bluuebunny/crossref_metadata_2025 huggingface dataset:
222. A new role for activin in endometrial restoration after menses
Authors: Kaitu'u-Lino T. J. , Phillips D. J. , Morison N. B. , Salamonsen L. A.
Published: November 2010

10% of Australian women suffer from abnormal uterine bleeding (AUB). To stop endometrial bleeding after menstruation, the endometrium must repair adequately. We propose that endometrial restoration after menstruation has characteristics of wound healing and that inadequate endometrial repair may result in AUB. In vivo studies support a contribution of activins to skin wound healing: in mice overexpressing activins' natural inhibitor, follistatin, wound healing is significantly delayed (1). We hypothesised that activin would enhance endometrial repair and examined its contribution using an in vitro wound healing model and our well characterised in vivo mouse model of endometrial breakdown and repair (2). For the in vitro model, confluent human endometrial epithelial cells (ECC-1 cell line) were wounded and treated with carrier protein (control, 0.1% BSA), activin A (50ng/mL) or EGF (positive control: 50ng/mL). Wound areas were quantitated daily for 6 days. For the in vivo study, serum follistatin levels were measured by ELISA in follistatin overexpressing mice (FS) (2) and wild-type (WT) littermates. Mice were induced to undergo endometrial breakdown and repair (mimicking menstruation in women). Activin βA was immunolocalised during endometrial repair, and extent of repair assessed using our morphological scoring system (2). ECC-1 wound repair was significantly (P < 0.05) enhanced by activin A treatment v. control from days 2–6 of culture. In WT mice, activin βA localised to areas of endometrial repair. Serum follistatin was significantly elevated in FS mice v. controls (33.3 ± 3.8 v 7.07 ± 1.8 ng/mL, P < 0.01). In FS mice (n = 8) only 50% of uterine sections showed complete repair after endometrial breakdown, significantly less than those from WT animals (n = 15, P < 0.05) where 85% of sections demonstrated complete repair. These results demonstrate for the first time that activin A functions to promote endometrial restoration following menses and that this can be delayed under physiological conditions: such studies indicate potential treatments for AUB.
(1) Wankell et al. (2001) EMBO J 20:5361–5372
(2) Kaitu'u-Lino et al. (2007) Endocrinology 148:5105–5111
223. Interleukin-11 inhibits human trophoblast invasion via STAT-3 and not MAPK, indicating a likely role in the decidual restraint of trophoblast invasion during placentation
Authors: Paiva P. , Salamonsen L. A. , Manuelpillai U. , Dimitriadis E.
Published: November 2010

Successful pregnancy depends on the precise regulation of extravillous trophoblast (EVT) invasion into the uterine decidua, primarily by decidua-derived factors. In humans, during early pregnancy, interleukin (IL)-11 is maximally expressed in the decidua1, with its receptor, IL-11-receptor α (Rα) also identified on invasive EVT in vivo2. While a role for IL-11 in EVT migration has been established2, whether it also plays a role in regulating EVT invasion is unknown. We investigated whether IL-11 influences human EVT invasion and the signalling pathways and underlying mechanisms involved using the HTR-8/SVneo immortalised EVT cell-line and primary EVT as models for EVT. The effect of IL-11 on tyrosine phosphorylation (p) of signal transducer and activator of transcription (STAT)-3 was determined by Western Blot. EVT invasion was assessed using in vitro Matrigel invasion assays. To elucidate the mechanisms by which IL-11 may influence EVT invasion, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA) activity were assessed by gelatin and plasminogen zymography / uPA activity assay respectively. Tissue inhibitor of MMPs (TIMPs)-1 and –2, plasminogen activator inhibitor (PAI)-1 and –2 and uPA receptor (uPAR) were assessed by ELISA whereas TIMP-3 was assessed by Western Blot. EVT adhesive properties and integrin expression were assessed by in vitro adhesion assays. IL-11 (100 ng/mL) significantly inhibited invasion of EVT cells by 40–60% (P < 0.001). This effect was abolished by inhibitors of STAT-3 but not of mitogen-activated protein kinase pathways. IL-11 (100 ng/mL) had no effect on MMP-2 and –9, TIMP 1–3, uPA, uPAR, PAI-1 and –2 in EVT conditioned media and / or cell lysates. IL-11 (100 ng/mL) also did not regulate EVT cell adhesion or integrin expression. These data demonstrate that IL-11 inhibits human EVT invasion via STAT-3 indicating an important role for IL-11 in the decidual restraint of EVT invasion during normal pregnancy.
(1) Dimitriadis et al. (2003) Reprod Biol Endocrinol. 1, 34–38
(2) Paiva et al. (2007) Endocrinol. 148, 5566–72
224. Disruption of bi-directional oocyte-cumulus paracrine signalling during oocyte in vitro maturation reduces subsequent mouse fetal survival
Authors: Yeo C. X. , Gilchrist R. B. , Lane M.
Published: November 2010

