bluuebunny/crossref_metadata_2025 huggingface dataset:
272.Decreased expression of oestrogen receptor β in the reproductive tract of pregnant relaxin-deficient (Rlx - / - ) mice
Authors: McGuane J. T. , Gehring H. M. , Parry L. J.
Published: November 2010

The peptide hormone relaxin (RLX) is reported to directly affect uterine oestrogen receptors (ERs) in the rat (1). Treatment of immature ovariectomised rats with porcine RLX causes a decrease in uterine ERβ mRNA levels within 6 h. However, RLX has no effect on ERα expression. As both ERβ1 and ERβ2 inhibit ER-mediated transcriptional activity, this RLX-induced downregulation in ERβ could be a prerequisite for oestrogen to exert its effects on target tissues. The aim of the current study was to use relaxin-deficient (Rlx–/–) pregnant mice to investigate if relaxin deficiency results in alterations in either ERβ or ERα mRNA expression in reproductive tissues. Cervix and vagina tissues were obtained from adult C57/Blk6J wild-type mice at five stages of gestation (Days 7.5, 10.5, 14.5, 17.5, 18.5 pc) and Rlx–/– littermates on Days 7.5, 14.5 and 18.5 pc. Q-PCR with TaqMan probes in the Opticon 2 thermal cycler (MJ Research, GeneWorks) was used to quantify ERα and ERα gene expression. ERα mRNA levels were significantly (P < 0.05; ANOVA) increased in the cervix/vagina on Days 17.5 and 18.5 pc in Rlx+/+ mice. The increase in ERα in Rlx+/+ mice was negatively correlated with a significant decrease in ERβ expression from Day 14.5 pc. In contrast, there was no decrease in ERβ expression in the cervix/vagina of Rlx–/– mice; ERβ mRNA levels were significantly (P < 0.05) higher compared to Rlx+/+ mice on Days 14.5 or 18.5 pc. However, there was no corresponding reduction in ERα expression in the cervix/vagina of the Rlx–/– mice, so that ERα mRNA levels were still elevated at term despite the maintenance of high ERα expression. In summary, these data show changes in ERα expression in the cervix/vagina of relaxin-deficient mice, which may subsequently affect ERα-mediated transcriptional activity.
(1) Pillai et al. (2002) Biol. Reprod. 67, 1919–1926.
274.Semen exposure in early pregnancy impacts fetal and neonatal growth
Authors: Bromfield J. J. , Roberts C. T. , Robertson S. A.
Published: November 2010

Optimal uterine receptivity is critically important to embryo implantation and subsequent development of the fetus and placenta. Modulators of the uterine cytokine and immune environment are present in seminal plasma. To determine the extent to which seminal plasma programs uterine receptivity and subsequent fetal growth, an embryo transfer model was developed. Embryos were transferred to female recipients prepared by mating with either vasectomised (vas) males or vasectomised males from which seminal vesicles, the major source of seminal plasma, were removed (vas+svx). Placental and fetal parameters were measured at Day 18 of pregnancy, and an additional cohort of embryo recipients progressed to birth to allow growth trajectories and body composition measurement in progeny. At Day 18 of pregnancy, the number of viable implantation sites was unaffected but fetal weight was reduced by 4% (P = 0.05) when females were mated to vas+svx males compared with control pregnancies. Placental weight was not affected. Histological analysis of Day 18 placentas showed decreases in the total mid-sagittal cross sectional area and that of labyrinthine (nutrient exchange) tissue of placentas derived from vas+svx mated females. In term experiments, neonates from matings with vas+svx males were 12% smaller 24 h after birth (P < 0.001) and 14% smaller at 8 days (P < 0.001). Mice derived from vas+svx matings exhibited lower weights compared to those from vas matings until 14 weeks of age. The data demonstrates a critical role for semen in preparing the pre-implantation uterine environment so as to optimise subsequent growth of the fetus and neonate. The effects of seminal plasma are likely to be mediated through the maternal immune response and cytokine expression which influence placental morphogenesis and nutrient transfer function. Our findings have relevance to assisted reproduction programs, where pregnancies are routinely initiated in the absence of female tract exposure to semen.
277.The role of pp60c-src tyrosine kinase in sperm capacitation
Authors: Cunningham-Smith K. A. , Witherdin R. , Hetherington L. , Baker M. A. , Aitken R.
Published: November 2010

