mRNA Transcript Abundance Research Articles

Effects of non-esterified fatty acids on relative abundance of prostaglandin E2 and F2α synthesis-related mRNA transcripts and protein in endometrial cells of cattle in vitro

Cows nearing parturition have a negative energy balance (NEB), which is closely associated with lesser fertility. The NEB results in greater fat mobilisation and production of a large amount of non-esterified fatty acid (NEFA). Prostaglandins (PG), especially prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α), have important functions in regulating reproductive function. There, however, is little known about how the synthesis and release of PG are affected by NEFA. In this study, there was a focus on effects of NEFA on PG secretion as well as relative abundances of mRNA transcript and protein for PG synthetases and PG receptors in bovine endometrial (BEND) cells. Proliferation rate of BEND cells decreased in a concentration-dependent manner as NEFA increased in the media. The concentrations of PGE2 and PGF2α in NEFA treatment groups also decreased, while the ratio of PGE2/PGF2α and the relative abundances of proteins and mRNA that regulate PG synthesis and PG receptor mRNA transcripts and protein were greater as the NEFA concentration increased. Collectively, when there were large NEFA concentrations in the medium, there was a lesser release of PGE2 and PGF2α, however, there was a greater ratio of PGE2/PGF2α and relative abundances of mRNA transcripts and protein for PG synthetases and PG receptors in BEND cells, which changed the internal milieu and physiological function of the uterus with possible effects on fertility after calving. These findings provide important information that will help for further investigation of associations between NEB and fertility in dairy cows during the non-lactation to lactation-transition period.

Abundance of insulin-like growth factors 1 and 2, and type 1 insulin-like growth factor receptor in placentas of dogs

Insulin-like growth factors (IGFs) are among the primary compounds regulating placental development. In bitches, relative abundance of IGF1, IGF2 and IGFR1 mRNA transcripts have been studied in the pre-implantation uterus and early endotheliochorial placentas. The IGF2 and IGFR1 distribution has also been previously described in the uterus before embryo implantation. The aim of this study was to detect, characterize, and localize the presence of IGF1, IGF2, and IGFR1 in early-developing and mature placentas of dogs. Placentas of 15 bitches were analyzed using immunohistochemistry. The IGFs were located in endometrial epithelium and glands, with the staining pattern and intensity being less in mature placentas. Cytotrophoblast cells (CTB) and syncytiotrophoblast (STB) cells contained both IGFs; the labeling was greater in CTB of the early-developing than mature placentas. The maternal endothelium was positively stained for both IGFs, while the vascular endothelium of the chorioallantoic membrane were only stained for IGF2. The IGFR1 was detected in all cell populations evaluated. Results regarding trophoblastic IGF are quite consistent with those reported in human placentas. Spatiotemporal IGFs/IGFR1 pattern might reflect the occurrence of autocrine and paracrine signaling during placentation in bitches, and the involvement in early placental developmental processes. Furthermore, it is hypothesized that, besides hemotrophic actions of plasma IGFs, endometrial secreted IGFs may promote early placental development through histotrophic signaling.

Differential impacts of carp and salmon pituitary extracts on induced oogenesis, egg quality, molecular ontogeny and embryonic developmental competence in European eel.

Low egg quality and embryonic survival are critical challenges in aquaculture, where assisted reproduction procedures and other factors may impact egg quality. This includes European eel (Anguilla anguilla), where pituitary extract from carp (CPE) or salmon (SPE) is applied to override a dopaminergic inhibition of the neuroendocrine system, preventing gonadotropin secretion and gonadal development. The present study used either CPE or SPE to induce vitellogenesis in female European eel and compared impacts on egg quality and offspring developmental competence with emphasis on the maternal-to-zygotic transition (MZT). Females treated with SPE produced significantly higher proportions of floating eggs with fewer cleavage abnormalities and higher embryonic survival. These findings related successful embryogenesis to higher abundance of mRNA transcripts of genes involved in cell adhesion, activation of MZT, and immune response (dcbld1, epcam, oct4, igm) throughout embryonic development. The abundance of mRNA transcripts of cldnd, foxr1, cea, ccna1, ccnb1, ccnb2, zar1, oct4, and npm2 was relatively stable during the first eight hours, followed by a drop during MZT and low levels thereafter, indicating transfer and subsequent clearance of maternal mRNA. mRNA abundance of zar1, epcam, and dicer1 was associated with cleavage abnormalities, while mRNA abundance of zar1, sox2, foxr1, cldnd, phb2, neurod4, and neurog1 (before MZT) was associated with subsequent embryonic survival. In a second pattern, low initial mRNA abundance with an increase during MZT and higher levels persisting thereafter indicating the activation of zygotic transcription. mRNA abundance of ccna1, npm2, oct4, neurod4, and neurog1 during later embryonic development was associated with hatch success. A deviating pattern was observed for dcbld1, which mRNA levels followed the maternal-effect gene pattern but only for embryos from SPE treated females. Together, the differences in offspring production and performance reported in this study show that PE composition impacts egg quality and embryogenesis and in particular, the transition from initial maternal transcripts to zygotic transcription.

