Serum omega-3 fatty acids and treatment outcomes among women undergoing assisted reproduction.

STUDY QUESTION: Are serum polyunsaturated fatty acids (PUFA) concentrations, including omega-3 (ω3-PUFA) and omega-6 (ω6-PUFA), related to ART outcomes? SUMMARY ANSWER: Serum levels of long-chain ω3-PUFA were positively associated with probability of live birth among women undergoing ART. WHAT IS KNOWN ALREADY: Intake of ω3-PUFA improves oocyte and embryo quality in animal and human studies. However, a recent cohort study found no relation between circulating ω3-PUFA levels and pregnancy rates after ART. STUDY DESIGN SIZE, AND DURATION: This analysis included a random sample of 100 women from a prospective cohort study (EARTH) at the Massachusetts General Hospital Fertility Center who underwent 136 ART cycles within one year of blood collection. PARTICIPANTS/MATERIALS, SETTING, METHODS: Serum fatty acids (expressed as percentage of total fatty acids) were measured by gas chromatography in samples taken between Days 3 and 9 of a stimulated cycle. Primary outcomes included the probability of implantation, clinical pregnancy and live birth per initiated cycle. Cluster-weighted generalized estimating equation (GEE) models were used to analyze the association of total and specific PUFAs with ART outcomes adjusting for age, body mass index, smoking status, physical activity, use of multivitamins and history of live birth. MAIN RESULTS AND ROLE OF CHANCE: The median [25th, 75th percentile] serum level of ω3-PUFA was 4.7% [3.8%, 5.8%] of total fatty acids. Higher levels of serum long-chain ω3-PUFA were associated with higher probability of clinical pregnancy and live birth. Specifically, after multivariable adjustment, the probability of clinical pregnancy and live birth increased by 8% (4%, 11%) and 8% (95% CI: 1%, 16%), respectively, for every 1% increase in serum long-chain ω3-PUFA levels. Intake of long-chain ω3-PUFA was also associated with a higher probability of life birth in these women, with RR of 2.37 (95% CI: 1.02, 5.51) when replacing 1% energy of long-chain ω3-PUFA for 1% energy of saturated fatty acids. Serum ω6-PUFA, ratios of ω6 and ω3-PUFA, and total PUFA were not associated with ART outcomes. LIMITATIONS REASONS FOR CAUTION: The generalizability of the findings to populations not undergoing infertility treatment may be limited. The use of a single measurement of serum fatty acids to characterize exposure may lead to potential misclassification during follow up. WIDER IMPLICATIONS OF THE FINDINGS: Serum ω3-PUFA are considered biomarkers of dietary intake. The association of higher serum long chain ω3-PUFA levels with improved ART outcomes suggests that increased intake of these fats be may be beneficial for women undergoing infertility treatment with ART. STUDY FUNDING/COMPETING INTERESTS: NIH grants R01-ES009718 from the National Institute of Environmental Health Sciences, P30-DK046200 and T32-DK007703-16 from the National Institute of Diabetes and Digestive and Kidney Diseases, and L50-HD085359 from the National Institute of Child Health and Human Development, and the Early Life Nutrition Fund from Danone Nutricia US. Dr Rueda is involved in a patent 9,295,662, methods for enhancing, improving, or increasing fertility or reproductive function (http://patents.com/us-9295662.html). This patent, however, does not lead to financial gain for Dr Rueda, or for Massachusetts General Hospital. Dr Rueda does not own any part of the company nor does he have any equity in any fertility related company. As Dr Rueda is not a physician, he does not evaluate patients or prescribe medications. All other coauthors have no conflicts of interest to declare.

Invasive treatment trends in urinary calculi in a third level hospital.