A protein kinase A-dependent tyrosine phosphorylation pathway in mammalian spermatozoa has been demonstrated to exist, and is unique to this cell type. As PKA is incapable of directly phosphorylating substrates on tyrosine residues, much research has focused on the identification of an intermediary tyrosine kinase which can be activated by serine/threonine phosphorylation via PKA. Inhibitory studies using genistein, tryphostin, erbstatin and herbimycin A, have demonstrated that the src family of kinases may be responsible for the tyrosine phosphorylation events seen during capacitation (1). Although one src family member, c-yes, has been implicated as the kinase responsible for these events (2), this has since been disputed (3). Another src family member, pp60c-src, can be activated by phosphorylation on Ser-17 by cAMP-dependent protein kinase and Ser-12 by calcium-phospholipid dependent protein kinase C (4), and may be the intermediary tyrosine kinase of interest. Western blot analysis demonstrated the presence of pp60c-src in rat sperm samples isolated from the caput and cauda epididymis. Furthermore, co-immunoprecipitation studies revealed a number of pp60c-src-associated proteins including outer dense fibre 2 (ODF2) and A-kinase anchoring protein 4 (AKAP4). Interestingly, both of these proteins become phosphorylated during capacitation of mouse sperm (data not shown) and AKAP4 is tyrosine phosphorylated in capacitated human sperm (5). These data implicate pp60c-src kinase activity in the phosphorylation of a number of sperm midpiece proteins, which may regulate hyperactivation during capacitation. Further research focusing on the activity of pp60c-src in non-capacitated and capacitated sperm will be conducted.
(1) Yaciuk P., et al. (1989) Mol. Cell Biol. 9, 2453–2461. (2) Baker M., et al. (2003) J. Cell Sci. 117, 211–222. (3) Leclerc P., Goupil S (2002) Biol. Reprod. 67, 301–307. (4) Bajpai M., et al. (2003) Arch. Androl. 49, 229–246. (5) Ficarro S., et al. (2003) J. Biol. Chem. 278, 11�579–11�589.
278.Bioactive activin and its effects on murine testis development
Authors: Mendis S. H. S. , Meachem S. , Brown C. , Loveland K. L.
Published: November 2010

Activin is a member of the TGF β (Transforming Growth Factor β) superfamily of ligands which influence many aspects of male germ cell development. Formed by the linkage of two common β subunits, activin A (a βA βA dimer) has been reported to (1) cause apoptosis of primodial germ cells, (2) inhibit the cellular transition of gonocytes into undifferentiated spermatogonia, and (3) enhance FSH-mediated stimulation of Sertoli cell proliferation in the newborn rat testis. Although closely related, activin βA and activin βB (a βB:βB dimer) differ in that activin βB is less bioactive. In this study we examined the role of activin during the first wave of spermatogenesis using knockout (Inhba–/–) and transgenic (InhbaBK/BK; two copies of the βB subunit gene coding sequence replace the βA coding sequence) mouse models with reduced levels of bioactive activin. Absolute gonocyte and Sertoli cell numbers were significantly elevated in the absence of activin A in newborn Inhba–/– testes compared to wild type, as determined by optical disector analysis. As the Inhba–/– mice die at birth, we next studied the BK/BK mice to examine postnatal effects of reduced activin bioactivity. Surprisingly, both body weight and testis weight were lower in the BK/BK compared to wild type mice at Day 7 and 14, but testis growth in proportion to body weight was significantly reduced between 7 and 14 days. At 2 weeks of age, the BK/BK animal displays a significant reduction in Sertoli cells and specific subpopulations of germ cells, the latter of which was evident only in heterozygote animals. Examination of these two models has identified that lower levels of bioactive activin affect Sertoli cells and germ cells at different stages of testicular development. Our ongoing studies involving RNA analyses of various candidate target genes will facilitate a greater understanding of the molecular basis for these observations.
279.Infertility in mice with null mutation of the Egr-1 transcription factor
Authors: Russell D. L.
Published: November 2010

Female infertility has been reported in two lines of mice with mutation of the Egr-1 gene. One underlying cause of this defect is deficient LH production by pituitary gonadotropes. However, Egr-1 is also acutely regulated by both FSH and LH in ovarian granulosa cells (1). A role for this transcription factor in regulating gonadotrophin responsive target genes and ovarian function is hypothesised. Indeed the LH-receptor is a proposed target of Egr-1 regulation, but this has not been investigated in detail in vivo and is difficult to reconcile with the pattern of Egr-1 expression.
In this study, the role of Egr-1 within the ovarian follicle was investigated using exogenous gonadotropin replacement in Egr-1–/– mice . Adult Egr-1–/– female mice superovulated by sequential PMSG and hCG stimulation and mated with proven male breeders failed to produced offspring while 90% of heterozygous females got pregnant and produced litters (7.4 � 2.9 pups per litter) within 22 days of stimulation. Recovery of oocytes from oviducts of immature superovulated mice revealed a reduced ovulation rate in null females (6.3 � 3.8 oocytes) compared to their heterozygous (18.0 � 6.5) and WT (17.8 � 6.8) littermates. Gross morphology and histology of exogenously stimulated ovaries were indistinguishable from their heterozygous or WT counterparts. Surprisingly, no alteration was detectable in the mRNA expression of previously reported direct Egr-1 responsive genes, namely LH-receptor and membrane prostaglandin E synthase (mPGES). Nor were mRNA for two critical ovulatory genes with putative Egr-1 response elements, ADAMTS-1 or versican V1 altered. Temporal and spatial expression of genes involved in ovarian steroidogenesis, P450scc and Cyp17 and LH-receptor, were indistinguishable from normal littermates during exogenously controled follicular development.
Combined observations of acute Egr-1 induction by gonadotropins, reduced ovulation and complete infertility suggest an important role for Egr-1 in ovarian function. However, genes identified as targets of Egr-1 regulation in other studies proved to be Egr-1 independent in this model.
(1) Russell et al. (2003) Mol. Endo. 17, 520.
280.Reproductive consequences of circadian dysfunction: fertility in the Bmal1 null mouse
Authors: Boden M. J. , Kennaway D. J.
Published: November 2010

