Prostaglandins have been implicated in several neovascular diseases. In the present study, we fou... more Prostaglandins have been implicated in several neovascular diseases. In the present study, we found elevated FP receptor and vascular endothelial growth factor (VEGF) expression colocalized in glandular epithelial and vascular cells lining the blood vessels in endometrial adenocarcinomas. We inves- tigated the signaling pathways activated by the FP receptor and their role in modulating VEGF expression in endometrial adenocarcinoma (Ishikawa)
Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of... more Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of oocytes derived from abattoir material. For application of IVF technology to captive endangered species, however, noninvasive techniques are required which allow repeated collection of oocytes from live females. The aim of this study was to develop a method for embryo production from mature oocytes collected laparoseopically from red deer hinds. Follicular development was synchronized in red deer hinds by the insertion of intravaginal progesterone-releasing devices for 10 d, and ovarian stimulation was induced with 1000 IU, im PMSG 48 h before progesterone device removal. Oocytes were harvested by laparoscopy under xylazine/ketamine sedation 24 h after progesterone device removal and then co-incubated with frozen-thawed red deer spermatozoa for 24 h. In Experiment 1, oocytes and embryos were fixed and stained at different developmental timepoints. Their external morphological changes (cumulus expansion, extrusion of the second polar body and cytokinesis) paralleled their nuclear developmental changes (formation of the 2nd metaphase spindle of meiosis, pronuclear formation and nuclear division, respectively). In Experiment 2, embryos were maintained in vitro until they ceased to undergo cell division. A total of 39 aspiration procedures was carried out on 14 red deer hinds. Forty-four cumulus-oocyte complexes (COC) were aspirated from 95 large Graafian follicles; of these, 27 were classed as mature/nondegenerated on the basis of cumulus/cytoplasmic morphology. Seventeen oocytes cleaved following in vitro fertilization, yielding six 2-cell embryos, six 4-cell embryos, four 8cell embryos and one 16-celt embryo. The results indicate that laparoscopic aspiration of mature oocytes from hormone-treated females offers a valuable source of genetic material for assisted deer breeding programs.
Superovulation has proved to be a problematic part of embryo transfer in red deer as in other dom... more Superovulation has proved to be a problematic part of embryo transfer in red deer as in other domestic ruminants. Use of PMSG in red deer hinds has been associated with disturbance of follicular development and the endocrine environment. In this study, the effects of PMSG neutralization with a monoclonal antibody on the ovulation rate and the associated LH surge were investigated. Thirty mature hinds were treated during the breeding season with intravaginal progesterone impregnated CIDR devices and divided into 6 treatment groups. Group 1 received no further treatment and served as controls. All other groups were treated with 1200 IU PMSG at-72 h (CIDR withdrawal=time 0). Group 2 received no further treatment. Groups 3 through 6 received a single intravenous injection of a monoclonal antibody to PMSG at-72, 0, 12 and 24 h respectively. Blood samples were taken throughout the experiment and for a 72 h intensive sampling period following CIDR device withdrawal and analyzed for progesterone and LH, respectively. Ovarian response was assessed at laparoscopy on Day 7. The proportion of animals with more than 1 corpus luteum was higher in PMSG-only and +24 h groups than in control,-72, 0 and +12 h groups (P<O.O5). Compared with the PMSG-only treated animals, ovulation rate in PMSG-treated hinds was reduced following treatment with the antibody at-72, 0 and +12 h (PcO.05) and the number of large Graafian follicles was reduced in all antibodytreated groups. The incidence of a LH surge was lower in the 0 and +12 h than in the PMSGonly and +24 h groups (P<O.OS). Peak progesterone concentrations in hinds treated with PMSG only and antibody at +24 h were higher than in hinds of the other treatment groups (P<O.OS). Two hinds treated with PMSG alone underwent premature luteal regression, evinced by a sharp decline in progesterone after Day 7. These results suggest that passive immunization neutralizes the biological action of PMSG in red deer, and may provide a means of overcoming some of the adverse effects of the hormone. However, the timing of the antibody treatment is crucial to ensure lack of interference with the endogenous LH surge and subsequent ovulation.
An alarming worldwide extinction of animal species is taking place as a result of the activities ... more An alarming worldwide extinction of animal species is taking place as a result of the activities of the increasing global human population. The original ranges of many animal species are being reduced and fragmented and, in some cases, they have been reduced to perilously small relict populations. The adverse genetic consequences of these restrictions are becoming clear, as are possible methods for their alleviation. The concept of ex situ genetic management of small captive populations of endangered species with a view to re‐introducing them into the wild is attracting increasing interest. Modern reproductive techniques will play an important role in such programmes, and it is likely that an increasing number of veterinarians will become involved. However, the literature describing the aims and methods of reproductive genetic management is scattered and often not readily available to interested veterinary surgeons. The aim of this review is to deal with this problem by describing s...
In order to investigate the effectiveness of ovine FSH as a superovulatory gonadotrophin in sheep... more In order to investigate the effectiveness of ovine FSH as a superovulatory gonadotrophin in sheep, three batches (oFSH-1, oFSH-2 and oFSH-3) of crude follicle stimulating hormone (FSH) extracts were prepared from ovine pituitaries by protein fractionation. The extracts were characterized in radioimmunoassays (RIA), radioreceptor assays (RRA) and bioassays. A FSH:LH (luteinizing hormone) ratio of 1:0.71 and 1:0.18 was calculated from oFSH-1 in RIA and RRA respectively. oFSH-1 was more potent than NIH-oFSH-S12 in RRA, with a potency ratio of 1:0.65 calculated at 50% binding. In mouse bioassay, oFSH-2 was effective in inducing folliculogenesis. The potency of oFSH-3 as an ovarian stimulatory hormone was tested in a field trial on sheep. Thirty-one mature Merino ewes were treated in the breeding season with intravaginal progestagen pessaries and 6, 12 or 24 mg doses of oFSH-3 were injected twice daily for 3 days (n = 10 or 11). Each ewe was inseminated in the uterus with fresh semen 24 ...
The prostaglandin F2 alpha analogue cloprostenol stimulates ovarian secretion of oxytocin in red ... more The prostaglandin F2 alpha analogue cloprostenol stimulates ovarian secretion of oxytocin in red deer hinds and Pere…
Two experiments were conducted with mature Merino ewes to investigate the effects of time and sit... more Two experiments were conducted with mature Merino ewes to investigate the effects of time and site of insemination of fresh and frozen-thawed semen on the fertility of superovulated ewes. In Experiment 1, each ewe was treated with an intravaginal progestagen sponge and PMSG and/or FSH. They were inseminated in the uterus with fresh or frozen-thawed semen (approximately 100 x 10(6) motile sperm) at 24, 44 or 64 h after sponge withdrawal. Ova were recovered at 88 h after sponge withdrawal and classified as fertilized if they had pronuclei or had cleaved. Mean fertilization rates of recovered ova were 60.0, 93.7 and 87.8% for fresh semen and 46.1, 98.2 and 26.1% for frozen-thawed semen at each of the insemination times (24, 44 and 64 h) respectively. Overall, fertilization rates were higher for fresh semen than for frozen-thawed semen (P less than 0.01), but there was an interaction with time of insemination (P less than 0.01). Following insemination with frozen-thawed semen at 64 h, o...
