bluuebunny/crossref_metadata_2025 huggingface dataset:
127. An α6β1-integrin/focal adhesion kinase complex may regulate spermiation and spermiation failure
Authors: Beardsley A. J. , Robertson D. M. , O'Donnell L.
Published: November 2010

Spermiation is the final step of spermatogenesis (sperm production) where mature spermatids are released from the somatic Sertoli cells. Spermiation is hormone sensitive; testosterone (T) and FSH withdrawal causes a disruption to the disengagement of spermatids, which are instead retained by Sertoli cells. The mechanisms involved with spermatid release and retention are not understood. We showed previously that an unknown adhesion junction containing β1-integrin persisted on retained spermatids suggesting that a defect in this adhesion complex at disengagement may underlie spermiation failure. The aim of this study is to identify the α-integrin dimerised with β1-integrin and investigate the role of phosphorylated FAK, a kinase that is involved with integrin-mediated cell adhesion, during spermiation and spermiation failure. Four adult Sprague-Dawley rats received T and oestradiol implants and FSH antibody for 7A days to suppress testicular T and FSH and induce spermiation failure. Using immunohistochemistry, α6-integrin (but not α4-integrin) and FAK-Tyr397 were localised on the Sertoli cell plasma membrane adjacent to mature spermatids. This localisation was observed until the point of spermatid release and remained on the Sertoli cell that surrounded retained spermatids after hormone suppression. A similar localisation has been previously observed with β1-integrin, suggesting that all three form a complex at the site of disengagement. To look at the function of FAK-Tyr397, comparative Western blot analysis is currently being undertaken on seminiferous tubules specific for spermiation from control and treated animals. Preliminary studies suggest that FAK-Tyr397 remains phosphorylated during spermiation failure, suggesting that FAK dephosphorylation may be important for the function of spermatid-associated adhesion complexes, as has been demonstrated in other adhesion systems. In conclusion, α6β1-integrin/FAK-containing adhesion complexes are associated with spermatids during spermiation, and the function of such complexes are likely to be perturbed during spermiation failure.
158. Effects of phytoestrogens on the ovarian and pituitary phenotypes of oestrogen deficient female aromatase knockout mice
Authors: Britt K. L. , Simpson E. R. , Findlay J. K.
Published: November 2010

Phytoestrogens can induce both estrogen agonistic and antagonistic effects, depending on the tissue, estrogen receptor content and endogenous levels of estrogen. Dietary phytoestrogens are promoted as alternatives to synthetic estrogens for hormone replacement therapy, however their effects on the reproductive axis have not been exhaustively studied in vivo. Female aromatase knockout mouse (ArKO) mice are estrogen-free, and anovulatory with a block in folliculogenesis, hemorrhagic cysts and development of Sertoli cells within their ovaries. We evaluated the ArKO mouse as a model to test the effects of phytoestrogen-supplemented diets on the reproductive organ weights, ovarian morphology, gonadotropin levels and the transcript levels of ovarian somatic cell and steroidogenic markers. The extent to which phytoestrogens either as soyfree or soymeal diet or genistein added to a soyfree diet, exert estrogenic effects varied with the type of phytoestrogen and the parameter being tested. The genistein diet significantly increased uterine and ovarian weights of ArKO compared to wildtype mice, whilst both the soy, and to a larger extent the genistein diet, improved ovarian morphology compared to the soyfree diet. Transformation to morphological Sertoli cells in ArKO mice was decreased by both phytoestrogen diets, whilst the gene expression of Sertoli cell markers was not affected. The soy diet increased both LH and FSH in both genotypes compared to animals on the soyfree diet. The genistein diet reduced FSH levels in ArKO mice, correlating with increased ovarian inhibin subunit expression. In conclusion, phytoestrogens are estrogenic in ArKO mice. Specifically, they can affect serum gonadotropin levels, and offset the development of Sertoli cells and hemorrhagic cysts within the ovaries, depending on the type of dietary phytoestrogen. Further studies are required to determine the effective doses and treatment regimes for phytoestrogens as endocrine modulators. Supported by NH&MRC Reg Key#198705.
161.Measurement of total testosterone in women: comparison of a direct radioimmunoassay versus radioimmunoassay after organic solvent extraction and celite partition column chromotography
Authors: Davison S. L. , Bell R. J. , Montalto J. G. , Sikaris K. , Donath S. , Stanczyk F. Z. , Simpson E. R. , Davis S. R.
Published: November 2010

Testosterone (T) has multiple significant physiological effects in women. To date no rapid, simple assay of total T has been shown to produce reliable results in women at the low end of the normal female range. The aim of this study was to evaluate the accuracy of a direct radioimmunoassay (dRIA) for total T by comparing values for total T measured by this assay with values determined by a conventional RIA (cRIA) method that utilizes extraction and chromatographic steps prior to quantification. Methods: Fasting serum samples were obtained from a sub-group of 259 healthy women, aged 18-75 years, randomly recruited from the community and stored at -80�C. Total T was measured by the dRIA method using antibody coated tubes and iodine-labeled T tracer. For comparison, total T levels were also measured using the cRIA after organic solvent (ethylacetate�:�hexane (3�:�2)) extraction and Celite column partition chromatography prior to RIA. Results: The mean T level by dRIA was 0.76 nmol/L (median 0.70, SD 0.54, min 0.10, max 3.2). The mean difference between the two measurements (dRIA-cRIA) was –0.28 (SD 0.3). The limits of agreement using the Bland-Altman approach on log transformed data showed that, on average, the dRIA value was 63% of the cRIA value and that 95% of the time the dRIA estimate lay between 26% and 155% of the cRIA estimate. However, with respect to clinical application, for classification of values in the lowest 10th centile, agreement between assays was seen in 245/259 women (Kappa�=�0.68) Conclusion: The dRIA is a clinically useful assay that provides precise measurements of total T in women, particularly when values are low, and is appropriate for the study of the issue of 'low' T within the female population.
201.Regulation of mouse mural granulosa cell progesterone synthesis by oocyte paracrine factors
Authors: Ritter L. J. , Gilchrist R. B.
Published: November 2010