OBJECTIVE: In the following study, we observe the progress of various invasive calculi treatments that have taken place in our hospital in the last 15 years. MATERIAL AND METHOD: We extracted data from our hospital database on patients who underwent extracorporeal shock wave lithotripsy (ESWL), endoscopic surgery and open surgery. We analyzed how the incidence of these treatments has evolved over the last 15 years. We also studied the number of publications in PubMed that reference invasive calculi treatments. RESULTS: From January 1998 to December 2012, a total of 10,947 patients were treated instrumentally for lithiasis, 9,695 of whom (90.4%) underwent ESWL and 1,034 of whom underwent endoscopic or open surgery (9.6%). The incidence of lithotripsy treatments reached its maximum in 2006, with a progressive reduction thereafter. The incidence of endoscopic surgery increased progressively until 2009 and then leveled off. We can see how in recent years there has been a clear increase in the number of studies that have covered endoscopic surgery, with a decreasing number covering ESWL. CONCLUSIONS: In our community, ESWL remains the most widely used invasive treatment for calculi. In recent years, there has been a reduction in the number of ESWL treatments and an increase in the number of endoscopic treatments, with open surgery showing a clearly decreasing trend.

Identification of LMX1B as a novel oncogene in human ovarian cancer.

Ovarian cancers are thought to result from the accumulation of multiple genetic aberrations that transform ovarian and/or fallopian tube surface epithelial cells, allowing for their abnormal growth, proliferation and metastasis. In the report presented here, we carried out genome-wide copy-number analysis using comparative genomic hybridization on a panel of mouse ovarian cancer (OVCA) cell lines previously established in our laboratory. We identified a recurrent focal amplification on mouse chromosomal region 2qB, which contains the LIM-homeodomain-containing transcription factor 1B (Lmx1b) gene. LMX1B is not expressed in normal human ovary, but is expressed in many human OVCA cell lines and primary tumors. High expression of LMX1B correlates with poor outcome. To clarify the role of LMX1B in ovarian carcinogenesis, we transduced LMX1B into a panel of mouse and human OVCA cell lines and demonstrated that LMX1B strongly promotes migration of cancer cells in culture and promotes xenograft growth in nude mice. Conversely, knockdown of LMX1B in a human cell line with endogenous high expression of LMX1B inhibits cell migration in vitro and tumor growth in vivo. Microarray analysis of cells overexpressing LMX1B identified the nuclear factor (NF)-κB pathway as a potential mediator of tumor progression and subsequent treatment of NFκB inhibitor decreased the migratory capacity of these cells. Thus, our data demonstrate that LMX1B is a novel oncogene in OVCA pathogenesis.

A rare polymorphism in the gene for Toll-like receptor 2 is associated with systemic sclerosis phenotype and increases the production of inflammatory mediators.

OBJECTIVE: To investigate whether polymorphisms in Toll-like receptor (TLR) genes, previously reported to be associated with immune-mediated diseases, are involved in systemic sclerosis (SSc). METHODS: We genotyped 14 polymorphisms in the genes for TLRs 2, 4, 7, 8, and 9 in a discovery cohort comprising 452 SSc patients and 537 controls and a replication cohort consisting of 1,170 SSc patients and 925 controls. In addition, we analyzed 15-year followup data on 964 patients to assess the potential association of TLR variants with the development of disease complications. We analyzed the functional impact of the associated polymorphism on monocyte-derived dendritic cells. RESULTS: In the discovery cohort, we observed that a rare functional polymorphism in TLR2 (Pro631His) was associated with antitopoisomerase (antitopo) positivity (odds ratio 2.24 [95% confidence interval 1.24-4.04], P=0.003). This observation was validated in the replication cohort (odds ratio 2.73 [95% confidence interval 1.85-4.04], P=0.0001). In addition, in the replication cohort the TLR2 variant was associated with the diffuse subtype of the disease (P=0.02) and with the development of pulmonary arterial hypertension (PAH) (Cox proportional hazards ratio 5.61 [95% confidence interval 1.53-20.58], P=0.003 by log rank test). Functional analysis revealed that monocyte-derived dendritic cells carrying the Pro63His variant produced increased levels of inflammatory mediators (tumor necrosis factor α and interleukin-6) upon TLR-2-mediated stimulation (both P<0.0001). CONCLUSION: Among patients with SSc, the rare TLR2 Pro631His variant is robustly associated with antitopoisomerase positivity, the diffuse form of the disease, and the development of PAH. In addition, this variant influences TLR-2-mediated cell responses. Further research is needed to elucidate the precise role of TLR-2 in the pathogenesis of SSc.

The FAS -670A>G polymorphism influences susceptibility to systemic sclerosis phenotypes.

