bluuebunny/crossref_metadata_2025 huggingface dataset:
240.Fertilisation in vitro causes precocious activation of transcription from the zygotic genome
Authors: O'Neill C. , Stojanov T.
Published: November 2010

In vitro fertilisation (IVF) may have long-term adverse effects on progeny. Infants conceived by in vitro fertilisation are more likely to be born small for dates and, in a mouse model, culture in vitro caused changes in neurological performance of progeny. Changes in the pattern of gene expression in IVF embryos have been detected, and these may be one cause of the long-term effects. This study investigates the effect of IVF on the ontogeny of onset of expression from the embryonic genome in the mouse.
The expression of two markers for the onset of transcription (the transcription requiring complex (TRC) and hsp 70.1) was assessed in 2-cell embryos produced by IVF or fertilisation in situ (ISF). It was confirmed that the time from fertilisation to first cleavage was not different for IVF and ISF. Zygotes were cultured and at 1-hourly intervals those cleaved were 'picked off' (time 0 h after cleavage) and placed in groups of 10 in 10 μL of modified-HTF. The expression of the gene products was assayed at times after 'pick-off'.
The proportion of embryos expressing TRC increased with time after cleavage (P�<�0.001). IVF embryos expressed it significantly earlier (P�<�0.01) than ISF embryos. Some IVF embryos expressed TRC immediately after cleavage and this was never found for ISF embryos. All IVF embryos were TRC-positive by 2.5 h after cleavage, while this did not occur until 4.5 h post cleavage for ISF. Hsp70.1 transcripts were first detected in IVF embryos 2 h after cleavage but not until 6 h after cleavage in ISF embryos (P�<�0.01).
The onset of transcription at the 2-cell stage is currently thought to reflect major reorganization of the nucleosomal structure of DNA. Evidence for precocious onset of transcription may indicate that this fundamentally import process is changed following IVF, and warrants further investigation.
242.Stress applied to ewes at Day 2 and 3 of gestation increases Day 6 embryo cell counts
Authors: Butler M. , Hinch G. , Hill J. R.
Published: November 2010

The effect of stress during pregnancy on fetal development has been demonstrated in studies such as the administration of glucocorticoids at 4 weeks of gestation which then 'programs' arterial blood pressure to be elevated during subsequent adult life (1). In the current experiment we have explored the effect of an acute stressful event applied to pregnant ewes at Day 2 and 3 of gestation.
The morphology and cell counts of Day 2 and 3 embryos were evaluated following surgical collection from 15 superovulated ewes. Seven of these ewes were subjected on Days 2 and 3 after insemination to a 15 min period of shearing (partial), isolation and confinement adjacent to a working dog. These stresses were intended to produce a short-term cortisol peak at the time embryos were at the 4–8 cell stage.
Embryos recovered from the 7 treated and 8 control ewes were graded according to IETS guidelines using a Nikon TE 300 inverted microscope. Cell counts were performed following staining of embryos with Hoechst 33342. Only embryos with >8 nuclei were included in the analysis to exclude embryos that were either unfertilised or died prior to the stressful events.
Subjective grading showed no difference in grading embryos as transferable (stage of compact morula or later with quality grade of 1 or 2) between embryos derived from stressed and non-stressed ewes (45/63 v. 45/64). However cell counts were dramatically different as stressed ewes produced embryos with higher cell counts (74.7 � 32.0 v. 43.3 � 22.9; P�<�0.001 using one-way ANOVA).
As it is presumed that cell counts are directly related to embryo quality, our results suggest that stress may be manipulated to increase embryo cell numbers and thus embryo quality. The apparently beneficial effect of stress is surprising as it is commonly stated that minimising stress during commercial animal embryo transfer programs results in improved results. We look forward to the opportunity to follow the in vivo development of embryos derived from ewes stressed at Day 2 and 3 of gestation.
(1) Dodic M. et al. (1998) An early prenatal exposure to excess glucocorticoid leads to hypertensive offspring in sheep. Clin. Sci (Lond.) 94, 149–155.
243.Nuclear transport of Gli transcription factors during spermatogenesis
Authors: Szczepny A. , Jans D. A. , Loveland K. L. , Dias M.
Published: November 2010

Development is highly regulated by complex signalling cascades. One such pathway is the Hedgehog (Hh) signalling pathway which plays an essential role in spermatogenesis. The Gli family of zinc finger TFs, consisting of Gli1, Gli2 and Gli3, are mediators of the Hh signalling cascade. Gli1 is an activator of Hh target genes, whereas Gli2 and Gli3 can undergo proteolytic cleavage and function as both activators and repressors. Little is known regarding the nuclear import pathway of these TFs. In this study, the mRNA expression pattern of all Gli family members in the developing mouse testis was compiled by in situ hybridisation and shown to have unique expression patterns. In the adult mouse testis, Gli1 mRNA was detected in spermatogonia through to round spermatids whereas Gli2 was only found in spermatogonia and spermatocytes. Very low levels of Gli3 mRNA were detected in all ages and cell types. Since little is known regarding the import pathway for Gli1, expression vectors containing different fragments of the N-terminus of Gli1 were created and used to perform transfection experiments and generate vectors for bacterial GFP-fusion protein expression. Transfection experiments into African green monkey kidney Cos-7 cells, and the murine spermatogenic cell lines, Gc-1 and Gc-2 using 3 different constructs localised the NLS(s) required to target Gli1 to the nucleus in the zinc finger DNA-binding domain of Gli1. Preliminary results for in vitro binding of bacterially expressed Gli1 indicated no binding by importin β 1 or β3 but a weak interaction with the importin α/β heterodimer. This can be seen as the first step towards defining the nuclear import pathway for Gli1. The mechanisms by which Gli activity is modulated remain unanswered and the regulation of its nuclear entry may be an important means of doing so.
244.Interaction between bone morphogenetic protein 4 and retinoid signalling in mouse spermatogenesis
Authors: Baleato R. M. , Aitken R. J. , Roman S. D.
Published: November 2010

