bluuebunny/crossref_metadata_2025 huggingface dataset:
247. Expression of components of the hedgehog signalling pathway during murine spermatogenesis
Authors: Szczepny A. , Jans D. A. , Hime G. , Loveland K. L.
Published: November 2010

Hedgehog (Hh) signalling is best known for its involvement in regulating patterning, driving cell proliferation, promoting cell survival and directing differentiation during embryonic development.1 The role that Hedgehog signalling plays in the testis is not yet clearly defined, though deletion of one Hedgehog ligand, Dhh, leads to male infertility. The Gli family of zinc finger TFs, consisting of Gli1, Gli2 and Gli3, are mediators of the Hh signalling cascade in vertebrates. We have previously shown that the mRNA transcripts encoding all three Glis in the adult mouse testis are expressed highly in spermatogonia, spermatocytes and to a lower extent in the round spermatids. To understand the potential sites of action of Hh proteins in spermatogenesis, we have extended our analysis to other genes involved in the Hh signalling pathway in the adult mouse testis. Using in situ hybridization, Patched2, a transmembrane receptor for Hh, was detected in spermatogonia and spermatocytes, with an apparently lower expression in the round spermatids. The mRNA of Smoothened, another transmembrane protein which forms a membrane receptor complex with Patched, is highly expressed in spermatogonia and spermatocytes, again showing lower expression in round spermatids and interstitial cells. Fused, a positive regulator of Hh signalling, is highly expressed in spermatogonia and spermatocytes with slightly lower expression in round spermatids. SuFu is a negative regulator of Hh signalling, known to repress Gli1 function in part by tethering it in the cytoplasm. The mRNA encoding SuFu is absent from spermatogonia, detected in spermatocytes and persists in round spermatids where its expression appears highest, suggesting that the SuFu protein may be acting to switch off Hh signalling at that stage of spermatogenesis. Overall, the regulated expression pattern of these genes in the adult mouse testis suggests a role for Hh signalling in the regulation of spermatogenesis.
(1)Ruiz i Altaba A. (1999) Trends Genet. 15(10), 418–425.
249. Fibroblast growth factor receptor-1 (FGFR-1) is essential for spermiogenesis, capacitation and male fertility
Authors: Cotton L. M. , Gibbs G. M. , De Kretser D. M. , O'Bryan M. K.
Published: November 2010

Male infertility is often a result of irregular sperm development/function. The identification of snt-2 (Suc-1 associated Neurotrophic Factor Target 2) and Fgfr-1 to the sperm tail, lead to the hypothesis that Fgf signalling through snt-2 is involved in sperm tail development/function. To test this hypothesis, transgenic mice carrying a dominant-negative variant of Fgfr-1, driven by the protamine 1 promoter (haploid specific) were created. Breeding experiments confirmed male fertility; however, one line was significantly sub-fertile and demonstrated a significantly reduced daily sperm production (DSP, 30%↓). Transgene expression levels were up to 70 times above native mRNA levels in wt mice; however, there was a concurrent upregulation of the native receptor in transgenic mice, resulting in only a 6× over-expression in transgenic:native mRNA. To increase transgene expression, independent lines were crossed (double heterozygous, DH). DH transgene expression levels were up to 120 times above the native mRNA in wild type mice, resulting in a 20× over-expression in transgenic:native mRNA. Breeding experiments showed males from 1 cross were significantly subfertile with DSPs further reduced (41%↓). Collectively this data shows Fgfr-1 signalling is required for quantitatively normal spermiogenesis. Given the millions of sperm that mice produce, a 40%↓ in DSP is unlikely to be responsible for the sub-fertility observed i.e. 2 v. 9 pups/litter. Therefore, a disruption of Fgfr-1 signalling may also induce a post-testicular phenotype. Western blot analysis, using tyrosine phosphorylation as a surrogate marker of sperm capacitation, showed transgenic mice had a significantly attenuated ability to initiate capacitation. As capacitation is an absolute requirement for fertilisation, the absence of capacitating capability is probably the major contributor to the sub-fertility seen in the transgenic mice. This research demonstrates for the first time that the Fgfr-1 signalling cascade is one of several pathways associated with sperm development and function.
250. Adult exposure to dietary phytoestrogens reduces fertility of male rats
Authors: Glover A. , Assinder S. J.
Published: November 2010

Phytoestrogens are plant-derived compounds that are particularly abundant in soy-based foods. Exposure to exogenous oestrogenic chemicals has been implicated in declining male fertility. The aim of this study is to deduce whether adult phytoestrogen exposure affects the reproductive function of male rats, and by what mechanisms phytoestrogens may be acting.
Six male rats were transferred from a low soy diet (control) to an experimental high soy diet, while nine males remained on the control diet. On days 3, 6 and 12 all males were mated and litter sizes recorded. A second group of male rats kept on the same dietary regimen were killed after 3, 6 or 12 days on the diets. The epididymides were collected from the rats. Real-time PCR was performed to measure mRNA quantities of oestrogen receptors alpha (ERα) and beta (ERβ), and androgen receptor (AR). The TBARS assay for lipid peroxidation was performed on epididymal sperm samples from rats fed the high or low phytoestrogen diet for 3 days.
The average litter size following 3 days on the high soy diet was significantly lower than that for rats maintained on the control diet. Litter sizes returned to control levels by day 12. ERα and AR expression decreased in the cauda region of the epididymis following 3 days on the high soy diet, but returned to control levels by day 6. Lipid peroxidation of epididymal sperm was increased in rats fed the high phytoestrogen diet for 3 days.
Short-term exposure to high phytoestrogen levels transiently reduces male fertility, and alters hormone receptor expression. Endocrine disruption may impair fertility by reducing antioxidant protection of sperm stored in the epididymis.
251. Growth differentiation factor 9 signalling systems regulate marmoset monkey granulosa cell proliferation
Authors: Ritter L. J. , Schulz S. J. , Grupen C. G. , Armstrong D. T. , Gilchrist R. B.
Published: November 2010