OBJECTIVE: To investigate the possible role of the FAS -670A>G functional polymorphism in the genetic predisposition to systemic sclerosis (SSc) susceptibility or clinical phenotype. METHODS: A total of 2,900 SSc patients and 3,186 healthy controls were included in this study. We analyzed the genotype and allele frequencies of the FAS -670A>G polymorphism in 9 distinct ethnic cohorts, including 6 cohorts of European ancestry (a Spanish cohort of 228 SSc patients and 265 controls, a Dutch cohort of 203 SSc patients and 277 controls, a German cohort of 313 SSc patients and 247 controls, an Italian cohort of 323 SSc cases and 89 controls, a British cohort of 269 SSc patients, and a Swedish cohort of 182 patients) and 3 distinct ethnic cohorts from the US (a cohort of 1,047 white patients and 692 controls, a cohort of 159 Hispanic patients and 137 controls, and a cohort of 176 black SSc patients and 194 controls). Genotyping was performed using a TaqMan 5' allelic discrimination assay. RESULTS: In the British, Italian, and American white cohorts we observed an association of the FAS -670G allele with limited cutaneous SSc (lcSSc) (odds ratios [ORs] 1.25, 1.43, and 1.18, respectively). A meta-analysis comprising all 9 cohorts revealed an association of both the FAS -670G allele (OR 1.10) and the FAS -670GG genotype (OR 1.13) with the lcSSc phenotype. In a meta-analysis including only white subjects, both the FAS -670G allele and the FAS -670GG genotype remained associated with lcSSc (allele OR 1.12; genotype OR 1.16). In addition, a recessive model of the -670GG genotype exhibited a strong association with SSc, lcSSc, and anticentromere antibody-positive lcSSc (OR 1.23, OR 1.33, and OR 1.45, respectively). CONCLUSION: Our data show that the FAS -670A>G polymorphism plays a role in lcSSc susceptibility. A similar trend has been observed in other autoimmune diseases.

Preemptive therapy is not adequate for prevention of cytomegalovirus disease in pancreas-kidney transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) remains the most common viral infection after pancreas-kidney transplantation (PKT). Comparative studies about CMV prophylaxis in PKT have not been developed. METHODS: We analyzed CMV disease in a cohort of 84 PKT recipients. All received intravenous ganciclovir during treatment with anti-thymocyte globulin and later one of the following options for pre-transplant CMV-seropositive recipients: (a) no prophylaxis (n=10 patients), (b) preemptive therapy (PT) (n=13), or (c) continuous prophylaxis (CP) for 12 weeks (n=29). Pre-transplant CMV-seronegative recipients received CP (n=21). RESULTS: Eleven patients were excluded because of organ explantation in the first 15 days. Incidence of CMV disease in seropositive recipients was 30% under no prophylaxis, 23% under PT, and 6.9% under CP. Incidence of CMV disease under CP was 33.3% in seronegative recipients. Six of 9 episodes of CMV disease under CP occurred after finishing prophylaxis. Under CP, the incidence of CMV disease was significantly higher in seronegative than in seropositive recipients (P<0.05). CONCLUSION: According to the results of our study, for CMV-seropositive PKT recipients, CP is a better strategy than PT. For CMV-seronegative recipients, 3 months of CP is an inadequate strategy.

Contribution of MHC class I region to genetic susceptibility for giant cell arteritis.

