bluuebunny/crossref_metadata_2025 huggingface dataset:
275. Neuronal regeneration peptide (NRP): a novel trophoblast migration and survival enhancing factor
Authors: Singh A. , Keelan J. , Sieg F.
Published: November 2010

Autocrine and paracrine factors regulate survival, proliferation, migration and invasion of placental cytotrophoblasts cells. While trophoblast migration appears to be tightly controlled, the nature of the chemoattractive factors that facilitate and direct trophoblast invasion remains undefined. Our group recently discovered a chemottractive factor (NRP) that exerts its biological activities on the CNS. Studies of NRP actions reveal extensive influences on postnatal neuronal migration, differentiation and survival. Based on the neuronal activities of NRPs and parallels between neuronal and placental cell behavior patterns, we speculated that NRPs could be involved in placental development.
Migration assays were performed over 22 h using Boyden chambers pre-coated with NRP and laminin (n = 6 chambers per condition), to test the effect of NRPs on trophoblast migration. CTBs were isolated from term placentas by trypsin digestion and Percoll purification, and experiments were conducted within 6 h. Trophoblasts were seeded into the inner chamber (50000 cells/well) in M199 media supplemented with 10% FBS and antibiotics. Total number of cells migrating was counted. Migration was increased by 95 ± 14 % (mean ± SEM) in the presence of 100 fM NRP (P = 0.0016, t-test) compared to controls (bovine serum albumin). Survival assays were also performed using both primary trophoblasts and Jar choriocarcinoma cells. Apoptosis over 48 h, induced by treatment with TNF-α (5 ng/mL) and IFN-γ (100 U/mL), was completely abrogated by 10 pM NRP in Jar cells and 1pM in primary trophoblasts, as judged by MTT assay (mitochondrial activity).
Inadequate placentation is implicated in the pathogenesis of a number of serious pregnancy disorders such as preeclampsia. Our findings suggest important roles for NRPs in regulating trophoblast migration and survival. The possibility that defective NRP actions may be involved in various placental pathologies, or that NRPs could be used pharmacologically to augment placentation, remain to be explored.
276. Sex difference in the effect of cortisol on the LH response of the pituitary to exogenous GnRH in hypothalamo-pituitary disconnected gonadectomised sheep
Authors: Stackpole C. A. , Clarke I. J. , Turner A. I. , Tilbrook A. J.
Published: November 2010

We have used the hypothalamo-pituitary disconnected (HPD) sheep model to investigate direct pituitary actions of cortisol to suppress LH secretion in response to exogenous GnRH. We previously observed that, during the non-breeding season, treatment with cortisol did not suppress the LH response to GnRH in HPD gonadectomised rams or ewes.1 In the present experiment, we tested the effect of cortisol on the LH response to exogenous GnRH in gonadectomised HPD sheep during the breeding season. Using a cross-over design, HPD gonadectomised Romney Marsh rams (n = 6) and ewes (n = 5) received a saline or cortisol (250 μg/kg/h) infusion for 30 h on each of two days, one week apart. All animals were treated with 125 ng i.v. injections of GnRH every 2 h during a 6h control period preceding the infusion and during the infusion. Jugular blood samples were taken during the control period and the first 6 h and last 6 h of the infusion (over 3 LH pulses). Mean plasma concentrations of LH and LH pulse amplitudes, driven by programmed GnRH injections, were similar in gonadectomised rams and ewes and there were no significant effects of saline infusion between the control periods or the saline infusion in either sex. The amplitude of LH pulses was significantly (P < 0.05) reduced in rams during the first 6 h of the cortisol infusion compared to the control period, but there were no effects of the cortisol infusion in ewes. These data show that, in the absence of sex steroids, there is a sex difference in the mechanism by which cortisol acts at the pituitary to reduce LH secretion in response to exogenous GnRH in HPD gonadectomized sheep during the breeding season. We conclude that the effect of cortisol to reduce secretion of LH involves an action on the pituitary, at least in gonadectomised rams.
(1)Stackpole CA, Turner AI, Clarke IJ and Tilbrook AJ (2003) Biology of Reproduction 36(Supplement 1), 288.
277. Is the high level of circulating FSH in Booroola ewes due to the Bmp R1b mutation?
Authors: Young J. , Edwards S. , Dearden P. , Wilson T.
Published: November 2010

Booroola ewes are characterised by having high ovulation rate and litter size caused by the action of the Booroola fecundity gene (FecB), which encodes the bone morphogenetic protein receptor (BMPR) type 1b mutation (Q249R). The product of the FecB gene influences both pituitary and ovarian activity. In both neonatal and adult Booroola ewes carrying a double copy of the FecB allele, plasma concentrations of the pituitary-derived follicle stimulating hormone (FSH) are higher than in non-carrier females. BMPR-1b is expressed in the sheep pituitary gland and recent reports have shown that in addition to GnRH, TGF-β family members such as BMP 6, 7, and 15 induce pituitary FSHβ expression. Thus BMPs may function in regulating FSH synthesis in the pituitary. We are investigating whether we can detect induction/secretion of FSH in response to BMP treatment of ovine primary pituitary cell cultures, with the aim of elucidating whether the Booroola mutation is the cause of the increased FSH secretion. Due to the high variance between cultures from different animals, and between replicates from the same animal, we are attempting to determine the best method for approaching the question. Some results from these investigations will be presented.
279. A dynamic model of the control of pulsatile luteinizing hormone secretion by gonadotrophin-releasing hormone
Authors: Ferasyi T. R. , Barrett H. , Blache D. , Martin G. B.
Published: November 2010