This study was conducted to characterize the receptor/signalling system utilized by the oocyte-secreted growth differentiation factor 9 (GDF9) to promote granulosa cell (GC) growth in the marmoset monkey. Seven adult female marmosets were primed with hFSH for 6 days, whole ovaries were removed on day 7, follicles manually excised, and GC collected from 3 size classes: periantral (PA; 0.42–0.66mm), small antral (SA; 0.66–1.5mm) and large antral (LA; >1.5mm). RNA was extracted from oocytes and GC and subjected to RT-PCR using human primers. In all follicle size classes oocytes expressed GDF9 mRNA and GC expressed mRNA for key GDF9 signalling molecules; bone morphogenetic protein receptor II, activin receptor-like kinase (ALK) 5 and Smad 3. To examine the intracellular response to GDF9, cultured GC from LA follicles were transfected with luciferase reporter constructs and treated with growth factors. CAGA-luciferase (Smad 3 pathway) in transfected GC was stimulated by TGFβ1 (18× above control), GDF9 and mouse oocytes (both ~5×), but not by BMP7. Conversely, neither TGFβ1, GDF9 nor oocytes activated BRE-luciferase (Smad 1/5/8 pathway), which was stimulated 30× by BMP7. 3H-thymidine incorporation was used to determine the effects of GDF9 on GC proliferation. Basal incorporation was highly dependent on follicle size, with PA follicles ~10× higher than SA and ~30× higher than LA follicles. GDF9 stimulated 3H-uptake in GC from all sized follicles, most potently in PA and SA cells. The mitogenic effect of GDF9 was amplified by IGF1; ~3× in SA GC and ~5× in LA GC. In contrast, in the presence of FSH or FSH+IGF1, GDF9 did not stimulate GC proliferation. Treatment of GC with an ALK4/5/7 kinase inhibitor, SB431542, antagonized both GDF9 and GDF9+IGF1 mitogenic effects, in a dose-dependent manner. Thus, GDF9 potently stimulates primate GC proliferation utilizing components of the TGFβ signalling system initially identified in rodents.
253. Calcium ionophore induction of marmoset oocyte activation
Authors: Grupen C. G. , Gilchrist R. B. , Schulz S. J. , Ritter L. J. , Armstrong D. T.
Published: November 2010

The marmoset monkey (Callithrix jacchus) is a valuable model for developing assisted reproductive technologies in humans and endangered primate species. Calcium ionophore treatments have been used to induce parthenogenetic activation in a number of species, but the effectiveness of this reagent in initiating marmoset embryo development has not yet been reported. The aim of this study was to determine the developmental potential of in vitro matured (IVM) marmoset oocytes, following treatment with calcium ionophore. Immature oocytes from large (LA; >1.5 mm) and small (SA; 0.67–1.5 mm) antral follicles were isolated from the ovaries of FSH-primed animals and cultured in modified G2 medium for 26–30 h at 37.0°C in 6% CO2 in air. Meiotically mature oocytes were sequentially incubated with 5 μM ionomycin for 5 min and 2 mM 6-dimethylaminopurine for 3 h and cultured in G1/G2 sequential medium at 37.0°C in 5% O2, 6% CO2, 89% N2 for 10 days. Cumulus cell expansion associated with LA oocytes (n=118) was greater than that of SA oocytes (n=212), as determined using well established classification criteria (2.7±0.1 v. 1.8±0.2; P<0.01). A greater proportion of LA oocytes completed meiosis to the metaphase-II stage compared with SA oocytes (85±7% v. 63±7%; P<0.05). Pronuclear formation was induced at similar rates in mature oocytes of both groups, but the rate of cleavage was higher for LA oocytes compared with SA oocytes (93±6% v. 66±5%; P<0.05). The number of cells per embryo was not different between the groups.This is the first study to demonstrate that calcium ionophore effectively induces parthenogenetic activation in IVM marmoset oocytes. However, the development of parthenotes was limited beyond the 8-cell stage. Further studies are needed to determine the cause of the developmental block.
254. Effect of donor age and follicle size on oocyte developmental competence in the pig
Authors: Bagg M. A. , Nottle M. B. , Grupen C. G. , Armstrong D. T.
Published: November 2010

Oocytes utilised for in vitro embryo production (IVP) are typically derived from 3–8 mm ovarian follicles of slaughtered pre-pubertal pigs. Following in vitro maturation (IVM), pre-pubertal oocytes display lower developmental competence (DC) than adult oocytes. The aim of this study was to determine the proportion of follicles 3, 4, and 5–8 mm in diameter on the surface of pre-pubertal and adult ovaries, and assess DC of corresponding oocytes. Oocytes were matured for 46 h in modified medium 199. Mature oocytes from the three follicle size cohorts were activated with calcium ionophore to assess blastocyst embryo formation rate. Data were subjected to arcsine transformation, ANOVA and the Tukey post-hoc test. Compared with adult ovaries, pre-pubertal ovaries contained a higher proportion of 3 mm follicles (46 ± 4 v. 72 ± 4%, P<0.01), but a lower proportion of 4 mm (33 ± 3 v. 22 ± 3%, P<0.01) and 5–8 mm follicles (21 ± 5 v. 6 ± 2%, P<0.01). Adult oocytes from the three follicle sizes displayed similar DC (41±2% to 47±3%). DC of pre-pubertal oocytes improved with increasing follicle size (3 mm < 4 mm < 5–8 mm; 12±4%, 27±8% and 50±8%, respectively; P < 0.05). In conclusion, the predominance of 3 mm follicles accounts for the low DC of oocytes from pre-pubertal donors compared with adult donors. Further research is required to understand DC acquisition in pre-pubertal pig oocytes from the smaller follicles <5mm in diameter.
255. In vitro maturation of bovine oocytes in serum-free media
Authors: Zhang S. , French A. J. , Tecirlioglu R. T.
Published: November 2010

