bluuebunny/crossref_metadata_2025 huggingface dataset:
304. Murine HIF-1α localisation by immunohistochemistry in a mouse reproductive tissue
Authors: Camp-Dotlic E. , Froiland D. , Kind K. L. , Irving-Rodgers H. , Thompson J. G. , Russell D. L.
Published: November 2010

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors consisting of an α and β subunit. The level of O2 within cells regulates the stability of HIF-1α and HIF-1 is considered the primary mediator of cellular responses to hypoxia, helping restore O2 homeostasis by promoting the expression of hypoxia-sensitive genes involved in cell survival, angiogenesis, cell proliferation and metabolism.
There are few published studies investigating the role of HIF-1 protein in mouse tissues using immunohistochemistry, due to the lack of a reliable protocol and the inability of many commercially available antibodies to detect murine HIF-1α protein. We have developed a protocol that has allowed us to analyse the presence and location of HIF-1α protein in the mouse epididymis and found that it was predominantly located in the nucleus of discrete principal cells in the epididymis of mice housed under normoxic conditions and sacrificed by cervical dislocation. Interestingly, a 2.5× increase (P < 0.05) of HIF-1α protein staining intensity in both the nucleus and cytoplasm of principal cells in the epididymis was detected in mice housed under normoxic conditions but sacrificed with CO2 gas, compared to mice sacrificed by cervical dislocation. HIF-1α protein detection was 3-fold increased in the nucleus and cytoplasm of principal cells when mice were exposed to hypoxic conditions (6% O2 for 1 h). Our results demonstrate that murine HIF-1α can be detected in discrete cells under normoxic conditions, suggesting local differences in O2. Acute hypoxic responses, via deliberate exposure or even CO2 euthanasia can significantly upregulate HIF-1α protein levels. Further studies will investigate the role of HIFs and hypoxia in male and female reproductive function.
305. Effects of moderate spinal cord injury on the expression of a barrier marker in endothelial cells of the testis and in the prostate of rats
Authors: Ghabriel M. N. , Lu J. J. , Lim W. H. , Setchell B. P.
Published: November 2010

It was recently shown that the endothelial barrier antigen (EBA), previously thought to be specific to endothelial cells in the central nervous system, was also present in endothelial cells in the testis and in epithelial cells in the dorsolateral prostate of adult rats.1 In the present study, we examined the effect of moderate spinal cord injury (SCI), produced by compression for 5 min of the cord at T 10/11.
There was a slight reduction in EBA in the testis and prostate 24 h after SCI, and this became more obvious after 3days. EBA was completely absent from the prostate and testis at 1 week. By 2 and 4 weeks some expression of EBA returned, and at these times EBA was also detected in the ventral prostate. Brain endothelial cells remained positive throughout.
We cannot yet say whether these changes are due directly to interference with the nerve supply, or involve changes in androgen status.
(1)Ghabriel MN, Lu JJ, Hermanis G, Zhu C, Setchell BP (2002) Reproduction 123, 389–397.
307. The effect of oxytocin on cell growth and steroid production in normal human prostate cells in vitro
Authors: Hogarth K. J. , King K. , Nicholson H. D.
Published: November 2010

Oxytocin (OT) is present in reproductive tissues of male mammals including human prostate tissue. OT increases prostatic muscle tone and prostatic growth. OT is increased in benign prostatic hyperplasia (BPH), an androgen dependent condition that develops with age. Dihydrotestosterone (DHT) is the active hormone in the prostate and is converted from testosterone by the enzyme 5 a reductase. Conversion has been shown to be augmented in the presence of OT. The aim of this study was to investigate the effect of oxytocin on cell growth and steroid production in cultured normal human prostate cells.
Normal human prostate stromal and epithelial cells (Clonetics) were cultured with OT, oxytocin antagonist (OTA) or oxytocin/oxytocin antagonist combination (10 ng/mL, 1 ng/mL or 0.1 ng/mL) in media containing 10 nmol of testosterone. Media was changed daily over the 5 day growth period and frozen. Cell proliferation assay was performed at harvest on day 5 to ascertain cell numbers. Media from days 1, 3 and 5 were extracted and radioimmunoassayed for testosterone and DHT.
OT increased stromal cell number in a dose dependent manner (P < 0.001). Treatment with OT or OTA had no significant effect on epithelial cell numbers. In stromal cell media from Day 1, DHT concentrations were higher in cells treated with OT than control cells (P < 0.05). By Day 5 the concentration of DHT was low in all treatment groups except OT (10 ng/mL). No effect of OT or OTA was seen on DHT concentrations of media from epithelial cells.
OT may increase cell growth in prostate stromal cells but not epithelial cells grown in vitro. This effect may be related to the conversion of testosterone to DHT and DHT to its metabolites. These results demonstrate that OT may play a role in the regulation of cell growth, steroid production and steroid metabolism in the human prostate.
309. Nutrition, insulin, leptin and puberty in Merino ram lambs
Authors: Santiago L. Lomas , Blache D. , Blackberry M. A. , Martin G. B. , Mâncio A. B.
Published: November 2010

