bluuebunny/crossref_metadata_2025 huggingface dataset:
113. DEVELOPMENT OF A METHOD OF SEMINIFEROUS TUBULE TRANSFECTION IN VITRO TO DEFINE POSTMEIOTIC GENE REGULATION
Authors: Danner S. , Kirchhoff C. , Ivell R.
Published: November 2010

Postmeiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. Here we report the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation. Culture conditions were developed which allowed the continued incubation of isolated rat seminiferous tubules for up to 48h without obvious cell death and loss of post-meiotic cells. Transfection of intact seminiferous tubules by microinjection and electroporation was optimized to achieve high expression efficiencies of control plasmids, using either fluorescent protein or luciferase as reporters, thereby allowing both morphological as well as quantitative assessment. Successful transfection was achieved into all cell types except for mature spermatozoa. However, there appeared to be only limited cell-type specificity for the promoters used, even though these had appeared to be specific when used in transgenic animals. We have devised a methodology which allows relatively high throughput analysis of post-meiotic gene promoters into primary cells of intact seminiferous tubules. An apparent lack of cell-type specificity suggests that the gene fragments used do not contain sufficient targeting information, or that the transient episomal expression of the constructs does not encourage appropriate expression specificity. The results also highlight the doubtful interpretation of many studies using heterologous transfection systems to analyse post-meiotically expressed genes.
115. WHOLE BODY HEAT EXPOSURE INDUCES APOPTOSIS IN MOUSE CAUDAL EPIDIDYMAL SPERMATOZOA
Authors: Wechalekar H. , Setchell B. P. , Breed W. G. , Ricci M. , Leigh C. , Peirce E.
Published: November 2010

Introduction: The aim of the present study was to determine the immediate effects of whole body heating on sperm numbers, motility and apoptosis. Material and Methods: C57BL/6 mice (n=7) were exposed to 37-38oC (8 hours/day), for three consecutive days while control mice (n=7) were kept at 23-24oC. Caudal epididymal spermatozoa were collected from control and heat treated mice 16 hours after the last heat treatment to determine the sperm number and motility using a Neubauer haemocytometer and sperm apoptotic changes by dual colorflow cytometry using Annexin V/PE (Annexin V conjugated with phycoerythrin) and 7AAD (7-amino-actinomycin D) stains. Results: There were no significant differences (p>0.05) in sperm numbers between heat treated and control mice, however heating did result in a significant reduction in sperm motility (p<0.05). Apoptosis staining identified four different subpopulations of spermatozoa: (a) live spermatozoa (Annexin V-/7AAD-), (b) early apoptotic spermatozoa with exteriorized phosphotidylserine (PS) receptor and intact plasmalemma (Annexin V+/7AAD-), (c) late apoptotic spermatozoa with PS receptor translocation and leaky plasmalemma (Annexin V+/7AAD+) and (d) dead spermatozoa with damaged plasmalemma with no detectable PS receptor (Annexin V-/7AAD+). Heating resulted in significant reduction in the percentage of live spermatozoa (p<0.05), an increase in early apoptotic (p<0.05), late apoptotic (p<0.05), and dead spermatozoa (p<0.05). Conclusion: This study shows that mice exposed to whole body heat exposure of 37-38oC for 8 hours per day for three consecutive days exhibited early and late apoptotic changes to epididymal spermatozoa. These findings suggest possible adverse effects of exposure to high temperature on the viability of human spermatozoa in the epididymides. In addition, these findings reinforce the importance of temperature during sperm preparation procedures in infertility clinics, and research laboratories.
116. EFFECTS OF SUPPLEMENTAL VITAMIN ON THE SEMEN CHARACTERISTICS OF MARKHOZ GOATS DURING NON-BREEDING SEASON
Authors: Towhidi A. , Farshad A. , Dolatpanah M. , Salehi R.
Published: November 2010

The aim of the study was to investigate the effect of dietary supplementation of vitamin E on the semen characteristics of the Markhoz goats. Eighth bucks were randomly allocated into two groups, and received two different diets: unsupplemented diet (control) and supplemented diet with vitamin E (0.30 g/Kg DM). All experimental diets were formulated according to AFRC 1998. Semen was collected at 14-days intervals from June 17, 2006 to September 2, 2006 (non-breeding season) using artificial vagina. Semen characteristics were evaluated including semen volume, sperm concentration, live sperm percentage, percentage of motility and progressive motility. This characteristics were evaluated at two weeks intervals the trial. The total number of spermatozoa per ejaculation was calculated by multiplication of the semen volume with sperm concentration. Sperm motility was also analyzed by placing a sample on pre-warmed (37 º C) microscope slide covered with a cover slip, and examined under a high power microscope at a magnification × 200. Data was analyzed using proc MIXED in SAS program. Significant effect (P≤0.05) of the week (sampling time) was observed for all the parameters except for semen volume. Vitamin E supplementation significantly improved (P≤0.05) total number (Control: 263.7 ×107±17.506 vs. Vit E: 320.95 ×107±17.506) and sperm concentration (Control: 301.79 ×107±13.657 vs. Vit E: 386.57 ×107± 13.657), motility (Control: 77.27% ±0.89 vs. Vit E: 82.6% ±0.89) and progressive motility (Control: 4.208 ±0.138 vs. Vit E: 4.229 ±0.138), percentage of viability (Control: 80.57% ±0.89 vs. Vit E: 85.9% ±0.89). The results suggested that the supplemental Vitamin E may improve the semen quality and fertility in the Markhoz goats.
117. THE COMPARISON OF SPERM FREEZABILITY USING TWO EGG YOLK-FREE DILUENTS IN ZANDI RAM
Authors: Ardebili R. , Towhidi A. , Zeinodini S. , Bahreini M.
Published: November 2010