Circadian rhythms are generated by a suite of genes called clock genes that are expressed in the brain and also in many peripheral tissues. In the peripheral tissues, these genes assist in regulating the expression of many genes involved in cell growth, angiogenesis and development. Bmal1 is a critical gene involved in circadian rhythm generation. Here we report on the fertility and fecundity of Bmal1 knockout mice (Bmal1–/–). Male Bmal1–/– mice have impaired fertility compared to controls [(litters produced/number of animals) wild type (5/5), CBA controls (5/5), Bmal1–/– (1/15)]. Fifty percent of male Bmal1–/– mice had defective caudal sperm, showing sperm that was both non-motile and malformed. Seminal vesicle weight was significantly reduced in the Bmal1–/– mice (50% reduction) in males at both 4 and 5.5 months old. Female Bmal1–/– mice had irregular oestrus cycles and failed to maintain a pregnancy to term following natural mating [(litters produced/number of animals) wild type (5/5) CBA controls (5/5) Bmal1–/– (0/5)]. When embryos were flushed from the uterus 4 days after natural mating, there was a reduced number of released oocytes and a reduced development to blastocysts in the Bmal1–/– female mice. Following a standard PMSG/HCG super ovulation protocol, Bmal1–/– mice showed both a reduction in ovulation rate as well as a slowed progression of embryos to blastocyst stage (Table 1, see PDF file).
These results suggest that disruption of a key clock gene has detrimental consequences on fertility in the mouse. Further, this reduction in fertility appears to be acting at multiple levels. Continued investigation into the importance of rhythm genes in reproductive function is required.
283.Effects of prostaglandins on SOCS expression in T-47D breast cancer cells
Authors: Barclay J. L. , Tongnkok P. , Anderson S. T. , Waters M. J. , Curlewis J. D.
Published: November 2010

There is increasing evidence to suggest that prostaglandins can upregulate suppressor-of-cytokine-signalling (SOCS) expression and so modify cellular responses to cytokines. Here we examined this possibility in two breast cancer cell lines. Initially we characterised prostaglandin receptor expression by reverse transcription-PCR, and found that T-47D cells express EP2, EP3 and EP4 receptors but not FP or EP1 receptors whereas MCF-7 cells expressed EP1 and EP4 receptors. A range of prostaglandin agonists were then used to elucidate whether prostaglandins affect SOCS expression and the receptor subtypes involved. SOCS 1-3 and CIS expression were measured by Real-Time PCR. In MCF-7 cells, PGE2 caused only minor increases in SOCS1 and SOCS3 expression. However in T-47D cells, PGE2 strongly induced SOCS3 expression with 2- and 5-fold increases in mRNA at 30 and 60 min respectively, returning to baseline at 120 min. SOCS1 was also upregulated at 30 and 60 min (3- and 5-fold respectively) and remained elevated (6-fold) at 120 min. In contrast, CIS and SOCS2 were not induced. Cloprostenol, Butaprost, Latanoprost and Sulprostone had no effect on SOCS expression, suggesting that the PGE2 response is mediated via the EP4 receptor. The induction of SOCS expression by PGE2 was not due to increased STAT3- or STAT5-tyrosine phosphorylation, and indeed we observed a decrease in STAT5 tyrosine phosphorylation 10 min and 1 h after PGE2, as determined by ip/western blotting. In summary, we propose that prostaglandin-induced SOCS1 and 3 expression in T-47Ds could constitute a means whereby cellular resistance to PRL is induced. Supported by the ARC.
285.Increased perinatal mortality following restriction of placental and fetal growth
Authors: Owens J. A. , De Blasio M. J. , Fielke S. , Robinson J. S. , Gatford K. L.
Published: November 2010

Intrauterine growth restriction and subsequent low birth weight in humans are associated with increased perinatal mortality and morbidity. Impaired placental function is a major cause of IUGR in humans, but its impact on perinatal survival has not been clearly defined. We have therefore investigated the effect of restriction of placental and fetal growth on perinatal survival and behaviour in the neonatal lamb. Placental growth was surgically restricted (PR) by removal of the majority of endometrial implantation sites prior to pregnancy, leaving either 6 to 7 (moderate PR) or 3 to 4 (severe PR) visible caruncles in each uterine horn, and ewes were mated following at least 10 weeks recovery. Perinatal outcomes (stillbirths and neonatal death before 3 days of age) were recorded in a cohort of 48 control (30 singleton, 18 twin) and 28 moderate PR (14 singleton, 14 twin), and 21 severe PR (11 singleton, 10 twin) lambs, and effects of PR or twinning were evaluated by Chi-square analysis. Rates of stillbirth (P = 0.006) and total perinatal deaths (P < 0.001) were higher in severe PR pregnancies than in control or moderate PR lambs, overall (see Table in PDF file).
Similarly, severe PR increased stillbirths and perinatal deaths in twins alone (P = 0.003 and P = 0.015 respectively), but the effects of PR were not significant in singletons (P = 0.10, P = 0.26 respectively). Twinning increased stillbirths and perinatal deaths overall (P = 0.002, P = 0.001) and in control lambs alone (P = 0.038, P = 0.017). Restricted fetal growth due to twinning or severe surgical restriction of placental growth thus decreases perinatal survival, due to increased stillbirths. We will further investigate the characteristics of neonatal morbidity following PR by recording neonatal behaviour, including time taken to stand and suckle, in a subsequent cohort of control and PR lambs.
001.Writing scientific papers
Authors: Lindsay D.
Published: November 2010