Oocytes secrete soluble paracrine factors that play an important role in the growth and development of surrounding follicular cells. It is known that oocytes suppress FSH-induced progesterone production by mural granulosa cells (MGC), however it is unclear which growth factor(s) are involved. Some candidate molecules include growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and BMP6. The aim of this study was to examine the role of these factors in the regulation of FSH-induced MGC progesterone production. Prepubertal 129/SV mice were primed with eCG and ovaries were collected ~46�h later. MGC from large antral follicles were cultured with either denuded oocytes (DO; 0.25/μl), GDF9 or BMP15 (0.25-4% v/v) or BMP6 (10-200ng/ml), in the absence or presence of FSH. Cells were cultured for 18�h followed by a further 6�h pulse of 3H-thymidine. After 24�h cells and media were harvested for assessment of MGC DNA and progesterone synthesis, respectively. Treatment with FSH increased MGC progesterone production 9-fold, which, as expected, was antagonised by coculture with DO (by 73%). GDF9 and BMP15 both decreased FSH-induced MGC progesterone in a dose dependent manner, significantly reducing control levels (100%) to 17% and 30%, respectively, at doses of 2%v/v. All doses of BMP6 abolished FSH-stimulated progesterone. Even though all treatments inhibited progesterone production, only two of these, GDF9 (0.25%v/v) and DO (0.25DO/μl), stimulated MGC DNA synthesis (2.1 and 3.3 fold above controls, respectively). The BMP receptor type-II (BMPR-II) is a known receptor of several oocyte factors. Treatment with a BMPR-II ectodomain completely antagonised DO- and GDF9-stimualted MGC DNA synthesis, but progesterone levels were only partially restored (by 50%). These data indicate that BMP15 and BMP6 mimic the progesterone-regulating, but not the growth-promoting, activities of oocytes, whereas GDF9 does both. Although the BMPR-II ECD antagonises these oocyte factors, this receptor-signalling system may not necessarily be the means by which oocytes regulate MGC progesterone synthesis.
202.Estrogen actions on follicle formation and early follicle development
Authors: Britt K. L. , Saunders P. K. , McPherson S. J. , Misso M. L. , Simpson E. R. , Findlay J. K.
Published: November 2010

Estradiol 17 beta (E2) effects late follicular development whilst primordial follicle formation and early activation are thought to be independent of E2. To test this hypothesis we compared numbers of primordial and primary follicles in wildtype and E2 deficient ArKO mice, and the immunohistochemical staining or mRNA expression of Mullerian inhibiting substance (MIS), Wilms tumour 1 (WT-1), and growth differentiation factor (GDF9), known to effect early follicular differentiation. Proliferating cell nuclear antigen (PCNA) staining was a marker of proliferative index. The effects of E2 replacement for 3 wk in 7 wk old ArKO and wildtype mice on these parameters were also tested. We used unbiased, assumption-free stereological methods for quantification of early follicular numbers in the mouse ovary (1). ArKO mice had reduced numbers of primordial and primary follicles compared to wildtype (63%, p<0.001 and 60%, p=0.062 of Wt respectively). This reduction was not corrected by E2 treatment, suggesting that E2 effects the initial formation or activation of primordial follicles. There was a significant increase in the diameters of the oocytes in primordial follicles of ArKO mice compared to wildtype. There were no differences in the immunostaining of MIS, WT-1 and PCNA in primordial and primary follicles between wildtype and ArKO mice. The only difference was as a consequence of Sertoli and Leydig cells in ovaries of ArKO mice. GDF9 mRNA expression was markedly increased in ArKO ovaries. E2 treatment restored the ovarian follicular morphology, and consequently the immunostaining patterns, but had no effect on early follicle numbers. In conclusion, E2 has a role in controlling the size of the oocyte and primordial follicle pools in mice. Supported by NH&MRC RegKey #241000 and 198705.
(1) Britt and Myers (2004) Reproduction 127,:569–580.
203.Studies of the activin pathway in dominant and subordinate bovine follicles before, during and after selection
Authors: Sisco B. C. , Shelling A. N. , Pfeffer P. L.
Published: November 2010