Thirty-four European fallow deer (Dama dama dama) were randomly allocated into embryo donor (n = ... more Thirty-four European fallow deer (Dama dama dama) were randomly allocated into embryo donor (n = 12) or embryo recipient (n = 22) groups. All does were treated with controlled internal drug release (CIDR) devices for 14 days. Animals in the embryo donor group were further treated with 200 I.U. pregnant mare serum gonadotrophin (PMSG) and 0.5 units ovine follicle-stimulating hormone (FSH). PMSG was administered 72 h before withdrawal of CIDR devices and FSH was given in eight 0.063 unit injections at 12-hourly intervals starting at the time of PMSG administration. All embryo donor animals were inseminated, by laparoscopy in both uterine horns, 36 h after withdrawal of CIDR devices with 25 x 10(6) fresh spermatozoa collected from Mesopotamian fallow deer (Dama dama mesopotamica). Embryos were recovered by laparotomy on Day 3 (n = 6) or Day 6 (n = 6) after withdrawal of CIDR devices and the ovarian response was determined. In total, 22 embryos were transferred into the oviduct (2-4-cel...
Administration of gonadotrophin releasing hormone (GnRH) 24 h after sponge withdrawal did not aff... more Administration of gonadotrophin releasing hormone (GnRH) 24 h after sponge withdrawal did not affect the numbers of corpora lutea (CL) or persistent large follicles (LF) in ewes superovulated with 400 I.U. pregnant mare serum gonadotrophin and 12 mg follicle stimulating hormone in spring (11.6 +/- 0.9 v. 13.0 +/- 0.9 CL and 0.8 +/- 0.9 v. 0.9 +/- 0.3 LF, for +GnRH and -GnRH ewes, respectively). However, it did increase the ovulatory response of ewes superovulated in autumn (15.8 +/- 1.2 v. 11.8 +/- 1.1 CL). The incidence of ewes with prematurely regressed CL was also greater in autumn than in spring (21/89 v. 5/88). Supplementary feeding with lupin grain in autumn had no effect on numbers of CL but did increase the incidence of ewes with LF (18/48 v. 7/46) and caused a marked reduction in the incidence of ewes with regressed CL (1/44 v. 20/45). For ewes treated in autumn, there were no effects of lupin supplementation or GnRH administration on peak oestradiol-17 beta (E2) or peak lu...
Six conceptuses were collected from red deer hinds on day 22 after synchronization of oestrus wit... more Six conceptuses were collected from red deer hinds on day 22 after synchronization of oestrus with intravaginal progesterone-releasing devices (removal of device = day 0). Within 24 h of culture in vitro, the supernatant from five of six conceptuses showed detectable antiviral activity. Interferon \g=a\(IFN-\g=a\)receptors were identified by immunohistochemistry on the luminal surface of the endometrium, in the neurohypophysis and paraventricular hypothalamus, but not in the ovaries of the hinds from which the conceptuses were collected. Another 16 intact hinds were synchronized as above. Injection of 4 mg IFN i.m. twice a day on days 13\p=n-\15had no effect on cloprostenol-induced oxytocin secretion on day 15 and did not prevent cloprostenol\x=req-\ induced luteal regression. Sixteen ovariectomized hinds received a protocol of steroid treatment to mimic ovarian hormone secretion during the normal oestrous cycle. On day 16, hinds showed undulant oxytocin secretion that showed a degree of temporal association with uterine PGF2\g=a\ release. Treatment with 4 mg IFN-\g=a\11twice a day on days 13\p=n-\16had no effect on this spontaneous oxytocin secretion, but reduced the magnitude of cloprostenol-induced oxytocin secretion on day 17 (P < 0.05). These results indicate that red deer conceptuses secrete an anti-luteolytic IFN to which the endometrium expresses a receptor during early pregnancy. The presence of IFN receptors in the hypothalamus and posterior pituitary and the IFN-induced suppression of extra-ovarian oxytocin secretion provides tentative evidence of an involvement of the central nervous system in maternal recognition of pregnancy in deer.
Three experiments were carried out to investigate the secretion of luteolytic hormones in red dee... more Three experiments were carried out to investigate the secretion of luteolytic hormones in red deer hinds during the oestrous cycle, early pregnancy and after administration of interferon, the putative pregnancy recognition signal. Three groups of hinds (n = 8\p=n-\9 per group) were treated with progesterone-impregnated intravaginal controlled internal drug releasing (CIDR) devices for 13 days (device withdrawal = day 0). Group 1 (n = 9) served as controls; Group 2 (n = 8) received injections of 4 mg recombinant bovine interferon-\g=a\11 twice a day on days 13\p=n-\18; Group 3 (n = 9) were run with a fertile stag on days 0\p=n-\3.Plasma samples collected each day on days 16\p=n-\23were analysed for progesterone. Plasma samples collected each hour for 16 h on days 4, 10, 16, 18 were analysed for oxytocin and the prostaglandin F2\ g=a\(PGF2\g=a\) metabolite (PGFM). Plasma progesterone concentrations declined to < 1 ng ml\m=-\1 between days 18 and 25 in control hinds indicating that luteolysis had occurred, whereas there was no endocrine evidence of luteolysis in interferon-treated or pregnant hinds. Control hinds (6/9) exhibited synchronous pulses of oxytocin and PGF2\g=a\ secretion on day 18, a greater proportion than on any other day in these hinds or on any day in the interferon-treated or mated hinds (P<0.05). In a second experiment, close synchrony in secretion of oxytocin and PGF2\g=a\pulses was evident in an unmated hind when samples were collected every 12 min on day 18. In a third experiment, oxytocin-induced PGF2\g=a\ secretion was potentiated by oxytocin administration at an interval of 1 h and inhibited by administration at a 6 h interval (P < 0.05). These results suggest that synchronous pulsatile secretion of oxytocin and PGF2\g=a\ induces luteolysis and is suppressed by pregnancy or administration of interferon. Oxytocin-induced alterations in uterine oxytocin sensitivity may underlie the pulsatile nature of luteolytic hormone secretion in red deer.
