bluuebunny/crossref_metadata_2025 huggingface dataset:
513. LOSS OF BETAGLYCAN DISRUPTS SOMATIC CELL DIFFERENTIATION IN THE FETAL MOUSE OVARY
Authors: Sarraj M. A. , Escalona R. M. , Umbers A. , Findlay J. K. , Stenvers K. L.
Published: November 2010

Betaglycan (TGFBR3) is an accessory receptor that modulates the activities of members of the TGF-beta superfamily of growth factors which regulate many aspects of reproductive biology. We reported the presence of betaglycan on somatic and germ cells in fetal murine ovary from 12.5 dpc [1]. However, the role of this receptor in early ovarian development is poorly understood. We hypothesised that, given its expression pattern, betaglycan could serve as a TGFb accessory receptor on both oogonia and somatic cells during early ovarian development. In the current study, we examined the Tgfbr3 (betaglycan) null and wildtype ovary from 11.5–14.5 dpc. During this window of development, no overt morphological phenotype was detected, and cell proliferation (PCNA immunostaining) and apoptosis (active caspase 3 immunostaining) were unchanged in the null ovary. To determine whether somatic and/or germ cell markers were altered in the absence of betaglycan, quantitative real time PCR analysis was conducted using total RNA derived from 11.5–14.5 dpc betaglycan wildtype, heterozygous, and null ovaries (n=3 at each age/genotype). From 12.5 dpc, genes associated with the differentiation of the female somatic lineages Wnt4, Fst, Bmp2 were significantly decreased in the null ovary by 30%–50% (P<0.05) while germ cell markers (Oct4, Mvh) showed no significant changes compared to wildtype ovary. Genes associated with the testis-differentiation pathway (e.g. Sox9, Cyp11a1, Cyp17a1, Hsd3b, Insl3, Dhh) were not aberrantly expressed in betaglycan null or heterozygous ovary, with expression levels very low to undetectable. Collectively, these data suggest that while betaglycan does not appear to play a key role in establishing the structure of the ovary, loss of this receptor results in a disruption to the differentiation of ovarian somatic cells.
514. THE REPRODUCTIVE PHENOTYPE OF THE IKKβ CONDITIONAL KNOCKOUT MOUSE
Authors: Drummond A. , Kuyznierewicz I. , Fuller P. J.
Published: November 2010

Nuclear factor-κB (NF-κB) designates a family of transcription factors that have been shown to modulate antiviral, inflammatory and immune responses. Activation of NF-κB is dependent on IKKβ a component of the IκB kinase (IKK) complex which promotes degradation of IκB inhibitory proteins and allows nuclear translocation of NF-κB. Our studies in ovarian granulosa cell tumour cell lines (COV434 and KGN) indicate that NF-κB signalling is constitutively activated. FSH has been reported to increase XIAP expression through NFκB activity in granulosa cells, but beyond that the role of NFκB signalling in folliculogenesis has not been elucidated. To establish the significance of NF-κB signalling in the ovary (and testis), we have generated a gonadal specific IKKbeta conditional knockout mouse. A transgenic mouse line containing floxed IKKβ alleles (gift of M Karin, UCSD) was crossed with a cre mouse line (gift of M Matzuk, BCM) expressing the recombinase in anti-Müllerian hormone receptor expressing cells (granulosa cells or Sertoli cells). The resulting mice will not express IKKβ in granulosa cells or Sertoli cells and thus cannot activate the classical NFκB signalling pathway. On histological assessment, the ovaries and testes from flox x cre (heterogenous) mice appear normal with follicles of all developmental stages and corpora lutea. Preliminary data suggests that breeding with the heterogenous females resulted in increased litter sizes. The histology of the testes is also unremarkable. The mice homozygous for the deletion of IKKβ in the granulosa cells appear healthy and a preliminary assessment does not reveal gross morphological abnormalities of the ovaries. The results of detailed histological and overall assessment of these mice will be presented. These IKK conditional knockout mice should provide insights into the role of NFκB signalling in gonadal function.
515. EFFECTS OF INHIBITION OF HEPERANASE BY SULPHATED MALTOHEXAOSE ON OVULATION, CORPORA LUTEA FORMATION AND PREGNANCY
Authors: Rodgers R. J. , Murdiyarso L. S. , Irving-Rodgers H. F. , Bonner W. , Freeman C.
Published: November 2010

Heparan sulphate proteoglycans such as perlecan and collagen XVIII, are present in the follicular basal lamina, focimatrix and sub-endothelial basal laminas in follicles, but following the LH surge the follicular basal lamina and focimatrix are degraded. Heparanase, which degrades the glycosaminoglycan heparan sulphate, is upregulated in granulosa cells during ovulation. We examined the effects of heparanase inhibition by maltohexaose sulphate treatment on ovulation, formation and vascularisation of corpora lutea, and the establishment of pregnancy. Female twenty-five days old mice were treated with 0.3 mg (>25 mg/kg) maltohexaose sulphate or saline daily for four days from time 0 h and subjected to induction of ovulation by treatment with PMSG (5 IU at 0 h) and hCG (5 IU at 48 h) and mated with a fertile male. Mice were sacrificed at time 0 h, 48 h, 120 h and day 11 (relative to the first injection of PMSG) (n = 20 per group), and ovaries were collected and pregnancy status examined. One ovary was stained with haematoxylin and eosin to measure numerical densities of antral follicles and corpora lutea in the ovary, as well as volume density of luteal tissue. Immunohistochemistry for CD34 (endothelial cell marker) and perlecan (localized to sub-endothelial basal lamina) was conducted to quantify the degree of vascularisation of the corpora lutea. In control mice there was a reduction in the number of antral follicles with increasing age, but no effect of treatment with sulphated maltohexaose was observed on antral follicle development. Nor was there any effect of sulphated maltohexaose on corpora lutea formation, or embryo implantation. In addition, this treatment did not have any effect on the vascularisation of the corpora lutea. These findings suggest that heparanase activity may not be critical during the later stages of follicle growth, ovulation and formation of corpora lutea.
517. RELATIONSHIPS OF LUTEAL PHASE VARIABLES (PRIOR TO AI) WITH FOLLICULAR WAVES IN DAIRY COWS
Authors: Dirandeh E. , Kohram H. , Aryanezhad M.
Published: November 2010