If you haven't written about your scientific work you might as well not have done it. But, if you haven’t written it well, you won’t have done much better because your scientific colleagues probably won’t read it. This short workshop illustrates an approach to structuring scientific papers that encourages readers to find and retain the important information that you wish them to read. It demonstrates that logical structure is even more important than style and shows how to organize the structure of a paper around the scientific hypothesis on which the work was based. The hypothesis is a statement of what might plausibly have been expected from the experiment before it was done and that expectation can be used to describe the experiment in a way that induces readers to anticipate what they are about to read. This anticipation not only persuades them to read on but enhances their chances of retaining the information that they pick up when they do.
002.Sertoli cell terminal differentiation: doing a 'U' turn on a one way street
Authors: Meachem S. J.
Published: November 2010

The concept of terminal differentiation of Sertoli cells has been challenged and this new information has important implications for male fertility. The mammalian Sertoli cell has two distinct functions: (i) formation of the seminiferous cords and (ii) provision of nutritional and structural support to the developing germ cells. For these to occur successfully, Sertoli cells must undergo numerous maturational changes between foetal and adult life, the main switches occur around the onset of puberty, coincident with the rise in serum gonadotrophins. These switches include the loss of proliferative activity and the formation of the blood testis barrier. Follicle stimulating hormone (FSH) plays a key role in supporting Sertoli cell proliferation in early postnatal life and thus is critical in establishing sperm output in adulthood. After puberty, the size of the Sertoli cell population is considered to be stable and unmodifiable by hormones. This accepted view has been contested as data shows that the size of the adult Sertoli cell population is modifiable by hormone suppression, and that Sertoli cells can regain proliferative activity when stimulated by FSH in the Djungarian hamster1. The molecular mechanism(s) by which Sertoli cells re-enter proliferation are not known in this model however a study demonstrated that helix-loop-inhibitor of differentiation proteins can induce terminally differentiated Sertoli cells to re-enter the cell cycle and proliferate2. Thyroid hormone and testosterone may be involved in the cessation of Sertoli cell proliferation. Gonadotrophin suppression in the adult Djungarian hamster also results in the disruption of the blood testis barrier and spatial organisation of the inter Sertoli cell tight junction proteins and as a consequence the loss of all germ cells that reside inside the blood testis barrier. FSH restores the organisation of these tight junction proteins, which is associated with the appearance of more mature germ cells. It is expected that the integrity of the blood testis barrier is also re-established. It is suggested that this demonstrated plasticity of the adult Sertoli cell may be relevant in clinical settings, particularly to some types of infertility and testicular malignancies where Sertoli cells have failed to undergo these important maturational switches.
(1)Chaudhary et al. (2005) Biol. Reprod. 72, 1205. (2)Meachem et al. (2005) Biol. Reprod. 72, 1187.
004.Control of skeletal muscle cell proliferation and differentiation
Authors: Grounds M.
Published: November 2010

Skeletal muscle is formed by mononucleated precursor cells (myoblasts) that cease cell proliferation to start differentiation; this results in fusion between the myoblasts to form multinucleated cells (myotubes) that continue to differentiate (and fuse with more muscle cells) and mature into myofibres. Myogenesis has been widely used as a model to study in vitro factors controlling cell proliferation and differentiation. Condition in vitro may not reflect what happens in the more complex in vivo environment. Some of the key issues are what activates quiescent myoblasts in mature skeletal muscle in vivo, and what controls the switch between proliferation and differentiation? The role of the matrix, and molecules such as MyoD, p53, NFAT and IGF-1 will be considered.
007.Recent advances in understanding and preventing pre-term birth - the oral health connection
Authors: Newnham J. P. , Shub A. , Doherty D. , Sloboda D. M. , Nitsos I. , Moss T. J. M.
Published: November 2010

Pre-term birth remains one of the major unsolved problems in human health. The incidence is increasing in many Western countries, despite several decades of research studies aimed at findings ways that early birth may be prevented. Nearly two-thirds of very early pre-term births are associated with features of inflammation in the newborn, suggesting that infection may be the origin. Many studies have focussed on the possibility that pre-labour rupture of membranes or the early onset of uterine activity may result from infection spreading upward from the vagina. Unfortunately, trials designed to identify potential pathogens in the genital tract followed by appropriate use of antibiotics have failed to prevent prematurity.
The strong association between features of intra-uterine inflammation and pre-term birth, and the ineffectiveness of antibiotics to prevent the problem, suggest that the source of inflammation may be from a distant site. We are addressing the possibility that the site may be the gums. Periodontal disease affects 15% of the adult Australian population, is often undiagnosed and is not responsive to systemic antibiotic therapy. In our pregnant population, we have shown that periodontal disease is strongly associated with low birth weight. Our sheep studies have taught us that the lipopolysaccharides (LPS) from periodontal pathogens, when injected into the amniotic cavity, have much greater lethality than enteric LPS, and similar effects in inducing inflammation. We are now investigating the effects of treating periodontal disease during mid-pregnancy in a randomised controlled trial, which aims to screen approximately 5000 pregnant women and allocates those with periodontal disease to treatment during pregnancy or soon after. This study is known as the Smile Study and commenced in February 2005. Improving oral health is an exciting, but yet unproven, strategy by which a major health problem may be prevented by a relatively simple and community-based intervention.
008.Placental inflammation and preterm labour: studies of pathophysiology and intervention using human ex-vivo models
Authors: Keelan J. A. , Mitchell M. D.
Published: November 2010

