalian oocytes are arrested at the dictyate stage of prophase I in the ovary. In growing follicles, oocytes can become responsive to Luteinising Hormone and will undergo meiotic resumption just before ovulation. During the first meiotic division, homologous chromosomes are segregated, a process that is very error prone in human oocytes. By ovulation the oocyte has extruded its first polar body and has re-arrested at metaphase of the first meiotic division. Recent work from our lab has established that the protein Cdh1 is involved uniquely in both in the process of prophase I arrest and the correct segregation of homologs in meiosis I. Thus in cultured oocytes, in vitro antisense knockdown of Cdh1 induces both meiotic resumption and high rates of aneuploidy as a result of non-disjunction during first meiosis. Cdh1 causes prophase I arrest by inducing cyclin B1 degradation and maintaining low levels of the kinase CDK1, whose activity induces meiotic resumption. Cdh1 is an activator of the Anaphase-Promoting Complex (APC), a ubiquitin ligase that earmarks proteins such as cyclin B1 for proteolysis. Cdh1 prevents aneuploidy by causing the degradation of Cdc20, a protein that is responsible for activating the APC once all homologs are correctly aligned at metaphase. Thus loss of Cdh1 seems to prematurely activate APC(Cdc20) activity. It is interesting that a single protein can affect two important meiotic transitions in oocytes. However to explore its functions more fully, and confirm that an in vitro knockdown is faithfully replicated by in vivo loss, a targeted knockout of Cdh1 is needed. Therefore we have generated an oocyte specific Cdh1 knockout by ZP3 promoter driven Cre- recombinase activity in oocytes carrying loxP insertions in the single copy Cdh1 gene. This talk will therefore focus on the effects of an in vivo Cdh1 knockout.
033. IMPORTIN α2 MEDIATES SUBNUCLEAR TARGETING OF THE CAJAL BODY COMPONENT COILIN; A KEY ROLE IN SPERMATOGENESIS?
Authors: Ly-Huynh J. D. , Efthymiadis A. , Whiley P. , Richards E. , Lescesen H. , Meier I. , Matera G. A. , Loveland K. L. , Jans D. A.
Published: November 2010

Spermatogenesis, the progressive maturation of immature germ cells to form spermatozoa, requires nucleocytoplasmic shuttling of nuclear factors to implement changes in gene transcription, as well as the storage and alternative splicing of mRNA transcripts in the nucleus that is vital for fertility. The key cellular mediators of nuclear entry are members of the importin (IMP) superfamily, of which the five different IMPα proteins in mouse testis are expressed dynamically throughout spermatogenesis, consistent with roles in transporting distinct, specific cargoes critical to gamete maturation. We identified the central Cajal body (CB) component Coilin as a specific testicular binding partner of IMPα2 in a yeast 2-hybrid screen and confirmed this interaction by coimmunoprecipitation from testis lysates. CBs are small nuclear inclusions that can associate with histone genes and facilitate histone pre-mRNA processing by recruiting RNA processing factors; intriguingly, expression of IMPα2 but not other IMPαs can regulate the number and size of CBs, as shown in cell culture experiments. Immunohistochemical analysis revealed that Coilin is predominantly in spermatocytes and in round and elongating spermatids in the rodent testis. The physiological importance of its role is indicated by the fact that Coilin knockout mice have reduced fertility, smaller testes and aberrant spermatogenesis. Our future work will focus on the testicular functions of Coilin and IMPα2 during spermatogenesis, and their roles in coordinating the pre-assembly, storage and targeting of transcription complexes to RNA processing machinery in the nucleus.
042. THE EFFECT OF THE INSECTICIDE PYRIPROXYFEN ON OVARY SYNTHESIS IN THE CHRISTMAS ISLAND RED CRAB, GECARCOIDEA NATALIS; A POSSIBLE CASE OF ENDOCRINE DISRUPTION?
Authors: Linton S. M. , Barrow L. , Davies C. , Harman L.
Published: November 2010

The yellow crazy ant, Anopholepis gracilipes is an invasive species on Christmas Island, Indian Ocean whose population needs to be controlled before there is irrevocable environmental damage. The insecticide, pyriproxyfen, a compound which mimics juvenile hormone of insects, has been proposed to do this. Before it can be used in the field, its effects on non target species such as the endemic red crab, Gecarcoidea natalis, need to be investigated. Land crabs, like all decapods, may utilise a similar hormone called methyl farnesoate which is thought to be involved in controlling early ovary development. Pyriproxyfen may also mimic methyl farnesoate and thus disrupt this process. The effect of pyriproxyfen on early ovary synthesis in G. natalis was investigated by feeding crabs a mixture of leaf litter and bait containing 0.5% pyriproxyfen (experimental groups) or a mixture of leaf litter and bait containing no pyriproxyfen (control groups) at simulated baiting doses (2 kg ha–1 and 4 kg ha–1). Two additional groups of crabs were fed ad libitum, either bait containing 0.5% pyriproxyfen or the control bait. Experiments were conducted from July to September of 2007. Red crabs synthesise their ovaries annually over two months (July to September) in the dry season. This situation of high nitrogen demand is funded from small excesses of nitrogen assimilated from a mainly leaf litter diet. Pyriproxyfen affected early ovary development. Ovaries from crabs in the experimental groups at all baiting levels had a higher total nitrogen content and dry mass than that of the control animals. The ovaries from the experimental animals were also more mature; they contained more previtellogenic and early vitellogenic oocytes, of a larger diameter, than ovaries of the control animals. Significant amounts of pyriproxyfen were accumulated in the target tissues, the midgut gland and ovary. Minor amounts of pyriproxyfen were accumulated in muscle, a non-target tissue. By mimicking methyl farnesoate, pyriproxyfen may have caused endocrine disruption in G. natalis. In particular it may have stimulated early ovary development and synthesis of yolk protein.
054. TROPHOBLAST, PLACENTA AND EARLY EMBRYO: HOW THE MARSUPIAL DEVELOPS
Authors: Renfree M. B. , Frankenberg S. R. , Freyer C.
Published: November 2010

