Sperm long non-coding RNAs as markers for ram fertility.

It is critical in sheep farming to accurately estimate ram fertility for maintaining reproductive effectiveness and for production profitability. However, there is currently a lack of reliable biomarkers to estimate semen quality and ram fertility, which is hindering advances in animal science and technology. The objective of this study was to uncover long non-coding RNAs (lncRNAs) in sperm from rams with distinct fertility phenotypes. Mature rams were allocated into two groups: high and low fertility (HF; n = 31; 94.5 ± 2.8%, LF; n = 25; 83.1 ± 5.73%; P = 0.028) according to the pregnancy rates sired by the rams (average pregnancy rate; 89.4 ± 7.2%). Total RNAs were isolated from sperm of the highest- and lowest-fertility rams (n = 4, pregnancy rate; 99.2 ± 1.6%, and 73.6 ± 4.4%, respectively) followed by next-generation sequencing of the transcripts. We uncovered 11,209 lncRNAs from the sperm of rams with HF and LF. In comparison to each other, there were 93 differentially expressed (DE) lncRNAs in sperm from the two distinct fertility phenotypes. Of these, 141 mRNAs were upregulated and 134 were downregulated between HF and LF, respectively. Genes commonly enriched for 9 + 2 motile cilium and sperm flagellum were ABHD2, AK1, CABS1, ROPN1, SEPTIN2, SLIRP, and TEKT3. Moreover, CABS1, CCDC39, CFAP97D1, ROPN1, SLIRP, TEKT3, and TTC12 were commonly enriched in flagellated sperm motility and sperm motility. Differentially expressed mRNAs were enriched in the top 16 KEGG pathways. Targets of the differentially expressed lncRNAs elucidate functions in cis and trans manner using the genetic context of the lncRNA locus, and lncRNA sequences revealed 471 mRNAs targets of 10 lncRNAs. This study illustrates the existence of potential lncRNA biomarkers that can be implemented in analyzing the quality of ram sperm and determining the sperm fertility and is used in breeding soundness exams for precision livestock farming to ensure food security on a global scale.

Risk Factor Analysis and Genetic Parameter Estimation for Pre-Weaning Mortality Traits in Boer, Spanish, and Crossbred Goat Kids.

The objectives of this study were to evaluate fixed risk factors associated with PWM and to estimate genetic parameters for PWM. A total of 927 birth records from a mixed population of purebred and crossbred Boer and Spanish goats born between 2016 and 2023 at the International Goat Research Center (IGRC) were used for this study. Four binary traits were studied: D0-3 (death within 3 days after birth), D4-60 (death between 4 and 60 days), D61-90 (death between 61 and 90 days), and D0-90 (death within 90 days). Logistic regression models were used to evaluate the risk factors associated with PWM traits. Bayesian threshold models and Gibbs sampling were used to estimate the genetic parameters. Birth weight, season, litter size, sex, dam age, breed, and heterosis were found to be significantly associated with at least one of the PWM traits. Heritability estimates were 0.263, 0.124, 0.080, and 0.207, for D0-3, D4-60, D61-90, and D0-90, respectively. The genetic correlations between the studied traits ranged from 0.892 (D0-3 and D0-90) to 0.999 (D0-3 and D61-90). These results suggest that PWM in goats is influenced by both non-genetic and genetic factors and can be reduced by management, genetic selection, and crossbreeding approaches.

Expression of circulating oar-miR-485-5p and oar-miR-493-5p during the estrous cycle and early pregnancy in ovine plasma.

