bluuebunny/crossref_metadata_2025 huggingface dataset:
119. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA
Authors: Redgrove K. A. , Nixon B. , McLaughlin E. A. , O'Bryan M. K. , Aitken R. J.
Published: November 2010

A unique characteristic of mammalian spermatozoa is that upon ejaculation, they are unable to recognise and bind to an ovulated oocyte. These functional attributes are only realised following the sperms ascent of the female reproductive tract whereupon they undergo a myriad of biochemical and biophysical changes collectively referred to as ‘capacitation’. Since spermatozoa are both transcriptionally and translationally quiescent cells, this functional transformation must be engineered by a combination of post-translational modification and spatial reorganisation of existing sperm proteins. Indeed, evidence from our laboratory suggests that a key attribute of capacitation is the remodeling of the sperm surface architecture leading to the assembly and / or presentation of multimeric sperm-oocyte receptor complex(es). Through the novel application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have secured the first direct evidence that human spermatozoa express a number of these protein complexes on their surface. Furthermore, we have demonstrated that a subset of these complexes harbour putative zona adhesion proteins and display strong affinity for solubilised zona pellucidae. In this study, we have extended our findings through the characterisation of one such complex containing arylsulfatase A (ASA), a protein with recognised affinity for sulfated ligands present within the zona pellucida. Through the application of immunohistochemistry and flow cytometry we revealed that ASA undergoes a capacitation-associated translocation to become expressed on the apical region of the human sperm head, a location compatible with a role in the mediation of sperm-zona pellucida interactions. This dramatic relocation was completely abolished by incubation of capacitating spermatozoa in exogenous cholesterol, suggesting that it may be driven in part by alteration in the membrane fluidity characteristics. Our current research is focused on confirming the role of ASA in human sperm-zona pellucida adhesion and elucidating the precise cellular mechanisms that underpin the proteins translocation to the cell surface.
120. SPRASA A POTENTIAL CONTRACEPTIVE VACCINE TARGET?
Authors: Chamley L. W. , Wagner A. , Shelling A. N.
Published: November 2010

A number of attempts have been made to develop immunocontraceptive vaccines for use in humans or animals. We have reported the discovery of a sperm-specific protein, SPRASA, as the antigen for antisperm antibodies in some infertile men [1]. More recently we found that SPRASA is also expressed by oocytes. The expression of SPRASA by both sperm and oocytes makes it a potential target for an immunocontraceptive vaccine that might function in both males and females. We undertook this study to investigate whether immunising mice against SRPASA would affect fertility. Ethical approval was obtained from the Animal Ethics Committee. Two groups of five female CD1 mice were immunised, up to four times, with recombinant human SPRASA or with keyhole limpet haemocyanin (KLH, controls). After immunisation, the females were date-mated with males of proven fertility and monitored for coital plugs. Weights were monitored for 12 days post mating to confirm pregnancy or lack of pregnancy. Mice that were not pregnant were again dated-mated. Mice that were pregnant were euthanased and their litters examined. Ovaries from three SPRASA and three control immunised mice were sectioned to completion and examined for follicles of various stages. All of the control KLH immunised female mice became pregnant within two matings. In contrast, the SPRASA-immunised females were profoundly infertile and none of them became pregnant despite being mated no fewer than five times each. There were no significant differences in the numbers of primary, early antral, antral or pre-ovulatory follicles between the SPRASA and control mice but there were fewer primordial in the SPRASA immunised females (P = 0.0028). Antisperm (and antioocyte) antibodies have long been known to be associated with infertility. Our results show SPRASA appears to be crucial for fertility and which may be a useful target for a contraceptive vaccine.
(1) Chiu, W.W., et al., SPRASA, a novel sperm protein involved in immune-mediated infertility. Hum Reprod, 2004. 19(2): 243–9.
121. SHORT TERM XENOBIOTIC EXPOSURE COMPROMISES LONG TERM OOCYTE VIABILITY
Authors: Sobinoff A. P. , Pye V. , Nixon B. , Roman S. D. , McLaughlin E. A.
Published: November 2010

Mammalian females are born with a finite number of non-renewing primordial follicles, the majority of which remain in a quiescent state for many years. These follicles serve as the primary source of all developing oocytes in the ovary, and cannot be regenerated post fetal development. Due to their non-renewing nature, these “resting” oocytes are particularly vulnerable to environmental and toxic insults, especially to those which are capable of inducing oxidative stress. Recent evidence suggests that certain synthetic chemical compounds, known as xenobiotics, have the potential to generate oxidative stress through the production of free oxygen radicals (ROS) as a byproduct of the cell’s detoxification process. Given the redox sensitive nature of the mammalian oocyte, we hypothesise that xenobiotic exposure may have adverse effects on long term oocyte viability. In this study, we attempted to identify the effects of short term xenobiotic exposure on long term oocyte viability. Female Swiss neonatal mice (day 4) were administered 7 daily consecutive doses of 4-Vinylcyclohexene diepoxide (40mg/kg/daily; 80mg/kg/daily) Methoxychlor (50mg/kg/daily; 100mg/kg/daily) or Menadione (7.5mg/kg/daily; 15mg/kg/daily). Mice were then superovulated at 6wks and their oocytes collected for analysis. Sperm-egg fusion assays revealed a significant decrease (P < 0.01) in sperm egg binding (1.4–7 fold) and fusion (4–20 fold) in a dose dependent manner for all three xenobiotic treatments in vivo, signifying a decrease in oocyte membrane fluidity. Follow-up lipid peroxidation analysis on xenobiotic cultured oocytes also showed a significant (P < 0.01) dose dependent increase (1.3–2.5 fold) in membrane lipid peroxidation for each xenobiotic compared to the control. These results provide some of the first evidence of short term xenobiotic exposure causing long term oocyte dysfunction, possibly interfering with the fluidity and/or elasticity of the oocyte plasma membrane through xenobiotic ROS induced lipid peroxidation.
122. PRODUCTION OF EMBRYOS IN SUPEROVULATED EWES USING FROZEN - THAWED, SEX-SORTED AND REFROZEN - THAWED SPERM
Authors: Beilby K. H. , Kaurivi Y. B. , Maxwell W. M. C. , Evans G. , De Graaf S. P. , Grupen C. G.
Published: November 2010