Merino sheep developed in Mediterranean regions so are well adapted to acute changes in food availability. However, restricted intake during pregnancy, especially in animals that are pregnant over the dry summer, could limit the positive effects of a winter rainy season on fetal development. In this study, we tested whether the level of nutrition during pregnancy and during pre-pubertal development affected blood concentrations of insulin and leptin, scrotal circumference and age of puberty in male Merino lambs fed with pasture. During dry weather, pregnant sheep were supplemented ad libitum with hay and lupin grain (Lupinus angustifolius) to compensate for decreases in pasture supply. Puberty was detected using a standardised behavioural test with oestrous ewes. Lambs were considered pubertal if they displayed mounting in two successive weekly tests. There were no differences in plasma concentrations of insulin or leptin. The values for both hormones simply displayed the same pattern, with a rise after feeding and a fall during non-feeding periods. There was no difference among treatments in either scrotal growth or age to puberty (Table 1). This might be because the dietary treatments, being administered by food restriction under field conditions, would not have the same effects as severe undernutrition that has been used in laboratory studies. Alternatively, Merino sheep may have a greater capacity to cope with mild nutritional stress.
310. Oestrogen receptor alpha and beta in the prostate of the brushtail possum (Trichosurus vulpecula)
Authors: Fink J. W. , McLeod B. J. , Nicholson H. D.
Published: November 2010

Oestrogen plays a physiological role in the growth of the prostate and has been implicated in the development of prostate disease in man. Both oestrogen receptors are present in the normal human prostate with oestrogen receptor alpha (ERα) localised in stromal cells and oestrogen receptor beta (ERβ) in epithelial cells. However, in benign prostatic hyperplasia (BPH) ERα is found in both stromal and epithelial cells. The study of human prostate growth is complicated by the lack of a suitable animal model. The brushtail possum (Trichosurus vulpecula), however, may provide a novel model for such studies. It has a prostate that is structurally similar to that in man and also exhibits seasonal hyperplasia and regression. This study investigated the localisation of ERα and ERβ in the possum prostate. Male possums were killed throughout the year including the breeding season when the prostate is enlarged and the non-breeding season. Each prostate was divided into central and posterior regions and either fixed in Bouins fluid or snap frozen. The presence of both ERα and ERβ were determined using Western blotting with the Novocastra antisera NCL-ER-6F11 (ERα) and NCL-ER-beta (ERβ). Immunohistochemical localisation of the receptors was determined using the same antisera for ERα on paraffin sections, and for ERβ on frozen sections. Western blotting demonstrated immunoreactive bands of ~60 kDa for ERα and ERβ protein in the central and posterior prostate in the breeding and non-breeding periods. ERα was localised to the nuclei of both epithelial and stromal cells in central and posterior regions of the prostate throughout the year. ERβ was localised to the nuclei of stromal cells in the central and posterior prostate throughout the year. This study confirms the presence of ERα and ERβ in the possum prostate. The distribution of ERα is similar to that seen in prostatic tissue from men with BPH.
311. Adult Sertoli cells proliferate in response to exogenous follicle stimulating hormone in the adult photo-inhibited Djungarian hamster
Authors: Tarulli G. A. , Stanton P. G. , Meachem S. J.
Published: November 2010

Sperm production relies on nutritional and structural support from Sertoli cells. Sertoli cells undergo maturational changes (e.g. cessation of proliferation and formation of the blood–testis barrier) around the onset of puberty in higher mammals1 and maturational failure has been associated with some infertility syndromes and testicular malignancies2. The Sertoli cell population is considered to be stable and unmodifiable by hormones after puberty in mammals, although recent data using the adult Djungarian hamster showed that Sertoli cell numbers decreased by 35% in the absence of serum gonadotrophins, and returned to control levels by short-term replacement of follicle stimulating hormone (FSH)3. Therefore, the aims of this study were to (i) quantify the proliferative activity of Sertoli cells in the hormonally manipulated Djungarian hamster, and (ii) examine the localisation of several tight junction proteins as markers of the blood–testis barrier. Long day (LD) photoperiod (16L : 8D) adult hamsters were exposed to short day (SD) photoperiod (8L : 16D) for 11 weeks to suppress gonadotrophins and then received FSH for up to 10 days. Sertoli cell proliferation was assessed immunohistochemically by the colocalisation of GATA-4 and PCNA, and quantified by stereology. Tight junction proteins (occludin and ZO-1) were colocalised using confocal microscopy. Sertoli cell proliferation in both the LD and SD controls was minimal; however, in response to FSH treatment proliferation was upregulated within 4 days compared with SD controls (98% v. 2%, P < 0.001, respectively). Tight junction proteins colocalised at the blood–testis barrier in LD hamsters, but were disorganised within the Sertoli cell cytoplasm in SD animals. FSH treatment restores colocalisation in a time-dependent manner. It is concluded that FSH contributes to the regulation of Sertoli cell proliferation and tight junction formation in the adult Djungarian hamster. This data provides definitive evidence that the adult Sertoli cell population in this model is modifiable by hormones.
(1)Meachem et al. (2005). Biol Reprod 72, 1187.(2)Allan et al. (2004). Endocrinol 145, 1587.(3)Russell and Peterson (1985). Int Rev Cytol 94, 177.
312. Testosterone secretion in the Australian sea lion
Authors: Atkin C. L. , D'Occhio M. J. , Johnston S. , Harrison K.
Published: November 2010