This study was done to consider relationships of luteal phase variables (prior to AI) with follicular waves. The estrous cycles of 10 cows were synchronized with 2 im injections of prostaglandin F2α given 11 d apart. The cows were randomly assigned to 1 of 2 treatments. Cows in the control treatment received no treatment, whereas GnRH6 cows received a GnRH injection on d 6 of the estrous cycle (estrus = d 0). Daily, from estrus d 0 to the next estrus d 23, cows had their ovaries scanned by ultrasound. Blood samples were collected by tail following each ultrasound examination from estrus until next estrus (estrus = d 0). Concentrations of plasma progesterone were determined by radioimmunoassay kit. The limit of detection of the assay was 0.125 ng/mL and the intra- and interassay coefficients of variation were 8.4% and 10.2%, respectively. Data that were analyzed by using PROC GLM of SAS. For comparisons between groups, the 2-sample t-test was used for continuous traits, such as size of cl or hormone concentrations. Prospective comparisons of indices of progesterone indicated that the length of luteal lifespan was longer in three-wave than in two-wave cows (P<0.01). Plasma progesterone concentrations were similar at peak and measured as area under the curve on day 5 through 17 preceding insemination in two-wave (6.70±0.30 ng/ml) and three-wave cows (7.30±0.50 ng/ml). Length of the luteal phase (defined as from the day of estrus until the last day on which plasma progesterone remained greater than 2 ng/ml) was <2 days shorter in two-wave cows than in three-wave cows (16.7±0.30 vs. 18.40±0.50 d; P<0.05). In addition, the day of peak progesterone occurred earlier in two-wave cows (13.50±0.40 vs. 16.30±0.70 d; P<0.05).
518. RELATIONSHIP BETWEEN THE OVARIAN STATUS ON THE INITIATION DAY OF SUPEROVULATION AND SUPEROVULATORY RESPONSES IN CATTLE
Authors: Kohram H. , Moakhar H. Kermani
Published: November 2010

The purpose of this experiment was to determine the influence of the ovarian status on the initiation day of superovulation (d 0) on superovulatory responses in terms of the number of large follicles (≥7mm) at estrus (d 4) and CL 7 days after estrus (d 11). Ultrasonography was performed on d 0, 4 and 11of the experiment. Animals conventionally superovulated (400 mg Folltropin-V) between d 8 to 12 of the estrous cycles and 102 superovulation cycles were considered in the present experiment. Data were analyzed using GLM procedure of the SAS. In these cases, the number of follicles was determined in each of the 2 studied classes (SF, 4 to 6 mm; and LF, ≥7mm) on d 0. Subsequently, each of these classes was divided into two subgroups (Low, High) based on the number of follicles observed on d 0 (Table 1). The number of LF at estrus in each of the 2 studied classes (Low vs. High) were (13.02±1.02 vs. 19.7±1.69, p<0.01; and 16.23±1.59 vs. 14.75±1.48, p>0.1) and the number of CL on d 11 were (9.75±0.57 vs. 12.75±0.98, p<0.01; 10.91±0.8 vs. 10.85±0.65, p>0.1). Therefore, the ovarian status on d 0 in terms of the number of SF affected the number of LF (d 4) and CL (d 11). In addition, the number of superovulatory cycles with low number of SF was 2 times more than the ones with a high number of SF on d 0; also 49 out of 102 superovulatory cycles had at least 2 LF on the initiation day of superovulation (Table 1). These results suggested that follicular puncture 1–2 days before a conventional superovulation treatment may increase the superovulatory responses.
519. INFLUENCE OF THE DURATION OF CIDR INSERTION ON OVARIAN RESPONSES IN AN eCG PROGRAM DURING NON-BREEDING SEASON IN EWES
Authors: Salehi R. , Kohram H.
Published: November 2010

The aim of this study was to investigate the effect of 14 versus 7 days CIDR insertion before eCG treatment on superovulatory responses during non-breeding season in ewes. 10 Iranian Shall ewes, between 2 and 3 years old were kept inside during the experiment period and were fed a live weight maintenance ration. Ewes were randomly assigned to 1 of 2 groups. CIDR was inserted to the ewes of group 1 for 14 days and for 7 days to group 2. Each ewe received 1500 IU eCG on the day of CIDR removal. Ovarian follicular activity was monitored by transrectal ultrasonography on the days of eCG treatment, estrus, and 7 days after estrus to monitor the number of corpus lutium. During examination the animals were held in a standing position. Scanning of both ovaries was recorded using a MP4 player. The number of medium and large follicles (≥3 mm in diameter) 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 shows that the ovarian responses in terms of the number of ≥3 mm follicles at estrus is greater (P<0.05) in group 1 than group 2 ewes. The mean number of CL after eCG injection in group 1 and 2 were also significantly different (3.6±0.24 vs 2.4±0.51; P<0.05). The results showed that the 14 days synchronization before eCG treatment during non-breading seasons in ewes had the beneficial effects on ovarian responses.
520. RELATIONSHIP BETWEEN THE OVARIAN STATUS ON THE INITIATION DAY OF SUPEROVULATION AND THE OVULATORY FOLLICLES AT ESTRUS IN SHALL IRANIAN EWES
Authors: Moakhar H. Kermani , Kohram H. , Shahne A. Zare
Published: November 2010