Culture medium supplemented with sera is commonly used for the in vitro production (IVP) of livestock embryos. However, serum induced complications including batch variation, the potential risk of virus and mycoplasma contamination and the implication in the large offspring syndrome in domestic animals impels the development of a serum-free culture system. In this study, we investigated whether replacement of fetal bovine serum (FBS) with bovine serum albumin (BSA) in three maturation media, tissue culture medium-199 (TCM-199), a modified synthetic oviduct fluid (mSOF) routinely used in our laboratories and a commercially available SOF-VC (Vitro Cleave, Cook Australia). Harvested oocytes were matured, parthenogenetically activated and in vitro cultured (Day 7) to measure maturation efficiency, embryo development and quality with the aim of developing a simplified and defined culture medium for the in vitro production of bovine embryos. Abattoir derived cumulus oocyte complexes were matured in TCM-199, mSOF and SOF-VC media supplemented with LH and beta-estradiol in the presence of 15% FBS or 0.08% BSA at 39ºC in 5% CO2 in air. Polar body extrusion was assessed twenty-two hour post maturation and selected MII occytes were activated using calcium ionophore/6-dimethylaminopurine and cultured for seven days in SOF medium supplemented with 0.8% BSA. On day seven, blastocyst development was assessed and randomly selected blastocysts were stained to determine inner cell mass (ICM), trophectoderm (TE) and total cell numbers (TCN). Supplementation with either BSA or FCS did not significantly affect the maturation efficiency, blastocyst rates or differential cell numbers within each maturation media tested. However, maturation efficiency and blastocyst rates were significantly lower (P < 0.01) when oocytes were matured in either mSOF or SOF-VC regardless of FBS or BSA supplementation. From this study, we conclude that BSA effectively replaces FCS and TCM-199 is superior to SOF (mSOF or SOF-VC) in terms of oocyte maturation regardless of protein source. Once matured SOF and TCM-199 parthenogenetically blastocysts were equivalent in terms of embryo development and quality.
256. Bovine oocyte vitrification in sodium free medium
Authors: Tecirlioglu R. T. , French A. J.
Published: November 2010

Vitrification has been widely developed for the cryopreservation of mammalian embryos. Despite considerable effort, the vitrification of immature bovine oocytes is less successful with poor survival post thawing and low fertilization rates and development to offspring. The MII oocyte is highly sensitive to temperature changes with cryopreservation resulting in high incidence of aneuploidy after fertilization. Recently the concentration of sodium ions has been implicated in vitrification-induced damage. This study investigated the effect of a low-sodium choline-based medium (CJ2) supplemented with either 10% bovine fetal serum (FBS), 0.8% bovine serum albumin (BSA) or 1% polyvinylalcohol (PVA) on the ability of in vitro matured metaphase II (MII) oocytes to survive vitrification. Cumulus removed MII matured oocytes were equilibrated in 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in CJ2 containing FBS, BSA or PVA for 5 minutes at 37ºC and then transferred into a vitrification solution composed of 20% EG, 20% DMSO and 0.6 M sucrose in CJ2. Twenty MII stage oocytes at a time were aspirated into the Gel Loader tip (GL-tip) with approximately 20 µL of vitrification solution, equilibrated 30 sec before plunging directly into liquid nitrogen and stored for 2 h. Oocytes were thawed rapidly and cryoprotectants removed by step-wise dilution in 0.25 M, 0.15 M, 0 M sucrose in TCM-199, 5 min each. Oocytes were then incubated in TCM-199 for 2 h before being stained with Hoechst-33342 and viewed under epi-florescence to determine survival. Developmental competence was determined by parthenogenetically activating (PA) surviving oocytes using calcium ionophore/6-dimethylaminopurine and cultured for 7 days in mSOF medium supplemented with 0.8% BSA. The recovery from vitrification procedure, survival post thawing and PA rates are summarized in the Table below. Vitrification in GL-tips allows efficient processing of MII oocytes with high rates of recovery. Overall, blastocyst development in this experiment was low in the control, sham groups and treated groups and that results may have been influenced by other factors (i.e. oocyte quality). Nevertheless, results demonstrate that reducing or eliminating sodium ions from the vitrification medium may protect the immature oocytes during vitrification or thawing and allow oocytes to be cryopreserved more effectively.
257. Addition of glycine to vitrification solutions protects oocyte and embryo physiology and health
Authors: Cashman K. S. , Froiland D. A. , Thompson J. G. , Lane M.
Published: November 2010

