bluuebunny/crossref_metadata_2025 huggingface dataset:
425. Expression of mammalian cysteine-rich secretory proteins in the mouse model
Authors: Reddy T. , Gibbs G. M. , Merriner D. J. , Kerr J. B. , O.'Bryan M. K.
Published: November 2010

Mammalian cysteine rich secretory proteins are a family of four proteins exhibiting a high amino acid sequence similarity and belonging to the CAP (Cysteine rich secretory proteins, Antigen-5 proteins and the plant Pathogenesis related-1 proteins) superfamily of proteins. They are designated CRISP 1, 2, 3 and 4. Structurally, mammalian CRISP’s are characterised by 16 cysteine residues involved in intra-molecular di-sulfide bonds and the formation of 2 domains, ie., the CRISP domain (CD) and CAP domain. Whilst studies on mouse CRISP2 suggest that the CD is involved in ion channel regulation, studies on non-mammalian CAP superfamily members suggest that the CAP domain is involved in proteolytic activity.They are predominantly expressed and localised in the male reproductive tract, however, the EST expression databases suggest that mammalian CRISPs are expressed more widely than in the male reproductive tract. The objective of this study was therefore to conclusively define the expression and localisation of each CRISP protein in a mammalian system.A reverse transcription PCR expression profile and immunohistochemical analysis of 16 mouse tissue was conducted to establish the expression and localisation of each of the four CRISPs. These data showed that although the CRISPs have a strong expression and localisation bias to the male reproductive tract, they are widely distributed throughout the body in mice, including the ovary, uterus, and mammary gland. Whilst each CRISP has a clear expression profile, there was a striking localisation of androgen regulated CRISPs (1, 3, 4) in immune tissue including the spleen and thymus. Such a localisation raises the spectre of a role for CRISPs in the normal physiology and disease of several organs.
426. The role of sperm mitochondria in the process of epididymal maturation
Authors: Lee Y. H. , Lin M. , Baker M. A. , Aitken R. J.
Published: November 2010

On leaving the testis, spermatozoa can neither swim nor achieve fertilisation of the oocyte. These functional properties are acquired as spermatozoa engage a process of post-testicular maturation in the epididymis. Research into the biochemical basis of sperm maturation has revealed that this process is associated with activation of sperm mitochondria. Immature spermatozoa from the caput epididymis displayed a low mitochondrial membrane potential (MMP) whereas mature spermatozoa from the caudal epididymis actively maintained a high MMP. Moreover mitochondrial generation of reactive oxygen species could be triggered by antimycin in mature caudal epididymal spermatozoa but not in immature cells recovered from the caput epididymis. The molecular mechanisms responsible for regulating mitochondrial function were reversible since washing the cells free of epididymal fluid allowed immature spermatozoa to acquire a high MMP while incubating mature caudal cells in caput epididymal fluid, suppressed MMP. These results strongly suggested that fluid from the caput epididymis contains a mitochondrial inhibitor and that activation of mitochondrial activity is due to the removal or inactivation of this inhibitor during epididymal transit. The causative factor is not species specific because incubation of ejaculated human spermatozoa in murine epididymal fluid effectively suppressed their MMP. Caput epididymal fluid was fractionated using FPLC and each of the fractions was tested for their bioactivity. A major protein band with a molecular mass ~150 kDa was present in the active fraction. Further characterisation of this reversible mitochondrial inhibitor may reveal the mechanisms by which epididymal spermatozoa control mitochondrial function during maturation. It may also contribute to our understanding of human male infertility and potentially serve as a novel target for male fertility regulation.
433. Identifying novel biomarkers predictive of impending human labour
Authors: Heng Y. J. , Di Quinzio M. K. W. , Permezel M. , Rice G. E. , Georgiou H. M.
Published: November 2010

Human labour is characterised by structural remodelling of the cervix and overlying fetal membranes, myometrial activation and parturition. We hypothesise that temporal biochemical alterations of the cervix and supracervical fetal membranes associated with impending labour may be reflected in the cervico-vaginal fluid (CVF). 2D PAGE proteomic analysis performed on serial CVF samples collected from women (n = 9) during late pregnancy and in spontaneous labour demonstrated 9 significantly altered protein spots (P < 0.05) in association with term labour. Seven different proteins were identified using electrospray ion-trap mass spectrometry (interleukin-1 receptor antagonist (IL-1ra), cystatin-A, glutathione S-transferase P, peroxiredoxin-2, thioredoxin, Cu,Zn superoxide dismutase, and epidermal fatty-acid binding protein). These proteins are involved in anti-inflammatory activity, protease inhibition, and oxidative stress defence. Validation of these potential biomarkers using ELISA is currently underway. Findings for the anti-inflammatory cytokine, IL-1ra are discussed. CVF was collected weekly from 106 women at 36 weeks' gestation up to and including spontaneous labour. The concentration of IL-1ra was 4-fold lower during labour compared with 15–21 and 22–28 days from labour (P < 0.05) and was also significantly lower (P < 0.05) at 0–7 days compared with 15–21 days before labour. After subdividing the women, the concentration of IL-1ra at 8–14 and 15–21 days before labour was 6-fold lower in women who had prelabour rupture of membranes at term followed by regular contractions compared with women who had spontaneous labour with intact membranes. Receiver-operator characteristic curve analysis indicated that IL-1ra best predicted term labour within 3 days of sampling with a cut-off value of 0.4 m g/mL (sensitivity 50.7%, specificity 72.2%, positive predictive value 36.1%, negative predictive value 82.6%). This IL-1ra validation study suggests that the decrease in IL-1ra in CVF during spontaneous term labour may be associated with proinflammatory-mediated remodelling of the fetal membranes leading to their rupture.
(1) Di Quinzio et al. 2008, J. Proteome Res, 7:1916–1921.
(2) Heng YJ et al. 2008, Am J Obstet Gynecol, In Press.
443. The fibroblast - smooth muscle cell relationship is altered in uterine leiomyoma
Authors: Zaitseva M. , Vollenhoven B. J. , Rogers P. A. W.
Published: November 2010