Vitamin A (retinol, or ROL) is also essential for normal spermatogenesis in the rat and mouse. Vitamin A-deficient (VAD) rodents suffer various disorders including blindness and male infertility. The molecular mechanisms leading to infertility in vitamin A deficient rodents have never been fully elucidated. Following prolonged vitamin A withdrawal the only germ cells remaining in the VAD rodent testis are stem cell spermatogonia, type A1 spermatogonia, and a few preleptotene spermatocytes. Supplementing the diet of these animals with retinoic acid (RA) alleviates all symptoms of vitamin A deficiency, with the exception of sight and spermatogenesis. It is not until VAD animals are re-administered ROL through the diet, or RA is injected in repeated high doses directly into the testis, that normal spermatogenic function is restored. Here we report an interaction, in germ cells, between the Bone Morphogenetic Protein (BMP) 4 and retinoid signalling pathways that may help explain the molecular mechanics of vitamin A deficiency. We localised BMP4 gene expression to adult germ cells, in particular spermatogonia, at both the mRNA and protein level. We generated VAD mice and found that in the absence of retinoids in vivo, bmp4 gene expression was significantly upregulated in the testis. We also observed that the expression of bmp4 is downregulated by retinoid treatment in germ cells isolated from vitamin A sufficient mice. Expression of bmp4 mRNA in isolated spermatogonia was more sensitive to ROL rather than RA. Our results may reflect a direct requirement for ROL by germ cells for the resumption of spermatogenesis in VAD animals that involves the regulation of BMP4 expression. Furthermore our observations suggest that retinoid signalling in germ cells is different to that observed in somatic cells, and may provide insights into the role of retinoids in spermatogenesis.
245.Characterisation of prostaglandin production in the normal and inflamed rat testis
Authors: Winnall W. , Muir J. , Hirst J. , Hedger M.
Published: November 2010

Prostaglandins E2 (PGE2) and F2α (PGF2α) play a role in Leydig cell function and in suppression of macrophage inflammatory functions. We predict that PGs also may play a role in interstitial fluid (IF) formation in the testis. Prostaglandin synthesis involves one of two distinct forms of cyclooxygenase (COX): constitutively expressed COX-1 and inducible COX-2. We recently demonstrated expression of both enzymes in macrophages, somatic and germ cells of the adult rat testis, and that COX-2 may the more important form in this organ. Adult male Sprague-Dawley rats were maintained on normal feed or 0.15% celebrex, a specific COX-2 inhibitor, for 5 weeks. Rats were subsequently treated with saline, or lipopolysaccharide (LPS; 0.1 mg/kg or 5 mg/kg), 6 h prior to collection of tissues. PGE2 was measured by RIA in medium of cultured testis fragments and testicular cells from normal rats (� 10 μg/mL LPS, 24 h, 37�C), and in testicular interstitial fluid. PGE2 was constitutively produced by whole testis, Sertoli cells, Leydig cells and round spermatids, but not by resting macrophages or pachytene spermatocytes in culture. Stimulation with LPS upregulated PGE2 in macrophage cultures, but not in other cells or whole testis. Normal PGE2 levels in IF were 16–20 ng/mL; levels were not altered by low-dose LPS, but were reduced by high-dose LPS. Celebrex caused a reduction in IF PGE2 levels in both the normal and low-dose groups, but not in the high-dose group. Celebrex elevated IF volume (25–50%) in all groups. Our experiments show cell-type specific regulation of PGE2 production in the rat testis, and predict a role for COX-2 elicited PGs in the IF regulation and in post-meiotic cell function. Paradoxically, low-level inflammation does not alter testicular PGE2 levels, as somatic and germ cells, which do not respond to LPS, appear to contribute most to the local levels of PGE2.
246.The effect of testosterone and season on prodynorphin mRNA expression in the preoptic area hypothalamus of the ram
Authors: Scott C. J. , Clarke I. J. , Tilbrook A. J.
Published: November 2010

Progesterone stimulates prodynorphin mRNA expression in the hypothalamus of the ewe (1), but whether testosterone regulates dynorphin gene expression in the ram is unknown. In a previous study (2), we showed that both testosterone and season influence mRNA expression of another opioid, enkephalin, in the preoptic area and hypothalamus of rams. Using tissue from the same study, we tested the hypothesis that testosterone and/or season modulate prodynorphin mRNA expression in specific areas of the hypothalamus in the ram. Adult Romney Marsh rams were castrated either during the 'breeding' season or 'non-breeding' season and 1 week later received intramuscular injections of either peanut oil (vehicle) or testosterone propionate (8 mg/12 h for 7 days) (5/group). Blood samples taken every 10 min for 12 h were assayed for plasma LH and testosterone. Prodynorphin mRNA expression was quantified in hypothalamic sections by in situ hybridisation using a 35S-labelled riboprobe and computer-aided image analysis. Plasma testosterone levels were higher in testosterone propionate-treated than oil-treated sheep. Mean plasma LH concentrations were reduced and the interpulse interval for LH pulses was greater in testosterone propionate-treated wethers compared to oil-treated wethers, with no change in LH pulse amplitude. Testosterone propionate treatment increased prodynorphin mRNA expression in the supraoptic nucleus and the bed nucleus of the stria terminalis, but only during the breeding season. Proenkephalin mRNA expression was also higher in the 'breeding' season than in the 'non-breeding' season in the caudal preoptic area and paraventricular nucleus. No differences were observed between treatments in five other regions of the hypothalamus. We conclude that testosterone and season regulate preproenkephalin mRNA levels in the preoptic area/hypothalamus in the ram in a region-specific manner.
(1) Foradori et al. (2002) Proc. Soc. Neurosci. A572.9. (2) Scott et al. (2003) Biol. Reprod. 69, 2015–2021.
250.Chemokine profiling in endometriosis using laser capture microdissection
Authors: Chand A. L. , Murray A. , Jones R. L. , Salamonsen L. , Rombauts L.
Published: November 2010