In the current study, we aimed to assess the expression levels of two circulating microRNAs (miRNA) (oar-miR-485-5p and oar-miR-493-5p) in the ovine plasma during the peri-implantation. After mating, we collected the plasma samples from a total of 8 ewes on day 22 of pregnancy (P22; n = 4) and day 22 of the estrous cycle (C22; n=4). We used mature miRNA sequences for oar-miR-485-5p and oar-miR-493-5p out of one hundred fifty, which were retrieved from our microarray results of previous study. We showed that the miRNA expression of oar-miR-485-5p and oar-miR-493-5p were upregulated in P22 (P<0.05) when compared to C22. Those two miRNAs targeted 311 target genes in the peri-implantation period of pregnancy. Furthermore, we revealed 151 GO/pathway terms in biological process (BP) and 25 GO/pathway terms in molecular function (MF), while we demonstrated 13 GO/pathway terms in cellular component (CC). We revealed three hub genes as interleukin 2 (IL2), interleukin 18 (IL18), and C-X-C Motif Chemokine Ligand 10 (CXCL10). In conclusion, both miR-485-5p and oar-miR-493-5p have the potential to be a biomarker to understand peri-implantation of the ovine pregnancy in the aspect of pregnancy-reflected changes in maternal plasma.

Altered luteal expression patterns of genomic and non-genomic progesterone receptors in bitches at different reproductive states.

The binding of steroid hormones to their specific receptors is necessary to exert their effects on target cells. Progesterone (P4), a steroid hormone, carries out its effects through both genomic and non-genomic (the cell membrane-associated) receptors. This study aimed to ascertain luteal expression patterns of genomic and non-genomic progesterone receptors in bitches in physiological (early dioestrus and early pregnant) and pathological (pyometra) reproductive states. Luteal tissue was collected from the bitches at early dioestrus (ED, n = 5), early pregnant (EP, n = 5), and pyometra (PY, n = 5). The expression profiles of Steroidogenic Acute Regulator Protein (STAR), Progesterone Receptor (PGR), Membrane Progestin Receptors (PAQR5, PAQR7 and PAQR8), and Progesterone Membrane Components (PGMRC1 and PGMRC2) were examined at the mRNA levels using Real-Time Polymerase Chain Reaction (RT-PCR). Protein levels of PGR, PGMRC1 and PGMRC2 were detected by western blotting (WB). The STAR expression was found in all groups, with a statistical difference observed between EP and PY groups (P < 0.05). The protein level of PGR was determined to be highest in the EP group and lowest in the PY group. The expression of PAQR8 increased in the EP group (P < 0.05). The PAQR5 exhibited high expression in the EP group and low expression in the PY group (P < 0.05). PGRMC1 was more elevated in the EP group and lower in the PY group (P < 0.05). Protein levels of PGMRC1 and PGMRC2 were also observed at the highest expression in EP group. According to the altered expression profiles for examined receptors, we suggest that those progesterone receptors have roles in early pregnancy or pyometra in bitches.

Effects of essential oil mixtures on expression of genes involved in lipogenesis and fatty acid oxidation in geese (Anser anser).

The use of essential oils has recently increased in the poultry sector. The aim of this study was to investigate the effects of essential oil mixture (juniper, mint, oregano and rosemary oil) on fatty acid oxidation and lipogenic gene expression in geese. Research groups were formed as C (control; no additives), EK1 (0.4 ml/l essential oil mixture supplemented) and EK2 (0.8 ml/l essential oil mixture supplemented). Relative expression levels of genes included in lipogenesis (ACCα, ChREBP, FASN, LXRα and SREBP-1) expression levels of genes included in fatty acid oxidation (ACOX1, CPT1, CPT1A, PPARα and PPARγ) were measured using RT-qPCR. Group EK1 upregulates the mRNA expression levels of genes involved in lipogenesis such as ACCα, ChREBP and SREBP-1, while it downregulates the mRNA expression in levels of all genes involved in fatty acid oxidation. Group EK2 increases the mRNA expression levels of genes involved in lipogenesis such as ACCα, FASN and SREBP-1, while it decreased mRNA expression at the levels of all genes involved in fatty acid oxidation, as in the other group. In the study, adding an essential oil mixture to drinking water is predicted to increase fatty liver because it upregulates genes related to fat synthesis (lipogenesis) and downregulates genes related to fat degradation (fatty acid oxidation).

Sperm signatures of fertility and freezability.

