Formation of the early canine CL and the role of prostaglandin E2 (PGE2) in regulation of its function: an in vivo approach.

The mechanisms governing corpus luteum (CL) function in domestic dogs remain not fully elucidated. The upregulated expression of cyclooxygenase 2 and prostaglandin (PG) E2 synthase (PGES) at the beginning of the canine luteal phase indicated their luteotrophic roles, and the steroidogenic activity of PGE2 in the early canine CL has been confirmed in vitro. Recently, by applying a cyclooxygenase 2 (COX2)-specific inhibitor (firocoxib [Previcox]; Merial) from the day of ovulation until the midluteal phase, the luteotrophic effects of PGs have been shown in vivo. This is a follow-up study investigating the underlying endocrine mechanisms associated with the firocoxib-mediated effects on the canine CL. Experimental groups were formed with ovariohysterectomies performed on Days 5, 10, 20, or 30 of firocoxib treatments (10 mg/kg bw/24h; TGs = treated groups). Untreated dogs served as controls. A decrease of steroidogenic acute regulatory (STAR) protein expression was observed in TGs. The expression of PGE2 synthase was significantly suppressed in TGs 5 and 10, and both PGE2 and PGF2α levels were decreased in luteal homogenates, particularly from CL in TG 5. Similarly, expression of the prolactin receptor (PRLR) was diminished in TGs 5 and 20. The expression of PGE2 receptors PTGER2 (EP2) and PTGER4 (EP4), the PG- transporter (PGT), and 15-hydroxy PG dehydrogenase (HPGD) was not affected in TGs. Our results substantiate a direct luteotrophic role of PGs in the early canine CL, i.e., by upregulating the steroidogenic machinery. Additionally, the possibility of an indirect effect on PRL function arises from the increased prolactin receptor expression in response to PGE2 treatment in canine lutein cells observed in vitro.

The role of toll-like receptors 2 and 4 in the pathogenesis of feline pyometra.

Pyometra is the most common uterine disease in queens. To protect itself from infection, the female reproductive tract possesses several immune mechanisms that are based on germline-encoded pattern recognition receptors (toll-like receptors [TLRs]). The aim of our study was to examine endometrial immunolocalization of TLR2/4, study the influence of lipopolysaccharide (LPS) and tumor necrosis factor (TNF) α on messenger RNA expression of both receptors in pyometric queens, and compare these patterns between estrous cycling queens and those hormonally treated with medroxyprogesterone acetate (MPA). Thirty-six queens, ranging in age from 7 months to 11 years, were allocated into seven groups (anestrus, estrus, mid-diestrus and late diestrus, short-term and long-term hormonally treated queens, and pyometric queens). At the messenger RNA level, the real-time polymerase chain reaction was applied, whereas at the TLR2/4 protein level, the expression was tested by immunohistochemistry. In queens at estrus, gene expression of TLR2 was upregulated after stimulation of endometrial explants by TNF (P < 0.001) and by TNF together with the LPS (P < 0.01). Moreover, gene expression of TLR2 was significantly upregulated after stimulation by TNF (P < 0.001) and LPS (P < 0.01) explants derived from queens that had been long-term hormonally treated with MPA. Endometrial gene expression of TLR4 was significantly upregulated after incubation of explants with TNF (P < 0.001) in queens at estrus and with LPS (P < 0.05) in queens short-term hormonally treated with MPA. Immunolocalization reported that TLR2/4 receptors are mainly localized in the surface and glandular epithelia. These data show that short-term and especially long-term administration of progesterone derivatives impairs TLRs in the endometrial epithelium, presumably enabling pathogens to break through this first natural barrier and thereby increase the risk of pyometra development.

Prostaglandin endoperoxide synthase 2 (PTGS2) and prostaglandins F2α and E2 synthases (PGFS and PGES) expression and prostaglandin F2α and E2 secretion following oestrogen and/or progesterone stimulation of the feline endometrium.

