Estrogen promotes fetal skeletal muscle mitochondrial distribution and ATP synthase activity important for insulin sensitivity in offspring.

PURPOSE: We previously showed that offspring delivered to baboons in which levels of estradiol (E2) were suppressed during the second half of gestation exhibit insulin resistance. Mitochondria are essential for the production of ATP as the main source of energy for intracellular metabolic pathways, and skeletal muscle of type 2 diabetics exhibit mitochondrial abnormalities. Mitochondria express estrogen receptor ß and E2 enhances mitochondrial function in adults. Therefore, the current study ascertained whether exposure of the fetus to E2 is essential for mitochondrial development. METHODS: Levels of ATP synthase and citrate synthase and the morphology of mitochondria were determined in fetal skeletal muscle obtained near term from baboons untreated or treated daily with the aromatase inhibitor letrozole or letrozole plus E2. RESULTS: Specific activity and amount of ATP synthase were 2-fold lower (P < 0.05) in mitochondria from skeletal muscle of E2 suppressed letrozole-treated fetuses and restored to normal by treatment with letrozole plus E2. Immunocytochemistry showed that in contrast to the punctate formation of mitochondria in myocytes of untreated and letrozole plus E2 treated animals, mitochondria appeared to be diffuse in myocytes of estrogen-suppressed fetuses. However, citrate synthase activity and levels of proteins that control mitochondrial fission/fusion were similar in estrogen replete and suppressed animals. CONCLUSION: We suggest that estrogen is essential for fetal skeletal muscle mitochondrial development and thus glucose homeostasis in adulthood.

Estrogen promotes fetal skeletal muscle myofiber development important for insulin sensitivity in offspring.

Using our nonhuman primate baboon model, we showed that offspring born to mothers deprived of estrogen during the second half of gestation exhibited insulin resistance and a deficit in first phase insulin release. Although insulin resistance was not due to an impairment of fetal or offspring growth, nor to an alteration in adipose or hepatic sensitivity to insulin, skeletal muscle microvacularization critical for delivery of nutrients/insulin was significantly reduced in fetuses and offspring deprived of estrogen in utero. Skeletal muscle myofiber maturation occurs in utero and estrogen modulates myofiber growth in adults. Therefore, the current study determined whether fetal skeletal muscle development was altered in baboons in which estradiol levels were suppressed/restored during the second half of gestation by maternal treatment with letrozole ± estradiol benzoate. In estrogen-suppressed animals, fetal skeletal muscle fascicles were structurally less organized, smaller, and comprised of slow type I and fast type II fibers, the size, but not the number of which were smaller than in untreated baboons. Moreover, the proportion of non-muscle fiber tissue was greater and that of muscle fibers lower in estrogen-deprived fetuses. Thus, the maintenance of fetal body weight in estrogen-deprived animals was maintained at the expense of muscle fibers and likely reflected increased deposition of non-muscle proteins. Importantly, fetal skeletal muscle development, including fascicle organization, myofiber size and composition was normal in baboons treated with letrozole and estradiol benzoate. Collectively, these and our previous findings support our proposal that exposure of the fetus to estrogen is important for fetal skeletal muscle development and glucose homeostasis in adulthood.

Application of Interphase Fluorescent in Situ Hybridization: a Screening Tool for the Diagnosis of Microdeletion Syndrome.