Uterine leiomyoma (fibroids) are benign neoplasms of the myometrium. Their aetiology is still poorly understood, but they are known to be sex steroid hormone dependant. Although these tumours are thought to consist of smooth muscle cells (SMC), fibroblasts constitute a major cellular component. Genes that are central to fibroblast activation and function, including transforming growth factor-β (TGFβ), as well as many extracellular matrix (ECM) genes have altered expression in leiomyoma. Despite this, the role of fibroblasts in these tumours has not been investigated in leiomyoma before. The aims of the present study were to isolate fibroblasts from myometrium (MAFs) and leiomyoma (LAFs); and to compare gene expression of fibroblasts and SMC from these 2 different sources. Myometrium and leiomyoma were dissociated into single cell suspensions, stained with fluorescently labelled CD31, CD90 and 1B10 antibodies and separated into different cell populations using fluorescence- activated cell sorting. Endothelial cells were removed from the analysis; remaining cells were sorted into 1B10-CD90 +ve (fibroblasts) and 1B10-CD90 –ve (SMC) populations and used for RNA isolation and quantitative real-time PCR. Significantly higher levels of expression of aldehyde dehydrogenase1 (ALDH1), cellular retinoic acid binding protein 2 (CRABP2), oestrogen receptor (ERα) and progesterone receptor (PR) were observed in MAFs compared with MSMC, while no equivalent differences were observed between LAFs and LSMC; and lower levels of TGFβ were observed in fibroblasts compared with SMC in both tissues. Results for ALDH1, ERα and PR were also confirmed using immunohistochemistry. This is the first study to successfully isolate LAFs and MAFs and demonstrate an altered fibroblast-SMC relationship in uterine leiomyoma. This work provides the first evidence that LAFs might play an important role in leiomyoma pathophysiology. Investigation of fibroblast function in this disease may provide important new fundamental knowledge on its aetiology, ultimately leading to the development of new medical treatments.
444. Embryonic survival in prolific ewes
Authors: O.'Connell A. R. , McNatty K. P. , Hurst P. R. , Phillips D. J. , Dodds K. G. , Davis G. H.
Published: November 2010

Introduction: A significant proportion of potential lambs are lost (commonly 15–20%) between ovulation and birth. Little is currently known about factors associated with multiple birth capacity of the uterus which are essential to convert gains in ovulation rate to live lambs. The relationship between maternal uterine and hormonal environment as well as the heritability of embryonic survival (ES) in prolific ewes is investigated. Methods: Litter size (LS) from known ovulation rate (OR) records (n = 6393) collected over 16 years were assessed for heritability with ASREML analysis and to identify the pedigree of outliers. From this flock, closely related high ovulation rate ewes with significantly different litter sizes (High ES; OR2.6/LS2.4 v. Low ES; OR2.9/LS1.6) were selected. Uterine anatomy collected from Day 14 cyclic (n = 5 High and n = 5 Low ES) and Day 16 pregnant ewes (n = 14 high and n = 10 Low) as well as systemic concentrations of hormones indicative of uterine (activin-A, follistatin) and ovarian (inhibin-{α}, progesterone) function were compared. Results and Discussion: ASREML analysis reported ES to be a trait of low repeatability (r = 0.10) and even lower heritability (h2 = 0.04). However, pedigrees of outlier animals indicated a segregation pattern consistent with a single autosomal gene with a major effect on enhanced ES. No anatomical differences between high and low ES ewes were discerned. However, significant differences for circulating levels of activin-A (depressed), time of activin-A peak (earlier), follistatin (elevated), progesterone (early rise) and inhibin-{α} (depressed) concentrations were found around the time of oestrus in High v. Low ES ewes. Given the involvement of activin-A in injury-induced inflammation, these results suggest a role for the activin-follistatin system, possibly through increased residency of immune cells and cytokine activation, for increasing ES in prolific ewes.
Acknowledgements; Norma Hudson and Stan Lun for progesterone and inhibin-{α} assays.
446. Ezrin and EBP50 relocate apically in rat uterine epithelial cells during contact with opposing cells, except cells contacting the blastocyst
Authors: Venuto L. , Lindsay L. A. , Murphy C. R.
Published: November 2010