Sex steroids in synergy with prostaglandins (PG) are involved in the regulation of cyclic ovarian function. In this study, we investigated the mRNA expression of three genes involved in arachidonic acid (AA) metabolism and hence PG production in domestic cats: PG-endoperoxide synthase (PTGS2), PGF(2α) synthase (PGFS) and PGE(2) synthase (PGES). Feline endometria (n = 16) were collected at oestrus and mid and late phases of pseudopregnancy. In addition, the effects of E(2) and/or P(4) on PG secretion and gene expression on endometrial explants were studied in an in vitro culture system. Expression levels of all examined genes were up-regulated at the mid phase of pseudopregnancy. The effects of E(2) and/or P(4) treatment on both PG secretion and expression of the genes were observed after 12 h of culture. Expression of PGES was significantly up-regulated by E(2) plus P(4) at oestrus and the mid phase of pseudopregnancy and was also up-regulated by a single treatment with P(4) at late pseudopregnancy (p < 0.05). Simultaneous incubation with E(2) and P(4) up-regulated PTGS2 gene expression at oestrus and mid-luteal phase (p < 0.05). Progesterone plus E(2) significantly increased PGE(2) secretion at oestrus and the mid phase of pseudopregnancy. However, treatment with E(2) and/or P(4) affected neither PGF(2α) secretion nor PGFS expression at any phase after 12 h of culture. The overall findings indicate that genes involved in PG synthesis are up-regulated at the mid phase of pseudopregnancy. An increase in PGE(2) secretion and up-regulation of PGES and PTGS2 are the main responses of the endometrium to treatment with E(2) and P(4) at oestrus and the mid phase of pseudopregnancy in the cat. These data support the hypothesis that ovarian sex steroids via endometrial PGE(2) are involved in endocrine homoeostasis, especially at oestrus and the mid, but not the late, phase of pseudopregnancy in cats.

Lipopolysaccharides, cytokines, and nitric oxide affect secretion of prostaglandins and leukotrienes by bovine mammary gland epithelial cells.

The aims of this study were to determine the effects of lipopolysaccharides (LPS), tumor necrosis factor (TNF), interleukin 1 alpha (IL-1α), nitric oxide donor (NONOate), or the combination of TNF + IL-1α + NONOate on the following: (i) secretion of prostaglandin (PG)-F(2α), PGE(2), leukotriene (LT)-B(4), and LTC(4) by epithelial cells of the teat cavity and lactiferous sinus of bovine mammary gland; (ii) messenger RNA (mRNA) transcription of enzymes responsible for arachidonic acid (AA) metabolism (prostaglandin-endoperoxide synthase 2 [PTGS2], prostaglandin E synthase [PTGES], prostaglandin F synthase [PGFS], and arachidonate 5-lipooxygenase [ALOX5]); and (iii) proliferation of the cells. The cells were stimulated for 24 h. Prostaglandins and LT were measured by enzyme immunoassay, mRNA transcription of enzymes was determined by real-time reverse transcription polymerase chain reaction, and the cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. All factors increased PG secretion, but the highest stimulation was observed after TNF and IL-1α (P < 0.001). Tumor necrosis factor, NONOate, and TNF + IL-1α + NONOate increased LTB(4) production (P < 0.01), whereas LTC(4) was increased by LPS, TNF, and IL-1α (P < 0.01). Lipopolysaccharides, TNF, IL-1α, and the reagents combination increased PTGS2, PTGES, and PGFS mRNA transcription (P < 0.01), whereas ALOX5 mRNA transcription was increased only by TNF (P < 0.001). Lipopolysaccharides, TNF, IL-1α, NONOate, and the combination of reagents increased the cell number (P < 0.001). Mediators of acute-clinical Escherichia coli mastitis locally modulate PG and LT secretion by the epithelial cells of the teat cavity and lactiferous sinus, which might be a useful first line of defense for the bovine mammary gland. Moreover, the modulation of PG and LT secretion and the changing ratio of luteotropic (PGE(2), LTB(4)) to luteolytic (PGF(2α), LTC(4)) metabolites may contribute to disorders in reproductive functions.

mRNA transcription of prostaglandin synthases and their products in the equine endometrium in the course of fibrosis.

