bluuebunny/crossref_metadata_2025 huggingface dataset:
246. Developmental switches in male sex determination and spermiogenesis; importin-chromatin remodeling factor interaction
Authors: Jans D. A. , Ly-Huynh J. , Kaur G. , Efthymiadis A. , Loveland K. L.
Published: November 2010

Spermatogenesis, the complex process of generating haploid sperm capable of fertilising the female gamete, requires precisely scheduled transport into the nucleus of transcription factors and chromatin remodelling factors to implement changes in nuclear gene expression, as well as genome compaction during sperm formation (spermiogenesis). This transport is mediated by members of the importin (IMP) superfamily, which display distinct expression patterns in the rodent testis, consistent with the idea that they may carry specific cargo(es) at discrete stages of testis development. A key cargo during fetal testis development is the sex determining chromatin remodelling factor SRY, whose role in the nucleus in modulating the expression of male-specific genes such as SOX9 is critically dependent on the efficiency of its nuclear import by the specific transporter IMPbeta1; specific mutations in SRY that reduce binding by IMPbeta1 result in XY female Swyer syndrome individuals. A second cargo of significance is Cdyl (Chromodomain Y chromosome-like), involved in the histone H4 hyperacetylation which precedes the replacement of histones with protamines during spermiogenesis. We recently identified IMPalpha2, together with IMPbeta1, as Cdyl’s specific nuclear transporter. Using site-directed mutagenesis to perturb Cdyl recognition by IMPalpha2, IMP/Cdyl cotransfection approaches and quantitative confocal laser miscroscopic analysis, we established that the efficiency of Cdyl nuclear import is critical to its function in facilitating histone H4 acetylation, supporting the idea that one of the specific roles of IMPalpha2 is to localise Cdyl in the nucleus of elongating spermatids. Our findings are consistent with precisely scheduled, efficient IMP-mediated nuclear import of key chromatin remodelling factors being critical to testis development, reflecting an emerging paradigm for developmental processes in general.
247. BMP signalling in the induction of germline precursors from mouse embryonic stem cells in vitro
Authors: Dias V. L. , Young J. C. , Loveland K. L.
Published: November 2010

BMP signalling is critical for germline lineage establishment during mouse embryogenesis. To assess its importance in the induction of germline precursors in vitro, a mouse embryonic stem (ES) cell line harbouring an Oct4 promoter-EGFP reporter construct was used to derive embryoid body (EB) aggregates cultured in the absence or presence of combinations of BMP2, BMP4 and BMP8b for 3 to 10 days. At both day 5 and 10 of culture, clearly defined clusters of Oct4-EGFP were observed in EBs cultured with BMPs, while these clusters were minimal to absent in untreated EBs. Quantitative mRNA analysis of day 3 to day 10 EBs revealed a significant elevation in the expression of genes associated with primordial germ cell specification in EBs grown in the presence of BMPs. Moreover, a transient elevation of early germ cell markers Blimp1, Fragilis and Stella was detected in day 3–4 EBs cultured with BMPs, followed their decline by day 5. In contrast, levels of the pluripotency markers, Oct3/4 and Nanog, and the later germ cell markers, Dazl and Vasa, progressively increased from day 3 to day 5. Levels of TGFβ superfamily signalling components ALK2, Smad1 and Smad5 remained relatively constant during this period. Wholemount immunofluorescence of day 5 Oct4-EGFP EBs exposed to BMP4 demonstrated co-localisation of primordial germ cell markers Oct3/4, Stella, and the cell surface antigen SSEA-1 with EGFP+ clusters. These results demonstrate that signalling by BMP2 and 4 enhances germ cell marker expression within EBs and identifies the day 3 to 5 timeframe in EB differentiation as a critical window when putative germ cells are first specified in vitro.
248. Oxidative stress and DNA damage in human spermatozoa
Authors: De Iuliis G. N. , Finnie J. M. , Aitken R. J.
Published: November 2010

Unusually high levels of DNA damage in the male germ line are, unfortunately, characteristic of our species. A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the mechanisms responsible for creating this stress have not yet been elucidated. We have undertaken a detailed analysis of the ability of estrogens, electromagnetic radiation and xenobiotics including metal ions to trigger reactive oxygen species (ROS) production and/or DNA damage in human spermatozoa in vitro. This investigation was completed using a range of techniques validated for use in these highly specialised cells. DNA integrity was assessed using the Comet and TUNEL assays, oxidative DNA adducts were detected by an anti-8-oxo-dG assay and cross-linking adducts were characterised by mass spectrometry. Intracellular redox activity was monitored using dihydroethidium as the probe. Of the factors evaluated, catechol estrogens, certain transition metal ions, radio frequency electromagnetic radiation and heat were all capable of stimulating ROS production in human spermatozoa. The oxidative stress created by exposure to such factors lead to the induction of significant DNA damage. Generally, redox inert compounds including non-catechol estrogens and xenobiotics such as phthalate esters did not lead to ROS production or measurable DNA damage. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin. These findings raise fundamental questions about the importance of xenobiotics, environmental factors as well endogenous compounds in creating oxidative stress and DNA damage in the male germ line.
249. Regulated expression of KIT protein in juvenile and adult germ cells of the rodent testis
Authors: Mithraprabhu S. , Brown C. W. , Matzuk M. M. , Loveland K. L.
Published: November 2010