The aim of this study was to investigate the effect of two kinds of commercial egg yolk-free extenders: Bioxcell and AndroMed on in vitro sperm function of sheep. A total of 10 ejaculates were collected from 5 adult Iranian Zandi rams using artificial vagina. Semen samples were mixed and diluted with Bioxcell or AndroMed. Freezing was conducted using imv semi-automatic equipment. The percentage of motility and progressive motility of sperm were evaluated before freezing (at 37°C), after refrigeration (at 5°C) and thawing, and recovery rate was calculated. Data was analyzed using proc GLM of SAS. The effect of extender on sperm qualitative characteristics after thawing was significant. Mean percentage of motility, progressive motility and recovery rate were higher (P≤0.01) in AndroMed than Bioxcell. Results suggested that AndroMed in comparison with Bioxcell had more ability to preserve sperm quality during freezing process.
118. CHARACTERISATION OF HEPARAN SULPHATE PROTEOGLYCANS IN THE MATURING CUMULUS OOCYTE COMPLEX
Authors: Watson L. N. , Sasseville M. , Gilchrist R. B. , Russell D. L.
Published: November 2010

Many growth factors including members of the transforming growth factor beta (TGFβ) superfamily and epidermal growth factor (Egf)-like ligands signal via interactions with heparan sulphate proteoglycans (HSPGs). Cell surface HSPGs can act by sequestering ligands at their site of action, by presenting a ligand to its signalling receptor, or by preventing ligand-receptor interaction. The oocyte secreted factors (OSF) growth differentiation factor 9 and bone morphogenetic protein 15 are members of the TGFβ superfamily that act selectively on cumulus cells. Conversely Egf-like ligands are secreted by mural granulosa cells and transmit LH-induced signals to cumulus cells. We investigated the possibility that HSPGs contribute to the spatially restricted responses these signals exert on cumulus cells. Syndecan-1 and Glypican-1 are cell surface HSPGs that are involved in numerous biological processes, including growth factor regulation, cell proliferation and differentiation. Microarray analysis showed Syndecan-1 and Glypican-1 mRNA expression induced 6-fold (P=10-9) and 3-fold (P=10-7) respectively in Egf+FSH stimulated cumulus oocyte complexes (COCs). Furthermore, Syndecan-1 and Glypican-1 mRNA were induced 27- and 16-fold respectively in COCs after hCG treatment of mice. Syndecan-1 and Glypican-1 protein was localised specifically to the COC through immunohistochemical analysis. In Vitro Maturation (IVM) of oocytes is a valuable alternative to gonadotropin mediated superovulation, but IVM COCs are less competent than those matured in vivo. Several components of the COC have been shown to be altered in IVM, including the chondroitin sulphate proteoglycan Versican. COCs from mice that underwent IVM in the presence of Egf+FSH and cilostamide for 16 hours had >16 fold reduced mRNA for Syndecan-1 when compared with In Vivo matured COCs. The lack of Syndecan-1 in IVM COCs could reduce signalling capacity of growth factors including OSFs. This may contribute to the reduced capacity of IVM oocytes to fertilise and produce a healthy embryo, and ultimately, a healthy offspring.
119. THE EFFECT OF BMPR-IB IMMUNIZATION IN IMMATURE FEMALE MICE
Authors: Ciller I. M. , McFarlane J. D. , McFarlane J. R.
Published: November 2010

The female reproductive system is regulated by well known endocrine signaling components and the less well understood autocrine and paracrine signaling components by growth factors and their receptors. The type I Bone Morphogenetic Protein Receptor IB (BMPR-IB) is a serine/threonine kinase that facilitates the signaling of various TGF-β family members. BMPR-IB is believed to have a substantial role in reproduction as demonstrated by the hyperprolific effect on ovulation in Booroola ewes which harbor a natural BMPR-IB receptor mutation, as well as the sterility induced by the BMPR-IB knockout in female mice. Maturation of the ovary and enhancement of follicular growth are known to be dependent on gonadotrophin stimulation from the pituitary. This study aims to elucidate the role of BMPR-IB in the female reproductive system by passive immune neutralization. Immature female mice (21 days old) were passively immunized against BMPR-IB via subcutaneous injections of 100 μl PBS containing anti BMPR-IB Ig in the absence and presence of equine chorionic gonadotrophin (eCG). The preparations where administered on day one and three of a four day experiment. On day four mice were asphyxiated with CO2 and the ovaries were removed and weighed. In immature mice immunization against BMPR-IB ovarian weights were not different from control, while eCG significantly increased ovarian weight compared to the controls. In combination immunization against BMPR-IB further augmented the gonadotrophin-stimulated increase in ovarian weight to a significant degree. The suppressive effect of BMPR-IB signaling on follicular development in response to gonadotrophin stimulation indicates a possible developmental role of this receptor in the initiation of follicle growth in response to increased circulatory gonadotrophins and prevention of precocious puberty.
120. OXYGEN REGULATED GENE EXPRESSION IN MOUSE CUMULUS CELLS
Authors: Tam K. , Banwell K. , Froiland D. , Russell D. , Kind K. , Thompson J.
Published: November 2010