In marsupials, the blastocyst forms as a single cell layer of cells. The marsupial blastocyst has no inner cell mass, so the 80–100 cell tammar embryo remains in diapause as a unilaminar blastocyst. All marsupials have a unilaminar stage, but what is unusual is that in the tammar the total cessation of cell division and cell metabolism lasts for 11 months each year. Marsupials are placental mammals. The yolk sac forms the definitive placenta up to birth. Only very few marsupials, such as the bandicoot, have a chorio-allantoic placenta, which supplements the placental functions of the yolk sac. However, the understanding how the unilaminar layer of trophoblast cells of the diapausing blastocyst become specified into placental and embryonic tissues has been an ongoing puzzle. To identify genes that do become differentially expressed in tammar development, we targeted the stage of the earliest appearance of the embryonic disc, at which the remainder of the blastocyst is then defined as trophoblast, as well as early cleavage stages. Intriguingly, we found no evidence for early differential expression of the canonical pluripotency genes POU5F1, SOX2 and NANOG, or of CDX2. By contrast, we found overt differential expression of GATA3, the closely related gene GATA2, and FGF4. This expression profile suggests that in the tammar, mechanisms regulating trophoblast- and pluriblast-specific expression of POU5F1, SOX2, NANOG and CDX2 are temporally secondary to those regulating GATA2 & -3 and FGF4 expression. Together, our results may signify the evolution of alternative mechanisms of early lineage specification in marsupials, or alternatively reveal a general hierarchy of signalling mechanisms that are masked in the relatively rapid and ‘compressed’ development of mice. The results of our ongoing study have important implications for understanding not only marsupial stem cells but the early development of all therian mammals.
055. EVEN REPTILES DO IT, THE STRUCTURE AND FUNCTION OF PLACENTAE IN VIVIPAROUS LIZARDS
Authors: Thompson M.
Published: November 2010

Live birth (viviparity) has evolved independently more than 100 times in squamate reptiles (lizards and snakes). Most viviparous lineages are characterised by a simple placenta with squamous epithelia on both maternal and embryonic sides, and the remnants of an eggshell persist for some, if not all, of pregnancy. The embryos are predominantly lecithotrophic, with maternal-embryonic exchanges being limited mostly to inorganic ions, water and respiratory gases. Nevertheless, there is differentiation of a chorioallantoic placenta and a yolk sac (omphalo) placenta in all species. Complex placentae have evolved in the Squamata in only four or five lineages, all in the lizard family Scincidae (skinks). In species with complex placentation, the uterus differentiates to allow different functions in association with each embryonic membrane type. Species with complex placentae are characterised by a hypertrophy of maternal and embryonic cells, elaboration of the maternal surface, a reduction in yolk with a concomitant increase in placentotrophy and, in some, regional differentiation of the choriallantois into a placentome and a paraplacentome. Both the placentome and omphaloplacenta are organs of embryonic nutrition, but they transport nutrients by different mechanisms; the paraplacentome is a specialised gas exchange organ. The most placentotrophic species are in the South American genus Mabuya, where the females produce micro-lecithal eggs and the placenta is more complex than in any mammal, with four specialised structures for nutrient exchange and one for gas exchange. The number of independent origins of viviparity and the range of placental complexities exhibited by skinks enables us to infer the evolutionary trajectories that have resulted in microlecithal eggs and an almost complete reliance of placentotrophy from oviparous ancestors.
056. EUTHERIAN MAMMALS DO IT DIFFERENTLY: PLACENTAL ENDOCRINE STRATEGIES FOR THE MAINTENANCE OF PREGNANCY IN RODENTS AND PRIMATES
Authors: Waddell B. J.
Published: November 2010