Intrafollicular injection of nonesterified fatty acids impaired dominant follicle growth in cattle

Dairy cows frequently undergo a state of negative energy balance (NEB) after parturition and some have impaired ovarian functions that result in delayed resumption of estrous cyclicity and development of follicles without ovulation occurring. During the postpartum period, cows undergo body-fat store losses, hormonal changes, fat mobilization and increases in nonesterified fatty acid (NEFAs) concentrations in blood and follicular fluid. The effect of NEFAs on follicular development and function of follicular cells, however, is not fully understood. The aim of this study, therefore, was to study the effect of an intrafollicular injection of a mixture of oleic, stearic and palmitic NEFAs on dominant follicle development and function of granulosa cells in cows that were not in a NEB state. Follicular size was less at 24 and 48 h after administration of NEFAs compared to that of control follicles injected with vehicle only. At 24 h after intrafollicular injection, the relative mRNA transcript abundance for proteins involved in steroidogenesis (CYP19A1, 3BHSD, STAR, FSHR), metabolism (GLUT1, GLUT3, INSR, IRS1, IRS2, SLC27A1, PPARG), and cell proliferation and apoptosis (CCND2; XIAP) in granulosa cells, as well as estradiol concentrations in follicular fluid were similar in control and NEFA-treated follicles. In conclusion, the results of this study indicate increased intrafollicular concentrations of NEFAs in cows that are not in a NEB state has a detrimental effect on follicle development. We propose intrafollicular injection is a useful approach to further investigate the local effects of NEFAs on the function of follicular cells.

Relative abundance of chemerin mRNA transcript and protein in pituitaries of pigs during the estrous cycle and early pregnancy and associations with LH and FSH secretion during the estrous cycle

Adipokines such as chemerin affect metabolic status and reproductive function in many species. The hypothesis in the present study was that there were chemerin mRNA transcript and protein in the pituitary of pigs and that relative abundances fluctuate during the estrous cycle and early pregnancy. Chemerin is thought to modulate luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion during the estrous cycle. Changes in the relative abundance of chemerin mRNA transcript and protein in anterior (AP) and posterior (PP) pituitaries of pigs were investigated, for the first time in the present study, during four phases of the estrous cycle and four periods of early pregnancy. Chemerin protein was localized in gonadotrophs, thyrotrophs and somatotrophs during the estrous cycle and early gestation. Chemerin treatments affected both basal, GnRH- and/or insulin-induced LH and FSH production, with there being variations with phase of the estrous cycle when tissues were collected. These findings indicate chemerin may be produced locally in the pituitary and may affect female reproductive function by controlling the release of LH and FSH from AP cells.

Functions of interleukin-6 in ovulation of female climbing perch, Anabas testudineus

In mammals, interleukin 6 (IL-6) has an important function during ovulation, however, the functions of IL-6 in fish have not been elucidated. In the present study, there was quantification of de novo synthesis of ovarian IL-6 and tumor necrosis factor-alpha (TNFα) in control and hCG-treated fish and results were compared with those from an in vitro study where there was evaluation of the regulatory functions of gonadotropins and TNFα of IL-6 secretions. Relatively greater concentrations of ovarian IL-6 at the post-GVBD (post-germinal vesicle breakdown) stage indicates IL-6 modulates ovulatory processes. The hCG-induced increase in relative abundance of IL-6 (in vitro) mRNA transcript and secretion from the ovary were attenuated when there was administration of the inhibitor of TNFα secreting enzyme, TAPI-I, which indicates TNFα modulates IL-6 secretion. Treatments with IL-6 induced a marked increase in ovulation rate in vitro when there was induction of activating matrix metalloproteinase (MMP). Furthermore, treatment with IL-6 resulted in production of prostaglandin as indicated by the IL-6 induced increase in the abundance of ptgs2 mRNA transcript in the ovary of Anabas testudineus. Furthermore, results indicate the source of IL-6 in the ovary is the granulosa cells with secretion of IL-6 being induced by the additions of hCG and TNFα in the medium. There was also an IL-6-induced increase in abundance of receptors (IL-6 Rα and gp130) to which it binds indicating IL-6 autoregulates this population of receptors. Results from this study, for the first time, elucidate the reproductive functions of IL-6 in a teleost fish.

