bluuebunny/crossref_metadata_2025 huggingface dataset:
003.Expression of secreted frizzled related protein-4 (sFRP-4) and associated Wnt signalling in cancer and apoptosis
Authors: Dharmarajan A. , Zeps N. , McLaren S.
Published: November 2010

We examined the interplay between Wnt and secreted frizzled related protein-4 (sFRP4) in estradiol induced cell growth in breast cancer cells (MCF-7), and also determined the in vivo distribution of sFRP-4 in human breast cancer. MCF-7 cells were treated with estradiol, sFRP-4 conditioned media and a combination of the two. Real-time RT-PCR and Western blot analysis were used to determine the expression of the sFRP-4 and its associated Wnt signalling molecules following treatment. Immunohistochemistry was performed to examine sFRP-4 expression patterns in human breast cancers. Estradiol treatment up-regulated the expression of the Wnt signalling genes Wnt-10b, beta-catenin and fz-4 (P < 0.001 for all genes). This up-regulation was not associated with an increase in the Wnt signalling pathway as measured by the levels of active beta-catenin. sFRP-4 conditioned media reduced MCF-7 cell proliferation, down-regulated the Wnt signalling genes beta-catenin and fz-4 as well as down-regulating wnt signalling activity. sFRP-4 was able to reduce the proliferation of estradiol stimulated MCF-7 cells. Cytoplasmic sFRP-4 protein was expressed in all breast tumours examined, with intense staining evident in the lobular carcinoma in situ and the ductal carcinoma. These data demonstrate that sFRP-4 is a potent inhibitor of the Wnt signalling pathway in MCF-7 cells, acting not only to down-regulate the activity of the wnt signalling pathway, but also down-regulate the transcription of Wnt signalling genes. The results of these in vitro and immunohistochemical experiments warrant further investigation as to whether sFRP-4 expression can be indicative of prognosis in human breast cancer. In addition to breast cancer, we have also examined the role of sFRP-4 in other cancers such as ovarian and prostate.
005.Efficiency of nuclear import of the chromatin-remodelling factor SRY is critical for sex determination
Authors: Jans D. A. , Kaur G. , Poon I. K. H. , Delluc-Clavieries A. , Wagstaff K. M.
Published: November 2010

15% of cases of human XY sex reversal are due to mutations in SRY (sex determining region on the Y chromosome), many of which map to one of SRY’s two independently acting nuclear localization signals (NLSs) flanking its DNA binding domain. The C-terminal NLS (C-NLS) targets SRY to the nucleus through a ‘conventional’ pathway dependent on the nuclear import receptor importin-β (Imp-β). No importin has been shown to bind the N-terminal NLS (N-NLS), but it is known to interact with the Ca2+-binding protein calmodulin (CaM). We examined seven distinct missense mutations in the SRY NLSs from XY sex-reversed human females for effects on nuclear import and ability to interact with CaM/Imp-β1. All mutations were found to result in reduced nuclear localization in transfected testicular cells compared to wild type. The CaM antagonist, calmidazolium chloride (CDZ), was found to significantly reduce SRY nuclear accumulation, indicating a dependence of SRY nuclear import on CaM. Intriguingly, N-NLS mutants were resistant to CDZ’s effects, implying a loss of interaction with CaM; this was confirmed directly by in vitro binding experiments using recombinantly expressed protein. Either impaired CaM or Imp-β1 binding can thus be the basis of sex-reversal in human patients. Our results implicate a CaM-dependent nuclear import pathway for SRY mediated by the N-NLS that, together with the C-NLS, is required to achieve threshold levels of SRY in the nucleus for male sex determination.
027. In vitro growth and maturation: how does this technology fit for clinical application?
Authors: Lane M. , Yeo C. , Cashman K. S. , Hamilton H. M.
Published: November 2010

Superovulation protocols used in IVF result in multiple eggs that can be fertilized and grown in the laboratory to allow for selection of the best embryo for return to the mother, thereby increasing the chances for a successful pregnancy. However, there are many side effects of these superovulation drug protocols, such as deep vein thrombosis and hyperstimulation. The latter is of particular concern for women with polycystic ovary syndrome. Furthermore, the use of gonadotrophins has been reported to compromise both oocyte quality and the uterine environment and may contribute to the low success rates of IVF. Therefore the ability to collect large numbers of oocytes from women and mature them in vitro is an attractive alternative. However, although there are reports in the literature on extended maturation/culture periods of human oocytes the pregnancy rates are significantly lower than that observed after in vivo maturation. The ability to offer such technology is currently limited by the lack of understanding of how the conditions for in vitro maturation affect the quality of the oocyte and the resulting embryo. Our research is concentrated on establishing the role of metabolic balance in the oocyte for the maintenance of subsequent viability. We have determined that disruptions to the balance between mitochondrial and cytoplasmic metabolism in animal oocytes have significant adverse consequences for the resultant embryo. Changing conditions for in vitro maturation were also found to alter the establishment of the metabolic settings of the oocyte. The ability to determine the role of such parameters in maturing human oocytes will be important for the prospect of adoption of this technology for routine clinical practice.
028. Cytokine networks and regulation of spermatogenesis - what should we really believe?
Authors: Hedger M. P.
Published: November 2010