Endometriosis is an inflammatory condition with elevated leukocyte infiltrate; defined as the ectopic growth of endometrium-like tissue, characterised by epithelial glands, outside the uterus. Chemokines selectively regulate influx and activation of leukocyte subpopulations. Their non-immune functions during tissue remodelling and disease pathogenesis include up-regulation of adhesion molecules, stimulation of inflammatory mediators, angiogenesis, cell proliferation and motility (1). The aims of the present study were to compare the chemokine mRNA profiles expressed by the epithelial glands of: eutopic endometrium from patients with / without endometriosis. Tissue heterogeneity in the endometrium and in ectopic lesions hinders precise study of the contribution of cell-specific inflammatory responses. Laser Capture Microdissection (LCM) was therefore utilised. Frozen eutopic curettings and ectopic endometriosis lesions were sectioned, H&E stained and glandular epithelium laser captured. 327 captured glands yields approximately 27 ng of RNA from each endometrial sample. To obtain enough RNA for gene array analysis and verification studies, cellular mRNA was amplified. Two rounds of linear mRNA amplification provided a sufficient yield of >1.8 μg from 1 ng of RNA. RNA from 4 patients and 4 controls were pooled, amplified and probed on gene arrays to build a chemokine profile. We identified 45 chemokines / receptors that are specifically abundant in glandular epithelium. 39 were highly upregulated (2- to 60-fold) in women with endometriosis compared to controls. In contrast, only 2 genes were downregulated more than 2-fold in endometriosis patients. Most genes have not been previously studied for their roles in endometriosis. Verification studies are currently being carried out. This is a novel study providing evidence of a distinct profile of the chemokine/receptors collectively in women with endometriosis.
(1) J. Immunol. (2002) 168, 4301.
251.Regulation of mouse cumulus expansion by oocyte-secreted growth differentiation factor-9 (GDF-9)
Authors: Dragovic R. A. , Ritter L. J. , Amato F. , Scott S. J. , Cranfield M. , Groome N. P. , Armstrong D. T. , Gilchrist R. B.
Published: November 2010

Oocyte paracrine signalling is necessary for mouse cumulus cell expansion, an important preovulatory process. The oocyte-secreted factor growth differentiation factor-9 (GDF-9) signals through the bone morphogenetic protein receptor-II (BMPR-II) and is currently the primary candidate molecule for the cumulus expansion enabling factor (CEEF). The present study was conducted to determine whether in the mouse GDF-9 is the CEEF. Cumulus oocyte complexes (COC) were collected from eCG-primed mice and the oocyte was microsurgically removed to generate an oocytectomised complex (OOX). An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P�<�0.05) induced by either recombinant mouse GDF-9 (score; mean +/– SEM: 2.7 +/– 0.1), recombinant TGF-μ1 (score: 2.6 +/– 0.2) or co-culture with oocytes (score: 2.3 +/– 0.2). A GDF-9 neutralising antibody mAb-53, raised against hGDF-9, was effective in neutralising the response of OOX complexes to GDF-9 (score: 0.1 +/– 0.1), but had no significant effect on the expansion of OOX complexes co-cultured with oocytes (score: 2.3 +/– 0.2). Likewise, a TGF-μ antagonist neutralised (P�<�0.05) TGF-μ-induced, but not oocyte-induced, expansion of OOX complexes. A soluble portion of the BMPR-II ectodomain, a known GDF-9 antagonist, failed to neutralise oocyte-induced cumulus expansion (P�>�0.05) at the highest dose implying that BMPR-II is not a critical receptor involved in regulating cumulus expansion. Using real-time RT-PCR, hyaluronan synthase-2 (HAS2) mRNA expression by OOXs was upregulated 6- to 7-fold by oocytes and GDF-9. The GDF-9 neutralising antibody mAb-53, partially neutralised GDF-9-induced OOX HAS2 expression, but not oocyte-induced HAS2 expression. This study provides evidence that like TGF-μ1, GDF-9 can enable FSH-induced cumulus expansion, however more importantly demonstrates that neither GDF-9 nor TGF-μ1 alone account for the crucial oocyte-secreted factor regulating cumulus expansion in the mouse.
252.Oocyte-secreted factor(s) regulate apoptosis of bovine cumulus cells
Authors: Hussein T. S. , Froliand D. A. , Thompson J. G. , Gilchrist R. B.
Published: November 2010