BACKGROUND: Most laboratories adopt the results of metaphase fluorescent in situ hybridization (FISH) for the diagnosis of microdeletion syndromes. To investigate the discrepancy between the results of interphase and metaphase, we compared the quantitative results of FISH for 5 kinds of microdeletion syndrome and gender determination disorders (SDD). METHODS: A total of 282 (135 for DiGeorge syndrome, 20 for Kalmann syndrome, 7 for Miller-Dieker syndrome, 38 for Prader Willi/Angelman syndrome, 62 for Williams syndrome, and 20 for SDD (SRY FISH)) were enrolled. For SRY FISH, we artificially mixed fresh blood of male and female with various ratios and then compared the results of metaphase and interphase SRY FISH. Using a bio-cell chip, we performed interphase FISH in 168 patients with microdeletion syndromes and compared the results with manual interphase. RESULTS: The concordance rate between the results of metaphase and interphase was 100% in microdeletion syndrome. In the disorders of gender development, SRY FISH showed 100% concordance between interphase and metaphase when we counted 50 metaphase cells and 100 interphase cells. Comparison with mixtures of male and female blood at various ratios also showed 100% concordance. The results of bio-cell chip showed 100% concordance between previous interphase FISH results. CONCLUSIONS: Considering the complete concordance between interphase and metaphase in microdeletion syndrome, the application of interphase FISH without performing metaphase FISH can be a screening test for microdeletion syndrome. Confirmation by metaphase FISH can be performed only in cases with abnormal results by interphase FISH.

Effect of Case-Based Small-Group Learning on Care Workers' Emergency Coping Abilities.

This study aimed to develop and implement an emergency coping education program using a case-based small-group learning method and verify its effect on care workers' emergency coping abilities. The study was conducted with 72 care workers in older adult care facilities and home care centers. Using a nonequivalent control group pretest-posttest design, 36 participants were assigned to each group (i.e., experimental and control groups). The collected data were analyzed through χ²-test and independent t-test using SPSS for Windows, version 25.0. Compared to the control group, a statistically significant increase in knowledge and performance levels in emergencies, emergency coping abilities, self-efficacy in coping with emergencies, and confidence in communication was observed in the experimental group. This study was able to verify the effectiveness of the emergency coping education program in care workers and recommends its use. To maximize the learning effects of educational programs, future research should develop and apply programs that incorporate simulation education.

Association of work environment and resilience with transition shock in newly licensed nurses: A cross-sectional study.

AIMS AND OBJECTIVES: To examine transition shock in newly licensed nurses and the association of work environment and resilience with nurses' transition shock. BACKGROUND: Although work environment is related to transition shock in newly licensed nurses, little is known about the factors of nursing work environment associated with transition shock. Furthermore, resilience is known to help nurses positively face workplace challenges; however, there is little evidence on the associations between resilience and transition shock in new nurses. DESIGN: A cross-sectional, descriptive study. METHODS: Data from 163 new nurses with <12 months of work experience in the current hospital since graduation were analysed. Participants' characteristics, work environment, nurse resilience and transition shock were self-reported. Multivariable linear regressions were performed in three steps (following the STROBE checklist). RESULTS: The highest mean score of transition shock was obtained for the item 'I perceive the limitations of my professional knowledge in nursing care'. In the regression analysis adjusted for all variables, two factors of work environment-'nurse staffing and resource adequacy' and 'collegial nurse-physician relationships'-were associated with transition shock. Meanwhile, resilience was not related to transition shock. CONCLUSIONS: The gap between newly licensed nurses' theoretical and practical knowledge continues to exist. Adequate nurse staffing and a positive relationship with physicians, rather than nurse resilience, were more likely to associate with new nurses' transition shock. RELEVANCE TO CLINICAL PRACTICE: Hospitals need to provide ward-based simulations and case-based learning methods to enhance nurses' transition to professional practice. For nurses' transition, hospitals should make efforts to provide adequate nurse staffing and resources. Furthermore, hospitals should provide communication opportunities to build a positive collaborative culture between nurses and physicians. Unit nurse managers need to assess newly licensed nurses' perception of nurse-physician professional relationship and create an atmosphere of respect and understanding for each other.

Beneficial Effects of Breastfeeding on the Prevention of Metabolic Syndrome Among Postmenopausal Women.