Prostaglandin (PG) E 2 E-series prostanoid-2 (EP2) receptor is elevated in numerous carcinomas in... more Prostaglandin (PG) E 2 E-series prostanoid-2 (EP2) receptor is elevated in numerous carcinomas including the endometrium and has been implicated in mediating the effects of PGE 2 on vascular function. In this study, we investigated the intracellular signaling pathways that are activated by the EP2 receptor and their role in regulation of the expression of vascular endothelial growth factor in endometrial adenocarcinoma (Ishikawa) cells. Ishikawa cells were stably transfected with EP2 receptor cDNA in the sense or antisense directions. Treatment of Ishikawa cells with PGE 2 rapidly induced transactivation of the epidermal growth factor receptor (EGFR) and activation of ERK1/2 via the EP2 receptor. Preincubation of cells with chemical in
The present study investigated the pattern and site of expression of the prolactin receptor gene ... more The present study investigated the pattern and site of expression of the prolactin receptor gene in the testis of the seasonally breeding Soay sheep. In experiment 1, Northern blot analysis confirmed expression of the prolactin receptor gene in the testis which was encoded by RNA transcripts of approximately 3.6, 11.2, 12.6, and 14.1 kb. In situ hybridisation localised expression of the receptor within the interstitial and seminiferous tubule compartments of the testis and immunohistochemistry localised expression of the receptor to Leydig cells and to pachytene spermatocytes, round and elongating spermatids. In experiment 2, phosphorylation of Jak2, Stat1 and Stat5 proteins in response to prolactin was investigated by Western blotting following incubation of testicular samples with 100 ng/ml ovine prolactin. Jak2 and Stat1 phosphorylation were induced by prolactin within 10 min and Stat5 within 30 min. In experiment 3, intact and hypothalamo-pituitary disconnected (HPD) rams were transferred from a short to a long day photoperiod regimen for a period of 8 weeks. By week 8, testicular diameter had declined in intact rams (52.71+/-1.06 cm vs. 48.00+/-0.49 cm for weeks 1 and 8 after transfer respectively, P&amp;amp;lt;0.01, n = 3) and increased in HPD rams (27.00+/-0.45 cm and 29.66+/-0.99 cm for weeks 1 and 8 after transfer respectively, P&amp;amp;lt;0.05, n = 3). RT-PCR using RNA extracted from intact and HPD rams confirmed expression of the prolactin receptor in the testis of both groups. Immunohistochemistry localised prolactin receptor expression in Leydig cells and in pachytene spermatocytes, round and elongating spermatids of intact sheep testis. In HPD rams, prolactin receptor expression was localised in Leydig cells and germ cells which were arrested predominantly at the pachytene spermatocyte stage. These data demonstrate expression of functional prolactin receptors in the testis of Soay rams. The site and pattern of expression of the receptor gene suggest a role for prolactin in the regulation of steroidogenesis and spermatogenesis.
ABSTRACT A total of 121 European fallow deer does, being either parous (n= 15) or nulliparous (n=... more ABSTRACT A total of 121 European fallow deer does, being either parous (n= 15) or nulliparous (n= 106), were treated with intravaginal progesterone impregnated controlled internal drug release (CIDR) devices for 14 days. The does were divided into three treatment groups and inseminated in utero by laparoscopy, at approximately 65 hours after CIDR device removal, with 25 × 106 fresh Mesopotamian (n= 40), 25–35 × 106 frozen-thawed Mesopotamian (n= 41) or 30–32.5 × 106 frozen-thawed European (n= 40) fallow deer spermatozoa. The semen used had been collected, from two Mesopotamian and two European fallow deer bucks, by electroejaculation under general anaesthesia. Pregnancy was diagnosed by rectal ultrasonogrdphy on Day 50 after insemination. There were no apparent differences in the quality of ejaculates between the two subspecies of fallow deer. The volume of semen and the total number of spermatozoa ranged between 0.6–1.2 ml and 2.11–4.95 × 109 per ml of semen, respectively. Evaluation of frozen-thawed spermatozoa revealed post-thaw motility rates between 50–70%. The overall conception rate was 65.3%. A higher conception rate was observed following insemination with European than Mesopotamian frozen-thawed spermatozoa (75% vs. 53.7%, respectively, P &lt; 0.05). Insemination with fresh Mesopotamian spermatozoa increased the conception rate to a level not significantly different from that observed following insemination with European frozen-thawed spermatozoa (67.5% vs. 75%, for fresh Mesopotamian and frozen-thawed European semen, respectively).
The Journal of Clinical Endocrinology & Metabolism, 2002
A role for prostaglandins (PGs) in the regulation of endometrial functions, such as menstruation,... more A role for prostaglandins (PGs) in the regulation of endometrial functions, such as menstruation, has been established, although the mechanisms by which this is achieved are not fully elucidated. In the present study, cDNA array analysis has identified endothelial monocyte-activating polypeptide II (EMAP II) as a PGE 2-regulated gene in endometrial epithelial cells. Incubation of endometrial epithelial cells with 100 nM PGE 2 for 4 and 24 h resulted in a 2.3-and 16-fold decrease in EMAP II expression, respectively. In endometrial tissue collected across the menstrual cycle, a significant increase in EMAP II mRNA was observed during the late secretory phase, compared with the proliferative and early-midsecretory phases. The temporal pattern of EMAP II expression was confirmed further by Western blotting; EMAP II protein expression was detected as a 43-kDa band. In situ hybridization and immunohistochemistry localized EMAP II mRNA and protein expression in glandular epithelial, endothelial, and stromal cells in the functionalis and basalis layers of the endometrium. Finally, the role of PGE 2 in the regulation of EMAP II expression in the endometrium was assessed. Incubation of fresh endometrial tissue (n ؍ 5) with 3 g/ml indomethacin resulted in an increase in EMAP II protein expression, compared with control untreated tissue. However, cotreatment of the cells with 100 nM PGE 2 resulted in a significant decrease in EMAP II protein expression, compared with tissue incubated with indomethacin alone (P < 0.05). These data confirm temporal variation in EMAP II expression in the human endometrium across the menstrual cycle and localize expression to glandular epithelial, endothelial, and stromal cells. Moreover, EMAP II expression is negatively regulated by PGE 2 .
The Journal of Clinical Endocrinology & Metabolism, 2004
Prostaglandin (PG) E 2 promotes tumor growth via interaction with its G protein-coupled receptors... more Prostaglandin (PG) E 2 promotes tumor growth via interaction with its G protein-coupled receptors and activation of intracellular signaling. Tuberous sclerosis 2 (tuberin) is a tumor suppressor, which negatively regulates cell growth. Its phosphorylation results in its inactivation and targeted downregulation, thus lifting the growth inhibition effects. This study investigated the expression and localization of tuberin in neoplastic and normal endometrium and the effect of PGE 2 on phosphorylation of tuberin via the Akt pathway. Quantitative RT-PCR and Western blot analysis demonstrated reduced expression of tuberin in neoplastic tissue, compared with normal endometrial tissue. Tuberin expression was localized by immunohistochemistry to the glandular epithelial compartment in neoplastic and normal endometrium. We investigated the effect of PGE 2 on phosphorylation of tuberin via the Akt pathway. Treatment of neoplastic and normal endometrium with 100 nM PGE 2 enhanced phosphorylated tuberin immunoreactivity in the glandular epithelium. PGE 2 also phosphorylated Akt and tuberin in Ishikawa endometrial adenocarcinoma cells, leading to a reduction in expression of total tuberin protein. Cotreatment of cells with wortmannin or LY294002 inhibited the PGE 2-induced phosphorylation of Akt and tuberin. These data suggest that PGE 2 signaling may promote endometrial tumorigenesis by inactivation of tuberin after its phosphorylation via the Akt signaling pathway.
