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Extracellular vesicles (EV) have been identified in uterine fluid (UF), however the bovine UF-EV profile during different phases of the oestrous cycle has not yet been established. Therefore, we compared the UF-EV, and their protein profile at follicular and luteal phases of the oestrous cycle. UF samples were collected from healthy uteri of six live and six slaughtered cows at follicular or luteal phases. Isolation of EV was performed using tangential flow filtration followed by size exclusion chromatography. EV were characterized by nanoparticle tracking analysis (NTA), fluorescence NTA, zeta potential, and transmission electron microscopy. Mass-spectrometry was used to evaluate EV protein profile from live cows. Particle concentrations (mean ± SD) were higher (P 8 ± 1.66 × 107 vs 7.56 × 107 ± 1.80 × 107, respectively) and slaughtered cows (1.17 × 108 ± 2.34 × 107 vs 9.12 × 107 ± 9.77 × 106, respectively). The proportion of fluorescently labelled EV varied significantly between follicular and luteal phases across live (28.9 ± 1.9% vs 19.3 ± 2.8%, respectively) and slaughtered cows (26.5 ± 6.3% vs 27.3 ± 2 .7%, respectively). In total, 41 EV proteins were differentially expressed between the phases. Some of the proteins were involved in reproductive processes, cell adhesion and proliferation, and cellular metabolic processes. The results indicated differences in bovine UF-EV concentration and protein profile at follicular and luteal phases, which would suggest that EV modulate uterine microenvironment across the oestrous cycle. Further research is needed to understand the effect of EV changes throughout the oestrous cycle.

The oviduct provides optimum physiological and biochemical milieu essential for successful fertilization, early embryo development and facilitates functional maturation of spermatozoa. A study has revealed that spermatozoa alters the gene expression in bovine oviductal epithelial cells (BOECs) remotely via bio-active particles, thus acting as a cue to the oviduct prior to their arrival. However, very little attention has been paid to the question of whether spermatozoa could alter the cargo of extracellular vesicles (EVs) derived from BOECs. Therefore, the aim of this study was to investigate the alterations in small non-coding RNAs in EVs cargo derived from BOECs when incubated with spermatozoa in contact and non-contact co-culture models. After 4 h of incubation the EVs were isolated from the conditioned media, followed by small non-coding sequencing of the BOEC derived EVs. Our results revealed that EVs from both co-culture models contained distinct cargo in form of miRNA, fragmented mRNA versus control. The pathway enrichment analysis revealed that EV miRNA from direct co-culture were involved in the biological processes associated with phagocytosis, macroautophagy, placenta development, cellular responses to TNF and FGF. The mRNA fragments also varied within the different groups and mapped to the exonic regions of the transcriptome providing vital insights regarding the changes in cellular transcriptome on the arrival of spermatozoa. The findings of this study suggest that spermatozoa, in contact as well as remotely, alter the EV cargo of female reproductive tract epithelial cells which might be playing an essential role in pre and post-fertilization events. Graphical abstract

Psoriasis vulgaris (PsV) and psoriatic arthritis (PsA) are inflammatory diseases with unresolved pathophysiological aspects. Extracellular vesicles (EVs) play an important role in intercellular communication. We compared the miRNA contents and surface proteome of the EVs in the blood serum of PsV and PsA patients to healthy controls. Size-exclusion chromatography was used to isolate EVs from the blood serum of 12 PsV patients, 12 PsA patients and 12 healthy control subjects. EV samples were characterized and RNA sequencing was used to identify differentially enriched EV-bound miRNAs. We found 212 differentially enriched EV-bound miRNAs present in both PsV and PsA groups—a total of 13 miRNAs at FDR ≤ 0.05. The predicted target genes of these miRNAs were significantly related to lesser known but potentially disease-relevant pathways. The EV array revealed that PsV patient EV samples were significantly enriched with CD9 EV-marker compared to controls. Analysis of EV-bound miRNAs suggests that signaling via EVs in the blood serum could play a role in the pathophysiological processes of PsV and PsA. EVs may be able to fill the void in clinically applicable diagnostic and prognostic biomarkers for PsV and PsA.

The process of luteal regression is tightly regulated by the immune system and chemokines-small cytokines responsible mostly for the activation and migration of immune cells. The role of chemokines in porcine corpus luteum (CL) function is still not well understood. The aim of this study was to investigate the expression profile and distribution of CC chemokines in the porcine CL during the natural oestrous cycle and early pregnancy. Additionally, the effect of PGF2α on the expression of selected chemokines and their luteotropic and apoptotic influence on CL cells were studied in vitro. The expression levels of the chemokines CCL2, CCL4, and CCL5 and the chemokine receptor CCR5 were time-dependent (low on Days 8-10 and high on Days 12-14 of the oestrous cycle). Moreover, CCL8 and CCR2 transcript levels were also elevated during the period of luteolysis. The immunolocalization of CCL2, CCL4, CCL5, CCR1, CCR2 and CCR5 was determined using CL sections obtained from cycling and pregnant pigs. The immunofluorescence signals were localized mainly in luteal cells. PGF2α treatment of CL cells caused increased mRNA expression of CCL2 and CCR1. CCL2 treatment alone upregulated the expression of genes BAX, BCL2 and StAR in CL cells in vitro, but additional experiments showed that the chemokines CCL2, CCL4 and CCL5 alone do not cause apoptosis in a mixed population of CL cells. The chemokine CCL4 increased the transcript levels of StAR and HSD3-β1. Additionally, CCL5 led to the inhibition of BAX gene expression. The differential spatiotemporal expression of CCL2, CCL4, CCL5 and CCR5 throughout the oestrous cycle and the direct but aberrant effect of these three chemokines on genes associated with apoptosis and progesterone synthesis indicate the complicated involvement of these factors in the regulation of luteolysis in pigs.