Flow cytometric sex-sorting of sperm that has previously been cryopreserved allows sex-sorting technology to be applied more widely. While offspring have been produced following artificial insemination of synchronised ewes with frozen-thawed, sex-sorted and refrozen-thawed (FSF) sperm (1), the fertility of FSF-sperm in superovulated ewes has not been reported. The aim of this study was to determine the effect of cryopreservation prior to sperm sex-sorting and freezing on embryo production in superovulated ewes. Several ejaculates from 2 rams were either frozen-thawed, then sex-sorted and re-frozen (FSF X- and Y-chromosome enriched sperm), or immediately sex-sorted before freezing (SF X- and Y-chromosome enriched sperm). A portion of each ejaculate was also cryopreserved without sex-sorting (control). Thirty-one ewes were superovulated and inseminated (15 ×106 sperm per insemination dose) with either SF X, SF Y, FSF X, FSF Y or control sperm as previously described (2). Embryos were recovered 6 d after insemination and assessed. The superovulatory response (mean number of corpora lutea per ewe: 11.8 ± 1.3) and the embryo recovery rates (72.0 ± 5.9%) did not differ significantly among the groups. The fertilisation rates tended to differ (P=0.068) as a result of sperm treatment (control: 33%; SF: 54%; FSF: 18%) and were unaffected by sperm sex (X: 33%; Y: 37%). Of the embryos that were recovered, those derived from FSF-sperm were predominantly at the blastocyst stage (65%), whereas those derived from SF-sperm were evenly distributed among the blastocyst (30%), morula (38%) and arrested (32%) stages, suggesting that fertilisation lifespan of SF-sperm was greater than that of FSF-sperm. A greater proportion of embryos derived from Y-sperm were at the blastocyst stage compared with embryos derived from X-sperm (53% vs. 26%; P < 0.05). This study is the first to demonstrate that FSF-sperm is capable of fertilising oocytes of superovulated ewes.
(1) de Graaf et al (2007) Theriogenology 67: 391–8.(2) de Graaf et al (2007) Theriogenology 67: 550–5.
123. THE EFFECT OF PATERNAL DIET INDUCED OBESITY ON SPERM CAPACITATION, ACROSOME REACTION, BINDING AND FERTILISATION IN MOUSE MODEL
Authors: Bakos H. W. , Palmer N. O. , Lane M.
Published: November 2010

While the effects of obesity on male fertility are emerging, the direct effects on sperm function are less clear. The aim of this study was to determine the effects of diet-induced obesity on sperm capacitation, acrosome reaction, oocyte binding and fertilisation. C57/Bl6 male mice (n = 12) were randomly allocated to two groups; group 1 received a control diet (6% fat) while group 2 received a high fat diet (HFD, 22% fat) for up to 14 weeks. Mice were sacrificed and spermatozoa obtained. Capacitation and acrosome reaction were measured using Arachis hypogaea (peanut) agglutinin. Sperm binding to oocytes was assessed by co-incubation of sperm with superovulated cumulus-enclosed oocytes for 4 hrs. Fertilisation rates were expressed as the percentage of oocytes with 2 pronuclei from the total number inseminated 6 hrs post insemination. The percentage of non-capacitated sperm in males fed a high fat diet was significantly lower compared to males fed a control diet (12.3% vs 21.1%; P < 0.01). The percentage of acrosome reacted sperm did not differ between the groups. Following 4 hrs of co-incubation with cumulus-enclosed oocytes, the number of sperm bound to each oocyte was significantly lower in the HFD group compared to controls (41.14±2.5 vs 58.39±2.4; P < 0.01). Moreover, the percentage of fertilized oocytes was significantly lower in the HFD group compared to controls (25.9% vs 43.9; P < 0.01). This study demonstrates that males fed a HFD to induce obesity have impaired spermatozoa as evidenced by lower levels of capacitation and reduced ability to bind and fertilise an oocyte. These data therefore provide direct evidence that the metabolic health of the male can have a significant impact on sperm function parameters that are associated with infertility.
124. THE ROLE OF CALCIUM ACTIVATED CHLORIDE CHANNELS AT FERTILISATION
Authors: Dalati S. , Day M. L.
Published: November 2010

