Steroids affect gene expression, ciliary activity, glucose uptake, progesterone receptor expression and immunoreactive steroidogenic protein expression in equine oviduct explants in vitro.

The oviduct undergoes dramatic functional and morphological changes throughout the oestrous cycle of the mare. To unravel the effects of steroids on the morphology, functionality and gene expression of the equine oviduct, an in vitro oviduct explant culture system was stimulated with physiological concentrations of progesterone and 17ß-oestradiol. Four conditions were compared: unsupplemented preovulatory explants, preovulatory explants that were stimulated with postovulatory hormone concentrations, unsupplemented postovulatory explants and postovulatory explants that were stimulated with preovulatory hormone concentrations. The modulating effects of both steroids on oviduct explants were investigated and the following parameters examined: (1) ciliary activity, (2) glucose consumption and lactate production pattern, (3) ultrastructure, (4) mRNA expression of embryotrophic genes, (5) steroidogenic capacities of oviductal explants and (6) progesterone receptor expression. The present paper shows that the equine oviduct is an organ with potential steroidogenic capacities, which is highly responsive to local changes in progesterone and 17ß-oestradiol concentrations at the level of morphology, functionality and gene expression of the oviduct. These data provide a basis to study the importance of endocrine and paracrine signalling during early embryonic development in the horse.

Progesterone, estradiol, arachidonic acid, oxytocin, forskolin and cAMP influence on aquaporin 1 and 5 expression in porcine uterine explants during the mid-luteal phase of the estrous cycle and luteolysis: an in vitro study.

BACKGROUND: The cell membrane water channel protein, aquaporins (AQPs), regulate cellular water transport and cell volume and play a key role in water homeostasis. Recently, AQPs are considered as important players in the field of reproduction. In previous studies, we have established the presence of AQP1 and 5 in porcine uterus. Their expression at protein level altered in distinct tissues of the female reproductive system depending on the phase of the estrous cycle. However, the regulation of aquaporin genes and proteins expression has not been examined in porcine uterine tissue. Therefore, we have designed an in vitro experiment to explain whether steroid hormones, progesterone (P4) and estradiol (E2), and other factors: oxytocine (OT), arachidonic acid (AA; substrate for prostaglandins synthesis) as well as forskolin (FSK; adenylate cyclase activator) and cAMP (second messenger, cyclic adenosine monophosphate) may impact AQPs expression. METHODS: Uterine tissues were collected on Days 10-12 and 14-16 of the estrous cycle representing the mid-luteal phase and luteolysis. Real-time PCR and Western blot analysis were performed to examine the expression of porcine AQP1 and AQP5. Their expression in the uterine explants was also evaluated by immunohistochemistry. RESULTS: The results indicated that uterine expression of AQP1 and AQP5 potentially remains under control of steroid hormones and AA-derived compounds (e.g. prostaglandins). P4, E2, AA, FSK and cAMP cause translocation of AQP5 from apical to the basolateral plasma membrane of the epithelial cells, which might affect the transcellular water movement (through epithelial cells) between uterine lumen and blood vessels. The AC/cAMP pathway is involved in the intracellular signals transduction connected with the regulation of AQPs expression in the pig uterus. CONCLUSIONS: This study documented specific patterns of AQP1 and AQP5 expression in response to P4, E2, AA, FSK and cAMP, thereby providing new indirect evidence of their role in maintaining the local fluid balance within the uterus during the mid-luteal phase of the estrous cycle and luteolysis in pigs.

Steroids in the equine oviduct: synthesis, local concentrations and receptor expression.

Steroids play an important role in mammalian reproduction and early pregnancy. Although systemic changes in steroid concentrations have been well documented, it is not clear how these correlate with local steroid concentrations in the genital tract. We hypothesised that, in the horse, the preimplantation embryo may be subjected to high local steroid concentrations for several days. Therefore, we measured progesterone, 17-hydroxyprogesterone, 17?-oestradiol, testosterone and 17?-testosterone concentrations in equine oviductal tissue by ultra-HPLC coupled with tandem mass spectrometry, and progesterone, 17?-oestradiol, oestrone and testosterone concentrations in oviduct fluid by radioimmunoassay, with reference to cycle stage and side of ovulation. Progesterone concentrations were high in oviductal tissue and fluid ipsilateral to the ovulation side during dioestrus, whereas other steroid hormone concentrations were not influenced by the side of ovulation. These results suggest that the high ipsilateral progesterone concentration is caused by: (1) contributions from the follicular fluid in the oviduct and diffusion of follicular fluid steroids after ovulation; (2) local transfer of steroids via blood or lymph; (3) local synthesis of progesterone in the oviduct, as evidenced by the expression of steroidogenic enzymes; and (4) a paracrine contribution from follicular cells. These data provide a basis for the study of the importance of endocrine and paracrine signalling during early embryonic development in the horse.

Transcriptomic analysis of the myometrium during peri-implantation period and luteolysis--the study on the pig model.