Paracrine factors secreted by the oocyte affect cumulus cell proliferation and differentiation. These factors may also act in an anti-apoptotic manner, maintaining the low incidence of cellular apoptosis within cumulus cells. The purpose of this study was to determine whether the incidence of apoptosis within cumulus cells is regulated by oocyte-secreted factors (OSF). Bovine cumulus-oocyte complexes (COC) aspirated from abattoir-derived ovaries were randomly allocated to 3 treatments: (1) groups of 5 intact COC; (2) groups of 5 oocytectomised complexes (OOX), where oocytes were removed microsurgically; and (3) groups of 5 OOX co-cultured with 25 denuded oocytes (DO). To examine a dose effect of OSF, OOX were also cultured with increasing numbers of DO (OOX+5DO, OOX+25DO, OOX+50DO). In both experiments, complexes were cultured in their respective treatments for 24 h in 50 �l of oocyte maturation medium (+/– rFSH; 0.1 IU/mL). Apoptosis was assessed using TUNEL, with all nuclei counterstained with propidum iodide (PI). Nikon TE2000 laser confocal scanning microscopy was used to visualise and quantify the incidence of apoptosis (TUNEL/PI). The proportion of apoptotic cells was determined by image analysis. Oocyte removal lead to a significant increase in cumulus cell apoptosis (OOX, 67% apoptotic; COC, 15%; P�<�0.001). However, the incidence of apoptosis in OOX was restored to COC levels when co-cultured with DO (18%; P�>�0.05, COC v. OOX+DO). FSH prevented apoptosis in all treatment groups (P�<�0.05) decreasing the incidence by 27% in OOX and by 12% in COC. Cumulus cell apoptosis within OOX (+/– FSH) was reduced in a dose dependent manner by treating with increasing numbers of DO. These results indicate for the first time that oocyte-secreted factor(s) regulate the incidence of apoptosis within cumulus cells.
253.Follicle stimulating-hormone (FSH) withdrawal induces germ cell apoptosis in the mature rat testis in vitro
Authors: Degen S. M. , Stanton P. G. , Loveland K. L. , Meachem S. J.
Published: November 2010

FSH is a key determinant of adult sperm output influencing both Sertoli and germ cell development. The aim of this study was to assess the impact of FSH action on Sertoli and germ cell proliferation and survival in vitro, and to identify FSH-regulated genes that may underpin these responses. Testis fragments from 17-day-old rats were cultured with recombinant human FSH for 2 or 24 h and then labelled with bromodeoxyuridine (BrdU) to identify proliferating cells. The testis fragments were then processed for analysis of cell numbers by stereology, BrdU incorporation by immunohistochemistry, and apoptosis by TUNEL. The TUNEL assay revealed that without FSH, spermatogonial apoptosis was induced to 195% and 179% (P�<�0.05) compared to fragments with FSH after 2 and 24 h, respectively. No difference in apoptosis was observed in spermatocyte or Sertoli cell populations at these time points. No differences in Sertoli or germ cell proliferation were observed with or without FSH. To understand how FSH mediates spermatogonial apoptosis the response of 5 testicular genes of interest was examined. Expression of cyclin D2 (cell cycle, G1-S), N-cadherin (N-Cad; adhesion molecule), Bax (pro-apoptotic), Bcl-w (anti-apoptotic), and stem cell factor (SCF; pro-apoptotic and other functions) was elevated to 151%, 348%, 209%, 258%, and 198%, respectively (all P�<�0.001), in fragments cultured without FSH for 24 h, compared to fragments with FSH. No gene expression differences were observed at 2 h, except for SCF, which was elevated to 135% (P�<�0.01). In conclusion, these studies have examined apoptosis and proliferation activities simultaneously in testis fragments in vitro, and demonstrated that FSH withdrawal induces both spermatogonial apoptosis and expression of testicular genes known to be involved in cell survival. This model will now be used to further investigate FSH-mediation of Sertoli and germ cell development.
254.Interleukin-11 enhances endometrial stromal cell decidualisation via activation and inhibition of target genes
Authors: White C. A. , Dimitriadis E. , Sharkey A. , Stoikos C. J. , Salamonsen L. A.
Published: November 2010

Differentiation of endometrial stromal cells into decidual cells is essential for successful embryo implantation. Interleukin (IL)-11 signalling is required for decidualisation in the mouse (1,2) and the expression pattern of IL-11 and its receptors during the menstrual cycle suggests a role for IL-11 in human decidualisation (3). Exogenous IL-11 has been shown to enhance hormone-induced decidualisation of human endometrial stromal cells in culture (4). This study aimed to determine the effects of IL-11 on downstream gene expression in endometrial stromal cells following 12 days of progesterone-induced decidualisation, and to examine the expression and functional significance of IL-11 target genes during this process. Stromal cells isolated from endometrial biopsies (n = 6) were decidualised with 17β-oestradiol and medroxyprogesterone acetate (EP) or EP with 100 ng/mL recombinant human IL-11. Medium was changed every 48 h, and total RNA extracted on Day 12 for gene expression analysis using custom-made 15K cDNA microarrays. Quantitative real-time RT-PCR was performed on the same samples to confirm gene expression levels. In subsequent experiments (n = 2), cells were cytocentrifuged onto glass slides for immunocytochemistry using specific antibodies. Microarray analysis revealed 16 upregulated and 11 downregulated cDNAs in EP + IL-11 compared to EP treated cells. Among these were IL-1β (6.1-fold upregulated) and insulin-like growth factor binding protein (IGFBP)-5 (3.6-fold downregulated). Using real-time RT-PCR, IL-11 was confirmed to increase IL-1β (fold change 1.3–107.1) and decrease IGFBP-5 (fold change 2.8–469.0) transcript abundance in 6 patients. Immunolocalisation of IL-1β in EP and EP + IL-11 treated cells revealed more intense vesicular cytoplasmic staining with IL-11 treatment, while staining intensity for IGFBP-5 was not affected. Interactions between IL-11 and its downstream targets IL-1β and IGFBP-5 are likely to have functional importance in early pregnancy, and may provide novel targets for the manipulation of human fertility.
(1) Robb L, Li R, Hartley L, Nandurkar HH, Koentgen F, Begley CG (1998) Nat. Med. 4, 303–308. (2) Bilinski P, Roopenian D, Gossler A (1998) Gene Dev. 12, 2234–2243. (3) Dimitriadis E, Salamonsen LA, Robb L (2000) Mol. Hum. Reprod. 6, 907–914. (4) Dimitriadis E, Robb L, Salamonsen LA (2002) Mol. Hum. Reprod. 8, 636–643.
255.Dnmt3L: a coordinator of epigenetic modifications during spermatogenesis
Authors: Webster K. E. , O'Bryan M. K. , Aapola U. , Crewther P. E. , Lyle R. , Meachem S. J. , Antonarakis S. E. , Hedger M. P. , Peterson P. , De Kretser D. M. , Scott H. S.
Published: November 2010