Simple Summary Before the union of an egg and spermatozoon, several vital processes occur for fertilization in the female reproductive system. One of these processes is the maturation of spermatozoa which occurs in the female reproductive tract. Spermatozoa not undergoing maturation in the female reproductive tract are unable to penetrate the egg. Many reports have suggested the involvement of different factors in mediating the functional maturation of spermatozoa. Follicular fluid (FF) is named as one of those factors. FF is an ovarian fluid that plays an essential role in egg maturation and sources extracellular vesicles (EVs). EVs are nano-containers that are released from different cells and are present in all body fluids. Several studies have reported that FF supports the functional maturation of spermatozoa. Therefore, we hypothesized that FF EVs might have a role in inducing functional maturation in spermatozoa. Surprisingly, the FF-derived EVs were able to aid vital functional parameters of spermatozoa and the effects from EVs were species- and source-specific. Therefore, deciphering the cargo of FF EVs responsible for modulating spermatozoa’s functions can potentially prove beneficial in diagnosing and treating male infertility and improving the current assisted reproductive technology protocols. Abstract While follicular fluid (FF) is known to enhance the functional properties of spermatozoa, the role of FF-derived extracellular vesicles (EVs) in this respect is unknown. We hypothesized that bovine FF EVs convey signals to spermatozoa supporting sperm viability, inducing sperm capacitation and acrosome reaction. In this study, the effects of bovine FF EVs on sperm functions are evaluated. Irrespective of the size of the follicles which FF EVs had originated from, they were capable of supporting sperm viability, inducing capacitation and acrosome reaction. These effects were specific to the source of bovine FF EVs, as human-cell-line-derived or porcine FF EVs did not affect spermatozoa viability or induced capacitation and acrosome reaction. A minimum of 5 × 105 EVs/mL was adequate to maintain sperm viability and induce capacitation and acrosome reaction in spermatozoa. Interestingly, with FF EV trypsin treatment, FF EVs lost their ability to support sperm functions. In conclusion, this study demonstrates that bovine FF EVs can support spermatozoa function and may contribute to a favorable periconceptional microenvironment. This is an important aspect of the interactions between different sexes at the earliest stages of reproduction and helps to understand molecular mechanisms modulating processes such as sperm competition and female cryptic choice.

Effective communication between the maternal reproductive tract, gametes and the pre-implantation embryo is essential for the successful establishment of pregnancy. Recent studies have recognised extracellular vesicles (EVs) as potent vehicles for intercellular communication, potentially via their transport of microRNAs (miRNAs). The aim of the current investigation was to determine the size, concentration and electrical surface properties (zeta potential) of EVs secreted by; (1) primary cultures of porcine oviductal epithelial cells (POECs) from the isthmus and ampullary regions of the female reproductive tract; (2) Ishikawa and RL95-2 human endometrial epithelial cell line cultures; and (3) the non-reproductive epithelial cell line HEK293T. In addition, this study investigated whether EVs secreted by POECs contained miRNAs. All cell types were cultured in EV-depleted medium for 24 or 48 h. EVs were successfully isolated from conditioned culture media using size exclusion chromatography. Nanoparticle tracking analysis (NTA) was performed to evaluate EV size, concentration and zeta potential. QRT-PCR was performed to quantify the expression of candidate miRNAs (miR-103, let-7a, miR-19a, miR-203, miR-126, miR-19b, RNU44, miR-92, miR-196a, miR-326 and miR-23a). NTA confirmed the presence of EVs with diameters of 50–150 nm in all cell types. EV size distribution was significantly different between cell types after 24 and 48 h of cell culture and the concentration of EVs secreted by POECs and Ishikawa cells was also time dependent. The distribution of EVs with specific electrokinetic potential measurements varied between cell types, indicating that EVs of differing cellular origin have varied membrane components. In addition, EVs secreted by POECs exhibited significantly different time dependant changes in zeta potential. QRT-PCR confirmed the presence of miR-103, let-7a, miR-19a, miR-203, miR-126, and miR-19b in EVs secreted by POECs (CT ≥ 29). Bioinformatics analysis suggests that these miRNAs are involved in cell proliferation, innate immune responses, apoptosis and cellular migration. In conclusion, reproductive epithelial cells secrete distinct populations of EVs containing miRNAs, which potentially act in intercellular communication in order to modulate the periconception events leading to successful establishment of pregnancy.

Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR &lt, 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients (p = 0.04) containing eight differentially expressed microRNAs (p &lt, 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.

Extracellular vesicles (EVs), including exosomes and microvesicles (

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.

While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.

Contents Uterine inflammatory response is mediated by inflammatory mediators including eicosanoids and cytokines produced by immune and endometrial cells. Interactions between lipopolysaccharide (LPS) and cytokines, and leukotrienes (LTs) in endothelium, important for the host defence during the inflammation, are unknown. We studied the effect of LPS, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-4 and IL-10 on 5-lipooxygenase (5-LO), LTA4 hydrolase (LTAH) and LTC4 synthase (LTCS) mRNA and protein expression, LTB4 and LTC4 release from porcine endometrial endothelial cells, and cell viability. For 24 hr, cells were exposed to LPS (10 or 100 ng/ml of medium) and cytokines (each 1 or 10 ng/ml). 5-LO mRNA/protein expression augmented after incubation with larger doses of LPS, TNF-α, IL-4 and IL-10 and smaller dose of IL-1β. Larger dose of TNF-α, smaller doses of LPS and IL-1β and both doses of IL-10 increased LTAH mRNA/protein expression. LTAH protein content was up-regulated by larger dose of LPS, but it was reduced in response to both doses of IL-4. LTCS mRNA expression was elevated by larger doses of LPS, IL-4 and IL-10 or both doses of TNF-α and IL-1β. LTCS protein level increased after treatment with both doses of IL-1β, IL-4 and IL-10, smaller dose of LPS and larger dose of TNF-α. Both doses of LPS and larger doses of TNF-α and IL-10 increased LTB4 release. LPS, IL-1β and IL-10 at smaller doses, or TNF-α and IL-4 at larger doses stimulated LTC4 release. Smaller doses of TNF-α and IL-1β or both doses of IL-4 enhanced the cell viability. This work provides new insight on the participation of LPS, TNF-α, IL-1β, IL-4 and IL-10 in LTB4 and LTC4 production/release from porcine endometrial endothelial cells, and the effect of above factors on these cells viability. The used cellular model gives the possibility to further establish the interactions between inflammatory mediators.

Background Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA. Methods We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR. Results We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication. Conclusion Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.