Cryopreservation procedures for oocytes result in a significant reduction in viability. Although cryopreservation procedures cause dehydration and therefore osmotic stress, the role of osmolytes in solutions has not been considered and they have therefore not been included for routine use. The aim of this study was to assess the effects of the addition of the osmolyte glycine to vitrification solutions on the health and developmental competence of mouse oocytes. Oocytes were collected from F1 female mice and cryopreserved using cryoloop vitrification with or without glycine, with fresh oocytes examined as controls (n = 2086). Mitochondrial distribution and membrane potential as well as the morphology of the spindles and chromosomes were assessed. Oocytes were fertilised to assess their ability to develop into blastocysts, which were then assessed for their expression of Glut1, Glut3 and IGF2 by real-time RT-PCR. Statistical analysis was performed using a generalised linear model followed by multiple comparisons using an LSD test. Vitrification without glycine perturbed mitochondrial distribution (mean pixel intensity of outer region:inner region, 1.58±0.20, P<0.01) and mitochondrial membrane potential (mean pixel intensity 0.56±0.01, P<0.01) compared to control oocytes (2.34±0.24 and 0.52±0.01, respectively). The addition of glycine prevented these changes (1.97±0.16 and 0.53±0.01, respectively). Vitrification without glycine resulted in 52% of spindles and chromosomes appearing normal while this was increased to 69% with the addition of glycine, however in both treatments these abnormalities appeared to recover after culture for 2 h. Vitrification did not affect fertilisation and blastocyst development however expression of Glut3 was decreased 2.9 fold in blastocysts resulting from oocytes vitrified in the absence of glycine (P<0.01). The data presented suggests that the addition of glycine results in fewer perturbations in oocyte physiology and gene expression of the subsequent blastocysts and should therefore be considered for routine inclusion in solutions for the cryopreservation of oocytes.
258. Characterisation of ovarian follicular growth in the brushtail possum
Authors: Mester B. , Thomson B. P. , Eckery D. C.
Published: November 2010

The number and size of follicles selected for ovulation differ between species. The aim of this study was to characterise antral follicular growth and determine the size when selection of the ovulatory follicle occurs in the monovular brushtail possum. For this study, antral follicles ≥ 1 mm were dissected from the ovaries of 31 adult female possums at different reproductive states and follicular fluid and granulosa cells were harvested from each individual follicle. Selection of the ovulatory follicle in the brushtail possum occurred when follicles reached between 2.5 and 2.8 mm in diameter. Based on the analysis of steroids in follicular fluid, before selection, most follicles produced varying amounts of oestradiol (E2), but very few if any produced progesterone (P4). After selection, the selected follicle continued to produce increasing amounts of E2 and P4, whereas most other follicles were steroidogenically inactive. Near the time of ovulation, presumably after the LH surge, P4 became the predominant steroid produced by the selected follicle and most other follicles once again produced varying amounts of E2. The number of granulosa cells per follicle was highly variable, but tended to increase with increasing diameter. Cell viability was very high, averaging about 95%. Interestingly, the morphology of granulosa cells changed markedly after selection becoming larger and granular in appearance. The weights of the vaginal cul-de-sac and uteri correlated well with the presence of a selected follicle. In ovaries from pregnant animals (n = 3), follicles grew up to 3.5 mm, and although they reached the size of a selected follicle during the follicular phase, E2 production by the other follicles was not suppressed and the weights of the cul-de-sac were less than those from non-pregnant animals with similar sized follicles. During anoestrus (n = 4), follicles did not grow beyond 2 mm and produced very little steroids.
260. Effects of diet-induced obesity on ovarian function and female fertility
Authors: Minge C. E. , Bennett B. D. , Tsagareli V. , Norman R. J. , Lane M. , Robker R. L.
Published: November 2010

Obesity and its related complications (metabolic syndrome, Type II diabetes and polycystic ovary syndrome) are increasingly associated with female infertility. Our research is focused on understanding how diet-induced obesity, which triggers insulin resistance and symptoms of chronic inflammation, directly impacts ovarian function and female fertility. Female mice were maintained on a “Western style” diet (22% fat, 0.15% cholesterol) or a matched control diet. Body weights were monitored weekly and after 16 weeks fasting insulin levels and glucose tolerance were assessed. Mice were then paired with males and tissues collected on day 1 on pregnancy. Blood samples were taken to determine levels of progesterone, metabolites (glucose, HDL/LDL) and inflammatory cytokines. Tissue weights (fat pads, liver, kidney, spleen, pancreas, ovary and uterus) were recorded and the reproductive tissues were fixed for analysis of histology and gene expression. Zygotes were isolated from the oviduct, cultured in vitro and scored for on-time development and differentially stained to assess blastocyst quality. Indices of ovarian function, including ovulation rate, steroid production and oocyte quality/blastocyst development will then be correlated with degrees of insulin resistance, dyslipidemia and inflammation. Five strains of mice were tested (CBA, Balb/c, C57, SV129 and Swiss) and showed significant differences in susceptibility to diet-induced obesity and insulin resistance. In CBA mice, the first group to be completed, the high fat diet significantly increased body weight, but did not result in overtly impaired glucose tolerance. The number of days to mating was slightly extended compared to mice on the control diet. Interestingly, the high fat diet did not affect ovulation rate but resulted in dramatically impaired blastocyst development. The results of this study will reveal how ovarian folliculogenesis, oocyte competence and ovulation are affected by obesity-induced metabolic changes, which are increasingly affecting women of reproductive age.
261. Resistance to GH signalling through stat5 is an early event in PGF2α induced luteolysis in the ewe
Authors: Anderson S. T. , Kusters D. H. L. , Barclay J. L. , Curlewis J. D.
Published: November 2010