KIT receptor is an established marker of differentiating spermatogonia and its activation is required to trigger spermatogonial maturation. KIT mRNA, however, can be detected in undifferentiated spermatogonia in the absence of protein expression, as previously established by us in the irradiated adult rat testes [1]. This differential regulation of mRNA and protein is presumably modulated by either local hormone action or by cues from the adult testicular microenvironment. Endogenous regulatory factors known to stimulate KIT synthesis in juvenile male germ cells in vitro are bone morphogenetic protein 4 (BMP4) and retinoic acid (RA), while factors known to suppress KIT at the onset of spermatogenesis have not yet been identified. Activin A, implicated in KIT downregulation in a murine erythroleukemia cell line [2], is produced within the juvenile mammalian testis and influences activities of spermatogonia and Sertoli cells. We hypothesised that activin acts to repress KIT expression in spermatogonia and therefore modulate spermatogonial behaviour. Evidence for this was first derived from Sertoli and germ cell co-cultures of day 8 wild type mouse testes in which exogenous activin addition caused a dose-dependent reduction of KIT mRNA. Whole testes mRNA analyses of two activin transgenic mouse models, the newborn Inhba−/− (lacking activin A) and postnatal InhbaBK/BK (decreased bioactive activin), revealed a significant elevation in KIT expression relative to wild type littermates. In the postnatal day 7 InhbaBK/BK testes, an elevated proportion of differentiated spermatogonia, increased cell surface KIT protein levels, enhanced mRNA levels of a known downstream target of KIT signalling pathway, cyclind3 and a meiotic marker, Sycp3, were observed. These data provide the first comprehensive evidence for activin modulation of KIT expression at spermatogenesis onset, in germ cells of the juvenile testis. This finding is of fundamental importance to other KIT-dependent processes.
(1) Prabhu, S.M., et al. Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis. Reproduction, 2006. 131(3): p. 489–99.
(2) Hino, M., et al. Down-modulation of c-kit mRNA and protein expression by erythroid differentiation factor/activin A. FEBS Lett, 1995. 374(1): p. 69–71.
250. Importin α2-recognised testis cargoes; relevance to spermatogenesis
Authors: Ly-Huynh J. D. , Efthymiadis A. , Loveland K. L. , Jans D. A.
Published: November 2010

Spermatogenesis, the morphogenetic process of generating haploid sperm capable of fertilising the female gamete, requires transport into the nucleus of transcription and chromatin remodelling factors to implement genome compaction. This transport is mediated by members of the importin (IMP) superfamily, which exhibit distinct expression patterns during male germ cell development, consistent with the idea that each IMP carries specific cargo(es) at discrete stages of spermatogenesis. To identify IMP cargoes in the testis to help identify potential developmental switches critical to the spermatogenic process, we performed a yeast two-hybrid screen using full length IMPα2 as bait and an adult mouse testis library. Binding partners identified included coilin, Hop2, Chrp (cysteine and histidine rich protein), TAF9 (TATA Binding Protein Associated Factor 9) and Cdyl (Chromodomain Y chromosome-like). These proteins may be important in cell cycle regulation, homologous chromosome pairing and recombination, transcriptional regulation, splicing, mRNA storage or histone-protamine exchange during spermatogenesis. IMPα2 interaction with these cargoes was verified by binding assays, coimmunoprecipation and cotransfection approaches, while immunohistochemical staining of rodent testis sections indicated their co-expression with IMPα2 in specific testicular cell types. A key cargo in this context is the chromatin remodelling component, Cdyl where we could show that interaction with IMPα2 (and not IMPα4 or IMPα6 which are known to be expressed in the same testicular cell types as IMPα2) is necessary for its efficient nuclear import and function in facilitating histone H4 acetylation, the prerequisite step for incorporation of protamines during the final stages of spermatogenesis. A similar approach is currently in progress to demonstrate the importance of IMPα2 for the efficient nuclear transport of the other cargoes identified for their function(s) during spermatogenesis. IMPα2-mediated nuclear localisation of specific cargoes would thus seem to represent a critical developmental switch during spermatogenesis.
251. Production of donor-derived live lambs following testis germ cell transplantation
Authors: Herrid M. , Jackson M. , Suchowerska N. , Stockwell S. , Hutton K. , Davey R. , Olejnik J. , Hope S. , Hill J. R.
Published: November 2010