Successful embryo implantation and its further development depends on appropriate endometrial remodelling. Porcine early pregnancy is associated with intensive endometrial angiogenesis and establishment of an immunotolerant environment for the embryo. An increasing number of factors are believed to participate in endometrial remodelling. The aim of this study was to elucidate the involvement of selected chemokines at the porcine maternal-foetal interface during the peri-implantation period. Real-time PCR analysis revealed several upregulated chemokines during the time of implantation, and Western blot/ELISA analyses and immunohistochemical staining confirmed their presence at the protein level. The gene expression of several chemokines and receptors was also confirmed in early porcine trophoblasts. The results indicated that IFNG, a porcine trophoblast signal, positively influenced the expression of some chemokines in endometrial cells. In conclusion, we suggest that some of the examined chemokines may be involved in endometrial communication with the trophoblast (CCL2, CCL5, CCL11, CXCL12), whereas others are implicated in the recruitment of immune cells and establishment of an immunotolerant environment for the embryo (CXCL9, CXCL10).

The influence of induction of ovulation and superovulation with eCG and hCG on LH and FSH receptor levels in porcine oviducts on day 3 postcoitum was studied. In experiment I, gilts were assigned into cyclic (control; n = 5) and inseminated (n = 5) groups. In experiment II, there were 3 groups of animals: inseminated (n = 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n = 5) and superovulated/inseminated (1500 IU eCG, 1000 IU hCG; n = 5) gilts. Oviduct tissues were collected 3 d after insemination or PBS infusion. The messenger RNA (mRNA) expression of FSH receptor (FSHR) and luteinizing hormone/chorionic gonadotropin receptor (LH/CGR) was measured by real-time reverse transcription PCR and protein levels using Western blots. Localization of LH/CGR and FSHR-positive cells was studied by immunohistochemical staining. Insemination by itself did not influence mRNA and protein levels of LH/CGR. However, FSHR mRNA expression in the isthmus and ampulla of the oviduct was affected by insemination (P < 0.05). Similarly, insemination decreased FSHR protein level in the isthmus (P < 0.05). Stimulation with hCG and eCG did not affect LH/CGR and FSHR mRNA expression, either in the isthmus or in the ampulla. Nevertheless, superovulation decreased LH/CGR protein level in the oviductal ampulla (P < 0.05) in comparison with inseminated gilts. Similarly, protein levels of FSHR in the oviductal ampulla decreased after superovulation (P < 0.05). LH/CGR-positive cells were observed in the mucosa as well as in smooth muscle cells of both parts of the oviduct. Follicle-stimulating hormone receptor–positive cells were observed in smooth muscle cells and blood vessels of the isthmus. In the ampulla, FSHR-positive cells were observed in the smooth muscle as well as in the mucosa. Summarizing, the present study revealed for the first time that stimulation with eCG and hCG, especially in high doses, can change LH/CGR and FSHR levels in porcine oviducts. This may in turn alter many signaling pathways, eg, PGs or vascular endothelial growth factor synthesis, and consequently disturb the oviductal environment, with possible detrimental effects on fertilization and/or embryonic development.

The endometrium undergoes cyclical changes during the estrous cycle and pregnancy. These alterations are controlled by various factors, including cytokines. The present study aimed to screen the effect of several chemokines (CCL2, CCL4, CCL5, CCL8, CXCL2, CXCL8, CXCL9, CXCL10, and CXCL12) on endometrial stromal and endothelial cells. Real-time PCR analysis revealed mRNA expression of all examined chemokines and their receptors in primary stromal cells and undetectable levels of CXCL9, CXCL10, and CXCR3 in endothelial cells. Immunocytochemical staining showed variable distribution of chemokine receptors in stromal and endothelial cells. All examined chemokines enhanced stromal cell proliferation, and CCL2 and CXCL12 also increased the migratory potential of these cells. The evaluation of a possible indirect effect of chemokines on angiogenesis and lymphangiogenesis demonstrated that CXCL12 may potentially negatively affect lymphatic vessel creation. Downregulation of VEGFC mRNA and protein expression was noticed after CXCL12 stimulation. Among all examined chemokines, CCL4 and CCL8 positively affected the proliferation and migration of endothelial cells. The number of capillary-like structures was significantly reduced after CXCL8, CXCL10, and CXCL12 stimulation. In conclusion, among all examined chemokines, CCL2 is thought to act as the modulator of stromal cell functions, whereas CCL4 and CCL8 are suggested to be potent factors directly stimulating blood vessel creation.

Early pregnancy is associated with morphological and functional changes within the uterus, accompanied by angiogenesis, increased vascular permeability and activation of immune tolerance. Intensive angiogenesis leads to accelerated vascular leakage and accumulation of interstitial fluid in endometrium. To protect the trophoblast from the harmful effect of extracellular fluid, process known as lymphangiogenesis is crucial. These studies are focused on VEGF-C, factor responsible of lymphatic vessels creating, and its receptors: Flk1 (VEGFR2) and Flt4 (VEGFR3) during the time of implantation as well as the effect of trophoblast signals (IFNG and E2) on VEGF-C production. Endometrial samples were collected from mature gilts from days 8, 10, 12, 14 of estrous cycle and pregnancy. Real-Time PCR analysis revealed increased mRNA expression of VEGF-C on days 10, 12, 14 of pregnancy compared to corresponding days of estrous cycle. The highest VEGF-C mRNA expression was observed on 14 day of pregnancy (p 0.05). Increased mRNA expression of Flk1 and Flt4 was noticed on day 14 of pregnancy in comparison to day 10. Enhanced Flk1 mRNA expression during 14 day of pregnancy was observed compared to corresponding day of estrous cycle (p 0.05). No significant difference on the protein level was revealed. VEGF-C and its receptors were localized mainly in luminal and glandular epithelial cells, but their presence were confirmed also in endothelial cells of blood and lymphatic vessels and 14 d trophoblasts. In vitro studies revealed positive effect of IFNG on VEGF-C mRNA expression in stromal cells and protein content in medium after stromal cells culture (p 0.05). Our studies demonstrated the presence of VEGF-C system in porcine endometrium and indicated its possible important role during the time of implantation.