Inflammatory processes and mediators are an integral aspect of the mechanics of parturition. Leukocyte infiltration/activation of the extraplacental membranes, cervix and uterus occurs prior to term labour and is accompanied by increased cytokine expression, eicosanoid production, and extracellular matrix degradation. These processes are propagated during labour to ensure progression through to delivery, and in normal parturition they occur in concert in a timely fashion. In contrast, pathological intrauterine inflammation appears to be a causative factor in a significant proportion of preterm births. While the presence of an infective organism in the amniotic cavity is confirmed in about half of pregnancies delivered with chorioamnionitis, in the remainder the cause of the excessive inflammatory response remains undetermined.
We have established and applied various models employing human placental tissues to study the processes involved in triggering intrauterine inflammatory activation and potential pharmacological approaches for intervention. Of these, an ex-vivo fetal membrane perfusion model has been the most powerful, allowing multi-aspect analysis of membrane gene expression, inflammatory mediator production, histological and structural integrity in response to maternal challenge with endotoxin (lipopolysaccharide). Using a variety of techniques including oligonucleotide and protein arrays we have determined that a robust and rapid inflammatory response is manifested in both the maternal and fetal compartments after maternal (decidual) exposure to lipopolysaccharide; this is accompanied by a marked increase in apoptosis in the chorionic membrane, but no detectable changes in membrane integrity.
We have evaluated in this model several anti-inflammatory drugs that inhibit the NF-κB pathway as potential pharmacologics for treating inflammation-induced preterm labour. Surprisingly, most were completely ineffective in suppressing lipopolysaccharide-induced cytokine production. Sulfasalazine, however, administered to the maternal face, effectively and rapidly abrogated the lipopolysaccharide response in both maternal and fetal compartments, with modest membrane transfer of drug. However, chorionic apoptosis was doubled in sulfasalazine-treated membranes, raising concerns over possible toxicity. Studies using these models are continuing to further evaluate the potential of novel and existing pharmacotherapies for the prevention of inflammation-associated preterm birth.
009. The effect of maternal asthma during pregnancy on placental function, fetal growth and childhood development
Authors: Clifton V. L.
Published: November 2010

Fetal growth and neonatal birth weight are significant contributing factors to the development of adult disease states in later life. In human pregnancy, we have identified sexually dimorphic differences in fetal growth with the female fetus reducing growth in response to maternal asthma and the male fetus continuing to grow at a normal rate but being at an increased risk of in utero death. The physiological mechanisms that confer sex-specific differences in the fetal response to maternal asthma are unknown. However our research has identified differences in mechanisms associated with fetal glucocorticoid regulation, which are also associated with changes in childhood growth patterns. Asthmatic and control pregnant women were recruited at their first antenatal visit and followed through to delivery. Subjects were assessed for severity of asthma and their use of medication, including glucocorticoid therapy, was recorded. In addition to routine antenatal care, fetal growth was determined using Doppler ultrasound. Following delivery placentas and cord blood were collected. The children of the women followed during the study were examined by a paediatrician at 6 months of age and every 12 months after that initial visit. Our data shows that in response to maternal asthma, the female fetus has an increase in cortisol, which downregulates placental GR expression, immune and hypothalamic-pituitary-adrenal function and is associated with decreased growth. The male fetus responds to increased cortisol with an increase in GR expression and no change in HPA or immune function or growth. These data indicate that the male and female fetus have different strategies to control growth and in their response to a maternal stress, such as asthma.
010. Oocyte control of granulosa cell development and function
Authors: Eppig J. J. , Sugiura K.
Published: November 2010