Infusion of GnRH in a continuous manner or with a very high pulse frequency initially stimulates but then downregulates LH secretion.1,2 This phenomenon is caused by the slow rate of internalisation of the GnRH receptor (GnRH-R) and the subsequent slow return of receptors to the plasma membrane of the gonadotroph.3 Pulsatile release of GnRH overcomes this problem by allowing a delay between successive stimulations. It is difficult to determine the relative importance of critical control points in this process in an animal model because GnRH activity reflects integrated inputs from many internal and external factors. We are therefore using SAAM II software to develop a compartmental model of the relationship between GnRH-R availability and LH responses following changes in GnRH pulse frequency. The model has three receptor states (free, bound, and internalised) and one LH compartment (Fig. 1). We assumed LH release is a function of the amount of receptor that binds GnRH. Following GnRH binding, receptors are rapidly lost as they enter the internalised state and then slowly returned to the membrane surface. We further assumed that the slow rate of receptor return explains the decrease in LH response with very high frequencies of GnRH pulses. The values for parameters were based on data obtained from experiments with sheep. In our current version of the model, downregulation is observed when gonadotrophs are stimulated with GnRH pulses every 15 min (Fig. 1), but not with pulses every 30 or 60 min, at a slow recycling rate (0.004 min–1). In contrast, LH secretion increases when GnRH is pulsed every 15 min and recycling rate is increased to 0.04 min–1. This suggests that, in sheep, a recycling rate between 0.004 and 0.04 min–1 is a critical aspect of the intracellular control of the process. Future work will include steroid feedback loops.
281. The role of insulin in milk protein synthesis
Authors: Menzies K. K. , Macmillan K. L. , Nicholas K. R. , Lefevre C. , Ormandy C.
Published: November 2010

The mammary explant culture model has been frequently used to mimic lactation and to examine the endocrine control of milk protein gene expression. Studies in the mouse show the expression of the milk protein genes in explants requires insulin in the presence of prolactin and cortisol. The role of insulin in milk protein synthesis in the dairy cow is not as clear. The bovine mammary explant culture model has been utilised to show that insulin is essential for alpha-s1-casein gene expression and the synthesis of the casein proteins. In addition, mouse culture experiments were undertaken to provide an insight into the underlying molecular mechanisms of insulin action in hte mammary gland. A global analysis of the genes induced in the cultured explants was done using Affymetrix microarray and showed 132 genes, including the major milk protein genes, required the complement of insulin, cortisol and prolactin for maximal expression. Twenty-seven genes showed a 3-fold change in gene expression in response to insulin. The function of these genes can be largely categorised into maintenance of cell integrity, signal transduction, transport mechanisms, cellular metabolism and a direct effect on gene transcription in the nucleus. The requirement for insulin in milk protein synthesis is highlighted by its role in inducing the STAT5 gene, known to be a key transcription factor for the milk protein genes. Interestingly, dairy cows of high genetic merit have unusually low serum concentrations of insulin. This has occured in association with a high selection pressure for milk volume that has altered the regulation of blood glucose homeostasis. Our study indicates that this intensity of selection for high milk volume could be compromising the dairy cow’s potential for milk protein production: Has selecting for milk volume in many populations of dairy cows been achieved by lowering circulating insulin levels with consequent effects on the efficiency for milk protein yield as well as compromised reproductive performance.
282. Anti-apoptotic effects of glucocorticoids and progesterone in a human mammary epithelial cell line
Authors: Berg M. N. , Waddell B. J. , Dharmarajan A. M.
Published: November 2010

Previous studies in the lactating rat mammary gland have shown that removal of both progesterone and glucocorticoids can induce apoptosis, and that replacement with either steroid alone can prevent this effect.1 These results raised the possibility that progesterone may exert its anti-apoptotic effects in the mammary gland via the glucocorticoid receptor (GR). A similar mechanism has previously been proposed to account for the action of progesterone in the corpus luteum.2 To assess this possibility we examined the effects of glucocorticoids and progesterone on apoptosis in the non-tumorigenic human mammary epithelial cell line, MCF-10A.
Initial PCR analysis demonstrated that MCF-10A cells do not express progesterone receptor mRNA (PR-A or PR-B). MCF-10A cells were incubated in serum-free DMEM:F12 medium with additives in the presence or absence of cortisol (10–6M) or progesterone (10–5M to 10–8M). DNA fragmentation (an index of apoptosis) was quantitated by either 3′-end labelling or by a novel method of image analysis of SYBR® gold-stained gels. DNA fragmentation was clearly increased after 24 h in the absence of cortisol, consistent with previous reports for this cell line.3 In a separate experiment, replacement of cortisol with progesterone also reduced apoptosis by 48 h, although this protection was evident only at the two highest progesterone doses (10–5M and 10–6M).
These results suggest that despite the absence of a progesterone receptor, progesterone is able to prevent apoptosis in MCF-10A cells, albeit at a physiologically high doses. This supports the hypothesis that some actions of progesterone may be mediated via the GR. Further studies are required to determine the precise molecular pathways by which cortisol and progesterone prevent apoptosis.
(1)Berg MN et al. (2002) Endocrinology 143, 222–227.(2)Sugino N et al. (1997) Endocrinology 138, 4497–4500.(3)Moran TJ et al. (2000) Cancer Res 60, 867–872.
283. Regulated expression of the c-fms receptor characterises distinct ovarian macrophage populations
Authors: Van der Hoek K. H. , Minge C. E. , Norman R. J. , Robker R. L.
Published: November 2010

