bluuebunny/crossref_metadata_2025 huggingface dataset:
219. Prevalence of sperm chromatin instability amongst bulls in a subtropical environment: a preliminary investigation
Authors: Hengstberger K. J. , Sester D. , Tutt D. , Holroyd R. G. , Fordyce G. , Johnston S. D. , D'Occhio M. J.
Published: November 2010

During mammalian spermiogenesis nucleosomal histone proteins bound to DNA are replaced by protamines. The integrity and stability of the protamine-DNA association in sperm can be measured using the sperm chromatin structure assay (SCSA). The recent emergence of compelling information from assisted reproduction in humans of a relationship between sperm chromatin stability and the establishment and maintenance of pregnancy has led to considerable interest in production and recreational animals.1 The aim in the present study was to determine the incidence of sperm chromatin instability amongst bulls in a subtropical region of northern Australia, as a first step in evaluating whether sperm chromatin instability is a contributing factor to reproductive wastage in cattle. Semen was obtained from 565 Bos indicus and Bos indicus x taurus bulls from northern and central Queensland aged between 20months and 10years. Samples were subjected to standard semen evaluation2 and aliquots stored in liquid nitrogen until chromatin integrity was determined using the SCSA.3 Samples exposed to the SCSA for 0.5min revealed 4.9% of bulls had a DFI >27% and this increased to 11.5% of bulls with DFI >27% when samples were exposed to the SCSA for 5 min. DFI was significantly correlated with sperm density, mass activity, motility and morphology. Location appeared to have a greater influence on DFI than genotype. Preliminary data from a small sample of bulls would suggest that a relatively high DFI can be repeatable for individual bulls. These findings indicate that sperm chromatin instability occurs in bulls in northern Australia although the prevalence might be considered to be relatively low. The relationship of sperm chromatin instability to the contribution of bulls to embryonic mortality requires further study and likewise the impact on reproductive wastage remains to be determined.
(1)Boe-Hansen GB et al. (2005). Theriogenology 63, 1789.(2)Fitzpatrick LA et al. (2002). Anim. Reprod. Sci. 71, 39.(3)Evenson D and Jost L (2000). Methods Cell Sci. 22, 169.
221. SPRASA, a sperm protein with a post-fertilization function?
Authors: Wagner A. , Shelling A. , Chamley L.
Published: November 2010

Objectives: SPRASA is a highly conserved sperm protein that is localised to the inner acrosome membrane, and shows high homology to the alpha-lactalbumin/C-type lysozyme family.1,2 We have previously shown that SPRASA is the antigen for antisperm antibodies in some infertile patients.1 To date, the function of SPRASA is unknown but in vitro, antibodies reactive with SPRASA inhibit sperm-oocyte binding in a zona-free hamster oocyte binding assay2. Based on this preliminary data, we postulated that SPRASA plays an integral role in fertilization, and that binding of antibodies to SPRASA may inhibit its function leading to infertility. In this study we investigated the effect of inhibiting bovine SPRASA, in vitro, on fertilization and embryonic development. Methods: Viable, motile sperm was prepared from frozen-thawed bovine sperm by swim up. Bovine oocytes were obtained from slaughter-house killed animals and matured in vitro. Three different treatments were investigated. (1) Sperm were incubated for an hour with a SPRASA-reactive antiserum, washed and used to fertilize 100 oocytes in insemination droplets. (2) One hundred in vitro matured oocytes were incubated for an hour with the SPRASA-reactive antiserum, washed, and fertilized with untreated swim-up sperm in insemination droplets. (3) Sperm and oocytes (n = 100) were coincubated with the SPRASA-reactive antiserum in insemination droplets. Controls consisted of similar numbers of sperm and/or oocytes incubated with an irrelevant antiserum. Results: There was a significant reduction in the number of embryos that reached morula (P = 0.03) or blastocyst (P=0.01) when oocytes were pre-treated with the SPRASA antiserum (treatment 2) or when sperm and oocytes were co-incubated with the antiserum (P=0.05 morula; P=0.01 blastocyst; treatment 3). However, there was no significant difference in the rates of embryos reaching earlier developmental stages in any of the treatment groups. Conclusion: These data suggest that SPRASA may be expressed by oocytes and/or preimplantation embryos.
(1)Chiu WW, Erikson EK, Sole CA, Shelling AN, Chamley LW. (2004). SPRASA, a novel sperm protein involved in immune-mediated infertility. Human Reproduction 19(2), 243–249.(2)Mandal A, Klotz KL, Shetty J, Jayes FL, Wolkowicz MJ, Bolling LC, Coonrod SA, Black MB, Diekman AB, Haystead TA, Flickinger CJ, Herr JC. (2003). SLLP1, a unique, intra-acrosomal, non-bacteriolytic, c lysozyme-like protein of human spermatozoa. Biology of Reproduction 68(5), 1525–1537.
222. Sperm morphology within the testis and cauda epididymis of a Tasmanian devil (Sarcophilus harrisii)
Authors: Matson P. L. , Eden P. A. , Vitali S. D. , Lawrence C. A. , Robertson H. M.
Published: November 2010