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that mediate the expression of a range of genes in response to low oxygen. Previously we showed that subsequent developmental outcomes were influenced by oxygen levels during in vitro maturation. The aim of the current study was to examine the effects of varying oxygen concentration during in vitro maturation of mouse COCs on expression of HIF target genes in the cumulus cells. I mmature COCs were collected from the ovaries of eCG-stimulated CBAB6F1 females (21 d) and cultured for 17-18 h under 2, 5 or 20% O2. Hyaluronidase-treated and recovered cumulus cells were collected and mRNA extracted for analysis. A microarray approach (Affymetrix 430_2) was used to identify genes in cumulus cells that were differentially expressed under varying oxygen concentrations (2, 5, 10 and 20%). This revealed 218 differentially expressed probes, of which 34 were up-regulated with decreasing oxygen levels. The great majority of these were classified as HIF-regulated genes. Specific analysis from real time RT-PCR of HIF regulated target genes Slc2a1, Ldha, Pgk1, Eno1, Ndrg1, Bnip3 were all significantly up-regulated (by at least 5–fold) when cells were cultured at 2% or 5% oxygen, when compared to 20% oxygen. Hif-1a mRNA decreased when cumulus cells were cultured in 2%, compared to 20% oxygen. This study demonstrates that cumulus cell gene expression is influenced by oxygen concentration, and suggests that these effects are mediated by the HIF transcription factors.
121. EXTRACELLULAR ROLES FOR PROREGIONS OF MOUSE BMP15 AND GDF9 IN VIVO
Authors: McIntosh C. J. , Lawrence S. , Juengel J. , McNatty K.
Published: November 2010

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are essential for normal follicular development and ovulation in mammalian species. As TGF-beta family proteins, BMP15 and GDF9 are expressed as pre-pro-mature proteins, with a signal region prompting extracellular secretion, a proregion that is post-translationally cleaved, and a mature protein that is known to be biologically active as a non-covalently interacting dimer. Until recently, the proregion was thought primarily to facilitate correct folding and dimerization of the mature proteins within the oocyte prior to processing and cleavage. However, our in vitro work with recombinant mouse BMP15 and GDF9 has shown that each of the proregion, mature protein and the non-processed promature protein are secreted from transfected 293H cells. We showed non-covalent interactions existing between the proregion and mature protein of each of BMP15 and GDF9, and between the BMP15 proregion and GDF9 mature protein. Importantly, a mouse BMP15 proregion antibody was able to abolish cooperative BMP15 and GDF9 bioactivity measured using a granulosa cell thymidine incorporation bioassay, providing strong evidence for an extracellular role for the mouse BMP15 proregion. Currently, to find out whether BMP15 and GDF9 proregions have extracellular roles in vivo, our investigation has utilised knock-down of BMP15 and GDF9 proregion proteins by mouse immunisation. Ovaries of mice immunised with the GDF9 proregion had significantly increased numbers of corpora lutea (p<0.005), while ovaries from mice immunised with BMP15 proregion peptides had significantly fewer corpora lutea (p<0.005). These findings provide the first evidence that the proregions of mouse GDF9 and BMP15 have different physiological roles outside the oocyte. Our future aim is to elucidate the function of these proregions, how these may differ between BMP15 and GDF9, and whether these aspects differ between species with different ovulation quota.
123. ACTIVIN A AND OVARIAN FOLLICLE DEVELOPMENT
Authors: Cossigny (Rosairo) D. A. , Findlay J. K. , Drummond A. E.
Published: November 2010

A significant developmental stage in ovarian folliculogenesis is the acquisition of gonadotropin sensitivity by ovarian follicles. Activin has previously been suggested to be involved in the responsiveness of granulosa cells to FSH (1). Therefore, the role of activin was investigated using a ‘physiological’ culture system to determine if pathways exist to transduce activin signals within the postnatal rat ovary. Organ cultures with day 4 whole ovaries were employed in order to assess the potential impact of Activin A on follicle growth and transition from the primordial through to the primary and later preantral stages of development. Ovaries were isolated and cultured for 10 days with the addition of supplemented DMEM/Hams F-12 media (2)and either FSH (100ng/ml), Activin A (50ng/ml), or a combination of the two. Media and treatments were refreshed every alternate day. At the end of the culture period, ovaries were fixed and sectioned, or placed immediately into Ultraspec for RNA extraction for future real-time PCR. Sections were used for morphological assessment and ovarian follicle counting of primordial, primary and preantral follicles. An evaluation of atresia by the detection of apoptotic cells was undertaken using terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick-end labeling (TUNEL). Primary follicle numbers increased significantly (P<0.05) in the combined treatment group whereas, preantral follicle numbers increased significantly (P<0.0001) when treated with Activin A alone. This is consistent with a morphological appraisal of atresia where a decrease in atresia was found in primordial and primary follicles, supporting the primary follicle development data and Activin A treatment alone resulted in more healthy primary and preantral follicles than atretic ones. Therefore, a stimulatory role for Activin A both in the presence of FSH (primary follicle development) or alone (preantral follicle development) has resulted in more follicles making the transition from the primordial to primary stages, as well as to the later preantral stages.
125. REGULATION OF STRESS PROTEIN GENES DURING PRE-IMPLANTATION EMBRYO DEVELOPMENT IN MICE BY GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF)
Authors: Chin P. Y. , Macpherson A. M. , Thompson J. G. , Lane M. , Robertson S. A.
Published: November 2010