Sperm entry into the oocyte triggers a signal transduction pathway resulting in intracellular calcium [Ca2+] oscillations that coincide with hyperpolarisations in membrane potential (Em). Ca2+ oscillations have been previously described and found to be important for embryo development, yet Em hyperpolarisations and their importance at fertilisation still remains unclear. Thimerosal, a sulfhydryl reagent, has been shown to mimic the physiological changes caused by sperm following fertilisation. It does this by direct sensitisation of the inositol 1,4,5-triphosphate receptor-1 to basal levels of inositol 1,4,5-triphopshate. Previous patch clamp analysis of unfertilised mouse oocytes has shown that thimerosal elicits simultaneous Em hyperpolarisations and Ca2+ oscillations. These results have lead us to hypothesise that hyperpolarisations in Em may be due to the activation of a Ca2+ activated Cl- channel (CaCC) present in the membrane of mouse oocytes. The present study aims to identify this CaCC and assess its role in early development following fertilisation. Hyperpolarisations induced by thimerosal were inhibited by niflumic acid, a selective blocker of CaCCs. The inhibition of Em hyperpolarisations suggests that a CaCC is present and plays an active role in initiating hyperpolarisations. To identify the function of the CaCC at fertilisation, in vitro fertilisation was performed in the presence of niflumic acid. Niflumic acid inhibited polar body extrusion and pronuclei formation; two events that are indicators of fertilisation. Furthermore, Ca2+ imaging experiments with the calcium sensitive dye fura 2-AM, demonstrated that in the presence of niflumic acid, Ca2+ oscillations induced by thimerosal are reduced in size, number and duration. Taken together these data suggest that the activation of a Ca2+ activated Cl– channel in the mouse oocyte may play an important role in the events occurring at fertilisation.
126. JAK/STAT SIGNALLING IN FOLLICULOGENESIS
Authors: Sutherland J. M. , Keightley R. , Robker R. L. , Russell D. L. , McLaughlin E. A.
Published: November 2010

Primordial follicle activation marks the first stage of pre-pubertal ovarian folliculogenesis, and is therefore fundamental to female fertility. Entry into development is initiated by a group of pleiotropic cytokines and growth factors, originating in and acting upon both the oocyte and granulosa support cells of the ovarian follicle through the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling pathway. Pivotal to this process is the transcriptional regulation of target genes via STAT complexes and negative regulation by the Suppressors of Cytokine Signalling (SOCS) family of proteins. Preliminary evidence indicates that STAT3 facilitates the activation of primordial follicles, while SOCS4 counterbalances the activity of STAT3, mediating the controlled release of primordial follicles into the growing pool throughout reproductive life. Leukemia Inhibitory Factor (LIF) has been previously demonstrated as a key granulosa cell derived cytokine involved in inducing primordial follicle activation. Through both quantitative gene expression (qPCR) and immunoblotting we have demonstrated that LIF can significantly upregulate STAT3 mRNA production (~2-fold) as well as increase STAT3 protein phosphorylation within neonatal mouse ovarian explants culture. Furthermore, through the generation of a recombinant SOCS4 protein construct, and its use in subsequent protein-protein pull-downs, we were able to multiple targets involved in oocyte maturation, STAT3 interactions, and JAK/STAT signaling. These targets were also found to be significantly upregulated via qPCR analysis in neonatal mouse ovaries treated with LIF. These results support our current model for the involvement of STAT3 and SOCS4 in a basic negative feedback loop within the JAK/STAT signalling pathway that results in the regulation of primordial follicle activation and development.
127. PUMA MEDIATES GERM CELL DEATH DURING OVARIAN DEVELOPMENT AND DETERMINES INITIAL PRIMORDIAL FOLLICLE NUMBER IN MICE
Authors: Morgan F. , Hutt K. J. , Scott C. L. , Cook M. , Strasser A. , Findlay J. K. , Kerr J. B.
Published: November 2010

The proteins that control the number of primordial follicles initially established within the ovary are largely unknown. Here we investigated the hypothesis that PUMA, a pro-apoptotic protein belonging to the Bcl-2 family, regulates germ cell death during ovarian development and thereby determines the number of primordial follicles that make up the ovarian reserve. Ovaries were obtained from embryonic day 17.5 (E17.5) and post-natal day 10 (PN10) wild-type (wt) and puma–/– mice and subjected to morphological, molecular and stereological characterisation (n = 3-6 mice/genotype/age). At E17.5, ovaries were densely populated with germ cells and early meiotic oocytes. Immunostaining for MVH and PCNA confirmed the identity of germ cells and proliferating germ cells, respectively. Pyknotic nuclei and TUNEL positive germ cells were rarely detected, suggesting that cell death was uncommon at this age. At PN10, primordial follicle assembly was complete for both genotypes, as confirmed morphologically and by immunostaining for oocyte markers GCNA and MSY2. The number of germ cells in E17.5 wt and puma–/– ovaries was comparable (p=0.81, See Table 1). However, PN10 puma–/– ovaries contained significantly more primordial follicles than wt ovaries (P < 0.001, See Table 1), revealing an over-endowment of primordial follicles in the absence of PUMA. These data show that PUMA regulates the developmentally programmed death of germ cells between E17.5 and PN10 in the mouse and thereby determines the number of primordial follicles that make up the initial ovarian reserve.

This work was supported by the NHMRC (Program Grants #494802 and #257502, Fellowships JKF (#441101), KJH (#494836), CLS (#406675), AS (#461299)); the Leukemia and Lymphoma Society (New York; SCOR grant#7015), the National Cancer Institute (NIH, US; CA80188 and CA43540) and Victorian Government Infrastructure Funds.
128. STRUCTURAL ANALYSIS OF GDF-9 MUTATIONS ASSOCIATED WITH PREMATURE OVARIAN FAILURE AND TWINNING
Authors: Simpson C. M. , Walton K. L. , Stanton P. G. , Harrison C. A.
Published: November 2010