Testes germ cell transplantation in livestock has the potential for amplification of transgenic genotypes and for use as an alternative to artificial insemination. This study investigated a workable protocol for testis germ cell transplantation in sheep between animals of the same breed and different breeds. Testes of two groups of recipients at the stage of pre-pubertal (transition from gonocytes to spermatogonia, n = 2) or peri-pubertal (spermatogenesis initiated, n = 2) were treated with a single dose of 9 grey (Gy) or 15 Gy with a 6MV photon beam irradiation, respectively. In the first experiment, using pre-pubertal irradiated animals, testis germ cell transplantation between the same breed was performed at 16 weeks post irradiation. The left testes of recipient rams were injected with donor cells labelled with fluorescent dye PHK26, while the right testes were given unlabelled cells. The left testes of recipients were removed by castration after 2 weeks following transplantation to evaluate the location of the transferred cells, while the right testes were kept in place for long-term assessment of sperm output. In cryosections of the left testes, PKH26 positive cells were found both on the basement membrane as single cells or in the interstitial area. In the second experiment, animals irradiated at the peri-pubertal stage, received donor cells at 5 weeks post irradiation and animals were kept intact for semen production. For a period of two years after transplantation, semen samples were collected routinely from two groups of rams and analysed using microsatellite markers. Two recipients (50%) demonstrated the presence of donor DNA in their ejaculates. In order to investigate the fertility of the donor-origin sperm in recipient ejaculates, 99 ewes were artificially inseminated with semen from two positive rams. Four lambs (8%) have been identified as being sired by donor-derived sperm produced in the recipient ram that received a Merino to Merino transplantation, while no donor-derived offspring was obtained from the recipient with Border Leicester to Merino transplantation. This study represents the first report of the production of live progeny following testis germ cell transplantation in sheep.
252. Regulated nuclear import of TATA binding protein associated factor 9 (TAF9) in spermatogenesis
Authors: Prakash S. B. , Ly-Huynh J. D. , Loveland K. L. , Jans D. A.
Published: November 2010

Spermatogenesis, the differentiation process resulting in the production of haploid germ cells able to fertilise an oocyte, is driven by nuclear transcription factors, and changes in nuclear morphology and function itself. Signal-dependent transport into and out of the nucleus is mediated by members of the importin (IMP) superfamily of transporters, which we have previously shown to change dynamically in expression profile during spermatogenesis. One IMP family member, IMPα2, is expressed during mid to late phase spermatogenesis, implying that it may transport specific cargoes important for these stages, with a potential cargo of significance identified in a yeast 2-hybrid screen being TAF9 (TATA-Binding Protein Associated Factor 9). TAF9 is an integral part of transcription initiation for many genes, such as those with a Downstream Promoter Element in the core promoter and those with activators such as SOX18, p53, HSF1, NF-IL6 or NF-κB. In addition it participates in histone acetylation complexes which have been previously described to be important for the completion of spermatogenesis. Our preliminary data confirm the interaction of IMPα2 and TAF9 using a cotransfection approach. We have also shown that the expression of these proteins in the testis is correlated using both publically available Affymetrix data, and immunohistochemistry staining. TAF9, like IMPα2, is expressed in the nucleus of elongating spermatids of the adult rodent testis. Our data are thus consistent with the idea that specific nuclear import of TAF9 by IMPα2 may be a critical step in the later stages of spermatogenesis.
254. Evidences for a novel cAMP-phosphodiesterase expressed in the bovine ovarian follicle
Authors: Sasseville M. , Albuz F. K. , Richard F. J. , Gilchrist R. B.
Published: November 2010

3′5’-Cyclic adenosine monophosphate (cAMP) is an important second messenger in the mammalian ovarian follicle implicated in gonadotrophin signalling as well as oocyte meiotic arrest. Cyclic AMP-degrading phosphodiesterases (PDE) modulate cAMP levels in the ovarian follicle, but the specific PDE subtypes responsible for this degradation in the different cellular compartments within the bovine follicle remain unknown. The current dogma, established principally in rodent, presents PDE3A as the ‘oocyte PDE’, while PDE4D is the ‘granulosa/cumulus PDE’. Our PDE activity measurements suggested that a PDE3 (cilostamide-sensitive, 10µM) was representing 79% of the total cAMP-PDE activity in the bovine oocyte, in agreement with the dogma. However, our results suggested that PDE4 (rolipram-sensitive, 10µM) is representing only 19% of the cAMP-PDE activity in the cumulus cells, while 65% of the activity was due to PDE8 (IBMX-insensitive, 500µM), a result in direct opposition with the accepted PDE distribution in the ovarian follicle. Mural granulosa cells were displaying equal amounts of PDE4 (31%) and PDE8 (30%) cAMP-PDE activities. Interestingly, cAMP-PDE activities were not varying during the first 9 h of IVM in the bovine cumulus-oocyte complexes (COC), as seen in rat. COCs treated with an adenylyl cyclase stimulator (forkolin 100µM) in combinaison with the only known inhibitor for the PDE8 family, dipyridamole, are showing a dose-dependant increase of cAMP levels and a significant delay nuclear maturation, whereas a potent PDE4 inhibitor, rolipram (up to 100µM), was ineffective. This study provides the first insight into subtype-specific PDE cAMP degrading activities in the bovine ovarian follicle, especially around oocyte nuclear maturation. It demonstrates dramatic differential PDE subtype compartmentalisation between ovarian somatic cells and the germ cell, including the important contribution of a new PDE family member in the ovarian follicle, PDE8. PDE8 could be a novel pharmacological target to improve bovine oocyte IVM conditions and to increase developmental competence.
255. Hormonal manipulation on the phenotype of ArKO female mice
Authors: Liew S. H. , Drummond A. E. , Margaret J. E. , Findlay J. K.
Published: November 2010