Macrophages represent a major immune cell type in reproductive tissues and are thought to regulate multiple aspects of reproduction, including ovarian function. We have previously shown a distinctive phenotype of ovarian thecal macrophages present around the preovulatory follicle, such that secreted cytokines are uniquely regulated within these cells across the oestrus cycle. C-fms is a macrophage-specific gene that encodes the receptor for colony-stimulating factor-1 (CSF-1), and that regulates macrophage proliferation, differentiation and migration, as well as pro-inflammatory responses.1,2 We acquired transgenic mice (from DA Hume, Institute for Molecular Bioscience, University of Queensland) that express green fluorescent protein (GFP) exclusively in macrophages under direction of the c-fms gene promoter.3 In ovaries from these animals we have previously reported that macrophages constitutively positive for macrophage markers, F4/80 and MHCII, exhibited spatially regulated expression of GFP (c-fms); being GFP+ within the stroma surrounding small follicles, particularly atretic follicles, but GFP– in theca surrounding preovulatory follicles and healthy corpora lutea (CL), further reinforcing the concept that these macrophages are not classically activated but have a unique resident phenotype. Further examination of the GFP+ ovarian macrophage population has revealed that the highest levels of GFP expression were in macrophages associated with TUNEL+ regressing CL and, even though CSF-1 typically induces proliferation, the GFP+ macrophages within the regressing CL did not incorporate BrdU label nor express cyclin D1. This indicates that in the murine ovary c-fms expression may not regulate ovarian macrophage proliferation or migration but more likely represents a subset of classically activated ovarian macrophages that are actively differentiating or phagocytically active.
(1)Fixe P and Praloran V (1998) Cytokine 10, 32–37.(2)Pixley FJ and Stanley ER (2004) Trends in Cell Biology 14, 628–38.(3)Sasmono RT et al. (2003) Blood 101, 1155–63.
284. Leukocyte trafficking in the ovary of mice immunised with recombinant murine cyclomegalovirus expressing murine zona pellucida 3
Authors: O'Leary S. , Lloyd M. L. , Shellam G. R. , Maddocks S.
Published: November 2010

Inoculation of female BALB/c mice with recombinant murine cytomegalovirus encoding murine zona pellucida antigen (MCMV-ZP3) confers infertility characterised by depletion in ovarian tertiary follicles by day 21 post inoculation followed by a progressive depletion in primordial follicles.1 Cell mediated immune responses begin as early as day 10 post immunisation with MCMV-ZP32 with the recruitment of leukocytes before serum antibody can be clearly detected in mice. The physiological mechanisms leading to infertility in inoculated mice are being progressively delineated with the role of leukocyte subsets implicated in early pathological changes in ovarian architecture. The aim of this study was to investigate the effect of MCMV-ZP3 infection on leukocytes including T cells recruited into the ovary following infection with recombinant virus. Fifteen BALB/c female mice were randomly allocated into three groups of five animals at 6 weeks of age. Group one received an injection of PBS, group two and three received intraperitoneal inoculations of 2 × 104 pfu of MCMV and MCMV-ZP3 respectively. Ovaries were retrieved at day 10, 21 and 35 post inoculation and one ovary from each mouse was sectioned for immunohistochemical analysis of resident leukocytes using mAb CD45 reactive with all leukocyte lineages and mAb for CD4 and CD8 positive T cells. MCMV-ZP3 inoculation increased the abundance of ovarian leukocytes including CD4 and CD8 positive T cells for all time points post immunisation except for CD8 positive T cells 21 days post infection (Table 1).
These results suggest that leukocytes, including T cells, are involved in causing early changes in the ovary post infection with MCMV-ZP3 that lead to the depletion of existing ovarian follicles leading to life long infertility in mice. Further experiments are underway to investigate the role of antibody and changes in leukocyte populations in the ovary as the course of infection with recombinant virus progresses.

This study is funded by the Cooperative Research Centre for Pest Animal Control.

(1)Lloyd ML, et al. (2003). Biol. Reprod.
68, 2024–32.(2)O’Leary S, et al. (2004). Reprod. Fertil. Devel.
16(Supplement), 77.
285. Endometrial lymphatics are reduced in the functionalis compared to basalis and myometrium
Authors: Donoghue J. F. , Lederman F. L. , Rogers P. A. W.
Published: November 2010