PURPOSE: This study aims to determine whether breastfeeding may have any beneficial effects on metabolic syndrome in a cohort of postmenopausal Korean women. METHODS: A cross-sectional study with secondary data analysis was conducted using the cohort in the Korean Genome and Epidemiology Study. Data from 1,983 postmenopausal women were analyzed by logistic regression analysis. Controlled covariates were chosen based on a biopsychosocial model and included age, family history of hypertension; type 2 diabetes mellitus; and cerebro-cardiovascular diseases, body mass index, age of menarche, parity, socioeconomic status of family, educational level, past or current smoking experience, and current alcohol consumption experience. RESULTS: Breastfeeding experience and duration were not significantly associated with a decreased likelihood of metabolic syndrome among postmenopausal women. However, breastfeeding experience (adjusted odds ratio [AOR]: 0.52 [p = .010]) and a total duration of breastfeeding exceeding 3 months were significantly associated with decreased likelihood of abdominal obesity (≥3 and < 6 months: AOR: 0.49 [p = .014]; ≥6 and < 12 months: AOR: 0.51 [p = .009]; ≥12 months: AOR: 0.56 [p = .024]). CONCLUSION: Our findings indicate that breastfeeding might have beneficial effects on reducing abdominal obesity in postmenopausal women. Health-care providers should publicize beneficial long-term effects of breastfeeding on the prevention of abdominal obesity, a component of metabolic syndrome.

Biological control of tomato bacterial wilt by oxydifficidin and difficidin-producing Bacillus methylotrophicus DR-08.

Bacillus methylotrophicus DR-08 exhibited strong antibacterial activity against Ralstonia solanacearum, a causal agent of tomato bacterial wilt. This study aimed to identify the antibacterial metabolites and evaluate the efficacy of the strain as a biocontrol agent for tomato bacterial wilt. A butanol extract of the DR-08 broth culture completely inhibited the growth of 14 phytopathogenic bacteria with minimum inhibitory concentration (MIC) values of 1.95-500 µg/mL. R. solanacearum was highly sensitive to the DR-08 extract, with an MIC value of 12.62 µg/mL. Two antibacterial metabolites were isolated and identified as difficidin and oxydifficidin derivatives through bioassay-guided fractionation and instrumental analyses. Both metabolite derivatives inhibited the growth of most of the phytopathogenic bacteria tested and the oxydifficidin derivatives generally presented a stronger antibacterial activity than the difficidin derivatives. A 30% suspension concentrate of DR-08, at a 500-fold dilution, effectively suppressed the development of tomato bacterial wilt in pot and field experiments. It also effectively reduced the development of bacterial leaf spot symptoms on peach and red pepper. The results of this study suggests that B. methylotrophicus DR-08 can be utilized as a biocontrol agent for various bacterial plant diseases including tomato bacterial wilt.

[Factors Associated with Pressure to Eat as a Feeding Practice among Mothers with Infants]. / 영아 어머니의 과도한 ìˆ˜ìœ ê´€ë ¨ 요인.

PURPOSE: This study aimed to identify factors-both infant-related and maternal-associated with pressure to eat as a feeding practice among mothers with infants. METHODS: This study used a cross-sectional design and included 163 mothers of infants aged 2~12 months. Of the 180 self-reported questionnaires that were distributed, 163 (91%) were included in the data analysis. Multiple regression analysis was used to identify the factors associated with pressure to eat as a feeding practice among the mothers. RESULTS: Infant's temperament (ß=-.17, p=.035), mother's body mass index (ß=-.16, p=.048), and concern about the infant being underweight (ß=.30, p=.001) were associated with pressure to eat as a feeding practice among mothers. The explanatory power of these variables in the predictive model was 19.2%. CONCLUSION: Educational programs should be developed for improving mother's awareness of cues from infants with difficult temperament. In addition, educational interventions regarding the correct evaluation of infant's weight are needed to relieve mother's concern about their infant's being underweight. These interventions might be helpful to reduce the prevalence of pressure to eat as a feeding practice among mothers with infants.

Emergencies in Long-Term Care Services for the Elderly in Korea: A Mixed-Methods Study.

This mixed-methods study explored ways to enhance the emergency response abilities of workers in long-term care services for the elderly. Based on different service types, we identify emergency situations and the response abilities of workers in long-term care services. Results indicated that there are more emergency situations in care facilities than in home care services. However, 71.3% of respondents in facilities said emergency response abilities were low compared to 44.2% of workers in home care services. Qualitative research identified six categories and 16 themes based on emotions experienced during emergencies and the challenges in determining solutions. The study confirms that there is a difference in emergency incidences and the coping abilities of workers in facilities and home services with high emergency incidence rates. Developing and applying guidelines for emergency response management by service type is recommended.