Replacement of dietary fishmeal with Sargassum ilicifolium meal on growth, innate immunity and immune gene mRNA transcript abundance in Lates calcarifer juveniles

Replacement of dietary fishmeal with <i>Sargassum ilicifolium</i> meal on growth, innate immunity and immune gene mRNA transcript abundance in <i>Lates calcarifer</i> juveniles

Expression of Insulin-Like Growth Factor 1 (IGF-1) and Epidermal Growth Factor (EGF) Receptors and the Effect of IGF-1 and EGF on Androgen and Estrogen Release in the Myometrium of Pigs-In Vitro Study.

Simple SummaryThe uterus of pregnant and nonpregnant females produces steroid hormones. The activity of uterine steroidogenesis is altered by the female physiological status, but its regulation is not well understood. This study tested whether growth factors may regulate steroid hormone production in the myometrium. To do so, myometrial slices were isolated from pigs during days 10 or 11, 12 or 13 and 15 or 16 of the estrous cycle and early pregnancy and it was determined whether the tissue possesses potential to respond insulin growth factor 1 (IGF-1) or epidermal growth factor (EGF) and whether they affect androstenedione (A4), testosterone (T), estrone (E1) and estradiol-17β (E2) production in the myometrium. The results showed that myometrial receptivity to IGF-1 and EGF was altered in pregnant and nonpregnant pigs and IGF-1 and EGF affect steroid hormone release in the myometrium. The observed alterations were limited to specific days of the estrous cycle and pregnancy. In conclusion, IGF-1 and EGF may be considered as specific regulators of myometrial steroid hormone production.Porcine myometrium possesses steroidogenic activity but its regulation is not well understood. It was hypothesized that the regulators of myometrial steroidogenesis are insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF), which were found to modulate the steroidogenic activity of the endometrium and embryos. Myometrial slices were collected from gravid and nongravid pigs on days 10 or 11, 12 or 13 and 15 or 16 and studied for: (1) the relative abundance of IGF-1R and EGFR mRNA transcripts and proteins, to determine myometrial readiness to response growth factors treatment and (2) the effect of IGF-1 or EGF on the myometrial release of androstenedione (A4), testosterone (T), estrone (E1) and estradiol-17β (E2). The results showed that the relative expression and abundance of IGF-1R and EGFR in the myometrium were altered regarding the female reproductive status. During the estrous cycle, EGF increased myometrial release of A4 on days 12–13 and E2 on days 15–16. In gravid pigs (days 15–16), IGF-1 and EGF increased the E1 release. In conclusion: (1) porcine myometrium possesses the potential to respond to IGF-1 and EGF treatment, (2) EGF significantly increases myometrial A4 and E2 release in cyclic pigs, while IGF-1 and EGF increase the E1 release in gravid pigs.

Consequences of electromagnetic field (EMF) radiation during early pregnancy - androgen synthesis and release from the myometrium of pigs in vitro

An electromagnetic field (EMF) has been found to affect reproductive processes in females. The aim of this study was to determine the effect of low, non-ionizing EMF radiation on the steroidogenic activity of myometrium collected from pigs during the fetal peri-implantation period. Myometrial slices were treated with an EMF (50 and 120 Hz, 2 and 4 h of incubation) and examined for the aromatase cytochrome P450 17α-hydroxylase/C17-20lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (HSD3B1) mRNA transcript abundance, cytochrome P450c17 and 3βHSD protein abundance and the secretion of androstenedione (A4) and testosterone (T). To determine whether progesterone (P4) functions as a protectant from EMF radiation, the selected slices were treated with P4. In slices incubated without P4, EMF at 50 Hz altered cytochrome P450c17 protein abundance (4 h), HSD3B1 mRNA transcript abundance (4 h) and A4 release (2 h) as well as T release (2 h) in P4-treated slices. The EMF at 120 Hz in non P4-treated slices altered A4 release (2 and 4 h) whereas in P4-treated slices altered CYP17A1 mRNA transcript abundance (4 h), 3βHSD protein abundance (4 h), A4 (4 h) and T release (2 h). In conclusion, EMF radiation in the myometrium collected during the peri-implantation period alters the CYP17A1 and HSD3B1 mRNA transcript and encoded protein abundance, and androgen release due to the time of treatment and P4 presence or absence. The P4 did not function directly as an obvious protector against EMF radiation in the myometrium of pigs during the fetal peri-implantation period.