Gonadotrophins and steroid hormones are vital in controlling the cyclical pattern of ovarian follicular development essential for fertility. Previous studies have shown that ArKO (aromatase knockout) female mice are infertile due to the absence of oestrogen, elevated levels of circulating gonadotrophins and testosterone and folliculogenic disruption. Therefore, the aim of this study was to determine the effects of E2 (oestradiol-17β) replacement, Acyline (GnRH antagonist) and Flutamide (anti-androgen) treatment on ArKO female mice. WT and ArKO female mice (C57B6/J129; 16 weeks old; n = 6–8/grp) were assigned into three main groups: group 1 - received either E2 (0.05 mg) pellet or placebo, group 2 - received either a single s.c. injection of acyline (1.5 mg/kg/week) or placebo and group 3 – received either flutamide (25 mg) pellet or placebo for 3 weeks. Mice were subjected to daily vaginal smears. The ovaries and uterine horns were collected and weighed. One ovary and the uterine horns were fixed in formalin for histological assessment, while the other ovary was snap frozen in Ultraspec solution for RNA isolation and gene expression studies. Serum was collected for hormone measurements. All female ArKO mice exhibited an abnormal cycle that alternated between diestrus and early oestrus. E2 replacement restored the oestrus cycle in ArKO female mice but acyline and flutamide treatment did not. Histologically, hemorrhagic cystic follicles were present in all placebo, acyline and flutamide treated ArKO ovaries, however, E2 replacement improved the ovarian and uterine phenotypes. E2 replacement and acyline treatment also led to a decrease in serum gonadotropin levels in ArKO mice. In summary, E2 replacement could reverse the abnormal reproductive phenotype of the ArKO female mice. This study suggests that the reproductive phenotype of the ArKO female mouse is due to the direct effect of oestrogen and not due to the elevated circulating levels of gonadotrophins and testosterone.
Supported by NH&MRC (Regkeys 241000, 338510 and 198705)
256. Ovarian lymphatic vascular development is hormonally regulated and Adamts1-dependent
Authors: Brown H. M. , Robker R. L. , Russell D. L.
Published: November 2010

The lymphatic system is important for return of extra-vascular fluid to the blood circulation, conductance of hormones and immune cell trafficking. Delicate hormonal control of fluid conductance during reproductive cycles is exemplified by the ovarian hyperstimulation syndrome, a dangerous condition of hypovolemia caused by fluid accumulation in the abdomen and reproductive tissues, in response to hormonal hyperstimulation. This study is the first to investigate the relationship between ovarian lymphatic development and follicle growth. Quantitative morphometric analysis of vessel size and number in mouse ovary revealed, for the first time, that the ovarian lymphatic vasculature develops postnatally and in synchrony with the induction of ovarian CYP19a1 (Aromatase); the time when secondary follicles become FSH-responsive and estrogenic. Mechanistically, we found that the FSH-analogue eCG mediates induction of lymphatic vascular endothelial growth factor Vegfd and the receptor Vegfr3 (Flt4) in granulosa cells. Importantly, stimulation with eCG also enhanced ovarian lymphatic vessel number and size. However, formation of ovarian lymphatics also required the matrix-remodelling protease Adamts1, since ovaries from Adamts1−/− mice failed to undergo normal lymphatic vascular development. Treatment of Adamts1 null mice with eCG significantly increased the number and size of ovarian lymphatic vessels, however, the vessels were still smaller and fewer in number than wildtypes. These combined results indicate that the ovarian lymphatic system develops in response to hormonal signals, which promote folliculogenesis, through induction of lymphangiogenic factors in granulosa cells; as well as involving Adamts1-dependent mechanisms. This study is the first demonstration of the novel principle of hormonal regulation of lymphangiogenesis in any tissue and suggests a requirement for functional lymphatics during normal folliculogenesis. In addition our results inform the elucidation of the tightly regulated processes that control fluid dynamics and immune cell surveillance within reproductive tissues.
259. Pluripotency genes in a marsupial, the tammar wallaby
Authors: Frankenberg S. , Pask A. J. , Renfree M. B.
Published: November 2010

Markers of pluripotency and early differentiation in the early embryo have been extensively characterised in eutherian species, most notably the mouse. By comparison, mechanisms controlling pluripotency and early lineage specification have received surprisingly little attention in marsupials, which represent the second major infraclass of mammals. Early marsupial embryogenesis exhibits overt morphological differences to that of eutherians, however the underlying developmental mechanisms may be conserved. In order to characterise early marsupial development at the molecular level, we have identified, cloned and analysed expression of orthologueues of several eutherian genes encoding transcription factors and signalling molecules involved in regulating pluripotency and early lineage specification. These genes include POU5F1 (OCT4), SOX2, NANOG, FGF4, FGFR2, CDX2, EOMES, TEAD4, GATA6 and KITL and are all expressed at early stages of development in the tammar. In addition, we have identified and cloned tammar POU2, which has orthologueues in non-mammalian vertebrates. POU2 is a paralogue of POU5F1 – a master regulator of pluripotency in eutherians. Genomic analysis indicates that POU5F1 arose via gene duplication of POU2 before the monotreme-therian divergence. Both genes have persisted in marsupials and monotremes, while POU2 was lost early during eutherian evolution. Similar expression profiles of tammar POU5F1 and POU2 in early embryos and gonadal tissues suggest possible overlapping roles in the maintenance of pluripotency.
260. Synergistic effects of cAMP modulating agents in pre-IVM and in IVM on bovine cumulus and oocyte functions
Authors: Albuz F. K. , Sasseville M. , Armstrong D. T. , Thompson J. G. , Gilchrist R. B.
Published: November 2010