Uterine epithelial cells are important in constantly maintaining a tissue protective barrier, and only under the specific hormonal conditioning of pregnancy which involves a remodelling of the cell ultrastructure termed the 'plasma membrane transformation'. This allows for the successful invasion of the blastocyst. Indirect immunofluorescence microscopy in rat uterine epithelial cells during pregnancy shows that ezrin and EBP50 are relocated to the apical membrane upon lumen closure at the time of implantation, and on average results in 90% colocalisation. Ezrin and EBP50 function as a linked protein complex at the time of implantation seen through immunoprecipitation results from day 6 of pregnancy. The ezrin-EBP50 complex is also associated with the membrane, shown using cell fractionation and western blotting analysis in which ezrin increased dramatically in the membrane concentrated fraction, and correspondingly decreased in the cytosolic fraction leading up to implantation. At the apical membrane these proteins are likely associating with intra-membranous signalling molecules which allow communication between contacting cells. The same protein complex is also relocated to the apical membrane when cells contact an inanimate filament inserted into the uterus of a non-pregnant rat. The only unique contacting circumstance in which these proteins are not seen at the apical membrane is within the implantation chamber itself, more specifically the cells in direct contact with the implanting blastocyst. These results highlight the unique situation that is implantation, which involves specific blastocyst signalling and influence upon the uterine epithelial cells lining the implantation chamber. It may be that the ezrin-EBP50 protein complex is critical at the earlier stage of apposition, and through blastocyst influence, are subsequently lost from the apical membrane to allow for successful invasion.
020. IN VITRO MATURATION OF FARM ANIMALS OOCYTES: A USEFUL TOOL FOR INVESTIGATING THE MECHANISMS LEADING TO FULL TERM DEVELOPMENT
Authors: Gandolfi F. , Brevini T.
Published: November 2010

Due to logistical and economical reasons assisted reproduction of domestic animals has been based mostly on the use of oocytes isolated from ovaries collected at the slaughterhouse. In order to propagate valuable or rare genetic material, perform somatic cell nuclear transfer, generate genetically modified animals it was essential to obtain fully competent oocytes that would allow full term development of the in vitro produced embryos. Such demanding need has soon made clearly evident the crucial role played by oocyte quality, how easy it is to compromise its developmental potential and the fact that it is impossible to restore it once it has been lost. Almost three decades after the first bovine, sheep, goat, horse and pig in vitro generated offsprings were born, a large body of information has accumulated on the mechanisms regulating oocyte competence and on how the latter may be preserved during all the required manipulations. The amount of knowledge is far from being complete and many laboratories are actively working to further expand it. In this review we will highlight the aspects of the ongoing research in which we have been actively involved.
100. IN VIVO DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS TO UTERINE TISSUE
Authors: Ye L. , Mayberry R. , Stanley E. , Elefanty A. , Gargett C.
Published: November 2010

The endometrium undergoes cyclic regeneration. This regeneration has been attributed to adult stem progenitor cells and developmental mechanisms [1, 2]. A better understanding of human endometrial development may shed light on the mechanisms involved in endometrial regeneration and on early origins of adult endometrial disease. The lack of human fetal endometrial tissue has impeded research in early human endometrial development. We hypothesized that directed differentiation of human embryonic stem cells (hESC) to human endometrial tissue by neonatal mouse uterine mesenchyme represents a novel system to study early development of human endometrium. Recent studies have shown that the neonatal mouse uterine mesenchyme is extremely inductive and undergoes reciprocal signalling with human endometrial epithelial cells [3]. Our aim is to establish a xenograft tissue recombination protocol based on a model for human prostate tissue differentiation using hESC [4]. Our method involved formation of embryoid body (EB) with GFP labelled hESC (ENVY) [5] for recombination with 2x0.5mm pieces of epithelial-free uterine mesenchyme from postnatal day 1 mice. Upon fusion in culture, the recombinant tissue is grafted under the kidney capsule of NOD/SCID mice for 4-12 weeks and monitored by in-vivo imaging. Immunohistochemical analysis of recombinant grafts 4 weeks post transplantation (n=4) revealed immature CK8+CK18+Hoxa10+ human epithelial cells surrounded by mouse mesenchymal cells suggesting differentiation of hESC to epithelial cells possibly of endometrial lineage. The ER+PR+SMA+Hoxa10+ mouse mesenchymal cells surrounding human glands differentiated into SMA+ cells possibly via reciprocal signalling from human epithelial cells. At 8 weeks, we found several CK18+/Hoxa10+ human glands co-expressing CA125. These glands are supported by Hoxa10+ human stromal cells. Further experiments are underway to induce the expression of ER and PR in Hoxa10+ epithelial cells which will be crucial in revealing their endometrial lineage.
103. ROLE OF CANDIDATE STEM/PROGENITOR CELLS IN A MOUSE MODEL OF ENDOMETRIAL MENSTRUAL BREAKDOWN AND REPAIR
Authors: Kaitu'u-Lino T. J. , Ye L. , Gargett C. E.
Published: November 2010