Apart from traditional semen examination parameters, there is not yet a set of functional markers for accurate determination of bull fertility and sperm freezability or cryopreservability, which are vital for production of food animals to feed the world. Therefore, reliable biomarkers are needed to objectively analyze semen quality and predict male fertility. Rapid developments in animal biotechnology have led to significant progress in developing science-based solutions for global problems in food animal production. Although andrology studies employing genomic and functional genomics (transcriptomics, proteomics, and metabolomics) approaches have elucidated some molecular aspects of sperm, there is also a need for additional mechanistic studies to ascertain the functional underpinnings. Biomarkers discovered through applying various -omics technologies using sperm from bulls with varying fertility phenotypes are valuable for semen evaluation and fertility prediction.

Circulating miRNAs in maternal plasma as potential biomarkers of early pregnancy in sheep.

MicroRNA (miRNA) plays an important role in the control of gene expression and is implied in many biological functions, including embryo implantation and development. The aim was to assess plasma miRNA profiles during the peri-implantation and ascertain potential candidate miRNA markers for early pregnancy diagnosis in ovine plasma. The plasma samples were obtained from a total of 24 ewes on days 12 (pre-implantation; P12, n = 4), 16 (implantation; P16, n = 4) and 22 (post-implantation; P22, n = 4) after mating, and on their corresponding days of 12 (Pre-C; C12, n = 4), 16 (Imp-C; C16, n = 4) and 22 (Post-C; C22, n = 4) of the estrous cycle. The miRNA profiles in plasma were assessed by microarray technology. We detected the presence of 60 ovine-specific miRNAs in plasma samples. Of these miRNAs, 22 demonstrated a differential expression pattern, especially between the estrous cycle and early pregnancy, and targeted 521 genes. Two miRNAs (oar-miR-218a and oar-miR-1185-3p) were confirmed using RT-qPCR in the ovine plasma samples. Protein-protein interaction (PPI) network of target genes established six functional modules, of which modules 1 and 3 were enriched in the common GO terms, such as inflammatory response, defense response, and regulation of immune response. In contrast, module 2 was enriched in the developmental process involved in reproduction, embryo development, embryonic morphogenesis, and regulation of the developmental process. The results indicate that miRNAs profiles of plasma seemed to be modulated during the peri-implantation stage of pregnancy in ewes. Circulating miRNAs could be promising candidates for diagnosis in early ovine pregnancy.

Expression pattern of microRNAs in ovine endometrium during the peri-implantation.

MicroRNA (miRNA), acting as the transcriptional regulator of gene expression, has been widely demonstrated to be involved in many biological functions, including embryo implantation and development. The objective of the current study was to illuminate the expression pattern of microRNAs (miRNAs) in the endometrium during the peri-implantation in ewes. Intercaruncular endometrial samples was obtained from a total of 24 ewes on days of 12 (pre-implantation, n = 4), 16 (implantation, n = 4) and 22 (post-implantation, n = 4) of pregnancy following mating, and on their corresponding days of 12 (n = 4), 16 (n = 4) and 22 (n = 4) of the estrous cycle. The miRNA profiles were examined in the endometrium by microarray technology. We detected 116 ovine specifics miRNAs in the endometrium. Of these, nineteen were differentially expressed in early pregnancy. Four miRNAs (oar-miR-370-3p, oar-miR-411b-5p, oar-miR-379-3p and oar-miR-411a-3p) that had the most differential fold change were confirmed by RT-qPCR in ovine endometrium. The differentially expressed miRNAs targeted a total of 315 genes, resulting in 39 GO terms in molecular function, 353 in biological process, and 17 in the cellular component. The construction of the PPI network of target genes established two functional modules mostly enriched in the innate immune system, toll receptor cascades in module 1, whereas genes in module 2 were associated with GMCSF-mediated signaling events, insulin pathway, and mTOR signaling pathway. Based on the results, we may imply that miRNAs modulate ovine endometrium during the peri-implantation.

Expression pattern and cellular localization of two critical non-nuclear progesterone receptors in the ovine corpus luteum during the estrous cycle and early pregnancy.