In vitro culture has been shown to be detrimental for pre-implantation embryo development and this has been associated with culture stress and elevated expression of apoptotic genes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to promote development and survival of both human and mouse pre-implantation embryos. To investigate the mechanism of action of GM-CSF in mouse embryos, gene expression was examined in in vitro cultured blastocysts with and without recombinant mouse GM-CSF (rmGM-CSF) and in vivo blastocysts flushed from Csf2 null mutant and wild-type mice. Microarray analysis of the effect of GM-CSF on transcription profile implicated apoptosis and stress response gene pathways in blastocyst responses to rmGM-CSF in vitro. Groups of 30 blastocysts were collected from in vitro cultured and in vivo developed blastocyst were analysed using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR analysis of in vitro blastocysts revealed that addition of rmGM-CSF causes differential expression of several genes associated with apoptosis and cellular stress pathway, including Cbl, Hspa5, Hsp90aa1, Hsp90ab1 and Gas5. Immunocytochemical analysis of common proteins of the apoptosis and cellular stress response pathways BAX, BCL2, TRP53 (p53) and HSPA1A/1B (Hsp70) in in vitro blastocysts revealed that HSPA1A/1B and BCL2 proteins were less abundant in embryos cultured in rmGM-CSF, but BAX and TRP53 were unchanged. In in vivo developed blastocysts, Csf2 null mutation resulted in elevated levels of only the heat shock protein Hsph1, suggesting that in vivo, other cytokines can compensate for GM-CSF deficiency as the absence of GM-CSF has a lesser effect on the stress response pathway. We conclude that GM-CSF is a regulator of the apoptosis and cellular stress response pathways influencing mouse pre-implantation embryo development to facilitate embryo growth and survival, and the effects of GM-CSF are particularly evident in in vitro culture media in the absence of other cytokines.
126. IMPLICATIONS FOR FETAL AND PLACENTAL DEVELOPMENT FOLLOWING MITOCHONDRIAL PERTURBATION IN THE EMBRYO
Authors: Wakefield S. , Lane M. , Mitchell M.
Published: November 2010

The environment an embryo is exposed to can profoundly influence peri- and post-natal development despite having some capacity to adapt. Whilst the mechanisms responsible remain largely unknown, mitochondria are a likely target. In this study we deliberately perturbed mitochondrial function in the mouse embryo, using a model we have established that shows step-wise changes in embryo metabolism and development. The aim of this study was to provide direct evidence implicating mitochondrial dysfunction in the embryo with perturbed fetal and placental development. Zygote stage embryos were recovered from superovulated female mice and cultured in control conditions to the 2-cell stage. Embryos were then allocated to one of three treatments; control media (0μM-AOA), 5μM or 50μM of the known mitochondrial inhibitor, Amino-Oxyacetate, in the absence of pyruvate (5μM-AOA, 50μM-AOA). Embryos were cultured to the blastocyst stage, then transferred to pseudopregnant recipients, with fetal and placental parameters measured on day 18 of pregnancy. Implantation rates and fetal survival for both 5μM-AOA and 50μM-AOA was comparable to control embryos. For 5μM-AOA there was a significant reduction in placental weight (P=0.02) but normal fetal weight, and a significant increase in fetal: placental weight ratio (P=0.002) relative to the control, suggesting increased placental efficiency. When mitochondria were further perturbed (50μM-AOA), the fetuses and placentas were both considerably compromised: that is, decreased fetal and placental weights (P=0.002), reduced placental diameter (P=0.03) and decreased fetal crown rump length (P=0.07). This study demonstrates that mitochondrial function in the embryo impacts on peri-natal development, providing compelling evidence for mitochondrial function involvement in the mechanisms underpinning “embryo programming”. This data suggests a threshold effect, whereby embryos can only adapt up until a point after which development is compromised. Further elucidating these mechanisms is important for understanding how maternal environments and embryo culture systems determine development of future offspring.
127. EPIGENETIC REPROGRAMMING IN ZYGOTES INVOLVES THE GLOBAL CYTOSINE DEMETHYLATION OF BOTH THE PATERNAL AND MATERNAL GENOMES
Authors: Morgan H. D. , Li Y. , O'Neill C.
Published: November 2010

Epigenetic reprogramming is essential for normal development and has been held to occur in a different manner for the paternally and maternally inherited genomes. The current paradigm implicates active global demethylation of the paternal pronucleus soon after fertlization, but passive demethylation of maternally-derived genome over many cell-cycles. This parent-of-origin difference has been difficult to reconcile with other biological processes prompting us to re-examine this evidence. DNA methylation levels were examined in mouse zygotes by immunolocalization with methylcytosine specific antibodies. Zygotes were isolated from the oviduct at times after hCG and staged for pronuclei maturity (PN1-5, least to most mature) or metaphase commencement. We found methylation levels to be high in PN1-2 stage pronuclei but then progressively declined. By PN5 stage methylcytosine staining was greatly diminished. Yet, contrary to the current paradigm, demethylation generally occurred in both the male and female pronucleus. We found no methylcytosine staining in any metaphase chromosomes. The contrast of our results with those widely cited prompted us to review the methodology previously used. In previous studies zygotes that had been collected after fertilization and then cultured in vitro, or produced by IVF and then cultured were used. When we prepared zygotes by these methods we found that many PN5-stage cultured zygotes displayed relatively more demethylation of the male pronucleus than the female. When zygotes were generated by IVF this asynchrony was further exacerbated. In contrast to the zygotes collected directly from the reproductive tract, metaphase chromosomes in cultured post-syngamal zygotes commonly showed extensive methylcytosine staining. Our results show that the normal process of epigenetic reprogramming in the mouse involves global demethylation of both the paternal and maternal genomes. This was variably perturbed (particularly in the female pronucleus) by IVF and zygote culture.
128. A ROLE FOR SIRTUIN 3 IN THE DEVELOPING MAMMALIAN EMBRYO
Authors: Filby A. , Mitchell M. , Georgiou A. , Lane M.
Published: November 2010