Recently we have shown that prostaglandin-induced luteolysis in pregnant rats involves resistance to prolactin-receptor signalling through the JAK2/STAT5 pathway.1 In the present study, we investigate whether PGF2alpha acts similarly to inhibit GH signalling in the ovine corpus luteum. The oestrous cycle of ewes was synchronised using cloprostenol and CIDR-G devices with oestrus detected by testosterone treated wethers with raddles. Twelve days after the first recorded oestrous mark, ewes were given an intramuscular injection of either saline or cloprostenol (125 µg), followed 1 h later with an intravenous injection (jugular vein) of either vehicle or 1.5 mg recombinant bovine GH (rbGH, Monsanto). After a further 15 min ewes were killed by pentobarbitone overdose and the corpus luteum removed. Tyrosine phosphorylation of STAT5 (STAT-P) in the corpus luteum was determined by immunoprecipitation and Western blot (n = 4 ewes/treatment). STAT5-P levels were relatively low in all ewes that were not treated with rbGH. Treatment with rbGH significantly (P < 0.01) increased STAT5-P in the corpus luteum of ewes pretreated with saline, compared to both control groups. However the STAT5-P response to rbGH was significantly (P < 0.01) reduced by the pretreatment with cloprostenol, although the response remained significantly (P < 0.05) higher than both control groups. In summary we have shown that (1) as expected, the GH-receptor signals through STAT5 in the ovine corpus luteum and (2) cloprostenol induces resistance to this GH-receptor signalling pathway.
(1)Curlewis et al. (2002). Endocrinology 143, 3984–3993.
262. Towards derivation of primordial germ cells from murine embryonic stem cells
Authors: Young J. C. , Dias V. L. , Holland M. , Loveland K. L.
Published: November 2010

The process of primordial germ cell (PGC) specification begins at the earliest stages of murine embryogenesis. The mechanisms underlying this process are the strong instructive cues generated by the extra-embryonic ectoderm, which, via ligand-receptor signalling in the visceral endoderm, activate pathways in the proximal epiblast to induce the PGC phenotype. We have subjected murine embryonic stem (ES) cells to similar cues in order to drive PGC lineage specification in vitro. ES cells were differentiated as aggregates (embryoid bodies (EBs)), a process that is thought to recapitulate the early stages of embryogenesis by providing an environment conducive to the spontaneous emergence of multiple cell lineages. To date, we have shown that EBs can also support the spontaneous emergence of cells expressing PGC markers. Expression analysis was performed on EBs from 1 to 30 days in culture. PGC markers, including nanog, dazl, fragilis, stella and SSEA1, are expressed in undifferentiated ES cells, but rapidly become undetectable in EBs as the constituent ES cells undergo differentiation. The spontaneous emergence of cells expressing these markers occurred only following long-term EB culture. This indicates a lag in the signalling normally required for PGC specification. In vivo, the lack of BMP4, its receptor (ALK-2) or downstream signalling molecules (Smad 1 and 5), results in the absence of PGCs in embryos. Therefore, in order to enhance PGC specification in our in vitro system, we have added BMP4 into the culture media. Under these conditions, the emergence of cells expressing PGC markers occurs at both an apparently higher efficiency and in a shorter time period. This suggests that BMP4 response pathways are present within the EB context and, when activated, can direct PGC specification. Thus, by recapitulating an in vivo physical and biochemical environment, we are able to direct PGC lineage specification in vitro.
264. Proteomic approaches to the study of conceptus fluids from first trimester bovine pregnancies
Authors: Riding G. A. , Lehnert S. A. , French A. J. , Hill J. R.
Published: November 2010

Advanced reproductive technologies using in vitro production systems in cattle and other livestock species offer significant opportunities for improving the reproductive performance of the female. Impeding the development is the overall inefficiency and significant costs surrounding the high incidence of embryo wastage, particularly during early pregnancy and the occurrence of foetal developmental abnormalities, placentation irregularities and late term losses.
Effective molecular interchange and communication between the conceptus and its mother is of utmost importance for the development, growth and continued viability of the foetus. We are using a proteomics approach to determine the protein profile (proteome) of the conceptus fluids (amniotic and allantoic) during the first trimester of normal, IVF and NT pregnancies. Proteomic analysis and comparison of the proteins present in these fluids may lead to the discovery of novel biomarkers which can be used to determine foetal viability status. Collections of foetal fluids from abattoir collections of Day 35 to Day 100 pregnancies were characterised with respect to volume and total protein estimates and subjected to various fractionation procedures. Protein fingerprints of conceptus fluid fractions have been generated by two-dimensional electrophoresis and the identity of proteins determined by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and database searches. Enrichment of the low-abundance and low molecular weight proteins has been achieved by ultrafiltration under denaturing and reducing conditions.
We were able to establish the identity of the majority of high-abundance proteins and a significant proportion of low-abundance proteins in both amniotic and allantoic fluids using this method. Searching of MSDB or NCBInr mammalian protein sequence databases coupled with the putative protein database deduced from the first draft assembly of the bovine genome sequence, was achieved using Mascot software.
265. Gonadotrophic hormones affect the uterus, implantation and fetal development in mice
Authors: Kelley R. L. , Kind K. L. , Lane M. , Robker R. L. , Thompson J. G. , Edwards L. J.
Published: November 2010

