Biol Reprod Research Articles

Effect of dietary carbohydrate challenge on activation of 5’-adenosine monophosphate activated protein kinase (AMPK) in liver, skeletal muscle, and digital laminae of lean and obese ponies

Effect of dietary carbohydrate challenge on activation of 5’-adenosine monophosphate activated protein kinase (AMPK) in liver, skeletal muscle, and digital laminae of lean and obese ponies

Adipokine, chemokine, and cytokine expression profiles in adipose tissue depots of lean and overweight ponies

Adipokine, chemokine, and cytokine expression profiles in adipose tissue depots of lean and overweight ponies

The interplay between RPGR, PDEδ and Arl2/3 regulate the ciliary targeting of farnesylated cargo

Defects in primary cilia result in human diseases known as ciliopathies. The retinitis pigmentosa GTPase regulator (RPGR), mutated in the most severe form of the eye disease, is located at the transition zone of the ciliary organelle. The RPGR-interacting partner PDEδ is involved in trafficking of farnesylated ciliary cargo, but the significance of this interaction is unknown. The crystal structure of the propeller domain of RPGR shows the location of patient mutations and how they perturb the structure. The RPGR·PDEδ complex structure shows PDEδ on a highly conserved surface patch of RPGR. Biochemical experiments and structural considerations show that RPGR can bind with high affinity to cargo-loaded PDEδ and exposes the Arl2/Arl3-binding site on PDEδ. On the basis of these results, we propose a model where RPGR is acting as a scaffold protein recruiting cargo-loaded PDEδ and Arl3 to release lipidated cargo into cilia.

Combined Inhibitory Effects of Pyruvate and Low Temperature on Postovulatory Aging of Mouse Oocytes1

This study tested the hypothesis that oocyte aging could be prevented for a longer time by reducing the culture temperature while supplementing the culture medium with more pyruvate. Newly ovulated mouse oocytes were cultured at various temperatures for various times in HCZB medium (Kimura and Yanagimachi, Biol Reprod 1995; 52:709-720) containing various concentrations of pyruvate before examining for aging parameters and developmental potential. The increase in susceptibility to activating stimuli was efficiently prevented when oocytes were cultured in HCZB with 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 96 h, and 5°C for 48 h. Satisfactory blastocyst development of both parthenotes and fertilized zygotes was achieved after oocyte culture in HCZB containing 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 36 h, and 5°C for 24 h. Transfer of two-cell embryos or blastocysts showed no difference between newly ovulated control oocytes and oocytes cultured at 15°C for 36 h in either term pregnancy, live young per pregnant recipient, live young/transferred embryos, or birth weight of young. Oocytes with impaired developmental potential after culture at 15°C for 96 h and at 5°C for 48 h showed unrecoverable decreases in the content of glutathione, the glutathione/oxidized glutathione ratio, the BCL2 content, and in the numbers of oocytes with normal spindles and cortical granule distribution, suggesting induction of oxidative stress, which caused oocyte apoptosis and cytoskeleton alterations by downregulating BCL2. Because oocytes cultured at 15°C for 36 h were activated or fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 42 h without impairing their developmental potential.

Unexplained Recurrent Upper and Lower Respiratory Tract Infections in Otherwise Young Healthy Male

SESSION TYPE: Airway Global Case Report Posters

Localization and Regulation of Murine Esco2 During Male and Female Meiosis1

Meiosis is essential for generation of healthy gametes in both sexes and involves recombination and segregation of homologous chromosomes to produce haploid gametes. The initiation of meiosis in both sexes relies upon retinoic acid (RA) (Griswold MD, Hogarth CA, Bowles J, Koopman P. Initiating Meiosis: The Case for Retinoic Acid. Biol Reprod 2012; 86(35):1-7). Previous studies have demonstrated that the stimulated by retinoic acid gene 8 (Stra8) was required for meiotic progression in both the mouse ovary and postnatal testis. To identify additional candidates that may play a role during meiosis, we used microarray databases to generate lists of transcripts with expression profiles similar to that of Stra8 in the embryonic ovary and postnatal testis. One such gene, establishment of cohesion 1 homolog 2 (Saccharomyces cerevisiae) (Esco2), has been described as a regulator of sister chromatid cohesion during mitosis. This study describes the first in-depth analysis of ESCO2 localization and regulation during meiosis in both males and females. ESCO2 colocalized with the gamma H2A histone family member X (H2AFX) in pachytene spermatocytes, indicating that ESCO2 is a component of the XY body. In pachytene cells of the embryonic ovary, ESCO2 colocalized with H2AFX, which is consistent with the presence of ESCO2 in areas of double-stranded breaks. In addition, the expression of Esco2 was found to be regulated by RA in the postnatal testis. These data indicate that ESCO2 may play a vital role in meiosis in both males and females.