Information on uterine lymphatics is limited. The aim of this study was to characterise uterine lymphatic vessels and the corresponding growth factors in the endometrium and myometrium across the normal human menstrual cycle. Uterine tissues were collected from patients undergoing hysterectomy. Lymphatic and microvascular density (MVD/mm2) was determined on serial sections of full thickness uterus (n = 45) with antibodies to D2-40, CD31, CD34 and FVIII. Lymphangiogenic growth factors VEGF-C and VEGF-D immunolocalisation was also determined on serial sections. VEGF-C, VEGF-D and lymphatic endothelial cell markers VEGF-R3 and D2-40 protein expression was determined on protein extracted from myometrium and endometrium and separated by SDS-PAGE from proliferative (n = 5) and secretory (n = 5) hysterectomy specimens. The lymphatic vessels were closely associated with smooth muscle cells of spiral arterioles and VEGF-C and VEGF-D were primarily localised in the endometrial glands, luminal epithelium and myometrial smooth muscle bundles. The lymphatic MVD was significantly reduced in the functionalis (15.1 ± 2.3mm2) compared to basalis (80 ± 11.4mm2) and myometrium (63 ± 9.2mm2). Overall, lymphatics constituted 12% of all vessels in the functionalis, 60 % in the basalis and 30% of the myometrium. D2-40 positive uterine lymphatics showed considerable heterogeneity, with 88% co-localisation with the blood vessel marker CD31, but only 46% expressing CD34 and 31% with FVIII. Protein expression of VEGF-C, VEGF-D, VEGF-R3 and D2-40 were significantly reduced during the proliferative phase compared to the secretory phase and were also significantly reduced in the endometrium compared to the myometrium across the cycle (P ≤ 0.05). Endometrial functionalis lymphatics are reduced in conjunction with a reduction in lymphangiogenic growth factors compared with the myometrium.
286. The role of uterine natural killer cells in causing irregular bleeding in HT users
Authors: Hickey M. , Crewe J. M. , Doherty D. , Fraser I. S. , Salamonsen L. A.
Published: November 2010

Menopausal hormone therapy (HT) causes irregular bleeding in up to 60% of user. This is extremely unpopular with patients, and commonly leads to invasive and expensive investigations to rule out underlying pelvic pathology. In most cases no cause is found. The aim of this study was to further elucidate the mechanisms of vascular fragility. Uterine NK cells are known to increase vascular fragility during the normal menstrual cycle. We hypothesise that HT is associated with an increase in uterine natural killer (uNK) cells. Eighty six endometrial biopsies were obtained from 59 postmenopausal users of continuous combined HT. Uterine NK cells were identified using immunohistochemistry as being CD56+. Image analysis was used to identify absolute number of CD56+ cells and their distribution within the stroma. Endometrial histology was classified using Noyes criteria. A statistically significant increase in endometrial uNK cell density was observed in HT users compared to postmenopausal women not using HT (P < 0.001). uNK cell populations were more marked in biopsies taken during a bleeding episode compared to those HT users with amenorrhoea (P = 031). uNK cells are a major regulator of endometrial vascular integrity and are known to be disrupted in irregular bleeding with progestin only contraceptives. This is the first study to report the presence of uNK cells in postmenopausal endometrium and the first to report a significant association between bleeding patterns and uNK cell density. We postulate that HT induces an increase in endometrial uNK cell populations and that their presence stimulates endometrial vascular fragility leading to bleeding.
287. LPS introduced at mating induces KC production in the murine uterus during early pregnancy
Authors: Glynn D. J. , Robertson S. A.
Published: November 2010

An inflammatory cascade is elicited in the female reproductive tract following mating in mice. The recruited leukocytes and cytokines have roles in facilitating implantation through activating immunological tolerance and endometrial tissue remodelling. We have previously shown that seminal plasma acts to induce synthesis of GM-CSF and IL-6 in the female reproductive tract in response to TGFβ1 present in seminal fluid. Recently we have shown that chemokines, specifically the neutrophil chemoattractant KC, is dramatically upregulated after mating. The purpose of this study was to identify the active constituent of semen responsible for KC induction. Female mice were mated with either intact, seminal vesicle deficient or vasectomized males and uterine flushings were collected approximately 8 h later, when KC content was measured by specific ELISA. KC production was increased 13-fold, 6-fold and 10-fold respectively, indicating that neither the seminal plasma nor the sperm fraction of semen was necessary for induction. To investigate more precisely the identity of the KC inducing factor, an in vitro primary uterine epithelial cell culture system was employed. Uterine epithelial cells were harvested from estrous female mice and exposed to a range of doses of seminal vesicle fluid, TGFβ1or lipopolysaccharide (LPS). Following addition of seminal vesicle fluid or TGFβ1 KC production was decreased by 100% and 58% respectively whereas it was increased ~2-fold in response to LPS. Together these data indicate that LPS derived from the male or lower female reproductive tract accessing the uterus after insemination is required for KC induction, and implicate commensal bacteria as having a key regulatory role in the cellular and molecular quality of the uterine immune environment during early pregnancy.
288. The post-insemination inflammatory response in the ewe
Authors: Scott J. L. , Ketheesan N. , Summers P. M.
Published: November 2010

Insemination causes an inflammatory response in the female reproductive tract of many species. The cytokine/leukocyte network initiated during this reaction is believed to enhance reproductive success.1 This study investigated the post-insemination inflammatory response in the ewe. Fifteen nonparous ewes were mated with the same ram for 1 h and their reproductive tracts were collected 3, 6, 18, 24 or 48 h later. Another fifteen ewes were used as controls. Tissue samples and luminal mucus were collected from 10 sites in each reproductive tract and stained with haematoxylin and eosin, Diffquik and immunohistochemically using a monoclonal CD68 antibody to quantify neutrophils, eosinophils and macrophages. Presence of interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated using immunohistochemistry and enzyme-linked immunosorbent assay. Neutrophils and macrophages increased in reproductive tissues following insemination. Mean cell counts in 1.5-mm2 tissue of mated (M) and control (C) ewes demonstrated a peak in neutrophils at 6–18 h post-insemination with significant differences (P < 0.05) between mated and controls in the posterior cervix (M = 23.7; C = 4.1) and uterine body (M = 34.5; C = 11.5). Macrophages peaked at 18–24 h with significant differences (P < 0.05) between mated and controls in the vagina (M=13.4; C = 4.6), posterior cervix (M = 10.4; C = 2.7), mid-cervix (M = 8.5; C = 3.0) and ipsilateral mid-uterine horn (M = 14.2; C = 7.9). Neutrophils increased in the lumen of the cervix and uterine body following insemination but macrophage numbers did not change. Insemination did not affect eosinophils. IL-8 and GM-CSF were detected in endometrial epithelial cells in mated and non-mated ewes. Highest concentrations of IL-8 were found in vaginal mucus. Small quantities of GM-CSF were detected in occasional mucus samples. No difference between mated and non-mated ewes was demonstrated for either cytokine. In conclusion, the post-insemination inflammatory reaction in the ewe involves an increase in neutrophils and macrophages in reproductive tissues, with neutrophils crossing the epithelium into the lumen. There was no apparent increase in IL-8 or GM-CSF in response to insemination.
(1)Robertson SA et al. (1997) American Journal of Reproductive Immunology 37, 438–442.
290. Calmodulin-dependent protein kinase II, and not protein kinase C, transduces the Ca2+ signal at fertilization
Authors: Jones K. T. , Levasseur M. , Chang H. , Madgwick S.
Published: November 2010