Although gonadotrophin stimulation using equine chorionic gonadotrophin (eCG) adversely influences pregnancy and fetal development, the effects of stimulation using recombinant human follicle stimulating hormone (rhFSH) are largely unknown. Evidence from human studies indicates that rhFSH may also be detrimental to the endometrium and implantation. We investigated the effect of gonadotrophin stimulation on ovarian hormones and uterine characteristics in the peri-implantation period, and pregnancy outcomes in mice. Adult female mice were stimulated with 2.5 IU or 10 IU rhFSH or 5 IU eCG, followed by 5 IU human chorionic gonadotrophin (hCG). Control mice received saline injections. On day 4 of pseudopregnancy mice either had embryos transferred to the uterus or were sacrificed and blood and uterine samples collected. Plasma progesterone and estradiol concentrations were determined by radioimmunoassay. Uterine mRNA levels of the estrogen and progesterone receptors (ERa and PR), leukaemia inhibitory factor (LIF), homeobox gene Hoxa10 and vascular endothelial growth factor (VEGF) were determined by real-time RT-PCR. Uterine protein distribution of PR was determined by immunohistochemistry. Embryo transfer recipients were sacrificed on day 15 to assess pregnancy outcomes. Gonadotrophin stimulation increased plasma progesterone concentration compared to controls, while estradiol concentrations were not affected. Stimulation also reduced total LIF mRNA and altered the spatial distribution of PR protein in the uterus on day 4. Embryo transfer recipients administered eCG or 10IU rhFSH had lower pregnancy rates compared to controls (11, 35 and 75% respectively) and fetuses from the rhFSH group had reduced mean weight (94 ± 6 v. 176 ± 8 mg), length (11 ± 0.2 v. 12 ± 0.1 mm) and maturity (14.1 ± 0.09 v. 14.6 ± 0.05 days) compared to controls. These results demonstrate that gonadotrophin stimulation induces changes to the maternal reproductive tract prior to implantation that have consequences for the establishment of pregnancy and fetal development in the mouse.
266. Supressor of cytokine signalling 3 regulates IL-11 induced human endometrial stromal cell decidualization
Authors: Dimitriadis E. , Stoikos C. , Salamonsen L. A.
Published: November 2010

Decidualization of endometrial stromal cells is critical for embryo implantation and establishment of pregnancy. Locally produced cytokines such as interleukin (IL)-11 enhance decidualization of human endometrial stromal cells (HESC). IL-11 signaling is negatively regulated by suppressor of cytokine signaling (SOCS) proteins. IL-11 stimulates SOCS3 in human pituitary cells. The aim of this study was to examine the role of SOCS3 on IL-11 induced HESC decidualization. Decidualization of HESC was assessed using an in vitro model in which estrogen (E)+progesterone (P) or cAMP was administered for 8 days to cells. Medium was collected for prolactin (PRL) assay (a decidual marker). Cellular protein was extracted for Western analysis and cellular RNA for real-time RT-PCR analysis. SOCS3 was overexpressed in HESC cells and the effect on decidualization assessed. HESC treated with E+P or cAMP secreted PRL from day 6. Treatment of HESC with E+P or cAMP increased the abundance of SOCS3 protein, coinciding with an increase in PRL secretion. cAMP maximally stimulated SOCS3 protein and mRNA during decidualization. Antiprogestin (onapristone) added to E+P or cAMP treated cells at day 6 reduced PRL secretion but had no influence on SOCS3 abundance suggesting that SOCS3 protein was not regulated via the P-receptor pathway. Addition of IL-11 to HESC increased SOCS3 abundance from 1 h. SOCS3 abundance returned to control levels following treatment of cells with IL-11 and IL-11 neutralising antibody. SOCS3 overexpression in HESC treated with cAMP reduced PRL secretion compared to mock- or non-transfected HESC. Furthermore, IL-11 mediated decidualization was diminished by SOCS3 overexpression. We have demonstrated for the first time that SOCS3 regulates IL-11 induced decidualization and that SOCS3 overexpression in HESC disrupts decidualization. This knowledge is important in understanding the mechanisms by which IL-11 promotes decidualization of HESC and thus the formation of decidua, an essential component of a functional placenta.
267. Interleukin-10 inhibits TNFα synthesis and protects against LPS-induced miscarriage and preterm labour
Authors: Robertson S. A. , Skinner R. J. , Care A. S.
Published: November 2010

The immune-deviating and anti-inflammatory cytokine interleukin-10 (IL-10) is expressed throughout pregnancy in the decidual and placental tissues. Mice with a null mutation in the IL-10 gene mice are fertile with no reduction in litter size, although fetal growth trajectories and placental structure are altered. IL-10 is known to terminate inflammatory responses and to limit inflammation-induced tissue pathology by inhibiting macrophage synthesis of tumor necrosis factor-α (TNFα). To investigate the anti-inflammatory role of IL-10 in pregnancy, the susceptibility of null mutant mice to low dose LPS-induced miscarriage and preterm labour has been evaluated. When IL-10 null mutant C57Bl/6 (IL-10–/–) and control (IL-10+/+) mice were given low dose E.coli LPS on d10 of pregnancy, IL-10 deficiency was associated with greater fetal loss with fewer mated IL-10–/– mice carrying viable fetuses at day 18 and increased rate of fetal resorption. In mice treated with LPS on day 17, preterm delivery within 24 h occurred in a higher proportion of IL-10–/– mice than IL-10+/+ mice. LPS induced very high and sustained TNFα and IL-6 content in serum, uterine and placental tissue in IL-10–/– mice, associated with upregulated mRNA expression of both cytokines in gestational tissues. These data show that IL-10 modulates placental resistance to inflammatory stimuli by down-regulating expression of the pro-inflammatory cytokines TNFα and IL-6. We conclude that IL-10 has a dual role in pregnancy, acting to regulate placental morphogenesis and fetal growth trajectory, and to protect against inflammation-induced miscarriage and preterm labour.
268. Experimentally induced hypoglycemia: a model to examine the effects of lactation on reproductive function in dairy cows?
Authors: Meier S. , Gore P. J. S. , Barnett C. M. E. , Cursons R. T. , Phillips D. E. , Watkins K. A. , Verkerk G. A.
Published: November 2010