Factors Affecting Care Workers' Coping Abilities in Emergencies to the Korean Elderly.

This study provides basic data for enhancing coping abilities in emergencies concerning direct long-term care (LTC) workers, which is necessary for providing safe care for elderly patients living in facilities and at home. A survey was conducted including 327 care workers who officially qualified as long-term providers for elderly patients through elderly care facilities and a domiciliary service center. The majority (91.4%) of the care workers surveyed experienced an emergency, but of them, only 36.4% performed first aid and 56.8% failed to perform first aid, for which the emergency was reported to nurses. The average score regarding first aid knowledge was 8.40 out of 21, and the mean scores for the subtopics of basic life support and general first aid were low (3.56 out of 7 and 5.84 out of 14, respectively). Nearly three-quarters (72.5%) responded that they were unaware of emergency coping methods, and the score for coping abilities in emergencies was also low (52.93 out of 100). The results indicate that factors affecting coping abilities in emergencies were related to the size of the workplace and first aid experience. We propose the development and implementation of an emergency coping training program focusing on case studies for direct LTC workers.

Lentiviral infection of proliferating brain macrophages in HIV and simian immunodeficiency virus encephalitis despite sterile alpha motif and histidine-aspartate domain-containing protein 1 expression.

OBJECTIVE: HIV-1 infection of the brain and related cognitive impairment remain prevalent in HIV-1-infected individuals despite combination antiretroviral therapy. Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is a newly identified host restriction factor that blocks the replication of HIV-1 and other retroviruses in myeloid cells. Cell cycle-regulated phosphorylation at residue Thr592 and viral protein X (Vpx)-mediated degradation of SAMHD1 have been shown to bypass SAMHD1 restriction in vitro. Herein, we investigated expression and phosphorylation of SAMHD1 in vivo in relation to macrophage infection and proliferation during the neuropathogenesis of HIV-1 and simian immunodeficiency virus (SIV) encephalitis. METHODS: Using brain and other tissues from uninfected and SIV-infected macaques with or without encephalitis, we performed immunohistochemistry, multilabel fluorescence microscopy and western blot to examine the expression, localization and phosphorylation of SAMHD1. RESULTS: The number of SAMHD1 nuclei increased in encephalitic brains despite the presence of Vpx. Many of these cells were perivascular macrophages, although subsets of SAMHD1 microglia and endothelial cells were also observed. The SAMHD1 macrophages were shown to be both infected and proliferating. Moreover, the presence of cycling SAMHD1 brain macrophages was confirmed in the tissue of HIV-1-infected patients with encephalitis. Finally, western blot analysis of brain-protein extracts from SIV-infected macaques showed that SAMHD1 protein exists in the brain mainly as an inactive Thr592-phosphorylated form. CONCLUSION: The ability of SAMHD1 to act as a restriction factor for SIV/HIV in the brain is likely bypassed in proliferating brain macrophages through the phosphorylation-mediated inactivation, not Vpx-mediated degradation of SAMHD1.

Vascular endothelial growth factors C and D may promote angiogenesis in the primate ovulatory follicle.