During folliculogenesis, oocyte to cumulus cell (CC) bi-directional communication is essential for normal development of the oocyte. We recently showed that addition of recombinant oocyte paracrine factor growth differentiation factor 9 (GDF9) during mouse oocyte in vitro maturation (IVM) increased fetal viability. GD.F. 9 signals through SMAD 2/3. Hence the effects of disrupting SMAD2/3 signalling and its interaction with FSH/EGF during IVM on oocyte development and subsequent fetal outcomes were investigated. Cumulus-oocyte complexes (COCs) from antral follicles (n = 400–500) of eCG treated pre-pubertal (C57BL/6xCBA F1 hybrid) mice were cultured for 18 h in Waymouth's medium+5% serum, with or without 50 mIU/mL FSH and 10ng/mL EGF, SMAD2/3 inhibitor SB-431542 (4µM), or its 0.04% DMSO control. Meiotic maturation was assessed by first polar body (PB1) extrusion immediately after culture. COCs were fertilised and cultured to the blastocyst stage in G1.2/G2.2 media at 37°C in 6%CO2:5%O2:89%N2. Blastocysts were either transferred to pseudo-pregnant Swiss females or differentially stained. Pregnancy outcome was assessed on Day 18 of pregnancy. Inhibition of SMAD 2/3 signalling did not alter meiotic maturation. No differences were observed in the percentage of blastocysts or hatching blastocysts from cleaved embryos with SMAD2/3 inhibition or the absence of FSH/EGF. However, IVM with SB-431542 or without FSH/EGF significantly decreased (P < 0.001) blastocyst inner cell mass percentages (26% v. 35% control;18% v. 28% control respectively). Fetal survival (fetuses per embryo transferred) of oocytes matured with SB-431542 was significantly decreased (30% v. 50% controls; P < 0.05) although implantation rates and subsequent fetal weights were unaffected. These findings demonstrate the importance of oocyte-CC communication throughout IVM. Inhibition of oocyte signalling through SMAD2/3 resulted in reduced blastocyst quality and fetal survival; outcomes similar to that of oocytes matured without FSH/EGF. Oocyte–cumulus cell bi-directional communication is thus an important feature of oocyte viability and has a substantial impact on subsequent fetal outcomes.
225. Characterisation of a diverse secretome generated by the mouse preimplantation embryo
Authors: O.'Neill C. , Beardsley A.
Published: November 2010

This study investigated the suitability of surface-enhanced laser desorption and ionisation time-of-flight (SELDI-TOF) and electro-spray ionisation (ESI) mass spectrometry for the analysis of the proteins released by the mouse preimplantation embryo in vitro. SELDI-TOF analysis with CM10 or IMAC30 (but not Q10) chips detected a protein peak at m/z 8570 released by both C57BL6 and hybrid embryos. No other peaks unique to the embryo were identified with this method. ESI mass spectrometry of tryptic digests of embryo conditioned media identified a total of 20 proteins released during development from the zygote to blastocysts stage. Four proteins were expressed in at least 7 out of 8 cultures tested, one of these (lactate dehydrogenase B) was in all cultures. A further five proteins were in at least half of the cultures and 11 more putative proteins were detected in at least one culture. The pattern of protein secretion was not obviously different for C57BL6 or hyrid embryos. The expression of two of these proteins is essential for preimplantation embryo development (NLR family, pyrin domain containing 5 protein and peptidyl arginine deiminase, type VI). A further four proteins detected have roles in redox regulation of cells, and three others are capable of inducing post-translational modification of proteins. This study shows the feasibility of ESI mass spectrometry and the limitations of SELD-TOF mass spectrometry for identifying the proteins scereted by the preimplantation embryo in vitro. This analysis identifies a range of targets that now require detailed functional analysis to assess whether their release by the embryo is an important property of the early embryo, or an artefact of in vitro culture.
226. Characterisation of a putative mouse sperm - zona pellucida receptor complex
Authors: Dun M. D. , Nixon B. , Aitken R. J.
Published: November 2010

Mammalian spermatozoa acquire the ability to fertilise an oocyte as they ascend the female reproductive tract. This process is characterised by a complex cascade of biophysical and biochemical changes collectively known as capacitation. The attainment of a capacitated state is accompanied by a dramatic reorganisation of the surface architecture which renders the spermatozoa competent to recognise the heterogenetic matrix of the zona pellucida surrounding the oocyte and initiate fertilisation. Emerging evidence from our laboratory indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, to date the presence and composition of such a complex has yet to be described. Through the novel application of blue native polyacrylamide gel electrophoresis (BN-PAGE), we have provided the first evidence that capacitated mouse spermatozoa express high molecular weight, multimeric protein complexes on their surface. Interestingly, at least two of these complexes contain heat shock protein 1 (HSPD1), a molecular chaperone that has previously been implicated in sperm-zona pellucida interaction. Furthermore, we were able to demonstrate that one of these complexes also possessed an affinity for solubilised zona pellucida as determined by Far-western blotting. 2D BN-PAGE was employed to further delineate the individual constituents of this high molecular weight complex, with several other chaperonin proteins not previously reported in functional sperm indentified. Collectively, these results support the notion the sperm-zona pellucida interaction are mediated by a multimeric receptor complex. Our current work is focussed on the identification of the key zona adhesion molecules that comprise this complex.
227. Eggs with a surprise: the 'sperm-specific' protein SPRASA is also expressed in the oocyte
Authors: Chamley L. W. , Wagner A. , Prendergast D. , Woad K. J. , Shelling A. N.
Published: November 2010