The metabolic changes subsequent to lactogenesis have been associated with poor reproduction in high-producing dairy cows.1,2 Periods of hypoglycaemia reflect severe energy deficit and are associated with changes in plasma levels of growth hormone (GH), insulin-like growth hormone-I (IGF-I) and insulin-like growth hormone-II (IGF-II). Somatotropic activity has been shown to influence reproductive functions.3–5
This study evaluated the effects of experimentally induced hypoglycaemia in seven non-lactating cows, over a 7-day period. Phloridizin treatment (8 g/d) resulted in urinary glucose loss (control: 3.5 ± 1.0 g/d and phloridizin: 468 ± 46 g/d) and decline in plasma glucose (control: 60.6 ± 0.6 mg/dL and phloridizin: 71.8 ± 0.4 mg/dL; P < 0.001). Treatment increased plasma beta hydroxybutyrate (BOH), non-esterified fatty acids (NEFA) and IGF-I concentrations (P < 0.001). Plasma insulin and GH concentrations did not differ. During treatment, expression of mRNA for total growth hormone receptor (GHR(tot); P = 0.012) and GHR(1A) (P < 0.001) in liver tissue declined. Luteal and follicle diameters in ovaries recovered after treatment did not differ. Expressions of mRNA for IGF-I (P = 0.052) and interleukin-1β (IL-1β) in corpus luteum and for 3β-hydroxysteroid dehydroxyenase (3β-HSD) within dominant follicles were significantly elevated, while mRNA for GHR(tot), cytochrome P450 cholesterol side chain cleavage enzyme (P450-SCC), and steroidogenic acute regulatory protein (StAR) tended to increase (P < 0.1) with treatment.
The treatment resulted in changes similar to those of nutritional stress or the initiation of lactogenesis. Phloridizin-induced hypoglycaemia may be a model to investigate mechanisms linking glucose metabolism, and the somatotropic axis to reproductive function. The advantages of such a model, is that it allows for strict control of the level of hypoglycaemia. The use of non-lactating cows also removes the feedback mechanisms that modulate mammary gland requirements, and therefore will minimize the between cow variance when using lactating cows.
This work was completed with the help from Dexcel Farms and the Dairy Cattle Fertility team. This study was funded by the New Zealand Foundation for Research, Science and Technology (DRCX 0202).
(1)Roberts et al. (1997) Journal of Animal Science 75, 1909–1917.(2)Thatcher et al. (1996) Reproduction, Fertility and Development 8, 203–217.(3)Royal et al. (2000) Animal Science 70, 487–501.(4)Butler (2000) Animal Reproduction Science 60, 449–457.(5)Lucy (2000) Journal of Dairy Science 84, 1635–1647.
269. Optimal testicular size of donor and recipient for testicular germ cell transplantation in the bovine
Authors: Herrid M. , Davey R. , Vignarajan S. , Hutton K. , Hill J.
Published: November 2010

The maturity status of donor and recipient testis appears to be important in the efficiency of testicular germ cell transplantation. When neonatal mice were used as recipients, they show 9.4 times greater colonization and 4 times larger colony size than in the adult.1 The objective of this study in cattle was to investigate the effect of testicular maturity of donor and recipient calves on success of the testis cell transplant procedure. Testicular maturity was measured indirectly by scrotal circumference and donor testicular cells were enzymatically isolated from 8 Angus calves aged 5–7 months (scrotal circumference, SC of 18–22cm) and injected into both testes of 12 recipient calves aged 4–6 months (SC 15–21cm. Donor cells were labeled with the red fluorescent dye PKH26 then transferred into the rete testis under ultrasonographic guidance. Castration of recipients was performed 2–6 months following injection and then frozen sections were used to localize the PKH26 positive donor cells. Five sections from different 5 areas in each testis were prepared and 100 tubules were counted.
In 15 of the 24 (63%) testes, PKH positive donor cells were identified. There was no correlation between colonization rate and maturity of donor animal testis for the range of testis sizes studied. Testis cells from donors of SC 18-20 cm or of SC 21-22 did not result in different number of recipient testis with positive cells (7/10 (70%) v. 8/14 (57%)) or the number of positive cells per testis (1.92 ± 0.67% v. 2.5 ± 1.01%). Recipient maturity (SC of 15–18 cm v. SC of 19–21 cm) had no effect on the colonization rate (7/11 (64%) v. 8/13 (62%)); however, there were significantly more positive cells per testis in less mature (SC of 15–18) recipients (3.18±1.21% v. 1.52 ± 0.64% P < 0.05)). In summary we have demonstrated successful testicular germ cell transplantation between calves and while donor testis cell age appeared to have little effect on the efficiency of colonization, less mature testis provided more suitable conditions for colonization.
(1)Shinohara T, Orwig KE, Avarbock MR and Brinster RL. (2001) Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. PNAS 98(11), 6186–6191.
270. Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells
Authors: Hickey T. E. , Marrocco D. L. , Amato F. , Ritter L. J. , Norman R. J. , Gilchrist R. B. , Armstrong D. T.
Published: November 2010