In monovulatory species such as cattle, one of a cohort of developing follicles assumes dominancy and continues to grow in each follicular wave. After dominant follicle selection, pituitary-derived FSH levels decrease through a negative feedback loop mediated by oestradiol and inhibin A produced by the dominant follicle. The dominant follicle itself only requires very low basal levels of FSH, thus escaping atresia which is the fate of the subordinate follicles. The mechanisms involved in dominant follicle (DF) selection remain unclear. Most studies have focused on the stages following selection. To investigate what roles activin and inhibin play in DF selection we looked at the quantitative changes in the expression of the genes coding for the activin/inhibin subunits (Inhibin α, βA and βB) as well as other genes in the activin pathway (SMAD2, ActRIIA/B, follistatin (FST), FSHR). We examined mRNA levels in follicular granulosa cells (GCs) before (d1.5), during (d2.5) and after (d3.5 and 7) DF selection using real-time RT-PCR. Prior to DF selection, highest levels of inhibin βA, FST and SMAD2 transcripts converged on the largest follicles. Inhibin α, ActRIIA/B and FSHR levels did not correlate with follicular size at this stage. At Day 2.5, highest levels of inhibin βA, inhibin α, FST and SMAD2 transcripts were seen in a single putative DF. ActRIIA/B and FSHR did not show any difference between follicles. By Days 3.5 and 7, a dramatic difference in expression levels of inhibin βA, inhibin α and FST were seen in DF compared to SF. Yet in absolute terms inhibin βA levels decreased after selection, whereas inhibin α levels increased. Inhibin βB expression was only detected in Day 7 GCs and was significantly higher in the DF. These results suggest a shift from an activin environment during the pre and peri-DF selection period, to an inhibin environment following DF selection. Inhibin/activin protein levels in the follicular fluid using western ligand blotting confirmed this. We postulate that the higher activin activity within DF influences the selection mechanism as activin and inhibin have been shown to play a role in gonadotropin regulation in the ovary around the time of selection.
205.The effect of FSH concentration during IVM and gamete co-incubation length during IVF on the development of unstimulated prepubertal ewe oocytes
Authors: Morton K. M. , Maxwell W. M. C. , Evans G.
Published: November 2010

The developmental competence of prepubertal oocytes can be increased by the administration of gonadotrophins prior to oocyte collection (1); but this is not possible with abattoir-sourced oocytes, and modifications to the IVP system may increase in vitro development. Experiments were conducted to determine the effects of FSH concentration (10, 20 or 60 μg mL-1) during IVM (5 replicates) and gamete co-incubation length (short: 2-3 h, long: 18-20 h) during IVF (6 replicates) on subsequent embryonic development. For both experiments ovaries were collected from prepubertal lambs (16-24 weeks) slaughtered at an abattoir and embryos produced in vitro (1). Data were analysed by chi-squared test. Oocyte cleavage at 48 hours post-insemination (hpi) was higher for oocytes matured in medium containing 20 (60/77; 77.9%) and 60 (56/73; 76.7%) than 10 μg mL-1 (40/67; 59.7%) FSH. Blastocyst formation (% cultured oocytes) on Day 7 (Day 0 = IVF) was higher for oocytes matured with 20 (31/77; 40.3%) than 10 (16/67; 23.9%) or 60 μg mL-1 (20/73; 27.4%). Oocyte cleavage at 48 hpi was reduced for short (36/57; 63.2%) compared with long (49/55; 89.1%) co-incubation, although blastocyst formation (% cultured oocytes; Day 7) did not differ between groups (22/57; 38.6% and 23/55; 41.8%, respectively). These results demonstrate that increasing the FSH concentration above normal levels during IVM of prepubertal lamb oocytes improves development in vitro. Gamete co-incubation length did not influence the proportion of oocytes progressing to the blastocyst stage.
(1) Morton et al. (2003) Proc. Soc. Reprod. Fert. P18.
207.Oolemmal proteomics: identification of oocyte cell surface protein complexes involved in sperm - egg interaction
Authors: Paul J. W. , McLaughlin E. A. , Aitken R. J.
Published: November 2010

While the molecular basis of sperm-oolemma interaction is of considerable biological significance, the protein(s) involved in this process are yet to be identified. In particular, the point at which developing mammalian oocytes acquire the capacity to bind to, and fuse with, capacitated, acrosome-reacted spermatozoa is yet to be clearly defined. Previous reports suggested that mature metaphase II (MII) mouse oocytes possessed the capacity to bind and fuse with spermatozoa while, in contrast, immature germinal vesicle (GV) phase oocytes did not. In this study oocytes were cultured in α-MEM containing 5% fetal calf serum. For GV oocytes, α-MEM was also supplemented with 1 μM Milrinone to prevent GV breakdown. Standard murine IVF was performed in 100 μL α-MEM media droplets containing 10 to 15 oocytes and 2.5×104 capacitated spermatozoa.
These studies found that no significant difference existed in the levels of sperm-oocyte binding or fusion between freshly isolated GV oocytes (14 sperm/egg bound, 7 sperm/egg fused), cultured arrested GV oocytes (16 sperm/egg bound, 7 sperm/egg fused) or cultured MII phase oocytes (17 sperm/egg bound, 8 sperm/egg fused). Interestingly, upon fusion MII oocytes commenced sperm nuclear decondensation whereas arrested GV oocytes did not.
Comparison of biotinylated oolemmal surface proteins revealed markedly different protein profiles between the GV oolemma and the MII oolemma. The MII oolemma revealed a multitude of proteins ranging in size from approximately 20 to 200 kDa, whilst the GV oolemma revealed only six middle range molecular weight proteins ranging from approximately 50 to 90 kDa. The protein(s) implicated in sperm-egg interaction must be one or more of those proteins found in both oocyte populations. Comparison of these two profiles has greatly reduced the number of possible candidates, allowing possible identification of the proposed GPI-linked sperm receptor.
208.Zona pellucida vaccines for fertility control of brushtail possums in New Zealand
Authors: Duckworth J. A. , Cui X. , Molinia F. C. , Lubitz W. , Cowan P. E.
Published: November 2010