Adult stem/progenitor cells (SPC) identified in human and mouse endometrium may be responsible for its remarkable regenerative capacity (1), however a functional role for SPC in menstruation is yet to be established. This study aimed to identify label retaining cells (LRC) as candidate epithelial SPC involved in the rapid re-epithelisation of the uterine surface in a mouse model; tissue SPCs are quiescent and will retain label (BrdU), while label is diluted out as transit amplifying cells proliferate. Mice were ovariectomised and endometrial breakdown and repair induced, mimicking menstruation in women (2). BrdU (50µg/g) was administered intraperitoneally 8.5 days before endometrial repair. Tissue was collected to assess initial labelling, and following four chase periods prior to and during endometrial repair (n=3-5 animals/group) and immunostained for BrdU. LRC were categorised as minimal (<50% nuclear label), partial (50-75%) and full (100%) and counted in the luminal (LE) and glandular (GE) epithelial compartments for each group. The majority of LE (91.4±1.9%; mean±SEM) and 35±3.8% of GE were initially labelled. During breakdown and repair the percentage of full LE LRC (38±13.1% vs 1.3±1.1%) and partial LE LRC (49.1±4.3% vs 1.8±0.7%) significantly decreased (p≤0.01) whilst minimal LRC significantly increased (12.9±3.5% vs 85.2±4.3%; p≤0.001) indicating dilution of BrdU label as cells proliferated. In contrast full, partial and minimal GE LRC did not significantly change throughout endometrial breakdown and repair. The rapid dilution of BrdU label in the LE suggests that the transit amplifying population are responsible for the rapid proliferation observed, whilst the lack of change of BrdU in the GE suggests that GE may be a source of SPC. Double immunofluorescence and confocal microscopy are currently underway to further characterise the LRC population in this model. This study provides some of the first insights into the contribution of candidate SPC to endometrial repair.
104. PROLIFERATION ABILITY, TELOMERASE ACTIVITY AND MOLECULAR CHARACTERIZATION OF PLURIPOTENT CELL LINES FROM IVF AND PARTHENOGENTIC PIG EMBRYOS
Authors: Brevini T. A. L. , Pennarossa G. , Attanasio L. , Gasparrini B. , Gandolfi F.
Published: November 2010

Porcine pluripotent ES cell lines are a promising tool for biotechnology, biomedical and developmental biology studies. However, no conclusive results have been obtained to derive genuine ES cells in the pig. Here we compare derivation efficiency of putative ES cells from IVF versus parthenogenetic pig embryos. We describe proliferation ability and doubling time, we study pluripotency markers and telomerase activity (TA) of the cell lines obtained. Pig oocytes were either fertilized in vitro or parthenogenetically activated. Blastocysts were subjected to immuno-surgery. Inner cell mass were plated and outgrowth expansion was monitored daily. Self renewal molecules were studied by RT-PCR and/or immunocytochemistry for up to 42 passages. TA was measured every five passages. The results obtained indicate that stable cell lines can be generated from IVF and parthenogenetic embryos. The latter appeared less resilient to immuno-surgery but demonstrated a higher ability to produce outgrowths. 77% of the parthenogenetic lines vs only 33% of the IVF ones expressed pluripotency markers and displayed high TA. Regardless to their origin, colonies showed a latency growth period in the 48 hours after plating, they grew exponentially between day 3 and 6 and then, proliferation rate was greatly reduced. Doubling time was estimated to be 31.5 hours. In both IVF and parthenogenetic cell lines, positivity for Oct-4, Nanog, Sox-2, Rex-1, SSEA-4, Alkaline phosphatase, TRA-1-81 and STAT3 was detected; no signal for LIF-Receptor beta and gp130 was shown. These results indicate that the main pluripotency network related molecules are expressed in the porcine species, while a classical LIF-Receptor beta- gp130-STAT3 activation pathway does not appear to be involved in the maintenance of self renewal. Finally, every cell lines expressed high TA, which was turned down once cells were induced to differentiate, indicating a physiologically normal control of TA in these cells.
114. HUMAN CUMULUS - OOCYTE COMPLEXES SECRETE CUMULUS EXPANSION ENABLING FACTOR(S)
Authors: Hussein T. S. , Filby A. N. , Gilchrist R. B. , Lane M.
Published: November 2010

Interactions between the oocyte and its companion somatic cells are crucial to establish and maintain a highly specialized microenvironment required for oocyte viability. Specifically, cumulus cell expansion in the mouse is reliant on oocyte-secreted factors (OSF). Little is know about factors secreted by the human oocyte and how they may interact with cumulus cells. Therefore, the aim of this study was to establish whether human cumulus oocyte complexes (COC) produce OSF that induces cumulus expansion. COC of patients undergoing routine clinical IVF were cultured individually for 6h following oocyte retrieval. The human oocyte conditioned medium (HOCM) was collected. The bioactivity of OSF in the HOCM was assessed using an established assay of cumulus expansion of mouse oocytectomized complexes (OOX). Cumulus expansion was assessed blinded using the scoring system; 1 (no expansion) to 4 (maximally expanded) and gene expression was assessed by real time RT-PCR. Culture of OOX in control media with or without FSH did not induce expansion. Similarly, OOX cultured in HOCM without FSH did not expand. However, culture of OOX in HOCM with FSH significantly induced expansion (2.4±0.1 compared with control 1.1±0.04, P<0.05). Furthermore, this expansion was not different to OOX co-cultured with human (2.9±0.1) or mouse (2.6±0.1) denuded oocytes. Cumulus/OOX gene expression of hyaluronan synthase-2 and cyclooxygenase-2 was significantly up-regulated 4-5 fold when OOX were cultured in HOCM compared to control (P<0.05). Interestingly, different patients produced HOCM which resulted in different levels of expansion (range from 1.5-3.7). This study has established that human COC secrete paracrine factor(s) that enable cumulus expansion. This expansion was dependent on the presence of FSH. The identity of these factor(s) are currently unknown however it appears that COC from different patients produce differing levels of these cumulus expansion enabling factor(s).
122. REGULATION OF GDF-9 AND GDF-9B BY FSH IN PREANTRAL FOLLICLE CULTURES
Authors: Haidari K. , Kuyznierewicz I. , Findlay J. K. , Drummond A. E.
Published: November 2010