SPRASA is a newly identified protein which in silico analysis suggests is not expressed in other tissues. Antibodies reactive with SPRASA have been identified in some infertile men and an antiserum reactive with recombinant SPRASA prevented human sperm binding to hamster oocytes in vitro, indicating an important role in sperm/oocyte recognition. The aim of this study was to investigate the spatial and temporal expression of SPRASA in reproductive and other tissues. Brain, thymus, heart, spleen, kidney, liver and the reproductive organs from duplicate female and male Balb/C mice were collected at several postnatal timepoints. RNA was extracted, reversed transcribed and analysed by quantitative real time PCR for SPRASA expression. Abattoir-derived, in vitro matured, bovine oocytes were examined for SPRASA expression by fluorescent immunochemistry. To examine SPRASA binding sites on oocytes, matured bovine oocytes were exposed to biotinylated recombinant human SPRASA or biotinylated α-lactalbumin (control), then visualised by confocal microscopy using DTAF-conjugated streptavidin. We found SPRASA mRNA was expressed in the reproductive organs of both females and male mice from postnatal day 10. Fluorescent immunochemistry indicated SPRASA was expressed on the oolemmal membrane and in the few cumulus cells remaining attached to zona-intact oocytes. Control preimmune serum did not stain the oocytes or cumulus cells. Recombinant human SPRASA bound to the oolemmal membrane of both zona intact and zona free bovine oocytes. To date the expression of SPRASA has only been reported in the testes/sperm with an additional single EST identified in brain. Our quantitative real-time PCR analysis demonstrated SPRASA is also expressed in the female reproductive organs. This was confirmed by our immunoassays which show oocytes and possibly cumulus cells express SRPASA while the oolemmal membrane has the ability to bind (sperm-derived) SPRASA. That SPRASA expression is restricted sperm and oocytes confirms the likely function of this protein in reproduction.
228. Proteasomal activity during mouse preimplantation development
Authors: McCue K. , Pantaleon M. , Kaye P. L.
Published: November 2010

Function of the 26S proteasome, a proteolytic organelle directed at proteins targeted for turnover by polyubiquitination, in preimplantation embryos is unclear. But it is well known to play a role in regulating meiosis. This paper reports the distribution of the proteasome and assessment of its functional importance in preimplantation development. Embryos from superovulated mice were either paraformaldehyde fixed for immunolabelling with a rabbit polyclonal antibody against the 20S proteasome core or cultured in KSOM medium with and without reversible (MG132) or irreversible (β-lactone) proteasomal inhibitors. Morphology, cell number, apoptosis and proteolysis were measured. Although diffuse throughout embryonic cytoplasm, there were distinct proteasomal concentrations in pronuclei, nuclei and cortical cytoplasm. When β-lactone was used to block blastocyst proteasomal proteolysis, ~25% of protein degradation was found to be proteasome-specific. Treatment of 2-cell embryos for more than 3 h with MG132 blocked blastocyst formation completely, even after washout, whilst both inhibitors reduced cell proliferation over the ensuing 48 h. Two hours exposure to MG132 tripled the proportion of apoptotic cells in expanded blastocysts 96 h post hCG. The nuclear concentration of proteasomes suggests a particular role in nuclear protein degradation possibly including the timed destruction of cell-cycle regulators and anti-apoptotic factors. This is supported by the loss-of-function studies which show that cell proliferation as well as morphogenesis require proteasomal activity at the late 2-cell stage and that without it apoptosis is dramatically increased. The mechanisms involved in the activation of apoptosis as a result of proteasomal inhibition in the early embryo are unknown but may include JNK signalling although this is controversial. More intriguing however is the identity of the proteasomal targets in the 2-cell embryo that must be degraded to permit continued morphogenesis.
229. Identification of transforming growth factor beta2 (TGF-β2) and its receptors TGF-βRI and TGF-βRII in the possum (Trichosurus vulpecula) prostate: evidence of seasonal changes
Authors: Martyn H. , Pugazhenthi K. , McLeod B. , Nicholson H. D.
Published: November 2010

Benign Prostatic Hyperplasia is an enlargement of the prostate affecting the ageing male population. The common Brushtail possum (Trichosurus vulpecula) has been identified as a possible model to study factors regulating prostate growth because its prostate grows and regresses seasonally. Transforming growth factor Beta 2 (TGF-β2) is present in human prostatic tissue. In vitro, TGF-β inhibits epithelial cell, but stimulates stromal cell proliferation (Mori et al. 1990). TGF-β2 binds to TGF-β receptor II (TGF-βRII), which then recruits the type 1 receptor (TGF-βRI) (Saez et al. 1998) The aim of this study was to identify any seasonal changes in expression of TGF-β2 and its receptors in the possum prostate. Six wild-caught possums were sacrificed in each of the months of January, March, May, July, September and November. The prostates were divided into a cranial and caudal region and immunohistochemistry and Western Blot analysis performed. In each animal the glandular and periurethral areas of the caudal and cranial prostates were examined separately. Immunohistochemistry identified the presence of TGF-β2 in both the stromal and epithelial cells of the glandular and periurethral areas of the cranial and caudal regions. In the cranial tissue, more immuno-positive stromal cells than epithelial cells were present, whereas in the caudal tissue immuno-reactivity was predominantly localised to the epithelial cells. Analysis of the western blots suggested that TGF-β2 expression was lowest immediately before and during the breeding season (March, May). Both TGF-βRI and TGF-βRII were identified in all regions of the prostate. Furthermore, immunohistochemistry revealed that the receptors were co-localised in the epithelial and stromal cells in all areas. TGF-β2 and its receptors are present in the possum prostate. TGF-β2 localisation varies between the caudal and cranial regions and as predicted from in vitro experiments TGF-β2 expression decreases during prostate growth.
(1) Mori H. et al. (1990). The Prostate, 16, 71 - 80.
(2) Saez C. et al. (1998). The Prostate, 37, 84 - 90.
230. Megalin, RAP and Nkx3.1 expression in the developing reproductive tract of a marsupial, the tammar wallaby
Authors: Gamat M. , Renfree M. B. , Pask A. J. , Shaw G.
Published: November 2010