Clear cell carcinoma arising in the abdominal wall: two case reports and literature review

Clear cell carcinoma originating in the abdominal wall is rare and usually develops within endometriotic implants in the scar. We describe 2 patients: a 42 year old with a 15 cm mass on the abdominal wall treated with neoadjuvant chemotherapy and excision and a 51 year old with a 6 cm abdominal mass treated with excision and adjuvant radiotherapy.

Statement of Retraction: Salama N, Kishimoto T, Kanayama HO, Kagawa S. “The mobile phone decreases fructose but not citrate in rabbit semen: a longitudinal study”

The editor and publisher would like to inform the readers the following article has been retracted from publication in Systems Biology in Reproductive Medicine:Salama N, Kishimoto T, Kanayama HO, Kagawa S. The mobile phone decreases fructose but not citrate in rabbit semen: a longitudinal study. Syst Biol Reprod Med. 2009 Dec;55(5-6):181-7. PubMed PMID: 19938952.This article has been found to reproduce content to a high degree of similarity (in the form of a duplicate figure and data within tables), without appropriate attribution or acknowledgment by the authors, from the following original articles:Salama N, Kishimoto T, Kanayama HO, Kagawa S. Effects of exposure to a mobile phone on sexual behavior in adult male rabbit: an observational study. Int J Impot Res. 2010 Mar-Apr;22(2):127-33. Epub 2009 Nov 26. PubMed PMID: 19940851.Salama N, Kishimoto T, Kanayama HO. Effects of exposure to a mobile phone on testicular function and structure in adult rabbit. Int J Androl. 2010 Feb;33(1):88-94. Epub 2009 Dec 2. PubMed PMID: 19076254.Independent statistical analysis confirmed that the data published in all three articles was essentially from the same study.The journal contacted the authors to investigate and have concluded the fidelity of the data that was captured during the course of the experiments, and used for analysis, cannot be assured. The journal also received a request by the co-authors (Kishimoto, Kanayama, and Kagawa) from the host institution to have their names be removed, this further lessens confidence in the data as reported.The journal's policy in this respect is clear: Systems Biology in Reproductive Medicine considers all manuscripts on the strict condition that they have been submitted only to Systems Biology in Reproductive Medicine, that they have not been published already, nor are they under consideration for publication or in press elsewhere.Systems Biology in Reproductive Medicine published this article in good faith, and on the basis of signed statements of the corresponding author regarding the originality and ethical reliability of their work. The article is withdrawn from all print and electronic editions.Stephen A. Krawetz (Editor-in-chief)Anna Heinink (Head of Journals Publishing, Informa Healthcare)

199 PREMATURATION OF BOVINE OOCYTES WITH BUTYROLACTONE I AND/OR CILOSTAMIDE: EFFECTS ON MEIOSIS PROGRESSION, CYTOPLASMIC MATURATION AND GENE EXPRESSION