Spermatogenesis is a complex yet highly organised process involving intimate interactions between the supporting Sertoli cells and germ cells at various stages of development. The repeating pattern of the cycle of the seminiferous epithelium is due to the fact that spermatogonia enter spermatogenesis at regularly spaced intervals and proceed through the process at a species-specific rate. How this degree of coordination is maintained remains poorly understood, but recent evidence has focussed attention on the role of growth factors produced by the Sertoli cells and germ cells. Several of these growth factors, such as interleukin-1α (IL-1α), IL-6, tumour necrosis factor (TNFα) and activin A, are also inflammatory cytokines. This has led some researchers to question the physiological significance of these data with respect to normal testicular function. For example, in spite of the fact that IL-1α is produced by the Sertoli cell and regulates spermatogonial proliferation and development in vitro, mice lacking the IL-1R, and hence unresponsive to IL-1α, possess relatively normal fertility. So what role, if any, do these cytokines play in the normal testis, or are they only important during inflammation? It is quite evident that these cytokines have stimulatory and/or inhibitory effects on spermatogonial and spermatocyte development. These cytokines also interact at multiple levels within each other’s signalling pathways and have considerable redundancy of action. Moreover, expression of these cytokines varies across the cycle of the seminiferous epithelium, with major changes in production coinciding with two key events within the cycle: the release of sperm from the epithelium, and the major peaks of DNA synthesis by spermatogonia and preleptotene spermatocytes. It is therefore possible to hypothesise that release of sperm and resorption of the residual cytoplasm triggers a self-regulating inflammatory cascade within the epithelium that initiates and then modulates the next round of spermatogenic development, ensuring that spermatogonia enter the process at the appropriately spaced intervals.
036. Nutritional programming, fetal growth and competence for life after birth
Authors: McMillen C. , Duffield J. A. , Muhlhausler B. S.
Published: November 2010

During the past decade there has been a world-wide series of epidemiological and clinical studies, which have demonstrated that there are associations between prenatal growth restriction and the risk of insulin resistance, central obesity, hypertension, type 2 diabetes and cardiovascular disease in adult life. More recently there has also been increasing interest in the consequences of exposure of the fetus to increased levels of maternal nutrition and whether maternal ‘overnutrition’ may program an ‘intergenerational cycle of obesity’. In this presentation, we review recent experimental evidence that highlights the impact of varying levels of fetal nutrition on the structural and functional development of the adipocyte, and on expression of a range of appetite regulatory peptides within the developing brain. The importance of the timing of nutritional perturbations and the different consequences of fetal undernutrition and overnutrition on subsequent gene expression within different fat depots and on the expression of appetite stimulatory and inhibitory neuropeptides will be reviewed. The importance of defining those critical windows during an individual’s lifespan when nutritional or other intervention strategies will have the maximum benefit in preventing the development of obesity and cardiovascular disease will also be considered.
209. P-glycoprotein limits activation of the glucocorticoid receptor in placental BeWo cells
Authors: Mark P. J. , Waddell B. J.
Published: November 2010

Inadequate placental growth and function are key determinants of fetal growth retardation. Glucocorticoids potently inhibit fetal and placental growth via activation of the glucocorticoid receptor (GR). Placental and fetal glucocorticoid exposure is minimised by the ‘placental glucocorticoid barrier’, which consists primarily of placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) converting maternal glucocorticoids to inactive metabolites. Recent studies in the rodent brain show that P-glycoprotein (P-gp) is also an important physiological regulator of glucocorticoid access to the GR in target cells. Therefore, we hypothesised that placental P-gp may serve to exclude maternal glucocorticoids from the placenta and fetus, and thereby augment the barrier. We have used the placental choriocarcinoma cell line BeWo, and MDR-BeWo, a daughter cell line virally transduced with P-gp, to assess whether P-gp regulates access of dexamethasone to the GR.
Quantitative PCR analysis showed that MDR-BeWo cells express ~10-fold higher levels of P-gp mRNA than BeWo cells. Syncytialisation of BeWo and MDR-BeWo cells with 20μM forskolin also increased P-gp mRNA by ~7-fold in each cell line. The elevated P-gp expression in MDR-BeWo cells resulted in a reduced activation of the GR with 1μM dexamethasone by ~50% (P<0.001) in comparison to BeWo cells. Accordingly, dexamethasone-induced apoptosis was reduced in MDR-BeWo cells, as indicated by a lack of induction of cleaved caspase 3 protein. Additionally, the P-glycoprotein inhibitor cyclosporin A (10μM) did not increase the level of dexamethasone-induced GR activation in the low P-gp expressing BeWo cells, but potentiated GR activation by ~2-fold in the MDR-BeWo cells, to a level comparable to that in BeWo cells.
These data support the hypothesis that P-glycoprotein contributes to the placental glucocorticoid barrier. Thus, 11β-HSD2 and P-glycoprotein are likely to act in unison to reduce fetal and placental exposure to maternal glucocorticoids and minimise their growth inhibitory actions.
210. Localisation of insulin-like growth factor-II (IGF-II) and its receptor in early murine pregnancy: a role in placentation and angiogenesis in the decidua?
Authors: Pringle K. G. , Roberts C. T.
Published: November 2010