GDF-9 and -9B (BMP-15) are oocyte-derived members of the TGF-β superfamily. In the mouse ovary, the absence of GDF-9 leads to an arrest of follicle development at the primary/preantral stage. As a result GDF9 deficient mice are infertile1 because follicle development does not reach a stage where ovulation and oocyte release can occur. In contrast, GDF-9B knockout mice are subfertile2. GDF-9 was shown to act via TGFβRI and BMPRII and GDF-9B via BMPRIB and BMPRII. We have much to learn about what regulates the production of GDF-9, GDF-9B and the expression of its receptors. These studies investigated whether FSH, an important mediator of folliculogenesis, plays a role in this regulation. Preantral follicles (110–135 µm in diameter) were isolated from 18 day old C57BL/6 mice using fine needles. Follicles were cultured (30–35 per well) for 7 days with varying doses of FSH (0–100ng/ml). RNA was extracted, reverse transcribed and real time PCR was carried out with primer sets for GDF-9, GDF-9B, TGFβRI, BMPRII and GAPDH. Immunohistochemistry was conducted on sections of formalin-fixed 18 day old mouse ovary using antisera directed at TGFβRI and BMPRII. GDF-9 and GDF-9B mRNAs were downregulated by FSH treatment (compared to untreated control). There was no effect of FSH on the expression of either receptor. TGFβRI and BMPRII receptor proteins were localised to the cytoplasm of oocytes and granulosa cells in 18 day old mouse ovary. Both were mostly localised to secondary follicles, lighter TGFβRI staining was found in less mature follicles. Receptors for GDF9 signalling were both present consistent with direct effects of GDF9 on ovarian function. GDF9B might also have an effect although it remains to be seen if the type 1 receptor is localised to the mouse ovary. Further studies are required to investigate receptor regulation.
124. DNA METHYLATION IN THE 2-CELL MOUSE EMBRYO IS THE RESULT OF DNMT ACTIVITY DURING DEVELOPMENT FROM THE ZYGOTE TO THE 2-CELL STAGE
Authors: Li Y. , Morgan H. D. , Ganeshan L. , O'Neill C.
Published: November 2010

In an accompanying abstract we show for the first time that global demethylation of both paternally- and maternally-derived genomes occurs prior to syngamy. It is commonly considered that new methylation of the genome does not commence until late in the preimplantation stage. Yet embryos during cleavage stage are known to show DNA methylation. This creates a paradox, if global demethylation occurs by the time of syngamy yet remethylation does not occur until the blastocysts stage, how can cleavage stage embryos possess methylated DNA. We examined this paradox. We examined DNA methylation in 2-cell embryos by confocal microscopy of anti-methylcytosine immunofluorescence and propidium iodide co-staining of whole mounts. We confirmed that DNA in late zygotes was substantially demethylated in both the male and female pronuclei. By the 2-cell stage, embryos collected direct from the oviduct showed high levels of cytosine methylation. We assessed whether this accumulation of cytosine methylation during the early 2-cell stage was a consequence of DNA methyltransferase (DNMT) activity. This was achieved by treating late stage zygotes with the DNMT inhibitor RG108 (5 μM) for the period of development spanning pronuclear stage 5 to early 2-cell stage. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (P<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. Our results show for the first time that de novo methylation of the genome occurs as early as the 2-cell stage of development and that this is mediated by a RG108-sensitive DNMT activity. The results substantially change our understanding of epigenetic reprogramming in the early embryo.
132. IMMUNE-DEVIATING CYTOKINES DETERMINE THE MATERNAL T CELL RESPONSE DURING PREGNANCY AND TOLERANCE OR REJECTION OF THE CONCEPTUS
Authors: Moldenhauer L. M. , Hayball J. D. , Robertson S. A.
Published: November 2010

In healthy pregnancies the maternal immune system establishes paternal antigen-specific tolerance allowing survival of the semi-allogeneic conceptus. The cytokine environment is a key factor in determining the phenotype of antigen-specific lymphocytes, influencing the development of either cytotoxic or tolerogenic cells. We hypothesized that the cytokine environment at the time of priming to paternal antigens influences the phenotype of the maternal T cell response and pregnancy outcome. Transgenic Act-mOVA male mice expressing chicken ovalbumin (OVA) ubiquitously provided OVA as a model paternal antigen. OVA is present within the semen of Act-mOVA mice and is inherited and expressed by the conceptus tissue. OVA-reactive CD8+ OT-I T cells were activated with OVA in the presence of various immune-deviating cytokines in vitro, before transfer at 3.5 dpc to C57Bl/6 (B6) females gestating OVA-expressing fetuses. Pregnant mice received either naïve OT-I T cells, cytotoxic OT-I T cells stimulated in vitro in the presence of IL-2 or OT-I T cells stimulated in vitro in the presence of TGFβ1 and IL-10, two factors present in the uterus and associated with immune tolerance. Immunohistochemistry was utilized to demonstrate that OT-I T cells infiltrate into the implantation site. Cytotoxic OT-I T cells caused fetal loss, while OT-I T cells activated in vivo or in vitro with TGFβ1 and IL-10 did not cause fetal loss. Additionally, cytotoxic OT-I T cells did not affect B6 x B6 matings, demonstrating the antigen-specific nature of the T cell-mediated fetal loss. Collectively these experiments show that maternal antigen-reactive T cells activated in vivo in the cytokine environment of the mated uterus are tolerogenic, not cytotoxic, and implicate TGFβ1 and IL-10 as key elements of that environment. We conclude that the cytokine environment at the time of priming to paternal antigens influences the T cell phenotype and impacts upon maternal immune tolerance and fetal survival.
137. ELUCIDATION OF THE MOLECULAR MECHANISMS THAT UNDERPIN CAPACITATION-ASSOCIATED SPERM SURFACE REMODELLING
Authors: Nixon B. , Skinner B. , Aitken R. J.
Published: November 2010