A male Tasmanian devil, aged 6.5 years, was euthanased with intravenous pentobarbitone overdose due to the diagnosis of squamous cell carcinoma. The testes and cauda epididymes were removed immediately after euthanasia and placed into TALP-HEPES medium containing 3% BSA and dissected. Smears of the isolated sperm were made, air-dried and stained using Diff-Quik. Sperm were classified using a modification of a system originally applied to the brush-tailed possum1 as follows:
(a) Immature I – head is perpendicular or greater than 45° to the tail.(b) Immature II – head forms an acute angle of 45° or less with the tail.(c) Immature III – head and tail lie in a straight line but the sperm head still has a partly expanded acrosome.(d) Mature – the head is aligned with and has a diameter similar to the tail.
No motility was seen in the testicular sperm, but >60% of sperm from the cauda epididymes were motile. The stained preparations showed a significant difference in the distribution of sperm between the testis and epididymis (χ2 = 150.45, df = 3, P < 0.001), with sperm within the testis being skewed towards the earlier stages of development whereas the epididymis was skewed towards the later stages of development (Table 1).
These results confirm the similarity with several other marsupials in that sperm are formed in the testis with the head at right angles to the tail but rotation of the head to become aligned with the tail is completed during epididymal transport. Work is now required to identify optimum conditions for the culture, cryopreservation and insemination of sperm from this species.
(1)Cummins JM. (1976). Epididymal maturation of spermatozoa in the marsupial Trichsurus vulpecula: changes in motility and gross morphology. Aust. J. Zool. 24, 499–511.
223. The epididymis secretes proteins involved in sperm competition: evidence from the echidna
Authors: Dacheux J. L. , Belghazi M. , Dacheux F. , Nixon B. , Jones R. C.
Published: November 2010

Although there is general recognition that the mammalian epididymis is unique among the vertebrates and is essential for male fertility, there is relatively little understanding of its role in the process of reproduction. It has been suggested that it plays an important role in the competition between males to achieve paternity (sperm competition) by accumulating and storing sperm.1 We now provide evidence that specific protein synthesized and secreted by the epididymis of the echidna plays a major role in sperm competition by binding spermatozoa into bundles of about a 100 sperm and consequently greatly enhancing their rate of motility, particularly in viscous medium. The epididymis of the echidna is structurally differentiated into a large caput epididymidis that is structurally similar to the ‘initial segment’ of other mammals, and a small cauda epididymidis. Using radiolabelling and 2-D electrophoresis we have shown that the caput epididymidis synthesises and secretes a similar pattern of proteins along most of its length where sperm are not associated with one another. The sperm form into bundles as they enter the cauda epididymidis where the pattern of protein secretion changes considerably, being characterised by a new band of about 80 kDa in reduced gels. Electron microscopy of the bundles shows electron dense material binding the sperm together in the bundles. The bundles persist for more than an hour after sperm are released into physiological media.
(1)Jones, RC (1998). Evolution of the vertebrate epididymis. J. Reprod. Fertil. Suppl. 53, 163–182.
224. Platelet derived growth factors and receptors in the rat ovary contribute towards preantral follicle growth
Authors: Sleer L. S. , Taylor C. C.
Published: November 2010

In this study, the family of platelet derived growth factors (PDGF) and receptors were identified and characterized in the rat ovary and a role in contributing towards growth of preantral follicles was revealed. Real-time polymerase chain reaction revealed the presence of mRNA for all platelet derived growth factors (PDGF-A, PDGF-B, PDGF-C and PDGF-D) and receptors (PDGF-Rα and PDGF-Rβ). In situ hybridization identified oocytes of primordial/primary follicles and cells of the theca layer as a source of PDGF-B, PDGF-C and PDGF-D mRNA. Protein expression was explored through immunohistochemistry. In rats aged days 0 and 4, PDGF-Rα, PDGF-A and PDGF-C immunoreactivity was observed within oocyte clusters, and PDGF-Rβ and PDGF-B immunoreactivity in cells surrounding oocyte clusters. In primordial follicles, PDGF-Rβ and PDGF-C was observed in the oocyte, and PDGF-Rα and A in the either the oocyte or pregranulosa cells. In primary follicles, PDGF-A, PDGF-C, PDGF-Rα and PDGF-Rβ are expressed in the oocyte. PDGF-Rβ is also expressed in cells surrounding primordial and primary follicles, possibly the precursors to theca cells. In secondary and antral follicles, all four PDGF isoforms and both receptors are expressed in either theca or vascular cells of the theca layer, and PDGF-Rα and A are also expressed in some granulosa cells in rats aged day 20 and older. A role in preantral follicle growth was identified by in vitro culture of preantral follicles. Preantral follicles cultured in serum free medium increased in diameter by 11.0 ± 1.57% over 5 days. Addition of PDGF-AA, PDGF-AB or PDGF-BB to the medium resulted in increases in follicle diameter after 5 days of 18.32 ± 2.18%, 17.72 ± 2.3% and 17.6 ± 1.81%, respectively, representing a significant increase over control diameters. In summary, this study has identified and characterized the presence and localization of all members of the family of platelet derived growth factors and receptors in the rat ovary and revealed a role for these growth factors in positively influencing early follicle growth.
225. Early ovarian follicle dysgenesis in a disintegrin and metalloproteinase with thrombospondin motifs type 1 (ADAMTS-1) null mice
Authors: Brown H. M. , Pritchard M. , Russell D. L.
Published: November 2010