Androgens induce the differentiation of the urogenital sinus (UGS) to form a prostate. An early marker of this response is upregulation of the transcription factor Nkx3.1 in the urogenital epithelium in the precursors of prostatic buds. In tammars, prostate differentiation begins ~3 weeks after birth and after the time the testis starts to secrete androgens, and 2 weeks after androgen stimulated Wolffian duct differentiation. The reason for this delay in prostate differentiation is unexplained. Androgen receptors are present in the UGS, and the potent androgen, androstanediol, induces prostatic development in females. Whilst androgens may diffuse into cells by across the cell membrane, there is increasing evidence that steroids are also internalised actively via the cell-surface transport molecule Megalin. We are exploring the possibility that the delay may be related to the establishment of a Megalin-mediated pathway. Megalin is a cell surface receptor expressed on epithelia and mediates the endocytosis of a wide range of ligands, including SHBG-bound sex steroids. Megalin action is regulated by Receptor Associated Protein (RAP), which acts as an antagonist to Megalin action. This study cloned partial sequences of Megalin, RAP and Nkx3.1 and examined their expression in the developing urogenital sinus of the tammar wallaby using RT–PCR. The cellular distribution of Megalin protein in the developing UGS was examined using immunohistochemistry. Megalin, RAP and Nkx3.1 in the tammar were all highly conserved with eutherian orthologueues. Megalin and Nkx3.1 transcripts were detected in the liver, kidney, ovary, testis and developing urogenital sinus of male and female tammars. In the developing UGS of the tammar, there was strong staining for Megalin protein in the urogenital epithelium with some diffuse staining in the surrounding mesenchyme. Together, these results suggest that Megalin could be a key gene in the mediation of androgen action in prostatic development in the tammar wallaby.
231. Sertoli cells de-differentiate in men after chronic gonadotrophin suppression
Authors: Tarulli G. A. , Stanton P. G. , Loveland K. , De Meyts E. Rajpert , McLachlan R. I. , Meachem S. J.
Published: November 2010

Recent studies suggest that adult Sertoli cells are not part of a homogenous and terminally differentiated population in phenotypes infertility1–4. The aims of this study were to compare the differentiation status of Sertoli cells from normal men undergoing gonadotrophin suppression by hormonal-based contraception (androgen plus progestin), as well as in pre-malignant and malignant testicular cancer. Confocal microscopy was performed to assess the expression of markers of cell proliferation (PCNA) and differentiation (androgen receptor) in Sertoli cells in all tissues. As additional markers of differentiation, Sertoli cell tight junction (claudin-11, JAM-A) and associated proteins (ZO-1) were assessed in men with testicular cancer. Samples from five different men were assessed in each group. In normal men, Sertoli cells exhibited a differentiated phenotype (i.e. PCNA negative, androgen receptor positive). However, after gonadotrophin suppression, 1.7 ± 0.6% of Sertoli cells exhibited intense PCNA reactivity and a reduction in androgen receptor immunoreactivity, demonstrating an undifferentiated phenotype. PCNA-positive Sertoli cells were never observed in pre-malignancy, and were only rarely observed in malignant testicular cancer, indicating a potential change in differentiation. Tight junction protein localisation was disrupted in pre-malignant cancer, with a reduction in JAM-A reactivity in Sertoli cells from pre-malignancy and strong JAM-A reactivity in malignant cancer; suggesting a potential role for JAM-A expression in the progression of testicular cancer. We conclude that Sertoli cells are not a homogenous and terminally differentiated population in men and their differentiation is modifiable by hormones and in the disease state.
(1) Meachem et al. 2005, Biol Reprod. 72:1187–93
(2) Tarulli et al. 2006, Biol Reprod. 74:798–806
(3) Donner et al. 2004, APMIS. 112:79–88
(4) Brehm et al. 2006, Anat Embryol (Berl). 211(3):223–36
233. A role for activin/inhibin in mouse gonocyte relocation and proliferation
Authors: Tubino L. , Barakat B. , Prahbu S. , Meachem S. , Nagaraja A. , Matzuk M. , Loveland K. L.
Published: November 2010

Gonocytes in the testis resume proliferation after birth and relocate to contact the basement membrane of the seminiferous cords where they become spermatogonia. A previous in vitro study indicated that activin can increase gonocyte numbers on day 3 postpartum (dpp) rat testis, while the activin antagonist, follistatin, together with FSH, increased the number of spermatogonia (Meehan et al. 2000). The aim of this study was to understand how FSH, activin and inhibin, a potent activin antagonist, interact to influence gonocyte proliferation and relocation in the newborn mouse testis using in vivo and in vitro approaches. Two mouse models were analysed, the inhibin α knock out (inh a −/−) mouse and the InhbaBK mouse. The Inh a −/− mouse lacks inhibin, and thus activin acts unopposed by its most potent antagonist (Matzuk et al. 1992). The InhbaBK mouse has the Inhbb allele inserted into the Inhba locus, thus directing the expression of the less bioactive activin βB, in the spatiotemporal pattern of activin βA (Brown et al. 2000). In addition, an in vitro model was developed in which 1dpp wild type testis fragments were cultured in hanging drops for 24 h with the addition of combinations of activin, inhibin and FSH. Gonocyte proliferation in inh a −/− was assessed using proliferating cell nuclear antigen (PCNA). A significant increase in germ cell proliferation and relocation to the basement membrane was measured in 0dpp inh a −/−, while no difference was observed at 4dpp. The opposite was observed in InhbaBK mice, with reduced gonocyte migration in mutant animals at 0dpp. In vitro, inhibin seemed to inhibit proliferation and reduce the percentage of relocated gonocytes while FSH showed a tendency for the opposite effect on gonocyte migration. These findings show that inhibin levels affect germ cell development during early postnatal development in mouse testis influencing both cell maturation and proliferation.
(1) Meehan T, Schlatt S, O'Bryan MK, de Kretser DM, Loveland KL. Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH. Dev Biol. 2000 Apr 15;220(2):225–37.
(2) Matzuk MM, Finegold MJ, Su JG, Hsueh AJ, Bradley A. Alpha-inhibin is a tumour-suppressor gene with gonadal specificity in mice. Nature. 1992 Nov 26;360(6402):313–9.
(3) Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM. Insertion of Inhbb into the Inhba locus rescues the Inhba null phenotype and reveals new activin functions. Nat Genet. 2000 Aug;25(4):453–7.
234. Hedgehog signalling components in adult rat testis spermatogonial cells
Authors: Sahin Z. , Meistrich M. , Szczepny A. , Loveland K.
Published: November 2010