Oocytes orchestrate the rate of follicular development and the patterns of gene expression by granulosa cells (GCs). There are two populations of GCs in large antral follicles: mural granulosa cells (MGCs) that line the ovarian follicle wall, and cumulus cells (CCs) closely associated with the oocyte. Subtraction hybridization was used to find transcripts more highly expressed in CCs than MGCs. Among the genes expressed more highly in CCs was one encoding an amino acid transporter (Slc38a3). Slc38a3 mRNA was not detected in oocytes. Expression of Slc38a3 mRNA was reduced in the CCs after removal of the oocyte and restored by co-culturing CCs with fully grown oocytes (FGOs). Alanine is one of the amino acids transported by SLC38A3. This amino acid is poorly transported across the oocyte plasma membrane, but gains access to the oocyte from the cumulus cells via gap junctional communication. Alanine transport into cumulus cells was promoted by paracrine factors secreted by FGOs, but not by growing oocytes (GOs) from preantral follicles. Thus FGOs promote the transport of alanine into CCs, and this amino acid is then passed on to the oocyte via gap junctions. Transcripts encoding enzymes in the glycolytic pathway were also more highly expressed in CCs than MGCs. FGOs, but not GOs, promote elevated expression of some of these transcripts. Likewise, FGOs promote both glycolysis and oxidative phosphorylation by isolated CCs and MGCs. Oocytes do not effectively utilize glucose as an energy source, and oocytes require the presence of CCs to resume meiosis when glucose is the only energy source present. In contrast, oocytes can resume meiosis in the absence of CCs when pyruvate is the sole energy source. Thus oocytes apparently promote glycolysis by their companion granulosa cells to provide energy for their own development. In addition, this may be one way that oocytes coordinate their development with that of follicular somatic components.
Supported by Grants HD23839 and HD44416 from the NICHD.
020. Oocyte signalling molecules and their effects on reproduction in ruminants
Authors: McNatty K. P.
Published: November 2010

Sheep (Ovis aries) are a highly diverse species with more than 900 different breeds that vary significantly in their physiological characteristics including ovulation rate and fecundity. From examination of inherited patterns of ovulation rate in sheep, several breeds have been identified with point mutations in two growth factor genes that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9b) gene and one in GDF9. Animals heterozygous for the GDF9 and/or the BMP15 mutations have higher ovulation rates (i.e. +0.6 to +5.0) than their wild-type contemporaries. In contrast, those homozygous for any of the aforementioned BMP15 or GDF9 mutations are sterile due to abnormal follicular development from the primary stage of growth. In bovine and ovine ovaries, GDF9 is expressed exclusively in oocytes throughout follicular growth from the primordial stage of development, whereas in sheep BMP15 is expressed exclusively in oocytes from the primary stage: no data for BMP15 are available for the cow. In vitro, ovine GDF9 (oGDF9) has no effect on 3H-thymidine incorporation by either bovine or ovine granulosa cells, whereas oBMP15 has modest (1.2 to 1.6-fold; P < 0.05) stimulatory effects. GDF9 or BMP15 alone inhibited progesterone production by bovine granulosa cells, whereas with ovine cells only GDF9 was inhibitory. The effects of GDF9 and BMP15 together were often cooperative and not always the same as those observed for each factor alone. Active immunisation of ewes with BMP15 and/or GDF9 peptides affected ovarian follicular development and ovulation rate. Depending on the GDF9 and/or BMP15 vaccine formulation, ovulation rate was either increased or suppressed. For example, immunisation of ewes with a BMP15 peptide in a water based adjuvant has led to a 25% increase in lambs born per ewe lambing. Collectively the evidence suggests that oocyte signalling molecules have profound effects on reproduction in mammals including rodents, humans and ruminants. Moreover, that in vivo manipulation of these oocyte signalling molecules provides a new approach to managing the fertility of ruminants.
021. Current progress into testis cell transfer between cattle breeds
Authors: Hill J. , Davey R. , Herrid M. , Hutton K. , Kelley B. , Olejnik J. , Stockwell S. , Vignarajan S. , Brownlee A.
Published: November 2010

Male germline cell transfer has produced offspring in mice (Brinster and Zimmermann 1994). Recently the first livestock animal, a goat, was produced (Honaramooz et al. 2003), while early results in cattle are promising (Oatley et al. 2002; Izadyar et al. 2003). There is an opportunity to develop this technology for the beef industry by transferring male germ line stem cells between breeds to improve the genetics of extensive Australian beef herds. This project is a part of the CSIRO National Research Flagship program that combines expertise and facilities in divisions with complementary expertise at Monash University and the University of Sydney. The environmental constraints of Northern Australia dictate that Brahman type animals show far better survival than Bos taurus cattle, although the carcass value of Brahmans is lower than Bos taurus animals. Artificial insemination is impractical in Northern Australia and thus we aim to develop testis cell transfer technique in cattle to permit Brahman bulls to deliver semen from elite Bos taurus or composite bulls, thereby significantly increasing the growth rate, yield and meat quality of the northern beef herd. Experiments using cattle were performed to determine the applicability of techniques used in the mouse. Initial proof of concept has been achieved that germ cell transfer can result in the donor cells successfully colonizing areas of recipient testis. The viability of isolated testis cells following short term (24 h) culture has been demonstrated through transfer into recipient calves. We have completed >50 male germ cell transfers into recipient calves, using ultrasonographic guided injection into the rete testis. Success of this procedure has been demonstrated by persistence of PKH26 dyed donor cells in the seminiferous tubules of a majority of recipients >2 months after transfer. These recipient male calves have not been depleted of their endogenous spermatogonial populations and we thus expect the efficiency of the procedure to increase as depletion procedures (ongoing) are established. Concurrent with these developments has been research into large scale culture of male germ line stem cells to provide large numbers of stem cells for transplant. Culture of testis cell suspensions has demonstrated survival of enriched testis cells under varying media and culture conditions. Initial passaging of testis cell colonies has revealed mixed cell populations (immunohistochemistry positive for spermatogonia and somatic cells). Further studies will aim to demonstrate that these cultured donor cells are able to undergo spermatogenesis in the recipient animals.
022. Isolation of stem cells from embryos and adult bovine tissues
Authors: Verma P. J. , Upton K. , Mc Connell H. , Vassiliev I.
Published: November 2010