Premature Ovarian Failure (POF) is a disorder associated with female infertility affecting 1% of women under the age of 40 years [1]. It is characterised by amenorrhea associated with an increase in plasma gonadotrophins and decreased estrogen [2, 3]. The major consequence of POF is the loss of fertility, however women also have increased risk of osteoporosis [4] and cardiovascular disease [5]. The causes of POF are unknown; in recent times genetic factors have been considered as etiological components. Genetic screening of women with POF has identified three mutations in Growth and Differentiation Factor-9 (GDF-9), an oocyte-secreted factor critical for folliculogenesis. These are S186Y, V216M and T238A, which are all located within the GDF-9 prodomain [2]. Mutations in GDF-9 may also contribute to the earlier menopause observed in mothers of dizygotic twins, suggestive of a direct relationship to women with POF [6]. The GDF-9 mutations associated with dizygote twinning (DZ) are; P103S located in the prodomain, and P374L and R454C located within the mature domain [6, 7]. The aim of this study is to understand the consequence of these mutations on the synthesis, secretion and biological activity of GDF-9. Utilising site-directed mutagenesis, all six mutations were individually introduced into GDF-9 cDNA and the impact on protein synthesis and secretion was assessed following transfection in HEK293T cells. Mutants S186Y and T238A appeared to have no affect on mature GDF-9 secretion; mutant R454C resulted in misfolded GDF-9; while mutants V216M, P103S and P374L resulted in a significant decrease of mature GDF-9. As the prodomain is necessary for correct folding, dimerisation and secretion of the biologically active mature protein [8], this decrease is most likely due to the impaired posttranslational processing of the prodomain. In vitro biological activity of these mutants is currently being assessed using a HEK293T cell luciferase reporter assay. Preliminary data suggests that mutants V216M and T238A lead to increased biological activity. It is concluded that GDF9 mutations associated with POF and DZ alter synthesis, secretion and bioactivity of GDF-9. It is anticipated that this study will provide structural basis for the impact of heterozygous GDF-9 mutations on ovarian insufficiency in humans.
(1) Dixit, H., et al., Menopause, 2005. 12(6): 749–754.(2) Laissue, P., et al., European Journal of Endocrinology, 2006. 154: 739–744.(3) Pasquale, E.D., et al., The Journal of Endocrinology and Metabolism, 2006. 91(5): 1976–1979.(4) Chand, A.L., et al., Fertility and Sterility, 2006. 86(4): 1009–1012.(5) Chand, A.L., C.A. Harrison, and A.N. Shelling, Human Reproduction Update, 2009: 1–12.(6) Hoekstra, C., et al., Human Reproduction Update, 2008. 14(1): 37–47.(7) Palmer, J.S., et al., The Journal of Clinical Endocrinology and Metabolism, 2006. 91: 4713–4716.(8) Walton, K.L., et al., The Journal of Biological Chemistry, 2010. 285(22): 17 029–17 037.
130. CONDITIONAL TARGETED DELETIONS OF STAT3 TO IDENTIFY ITS ROLE IN OOCYTESAND GRANULOSA CELLS
Authors: Watson L. N. , Robker R. L. , Dunning K. R. , McLaughlin E. A. , Russell D. L.
Published: November 2010

Signal transducers and activators of transcription-3 (STAT3) is a transcription factor activated by JAK kinases after cytokine-receptor binding. We have identified active phospho-STAT3 in granulosa cells of ovarian follicles from the very early activated stage. Abundant STAT3 has also been identified by us and others in oocytes, cumulus and granulosa cells of mature preovulatory follicles. Further, we have found the STAT responsive gene product SOCS4 is present in granulosa cells of early activated and growing follicles (1). To determine the role of STAT3 in follicle growth and ovulation we have generated three unique lines of conditional STAT3 null mice with STAT3 deletion in granulosa cells or oocytes. Mice with STAT3 gene sequenced flanked by LoxP elements (STAT3fl/fl) were crossed with lines expressing Cre-recombinase in granulosa cells (AmhR2-Cre, FSHR-Cre) or oocytes (ZP3-Cre). Fertility analysis of STAT3fl/fl;Zp3-Cre females crossed with wildtype males showed no effect on fertility from STAT3 deletion in the oocyte. In contrast, both FSHR-Cre and AmhR2-Cre mediated granulosa deficient STAT3 lines had significantly reduced litter sizes; 1.4-fold and 1.9-fold lower respectively compared to control littermates (STAT3fl/+;Cre+ or STAT3fl/fl;Cre-). Furthermore AmhR2-Cre mediated STAT3 deletion resulted in a significant 1.9-fold reduction in litters per month and an increased time to first litter. Surprisingly we did not find any difference in ovulation rate after eCG+hCG stimulated superovulation, nor in naturally cylcing mice. Zygotes flushed the morning after mating and placed into culture showed no deficit in cleavage of 2-cell embryos or development to blastocyst. A potential uterine deficiency was investigated and gross morphological observations indicate a defect in the uteri which is consistent with the recent report of Cre-recombinase expression in uterine cells derived from the Mullerian duct mesenchyme (2). Thus STAT3 deficiency in oocytes does not lead to infertility while AmhR2-Cre and FSHR-Cre mediated STAT3 deficiency leads to a sub-fertile phenotype.
(1) Keightley RA, et al. 2009 Reprod, Fertil Dev 21(Suppl.): 509.(2) Gonzalez G, Behringer RR. 2009 Mol Reprod Dev 76: 678–688.
131. SIRT3 IN OVARIAN CELLS IS ALTERED BY MATERNAL AGE AND OVARIAN RESERVE
Authors: Pacella L. , Zander-Fox D. , Hussein T. , Fullston T. , Lane M.
Published: November 2010