ADAMTS-1 is a multi-domain, multi-functional matrix metalloproteinase expressed in the mouse ovary during folliculogenesis and ovulation. Adult ADAMTS-1 null female mice are infertile; however, the exact mechanism responsible for their reproductive failure is yet to be explained.
ADAMTS-1 null ovaries prior to and following ovulation were examined and a defect in ovarian follicle development was identified. A classification system was devised and used to identify morphological differences between normal and ADAMTS-1 null ovaries. The phenotype indicated a progressive loss of follicle structural integrity ranging from a slight loss of shape, to full structural dysgenesis. As a result, large numbers of oocytes with healthy appearance were found with no surrounding follicle structure. The numbers of preantral follicles was not altered, but type 6 (early antral) follicles in the ADAMTS-1 null ovaries were significantly reduced (P < 0.05) when compared to wild-type ovaries suggesting initiation of follicular degeneration coincident with antrum formation. There was also a significant decrease (P < 0.05) in the number of periovulatory follicles (type 7 and 8) in the ADAMTS-1 null ovaries. This suggests that late folliculogenesis, in the period of rapid growth and expansion is disrupted when ADAMTS-1 is not present and results in fewer follicles available for ovulation. Further, we have demonstrated that the active form of ADAMTS-1 is present in the thecal-granulosa boundary of the ovarian follicle suggesting a role in extracellular matrix remodeling at this boundary during follicle growth. Analyses of the basement membrane at this boundary both in growing and ovulating follicles indicate that ECM remodeling in this region is indeed disrupted.
These data demonstrate that remodeling of surrounding structural matrix is crucial to follicle growth and structural integrity. Functional ADAMTS-1 is important for matrix remodeling during the growth of the follicle, particularly during antrum formation.
226. Expression of matrix metalloproteinases in bovine thecal cells
Authors: Harland L. , Irving-Rodgers H. F. , Morris S. E. , Rodgers R. J.
Published: November 2010

As follicles grow the thecal layers expand. It is likely that extracellular matrix is remodelled in this process and possibly by matrix metalloproteinases (MMPs). A promising candidate to regulate MMPs is insulin-like factor 3 (INSL3). It is produced by thecal cells, its receptor, LGR8, is expressed in the theca interna (unpublished) and a related molecule, relaxin, regulates turnover of matrix in a number of tissues. For this reason we sort to examine the role of INSL3 in matrix turnover. However, in all thecal cell culture systems examined LGR8 receptors appear to be down regulated within 24 h. We therefore examined the effects of second messenger pathway activators. Thecal and granulosa cells were isolated and cultured and the levels of RNA for MMP2 and 9 quantitated by RT-PCR. MMP2 mRNA levels in thecal tissues were >10 fold higher than in granulosa cells (n = 19 follicles >10 mm). MMP2 levels were substantially greater than MMP9. At 12 h phorbol ester (100 nM phorbol 12,13-didecanoate) increased thecal expression of MMP 9 mRNA levels 11.5 fold (P < 0.001) and at 48 h MMP2 mRNA was increased 5 fold (P < 0.01). Pieces of whole follicle wall [follicles <5 mm in diameter, classified as healthy (n = 12) or atretic (n = 6)] were cultured in serum free media. Expression of the steroidogenic enzymes 17β HSD and P450scc but not 3β HSD were detected by immunohistochemistry even after 10 days. MMP activity on day 2 was analysed by gelatin zymography. Treatment with phorbol ester increased active MMP9 19 fold (P < 0.001). Treatment of thecal cells or follicle walls with 1 mM dibutyryl cAMP induced additional MMP activities at sizes of 110 and 122kDa. No effects on MMP2 activity were observed. In conclusion whilst we do not know the ligand inducers of the synthesis and activator of MMPs in thecal cells they can be regulated. Hence MMPs are candidates for remodelling the extracellular matrix of thecal layers.
229. Effect of follicular status on sheep embryo yields is mediated by changes in the preovulatory LH surge
Authors: Veiga-Lopez A. , Cocero M. J. , Dominguez V. , McNeilly A. S. , Gonzalez-Bulnes A.
Published: November 2010

Follicular status at the start of superovulatory treatment (presence/absence of a dominant follicle/corpus luteum) has been reported to affect subsequent embryo yields.1 The aim of the current study was to assess if this effect may be exerted through changes in the occurrence, or characteristics, of the preovulatory LH surge. Forty-three Manchega ewes were superovulated with eight decreasing doses of oFSH (OVAGENTM), starting on Day 12 after the insertion of a progestagen intravaginal sponge. At the first two FSH doses, diameter of both largest follicles (LF1 and LF2, respectively) were determined by ultrasonography. Blood sampling for LH determination was performed from 18 to 60 h after progestagen withdrawal. Ewes were also mated between 18 and 60 h after sponge withdrawal and embryos were recovered and evaluated on Day 21, being in vitro cultured until hatching. Moreover, the effect of the LH surge on superovulatory yields showed that ewes showing LH surges either with later onset or shorter duration showed higher rates of embryo viability after in vitro culture (r = 0.461, P < 0.005 and r = 0.980, P < 0.0005). In addition, earlier LH surges were related to a decreased number of non-viable embryos at recovery (r = 0.777, P < 0.05), due to a higher number of degenerated embryos, and to a lower viability rate after in vitro culture (r = 0.521, P < 0.05 and r = 0.562, P < 0.0005). Finally, the current study showed that ewes with a higher difference between LF1 and LF2 displayed earlier LH surges (r = 0.420, P < 0.05), which is related to decreased embryo yields (r = 0.777, P < 0.05).
(1)Gonzalez-Bulnes et al. (2002). Theriogenology 58, 1607–1614.
230. Altered matrix composition of cumulus oocyte complexes following in vitro maturation
Authors: Dunning K. R. , Yeo C. X. , Russell D. L.
Published: November 2010