The objectives in this study were to characterize the pattern of testosterone secretion over 24 h in the captive Australian sea lion and to establish whether exogenous hormone stimulation could be used to obtain an index of testosterone secretion in this species. Blood samples were collected from three males at 3-h intervals for 24 h on three occasions at 2-week intervals. Exogenous GnRH, hCG and LH were administered at the end of each 24-h sampling period and changes in circulating testosterone monitored over 5 h. Circulating concentrations of testosterone during 24-h sampling periods were greater (P < 0.01) during darkness than during daylight. Exogenous hCG administered induced a significant rise in circulating concentrations of testosterone. In the 5 h following GnRH, hCG and LH administration, circulating testosterone concentrations reached similar levels to the highest concentrations observed during the respective 24-h sampling periods. These findings have provided evidence of a 24-h pattern of testosterone secretion in the Australian sea lion. This study has also shown that the testis response to exogenous GnRH, hCG and LH in the Australian sea lion can provide an index of the prevailing testosterone biosynthetic capacity of the testes.
201. Homeobox gene DLX3 regulates forskolin induced trophoblast differentiation
Authors: Chui A. , Kalionis B. , Brennecke S. , Murthi P.
Published: November 2010

Trophoblast cells carry out important functions required for the development of the normal placenta. Disruption of these functions is associated with significant pregnancy disorders such as fetal growth restriction and pre-eclampsia. Transcription factors regulate trophoblast functions. We are interested in one such class of transcription factors known as homeobox genes. The homeobox gene Distal-less 3 (DLX3) plays a vital role in the development of the mouse placenta (Morasso, Grinberg et al. 1999) and increased levels of DLX3 have been found in placentae affected by human fetal growth restriction (Murthi and Chui, unpubl. data). However, the function of DLX3 in the human placenta is not well established. Here, we investigated whether DLX3 regulates trophoblast differentiation using a plasmid construct to overexpress DLX3 in the human trophoblast cell line, BeWo. Real-time PCR showed a significant increase in DLX3 mRNA (3.1 ± 0.1 v. 1.0 ± 0.2 control, P < 0.05, n = 3), as well as the mRNA of two known markers of differentiation 3-β-hydroxysteroid dehydrogenase (3β-HSD) (8.1 ± 1.8 v. 1.2 ± 0.1 control, P < 0.05, n = 3) and β-human chorionic gonadotropin (β-hCG) (54.9 ± 0.9 v. 49.2 ± 1.6 control, P < 0.05, n = 3). Furthermore, forskolin mediated trophoblast differentiation was verified in BeWo cells. Following forskolin induction, real-time PCR showed a significant increase in the expression of DLX3 mRNA (12.9 ± 1.2 v. 3.8 ± 0.9 control, P < 0.05, n = 4), as well as a significant increase in the mRNA expression of the differentiation markers previously tested, 3β-HSD (28.3 ± 2.4 v. 1.0 ± 0.08 control, P < 0.05, n = 4) and β-hCG (2.3 ± 1.9 v. 30.9 ± 0.08 control, P < 0.001, n = 3). The expression of an additional differentiation marker, syncytin, was also significantly increased (4.0 ± 1.9 v. 1.0 ± 0.08 control, P < 0.05, n = 4). Thus, we have shown that DLX3 is a regulator of human trophoblast cell differentiation, and that forskolin acts through DLX3 to induce trophoblast differentiation.
(1) Morasso, M. I., A. Grinberg et al. (1999).
202. Differential dietary regulation of placental and muscle myostatin in a transgenerational rat model of maternal under-nutrition
Authors: Peiris H. N. , Tovey S. , Mitchell M. D. , Green M. P. , Ponnampalam A. P.
Published: November 2010

Myostatin, a negative regulator of muscle growth, affects glucose uptake independent of insulin and also regulates placental glucose uptake in vitro in the human placenta. Maternal under-nutrition during early gestation has been associated with fetal programming for increased risk of metabolic disorders in later life. The aim of this project was to investigate changes in myostatin expression in placental and muscle samples in an established rat model of fetal programming. Pregnant rats were fed either a standard diet ad libitum (AD) or 30% of ad libitum (UN) throughout gestation. Female F1 offspring were in turn subjected to AD or UN conditions throughout pregnancy, giving three groups of F2 offspring: AD-AD, AD-UN and UN-AD. The F2 rats were then fed either a chow or a high fat (HF, 65% kcals fat) diet. Muscle and placental samples were collected at various time-points for analysis of myostatin and pro-myostatin expression. Myostatin mRNA expression was found to be significantly higher in placentae compared with skeletal muscle samples, and in UN-AD muscle compared with AD-AD muscle. At the protein level, expression of both myostatin dimer and precursor varied with time and across groups: particularly, placental myostatin dimer expression was significantly higher in UNAD females compared with ADUN and ADAD groups; muscle myostatin precursor expression was reduced in AD-UN and UN-AD females compared with the AD-AD group at embryonic day20; while at day140, there was a trend towards an increase in muscle myostatin dimer expression in UN-AD HF fed compared with UN-AD CHOW animals. Our data demonstrate that in response to maternal diet myostatin is differentially regulated in placentae compared with skeletal muscle, with both myostatin dimer and myostatin precursor levels affected. Modulation of placental myostatin activity through changes in relative expression of myostatin dimer and myostatin precursor in response to maternal diet may contribute to aberrant nutritional uptake by the fetus.
203. Partial progesterone withdrawal during late gestation increases placental expression of 11β-HSD1 in the rat
Authors: Mark P. J. , Augustus S. , Hewitt D. P. , Waddell B. J.
Published: November 2010