The Journal of Clinical Endocrinology & Metabolism, 2003
Prostaglandin (PG) F 2␣ , a member of the prostanoid bioactive lipid family, is secreted by human... more Prostaglandin (PG) F 2␣ , a member of the prostanoid bioactive lipid family, is secreted by human endometrium throughout the menstrual cycle and is present in both menstrual fluid and medium of endometrial explants in culture. PGF 2␣ mediates its effects through a seven-transmembrane G-protein-coupled receptor (FP). The aim of this study was to examine the temporal expression, signaling, and role of FP receptor in the human endometrium. Quantitative RT-PCR analysis demonstrated highest expression of FP receptor in the mid-to lateproliferative phase, compared with early-proliferative and secretory phase endometrium. In situ hybridization studies localized FP receptor mRNA expression to the epithelial cell compartment during the mid-to late-proliferative phase. Moreover, treatment of endometrial tissue with 1-100 nM PGF 2␣ induced a concentration-dependent increase in inositol phosphate mobilization, indicating functional FP receptor expression. The Ishikawa human endometrial epithelial cell line was used to investigate further the signaling and role of PGF 2␣ in endometrial epithelial cells. Ishikawa cells endogenously express the FP receptor, and treatment with 1-100 nM PGF 2␣ elicits a concentration-dependent increase in inositol phosphate release. Moreover, treatment of Ishikawa cells with 100 nM PGF 2␣ induced phosphorylation of ERK1/2 that was abolished when cells were cotreated with 50 M PD98059 (MAPK kinase inhibitor) or 10 M U73122 [phospholipase C (PLC) inhibitor]. Treatment of Ishikawa cells with PGF 2␣ for 24 h induced a significant concentration-dependent increase in Ishikawa cell proliferation. Coincubation of the cells with 50 M PD98059 or 2 M U73122 demonstrated that PLC inhibition significantly reduced PGF 2␣-induced proliferation, whereas MAPK kinase inhibition had no effect. In summary, these studies demonstrate increased FP receptor expression in endometrial epithelial cells during the proliferative phase of the menstrual cycle and identify a role for PGF 2␣ in epithelial cell proliferation via a PLC-dependent pathway.
BACKGROUND: Although the mechanisms underlying the causes of heavy menstrual blood loss (MBL) rem... more BACKGROUND: Although the mechanisms underlying the causes of heavy menstrual blood loss (MBL) remain to be elucidated, prostaglandins have been previously implicated. This study was initiated to elucidate a pattern of expression of the various components of the cyclooxygenase (COX)-prostaglandin signalling pathways present in the endometrium of women with normal and heavy MBLs. METHODS: Endometrial biopsies were collected at different stages of the menstrual cycle from women who underwent measurement of MBL. Tissue was divided for either examination of gene expression by quantitative RT-PCR analysis or in vitro culture experimentation. RESULTS: Analysis of gene expression demonstrated a significant elevation in expression of COX-1 and COX-2 mRNA in endometrium obtained from women with heavy MBL when compared with endometrium obtained from women with normal MBL. Tissue culture with PGE 2 stimulation caused a significantly elevated production of cyclic AMP (cAMP) by endometrium of women with heavy MBL when compared with normal MBL. Expression of phosphodiesterase 4B, an enzyme involved in cAMP breakdown, was reduced in these same endometrial samples obtained from women with heavy MBL. CONCLUSIONS: These data identify the E series prostaglandin receptors and their signalling pathways as potential therapeutic targets in the treatment of heavy menstruation.
BACKGROUND: Prostaglandin E 2 (PGE 2) has been shown to modulate angiogenesis and tumour progress... more BACKGROUND: Prostaglandin E 2 (PGE 2) has been shown to modulate angiogenesis and tumour progression via the E-series prostanoid-2 (EP2) receptor. Endometrial adenocarcinomas may be exposed to endogenous PGE 2 and exogenous PGE 2 , present at high concentration in seminal plasma. METHODS: This study investigated fibroblast growth factor 2 (FGF2) mRNA expression and cell signalling in response to seminal plasma or PGE 2 , using an endometrial adenocarcinoma (Ishikawa) cell line stably expressing the EP2 receptor (EP2 sense cells) and endometrial adenocarcinoma explants. RESULTS: Seminal plasma and PGE 2 induced a significant up-regulation of FGF2 expression in EP2 sense but not parental untransfected Ishikawa (wild-type) cells (P < 0.05). These effects were inhibited by cotreatment with EP2 receptor antagonist or inhibitors of protein kinase A, c-Src, epidermal growth factor receptor (EGFR) kinase or extracellular signal-regulated kinase (ERK) signalling. The treatment of EP2 sense cells with seminal plasma induced cAMP accumulation and phosphorylation of c-Src, EGFR kinase and ERK via the EP2 receptor. Finally, seminal plasma and PGE 2 significantly increased FGF2 mRNA expression in endometrial adenocarcinoma tissue explants via the EP2 receptor (P < 0.05). CONCLUSIONS: Seminal plasma and PGE 2 can similarly activate FGF2 expression and EP2 receptor signalling in endometrial adenocarcinoma cells. These data highlight the potential for seminal plasma exposure to facilitate tumorigenesis-angiogenesis in endometrial adenocarcinomas in vivo.