The luteinizing hormone (LH) surge initiates cumulus expansion, through synthesis of hyaluronan and cross-linking proteins including versican, which stabilise the cumulus oocyte complex (COC) matrix. Versican is a substrate for the protease ADAMTS-1 and mRNA for each are localised to granulosa cells (GCs) and greatly induced following the LH surge.
In humans, the use of in vitro maturation (IVM) of oocytes is an appealing option, reducing costs and risk of side effects associated with in vitro fertilisation. IVM oocytes are of poorer quality, likely resulting from altered gene expression and environmental conditions during oocyte maturation.
Real-time PCR showed that IVM and immature COCs from Balb/c mice have 12 and 13 fold-reduced levels of ADAMTS-1 and versican expression respectively compared to in vivo matured COCs (PMSG+hCG 12h). Ovulated COCs (PMSG+hCG 15h) had similar low levels of ADAMTS-1 and versican. Samples isolated from F1 C57Bl/6xCBA mice showed similar reduced versican and ADAMTS-1 mRNA. Western blot analysis revealed that full length and cleaved versican, from ADAMTS-1/4 activity, was not detected in immature COCs, was present in in vivo matured COCs isolated from follicles, but strongest in ovulated COCs. IVM COCs had no detectable versican protein, supporting the mRNA data. Full-length versican was also present in GCs after PMSG+hCG 12h or 15h. ADAMTS-1 protein was most abundant in in vivo matured COCs with reduced levels seen in ovulated COCs, but was absent from IVM and immature COCs.
These results indicate that ADAMTS-1 and versican are secreted products of granulosa cells that bind and incorporate into the COC matrix. The presence of versican and ADAMTS-1 is not essential for cumulus matrix expansion in vitro, but may contribute to oocyte maturation, ovulation of the COC and/or interaction with sperm during fertilisation.
231. Mouse oocyte paracrine signalling to cumulus cells by TGF-β superfamily molecules is indispensable for cumulus expansion
Authors: Dragovic R. A. , Ritter L. J. , Schulz S. J. , Armstrong D. T. , Gilchrist R. B.
Published: November 2010

Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex (COC), which is necessary for ovulation. Members of the transforming growth factor-β (TGF-β) superfamily are prime candidates for the mouse cumulus expansion-enabling factor (CEEF), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. This study was conducted to examine TGF-β superfamily processes regulating cumulus expansion. COCs were collected from eCG-primed mice and the oocyte microsurgically removed to generate oocytectomised (OOX) complexes. An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P < 0.05) induced by either GDF9 (score: mean ± SEM, 3.7 ± 0.1), activin A (2.6 ± 0.1), or co-culture with oocytes (3.2 ± 0.2). The type-I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively. We tested the ability of the ALK4/5/7 kinase inhibitor, SB431542, to neutralise cumulus expansion. SB431542 completely neutralised (P < 0.05) the response of OOX complexes to GDF9, activin and oocyte-induced cumulus expansion. SB431542 also neutralised (P < 0.05) COC expansion in a dose dependent manner. Follistatin, an activin antagonist was effective at neutralising the response of OOX complexes to activin (score: 0), but had no significant effect (P > 0.05) on the expansion of OOX complexes co-cultured with oocytes (score: 2.7 ± 0.2). This study provides evidence that activin is not the sole CEEF, but signalling through the ALK4/5/7 pathway is indispensable for mouse cumulus expansion.
232. Oocytes prevent bovine cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins
Authors: Hussein T. S. , Froiland D. A. , Thompson J. G. , Gilchrist R. B.
Published: November 2010

Paracrine factors secreted by the oocyte regulate a broad range of cumulus cell (CC) functions. Previously we have shown that the low incidence of apoptosis in CCs is due to unidentified oocyte-secreted factors (OSF) acting in an anti-apoptotic manner. Here we examine the nature of the paracrine network of oocyte BMP growth factors and their binding proteins regulating CC apoptosis. Bovine cumulus–oocyte complexes (COC) were aspirated from abattoir-derived ovaries and oocytes microsurgically removed to create oocytectomized (OOX) complexes. OOX were treated with denuded oocytes (DO) or various growth factors for 24 h, then CC apoptosis was assessed using TUNEL together with confocal microscopy plus image analysis and by Western blotting for Bcl-2 and Bax. CC apoptosis was significantly (P < 0.001) reduced by DO, bone morphogenetic protein 15 (BMP15), BMP6 or BMP7 as assessed by TUNEL. Accordingly, expression of anti-apoptotic Bcl-2 was high in OOX+DO and OOX+BMP15, and low with OOX+GDF9 and OOX alone, whereas the reverse was observed for pro-apoptotic Bax. Combined treatment of OOXs with BMP6 and BMP15 did not further decrease apoptosis levels beyond that of BMP15 alone (P > 0.05), suggesting no additive effect of these two BMPs. Follistatin (FS) effectively antagonized BMP15 anti-apoptotic effects, and likewise, a BMP6 neutralizing antibody (NAb) antagonized the inhibitory effect of BMP6. Gremlin blocked BMP7 anti-apoptotic effects on CCs, but had no significant effect on BMP15. FS or BMP6. NAb antagonized ~50% of the anti-apoptotic activity of oocytes; however, these effects were not additive suggesting the additional involvement of other OSF. These results indicate for the first time that OSF (BMP15 and BMP6 in particular) maintains the low incidence of CC apoptosis by establishing a localized morphogenic gradient of bone morphogenetic proteins.
234. The ubiquitin-proteasome pathway in bovine and murine oocytes undergoing maturation
Authors: Edgecumbe P. C. , D'Occhio M. , Kaye P. L. , Pantaleon M.
Published: November 2010