Meiotic block during a prematuration culture (pre-IVM) before in vitro maturation (IVM) is suggested as a way to provide additional time to synchronize oocyte-somatic cell communication, leading to improved cytoplasmic maturation and nuclear meiotic competence and the acquisition of critical cellular functions necessary for developmental competence. This study was designed to evaluate the effects of the inhibitors butyrolactone I (Bl-I) and cilostamide on oocyte nuclear and cytoplasmic maturation. For that, 2 well-established methods of pre-IVM and IVM for cilostamide (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011) or Bl-I (Hashimoto et al. 2002 Biol Reprod. 66, 1696–1701) were used. Abattoir-collected oocytes were IVM in maturation medium (MM: mSOF with 0.8% BSA and hormones) for 24 h, without a previous pre-IVM culture (control). Cilostamide-group oocytes were treated for the first 2 h in vitro (pre-IVM) with 100 μM of the adenylate cyclase activator forskolin and 500 μM IBMX (nonspecific phosphodiesterase inhibitor) and then oocytes underwent extended IVM for 30 h in the presence of 20 μM cilostamide (type 3 phosphodiesterase inhibitor; pre-IVM 2 h + IVM 30 h). The Bl-I-group oocytes were pre-IVM for 24 h with 100 μM Bl-I diluted in TCM-199 supplemented with 0.2 mM pyruvate and then were IVM in MM for 20 h (pre-IVM 24 h + IVM 20 h). The Bl-I + Cilost group was a combination of both procedures: oocytes were first pre-IVM with Bl-I for 24 h and then cultured as described for the cilostamide group (pre-IVM 24 h + IVM 30 h). Cultures were carried out at 38.5°C in 5% CO2 in humidified air. After IVM, oocytes were stained with 500 nM mitotracker red to assess the mitochondrial membrane potential (Δψm) and with 10 μg mL–1 of Hoescht 33342 to evaluate the nuclear maturation (n = 207). Images were captured by an Olympus confocal microscope and analyzed with Fluoview software. The TUNEL assay was used to detect oocyte DNA fragmentation (n = 74). Relative amounts of mRNA for apoptotic-related genes were quantified after IVM in individual oocytes using real-time PCR (Kameyama et al. 2007 Reproduction 133, 423–32). Means were compared by ANOVA and Tukey's test or by chi-square (P ≤ 0.05). The percentage of oocytes reaching metaphase II after IVM did not differ among groups (73.8 to 90.4%; P ≥ 0.05), indicating that in vitro meiotic resumption was normal. The Δψm, expressed in arbitrary units of fluorescence, was 1.0 ± 0.1a (control), 2.7 ± 0.4b (Bl-I), 3.2 ± 0.5b (Cilost) and 2.1 ± 0.3ab (Bl-I + Cilost). The percentage of TUNEL-positive oocytes (18.8–41.3%) did not differ among groups (P ≥ 0.05). The relative abundance of BAX (1.0 ± 0.4 to 2.3 ± 0.4) and BCL-XL (1.0 ± 0.3 to 0.3 ± 0.1) transcripts was unaffected by pre-IVM and IVM (P ≥ 0.05). In conclusion, except for an increase in mitochondrial activity, pre-IVM with cilostamide and/or Bl-I did not affect cytoplasmic and nuclear oocyte maturation. However, oocyte developmental potential needs to be better evaluated in a future study through assessment of embryonic development. We acknowledge FAPESP and NSERC.

Clinical characteristics and outcomes of critically ill obstetric patients: a ten-year review

BACKGROUND AND OBJECTIVES:Pregnancy and delivery can involve complications that necessitate admission to critical care facilities. The objective of our study was to assess the incidence, indications, and outcomes of obstetric patients requiring admission to an intensive care unit (ICU) in a tertiary care hospital, in Saudi Arabia.DESIGN AND SETTING:Retrospective cohort study of consecutive obstetric admissions to the ICU at the King Abdulaziz Medical City over a 10-year period.PATIENTS AND METHODS:We collected baseline demographic data and acute physiology and chronic health evaluation II (APACHE II) scores. ICU mortality was the primary outcome.RESULTS:Over 10 years, 75 obstetric patients were admitted to the ICU, and 59 of these patients (78.6%) were admitted during the antepartum period. The main obstetric indication for ICU admission was pregnancy-induced hypertension (21 patients, 28%) and the leading non-obstetric indication was sepsis (12 patients, 16%). The APACHE II score was 19.59 (15.05). The predicted mortality rate based on the APACHE II score was 21.97%; however, there were only six maternal deaths (8%) among the obstetric patients admitted to the ICU.CONCLUSION:The overall mortality was low. A team approach facilitated the application of optimal care to these patients. Obstetric patients had better outcomes than those predicted by the APACHE II scores. Appropriate antenatal care is important for preventing obstetric complications.