Maternal age and reduced AMH levels affect the follicular environment and consequently oocyte viability. The Sirtuin family of protein deacetylases are able to regulate various cellular functions involved in the ageing process in other tissues. In particular, SIRT3 is related to longevity in several cell types and regulates mitochondrial function, however, its presence and role in ovarian cells remains unknown. This study therefore, investigated the presence of SIRT3 in granulosa and cumulus cells, from patients undergoing IVF, and determined the impact of maternal age and low AMH on SIRT3 levels. Granulosa and cumulus cells were collected from women (n = 36), after informed consent, and classified into 3 groups; A (<35 years, normal AMH), B (>40 years (advanced maternal age), normal AMH) and C (<35 years, low AMH). The presence of SIRT3 was determined by q-PCR (expressed as fold-change) or immunohistochemistry. SIRT3 was present in the ovarian cells of all patients analysed. SIRT3 gene expression was reduced in granulosa cells from women with low AMH (0.67 ± 0.17) compared to women with normal AMH (1.00 ± 0.23; P < 0.05). In cumulus cells, levels were reduced with advanced maternal age (0.81 ± 0.08) compared to women <35 years (1.00 ± 0.22; P < 0.05). SIRT3 protein co-localised with mitochondria in the ovarian cells, confirming previous findings for other cell types. In comparison to women <35 years with normal AMH, image analysis determined that SIRT3 protein levels were significantly reduced in the granulosa and cumulus cells from women of advanced maternal age by 21.4% and 31.8% and in women with low AMH by 34.1% and 47.2% respectively. This is the first study to demonstrate SIRT3 presence in human ovarian cells. The observation that SIRT3 levels are altered by advanced maternal age or low AMH (reduced ovarian reserve) implicate its role in ovarian ageing and plausibly in the decrease in oocyte viability observed in these women.
132. ACTIVIN A HAS A STIMULATORY EFFECT IN VITRO ON EARLY FOLLICLE DEVELOPMENT IN RAT OVARIES
Authors: Cossigny D. A. , Findlay J. K. , Drummond A. E.
Published: November 2010

Activins are dimers of inhibin β subunits and are growth and differentiation factors belonging to the transforming growth factor-β (TGF-β) superfamily (1). Both βA and βB subunits are highly expressed in rat granulosa cells, while theca cells express little or no β subunit mRNAs (2). Oocytes lack expression of either subunit (3, 4). Activin is suggested to facilitate the responsiveness of granulosa cells to FSH (5). We hypothesized that activin, with or without FSH, could enhance the transition from the primordial to later preantral stages of follicle development. In two independent experiments, day 4 rat ovaries (n = 3 from different rats per treatment) were randomly assigned and cultured (6, 7) for 10 days in DMEM/Hams F-12 media with either no additives, FSH (100 ng/mL), activin A (50 ng/mL), or both. Day 4 fresh ovaries were also used as controls. Media and treatments were refreshed every alternate day. Ovaries were fixed andsectioned, or placed into Ultraspec for RNA extraction and real-time PCR analysis. Follicle numbers were counted as described previously (7). The proportion of atretic follicles (TUNEL staining) was determined in 3 randomly selected sections per ovary. Primordial follicles in all treatment groups were approximately 20% of those in Day 4 fresh ovaries. Primary follicles increased significantly (P < 0.05) only in the combined treatment group, where preantral follicles increased significantly (P < 0.0001) only when treated with activin A alone. Activin A alone decreased the proportion of atretic follicles in the primary and preantral classes, where the combined treatment increased the proportion of atretic preantral follicles. Real-time analysis revealed that expression levels of follistatin, FSH receptor and activin βA and βB subunits were all expressed at significantly higher levels in the Activin A-only treated group (P < 0.05). In summary, there was no effect on primordial follicle activation by any treatment. Activin alone had a stimulatory effect in vitro on subsequent folliculogenesis, but in the presence of FSH its effect was counteracted shown by an increase in atresia. Reasons for an increase in atretic preantral follicles in the combined treatment group are unclear. These studies support a stimulatory role for activin A in early follicle development and confirm the in vivo effects of activin on folliculogenesis (4).
NHMRC program grant # 494802 and Fellowship (# 441101) provided financial support.
(1) Vale W et al. 1986. Nature 321: 776–779.(2) Meunier H et al. 1988. Proc Natl Acad Sci USA 85: 547–251.(3) Roberts V et al. 1993. Journal of Clinical Endocrinology & Metabolism 7: 1402–1410.(4) Sidis Y et al. 1998. Biology of Reproduction 59(4): 807–812.(5) Drummond A et al. 2002. Endocrinology 143 (4): 1423–1433.(6) Nilsson E et al. 2001. Molecular and Cellular Endocrinology 182 (2): 145–155.(7) Rosairo D et al. 2008. Reproduction 136: 799–809.
133. LESION WEIGHT AND GLANDULAR DEVELOPMENT ARE SUPPRESSED IN A TGFB1 DEFICIENT MOUSE MODEL OF ENDOMETRIOSIS
Authors: Johan M. Z. , Ingman W. V. , Robertson S. A. , Hull M. L.
Published: November 2010

Endometriosis causes subfertility, pelvic pain and dysmenorrhea, and affects 10% of women of reproductive age globally. The pathology of endometriosis is still poorly understood; however microarray data from a mouse model revealed transforming growth factor beta 1 (TGFB1) as central component in molecular pathways that promote tissue remodelling of ectopic endometrial tissues [1]. We hypothesised that a host deficiency of TGFB inhibits the growth of endometriotic lesions and changes the cellular composition of the tissues. To test this hypothesis, human eutopic endometrial tissue was implanted into Tgfb1–/– mice with Tgfb1+/+ wildtype mice were used as controls (n = 8 and 19 respectively). All mice were on a background of severe combined immunodeficiency to prevent graft rejection. The weight and volume fraction of the glandular and stromal compartments of the resulting lesions were evaluated and the sections were stained with BrdU as a marker of proliferating cells. Sixty percent of mice developed ectopic endometrial lesions in both groups. The median weight of the xenografts from Tgfb1+/+ wildtype mice was 11-fold higher than the Tgfb1–/– mice (Mann-Whitney U test, P = 0.0275). The glandular volume fraction in endometriosis-like lesion from Tgfb1–/– mice was 0.35 and was 33% lower than in lesions from the control mice (volume fraction of glands = 0.52) (independent t-test, P = 0.0415). These studies show that TGFB1 is critical for normal endometriosis-like lesion development and a host deficiency of TGFB1 is associated with reduced weight and glandular volume fraction of xenografts. Targeted suppression of TGFB1 in the host response could be a successful therapeutic strategy for women with this disease.
(1) Hull ML et al., Endometrial–peritoneal interactions during endometriotic lesion establishment. Am J Pathol, 2008. 173(3): 700–715.
134. DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS TO MULLERIAN TISSUE
Authors: Ye L. , Mayberry R. , Stanley E. , Elefanty A. , Gargett C.
Published: November 2010