Introduced marsupial brushtail possums (Trichosurus vulpecula) are a major pest in New Zealand because of their impacts on conservation values and agricultural production. Immunologically-based fertility control (immunocontraception) offers an effective and humane alternative approach to possum management. The zona pellucida (ZP) is an extracellular coat around all mammalian eggs and an attractive target for the development of immunocontraceptive vaccines. Antibodies against ZP are ovary-specific and act by preventing sperm from binding and penetrating the ova and/or by disrupting the development of follicles or early embryo. The aim of these studies was to test the efficacy of possum-derived ZP antigens for their ability to elicit sustained immune responses and cause infertility, and to assess a range of options for development of a bait-delivered contraceptive vaccine. Alloimmunisation with possum ZP2 and ZP3 proteins showed that self-ZP antigens elicited strong humoral immune responses and reduced the fertility of female possums by 72-80%. Several potentially possum-specific immunocontraceptive peptides have been identified by linear epitope mapping and amino acid alignment and are being tested for their ability to reduce fertility. Recent trials have demonstrated that possums mount immune responses against ZP antigens delivered in transgenic plants and bacterial ghosts. Research on antigen and specific peptide identification, non-target effects and delivery systems is ongoing. Research supported by NZ Foundation for�Research Science & Technology, Marsupial CRC and NZ Animal Health Board.
209.A proteomic analysis of rat caput and cauda sperm using difference in 2D-gel electrophoresis
Authors: Baker M. A. , Witherdin R. , Hetherington L. , Cunningham-Smith K. , Aitken J.
Published: November 2010

When testicular spermatozoa migrate to the epididymis they are still functionally incompetent. Having lost the cellular machinery to support gene transcription and protein translation, these cells acquire the gamut of biological functions needed to achieve fertilization under the influence of factors provided by the epididymal microenvironment. Although the biological changes exhibited by spermatozoa during epididymal transit have been well established, the molecular basis for these changes is still poorly understood. Difference in 2D Gel electrophoresis (DIGE) is a powerful new technology for comparing up to three different protein samples in the same 2D gel, thus eliminating the variation that occurs with more traditional proteomic approaches. Using a combination of this procedure and matrix assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry, we have unambiguously identified 8 proteins that change significantly during epididymal maturation including a-enolase, hsp60, endoplasmin, phosphatidyl-ethanolamine binding protein, testis lipid binding protein and the b-subunit of the F1 ATPase. The nature of these changes (80 kDa mass shift and increase electronegative charge) suggested a series of phosphorylation events. In order to further characterize these changes, Western blot studies were conducted using anti-phosphoserine antibodies. This analysis revealed a dramatic increase in serine phosphorylation for two major proteins (54 and 73 kDa) during epididymal transit, one of which (54 kDa) and confirmed by MALDI-TOF analysis to be the b-subunit of the mitochondrial ATPase. The phosphorylation of this protein was associated with a 3-fold increase in the ATP content of epididymal spermatozoa as they pass from the caput to the cauda epididymis.� This change clearly identifies mitochondrial ATP production as a key component of the epididymal changes that lead to the generation of vigorously motile functional spermatozoa. The kinase responsible for the F1-ATPase phosphorylation appears to be PKA regulated and a clear potential target for contraceptive intervention.
210.Regulated expression of mRNAs encoding nuclear transport proteins during spermatogenesis
Authors: Hogarth C. A. , Jans D. A. , Loveland K. L.
Published: November 2010

During spermatogenesis, the germ line stem cells undergo a complex series of cellular transitions to form mature male gametes. These transitions require regulated nucleocytoplasmic shuttling of transcription factors and cell cycle regulators, mediated in part by proteins known as importins. The importins bind to specific cargo proteins in the cytoplasm and transport them into the nucleus via interactions with components of the nuclear pore. There are two families of importins, termed the αs and the βs, with five different importin α members identified in the mouse genome. In this study, we examined the mRNA expression patterns of importin αs in the rodent testis using in situ hybridization and Northern blotting. Each importin α displayed a distinct expression pattern in the adult mouse testis. Imp α1 mRNA was detected in spermatogonia through to early pachytene spermatocytes. Imp α1 mRNA was detected in pachytene spermatocytes, α6 in round spermatids and α2 in both of these cell types. Northern blotting with in situ hybridization probes on total testis RNA from adult rat and mouse and 10 dpp (days postpartum) rat revealed distinct transcript sizes for imp α1, 2, 4 and 6. For all importin αs, the mRNA signal level in the 10 dpp rat sample was lower than in the corresponding adult sample. The distinct expression patterns for each importin α family member in germ cells of the adult rodent testis suggests these importins are required to carry specific cargo at distinct stages of spermatogenesis. These data extend our previous analysis of importin β1 and β3 expression in the fetal and adult testis, which also demonstrate developmentally regulated importin expression. Ongoing studies are examining the cellular localization of importin α proteins and investigating their specific functions. We predict they each carry cargo required for distinct transitions in spermatogenesis.
211.Subcellular localisation of testis forms of MAP2 is a product of competing targeting signals/domains
Authors: Lo C. Y. , Barton C. L. , Jans D. A. , Loveland K. L.
Published: November 2010