In pigs, implantation begins with the attachment of embryos to the endometrium. As the process is regulated by the expression of numerous genes, endometrial transcriptomic profiles have been extensively studied in early gravid pigs. However, the myometrium, a secretory tissue, should not be neglected, as it can also participate in the regulation of implantation in early pregnant pigs. To clarify this issue, the transcriptomic profile of the porcine myometrium during the peri-implantation period (i.e. on days 15 to 16 of pregnancy) was compared with the profile observed during luteolysis (i.e. on days 15 to 16 of the oestrous cycle) with an Agilent's Porcine (V2) Two-Colour Gene Expression Microarray 4 × 44 (Agilent, USA). Analysis of the microarray data revealed that of 526 unique, accurately annotated genes, the expression of 271 unique genes was upregulated, while the expression of 255 genes was downregulated in pregnant versus cyclic myometrium. The in-depth data analysis revealed differential expression of genes encoding for factors involved in immunomodulation, tissue growth and differentiation, and prostaglandin and steroid biosynthesis and action. Moreover, the comparison of the obtained data on the myometrial transcriptome with our previously published results on the endometrial transcriptome allowed us to determine substantial differences in the regulatory function of both tissues. The new insights into the function of the myometrium of early pregnant pigs obtained here are in agreement with our previous results that suggest that this tissue plays an important role in providing optimal conditions for developing embryos. Therefore, the importance of the myometrium as an active embryo signal-responsive tissue during early pregnancy cannot be underestimated.

The activity and localization of 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase and release of androstenedione and progesterone by uterine tissues during early pregnancy and the estrous cycle in pigs.

Steroid hormones are produced by the porcine uterus. We hypothesized that the uterus in pigs possesses active 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3ß-HSD) responsible for progesterone and androstenedione production, that uterine steroids may supplement the amount of steroid hormones produced by embryos and corpus luteum and that these steroids are necessary for maintenance of pregnancy. In this study, we examined 1) endometrial and myometrial expression of 3ß-HSD mRNA, 2) uterine 3ß-HSD protein activity and 3) in vitro production of A(4) and P(4) by uterine slices harvested from pigs on days 10 to 11, 12 to 13 and 15 to 16 of pregnancy and the estrous cycle. The expression of 3ß-HSD and the presence and activity of 3ß-HSD protein were different in the endometrium and the myometrium during the examined periods of pregnancy and the estrous cycle. Production of A(4) by the endometrium and myometrium was highest on days 12 to 13 of pregnancy and the estrous cycle. Endometrial secretion of P(4) did not differ in the course of early pregnancy and on the respective days of the estrous cycle. The gravid myometrium was the highest source of P(4) in pregnant pigs on days 12 to 13. The release of P(4) by the cyclic myometrium rose during the examined days of the estrous cycle. The steroidogenic activity of the uterus, as described in this study, may support early pregnancy or the luteal phase of the estrous cycle in pigs.

Novel aspects of cytokine action in porcine uterus--endometrial and myometrial production of estrone (E1) in the presence of interleukin 1beta (IL1beta), interleukin 6 (IL6) and tumor necrosis factor (TNFalpha)--in vitro study.

Interleukin 1beta (IL-1beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFalpha) increased (P < 0.05) estrone (E1) release from endometrial explants of pregnant pigs on days 10 to 11 after 12 h of tissue incubation in vitro with cytokines and on days 12 to 13 after 6 h of incubation. After 12 h of incubation on days 12 to 13 and 15 to 16 of pregnancy only IL6 increased E1 release. In non-gravid pigs IL1beta, IL6 and TNFalpha increased endometrial E1 release only on days 12 to 13 of the estrous cycle. The cytokines did not affect myometrial E1 release on days 10 to 11 and 15 to 16 ofpregnancy. On days 12 to 13 of pregnancy myometrial release of E1 was markedly increased in response to IL 1beta and IL6. In cyclic pigs only IL6 after 6 h of in vitro incubation increased myometrial E1 release on days 12 to 13 and 15 to 16. Progesterone (P4) increased both endometrial and myometrial release of E1 during the studied days of pregnancy and the estrous cycle, except for endometrial release on days 10 to 11 and 15 to 16 of the estrous cycle after 6 h of in vitro incubation. The results demonstrated that these cytokines may regulate the release of E1 both from the endometrium and myometrium harvested from gravid and non-gravid pigs. The results showed a pivotal role of IL 1beta, IL6 and TNFalpha in the regulation of E1 release in the porcine uterus in vitro.

Are KIF6 and APOE polymorphisms associated with power and endurance athletes?