The prostacyclin (PGI2) signaling pathway plays an important role during early pregnancy in rodents and ruminants. Abundant concentrations of PGI2 were also found in the endometrium and uterine lumen of gilts during the period of implantation. The present study was designed to examine (1) the expression of PGI2 receptor (PTGIR) messenger RNA (mRNA) and protein in the endometrium of cyclic and early-pregnant gilts; (2) possible regulation of endometrial PTGIR gene expression by conceptus products, estradiol and cytokines; (3) the effect of iloprost (a PGI2 analogue) on cAMP formation and the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF; isoform 164) mRNA in luminal epithelial (LE) and stromal (ST) cells. Increased PTGIR mRNA expression in the endometrium was detected on Days 11 to 12 and 18 to 20 of pregnancy compared with respective days of the estrous cycle (P < 0.05). Moreover, greater PTGIR protein level was observed in pregnant than in cyclic gilts on Days 11 to 12 after estrus (P < 0.05). Gilts with unilateral pregnancy revealed abundant PTGIR expression in the endometrium collected from the gravid uterine horn of Day-11 pregnant gilts compared with the respective horn of cyclic animals (P < 0.01). Both LE and ST cells of the endometrium possess PTGIR protein. Moreover, IL1β, IFNγ, and conceptus-exposed medium, but not estradiol, stimulated PTGIR mRNA expression in LE and ST cells in vitro. Activation of PTGIR by incubation of LE and ST cells with iloprost resulted in greater cAMP generation (P < 0.01). Moreover, iloprost increased FGF-2 and VEGF164 mRNA expression in ST (P < 0.05), but not LE cells. In conclusion, this study revealed increased expression of PTGIR in the porcine endometrium during the peri-implantation period and reported a possible regulation of PTGIR abundance by conceptus-derived factors. Moreover, besides its important role in vascular system, PGI2 may promote the expression of proangiogenic genes in the uterine stroma.

Gametogenesis and the temporal changes occurring in the ovaries and testes throughout the reproductive cycle in the invasive alien bivalve, Chinese pond mussel Sinanodonta woodiana (Lea), from the heated Konin lakes system (central Poland) were studied using histological techniques. S. woodiana was confirmed to be a gonochoristic species with overall sex ratio of 1:1. The examined morphological parameters of Chinese pond mussel spermatozoa, i.e. 42 μm mean total length; 4.3 μm mean head length and the maximum size of previtellogenic (34-43 μm) and vitellogenic oocytes (75-83 μm) are consistent with values established for closely related members of the Unionidae family. Our results suggest that S. woodiana in the Konin lakes system are able to spawn throughout March to October, with a season of higher reproductive activity in females extending from March to April. This type of reproductive biology may contribute to the Chinese pond mussel's success in thriving in freshwater ecosystems.

The studies on the acquisition of luteolytic sensitivity have been focused mainly on molecular changes induced in the luteal tissue after treatment with exogenous PGF2α or on physiological changes occurring during the estrous cycle. The comparison of changes leading to the acquisition of luteolytic sensitivity after Day 12 of the estrous cycle and corresponding days of pregnancy has not been investigated in the pig. The present study was undertaken to evaluate (1) apoptosis measured as the proportions of early apoptotic, late apoptotic, and viable cells; (2) expression of factors involved in the extrinsic (TNFA/TNFα, TNFRSF1A/TNFR1, TNFRSF1B/TNFR2, FAS/Fas, and FASLG/FasL) and intrinsic (CASP3/Casp3, TP53/p-53, BAX/Bax, and BCL2/Bcl-2) apoptotic pathways, with two components of the activating protein-1 complex, i.e., FOS/Fos and JUN/Jun and IFNG/IFNγ; and (3) concentrations of luteal and blood plasma progesterone (P4) throughout the luteal phase of the estrous cycle and early pregnancy. Corpora lutea (CL) were collected postmortem on Days 8, 10, 12, and 14 of the estrous cycle and the corresponding days of pregnancy. The luteal tissue was subjected to RNA and/or protein isolation and disaggregation of CL cells followed by flow cytometry analysis aimed to determine apoptotic changes. Luteal and blood plasma P4 concentrations decreased on Day 14 of the estrous cycle versus pregnancy (P < 0.05 and P < 0.001, respectively). A significant increase in the number of early apoptotic cells and a decrease in the number of viable cells were observed on Day 14 of the estrous cycle (P < 0.001 and P < 0.05, respectively). Increase (P < 0.05) of TNFA messenger RNA (mRNA) level coincided with that of IFNG on Day 12 of the estrous cycle but not on the corresponding day of pregnancy. The content of FAS mRNA and protein increased on Day 14 of the estrous cycle versus pregnancy (P < 0.05). The mRNA expression of CASP3, BCL-2 and BAX was unchanged in cyclic and pregnant CL, while level of TP53 increased (P < 0.05) on Day 12 of the estrous cycle versus Day 8. The level of FOS and JUN mRNA increased (P < 0.05) on Day 14 of the estrous cycle versus the remaining days. The level of FOS and JUN mRNA was significantly higher (P < 0.001 and P < 0.05, respectively) on Day 14 of the estrous cycle than that on the corresponding day of pregnancy. In summary, the simultaneous increase of TNFA and IFNG transcript in cyclic CL suggests the crucial role of both cytokines in sensitization of porcine CL to further luteolytic action of PGF2α. The upregulated expression of FAS, FOS, and JUN mRNA in the late luteal phase in cyclic CL can indicate their involvement in structural luteolysis. The increased viability of luteal cells and elevated P4 concentrations in pregnant CL confirm the protective role of luteal P4 against apoptosis.

During early pregnancy, uterine epithelial cells undergo major transformations in their cytoskeleton that make the endometrium receptive for conceptus attachment. Actin binding proteins (ABPs) such as cofilin, gelsolin, and vinculin are involved in regulating actin polymerization, severing or crosslinking actin to integrins. However, whether ABPs are involved in epithelial remodeling or embryo adhesion in pigs is unknown. Therefore, the expression and distribution of these proteins were investigated in porcine endometrium on Days 10 and 13 (pre-implantation period), and 16 (attachment phase) of the estrous cycle or pregnancy. While day and pregnancy status had no effect on ABP gene expression, the protein abundance of vinculin was significantly higher on Day 13 than on Day 10 (p