Mouse eggs arrest at metaphase II following ovulation and are only triggered to complete meiosis when fertilized. Sperm break the cell cycle arrest by a long-lasting series of Ca2+ spikes that lead to an activation of the anaphase-promoting complex. The signal transduction pathway is not fully resolved but both protein kinase C (PKC) and calmodulin-dependent protein kinase II (CamKII) activities increase at fertilization and previous pharmacological studies have implicated both in cell cycle resumption. Here we used a combination of pharmacological inhibitors and constitutively-active cRNA constructs of PKCα and CamKII microinjected into mouse eggs, to show that it is CamKII and not PKC that is the sufficient trigger for cell cycle resumption from metaphase II arrest.
Constitutively active PKC constructs had no effect on meiotic resumption but caused an immediate and persistent elevation in intracellular Ca2+ when store-operated Ca2+ entry was stimulated. With respect to meiotic resumption, the effects of constitutively-active CamKII on eggs were the same as sperm. Eggs underwent second polar body extrusion and pronucleus formation with normal timings; while both securin and cyclin B1 destruction, visualised by coupling to fluorescent protein tags, were complete by the time of polar body extrusion. Induction of a spindle checkpoint by overexpression of Mad2 or by spindle poisons blocked CamKII-induced meiotic resumption but the Ca2+ chelator BAPTA did not. Furthermore direct measurement of Ca2+ levels showed that CamKII did not induce exit from metaphase II arrest by raising Ca2+. Therefore we conclude that PKCs may play an important role in maintaining Ca2+ spiking at fertilization by promoting store-operated Ca2+ entry, while CamKII transduces cell cycle resumption, and lies downstream of sperm-induced Ca2+ release but upstream of a spindle checkpoint. These data, combined with the knowledge that CamKII activity increase at fertilization, suggest that mouse eggs undergo cell cycle resumption through stimulation of CamKII.
291. Potential role of Glycodelin for fertilization success in ART
Authors: Keck G. , Reichel N. , Zimmermann A. , Trinkaus I. , Schultz A. , Distler W.
Published: November 2010

Background: Glycodelin (Gd), a dimeric glycoprotein, appears in the female and male reproductive tract in isoforms like GdA from amniotic fluid and endometrium, GdF from follicular fluid, GdS from seminal plasma and on sperm surface as well. Little is known about the role of Gd in IVF. The aim of our study was to investigate the influence of the Gd-amount in supernatants of IVF-cultures to fertilization success. Methods: Employing monoclonal antibody (mAb) M4f8 soluble Gd levels were evaluated by an enzyme-linked immunosorbent assay (ELISA). We assayed 107 supernatants after 18 h in-vitro fertilisation of single cultured oocytes (S18) with 100000 motile spermatozoa derived from 11 couples undergoing conventional IVF procedure. The results have been compared to blanks comprised of sperm suspensions incubated without any oocytes. The Gd-content in S18 were correlated to fertilization success and characterized by PN-scoring 18 h after insemination. Furthermore we evaluated supernatants (n = 21) of oocytes after ICSI. Total protein (TP) of all supernatants was assessed for calculating the Gd/TP-ratio. Results: The soluble Gd values were calculated for the cultures belonging to each couple. The levels of Gd differed interindividually by a wide range from 4.9 up to 69.2 ng/mL (0.1–3.0% Gd/TP) and in the blanks 2.0–59.5 ng/mL (0.1–4.5% Gd/TP) as well. We associated a couple specific Gd level from 10.0–60.0 ng/mL with a high fertilisation rate (FR = 88%). Both a soluble Gd at a lower level in sperm–oocyte suspensions and a S18 > 60.0 ng/mL were accompanied by a decreased FR (25%). The supernatants (n = 21) after ICSI (FR = 14%) were found Gd-free. Conclusions: Gd-amounts in S18 fluctuated both between the different patients and within their individual sperm–egg cultures. Therefore the protein is suggested not being introduced by spermatozoa only but rather by cumulus cell effects. Beside other parameters one of the fertilisation-dependant factors may be the patient-specific Gd concentration in the surrounding medium.
292. Influence of the in vitro environment on rat gametes and pre-implantation embryos
Authors: Borg N. R. , Holland M. K.
Published: November 2010