Pre-implantation embryo development relies critically on the balance between cytoplasmic and mitochondrial metabolism for the generation of metabolic intermediates such as NAD+. SIRT3 is a mitochondrial sirtuin with NAD+-dependant deacetylase activity that, targets glutamate dehydrogenase (GDH). In this study we characterised SIRT3 mRNA, protein and activity through pre-implantation development and determined whether modulation of SIRT3 activity influenced GDH activity. Embryos (zygotes, 2-cell, 8-cell and blastocyst stages) were recovered from female CBA/C57Bl6 mice following ovarian stimulation and mating with CBA/C57Bl6 males. Expression of SIRT3 mRNA was measured using real-time RTPCR, protein localisation examined using immunohistochemistry and SIRT3 activity measured using a Fluor-de-Lys SIRT3 fluorescentassay. Functional GDH activity was assessed in 2-cell embryos indirectly by measuring glutamine oxidation, following culture from zygote to 2-cell in the presence of nicotinamide, (a sirtuin inhibitor), G1.2 media, or simpleG1 media, compared to in vivo controls. SIRT3 mRNA was detected at all stages of development, with significantly greater levels expressed in the blastocyst. SIRT3 protein was localised predominantly around the nucleus of zygote and 2-cell embryos, and was mainly cytoplasmic in 8-cell embryos and blastocysts. SIRT3 activity remained constant throughout pre-implantation development, and tended to increase at the blastocyst stage. Glutamine oxidation was reduced for embryos cultured in G1.2 media relative to in vivo controls (0.14 pmol/e/hr vs 0.21pmol/e/hr), and this was further reduced by the addition of nicotinamide (0.07pmol/e/hr). Embryo culture in perturbing simpleG1 increased glutamine metabolism (0.33pmol/e/hr). In conclusion, SIRT3 mRNA, protein and activity was detected throughout pre-implantation development. Modulation of sirtuins by nicotinamide decreased glutamine metabolism, likely as a result of decreased deacetylation, thus decreased activity of GDH. SIRT3 can translocate to the mitochondria during cellular stress, thus the increased glutamine metabolism in simpleG1 conditions may be caused by translocation of SIRT3 to mitochondria, potentially increasing GDH deacetylation and enzymatic activity.
129. REDUCED OOCYTE DEVELOPMENTAL COMPETENCE DURING THE PERIOD OF SEASONAL INFERTILITY IN PIGS
Authors: Bertoldo M. , Holyoake P. K. , Evans G. , Grupen C. G.
Published: November 2010

Reduced farrowing rate due to early pregnancy loss is a manifestation of seasonal infertility in pigs. It has been hypothesised that the early disruption of pregnancy is due to poor oocyte developmental competence. The aim of this study was to determine if there are seasonal differences in oocyte developmental competence. Ovaries were collected from sows slaughtered 4 days after weaning. Cumulus-oocyte complexes (COCs) recovered from small (3–4mm) and large (5–8mm) antral follicles were morphologically graded and subjected to parthenogenetic activation following in vitro maturation (IVM) during the winter (n = 1419) and summer (n = 2803). Cumulus expansion was assessed subsequent to IVM. Data were analysed using a generalised linear mixed model in GenStat release 10. There was an effect of season on oocyte grade, with a larger proportion of oocytes collected in summer being graded suitable for IVM, compared with winter oocytes (P<0.05). A larger proportion of COCs had expanded cumulus during the winter than in the summer, which suggested that the preovulatory LH surge had already occurred. There was a season x follicle size interaction affecting cumulus expansion (P<0.05). There were no seasonal effects on the proportion of oocytes reaching metaphase II or cleaving after parthenogenetic activation. However, the proportion of oocytes from large follicles that developed to the blastocyst stage was higher in winter than in summer (55% vs 23%; P<0.05). There was no effect of season on the proportion of oocytes developing to the blastocyst stage from small follicles. However, during summer there was a reduction in the cell number of blastocysts derived from small follicles (P<0.05). Our results suggest that porcine oocytes are less able to reach their full developmental potential during the period of seasonal infertility, and that the associated pregnancy losses are due to reduced oocyte developmental competence.
130. MICROARRAY ANALYSIS OF FOETAL MOUSE BRAIN FOLLOWING INDUCTION OF MITOCHONDRIAL DYSFUNCTION DURING PRE-IMPLANTATION EMBRYO DEVELOPMENT
Authors: Fullston T. , Mitchell M. , Wakefield S. , Filby A. , Lane M.
Published: November 2010