The placenta of rats and humans share important anatomical similarities, each with a chorio-allantoic, single discoid, haemochorial structure that facilitates highly efficient nutrient transport. Importantly, however, these similarities reflect convergent evolution and conceal markedly different developmental trajectories and endocrine functions. Placental endocrine signals are essential to drive maternal adaptations that facilitate fetal development and ultimately successful birth. Central to these adaptations is a sustained increase in production of the sex steroids progesterone and oestrogen, each driven by very different placental signalling in rodents and primates. Specifically, while the rat placenta supplies androgen precursors for ovarian (luteal) oestrogen synthesis, in humans and closely-related primates the fetal adrenal cortex supplies androgen precursors for placental oestrogen synthesis. In both cases the resultant increase in oestrogen provides a local stimulus to ovarian (rat) and placental (primate) progesterone synthesis. This shift from a placental-ovarian to a feto-placental unit for oestrogen synthesis in primates may have evolved to ensure greater fetal influence over maternal adaptations. Placental regulation of maternal physiology is also mediated via a third steroid group, the glucocorticoids, which promote a successful pregnancy outcome via effects on maternal metabolism and fetal organ maturation. Glucocorticoids are produced within the HPA axis, activity of which is enhanced by the placenta (eg, via oestrogen in rodents and CRH in primates). Moreover, the placenta regulates access of maternal glucocorticoids to the fetus via expression of the 11 b -HSD enzymes which constitute the placental glucocorticoid barrier. Intriguingly, this barrier effectively disappears during late fetal life in rodents but increases markedly in primates (notably baboons and humans). We hypothesise that this opposite developmental change is due in part to the evolution of the feto-placental unit for oestrogen synthesis in these primate species, and the associated need to prevent suppression of the fetal HPA axis by maternal glucocorticoids in late gestation.
058. GESTATIONAL DIABETES AND TYPE 2 DIABETES IN PREGNANCY IN AUSTRALIA
Authors: Cheung N. W.
Published: November 2010

In the last 30 years, there has been a dramatic increase in the incidence of gestational diabetes (GDM) in Australia. GDM has become a significant population health issue and Australia has been at the forefront of international research into its significance and management. More recently, the tsunami of GDM has been followed by a growing wave of type 2 diabetes in pregnancy. Type 2 diabetes is becoming more prevalent than type 1 diabetes in pregnancy, and adverse pregnancy outcomes are more common. However, diabetes itself is but one factor influencing outcomes in this group of women, with obesity, cultural issues and socioeconomic disadvantage being other significant issues. The research of our group has focused on examining traditional and non-traditional risk factors for GDM, and for the progression from GDM to type 2 diabetes in Australia. Our research has also been directed towards breaking the nexus between GDM and type 2 diabetes. The identification of women with GDM is an opportunity to institute interventions to prevent both GDM and type 2 diabetes. Unfortunately there are numerous barriers to improving lifestyle and reducing diabetes risk in this population. The National Diabetes Services Scheme has provided the opportunity to start translating some of our research into health promotion activities. The NDSS has greatly aided the management of diabetes and GDM by providing subsidised diabetes related products. It has also been established to provide information and services to people with diabetes. As part of this charter, the NDSS has recently started health promotion activities in the area of diabetes in pregnancy. It will underpin a national recall and screening program for diabetes after GDM, and forms the basis for other public health initiatives such as providing information to women with diabetes in pregnancy, facilitating the prevention of diabetes after GDM.
101. THE RELATIONSHIP BETWEEN SERTOLI CELL STATUS AND IDIOPATHIC MALE INFERTILITY
Authors: Haverfield J. T. , Stanton P. G. , Meachem S. J.
Published: November 2010

The cornerstone of normal adult testicular function is a mature Sertoli cell population. The maturational switch for Sertoli cells occurs at puberty, where immature Sertoli cells differentiate into a mature population that hold the necessary architectural and functional characteristics to regulate spermatogenesis (1). Data from rodent models (2, 3) suggest a relationship between Sertoli cell immaturity and infertility, however clinical data confirming this relationship is limited. We postulate that adult Sertoli cells in the infertile human testis display an immature status, with more severe disruptions of spermatogenesis associating with a greater extent of Sertoli cell immaturity. Using testicular biopsy samples obtained from fertile men (n = 3) and infertile patients (n = 6/group) displaying meiotic arrest (MA) and Sertoli cell only (SCO) syndrome, we sought to survey the status of Sertoli cell populations. All samples were immunofluorescently probed for three hallmark features of adult Sertoli cell maturation; organisation of the inter-Sertoli tight junctions, expression of the androgen receptor and proliferative ability. Differences between groups were quantified using stereology. The results show that the majority of infertile patients display highly disorganised tight junctions, a feature not seen in fertile men, however surprisingly no difference in the extent of tight junction disruption was observed between MA and SCO. Preliminary data also show that some component of the Sertoli cell population in MA and SCO patients was non-functional and proliferative. These results suggest that the Sertoli cell population in men suffering from idiopathic infertility present an abnormal maturational status that is independent of the extent of spermatogenic disruption. Moreover, this study supports the growing body of evidence proposing that the adult Sertoli cell population is not a homogenous, terminally differentiated population, and suggests that the failure of Sertoli cells to reach or maintain their mature status may be the cornerstone of abnormal adult testicular function.
(1) Sharpe R.M. et al., 2003, Reproduction, 125: 769.(2) Tarulli G.A. et al., 2006, Biology of Reproduction, 74: 798–806.(3) Tarulli G.A. et al., 2008, Reproduction, 135: 867–877.
103. THE EFFECT OF PATERNAL OBESITY IN MICE ON REPRODUCTIVE AND METABOLIC FITNESS OF F1 MALE OFFSPRING
Authors: Mitchell M. , Fullston T. , Palmer N. O. , Bakos H. W. , Owens J. A. , Lane M.
Published: November 2010