Redundant and Differential Roles of Transcription Factors Gli1 and Gli2 in the Development of Mouse Fetal Leydig Cells1

Appearance of mouse fetal Leydig cells requires activation of the Hedgehog pathway. Upon binding to the membrane-bound receptor patched, Hedgehog ligands induce intracellular responses via a combined effect of Gli transcription factors. Szczepny et al. (Biol Reprod 2009; 80:258-263) found that Gli1, one of the three Gli transcription factors, is present in the fetal testis and that its expression is suppressed by the Hedgehog inhibitor cyclopamine. In this study, we investigated the involvement of the Gli1 and Gli2 factors in mouse fetal Leydig cell differentiation. The Gli1 and Gli2 transcription factors showed an overlapping expression pattern in the testis interstitium at the time when fetal Leydig cells appear. Despite their similar expression, Gli1 and Gli2 patterns were differentially regulated. Initial Gli1 and Gli2 expression depends upon an active Hedgehog pathway; however, maintenance of only Gli1, but not Gli2, expression requires activation of the pathway. Inactivation of either the Gli1 or Gli2 gene did not affect fetal Leydig cell development and testis morphology, suggesting a functional redundancy. When the transcriptional activity of both GLI1 and GLI2 was suppressed by a selective inhibitor, GANT61, in cultured fetal testes before the appearance of fetal Leydig cells, Gli1 and Gli2 expression and steroidogenic marker activity were completely abolished. However at later stages when Leydig cells were already present, GANT61 treatment inhibited Gli1 expression but had no effects on Gli2 expression and fetal Leydig cell appearance. Our results reveal overlapping and redundant Gli1 and Gli2 roles in fetal Leydig cell differentiation and a novel regulation of Gli2 expression in the fetal testis.

Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Localization of a Novel RNA-binding Protein, SKIV2L2, to the Nucleus in the Round Spermatids of Mice

In our previous study (Kawashima et al., Biol Reprod 2009; 80: 1293-1304), we suggested that the first cycle of spermatogenesis recovered from busulfan-induced temporary arrest was a good model system to analyze the proteins expressed at the specific stages of spermatogenesis in the mouse, and this has been confirmed in the present paper. Namely, six-week-old mice were injected with busulfan at 20 mg/kg body weight. The germ cells except for the undifferentiated spermatogonia disappeared by 32 days after injection. The surviving spermatogonia started to proliferate, and spermatogenesis was entirely recovered about 77 days after injection. By proteome analysis of the busulfan-treated testis during the process of recovery of spermatogenesis, we identified a protein that was expected to be expressed in the spermatogenic cells as Superkiller viralicidic activity-2-like-2 (SKIV2L2). Skiv2l2 mRNA was found in the kidney, epididymis and heart as well as the testis. In the testis, Skiv2l2 mRNA was shown to be highly expressed in the spermatocytes at stages I to VI. On the other hand, SKIV2L2 protein was found to be predominantly localized in the nuclei of round spermatids. In accordance with the fact that SKIV2L2 belongs to the Ski2 family within the superfamily 2 of RNA helicases, it has been shown that SKIV2L2 has both the RNA-binding and ATPase activities.

Interleukin-10 Promoter Microsatellite Polymorphisms Influence the Immune Response to Heparin and the risk of Heparin-Induced Thrombocytopenia.