Fetal glucocorticoid excess programs adverse outcomes in adult offspring, including hypertension, obesity and insulin resistance. Access of maternal glucocorticoids to the fetus is regulated by the placental glucocorticoid barrier which consists of the 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) enzyme and P-glycoprotein (Abcb1). Both proteins act to reduce fetal and placental exposure to active, circulating glucocorticoids. In addition, placental expression of 11β-HSD1 is thought to limit the effectiveness of the barrier by local reactivation of inert glucocorticoids. The present study measured expression of placental 11β-HSD1 and 11β-HSD2 in normal rat pregnancy and after either partial progesterone withdrawal or treatment with dexamethasone, both of which reduce fetal growth. Placentas were collected and dissected into their morphologically- and functionally-distinct zones (junctional and labyrinth) on days 16 and 22 of normal pregnancy (term = 23 days) and after either dexamethasone treatment (0.75 μg/mL in drinking water from day 13) or ovariectomy (day 16) plus full oestrogen and partial progesterone replacement (to approximately one-third of day 22 levels). Junctional and labyrinth zone expression of 11β-HSD1 and 11β-HSD2 mRNA were determined by qRT–PCR. Labyrinthine expression of 11β-HSD1 increased markedly between days 16 and 22 and there was a concomitant decrease in labyrinthine 11β-HSD2 expression. Dexamethasone administration had no effect on the expression of either 11β-HSD isoform in either placental zone. Partial progesterone withdrawal increased 11β-HSD1 expression in both placental zones (1.9 and 3.1-fold in LZ and JZ respectively, P < 0.05), but had no effect on 11β-HSD2 levels. In conclusion, these data confirm the pattern of placental 11β-HSD isoform expression in late rat pregnancy and suggest that that labyrinth zone 11β-HSD1 is normally suppressed by progesterone. Thus, the normal pre-partum decline in circulating progesterone may provide a key stimulus for the marked rise in labyrinth zone 11β-HSD1 that occurs between days 16 and 22 of pregnancy.
204. Proteomic analysis of the effluent from perfused placental cotyledons identifies proteins associated with pre-eclampsia
Authors: Crawford K. E. , Wong M. H. , Stevenson J. L. , Gude N. M.
Published: November 2010

Pre-eclampsia (PE) is proposed to involve maternal systemic endothelial cell dysfunction which may be caused by excessive shedding of syncytiotrophoblast microparticles (STBM) from the placenta into the maternal circulation. We have examined STBM harvested by perfusion of placental cotyledons from PE and gestation-matched control (GMC) pregnancies. Following proteomic analysis, the proteins Calreticulin, Chloride intracellular channel 3 (CLIC3), Valosin-containing protein (VCP), Protein disulfide-isomerase A3 (ERp57) and Protein kinase C inhibitor protein 1 (14–3-3) were identified as having increased expression in PE samples. This result suggests that there may be increased rate of release of these proteins from the placenta into the maternal circulation. The aims of this work are to measure the expression of these proteins in placental extracts and maternal plasma, and to compare expression between PE and GMC using western blots and/or ELISA with antibodies specific to each protein. Placental expression was confirmed for all five proteins, however, CLIC3 was the only protein significantly increased in the placenta in PE (17.858µg+1.62, n = 27, t-test P < 0.05) compared with GMC (10.478µg+0.76, n = 25). western blot determined there was a significant increase in calreticulin expression in plasma from PE women compared with GMC (as shown by Gu et al. 2008). Likewise, VCP was also increased in PE plasma (1810+280 density/0.25µl, n = 10) compared with GMC (884+260, n = 9). In contrast, the protein 14–3-3 was decreased in PE plasma (4 out of 27 PE plasma samples tested expressed 14–3-3) compared with GMC (10 out of 27 control plasma samples expressed 14–3-3). One of the western blots determined that there was a significant decrease in 14–3-3 expression in plasma from PE women (199.24+186 density/2.5µl, n = 10, un-paired t-test P < 0.05) compared with GMC (2174.91 ± 723.29, n = 11). Neither ERp57 nor CLIC3 was detected in maternal plasma. Those proteins found to be significantly different in PE compared with GMC may be involved in the pathophysiology of PE. These results demonstrate that proteomic analysis of the effluent from perfused placental cotyledons is a reliable screening method to successfully identify proteins that have altered expression with PE.
(1) Gu et al. Mole. Human Repro. April 16 2008
205. Homeobox gene TGIF is increased in human idiopathic fetal growth restriction
Authors: Murthi P. , Pathirage N. , Borg A. , Brennecke S. , Kalionis B.
Published: November 2010

Fetal Growth Restriction (FGR) is a clinically significant pregnancy disorder in which the fetus fails to achieve its full growth potential in utero. Recently, we identified a novel homeobox gene TGIF, in the placenta using microarray expression profiling (1). Targeted mutation of tgif in mouse results in placental dysfunction (2). In this study, we have investigated TGIF expression levels in idiopathic FGR. FGR-affected placental samples were collected based on strict clinical criteria to ensure inclusion of cases at the severe end of the spectrum of the disease. TGIF mRNA expression was analysed in placentae obtained from pregnancies complicated by idiopathic FGR and gestation-matched control pregnancies (n = 25 each). Real-time PCR showed a significant increase in TGIF mRNA levels in FGR-affected placentae and gestation-matched controls [1.29 ± 0.06 FGR v. 0.78 ± 0.04 Control, P < 0.001]. western blotting using a TGIF polyclonal antibody revealed significantly increased levels of TGIF protein in term FGR-affected placentae compared with term controls [3970 ± 1101 (n = 10) v. 2323 ± 644 (n = 10), P < 0.05]. The spatial distribution of TGIF protein by immunohistochemistry revealed immunoreactive TGIF protein in residual cytotrophoblast cells, syncytiotrophoblast cells, microvascular endothelial cells and in stromal cells. We conclude that increased expression of homeobox gene TGIF may be a contributing factor to the developmental abnormalities seen in the FGR-affected placentae.
(1) Murthi P, Hiden U, Rajaraman G, Kalionis B. Placenta May 29, [Epub ahead of print].
(2) Bartholin L, Melhuish TA et al. Dev Biol. 2008 May 2. [Epub ahead of print].
208. Absence of GH-R exon 3 in marsupials and monotremes argues for a eutherian specific origin and fetal specific purpose of this domain
Authors: Menzies B. R. , Shaw G. , Fletcher T. P. , Pask A. J. , Renfree M. B.
Published: November 2010