The study aimed to investigate the expression and cellular localization of two critical non-nuclear progesterone receptors, including membrane-associated-progesterone-receptor-component-1 (PGRMC1) and progestin and adipoQ receptor family member 7 (PAQR7) throughout the estrous cycle and early pregnancy in ovine corpus luteum (CL). Ewes were randomly grouped into cyclic (C, n = 4 per group) or pregnant (P, n = 4 per group) groups. Following slaughtering, the CL was obtained from both cyclic and pregnant ewes on days 12 (C12 and P12), 16 (C16 and P16), and 22 (C22 and P22). Western blotting and RT-qPCR were utilized to assess the expression levels of PGRMC1 and PAQR7, whereas immunohistochemistry was performed to determine the localization of PGRMC1 and PAQR7 in CL. Data were evaluated by one-way ANOVA, and the P < 0.05 was considered a significant difference. PGRMC1 was shown to be expressed in both small and large luteal cells and endothelial cells in CL, while PAQR7 expression was only found in small and large luteal cells. Compared to cycle days, pregnancy increased the expression of PGRMC1. PAQR7 did not differ during early pregnancy but reduced during the functional luteolysis stage (C16). mRNA and protein expression patterns for PGRMC1 and PAQR7 were similar on the studied days. This is the first study that demonstrates the expression and cellular localization of PGRMC1 and PAQR7 in ovine CL. We suggest that these receptors could execute a significant role in the ovine CL life span in both cyclic changes and the establishment of pregnancy.

Expression patterns of genes in steroidogenic, cholesterol uptake, and liver x receptor-mediated cholesterol efflux pathway regulating cholesterol homeostasis in natural and PGF2α induced luteolysis as well as early pregnancy in ovine corpus luteum.

The aim was to evaluate the expression of genes of steroidogenic, cholesterol uptake, and liver X receptor (LXR) mediated cholesterol efflux pathway in ovine corpus luteum (CL) during natural and prostaglandin F2α (PGF2α) induced luteolysis and early pregnancy. For this study, two experiments were carried out 1); ewes were grouped into two sub-groups as cyclic 12 (C12, n = 4) and 16 (C16, n = 4) and pregnant 12 (P12, n = 4), 16 (P16, n = 4), and 22 (P22, n = 4). Additionally, 2) ewes were grouped into four groups following treatment of PGF2α, the duration of PGF2α challenge at 1 (PG1, n = 4), 4 (PG4, n = 4), and 16 (PG16, n = 4) hours on day 12 of the cycle was compared with 0 h. The corpus luteum tissue samples were collected on the corresponding estrus cycle and pregnancy days, and RNA was extracted using Trizol. mRNA expression levels of the steroidogenic (StAR, CYP11A1, and HSD3B1) and cholesterol uptake receptors (SCARB1 and LDLR) and LXR pathway (NR1H3, NR1H2, ABCA1, and ABCG1) were assessed using quantitative PCR (qPCR), and protein of LXR pathway was investigated using western blot. In-situ hybridization was used to detect mRNA localization. Steroidogenic and cholesterol uptake mRNAs were decreased in C16, while NR1H2 and ABCG1 were increased in C16, compared to C12. Steroidogenic and cholesterol uptake mRNA was greater in P16 than in C16. NR1H2 and ABCA1 protein expression were higher in P16 than in C16. LDLR mRNA was higher in P22 than in P12, while SCARB1 was higher in P16 than in P12. NR1H2 mRNA was greater in P22 than in P12. Steroidogenic and cholesterol uptake mRNA were decreased in PGF2α-induced luteolysis groups against C12. ABCG1 mRNA was higher in PG16 than in PG4 and PG1. The reduction of lipoprotein receptors rather than LXR-mediated reverse transport may contribute to the decline in progesterone (P4) in natural and functional luteolysis.

Cell-Specific Expression Pattern of Toll-Like Receptors and Their Roles in Animal Reproduction.