Environmental stress can disrupt mitochondrial function in the pre-implantation embryo, subsequently hindering embryo viability. Brain tissue is also sensitive to developmental perturbations, and we have previously discovered genes involved in neurological function and epigenetic modification are differentially expressed in blastocysts following mitochondrial dysfunction by amino-oxyacetate (AOA). In this study CBAxC57Bl6 2 cell stage mouse embryos were cultured in 5μM-AOA without pyruvate for 72h to induce mitochondrial dysfunction. Blastocyst stage embryos were then transferred to pseudopregnant recipients and the expression profile of day 18 foetal brains was interrogated using microarray. mRNA from mouse whole brain (4 per treatment) was extracted and analysed using an Affymetrix gene array. Ingenuity Pathway Analysis software identified persistent alterations in gene expression pathways in foetal brain after AOA treatment during embryo culture, that were subsequently confirmed by qPCR. Expression was significantly increased by both array and qPCR (>1.5 fold, p<0.05) for; 1) Eomes (1.9, 2.9 fold respectively), a T-box transcription factor involved in differentiation, cell death and development, 2) Nr4a3 (1.8, 2.2 fold respectively), a steroid hormone receptor and putative transcriptional activator and 3) Nola3 (1.7, 1.9 fold respectively), a small nucleolar ribonucleoprotein involved in rRNA processing. Neurological disease, behavioural disorders, carbohydrate metabolism, cellular growth and proliferation, cell death, DNA replication, recombination and repair pathways also showed altered gene expression (>1.25 fold). qPCR was performed on 28 genes exhibiting the greatest change in expression. 24/28 genes confirmed the array data, and of the 4 genes that did not; two had expression not detected by qPCR (Snhg1, Speer6-ps1), and two contradicted array results (Atp1b3 p=0.05, Stk38l p=0.06). This study links mitochondrial dysfunction during early embryo development and persistent molecular changes in the developing foetal brain. This indicates that insults incurred during early embryo development can cause permanent changes that we predict results from aberrant epigenetic modification.
131. INDUCED OOCYTE IN VITRO MATURATION (IVM) SUBSTANTIALLY IMPROVES EMBRYO YIELD AND PREGNANCY OUTCOMES
Authors: Albuz F. K. , Sasseville M. , Armstrong D. T. , Lane M. , Thompson J. G. , Gilchrist R. B.
Published: November 2010

Oocyte maturation in vivo is a highly orchestrated, induced process, whereby 3'-5'-cyclic adenosine monophosphate (cAMP)-mediated meiotic arrest is overridden by the gonadotrophin surge prior to ovulation. However, in vitro matured (IVM) oocytes resume maturation spontaneously hence compromising developmental competence. Hence we hypothesized that establishing an induced system in vitro would synchronise oocyte-somatic cell communication leading to improved oocyte quality. Bovine or mouse oocytes were treated for the first 1-2 h in vitro with the adenylate cyclase activator forskolin and a non-specific phosphodiesterase (PDE) inhibitor, IBMX, which substantially increased cumulus-oocyte complex (COC) cAMP (180 vs. 2 fmol/ COC , treated vs. control, P<0.001) to in vivo physiological levels. To maintain oocyte cAMP levels and prevent precocious oocyte maturation, oocytes were then cultured with an oocyte-specific (type 3) PDE inhibitor, cilostamide. The net effect of this system (“Induced IVM”) was to increase oocyte-somatic cell gap-junctional communication (bovine: 1000±148 vs. 340±73 units; treated vs. control, p<0.05) and to slow meiotic progression through prophase I to metaphase II, extending the normal IVM interval (bovine: 30 vs. 24h, mouse: 22 vs. 18 h; treated vs. control). These effects on oocyte and somatic cell functions had profound consequences for oocyte developmental potential. In bovine, Induced IVM more than doubled embryo yield (69% vs. 27%; treated vs. control, p<0.05). In mouse, Induced IVM increased blastocyst rate (86% vs. 55%; treated vs. control, p<0.05), implantation rate (51 vs. 25%), fetal survival rate (29% vs. 5%) and fetal weight (0.9g vs. 0.5g, p<0.01). All these developmental outcomes in mice were restored, by using Induced IVM, to levels obtained from in vivo matured control oocytes (conventional IVF). In conclusion, Induced IVM mimics some of the characteristics of oocyte maturation in vivo and substantially improves oocyte developmental outcomes. This should lead to an increase in the use of this technique in reproductive biotechnologies.
133. SPATIAL REGULATION OF APCCdh1 INDUCED CYCLIN B1 DEGRADATION MAINTAINS GV ARREST IN MOUSE OOCYTES
Authors: Holt J. E. , Weaver J. L. , Jones K. T.
Published: November 2010

Within the mammalian ovary, oocytes remain prophase I arrested until a hormonal cue triggers meiotic resumption. The E3 ubiquitin ligase, Anaphase-Promoting Complex with its co-activator Cdh1 (APCCdh1) is known to be essential for this process by promoting cyclin B1 degradation via the 26S proteasome. Cyclin B1 is the regulatory subunit of Maturation-Promoting Factor, forming a heterodimer with CDK1, which is essential for nuclear envelope breakdown (NEB). Here we describe the intracellular partitioning that would explain how Cdh1 activity is fine-tuned, such that cyclin B1 is maintained at sufficient levels to allow oocytes to resume meiosis but not so high as to cause premature meiosis re-entry. Using RT-PCR we detected only one splice variant in mouse oocytes, Cdh1α, which possessed a nuclear localisation signal. By immunofluorescence we confirmed the nuclear location of Cdh1 and degradation machinery components including essential subunits of the APC and 26S proteasome. In all systems studied, cyclin B1 shuttles between the cytoplasm and nucleus, with nuclear localisation occurring just before NEB. We reasoned therefore that the nuclear localisation of the APCCdh1 and 26S proteasome would aid in maintaining low nuclear levels of cyclin B1. Using two GFP-coupled cyclin B1 mutants, which differed in their intracellular location we found that nuclear accumulation of cyclin B1 was necessary in order for it to promote meiotic resumption because over-expression of nuclear-cyclin B1 accelerated entry into meiosis, whereas cytoplasmic-cyclin B1 did not. However, in milrinone-arrested GV oocytes rates of nuclear-cyclin B1 degradation were 5 fold higher than cytoplasmic-cyclin B1. Therefore we conclude that in oocytes, an increase in the nuclear-cytoplasmic ratio of cyclin B1 is an essential step in meiotic resumption, and that nuclear APCCdh1 activity guards against early meiotic resumption, until the degradation machinery is overwhelmed by cyclin B1 translocation.
134. EPIDERMAL GROWTH FACTOR RECEPTOR/MAPK3/1 PATHWAY CROSS-TALK ENABLES GROWTH DIFFERENTIATION FACTOR 9 TO SIGNAL THROUGH SMAD2/3 IN MOUSE GRANULOSA CELLS
Authors: Sasseville M. , Ritter L. J. , Nguyen T. , Mottershead D. G. , Russell D. L. , Gilchrist R. B.
Published: November 2010