OBJECTIVE: The aim of this study was to assess the potential contribution of HLA-class I MICA and HLA-B gene polymorphisms towards the pathogenesis of giant cell arteritis (GCA). METHODS: Ninety-eight biopsy-proven GCA patients and 225 ethnically matched controls from Lugo, Northwest Spain, were genotyped for the MICA-TM microsatellite polymorphism using a polymerase chain reaction (PCR)-based method. Genotyping of HLA-B was performed using PCR and detection with a reverse sequence-specific oligonucleotide (SSO) probes system. RESULTS: A significant difference in the distribution of the alleles of MICA between patient and control groups (P = 0.005) was found. This was due to an increased frequency of the MICA A5 allele in GCA patients compared with controls (26 vs 13.6%; P = 0.0001; P(C) = 0.0005; OR 2.2, 95% CI 1.4-3.4). In addition, the HLA-B*15 allele showed a higher frequency in GCA patients compared with controls (P = 0.004; P(C) = 0.04; OR 2.7, 95% CI 1.3-5.7). Interestingly, the association observed with the MICA A5 allele seems to be independent of linkage disequilibrium with HLA-B, as well as independent of that previously described with HLA-DRB1*04. Remarkably, simultaneous presence of MICA A5 and HLA-B*15 or HLA-DRB1*04 genetic markers leads to an increase in the OR obtained for each individual genetic marker (MICA A5 + B*15 OR 3.2; MICA A5 + DRB1*04 OR 5.8). CONCLUSIONS: Our results provide the first evidence that the MICA and HLA-B genes are independently associated with the genetic susceptibility to GCA, and suggest that several genes within the MHC might have independent effects in the susceptibility to this systemic vasculitis.

Genetic basis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a clinically heterogeneous condition with a complex aetiology in which environmental and genetic factors are implicated. The contribution of human leukocyte antigen (HLA) genes, particularly the HLA-DRB1 gene, to RA genetic predisposition was the first described, and remains as the best characterised single genetic risk factor contributing to RA. However, it has been estimated that only 30% of the genetic contribution to RA can be attributed to HLA genes and it is suggested that other non-HLA genes may play a relevant role in RA susceptibility. Linkage studies and association studies are the two main strategies used in the investigation of genetic factors contributing to complex genetic traits. In this work we review the progress made in the field of RA genetics, focusing mainly on the contribution of candidate gene association studies to the dissection of RA genetic risk factors.

CTLA4/CT60 polymorphism is not relevant in susceptibility to autoimmune inflammatory intestinal disorders.

The aim of this work was to investigate the possible influence of the recently described CT60 A/G dimorphism of the CTLA4 (cytotoxic T-lymphocyte antigen 4) gene in the susceptibility to two different autoimmune inflammatory intestinal disorders, inflammatory bowel disease (IBD) and celiac disease. We analyzed a case-control cohort composed of 528 Spanish patients with IBD (284 with Crohn disease and 244 with ulcerative colitis) and 454 unrelated healthy individuals, and additionally a group of 90 celiac disease families. CT60 genotyping was performed with a TaqMan 5' allelic discrimination assay. After comparing patients with IBD with the control population, we found no significant deviation in the distribution of the alleles or genotypes of CTLA4/CT60 dimorphism. In addition, by means of familial and case-control analysis, no evidence for a statistically significant association was observed between CTLA4/CT60 and celiac disease susceptibility. Therefore, our results suggest that the CTLA4/CT60 polymorphism does not play a major role in inflammatory intestinal disorders.

Leptin serves as an upstream activator of an obligatory signaling cascade in the embryo-implantation process.

Leptin is essential for mouse reproduction, but the exact roles it serves are yet to be determined. Treatment of cultured endometrial cells with leptin increases the level of beta3-integrin, IL-1, leukemia inhibitory factor, and their corresponding receptors. These leptin-induced effects are eliminated by inhibitors of leptin receptor (OB-R) signaling. Herein the impact of blocking leptin/OB-R signaling in the mouse endometrium was assessed. Intrauterine injection of either leptin peptide antagonists (LPA-1 or -2) or OB-R antibody on d 3 of pregnancy impaired mouse implantation in comparison to intrauterine injection of scrambled peptides (LPA-Sc) or species-matched IgGs. Significant reduction in the number of implantation sites and uterine horns with implanted embryos was found after intrauterine injection of LPA-1 (1 of 22) vs. LPA-1Sc (11 of 15) and LPA-2 (3 of 17) vs. LPA-2Sc (14 of 16). The impact of disruption of leptin signaling on the endometrial expression of several molecules in pregnant mice was assessed by Western blot, immunohistochemistry, and confocal microscopy. Disruption of leptin signaling resulted in a significant reduction of IL-1 receptor type I, leukemia inhibitory factor, vascular endothelial growth factor receptor 2, and beta3-integrin levels. The levels of colony stimulating factor-1 receptor and OB-R were unaltered after treatment with LPAs compared with controls. Expression of OB-R protein was pregnancy dependent and found only in glandular epithelium after implantation occurred. Our findings support previous observations that leptin signaling is critical to the implantation process and suggest that molecules downstream of leptin-activated receptor may serve obligatory roles in endometrial receptivity and successful implantation.