Toll-like receptors (TLRs), a part of the innate immune system, have critical roles in protection against infections and involve in basic pathology and physiology. Secreted molecules from the body or pathogens could be a ligand for induction of the TLR system. There are many immune and non-immune types of cells that express at a least single TLR on their surface or cytoplasm. Those cells may be a player in a defense system or in the physiological regulation mechanisms. Reproductive tract and organs contain different types of cells that have essential functions such as hormone production, providing an environment for embryo/fetus, germ cell production, etc. Although lower parts of reproductive organs are in a relationship with outsider contaminants (bacteria, viruses, etc.), upper parts should be sterile to provide a healthy pregnancy and germ cell production. In those areas, TLRs bear controller or regulator roles. In this chapter, we will provide current information about physiological functions of TLR in the cells of the reproductive organs and tract, and especially about their roles in follicle selection, maturation, follicular atresia, ovulation, corpus luteum (CL) formation and regression, establishment and maintenance of pregnancy, sperm production, maturation, capacitation as well as the relationship between TLR polymorphism and reproduction in domestic animals. We will also discuss pathogen-associated molecular patterns (PAMPs)-induced TLRs that involve in reproductive inflammation/pathology.

The effect of medicarpin on PTEN/AKT signal pathway in head and neck squamous cell carcinoma.

Background/Aim: We aimed to investigate the in vitro modulating effects of medicarpin on the PI3K/AKT signal pathway gene expressions in head and neck squamous cell carcinoma (HNSCC). Materials and Methods: The effect of medicarpin on PTEN and other associated genes in the PTEN/AKT signal pathway was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, real-time quantitative polymerase chain reaction, and Western blot analysis in the SCCL-MT1 (HNSCC) and control (HEK-293) cell lines. Results: The IC50 dose was 80 µM as a result of medicarpin treatment on HNSCC cells (P = 0.0006). It was found that PTEN and AKT gene expressions increased after the medicarpin administration while PDK1 gene expression was decreased in SCCL-MT1 cells (P = 0.0002, P = 0.0003, and P = 0.05, respectively). Protein expression results showed that medicarpin-treated cells significantly increased in pAKT (P = 0.024), pPTEN (P = 0.032), and decreased pPDK1 (P = 0.059) in SCCL-MT1. Conclusions: Our data show that medicarpin modulates HNSCC cells by increasing the PTEN and decreasing PDK1 expressions. PDK1 gene expression effects mTOR pathway which may increase AKT gene. Our study suggests that both medicarpin extracts combination with the HNSCC drug may be more effective in cancer treatment. Future prospective studies that integrate molecular and pharmacogenetic studies are crucial for revealing the mechanism and preventive medical efforts.

Proteomic fertility markers in ram sperm.

Precise estimation of ram fertility is important for sheep farming to sustain reproduction efficiency and profitability of production. There, however, is no conventional method to accurately predict ram fertility. The objective of this study, therefore, was to ascertain proteomic profiles of ram sperm having contrasting fertility phenotypes. Mature rams (n = 66) having greater pregnancy rates than average (89.4 ± 7.2%) were assigned into relatively-greater fertility (GF; n = 31; 94.5 ± 2.8%) whereas those with less-than-average pregnancy rates were assigned into a lesser-fertility (LF; n = 25; 83.1 ± 5.73%; P = 0.028) group. Sperm samples from the outlier greatest- and least-fertility rams (n = 6, pregnancy rate; 98.4 ± 1.8% and 76.1 ± 3.9%) were used for proteomics assessments utilizing Label-free LC-MS/MS. A total of 997 proteins were identified, and among these, 840 were shared by both groups, and 57 and 93 were unique to GF and LF, respectively. Furthermore, 190 differentially abundant proteins were identified; the abundance of 124 was larger in GF while 66 was larger in LF rams. The GF ram sperm had 79 GO/pathway terms in ten major biological networks while there were 47 GO/pathway terms in six biological networks in sperm of LF rams. Accordingly, differential abundances of sperm proteins between sperm of GF and LF rams were indicative of functional implications of sperm proteome on male fertility. The results of this study emphasize there are potential protein markers for evaluation of semen quality and estimation of ram sperm fertilizing capacity.

Sperm Functional Genome Associated With Bull Fertility.