Angiogenesis in the ovary occurs rapidly as the ovarian follicle transforms into a mature corpus luteum. Granulosa cells produce vascular endothelial growth factor A (VEGFA) in response to the ovulatory gonadotropin surge. VEGFA is established as a key mediator of angiogenesis in the primate ovulatory follicle. To determine if additional VEGF family members may be involved in angiogenesis within the ovulatory follicle, cynomolgus monkeys (Macaca fascicularis) received gonadotropins to stimulate multiple follicular development, and human chorionic gonadotropin (hCG) substituted for the luteinizing hormone surge to initiate ovulatory events. Granulosa cells of monkey ovulatory follicles contained mRNA and protein for VEGFC and VEGFD before and after hCG administration. VEGFC and VEGFD were detected in monkey follicular fluid and granulosa cell-conditioned culture media, suggesting that granulosa cells of ovulatory follicles secrete both VEGFC and VEGFD. To determine if these VEGF family members can stimulate angiogenic events, monkey ovarian microvascular endothelial cells (mOMECs) were obtained from monkey ovulatory follicles and treated in vitro with VEGFC and VEGFD. Angiogenic events are mediated via three VEGF receptors; mOMECs express all three VEGF receptors in vivo and in vitro. Exposure of mOMECs to VEGFC increased phosphorylation of AKT, while VEGFD treatment increased phosphorylation of both AKT and CREB. VEGFC and VEGFD increased mOMEC migration and the formation of endothelial cell sprouts in vitro. However, only VEGFD increased mOMEC proliferation. These findings suggest that VEGFC and VEGFD may work in conjunction with VEGFA to stimulate early events in angiogenesis of the primate ovulatory follicle.

Adipose and Liver Function in Primate Offspring with Insulin Resistance Induced by Estrogen Deprivation in Utero.

PURPOSE: We recently demonstrated that offspring delivered to baboons deprived of estrogen during the second half of gestation exhibited insulin resistance. Therefore, because skeletal muscle accounts for >80% of insulin dependent glucose disposal, we suggested that estrogen in utero programs factors in fetal skeletal muscle important for insulin sensitivity in offspring. However, liver and adipose are also sites of insulin action and adipose insulin resistance can increase serum free fatty acid (FFA) levels and thereby reduce skeletal muscle insulin sensitivity. Therefore, in the current study we determined whether estrogen-deprived offspring exhibit normal adipose and hepatic function. RESULTS: The fasting serum levels of adiponectin, leptin, glucose, and analytes of liver function as well as the basal levels of serum FFA were similar in offspring of estrogen replete/suppressed baboons. Moreover, the normal glucose-induced decline in serum FFA levels measured in untreated offspring was also measured in offspring of letrozole-treated baboons. Fetal serum levels of adiponectin and leptin in late gestation also were similar and expression of nitrotyrosine negligible in fetal liver and adipose of untreated and letrozole-treated animals. CONCLUSIONS: These results indicate that offspring of letrozole-treated baboons have normal adipose and liver function and do not exhibit adipose insulin resistance. Therefore, we suggest that the insulin resistance observed in estrogen-deprived offspring primarily reflects a decline in insulin-stimulated glucose clearance by skeletal muscle and which supports our original suggestion that estrogen in utero programs factors in fetal skeletal muscle that promote insulin sensitivity in offspring.

Insulin resistance elicited in postpubertal primate offspring deprived of estrogen in utero.

We recently demonstrated that offspring delivered to baboons deprived of estrogen during the second half of gestation exhibited insulin resistance prior to onset of puberty. Because gonadal hormones have a profound effect on insulin action and secretion in adults, we determined whether insulin resistance is retained after initiation of gonadal secretion of testosterone and estradiol. Glucose tolerance tests were performed in postpubertal baboon offspring of untreated and letrozole-treated animals (serum estradiol reduced >95 %). Basal fasting levels of insulin (P < 0.05) and peak 1 min and 1 + 3 + 5 min levels of glucose after glucose tolerance tests challenge (P < 0.03) were greater in offspring delivered to letrozole-treated, estrogen-deprived baboons than untreated animals. Moreover, the value for the HOMA-IR, an accepted index of insulin resistance, was 2-fold greater (P < 0.05) in offspring delivered to baboons treated with letrozole than in untreated animals. Collectively these results support the proposal that estrogen normally has an important role in programming mechanisms in utero within the developing fetus that lead to insulin sensitivity after birth.

Mapping PTGERs to the Ovulatory Follicle: Regional Responses to the Ovulatory PGE2 Signal.