This study was performed to determine the influence of insemination as well as treatment with human chorionic gonadotropin (hCG) and equine chorionic gonadotropin (eCG) on expression of the vascular endothelial growth factor (VEGF) system in porcine oviducts. In the first experiment, 10 gilts were assigned to 2 groups: cyclic (treated with phosphate-buffered saline; n = 5) and inseminated (n = 5). In experiment II, 15 gilts were assigned to 3 groups: inseminated (control; n = 5), induced ovulation and inseminated (750 IU eCG, 500 IU hCG; n = 5), and superovulated and inseminated (1500 IU eCG, 1000 IU hCG; n = 5). Oviducts (isthmus and ampulla) were collected 3 days after phosphate-buffered saline treatment (experiment I) or insemination. Blood samples were collected during slaughter for E2 (estradiol) and P4 (progesterone) analysis. Levels of messenger RNA (mRNA) of the VEGF system were analyzed by real-time polymerase chain reaction and protein by Western blot and E2 and P4 using radioimmunoassays. Insemination by itself decreased VEGF120 mRNA expression and VEGF-A protein level in the oviductal isthmus (P0.05) but did not alter VEGF164 mRNA. Expression of Flt-1 (c-fms-like tyrosine kinase VEGFR-1) mRNA increased in the isthmus of inseminated relative to cyclic gilts (P0.05), whereas KDR (fetal liver kinase-1 VEGFR-2) mRNA levels decreased in both the oviductal isthmus (P0.05) and ampulla (P0.001). Superovulation decreased VEGF120 and VEGF164 mRNA expression in the isthmus compared with the inseminated group (P0.05), and lowered protein levels of VEGF-A in the isthmus of both stimulated groups (P0.001). Expression of Flt-1 mRNA was affected by hCG and eCG treatment in both gonadotropin-stimulated groups in the isthmus as well as in the ampulla (P0.001) and protein levels in the ampulla of superovulated gilts (P0.05). Protein levels of KDR were reduced in the oviductal ampulla of gilts in both the induced ovulation and superovulated groups (P0.05). The concentrations of both E2 and P4 increased significantly in superovulated group of gilts (P0.01 and P0.05 for E2 and P4, respectively). Our study showed that insemination alone as well as ovarian stimulation affected the mRNA and protein profiles of the VEGF system in the porcine oviduct. Disrupted VEGF system expression may be crucial to many events occurring during the periovulatory period and consequently could lead to deprivation of VEGF-dependent factors that are necessary for proper fertilization, gamete transport, and embryo development.

The overall objective of this study was to examine the effect of vaginal administration of prostaglandin E2(PGE2) and/or17β-estradiol (E2) on luteal function maintenance and histological properties of the porcine cervix. For this purpose, crossbred gilts were divided into three groups (n=5 per group) supplied on days 11-16 of the estrous cycle with suppositories containing: (1) placebo (Group I, Control); (2) 0.4 mg of E2(Group II); (3) 0.4 mg of E2and 2 mg of PGE2(Group III). Blood samples were collected on days 11-19 of the estrous cycle to determine the concentration of progesterone (P4). Additionally, to examine local effects of the hormones applied, segments from the uterine and vaginal parts of the cervix and from the ovaries were collected post-mortem. Prolonged luteal function and extended synthesis of P4were observed in 2 of 5 gilts receiving PGE2 and E2 simultaneously (Group III). Then, these gilts were subdivided into Group IIIA (n=2; presence of corpora lutea on the ovaries) and Group IIIB (n=3; lack of corpora lutea). Increased levels of plasma P4were observed in Group IIIA on days 15-19 compared to Group IIIB and on days 16-19 compared to Group I and Group II (P2and PGE2applied intravaginally. These results are significant considering that very low doses of E2were used when compared to previous attempts. Despite the inadequate response to treatments in some of the gilts, the local effects of these hormones on the histological properties of the porcine cervix suggest that further improvements in the vaginal administration route might help to elaborate new methods for enhancing the luteal function in the pig.

Contents The oviduct plays a crucial role in fertilization, gamete maturation and embryo transport. Prostaglandins are some of the main factors determining its roles. The present study investigated the influence of oestrus synchronization and superovulation on prostaglandins synthesis in the porcine oviduct. Mature cross-bred gilts after exhibiting oestrous cycles were divided into four groups: I, cyclic; II, inseminated; III, synchronized and inseminated; and IV, superovulated and inseminated. Oviducts were collected on the third day of the oestrous cycle or after insemination and divided into isthmus and ampullary parts. This study demonstrated lower mRNA (in the isthmus and ampulla; p

Two independent experiments were performed on cyclic (Experiment I) and pregnant (Experiment II) gilts to examine the effect of human Chorionic Gonadotropin (hCG) administration on day 12 of the estrous cycle/pregnancy on ovarian and endometrial secretory function. Animals were divided into hCG Group (injection of 750 IU hCG) and Control Group (injection of saline). In Experiment I, the prolonged lifespan of the corpus luteum (CL), extended progesterone (P4) production (P

Nerve growth factor (NGF) is a neurotrophin implicated in the pathophysiology of allergy, diseases of the immune system and inflammation. Expression of NGF and its receptors (TrkA and p75) has not been examined in inflamed uterine tissue. The aim of the present study was to evaluate the patterns of NGF, TrkA and p75 expression in normal and inflamed porcine uteri using real-time reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and immunohistochemistry. On day 3 of the oestrous cycle, 50 ml of saline or 50 ml of Escherichia coli suspension containing 10(9) colony forming units/ml, was injected into both uterine horns of control (n = 10) and experimental (n = 10) gilts, respectively. Infected animals developed moderate or severe acute endometritis after 8 days and moderate acute or subacute endometritis after 16 days. On day 8 of the study, the expression of NGF and TrkA mRNA and protein was higher in the endometrium of gilts with endometritis than in normal tissue. NGF protein was also more prominently expressed in the endometrium of those animals with endometritis 16 days post infection. There was no difference in endometrial p75 mRNA and protein expression on either day. The myometrium of animals in both groups showed no difference in expression of any of the three molecules. Immunohistochemically, on day 8 there was greater expression of NGF and TrkA by the luminal epithelium and some glandular and endothelial cells as well as the myometrium of the uteri of gilts with endometritis compared with normal tissues. At day 16, there was greater expression of NGF by the luminal epithelium and of TrkA by some endothelial cells and the myometrium, compared with control tissue. There is therefore, up-regulation of NGF and TrkA mRNA and protein expression in the porcine uterus when affected by E. coli-induced inflammation. NGF and TrkA may have roles in this inflammatory process and/or in the innervation of the uterus.