We know relatively little of the consequences of male obesity for reproductive success compared to female obesity. Conflicting evidence exists in both humans and rodents regarding whether paternal obesity alters sperm motility and concentration. However, we have described impaired embryo and fetal development, and implantation, in rodents as a consequence of paternal obesity. This study investigated whether founder male obesity influenced the reproductive and metabolic fitness of males of the subsequent F1 generation. C57BL/6 founder male mice were fed a standard chow (CD) or a high-fat diet (HF) for 8wks. This increases adiposity in the absence of changes in fasting glucose levels. Males were mated to female C57BL/6 mice, and subsequent m ale F1 offspring from HF (HF-F1) or CD (CD-F1) founders were weighed weekly and maintained on standard chow. At 8 weeks and 14 weeks glucose tolerance tests were performed and following euthanasia, tissues and sperm collected. Sperm reactive oxygen species (ROS) and DNA damage levels were determined, and various organs weighed. HF-F1 male pups were significantly heavier relative to CD-F1 males (P < 0.05) although adult bodyweight did not differ significantly. Despite this, liver, pancreas, testes and epididymis weight was significantly elevated for HF-F1 males at 17wks of age (P < 0.05). At both 8wks and 14wks of age HF-F1 males were hypoglycaemic and had impaired glucose metabolism. Sperm analysis of HF-F1 males indicated a significant increase in ROS levels (P < 0.05), DNA damage (P < 0.05) and a decrease in fertilization rates in vitro (P < 0.05). This data indicates significant physiological changes and perturbed sperm parameters in F1 males as a consequence of founder male obesity. It supports further interrogation of male and female F1 offspring, and warrants examination of potential effects for a subsequent F2 population.
105. MEIOTIC ACROBATS: MONOTREME SEX CHROMOSOME ORGANISATION DURING SPERMATOGENESIS
Authors: Grutzner F. , Casey A. , Daish T.
Published: November 2010

Monotremes feature an extraordinarily complex sex chromosome system which shares extensive homology with bird sex chromosomes but no homology to sex chromosomes of other mammals (1,2,3). At meiotic prophase I the ten sex chromosomes in platypus (nine in echidna) assemble in a sex chromosome chain. We previously identified the multiple sex chromosomes in platypus and echidna that form the meiotic chain in males (1,2,4). We showed that sex chromosomes assembly in the chain in a specific order (5) and that they segregate alternately (1). In secondary spermatocytes we observed clustering of X and Y chromosomes in sperm (6). Our current research investigates the formation of the synaptonemal complex, recombination and meiotic silencing of monotreme sex chromosomes. Meiotic sex chromosome inactivation (MSCI) has been observed in eutherian mammals, marsupials and birds but has so far not been investigated experimentally in monotremes. We found that during pachytene the X5Y5 end of the chain closely associates with the nucleolus and accumulates repressive chromatin marks (e.g. histone variant mH2A). In contrast to the differential accumulation of mH2A we observe extensive loading of the cohesin SMC3 on sex chromosomes in particular during the pachytene stage of meiotic prophase I. We have also used markers of active transcription and gene expression analysis to investigate gene activity in platypus meiotic cells. I will discuss how these findings contribute to our current understanding of the meiotic organisation of monotreme sex chromosomes and the evolution of MSCI in birds and mammals.
(1) Grützner et al. (2004), Nature 432: 913–917.(2) Rens et al. (2007), Genome Biology 16;8(11): R243.(3) Veyrunes et al. (2008), Genome Research, 18(6): 995–1004.(4) Rens et al. (2004), Proceedings of the National Academy of Sciences USA. 101 (46): 16 257–16 261.(5) Daish et al. (2009), Reprod Fertil Dev. 21(8): 976–84.(6) Tsend-Ayush et al. (2009), Chromosoma 118(1): 53–69.
106. NUCLEOSOME RETENTION DURING CHROMATIN PACKAGING IN HUMAN SPERMATOZOA
Authors: Roman S. D. , Reid A. T. , McEwan K. , Campbell D. M. , Jans D. A.
Published: November 2010

In contrast to the histone packaging of somatic cells, spermatozoa are predominantly packaged by the protamine proteins. However, human spermatozoa retain ~15% histone packaging. Regions that are left nucleosome bound could be: either genes that are active shortly after fertilisation or genes that are transcribed late during spermatogenesis. DNA damage at histone bound loci would be of consequence to spermatogenesis and/or to a resulting embryo post-fertilisation. Western blot analysis confirmed the presence of acetylated H3 and H4 in sperm. Interestingly, we identified the presence of these modified histones in isolated good quality sperm considered to have complete packaging. This indicates that histone retention is not a consequence of aberrant chromatin remodelling. Using a combination of ChIP (chromatin immunoprecipitation) and tiling arrays we have identified regions bound by acetylated histone 3 (H3). ChIP-PCR confirmed histone retention at several of these loci. For the previously identified loci in exon 1 of the TNP-2 gene we were able to narrow the region bound to the size of one nucleosome only. Using a modified form of ChIP known as carrier ChIP we examined histone retention in individual ejaculates. We demonstrate that humans consistently retain acetylated H3 at the same loci. Retention of one nucleosome at the TNP-2 loci was maintained across ejaculates and donors. In conclusion, retention of acetylated H3 is consistently maintained during packaging of the genome in spermiogenesis.
107. RESPONSES OF THE PREIMPLANTATION EMBRYO TO DEFINED GENOTOXIC STRESSES
Authors: O'Neill C. , Mu X. , Farnham M. , Jin X. L.
Published: November 2010