Effects in broiler hens of genetic lines differing in fertility, biotin supplementation, and age on relative abundance of oviductal transforming growth factor-β and carbonic anhydrase mRNA transcripts

There was evaluation of effects of biotin administration on oviductal abundance of transforming growth factor-β (TGF-β) and carbonic anhydrase (CA) mRNA transcript in younger and older broiler hens of relatively lesser and greater fertility lines. Additionally, effects of biotin supplementation on attenuation of age-related subfertility were evaluated. Hens from the relatively greater (Line D, n = 60) and lesser (Line B, n = 60) fertility rate line were randomly assigned to three treatment groups. Biotin was not or was administered in drinking water from 30 to 33 (younger age) and 53 to 56 (older age) wk of age to have access to no biotin (T0), or 0.3 (T1), or 0.45 (T2) mg/L of biotin. There was assessment the relative oviductal abundances of TGF-β and CA mRNA transcript abundances. Supplemental biotin and age had no effect on the relative abundance of oviductal TGF-β mRNA transcript in hens of Line D. There, however, was a ten-fold greater abundance of TGF-β in hens of the T0 group of Line B compared with Line D. Relative abundance of TGF-β mRNA transcript was greater in younger hens of Line B; however, biotin supplementation of older hens of the T2 group of Line B resulted in a similar TGF-β abundance to that of younger hens. Inconstant with the TGF-β abundance, CA abundance in hens of Line B was not affected by supplemental biotin or bird age. Overall, differences in TGF-β or CA abundances did not affect fertility of broiler hens.

Persian onager (Equus hemionus onager) endometrial explant cryopreservation and in vitro culture

Assisted reproduction of endangered equids, such as Persian onagers (Equus hemionus onager), is vital for species conservation. Little is known about Persian onager reproductive functions, including functions of the uterine endometrium. Recently, successful cryopreservation of the domestic mare endometrium was reported, but there is no information on cryo-sensitivity or in vitro culture of endometrial tissues of any non-domestic equid. In the present study, endometrial explants from Persian onagers were cryopreserved and cultured in vitro for 5 days. There was no difference between endometrial explants when 10% and 20% dimethyl sulfoxide (DMSO) was used for cryopreservation. Cell viability and structural integrity were comparable to fresh tissue. Abundance of estrogen receptor-α (ESR1) and progesterone receptor (PGR) mRNA transcript in endometrial explants was less in most treatment groups compared to the fresh tissue control. There was variation in E-cadherin mRNA abundance in endometrial explants among treatment groups with some treatment groups having a lesser abundance compared to the control group. The abundance of Ki67 mRNA transcript of endometrial explants was not different among treatment groups compared to the control group. Results indicate that DMSO is a suitable cryoprotectant for the Persian onager endometrium, and in vitro culture in a liquid-gas interface can maintain Persian onager endometrial explants for as long as 5 days. Findings allow for a greater understanding of reproductive mechanisms in vitro for this endangered species and other domestic equids including donkeys.

Pituitary porcine FSH, and recombinant bovine and human FSH differentially affect growth and relative abundances of mRNA transcripts of preantral and early developing antral follicles in goats