The highly invasive activity of the human placenta is tightly regulated by a variety of growth factors and other molecules. Contrary to the dominant view, recent data suggests that IGF-II, upon binding to the IGF2R, can stimulate an intracellular signalling pathway.1 Evidence in humans and mice suggests that IGF-II and the IGF2R are important regulators of placental growth; however, to date they have not yet been localised to early murine implantation sites. This study provides a photo micrographic account of early placental development and the decidual vasculature in the mouse, and localises IGF-II and the IGF2R from days 5.5 to 10.5 of pregnancy. During early pregnancy, the decidua displays a paucity of blood vessels, which appear to undergo angiogenesis, so that by day 10.5 the decidua has become a highly vascularized structure, with an extensive network of dilated vessels that presumably enable maximal blood supply to the placenta. Unlike humans, murine trophoblast cells do not invade the endometrium individually, but remain in close contact with the main giant cell layer. The trophoblast giant cells (TGCs) are the outermost cell type of the murine placenta and maternal blood spaces beneath this layer are not lined by endothelium. Due to their location, TGCs appear to play a direct role in displacing this endothelium and therefore may play a role in the transformation into trophoblast lined maternal blood spaces. IGF-II and its receptor were present throughout early pregnancy in the conceptus and maternal decidua supporting their role as regulators of fetal and placental development. Most interesting, however, was their association with the developing maternal blood vessels in the mesometrial decidua. It seems likely that in mice the maternal vessels are remodelled by a variety of locally derived molecules. By association, IGF-II and its receptor are likely candidates.
(1)McKinnon T, et al. (2001). Stimulation of human extravillous trophoblast migration by IGF-II is mediated by IGF type 2 receptor involving inhibitory G protein(s) and phosphorylation of MAPK. J. Clin. Endocrinol. Metab. 86(8), 3665–3674.
216. c-kit Expression study: timing of onset in rodent testis and irradiated rat testis model
Authors: Prabhu S. Mithra , Meistrich M. L. , Mendis S. , McLaughlin E. A. , Loveland K. L.
Published: November 2010

Primordial germ cell and spermatogonial cell function is essential for normal male fertility. These cells require Sertoli–germ cell interactions, specifically somatic cell-derived stem cell factor (SCF) that acts through the c-kit receptor to govern primordial germ cell migration in the foetus, spermatogonial differentiation during puberty and adulthood, and Leydig cell steroidogenesis. We performed a comprehensive study of the c-kit mRNA expression profile in the pre- and post-pubertal mouse and rat testes by in situ hybridisation. Expression of c-kit mRNA was first visualised in germ cells after birth, with the levels concordant with the number and appearance of the differentiated spermatogonial subtypes in both the rat and the mouse. We also studied c-kit expression in the irradiated adult rat testis, in which only undifferentiated spermatogonia are present. After treatment with Cetrorelix, GnRH antagonist (3 days, 1, 2 and 4 weeks) germ cell maturation is re-initiated. Expression of c-kit messenger RNA was observed in the undifferentiated spermatogonia in both untreated and treated testes sections. In contrast, c-kit protein expression was undetectable until 4 weeks of hormone treatment. This suggests that c-kit mRNA and protein expression are differentially regulated and that protein expression relates to somatic cell function.
218. Expression of Wsb2 in the mouse testis
Authors: Sarraj M. , McClive P. J. , Loveland K. L. , Sinclair A. H.
Published: November 2010

We present a detailed study on the expression pattern of Wsb2 in the mouse foetal and adult gonad. Wsb2 expression was analysed during mouse embryogenesis by whole-mount, section in situ hybridisation and immunohistochemistry. Wsb2 was found to be expressed in the developing mouse gonads from 11.5 dpc to 16.5 dpc. Expression is initially equal in both sexes from 10.5 dpc until 12.0 dpc, then it persists in the male gonad. Wsb2 expression was confined to the cords in both Sertoli cell and germ cells. Other sites of Wsb2 embryonic expression were the somites, dorsal root ganglia and the lateral mantle layer of the neural tube.
mRNA encoding Wsb2 and Wsb2 protein has been detected in the newborn testis in both gonocytes and Sertoli cells. Wsb2 mRNA in the adult mouse testis was observed in Sertoli cells, spermatogonia, spermatocytes and the corresponding Wsb2 protein expression was in pachytene spermatocytes, round and elongated spermatids, Sertoli cells and Leydig cells.
The differential expression of Wsb2 in male versus female embryonic gonads suggests it may play a role in mammalian sex determination during embryonic development and its expression in the first wave of spermatogenesis and in the adult suggests a later role in spermatogenesis.
220. Equine growth hormone enhances motility and extends longevity of ram spermatozoa in vitro
Authors: King B. J. , Da Silva V. V. , Harper J. D. , Scott C. J. , Sillence M. N.
Published: November 2010