Rat in vitro fertilization (IVF) and culture (IVC) is attempted by few because of its reputation for difficulty. Currently very few functional rat in vitro systems (IVS) exist for sperm–oocyte interaction research. Successful fertilization of rat metaphase II (MII) oocytes was achieved with two different media, Enriched Krebs Ringer Bicarbonate (EKRB) (70.2%) and M16 (57.4%). Using this IVS we have shown that the rat germinal vesicle-intact (GV-i) oocyte lacks the necessary maturity to interact with capacitated caudal epididymal spermatozoa, whether zona pellucida intact (ZP-i) or free (ZP-f). Proteomic analysis of the protein profile of the oolemma from the GV-i stage through to the MII stage in oocytes is being conducted to characterize any maturational changes that may occur. In addition we provide initial evidence to suggest that an acrosome-intact spermatozoa can fuse with the oolemma of a ZP-f MII oocyte during IVF. Although high percentages of polyspermic embryos in ZP-f IVF (64.8–100%) were observed, the possibility that the rat oolemma may undergo a post-fusion block to polyspermy was implied by a small proportion of normally fertilized embryos (3.8–17.0%) in M16 supplemented with different ratios of hyperactived spermatozoa. Despite successful culture to the blastocyst stage for in vivo fertilized zygotes (33.73%) and 2-cell stage embryos (79.3%), IVF embryos have repeatedly failed to develop in culture. Two dimensional analyses of the protein profile of oocytes/embryos immediately prior to fertilization (MII oocyte–101 spots) and the maternal to zygotic transition (MZT) (zygotes–59 spots and 2-cell embryos–84 spots) has shown a difference in patterns of protein expressed. Comparison of IVF zygotes (41 spots) obtained from EKRB displayed reduced protein expression suggesting that nuclear maturation and/or MZT is not being adequately supported. These data illustrate that rat IVF and IVC require suitable media if its problematic reputation is finally to be shed.
293. Gametes alter the oviductal secretory proteome in vivo
Authors: Georgiou A. S. , Sostaric E. , Wong C. H. , Snijders A. P. L. , Wright P. C. , Vazquez J. M. , Roca J. , Martinez E. A. , Moore H. D. , Fazeli A.
Published: November 2010

We sought to identify altered oviductal protein secretions in response to the presence of gametes in the oviduct in vivo at the time of ovulation. Experiments were designed to compare oviductal fluid from a gamete-stimulated oviduct to a non-gamete-stimulated oviduct within the same animal. Clips were introduced at the infundibulum of both oviducts to prevent oocytes from entering the oviducts and one uterine horn was cut to prevent sperm access to that oviduct. Sows were artificially inseminated the next day with diluted boar semen. Control sows that had undergone the same surgical procedures were inseminated with diluent (no sperm). The day after insemination oviducts were removed and oviductal fluid was collected. Two-dimensional gel electrophoresis, isotope coded affinity tag (ICAT) technology and LC-ESI-MS/MS were used to identify and quantify proteins regulated by presence of spermatozoa in the oviduct. To identify the effect of oocyte presence in the oviduct, only one oviduct was clipped at the infundibulum. This prevented oocytes from entering that oviduct. Sows were monitored for ovulation using sonography and 24 h after ovulation oviducts were removed and oviductal fluid was analysed as described above. Results indicated that in vivo, the oviduct responded to the presence of spermatozoa and oocytes by altering its secretory proteomic profile. Our surgical interventions were not responsible for any of these alterations. Many of the identified proteins are known to be involved in oocyte maturation, maintenance of sperm viability, fertilisation, and embryonic development. Our findings suggest that the oviduct responds to gametes by providing specific molecules to sustain, regulate or enhance events preceding and during fertilisation, and early embryonic development. Furthermore, it seems a gamete recognition system is present in the oviduct.
Supported by grants from the BBSRC and Sygen International PLC.
294. Insulin receptor internalization in mouse preimplantation embryos
Authors: Hung F.-C. , Pantaleon M. , Kaye P. L.
Published: November 2010

The insulin receptor (IR) mediates the actions of insulin and insulin-like growth factors (IGF-I and II). Two IR isoforms result from alternate splicing of exon 11, IR-A (without exon 11) and IR-B (with exon 11). Exon 11 is 36 bp and encodes 12 amino acids (717–729) in the COOH-terminus of the IR alpha-subunit. IR-A has higher binding affinity for insulin and IGF-II than IR-B. Interestingly, IR-A is predominantly expressed in fetal tissues, peripheral nerve, brain and tumours whilst IR-B is expressed primarily in classical insulin sensitive tissues such as adult liver and muscle. Our previous studies showed that in mice, like other species, the IR is expressed throughout preimplantation development. IR-B is expressed throughout the preimplantation period, whilst IR-A is expressed following compaction. Immunofluorescent confocal microsopy using an exon11 specific antiserum revealed IR-B immunoreactivity in cell membranes of zygotes and embryos to the morula stage and concentrated in the trophectoderm of blastocysts. Previous studies have shown that insulin can have proliferative effects prior to compaction.1 Consistent with a functional IR at the 2-cell stage, insulin treatment rapidly increased cytoplasmic staining for IR-B within 5-15 min suggesting IR internalization on binding of insulin, which may be either trafficking to the nucleus for regulation of transcription or bound for degradation. Further investigations are underway to address these two options.
(1)Gardner, H. G., and Kaye, P. L. (1991). Insulin increase cell numbers and morphological development in mouse pre-implantation embryos in vitro. Reprod Fertil Dev 3, 79–91.
297. Reproduction in the arrhythmic Bmal1 knockout mouse
Authors: Boden M. J. , Kennaway D. J.
Published: November 2010