Growth hormone receptor (GH-R) plays a critical role in the control of growth and metabolism in all vertebrates. GH-R consists of 9 coding exons (2–10) in all eutherian mammals, while the chicken only has 8 coding exons, and does not have an orthologous region to eutherian exon 3. To further understand the evolutionary origins of exon 3 of the GH-R we have cloned the full-length GH-R sequence in a marsupial, the tammar wallaby to determine whether exon 3 was present or absent in marsupial liver cDNA. There was no evidence for the presence of an exon 3 containing mRNA in sequence of tammar pouch young and adult livers. We next examined the genomes of the platypus (a monotreme mammal) and the grey short-tailed opossum (another marsupial). Like the tammar, the GH-R gene of neither species contained an exon 3. GH receptor can obviously function in the absence of this exon, raising speculation about the function of this domain, if any, in eutherians. A comparison of exon 3 protein sequences within 16 species of eutherian mammals showed that there was ~75% homology in the domain despite only 3 residues being identical (Leu12, Gln13 and Pro17). Interestingly, we detected greater evolutionary divergence in exon 3 sequences from species that have variants of GH or prolactin (PRL) in their placentas. These data show that exon 3 was inserted into the GH-R after the divergence of marsupial and eutherian lineages at least 130 million years ago.
210. Fibroblast growth factor 9 (FGF9) in the developing marsupial
Authors: Chung J. , Pask A. , Renfree M.
Published: November 2010

The testicular differentiation pathway in mammals is controlled by a complex array of factors acting in multiple signalling pathways. An important group of factors that regulates this process are the fibroblast growth factors (FGFs). In particular, fibroblast growth factor 9 (FGF9) in mice is critical for normal sexual differentiation. Fgf9 is thought to be important in tipping the balance of development in the bipotential gonad down the testis differentiation pathway. In the developing mouse testis, Fgf9 is antagonistic to the expression of the ovarian differentiation factor Wnt4. In order to examine the conserved role of FGF9 in mammalian testis formation, we cloned FGF9 in a marsupial, the tammar wallaby. Tammar FGF9 nucleotide and protein sequences are highly homologous to the human and chicken orthologues. FGF9 is expressed during the period of testicular differentiation and our data shows that FGF9 is upregulated during testicular differentiation in an opposite but complementary manner to that of WNT4 expression. FGF9 protein was widely distributed in the bipotential gonads but become restricted only to the testis as seminiferous cords formed and was markedly downregulated in the ovary as differentiation proceeded. This strongly suggests that FGF9 has a highly conserved and crucial role in male sexual differentiation similar to the mouse, and supports the suggestion that FGF9 is involved with the control of WNT4 expression in a balancing act to direct the developmental outcome of the gonad.
211. Proteomic identification of caldesmon as one of the physiological substrates of proprotein convertase 6 during decidualisation
Authors: Hardman B. M. , Kilpatrick L. M. , Stephens A. N. , Chen J. I. C. , Stanton P. , Salamonsen L. A. , Nie G.
Published: November 2010

We have previously demonstrated that proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is a critical endometrial factor for implantation. PC6 is upregulated in the endometrium specifically at implantation in association with epithelial differentiation (in human and monkey) and stromal cell decidualisation (in the mouse, human and monkey). Knockdown of endometrial PC6 during early pregnancy in mice in vivo led to complete failure of implantation, while blocking of PC6 production in human endometrial stromal cells in vitro inhibited decidualisation. PCs convert a range of precursor proteins of important functions into their bioactive forms; they are thus regarded as critical ‘master switch’ molecules. We hypothesise that PC6 exerts its roles in the endometrium by regulating proteins of diverse functions essential for implantation. In this study, we utilised proteomic technology and aimed to identify proteins that are specifically cleaved by PC6 in human endometrial stromal cells (HESC) during decidualisation. HESC were decidualised with cyclic AMP, the cell lysates were treated with and without recombinant human PC6-A (rPC6-A), and the 2D Differential in Gel Electrophoresis (2D DiGE) protein profiles were compared between the two treatments. We identified several proteins which were differentially cleaved following the addition of rPC6-A. Mass spectrometric analysis confirmed that the most abundant of these were caldesmon, tropomyosin-2, tropomyosin-4, hypoxia Inducible factor-1 and chloride intracellular channel-1. These proteins showed spot shifts in hPC6-A treated HESC lysates consistent with hPC6-A cleavage. western blot analysis confirmed the specific cleavage of caldesmon by PC6 in HESCs, and immunohistochemical analysis showed co-localisation of caldesmon and PC6 in decidual cells in human endometrial tissue. Given that caldesmon is a structural protein previously found to be involved in actin filament reorganisation, our results strongly suggest that PC6 is a mediator of structural remodelling of stromal cells during decidualisation in the endometrium.
212. Specific targeting of uterine proprotein convertase 6 (PC6) facilitates the development of dual function contraception
Authors: Borg N. , Hardman B. , Salamonsen L. , Nie G.
Published: November 2010