Three different sources of FSH (porcine pituitary, pFSH; recombinant bovine, rbFSH; and recombinant human, rhFSH) were compared during in vitro culture of preantral and early antral follicles of goats for 18 days. Treatments were: base medium supplemented with no FSH (control), 10, 50, or 100 mIU/mL pFSH (pFSH10, pFSH50, and pFSH100, respectively), 100 ng/mL rbFSH (rbFSH), and 50 mIU/mL rhFSH (rhFSH). There were evaluations of follicle morphology, antrum formation, growth rate, estradiol production, oocyte viability and chromatin configuration, and follicle wall relative abundance of mRNA transcript for MMP-9, TIMP-2, CYP17, CYP19A1, FSHR, Insulin-R, and BAX/BCL-2 ratio. Follicle degeneration rates were similar among all treatment groups at the end of culturing. When there were treatments with pFSH, however, there was a lesser (P < 0.05) percentage of intact follicles and estradiol production, and greater (P < 0.05) extrusion rates. Furthermore, with only pFSH10 (antral follicles) and pFSH100 (preantral and antral follicles) treatments, there was a lesser (P < 0.05) follicle growth. For preantral follicles, when there was addition of pFSH10, pFSH100, and rhFSH there was lesser (P < 0.05) oocyte meiotic resumption compared to control and rbFSH treatments. For antral follicles, when there were treatments with rhFSH and pFSH10 there was greater (P = 0.08 – P < 0.05) oocyte maturation. In conclusion, the source of FSH differentially affected gene expression, as indicated by mRNA abundances, and follicular dynamics of preantral and antral follicles in vitro. Addition of FSH during the in vitro culture improved the developmental outcomes only for antral follicles.

Identification of cadaveric liver tissues using thanatotranscriptome biomarkers

Thanatotranscriptome studies involve the examination of mRNA transcript abundance and gene expression patterns in the internal organs of deceased humans. Postmortem gene expression is indicative of the cellular status of a corpse at the time of death, a portion of which may represent a cascade of molecular events occasioned by death. Specific gene biomarkers identify perceptible transcriptional changes induced by stochastic responses to the cessation of biological functions. Transcriptome analyses of postmortem mRNA from a tissue fragment may determine unique molecular identifiers for specific organs and demonstrate unique patterns of gene expression that can provide essential contextual anatomical information. We evaluated the impact of targeted transcriptome analysis using RNA sequencing to reveal global changes in postmortem gene expression in liver tissues from 27 Italian and United States corpses: 3.5-hour-old to 37-day-old. We found that our single blind study using eight liver tissue-specific gene biomarkers (e.g. AMBP and AHSG) is highly specific, with autopsy-derived organ samples correctly identified as tissues originating from postmortem livers. The results demonstrate that 98–100% of sequencing reads were mapped to these liver biomarkers. Our findings indicate that gene expression signatures of mRNA exposed up to 37 days of autolysis, can be used to validate the putative identity of tissue fragments.

17β-estradiol regulates prostaglandin E2 and F2α synthesis and function in endometrial explants of cattle

Prostaglandins (PG) have primary functions in the reproductive tract, however, the mechanism of regulation of PG secretion in the endometrium is unclear. Estrogen as a predominant regulator of uterine functions during the mammalian estrous cycle and effects of estrogen on synthesis of PG and function in uterine tissues of cattle are not fully understood. In this study, there was evaluation of the concentration- and time-effects of 17β-estradiol on PG synthesis in endometrial explants of cattle, focusing on the secretion of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) as well as relative abundance of mRNA transcript and protein for both the enzymes responsible for PGE2 and PGF2α synthesis, including prostaglandin-endoperoxide synthase 1 and 2 (PTGS1, PTGS2), PGE2 synthase (PGES), PGF2α synthase (PGFS), and carbonyl reductase (CBR1), and the receptors responsible for downstream PGE2 (PTGER2, PTGER4) and PGF2α (PTGFR) signaling. Results indicated that 17β-estradiol increased PGE2 and PGF2α production at concentrations ranging from 10-11 to 10-8 M. Furthermore, abundances of PTGS1, PTGS2, PGES, PGFS, PTGER2, PTGER4, and PTGFR mRNA transcripts and protein were greater immediately after 17β-estradiol treatment at almost all the concentrations, while these CBR1 abundances were less as a result of treatments with 17β-estradiol. These data support the hypothesis that estradiol modulates the synthesis and function of PG in the endometrium of cattle.

Crowding stress during the period of sex determination causes masculinization in pejerrey Odontesthes bonariensis, a fish with temperature-dependent sex determination