The isolation of stem cells has become an area of increasing interest due to their potential uses in animal reproduction, somatic cell nuclear transfer and cell therapies. The most attractive options are the isolation of stem cells from individual embryos or adult somatic tissues. In addition, for cell therapy, the use of autologous stem cells is considered to have an advantage over heterologous cell based therapies in that immune rejection issues would be circumvented. Here we report on our attempts to isolate stem cells from both these sources in a bovine model.
Bovine ES-like (bES) cells were successfully isolated from embryos and maintained in vitro for up to six passages. These cells retained the morphology characteristic of bES cells: small cytoplasmic/nuclear ratio, nuclei with multiple nucleoli, and multiple lipid inclusions in cytoplasm. bES cell colonies grew as monolayers, as islands of ES cells surrounded by trophectoderm (TE) cells. Immunohistochemical detection of SSEA-1 and SSEA-4 demonstrated expression of these markers in bES cells but not in TE cells. Further, the expression of the pluripotent markers Oct-4, Rex-1 and SSEA-1 by RT-PCR was also detected in bES cells but not in TE cells. On spontaneous differentiation, these cells were able to form a variety of cell types including beating muscle with the cells displaying a propensity to differentiate in a manner reminiscent of human ES cells. (2) We also report the isolation of putative stem cells from adult bovine skin biopsies, which express the stem cell markers Oct-4 and SSEA-1 analysed by RT-PCR and are capable of forming 3-dimensional colonies. These cells are obtained from a skin biopsy, a relatively non-invasive technique that makes them useful as donors for therapeutic applications.
In summary, we have identified populations of stem cells from embryonic and adult bovine tissues, which are readily isolated. Further characterization of the differentiation potential of these cells is needed to identify the suitability of this population for use in autologous stem cell therapies.
023. Biotechnology and reproduction in mainstream animal industry - a perspective
Authors: Martin G. B.
Published: November 2010

This paper considers strategies to improve the reproductive performance of small ruminants in ways that lead to production systems that are ‘clean, green and ethical’. This view arises from feedback from consumers, particularly in attractive export markets, and from a need to refocus on the needs of Australian producers, most of whom operate large, extensive enterprises. These people cannot use ‘high-tech’ systems but need low-cost, low-labour solutions to their problems. First, to control of the timing of reproductive events, they can use the socio-sexual inputs of the ‘male effect’ to induce synchronised ovulation in females that would otherwise be anovulatory (seasonal, lactational, prepubertal). Second, they can use nutritional stimuli for ‘focus feeding’, in which short periods of nutritional supplements are precisely timed and specifically designed for individual events in the reproductive process: gamete production, embryo survival, ‘fetal programming’ and colostrum production. Third, they can use simple behavioural observations to genetically select for temperament – this will maximize offspring survival, product quality and animal welfare. All of these approaches involve non-pharmacological manipulation of the endogenous control systems of the animals and complement the detailed information from ultrasound that is now becoming available.1 The use of such clean, green and ethical tools in the management of our animals can be cost-effective, increase productivity and, at the same time, greatly improve the image of meat and milk industries in society and the marketplace. This does not mean, however, that they will not benefit from the opportunities that evolve from breakthroughs in reproductive technology or gene research. On the contrary, if this ‘high-tech’ research is done within the context of the needs of a ‘clean, green and ethical’ industry, first class science can have very direct and immediate benefits to our livestock industries.
(1)Martin GB, Milton JTB, Davidson RH, Banchero Hunzicker GE, Lindsay DR and Blache D. (2004). Natural methods of increasing reproductive efficiency in sheep and goats. Anim. Reprod. Sci. 82–83, 231–246.
025. Prolactin signaling through the short form of its cognate receptor causes severe ovarian defect
Authors: Gibori G. , Halpern J. , Stocco C. , Kelly P. , Binart N.
Published: November 2010