Bull fertility is an important economic trait in sustainable cattle production, as infertile or subfertile bulls give rise to large economic losses. Current methods to assess bull fertility are tedious and not totally accurate. The massive collection of functional data analyses, including genomics, proteomics, metabolomics, transcriptomics, and epigenomics, helps researchers generate extensive knowledge to better understand the unraveling physiological mechanisms underlying subpar male fertility. This review focuses on the sperm phenomes of the functional genome and epigenome that are associated with bull fertility. Findings from multiple sources were integrated to generate new knowledge that is transferable to applied andrology. Diverse methods encompassing analyses of molecular and cellular dynamics in the fertility-associated molecules and conventional sperm parameters can be considered an effective approach to determine bull fertility for efficient and sustainable cattle production. In addition to gene expression information, we also provide methodological information, which is important for the rigor and reliability of the studies. Fertility is a complex trait influenced by several factors and has low heritability, although heritability of scrotal circumference is high and that it is a known fertility maker. There is a need for new knowledge on the expression levels and functions of sperm RNA, proteins, and metabolites. The new knowledge can shed light on additional fertility markers that can be used in combination with scrotal circumference to predict the fertility of breeding bulls. This review provides a comprehensive review of sperm functional characteristics or phenotypes associated with bull fertility.

Advancing Semen Evaluation Using Lipidomics.

Developing a deeper understanding of biological components of sperm is essential to improving cryopreservation techniques and reproductive technologies. To fully ascertain the functional determinants of fertility, lipidomic methods have come to the forefront. Lipidomics is the study of the lipid profile (lipidome) within a cell, tissue, or organism and provides a quantitative analysis of the lipid content in that sample. Sperm cells are composed of various lipids, each with their unique contribution to the overall function of the cell. Lipidomics has already been used to find new and exciting information regarding the fatty acid content of sperm cells from different species. While the applications of lipidomics are rapidly evolving, gaps in the knowledge base remain unresolved. Current limitations of lipidomics studies include the number of available samples to analyze and the total amount of cells within those samples needed to detect changes in the lipid profiles across different subjects. The information obtained through lipidomics research is essential to systems and cellular biology. This review provides a concise analysis of the most recent developments in lipidomic research. This scientific resource is important because these developments can be used to not only combat the reproductive challenges faced when using cryopreserved semen and artificial reproductive technologies in livestock such as cattle, but also other mammals, such as humans or endangered species.

Lipidomic markers of sperm cryotolerance in cattle.

The objective of the current study was to determine the fatty acid composition of sperm from Holstein bulls with different freezability (Good and Poor; n = 12). Fatty acids were extracted from frozen sperm in 1:2 (v/v) chloroform-methanol solvent, fractionated into neutral and polar fractions, and composition determined by gas chromatography-mass spectrometry. Thirty-four fatty acids were quantified and their concentrations and percentages within each lipid fraction were calculated. Overall, saturated fatty acids (SFA) were predominant, accounting for 71 to 80% of fatty acids in neutral and polar lipid factions. There were marked differences in fatty acid composition between the lipid fractions (P < 0.001). The branched chain fatty acid (BCFA) concentration (15 to 18 µg) was almost twice as much as polyunsaturated fatty acids (PUFA) concentration found in the polar lipid fraction (8 to 9 µg; P < 0.001). Sperm with different freezability phenotypes only had a few differences in 22:0, 18:1 cis 9, and 14:0 13-methyl fatty acids (P ≤ 0.011). These results are significant because they reveal key understandings of fatty acid composition of sperm membrane and lay a foundation for the manipulation of membrane integrity, fluidity, and stability to advance the assisted reproductive technologies.

Cellular and Functional Physiopathology of Bull Sperm With Altered Sperm Freezability.