Times of the ovulatory LH surge and characteristics Social interactions affect reproductive funct... more Times of the ovulatory LH surge and characteristics Social interactions affect reproductive function in a of the rise in circulating progesterone concentrations wide range of species. Among mammals there are nuafter ovulation in red deer hinds were investigated in merous examples of the suppression or delay of reprorelation to each animal's dominance status. Observaduction in subordinate individuals, notably among pritions were made during the 1992 (experiment 1) and 1993 mates (Abbott and Hearn, 1975; Dewsbury, 1982; Ber-(experiment 2) breeding seasons, while the same 12 covitch, 1993), carnivores (Creel and MacDonald, 1995) hinds were held in a pen in the absence of a stag. Ovulaand rodents (Jarvis, 1981; Pratt and Lisk, 1991). There tion was synchronized by administration of progesteris also an extensive literature on the ways in which one followed by luteolytic prostaglandin F 2a analogue. neuroendocrine mechanisms mediate effects of social Social status was determined for each hind by noting status on reproduction (Batty, Herbert, Keverne, and dyadic agonistic interactions during the period of pro
Prostaglandins have been implicated in several neovascular diseases. In the present study, we fou... more Prostaglandins have been implicated in several neovascular diseases. In the present study, we found elevated FP receptor and vascular endothelial growth factor (VEGF) expression colocalized in glandular epithelial and vascular cells lining the blood vessels in endometrial adenocarcinomas. We inves- tigated the signaling pathways activated by the FP receptor and their role in modulating VEGF expression in endometrial adenocarcinoma (Ishikawa)
Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of... more Most current protocols of in vitro fertilization in ruminants are based on in vitro maturation of oocytes derived from abattoir material. For application of IVF technology to captive endangered species, however, noninvasive techniques are required which allow repeated collection of oocytes from live females. The aim of this study was to develop a method for embryo production from mature oocytes collected laparoseopically from red deer hinds. Follicular development was synchronized in red deer hinds by the insertion of intravaginal progesterone-releasing devices for 10 d, and ovarian stimulation was induced with 1000 IU, im PMSG 48 h before progesterone device removal. Oocytes were harvested by laparoscopy under xylazine/ketamine sedation 24 h after progesterone device removal and then co-incubated with frozen-thawed red deer spermatozoa for 24 h. In Experiment 1, oocytes and embryos were fixed and stained at different developmental timepoints. Their external morphological changes (cumulus expansion, extrusion of the second polar body and cytokinesis) paralleled their nuclear developmental changes (formation of the 2nd metaphase spindle of meiosis, pronuclear formation and nuclear division, respectively). In Experiment 2, embryos were maintained in vitro until they ceased to undergo cell division. A total of 39 aspiration procedures was carried out on 14 red deer hinds. Forty-four cumulus-oocyte complexes (COC) were aspirated from 95 large Graafian follicles; of these, 27 were classed as mature/nondegenerated on the basis of cumulus/cytoplasmic morphology. Seventeen oocytes cleaved following in vitro fertilization, yielding six 2-cell embryos, six 4-cell embryos, four 8cell embryos and one 16-celt embryo. The results indicate that laparoscopic aspiration of mature oocytes from hormone-treated females offers a valuable source of genetic material for assisted deer breeding programs.
Superovulation has proved to be a problematic part of embryo transfer in red deer as in other dom... more Superovulation has proved to be a problematic part of embryo transfer in red deer as in other domestic ruminants. Use of PMSG in red deer hinds has been associated with disturbance of follicular development and the endocrine environment. In this study, the effects of PMSG neutralization with a monoclonal antibody on the ovulation rate and the associated LH surge were investigated. Thirty mature hinds were treated during the breeding season with intravaginal progesterone impregnated CIDR devices and divided into 6 treatment groups. Group 1 received no further treatment and served as controls. All other groups were treated with 1200 IU PMSG at-72 h (CIDR withdrawal=time 0). Group 2 received no further treatment. Groups 3 through 6 received a single intravenous injection of a monoclonal antibody to PMSG at-72, 0, 12 and 24 h respectively. Blood samples were taken throughout the experiment and for a 72 h intensive sampling period following CIDR device withdrawal and analyzed for progesterone and LH, respectively. Ovarian response was assessed at laparoscopy on Day 7. The proportion of animals with more than 1 corpus luteum was higher in PMSG-only and +24 h groups than in control,-72, 0 and +12 h groups (P<O.O5). Compared with the PMSG-only treated animals, ovulation rate in PMSG-treated hinds was reduced following treatment with the antibody at-72, 0 and +12 h (PcO.05) and the number of large Graafian follicles was reduced in all antibodytreated groups. The incidence of a LH surge was lower in the 0 and +12 h than in the PMSGonly and +24 h groups (P<O.OS). Peak progesterone concentrations in hinds treated with PMSG only and antibody at +24 h were higher than in hinds of the other treatment groups (P<O.OS). Two hinds treated with PMSG alone underwent premature luteal regression, evinced by a sharp decline in progesterone after Day 7. These results suggest that passive immunization neutralizes the biological action of PMSG in red deer, and may provide a means of overcoming some of the adverse effects of the hormone. However, the timing of the antibody treatment is crucial to ensure lack of interference with the endogenous LH surge and subsequent ovulation.
An alarming worldwide extinction of animal species is taking place as a result of the activities ... more An alarming worldwide extinction of animal species is taking place as a result of the activities of the increasing global human population. The original ranges of many animal species are being reduced and fragmented and, in some cases, they have been reduced to perilously small relict populations. The adverse genetic consequences of these restrictions are becoming clear, as are possible methods for their alleviation. The concept of ex situ genetic management of small captive populations of endangered species with a view to re‐introducing them into the wild is attracting increasing interest. Modern reproductive techniques will play an important role in such programmes, and it is likely that an increasing number of veterinarians will become involved. However, the literature describing the aims and methods of reproductive genetic management is scattered and often not readily available to interested veterinary surgeons. The aim of this review is to deal with this problem by describing s...
In order to investigate the effectiveness of ovine FSH as a superovulatory gonadotrophin in sheep... more In order to investigate the effectiveness of ovine FSH as a superovulatory gonadotrophin in sheep, three batches (oFSH-1, oFSH-2 and oFSH-3) of crude follicle stimulating hormone (FSH) extracts were prepared from ovine pituitaries by protein fractionation. The extracts were characterized in radioimmunoassays (RIA), radioreceptor assays (RRA) and bioassays. A FSH:LH (luteinizing hormone) ratio of 1:0.71 and 1:0.18 was calculated from oFSH-1 in RIA and RRA respectively. oFSH-1 was more potent than NIH-oFSH-S12 in RRA, with a potency ratio of 1:0.65 calculated at 50% binding. In mouse bioassay, oFSH-2 was effective in inducing folliculogenesis. The potency of oFSH-3 as an ovarian stimulatory hormone was tested in a field trial on sheep. Thirty-one mature Merino ewes were treated in the breeding season with intravaginal progestagen pessaries and 6, 12 or 24 mg doses of oFSH-3 were injected twice daily for 3 days (n = 10 or 11). Each ewe was inseminated in the uterus with fresh semen 24 ...
The prostaglandin F2 alpha analogue cloprostenol stimulates ovarian secretion of oxytocin in red ... more The prostaglandin F2 alpha analogue cloprostenol stimulates ovarian secretion of oxytocin in red deer hinds and Pere…
Two experiments were conducted with mature Merino ewes to investigate the effects of time and sit... more Two experiments were conducted with mature Merino ewes to investigate the effects of time and site of insemination of fresh and frozen-thawed semen on the fertility of superovulated ewes. In Experiment 1, each ewe was treated with an intravaginal progestagen sponge and PMSG and/or FSH. They were inseminated in the uterus with fresh or frozen-thawed semen (approximately 100 x 10(6) motile sperm) at 24, 44 or 64 h after sponge withdrawal. Ova were recovered at 88 h after sponge withdrawal and classified as fertilized if they had pronuclei or had cleaved. Mean fertilization rates of recovered ova were 60.0, 93.7 and 87.8% for fresh semen and 46.1, 98.2 and 26.1% for frozen-thawed semen at each of the insemination times (24, 44 and 64 h) respectively. Overall, fertilization rates were higher for fresh semen than for frozen-thawed semen (P less than 0.01), but there was an interaction with time of insemination (P less than 0.01). Following insemination with frozen-thawed semen at 64 h, o...