Genetic polymorphisms within physiologically relevant KIF6 and APOE genes were examined in the context of athletic performance. KIF6 and ApoE are involved in cardiovascular health, modulation of lipid level and neurotransmission amongst others. The aim of this study was to examine whether three polymorphisms, KIF6 rs20455T > C, APOE rs429358T > C and APOE rs7412 C > T, were associated with athletic status of an athlete defined as performance type (endurance or power). Genotyping was performed using real-time PCR on buccal genomic DNA from 204 Polish athletes including 104 endurance and 100 power athletes, and 161 sedentary individuals. APOE rs429358 genotype frequencies differed significantly between power athletes and sedentary individuals (p = 0.046). KIF6 rs20455 and APOE rs7412 were found to be epistatically associated with the power athletic status (p = 0.032). KIF6 rs20455, APOE rs429358 and APOE rs7412 were associated with athletic status of Polish athletes. In the future, these polymorphisms could contribute to predictive models aimed at assessment of an individual's athletic status.

Compound craniosynostosis, intellectual disability, and Noonan-like facial dysmorphism associated with 7q32.3-q35 deletion.

OBJECTIVE: Craniosynostosis (CS) is the premature fusion of the cranial sutures, occurring either in isolated or syndromic form. Syndromic CS, which was described in over 180 genetic syndromes, accounts for 15-30% of all CS cases and usually originates from mutations within the FGFR1, FGFR2, FGFR3, and TWIST1 genes. However, causative alterations in other genes, or rarely copy number variations (CNVs) were also reported. In this article, we describe a patient with Noonan-like facial dysmorphism accompanied by intellectual disability and compound CS, involving coronal, sagittal, and squamous sutures. METHODS: We applied karyotyping, copy number variations analysis using array comparative genomic hybridization, and microarray-based genes expresion analysis. RESULTS: We have shown that the index carried a large and rare heterozygous deletion, which encompassed 12.782 Mb and mapped to a chromosomal region of 7q32.3-q35 (HG38 - chr7:131837067-144607071). The aberration comprised 109 protein-coding genes, including BRAF, that encodes serine/threonine-protein kinase B-Raf, being a part of the RAS/MAPK signaling pathway. DISCUSSION: The RAS/MAPK pathway plays an essential role in human development; hence, its dysregulation not surprisingly results in severe congenital anomalies, such as phenotypically overlapping syndromes termed RASopathies. To our best knowledge, we report here the first CNV causing haploinsufficiency of BRAF, resulting in dysregulation of the RAS/MAPK cascade, and consequently, in the phenotype observed in our patient. To conclude, with this report, we have pointed to the involvement of the RAS/MAPK signaling pathway in CS development. Moreover, we have shown that the molecular analysis based on both DNA and RNA profiling, undoubtedly constitutes a comprehensive diagnostic and research strategy for elucidating a cause of genetic diseases.

Adapting SureSelect enrichment protocol to the Ion Torrent S5 platform in molecular diagnostics of craniosynostosis.

Obtaining reliable and high fidelity next-generation sequencing (NGS) data requires to choose a suitable sequencing platform and a library preparation approach, which both have their inherent assay-specific limitations. Here, we present the results of successful adaptation of SureSelect hybridisation-based target enrichment protocol for the sequencing on the Ion Torrent S5 platform, which is designed to work preferably with amplicon-based panels. In our study, we applied a custom NGS panel to screen a cohort of 16 unrelated patients affected by premature fusion of the cranial sutures, i.e. craniosynostosis (CS). CS occurs either as an isolated malformation or in a syndromic form, representing a genetically heterogeneous and clinically variable group of disorders. The approach presented here allowed us to achieve high quality NGS data and confirmed molecular diagnosis in 19% of cases, reaching the diagnostic yield similar to some of the published research reports. In conclusion, we demonstrated that an alternative enrichment strategy for library preparations can be successfully applied prior to sequencing on the Ion Torrent S5 platform. Also, we proved that the custom NGS panel designed by us represents a useful and effective tool in the molecular diagnostics of patients with CS.

Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal.

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.

Transcriptomic analysis of the porcine endometrium during early pregnancy and the estrous cycle.

The goal of this study was to describe the alterations in the transcriptome of the endometrium in pigs during the beginning of implantation (days 15-16 of pregnancy) compared to cyclic pigs during the onset of luteolysis (days 15-16 of the estrous cycle). The global expression of genes in porcine gravid and non-gravid endometria was investigated using the Porcine (V2) Two-color gene expression microarray, 4 × 44 (Agilent, USA). Analysis of the microarray data showed that, of 589 accurately annotated genes, the expression of 266 genes was up-regulated and expression of 323 was down-regulated in the endometrium harvested during early pregnancy compared with the endometrium during the estrous cycle. In pregnant pigs, genes with the most significantly altered expression were involved in the following biological processes: the metabolic process, cellular process, cell communication, immune system process, developmental process, cell adhesion, antigen processing and presentation, antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class II, immune response, and the polysaccharide metabolic process. In the pregnant endometrium, cell adhesion molecules and steroid hormone biosynthesis pathways were the most significantly enriched biological pathways. Analysis of the interaction network among selected genes showed that androgen receptor (AR) encoding genes interact with genes involved in important processes occurring during early pregnancy. The bioinformatic analysis revealed information about the meaning of differentially expressed genes. The data provided new insight into the dynamic changes of the endometrial gene expression profile during days 15-16 of pregnancy.