BAG-1 expression in normal and neoplastic endometrium.

OBJECTIVE: BAG-1 has anti-apoptotic actions and is known to bind BCL-2 and steroid receptors. High levels of BAG-1 have been implicated as a prognostic indicator in breast cancer. Whether this observation can be generalized to endometrial cancer remains unknown. METHODS: IRB permission was obtained for use of human discarded tissue. Immunohistochemical analyses were performed on: proliferative endometrium (PEM, 6), secretory endometrium (SEM, 28), "low-grade" neoplastic lesions (complex atypical hyperplasia and grade 1 endometrial adenocarcinomas) (19), and "high-grade" cancers (grade 2 and 3 endometrial adenocarcinomas) (13). The level of total BAG-1 and its isoforms was evaluated by Western blot in lysates from Ishikawa cells (grade 1), MFE 296 cells (grade 2), and SK-UT(2) cells (grade 3). RESULTS: The proportion of "high-grade" cancers with positive cytoplasmic staining for BAG-1 was higher than that of secretory endometrium (P = 0.006). Additionally, the proportion of specimens with positive staining for nuclear BAG-1 expression was significantly higher among high-grade carcinoma specimens compared to secretory specimens (P = 0.009). A high proportion (91%) of all specimens were positive for BCL-2, limiting the ability to subcategorize the other variables analyzed. There was no relationship between positive nuclear BAG-1 expression and either estrogen receptor (ER) or progesterone receptor (PR) expression. BAG-1 was expressed in the three cell lines evaluated and total BAG-1 level was not different among the different cell lines. CONCLUSION: BAG-1 is expressed in the endometrium. High-grade cancers stain more frequently than secretory endometrium for both cytoplasmic and nuclear BAG-1 expression, perhaps indicating an association between expression of BAG-1 and prognosis.

Leptin-induced increase in leukemia inhibitory factor and its receptor by human endometrium is partially mediated by interleukin 1 receptor signaling.

Leptin and leukemia inhibitory factor (LIF) have been implicated as important mediators of implantation. The present study was designed to investigate whether leptin can directly regulate the expression of LIF and its receptor (LIF-R) in human endometrial cells and/or whether leptin-induced effects are linked to, or regulated in part by IL-1 signaling. Primary endometrial cells and endometrial epithelial cell lines (HES and Ishikawa cells) were cultured for 24-48 h in a medium containing insulin (5 microg/ml) and leptin (3, 10, and 62 nm) or IL-1beta (0.6, 3, and 10 nm) in the presence or absence of cytokines and/or receptor antagonists. The endpoints included phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the relative levels of LIF, LIF-R, IL-1beta, IL-1 receptor antagonist (IL-1Ra) and IL-1 receptor type I (IL-1R tI) as determined by ELISA or Western blotting techniques. Leptin treatment increases the level of phosphorylated STAT3, LIF-R, and LIF. Leptin also increases the levels of IL-1 ligand, receptor, and antagonist as was previously reported. Blockade of OB-R with antibodies or with a specific OB-R inhibitor (leptin peptide antagonist-2) abrogated leptin-induced effects, suggesting that leptin binding to its receptor activates Janus kinase 2/STAT3 signaling. Treatment of endometrial cells with IL-1beta also results in elevated levels of LIF-R. Interestingly, the inhibition of IL-1R tI with a specific antibody or with IL-1Ra negatively affects both leptin-induced and IL-1-induced effects on LIF-R levels. Abnormal endometrial LIF expression has been associated with human infertility and leptin has profound effects on the levels of LIF, IL-1, and their cognate receptors in vitro. Thus, it is tempting to speculate that leptin's role in vivo could include the regulation of other key cytokines to be fundamental to endometrial receptivity during implantation (i.e. LIF and IL-1).

Endometrial cancer in women 40 years old or younger.