Sperm survival in vitro decreases with time at room temperature, but may be improved by treatment with recombinant bovine growth hormone in rats1, bulls2 and horses3. Two experiments investigated the effect of equine growth hormone (eGH) on the longevity of ram spermatozoa in vitro. The aim of experiment 1 was to determine if the addition eGH would improve the motility of ram spermatozoa after 24 h and identify any interaction with semen dilution rates used for ram semen preservation. Semen was collected from five mature Merino rams. Ejaculates were assessed for good quality and were diluted 1+50, 1+4, 1+3, 1+2 (semen+diluent) with a Tris-based cryoprotectant. Aliquots from each ram were mixed with eGH to achieve a final concentration of 100 ng/mL eGH and stored at 20°C for 24 h. Motility of spermatozoa was then assessed manually. eGH improved the motility of spermatozoa at all dilution rates compared to controls (P < 0.0001) but most markedly in the 1 + 3 and 1 + 2 samples (42.6 ± 0.8% eGH v. 22.7 ± 2.6% control; 40.5 ± 1.4% eGH v. 22.7 ± 2.2% control, respectively, P < 0.01). The aim of experiment 2 was to determine the optimum eGH concentration for improving sperm motility. eGH was added to aliquots of diluted semen (1+3 dilution rate) to produce samples with final concentrations of 1000, 100, 10 and 1 ng eGH/mL. The samples were placed in a water bath at 20°C for 24 h at which time the motility of sperm was assessed as before. Sperm motility was higher in the 100 ng/mL eGH sample (P < 0.05; 39.6 ± 0.7%) compared to other concentrations (1000 ng/mL 11.8 ± 0.7%, 10 ng/mL 21.5 ± 0.7% and 1 ng/mL 11.3±0.7%). We conclude that growth hormone is effective in promoting the longevity in vitro of ram spermatozoa stored at room temperature, and that this effect is concentration dependent.
(1)Breier et al. (1996). Endocrinology 137, 4061–4064.(2)Sauerwein et al. (2000). Dom. Anim. Endocrinol. 18, 145–158.(3)Champion et al. (2002). Theriogenology 57, 1793–1800.
227. FSH receptor expression in small human ovarian follicles
Authors: Quennell J. H. , Stanton J. L. , Hurst P. R.
Published: November 2010

Follicle-stimulating hormone (FSH) is pivotal in ovarian follicle development; the granulosa cells are the targets of FSH action in the ovary via FSH receptors. Granulosa cell growth and division mark initial follicle recruitment. The acquisition of FSH receptors on granulosa cells is regarded as a key event in hormone responsiveness and consequently follicle development. Due to the low abundance of FSH receptors and low expression of its mRNA it has been difficult to definitively characterise FSH receptor expression patterns. Here, localisation of FSH receptor in different follicle populations has been assessed with in situ hybridisation and real-time PCR of laser microdissected samples. We have used non-radioactive in situ hybridisation to investigate FSH receptor mRNA on a wide range of follicle stages. Biopsies from healthy fertile women (28–33 years) were frozen, embedded and cryosectioned at 10 µm. DIG-labelled RNA probes were designed to detect all splice variants. Hybridised probes were detected with NBT/BCIP in a colorimetric reaction. Secondly, follicles of different morphometric stages were isolated with a laser microscope. RNA extraction, reverse transcription and real-time PCR were used to confirm RNA presence and quantify relative expression. All follicle stages (from primordial to large antral) showed the presence of FSH receptor mRNA in their granulosa cells; sense controls were negative. Observations from real-time PCR indicate FSH receptor mRNA is present in all follicle stages observed and relative expression levels increase over early follicle development. These results challenge the existing doctrine that FSH receptor is absent in the smallest follicles. This suggests initial follicle recruitment may involve gonadotrophins. The use of sensitive molecular techniques will be crucial in elucidating this further.
228. In vivo evidence for a role of bone morphogenetic protein-4 in ovarian function
Authors: Tanwar P. S. , McFarlane J. R.
Published: November 2010

Bone morphogenetic proteins (BMPs) were first identified on the basis of their bone inducing capacity, and later shown to be members of the transforming growth factor β (TGF β) super family. Nilsson et al.1 studied the effect of BMP-4 on follicular development in rat ovaries and found that the addition of BMP-4 to whole ovary cultures led to more numbers of developing primary follicles but less numbers of primordial follicles. Their studies indicate that BMP-4 acts as a transition factor for the conversion of primordial follicles to primary follicles. To test this hypothesis in-vivo, we conducted passive immunization studies against BMP-4 in prepubertal female mice.
The mice were divided in to four groups (n = 5), and given daily SC injections of the following treatment: anti BMP-4 (50μg), PMSG (10 IU) (pregnant mare serum gonadotropin) with and without anti BMP-4 (0.5 mg/mL) and PBS for 3 days. All experimentation was approved by animal ethics committee, University of New England, Armidale, NSW. On the fourth day the mice were killed and the ovaries removed and weighed. The mice treated with anti BMP-4 had significantly smaller ovaries (4.1 ± 0.4 mg) than the control group (8.6 ± 0.9 mg). PMSG stimulated ovarian weight (21.0 ± 1.2 mg) but anti BMP-4 (23.2 ± 1.3 mg) did not significantly affect the weight of the stimulated ovaries. This data confirms BMP-4 is important in ovarian function; however, it is unclear whether this effect is on the ovary directly or via FSH.
(1)Nilsson, E. E., Skinner, M.K. (2003). Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development. Biology of Reproduction 69, 1265–1272.
233. Oxygen concentration during in vitro maturation of murine oocytes affects blastocyst cell lineage
Authors: Banwell K. M. , Lane M. , Russell D. L. , Kind K. L. , Thompson J. G.
Published: November 2010

Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% O2 and while low O2 has been shown to be beneficial to embryo development in many species, the effect of altering O2 concentration during IVM has not been adequately investigated. Here we investigated the effects of a range of O2 concentrations during IVM on meiotic maturation and subsequent embryo development after IVF. Ovaries from eCG-stimulated CBA F1 female mice (21 days) were collected and intact cumulus oocyte complexes (COCs) cultured for 17–18 h under 2, 5, 10 or 20% O2 (6% CO2 and balance of N2). Matured COCs were denuded of cumulus cells, fixed and stained (1% aceto-orcein) for visualisation of maturation status. No significant difference in maturation rates between treatment groups was observed. Following IVF (performed under 5% O2, 6% CO2 and balance of N2), no difference in fertilisation rates between treatment groups was observed in a randomly selected cohort 7 h post-fertilisation. There was also no significant difference in cleavage rates after 24 h or ability to reach blastocyst stage after 96 h, with a tendency (P = 0.079) for more blastocysts in 2% O2. However there was a significant increase in the number of trophectoderm cells present in the resulting blastocysts (P < 0.05) in the 2% O2 group (35 ± 2.1) compared to 20% O2 (25 ± 2.8). Our data suggests that O2 concentration during IVM does not influence nuclear maturation or subsequent fertilisation, cleavage and blastocyst development rates. However, maturation in 2% O2 significantly alters subsequent cell lineage within blastocysts to favour trophectoderm development. Such skewed trophectoderm cell number may influence embryo viability.
Funded by NHMRC and NIH.
240. Spatial and temporal expression pattern of furin in the human endometrium
Authors: Freyer C. , Kilpatrick L. , Salamonsen L. , Nie G.
Published: November 2010

Furin is a proprotein convertase (PC) implicated in the endoproteolytic maturation of inactive protein precursors of growth factors, hormones, receptors, and viral envelope glycoproteins.1 Two functionally active forms of furin, one membrane-bound containing a C-terminal transmembrane domain (TD) and a cytoplasmic tail (CT), and one soluble without the TD and CT, have been characterised. We have previously shown that PC6, one of the PCs closely related to furin, is expressed in the human endometrium and is closely associated with decidualization of stromal cells during implantation.2 Although furin is ubiquitously expressed, its expression in the human endometrium is unknown. In this study, we investigated the spatial and temporal expression pattern of furin in the human endometrium using RT-PCR and immunohistochemistry. While furin expression is detected throughout the menstrual cycle and during early pregnancy, lowest mRNA levels are seen during the proliferative phase. Using an antibody directed against the C-terminus of the membrane bound form, furin is detected in the stroma, glandular and luminal epithelium, as well as in endothelia and neutrophils throughout the menstrual cycle and during early pregnancy. In the stroma, highest levels of furin are present during menstruation (n = 3), they are also high during the proliferative phase (n = 4), but significantly lower levels are detected during the secretory phase (n = 10, P < 0.05, Tukey HSD). In the first trimester decidua, furin is present in well decidualised stromal cells. The overall expression pattern of furin is different to that of PC6; in particular, furin expression is associated only with well decidualized stromal cells whereas PC6 is involved in the initial stages of decidualization. These data suggest that furin and PC6 play different roles in the human endometrium, especially during embryo implantation.
(1)Nakayama K. (1997). Biochem. J. 327, 625–635.(2)Nie et al. (2005). Biol. Reprod. 72, 1029–1036.
245. Endometrial changes in a mouse model for long-term progestin exposure: implications for break-through bleeding
Authors: Morison N. B. , Shen J. , Zhang J. , Kaitu'u T. J. , Salamonsen L. A.
Published: November 2010

A mouse model of menstruation1 was modified to evaluate the effects of long-term progestagens on the endometrium. Many women using long-acting progestin (P)-only contraceptives experience break-through bleeding (BTB) and this is the main reason for discontinuation of their use by 30% of users. To understand the mechanisms involved in BTB, ovariectomised mice treated with a strict steroid hormone regime, had etonogestrel- or levonorgestrel-releasing implants inserted subcutaneously. A decidualizing stimulus (oil) was administered into one uterine horn; decidualisation was maximal 48 h later, designated day 0 (d0). The implant was left in place for a further 45 days. Uteri were collected at 5-day intervals and analysed for morphological changes, leukocyte infiltration, and matrix metalloproteinase (MMP) expression.
In uteri from mice treated with etonogestrel and examined at d0, decidualisation was maximal and very few leukocytes were present (neutrophils, NEU; Macrophages, Mac; uterine natural killer cells, uNK). Some loss of tissue integrity was seen at 5d, associated with some MMP-9 positive leukocytes. However, substantial stromal breakdown was observed at 10d and this progressed until 25d. A large infiltrate of NEU and uNKs, as well as increased MMP-9 expression, was observed at these times. At 25d there was evidence of re-epithelialisation and by 45d the tissue had fully repaired back to its pre-decidualised state. Re-epithelialisation was associated with a decline in NEU and uNKs, as well as MMP-9 positive immunostaining. The morphology of uteri from mice treated with levonorgestrel implants was compared to mice treated with etonogestrel. The changes observed were very similar with a loss of tissue integrity at 5d and tissue destruction at 10d.
This mouse model mimics the changes in leukocyte populations and MMP expression observed in endometria of some women using P-only contraceptives.
(1)Brasted M, White CA, Kennedy TG, Salamonsen LA. (2003) Biololgy of Reproduction 69(4), 1273–1280.
248. Oestrogen receptor beta is involved in the regulation of Leydig cell number in the mouse
Authors: Gould M. , Nicholson H. D.
Published: November 2010