The objective of this experiment was to determine the influence of the ovarian status on the initiation day of superovulation (day 14) on superovulatory response in terms of the number of ovulatory follicles (F, ≥4mm) at estrus (day 16). Estrous cycles of 50 Shall Iranian ewes were synchronized by CIDR. The day of CIDR insertion was considered as the starting point of the experiment (day 0). At the time of CIDR removal (day 14), 650 IU of eCG was administered to ewes. Ultrasonography was performed on days 14 and 16. Data were analyzed by means of the GLM procedure of the SAS. In these cases, the number of follicles were determined in each of the three studied classes (<2, 2 to 4, and ≥4 mm) on day 14. Subsequently, each of these classes was divided into two subgroups (Low, High) based on the number of follicles observed on day 14 (Table 1). The number of large follicles at estrus in each of the three studied classes were (3.96±0.28 vs. 4.78±0.26, p<0.05; 4.31±0.35 vs. 4.41±0.24, p>0.05; and 4.50±0.23 vs. 4.07±0.37, p>0.05) respectively. The number of medium and large follicles on day 14 did not affect the superovulatory response. However, the high number of small follicles on the initiation day of superovulation increased the number of ovulatory follicles at estrus. Since, 23 out of 50 ewes had a low number of small follicles on day 14 (Table 1), which may suggest that an injection of FSH 1-2 days before initiation of eCG treatment enhance the superovulatory response.
521. EFFECT OF KISSPEPTIN ON REPRODUCTIVE FUNCTION IN SEASONALLY ACYCLIC RED DEER HINDS
Authors: Barrell G. K. , Ridgway M. J. , Wellby M. , Smith J. T. , Clarke I. J.
Published: November 2010

A preliminary study was conducted to examine whether the marked inhibition of ovulation in seasonally acyclic red deer could be overcome by use of kisspeptin to stimulate gonadotrophin release in these animals. After 14 days progesterone priming with intravaginal CIDR-G devices, non-lactating and lactating hinds (n = 8 in both cases) received 0.50 µmol murine kisspeptin-10 in saline solution delivered intravenously in 30 hours, either continuously or as 4-hourly boluses, and lactating hinds (n = 8) received saline solution intravenously as controls. Kisspeptin treatment caused an elevation (P < 0.05) of the mean plasma LH concentration at 2 h (0.36 + 0.237 versus 0.11 + 0.006 ng/ml, treated versus controls, respectively). Two weeks later there was a tendency (Chi-square = 9.286, 4 d.f., P = 0.054) for an increase in occurrence of medium and larger sized ovarian follicles in treated hinds. However, no ovulations resulted from the infusions and there was no effect on timing of the onset of ovulatory activity during the subsequent breeding season, as determined from circulating progesterone concentrations. It is concluded that kisspeptin stimulates the reproductive axis in seasonally acyclic red deer hinds, however the dose or mode of administration required to produce ovulations may be higher than what is effective for sheep in an equivalent reproductive state.
522. THE ASSOCIATION OF FOLATE PATHWAY ENZYME POLYMORPHISMS AND PREGNANCY OUTCOME
Authors: Furness D. L. F. , Dekker G. A. , McCormack C. D. , Nowak R. C. , Thompson S. D. , Roberts C. T.
Published: November 2010

The folate, vitamin B12 and vitamin B6 (one-carbon) metabolic pathway is essential for the synthesis of precursors used in DNA synthesis, repair, and methylation. We hypothesise that single nucleotide polymorphisms in genes encoding enzymes in this pathway can disrupt these processes leading to adverse pregnancy outcomes. We investigated associations of six candidate polymorphisms in five genes related to one-carbon metabolism with risk for adverse pregnancy outcome in 586 nulliparous Caucasian couples with normal fertility. Chi-square analysis was used to compare genotype frequencies with pregnancy outcomes. Pregnancies were classified as healthy (n=261), preeclampsia (PE, n=38), gestational hypertension (GHT, n=32), small-for-gestational-age (SGA, n=60) and PE+SGA (n=22). Associations between maternal, paternal and neonatal genotypes with customised birthweight centiles and placental weight were determined using ANOVA with SIDAK post-hoc analyses. The maternal MTR 2756 G allele was associated with decreased placental weight (–87g, P=0.040). Both paternal and neonatal MTR 2756 G alleles were associated with lower birthweight (–12%, P=0.028 and –10%, P=0.039) while the latter was also associated with PE+SGA (P <0.000). Neonatal MTRR GG genotype was associated with GHT and PE with SGA (P=0.033, P=0.011). Neonatal MTHFD1 GG genotype was twice as frequent in PE and GHT (P=0.037; P=0.019) while neonatal TCN2 GG genotype doubled in SGA (P=0.042) compared with healthy pregnancies. Our findings indicate that genetic variation in multiple one-carbon metabolism genes may influence risk of adverse pregnancy outcome. MTR with cofactor vitamin-B12, catalyses the methylation of homocysteine to methionine. Formation of methionine through this pathway is important for synthesis of phospholipids, proteins, myelin, DNA, RNA and S-adenosyl methionine. TCN2 encodes the vitamin-B12 transport protein and MTHFD1 catalyses the conversion of one-carbon derivatives of tetrahydrofolate, which are substrates for methionine, thymine and purine synthesis and are important for healthy placental and fetal development. Larger studies are needed to further examine gene/gene and gene/diet interactions in this pathway.
524. SPECIES DIFFERENCES IN THE REQUIREMENT OF Smad2/3 AND MAPK SIGNALLING FOR CUMULUS CELL EXPANSION
Authors: Gilchrist R. B. , Ritter L. J.
Published: November 2010