Genotoxic cellular stressors induce damage to the structure of DNA. This potentially compromises the genetic integrity of the cell. Such damage is particularly dangerous in the early embryo since transmission of defects can affect most cells in the body. Canonical responses of cells to such stress include delaying or blocking mitosis to allow DNA repair to occur or induction of cell death (apoptosis) to ensure that damage is not propagated. To date there have been only limited studies of the response of the preimplantation embryo to genotoxic stress. Ultra violet (UV) irradiation typically induces single strand DNA breaks while drugs such as the chemotherapeutic cisplatin commonly induce double strand breaks. ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia and rad3 related) are checkpoint kinase that mediate early responses of the cell to DNA damage. ATM primarily responds to DNA double strand breaks while ATR typically responds to single strand breaks and stalled replication forks. However, ATM and ATR may have partially overlapping and complementary functions. Both kinases can exert P53 dependent and independent responses. ATR protein was detected by immunofluorescence in all preimplantation stages up to the morulae stage. ATM protein was detected in oocytes and all stages. Cisplatin or UV-irradiation at the 2-cell stage caused increased nuclear staining of both ATM and ATR. UV-irradiation of 2-cell embryos induced irreversible, ATR-dependent, P53-independent cell-cycle block without apoptosis. Cisplatin allowed cell-cycle progression with progressive ATR and ATM-dependent, P53-independent apoptosis over subsequent cell-cycles. UV-irradiation of morula caused an ATR and ATM-dependent, P53-independent block of blastocyst formation while the block caused by cisplatin was ATR and ATM-dependent and at least partially P53-dependent. The results demonstrate complexity and maturation of the cellular responses of the embryo to defined genotoxic stressors and help to define the nature of embryopathy under these circumstances.
108. INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER IS DEPENDENT ON COMPATIBLE CYTOPLASMIC FACTORS AND MITOCHONDRIAL DNA
Authors: Jiang Y. , Kelly R. , Peters A. , Fulka H. , Mitchell D. A. , St John J. C.
Published: November 2010

Interspecies somatic cell nuclear transfer (iSCNT) offers significant opportunities to analyze and understand nuclear-cytoplasmic interactions. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility, specifically mitochondrial DNA (mtDNA), on successful development. Transfer of somatic murine fetal fibroblasts into enucleated porcine oocytes resulted in extremely low blastocyst rates (0.4%); increased DNA strand breaks; deficient nuclear pore complex arrangements and increased aberrant karyokinesis than observed in porcine-porcine SCNT embryos. Using allele specific-PCR analysis, murine mtDNA was detected at ever-decreasing levels to the blastocyst stage, with peak levels being 0.14 ± 0.055% in 2-cell embryos. Furthermore, these embryos reduced total mtDNA copy number during preimplantation development in a manner similar to porcine embryos. Injecting mouse embryonic stem cell extract and mitochondria along with the murine donor cell into a mitochondria depleted porcine oocyte, increased blastocyst zona pellucida thinning and blastocyst rates significantly (0.4% vs 3.42%) compared to the non-supplemented iSCNT group. They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout preimplantation development. At later stages of preimplantation development, they possessed 48.00% ± 17.38% murine mtDNA and exhibited a mtDNA copy number profile similar to murine embryos. Overall, these data demonstrate that the addition of species compatible cytoplasmic factors and mitochondrial DNA improve developmental competence of iSCNT embryos.
110. DIMETHYL FORMAMIDE IMPROVES THE DNA INTEGRITY AND MOTILITY OF SEX-SORTED CRYOPRESERVED STALLION SPERMATOZOA
Authors: Gibb Z. , Morris L. H. A. , Maxwell W. M. C. , Grupen C. G.
Published: November 2010

The fertility of flow-cytometrically sex-sorted stallion spermatozoa is compromised by the additional stress of cryopreservation, a step which is essential for the wider application of this technology. The aim of this study was to compare the cryoprotective effects of dimethyl formamide (DMF) and glycerol at different concentrations. Sex-sorted and control spermatozoa were cryopreserved in the presence of 2, 3 or 4% DMF or glycerol. Post-thaw total motility (TM) was assessed using computer assisted sperm assessment and viability was assessed using Syto-16/propidium iodide staining and flow cytometry at 0, 45 and 90 min post-thaw. The DNA integrity (%DFI) was determined using the sperm chromatin structure assay immediately post-thaw. Compared to controls, se x-sorting caused a significant decrease in TM (34.6 vs. 9.4%) and viability (30.0 vs. 12.5), and a significant increase in %DFI (11.3 vs. 63.2%). The TM of control and sex-sorted spermatozoa did not differ at 0 min (29.2 vs. 22.0%), but were significantly different at 45 and 90 min (37.4 vs. 5.6% and 37.2 vs. 0.5% respectively). The viability of sex-sorted spermatozoa decreased significantly at each time point (20.9, 9.6 and 6.9% at 0, 45 and 90 min) though the viability of control spermatozoa did not decrease over time. DMF resulted in lower %DFI values than glycerol for sex-sorted, but not control spermatozoa (59.6 vs. 66.0). However, DMF reduced the viability of sex-sorted spermatozoa compared with glycerol (11.0 vs. 13.9%). DMF resulted in higher TM than glycerol for control spermatozoa only (37.9 vs. 31.3%). Increasing the cryoprotectant concentration caused a significant decrease to the viability of control spermatozoa (33.4, 30.8 and 25.9% for 2, 3 and 4% respectively), but had no effect on sex-sorted spermatozoa. The results of this study indicate that the cryoprotective effect of DMF on sex-sorted stallion spermatozoa was superior to that of glycerol.
112. FETAL CALF SERUM AFFECTS hESC METABOLISM AND GENE EXPRESSION LEADING TO DIFFERENTIATION IN CULTURE
Authors: Yeo C. X. , Rathjen J. , Gardner D. K.
Published: November 2010