The pejerrey is an atherinopsid species from South America that presents a combination of genotypic and environmental (temperature-dependent) sex determination whereby low and high temperatures induce feminization and masculinization, respectively. Masculinization involves a heat-induced stress response leading to increased circulating cortisol and androgens. We tested whether crowding would elicit a similar response as high temperature and affect the sex ratios of pejerrey. Larvae with XX and XY genotypes were reared at 15, 62 and 250 larvae/L in 0.4, 1.6, and 6.4 L containers during a period considered critical for sex determination at 25 °C, a mixed-sex promoting temperature. Fish were analysed at 3–7 weeks for whole-body cortisol and 11-ketotestosterone (11-KT) titer and hydroxy-steroid dehydrogenase (hsd11b2) mRNA transcript abundance, and after completion of gonadal sex differentiation (10–14 weeks) for determination of phenotypic and genotypic sex mismatches. Crowding was associated with depressed growth, higher cortisol and 11-KT titers, increased hsd11b2 transcription, and increased frequency of masculinization compared to intermediate and/or low rearing densities. Perceived crowding (by rearing in containers with mirror-finish, reflecting walls) also caused masculinization. These results suggest the possibility that other environmental factors besides temperature can also affect sex determination in pejerrey and that a stress response leading to increased cortisol and androgen levels, which is potentially perceived by the brain, may be a common feature among different forms of environmental sex determination in this species.

Temporal transcript abundance of clock genes, angiogenic factors and nutrient sensing genes in bovine placental explants

Recent research has shown expression of clock genes in peripheral tissue explants, targeting multiple pathways leading to the entrainment of circadian rhythms. Temporal variations are not solely regulated by photoperiod, but factors such as maternal feed availability can entrain fetal circadian clock. Currently, a paucity of information exists for clock gene expression and short-term temporal transcript abundance in the bovine placenta, which is essential for proper offspring development. Therefore, the objective of this study was to determine the effect of early to mid-gestational nutrient restriction on clock genes, angiogenic factors, and nutrient sensing genes mRNA transcript abundance in placental explants during a 24 h period. Placentomes from adequately fed and nutrient restricted heifers were collected via Cesarean section at day 180 of gestation; separated into caruncular and cotyledonary tissue and placed in culture media for a 24 h period. The mRNA transcript abundance of clock genes (ARNTL, CRY1, and PER2), angiogenic factors (HIF1A and VEGFA), and nutrient sensing genes (NAMPT and NR3C1) was determined every 4 h. Clock genes were expressed in caruncular and cotyledonary explant tissue. The caruncular explant transcript abundance of the clock genes was not influenced by time (P > 0.05); while ARNTL abundance decreased over time in the cotyledon explant (P < 0.05). A main effect of time was observed for HIF1A, VEGFA, and NR3C1 in the caruncular tissue (P < 0.05). Although, angiogenic factors and nutrient sensing genes in cotyledonary tissue displayed evident temporal variation in transcript abundance (P < 0.05). Nutrient restriction did not alter (P > 0.15) mRNA transcript abundance of clock genes, angiogenic factors, or nutrient sensing genes in either caruncular or cotyledonary tissue. Interestingly, these data may indicate limited transmission and synchronization of maternal and fetal temporal variations in transcript abundance. These findings demonstrate that multiple timepoint collections are needed in future studies due to the innate existence of temporal oscillations observed in the bovine placenta.

Relative abundance and localization of interferon-stimulated gene 15 mRNA transcript in intra- and extra-uterine tissues during the early stages of pregnancy in sheep

The aim of this study was to investigate relative abundance and localization of ISG15 mRNA transcript in intra-uterine (trophoblast, endometrium) and extra-uterine (hypothalamus, anterior pituitary, corpus luteum) tissues before and during the period of conceptus implantation. Multiparous ewes (n = 16) were randomly allotted into four groups: pregnant or estrous cyclic on days of 12 and 16 (n = 4 per group) following estrus. Relative abundances of ISG15 mRNA transcript were determined in the endometrium, corpus luteum, hypothalamus, and anterior-pituitary using real time quantitative PCR. Localization of ISG15 mRNA transcript was evaluated using in situ hybridization. The presence of ISG15 mRNA transcript was only visualized in intra-uterine tissues including the endometrium and trophoblast on day 12 of pregnancy. The ISG15 mRNA transcript was detected in all tissues evaluated on day 16 of pregnancy. The abundance of ISG15 mRNA transcript was greater in the endometrium on day 12 of pregnancy than at other days when evaluations occurred while in all other tissues except the hypothalamus there were large abundances of ISG15 mRNA on day 16 of pregnancy. It is concluded that the ISG15 mRNA transcript is only present in intra-uterine tissues before conceptus implantation. The ISG15 mRNA transcript, however, is present in extra-uterine tissues of ewes during implantation probably due to an increased amount of interferon-tau in blood circulation that is produced by the developing embryo. Results also indicate, for the first time, that pregnancy is associated with an intra-hypothalamus and anterior pituitary increased abundance of ISG15 mRNA transcript in ewes.

