Synthesis and reception of prostaglandins in corpora lutea of domestic cat and lynx.

Felids show different reproductive strategies related to the luteal phase. Domestic cats exhibit a seasonal polyoestrus and ovulation is followed by formation of corpora lutea (CL). Pregnant and non-pregnant cycles are reflected by diverging plasma progesterone (P4) profiles. Eurasian and Iberian lynxes show a seasonal monooestrus, in which physiologically persistent CL (perCL) support constantly elevated plasma P4 levels. Prostaglandins (PGs) represent key regulators of reproduction, and we aimed to characterise PG synthesis in feline CL to identify their contribution to the luteal lifespan. We assessed mRNA and protein expression of PG synthases (PTGS2/COX2, PTGES, PGFS/AKR1C3) and PG receptors (PTGER2, PTGER4, PTGFR), and intra-luteal levels of PGE2 and PGF2α Therefore, CL of pregnant (pre-implantation, post-implantation, regression stages) and non-pregnant (formation, development/maintenance, early regression, late regression stages) domestic cats, and prooestrous Eurasian (perCL, pre-mating) and metoestrous Iberian (perCL, freshCL, post-mating) lynxes were investigated. Expression of PTGS2/COX2, PTGES and PTGER4 was independent of the luteal stage in the investigated species. High levels of luteotrophic PGE2 in perCL might be associated with persistence of luteal function in lynxes. Signals for PGFS/AKR1C3 expression were weak in mid and late luteal stages of cats but were absent in lynxes, concomitant with low PGF2α levels in these species. Thus, regulation of CL regression by luteal PGF2α seems negligible. In contrast, expression of PTGFR was evident in nearly all investigated CL of cat and lynxes, implying that luteal regression, e.g. at the end of pregnancy, is triggered by extra-luteal PGF2α.

Comparison of the effect of lipopolysaccharide on tumor necrosis factor α (TNF-α) secretion and TNF and TNFR1 mRNA levels in feline endometrium throughout the estrous cycle during pyometra and after medroxyprogesterone acetate treatment.

Endotoxins released by Gram-negative bacteria are potent stimulators of tumor necrosis factor α (TNF-α) production. The objectives of this study were to evaluate plasma levels of TNF-α, TNF-α secretion, and mRNA levels of TNF and TNF-α receptor type 1 (TNFR1) following exposure to lipopolysaccharide (LPS). For this, we used cultured endometrial cells or organ cultures, throughout the estrous cycle, after hormone treatment with medroxyprogesterone acetate (MPA), and during pyometra. Plasma TNF-α concentrations were increased in animals at estrus (P < 0.05) compared to other groups. In the LPS-challenged endometrium, secretion of TNF-α by tissues collected during estrus increased (P < 0.001) compared to that of other groups. LPS, alone or combined with TNF-α, upregulated TNF gene expression in the feline endometrium at diestrus (P < 0.001 for both treatments), in queens treated short-term with MPA (P < 0.01 and P < 0.05, respectively) and in queens treated long-term with MPA (P < 0.01 and P < 0.001, respectively). During pyometra, TNF and TNFR1 mRNA were increased only after tissues were challenged with TNF-α and LPS (P < 0.001 and P < 0.01, respectively). When cultured endometrial cells were challenged with LPS, the concentration of TNF-α increased only in epithelial cells after 4 h and 12 h (P < 0.05 and P < 0.01, respectively). Since LPS did not affect stromal cells, but TNF-α increased its own transcript after 2 h (P < 0.01), 4 h (P < 0.05) and 12 h (P < 0.001), we assume that stromal cells are not directly involved in pathogen recognition, as was the case for epithelial cells.

Comparison of the biopsy and cytobrush techniques for diagnosis of subclinical endometritis in mares.

BACKGROUND: Endometritis is a major cause of infertility in the mare. Therefore, the diagnosis of this disease is very important in veterinary practice. The objective of this study was to compare bacteriological and cytological results obtained from the mare uterus using biopsy (EB) and cytobrush (CB) techniques and relating these findings to the presence of polymorphonuclear cells (PMNs) in endometrial tissue as the gold standard for detection of endometritis. In particular, we tested the hypothesis that endometrial cytology and microbiology data obtained from material collected using the EB and CB techniques are similar, so that the CB technique could preferentially be used to detect subclinical endometritis in clinical practice. METHODS: A total of 69 mares suspected of subclinical endometritis because of previous reproductive history and 15 maiden mares were enrolled in this study. Material collected from both EB and CB was smeared on sterile glass slides for cytological examinations and on culture media for microbiological examinations. Bacteriological cultures and cytological samples were classified as negative (no growth or mixed cultures of more than three microorganisms; <2% PMNs) or positive (pure growth of microorganisms; >2% PMNs) for endometritis. RESULTS: Positive growth was observed in 43% of CB samples and in 54% of EB samples (difference not significant). The growth of ß-hemolytic streptococci was always connected with positive cytology. This relationship was not observed for growth of E. coli or for non-pathogenic flora. The sensitivity of bacterial growth and cytology from EB was 0.63 and 0.73 respectively. The sensitivities of bacterial growth and cytology from CB were 0.50 and 0.71 respectively. CONCLUSION: Microbiological and cytological results obtained from CB are similar to those obtained from EB and based on these findings the CB technique may be recommended for collection of materials from the mare's uterus in clinical practice.