Cyclic AMP plays a crucial role in oocyte maturation. It has been shown that prolonging spontaneous maturation by modulating oocyte cAMP levels during in vitro maturation (IVM) improves developmental outcomes. This study sought to assess the effect of inclusion of cAMP modulators during a pre-IVM phase and during IVM on cumulus cell (CC) and oocyte function. Bovine cumulus-oocyte complexes (COCs) were precultured for 2 h in medium supplemented with/without cAMP modulators: 0.5 mM IBMX and 0.1 mM forskolin (FK). Cyclic AMP in control COCs dropped significantly from 15 ± 4 to 2 ± 1 fmol/COC within 30 min, whereas FK+IBMX during pre-IVM increased levels to 180 ± 19 fmol/COC, which had notable persistent effects on several COC functions throughout IVM. Cyclic AMP modulators delayed oocyte GV progression; after 2 h of pre-IVM, 67 ± 5% were at GVII stage compared (P < 0.0001) to 12 ± 3% in controls, 66 ± 5% of which had progressed to GVIII. This was associated with a significantly higher level of oocyte-CC gap junction mediated communication (1000 ± 148 v. 340 ± 73 units). We next assessed the interaction of cAMP modulators in pre-IVM and IVM (20uM cilostamide) phases. After 9 h of IVM, significantly more oocytes were arrested at GV stage when modulators were present in both pre-IVM and IVM phases, compared with in IVM alone (96 ± 1 v. 74 ± 2%, P < 0.05). Moreover, progression to MII was delayed by modulators in pre-IVM and IVM (24 ± 1, 75 ± 2, 92 ± 1% MII at 20, 24 and 28 h, respectively), compared with in IVM alone (78 ± 1, 95 ± 1, 98 ± 2% MII). After 24 h of IVM, intra-oocyte cAMP levels remained 15-fold higher (P < 0.05) with modulators in both phases compared with in IVM alone. These results show that the efficacy of cAMP modulators during IVM is substantially improved by management of COC cAMP levels during the pre-IVM phase. Such an approach has profound effects on CC and oocyte function, including increasing oocyte-CC communication and delaying meiotic resumption.
263. Substitution of skim milk with bovine serum albumin in a stallion semen diluent
Authors: Gibb Z. , Grupen C. G. , Morris L. H. A. , Evans G. , Maxwell W. M. C.
Published: November 2010

Skim milk has long been utilised as a source of protective proteins in stallion semen diluents. However, skim milk is also thought to contain components that are toxic to sperm and reduces the clarity of sperm suspensions, which impedes sperm assessments. This may also reduce the effectiveness of staining procedures used to process sperm for flow cytometric sex-sorting. The aim of this study was to ascertain the optimal concentration of bovine serum albumin (BSA) to replace skim milk in a traditional stallion semen diluent, Kenney's Modified Tyrode's (KMT) Medium1, for handling and processing stallion sperm before flow cytometric sex-sorting. Two ejaculates were collected from each of three pony stallions. Each ejaculate was divided into five aliquots and diluted in either KMT with skim milk or KMT supplemented with 0, 0.25, 0.5 or 1% BSA. Diluted samples were further divided into two aliquots and either stored at 15°C for 18 h before incubation and assessment, or incubated and assessed immediately upon arrival. Samples were incubated at 34°C and evaluated at 0, 45 and 90 min for objective motility and acrosome integrity. No interactions were observed between any treatments over time. There was a lower percentage of intact and a higher percentage of detached acrosomes for sperm incubated in KMT containing 0% BSA than all other treatments. A greater proportion of sperm incubated in KMT with skim milk had partial acrosome damage compared with other treatments. There was no difference in % total motility for sperm incubated in KMT with skim milk, and KMT containing 0.5 and 1% BSA (Table 1). These results indicate that BSA may be suitable as an alternative protein source in stallion semen diluents. Further studies are required to compare sex-sorting rates and sperm quality after sex-sorting, incubation and staining in skim milk compared with BSA-based media.
(1) Padilla, A W and Foote, R H 1991, ‘Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa’, Journal of Animal Science, vol. 69, no. 8, pp. 3308–331
264. Primordial germ cell specification in a marsupial, the tammar wallaby
Authors: Hickford D. , Pask A. , Shaw G. , Renfree M. B.
Published: November 2010

Primordial germ cells (PGCs) are the precursors of the gametes. In the mouse, PGCs are specified within the proximal epiblast in response to signals from the extraembryonic membranes during early gastrulation. Epiblast cells competent to form PGCs express Ifitm3. A subset of these cells then express Blimp1, a marker of PGC precursors. Once lineage-restricted, PGCs express Stella. Germ cells entering the gonads express VASA protein, which is a component of the germ plasm in animals in which germ cells are specified by the inheritance of maternal determinatives. Almost all of the research on mammalian PGC specification has used the mouse as a model and it is tacitly assumed that findings in the mouse will apply to mammals in general. We are using the tammar wallaby as a marsupial model for PGC specification. Eutherians and marsupials diverged 125–148 million years ago, so comparisons between the two will provide insights into the evolution of the control of mammalian PGC specification. There are IFITM clusters in both the human (chromosome 11) and mouse (chromosome 7). In the mouse, IFITM1, 2 and 3 are expressed in PGCs, whereas IFITM4 and 5 are not (1). Only one IFITM member, IFITM5, is annotated in the opossum Ensemble database. We have cloned one tammar IFITM member and identified at least one other putative member in the tammar trace archive database. We have also cloned tammar BLIMP1 and VASA, both of which show high sequence conservation with other mammals. RT–PCR profiles for both genes during tammar gastrulation are similar to those for the mouse. In contrast, no marsupial STELLA orthologueue has been identified in either the opossum or tammar genomes. These findings suggest that some but not all of the signals and mechanisms involved in eutherian PGC specification are also applicable to marsupials.
(1) Lange UC, Saitou M, Western P, Barton SC and Surani MA (2003) BMC Dev. Biol. Epub 2003 Mar 19
265. Increased FSH activity increases primordial follicle reserve and enhances preovulatory follicle survival in transgenic FSH female mice
Authors: McTavish K. J. , Walters K. A. , Handelsman D. J. , Allan C. M.
Published: November 2010