Fetal calf serum (FCS) has conventionally been used to support the growth and maintenance of human embryonic stem cells (hESCs). FCS however, is an undefined complex mixture containing factors which potentially alter the functionality of hESCs. Inclusion of FCS during embryo culture negatively impacts embryo metabolism and viability but comparative studies on hESCs have been hindered by the lack of serum and feeder independent culture systems. Using a recently available defined culture system, the effects of FCS on hESC metabolism and pluripotentcy were investigated. Mel2 hESCs were grown at 37°C in 5% CO2 for 3 days on matrigel (hESC-qualified) coated tissue culture wells in mTeSR1 medium. hESCs were then cultured in mTeSR1 (control) or in mTeSR1 supplemented with 20% FCS from different manufacturers or knockout serum replacement (KOSR), for 96 hours. Media was renewed daily. At the end of the culture period, spent media was collected and cells were trypsinised and counted and/or collected for gene expression analysis. FCS decreased cell survival and altered hESC morphology from densely packed colonies with distinct borders into non-uniform heterogeneous populations comprising of hESC-like cells and fibroblastic-like cells with high cytoplasmic to nuclear ratios. Media analysis revealed altered cell metabolism with increased glucose consumption rates per cell (P < 0.01) with FCS supplementation, compared to cells cultured in mTeSR1 alone. Gene expression analysis revealed that FCS, regardless of its manufacturer, decreased the expression of some pluripotent markers and increased differentiation markers. A decrease in pluripotent gene expression was also observed in hESCs cultured with KOSR compared to mTeSR1 alone. Maintenance of homogeneity in hESC populations is crucial for the advancement of hESC clinical therapies. This study demonstrates that FCS promotes heterogeneity and impacts the metabolic function and gene expression of hESCs thereby supporting the need for serum-free culture systems as standard practice in hESC culture.
113. VASCULAR ENDOTHELIAL GROWTH FACTOR GENE POLYMORPHISMS IN PLACENTAL IMPAIRMENT AND SMALL FOR GESTATIONAL AGE BIRTH
Authors: Andraweera P. H. , Dekker G. A. , Nowak R. C. , Thompson S. D. , McCowan L. M. E. , North R. A. , Roberts C. T.
Published: November 2010

Impaired placental angiogenesis is implicated in the pathophysiology of small for gestational age (SGA) infants. Placental expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, is reduced in SGA pregnancies. We aimed to evaluate the association of two single nucleotide polymorphisms (SNPs, VEGF-2578C/A and VEGF+936C/T) in VEGF gene which reduce VEGF expression, in SGA pregnancies andexamine their effects on first trimester placental VEGF expression. 3196 nulliparous pregnant women, their partners and babies were recruited in Adelaide and Auckland to a prospective multicentre cohort study (SCOPE Study). Amongst 2123 Caucasian women, 216 (11.9%) delivered a SGA infant defined as <10th customised centile. Uncomplicated Caucasian pregnancies served as controls (n = 1176). Uterine and umbilical artery Doppler velocimetry was performed at 20 weeks gestation. DNA extracted from peripheral blood from couples and cord blood from babies was genotyped using Sequenom MassARRAY. 74 first trimester placentae collected from elective terminations of pregnancy were genotyped for the same SNPs and the VEGF expression determined by RT-PCR. Neonatal VEGF+936 CT+TT genotypes associate with SGA (OR 1.6, 95%CI 1.1–2.3), lower birthweight (P = 0.005), customised birthweight centile (p=0.03), lower placental weight (P = 0.04) and an increased uterine artery resistance index (RI, P = 0.004). Maternal VEGF+936 CT+TT associate with bilateral notching of the uterine artery waveform (OR 1.4, 95%CI 1.0–1.8) and an increased umbilical artery RI (OR 1.5, 95%CI 1.1–2.1). VEGF+936 CT first trimester placentae have lower VEGF expression compared to CC (P = 0.045). Neonatal VEGF-2578 AA associates with bilateral uterine artery notching (OR 1.5, 95%CI 1.1–2.2) and increased umbilical artery RI (OR 1.6, 95%CI 1.0–2.6). Maternal VEGF-2578 CA+AA associate with increased umbilical artery RI (OR 1.5, 95%CI 1.0–2.2). VEGF polymorphisms reduce first trimester VEGF expression and associate with increased resistance in the placental circulation suggesting impaired placental function. VEGF+936 SNP confers increased risk for SGA.
114. HYPERTENSIVE DISORDERS OF PREGNANCY ARE ASSOCIATED WITH IMMUNOREGULATORY GENE POLYMORPHISMS
Authors: Highet A. , Thompson S. , Furness D. , Zhang V. , Dekker G. , Roberts C.
Published: November 2010