The process by which mitotic spermatogonia develop to form spermatozoa requires fine control of microtubule-based structures. Assembly, stabilization and disassembly of these structures are regulated by a heterogeneous group of proteins known as Microtubule Associated Proteins (MAPs). One of the best characterized MAPs is MAP2, which is found in both brain and testis. Different MAP2 isoforms arise from alternative splicing of the same gene. These have been categorized into high and low molecular weight (HMW and LMW), depending on the presence/absence of an extension arm encoded by exon 9. Exon 10 encodes for a nuclear localization signal (NLS), and in the brain, this exon is found exclusively on HMW isoforms, which localize to the cytoplasm of neurons. In the testis, however, exon 10 can be found on LMW isoforms, which localize within the nucleus of germ cells. Transfection of GFP-tagged MAP2 constructs containing exons 10 and 11 into mammalian cell lines (GC-1, GC-2 and COS-7) reveal that the NLS is capable of localizing the entire protein within the nucleus of transfected cells, and this effect is partially dependent on the presence of the protein's tubulin binding domains. Experiments utilizing an exon 10/11 specific probe to hybridise to MAP2 mRNA in mouse and rat testis sections show that mRNA containing exon 10/11 is present in both 15 day post partum (dpp) and adult testis in spermatogonia and spermatocytes. Expression of exon 10/11 MAP2 mRNAs were not detected at 5dpp, indicating that a switch in mRNA expression occurs sometime in the 5-15dpp interval. The expression pattern of exon 10/11 MAP2 mRNA and the NLS's ability to localize LMW MAP2 protein into the nucleus indicate a novel role for MAP2 in germ cell development.
213.Culture of mouse male germ cells for genetic manipulations
Authors: Itman C. M. , Barakat B. , Loveland K.
Published: November 2010

The ability to maintain germ cells in culture offers the opportunity to manipulate their differentiation and to deliver new genes through the germline. However the study of male germ cell biology is hindered by the lack of an in vitro culture system that supports germ cell maintenance and differentiation, and the lack of efficient gene transfer technologies. To address this, we developed a short term (1-6 day) in vitro germ cell-Sertoli cell co-culture system using testes of 6 day old mice, in which the only germ cell type present is the spermatogonium. To establish culture conditions favourable for maintaining germ cells in culture, we compared different substrates (plastic, laminin, poly-L-lysine, Matrigel) and cell culture additives (insulin/transferrin, retinoic acid, lactic acid, pyruvic acid). Using immunocytochemistry, we observed that germ cell numbers varied depending on the substrate, and was greatly improved on laminin or Matrigel. The culture additives tested had no effect. We noted a striking difference in germ cell growth depending on the mouse strain used, with C57BlXCBA F1 cross > Swiss > BalbC >> C57Bl/6J. To investigate gene transfer into germ cells, constructs were generated using promoters identified as exhibiting germ cell-specific expression in transgenic animals (EF1-α, Oct4, Stra8) with EGFP and lacz reporter genes. From this baseline, we have started gene transfer experiments. Initial transfection experiments were performed in mouse testis cell lines (GC1, GC2, TM3, TM4) and the P19 mouse embryonal carcinoma cell line. Several commercial transfection agents and a non-commercial product (1) were used, as was electroporation, however these did not yield reporter expression in germ cells. As these techniques require proliferating cells, we are now undertaking a study with the addition of growth factors in culture (BMP4, activin, GDNF, FSH) to enhance germ cell proliferation and therefore improve transfection efficiencies. We will also employ lentiviral transduction, as this is reportedly 100% efficient and independent of proliferation.
(1) Celebi et al. (2002) Mol. Reprod. Dev. 62,477–482.
215.Analysis of DNA damage induced by pro-oxidant treatment of mammalian spermatozoa in vitro
Authors: Bennetts L. E. , Aitken R. J.
Published: November 2010

Defects in the male genome produced as a consequence of oxidative insult have been associated with decreased fertility levels, an elevated incidence of childhood cancer and dominant genetic disease in the offspring (1). The objective of this study was to determine the relative susceptibility of sperm DNA of different mammalian species to oxidative injury. We applied a highly sensitive quantitative PCR assay (QPCR) to measure gene-specific DNA damage in nuclear and mitochondrial compartments of spermatozoa treated with H2O2. Human, murine and tammar wallaby (Macropus eugenii) spermatozoa were treated with H2O2 (0–5�mM) over a 1�h period. After DNA purification, DNA damage was assessed in a nuclear and a mitochondrial fragment of DNA by quantitative polymerase chain reaction assay (QPCR). DNA damage was detected as a decrease amplification of the target sequences. In murine and human spermatozoa, mitochondrial DNA exhibited greater sensitivity to oxidative damage than nuclear DNA. Doses ranging from 0.25–5�mM H2O2 induced DNA damage of up to 0.65 lesions/10�kb in the mouse, and 1.42 lesions /10�kb in the human. No significant effect on DNA damage was observed over this dose range in the nuclear DNA fragments investigated in these species. In contrast, tammar wallaby spermatozoa were susceptible to DNA damage at the 5�mM H2O2 dose in both nuclear (0.51 lesions/10�kb) and mitochondrial (0.55 lesions/10�kb) genomes. This study is the first to compare DNA damage in specific DNA sequences in spermatozoa of different mammalian species. Nuclear DNA of the metatherian species, the tammar wallaby, was more susceptible to oxidative damage than that of the eutherian species. A major difference between metatherian and eutherian spermatozoa is that, in general, the former possess protamines that are not stabilised by disulfide cross-linkage. These findings therefore suggest that sperm chromatin packaging affects the susceptibility of sperm DNA to oxidative damage.
(1) Sawyer and Aitken (2000) Reprod. Med. Rev. 8, 107–126.
216.The effects of Viagra on sperm function and early embryo development
Authors: Lewis S. E. M. , Glenn D. R. J. , McClure N.
Published: November 2010