Cumulus cell expansion is necessary for female fertility. In the mouse, cumulus expansion is dependent on activation of two cumulus signalling pathways: 1) mitogen-activated protein kinase (MAPK3/1) by FSH/EGF and 2) Smad2/3 signalling by oocyte-secreted factors. This study was conducted to determine the involvement of these two signalling pathways in pig cumulus expansion. Abattoir-collected pig COC and eCG-primed mouse COC were cultured for either 24 or 20 hours respectively in the presence of FSH and treated with increasing doses of either a MAPK 3/1 inhibitor (U0126), it's inactive analogue (U0124), or kinase inhibitors SB431542 and dorsomorphin that prevent phosphorylation of Smad2/3 and Smad1/5/8, respectively, or a vehicle control. Morphological cumulus expansion was measured using an established subjective scoring system. At a dose of 10µM, U0126 completely abolished mouse expansion (0±0, p<0.05) and caused severe impairment of cumulus expansion in porcine COC (1.23±0.09, p<0.05). U0124 and dorsomorphin were unable to inhibit cumulus expansion in either species. SB431542 was also ineffective at blocking pig cumulus expansion (2.6±0.16 p<0.05) despite using a dose 4 times higher (8µM) than that required to ablate mouse COC expansion. To determine if pig oocytes activate cumulus cell Smad2/3 or 1/5/8, a Smad-luciferase reporter assay was undertaken. In contrast to mouse oocytes which only activated the Smad2/3 pathway, pig oocytes activated both pathways. This suggests that the failure of SB431542 to prevent pig cumulus expansion is not due to inadequate oocyte activation of Smad2/3. These results demonstrate that while activation of both MAPK3/1 and Smad2/3 pathways are required for mouse cumulus cell expansion, pig COC expansion only requires the MAPK3/1 pathway. Neither species require Smad1/5/8 pathway activation. This study provides new insight into the differing signalling mechanisms required for mammalian cumulus cell expansion.
525. ROLES FOR HUMAN CHORIONIC GONADOTROPHIN IN EMBRYO-ENDOMETRIAL CROSS-TALK DURING BLASTOCYST IMPLANTATION
Authors: Paiva P. , Meehan K. , Salamonsen L. A. , Dimitriadis E.
Published: November 2010

Emerging evidence suggests an important role for the early embryo product human chorionic gonadotrophin (hCG) in embryo-endometrial interactions critical for successful embryo implantation1. The human endometrium is also a source of hCG, with maximal expression of hCG and its receptor, hCG/LHR, in endometrial epithelial cells during the window of implantation in vivo2,3, and in primary endometrial epithelial cells (EECs)3. Implantation is tightly regulated by growth and regulatory factors produced within the embryo-endometrial microenvironment. We hypothesise that embryo/endometrial-derived hCG mediates the molecular cross talk vital for successful implantation. The main objective of this study was to investigate the effect of hCG on the production of a selected cohort of 42 cytokines and growth factors by EECs. These included those with both known and previously unidentified roles during implantation. The secretory profile of cytokines/growth factors produced by EECs was also analysed. EECs (n=8 cultures) were isolated from biopsies collected from fertile cycling women. Cells were treated without or with recombinant hCG for 48 hr and conditioned media collected for quantitative analysis using LuminexTM multiplex technology. For the first time, a secretory profile of 42 cytokines and growth factors produced by EECs was established, as was the identification of fibroblast growth factor-2 (FGF-2) secretion by human endometrial epithelium. hCG (2 IU/ml) significantly increased the production of a number factors including those with known roles during trophoblast migration and adhesion (CX3CL1; 71±31%, CXCL10; 67±24%, CCL4; 87±12%), in trophoblast differentiation (IL-1α ; 68±31%) and with unidentified roles during implantation (CCL22; 78±40%, GM-CSF; 45±16%, FGF-2; 50±25%; all p<0.05). Upregulation of the known hCG regulated proteins, VEGF and LIF, validated this study. These findings clearly support roles for the embryo/endometrium via hCG in actively contributing to the molecular cross-talk during the early stages of implantation.
526. GENETIC POLYMORPHISMS IN INTERLEUKIN-1 FAMILY MEMBERS AND PREGNANCY OUTCOME
Authors: Nowak R. C. , Thompson S. D. , Zhang J. , Dekker G. A. , Roberts C. T.
Published: November 2010