AbstractAbstract 3682 Introduction.Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of platelet-activating IgG antibodies (Abs). However, the reasons explaining why only a subset of patients treated with heparin develop IgG to PF4/H and why most of immunized patients do not develop HIT have yet to be fully defined. The immune response in HIT involves both B and T cells, and we hypothesized that polymorphisms located in the promoter of the IL10 gene that encodes IL10, a major regulatory cytokine, have an influence on the risk of HIT. Aim of the study.To investigate whether IL10 promoter single nucleotide polymorphisms (SNPs) and variations in IL10R and IL10G microsatellites are associated with the synthesis of Abs to PF4 and HIT. Patients and Methods.A cohort of 82 patients with definite HIT (both PF4-specific ELISA and serotonin release assay were positive) and two control groups were studied. The first control group (Abneg) consisted of 85 patients who had undergone cardiopulmonary bypass (CBP) with high doses of heparin and who did not develop Abs to PF4 post-operatively. The second control group (Abpos) consisted of 84 patients who had also undergone cardiac surgery with CPB and had developed significant levels of PF4-specific antibodies but not HIT.The SNPs rs1800896 (-1082A>G), rs1800871 (-819C>T) and rs180072 (592A>C), and the polymorphic CA repeat microsatellites IL10 R [5325CA(11_15)] and IL10 G [8134CA(14_29)] (all previously identified in IL10 promoter) were screened. Genotyping of SNPs was achieved by allelic discrimination using a real-time PCR method (ICycler Bio-Rad). Twenty nanograms of purified gDNA were used for each experiment with TaqMan® Universal PCR Master Mix, using the two primers and two fluorescent probes of the TaqMan® SNP Genotyping Assays (Applied Biosystems). Genotyping of IL10 microsatellites was determined as previously described (Cochery-Nouvellon et al. Biol Reprod 80, 1115–20. 2009). Linkage disequilibrium analysis was assessed using HAPLOVIEW 4.1, and haplotype reconstruction was carried-out with PHASE 2.1. Statistical analyses were performed with StatView 5.0 and Abacus: logistic regression was used to calculate genetic associations, and ANOVA to determine genotype-phenotype correlations. The Chi-square test was used to compare genotype distributions between the different groups. Results.The genotypes and allele frequencies of the 3 SNPs studied were similar in HIT patients and controls, with very high linkage disequilibrium between the -592 A>C and -819 C>T polymorphisms. Fourteen different alleles in the IL10G microsatellite (A16 to A29) and 3 alleles in IL10R (A13 to A15) could be defined depending on the number of CA repeat elements identified in the promoter sequence. The short A20 allele of IL10G was more frequent in Abneg patients (8.2%) than in Abpos (2.3%) and HIT patients (3%), thereby appearing highly protective from the risk of developing Abs to PF4 (OR 0.29; 95% CI [0.12-0.70], p < 0.006). Moreover, the short IL10R A13 allele was also less frequent in HIT patients (70.1%) than in controls (75.9%)(OR 0.37; 95% CI [0.15-0.89], p=0.026). Combinations of the 3 SNPs with IL10G and IL10R variations (short or long alleles) defined nine different representative haplotypes (H1 to H9). Interestingly, the combined H1/H8 haplotype comprising the short alleles for both IL10G and IL10R (A13) was less represented in HIT patients (4.9% vs. 13.6% in controls)(OR 0.33; 95% CI [0.11-0.97], p=0.036). In addition, levels of Abs to PF4 measured by ELISA in Abpos patients were also lower in H1/H8 subjects (median OD 0.67 vs. 1.21 in non-H1/H8 individuals, p=0.019). Discussion-Conclusion.IL10 promoter microsatellite polymorphisms appear to have a significant influence on the immune response to heparin. The haplotypes combining short IL10G and IL10R alleles are associated with lower levels of antibodies to PF4 and a reduced risk of HIT. Disclosures:No relevant conflicts of interest to declare.

Predictive factors of failure in management of ectopic pregnancy with single-dose methotrexate: a general population-based analysis from the Auvergne Register, France

In a general population setting, the existence of a learning curve for treatment of ectopic pregnancy with single-dose methotrexate has been demonstrated, because a significant decrease in failure rate can be achieved over time. Despite this finding, the risk of single-dose methotrexate failure increases significantly in patients with initial hCG levels>1,300 IU/L and/or in women who report having ever used combined oral contraception before pregnancy.

Use of novel intravascular contrast reagents to define vascular parameters in rhesus macaque ovaries during controlled ovarian stimulation