Pregnancy is a controlled state of inflammation. Deregulation of cytokine networks can lead to adverse pregnancy outcomes including preeclampsia (PE). We aimed to identify single nucleotide polymorphisms in immunoregulatory genes that signify an increased risk of the gestational hypertensive disorders PE and gestational hypertension (GH). 1169 nulliparous pregnant women and their partners were recruited prospectively for the Adelaide SCOPE study. PE and GH were classified using strict guidelines. Uncomplicated pregnancies served as controls. Peripheral blood from couples and cord blood from neonates were collected. DNA was extracted and genotyped for Interleukin (IL)-6 rs1800795, IL-4 rs2243250, IL-10 rs1800896 and rs1800871, mannose binding lectin (MBL) rs1800450, transforming growth factor beta 1 (TGFβ-1) rs1800469 and cyclooxygenase (COX)-2 rs20417 & rs5275 and inducible nitric oxide synthase (NOS2A) rs1137933 using the Sequenom MassARRAY system. Genotypes for Caucasian PE (n = 75) and GH (n = 102) were compared with controls (n = 422) and analysed using Chi-Square. In neonates IL-6 G allele carriage was associated with PE (P = 0.011, OR=2.0, 95% CI = 1.2–3.7) and the CC genotype associated with GH (P = 0.002). Neonatal IL-10 RS180071 AA genotype associated with PE (P = 0.041) and IL-10 RS1800896 AA associated with GH (P = 0.022). Paternal NOS2A C allele was more frequent in PE (P = 0.03, OR = 2.1, 95% CI = 1.1–4.5), and maternal NOS2A CC more frequent in GH (P = 0.018). Increased neonatal carriage of MBL rs1800450 AA+GA genotypes associated with GH (P = 0.03, OR = 2.2, 95% CI = 1.1–4.5). No associations were observed between TGFβ-1 or COX2 genotypes and PE or GH. Associations between neonatal IL-6 G, which confers high placental IL-6 expression, and PE suggest a possible mechanism by which PE is a pro-inflammatory exacerbation of placental origin. Since placental IL-10 is important for maternal tolerance of the fetus, genotypes predisposing to low IL-10 expression in the neonate which associate with both PE and GH, suggest a role for decreased placental IL-10 in these disorders.
115. DEVELOPMENT OF POTENT AND STABLE PC6 INHIBITORS TO BLOCK EMBRYO IMPLANTATION FOR FEMALE CONTRACEPTION AND PREVENTION OF HIV
Authors: Ho H. , Nie G.
Published: November 2010

Proprotein convertase (PC) 6, a member of the PC family that activate precursor proteins into their active forms, is a critical endometrial factor for embryo implantation. Blocking PC6 production in mice inhibits decidualisation (a critical process of implantation) and inhibits implantation. PCs including PC6 also play a critical role in HIV infection through cleaving HIV envelope precursor protein gp160 into functional gp120 and gp41. PC inhibitors are demonstrated to inhibit HIV transmission via blocking gp160 cleavage. We hypothesised that PC6 is a potential target for the development of female contraception that could also provide protection from HIV infection. One key requirement to prove this concept in an animal model is a potent PC6 inhibitor that is stable in serum. Polyarginine peptide (polyR) is published to be a potent PC6 inhibitor that also inhibits HIV. We have confirmed that polyR inhibits PC6 in vivo and completely blocks decidualisation of human endometrial stromal cells in culture. However, polyR has short serum half-life. The aim of this current study was to generate polyR derivatives that are potent PC6 inhibitors, with increased serum stability. We modified polyR by either PEGylation with different sized PEG (polyethylene glycol) or cyclization, and tested their potency, stability and utility in vivo. Modifications at both terminals of polyR dramatically reduced its PC6 inhibitory potency. PEGylation at the C-terminal, regardless of the PEG size, had no effect. In silico docking experiments showed that N-terminal PEGylation or cyclization affected the binding of polyR to the PC6 active site, but not C-terminal PEGylation. One of the polyR derivatives, C-30kDa-PEG polyR was confirmed to be as potent as the parental peptide but much more stable in serum. Studies are currently in place to inhibit embryo implantation in mice using C-30kDa-PEG polyR and to determine its ability to inhibit HIV.
116. TROPHOBLAST ANTIPHOSPHOLIPID ANTIBODY INTERNALISATION BY A β2 GLYCOPROTEIN I-ANIONIC PHOSPHOLIPID-MEGALIN COMPLEX
Authors: Viall C. A. , Chamley L. W. , Chen Q.
Published: November 2010