WNTs (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. β-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17β-estradiol (E(2)) and progesterone (P(4)) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, β-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E(2) on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E(2) and P(4) level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, β-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.

Establishment of pregnancy in pigs requires continuous function of corpora lutea and endometrial preparation for embryo implantation. Progesterone regulates expression of many proteins necessary for endometrial remodelling and embryo-maternal communications. Attaining the uterine receptivity involves progesterone priming and loss of progesterone receptors in the uterine epithelium before days 10-12 after oestrus. Spermatozoa and oocytes in oviduct alter secretion of specific proteins that exert beneficial effect on gametes and embryos. Moreover, an appropriate leucocyte activation and maintenance of delicate cytokine balance within the oviduct and uterus are important for early pregnancy. This early local immune response is rather mediated by seminal plasma components. These components also influence prostaglandin (PG) synthesis in the oviduct that is important for gamete and embryo transport. Pregnancy establishment requires the biphasic pattern of oestrogen secretion by conceptuses on days 11-12 and 15-30. Conceptus affects lipid signalling system consisting of prostaglandins and lysophosphatic acid. PG synthesis is changed by conceptus signals in favour of luteoprotective PGE(2) . Additionally, existence of PGE(2) positive feedback loop in the endometrium contributes to increased PGE(2) /PGF(2α) ratio during the peri-implantation period. PGE(2) through endometrial PGE(2) receptor (PTGER2) elevates the expression of enzymes involved in PGE(2) synthesis. Higher PGE(2) secretion in uterine lumen coincides with the elevated expression of HOXA10 transcription factor critical for implantation. A stable adhesion between conceptus and endometrium requires reduction in mucin-1 on the apical surface of epithelium and integrin activation by extracellular matrix proteins. Furthermore, growth factors, cytokines and its receptors are involved in embryo-maternal interactions.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis. Mol. Reprod. Dev. 78:597–610, 2011. © 2011 Wiley-Liss, Inc.

The umbilical cord (UC) and the placenta are important organs through which respiratory gases, nutrients, wastes and biologically active substances are exchanged between the maternal and the foetal system. A rapid placental vascularization observed in the second half of pig pregnancy is positively correlated with the mRNA expression of the vascular endothelial growth factor (VEGF). Based on these findings, we hypothesized that VEGF may have a stimulatory effect in the dynamically growing UC. To further understand the role of the VEGF-VEGFR system during UC development, mRNA and protein expression as well as the cellular localization of VEGF-A, VEGFR-1 and VEGFR-2 in UC were examined on days 40, 60, 75 and 90 of pregnancy and after physiological delivery in the pig (day 114 of pregnancy). Real Time RT-PCR analysis showed an increase in the mRNA levels of VEGF120 and VEGF164 from day 90 of pregnancy. VEGFR-1 mRNA expression was significantly increased on day 75 of pregnancy. No significant changes in VEGFR-2 mRNA expression were detected. In turn, western blot analysis revealed an increase in VEGF-A protein expression on day 40, compared to the later days of pregnancy. A rapid increase in the VEGFR-1 protein level was noted on day 75 and 90 of gestation. No significant changes in VEGFR-2 protein expression were detected on any of the analysed days of pregnancy. Immunohistochemical staining enabled detection of VEGF-VEGFR system, in endothelial and tunica media cells of the umbilical vessels and in allantoic duct and amniotic epithelium on all analysed days of pregnancy. Positive reactions for VEGF-A and VEGFR-1, but not VEGFR-2, were also observed in myofibroblasts. In conclusion, this data shows that members of the VEGF-VEGFR system are temporally and spatially well localized for playing key roles during umbilical cord formation and its intensive growth observed after day 75 of pregnancy.

The present study was undertaken to determine the effect of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) on prostaglandin (PG)F2αand PGE2secretion as well as cyclooxygenase-2 (COX-2) protein expression in myometrium collected on days 25, 30 and 40 of pregnancy in pigs. Myometrial slices were incubated for 16 h with IL-1β, IL-6 and TNF-α (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of IL-1β and IL-6 on PGF2αand PGE2secretion from myometrium collected on all examined days of pregnancy, excepting of influence of IL-6 on release of PGF2αby tissue from day 30. In turn, TNF-α was able to stimulate only PGE2secretion by myometrium of 40-day-pregnant gilts. The three cytokines applied in combination augmented release of PGE2from myometrium collected on days 30 and 40 of pregnancy. Stimulation of PGE2secretion by cytokines used individually was more frequent than that of PGF2α. Moreover, an enhancement in PGF2αand/or PGE2release was accompanied by an increase of COX-2 protein expression. Our study shows the ability of cytokines to stimulate PGF2αand PGE2release by porcine myometrium from the first third of pregnancy. Obtained data suggest that locally PGs produced in myometrium influencing the uterine contraction activity may be important for the maintenance of myometrial quiescence during pregnancy and confirm also that the complex cytokine network is an important regulatory mechanism of PGs production during pregnancy.

The aim of the present study was to investigate the localization and expression of NADPH-diaphorase (NADPH-d) as well as endothelial (eNOS) and inducible (iNOS) nitric oxide synthase isoforms in the porcine uterus on Days 20, 30, 40, 60, 75 and 90 of gestation. The significant increase (p

SUMMARY The aim of the study was to examine: 1/ allantochorial concentrations of nitrate/nitrite (NOx) and 2/ plasma concentration of NO x in pigs on days 25, 35, 40 and 60 of pregnancy as well as 3/ the influence of estradiol-17β (E 2 ) and/or progesterone (P 4 ) on NO x production by porcine fetal membranes on the studied days of pregnancy. Total NO x concentration was determined using a microplate assay method based on the Griess reaction. Fetal membrane NO x content gradually increased from day 25 to day 60 of gestation. Blood plasma NO x concentration decreased from day 25 to 40, and then plasma NO x concentration significantly increased on day 60. In addition, the stimulatory effect of E 2 , P 4 and E 2 +P 4 on NO in vitro production by porcine fetal membranes was demonstrated. The stimulatory effect of steroid hormones on NO x release depended on steroid dose and day of pregnancy. It is possible that the observed differences in the strength of the stimulatory action of E 2 , P 4 and E 2 +P 4 on fetal membrane NO x production are associated with an activation of different isoforms of nitric oxide synthase.