Recent evidence suggests that oestrogen plays a physiological role in the testis. Both oestrogen receptor alpha and oestrogen receptor beta (ERb) are present in the testis and administration of oestrogen has been shown to inhibit the development of Sertoli, Leydig and germ cells. This study investigates the effect of ERb on the testis using ERb knockout mice (bERKO). Adult male bERKO mice (n=8) and their wild-type littermates (n=7) were killed at 11 weeks postpartum. One testis from each animal was fixed in Bouin’s fluid and embedded. Each testis was fractionated and thick sections cut and stained with PAS. The optical disector method was used to count the number of Leydig cells, Sertoli cells, spermatogonia, spermatocytes and spermatids in each testis. Trunk blood was collected and plasma testosterone concentrations measured by radioimmunoassay. No significant differences in body or testis weight were seen between the bERKO or wild-type mice. Similar numbers of Sertoli cells, spermatogonia, spermatocytes and spermatids were also observed between the two groups. The number of Leydig cells was significantly increased in bERKO mice compared with their wild-type littermates (P < 0.05). Despite the increased number of Leydig cells in the bERKO mice there was no significant difference in plasma testosterone concentrations in this group compared to the wild-type mice. Oestrogen has been reported to inhibit proliferation of adult-type Leydig cells and to inhibit steroidogenesis. This study suggests that the regulation of Leydig cell proliferation may be mediated by ERb. The presence of normal circulating testosterone concentrations in bERKO mice suggests that the effects of oestrogen on steroidogenesis are not brought about by ERbeta.
252. Morphometric and histological analysis of ovaries from sheep heterozygous for the prolific woodlands allele
Authors: Feary E. S. , Juengel J. L. , Smith P. , O'Connell A. R. , Davis G. H. , McNatty K. P.
Published: November 2010

Woodlands are a line of Coopworth sheep with a novel, imprinted X-linked fecundity allele resulting in ovulation rates about 0.40 higher than wild-type animals. Daughters of progeny tested sires with and without the gene were studied.
Previously, lambs heterozygous for the Woodlands allele were found to have larger ovaries and more antral (i.e. type 5) but not preantral (i.e. types 1–4) follicles than in wild-type contemporaries. The large ovary phenotype was found to be transient and was absent after puberty. However, based on follow-up studies it was evident that the large ovary phenotype was not strongly associated with the Woodlands fecundity allele. Thus, it was uncertain whether animals carrying the Woodlands gene had different follicular populations compared to wild-type controls. To address this question, follicular populations were compared in adult ewes heterozygous for the Woodlands allele with age-matched controls. Using standard morphometric methods and histological analysis, no differences were observed in the mean numbers of types 1, 1a, 2, 3 and 4 preantral follicles between the genotypes. Furthermore, no differences were observed between genotypes in follicular or oocyte diameters for any follicular type. The adult Woodlands carrier ewes had twice as many small type 5 follicles (< 1mm) when compared to wild-type contemporaries although no difference was seen in the numbers of antral follicles > 1mm in diameter. In addition, antrum formation occurred at a smaller follicular diameter in the heterozygous Woodlands animals.
Therefore, the increased number of antral follicles observed in both lambs and adult ewes suggests that this difference in pattern of follicular development is associated with the X-linked fecundity allele. This novel phenotype of early antrum formation and larger number of small preantral follicles differs from that observed in sheep with the Inverdale or Booroola mutations, suggesting that a different mechanistic pathway is involved.
Acknowledgements: The Marsden Fund, FRST and Ovita.
259. Reproductive phenotype of the female aromatase overexpressing mouse
Authors: Liew J. , Drummond A. E. , Jones M. E. , Poutanen M. , Findlay J. K.
Published: November 2010

Aromatase, the product of the Cyp 19 gene, converts androgens to estrogens. The role of estrogens within the ovary has recently been revisited; using the aromatase knockout (ArKO) mouse, we investigated the effect of estrogen deficiency on ovarian function. We now have an aromatase overexpressing (AROM+) female mouse model with elevated levels of estrogens. These mice were fertile and bred with FVB/N wildtype (WT) males, the AROM+ male being infertile. In this study we characterised the reproductive phenotype of the female AROM+ mouse.
5 WT and 10 AROM+ mice, 22–27 weeks of age were used in the study. The mice were subject to vaginal smears and killed during estrus. The ovaries, uterine horns and gonadal fat were collected and weighed. One ovary and the uterine horns were fixed in formalin for histological assessment, while the other ovary was snap frozen in Ultraspec solution for RNA isolation and gene expression studies. Serum was collected for hormone measurements.
All AROM+ mice exhibited an abnormal pattern of cycling that in general, alternated between estrus and post-estrus. AROM+ mice were significantly heavier than their WT counterparts (WT 35.28 ± 2.89 g v. AROM+ 43.38 ± 2.11 g, P < 0.05). Ovarian, uterine and gonadal fat pad weights were not significantly different between the 2 groups (ovary: WT 17.4 ± 1.14 mg v. AROM+ 17.9 ± 0.06 mg; uterine horns: WT 89.7 ± 11.40 mg v. AROM+ 92.1 ± 6.64 mg; gonadal fat pads: WT 2.47 ± 0.62 g v. AROM+ 3.46±0.26 g). Histological, gene expression and hormone analyses are in progress.
Our preliminary analyses indicated no significant effect of excess estrogen on ovarian, uterine and gonadal fat pad weights, despite the AROM+ mice being heavier. It remains to be determined as to whether the ovaries and uterine horns are histologically normal.
Supported by the NHMRC (Regkeys 241000, 338510, 198705)
263. The successful use of busulfan to deplete endogenous spermatogonia in ram testes
Authors: Olejnik J. A. , Herrid M. , Davey R. , Hutton K. , Hinch G. , Hill J.
Published: November 2010