The objective of this study was to ascertain the cellular and functional parameters as well as ROS related changes in sperm from bulls with varied sperm freezability phenotypes. Using principal component analysis (PCA), the variables were reduced to two principal components, of which PC1 explained 48% of the variance, and PC2 explained 24% of the variance, and clustered animals into two distinct groups of good freezability (GF) and poor freezability (PF). In ROS associated pathophysiology, there were more dead superoxide anion positive (Dead SO+) sperm in GF bulls than those in PF (15.72 and 12.00%; P = 0.024), and that Dead SO+ and live hydrogen positive cells (live H2O2+) were positively correlated with freezability, respectively (R 2 = 0.55, P < 0.0130) and (rs = 0.63, P = 0.0498). Related to sperm functional integrity, sperm from PF bulls had greater dead intact acrosome (DIAC) than those from GF bulls (26.29 and 16.10%; P = 0.028) whereas sperm from GF bulls tended to have greater live intact acrosome (LIAC) than those from PF bulls (64.47 and 50.05%; P = 0.084). Sperm with dead reacted acrosome (DRAC) in PF bulls were greater compared to those in GF (19.27 and 11.48%; P = 0.007). While DIAC (R 2 = 0.56, P = 0.0124) and DRAC (R 2 = 0.57, P < 0.0111) were negatively correlated with freezability phenotype, LIAC (R 2 = 0.36, P = 0.0628) was positively correlated. Protamine deficiency (PRM) was similar between sperm from GF and PF bulls (7.20 and 0.64%; P = 0.206) and (rs = 0.70, P = 0.0251) was correlated with freezability. Sperm characteristics associated with cryotolerance are important for advancing both fundamental andrology and assisted reproductive technologies across mammals.

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.

Expression profile of Toll-like receptor 4 in rat testis and epididymis throughout postnatal development.

Toll-like receptors (TLRs) belonging to pattern recognition receptors are involved in maintaining testicular and epididymal immune homeostasis. The purpose of the current study was to investigate TLR4 expression in rat testis and epididymis throughout postnatal development. Weak staining was detected in peritubular myoid cells and immature Sertoli cells while no staining was observed in gonocytes during prepubertal period. However, TLR4 expression began to appear in spermatocytes in pubertal period and gradually increased in spermatids. An intense staining was observed in steps 5-19 spermatids in post pubertal and mature periods. Similarly, TLR4 expression in the testes steadily increased from pubertal period to mature period. Puberty also caused a significant increase in TLR4 expression in epididymis. TLR4 expression in cauda epididymis was lower as compared to those of other epididymal segments. The majority of epididymal epithelial cells exhibited apical TLR4 expression, whereas basal cells showed intense intracytoplasmic immunoreaction. We detected an intense staining in epididymal smooth muscle cells. The expression levels of TLR4 showed dynamic changes in both spermatogenic cells, and entire testicular and epididymal tissues during postnatal development. These results suggest that TLR4 expression contributes not only to inflammation but also to the development of spermatogenic cells.

Mucus from different fish species alleviates carrageenan-induced inflammatory paw edema in rats

Objective: To determine the anti-inflammatory effects of mucus obtained from different fish species on the carrageenan-induced acute paw edema in rats. Methods: Forty-two rats were randomly divided into seven groups. Acute paw edema was induced by 0.1 mL of 1％ carrageenan, and a single dose of diclofenac and lyophilized mucus (25 mg/kg) of rainbow trout, brook trout, European sea bass, and gilthead sea bream were administered to rats through gastric gavage 1 h before carrageenan treatment. Rat paws were measured before and 1-4 h after carrageenan treatment. The mRNA expressions of cytokines (TNF-α, IL-1β, IL-6, IL-10, and TGF-β), antioxidant markers (catalase and superoxide dismutase), and COX-2 were investigated using quantitative polymerase chain reaction. The histopathological changes were evaluated by hematoxylin and eosin staining. Results: The inhibition percentage of carrageenan-induced paw edema by different fish mucus ranged from 52.46％ to 74.86％ at 4 h. Histopathological evaluation showed that all fish mucus diminished carrageenan-induced edema and inflammatory cell infiltration. The upregulation of IL-1β mRNA induced by carrageenan was decreased by the mucus of rainbow trout and gilthead sea bream while an increase in the expression of IL-6 mRNA was reduced by the mucus of rainbow trout, brook trout, and gilthead sea bream. In addition, the mRNA expression of superoxide dismutase was higher in the rainbow trout mucus group than the carrageenan group. Conclusions: Mucus obtained from different fish species may have anti-inflammatory effects.