Androgens, acting directly through the androgen receptor (AR), are thought to promote granulosa cell (GC) growth in vivo, but generally inhibit growth and promote GC differentiation in vitro. We hypothesised that the growth-promoting action of androgens on GC requires paracrine signalling from the oocyte. To test this hypothesis, we cultured mural GC from small antral (1–3mm) pre-pubertal pig follicles in the presence or absence of denuded oocytes (DO) from the same follicles to examine whether mitogenic responses, determined by uptake of tritiated thymidine, to combinations of FSH, insulin like growth factor 1 (IGF1) and dihydrotestosterone (DHT; 500 nM), were influenced by oocyte-secreted factors (OSFs). To further explore the identity of such factors, we performed the same experiments, substituting recombinant mouse growth differentiation factor 9 (GDF9), a known OSF, for the DO. Alone, DHT induced a small (<2-fold), but consistent increase in IGF1-stimulated DNA synthesis. OSFs stimulated DNA synthesis in all experimental combinations, most significantly in the presence of IGF1 (P < 0.0001), and DHT enhanced (P<0.05) the stimulatory effect of OSFs in all instances. Like OSFs, GDF9 substantially increased IGF1-stimulated DNA synthesis (P < 0.0001), and again, DHT enhanced (P > 0.01) this effect. In further experiments, two AR agonists, testosterone (10-1000nM) and DHT (5–500 nM), dose-dependently augmented the mitogenic effect of OSFs or GDF9 in the presence of IGF1. Only the highest doses of androgen had an independent stimulatory effect; lower doses required the presence of an OSF(s). Antiandrogen (hydroxyflutamide) treatment, used to block AR activity, antagonized the androgen X GDF9 interaction. In conclusion, androgens, via activation of the AR, stimulate porcine GC proliferation in vitro by potentiating the growth-promoting effects of oocytes or GDF9. These signalling pathway interactions are likely to be important regulators of folliculogenesis in vivo and may cause the excess follicle growth that is observed in androgen-treated female animals.
273. Seminal plasma influences pregnancy outcome through effects on both uterine receptivity and the pre-implantation embryo
Authors: Bromfield J. J. , Robertson S. A.
Published: November 2010

Optimal uterine receptivity and viability of the preimplantation embryo are key requirements for successful initiation of a pregnancy. The molecular and cellular events activated by insemination are thought to play a role in establishing an environment conducive to positive pregnancy outcomes. Previously, we have employed a blastocyst transfer model to demonstrate that lack of uterine exposure to seminal plasma at the time of conception results in fetal growth restriction and impaired postnatal development of progeny. Here we utilise a 2-cell embryo transfer technique to determine the effects of uterine seminal plasma exposure on embryo development and its subsequent effects on pregnancy outcomes at term in the mouse. Two-cell embryos were collected from donor mice and transferred into the oviduct of day 1 pseudopregnant recipient females primed either by mating with vasectomised (vas) males or vasectomised males from which the seminal vesicle glands, the major source of seminal plasma, had been surgically removed (vas+svx). At day 18 of pregnancy there was no difference in the number of total implantations, however fetal weight was reduced by approximately 5% (P < 0.05). Placental weight at day 18 was significantly increased, resulting in an increase in the fetal weight: placental weight ratio, an indicative measure of placental function. At term, progeny from pregnancies initiated in the absence of semen exhibited a 10% reduction in birth weight and remained smaller through the pre-weaning period. Fetal growth retardation was more severe when embryos were transferred at 2-cell stage than in comparable experiments where blastocysts were transferred after development in a normal donor tract. In combination, these experiments demonstrate that seminal plasma exposure at the time of conception impacts on pregnancy outcome through independent actions on both uterine receptivity and embryo development. These findings are important when considering current practises of assisted reproductive technologies, as well as having implications in pregnancy outcomes in the general population where seminal plasma signalling may be compromised.
274. Placental expression of secreted frizzled related protein (sFRP4) in the rat: association with β-catenin localization and regulation by glucocorticoids
Authors: Hewitt D. P. , Mark P. J. , Dharmarajan A. M. , Waddell B. J.
Published: November 2010

Wnt genes regulate a diverse range of developmental processes including placental development. Activation of the wnt pathway results in nuclear translocation of β-catenin and activation of the TCF/Lef family of transcription factors. The secreted frizzled related proteins (sFRPs) modulate wnt signaling by binding to either the wnt ligand or its transmembrane frizzled receptor. The current study examined the spatial and temporal expression of one of these secreted molecules, sFRP4, in the rat placenta over the final third of pregnancy, and whether associated changes occurred in the expression and localization of β-catenin. The expression of sFRP4 and β-catenin was also analysed in a model of glucocorticoid-induced placental growth restriction.
Analysis by quantitative RT-PCR over the final third of pregnancy demonstrated a dramatic increase in sFRP4 mRNA (14-fold, P < 0.001) specifically within the basal zone of the placenta near term. In situ hybridization and immunohistochemistry localized sFRP4 expression primarily to giant trophoblasts of the basal zone. In addition, sFRP4 protein was notably upregulated in association with a restricted nuclear translocation of β-catenin. Maternal dexamethasone treatment (1 μg/mL in drinking water; day 13–22) further increased the expression of sFRP4 mRNA in both the basal (120%, P < 0.05) and labyrinth (285%, P < 0.01) zones of the placenta at day 22 (term = 23 days) compared to untreated controls.
These data indicate that sFRP4 expression is increased in the basal zone of the rat placenta, the major site of apoptosis in late pregnancy, and is further stimulated by glucocorticoids. Moreover, the observed inhibition of β-catenin expression and its nuclear translocation suggest that sFRP4 inhibition of wnt signaling in the placenta may contribute to placental apoptosis and ultimately fetal growth restriction.