Spermatogenesis is a process with unique epigenetic requirements. The differentiation from diploid spermatogonia to haploid spermatozoa requires regulation of genomic imprint establishment, stage specific gene expression, meiotic division, and the histone-protamine transition. The methyltransferase regulator, Dnmt3L, is expressed during gametogenesis and is necessary for establishment of maternal methylation imprints in the oocyte. Targeted disruption of Dnmt3L does not appear to affect oogenesis, as mature oocytes are generated, however resultant heterozygous progeny die mid gestation due to biallelic expression of imprinted genes. Dnmt3L–/– males however show spermatogenic arrest. We found that this arrest occurs during prophase I of meiosis, with spermatocytes lost by both apoptosis and germ cell sloughing. A progressive degeneration ensues, resulting in a Sertoli cell phenotype. Electron microscopy of meiotic spermatocytes revealed that homologous chromosomes fail to align and form synaptonemal complexes. Furthermore, Dnmt3L–/– spermatocytes show abnormal methylation on paternally imprinted genes and abnormal global retention of histone acetylation, implicating Dnmt3L in histone deacetylase recruitment. Thus, during spermatogenesis, Dnmt3L is crucial for two distinct epigenetic modifications; imprint establishment and global histone deacetylation prior to homologous chromosome alignment. The latter defect is likely to affect the alignment of homologous chromosomes and trigger the pachytene checkpoint leading to spermatocyte death. Since Dnmt3L has no DNA methlytransferase or HDAC activity itself, we propose that Dnmt3L is essential for the coordination of epigenetic layers, at least during spermatogenesis.
257.Fibroblast growth factor receptor-1 (FGFR-1) is essential for spermiogenesis and male fertility
Authors: Cotton L. M. , Gibbs G. M. , De Kretser D. M. , O'Bryan M. K.
Published: November 2010

SNTs (Suc-1 associated Neurotrophic Factor Targets) are FGF signalling adaptors and crucial activators of the MAP/PI3 kinase pathways via FGFRs. Screening a rat testis library identified snt-2 as a potential ODF component. ODFs are a major constituent of the sperm tail that we hypothesise play an active role in motility. Using western blot analysis I have localised Fgfr-1 to the sperm tail. As such, I propose that Fgf signalling through snt-2 is involved in sperm tail development/function. To test this hypothesis, I created transgenic mice carrying a dominant-negative variant of Fgfr-1 driven by the protamine 1 promoter (haploid specific). Breeding experiments confirmed males were fertile, although one line showed a tendency towards reduced pup numbers. This effect was strengthened by Daily Sperm Production (DSP), showing significantly reduced DSP (30%↓) compared to wt mice. Transgene expression levels were expressed up to 70 times above native mRNA levels in wt mice; however there was a concurrent up-regulation of the native receptor in transgenic mice. Cumulatively this resulted in only a 6x over-expression in transgene: native mRNA, and illustrated the presence of a feedback mechanism controlling Fgfr-1 expression. To increase transgene expression, I crossed independent lines (double heterozygous, DH) males. Breeding experiments showed males from 1 cross were significantly subfertile (2 v. 10 in wt mice) . DSPs were further reduced, (41%↓) compared to wt mice. Collectively this data shows Fgfr-1 signalling is required for quantitatively normal spermiogenesis, but is also likely to have a post testicular role in sperm function. I hypothesise this is mediated via activation/regulation of motility through the MAP/PI3 kinase pathways. Further, these mouse models provide compelling evidence that infertility in Kallmann's Syndrome patients is composed of both hypothalamic and testicular components. These mice will provide valuable insights into the signal transduction mechanisms controlling sperm function and avenues for contraceptive development.
259.Identification of elevated levels of apoptosis among T-cells isolated from the rat testis
Authors: Crane M. , Hutchinson P. , Scott B. , De Kretser D. , Hedger M.
Published: November 2010