Mammalian oocyte maturation is governed by an interaction between protein kinases, phosphatases and proteasases. Maturation promoting factor (MPF) is a serine/threonine kinase heterodimer composed of a catalytic cdc2/cdk1 subunit and a regulatory cyclin B1 subunit.1 Cyclin B1 undergoes rapid turnover via degradation in the ubiquitin-proteasome pathway (UPP) followed by de novo synthesis. A high level of MPF causes metaphase arrest, then UPP degradation of cyclin B1 allows the oocyte to exit metaphase I (MI). Proteasomes have been localised in pig and rat oocytes. Although in both species they are associated with the MI and MII meiotic spindles, immunostaining at the germinal vesicle (GV) stage is different with perinuclear staining in rat GV and intra-GV staining in the pig. This may suggest different roles for proteasomes prior to GV breakdown (GVBD) in different species. This study used confocal laser scanning immunohistochemistry with a specific antiserum against the 20S proteolytic ‘core’ of the 26S proteasome to reveal proteasomes in murine oocytes undergoing maturation in vitro. In the mouse, proteasomes were associated with the meiotic spindle, similar to observations in pig and rat oocytes and preliminary studies in the bovine suggest a similar immunolocalisation. Cyclin B1 also accumulates around the spindle during meiosis.1 This suggests that proteasomes are prevented from degrading cyclin B1 until the MI-AI and MII-AII transitions. Immunolabelling showed Fam, a deubiquitinylating enzyme (also known as Usp9x) was localised at the spindle during MI and MII. This suggests a link between Fam, the UPP and the spindle assembly checkpoint to prevent cyclin B1 degradation until required.
(1)Huo LJ, Fan HY, Zhong ZS, Chen DY, Schatten H, Sun QY. (2004). Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation. Mech. Dev. 121, 1275–1287.
235. Abberant murine embryonic development following glucosamine exposure during IVM or embryo culture
Authors: Schelbach C. , Lane M. , Kind K. L. , Thompson J. G.
Published: November 2010

The hexosamine biosynthesis pathway is an alternate fate for glucose metabolism providing glycosylation moieties and is significantly upregulated by addition of glucosamine, a common dietary supplement. Here we determined the impact of glucosamine addition to cumulus oocyte complex (COC) maturation or during embryo culture on subsequent embryonic development. COCs were collected from 23-day-old mice 46 h post-eCG, and matured under several conditions prior to being fertilized and cultured: (1) 10mL/COC a MEM (5.56mM glucose) + 0, 1.25 or 5mM glucosamine; (2) 10mL/COC a MEM (20mM glucose) + 0, 1.25 or 5mM glucosamine; (3) 100mL/COC G2.3 (5mM glucose) + 0, 1.25 or 2.5mM glucosamine. One-cell embryos were also flushed from age-matched donors 24 h after mating and cultured in 0, 1.25 or 2.5mM glucosamine in G 1.3/2.3 sequential media. No differences in rates of embryonic development were detected between COCs matured in 10mL of media with 5.56mM glucose with glucosamine. However, blastocyst formation was significantly impaired (P<0.001) when COC maturation occurred in equivalent volumes of media that contained 20mM glucose + 1.25mM (49.98%) or 5mM glucosamine (44.7%) v. control (86.55%). Intriguingly, embryonic viability was significantly (P<0.001) reduced in COCs matured in 100mL G2.3 containing 5mM glucose + 1.25mM (44.6%) or 2.5mM glucosamine (40.1%) v. control (79.81%), suggesting a volume × glucose concentration interaction. In contrast, embryonic development was significantly reduced (34%, P < 0.002) and completely ablated when 1-cell embryos were cultured in media containing 1.25mM and 2.5mM glucosamine, respectively (control = 88.57%). These results suggest that glucosamine up-regulated hexosamine pathway activity in both COCs and early embryos impairs subsequent embryonic development by as yet undescribed mechanisms.
Funded by NIH and NHMRC
236. Characterising the stem cell activity of human endometrial epithelial and stromal cells
Authors: Gargett C. E. , Zillwood R. M. , Schwab K. E. , Naqvi S. Z.
Published: November 2010