The human uterus develops from the distal Mullerian Duct, a derivative of the mesoderm germ layer. Unlike other mammalian species (eg. mouse) the endometrium of the human uterus develops prenatally during gestation. Little is known about the developmental process involved. A better understanding of human endometrial development may shed light on the mechanisms involved in endometrial regeneration and pathogenesis of adult proliferative endometrial diseases. Mouse neonatal uterine mesenchyme (mNUM) is inductive and can maintain the phenotype of normal adult human endometrial epithelial cells [1]. Both adult human endometrial stroma and neonatal mouse endometrial mesenchyme secrete growth factors of the TGF-beta family including BMPs which have been shown to play an important role in differentiation of human embryonic stem cells (HESC) [2, 3]. Hypothesis: mNUM will direct differentiation of HESC to form Mullerian Duct-like epithelium. Aim: to investigate the role of mNUM in differentiating HESC in vitro and in vivo using A tissue recombination technique. Method: Embryoid bodies (EB) were formed from GFP labelled HESC (ENVY) and GFP-MIXL1 HESC reporter line [4, 5] and recombined with 2 × 0.5 mm pieces of day 1 epithelial cell-free mNUM. Recombinant tissues were either harvested for gene expression analysis or grafted under the kidney capsule of NOD/SCID mice. Results: We found by qRT-PCR that mNUM induces HESC to form mesendoderm/mesoderm progenitors in vitro, obligate intermediates of the developing Mullerian Duct. After further incubation in vivo under the guidance of mNUM, HESC differentiated to form duct-like structures comprising mesoepithelial cells that co-expressed several key developmental proteins of the Mullerian Duct including Emx2, Pax2, Hoxa10, CA125, and also intermediate filament markers such as CK8/18, Vimentin (n = 8). Conclusion: Our study demonstrated for the first time that mNUM can direct HESC to form a mesodermally derived epithelium that is Mullerian Duct-like, providing a novel model for studying human uterine development.
(1) Kurita T, et al., The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen. Differentiation, 2005. 73(6): 313–22.(2) Hu J, Gray CA, Spencer TE, Gene expression profiling of neonatal mouse uterine development. Biol Reprod, 2004. 70(6): 1870–6.(3) Stoikos CJ, et al., A distinct cohort of the TGFbeta superfamily members expressed in human endometrium regulate decidualization. Hum Reprod, 2008. 23(6): 1447–56.(4) Davis R, et al., Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood, 2008. 111(4): 1876–84.(5) Costa M, et al., The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat Methods, 2005. 2(4): 259–60.
135. GENOME-WIDE ASSOCIATION STUDY IDENTIFIES A LOCUS AT 7p15.2 ASSOCIATED WITH THE DEVELOPMENT OF MODERATE - SEVERE ENDOMETRIOSIS
Authors: Montgomery G. W. , Painter J. N. , Anderson C. A. , Nyholt D. R. , Macgregor S. , Lee S. H. , Visscher P. M. , Kraft P. , Martin N. G. , Morris A. P. , Treloar S. A. , Kennedy S. H. , Missmer S. A. , Zondervan K. T.
Published: November 2010

Endometriosis is a common gynaecological disease associated with severe pelvic pain and sub-fertility. There is considerable debate whether different endometriosis stages represent disease progression, or whether moderate-severe (rAFS III/IV) disease is pathological and minimal-mild (rAFS I/II) an epiphenomenon. We conducted a genome-wide association study using 540 082 SNPs in 3194 surgically confirmed endometriosis cases and 7060 controls from Australia and the UK. We used novel statistical methods to estimate the proportion of common variation explained by all markers and performed polygenic predictive modelling for disease stage, both showing significantly increased genetic loading among the 42% of cases with moderate-severe endometriosis. The strongest signals of association were also observed for moderate-severe disease. We subsequently genotyped 72 SNPs in an independent US dataset comprising 2392 endometriosis cases and 1646 controls. An association with rs7798431 on 7p15.2 for moderate-severe endometriosis (P = 6.0 × 10–8, OR = 1.34 (1.21–1.49)) was replicated, reaching combined genome-wide significance (P = 1.7 × 10–9; OR = 1.26 (1.17–1.35)). The implicated inter-genic region involves a 48 kb segment of high LD upstream of plausible candidate genes NFE2L3 and HOXA10. This locus is the first to be robustly implicated in the aetiology of endometriosis, with evidence of association limited to moderate-severe disease.
137. GENOMIC IMPRINTING IN THE MARSUPIAL MAMMARY GLAND
Authors: Stringer J. M. , Shaw G. , Pask A. , Renfree M. B.
Published: November 2010