OBJECTIVE: To evaluate the ability of ABLAVAR (Lantheus Medical Imaging), Feraheme (AMAG Pharm), and Prohance (Bracco Diagnostics) to define vascular parameters of ovaries after controlled ovarian stimulation (COS), with possible application to the study of Ovarian Hyperstimulation Syndrome (OHSS).DESIGN: Prohance, a low molecular weight contrast reagent (CR) is used to measure vascular permeability in women and rhesus monkeys, but has limited capabilities to measure blood volume. ABLAVAR which binds to albumin is used to measure the blood pool by MRI, but the vasculature loses protein during OHSS. Feraheme, a nano-sized iron particle, should be a true intravascular CR. Contrast-enhanced MRI and dynamic CE-MRI (DCE-MRI) were performed on macaques during the follicular phase and COS before and after an ovulatory bolus of hCG [Biol Reprod 81(1 Sup):613, 2009]. The ability of ABLAVAR and Feraheme to measure the blood pool was investigated.MATERIALS AND METHODS: Monkeys (n=4) were monitored for menses, and a subset (n=3) subjected to COS as reported [Biol Reprod 71:486-93, 2004]. Oocytes were aspirated 36 h post-hCG. Abdominal images were obtained using the Trio 3T system (Siemens) on the day of hCG (PreA), 3-4 days Post-hCG (PostA), and during the follicular phase (FP) as reported [Proc Int Soc Magn Reson Med 18:6052, 2010] with Prohance and/or Feraheme or ABLAVAR.RESULTS: Prohance identified extravascular areas associated with antral and non-luteinized follicles and corpora lutea both PreA and PostA. ABLAVAR showed minimal leakage of this CR during the FP and PreA. Systemic leakage of ABLAVAR was observed PostA and associated with abdominal fluid. Feraheme signal was limited to the blood pool both PreA and PostA.CONCLUSION: Prohance and ABLAVAR offer complementary data on vascular parameters of COS ovaries, with ABLAVAR indicating possible protein leakage from the vasculature during OHSS symptoms PostA. Feraheme appears to be a true intravascular agent in MRI scanning during COS. OBJECTIVE: To evaluate the ability of ABLAVAR (Lantheus Medical Imaging), Feraheme (AMAG Pharm), and Prohance (Bracco Diagnostics) to define vascular parameters of ovaries after controlled ovarian stimulation (COS), with possible application to the study of Ovarian Hyperstimulation Syndrome (OHSS). DESIGN: Prohance, a low molecular weight contrast reagent (CR) is used to measure vascular permeability in women and rhesus monkeys, but has limited capabilities to measure blood volume. ABLAVAR which binds to albumin is used to measure the blood pool by MRI, but the vasculature loses protein during OHSS. Feraheme, a nano-sized iron particle, should be a true intravascular CR. Contrast-enhanced MRI and dynamic CE-MRI (DCE-MRI) were performed on macaques during the follicular phase and COS before and after an ovulatory bolus of hCG [Biol Reprod 81(1 Sup):613, 2009]. The ability of ABLAVAR and Feraheme to measure the blood pool was investigated. MATERIALS AND METHODS: Monkeys (n=4) were monitored for menses, and a subset (n=3) subjected to COS as reported [Biol Reprod 71:486-93, 2004]. Oocytes were aspirated 36 h post-hCG. Abdominal images were obtained using the Trio 3T system (Siemens) on the day of hCG (PreA), 3-4 days Post-hCG (PostA), and during the follicular phase (FP) as reported [Proc Int Soc Magn Reson Med 18:6052, 2010] with Prohance and/or Feraheme or ABLAVAR. RESULTS: Prohance identified extravascular areas associated with antral and non-luteinized follicles and corpora lutea both PreA and PostA. ABLAVAR showed minimal leakage of this CR during the FP and PreA. Systemic leakage of ABLAVAR was observed PostA and associated with abdominal fluid. Feraheme signal was limited to the blood pool both PreA and PostA. CONCLUSION: Prohance and ABLAVAR offer complementary data on vascular parameters of COS ovaries, with ABLAVAR indicating possible protein leakage from the vasculature during OHSS symptoms PostA. Feraheme appears to be a true intravascular agent in MRI scanning during COS.

Abstract 4596: Restoring hypoxia-inducible factor-1α in MCF-7 breast cancer cells in vitro by preventing the excessive degradation induced by high oxygen unmasks strong estradiol induction of vascular endothelial growth factor expression