In normal tissues, Hedgehog-induced progenitor cell proliferation is transient and tightly regulated, preventing continuous regeneration. However, activation of constitutive Hedgehog signalling results in unregulated self-renewal of progenitor cells in association with several human cancers. Although the contribution of Hedgehog signalling to cancers is widely accepted, its impact on spermatogonial stem cells and impact on male fertility are unknown. In this study, we aimed to clarify the possible role of Hh signalling on normal spermatogenesis in the adult rat and in adult testicular stem cells in the irradiated model {1}. Adult male rats were obtained from Monash University Central Animal Service and killed by cervical dislocation before tissue removal and fixation in Bouins for routine histochemical procedures. For studies on irradiated testes, adult LBNF1 male rats (hybrids between Lewis and Brown–Norway) were purchased from Harlan Sprague–Dawley, Inc. (Indianapolis, IN, USA). Testes were irradiated with 6 Gy to deplete all maturing germ cell types. At 15 weeks after irradiation the animals were injected simultaneously with 1.5 mg each of Cetrorelix pamoate and Cetrorelix acetate. Testes were collected 1, 2 or 4 weeks after injection. In situ hybridisation combined with immunohistochemistry was performed using DIG-labelled cRNA probes to identify the cells in which Hedgehog signalling components are made {2}. Signals for mRNAs encoding t he transmembrane receptors Ptc2 and Smo are most intensely detected in spermatogonia and spermatocytes and are much less intense in the round spermatids. The mRNA for the cytoplamic regulator, Fused, is restricted to the earliest germ cell types, whereas expression of the negative cytoplasmic regulator, SuFu, only begins in the round spermatids and persists in elongating spermatids. Gli1 and Gli3 are expressed from spermatogonia through to round spermatids, whereas Gli2 is restricted to spermatogonia and spermatocytes. This pattern mimics what was reported for mouse {2}. Examination of the irradiated rat testis model revealed that Hedgehog signalling machinery is produced by resting spermatogonial stem cells but is turned off when they differentiate in response to hormones. This matches the emerging understanding of Hedgehog signals in cancer stem cells and provides the first demonstration that Hedgehog signalling may influence stem cells in the adult testis.
(1) Shuttlesworth G.A. et al. 2000. Endocrinology. 141: 37–49
(2) Szczepny A. et al. 2006. Dev Dyn. 235:3063–3070.
236. Effects of long term recombinant rat follicle-stimulating hormone replacement on the restoration of spermatogenesis after chronic suppression of gonadotrophins in adult rats
Authors: Ruwanpura S. M. , Stanton P. G. , Robertson D. M. , McLachlan R. I. , Makanji Y. , Meachem S. J.
Published: November 2010

Follicle stimulating hormone (FSH) in short-term rat studies supports spermatogenesis at multiple levels, notably spermatogonial development. The role of FSH in supporting full spermatogenesis in rats is still in question as long-term studies have not been possible due the development of neutralising antibodies to heterologous FSH preparations. This study sought to assess the effects of a homologous recombinant rat FSH (rr-FSH) preparation on the long-term restoration of spermatogenesis. Adult rats were GnRH-immunised (GnRH-im) for 12 weeks then, administered an anti-androgen; flutamide (flut), alone or together with rr-FSH (8µg/rat/daily) for 56 days (1 spermatogenic cycle). Germ and Sertoli cell numbers were quantified using an optical disector stereological method. Testis weight, serum FSH and inhibin B and Sertoli cell nuclear volume were significantly reduced to 15%, 13%, 25% and 57% of controls respectively, following GnRH-im+flut treatment. GnRH-im+flut treatment reduced A/I spermatogonial, type B spermatogonial+preleptotene, leptotene+zygotene and early pachytene spermatocyte numbers to 28%, 68%, 50% and 19% (P < 0.001) of controls respectively, with later germ cells rarely observed. After FSH treatment, no significant affect on testis weight, serum FSH and inhibin B or Sertoli cell number were observed. However, rr-FSH treatment significantly increased numbers of A/I spermatogonia, leptotene+zygotene and early pachytene spermatocytes from 28 = >42%, 50 = >69% and 19 = >27% of controls, respectively, while no differences were observed in later germ cell types. rr-FSH also increased (P < 0.05) the volume of Sertoli cell nuclei from 57 = >66% of control. In conclusion, FSH is unable to support full rat spermatogenesis; however, FSH can partially support germ cells notably spermatogonia through to early pachytene spermatocytes, despite the absence of androgenic support.
237. AR-mediated androgen actions are essential for normal mouse uterine growth and development but not implantation and embryo development
Authors: Walters K. A. , Jimenez M. , Salamonsen L. A. , Handelsman D. J.
Published: November 2010