Accurate regulation of the reproductive cycle and successful implantation depend on proper functioning of the endometrium. The aim of this study was to determine whether mRNA transcription of specific enzymes responsible for prostaglandin (PG) synthesis (prostaglandin-endoperoxide synthase, PTGS-2; prostaglandin F(2α) synthase, PGFS; and prostaglandin E(2) synthases, PGES) and PG concentrations in endometrial extracts would change in moderate (Kenney's Category II) and severe phases of fibrosis (Kenney's Category III; endometrosis), compared with healthy endometrium (Kenney's Category I), during the estrous cycle. Endometrial tissues samples were obtained from mares at the early (n = 12), mid (n = 12) and late (n = 12) luteal phases and the follicular phase (n = 12) of the estrous cycle. Additionally, all endometria were classified microscopically as belonging to Categories I and II or III according to the Kenney classification, resulting in allocation of 4 samples for each subcategory, e.g., mid luteal I, II and III. Relative mRNA transcription was quantified using Real-time PCR. Concentrations of PGE(2) and PGF(2α) in the endometrial extracts were determined using enzyme-linked immunosorbent assay (EIA). In Category I, PTGS-2 mRNA transcription was upregulated at the mid (P < 0.05) and late luteal phases (P < 0.001) and at the follicular phase (P < 0.05) compared to the early luteal phase. PGFS mRNA transcription as well as PGF(2α) concentrations increased at the mid (P < 0.01) and late (P < 0.05) luteal phases compared to the early luteal phase in Category I. PGES mRNA transcription was higher at the mid (P < 0.01) and late luteal phases (P < 0.05) compared to the early luteal and follicular phases in Category I. Prostaglandin E(2) concentration in Category I was higher at the mid luteal phase (P < 0.01) compared to all other phases of the estrous cycle. During incipient endometrosis (Category II) and under full endometrosis (Category III), PTGS-2, PGFS and PGES mRNA transcription and PG concentration were altered compared to the respective estrous phases in healthy endometria (P < 0.05). It may be concluded that serious changes in mRNA transcription of PG synthases and PG production that occur in the equine endometrium during the course of fibrosis in the estrous cycle could be responsible for disturbances leading to disorders of the estrous cycle and early embryo losses.

Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells.

Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2-5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10(-7)m) for 24 h. The concentrations of PGE(2) and PGF(2α) in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE(2) and PGF(2α) release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.

Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows.

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. ß-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in ß-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows.

Ovarian steroids modulate tumor necrosis factor-α and nitric oxide-regulated prostaglandin secretion by cultured bovine oviductal epithelial cells.

Ovarian steroids assure an optimum environment for the final maturation of oocytes, gamete transport, fertilization, and early embryonic development. The aim of experiment 1 was to examine the influence of ovarian steroids on tumor necrosis factor-α (TNF-α)- or nitric oxide (NO)-regulated prostaglandin (PG), and nitrite/nitrate (NO2/NO3) secretion by cultured bovine oviductal epithelial cells (BOECs). BOECs were pretreated with 17ß-estradiol (E2; 10⁻9 M) and/or progesterone (P4; 10⁻7 M) for 24 h. For the next 24 h, BOECs were treated with TNF-α (10 ng/mL) or spermine nitric oxide complex (NONOate; 10⁻5 M). Prostaglandin F(2α) and PGE2 secretion was measured in medium by ELISA. The pretreatment of cells with P4 (progesterone), E2 (17 ß-estradiol), or E2/P4 augmented TNF-α-induced PGF(2α) and PGE2 secretion (P < 0.01). The pretreatment of cells with E2 or E2/P4 increased NONOate-induced PGF(2α) and PGE2 secretion (P < 0.01). TNF-α induced NO2/NO3 production by BOECs. The pretreatment of cells with E2 augmented only TNF-α-induced NO2/NO3 production (P < 0.05). The aim of experiment 2 was to examine the influence of TNF-α, NO, and ovarian steroids on the protein content of enzymes specifically involved in PG and NO production, PG synthases, and NO synthases (NOSs). BOECs were treated with TNF-α (10 ng/mL) or NONOate (10⁻5 M). TNF-α increased the protein content of PGG/H synthase, PGF synthase, and PGE synthase (P < 0.05) and endothelial and inducible NOSs (P < 0.05). Nitric oxide increased the protein content of PGF synthase, PGE synthase, endothelial NOS, and inducible NOS (P < 0.05). These results show possible linkage between TNF-α and NO, modulated by ovarian steroids, in the regulation of PG synthesis by BOECs that may be important for triggering the process of oviductal contractions.