In an audit of UK fertility units, we have demonstrated that 42% prescribe Viagra to aid patient semen production. Viagra is a phosphodiesterase inhibitor (PDE5) and as non-specific PDEs have been shown to affect fertility, safety concerns have been raised. The aims of this study are to investigate the effects of Viagra on sperm function and early embryo cleavage. Human semen was incubated with and without Viagra (450�ng/mL sildenafil citrate, equivalent to plasma concentrations after 100�mg oral dose; Pfizer, UK). Aliquots were also prepared by a 90/45% density centrifugation gradient to separate good and poor subpopulations. All samples were analysed by computer assisted semen analysis (HTM-IVOS) up to 60 and 120�min. Prepared samples were also labelled with fluorescein isothiocyanate–peanut agglutinin to determine acrosome status. Male mice were gavaged with Viagra (equivalent dose/body wt) and mated with superovulating females. Twenty females were sacrificed 12�h later, their oviducts flushed and viable fertilized oocytes counted. Another 20 females were sacrificed 4�days after mating and their embryo numbers and cleavage stages determined. Viagra increased % progressive motility in semen (n�=�22) by 38%, VAP by 21%, VSL by 21% and VCL by 16% at 60�min (all P values <0.001). These effects were sustained at 120�min. Sperm isolated from 90% (n�=�57) and 45% (n�=�15) fractions showed similar increases. Viagra also increased the proportion of acrosome reacted sperm in the 90% (+79%, P�<�0.001) and 45% (+77%, P�<�0.001) fractions. Further, Viagra caused a reduction in both the numbers of fertilised oocytes (–35%, P�<�0.001) and those reaching blastocyst stage (85%, P�<�0.001). This study demonstrates that Viagra increases human sperm motility. However, Viagra induces human premature acrosome reactions and impairs mouse fertilisation and embryo cleavage. This study raises significant concerns for its use in assisted reproduction.
219.Analysis of ovarian macrophage populations using macrophage-specific green fluorescent protein (GFP) transgenic mice
Authors: Haynes C. E. , Norman R. J. , Robker R. L.
Published: November 2010

Macrophages represent a major immune cell type in reproductive tissues and are thought to regulate multiple aspects of reproduction, including ovarian function. We have previously shown distinctive phenotypes and functions of ovarian macrophages such that many immunological mediators, such as cytokines and hormone receptors, are uniquely regulated within these cells across the oestrus cycle. In order to isolate macrophages from ovarian tissue by fluorescence activated cell sorting (FACS), we acquired transgenic mice (from DA Hume, Institute for Molecular Bioscience, University of Queensland) which express GFP exclusively in macrophages (1). In these mice GFP is expressed under direction of the c-fms gene promoter, which encodes the receptor for colony-stimulating factor-1 (CSF-1R), a major macrophage growth factor. Using flow cytometry we confirmed that 95% of peritoneal macrophages express GFP and 88% co-express GFP and the classical macrophage marker F4/80. The distribution of GFP+ macrophages in tissues was co-localized with macrophage markers F4/80 and major histocompatibility complex class II (MHCII) by immunohistochemistry using phycoerythrin (PE)-labelled antibodies. The liver, uterus and oviduct exhibited many GFP+ cells in characteristic macrophage distributions. Furthermore, GFP fluorescence was tightly co-localized with PE fluorescence of either F4/80 or MHCII, indicating that CSF-1R is expressed in the macrophages of these tissues. In contrast, macrophages in the ovary were positive for F4/80 and MHCII, but rarely expressed GFP. Thus unlike macrophages of other reproductive tissues, ovarian macrophages do not consistently express CSF-1R. In ovaries from gonadotrophin-primed immature females, GFP was not expressed in macrophages (F4/80+/MHCII+) surrounding follicles but was detected in macrophages within the regressing corpus luteum. Thus CSF-1R is a hormonally regulated gene, expressed only in specific subsets of ovarian macrophages suggesting that CSF-1 controls functional activities of ovarian macrophages at specific stages of the ovarian cycle.
(1) Sasmono, R. T., et al. (2003) Blood 101, 1155–1163.
221.Effects of exogenous gonadotrophin stimulation on ovarian tissue grafts in the mouse
Authors: Yang H. , Cox S. , Shaw J. , Jenkin G.
Published: November 2010

Ovarian tissue grafts commonly contain only limited numbers of follicles. The functional life span and ability to retrieve as many mature oocytes as possible from ovarian grafts is important when grafting is used to restore fertility. This study aimed to determine whether ovarian grafts responded to exogenous hormones in a similar manner to that of in situ ovaries. Ovaries of C57BlxCBA F1 mice were cut in half and grafted to one of three different graft sites in females of the same F1 line; bursal capsule (BC, n = 12), kidney capsule (KC, n = 6), subcutaneous tissue (SC, n = 24). Three weeks after grafting, half of the graft recipients in each group were treated with 5IU PMSG followed by 5IU hCG 48 hours later. Oocytes were collected directly from the grafted ovaries 10 hours after the hCG injection and fertilized in vitro. Oocytes from the ovaries of superovulated normal mice (n = 4) of the same hybrid strain were used as controls. Two-cell embryos were transferred to pseudopregnant recipients and collected at day 15 of gestation or the animals were allowed to go to term. Mature fertilisable MII oocytes were retrieved from stimulated grafts from all graft sites, however, the number (BC 9, KC 5, SC 2 oocytes per ovary) and proportion of two-cell embryos in each grafted group (BC 52%, KC 32%, SC 32%) was significantly (P < 0.05) lower than in the in vivo matured control (16 oocytes, 85% two-cell). The fetal and placental weights of fetuses produced from graft-derived oocytes were not significantly different to the control group. Phenotypically normal pups were born in each of the graft and control groups. In conclusion, ovarian grafts treated with exogenous gonadotrophins produce significantly fewer mature oocytes and two cell embryos compared to in situ ovaries. Work supported by ARC and NIH RFA.
225.Expression of chemokines and their receptors at the human maternal - embryonic interface
Authors: Hannan N. J. , Jones R. L. , Salamonsen L. A.
Published: November 2010