Recently the androgen receptor (AR) has been shown definitively to play a role in female reproduction. We generated a homozygous AR−/− female mouse using Cre/LoxP recombination for an in-frame excision of exon 3, encoding the second zinc finger essential for DNA-binding, while allowing production of an exon 3 deleted mutant AR protein which is nonfunctional as a nuclear transcription factor. AR−/− females were sub-fertile due primarily to ovulatory dysfunction (1). However, the mechanism(s) of the observed sub-fertility remains to be fully defined. To evaluate the role of AR in uterine function we carried out a morphological and function analysis of the AR−/− uterus. Uterine weights did not differ, however, AR−/− females exhibited a significant increase in uterine horn length (P < 0.01), and a significant reduction in uterine diameter (P < 0.01), total uterine area (P < 0.01), endometrial area (P < 0.05) and myometrial area (P < 0.01), indicating a role for genomic AR-mediated actions in physiological uterine growth and development. Furthermore, during late pregnancy AR−/− females had significantly fewer implantation sites (P < 0.01), fetuses present in utero (P < 0.05) and a lower serum progesterone concentration (P < 0.01). In spite of these findings, AR−/− females had normal gestational length, parturition and pup weights, as well as similar pre- and post implantation losses compared with AR+/+ females. Therefore, although AR is not essential for normal uterine reproductive function, disrupting genomic AR signalling in the uterus leads to dysfunctional uterine development which may have important long-term functional consequences for hormone dependent uterine disorders such as endometrial hyperplasia and cancer.
(1) K. A. Walters et al. Endocrinology 148, 3674 (2007).
238. Endometrial vessel morphology is altered following progestin treatment in a mouse xenograft model
Authors: Donoghue J. F. , Girling J. E. , Rogers P. A. W.
Published: November 2010

Human endometrium undergoes cyclic changes under the influence of oestrogen and progesterone. When progestins are used for contraception, the endometrium regresses and breakthrough bleeding often occurs. The aim of this study was to investigate the short-term effects of progestin on human endometrium in a mouse xenograft model. Uterine tissue was placed subcutaneously into NOD/SCID mice (n = 12). Mice were given estradial valerate every fourth day for two weeks. Mice then received an implant containing medroxyprogesterone acetate (MPA) or an empty implant. After two weeks, mice were dissected and the xenografts formalin fixed and serially sectioned (5µm) for immunohistochemical analysis. Sections were double immunostained for α-smooth muscle actin and either FVIII (blood vessels) or D2–40 (lymphatic vessels). The endometrium from the progestin treated group contained decidual-like stroma cells and glandular epithelium with morphology ranging from squamous to columnar. The endometrium from the control group also contained fibroblast-like stromal cells and glandular epithelium with tall columnar epithelium. The endometrial blood vessel density was significantly reduced in the progestin-treated group (156.3 ± 13.4 vessel profiles/mm2) compared with the control group (273.5 ± 41.5 vessel profiles/mm2) (P = 0.02); there was no significant difference in lymphatic vessel density (progestin: 43.5 ± 5.9 v. control: 35.6 ± 9.6 vessel profiles/mm2). Blood vessel area was significantly increased in the progestin-treated group (3.7x10−4 ± 1.7x10−5 mm2) compared with controls (1.8x10−4 ± 1.2x10−5 mm2) (P = 0.0001) and the lymphatic v essel area was also significantly increased in the progestin-treated group (8.8x10−4 ± 7.8x10−5 mm2) compared with controls (2.9x10−4 ± 5.7x10−5 mm2) (P = 0.0001). This work has provided a model for the study of human endometrial vasculature, illustrating a significant increase in blood and lymphatic vessel size during progestin treatment. The increase in blood vessel size was associated with a significant reduction in blood vessel density in progestin treated samples.
239. Expression of PAF-R and p53 in the endometrium during entry into and reactivation from diapause in the tammar wallaby
Authors: Fenelon J. C. , Shaw G. , Renfree M. B.
Published: November 2010

Embryonic diapause is widespread amongst mammals, but is especially common in the kangaroos and wallabies. In the tammar, Macropus eugenii, the sequence of endocrine events leading to embryonic diapause and reactivation are well defined and the blastocyst can remain in diapause for up to 11 months without cell division or apoptosis occurring (Renfree and Shaw 2000). The ovarian hormones exert their effects on the blastocyst by alterations in the endometrial secretions, but the molecular cross-talk between the endometrium and blastocyst is unknown. One possible regulator of diapause is the phospholipid PAF, an embryotrophin that acts as a trophic/survival factor for the early embryo (O'Neill 2005) partly by inactivating the expression of p53, a cell cycle inhibitor, via the PI3-K pathway. PAF is released from the tammar endometrium around the time of reactivation from diapause (Kojima et al. 1993). This study examined the expression of PAF-R and p53 in the tammar endometrium at entry into, and reactivation from, diapause. PAF-R and p53 were highly conserved with orthologueues in human and mouse. PAF-R and p53 expression was assessed by RT–PCR and both genes were expressed in the endometrium at all stages examined. Quantitative PCR (QPCR) studies performed for PAF-R in the endometrium show that levels of PAF-R vary depending on the stage examined and appear to be increasing at entry into diapause and decreasing at exit from diapause. Immunohistochemical (IHC) studies are in progress to determine the cellular location of PAF-R in the endometrium and confirm the QPCR results. QPCR and IHC studies are in progress to determine if there is any change in levels of expression or cellular location of p53 between the stages examined and how this relates to PAF-R availability. These results suggest that the control of diapause in the tammar involves interactions between multiple factors.
(1) Renfree MB, Shaw G (2000) Diapause. Annu Rev Physiol 62, 353–375
(2) O'Neill C (2005) The role of Paf in embryo physiology. Human Reproduction Update 11, 215–228
(3) Kojima T et. al. (1993) Production and secretion of progesterone in vitro and presence of PAF in early pregnancy of the marsupial, Macropus eugenii. Reproduction Fertility Development 5, 15–25.
240. Reduced endometrial angiogenesis during early pregnancy in relaxin-deficient mice
Authors: Girling J. E. , Ng L. , Parry L. J.
Published: November 2010