The human endometrium has remarkable regenerative capacity. It contains rare clonogenic epithelial and stromal cells.1 The aims of this study were to compare the stem cell properties, self renewal, differentiation and proliferative potential, of endometrial cells initiating large clones (HPP-CFU, high proliferative potential–colony forming units) with those initiating small clones (LPP-CFU, low PP-CFU). Endometrial tissue obtained from 12 ovulating women undergoing hysterectomy was dissociated into single cell suspensions and epithelial and stromal cells were cultured at clonal density (8–20cells/cm2). Individual clones were harvested and serially recloned to measure self-renewal, serially passaged to assess proliferative potential and cultured in various media to assess differentiation. Secondary stromal clones were characterized for mesenchymal stem cell (MSC) markers by flow cytometry. Epithelial HPP-CFU serially cloned significantly more times than epithelial LPP-CFU (P<0.005, Table1) indicating greater self renewal capacity. Similarly, stromal HPP-CFU demonstrated greater self renewal activity compared with stromal LPP-CFU (P<0.005, Table1). Serially passaged epithelial HPP-CFU underwent significantly more population doublings (PD) before senescence than epithelial LPP-CFU (P<0.05, Table1). Likewise, stromal HPP-CFU underwent a higher number of PD compared with stromal LPP-CFU (P<0.005, Table1). Stromal HPP-CFU expressed CD29, CD44, CD73, CD90, CD105 and CD146, and were negative for haemopoietic and endothelial markers, CD45, CD34 and CD31. Stromal HPP-CFU differentiated into adipocytes, smooth muscle cells, osteoblasts and chondrocytes when cultured in appropriate differentiation media. Our studies of human endometrial cells demonstrate that the rare epithelial and stromal HPP-CFU exhibit self-renewal and high proliferative potential. Furthermore stromal HPP-CFU express markers typical of bone marrow MSC and differentiate into four mesenchymal lineages. These data suggest that epithelial and stromal HPP-CFU, but not LPP-CFU, have characteristic properties of adult epithelial stem cells and MSC respectively and are likely responsible for the remarkable cyclical, regenerative capacity of human endometrium.
237. Aquaporins in rat uterine epithelial cells during early pregnancy and in response to progesterone
Authors: Lindsay L. A. , Murphy C. R.
Published: November 2010

Implantation of the rat blastocyst is a highly regulated process, involving transformation of the uterine environment into one which is receptive to an implanting blastocyst. At the time of implantation, in response to progesterone, there is a dramatic decrease in the amount of uterine luminal fluid leading to close apposition between the luminal epithelium and trophoblastic cells. The rat blastocyst also always implants at the antimesometrial pole of the uterine lumen and currently mechanisms regulating this process are unknown. Aquaporins, a family of transmembrane water channels, are involved in the regulation of water movement across epithelial barriers. We investigated several aquaporins in the rat uterus during early pregnancy using reverse transcriptase PCR. Immunofluorescence and immunogold electron microscopy techniques were then used to investigate the localisation of particular aquaporins including AQP5 in the uterine epithelium during early pregnancy and in ovariectomised rats treated with progesterone. There was an increase in AQP5 molecules in the apical plasma membrane of luminal epithelial cells at the time of implantation, with a greater increase at the mesometrial compared to antimesometrial pole. A similar result was seen in luminal epithelial cells from ovariectomised rats treated with progesterone, however there was no differential concentration between mesometrial and antimesometrial poles, as there was during early pregnancy. It is suggested that the increase in AQP5 protein expression in the apical plasma membrane of luminal epithelial cells is involved in reabsorption of luminal fluid at the time of implantation. Furthermore, the differential concentration of AQP5 on luminal epithelial cells at the time of implantation could lead to the establishment of a fluid gradient within the uterine lumen and hence lead to the asymmetrical implantation position of the rat blastocyst.
238. Claudins and occludin in the rat endometrium
Authors: Nicholson M. D. O. , Murphy C. R.
Published: November 2010

Regulation of the uterine luminal environment is important for the successful attachment and implantation of the blastocyst. Tight junctions regulate the paracellular pathway between epithelial cells lining the uterine lumen and the uterine glands. The aims of this present study was firstly to establish the presence and cellular distribution of claudins and occludin in the luminal epithelia during early pregnancy using immunofluorescence microscopy and deconvolution, and secondly to determine the influence of ovarian hormones on their expression. Occludin and claudins -1, -3, and -4 were present in luminal epithelium. Occludin and claudin-4 showed increased expression in luminal epithelium at the time of implantation, whereas claudin-1 and -3 expression remained the same throughout early pregnancy. In ovariectomised rats administered ovarian hormones, occludin and claudin-4 showed increased expression in luminal epithelium in progesterone-dominant regimes and decreased expression when administered oestrogen alone. Expression of claudin-1 and -3 in luminal epithelium was not effected by ovarian hormones. Claudin-2 was not expressed during early pregnancy nor in ovariectomised rats. In conclusion, these results show that occludin and claudins -1, -3 and -4 are present in luminal and glandular epithelium, and provide the permeability properties needed to separate the luminal and the stromal environment at the time of implantation. Furthermore, occludin and claudin-4 expression is controlled by ovarian hormones being upregulated by progesterone.
239. Hormonal control of vascular mural cell recruitment in the mouse endometrium
Authors: Girling J. E. , Walter L. M. , Rogers P. A. W.
Published: November 2010