There is strong epidemiological evidence indicating that disruption of the endogenous circadian rhythms can cause a range of health problems ranging from metabolic and cardiovascular disorders to reproductive failure. Circadian rhythmicity is generated by a suite of genes called ‘clock genes’ that are cyclically expressed in the brain and peripheral tissues. The CLOCK and BMAL1 transcription factors regulate the expression of many genes involved in cell growth, angiogenesis and development. The Bmal1 knockout mouse provides an interesting model to analyse the impact of arrhythmicity on reproductive physiology. Female Bmal1–/– mice show a delay in the onset of puberty (WT = 32.7 d, KO = 38.6 d, n = 8–16). Female Bmal1–/– mice reproductive tissues are significantly smaller than in WT mice (Ovaries –40%, Oviduct –25%, Uterus –60%, n = 10). Female Bmal1–/– mice have essentially normal estrus cycles (cycle length WT = 4.2 d, KO = 4.8 d, n = 8) and are able to ovulate and mate but are unable to establish viable pregnancies. They are as responsive to a standard superovulation protocol as their wild type littermates (ovulated oocytes WT = 23.8, KO=22.8, n = 7–10), suggesting the ovaries are developmentally competent.
These results suggest disruption of circadian rhythmicity in the mouse affects fertility at multiple sites. Further investigation into the importance of rhythmicity, particularly post ovulation and post fertilisation is required.
299. Sensitivity of embryos to an environmental stressor, ammonium, is dependent on stage of temporal exposure
Authors: Zander D. L. , Thompson J. G. , Lane M.
Published: November 2010

Extended embryo culture in vitro may cause increased cellular perturbations resulting in poorer developmental outcomes. Exposure of embryos to ammonium throughout the entire pre-implantation period decreased cell number and ICM development, increased apoptosis and perturbs glucose metabolism. The aim of this study was to examine the relative susceptibility of the pre- and post-compaction stage embryo to these perturbations resulting from temporal exposure to ammonium. Mouse embryos (n = 350 per treatment) were collected from F1 female mice. Embryos were exposed to either control medium or medium with 300 μM ammonium for the entire culture period. Temporal treatments involved culture with or without ammonium, from the zygote to 2-cell stage, 2-cell to 8-cell stage, or the 8-cell to the blastocyst stage. At the blastocyst stage, ICM development, apoptosis, gene expression and glucose metabolism were assessed. Differences between treatments were determined using generalised linear modelling and LSD post-hoc tests. Exposure to ammonium at any stage did not affect blastocyst development. Exposure to ammonium pre-compaction significantly decreased both blastocyst and ICM cell number while these were unaffected when exposure occurred post-compaction. Levels of apoptosis were significantly increased when exposure to ammonium was continual to the blastocyst stage (6.5% compared to control 2.4%, P < 0.05) or from the zygote to the 2-cell stage (5.8%, P < 0.05). However, apoptosis was not altered during post-compaction exposure (2.8%). Glucose uptake was decreased by culture with ammonium at all stages of development (P < 0.001). Gene expression of GLUT1 in the blastocyst was not altered by ammonium while GLUT3 expression was significantly reduced by exposure at all stages of development (P < 0.01). The data presented suggests that the pre-compaction stage embryo is most susceptible to ammonium stress and the effects of this early stage exposure appear irreversible. Intriguingly, glucose uptake and GLUT3 expression at the blastocyst stage appear to be markers of ammonium exposure.
300. Differential expression patterns of genes with immune and developmental relevance in individual bovine preimplantation embryos produced by nuclear transfer
Authors: Moser R. , Bower N. , Reverter T. , Donaldson L. , Hill J. , Lehnert S.
Published: November 2010

The overall efficiency of cloning by somatic cell nuclear transfer (NT) remains low when compared to in-vitro fertilisation (IVF). Inefficient reprogramming of the donor nucleus and epigenetic effects are hypothesized to lead to aberrant gene expression in cloned embryos. In this work differences in gene expression, between blastocysts from IVF and NT derived pre-implantation bovine embryos, were explored using a bovine cDNA array. 439 array elements were identified as being differentially expressed. 102 elements showing upregulation and 337 elements downregulation in NT blastocysts in a pairwise comparison to IVF-derived blastocysts. A major subset (65 elements) of differentially expressed elements comprised immune-related genes, possibly a reflection of the immune-focused cDNA microarray used in this study. For example, several cytokine genes, as well as elements of the innate immune system, were showing elevated expression in NT blastocysts. In addition, two regulatory elements of the same endocrine pathway exhibited a remarkable expression pattern, where the gene for one inhibitor showed upregulation and the gene for another inhibitor showed downregulation in NT blastocysts. Of specific interest is the observation that genes belonging to two growth factor pathways are upregulated in NT embryos. These results suggest an important role for immune-related genes during embryogenesis and indicate that specific cell growth and differentiation factors are of significant interest as targets in defining the abnormalities of preimplantation NT embryo development.
301. Effect of exogenous transforming growth factor beta 1 on reproductive performance in male TGFβ1 null mice
Authors: McGrath L. J. , Robker R. , Robertson S. A.
Published: November 2010