Proprotein convertase 6 (PC6), is a key player during embryo implantation in humans and mice. We have previously shown that PC6 is essential for decidualisation in the mouse and knockdown of endometrial PC6 leads to implantation failure. The PC family of proteases, including PC6, are necessary for transmission of human immunodeficiency virus (HIV). It has been postulated that inhibition of PC activity could prevent HIV infection. We hypothesise that PC6 is a prospective target for the development of a dual role contraceptive for women to avoid pregnancy and protect from HIV infection. The aim of this study is to evaluate if a PC6 inhibitor that is capable of preventing HIV transmission can block implantation in mice. We used a generic PC peptide substrate to assess the potency of the inhibitor to block PC6 activity in vitro. The substrate releases a fluorochrome when cleaved by PC6; no fluorescent signals were observed in samples when inhibitor concentrations were 10μM or higher. We then gauged inhibitor uptake by the uterus over 24 h in mice by two delivery routes; intrauterine injection (IU) and vaginal delivery (VD) with a neutral gel. Uptake was tracked with a FITC-conjugated inhibitor at 50μM (IU) and 500μM (VD). Strong fluorescent signals were seen at 2, 4, 6 and 24 h at sites of endometrial PC6 activity in the IU and VD groups. Administration of a 50μM dosage (20μl) to the uterine lumen (IU) caused a significant reduction (P = 0.002) in the number of implantation sites compared with controls (saline only) when treated between 2000–2100 on E3.75. The inhibitor's ability to block uterine PC6 activity and implantation via VD was assessed and to date outcomes have suggested that correct timing is crucial to prevent implantation and decidualisation. These outcomes show the potential of the inhibitor to block implantation in mice.
214. Altered placental gene expression following disruption of mitochondrial metabolism in mouse embryos
Authors: Filby A. N. , Lane M. , Cashman K. S. , Mitchell M.
Published: November 2010

Metabolic changes in the preimplantation embryo are known to alter blastocyst viability. We used a mouse embryo model, inhibiting the malate-aspartate shuttle (MAS) which is required for energy metabolism in the early embryo, to examine the consequences on subsequent fetal and placental development, and placental gene expression. Mouse zygotes were obtained from C57BL6xCBA females after gonadotrophin stimulation. Following culture in G1.2 for 48h, 8-cell embryos were cultured to the blastocyst stage in either G2.2 control media (C), G2.2 media without pyruvate (-P), or -P with 0.5 mM aminooxyacetate, an inhibitor of MAS (-P+AOA). Blastocysts were transferred to day 4 pseudopregnant Swiss mice, and fetuses and placentas were harvested on day 18 of pregnancy. RNA was extracted from placentas for real time PCR expression analysis by ddCt relative to 18S. Expression of mitochondrial transcription factors (mTERF, mTFAM, Nrf-1, Nrf-2), glucose transporters (Glut1 and Glut3) and amino acid transporters (Slc38a2 and Slc38a4) were analysed. Following transfer, fetal development per implantation was significantly reduced when embryos were cultured in –P+AOA conditions (25.9%) relative to both C and –P conditions (57.5% and 68.8% respectively). Although placental weight did not differ between treatment groups, fetal weight was significantly reduced for –P and –P+AOA groups (P < 0.05), suggestive of altered placental function thus gene expression was examined. There were no differences in gene expression between placentas from C and –P conditions for any of the genes analysed. When compared with C placentas, -P+AOA conditions reduced the expression of Glut 1 (P = 0.055) and Glut 3 (P = 0.043). The –P+AOA placentas also tended to have significantly reduced expression of Glut 3 (P = 0.06) compared with placentas from –P conditions, but all other genes were expressed similarly. This data suggests that decreased glucose transport may be the cause of reduced fetal growth as a consequence of metabolic perturbations in the embryo.
216. Activin A regulates trophoblast cell adhesion: implications for uterine receptivity and embryo implantation
Authors: Stoikos C. J. , O.'Connor A. , Salamonsen L. A. , Dimitriadis E.
Published: November 2010

Embryo implantation involves blastocyst attachment to the endometrial luminal epithelium, followed by trophoblast invasion. This process involves a coordinated crosstalk between the implanting blastocyst and the endometrium. Adhesion molecules play an instrumental role during implantation and are regulated by a variety of factors including cytokines and growth factors. Activin A, a TGF-β superfamily member, has been detected in uterine washings,1 and its subunit, β A, is produced by endometrial glands during the secretory phase of the menstrual cycle.2 In endometriosis, a disease that associated with sub-fertility, β A immunostaining is increased in endometrial glands,3 suggesting higher levels of activin A secreted into the uterine lumen could contribute to sub-fertility observed in endometriosis. Therefore we hypothesised that activin A secretion into the uterine cavity affects the adhesive properties of the cells present at the maternal-fetal interface. The aims of the study were to measure and compare activin A secretion in uterine washings from women with and without endometriosis and to demonstrate whether activin A regulates adhesion to extracellular matrix (ECM) components. Uterine washings (5 mL of sterile saline) were collected from women with and without endometriosis during the secretory phase. Activin A was measured by ELISA. HTR8 (human trophoblast cell-line) cells were treated with rhActivin A (50 ng/mL) and assessed for binding to fibronectin, laminin, vitronectin, collagen I and IV. Activin A (>10pg/mL) was detectable in uterine washings from women with and without endometriosis and levels were elevated in endometriosis patients. Untreated HTR8 cells adhered maximally to fibronectin, collagen I and collagen IV with low binding to vitronectin and laminin. Following activin treatment, HTR8 cell binding to fibronectin, collagen I and IV was significantly decreased (n = 3, P < 0.05). These results suggest that activin A regulates the adhesive properties of the blastocyst during implantation. This study also implies that abnormalities in local activin A levels during endometrial receptivity may contribute to sub-fertility in women.
(1) Petraglia et., al. (1998) J Clin Endocrinol Metab.
(2) Jones et., al. (2000) Mol Hum Reprod.
(3) Rombauts et., al. (2006) Aust N Z J Obstet Gynaecol.
217. Inflammatory stimulation of human decidual cells by periodontopathic bacteria
Authors: Keelan J. A. , Wong P. , Bird P. S.
Published: November 2010