Thirty-four European fallow deer (Dama dama dama) were randomly allocated into embryo donor (n = ... more Thirty-four European fallow deer (Dama dama dama) were randomly allocated into embryo donor (n = 12) or embryo recipient (n = 22) groups. All does were treated with controlled internal drug release (CIDR) devices for 14 days. Animals in the embryo donor group were further treated with 200 I.U. pregnant mare serum gonadotrophin (PMSG) and 0.5 units ovine follicle-stimulating hormone (FSH). PMSG was administered 72 h before withdrawal of CIDR devices and FSH was given in eight 0.063 unit injections at 12-hourly intervals starting at the time of PMSG administration. All embryo donor animals were inseminated, by laparoscopy in both uterine horns, 36 h after withdrawal of CIDR devices with 25 x 10(6) fresh spermatozoa collected from Mesopotamian fallow deer (Dama dama mesopotamica). Embryos were recovered by laparotomy on Day 3 (n = 6) or Day 6 (n = 6) after withdrawal of CIDR devices and the ovarian response was determined. In total, 22 embryos were transferred into the oviduct (2-4-cel...
Administration of gonadotrophin releasing hormone (GnRH) 24 h after sponge withdrawal did not aff... more Administration of gonadotrophin releasing hormone (GnRH) 24 h after sponge withdrawal did not affect the numbers of corpora lutea (CL) or persistent large follicles (LF) in ewes superovulated with 400 I.U. pregnant mare serum gonadotrophin and 12 mg follicle stimulating hormone in spring (11.6 +/- 0.9 v. 13.0 +/- 0.9 CL and 0.8 +/- 0.9 v. 0.9 +/- 0.3 LF, for +GnRH and -GnRH ewes, respectively). However, it did increase the ovulatory response of ewes superovulated in autumn (15.8 +/- 1.2 v. 11.8 +/- 1.1 CL). The incidence of ewes with prematurely regressed CL was also greater in autumn than in spring (21/89 v. 5/88). Supplementary feeding with lupin grain in autumn had no effect on numbers of CL but did increase the incidence of ewes with LF (18/48 v. 7/46) and caused a marked reduction in the incidence of ewes with regressed CL (1/44 v. 20/45). For ewes treated in autumn, there were no effects of lupin supplementation or GnRH administration on peak oestradiol-17 beta (E2) or peak lu...
Six conceptuses were collected from red deer hinds on day 22 after synchronization of oestrus wit... more Six conceptuses were collected from red deer hinds on day 22 after synchronization of oestrus with intravaginal progesterone-releasing devices (removal of device = day 0). Within 24 h of culture in vitro, the supernatant from five of six conceptuses showed detectable antiviral activity. Interferon \g=a\(IFN-\g=a\)receptors were identified by immunohistochemistry on the luminal surface of the endometrium, in the neurohypophysis and paraventricular hypothalamus, but not in the ovaries of the hinds from which the conceptuses were collected. Another 16 intact hinds were synchronized as above. Injection of 4 mg IFN i.m. twice a day on days 13\p=n-\15had no effect on cloprostenol-induced oxytocin secretion on day 15 and did not prevent cloprostenol\x=req-\ induced luteal regression. Sixteen ovariectomized hinds received a protocol of steroid treatment to mimic ovarian hormone secretion during the normal oestrous cycle. On day 16, hinds showed undulant oxytocin secretion that showed a degree of temporal association with uterine PGF2\g=a\ release. Treatment with 4 mg IFN-\g=a\11twice a day on days 13\p=n-\16had no effect on this spontaneous oxytocin secretion, but reduced the magnitude of cloprostenol-induced oxytocin secretion on day 17 (P < 0.05). These results indicate that red deer conceptuses secrete an anti-luteolytic IFN to which the endometrium expresses a receptor during early pregnancy. The presence of IFN receptors in the hypothalamus and posterior pituitary and the IFN-induced suppression of extra-ovarian oxytocin secretion provides tentative evidence of an involvement of the central nervous system in maternal recognition of pregnancy in deer.
Three experiments were carried out to investigate the secretion of luteolytic hormones in red dee... more Three experiments were carried out to investigate the secretion of luteolytic hormones in red deer hinds during the oestrous cycle, early pregnancy and after administration of interferon, the putative pregnancy recognition signal. Three groups of hinds (n = 8\p=n-\9 per group) were treated with progesterone-impregnated intravaginal controlled internal drug releasing (CIDR) devices for 13 days (device withdrawal = day 0). Group 1 (n = 9) served as controls; Group 2 (n = 8) received injections of 4 mg recombinant bovine interferon-\g=a\11 twice a day on days 13\p=n-\18; Group 3 (n = 9) were run with a fertile stag on days 0\p=n-\3.Plasma samples collected each day on days 16\p=n-\23were analysed for progesterone. Plasma samples collected each hour for 16 h on days 4, 10, 16, 18 were analysed for oxytocin and the prostaglandin F2\ g=a\(PGF2\g=a\) metabolite (PGFM). Plasma progesterone concentrations declined to < 1 ng ml\m=-\1 between days 18 and 25 in control hinds indicating that luteolysis had occurred, whereas there was no endocrine evidence of luteolysis in interferon-treated or pregnant hinds. Control hinds (6/9) exhibited synchronous pulses of oxytocin and PGF2\g=a\ secretion on day 18, a greater proportion than on any other day in these hinds or on any day in the interferon-treated or mated hinds (P<0.05). In a second experiment, close synchrony in secretion of oxytocin and PGF2\g=a\pulses was evident in an unmated hind when samples were collected every 12 min on day 18. In a third experiment, oxytocin-induced PGF2\g=a\ secretion was potentiated by oxytocin administration at an interval of 1 h and inhibited by administration at a 6 h interval (P < 0.05). These results suggest that synchronous pulsatile secretion of oxytocin and PGF2\g=a\ induces luteolysis and is suppressed by pregnancy or administration of interferon. Oxytocin-induced alterations in uterine oxytocin sensitivity may underlie the pulsatile nature of luteolytic hormone secretion in red deer.