The human endometrium undergoes regular periods of growth and regression, including concomitant changes in the vasculature, and is one of the few adult tissues where significant angiogenesis (new blood vessel formation) and arteriogenesis (recruitment of vascular smooth muscle cells (VSMC) and pericytes) occurs on a routine, physiological basis. In this study, mouse models were used to investigate the effects of oestrogen and progesterone on endometrial vascular mural cell recruitment. The aim was to quantify changes in the proportion of vessels covered by α-smooth muscle actin (α-SMA, a marker of VSMC and pericytes) in hormone-treated ovariectomised mice. We hypothesised that relative vessel α-SMA coverage would increase following progesterone treatment (in conjunction with endothelial cell (EC) proliferation), but not following oestrogen treatment (when EC proliferation also occurs). Ovariectomised mice were given a single oestradiol (100 ng) or vehicle injection, before dissection 24 h later, or three consecutive daily injections of progesterone (1 mg) or vehicle. The percentage of vessel profiles with no, minimal, extensive or complete α-SMA coverage were quantified after CD31/α-SMA double immunostaining. There was a significant decrease in the percentage of vessel profiles with no α-SMA coverage following progesterone treatment (20±4.3 % [mean±SE] v. 57±4.6 %, t(7)=12.5, P<0.001), and a significant increase in the percentage of vessels with minimal or extensive α-SMA coverage (44±3.4 % v. 27±3.7%, t(7)=4.7, P<0.001 and 27±4.3% v. 5±0.5%, t(7)=5.8, P<0.001, respectively), in comparison to vehicle-treated mice. The percentage of vessels with complete α-SMA coverage, representing vessels with a coat of VSMC, did not change significantly in comparison to vehicle-treated mice (8±2.3% v. 10±1.2%, t(7)=0.6, P=0.55). There were no significant changes in the percentage of vessels with differing α-SMA coverage in oestrogen-treated mice. In continuing studies, we will quantify the proportion of proliferating α-SMA positive cells and examine mouse endometrial tissues using a pericyte-specific marker.
241. Changes in the expression of Annexin IV mRNA and protein in human endometrium
Authors: Ponnampalam A. P. , Rogers P. A. W.
Published: November 2010

In a previous study investigating global gene expression throughout the menstrual cycle,1 Annexin 4 (ANXIV) was identified as having significant cyclic changes in human endometrium. ANXIV belongs to a ubiquitous family of Ca2+-dependent phospholipid and membrane-binding proteins. The aims of this study were to investigate the cellular localization and regulation of ANXIV mRNA and temporal expression of ANXIV protein in human endometrium during the menstrual cycle.
mRNA Expression: The menstrual cycle was divided into seven stages by histological evaluation. Curettings of endometrium were collected from 60 cycling women. For cellular localization, tissues from eight endometrial curettings were dissociated with collagenase into single cells, separated into epithelial and stromal cell fractions and snap frozen. Total RNA was extracted and ANXIV mRNA was quantified by real-time PCR.
Immunohistochemistry: Full thickness endometrial tissue was collected from 50 reproductive age women undergoing hysterectomy. Tissue sections were formalin-fixed and paraffin-embedded. Goat polyclonal ANXIV antibody was used to localize ANXIV protein.
ANXIV mRNA was significantly upregulated in the whole tissue during mid-late secretory phase of the cycle, and was predominantly expressed in epithelial cells. ANXIV protein was detected in the luminal and glandular epithelium in high levels throughout the menstrual cycle except in early secretory (ES) phase. The intensity of immunostaining was stronger in the glands of the basalis compared to functionalis in early proliferative phase, however, by the late secretory phase the functionalis glands showed higher expression levels.
ANXIV mRNA data are consistent with a role for progesterone in upregulating the expression of ANXIV, although protein levels remain high through menstruation and into the proliferative phase. ANXIV can indirectly inhibit prostaglandin production, which is important for implantation. Hence the low levels of ANXIV protein at ES phase may relate to processes involved in implantation.
(1)Ponnampalam et al. (2004). Mol. Hum. Reprod. 10(12), 879–893.
242. LIF expression is induced in the mouse oviduct following activation by seminal factors
Authors: Jasper M. J. , Bromfield J. J. , Skinner R. , Robertson S. A.
Published: November 2010

A precisely regulated sequence of molecular and cellular changes occurs in the female reproductive tract in early pregnancy to facilitate development of the embryo and its successful implantation. Cytokine–leukocyte networks are integral in the tissue remodelling and immuno-regulatory processes underpinning successful implantation. Seminal factors are implicated in activating expression of cytokine genes and inflammatory leukocyte recruitment in the uterus, but whether semen-induced effects extend to the oviduct to influence blastocyst development has not been examined. The aim of this study was to quantitate the expression of mRNA encoding epidermal growth factor (EGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), insulin-like growth factor (IGFII), leukaemia inhibitory factor (LIF), tumour necrosis factor (TNFα), transforming growth factor (TGFα) and TGFβ from oviducts collected from mice at oestrus and on day 1 of pregnancy, after mating with intact, seminal plasma deficient (svx) and vasectomised (vas) mice. Total RNA was extracted, DNAse treated, reverse transcribed into cDNA, and quantified by real-time PCR using SYBR Green chemistry. All cytokine-specific primers were designed using GenBank sequences and data were normalised to β-actin mRNA expression. Expression of LIF mRNA was induced following mating with intact or vas males, but not svx males, showing that LIF mRNA is induced by factors present in seminal plasma. mRNAs encoding EGF, GM-CSF, HB EGF, IGFII, TNFα, TGFα and TGFβ were all detected in oviduct cDNA collected from oestrus and day 1 mice. These data support the proposal that cytokine-leukocyte networks shown previously to be operative in the uterus extend to the oviduct, and are influenced by exposure to seminal plasma at mating. The cytokines expressed in the oviduct during early pregnancy are likely to be key regulators of embryo growth and development.
243. In vitro culture significantly reduces differences in gene expression profiles between myometrial and fibroid smooth muscle cells
Authors: Zaitseva M. , Rogers P. A. W.
Published: November 2010