Protection of the developing gametes from attack by the immune system is essential for reproductive success. Autoimmune infertility represents a failure of this protection. Specific T-cell apoptosis is the main mechanism for control of antigen-specific immune responses. Studies were undertaken to investigate this regulatory process in adult rat testes. Flow cytometry was employed in conjunction with annexin-V/propidium iodide dual staining to identify apoptotic cells concurrent with CD3 staining to identify T-cells. CD3-positive cells isolated from the testicular interstitial tissue were shown to be 34.12���3.0% apoptotic (mean���s.e.m., n�=�3) at collection. This was consistently greater than the numbers of apoptotic CD3-positive cells isolated from lymph nodes (4.04���1.95%, n�=�2), spleen (16.77���4.73%, n�=�4) and peripheral blood (9.64���1.44%, n�=�2). These results also were confirmed by using T-cells purified with MACS microbeads against the pan T-cell marker OX52 to improve sample purity: 40% of isolated testicular T-cells and 3% lymph node T-cells were found to be undergoing apoptosis. The level of apoptosis among T-cells isolated from another non-lymphoid organ, the liver, was only 6%. It is hypothesised that the immunosuppressive milieu of the testis induces an increased level of apoptotic deletion among T-cells that gain entry into the testis and potentially threaten gamete viability. Further studies of the mechanism responsible for this elevated level of T-cell apoptosis in the testis will significantly enhance our knowledge of how testicular immune tolerance is maintained.
260.Sperm trains and mating behaviour in wood mice of the genus Apodemus
Authors: Dvorakova K. , Stopka P.
Published: November 2010

There is evidence of possible correlation between mating behaviour, sperm morphology and sperm behaviour in the female reproductive tract prior to fertilization. As shown in A. sylvaticus (1), cooperation between spermatozoa of an individual confers a significant advantage for fertilization, where inter-male sperm competition is intense. In our study we aimed to focus on three other species of rodent genus Apodemus; A. flavicolis, microps, agrarius versus A. sylvaticus, whose either promiscuous or monogamous mating behaviour has already been demonstrated (Stopka et al., unpublished). We placed spermatozoa from cauda epididymis of adult males of selected species into mouse in vitro fertilisation medium, and compared sperm behaviour of individual species. Surprisingly, despite monogamous or polygamous mating behaviour, sperm of all the above species rapidly aggregated into motile trains, as in A. sylvaticus, consisting of hundreds of cells. However, size and cell–cell coupling differed between each species. This may point to another aspect besides mating behaviour that may be involved in building up a mechanism for successful sperm delivery to the egg. These unique and organised aggregations can significantly increase sperm progressive motility. A detailed study of the main cytoskeletal proteins, using immunofluorescent methods, together with confocal microscopy, shows that in sperm-sperm binding especially protein actin plays an important role. According to in vitro experiments, we speculate that the viscous environment of the female reproductive tract may have played a crucial role in the evolution of sperm behaviour. Disintegration of motile sperm trains was associated with the majority of spermatozoa undergoing a premature acrosome reaction. However, the mechanism that triggers it remains to be determined. To conclude, it is likely that a myriad of changes in social behaviour of particular species were not accompanied by changes in sperm behaviour, but instead they retained original ancestral tactics typical for the genus Apodemus.
(1) Moore, H., Dvorakova, K., Jenkins, N., Breed, W. (2002) Exceptional sperm cooperation in the wood mouse. Nature 418, 174–177.
261.Probiotic Lactobacillus in seminal plasma
Authors: Sivaramakrishnan G. , Jasper M. J. , O'Leary S. , Robertson S. A.
Published: November 2010

Commensal bacteria of the Lactobacillus genus are implicated in beneficial 'probiotic' roles in the gut and other mucosal tissues. Their presence reduces the incidence of pathogenic infections, both passively and via production of antimicrobial substances, and through Toll-like receptor-mediated activation of cytokine expression in host tissues. Lactobacilli are present in the female reproductive tract but have not been examined in the male. This study aimed to investigate, by selective culture techniques and real-time quantitative PCR, the prevalence in boar seminal plasma of Lactobacilli compared with other pathogenic bacteria. Using acidified Rogosa Agar, Lactobacilli were cultured from 3/3 fresh semen samples and were found to be most prevalent in the first fraction of the ejaculate. For PCR, DNA was extracted from reference bacterial cultures and archived seminal plasma samples from 40 healthy boars. Bacterial species-specific primers targeting Lactobacillus 16s and 16s-23s rDNA sequences, and Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus-specific Sau3AI, oprL, and 16s rDNA genes respectively, were used in real-time PCR assays employing SYBRgreen (Applied Biosystems) technology. Lactobacilli were detected in 22/40 (55%) of seminal plasma samples, while pathogenic bacteria were detected in <10% of samples (Staphylococcus aureus, 1/40; Pseudomonas aeruginosa, 2/40; and Bacillus, 3/40). The Lactobacillus content of individual boars ranged from 1.5 to 15 × 106 cells/mL, and within boars, content varied within 30% of the mean value in successive samples over a 6-month period. We conclude that Lactobacilli are present in abundance in boar seminal plasma compared to other potentially pathogenic bacteria. These bacteria may protect the male tract from pathogen infection, and after ejaculation, may influence the female immune response to male antigens. Ongoing studies will investigate whether Lactobacilli abundance in seminal plasma correlates with boar fertility, and examine the potential value of improving reproductive performance in pigs and other species by administration of probiotic agents.
263.Effects of animal pairing on marmoset sperm collected by penile vibratory stimulation
Authors: Grupen C. G. , Scott S. J. , Gilchrist R. B.
Published: November 2010