Oocyte-secreted growth differentiation factor 9 (GDF9) plays a critical role throughout folliculogenesis. It has been shown to control many functions of granulosa cells, including gene expression, steroidogenesis and proliferation. This study investigates the cellular requirements that allow GDF9 to act on granulosa cells. Our results showed that GDF9 (20 ng/ml)-stimulated mouse granulosa cells 3H-thymidine incorporation was inhibited by a type 1 receptor Alk4/5/7 inhibitor (SB431542, 5 μM), by an epidermal growth factor (EGF) receptor inhibitor (AG1478, 5μM) and a MEK1 inhibitor (U0126, 10 μM). Interestingly, activin A- and TGFβ-stimulated 3H-thymidine incorporation shared similar inhibitor sensitivity. Moreover, when denuded oocytes were used as the mitogenic agent, SB431542, AG1478 and U0126 all prevented the increase in 3H-thymidine incorporation. Oocyte-stimulated 3H-thymidine incorporation in secondary follicles and cumulus-oocyte complexes were also sensitive to Alk4/5/7, EGF receptor and MEK1 inhibition. Basal and EGF-stimulated levels of phopho-MAPK3/1 were inhibited by using the EGF receptor inhibitor, but were not affected by inhibition of Alk4/5/7 or by adding GDF9 in granulosa cells. Using granulosa cells transfected with a SMAD3-luciferase reporter construct, GDF9-stimulated SMAD3 response could be inhibited by Alk4/5/7, EGFR and MEK1 inhibitors. Genes involved in cumulus cells expansion (Ptx3 and Has2) were upregulated in granulosa cells by co-culturing with denuded oocytes and that upregulation was inhibited by Alk4/5/7 as well as by EGF receptor inhibition. These results suggest that TGFβ superfamily members signalling through Smad2/3 share a common requirement of EGF receptor-dependant phospho-MAPK3/1 throughout folliculogenesis. These results strongly suggest that, apart from its role in the transmission of the ovulatory LH signal within the ovarian follicle, EGF receptor pathway might serve as modulators of GDF9 action on granulosa cells. Hence the interaction between endocrine and oocyte signalling may be mediated at the level of MAPK and Smad2/3 cross-talk in granulosa cells.
135. FATTY ACID OXIDATION IS ESSENTIAL FOR OOCYTE DEVELOPMENTAL COMPETENCE
Authors: Dunning K. R. , Cashman K. , Norman R. J. , Robker R. L.
Published: November 2010

Oocyte lipid composition and developmental competence are influenced by dietary fat yet whether lipids are metabolised by the oocyte or essential for subsequent embryo development is largely unexplored. Fatty acid oxidation (FAO) is largely overlooked as an energy source for the oocyte, despite generating several-fold more energy than glucose oxidation. FAO requires the rate-limiting enzyme carnitine palmitoyltransferase-1 (Cpt1) and the metabolite Carnitine to shuttle fatty acids into mitochondria for energy production. Analysis of Cpt1 mRNA during oocyte maturation showed that Cpt1 expression was hormonally induced (p<0.05) in the cumulus oocyte complex (COC), peaking at 10h following ovulatory hCG treatment. In contrast, Cpt1 was not hormonally regulated in granulosa cells (p>0.05). To investigate the role of Cpt1-mediated FAO during oocyte maturation we measured FAO in oocytes in the presence and absence of Carnitine and inhibited FAO to determine its importance for oocyte developmental competence. Levels of FAO in COCs were assessed as metabolism of the fatty acid 3H-palmitate. During oocyte maturation there was a 2.1-fold increase (p<0.0001) in FAO compared to immature COCs. Carnitine supplementation led to a further 3.7-fold increase (p<0.001), while inhibition of Cpt1 with Etomoxir resulted in a 6.5-fold decrease (p<0.0002) in FAO during oocyte maturation. FAO inhibition had no effect on cumulus expansion. However inhibition of FAO during oocyte maturation followed by IVF and embryo culture in the absence of inhibitor, resulted in significantly decreased numbers of embryos developing ‘on time' (p<0.002). This is the first identification of hormonal induction of Cpt1 and Cpt1 mediated FAO in the COC during oocyte maturation. Further, the results demonstrate that oxidation of fatty acids by the oocyte is essential for oocyte developmental competence and can be modulated by Carnitine. These findings provide a potential mechanism by which dietary fat, obesity or metabolic disorders including CPT deficiency lead to infertility.
136. SPECIFICITY STUDIES ON THE IMMUNOSUPPRESSIVE AND CYTOTOXIC ACTIVITIES OF LYSOPHOSPHATIDYLCHOLINES (LPCs) OF GONADAL ORIGIN
Authors: Crane M. , Foulds L. M. , Muir J. A. , Hedger M. P.
Published: November 2010