Human embryo implantation is a complex process involving attachment of the developing blastocyst to the receptive endometrial epithelium, and subsequent trophoblast invasion through decidua. This is regulated by crosstalk between the maternal and embryonic cells, however little is known about the factors involved in enabling and directing trophoblast invasion. Chemokines are cytokines that regulate leukocyte chemotaxis via stimulation of adhesion molecules and cell migration. We have previously shown that two chemokines, fractalkine and MCP-3, are produced by endometrial epithelial and decidual cells, maximally around the time of implantation and early pregnancy (1, 2). We hypothesized that endometrially derived fractalkine and MCP-3 are important for the attachment/invasion of fetal trophoblast cells during implantation. To investigate this, expression of fractalkine, MCP-3 and their receptors (CX3CR1, CCR1, CCR2, CCR3 and CCR5) were assessed in cell types present at the maternal-embryonic interface. RNA, extracted from three trophoblast cell lines (JEG-3 and two trophoblast-choriocarcinoma hybrids), a human epithelial cell line (HES), primary endometrial epithelial cells, mid-secretory endometrium and placental tissue, was subjected to RT-PCR for the chemokines and receptors. Both chemokines were produced by endometrial and placental cells. Chemokine receptor expression was more variable, CX3CR1, CCR1, 2 and 3 were expressed by one or more of the trophoblast cells lines while CX3CR1, CCR1, 2 and 5 were expressed by endometrial cells. Marked differences in expression patterns in the different cell lines highlight the importance of studies to select those cell lines of most physiological relevance: in this case, one that most closely resembles early invasive trophoblasts. These data confirm that chemokines are produced by maternal and embryonic cells during implantation and the strong expression of their receptors on trophoblast cells supports a role for chemokines in embryo implantation. Further, these studies have characterized a number of trophoblast cells for future trophoblast migration and attachment assays.
(1) Hannan, N., et al. JCEM (in press). (2) Jones, R., et al. JCEM (in press).
226.Regulated expression of adhesion and anti-adhesion molecules in mouse endometrium during early pregnancy
Authors: Jasper M. J. , Stocker A. , Robertson S. A.
Published: November 2010

To implant and establish the connections that are vital for further development, the early embryo must attach to and then breech the barrier posed by the epithelium of the maternal tract. Expression of adhesion and anti-adhesion molecules in the luminal epithelium of the endometrium are thought to fluctuate in a temporal pattern to 'frame' the implantation site, with their expression regulated by endocrine and paracrine factors. Anti-adhesion molecules, such as members of the mucin family, provide a barrier to implantation in sites or at times unsuitable for embryo development. Expression of adhesion molecules, or specific integrins, are thought to aid in the adhesion of the embryo, allowing it to induce changes in the underlying tissue promoting embryo invasion and pregnancy. The aim of this study was to quantitate the expression of mRNA encoding the integrins αυ, α4 and β3 and MUC1 and MUC4 from Day 0 (oestrous) to Day 4 of pregnancy (implantation) using quantitative real time RT-PCR. Uterine tissues were collected at oestrous and at Days 1, 2, 3 and 4 of pregnancy (Day 1 corresponding to the presence of a vaginal plug), total RNA was extracted, DNAse treated, reverse transcribed into cDNA, and quantified by real-time PCR using SYBR Green chemistry. All specific primers were designed using GenBank sequences and data were normalised to β-actin mRNA expression. Expression of MUC1 and MUC4 mRNAs was dramatically reduced, with mean values 20-fold and 100-fold less than at oestrous respectively, by Day 4 of pregnancy. In contrast, expression of mRNAs encoding integrins αυ, α4 and β3 was detected throughout early pregnancy. These data demonstrate that adhesion and anti-adhesion molecules are differentially expressed in the murine uterus during early pregnancy and may be key mediators in embryo implantation, promoting attachment of the embryo to the luminal epithelium in an environment conducive to embryo growth and development. Supported by a Clive & Vera Ramaciotti Project Grant to MJ Jasper.
228.Progestin-induced proprotein convertase 6 is necessary for decidualisation of human endometrial stromal cells in vitro
Authors: Okada H. , Nie G. , Salamonsen L. A.
Published: November 2010