OBJECTIVE: The aim of this study was to characterize endometrial cancer in women 40 years of age and younger, with special attention toward body-mass index (BMI). METHODS: A retrospective review of women age 40 and under with endometrial cancer was performed. Patients were identified via tumor registry data as well as a search of pathology department diagnoses over the dates 1980-1998. Data were abstracted regarding tumor grade and histology, stage, treatment, smoking, use of oral contraceptives, BMI, medical and family history, parity, and survival. Data were also collected with regard to uterine conservation and pregnancies following endometrial cancer diagnoses. RESULTS: Ninety-five patients were identified. The age range was 24-40 years (median 37) with BMI ranging from 17.5 to 63.6 (median 28.4). Forty-eight patients (52%) were not obese, with BMI < 30. Seventy-six patients (80%) had stage I disease and 60 patients (63%) had grade 1 disease. All but 4 patients had endometrioid histology. Women with BMI < 25 were more likely to have advanced disease (P = 0.04) and more likely to have high-risk histology (P = 0.02). Of the 4 patients with high-risk histology (clear cell or serous papillary), all had BMI < 25. Twelve patients were treated medically rather than surgically, and 4 patients achieved pregnancy, with 5 live births. CONCLUSION: Women under 40 who are not obese are at higher risk of both advanced disease and high-risk histology. Further study at the molecular and genetic level is ongoing in our laboratory to determine whether the mechanism of disease is different in slender woman.

[Bilateral testicular microlithiasis]. / Microlitiasis testicular bilateral.

OBJECTIVE: To report an uncommon case of testicular microlithiasis that was incidentally discovered. METHODS/RESULTS: A case of bilateral testicular microlithiasis in an 8-year-old boy that presented with a left scrotal cystic mass is described. Testicular microlithiasis was discovered on ultrasound evaluation. CONCLUSIONS: Bilateral testicular microlithiasis is a rare condition that requires control follow-up of tumor markers and by ultrasound since this condition has been associated with tumors.

Mediators of interferon gamma-initiated signaling in bovine luteal cells.

Interferon gamma (IFNgamma) has been implicated as a mediator of luteal steroidogenesis and cell fate. IFNgamma-initiated signaling events, although implied by studies in cell lines, have yet to be described in primary luteal cells. The objective of these studies was to begin to characterize IFNgamma-initiated signaling within luteal cells. Dispersed bovine luteal cell cultures were challenged with increasing levels of bovine recombinant IFNgamma (0-1000 U) or IFNgamma (200 U) in the presence or absence of tumor necrosis factor alpha (TNFalpha, 10 ng/ml) over time (short term, 0-60 min; long term, 0, 24, 48 h). Fractionated or total cell lysates were evaluated by the Western blotting technique to determine the changes in the levels of signal transducers and activators of transcription (STAT), interferon regulatory factor 1 (IRF-1), and I kappa B alpha (IkappaB-alpha). Utilizing antibodies that recognize the nonphosphorylated forms of STAT-1 and STAT-3, it was determined that levels of STAT-1 and STAT-3 in total cell lysates were constitutively expressed and did not change in response to treatment with IFNgamma or TNFalpha. In contrast, nuclear levels of STAT-1 and phosphorylated STAT-3 were elevated in a time-dependent manner in response to IFNgamma treatment. Furthermore, IFNgamma and TNFalpha treatment elevated levels of IRF-1 within 2 h. TNFalpha-induced increases in the levels of IRF-1 were transient, whereas the levels of IRF-1 in response to IFNgamma treatment remained elevated at 48 h. These data suggest that IFNgamma treatment can activate members of the STAT pathway, resulting in increased levels of IRF-1. TNFalpha treatment induced a rapid decrease in the levels of IkappaB-alpha. IFNgamma treatment did not alter the levels of IkappaB-alpha and failed to inhibit the TNFalpha-initiated decrease in the levels of IkappaB-alpha. The present experiment demonstrates that the steroidogenic cells of the corpus luteum have the capacity to respond to IFNgamma via activation of STAT and IRF-1, providing further evidence that IFNgamma may be involved in the luteolytic process. These data also suggest that IFNgamma does not signal through the nuclear factor kappa B cell survival signaling pathway.