The mammalian female reproductive lifespan is determined by the depletion rate of the finite ovarian follicle reserve established before or shortly after birth. Follicle formation, initiation and early growth are thought to be independent of follicle-stimulating hormone (FSH), whereas antral follicle development requires FSH stimulation. Rising serum FSH is one of the earliest signs of reproductive ageing in women, coinciding with declining fecundity and an accelerated decline in remaining follicle reserves, but whether or not increased FSH plays a direct or feed-forward role in accelerating reproductive ageing remains undetermined. We previously described transgenic (Tg) mice with rising serum human FSH that produced larger litter sizes <20 weeks of age, then rapidly declining litter size from 20–40 weeks old (wo) culminating in premature infertility1. Despite declining fertility, ageing TgFSH females maintained ovulation rates ~3-fold higher than wt females. Follicle quantitation revealed that ovarian antral follicle numbers at diestrus were equivalent in 26 wo TgFSH and wt females. The elevated ovulation rates in TgFSH females may reflect increased preovulatory follicle survival during proestrus, as ~70% of large antral follicles go on to ovulate in TgFSH females, compared with only 30% in wt females. In contrast to the view that higher FSH may increase follicle development and consequently accelerate follicle depletion, examination of follicle reserve revealed that subfertile or infertile 26–52 wo TgFSH females exhibited increased total ovarian primordial follicle numbers (60%, P < 0.05) with no significant change in primary follicle numbers compared with age-matched wt females. Therefore, increased FSH activity appeared to act as a survival factor for primordial follicles. Our current analysis of increased FSH actions in female mice suggests that FSH may enhance the survival of both early (primordial) and late (preovulatory) follicle populations.
(1) McTavish KJ et al. Endocrinology. 2007 Sep;148(9):4432–9.
266. Loss of betaglycan expression contributes to malignant properties of human granulosa tumour cells
Authors: Bilandzic M. , Chu S. , Wang Y. , Farnworth P. G. , Escalona R. , Fuller P. J. , Findlay J. K. , Stenvers K. L.
Published: November 2010

Betaglycan is a type III TGF-β receptor that binds to both inhibin and TGF-β with high affinity and determines cellular sensitivity to these ligands. Previous studies have suggested that betaglycan acts as a tumour suppressor in certain human epithelial cancers. However, the roles of betaglycan in ovarian granulosa cell tumours (GCTs) are poorly understood. The objective of this study was to determine whether human GCTs exhibit betaglycan expression, and if so, what impact this receptor has on tumour biology. Real-time PCR was used to quantify betaglycan transcripts in human GCTs (n = 18) and normal premenopausal ovaries (n = 11). This analysis established that GCTs exhibited a significant 2-fold reduction in mean betaglycan mRNA levels as compared with the normal ovary (P < 0.05). Similarly, two human GCT cell lines, the KGN and COV434, exhibited low betaglycan expression and poor responsiveness to TGF-β and inhibin in luciferase reporter assays. Stable transfection of GCT cell lines with a wildtype betaglycan (WT-BG) expression plasmid conveyed ligand responsiveness. FACS analysis was used to examine cell cycle progression and cell death in the GCT cell lines. This analysis revealed that WT-BG had no effect on the number of cells cycling or undergoing apoptosis. However, WT-BG significantly increased the adhesion of COV434 (P < 0.05) and KGN (P < 0.0001) cells to collagen IV and fibronectin, decreased cellular invasion through Matrigel COV434 (50%) and KGN (75%), and inhibited wound healing COV434 (70%) and KGN (80%). Collectively, the data establish that betaglycan is an important regulator of granulosa cell biology and suggest that a deficiency in betaglycan contributes to the pathogenesis of granulosa cell cancer. The work further suggests a role for betaglycan in the prevention of tumour invasion, possibly by increasing the adhesion of granulosa cells to matrix components. Supported by: the NHMRC of Australia (RegKeys 338516; 241000; 441101; 388904).
267. A role for transforming growth factor-β 1 (TGF-β1) during the establishment of folliculogenesis
Authors: Kuyznierewicz I. , Findlay J. K. , Drummond A. E.
Published: November 2010