Fibroids are benign neoplasms of the smooth muscle cells of the uterus. Cultured myometrial (M) and fibroid (F) smooth muscle cells (SMC) have been widely used as a model for the study of fibroid growth. Although it has been shown that FSMC can behave differently in culture to MSMC, it is not clear how relevant the cultured cells and their responses are to the in-vivo situation. The aim of the present study was to compare gene expression profiles of M and F tissue to cells isolated from the same tissue and cultured for up to 3 passages. M and F were collected from hysterectomy specimens (n = 6), part was snap frozen for RNA and the rest used to isolate SMC, which were cultured for 3 passages and RNA was collected at passage 0 (P0) and 3 (P3). 36 microarrays were performed on 8K human cDNA slides, 6 per each specimen (3 for M and 3 for F: tissue, cell at P0 and P3) against reference RNA. Analysis revealed significant differences between tissues and cultured cells. Independent clustering assigned tissues versus cells into two distinct groups based on their expression profiles. Parametric ANOVA with Benjamini-Hochberg correction and post-hoc testing was used to determine similarities and differences between tissues and cells. 128 genes were found to be statistically different between M and F tissue, 66 between MSMC and FSMC at P0, and only 9 at P3. More than 1100 genes were significantly changed between tissues and cultured cells, with 648 genes common between both M and F cells at P0 and P3. Similar numbers of genes were up regulated as were down regulated. Expression profiles of genes of interest including estrogen receptor α and progesterone receptor were also validated using real-time PCR. This is the first study to compare gene expression of in vivo and in vitro fibroid and myometrial SMC. The results demonstrate that large changes occur in SMC gene expression in culture, reducing differences between myometrial and fibroid cells. This study indicates that results of in vitro studies should be interpreted with caution as many genes have an altered gene expression profile in culture.
244. Effect of batimastat, a specific inhibitor of matrix metalloproteinases, on endometrial breakdown and repair in a mouse model
Authors: Kaitu'u T. J. , Morison N. B. , Salamonsen L. A.
Published: November 2010

Strong correlative evidence supports a role for matrix metalloproteinases (MMPs) in the tissue breakdown at menstruation. As menstruation occurs in very few species besides women, there is a lack of suitable and easily accessible animal models available to examine the functional significance of potential key mediators of this process. A mouse model of endometrial breakdown and repair has been developed,1 which morphologically resembles that of human endometrium at menstruation. Previous studies in our laboratory showed that the expression patterns of various MMPs in the mouse model closely resembled those seen in the human.2 Administration of doxycycline, a broad spectrum MMP inhibitor, decreased gelatinase activity, but had no effect on tissue breakdown in this model. The aim of the present study was to further examine the importance of MMPs in endometrial breakdown and repair via administration of batimistat, a highly potent and specific MMP inhibitor.
Batimistat was administered I.P to mice 24 h prior to the expected time of endometrial breakdown. The efficacy of batimistat within the uterus was proven using in situ zymography, which identifies MMP activity (rather than latent forms). This demonstrated that batimistat was reaching its target organ and effectively inhibiting MMP activities (both gelatinase and collagenase). Examination of gross uterine morphology revealed no apparent difference between groups, with batimistat treated uteri displaying a similar extent of tissue breakdown and repair to their control counterparts. Measurement of the breaking down area compared to total endometrial area revealed no difference between control and batimistat treatment, with the breaking down areas being 69 ±13% and 72 ± 9.8% of total endometrial cross-sectional area respectively. There was likewise no effect on endometrial repair.
The results of this study together with our previous study using doxycycline, indicate that MMPs are not the key mediators of endometrial breakdown in this model.
(1)Shen et al. (2004) Reprod. Fert. Dev. 16(Suppl), A265, p. 97.(2)Brasted et al. (2003) Biol. Reprod. 69, 1273.
246. A new look at menstrual repair: the role of CD34+ cells and CD56+ uterine NK cells
Authors: Garry R. , Crewe J. , Hickey M.
Published: November 2010

Endometrial regeneration occurs rapidly in a low estrogen environment without observable mitosis following menstruation. We have used a combination of hysteroscopy, scanning electron microscopy and immuno-histochemistry to re-examine this poorly understood process. Full thickness endometrial biopsies from 25 cycling women were used for immuno-histochemical analysis of the distribution of CD34+ and CD56+ cells.
Histological appearances show that immediately preceding menstruation, a small population of CD34+ stromal cells are located in the basalis but that their numbers increase dramatically following the onset of menstruation, becoming the dominant stromal cell type at this time. Mean surface stromal count during menstruation of CD34+ cells was 5847/mm2 (n = 8) compared with pre-menstrual 108/mm2 (n = 7). Conversely CD56+ uNK cells in the endometrial stroma were minimal in the proliferative phase, mean 228/mm2 (n = 6) reaching a mean 2709/mm2 (n = 7) immediately pre-menstrually. There is a significant inverse correlation (at 0.01 level (2-tailed)) between CD34+ cells and CD56+ uNK cells across the menstrual cycle. We present additional scanning electron microscope and histological images to support the hypothesis that uterine endometrial repair may occur via a blood borne supply of CD34+ stem cells which differentiate in to elements of the endometrium; surface epithelium, glandular epithelium and stromal uNK cells. This hypothesis has profound clinical implications for the mechanisms and management of common gynaecological conditions resulting from abnormal endometrial repair and is contrary to the current understanding that uterine endometrium is replaced from basal remnants.