Our research into germ cell transfer builds upon murine research1 and is aimed at using this technique in livestock species. To increase the efficiency of colonization of transplanted germ cells, the recipient testes must be depleted of endogenous spermatogonia, without affecting the supporting cells. Three depletion methods were investigated, heat, cold and chemotherapy.
Our first investigation looked at the direct cooling (0ºC) and heating (45ºC) of the testes of 4–6 week old ram lambs. Testes were collected 7 days post treatment.
The second investigation involved the systemic injection of busulfan to ram lambs aged 3-4 months. Busulfan is used for preparing recipient mice for testes cell transfer.1 At doses affecting the stem cells of the testes, busulfan will result in mylosuppression. Therefore a preliminary dose response trial was conducted at dose rates 4, 8 and 16mg/kg to determine the most effective dose, without threatening the survival of the animal. Testes were recovered after 3 and 6 weeks. All testes sections followed routine histology and immunochemistry with PGP 9.5 (Table 1).
For the heat and cold study, only gonocytes were present and there were no differences in testes weights, tubule diameters or gonocyte numbers in any of the treatment groups. For the busulfan study, dose rates of 8 and 16 mg/kg resulted in severe mylosuppression and euthanasia of 7 out of 8 animals between day 12 and 18, whereas animals in the 4mg/kg group showed only mild clinical effects, that were not life threatening.
These results indicate that busulfan reduced endogenous spermatogonia in the pre-pubertal ram. This effect is observed at systemic doses of 4 mg/kg or higher; however, doses of 8 mg/kg and above are lethal to the survival of the animal. The use of direct heat (45ºC) or cold (0ºC) to the testes does not affect gonocyte numbers in ram lambs; however, effects on more mature stages was not studied.
(1)Brinster RL, Zimmermann JW. (1994) Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA 91, 11298–11302.
271. Prospective isolation of human endometrial mesenchymal stem cells using CD146 and platelet-derived growth factor receptor-β
Authors: Schwab K. E. , Gargett C. E.
Published: November 2010

The human endometrium has an immense regenerative capacity. Previously we identified a small population of clonogenic endometrial stromal cells or mesenchymal stem cells (MSC).1 Prospective isolation of MSC allows for their characterisation. We hypothesise that the expression of MSC marker, CD146, and pericyte/fibroblast marker, platelet-derived growth factor receptor-β (PDGFRβ), will enable the prospective isolation of endometrial MSCs. The aims of this study were to (1) determine if CD146 and PDGFRβ will prospectively isolate endometrial MSCs with clonogenic activity, (2) identify their location in human endometrium, and (3) determine the differentiation capacity of CD146+PDGFRβ+ stromal cells.
Endometrial tissue from 13 ovulating women undergoing hysterectomy was digested with collagenase to produce single cell suspensions. Leukocytes and epithelial cells were removed. Stromal cells were analysed by flow cytometry, FACS sorted into enriched and depleted populations, and cultured for clonal analysis.1 Immunohistochemistry was performed on full thickness human endometrium. Sorted populations of stromal cells were passaged for culture in various differentiation media, and analysed for adipogenic, myogenic, chondrogenic or osteogenic differentiation by histological stains and RT-PCR.
A small, consistent population of CD146+ endometrial stromal cells was identified (7.8 ± 1.1%, n = 8). In contrast, PDGFRβ expression varied (34.1 ± 9.7%, n = 5), and 2.5% of cells were CD146+PDGFRβ+. Clonogenicity of CD146+ stromal cells was significantly higher than CD146- stromal cells, 2.5 ± 1.1% and 1.2 ± 0.6%, respectively (n = 6, P = 0.03). CD146+ stromal cells were located perivascularly, similar to bone marrow MSCs, whereas PDGFRβ weakly stained the stroma, with stronger staining observed around the blood vessels. CD146+ cells differentiated into adipocytes, smooth muscle cells, chondrocytes and osteoblasts.
This study identified CD146 as a marker of clonogenic endometrial stromal cells, and supports the perivascular location of endometrial MSCs. It also demonstrated that CD146+ cells can differentiate into four mesenchymal lineages. These data suggest that CD146 can be used for the prospective isolation of endometrial MSCs, which may be further enriched by PDGFRβ co-expression.
(1)Chan RW, Schwab KE and Gargett CE (2004) Biology of Reproduction 70, 1738.
272. Progesterone stimulates endothelial cell proliferation, but not stromal cell proliferation, in mouse endometrium
Authors: Walter L. M. , Rogers P. A. W. , Girling J. E.
Published: November 2010