The marmoset monkey (Callithrix jacchus) is an important model species for the development of reproductive technologies in humans and endangered primates. Obtaining sufficient sperm of high quality is a limiting factor in implementing marmoset IVF. A method for the collection of marmoset semen, using penile vibratory stimulation (PVS), has recently been described (1). Due to the high rate of copulation in marmosets, the pairing of males with females may affect semen collection. The aim of the present study was to determine whether the quantity and motility of sperm collected by PVS is affected by animal pairing. A total of 10 adult male marmosets were used, of which 3 were paired with another adult or juvenile male, and 7 were paired with an adult female. Semen was collected from each male on up to 5 separate occassions in sterile glass tubes. PVS involved the application of successive sequences of increasing vibration to the penis using a FertiCare personal vibrator. Immediately following collection, pre-warmed Hepes-buffered TALP medium (200 μl) was added to the ejaculate. Sperm suspensions were evaluated for total sperm count and sperm motility. Ejaculates were obtained from male-paired males on every attempt (12 of 12), whereas 5 of 23 attempts failed to yield an ejaculate from female-paired males. The number of sequences of stimulation needed to obtain an ejaculate differed between males but was unaffected by animal pairing. The ejaculates collected from female-paired males had lower total numbers of sperm (3.9���1.4�×�106 v. 10.1���2.2�×�106; P�<�0.05) with a lower percentage motile (35���9% v. 85 � 13%; P<0.01), compared with those from male-paired males. We conclude that housing males separately from females increases the quantity and motility of sperm collected by PVS.
(1) Schneiders, A., Sonksen, J., Hodges, J. K. (2004) J. Med. Primatol. 33, 98–104.
264.The effects of progesterone on endometrial angiogenesis in pregnant and ovariectomised mice
Authors: Walter L. M. , Rogers P. A. W. , Girling J. E.
Published: November 2010

In mice, early pregnancy is associated with an increase in endometrial angiogenesis in preparation for the implanting embryo. The aims of this study were to quantify endometrial angiogenesis in pregnant mice and to investigate the role of progesterone in promoting endothelial cell (EC) proliferation in ovariectomised mice; we hypothesised that EC proliferation would increase with increasing plasma progesterone concentrations in pregnant mice and that progesterone would stimulate EC proliferation in ovariectomised mice, but only following oestrogen priming. Uterine tissue from CBA x C57 mice was collected on Days 1–4 of pregnancy (n�=�4–5/day) when circulating progesterone concentrations are increasing but before implantation occurs. Prior to dissection, mice were injected with BrdU enabling proliferating EC to be quantified and localised within blood vessels by CD31/BrdU double staining immunohistochemistry. There was a significant increase in proliferating EC (Kruskal-Wallis statistic (KW) = 17.1, P = 0.002) on Day 3 of pregnancy (Days 1 and 2, no proliferation; Day 3,126.6 � 45.6 proliferating EC/mm2 (mean � s.e.)), when plasma progesterone also began to increase (as measured by radioimmunoassay). To determine if the EC proliferation was due to progesterone, a second experiment was performed on ovariectomised mice. One group of mice (n = 6) were treated with a single injection of 100 ng of estradiol on day eight after ovariectomy, followed by a day with no treatment and three consecutive daily injections of 1 mg progesterone. Other groups were treated with either the vehicle (n = 5), estradiol (n = 4) or progesterone (n = 5) injections only. All groups were dissected following BrdU injection on Day 13 following ovariectomy. Unexpectedly, mice treated with progesterone only had the highest amount of EC proliferation (114.7 � 30.9 proliferating EC/mm2); oestrogen priming was not required and actually significantly reduced progesterone induced EC proliferation (44.8 � 15.5 proliferating EC/mm2; KW = 13.8, P = 0.008). We are currently investigating the interaction between progesterone and VEGF using immunohistochemistry and inhibition studies.
266.Human endometrial cycle stages can be determined by global gene expression profiling
Authors: Ponnampalam A. P. , Weston G. C. , Trajstman A. C. , Susil B. , Rogers P. A. W.
Published: November 2010

Endometrium is a dynamic tissue which undergoes cyclic changes each month, under the overall control of oestrogen and progesterone. The aims of this study were to investigate the changing global gene expression profile of human endometrium during the menstrual cycle using microarray technology and to determine the correlation between histopathological evaluation and molecular profile of the samples. Curettings of endometrium were collected from 43 cycling women and immediately snap frozen. The menstrual cycle was divided into seven stages by histological evaluation. Standard two-color cDNA microarrays were performed on the 43 samples against a common reference, using a 10.5 K cDNA glass slide microarray. Expressed genes were identified using a Scanarray 5000 UV laser scanner. Quantarray software was used to quantify the relative gene expression values. Normalisation and visualisation of the gene expression changes were performed using the GeneSpring software package. Hierarchical clustering of all 43 samples was performed, based on the expression profile of 571 genes, which were identified as differentially expressed by parametric ANOVA with Benjamini-Hochberg correction. The 43 samples were sorted into nine groups which all agreed with histopathology by either being in the same group or an adjacent group apart from four samples. For further analysis, the four outliers were removed, one group was excluded due to lack of replicates and two groups were merged to get the final molecular classification of the cycle. The statistical analysis was repeated and 1452 genes were identified as differentially expressed at P d 0.05. The data were also independently analysed by a CSIRO algorithm called GeneRave and the results from both methods were comparable. mRNA expression profiles of the genes TGF a (Hs.170009), NCR3 (Hs.509513) and FUT4 (Hs.390420) were verified using real-time PCR. We have shown for the first time that endometrial cycle stage prediction is possible based on global gene expression profile.
269.Estrogen and progesterone receptor expression is significantly reduced in cultured myometrial and fibroid smooth muscle cells
Authors: Zaitseva M. , Rogers P. A. W.
Published: November 2010