Women with antiphospholipid antibodies (aPL) are at an increased risk of preeclampsia, recurrent miscarriage, stillbirth and intrauterine growth restriction. Antiphospholipid antibodies may predispose to these pathologies by damaging the placenta, although exactly how is not understood. Recently, a novel pathogenic mechanism was suggested by work which showed that aPL are specifically internalised by placental trophoblasts where they caused aberrant trophoblast death. Internalisation may occur via an endocytic receptor called megalin in a process that seems to involve at least one of the two components of the antigen for aPL, the anionic phospholipid-binding protein β2 glycoprotein I (β2GPI). However, whether internalisation is also dependent upon anionic phospholipids is unknown. Identifying the receptor pathway responsible for aPL internalisation may provide insight into the pathogenesis of aPL in the placenta. To investigate the process of aPL internalisation, first trimester placental explants were cultured with fluorescently-labeled monoclonal aPL, or a control antibody and/or β2GPI or acetylated β2GPI, which can not bind anionic phospholipids. The explants were then sectioned and the localisation of the aPL, β2GPI, or acetylated β2GPI was determined by confocal microscopy. The localisation of megalin expression in placental explants was determined by immunohistochemistry. Megalin was expressed throughout the syncytiotrophoblast but more strongly in some regions. After an overnight incubation, both aPL and β2GPI, but not control antibodies were co-localised in the cytoplasm of the syncytiotrophoblast. Acetylated β2GPI was not internalised and partially blocked aPL uptake. These results suggest that aPL are internalised into the synctiotrophoblast by a receptor-dependent mechanism involving β2GPI, anionic phospholipids and megalin. This work forms the first step to understanding how aPL are internalised by trophoblasts. Further investigation of this mechanism and the subsequent intracellular effects of aPL may lead to a new therapeutic strategy for aPL-positive pregnant women by preventing the pathogenic effect of aPL on the placenta.
117. THE ROLE OF FOCAL ADHESION PROTEINS AND THEIR HORMONAL REGULATION IN RAT UTERINE EPITHELIAL CELLS DURING EARLY PREGNANCY
Authors: Kaneko Y. , Day M. , Murphy C. R.
Published: November 2010

Uterine epithelial cells (UECs) undergo extensive alteration during early pregnancy followed by their removal in order for the implanting blastocyst to penetrate into the underlying endometrium. Focal adhesions (FAs) may play a role during this process as FAs provide adhesion between the cell and its underlying basal lamina. The present study investigated the distribution and expression of FA proteins in rat UECs at the time of implantation and their hormonal control as well as their expression in blastocyst stage embryos. Immunofluorescence microscopy showed that the principal focal adhesion proteins, talin and paxillin, were localised along the basal cell surface of UECs on day 1 of pregnancy, however they were markedly reduced from the site of FAs at the time of implantation. This is thought to be a critical process in the removal of UECs, which allows the invasion of the blastocyst into the underlying endometrium. Hormone treatments in ovariectomised rats showed that disassembly of talin and paxillin from the site of FAs were predominantly under the control of progesterone. Formation of FAs is initiated by the clustering of specific integrin subunits and both integrin beta1 and beta3 colocalised and interacted with talin at the site of FAs on day 1 of pregnancy. Integrin beta1 and beta3 disassembled from the site of FAs at the time of implantation, however integrin beta3 significantly increased along the apical membrane at this time suggesting a role in embryo attachment. In the rat blastocyst, integrin beta3 was concentrated around the nuclei of the trophoblast cells and once the blastocyst was placed onto a receptive endometrial cell line, Ishikawa cell line, integrin beta3 relocated to the apical membrane of the trophoblast cells. Taken together, our results show that disassembly of FA proteins plays a pivotal role in the removal of UECs in order to establish successful implantation and is tightly regulated by ovarian hormones.
118. THE PRORENIN RECEPTOR/PLZF PATHWAY IN HUMAN AMNION
Authors: Pringle K. G. , Conquest A. L. , Mitchell C. M. , Zakar T. , Lumbers E. R.
Published: November 2010

Prorenin, despite being inactive, is the major form of renin found in amniotic fluid and reproductive tissues. Prorenin becomes active if it binds to the novel prorenin receptor (ATP6AP2). The prorenin-ATP6AP2 complex has been found to stimulate translocation of Promyelocytic Zinc Finger (PLZF) protein to the nucleus where it increases expression of the p85α subunit of PI3 kinase (PI3K-p85α) and represses the expression of ATP6AP21. Progesterone and glucocorticoids have also been shown to stimulate PLZF2, 3. We aimed to find out if PLZF and the prorenin-ATP6AP2 pathway interact in human reproductive tissues. Human amnion was cultured for 24 h in media containing vehicle, dexamethasone, amniotic fluid or recombinant human (rh) prorenin. Total RNA was extracted using TRIZol® and converted to cDNA for quantitative real-time PCR using SuperScript III and random hexamers. mRNA abundances for PLZF, PI3K-p85α and ATP6AP2 were calculated relative to Alien RNA using the ΔΔCT method. Our preliminary data show that exposure of amnion explants to dexamethasone upregulates PLZF and PI3K-p85α mRNA but has no effect on ATP6AP2. Culture of amnion explants with amniotic fluid also increases PLZF but does not change PI3K or ATP6AP2. In contrast, culture of amnion explants with (rh) prorenin increases PI3K mRNA but not PLZF or ATP6AP2. As expected, dexamethasone affects PLZF expression, however in amnion there is no interaction with the ATP6AP2 pathway. In addition, we believe we have identified a novel prorenin/ATP6AP2 signalling pathway which acts on PI3K-p85α independent of PLZF. In contrast to these data, amniotic fluid increases PLZF but not PI3K-p85α mRNA levels suggesting that amniotic fluid contains other factors that oppose prorenin and glucocorticoid effects on PI3K-p85α.
(1) Schefe, J.H., et al. Circ Res, 2006. 99(12): 1355–1366.(2) Fahnenstich, J., et al. Mol. Hum. Reprod., 2003. 9(10): 611–623.(3) Conquest, A. et al. Fetal and Neonatal Physiology Workshop, 2010. Wellington, New Zealand.