Prostaglandin (PG) E 2 E-series prostanoid-2 (EP2) receptor is elevated in numerous carcinomas in... more Prostaglandin (PG) E 2 E-series prostanoid-2 (EP2) receptor is elevated in numerous carcinomas including the endometrium and has been implicated in mediating the effects of PGE 2 on vascular function. In this study, we investigated the intracellular signaling pathways that are activated by the EP2 receptor and their role in regulation of the expression of vascular endothelial growth factor in endometrial adenocarcinoma (Ishikawa) cells. Ishikawa cells were stably transfected with EP2 receptor cDNA in the sense or antisense directions. Treatment of Ishikawa cells with PGE 2 rapidly induced transactivation of the epidermal growth factor receptor (EGFR) and activation of ERK1/2 via the EP2 receptor. Preincubation of cells with chemical in
The present study investigated the pattern and site of expression of the prolactin receptor gene ... more The present study investigated the pattern and site of expression of the prolactin receptor gene in the testis of the seasonally breeding Soay sheep. In experiment 1, Northern blot analysis confirmed expression of the prolactin receptor gene in the testis which was encoded by RNA transcripts of approximately 3.6, 11.2, 12.6, and 14.1 kb. In situ hybridisation localised expression of the receptor within the interstitial and seminiferous tubule compartments of the testis and immunohistochemistry localised expression of the receptor to Leydig cells and to pachytene spermatocytes, round and elongating spermatids. In experiment 2, phosphorylation of Jak2, Stat1 and Stat5 proteins in response to prolactin was investigated by Western blotting following incubation of testicular samples with 100 ng/ml ovine prolactin. Jak2 and Stat1 phosphorylation were induced by prolactin within 10 min and Stat5 within 30 min. In experiment 3, intact and hypothalamo-pituitary disconnected (HPD) rams were transferred from a short to a long day photoperiod regimen for a period of 8 weeks. By week 8, testicular diameter had declined in intact rams (52.71+/-1.06 cm vs. 48.00+/-0.49 cm for weeks 1 and 8 after transfer respectively, P&amp;amp;lt;0.01, n = 3) and increased in HPD rams (27.00+/-0.45 cm and 29.66+/-0.99 cm for weeks 1 and 8 after transfer respectively, P&amp;amp;lt;0.05, n = 3). RT-PCR using RNA extracted from intact and HPD rams confirmed expression of the prolactin receptor in the testis of both groups. Immunohistochemistry localised prolactin receptor expression in Leydig cells and in pachytene spermatocytes, round and elongating spermatids of intact sheep testis. In HPD rams, prolactin receptor expression was localised in Leydig cells and germ cells which were arrested predominantly at the pachytene spermatocyte stage. These data demonstrate expression of functional prolactin receptors in the testis of Soay rams. The site and pattern of expression of the receptor gene suggest a role for prolactin in the regulation of steroidogenesis and spermatogenesis.
ABSTRACT A total of 121 European fallow deer does, being either parous (n= 15) or nulliparous (n=... more ABSTRACT A total of 121 European fallow deer does, being either parous (n= 15) or nulliparous (n= 106), were treated with intravaginal progesterone impregnated controlled internal drug release (CIDR) devices for 14 days. The does were divided into three treatment groups and inseminated in utero by laparoscopy, at approximately 65 hours after CIDR device removal, with 25 × 106 fresh Mesopotamian (n= 40), 25–35 × 106 frozen-thawed Mesopotamian (n= 41) or 30–32.5 × 106 frozen-thawed European (n= 40) fallow deer spermatozoa. The semen used had been collected, from two Mesopotamian and two European fallow deer bucks, by electroejaculation under general anaesthesia. Pregnancy was diagnosed by rectal ultrasonogrdphy on Day 50 after insemination. There were no apparent differences in the quality of ejaculates between the two subspecies of fallow deer. The volume of semen and the total number of spermatozoa ranged between 0.6–1.2 ml and 2.11–4.95 × 109 per ml of semen, respectively. Evaluation of frozen-thawed spermatozoa revealed post-thaw motility rates between 50–70%. The overall conception rate was 65.3%. A higher conception rate was observed following insemination with European than Mesopotamian frozen-thawed spermatozoa (75% vs. 53.7%, respectively, P &lt; 0.05). Insemination with fresh Mesopotamian spermatozoa increased the conception rate to a level not significantly different from that observed following insemination with European frozen-thawed spermatozoa (67.5% vs. 75%, for fresh Mesopotamian and frozen-thawed European semen, respectively).
The Journal of Clinical Endocrinology & Metabolism, 2002
A role for prostaglandins (PGs) in the regulation of endometrial functions, such as menstruation,... more A role for prostaglandins (PGs) in the regulation of endometrial functions, such as menstruation, has been established, although the mechanisms by which this is achieved are not fully elucidated. In the present study, cDNA array analysis has identified endothelial monocyte-activating polypeptide II (EMAP II) as a PGE 2-regulated gene in endometrial epithelial cells. Incubation of endometrial epithelial cells with 100 nM PGE 2 for 4 and 24 h resulted in a 2.3-and 16-fold decrease in EMAP II expression, respectively. In endometrial tissue collected across the menstrual cycle, a significant increase in EMAP II mRNA was observed during the late secretory phase, compared with the proliferative and early-midsecretory phases. The temporal pattern of EMAP II expression was confirmed further by Western blotting; EMAP II protein expression was detected as a 43-kDa band. In situ hybridization and immunohistochemistry localized EMAP II mRNA and protein expression in glandular epithelial, endothelial, and stromal cells in the functionalis and basalis layers of the endometrium. Finally, the role of PGE 2 in the regulation of EMAP II expression in the endometrium was assessed. Incubation of fresh endometrial tissue (n ؍ 5) with 3 g/ml indomethacin resulted in an increase in EMAP II protein expression, compared with control untreated tissue. However, cotreatment of the cells with 100 nM PGE 2 resulted in a significant decrease in EMAP II protein expression, compared with tissue incubated with indomethacin alone (P < 0.05). These data confirm temporal variation in EMAP II expression in the human endometrium across the menstrual cycle and localize expression to glandular epithelial, endothelial, and stromal cells. Moreover, EMAP II expression is negatively regulated by PGE 2 .
The Journal of Clinical Endocrinology & Metabolism, 2004
Prostaglandin (PG) E 2 promotes tumor growth via interaction with its G protein-coupled receptors... more Prostaglandin (PG) E 2 promotes tumor growth via interaction with its G protein-coupled receptors and activation of intracellular signaling. Tuberous sclerosis 2 (tuberin) is a tumor suppressor, which negatively regulates cell growth. Its phosphorylation results in its inactivation and targeted downregulation, thus lifting the growth inhibition effects. This study investigated the expression and localization of tuberin in neoplastic and normal endometrium and the effect of PGE 2 on phosphorylation of tuberin via the Akt pathway. Quantitative RT-PCR and Western blot analysis demonstrated reduced expression of tuberin in neoplastic tissue, compared with normal endometrial tissue. Tuberin expression was localized by immunohistochemistry to the glandular epithelial compartment in neoplastic and normal endometrium. We investigated the effect of PGE 2 on phosphorylation of tuberin via the Akt pathway. Treatment of neoplastic and normal endometrium with 100 nM PGE 2 enhanced phosphorylated tuberin immunoreactivity in the glandular epithelium. PGE 2 also phosphorylated Akt and tuberin in Ishikawa endometrial adenocarcinoma cells, leading to a reduction in expression of total tuberin protein. Cotreatment of cells with wortmannin or LY294002 inhibited the PGE 2-induced phosphorylation of Akt and tuberin. These data suggest that PGE 2 signaling may promote endometrial tumorigenesis by inactivation of tuberin after its phosphorylation via the Akt signaling pathway.