A group of structurally related proteins, known as the transforming growth factor-β (TGF- β) superfamily, have been implicated in the local regulation of ovarian function. It is unclear what role TGF-β1–3 plays in folliculogenesis during the period after birth in the rat. We investigated whether the TGF-β ligands and their receptors were present during this period of development and the effects of TGF-β1 on granulosa cell function (proliferation, apoptosis, steroidogenesis). Ovaries from rats 4, 8 and 12 days of age were isolated and RNA extracted and reverse transcribed for real-time PCR. The expression of the TGF-β ligands and TGFβRI and TGFβRII were measured. Granulosa cells isolated from DES treated immature rats were treated with FSH (100ng/mL) and TGF-β1 (1 or 10ng/mL) for 2hr, n = 4 replicates. The RNA was extracted and prepared for RT–PCR. The expression of cyclin D2, FKHR, SCC, 3βHSD and StAR were measured. TGFβRI and TGFβRII proteins were localised to postnatal rat ovary by immunohistochemistry. TGF-β1–3, TGFβRI and TGFβRII were present in rat ovaries as early as 4 days after birth. Expression of TGF-β1 mRNA increased 2-fold between day 4 and 12. TGF-β2 and TGF-β3 mRNAs declined between day 4 and 8 and remained low at day 12. The type I and II TGF-β receptors were differentially regulated with TGFβRI expression high at day 4, declining at day 8. In contrast, TGFβRII appeared to be ubiquitously expressed. Cyclin D2 mRNA expression was enhanced in the presence of both TGF-β1 and FSH, whereas FKHR mRNA expression declined. TGF-β1 had no impact on the steroidogenic mRNAs. TGFβRI and TGFβRII proteins were localised to the cytoplasm of oocytes, granulosa cells and theca cells. These studies indicate that TGF-β1 can exert effects on ovarian folliculogenesis as it is established during the postnatal period. Proliferation and apoptosis appear to be targets of TGF-β1 action.
Supported by the NHMRC of Australia (Regkeys 241000, 198705, 441101 & 465415)
268. Suppressors of cytokine signalling (SOCS): roles in ovarian follicle activation
Authors: Keightley R. , McLaughlin E. , Roman S. D. , Robker R. L. , Russell D. L.
Published: November 2010

Oocytes are sequestered in primordial follicles before birth and remain quiescent in the ovary for decades, until recruited into the growing pool throughout the reproductive years. Therefore activation of follicle growth is a major biological checkpoint that controls female reproductive potential. However we are only just beginning to elucidate the cellular mechanisms required, for either maintenance of the quiescent primordial pool, or initiation of follicle growth. Analysis of microarray data derived from neonatal mouse ovaries indicated that members of the Suppressors of Cytokine Signalling SOCS family of proteins may play pivotal roles in folliculogenesis. We undertook a detailed analysis of gene and protein expression patterns of the eight members of the SOCS family, namely CIS and SOCS1–7, within adult and neonatal mouse ovaries. Quantitative real time PCR and immunohistochemistry was performed to determine mRNA levels and cellular localisation in the ovaries of cycling and new born animals. SOCS proteins were expressed largely within the oocytes of developing follicles and in the granulosa cells of the larger preovulatory follicles. Expression of SOCS4 in the granulosa cells and SOCS5 within the oocyte was coincident with the activation of oocyte growth and the differentiation of squamous pregranulosa to cuboidal granulosa cells. Our investigation has identified a role for the SOCS family proteins within the ovary and SOCS4 and SOCS5 as major regulators of cytokine signalling pathways in follicle activation and development.
269. CD44 signaling in mouse ovulatory cumulus - oocyte complexes
Authors: Alvino E. R. , Robker R. L. , Russell D. L.
Published: November 2010

Oocytes grow and develop within ovarian follicles, providing a nurturing environment before their release (ovulation) into the oviduct for fertilisation. For ovulation to occur the ovarian follicle responds to LH from the pituitary, leading to a cascade of regulated gene expression and formation of the hyaluronan rich cumulus matrix around the oocyte. This COC (cumulus oocyte complex) matrix is composed of high concentrations of the ECM glycosaminoglycan hyaluronan (HA) cross-linked by several HA-binding proteins. Several of the COC matrix components are essential for ovulation, since null gene mutations in mice lead to ovulation defects. Mechanisms by which the COC matrix controls ovulation however, are unknown. We have investigated cellular signalling and cellular phenotypes that occur as part of the formation of the COC matrix. The transmembrane HA receptor CD44 was significantly upregulated in cumulus cells from 6 h after hCG (LH analogue) treatment (9.8 ± 1.5 fold) until ovulation at 12 h post hCG (11.8 ± 2.9-fold). In many cell types CD44 activates the intracellular Rho-family GTPase Rac1 and its activator, the guanine exchange factor Tiam1, pleiotropic regulators of cytoskeletal function, cell-cell adhesion and migration. We found both Rac1 and Tiam1 were strongly detected in cumulus cells, but not regulated by hCG. These observations show that at the time of ovulation a macro-molecular complex associated with cell motility is assembled through the extracellular interaction of the COC matrix and cell surface proteins. We investigated the migratory and invasive activity of COCs from hormonally stimulated mice. Migration of cumulus cells from hCG treated mice was significantly increased compared with untreated COCs. Furthermore the hCG-stimulated cumulus cells were able to invade a range of ECM substrates including collagen and laminin. These results suggest the cumulus cells in the expanded COC transition to a motile cell phenotype that may play a key role in promoting ovulation.
270. TGF-β and ovarian follicle development
Authors: Rosairo D. A. , Findlay J. K. , Drummond A. E.
Published: November 2010