The aim of the present study was to determine the immunoreactivity of vascular endothelial growth factor (VEGF-A) and its two receptors, viz., Flt-1 (fms-like tyrosine kinase) and Flk-1 (fetal liver kinase), on the surface of endothelial cells of the uterine artery and its branches and of the arcuate arteries in the area of the uterine broad ligament during various phases of the estrous cycle in the pig. We also investigated their expression to determine whether this was phase-related. The highest immunoreactivity for VEGF-A was observed in the uterine artery and arcuate arteries at the early luteal phase and in the branches of the uterine artery during the follicular phase of the estrous cycle. The strongest immunostaining intensity of Flt-1 was found in the uterine artery and its branches at the follicular phase and in arcuate arteries at the mid-luteal phase, whereas Flk-1 immunostaining was at its highest in the uterine artery at the mid-luteal phase and in the branches of the uterine artery and arcuate arteries at the follicular phase. Additionally, VEGF-A expression was assessed by semi-quantitative Western blot analysis, which revealed significantly higher levels of VEGF-A protein during the early luteal and the follicular phase of the estrous cycle (P < 0.001). The phase-related differences in the immunoreactivity and expression of VEGF-A and VEGF receptors suggest that these factors are hormone-dependent during the estrous cycle in the pig.

Prostaglandins are well-known mediators of crucial events in the female reproductive tract, eg, early embryo development and implantation. Prostacyclin (PGI2) is the most synthesized prostaglandin in the human oviduct during the postovulatory period, indicating its important role in supporting and regulating the oviductal environment. The present study was undertaken to determine the influence of insemination and ovarian stimulation with human chorionic gonadotropin (hCG)/equine chorionic gonadotropin (eCG) on PGI2 synthesis in the porcine oviduct on day 3 post coitus. Mature gilts (n = 25) were assigned into 2 experiments. In experiment I, gilts were divided into cyclic (control; n = 5) and inseminated (control; n = 5) groups. In experiment II, there were 3 groups of animals: inseminated (n = 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n = 5), and superovulated/inseminated (1,500 IU eCG, 1,000 IU hCG; n = 5) gilts. Parts of oviducts (isthmus and ampulla) were collected 3 days after phosphate-buffered saline treatment (cyclic gilts of experiment I) or insemination (all other groups). Expression of messenger RNA for PGI2 synthase (PGIS) and its receptor (IP) was measured by real-time reverse transcription polymerase chain reaction (real-time RT PCR) and protein levels using Western blots. Concentrations of the PGI2 metabolite 6-keto PGF1α were evaluated by enzyme immunoassay and localization of PGIS and IP in the oviductal tissues using immunohistochemical staining. Insemination by itself increased PGIS protein levels in the oviductal isthmus (P < 0.05) and IP protein expression in the ampulla (P < 0.05). The concentration of 6-keto PGF1α increased significantly in the oviductal ampulla after insemination (P < 0.05). Induction of ovulation decreased IP protein levels in the oviductal ampulla (P < 0.05), whereas superovulation reduced IP levels in both parts of the oviduct (P < 0.01). Synthesis of 6-keto PGF1α was reduced by induction of ovulation and by superovulation in the oviductal ampulla (P < 0.05). Immunohistochemical staining confirmed the presence of PGIS in the muscular layer of the isthmus and both mucosa and muscular layers of the ampulla. IP-positive cells were observed in both mucosal and muscular layers of the isthmus and ampulla. This study showed for the first time that PGI2 synthesis and IP expression are insemination dependent. Moreover, ovarian stimulation with hCG/eCG decreases IP expression and 6-keto PGF1α concentrations in porcine oviducts. Therefore, disturbances in PGI2/IP expression and synthesis may lead to disruption of the oviductal environment and, in turn, perturbed development of embryos and their transport to the uterus.

Basic characteristics of the European smelt (Osmerus eperlanus) sperm are reported here for the first time. Smelt spermatozoa had a bullet-shaped head (1.42 μm length), a short midpiece and a long flagellum (27.72 μm). Two mitochondria were located along the flagella. The volume of smelt sperm was small (30–60 μl) and the duration of sperm motility was short (22 s in distilled water and 41 s in 20 mM sodium bicarbonate solution). Sodium chloride at concentrations ranging from 0–120 mM did not influence the percentage of motile spermatozoa but caused a steady increase in the duration of sperm movement. Potassium ions clearly reduced the percentage of motile sperm at a concentration of 10 mM. Spermatozoa were motile through a broad range of pH with an optimum from 7.5 to 8.5. Testicular spermatozoa had a different motility pattern compared to stripped spermatozoa (the latter exhibiting a reduction of motile spermatozoa by 30%, lower ALH and VCL and higher LIN and VSL). These results indicate that maturation of smelt spermatozoa occurring in sperm ducts is related not only to an increase of the percentage of motile spermatozoa but also to changes in the sperm motility pattern. Maintaining males with females resulted in stimulation of milt production. Our results indicate that European smelt sperm characteristics are similar to those of ayu (Osmeridae).

Nitric oxide (NO), a highly reactive free radical is involved in vasodilation, neurotransmission, hormone secretion, and reproduction. Since all known nitric oxide synthase (NOS) isoforms possess NADPH-diaphorase (NADPH-d) activity, NADPH-d histochemistry was used as a commonly accepted procedure for NOS identification. The aim of our study was to determine the cellular localization of NADPH-d, eNOS, and iNOS in the porcine uterus and the correlation between NADPH-d and NOS activity in the early, middle, late luteal, and follicular phase of the estrous cycle. Light-microscopic observations of the sections revealed the differential expression of the NADPH-d in the analyzed stages of the estrous cycle. The most intense staining was observed in the luminal epithelium in the late luteal phase and in some groups of the endometrial glands in all studied stages. Positive reaction was also found in the endothelial cells of blood vessels and in the myometrium itself. Immunostaining for eNOS was observed in the luminal and glandular epithelium in all studied stages, but no clear fluctuations were observed. The endothelium of both endometrial and myometrial blood vessels displayed pronounced eNOS immunostaining. Strong iNOS staining was observed in the luminal epithelium in the late luteal and follicular phase and in selected groups of endometrial glands. Thus, only NADPH-d and iNOS undergo cyclic changes in the studied stages of the estrous cycle. The differential expression of NADPH-d/NOS in the porcine uterine horn during the estrous cycle suggests a role for NO in modulating uterine function.