Poor placental development is associated with a variety of common, potentially life-threatening pregnancy complications, including preeclampsia (PE) and small-for-gestational-age (SGA) babies. The placenta and fetus express a combination of maternal and paternal genes. Interleukin-1 alpha (IL-1α) and -1 beta (IL-1β) promote trophoblast invasion by upregulating expression of matrix metalloproteinases, while interleukin-1 receptor antagonist (IL-1RN) acts as a competitive inhibitor of IL-1 a and IL-1β by binding to the IL-1 receptor. We hypothesis that polymorphisms which decrease IL-1 activity, within IL-1α rs1800587 (C-allele) and rs17561 (T-allele), IL-1β rs16944 (C-allele) and IL-1RN rs454078 (A-allele) affect placental development and thus pregnancy outcome. To determine the effect of the IL-1 SNPs on pregnancy outcome, blood was collected prospectively from pregnant women at 15 weeks gestation and from their partners and babies at birth. DNA was extracted and genotyped by Sequenom MassArray. Pregnancies were fully characterised and classified into control or PE, PE+SGA, SGA after delivery by an experienced obstetrician. The IL-1α rs1800587 fetal genotype was associated with PE+SGA and smaller head circumference at birth. IL-1α rs17561 fetal genotype was associated with PE+SGA, and a smaller head circumference and body length at birth. The frequency of IL-1β C-allele was associated with PE in neonates, and in fathers the CC homozygote genotype was associated with decreased placental weight at birth. The IL-1RN rs454078 maternal genotype was associated with PE and maternal waist circumference while fetal AA genotype was associated with SGA and decreased placental weight at birth. Genotypes which decrease the production of IL-1α and IL-1β while increasing IL-1RN will reduce proinflammatory cytokines and thereby affect the invasive potential of placental trophoblasts. We have shown that these polymorphisms in both parents may be associated with poor pregnancy outcome. Suggesting that both partners contribute to placental differentiation and function.
527. EXTENSIVE LYMPHATIC REMODELLING OCCURS IN HUMAN PLACENTAL BED DURING THE FIRST 20 WEEKS OF PREGNANCY
Authors: Rogers P. A. W. , Volchek M. , Girling J. E. , Lash G. E. , Cann L. , Robson S. C. , Bulmer J. N.
Published: November 2010

The aim of this study was to describe the distribution of lymphatics in human placental bed from 6–20 weeks of pregnancy, with emphasis on the lymphatics surrounding the spiral arterioles as they undergo remodelling associated with trophoblast invasion. Placental bed biopsies were obtained following elective termination (n=41, 6–18 weeks gestation) and at term (n=5). Samples were routinely processed for serial sectioning at 3um to allow structural relationships to be compared in consecutive sections. Double immunohistochemistry protocols were used to identify lymphatic vessels in conjunction with one of: blood vessels, smooth muscle, epithelium and trophoblast, or proliferating cells. Immunostaining demonstrated that lymphatic vessels were present in abundance in the hypersecretory endometrium throughout all stages of gestation, and were prominent encircling spiral arterioles and adjacent to the endometrial glands. In contrast, the decidua was nearly always devoid of lymphatics, and in particular they were absent from the vicinity of spiral arterioles that were surrounded by decidual cells. In hypersecretory endometrium lymphatic vessel profiles varied greatly in architecture and size, and included focal accumulations of endothelial cells, through to fully formed lymphatic vessels. There was evidence of lymphatic vessel endothelial cell proliferation in all the different vessel types. The trophoblast appeared to have no overt influence on the structure or appearance of the lymphatic endothelium, with no evidence of incorporation into, or distortion of, the lymphatic vessel walls. Obvious increases were noted in the size of the lymphatic vessel profiles in hypersecretory endometrium as gestation progressed. The study provides the first detailed description of the placental bed lymphatics and their relationship to other endometrial structures during human pregnancy. We have shown that lymphatic vessels are absent from the decidua, apparently regressing as surrounding stromal cells decidualise. In contrast, the underlying hypersecretory endometrium contains abundant lymphatics which increase in size as pregnancy progresses.
531. Lyve-1 AND Vegfr-3 POSITIVE LYMPHATIC VESSELS ARE LARGELY RESTRICTED TO THE MYOMETRIUM IN THE MOUSE UTERUS
Authors: Girling J. E. , Lederman F. , Rogers P. A.W.
Published: November 2010

The human endometrium contains a distinct population of lymphatic vessels, with reduced numbers of vessel profiles in the functionalis relative to the basalis and underlying myometrium. The mechanisms restricting lymphatic vessel development within the functionalis and the consequences of this distribution for endometrial function have not been investigated. To determine how the distribution of endometrial lymphatics is regulated, an in vivo animal model is required for mechanistic studies. As an initial step, we described the distribution of lymphatic vessels and the expression of Vegf-C and Vegf-D within the mouse uterus using immunohistochemistry. Uterine tissues were collected from mice during the oestrus cycle (diestrus, proestrus and oestrus, n=7–9 per group), during early pregnancy (days 1-4, n = 4–5 per group), and from ovariectomised mice treated with vehicle, estradiol-17β or progesterone treatment (n=7–8 per group). Uterine sections were immunostained with antibodies against Lyve-1, Vegfr3, Vegf-C and Vegf-D. Lyve-1 positive lymphatic vessels were almost exclusively observed in the connective tissue between the longitudinal and circular muscle layers of the myometrium. Lymphatic vessel profiles were rare within the endometrium and were only observed on 24 % of the sections examined. The Vegfr3 immunostaining was less robust than Lyve-1 with considerable variation in non-lymphatic staining among the different sections (which did not relate to a particular reproductive state or treatment group). Despite the variability, the pattern of lymphatic vessels was as seen with the Lyve-1 antibody. Vegf-C and Vegf-D immunostaining was present in all uterine compartments (epithelium, stroma, myometrium), however, only minimal changes were noted in expression across the oestrus cycle, during early pregnancy, or in hormone treated mice. In conclusion, despite the presence of the key lymphangiogenic growth factors Vegf-C and Vegf-D, there are minimal lymphatic vessels present within the mouse endometrium. This implicates other factors in the mechanism restricting endometrial lymphatic vessel growth.
532. SOLUBLE MEDIATORS IN THE HUMAN UTERINE CAVITY: POTENTIAL ROLES IN EMBRYO IMPLANTATION
Authors: Hannan N. , Meehan K. L. , Rombauts L. J. F. , Salamonsen L. A.
Published: November 2010