Recent research from our laboratory has provided evidence that sperm-egg interaction is mediated by a multimeric sperm receptor complex. Furthermore, we have demonstrated that this complex is assembled on the sperm surface during the final phase of their maturation, a process known as capacitation. The mechanisms underpinning this capacitation-associated surface remodelling remain poorly understood and are the subject of our current investigation. Specifically we have focused on whether this process is driven by vesicle mediated, intracellular trafficking of proteins. For this purpose we have examined the presence and physiological significance of a key part of the molecular machinery necessary for this form of trafficking, namely the enzyme dynamin. Our studies revealed that sperm are endowed with at least two isoforms of dynamin (1 and 2) both of which reside within the peri-acrosomal region of the sperm head, a location compatible with a role in sperm membrane remodelling. Consistent with this putative role, it was demonstrated that dynamin 1 was phosphorylated during capacitation, a post-translational modification that has been causally linked to both its activation and to the capacitation-associated surface remodelling of mouse spermatozoa. Furthermore, it was demonstrated that pharmacological inhibition of dynamin activity led to a concomitant reduction in the ability of spermatozoa to bind to the zona pellucida of homologous oocytes. This suppression was also correlated with reduced surface expression of a number of proteins including a subset of putative sperm-zona pellucida receptors. Collectively these data support the novel hypothesis that dynamin does participate indirectly in sperm membrane remodelling events by virtue of its ability to mediate intracellular trafficking.
145. TRP53 REGULATES THE FORMATION OF A PLURIPOTENT INNER CELL MASS IN THE EARLY EMBRYO
Authors: Ganeshan L. , O'Neill C.
Published: November 2010

The developmental viability of the early embryo requires the formation of the inner cell mass (ICM) at the blastocyst stage. The ICM contributes to all cell lineages within the developing embryo in vivo and the embryonic stem cell (ESC) lineage in vitro. Commitment of cells to the ICM lineage and its pluripotency requires the expression of core transcription factors, including Nanog and Pou5f1 (Oct4). Embryos subjected to culture in vitro commonly display a reduced developmental potential. Much of this loss of viability is due to the up-regulation of TRP53 in affected embryos. This study investigated whether increased TRP53 disrupts the expression of the pluripotency proteins and the normal formation of the ICM lineage. Mouse C57BL6 morulae and blastocysts cultured from zygotes (modHTF media) possessed fewer (p < 0.001) NANOG-positive cells than equivalent stage embryos collected fresh from the uterus. Blocking TRP53 actions by either genetic deletion (Trp53–/–) or pharmacological inhibition (Pifithrin-α) reversed this loss of NANOG expression during culture. Zygote culture also resulted in a TRP53-dependent loss of POU5F1-positive cells from resulting blastocysts. Drug-induced expression of TRP53 (by Nutlin-3) also caused a reduction in formation of pluripotent ICM. The loss of NANOG- and POU5F1-positive cells caused a marked reduction in the capacity of blastocysts to form proliferating ICM after outgrowth, and a consequent reduced ability to form ESC lines. These poor outcomes were ameliorated by the absence of TRP53, resulting in transmission distortion in favour of Trp53–/– zygotes (p < 0.001). This study shows that stresses induced by culture caused TRP53-dependent loss of pluripotent cells from the early embryo. This is a cause of the relative loss of viability and developmental potential of cultured embryos. The preferential survival of Trp53–/– embryos after culture due to their improved formation of pluripotent cells creates a genetic danger associated with these technologies.
169. SERTOLI CELL-SPECIFIC DISRUPTION OF THE ANDROGEN RECEPTOR DNA-BINDING DOMAIN REVEALS DIFFERENTIAL TEMPORAL CONTROL OF DISTINCT ANDROGEN-REGULATED GENES
Authors: Allan C. M. , Robson M. , Harwood T. , Handelsman D. J.
Published: November 2010

Androgen receptor (AR) actions are vital for spermatogenesis. However, in postnatal development male germ cells do not express AR, highlighting its key role in testicular somatic cells. We recently used a Cre-loxP strategy to determine the in vivo requirement of AR DNA-binding in Sertoli cell (SC) function. Transgenic (Tg) mice with Cre expression targeted by SC-specific AMH or Abp promoters were crossed with floxed-Ar (Arflox) mice for Cre-loxP inframe deletion of Ar exon 3, which encodes a zinc finger essential for the DNA-binding domain (DBD). SC-specific mutated ARΔex3 (SCARΔex3) produced infertile AMH.SCARΔex3 and Abp.SCARΔex3 males. Testes from adult homozygous TgCre(+/+) AMH.SCARΔex3 or Abp.SCARΔex3 males were 30% of normal size and exhibited meiotic arrest, whereas testes from hemizygous TgCre(+/–) Abp.SCARΔex3 males were larger (47% normal) with more postmeiotic germ cell development. Despite marked Leydig cell hypertrophy, testicular expression of the adult Leydig marker Hsd3b6 (RT-PCR) and normal intratesticular testosterone levels (LC-MS/MS) in SCARΔex3 males indicated the presence of morphologically distinct but functional adult Leydig cells. SC-specific mutated AR Δex3 was predicted to disrupt classical AR-regulated pathways via loss of direct DNA interaction. Androgen-repressed testicular Ngfr expression (known to be via non-classical AR pathways) was not upregulated in SCARΔex3 testes, suggesting maintenance of a non-classical mechanism independent of AR-DBD. In contrast, SC-specific Rhox5 and Eppin transcription, regulated by divergent or classical androgen-response elements respectively, were both decreased in postnatal SCARΔex3 vs. control testes, demonstrating SC-specific AR function as early as postnatal day 5. However, Rhox5 expression declined dose-dependently, whereas Eppin expression increased, in adult TgCre(+/−) and TgCre(+/+) SCARΔex3 testes, revealing differential temporal control for distinct AR-regulated transcripts. Thus, our SCARΔex3 paradigm displayed dose-dependent TgCre-disruption of meiotic competence and post-meiotic development as well as gene expression, and represents a unique model to selectively differentiate AR-regulated genes.
170. TGFβ SIGNALING IN AN IN VITRO SEMINOMA MODEL
Authors: Young J. C. , Jaiprakash A. , Mithraprabhu S. , Itman C. , Kitazawa S. , Loveland K. L.
Published: November 2010