Tumor necrosis factor-alpha as a possible auto-/paracrine factor affecting estrous cycle in the cat uterus.

Tumor Necrosis Factor-alpha (TNFalpha) is a pleiotrophic cytokine, affects either normal or tumor cells, and influences cellular differentiation. TNFalpha role in female reproduction has been proven to be mediated through an influence on prostaglandin (PGs) synthesis and output. To evaluate the possible role of TNFalpha in an auto-/paracrine regulation in the cat uterus, mRNA expression coding for TNFalpha and its receptors (TNFR1 and TNFR2), and TNFalpha protein content at different stages of the estrous cycle were investigated. Additionally, TNFalpha involvement in PG secretion at different stages of the estrous cycle was investigated by in vitro tissue culture. Gene expressions coding for TNFalpha and TNFR1 were the highest at diestrus (P < 0.05). TNFalpha protein expression was the lowest at interestrus (P < 0.05). Nevertheless, TNFR2 was not affected by the estrous stage. TNFalpha at a dose of 1 ng/ml significantly increased PGF2alpha secretion at estrus (P < 0.01) and PGE2 secretion at diestrus (P < 0.001) after 12h incubation. Overall findings indicate that TNFalpha locally produced in the cat's uterus, stimulates PG secretion in an estrous cycle-related manner.

Apoptotic changes in the epithelium germinativum of the cat (Felis catus s. domestica, L. 1758) at different ages and breeding seasons.

Apoptosis (programmed cell death) could be considered as a physiological process that takes part in a healthy organism, which helps to maintain organism homeostasis. The visible deterioration of semen quality and the number of germ cells is accompanied by a seasonal decrease of the reproductive activity in some species. This post-effect cascade is caused by apoptosis, which is the primary mechanism responsible for the elimination of germ cells during spermatogenesis. The aim of our study was to assess apoptotic changes in the epithelium germinativum in cat testes at different ages. One hundred and two pairs of testes were obtained from domestic cats aged between 4 months and 10 years. The paraffin-embedded tissue sections were labelled using the Oncogene and Calbiochem Research Products DNA Fragmentation Detection Kit (Cat# QIA21; Darmstadt, Germany), which allows the recognition of apoptotic nuclei in tissue sections with Fragment End Labelling (FragEL) of DNA. The activity of apoptotic processes in cat testes collected from the spring-summer period compared with the autumn-winter season revealed that, 59.42% and 51.51%, respectively, males testes were characterized by insignificant changes. The obtained data revealed a distinctive apoptotic changes in the young animal testes before spermatogenesis onset. An intensification of programmed death cells in the epithelium germinativum in the elder cats (between 3-6 and 6-10 years) was not observed. Apoptotic changes slightly intensified in cats aged between 12 and 36 months.

Assessment of selected quality parameters of epididymal cat (Felis catus s. domestica, L. 1758) sperm using flow cytometry method and computer assisted sperm analyser.

The improvement of biotechnical methods connected with fast and precise semen quality assessment and its utilization in assisted reproductive techniques is an urgent necessity in felids. The aim of this study was to evaluate some quality parameters (i.e. the viability and share of cells with intact plasma membrane) of epididymal sperm of cats using the flow cytometry method and computer-assisted sperm analysis (CASA) examination. The material consisted of epididymal spermatozoa flushed from 22 pairs of epididymes after routine neutering procedures obtained from domestic cats aged between 8 and 36 months. The epididymes were cut and incubated with an extender without egg yolk. The samples were assessed for sperm viability (Live/Dead Sperm Viability Kit), percentage of subtle membrane changes (Apoptosis Detection Kit) and motility using FACScalibur flow cytometer and assisted sperm analyser HTM IVOS version 12.2. The flow cytometry method revealed 71.3% and 84.4% of live sperm using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit respectively. The population of early-apoptotic and late-apoptotic sperm were 0.8% and 1.1% respectively. The CASA examination found 51.5% of motile sperm. However, the motility examination under light microscope revealed 69.5% of motile sperm. The data revealed an indistinctive per cent of apoptotic cells and 18.9% and 15.6% of dead cells using Live/Dead Sperm Viability Kit and Apoptosis Detection Kit, respectively, which indicate that the sperm obtained after flushing the epididymis possess potential properties for further assisted reproduction techniques.