Periodontal disease is associated with increased risk of preterm birth, although a mechanistic connection has not yet been confirmed. We hypothesised that circulating endotoxins (e.g. lipopolysaccharide, LPS) from periodontopathic organisms might possess the ability to exert highly potent immunostimulatory effects in extraplacental membranes, thereby triggering inflammatory activation sufficient to precipitate preterm labour and birth in the absence of overt intrauterine infection. We therefore tested the stimulatory effects of LPS prepared from three periodontopathic bacteria [porphyromonas gingivalis (P.g), Aggregatibacter actinomycetemcomitants (A.a), Fusobacterium nucleatum (F.n)] in comparison with ‘standard' LPS from E.Coli (O55:B5). Human decidual cells were isolated by collagenase/dispase digestion with Percoll purification from term decidual membranes delivered before the onset of labour by Caesarean section. Cells were stimulated overnight with 0.02, 0.2 and 2 mg/L LPS or equivalent doses of whole cell non-viable bacteria. As an index of inflammatory stimulation, cytokine (TNF-α) production was measured by ELISA and normalised to cellular protein. The different LPS preparations all stimulated decidual cytokine production, with ranked potencies as follows: Ec > Aa > Fn > > Pg. Maximal stimulation of TNF-α production achieved was 15-, 4.5-, 23- and 7-fold above control by Ec, Aa, Fn and Pg, respectively. Overall, the LPS preparations were more potent stimulators than whole cell bacteria, achieving greater levels of stimulation at lower doses. However, whole cell Aa was notable for its inflammatory effects, generating a >60-fold increase in TNF-α levels relative to control at the highest dose tested (2 mg/L). These data highlight wide variability in the ability of periodontopathic bacteria to stimulate an inflammatory response in the human decidua, both in terms of their potency and efficacy. The potential significance of bacterial molecular patterns other than LPS as potential triggers of inflammation-driven preterm labour warrants further investigation.
218. Homeobox gene HLX is expressed in choriodecidua mesenchymal stem cells and regulates their ability to migrate
Authors: Qin S. , Murthi P. , Brennecke S. , Kalionis B.
Published: November 2010

Mesenchymal stem cells (MSCs) can be prepared from the placenta (PMSC) and the choriodecidua component of the fetal membranes (CDMSC). PMSCs and CDMSCs share basic stem cell properties with adult MSCs but differ in their gene expression profiles and ultrastructure, showing features of more primitive and metabolically quiescent stem cells (1). Homeobox gene transcription factors are critical markers for identifying stem cells and they regulate important stem cell functions. Our laboratory showed the homeobox gene HLX is expressed in the placenta and the choriodecidua component of the fetal membranes, and is a regulator of proliferation in placental cells (2). In this study, our aim was to determine whether HLX was expressed in CDMSCs and to use short interfering RNAs (siRNAs) to specifically inactivate HLX and determine the effect on CDMSC function. Complementary DNA was prepared from CDMSCs and RT–PCR using HLX-specific primers generated the expected band size of 485bp following agarose gel electrophoresis (n = 3). At the protein level, HLX expression was detected in the nuclei of CDMSCs using immunocytochemistry. The expected HLX protein product was detected at ~50kDa using western blotting (n = 3). Conditions were optimised for the use of short interfering RNAs (siRNA) to decrease HLX expression in CDMSCs with 5nM giving the most efficient downregulation. Two independent siRNAs were tested (HLXsi3–4) and of these, HLXsi4 resulted in significantly decreased HLX mRNA levels in CDMSCs as shown by real-time PCR (0.66 ± 0.08, P = 0.03, n = 3). Functional assays to measure stem cell migration were carried out in quadriplicates on two samples. 10000 cells were placed on one side of a filter and the number of cells that migrated to the other side of the filter was stained and densitometric analysis was carried out using Axiovision image analysis software. These results suggest that the HLXsi4-mediated decrease in HLX expression resulted in reduced CDMSC migration (2.6x103 ± 401 v. 1.3x103 ± 225 densitometric units, P = 0.02). Therefore, HLX may play a role in stem cell migration.
(1) Pasquinelli G, Tazzari P, Ricci F, Vaselli C, Buzzi M, Conte R, Orrico C, Foroni L, Stella A, Alviano F, Bagnara GP and Lucarelli E., Ultrastructural characteristics of human mesenchymal stromal.
(2) Rajaraman G, Murthi P, Quinn L, Brennecke SP, Kalionis B. Homeodomain protein HLX is expressed primarily in cytotrophoblast cell types in the early pregnancy human placenta. Reprod Fertil Dev. 2008.
(3) Rajaraman G, Murthi P, Leo B, Brennecke SP and Kalionis B. Homeobox gene HLX1 is a regulator of colony stimulating factor-1 dependent trophoblast cell proliferation. Placenta. 2007. 28(10):991–8.
219. Parkin co-regulated gene (Pacrg) is an axonemal protein involved in sperm tail and ependymal cell function and is a candidate primary ciliary dyskinesia gene
Authors: Wilson G. R. , Wang H. X. , Egan G. F. , Delatycki M. B. , O.'Bryan M. K. , Lockhart P. J.
Published: November 2010