The role TGF-β plays in ovarian follicular growth and differentiation was investigated using a ‘physiological' culture system. TGF- β ligand and receptors are present in the rat ovary from 4 days after birth. Therefore we established organ cultures with these ovaries in order to assess the potential impact of TGF- β1 on follicle growth and transition from the primordial through to the primary and preantral stages of development. Whole ovaries were isolated and cultured for 10 days on floating filters with the addition of supplemented DMEM/Hams F-12 media and either FSH (100ng/mL), TGF- β1 (10ng/mL), or a combination of the two. Media as well as treatments were refreshed every second day. At the end of the culture period, ovaries were fixed in 10% formalin, embedded in paraffin and sectioned at 5µm. Sections were used for morphological assessment and ovarian follicle counting with three serial sections mounted/slide and every alternate slide used for 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 labelling (TUNEL) via the ApopTag® Peroxidase in situ apoptosis detection kit. Results gathered from this study show preantral follicle numbers declined significantly when treated with the combination of FSH and TGF- β1, consistent with our morphological appraisal of atresia where the combined treatment appeared to produce more apoptotic follicles than healthy follicles, suggesting an increase in atretic primary and preantral follicles. These preliminary findings suggest an inhibitory role for TGF- β1 in the presence of FSH, resulting in fewer follicles making the transition from the primary to the preantral stage. Further studies are required to test the effects of other TGF-β superfamily members on follicle transition in vitro.
Supported by the NHMRC of Australia (Regkeys 241000, 441101, 465415, 198705)
271. Effect of season on sow ovarian morphology
Authors: Bertoldo M. , Holyoake P. K. , Evans G. , Grupen C. G.
Published: November 2010

Reduced farrowing rate caused by embryonic mortality is a manifestation of seasonal infertility in pigs. The ability of the oocyte to mature, be fertilised and sustain embryonic development is acquired gradually by the oocyte throughout folliculogenesis. This study was undertaken to determine if seasonal differences in ovarian morphology are associated with reduced reproductive performance displayed during seasonal infertility. Sows culled after weaning were sourced from two genetically distinct herds (Farms A and B). Pairs of ovaries were collected from sows 4 days post-weaning during winter (n = 131) and summer (n = 275). Ovarian weight (Farm A only) and the numbers of small (3–4 mm) and large (5–8 mm) follicles were assessed (Farms A and B). Data did not follow normality and was analysed using the Mann–Whitney test. Mean ovarian weight per sow during winter (20.0 ± 1.3 g) was significantly heavier than that during summer (15.3 ± 0.8 g; P < 0.05). Farm A ovaries had a greater total number of antral follicles in winter compared with summer, and a greater number of antral follicles in winter compared with Farm B ovaries (P < 0.05). In Farm A ovaries, the proportion of follicles that were large was greater in summer compared with winter (78% v. 66%; P < 0.05), but the follicular distribution did not change with season in Farm B ovaries. While the findings demonstrate that ovarian weight was greater in winter compared with summer, they suggest that this difference was not the result of changes in the number or distribution of surface antral follicles. The difference in ovarian weight is possibly due to differences in ovarian tissue resulting from regressed corpora lutea. Further studies are being undertaken to assess the effect of season on oocyte developmental competence and the steroid content of follicular fluid isolated from small and large follicles.
272. Signalling pathways involved in mouse GDF9 and BMP15 stimulated thymidine uptake by rat granulosa cells
Authors: Reader K. L. , McIntosh C. J. , Juengel J. L.
Published: November 2010

The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth and development. Understanding the molecular mechanisms of these factors could assist with the development of future products for fertility control. Thymidine uptake by rat granulosa cells is stimulated cooperatively by GDF9 and BMP15. Inhibitors of the activin receptor-like kinase (ALK) 4,5,7 and the nuclear factor kappaB (NFKB) second messenger pathways block ovine GDF9 and BMP15 stimulated thymidine incorporation. The ALK 4,5,7 receptor pathway is known to be essential for the cooperative effects of mouse (m)GDF9 and mBMP15 on thymidine incorporation but the role of other pathways has yet to be determined, which was the focus of this study. Inhibitors of NFKB (Sn50; 10µg/mL), ALK 2,3,6 receptor (Dorsomorphin; 1µM), p38 mitogen-activated protein kinase (p38 MAPK; SB239063; 5 µM) and c-Jun-N-terminal kinase (JNK; TAT-TI-JIP153–163; 5 µM) pathways were each cultured with recombinant mGDF9 (25 ng/mL) and mBMP15 (6 ng/mL) in a rat granulosa cell [3H]-thymidine bioassay. The p38 MAPK inhibitor caused partial inhibition of thymidine uptake but this appeared to be non-specific as a similar level of suppression was observed in the control cultures. Neither the ALK 2,3,6 receptor nor the NFKB pathway inhibitors had any effect on mGDF9 and mBMP15 stimulated thymidine uptake. The JNK inhibitor showed a 1.7-fold increase in stimulation above the mGDF9 and mBMP15 effect (P < 0.01) but a similar stimulation was also observed in some controls. This differs from the results observed with ovine GDF9 and BMP15 where thymidine uptake was completely blocked by the NFKB inhibitor and the JNK inhibitor had no effect. In conclusion, the molecular mechanisms of GDF9 and BMP15 function are dependent on the species of origin of the growth factor and therefore caution is needed when extrapolating findings from one species to another.