Extensive investigations from our laboratory have clarified the action and interaction of estradiol (E) and prolactin (PRL) on corpus luteum (CL) function. Our research has led us to discover and isolate a CL specific gene that encodes a protein we named PRAP, that associates with the intracellular domain of the short form (PRLRS) but not the long form (PRLRL) and whose expression is tightly regulated by E. Our laboratory and others have established that this protein, expressed in CL of every species investigated, is a novel 17 beta hydroxysteroid dehydrogenase (17bHSD-7) whose function is to catalyze the transformation of estrone to E. Our results with cells expressing only PRLRS revealed that PRL acting through PRLRS leads to phosphorylation of PRAP/17bHSD-7 (PRAP/17b) by JAK2 establishing for the first time that a steroidogenic enzyme can be phosphorylated through its association with a membrane bound protein. The association of PRAP/17b with the PRLRS and its phosphorylation leads to its stabilization. To further investigate the role of PRL signaling through PRLRS, we used PRLR(–/–) mice expressing the PRLRS as a transgene. The results obtained were totally unexpected and of great interest. The follicles of the ovaries, expressing PRLRS only, underwent premature development followed by severe granulosa and oocyte death leaving holes surrounding collapsed zona pellucida and premature ovarian failure. The observations that: (1) the expression of PRLRS in the ovaries of PRL null mice leads to inhibition in Foxo3a and of GALT, two proteins whose deletion/mutation causes similar premature ovarian failure; and (2) that GALT promoter activity is stimulated by Foxo3a transcription factor led us to hypothesize that PRL acting through PRLRS prevents the expression of Foxo3a, which normally stimulates GALT transcriptional activity. Absence of Foxo3a then leads to inhibition of GALT and increases in galactose and its metabolites, causing galactose toxicity and granulosa as well as oocyte cell death.
026. The cumulus matrix in ovulation: inert packaging or active delivery vehicle for the oocyte?
Authors: Russell D. L.
Published: November 2010

Preovulatory follicles respond to the LH-surge with a cascade of molecular events. The ovulatory signal initially impinges on the mural granulosa layers triggering rapid tissue morphogenesis and ultimately terminal differentiation of these cells. Mural granulosa cells transiently produce a suite of transcriptional regulators, EGF-like ligands as well the extracellular matrix (ECM) proteoglycan, versican and the protease ADAMTS-1. These act in concert with permissive oocyte signals to induce and organise a complex hyaluronan (HA) rich ECM surrounding the cumulus cells and oocyte. This expanded cumulus matrix is analogous in composition to an extensive form of pericellular matrices actively associated with cell migration. During ovulation the cumulus matrix becomes anti-adhesive to the intra-follicular environment but is strongly pro-adhesive for the oviductal fimbria. When the follicle apex is perforated the COC is released binds to the fimbria and transports into the oviduct where fertilisation occurs. Success of ovulation and fertilisation is sensitive to the appropriate production and assembly of cumulus matrix components that are in turn dependent on an appropriate balance of oocyte and granulosa derived signals. Production of these cumulus matrix components is thus a potential checkpoint that assures ovulation of competent oocytes. The HA matrix is cross-linked by organiser molecules and also is enriched in proteases ADAMTS-1, 4, 5. Although these have potentially redundant functionality, ADAMTS-1 null female mice are profoundly sub-fertile and have reduced ovulation rate. Specific components of the cumulus matrix are disorganised in ADAMTS-1 null mice and cleavage of versican in these cumulus complexes is reduced. Thus ADAMTS-1 and versican have unique roles in normal cumulus matrix expansion that is important for ovulation. Altered interaction of the cumulus complex with neighbouring tissues alters transport through the oviduct, while abnormal persistence of COC matrix structure after ovulation is also likely to impair sperm interaction and penetration. Evidence thus indicates that the expanded cumulus matrix plays several active roles in oocyte release, transport and sperm interaction.
034. Nutrient sensing by the early mouse embryo: hexosamine biosynthesis and glucose signalling during preimplantation development
Authors: Pantaleon M. , Kaye P. L.
Published: November 2010

Adequate nutrient supply prior to implantation is not only essential to early embryonic growth and development but has also been implicated in metabolic programming events that influence later stage development and the onset of adult disease. The molecular mechanisms involved in early embryonic nutrient sensing and subsequent programming however have not yet been determined.
Glucose can act as an essential molecular signal for metabolic differentiation and blastocyst formation.1,2 Our work demonstrates that propagation of this nutrient signal involves glucose metabolism through the hexosamine biosynthetic pathway, whose end-product, uridine 5′-diphospho-N-acetylglucosamine (UDP-Glc-NAc) acts as a donor substrate adding a single O-linked β-N-acetylglucosamine (O-GlcNAc) unit to serine and threonine residues of nucleocytoplasmic proteins. The number of proteins modified by this O-linked glycosylation is large and includes transcription factors, cytoskeletal components, metabolic enzymes and other cellular signaling components. This tightly regulated and dynamic modification operates in a functionally reciprocal relationship to the more familiar phosphorylation at the same sites hence altering the activity and/or stability of targeted proteins and providing a mechanism for modulating cellular physiology in response to nutrient availability.
We show that early embryonic glucose exposure, whilst not essential for energy generation during cleavage development, is nonetheless critical for the maintenance of cellular homeostasis with perturbations in glucose levels during early development leading to decreased levels of cell survival. Furthermore, using antisera specific for O-GlcNAc we have examined levels of O-glycosylated proteins in early mouse embryos in response to the presence or absence of glucose and find dramatically reduced global levels of O-linked glycosylation as well as altered nuclear levels of key transcription factors in embryos deprived of glucose.
We believe that this is the first demonstration of a nutrient effect on levels of transcriptional regulators in early development. Elucidation of the mechanisms by which the nutrient environment influences embryonic development is of fundamental importance to our understanding of the origins of adult disease.
(1)Pantaleon et al. (2001). Proc 32nd Annual SRB conference, Gold Coast, Qld. Abstract #42.(2)Martin and Leese (1995). Mol. Reprod. Dev. 40, 436–443.