Genomic imprinting is an epigenetic mechanism that differentially regulates the expression of certain genes, resulting in expression from only one parental allele. In mammals, genomic imprinting occurs in the placenta of both eutherians and marsupials, and plays an important role in regulating nutrition and growth of the developing fetus. The mammary gland also provides a critical source of nutrition for the neonate in all mammals, but there are few imprinting studies of this organ. Marsupials deliver tiny, altricial young that complete development during an extended lactation. INS (insulin) is paternally expressed in the eutherian and marsupial yolk sac and curiously is the only gene that is solely imprinted in this organ (1, 2). Insulin regulates carbohydrate metabolism, protein synthesis and cell growth. Insulin, (plus cortisol and prolactin) is required for the onset of lactation and the synthesis of milk (3). We characterised INS expression and examined its imprint status in the mammary gland of the tammar wallaby. INS mRNA is expressed in the mammary gland of the tammar from birth and throughout of lactation with highest expression at the initiation of lactation (Phase 1-2a) and around Phase 3 of lactation. Direct sequencing of 7 individuals at various stages of lactation confirmed that INS is imprinted in the mammary gland. Surprisingly, INS may also be imprinted in several other organs in the adult and juvenile wallaby. Preliminary bisulfite sequencing suggests there is a differentially methylated region located upstream of INS which may help to regulate INS expression. This is the first study to identify INS imprinting outside the yolk sac. As INS is critical for lactation, this is also the first indication that genomic imprinting may regulate lactation, suggesting that imprinting in the mammary gland may be as critical for post-natal survival as placental imprinting is for pre-natal development.
(1) Deltour LX, et al. (1995). Tissue- and developmental stage-specific imprinting of the mouse proinsulin gene, Ins2. Dev Biol 168(2): 686–688.(2) Ager EI, et al. (2007). Insulin is imprinted in the placenta of the marsupial, Macropus eugenii. Dev Biol 309: 317–328.(3) Bolander FF, et al. (1981). Insulin is essential for accumulation of casein mRNA in mouse mammary epithelial cells. Proc Natl Acad Sci USA 78(9): 5682–5684.
138. GENOME-WIDE LINKAGE SCAN FOR FAMILIAL DIZYGOTIC TWINNING
Authors: Painter J. N. , Willemsen G. , Nyholt D. R. , Hoekstra C. , Duffy D. , Henders A. , Wallace L. , Healy S. , Cannon-Albright L. A. , Skolnick M. , Martin N. G. , Boomsma D. I. , Montgomery G. W.
Published: November 2010

The tendency to conceive dizygotic (DZ) twins is a complex trait influenced by genetic and environmental factors. To search for new candidate loci for twinning we have conducted a genome-wide linkage scan in 525 families using microsatellite and single nucleotide polymorphism (SNP) marker panels. Non-parametric linkage analyses including 523 families containing a total of 1115 mothers of DZ twins (MODZT) from Australia and New Zealand (ANZ) and The Netherlands (NL) produced four linkage peaks above the threshold for suggestive linkage, including a highly suggestive peak at the extreme telomeric end of chromosome 6 with an exponential (exp)LOD score of 2.813 (P = 0.0002). Since the DZ twinning rate increases steeply with maternal age independent of genetic effects, we also investigated linkage including only families where at least one MODZT gave birth to her first set of twins before the age of 30. These analyses produced a maximum expLOD score of 2.718 (p = 0.0002), largely due to linkage signal from the ANZ cohort, however, ordered subset analyses indicated this result is most likely a chance finding in the combined dataset. Linkage analyses were also performed for two large DZ twinning families from the USA, one of which produced a peak on chromosome 2 in the region of two potential candidate genes. Sequencing of FSHR and FIGLA, along with INHBB in MODZTs from two large NL families with family-specific linkage peaks directly over this gene, revealed a potentially functional variant in the 5’ untranslated region of FSHR that segregated with the DZ twinning phenotype in the UT family. Work is continuing screening candidate genes. Our data provide further evidence for complex inheritance of familial DZ twinning.
139. PLACENTAL EXPRESSION OF microRNAs ALTERS WITH GESTATION IN THE GUINEA PIG
Authors: Grant P. A. , Kind K. L. , Sohlstrom A. , Gatford K. , Roberts C. T. , Owens J. A.
Published: November 2010

Functional development of the placenta ensures an adequate supply of nutrients for fetal growth throughout gestation. Placental nutrient transport capacity increases during gestation, through alterations in structure and abundance of its molecular determinants, including expression of Slc2a1 (glucose transporter type-1) and Slc38a2 (system A amino acid transporter), as well as Igf1and Igf2. Each of these genes are predicted targets of microRNAs. Non-coding RNAs can down-regulate, as well as activate translation, by interacting with complementary regions in the promoter, coding, or 3’UTR of target mRNAs.(1) MicroRNAs are present in the mammalian placenta,(2) but little is known about developmental changes in their expression and actions. We hypothesised that placental expression of microRNAs which target molecular mediators of nutrient transport changes during gestation. Expression of microRNAs in the guinea pig placenta was examined at D30 (n = 7) and D60 (n = 7) of gestation (term = D70) by Exiqon microarray. Gene expression was measured by real-time PCR. Predicted gene targets were identified using miRecords and networks and pathways by Ingenuity Pathway Analysis. Placental expression of 119 microRNAs was upregulated (P < 0.05), and that of 40 was down-regulated (P < 0.05), at late compared to early gestation. Of the 20 most abundant differentially up- or down-regulated microRNAs, 11 are predicted to target members of solute carrier families. This includes has-miR-26a (↑2X), which is predicted to target Slc38a2 mRNA and expression of has-miR-26a and Slc38a2 was positively correlated (P < 0.02). Alternate predicted targets of has-miR-26a include networks involving amino acid metabolism, molecular transport and small molecular biochemistry. These findings support the hypotheses that gestational changes in microRNA expression act to regulate functional development of the placenta, including expression of genes that mediate nutrient transport.
(1) Breving K, Esquela-Kerscher A. Int J Biochem Cell Biol 2009.(2) Barad O, et al. Genome Res 2004 14: 2486–2494.
140. GENES CONTROLLING PHALLUS DEVELOPMENT
Authors: Chew K. , Pask A. , Shaw G. , Renfree M. B.
Published: November 2010