Immunohistochemical localization and distribution of endothelin (ET-1) and nitric oxide synthase (eNOS) were investigated in precollector and collector lymphangions of lymphatic vessels leaving the ovary and were found in the vascular subovarian plexus (mesovarium) as well as in those emanating from the oviductal isthmus and uterine horn (mesosalpinx and mesometrium, respectively) forming the paraovarian lymphatic plexus in the broad ligament of the uterus during different phases of the estrous cycle in pigs. The polyclonal antibody for ET-1 and the monoclonal antibody for eNOS isoform were used for studies on the light-microscopic level. Immunoreactivities to both ET-1 and eNOS were observed in the endothelial cell cytoplasm of precollector and collector lymphangions and were not demonstrated in smooth muscle cells of the lymphatics examined. In the endothelium, the intensity of immunostaining for ET-1 and eNOS was found to be estrous phase-dependent and differed between precollector and collector lymphangions. In general, immunoreactivity to ET-1 was more intense in the endothelium of shrunken lymphangions, whereas that for eNOS was more intense in lymphangions with the large lumen. These results suggest that ET-1 and eNOS can play a role in mechanisms regulating the vascular contractile activity promoting lymph flow during the estrous cycle in the porcine broad ligament.

We studied the effect of lipopolysaccharide (LPS), proinflammatory cytokines (tumor necrosis factor α [TNF-α] and interleukin [IL]-1β), and anti-inflammatory cytokines (IL-4 and IL-10) on leukotriene (LT) A4 hydrolase and LTC4 synthase (LTCS) protein expression in, and LTB4 and LTC4 secretion from, an inflamed porcine endometrium. On day 3 of the estrous cycle (day 0 of the study), 50 mL of either saline or Escherichia coli suspension (10(9) CFU/mL) was injected into each uterine horn of gilts (n = 12 per group). Endometrial explants, obtained 8 and 16 days later, were incubated for 24 h with LPS (10 or 100 ng/mL of medium), TNF-α, IL-1β, IL-4, and IL-10 (each cytokine: 1 or 10 ng/mL of medium). Although acute endometritis developed in all bacteria-inoculated gilts, a severe form of acute endometritis was diagnosed more often on day 8 of the study than on day 16. The amount of the LTA4 hydrolase (LTAH) protein in the inflamed endometrium on day 8 was greater after applying the lower dose of TNF-α (P0.001) and both doses of IL-1β (P0.001) and IL-4 (1 ng, P0.01 and 10 ng, P0.001) than in the saline-treated uteri. A similar situation was observed in the case of the inflamed tissue on day 16 in response to LPS (100 ng, P0.01), TNF-α (10 ng, P0.05), and IL-4 (1 ng, P0.001). The content of LTC4 synthase in the inflamed endometrium on day 8 was reduced by LPS (100 ng, P0.05), IL-1β (10 ng, P0.05), IL-4 (1 and 10 ng, P0.05), and IL-10 (1 ng, P0.01) but increased after the application of LPS (100 ng, P0.05) and TNF-α (1 and 10 ng, P0.001), IL-1β, and IL-4 (1 ng, P0.05 and 10 ng, P0.001) on day 16. On day 8, endometrial secretion of LTB4 from the saline-injected and E coli-injected organs was similar in response to all of the used mediators. On the other hand, the contents of LTB4 in the medium decreased after incubating the inflamed tissues from day 16 with TNF-α (1 ng, P0.05 and 10 ng, P0.01), IL-1β (1 ng, P0.01), and IL-10 (10 ng, P0.05) compared with the saline-treated ones. Secretion of LTC4 from the inflamed uteri on day 8 was elevated by the lower doses of TNF-α (P0.01) and IL-10 (P0.05), whereas on day 16, such an effect occurred in response to the higher doses of IL-4 (P0.01) and IL-10 (P0.05). The obtained results show that pro- and anti-inflammatory mediators participate in the synthesis/secretion of LTs from an inflamed porcine endometrium. Our data suggest that inflammatory mediators may indirectly affect the processes regulated by LTs by influencing LT production.

Implementation of the swine umbilical vein endothelial cells (SUVECs) model in vitro can be instrumental in determining the biology of endothelial cells. We have generated an immortalized endothelial cell line, G-1410, using Simian virus 40 T-antigen (SV40 T-ag) primarily to overcome the short life span before the onset of senescence and high variability among enzymatically isolated cells of primary cultures. Fast proliferating cells were selected from cultures and, after a fifth passage, examined for the presence of the SV40 T-ag by PCR and immunocytochemistry. Phase contrast and transmission electron microscopy revealed that G-1410 cells did not differ morphologically from SUVECs. The G-1410 cells exhibited positive staining for vascular endothelial (VE)-cadherin and von Willebrand factor (vWF), and formed capillary-like tube structures on Matrigel. Despite the strong oncogenic signal provided by SV40 T-ag, these transformed G-1410 cells have remained karyotypically normal and non-tumorigenic. G-1410 cells also responded to stimulation with VEGF, FGF-2, and newborn calf serum. Moreover, G-1410 cells showed elevated expression of VEGF120, VEGF164 (VEGF-A), and FGF-2 at both mRNA and protein levels. In conclusion, based on the cytological and functional evaluation of the newly obtained immortalized cell line, it can be concluded that G-1410 cells provide a useful tool for studying the effects of VEGF and FGF systems, and other signal transduction pathways related to angiogenesis.

The objective of this study was to describe the morphometry and motility parameters of vendace (Coregonus albula) spermatozoa. Morphometric parameters of vendace sperm head and tail were of values similar to rainbow trout. The effects of pH, sodium, potassium and calcium ion concentrations on computer-assisted sperm analysis (CASA) sperm motility characteristics were tested. Vendace sperm was motile in a wide pH range of 6.0-10.5 with the optimum pH established at 9.0. Increases in potassium and calcium ions caused decreases in the percentage of motile sperm. The CASA parameters and erratic sperm movement pattern of vendace spermatozoa were similar to whitefish (C. lavaretus) sperm motility, suggesting that there is a coregonid-specific sperm motility pattern.