Interferon-tau suppresses prostaglandin F2alpha secretion independently of the mitogen-activated protein kinase and nuclear factor kappa B pathways.

Pregnancy is established in ruminants through inhibitory actions of interferon (IFN)-tau on the release of prostaglandin F2alpha (PGF), which allows the corpus luteum to survive and continue to produce progesterone. Experiments were designed to 1) delineate the signal transduction pathway coordinating the synthesis of PGF, 2) determine how rapidly recombinant bovine (rb) IFN-tau attenuated phorbol ester (PDBu)-induced secretion of PGF, and 3) establish the site at which rbIFN-tau attenuates the secretion of PGF in cultured bovine endometrial (BEND) cells. BEND cells were untreated (control) or treated for 5, 10, 60, 180, or 300 min with PDBu (100 ng/ml), rbIFN-tau (50 or 500 ng/ml), PDBu + rbIFN-tau, or PDBu + PD98059 (MEK-1 inhibitor; 50 microM). Secretion of PGF was induced (P < 0.0001) by PDBu within 180 min, but induction was inhibited 74% by the addition of rbIFN-tau (P < 0.0001) and was ablated completely by PD98059. Parallel results were obtained for cyclooxygenase (COX)-2 protein expression. PDBu induced (P < 0.05) activation of the Raf-1/MEK-1/ERK-1/2 pathway, which was obligatory for the expression of COX-2 and secretion of PGF but was not altered by cotreatment with rbIFN-tau. PDBu induced (P < 0.05) transcription of c-jun and c-fos mRNAs within 30 min; induction was inhibited (P < 0.05) by cotreatment with PD98059 but not by cotreatment with rbIFN-tau. Treatment of BEND cells with rbIFN-tau also did not attenuate PDBu-induced degradation of IkappaBalpha, suggesting that the IkappaBalpha/NFkappaB pathway is not a site of IFN-tau inhibition of PGF. However, rbIFN-tau did block transcription of the COX-2 gene induced by PDBu within 30 min. In conclusion, COX-2 expression and PGF secretion induced by PDBu is mediated through the Raf-1/MEK-1/ERK-1/2 pathway, but this pathway is not disrupted by rbIFN-tau. Because rbIFN-tau inhibits COX-2 mRNA within 30 min, we hypothesized that transcription factors activated by rbIFN-tau rapidly and directly attenuate COX-2 gene expression, thereby suppressing secretion of PGF.

Stress-induced mitogen-activated protein kinase signaling in the corpus luteum.

Current evidence suggests that stress-induced apoptosis is mediated through the activation of the mitogen-activated protein kinase (MAPK) signaling cascade. We hypothesize that stress-related signaling events documented in other cell lines may also occur in the corpus luteum. To test this, cultured bovine luteal cells were exposed to UV irradiation and harvested at different intervals (0, 30, 120, 240 and 360 min) for analysis of protein or apoptotic cell death. In response to UV treatment cellular levels of phosphorylated p38MAPK and jun-n-terminal kinase (JNK) were increased within 30 min and remained elevated over controls for the duration of the experiment. In contrast, the levels of the phosphorylated forms of p42MAPK and p44MAPK were dramatically reduced. The changes in MAPK signaling were similar to those observed in response to tumor necrosis factor alpha, a cytokine implicated in luteal regression. The UV-induced changes in MAPK phosphorylation were associated with an increase in caspase 3 activity and apoptotic cell death. Taken together, these data demonstrate that stress-induced signaling events in the corpus luteum are similar to those observed in unrelated cell types. Thus, stress-related signaling events may play a role in luteal regression.

Putative role of the phosphatidylinositol 3-kinase-Akt signaling pathway in the survival of granulosa cells.