Embryo implantation requires synchronized dialogue between a receptive endometrium and an activated blastocyst via locally produced soluble mediators. During the mid-secretory (MS) phase of the menstrual cycle there is increased glandular secretion into the uterine lumen. These secretions likely contain important mediators that modulate the endometrium and support the conceptus during implantation. Previously we identified that several chemokines were maximally produced during the MS phase by endometrial glandular epithelium (GE) (1, 2) and the presence of chemokine receptors on GE and human trophoblast (3). Furthermore recombinant human chemokines and endometrial epithelial cell-conditioned media stimulated trophoblast migration; this was attenuated by neutralizing specific chemokines (3). Chemokines also regulate a variety of adhesion and ECM molecules on trophoblast (4). Thus chemokines have important roles during embryo implantation. We hypothesized that chemokines are secreted into the uterine cavity and may act on the implanting blastocyst and the endometrium. This study aimed to identify chemokines in uterine fluid (collected by flushing the uterine cavity with 5mls of saline) from fertile women during the proliferative (non-receptive; n=4) and MS (receptive; n=4) phases of the cycle, and from women with unexplained infertility during the MS phase (n=4). Uterine fluid was analyzed using quantitative MilliplexTM Luminex® 42-plex cytokine/chemokine assays revealing the presence of IL-8, CCL2, CCL4, CCL7, CCL11, CCL22 and CX3CL1 in uterine fluid from all women. Importantly chemokine profiles were altered with both cycle phase and fertility; for example CCL4 and CCL22 levels were lower in the infertile cohort, where as CCL2 levels were higher in uterine fluid collected during the proliferative phase. Identifying the soluble mediators in human uterine fluid may provide potential markers of endometrial receptivity, insight into the unique microenvironment essential for pregnancy and a profile of maternal factors that influence the implanting blastocyst.
533. MODULATION OF THE MATERNAL IMMUNE SYSTEM DURING EARLY BOVINE PREGANCY
Authors: Walker C. G. , Meier S. , Mitchell M. D. , Roche J. R. , Littlejohn M. D.
Published: November 2010

Over the past three decades, there has been a significant decline in dairy cattle fertility. A large proportion of pregnancy losses are believed to occur during the pre-implantation period, when the developing embryo is elongating rapidly and signalling its presence to the maternal system. The molecular mechanisms that prevent progression of the estrous cycle and allow the allogenic embryo to survive within the maternal environment are not well understood. To gain a more complete picture of these molecular events, global transcriptional profiling was performed using endometrial tissue from reproductive day 17 pregnant and non-pregnant (cycling) Holstein-Friesian dairy cattle. Microarray analysis revealed 357 differentially expressed transcripts (with≥2 fold change in expression; P-value < 0.05) between pregnant and cycling animals in both caruncular and intercaruncular tissue. Additionally, 122 and 26 differentially expressed transcripts were identified only in either caruncular or intercaruncular endometrium, respectively. Pathway analysis of differentially expressed genes revealed enrichment for genes involved in interferon signalling and modulation of the immune response in pregnant animals. Many of these genes have already been described in relation to bovine reproduction. However, this analysis also revealed a number of genes not previously identified in this context, providing further insight into the mechanisms by which the embryo regulates the maternal immune response.
534. CO-ORDINATED GENE EXPRESSION IN BOVINE GRANULOSA CELLS PRECEDES FOLLICULAR DOMINANCE
Authors: Matti N. , Irving-Rodgers H. F. , Bonner W. M. , Hatzirodos N. , Sullivan T. R. , Rodgers R. J.
Published: November 2010

During growth of bovine follicles, one emerges as the largest and dominant follicle. What regulates dominance remains unknown, but candidates include oestradiol, TGFB1, and recently CYP11A1 and focal intra-epithelial matrix (focimatrix). The two to four largest follicles were dissected from pairs of bovine ovaries and follicular fluid collected. A portion of the follicle wall was histologically classified for follicle health or atretia, and granulosa cells harvested for quantitative RT-PCR. Messenger RNA levels of focimatrix (COL4A1, LAMB2, HSPG2), steroidogenic enzymes (CYP11A1, CYP19A1) and TGFB1 genes were measured. Follicular fluid progesterone and oestradiol concentrations were measured by RIA. Follicles were identified as pre-deviated (before size-deviation) if the largest two or more healthy follicles were of equal size (6.7±0.1 mm, n = 14 animals, 35 follicles), and as post-deviated (after size-deviation) if they differed in size by 0.5–1.0 mm (7.2±0.2 mm; n = 11 animals, 26 follicles). For analyses, pre-deviated follicles were grouped into either the highest (oestradiol, CYP11A1) or lowest (TGFB1) expression (n = 14) and compared to the remaining follicles (n = 21). Deviated follicles were classified into dominant (n = 12) and subordinate (n = 14) based on diameter. Dominant follicles did not differ from subordinate follicles in any parameters measured, but were significantly larger than subordinate or pre-deviated follicles (P<0.01). For pre-deviated follicles grouped on oestradiol no parameters differed significantly, and when grouped on TGFB1, LAMB2 (P<0.05), HSPG (P<0.05), CYP19A1 (P<0.05) and TGFB1 (P<0.01) differed but levels were lower, not higher as expected. When grouped on CYP11A1, COL4A1 (P<0.05), LAMB2 (P<0.01), HSPG2 (P<0.01) and CYP19A1 (P<0.001) were significantly elevated in the high CYP11A1 group. This suggests that CYP11A1 and focimatrix might be important in follicle dominance.
535. THE EFFECT OF ALTERING GLUCOSE LEVELS DURING COLLECTION AND MATURATION OF MOUSE OOCYTES ON SUBSEQUENT DEVELOPMENTAL COMPETENCE
Authors: Frank L. A. , Sutton-McDowall M. L. , Russell D. L. , Lane M. , Gilchrist R. B. , Thompson J. G.
Published: November 2010

