Gonadotropin-releasing hormone alters the T helper cytokine balance in the pregnant rat

The interactions between immune-endocrine and reproductive systems are heightened during pregnancy as an adaptive mechanism, and are regulated by a complex array of hormones and cytokines that control the survival of a semiallogeneic conceptus. GnRH can exert direct effects on the immune system via its receptor (GnRH-R) on lymphoid cells. In the present study, we employed in vitro, ex vivo, and in vivo approaches to investigate the role of GnRH in the modulation of T helper cytokines in pregnant rats undergoing termination of pregnancy. Day 8 pregnant rats were infused with a GnRH agonist (GnRH-Ag) for 24 h using an osmotic minipump. Sham control rats were infused with the vehicle, saline. Lymphocytes were isolated from sham and treated rats and polyclonally stimulated with immobilized anti-CD3 antibody. The levels of the signature T helper 1 (Th-1) cytokines (interferon-gamma [IFN-gamma] and interleukin-2 [IL-2]) and Th-2 cytokines (IL-4 and IL-10) were measured in culture supernatants. Using immunoflourescence confocal microscopy, we demonstrated for the first time the spatial localization of GnRH-R protein on the surface of lymphocytes. We observed a marked increase in IFN-gamma and inhibition of IL-4 production from lymphocytes of pregnant rats treated in vitro with different doses of GnRH-Ag. Further, the responsiveness of lymphocytes to produce IFN-gamma was markedly increased in cells cultured ex vivo from GnRH-Ag infused rats, whereas the capacity of lymphocytes to produce IL-4 was significantly inhibited. In addition, GnRH-Ag infusion in pregnant rats induced a shift toward Th-1 cytokines in the serum. We did not observe any significant difference in IL-2 and IL-10 production in response to GnRH-Ag. Our results suggest an additional function for GnRH as a Th-1 inducer and Th-2 inhibitor. GnRH can thus skew the cytokine balance to predominantly Th-1 type in pregnancy, leading to the termination of pregnancy in rats.     

Clinostat rotation induces apoptosis in luteal cells of the pregnant rat

Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.     

Novel conserved assembly factor of the bacterial flagellum

TP0658 (FliW) and its orthologs, conserved proteins of unknown function in Treponema pallidum and other species, interact with a C-terminal region of flagellin (FlaB1-3 in T. pallidum; FliC in most other species). Mutants of orthologs in Bacillus subtilis and Campylobacter jejuni (yviF, CJ1075) showed strongly reduced motility. TP0658 stabilizes flagellin in a way similar to FliS, suggesting that TP0658 is a conserved assembly factor for the bacterial flagellum.     

Expression of mRNA and proteins for GnRH I and II and their receptors in primate corpus luteum during menstrual cycle

The differential expression of mRNA and protein of GnRH I, II and their receptors (RI and RII) in the monkey corpus luteum (CL) were measured during different stages of the luteal phase of the menstrual cycle as an initial step towards considering the role and regulation of GnRH (I and II) system during luteinization and luteolysis in primates. RT-PCR confirmed the sequence identity of PCR products and real time PCR quantified specific mRNA expressions. Proteins were localized by immunohistochemistry (IHC). Changes in mRNA expression patterns of GnRH I and II (increased) and GnRH RII (decreased) were maximal at mid-late to late stages, that is, at CL regression, where as GnRH RI was low during the entire luteal phase. However, RT-PCR and IHC studies confirmed the presence of GnRH RI at both mRNA and protein levels, respectively. IHC results showed the presence of GnRH I, II and their receptors in steroidogenic cells (granulose-luteal cells and thecal-luteal cells) across the luteal phase. Hence, GnRH I and II systems may have a role on both luteinization (from early to mid stages of CL) and luteolysis (from mid-late to very-late stages of CL). These novel findings suggest that monkey luteal GnRH system may have a role in fertility regulation in paracrine and/or autocrine manner.     