Insulin-like growth factor-I (IGF-I) is an important differentiation and survival factor for granulosa cells. The purpose of this study was to test the hypothesis that IGF-I promotes survival of porcine granulosa cells by signaling through the phosphatidylinositol (PI) 3-kinase/Akt signal transduction pathway. Treatment with IGF-I (100 ng/mL) for 10 min stimulated PI 3-kinase and Akt protein kinase activity. IGF-I stimulated the phosphorylation and activation of Akt in a time- and concentration-dependent manner. The PI 3-kinase inhibitors wortmannin and LY294002 blocked IGF-I induced increases in PI 3-kinase activity and phosphorylation of Akt. Additionally, IGF-I treatment prevented apoptosis. The survival response to IGF-I was blocked by treatment with either wortmannin or LY294002. These data suggest that IGF-I-induced phosphorylation of Akt is mediated through PI 3-kinase and that inactivation of this pathway results in granulosa cell apoptosis. We conclude that the PI 3-kinase/Akt signaling serves as a functional survival pathway in the ovary.

Decreased progesterone levels and progesterone receptor antagonists promote apoptotic cell death in bovine luteal cells.

We tested the hypothesis that progesterone (P(4)) acts at a local level to inhibit luteal apoptosis. Initial experiments employed aminoglutethimide, a P450 cholesterol side-chain cleavage inhibitor, to inhibit steroid synthesis. Cultured bovine luteal cells were treated with aminoglutethimide (0.15 mM) +/- P(4) (500 ng/ml) for 48 h. Luteal cells were recovered and snap frozen for isolation and analysis of oligonucleosomal DNA fragmentation or fixed for morphological analysis. Medium was collected for analysis of P(4) levels by RIA. Aminoglutethimide inhibited P(4) synthesis by > 95% and increased the level of apoptosis as evidenced by (32)P-labeled oligonucleosomal DNA fragmentation (> 40%). P(4) supplementation inhibited the onset of apoptosis that was induced by aminoglutethimide. These data were further supported by morphological assessment of apoptotic cells utilizing a Hoechst staining technique and together strongly suggest that P(4) has anti-apoptotic capacity. Using reverse transcription-polymerase chain reaction, we were able to isolate a 380-base pair cDNA from the bovine corpus luteum (CL) that was 100% homologous to the progesterone receptor (PR) previously found in bovine oviductal tissue. Furthermore, PR transcripts were present in large and small luteal cells. Immunohistochemistry also revealed that PR protein was present in both large and small luteal cells. To determine whether the anti-apoptotic effect of P(4) was regulated at the receptor level, luteal cells were cultured in the presence of PR antagonists, RU-486 and onapristone, for 48 h. Both antagonists caused approximately a 40% increase in (32)P-labeled oligonucleosomal DNA fragmentation. Interestingly, there was no difference (P >/= 0.05) in P(4) levels after treatment with PR antagonists. These observations support the concept that P(4) represses the onset of apoptosis in the CL by a PR-dependent mechanism.

Ovine prostaglandin F2alpha receptor: steroid influence on steady-state levels of luteal mRNA.

Expression of the receptor for prostaglandin F2alpha (PGF2alpha) is decreased in the ovine corpus luteum during regression and increased in early pregnancy. This study was designed to evaluate the influence of progesterone and/or 17beta-estradiol (E2) on this regulation. Circulating progesterone (functional regression) and luteal PGF receptor mRNA decreased (p < 0.05) within 8 h of PGF2alpha-induced luteal regression in midluteal phase (day 10; d 10) ewes; however, internucleosomal DNA fragmentation (structural regression) was not yet increased. Additionally, luteal PGF receptor mRNA and circulating progesterone were greater (p < 0.05) in pregnant than in nonpregnant ewes on d 14, but not on d 12. Twelve hours following injection of d 10 ewes with E2, steady-state levels of mRNA for PGF receptor were decreased (p < 0.05), although circulating progesterone and DNA laddering were unchanged. Conversely, luteal mRNA for PGF receptor was increased (p < 0.05) by E2 treatment in hysterectomized ewes. These results provide evidence that (1) luteal PGF receptor expression parallels circulating progesterone levels during functional regression and in early pregnancy, but (2) expression of PGF receptor can be dissociated from alterations in circulating progesterone by injection with E2. Additionally, decreased PGF receptor expression initiated by E2 is uterine-dependent, whereas the direct luteal effect (hysterectomized ewes) of E2 is a stimulation of PGF receptor expression. These results collectively support the belief that the apparent downregulation of PGF receptor during luteal regression is associated with uterine-derived PGF2alpha and its intracellular effects rather than with alterations in ovarian steroid production.