Previous studies have suggested that progesterone stimulates stromal cell (SC) proliferation in the mouse endometrium1. However, these studies have not differentiated endothelial cells (EC) from other SC. In this study, we investigated the effects of progesterone on cellular proliferation in ovariectomised mouse endometrium. We hypothesised that progesterone would stimulate both SC and EC proliferation. One group of CBA × C57 mice (n = 6) were treated with a single injection of 100 ng of estradiol on day eight following 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 on day 13 after ovariectomy, 4 h following a BrdU injection. CD31/BrdU double staining immunohistochemistry allowed proliferating EC to be differentiated from proliferating SC. Mice treated with progesterone only had significantly higher EC proliferation in comparison to females treated with progesterone following oestrogen priming (P = 0.05) or vehicle only (P = 0.01) (progesterone only: median=97.3 proliferating EC (PEC)/mm2 [range = 60.8–203.4]; oestrogen plus progesterone: 41.0 PEC/mm2 [8.9–86.9]; vehicle only: 0.0 PEC/mm2 [0.0–3.1]). Unexpectedly, there was no significant difference in SC proliferation among the treatment groups (progesterone only: 50.1 PSC/mm2 [39.2–102.6]; oestrogen plus progesterone: 46.1 PSC/mm2 [12.6–120.8]; vehicle only: 44.8 PSC/mm2 [17.3–68.4]). To determine if VEGF had a role in the progesterone-induced EC proliferation, the previous experiment was repeated with the inclusion of mice treated with VEGF anti-serum. The addition of VEGF anti-serum significantly inhibited progesterone-induced EC proliferation (46.8 PEC/mm2 [38.9–128.0]; P = 0.04], but had no effect on SC proliferation (P = 0.3). These results demonstrate that progesterone stimulates endometrial EC proliferation, but not SC proliferation as reported by earlier studies1. Studies are currently underway to further investigate the role of VEGF in mediating progesterone effects on endometrial EC.
(1)Clarke, C.L. and Sutherland, R.L. (1990) Endocrine Reviews 11, 266–301.
278. Repeated 24-hour exposure of ewes to rams during the transition into breeding season compacts the mating of the flock
Authors: Hawken P. A. R. , Evans A. C. O. , Beard A. P.
Published: November 2010

Synchronisation of oestrus during the breeding season is currently only possible with exogenous hormones, but these practices are not sustainable with growing consumer demand for ‘clean, green and ethical’ products.1 Therefore non-pharmacological methods of oestrus control need to be developed. Exposure of anoestrous ewes to rams induces a synchronous ovulation, but this ‘ram effect’ is not thought to work in the breeding season. This experiment tested whether repeated 24-h exposure of ewes to rams during the transition into the breeding season would compact the subsequent distribution of mating. This experiment used two groups of ewes: Group VR (n = 103) was exposed to 3 vasectomised rams for 24 h on Days 0 (September 10th), 17 and 34 of the study, while Group VC (n = 106) remained isolated from rams. On Day 50, raddled entire rams were introduced (ERI) and marks recorded daily. Blood was sampled from a subgroup of 35 ewes per treatment twice weekly for progesterone. At mating, synchrony scores were calculated for each group (lower score = greater synchrony). VR ewes mated 5 days (median) after ERI whereas VC ewes mated after 6 days (interquartile range: 2–6 and 3–8; P < 0.001). VR ewes had a lower synchrony score than VC ewes (2.34 v. 3.08; P < 0.01) and less variance around the median time of mating (Levene’ test; P < 0.05) indicating a more compact mating (Fig. 1). The median onset of the breeding season was earlier in VR ewes (median: October 7th; interquartile range: 3rd–7th) than VC ewes (median: October 10th; interquartile range 7th–10th; P < 0.01). The enhanced synchrony in VR ewes was not solely due to a more synchronous onset of the breeding season. In conclusion, repeated short-term ram exposure during the transition into the breeding season is a potential non-pharmacological method of oestrus synchronisation during the breeding season.
280. Photo inhibited heat shock protein 108 gene expression in the chicken hypothalamus
Authors: Graham G. K. , Boswell T. , Li Q. , Wilson P. , Talbot R. , Downing A. , Sharp P. J.
Published: November 2010

In domestic juvenile chickens kept on short days, photoinduced luteinsing hormone (LH) release, and by inference gonadotrophin-releasing hormone (GnRH) release, are readily detectable within 4 days of photostimulation.2 The molecular mechanisms responsible for the rapid photoinduced release of LH and GnRH in avian species are unknown. It has been suggested that it might involve a cascade of gene expression associated with an increase in cfos in the basal hypothalamus and glial cells in the median eminence.1 A microarray was made consisting of known genes of interest and clones obtained from a hypothalamic short day/long day subtractive library. An experiment was undertaken to determine if this reproductive neuroendocrine microarray could detect new targets for study in the chicken model of photostimulated GnRH release. The microarray was interrogated with hypothalamic RNA from juvenile chickens showing an increase in plasma LH after 4 days of photostimulation. Six genes were identified as showing changes in expression after photostimulation on the microarray. However, only one gene, encoding heat shock protein 108 (HSP108), could be confirmed by quantitative competitive RT-PCR. The expression of this gene decreased both in the hypothalamus and the optic tectum. Treatment of short day juvenile chickens with thyroxine, to mimic the effects of photostimulation, resulted in LH release and depression of HSP108 expression in the anterior but not the basal hypothalamus. Immunocytochemical analyses showed that HSP108 is widely distributed in the brain including glial-like cells with terminals in the median eminence. HSP108 is suggested as a candidate protein involved in the regulation of gonadotrophin release from the median eminence by glial cells.
(1)Meddle SL and Follett BK (1995) Photoperiodic activation of fos-like immunoreactive protein in neurones within the tuberal hypothalamus of Japanese quail. Journal of Comparative Physiology [A] 176(1), 79–89.(2)Sreekumar KP and Sharp PJ (1998) Ontogeny of the photoperiodic control of prolactin and luteinizing hormone secretion in male and female bantams (Gallus domesticus). General and Comparative Endocrinology 109(1), 69–74.