The Journal of Clinical Endocrinology & Metabolism, 2003
Prostaglandin (PG) F 2␣ , a member of the prostanoid bioactive lipid family, is secreted by human... more Prostaglandin (PG) F 2␣ , a member of the prostanoid bioactive lipid family, is secreted by human endometrium throughout the menstrual cycle and is present in both menstrual fluid and medium of endometrial explants in culture. PGF 2␣ mediates its effects through a seven-transmembrane G-protein-coupled receptor (FP). The aim of this study was to examine the temporal expression, signaling, and role of FP receptor in the human endometrium. Quantitative RT-PCR analysis demonstrated highest expression of FP receptor in the mid-to lateproliferative phase, compared with early-proliferative and secretory phase endometrium. In situ hybridization studies localized FP receptor mRNA expression to the epithelial cell compartment during the mid-to late-proliferative phase. Moreover, treatment of endometrial tissue with 1-100 nM PGF 2␣ induced a concentration-dependent increase in inositol phosphate mobilization, indicating functional FP receptor expression. The Ishikawa human endometrial epithelial cell line was used to investigate further the signaling and role of PGF 2␣ in endometrial epithelial cells. Ishikawa cells endogenously express the FP receptor, and treatment with 1-100 nM PGF 2␣ elicits a concentration-dependent increase in inositol phosphate release. Moreover, treatment of Ishikawa cells with 100 nM PGF 2␣ induced phosphorylation of ERK1/2 that was abolished when cells were cotreated with 50 M PD98059 (MAPK kinase inhibitor) or 10 M U73122 [phospholipase C (PLC) inhibitor]. Treatment of Ishikawa cells with PGF 2␣ for 24 h induced a significant concentration-dependent increase in Ishikawa cell proliferation. Coincubation of the cells with 50 M PD98059 or 2 M U73122 demonstrated that PLC inhibition significantly reduced PGF 2␣-induced proliferation, whereas MAPK kinase inhibition had no effect. In summary, these studies demonstrate increased FP receptor expression in endometrial epithelial cells during the proliferative phase of the menstrual cycle and identify a role for PGF 2␣ in epithelial cell proliferation via a PLC-dependent pathway.
BACKGROUND: Although the mechanisms underlying the causes of heavy menstrual blood loss (MBL) rem... more BACKGROUND: Although the mechanisms underlying the causes of heavy menstrual blood loss (MBL) remain to be elucidated, prostaglandins have been previously implicated. This study was initiated to elucidate a pattern of expression of the various components of the cyclooxygenase (COX)-prostaglandin signalling pathways present in the endometrium of women with normal and heavy MBLs. METHODS: Endometrial biopsies were collected at different stages of the menstrual cycle from women who underwent measurement of MBL. Tissue was divided for either examination of gene expression by quantitative RT-PCR analysis or in vitro culture experimentation. RESULTS: Analysis of gene expression demonstrated a significant elevation in expression of COX-1 and COX-2 mRNA in endometrium obtained from women with heavy MBL when compared with endometrium obtained from women with normal MBL. Tissue culture with PGE 2 stimulation caused a significantly elevated production of cyclic AMP (cAMP) by endometrium of women with heavy MBL when compared with normal MBL. Expression of phosphodiesterase 4B, an enzyme involved in cAMP breakdown, was reduced in these same endometrial samples obtained from women with heavy MBL. CONCLUSIONS: These data identify the E series prostaglandin receptors and their signalling pathways as potential therapeutic targets in the treatment of heavy menstruation.
BACKGROUND: Prostaglandin E 2 (PGE 2) has been shown to modulate angiogenesis and tumour progress... more BACKGROUND: Prostaglandin E 2 (PGE 2) has been shown to modulate angiogenesis and tumour progression via the E-series prostanoid-2 (EP2) receptor. Endometrial adenocarcinomas may be exposed to endogenous PGE 2 and exogenous PGE 2 , present at high concentration in seminal plasma. METHODS: This study investigated fibroblast growth factor 2 (FGF2) mRNA expression and cell signalling in response to seminal plasma or PGE 2 , using an endometrial adenocarcinoma (Ishikawa) cell line stably expressing the EP2 receptor (EP2 sense cells) and endometrial adenocarcinoma explants. RESULTS: Seminal plasma and PGE 2 induced a significant up-regulation of FGF2 expression in EP2 sense but not parental untransfected Ishikawa (wild-type) cells (P < 0.05). These effects were inhibited by cotreatment with EP2 receptor antagonist or inhibitors of protein kinase A, c-Src, epidermal growth factor receptor (EGFR) kinase or extracellular signal-regulated kinase (ERK) signalling. The treatment of EP2 sense cells with seminal plasma induced cAMP accumulation and phosphorylation of c-Src, EGFR kinase and ERK via the EP2 receptor. Finally, seminal plasma and PGE 2 significantly increased FGF2 mRNA expression in endometrial adenocarcinoma tissue explants via the EP2 receptor (P < 0.05). CONCLUSIONS: Seminal plasma and PGE 2 can similarly activate FGF2 expression and EP2 receptor signalling in endometrial adenocarcinoma cells. These data highlight the potential for seminal plasma exposure to facilitate tumorigenesis-angiogenesis in endometrial adenocarcinomas in vivo.
Times of the ovulatory LH surge and characteristics Social interactions affect reproductive funct... more Times of the ovulatory LH surge and characteristics Social interactions affect reproductive function in a of the rise in circulating progesterone concentrations wide range of species. Among mammals there are nuafter ovulation in red deer hinds were investigated in merous examples of the suppression or delay of reprorelation to each animal's dominance status. Observaduction in subordinate individuals, notably among pritions were made during the 1992 (experiment 1) and 1993 mates (Abbott and Hearn, 1975; Dewsbury, 1982; Ber-(experiment 2) breeding seasons, while the same 12 covitch, 1993), carnivores (Creel and MacDonald, 1995) hinds were held in a pen in the absence of a stag. Ovulaand rodents (Jarvis, 1981; Pratt and Lisk, 1991). There tion was synchronized by administration of progesteris also an extensive literature on the ways in which one followed by luteolytic prostaglandin F 2a analogue. neuroendocrine mechanisms mediate effects of social Social status was determined for each hind by noting status on reproduction (Batty, Herbert, Keverne, and dyadic agonistic interactions during the period of pro
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Papers by Henry Jabbour