Prostaglandin E2 (PGE2) is a key intrafollicular mediator of ovulation in many, if not all, mammalian species. PGE2 acts at follicular cells via four distinct PGE2 receptors (PTGERs). Within the ovulatory follicle, each cell type (e.g., oocyte, cumulus granulosa cell, mural granulosa cell, theca cell, endothelial cell) expresses a different subset of the four PTGERs. Expression of a subset of PTGERs has consequences for the generation of intracellular signals and ultimately the unique functions of follicular cells that respond to PGE2. Just as the ovulatory LH surge regulates PGE2 synthesis, the LH surge also regulates expression of the four PTGERs. The pattern of expression of the four PTGERs among follicular cells before and after the LH surge forms a spatial and temporal map of PGE2 responses. Differential PTGER expression, coupled with activation of cell-specific intracellular signals, may explain how a single paracrine mediator can have pleotropic actions within the ovulatory follicle. Understanding the role of each PTGER in ovulation may point to previously unappreciated opportunities to both promote and prevent fertility.

Estrogen promotes luteolysis by redistributing prostaglandin F2α receptors within primate luteal cells.

Prostaglandin F2α (PGF2α) has been proposed as a functional luteolysin in primates. However, administration of PGF2α or prostaglandin synthesis inhibitors in vivo both initiate luteolysis. These contradictory findings may reflect changes in PGF2α receptors (PTGFRs) or responsiveness to PGF2α at a critical point during the life span of the corpus luteum. The current study addressed this question using ovarian cells and tissues from female cynomolgus monkeys and luteinizing granulosa cells from healthy women undergoing follicle aspiration. PTGFRs were present in the cytoplasm of monkey granulosa cells, while PTGFRs were localized in the perinuclear region of large, granulosa-derived monkey luteal cells by mid-late luteal phase. A PTGFR agonist decreased progesterone production in luteal cells obtained at mid-late and late luteal phases, but did not decrease progesterone production by granulosa cells or luteal cells from younger corpora lutea. These findings are consistent with a role for perinuclear PTGFRs in functional luteolysis. This concept was explored using human luteinizing granulosa cells maintained in vitro as a model for luteal cell differentiation. In these cells, PTGFRs relocated from the cytoplasm to the perinuclear area in an estrogen- and estrogen receptor-dependent manner. Similar to our findings with monkey luteal cells, human luteinizing granulosa cells with perinuclear PTGFRs responded to a PTGFR agonist with decreased progesterone production. These data support the concept that PTGFR stimulation promotes functional luteolysis only when PTGFRs are located in the perinuclear region. Estrogen receptor-mediated relocation of PTGFRs within luteal cells may be a necessary step in the initiation of luteolysis in primates.

Prostaglandin E2 (EP) receptors mediate PGE2-specific events in ovulation and luteinization within primate ovarian follicles.

Prostaglandin E2 (PGE2) is a key mediator of ovulation. All 4 PGE2 receptors (EP receptors) are expressed in the primate follicle, but the specific role of each EP receptor in ovulatory events is poorly understood. To examine the ovulatory events mediated via these EP receptors, preovulatory monkey follicles were injected with vehicle, the PG synthesis inhibitor indomethacin, or indomethacin plus PGE2. An ovulatory dose of human chorionic gonadotropin was administered; the injected ovary was collected 48 hours later and serially sectioned. Vehicle-injected follicles showed normal ovulatory events, including follicle rupture, absence of an oocyte, and thickening of the granulosa cell layer. Indomethacin-injected follicles did not rupture and contained oocytes surrounded by unexpanded cumulus; granulosa cell hypertrophy did not occur. Follicles injected with indomethacin plus PGE2 were similar to vehicle-injected ovaries, indicating that PGE2 restored the ovulatory changes inhibited by indomethacin. Additional follicles were injected with indomethacin plus an agonist for each EP receptor. EP1, EP2, and EP4 agonists each promoted aspects of follicle rupture, but no single EP agonist recapitulated normal follicle rupture as seen in follicles injected with either vehicle or indomethacin plus PGE2. Although EP4 agonist-injected follicles contained oocytes in unexpanded cumulus, the absence of oocytes in EP1 agonist- and EP2 agonist-injected follicles suggests that these EP receptors promote cumulus expansion. Surprisingly, the EP3 agonist did not stimulate any of these ovulatory changes, despite the high level of EP3 receptor expression in the monkey follicle. Therefore, agonists and antagonists selective for EP1 and EP2 receptors hold the most promise for control of ovulatory events in women.