Relaxin is a peptide hormone with important roles in the reproductive tract, including the growth and remodelling of endometrial vasculature. It has been shown to stimulate VEGF secretion from human endometrial stromal cells in vitro and increases endometrial vascularisation in ovariectomised steroid-primed primates in vivo. We have used mouse models to show that oestrogen and progesterone stimulate angiogenesis (new blood vessel growth) within the endometrium. Endometrial angiogenesis also occurs in the early stages of mouse pregnancy, which coincides with an increase in circulating progesterone. To date, no studies have investigated the effects of relaxin on endometrial angiogenesis in early pregnancy. Our aim was to test the hypothesis that endometrial angiogenesis would be reduced in relaxin-deficient mice (Rln−/−) in comparison to their wildtype (Rln+/+) counterparts. Uterine tissues were collected from Rln−/− and Rln+/+ mice on days 1 to 4 of pregnancy, before implantation. All mice were treated with BrdU before dissection to allow the number of blood vessel profiles containing proliferating endothelial cells (PVPs) to be quantified by double CD31/BrdU immunohistochemistry. Consistent with published studies, PVPs were first observed on days 3 and 4 of pregnancy. However, the percentage of PVPs was reduced in Rln−/− mice compared with Rln+/+ mice (Day 3: median = 4.4% v. 19.6%, Day 4: 9.6% v. 22.2%). We subsequently identified relaxin and relaxin receptors in the mouse endometrium in early pregnancy. Our data suggest that locally synthesised relaxin acts in synergy with progesterone to initiate endometrial angiogenesis in early pregnancy.
241. Relaxin and INSL3 receptors and their signaling in primary human myometrial cells
Authors: Heng K. , Ivell R. , Waagarachi P. , Anand-Ivell R.
Published: November 2010

In model species such as the pig or rat, the ovarian peptide hormone relaxin is able to induce uterine quiescence, suppressing natural or oxytocin-induced contractility via a cAMP-dependent mechanism. This is not observed in humans. The present study investigated the ability of relaxin to activate adenylate cyclase in primary human myometrial smooth muscle cells from non-pregnant tissue obtained at hysterectomy. We show that relaxin is indeed able to stimulate the generation of cAMP, and it does this in a manner which is dependent upon the mediation of a tyrosine phosphorylation activity as has been shown by us earlier in the endometrium and THP-1 cells, though likely not involving PI3-kinase. Furthermore, we identified transcripts for the relaxin receptor RXFP1 (formerly LGR7) as full-length variants, though a minor splice variant missing exon 2 was also present in low amounts. Interestingly human myometrial cells also express transcripts encoding the full-length receptor, RXFP2 (formerly LGR8), for the closely related peptide hormone, INSL3. This receptor can also respond to relaxin at high concentration. However, this receptor does not appear to function by contributing to the cAMP production in human myometrial cells, nor does INSL3 act as a functional agonist or antagonist of relaxin action. In conclusion, therefore, the well known inability of relaxin to inhibit contractility in human myometrial cells appears to be due to uncoupling events downstream of cAMP generation.
Research supported by NH&MRC project grant 349502.
242. Reproductive performance of Australia Cashmere goats supplemented with lupin grain
Authors: Maidin M. Shikh , Chadwick A. , Khaiseb P. C. , Hawken P. A. , Martin G. B.
Published: November 2010

The productivity of Cashmere goats depends on their reproductive performance, which, in turn, depends on their level of nutrition. Ovulation rate and pregnancy in sheep are both affected by nutrition, but little is known about the response of female goats (does) to supplementary feeding. The lupin group (n = 40) received 250 g lupin per head per day in addition to pasture whereas the control group (n = 40) received no nutritional supplement. Both groups were synchronised for 17 days with intravaginal progestagen pessaries. The supplement was fed for 21 days, commencing 7 days before the bucks were introduced and intravaginal pessaries were removed (Day –2). Does were expected to ovulate 2 days later on Day 0 and the bucks were removed on Day 3. Blood was sampled for progesterone every 3 days from buck removal (Day 3) until Day 18. Ovulation rate was assessed by trans-rectal ultrasonography on Day 13 and pregnancy was diagnosed by trans-abdominal ultrasonography on Day 61 of the experiment. Does supplemented with lupins had a numerically higher ovulation rate than does fed only on pasture, but this difference was not significant (1.76 ± 3.21 v. 1.52 ± 3.79; P > 0.05). Similarly, there was no difference in the numbers of does conceiving to the first service between the lupin and control group (89% v. 94%; P > 0.05). Progesterone concentrations on Day 12 were higher in does supplemented with lupins than does fed only pasture (6.29 ± 0.27 ng/mL v. 5.41 ± 0.27 ng/mL; lupin and control group; P < 0.05). In conclusion, lupin supplementation induced a numerical increase in ovulation rate but this difference failed to reach significance. Does supplemented with lupins had higher concentrations of progesterone during early pregnancy, which is the opposite effect to that previously reported in sheep.
243. Interleukin-6 is an essential regulator of parturition and perinatal viability in mice
Authors: Robertson S. A. , Dorian C.
Published: November 2010