Testicular cancer, the second most common malignancy in young men, has a 95% cure rate but can result in infertility or subfertility. Its incidence has increased significantly in recent decades (1). This cancer is thought to arise during embryogenesis, based on the persistence of embryonic germ cell markers such as Blimp1 (2), Oct3/4 (3) and Nanog (3) in adult seminoma cells. TCam2 cells are a recently characterised in vitro seminoma model (4). We show by Q-PCR and immunofluorescence that they also express these early germ cell markers. TGFβ signaling plays a key role during germ cell development, and is implicated in the development of testicular cancers (5, 6). To investigate this further, we first determined whether the pathway is active in TCam2 cells. By Q-PCR we demonstrate expression of the TGFβ downstream transcription factors Smad 2, 3 and 4, and Activin type I and II receptors. Importantly, ActRIIA, which is undetectable in adult testicular germ cells, but readily detected in human foetal germ cells (7) and clinical seminoma samples (6), is readily detectable at both the mRNA and protein level in TCam2 cells. Furthermore, 24 hour treatment with Activin (5 and 50ng/ml) or BMP4 (5 and 50ng/ml) induces a 3-4 fold increase in ActRIIA mRNA levels, but not ActRIA, ActRIB or ActRIIB. Strikingly, in TCam2 cells BMP4 and to a lesser extent retinoic acid, but not activin, support survival and proliferation of TCam2 cells in the absence of serum. This is consistent with known roles of BMP4 and retinoic acid in enhancing murine foetal germ cell proliferation/self-renewal and survival (8, 9), and activin inhibition of foetal murine germ cell proliferation (10). This study is the first to demonstrate a functional response in seminoma cells consistent with their foetal germ cell-like identity and forms the basis for future mechanistic analyses of the role of TGFβ signaling in human testicular cancer.
174. THE EPIDERMAL GROWTH FACTOR (EGF) FAMILY IN THE ENDOMETRIUM AND BLASTOCYST OF THE TAMMAR WALLABY, MACROPUS EUGENII DURING EMBRYONIC DIAPAUSE
Authors: Fenelon J. C. , Shaw G. , Renfree M. B.
Published: November 2010

Embryonic diapause, a suspension of cell division and growth at the blastocyst stage, is widespread amongst mammals, but is especially common in the kangaroos and wallabies. In the tammar, Macropus eugenii, the sequence of endocrine events leading to embryonic diapause and reactivation are well defined[1]. The blastocyst can remain in diapause for up to 11 months without cell division, measurable metabolism or apoptosis occurring [2]. The ovarian hormones, especially progesterone, exert their effects on the blastocyst by alterations in the endometrial secretions [3], but the molecular cross-talk between the endometrium and blastocyst is unknown. There is increasing evidence for the involvement of leukaemia inhibitory factor (LIF)but the epidermal growth factor (EGF) family of growth factors are also likely to be involved.This study examined the expression of EGF and HB-EGF as well as their receptors, ERBB1 and ERBB4, in the tammar endometrium and blastocyst at entry into, and reactivation from, diapause. The genes for these factors were highly conserved in the tammar with orthologues in human and mouse. Quantitative RT-PCR of all four factors in the endometrium showed that expression changed with stage. Although expression levels of both receptors did not change between diapause and reactivation, both HB-EGF and EGF levels increased at reactivation from diapause and levels of HB-EGF decreased at entry into diapause. All factors were immunopositive in the endometrium. Studies underway will determine whether the cellular location and quantity of these factors change with entry into or exit from diapause, and define the molecular interactions occurring between the blastocyst and endometrium. These results are consistent with a role for the EGF family of growth factors in the control of embryonic diapause in tammars.
500. THE MANIPULATION OF THE EPIGENETIC MARK HISTONE 3 LYSINE 9 TRIMETHYLATION IN DONOR CELLS AND ITS EFFECTS ON THE DEVELOPMENT OF CLONED MOUSE EMBRYOS
Authors: Antony J. , Oback F. , Broadhurst R. , Cole S. , Graham C. , Jenuwein T. , Chamley L. , Oback B. , Laible G.
Published: November 2010

To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.
502. COLD SHOCK DURING RAPID COOLING OF SPERM FROM A TROPICAL ANURAN, THE CANE TOAD, BUFO MARINUS
Authors: Fitzsimmons C. , Mahony M. J. , Clulow J.
Published: November 2010