Abnormalities of the limb and genital urinary tract are amongst the most prevalent congenital birth defects. The phallus and limb are both appendages controlled by similar patterning mechanisms during the early stages of differentiation. However, the phallus later undergoes an androgen dependent masculinisation phase whilst the limb does not. Marsupials deliver altricial young that complete their development after birth so are ideal models to investigate appendage development. In the tammar, development of the phallus occurs after birth during a period when the testicular androgen is low while the young is in the pouch, stages that normally occur in utero in mice. Exposure to androgens during development, but before differentiation of the phallus can induce sex reversal of the female phallus and hypospadias.1 However, the genes controlling development of the phallus and how they interact with the changing androgen environment have not been well studied as yet in any mammal. We are investigating the expression of several key genes, namely SHH, FGF8, BMP4 and DLX5, using RT-PCR, immunohistochemistry and in situ hybridization throughout the differentiation of the phallus and after treatment with androgens. Endogenous application of the potent androgen androstanediol to the phallus resulted in the downregulation of SHH, FGF8, BMP4 and DLX5. These genes are crucial for the correct development of both limb and phallus. However, the limb does not have an androgen dependant phase and it is not sexually dimorphic at these stages. SHH protein was localised in the epithelium of the phallus and in the urethral groove. The expression of the genes patterning the limb and phallus in the tammar therefore appear to be broadly conserved with those of the mouse. However, this is the first identification of androgen-regulated gene expression in the developing mammalian phallus.
(1) Shaw G, Renfree M, Short R, Experimental manipulation of sexual differentiation in wallaby pouch young treated with exogenous steroids. Development, 1988. 104(4): 689.
141. METHYLATION OF GENES OF THE RENIN ANGIOTENSIN SYSTEM (RAS) IN EARLY HUMAN AMNION
Authors: Mitchell C. M. , Zakar T. , Sykes S. D. , Pringle K. G. , Lumbers E. R.
Published: November 2010

The renin angiotensin system (RAS) genes angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1) and prorenin receptor (ATP6AP2) have CpG islands at their promoters, so these genes may be regulated by CpG island methylation as may the expression of two proteases implicated in prorenin activation (cathepsin D {CTSD} and kallikrein 1 {KLK1}). We measured CpG island methylation of 3 RAS genes and of CTSD and KLK1 in amnion using the Methyl-Profiler assay (SA Biosciences), which discriminates methyl-CpG density between hypermethylated, intermediately methylated and unmethylated CpG islands. DNA from human amnion collected between 10–17.8 weeks gestation, at elective caesarean section and after labour at term was analysed. The bulk (>80%) of CpG islands in all genes examined except for KLK1 was unmethylated and without intermediate methylation throughout gestation, while the rest was hypermethylated. There was no change in methylation density with labour (Table 1). In early gestation KLK1 methylation was greater than ACE, AGTR1 and ATP6AP2 (P < 0.05). KLKI methylation shifted towards the unmethylated state after labour (P < 0.05). There were correlations between methylation of ACE, AGTR1 and ATP6AP2 (P < 0.02) but not between RAS genes and KLKI. This suggests that the 3 RAS genes in amnion are not controlled by CpG island methylation. It is likely, however, that KLK1 is silenced in early gestation amnion partially by CpG island methylation, which is reduced by term. Since Kallikrein 1 can activate prorenin, the methylation status of this gene may regulate RAS activity in amnion during gestation.
142. microRNA AND EARLY MALE GERM CELLS
Authors: McIver S. C. , Roman S. D. , Nixon B. , McLaughlin E. A.
Published: November 2010

MicroRNAs are short regulatory noncoding RNA molecules that bind to the 3′ untranslated region of mRNA targets to control translation therefore influencing the abundance of many different protein molecules. Aberrant expression of miRNA is linked to many diseases and developmental abnormalities. Testicular Germ Cell Tumours (TGCT) develop from Carcinoma in situ cells which have been identified as dysfunctional gonocytes. Due to the continual rise in the rate of testicular cancer in the developed world, the molecular mechanisms underlying the failure of gonocytes to differentiate into spermatogonia is of great interest. Gonocytes from post natal day 1 testes and spermatogonia from day 7–9 mice were enriched by 2–4% BSA gradient sedimentation. Total RNA including microRNA was extracted and analysed in by Illumina miRNA microarray. Total RNA was also reverse transcribed using specific primers and analysed by qPCR. Three biological replicates were performed in both the microarray and qPCR experiments. Bioinformatic analysis with SAM (Significance Analysis of Microarrays) identified seven significantly different miRNA molecules between spermatogonia and gonocytes. qPCR analysis confirmed two miRNAs were significantly upregulated in spermatogonia (743a, 463*) and three miRNAs were significantly down regulated in spermatogonia (293, 290-5p, 291a-5p). Several miRNA molecules were selected for further study (293, 290-5p, 136, and 146a) and overexpression assays first in the P19 cell line, then in isolated spermatogonia will help determine their function. In the future the role of these molecules in human seminoma will be analysed using overexpression within a seminoma cell line. It is hypothesised that these miRNA molecules control genes involved in male development and differentiation, such as stella, nanog and oct3/4, and may also play a role in tumour development. In conclusion miRNA expression is significantly different between gonocytes and spermatogonia and we propose that this results in the initiation of differentiation and commencement of spermatogenesis.