The preconception environment is known to influence oocyte developmental competence. In particular, hyperglycaemic conditions during cumulus-oocyte complex (COC) maturation result in decreased oocyte quality. This is, in part, due to perturbations in O-linked glycosylation in the cumulus cells. In embryos, even a brief exposure to glucose during early cleavage can have significant impact on O-linked glycosylation and further development. The aim of this study was to determine the effect of altering glucose concentrations during the collection and maturation phases of COCs on oocyte developmental competence. COCs were collected and matured for 17h at 37°C in 6% CO2 with 0 or 10mM glucose in a 2 x 2 factorial design. A fifth group used standard concentrations of 0.5mM and 5.55mM glucose in the collection and maturation media respectively. Following maturation, oocytes were inseminated and cultured to the blastocyst stage. The average time for collection was 1 h. COCs exposed to 0mM glucose during collection and 10mM glucose during maturation had the greatest cumulus expansion despite no change in the proportion of COCs completing nuclear maturation. However, this same treatment group resulted in significantly lower blastocyst production than the control group (8.4% vs. 25.0%, P<0.05). These results show that glucose concentration in collection medium has a significant influence on maturation indices and oocyte developmental competence, as determined by blastocyst development rates. Our data further supports the concept that the conditions used for the collection of oocytes can have profound effects on subsequent development. We intend to investigate if these effects are related to perturbations in cumulus cell O-linked glycosylation.
001. ORIGINS OF DNA DAMAGE IN SPERMATOZOA
Authors: Aitken R. J.
Published: November 2010

DNA damage is frequently encountered in the spermatozoa of sub-fertile male mammals and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired chromatin remodelling during spermiogenesis. In light of these associations we propose a two step hypothesis for the origins of DNA damage in spermatozoa. In Step 1, a variety of intrinsic (diabetes, varicocele, testicular torsion, obesity) and extrinsic (radiofrequency electromagnetic radiation, heat, cigarette smoke, diet, environmental toxicants) factors collude to generate a state of oxidative stress in the testes. This stress impedes spermiogenesis resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells readily default to an apoptotic pathway comprising motility loss, caspase activation, phosphatidylserine exteriorization and the production of reactive oxygen species (ROS) by the mitochondria. In Step 2, these mitochondrial ROS attack the spermatozoa inducing lipid peroxidation and oxidative DNA damage, which then leads to DNA strand breakage and cell death. Nucleases activated and released during the apoptotic process are denied access to the sperm nucleus because the unique physical architecture of this cell prevents it. For this reason, a majority of the DNA damage encountered in human spermatozoa is oxidative. Given the importance of oxidative stress in the etiology of DNA damage, there should be a significant therapeutic role for antioxidants in the treatment of this condition. Furthermore, if oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.
004. A NEW ERA IN CONTRACEPTIVE DEVELOPMENT: NON-HORMONAL OPTIONS THAT ALSO TARGET SEXUALLY TRANSMITTED INFECTIONS
Authors: Dimitriadis E.
Published: November 2010

Despite huge increases in access to contraceptives globally more than 700 000 maternal deaths related to unintended pregnancies occurred between 1995 and 2000 mostly in developing countries. Over 80 million women have unintended or unwanted pregnancies annually. Remarkably, there have been no new methods of contraceptives developed in the last 50 years. The extremely high incidence of sexually transmitted infections (STIs) indicates that it is desirable to develop contraceptives that also target STIs. Two interleukin (IL) 6-type cytokines, leukemia inhibitory factor (LIF) and IL11, are obligatory for implantation in mice and are dysregulated in endometrium of some women with infertility. Both LIF and IL11 are also thought to have roles in Chlamydia-induced inflammation which can lead to a multitude of pathologies. LIF and IL11 antagonists were produced and their contraceptive efficacy tested in mice. Polyethylene glycol (PEG) was conjugated to LIF antagonist (LA) or IL11 antagonist (IL11A) to increase their serum half-life. PEGLA injected during the peri-implantation period blocked LIF action in the endometrium and totally prevented embryo implantation while having no embryo toxic effects.1 Similarly, injection of PEGIL11A blocked decidua formation resulting in pregnancy failure.2 In women, vaginally administered drugs preferentially localise to the uterus suggesting that vaginal administration of PEGLA is an appropriate delivery method for contraceptive purposes. Further, vaginally administered PEGLA may be useful as a ‘dual-role’ contraceptive to also block STIs. PEGLA administered via vaginal gel was shown to prevent implantation having minimal effects on non-uterine LIF targets. This is the first study to show the contraceptive efficacy of a PEGylated compound delivered vaginally. It further indicates that PEGLA may be useful as a dual-role contraceptive. Contraceptive trials in non-human primates are currently underway to determine the effect of PEGLA on implantation. If effective, this will offer new opportunities as pharmacological, non-hormonal dual-role contraceptives for women.
(1) White CA, Zhang JG, Salamonsen LA, Baca M, Fairlie WD, Metcalf D, Nicola NA, Robb L, Dimitriadis E (2007) Proc Natl Acad Sci USA 104: 19 357–62.(2) Menkhorst E, Salamonsen LA, Robb L, Dimitriadis E (2009) Biol of Reprod 80: 920–927.
006. HUMAN GERM CELL FORMATION AND DIFFERENTIATION FROM PLURIPOTENT STEM CELLS
Authors: ReijoPiera R. A.
Published: November 2010