A leading cause of male infertility is genetic variation in genes required for sperm formation or function. Considerable evidence suggests PACRG is involved in spermiogeneis. The loss of Pacrg function causes infertility in mice (Lorenzetti et al. 2004) and we have shown an association between variability in the 5′ untranslated region of PACRG and human male infertility (Wilson et al. in preparation). Evidence from studies in C.reinhardtii and T.brucei indicate Pacrg is crucial for axonome formation and microtubule stability. To assess this possibility in mammals, we generated and characterised Pacrg knockout (Quaking viable, Qkv), wildtype and Pacrg transgenic mice (Qkv-Tg). Using confocal and immunoelectron microscopy we showed that Pacrg was localised to the axonemal microtubule doublets of sperm, tracheal and ependymal cilia. The absence of Pacrg was associated with compromised sperm flagella formation and MRI analyses revealed the occurrence of hydrocephalus. Specifically, Qkv mice showed an inward expansion of the lateral ventricles, resulting in a significant reduction in distance between ventricles (1.0 ± 0.6 mm, mean ± s.d., n = 5) and a ~250% increase in ventricle area (70 ± 13 arbitrary units, mean ± s.d., n = 5) compared with wildtype littermates (1.38 ± 0.09 mm; area 26 ± 12, n = 3). Transgenic expression of Pacrg was necessary and sufficient to correct the hydrocephalus (1.45 ± 0.05 mm; area 26 ± 9, n = 2) and infertility phenotypes (evidenced by daily sperm counts and litter sizes). In conclusion, we have shown Pacrg is a novel axoneme associated protein in a subset of motile cilia/flagella and loss of Pacrg function results in spermiogenic defects and hydrocephalus in mice. Further, we have shown that variations in the human PACRG promoter are a risk factor in human male infertility. Collectively these data suggest PACRG is a candidate gene in the human syndrome of primary ciliary dyskinesia.
(1) Lorenzetti D, Bishop CE, Justice MJ. 2004. Deletion of the Parkin coregulated gene causes male sterility in the quaking (viable) mouse mutant. Proc Natl Acad Sci U S A 101(22):8402–8407
220. Homeobox gene HLX is a regulator of HGF/c-met mediated trophoblast migration
Authors: Rajaraman G. , Murthi P. , Brennecke S. P. , Kalionis B.
Published: November 2010

Homeobox gene transcription factors play critical roles in normal placental development and are expressed in specialised trophoblast cells. Abnormal trophoblast cell function is associated with pregnancy disorders including fetal growth restriction. Our studies show homeobox gene HLX expression in trophoblast cells (1) and that HLX is significantly decreased in fetal growth restriction (2). HLX gene inactivation in cultured trophoblast cells shows that HLX is a regulator of cytokine-dependent trophoblast proliferation (3). Hepatocyte growth factor (HGF) activates trophoblast cell migration in a paracrine fashion and its receptor, c-met, is expressed in trophoblast cells. This study investigates the regulation of HGF/c-met mediated trophoblast migration by HLX, in two human trophoblast cell lines SGHPL-4 and HTR-8/SVNeo. HGF stimulation significantly increased HLX mRNA expression (e.g. 43.2 ± 2.5, HGF v. 18.4 ± 1.7 control, densitometric units, P < 0.001, n = 3). siRNA-mediated inactivation of HLX resulted in significantly decreased trophoblast migration (e.g. 32 ± 4, siRNA v. 127 ± 12 control, migrated cells, P < 0.05, n = 4). When HLX was inactivated in the presence of HGF stimulation, migration remained significantly decreased (e.g. 112 ± 15, siRNA + HGF v. 368 ± 44 HGF, migrated cells, P < 0.05, n = 4). In order to determine if HGF is acting via the c-met receptor, the Met tyrosine kinase inhibitor, SU11274, was employed to inhibit c-met activity. c-met inhibition resulted in significantly reduced HLX mRNA expression (e.g. 2.1 ± 0.32, SU11274 v. 12.3 ± 1.4 control, densitometric units, P < 0.05, n = 3). HLX expression remained significantly reduced with HGF stimulation and SU11274 mediated c-met inhibition (e.g. 8.02 ± 1.3, SU11274 v. 38.3 ± 5.4 HGF, densitometric units, P < 0.05, n = 3). This is the first study to show that homeobox gene HLX is a downstream effector gene of HGF, that HLX regulates trophoblast migration and that HGF, via its receptor c-met, acts through HLX to control cell migration.
(1) Rajaraman G, Murthi P, Quinn L, Brennecke SP, Kalionis B. Homeodomain protein HLX is expressed primarily in cytotrophoblast cell types in the early human placenta. (2008) Reproduction, Fertility a
(2) Murthi P, Doherty V, Said J, Donath S, Brennecke SP, Kalionis B. Homeobox gene HLX1 expression is decreased in idiopathic human fetal growth restriction. (2006) Am J Pathol. 2006 Feb;168(2):511–8.
(3) Rajaraman G, Murthi P, Leo B, Brennecke SP, Kalionis B. Homeobox gene HLX1 is a regulator of colony stimulating factor-1 dependent cell proliferation. (2007) Placenta Volume 28, Issue 10, October