Amphibian sperm are not considered to be susceptible to cold shock injury during cooling and cryopreservation. In this study we investigated the susceptibility of the tropical bufonid, the Cane Toad (Bufo marinus) to sperm cold shock. Sperm from testes macerated in 2x Simplified Amphibian Ringer (inactivated state) were diluted 1:6 in various cryodiluents containing 10% sucrose and 10–20% glycerol or DMSO, or were used directly as undiluted controls. Samples were cooled at three cooling rates (1oC min–1, 5oC min–1 or placed directly on ice – rapid cooling) to 0oC and then either warmed to room temperature and their motility and viability assessed after activation by dilution, or cryopreserved. Cryopreserved samples were stored in liquid nitrogen for two days and thawed at room temperature before assessment of motility and sperm viability. Cooling rapidly to 0oC by directly placing samples on ice or cooling at 5oC min–1 before warming to room temperature resulted in a significant decline in motility (all means less than 40% of control motility after 30 min at room temperature) in comparison to samples cooled slowly at 1oC min–1 (all means greater than 80% motility; p < 0.05 to 0.01). Samples cryopreserved after cold shock (rapid cooling to 2oC by immediate exposure of straws to 2oC ambient temperature) versus samples cooled slowly (1oC min–1) to 2oC prior to cryopreservation had significantly lower mean post-thaw motilities (p<0.05; in the range of 40–60% motile versus 80–95% for non-cold shocked). These data together indicate that the sperm of B. marinus undergo cold shock injury prior to freezing, and that post-thaw recovery after cryopreservation of cold-shocked sperm is substantially reduced.
512. COMMERCIAL EQUINE CHORIONIC GONADOTROPHIN ISOFORM COMPOSITION, IMMUNOACTIVITY, AND BIOACTIVITY IN A MURINE MODEL USING OVARIAN AUGMENTATION AND TESTICULAR INTERSTITIAL CELL BIOASSAYS
Authors: Ciller U. A. , McFarlane J. D. , McFarlane J. R.
Published: November 2010

Equine chorionic gonadotrophin (eCG) is a placental glycoprotein hormone that is harvested from the plasma of pregnant mares for the formulation of commercial products which are used in a variety of assisted reproductive procedures in livestock. It is well documented that the bioactivity of gonadotrophin products is highly variable. The aim of this study was therefore to determine how different eCG products affected target tissues and cells. Isoforms of eCG were separated with iso-electric focusing and immunoactivity measured with RIA. For in vivo bioassay, dose-response eCG treatments were administered subcutaneously to immature female mice with follow-up treatment on day 2. On day 3 the mice were asphyxiated and ovaries dissected and weighed relative to standard. For in vitro bioassay, adult male mice were asphyxiated, testes removed, decapsulated, dispersed, strained and washed with DMEM:F12 0.1% BSA. The cell stock was counted and diluted for culture at 20,000 cells/well. Cell viability was determined using trypan blue. Five doses were tested for each eCG product. The cells were incubated at 32ºC for 3 hours in 5.0% CO2 in humidified conditions. Media was collected after 3 hours and immediately assayed for testosterone with RIA. Product eCG with ~90% isoforms with pH 3.0–3.7 and ~10% with pH 3.8–4.4, showed 29% greater biologically activity in the ovarian augmentation assay and 44.8% more immunoactivity then stated product bioactivity. Product eCG with ~70% isoforms with pH 3.0–3.7 and ~30% with pH 3.8–7.4 was 3.0% less effective in vivo and had 7.9% less immunoactivity then stated bioactivity, however, in vitro testosterone production was more effectively stimulated then with the previous more acidic eCG product. Our study shows a selective difference in commercial eCG biological activity that appears to depend on subtle isoform heterogeneity. Future studies aim to determine the specific actions of eCG isoforms in target tissues.
516. THE EFFECTS OF eCG INJECTION ON SUPEROVULATORY RESPONSES BEFORE VERSUS AFTER CIDR REMOVAL IN IRANIAN SHALL EWES
Authors: Salehi R. , Kohram H.
Published: November 2010

The aim of this study was to investigate the effects of eCG injection on ovarian responses 2 days before or immediately after CIDR removal in a superovulation program during non-breeding season in Iranian Shall ewes. 12 ewes were kept inside for the duration of the experiment and were fed a live weight maintenance ration. The ewes were synchronized by CIDR. The day of CIDR insertion was considered as the starting point of the experiment (day 0). The ewes received 1500 IU eCG on day 12. Ewes were randomly assigned to 1 of 2 groups, and CIDR removed on days 12 and 14 in groups 1 and 2, respectively. Ovarian follicular activity was monitored by transrectal ultrasonography daily from the initiation day of superovulatory stimulation (day 12) until the day of estrus (day 14), and once 7 days after estrus (day 21) to monitor the number of corpus lutium. During examination the animals were held in a standing position. Ultrasonographic scanning of both ovaries was recorded using a MP4 player. The number of medium and large follicles (≥3 mm) were recognized, measured and mapped to their location using printed images of both ovaries. Data were analyzed using GLM procedure of the SAS. The results presented in Table 1 showed that the ovarian responses in terms of the number of ≥3 mm follicles at estrus and CL 7 days later were greater (P<0.05) in group 1 ewes. The results indicated that the superior time for eCG injection is on the day of CIDR removal.