Fibroids are benign neoplasms of the smooth muscle cells of the uterus. Cultured myometrial and fibroid smooth muscle cells (MSMC & FSMC) have been widely used as a model for the study of fibroid growth. However, there is ongoing controversy regarding expression levels of estrogen and progesterone receptors (ER and PR) in cultured cells vs tissue. The aim of the present study was to measure levels of mRNA for ER� and PR in myometrium and fibroid, and in cultured MSMC & FSMC. Myometrium and fibroids were collected from hysterectomy specimens (n = 8). Part of the tissue was snap frozen and the rest was used to isolate SMC, which were cultured for 3 passages and collected for RNA at P0 (2 weeks in culture) and P3 (5 weeks in culture). ERα and PR levels were quantified using real-time PCR and normalized using 18S rRNA as an internal control. Both ERα and PR were detected in all samples. Large variability in receptor levels between different isolates was detected. Surprisingly, despite large differences between the means, none of comparisons of tissue v. P0 cells were significant by non-parametric tests. However, there was a statistically significant reduction in both ERα and PR expression between whole tissue and isolated cells at P3 (Table 1, see PDF file).
This is the first study to provide objective data to support a significant decline in ERα and PR expression in cultured MSMC and FSMC. Despite this decline, detectable levels of ERα and PR mRNA were present at both P0 and P3, potentially explaining why some published studies have been able to demonstrate in vitro response to steroids in these cells.
270.Macrophage inhibitory cytokine-1 at the maternal - fetal interface in early pregnancy
Authors: Marjono B. , Manuelpillai U. , Dimitriadis E. , Salamonsen L. , Breit S. , Wallace E.
Published: November 2010

Macrophage inhibitory cytokine-1 (MIC-1) is a transforming growth factor-β (TGF-β) superfamily member, first isolated from activated macrophages and subsequently localised in the human placenta. We previously reported that decreased circulating levels in very early pregnancy are associated with subsequent miscarriage. We undertook these current in vitro studies to investigate possible roles for MIC-1 in early pregnancy: (1) regulation of placental matrix metalloproteinase-2 and -9 (MMP-2 and -9); (2) effect on placental apoptosis; and (3) regulation of endometrial stromal cell decidualisation. (1) First trimester placental explant cultures were treated with 100–200 ng/mL MIC-1 � 1/1000 (v/v) anti-MIC-1 antibody. MMP-2 and -9 were measured by gelatin zymography. MMP activation via the plasminogen activation pathway was examined by measuring mRNA expression for urokinase plasminogen activator and its receptor (uPA, uPAR) and type-1 plasminogen activation inhibitor (PAI-1). (2) In first trimester trophoblast explants, apoptosis was induced in vitro with tumor necrosis factor-α (TNF-α) and interferon-β (IFN-β) � 200 ng/mL MIC-1. The pro-apoptosis factor caspase-3 was localised by immunohistochemistry. (3) Using an established model of oestrogen and progesterone induced endometrial stromal cell decidualisation, MIC-1 production was measured and correlated with morphological changes. Cultures were also treated with 20 ng/mL MIC-1. MIC-1 treatment inhibited activation of both MMP-2 and MMP-9 while treatment with anti-MIC-1 antibody blocked the inhibition. uPA, uPAR and PAI-1 mRNA did not change with either treatment. MIC-1 treatment mitigated TNF-α/IFN-β induced trophoblast apoptosis. MIC-1 production increased during induced decidualisation and MIC-1 treatment facilitates further decidualisation in this model. MIC-1 appears to have a number of potentially important functions in the human placenta and decidua consistent with physiological roles in normal placentation. Whether these functions are key to successful pregnancy remains to be studied.
271.Role of homeobox gene HLX expression in normal placental development
Authors: Murthi P. , Bates M. D. , Sankaran G. , Brennecke S. P. , Kalionis B.
Published: November 2010

In a screen for homeobox genes in the human placenta, we cloned and characterised HLX1 (also known as HB24) (1). Furthermore, we provided evidence that HLX1 may be a regulator of human placental develolpment (2). We have since shown that the mouse homologue of HLX1, called Hlx, is expressed in the murine placenta. In situ mRNA hybridisation studies and antibody localisation of Hlx revealed expression in the labyrinth layer (LL), secondary giant cells (GC) and in the spongiotrophoblast layer (STL) (3). The STL is required for structural support of the placenta. Targeted gene mutation of Hlx resulted in embryonic defects in the developing gut and the liver (4) but the effects on placental development were not investigated. Histological preparations of placental tissues collected from Days 10.5, 13.5 and 19.5 from Hlx mutant mice were investigated for morphological changes. Our preliminary observations reveal that by haematoxylin and eosin staining the STL of the mutant murine placenta is severely disrupted but the overlying GC layer appears to be unaffected. Endogenous alkaline phosphatase staining of the LL further confirmed that the highly vascularised LL where fetal- maternal exchange occurs, is disorganised and expands into the region normally occupied by the STL. These observations indicate that Hlx is essential for normal placental development.
(1) Quinn LM, Kalionis B. (1997) Gene 187, 55–61. (2) Quinn LM, Kalionis B. (1997) Repro. Dev. 9, 617–623. (3) Johnson B (1999) PhD Thesis, University of Adelaide. (4) Hentsch B, Harvey RP (1996) Gene Dev. 10, 70–79.