The immunosuppressive activity of ovarian follicular fluid and testicular interstitial fluid is due to the presence of several LPCs, but the specificity of this inhibition is a potential source of controversy, as these molecules also possess lytic activity. In the following study, we compared the immunosuppressive and cytotoxic activities of the two most abundant gonadal LPCs, 1-palmitoyl-sn-glycero-3-phosphocholine (16:0aLPC) and 1-oleoyl-sn-glycero-3-phosphocholine (18:1aLPC), together with a number of related lysophospholipids (LPs), using a T-cell activation inhibition assay and an ovarian granulosa cell viability assay. Both the immunosuppressive and cytotoxic activities of the LPCs were blocked by serum (>5%) and serum albumin (>5mg/ml) in vitro. In the absence of serum proteins, the most immunosuppressive LPCs were 16:0aLPC, 18:0aLPC, 18:1aLPC and platelet activating factor (PAF; 1-O-palmitoyl-2-O-acetyl-sn-glycero-3-phosphocholine) with IC50 values of 1.2-4.3 μM. Curiously, PAF was the LPC most cytotoxic to granulosa cells (IC50 10 μM). The other immunosuppressive LPCs exhibited cytotoxicity within the range of 40-50 μM, i.e. at doses 10–50-fold higher than their immunosuppressive concentrations. Comparison of LPs of different structures indicated that optimal immunosuppression is related to a phosphocholine, but not serine, ethanolamine or phosphate group, at sn-3, and an ester- or ether-linked fatty acid of chain length C16-C18 at sn-1. Acetylation of sn-2, as in PAF, had only minor effects on immunosuppressive activity, but greatly increased cytotoxicity. These data establish that inhibition of activated T-cells is not a direct consequence of the cytotoxicity of these molecules, although some structural features that contribute to lytic activity, such as fatty acid chain length, overlap with the ability to confer immunosuppression. On the basis of these data, we propose that the effects of LPCs on T-cell proliferation may not be mediated by a specific lock-and-key receptor, but rather by a direct interaction with the cell membrane at concentrations significantly below their lytic concentrations.
138. REGION- AND TIME-DEPENDENT CHANGES IN STRUCTURE AND CELLULAR TURNOVER IN ANDROGEN DEPRIVED MOUSE EPIDIDYMIS
Authors: Gao Y. , Simanainen U. , Handelsman D. J.
Published: November 2010

Epididymal maturation of spermatozoa including acquisition of motility and fertilizing ability depends on androgens both directly from testis and indirectly via the circulation. Androgen action via androgen receptor (AR) can cause both proliferative and anti-proliferative effects (1,2) so we have analysed changes in mouse epididymis following androgen deprivation either by orchidectomy or in prostate epithelial AR knockout (PEARKO) males with reduced androgen action also in epididymis (3). Structural changes (stereology), proliferation (PCNA) and apoptosis (TUNEL) were compared between mature intact males, orchidectomized males 3 day (3d) or 3 weeks (3w) after castration and PEARKO males (3) in the caput and cauda epididymis regions. In caput, epithelial volume decreased while stroma increased in castrates but not in PEARKO whereas lumen volume decreased only after 3 weeks of castration. Proliferating cells (per 100 tubules) were significantly increased 2.8-fold in PEARKO and 6.6-fold in 3d castrate group whereas in the 3wk castrate group proliferation was significantly decreased compared with intact controls. Apoptosis significantly increased by 3.3, 42 and 5.7-folds in PEARKO, 3d and 3wk castrate groups, respectively, compared with intact controls. In the cauda epididymis, castration significantly decreased the volume of lumen and increased stromal volume relative to intact controls. Epithelial proliferation was increased by 20-fold in 3d castrates compared with intact controls. Castration significantly increased apoptosis by 19 and 4.3-folds in 3d and 3wk castrates, respectively, compared with intact controls. We show that the androgen deprivation triggers changes in epididymis structure and cellular turnover in a region-specific and time-dependent manner. The results also reveal a role of testicular factors (presumably androgens) in suppression of epithelial proliferation in the epididymis.
139. THE REGULATION OF TRP53 IN SPERM
Authors: Mudaliar H. , O'Neill C.
Published: November 2010

TRP53 is a tumour suppressor protein that is a universal sensor of cell stress. Upon ejaculation, sperm undergo a process of capacitation which allows them to become fertile. We have previously shown that mouse and human sperm possess TRP53. In this study we analyzed the regulation TRP53 presence in sperm. Mouse sperm were collected from the epididymides and incubated for various times in fertilisation medium. TRP53 was detected by both Western blot analysis and immunolocalization. We found that sperm collected directly from the epididymis generally had little or no detectable TRP53. The level increased with time in culture over a period of 120 min. Most of the TRP53 was detected in the midpiece of sperm although it was also detected in the head of a small proportion of sperm. The increase in TRP53 with time accompanied the increase in the proportion of sperm undergoing the acrosome reaction. Yet, Trp53-null sperm still underwent the acrosome reaction at a normal rate. By contrast, sperm that were prevented from undergoing capacitation and the acrosome reaction (by the removal of albumin or calcium from media) showed a marked reduction in the amount of TRP53 detected. This shows that TRP53 may be dependent upon capacitation, but the reverse was not the case. Inhibition of protein synthesis by puromycin did not block the time-dependent increase in TRP53 in sperm. Canonically, the TRP53 level is controlled by its rate of degradation by MDM2-ubiquitin mediated proteolysis. We found that MDM2 was present in sperm and inhibition of MDM2 (by Nutlin-3) caused an acute increase in TRP53 detected in sperm. This study shows that TRP53 levels are acutely regulated in sperm during the time that sperm acquire the capacity to fertilise, yet sperm lacking TRP53 are capable of fertility. Identification of the role for this TRP53 in sperm is under investigation.