The COX-2 inhibitor meloxicam prevents pregnancy when administered as an emergency contraceptive to nonhuman primates.

OBJECTIVE: Cyclooxygenase-2 (COX-2) inhibitors reduce prostaglandin synthesis and disrupt essential reproductive processes. Ultrasound studies in women demonstrated that oral COX-2 inhibitors can delay or prevent follicle collapse associated with ovulation. The goal of this study was to determine if oral administration of a COX-2 inhibitor can inhibit reproductive function with sufficient efficacy to prevent pregnancy in primates. STUDY DESIGN: The COX-2 inhibitor meloxicam (or vehicle) was administered orally to proven fertile female cynomolgus macaques using one emergency contraceptive model and three monthly contraceptive models. In the emergency contraceptive model, females were bred with a proven fertile male once 2±1 days before ovulation, returned to the females' home cage, and then received 5 days of meloxicam treatment. In the monthly contraceptive models, females were co-caged for breeding with a proven fertile male for a total of 5 days beginning 2±1 days before ovulation. Animals received meloxicam treatment (1) cycle days 5-22, or (2) every day, or (3) each day of the 5-day breeding period. Female were then assessed for pregnancy. RESULTS: The pregnancy rate with meloxicam administration using the emergency contraception model was 6.5%, significantly lower than the pregnancy rate of 33.3% when vehicle without meloxicam was administered. Pregnancy rates with the three monthly contraceptive models (75%-100%) were not consistent with preventing pregnancy. CONCLUSIONS: Oral COX-2 inhibitor administration can prevent pregnancy after a single instance of breeding in primates. While meloxicam may be ineffective for regular contraception, pharmacological inhibition of COX-2 may be an effective method of emergency contraception for women. IMPLICATIONS: COX-2 inhibitors can interfere with ovulation, but the contraceptive efficacy of drugs of this class has not been directly tested. This study, conducted in nonhuman primates, is the first to suggest that a COX-2 inhibitor may be effective as an emergency contraceptive.

EP3 receptor isoforms are differentially expressed in subpopulations of primate granulosa cells and couple to unique G-proteins.

Prostaglandin E2 (PGE2) produced within the ovarian follicle is necessary for ovulation. PGE2 is recognized by four distinct G-protein-coupled receptors. Among them, PTGER3 (also known as EP3) is unique in that mRNA splicing generates multiple isoforms. Each isoform has a distinct amino acid composition in the C-terminal region, which is involved in G-protein coupling. To determine whether monkey EP3 isoforms couple to different G-proteins, each EP3 isoform was expressed in Chinese hamster ovary cells, and intracellular signals were examined after stimulation with the EP3 agonist sulprostone. Stimulation of EP3 isoform 5 (EP3-5) reduced cAMP in a pertussis toxin (PTX)-sensitive manner, indicating involvement of Gαi. Stimulation of EP3-9 increased cAMP, which was reduced by the general G-protein inhibitor GDP-ß-S, and also increased intracellular calcium, which was reduced by PTX and GDP-ß-S. So, EP3-9 likely couples to both Gαs and a PTX-sensitive G-protein to regulate intracellular signals. Stimulation of EP3-14 increased cAMP, which was further increased by PTX, so EP3-14 likely regulates cAMP via multiple G-proteins. Granulosa cell expression of all EP3 isoforms increased in response to an ovulatory dose of human chorionic gonadotropin. Two EP3 isoforms were differentially expressed in functional subpopulations of granulosa cells. EP3-5 was low in granulosa cells at the follicle apex while EP3-9 was high in cumulus granulosa cells. Differential expression of EP3 isoforms may yield different intracellular responses to PGE2 in granulosa cell subpopulations, contributing to the different roles played by granulosa cell subpopulations in the process of ovulation.

The colchicine derivative CT20126 shows a novel microtubule-modulating activity with apoptosis.

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 µM), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 nM, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.