IL-6 is an immune-regulatory cytokine which has functional overlap with LIF and IL-11 due to shared use of the gp130 receptor. IL-6 is synthesised abundantly in the uterus throughout pregnancy and at the time of parturition. Previously we have shown that mice with a null mutation in the Il6 gene (Il6−/−) have elevated rates of fetal resorption (45% v. 14% in WT controls) and a high incidence of postnatal death. Additionally, timing of birth is delayed ~24 h in Il6−/− mice (1), in association with altered uterine expression of several parturition-associated genes including PGHS-2, PTGFR and CX-43, and delayed progesterone decline (2). To investigate the effect of exogenous IL-6 replacement on perinatal parameters, Il6−/− and WT females were mated with males of the same genotype and on day 11.5 pc were surgically implanted subcutaneously with Alzet micro-osmotic pumps (7 day) containing 1 μg rhIL-6 (R&D Systems), or PBS+0.1% BSA carrier alone (n = 12 females per group). The mean time of parturition was advanced after rhIL-6 replacement in Il6−/− females from day 20.3 pc to day 19.6 pc (P < 0.05), but was unchanged in WT mice (day 19.5). As expected, the number of viable pups delivered by Il6−/− mice was less than in WT mice, but was unchanged in either group by IL-6 replacement. However rhIL6 replacement in Il6−/− mice substantially increased the proportion of neonates that survived to weaning (from 60% to 98%, P < 0.05), with no effect on postnatal survival in WT mice (76% v. 88%). Together these data provide further evidence supporting a central role for IL-6 in the events of parturition and postnatal survival, and indicate that exogenous IL-6 replacement in IL-6 deficient mice can improve perinatal outcomes.
(1) Robertson et al. ASRB 31 Abstract 97, 2000
(2) Robertson et al. SGI Abstract 729, 2008
244. Effects of relaxin treatment on hyaluronic synthase expression in the cervix of pregnant relaxin-deficient (Rln-/-) mice
Authors: Soh Y. M. , Vodstrcil L. A. , Parry L. J.
Published: November 2010

The cervical extracellular matrix (ECM) changes extensively in a process called cervical ripening in late pregnancy. This occurs through a complex series of processes leading to collagen fibre degradation and dispersal. We tested the hypothesis that relaxin increases cervical water content by stimulating hyaluronic synthase (HAS) enzymes that regulate hyaluronic acid (HA) synthesis. HA is thought to recruit water molecules into interstitial spaces between collagen fibrils, causing them to disperse. In the first study, cervices were collected from Rln+/+ and Rln−/− mice on days 14.5, 16.5, 18.5 and 19 gestation, and day 1 postpartum (PP) to compare has expression between the genotypes by qPCR analysis. In the second study, Rln−/− mice were implanted with Alzet osmotic minipumps on day 12.5 gestation to infuse either recombinant H2 human relaxin (200 μg/mL; BAS Medical Inc.) or 0.9% saline at a continuous flow rate of 0.5 μl/h. Cervices were collected after 4 or 6 days of infusion. has1 and has2 gene expression increased significantly at term and decreased immediately postpartum in the Rln+/+ mice. Although has1 and has2 were expressed in Rln−/− mice, there was no increase in expression on day 18.5 gestation and cervix wet weight did not increase compared with Rln+/+ mice. Relaxin infusion for 4 or 6 days in pregnant Rln−/− mice significantly increased cervical has2 but decreased has1 expression compared with saline-controls. Additionally, relaxin treatment caused a 6-fold increase in cervix wet weight, a 5% increase in water content and a significant decrease in relaxin receptor (Rxfp1) expression compared with saline-controls. These data suggest that relaxin promotes cervical hydration through an has2-mediated increase in HA, and may facilitate cervical ripening by causing collagen fibril dispersal in the ECM.
245. Aristaless-related homeobox gene is involved in early development and spermatogenesis in mammals
Authors: Yu H. , Hu Y. , Pask A. J. , Shaw G. , Renfree M. B.
Published: November 2010

The aristaless homeobox gene, ARX, belongs to a large family of homeodomain transcription factors with essential roles in forebrain, pancreas, muscle tissues and testes development in human and mouse. Mutation of ARX in humans results in mental retardation with or without ambiguous genitalia. We used comparative analyses to examine the evolutionary conservation of the mammalian ARX gene. We characterised ARX in a marsupial, the tammar wallaby, to determine if this gene is highly conserved in the homeodomain, aristaless domain, octapeptide motif and polyalanine tracts of all mammals. We further investigated the mRNA distribution in the developing head of tammar with in situ hybridisation, and found that it is expressed in forebrain and olfactory bulb as expected. Besides these regions, very strong expression was detected in the epithelium of the tongue and nasal pits. In the gonads, there is very strong staining in the interstitial cells and some of the germ cells in the developing ovary; strong staining was also seen in the cytoplasm of Sertoli cells and some of the germ cells, weak staining was also detected in the interstitium of the testis, possibly within the vessel endothelial cells and interstitial fibroblast-like cells. In addition, we investigated mRNA distribution in adult testes based on a very strong signal observed with northern blotting. Interestingly, mRNA expression was restricted to the round spermatids, and was not seen before or after this stage. In order to confirm this new role for ARX in the adult testis, we further investigated mRNA distribution of Arx in adult mouse testis, and found the same expression pattern, which implies a conserved function for ARX in spermatogenesis and may explain why humans with ARX mutations are infertile. This is the first report that ARX gene is involved in spermatogenesis in addition to its conserved roles in early mammalian development.