Decidualisation of human endometrium is an essential preparative event for successful establishment of pregnancy, and involves dramatic morphological and functional differentiation of the human endometrial stromal cells (ESCs). Proprotein convertase 6 (PC6) plays an important role in the processes of stromal cell decidualisation and embryo implantation in the mouse. PC6 is a member of the proprotein convertase family responsible for processing precursor proteins to their bioactive forms by selective proteolysis. In the present study we investigated the regulation of PC6 mRNA and protein expression in ESCs during decidualisation in vitro, and established a function for PC6 in decidualisation using morpholino antisense oligonucleotides (MOs). PC6 mRNA levels in ESCs during decidualisation were determined using quantitative real-time RT-PCR. 17β-oestradiol (E) plus medroxy-progesterone acetate (P) caused a significant increase in PC6 mRNA during decidualisation, whereas E alone did not increase PC6 mRNA expression. Consistent with the results of real-time PCR, much stronger PC6 immunostaining was observed in the cytoplasm of E plus P-treated ESCs (decidualised) compared to the E-treated ESCs (non-decidualised) on Day 12 of culture. This strong staining for PC6 was abolished by cotreatment with ZK 98299, a progesterone receptor antagonist. To investigate whether the induction of PC6 was necessary for decidualisation in vitro, MOs were used to block PC6 synthesis in cultured ESCs. PRL production, a typical marker for decidualisation, was significantly attenuated in decidualing ESCs following treatment with PC6 MOs in comparison to controls. These results suggest that PC6 plays a key role for decidualisation in human ESCs.
229.Seminal plasma TGFβ activates pro-inflammatory cytokine synthesis in human cervical epithelial cells
Authors: Sharkey D. J. , Robertson S. A.
Published: November 2010

Exposure to semen at intercourse in women elicits an inflammation-like response characterised by recruitment of inflammatory cells and expression of pro-inflammatory cytokines including GM-CSF, interleukin-6 (IL-6) and IL-8 (1). Studies in animal models have implicated TGFβ as the major active moiety in seminal plasma, and we have shown previously that TGFβ1 and TGFβ3 are present in high concentrations in human seminal plasma (>100 ng/mL), while TGFβ2 is less abundant. To investigate the physiological significance of each of the three TGFβ isoforms as pro-inflammatory agents in human seminal plasma, we have established in vitro model systems to measure human cervical cell cytokine synthesis. Primary cervical epithelial cells prepared from ectocervix of hysterectomy tissues or transformed Ect1 cells were incubated for 12 h with human recombinant TGFβ (isoforms 1, 2 or 3) or with seminal plasma in the presence or absence of isoform-specific TGFβ neutralising antibodies. Epithelial cell supernatants were recovered 24 h later and supernatants were analysed by commercial ELISA to quantify GM-CSF, IL-6 and IL-8 production. Each of the three TGFβ isoforms mimicked seminal plasma and were comparable in their capacity to stimulate >10-fold increases in both GM-CSF and IL-6 expression in a dose-responsive manner. In contrast, unlike seminal plasma none of the TGFβ isoforms induced IL-8 expression. Addition of neutralising antibodies to TFGβ1, TGFβ2 and TGFβ3 each effected >50% reduction in the ability of seminal plasma to induce GM-CSF and IL-6, but did not impair seminal plasma-stimulated IL-8 production. Together these data show that TGFβ1, TGFβ2 and TGFβ3 are major active constituents of seminal plasma, acting to elicit GM-CSF and IL-6 production in cervical epithelial cells. However, TGFβ does not fully account for the pro-inflammatory effects of human seminal plasma, and other active constituents remain to be identified.
(1) D. J. Sharkey et al. (2003) Proc. SRB.
234.Calcium involvement in glucose induced GLUT3 expression in preimplantation mouse embryos
Authors: Gardner K. , Pantaleon M. , Kaye P. L.
Published: November 2010

Despite their inability to utilise glucose for energy prior to compaction (E3), mouse embryos have a requirement for at least a brief glucose exposure to permit normal development. In the absence of this glucose pulse in vitro, we and others have found that embryos cleave to form morulae but fail to form blastocysts and subsequently degenerate. These embryos do not develop the capacity to utilise glucose preferentially and are unable to adapt to their nutrient environment and utilise alternate substrates (1). This inability to utilise glucose is due to failure to express GLUT3 at compaction (2). Brief glucose exposure prior to the 8-cell stage is sufficient to permit the embryo to undergo compaction, express GLUT3 and ultimately form a blastocyst, suggesting that glucose induces metabolic differentiation of the developing embryo. In this study we have explored the role of intracellular calcium in response to glucose given its central role in pancreatic glucose induced signalling events. Zygotes were cultured in the presence and absence of glucose and treated with either calcium mobilising agents, ethanol or ionomycin at 54�h post hCG or with the intracellular calcium chelator BAPTA-AM. Embryos were fixed and assayed for GLUT3 expression individually at 96�h post hCG using confocal immunofluorescence. Release of intracellular calcium by either ethanol or ionomycin, activated GLUT3 expression in a glucose like manner (P�<�0.01) suggesting that calcium transients may be involved in glucose sensing. Moreover, buffering of calcium with the calcium chelator BAPTA-AM interfered with the ability of glucose to activate GLUT3 expression (P�<�0.05), suggesting that glucose exposure does result in calcium transients that affect GLUT3 expression. It is unclear whether these calcium transients occur as a result of influx of extracellular calcium via voltage-gated ion channels or the release of calcium from intracellular stores via inositol triphosphate-gated calcium release channels in the endoplasmic reticulum.
(1) Martin and Leese (1995) Mol. Reprod. Dev. 40, 436–443. (2) Pantaleon et al. (2001) Proc 32nd Annual SRB Conference, Gold Coast, Qld. A42.