The transforming growth factor beta 1 (TGFβ1) family are potent cytokines that regulate tissue development, inflammation and immunity. Our studies in null mutant mice implicate a key role for TGFβ1 in male reproductive function. The TGFβ1 null mutation results in profound infertility due to inability to copulate successfully associated with reduced testosterone synthesis, although penile erection and sperm production do occur. To investigate whether fertility status can be improved in TGFβ1 null mutant mice by exogenous cytokine replacement, we used Alzet mini-pumps implanted subcutaneously to deliver a constant supply of recombinant latent TGFβ1 to TGFβ–/– mice (n = 7, 2.1µg/day over 2 weeks). Control TGFβ–/– mice (n = 6) and +/+ mice (n = 10) received pumps containing BSA carrier only. Circulating levels of TGFß1 were increased in TGFβ–/– mice and reached levels comparable to those seen in fertile heterozygote littermates. Increased circulating testosterone was evident in a proportion of TGFβ–/– mice after exogenous TGFβ replacement compared with untreated control mice. However, serum testosterone content was widely variable within all groups, so statistical significance was not achieved. Videotaping of nocturnal mating behaviour while caging treated males with normal receptive female mice showed that unlike TGFβ+/+ mice, which successfully mounted and intromitted, untreated TGFβ–/– mice failed to engage in normal mating behaviour. TGFβ–/– mice treated with exogenous cytokine were occasionally seen to intromit but less frequently than TGFβ+/+ controls. Ejaculation did not occur in any of the TGFβ–/– mice regardless of TGFβ replacement, compared with TGFβ+/+ mice where 8/10 mice ejaculated during the 2 h observation period. The trend towards improvement in both testosterone levels and copulation activity of the TGFβ1 null mice treated with exogenous cytokine suggests that systemic TGFβ1 availability may influence reproductive performance in male mice. However, since fertility was not restored by cytokine replacement, locally produced TGFβ in the reproductive tract and/or hypothalamic pituitary axis are also implicated in regulating fertility.
302. Characterisation of lipopolysaccharide (LPS) receptor expression and the inflammatory response of the rat testis
Authors: Winnall W. , Muir J. , Hutchinson P. , Hedger M.
Published: November 2010

Inflammation in the testis is disastrous for the developing spermatogenic cells, leading to temporary and sometimes permanent sterility. The majority of testicular macrophages display a unique protective phenotype whereby production of some key inflammatory mediators, specifically interleukin-1β (IL-1β), tumour necrosis factor-α (TNFα) and NO, in response to stimulation with LPS is relatively poor. Leydig cells and Sertoli cells also respond to high doses of LPS, producing the inflammatory cytokines, particularly IL-1α and IL-6. Although these data suggest that the LPS receptor (toll-like receptor 4, TLR4) and its associated binding proteins, CD14 and MD2, are expressed on several testicular cell types, expression of these proteins in the testis has not been described previously.
Using real-time PCR and Western blotting, we established that TLR4, CD14 and MD2 are all expressed by testicular macrophages, Leydig cells, Sertoli cells, spermatocytes and round spermatids. Unexpectedly, the spermatogenic cells displayed the highest level of TLR4 surface expression as determined by flow cytometry. There was no response of the spermatogenic cells to LPS stimulation in vitro, at least in terms of mRNA expression for the inflammatory cytokines, IL-1α, IL-1β, TNFα, IL-6, activin A and the chemoattractants, CXCL-1 and CXCL-2. Although production of several cytokines was relatively diminished, testicular macrophages responded to LPS with a significant increase in mRNA expression for all of these inflammatory regulators. These data indicate that the protective phenotype of the testicular macrophages is not due to insensitivity to LPS or absence of the receptor, but may involve downstream regulation of specific inflammatory responses. The data also suggest that spermatogenic cells are capable of responding to TLR4 ligands, although not by producing inflammatory mediators. The actual function of the LPS receptor on the spermatogenic cells remains to be discovered.
303. Further characterisation of lymphocyte-suppressing activity in gonadal fluids
Authors: Crane M. , Foulds L. , Muir J. , Aridi D. , Hutchinson P. , Scott B. , De Kretser D. , Hedger M.
Published: November 2010

Protection of the developing gametes from an autoimmune response within the testis and ovary is essential for reproductive success, and autoimmune infertility represents a failure of this protection. The gonads also represent favorable sites for grafts of foreign tissue, that is, they are ‘immunologically privileged’. The actual mechanisms responsible for testicular and ovarian immune privilege are poorly understood. However, it has been well established that testicular interstitial fluid and ovarian fluid have profound inhibitory effects on T-cell activation and proliferation in vitro. We have established previously that a partially purified preparation of the inhibitor, isolated from bovine follicular fluid, suppresses proliferation in an in vitro T-cell activation assay, through induction of T-cell anergy and/or atypical apoptosis. Addition of increasing doses of normal fetal calf serum and/or bovine serum albumin blocks the actions of the inhibitor and progressively increases the ED50 of the assay. It has also been shown that stimulating the T-cells with phorbol-12-myristate-13-acetate (PMA) in place of a polyclonal mitogenic stimulus such as phytohaemagglutinin bypasses the anergic effects of the inhibitor. These results suggest that the activity of the inhibitor may be negatively regulated in the circulation and tissues by serum-derived proteins and other factors. These data also indicate that the inhibitor’s activity is mediated through a specific cellular pathway, most likely involving protein kinase C isotypes, which are activated by PMA. Further work will delineate the molecular pathways and mechanisms of serum regulation of the gonadal lymphocyte-suppressing activity, which may be exploited in the treatment of autoimmune diseases and for prevention of transplant rejection.