Human embryo development begins with the fusion of egg and sperm, followed by reprogramming of the DNA, a series of cell divisions and activation of the embryo’s genome. As development continues, the germ cells (egg and sperm) must be set aside from other cell types. A major cause of infertility in men and women is quantitative and qualitative defects in human germ cell (oocyte and sperm) development. Yet, it has been difficult to study human germ cell development, especially features that are unique relative to model organisms. We have developed a system to differentiate human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to germ cells and to quantitate and isolate primordial germ cells (PGCs) derived from both XX- and XY-bearing hESCs and iPSCs. This allowed silencing and overexpression of genes that encode germ cell-specific cytoplasmic RNA-binding proteins (not transcription factors) and resulted in the modulation of human male and female germ cell formation and developmental progression. We observed that human DAZL (Deleted in AZoospermia-Like) functions in female and male PGC formation and maintenance, whereas closely-related family members, BOULE and DAZ, promote entry into meiosis and development of haploid gametes with sperm-specific methylation patterns at imprinted loci in the male. We also conducted critical proof-of-concept studies in mice that showed that phenotypes observed in germ cell development in vitro from wildtype, heterozygous, and Dazl–/– mutation-carrying mouse ESCs (mESCs) mirrored the phenotypes that were observed in vivo. Furthermore, transplantation of XX mESC-derived oocytes resulted in recruitment of somatic cells to form follicles. These studies comprised the first direct experimental analysis of the genetics of human germ cell development and set the stage for extensive exploration of complex genetic variants linked to infertility. Results are significant to the generation of gametes for developmental genetic studies and potential clinical applications.
010. PERICONCEPTIONAL UNDERNUTRITION: LIFE-LONG EFFECTS FOR THE OFFSPRING
Authors: Bloomfield F. H. , Oliver M. H. , Jaquiery A. L. , Hernandez C. , Challis J. R. G. , White A. , Harding J. E.
Published: November 2010

Poor nutrition in women of child-bearing age is common, even in Western countries. It has been estimated that approximately 30% of women of child-bearing age in affluent cities such as Sydney and Southampton are either actively dieting or have a nutritional intake that does not meet daily recommended requirements for all nutrients. We have investigated the effect of reduced maternal nutrition before and around the time of conception on fetal growth and development, and have followed offspring through to adulthood. In this paradigm, ewes were fed to lose 10–15% of their body weight and then to gain weight according to conceptus mass. Control ewes were well fed throughout. Different timing and duration of undernutrition in the periconceptional period were utilised to investigate the most critical window for fetal development. Periconceptional undernutrition resulted in accelerated development of the fetal hypothalamic-pituitary-adrenal (HPA) and glucose-insulin axes in late gestation, and preterm birth. Offspring of periconceptionally undernourished ewes demonstrated altered laterality and an altered response to isolation stress; HPA axis function was also suppressed. As offspring aged, glucose tolerance decreased and became significantly impaired by young adulthood compared with control offspring. The effects of maternal undernutrition on offspring were modified by offspring sex and also by being one of a twin pair. Interestingly, our data also demonstrate that conception as a twin, regardless of maternal nutritional status, also affects all these outcomes but in a different way to maternal undernutrition. Preliminary data suggest that epigenetic changes in feeding centres of the hypothalamus may play a role in the mechanism behind some of these effects. These studies suggest that even moderate maternal undernutrition in very early pregnancy has life-long effects. Should this also be true in humans, then health care messages for women may need to be targeted prior to pregnancy.
011. GESTATIONAL DIABETES - COMPLICATIONS, MANAGEMENT, OUTCOMES
Authors: Rowan J. A.
Published: November 2010

Gestational diabetes (GDM) is associated with increased maternal risks of hypertensive complications, caesarean section and later diabetes. The fetus is exposed to an excess nutrient load and risks of macrosomia, trauma at delivery, neonatal complications and later obesity and associated metabolic consequences. Perinatal outcomes are improved by treating GDM, but the effect on longer term outcomes is not clear. Can we improve pregnancy and later outcomes further by considering choice of medication and treatment targets? The metformin in gestational diabetes (MiG) trial demonstrated that pregnancy outcomes were not different between women randomized to metformin compared to insulin. A composite of neonatal complications was seen in 32.0% and 32.2% respectively, RR 0.99 (95% CI 0.80–1.23). Examining glucose control during treatment in tertiles, women who achieved a mean fasting capillary glucose level <4.9 mmol/L had the lowest risk of neonatal complications. Those achieving a postprandial capillary glucose mean <6.5mmol/L had lower rates of preeclampsia and birth weight >90th centile. Obesity was not a significant factor predicting outcomes (unlike diet-alone treated women). Preliminary analyses from the follow up of two year old children from the MiG trial, the offspring follow up (TOFU) show body composition, diet and activity assessments are similar, with small differences between the metformin and insulin groups, respectively in biceps (6.1 vs 5.6 mm P = 0.04) and subscapular skin folds (6.38 vs 6.10 mm P = 0.03) and upper arm circumference (17.3 vs 16.7 cm P = 0.003). Ratios of central to peripheral fat as measured by waist: hip circumference, suscapular:triceps skin folds and abdominal:thigh fat by DEXA were no different. Further analyses will be performed when the final data entries are completed and details of these will be presented.