Abstract Vascular endothelial growth factor (VEGF) is the most potent inducer of increased microvascular permeability known and the key mediator of angiogenesis, pivotal processes in development, reproduction, and tumor growth. VEGF expression is rapidly and strongly induced in normal uterine endometrial epithelial cells in vivo by 17β-estradiol (E2; Kazi et al. 2009 Biol Reprod 81:378). Recent studies indicate that E2-induced epithelial cell proliferation in the mammary gland is also dependent on VEGF. VEGF is also strongly up-regulated by E2 in rat mammary tumors in vivo (Nakamura et al. 1996 Endocrinology 137:5589). By contrast, numerous studies have found that E2 has little or no effect on VEGF expression by breast cancer cells in vitro. We have recently shown that the E2 induction of VEGF is mediated by both estrogen receptor α (ERα) and hypoxia-inducible factor 1α (HIF-1α; Kazi et al. 2005 Mol Endocrinol 19:2006; Molitoris et al. 2009 Endocrinology 150:5405). Cultured cells, however, usually lack HIF-1α due to excessive degradation caused by the abnormally high oxygen level (20%) to which they are exposed. To test the hypothesis that restoring HIF-1α would restore the ability of E2 to strongly induce VEGF expression in breast cancer cells, we treated MCF-7 cells with cobalt chloride (CoCl2; 12.5-100 µM), which prevents oxygen-induced HIF-1α degradation. HIF-1α was very low to undetectable in untreated MCF-7 cells grown in 20% O2, but increased in a dose-dependent manner in response to CoCl2. Although high doses of CoCl2 alone significantly stimulated VEGF mRNA expression, 12.5 µM did not do so. A modest increase in VEGF expression was also seen when MCF-7 cells were treated with E2 alone (∼ 2.5-fold). When cells were co-treated with E2 and 12.5 µM CoCl2, however, a much larger increase in VEGF expression (∼ 6-fold), similar to that induced by E2 in vivo, was seen. This finding is consistent with the hypothesis that HIF-1α is required for maximal E2 induction of VEGF expression in carcinoma cells. Interestingly, when cells were grown for 4 days before treatment, high levels of HIF-1α protein were present in untreated cells. The tendency for MCF-7 cells to express HIF-1α as they become crowded in vitro, even in the presence of high oxygen levels, may explain the ability of E2 alone to induce some VEGF expression in vitro as observed here and in a few previous studies. In conclusion, these results confirm that HIF-1 plays an essential role in E2-induced VEGF expression. The rapid induction of VEGF expression by E2 and the increased microvascular permeability that results may play a critical role in tumor growth by exposing cancer cells to circulating IGF-1, which synergizes with E2 to induce mitosis [Supported by NICHD SCCPIR U54 HD36207 and R01 HD047275]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4596.

159. EXTRACELLULAR MATRIX DYNAMICS IN SCAR-FREE ENDOMETRIAL REPAIR

Tissue destruction and repair occur simultaneously within the menstruating uterus. Whilst the factors that control menstruation are increasingly understood (1) the endometrial milieu which governs repair remains elusive. The extracellular-matrix (ECM) plays a dynamic role within the repairing endometrium, with roles suggested for fibronectin and certain integrins (2). We here use both an in vivo mouse model of breakdown and repair (3) and an in vitro model of re-epithelialisation to determine factors involved in repair of this tissue. Murine uterine horns were subjected to laser-capture microdissection of the repairing luminal epithelium (LE) and immediate sub-luminal stroma. Extracted RNA was processed for pathway-focused array that revealed expression of epithelial and leukocyte expressed integrins along with their ligands, proteases and protease inhibitors and basement membrane proteins as elevated in repairing uterine horns. Immunohistochemistry confirmed expression and localisation of integrins a 5 and b 1 in addition to integrin ligands VCAM-1 and fibronectin. Additionally, we demonstrated intimate association of neutrophils with repairing/recently repaired luminal epithelium. To confirm the relevance of these ECM interactions in a human model, human endometrial luminal epithelial cell (ECC-1) monolayers were wounded and treated with inhibitors of MMPs (doxycycline, GM6001) or integrin-fibronectin interactions (RGDS). Significant inhibition of in vitro re-epithelialisation was seen following treatment of wounded ECC-1 monolayers with RGDS, doxycycline or GM6001, suggesting a requirement for these ECM components in endometrial re-epithelialisation. We subsequently demonstrated that menstrual blood derived factors may play a role in the initial endometrial re-epithelialisation. Menstrual blood and peripheral blood were collected on the same day from volunteers. Treatment of wounded ECC-1 monolayers with menstrual blood plasma enhanced the rate of repair compared with peripheral blood plasma from the same woman. Enhanced understanding of the mechanisms governing scar-free repair in the endometrium may help resolve pathological endometrial bleeding disorders and may be applied to aid healing in other tissues. (1) Jabbour et al, 2006. Endocr Rev. 27(1): 17–46.(2) Cao et al, 2007. Hum Reprod. 22(12): 3223–31(3) Brasted et al, 2003. Biol Reprod. 69(4): 1273–80.

134. DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS TO MULLERIAN TISSUE

The human uterus develops from the distal Mullerian Duct, a derivative of the mesoderm germ layer. Unlike other mammalian species (eg. mouse) the endometrium of the human uterus develops prenatally during gestation. Little is known about the developmental process involved. A better understanding of human endometrial development may shed light on the mechanisms involved in endometrial regeneration and pathogenesis of adult proliferative endometrial diseases. Mouse neonatal uterine mesenchyme (mNUM) is inductive and can maintain the phenotype of normal adult human endometrial epithelial cells [1]. Both adult human endometrial stroma and neonatal mouse endometrial mesenchyme secrete growth factors of the TGF-beta family including BMPs which have been shown to play an important role in differentiation of human embryonic stem cells (HESC) [2, 3]. Hypothesis: mNUM will direct differentiation of HESC to form Mullerian Duct-like epithelium. Aim: to investigate the role of mNUM in differentiating HESC in vitro and in vivo using A tissue recombination technique. Method: Embryoid bodies (EB) were formed from GFP labelled HESC (ENVY) and GFP-MIXL1 HESC reporter line [4, 5] and recombined with 2 × 0.5 mm pieces of day 1 epithelial cell-free mNUM. Recombinant tissues were either harvested for gene expression analysis or grafted under the kidney capsule of NOD/SCID mice. Results: We found by qRT-PCR that mNUM induces HESC to form mesendoderm/mesoderm progenitors in vitro, obligate intermediates of the developing Mullerian Duct. After further incubation in vivo under the guidance of mNUM, HESC differentiated to form duct-like structures comprising mesoepithelial cells that co-expressed several key developmental proteins of the Mullerian Duct including Emx2, Pax2, Hoxa10, CA125, and also intermediate filament markers such as CK8/18, Vimentin (n = 8). Conclusion: Our study demonstrated for the first time that mNUM can direct HESC to form a mesodermally derived epithelium that is Mullerian Duct-like, providing a novel model for studying human uterine development. (1) Kurita T, et al., The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen. Differentiation, 2005. 73(6): 313–22.(2) Hu J, Gray CA, Spencer TE, Gene expression profiling of neonatal mouse uterine development. Biol Reprod, 2004. 70(6): 1870–6.(3) Stoikos CJ, et al., A distinct cohort of the TGFbeta superfamily members expressed in human endometrium regulate decidualization. Hum Reprod, 2008. 23(6): 1447–56.(4) Davis R, et al., Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood, 2008. 111(4): 1876–84.(5) Costa M, et al., The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat Methods, 2005. 2(4): 259–60.

168. TGFβ 2-BETAGLYCAN REGULATE FOETAL TESTIS DEVELOPMENT IN VITRO

Betaglycan is a co-receptor for the TGFβ superfamily, known to modulate TGFβ binding in target cells. We have previously found that betaglycan null murine testes at 12.5-13.5 dpc display poorly delineated seminiferous cords and disrupted Leydig cell development (1). Both TGFβs and inhibins are expressed by the fetal testis and it is currently unclear which regulate its development. We tested the hypothesis that loss of betaglycan compromises the functions of TGFβ2 in the differentiating fetal testis as TGFβ2 is known to bind poorly to its type II receptor in the absence of betaglycan. We tested the effect of TGFβ2 on betaglycan wildtype and null foetal gonad/mesonephros complexes using hanging drop or agar block culture methods. From each embryo, one gonad acted as a control; the other was treated. Gonads were cultured in the presence or absence of TGFβ2 (2.5-5 ng/mL) for 48 hours (n =3 pairs). In both culture methods, development in the absence of exogenous growth factor recapitulated normal cord development in wildtype testis and the disrupted cord phenotype in null testes. TGFβ2-treated cultures, 13.5 dpc wildtype mouse testes displayed a 14-35% reduction in total area compared to untreated cultures. Null testes exhibited significantly smaller reductions in gonadal area (2-13%; P &lt; 0.01), indicating that betaglycan null testes exhibit reduced sensitivity to TGFβ2-mediated growth inhibition. However, preliminary observations suggest that TGFβ2 treatment partly rescued cord formation in two of three betaglycan knockout testes in vitro, with testis morphology confirmed by laminin and AMH immunostaining. These data support the notion that TGFβ2 acts via betaglycan to regulate cord development during foetal testis development. Supported by the New Investigator NHMRC (AUS) grant #550915 to MS, JKF Fellowship (#441101, #550915, #338516;#241000) and Victorian Government infrastructure funds. (1) Sarraj et al., 2010. Biol Reprod; 82(1): 153–62.