Initiation of Conceptus Elongation Coincides with an Endometrium Basic Fibroblast Growth Factor (FGF2) Protein Increase in Heifers.

Fibroblast growth factors (FGF) play an important role during embryo development. To date, the role of FGF and the respective receptors (FGFR) during the preimplantation phase in cattle are not fully characterized. We examined FGF1, FGF2, FGFR1, FGFR2, and FGFR3 in cyclic and early pregnant heifers at Days 12, 15, and 18 after insemination (Day 0). Endometrial FGF1 mRNA transcript abundance in heifers varied significantly with respect to the day after insemination, the pregnancy status, and their interaction. The expression was higher in nonpregnant than in pregnant heifers at Day 18. The conceptus transcripts abundance of FGFR2 and FGFR3 were significantly lower at Day 15 than 18. In the endometrium, FGF1 protein abundance significantly decreased from Day 12 onwards and FGF2 protein abundance showed a minor, but a significant increase at Day 15 in comparison to Days 12 and 18. We concluded that the decrease in FGF1 mRNA expression in pregnant heifers at Day 18 points towards a potential contribution of FGF1 in the preimplantation process. Additionally, successful embryo elongation might require a spatiotemporal FGF2 protein increase in the endometrium.

Cortisol inhibits the Escherichia coli-induced endometrial inflammatory response through NF-κB and MAPK pathways in postpartum goats

Glucocorticoids have been widely used as anti-inflammatory therapies. The mechanisms of cortisol action in goat does with endometritis, however, have not been reported. The aim of this study was to investigate the mechanism of cortisol in modulation of effects of E. coli-induced endometritis in the does. Does (n = 24) were assigned to four groups (n = 6): control, E. coli, cortisol, and E. coli + cortisol groups. Does in the cortisol and E. coli + cortisol group were treated with cortisol from 3 days before E. coli inoculations occurred to 36 days post-partum. Does in the E. coli and inoculation groups were administered via intrauterine infusion E. coli O55 (109 CFU/mL) at 0 h. Physical indicators, macroscopic and microscopic changes in the endometrium, uterine secretion cytology and bacteriology were evaluated before (0 h) and at 6, 12, 24, 48, and 72 h after E. coli inoculation. The TLR4 and pro-inflammatory cytokine mRNA transcripts were detected using qPCR. The activations of NF-κB and MAPK signaling pathways were detected using Western blot procedures. As a result, cortisol inhibited the inflammatory response of does by reducing the clinical symptoms, morphological endometrial damage, % PMN in uterine secretions, relative abundance of inflammatory gene mRNA transcripts in the endometrium of does. Cortisol inhibited NF-κB activity by reducing MyD88 and IκB phosphorylation. Treatment with cortisol suppressed the phosphorylation of ERK1/2, p38MAPK, and JNK. These results indicate the anti-inflammatory effect of cortisol in the endometrium of does may be regulated by NF-κB and MAPK pathways.

Molecular characteristics and abundance of insulin-like androgenic gland hormone and effects of RNA interference in Eriocheir sinensis

Insulin-like androgenic gland hormone (IAG) has an important function in sexual differentiation and somatic growth in crustaceans. In this study, there was cloning of the full-length sequence of IAG from Eriocheir sinensis (Es-IAG). The full-length Es-IAG gene was 1392 base pairs long and encoded a protein of 151 amino acid residues. The precursor peptide included a signal peptide, and the protein was a protein that is secreted from the cell in which it is produced with no transmembrane domain. Amino acid sequence alignment indicated there was the greatest homology between E. sinensis and Chaceon quinquedens (47 %), followed by Callinectes sapidus (44 %). Results from analysis of the relative abundances of Es-IAG mRNA transcript at different developmental stages indicated that Es-IAG may have an important endocrine function in early embryonic development, and that Stages I through Ⅲ may be an important period for sexual differentiation in juvenile E. sinensis. In vivo treatment with siRNA-391 resulted in a 66.7 % lesser relative abundance of the Es-IAG mRNA transcript. Three treatments with siRNA-391 to inhibit Es-IAG production during Stages Ⅲ to Ⅴ period resulted in about 10 % of male crabs being transformed into “neo-females.” These results provide the basis for further research into the sexual differentiation mechanism and monosex breeding of E. sinensis.