Ability of high hydrostatic pressure treated plasmids and cells of Escherichia coli to genetically transform

The exposure of plasmid pUC18 and pBR322 DNA to high hydrostatic pressure increased the ability of plasmids to transform competent Escherichia coli cells. For pUC18 plasmid, a pressure of 400 MPa, and for pBR322, a pressure of 200 MPa was found to provide the highest transformation efficiency. The DNA duplexes of the two plasmids were found to be the most stable for melting conditions at these pressures. At pressures higher than these, both the stability of the duplex DNA and the transformation efficiency were affected. The stabilizing effect of high hydrostatic pressure on the hydrogen bond may be responsible for the observed increase in transformation efficiency of the pressure-exposed plasmid DNA. The possibility of pressure-induced changes in the structure and conformation of DNA was studied using various techniques. In agarose gel electrophoresis, pressure-treated plasmids (pUC18 at 400 MPa and pBR322 at 200 MPa) consistently showed visibly distinct higher mobility compared to untreated plasmids. Pressure-treated pUC18 as well as pBR322 DNA showed significant reduction in ethidium bromide binding as is evident from the reduced intensity of fluorescence of the dye bound pressure-treated DNA. Spectroscopic studies using circular dichroism and Fourier transform infrared (FTIR) spectroscopy also showed significant differences in the absorption profiles of pressure-treated plasmids as compared to an untreated control. These studies revealed that the pressure-induced changes in the conformation of these DNAs may be responsible for the observed increase in the transformation ability of the plasmids. On the other hand, the exposure of competent cells of E. coli to a high hydrostatic pressure of 50 MPa not only reduced their colony-forming ability but also drastically reduced their ability to take up plasmid DNA.     

Enterovirus D-68 Infection,Prophylaxis, and Vaccination in a Novel Permissive Animal Model,the Cotton Rat (Sigmodon hispidus)

In recent years, there has been a significant increase in detection of Enterovirus D-68 (EV-D68) among patients with severe respiratory infections worldwide. EV-D68 is now recognized as a re-emerging pathogen; however, due to lack of a permissive animal model for EV-D68, a comprehensive understanding of the pathogenesis and immune response against EV-D68 has been hampered. Recently, it was shown that EV-D68 has a strong affinity for α2,6-linked sialic acids (SAs) and we have shown previously that α2,6-linked SAs are abundantly present in the respiratory tract of cotton rats (Sigmodon hispidus). Thus, we hypothesized that cotton rats could be a potential model for EV-D68 infection. Here, we evaluated the ability of two recently isolated EV-D68 strains (VANBT/1 and MO/14/49), along with the historical prototype Fermon strain (ATCC), to infect cotton rats. We found that cotton rats are permissive to EV-D68 infection without virus adaptation. The different strains of EV-D68 showed variable infection profiles and the ability to produce neutralizing antibody (NA) upon intranasal infection or intramuscular immunization. Infection with the VANBT/1 resulted in significant induction of pulmonary cytokine gene expression and lung pathology. Intramuscular immunization with live VANBT/1 or MO/14/49 induced strong homologous antibody responses, but a moderate heterologous NA response. We showed that passive prophylactic administration of serum with high content of NA against VANBT/1 resulted in an efficient antiviral therapy. VANBT/1-immunized animals showed complete protection from VANBT/1 challenge, but induced strong pulmonary Th1 and Th2 cytokine responses and enhanced lung pathology, indicating the generation of exacerbated immune response by immunization. In conclusion, our data illustrate that the cotton rat is a powerful animal model that provides an experimental platform to investigate pathogenesis, immune response, anti-viral therapies and vaccines against EV-D68 infection.     

Regulated expression of inhibitor of apoptosis protein 3 in the rat corpus luteum

We sought to investigate the role inhibitor of apoptosis proteins (IAPs) play in the life cycle of the corpus luteum (CL) of the rat. We isolated two clones with amino acid homology to rat IAP2 (BIRC 3) and three to rat IAP3 (rIAP3; BIRC 4). The expression of rIAP3 mRNA was examined in the rat CL during and after pregnancy, in Day 8 pregnant rats after 24-h treatment of gonadotropin-releasing hormone-agonist (GnRH-Ag), and in a CL organ culture model of spontaneous apoptosis in the absence of tropic support with and without superoxide dismutase. We used real-time RT-PCR to quantitate rIAP3 mRNA expression. Interestingly, a significant reduction in rIAP3 levels was seen at the time of CL regression in the course of natural pregnancy and the GnRH-Ag model. Surprisingly, rIAP3 mRNA levels in the CL organ culture model of spontaneous apoptosis failed to show significant changes, although TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end-labeling) reaction showed 30%-40% of the cells undergoing DNA fragmentation after 2 h in culture. In situ hybridization revealed that rIAP3 expression was localized to the cytoplasm of luteal and granulosa cells. These data clearly demonstrate both the presence of IAPs in the rat CL and the regulation of rIAP3 during in vivo apoptotic cell death, indicating a role for IAPs in the maintenance of CL function and demise.     

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.