LPS-challenged TNFα production, prostaglandin secretion, and TNFα/TNFRs expression in the endometrium of domestic cats in estrus or diestrus, and in cats with pyometra or receiving medroxyprogesterone acetate.

Progesterone (P4) derivatives which are commonly used to block the cyclicity of domestic cats disturb the endocrine balance in the endometrium. The aims of this study were (i) to examine whether lipopolysaccharide (LPS) is responsible for enhancement of tumor necrosis factor-α (TNFα) secretion by the feline endometrial epithelial and stromal cells in vitro, (ii) to know whether immunolocalization of TNFα/TNFR1 and TNFR2 differs in cats at estrus or diestrus, receiving medroxyprogesterone acetate and suffering from pyometra, and (iii) to determine if TNFα-challenged prostaglandin secretion is stopped by prostaglandin synthases inhibitors. A total of 37 domestic adult cats in estrus or diestrus, receiving octane medroxyprogesterone or having clinical symptoms of pyometra, were enrolled in this study. The results obtained showed a distinct increase in LPS-challenged TNFα secretion in endometrial epithelial, but not stromal cells. TNFα augmented PG secretion was blocked by phospholipase A2 (PLA2) and cyclooxygeanase-2 (COX-2), but not by mitogen-activated protein kinase (MAPK) inhibitor. TNFα/TNFR1 and 2 protein expressions were limited mostly to the surface and glandular epithelium. TNFα/TNFRs protein was upregulated in the inflammatory uterus and hence may be involved in development of pathologic changes in the endometrial glands in cats receiving exogenous P4 as a hormonal contraceptive.

Placental origin of prostaglandin F2α in the domestic cat.

In the present study, the question was addressed whether the feline placenta can synthesize prostaglandin F2α (PGF2α). The PGFS protein was elevated, particularly at 2.5-3 weeks of pregnancy compared to 7-8 (P < 0.05) and 8.5-9 weeks (P < 0.001). Transcripts for PGFS were significantly upregulated at 2.5-3 weeks of pregnancy and then gradually declined towards the end of gestation (P < 0.001). Transcripts for PTGS2 were only upregulated in placentas from queens close to term (P < 0.001) compared with earlier phases. Staining of PTGS2 showed distinct positive signals in placentas obtained during the last week before labor, particularly in the strongly invading trophoblast surrounding blood vessels, and also in decidual cells. Shortly after implantation, signals for PGFS were localized in the trophoblast cells. Near term, PGFS staining was seen mainly in decidual cells. Both placental PGF2α and plasma PGFM were elevated towards the end of pregnancy (P < 0.001) compared with earlier weeks of pregnancy. The content of PGF2α in extracted placenta mirrored the PGFM level in plasma of pregnant females. During late gestation there is a significant increase in PGFM levels in maternal blood and of PGF2α levels in placental tissue concomitant with an upregulation of placental PTGS2.

Steroidogenic capacity of the placenta as a supplemental source of progesterone during pregnancy in domestic cats.

BACKGROUND: Until recently, the corpus luteum (CL) was considered to be the main source of progesterone (P4) during pregnancy in the domestic cat (Felis catus). However, other possible sources of P4 have not been ruled out. Although feline placental homogenates were found to be capable of synthesizing P4, expression of the respective steroidogenic enzymes has not been investigated at the molecular level. Therefore, in the present study, expression of the two major factors involved in the synthesis of P4 - 3beta-hydroxysteroid dehydrogenase (3betaHSD) and steroidogenic acute regulatory protein (StAR) - was investigated in the feline CL and placenta during the course of pseudopregnancy and pregnancy. METHODS: The mRNA levels of StAR and 3betaHSD were determined using Real Time PCR and their localizations were determined by immunohistochemistry. Placental P4 concentrations, after ethyl extraction, were measured by EIA. RESULTS: Luteal 3betaHSD and StAR mRNA levels were strongly time-dependent, peaking during mid-pregnancy. The placental 3betaHSD mRNA level was significantly upregulated towards the end of pregnancy. In the CL, 3betaHSD and StAR protein were localized in the luteal cells whereas in the placenta they were localized to the maternal decidual cells. Placental P4 concentrations were low in early pregnant queens, but increased along with gestational age. CONCLUSIONS: These results confirm that the placenta is an additional source of P4 in pregnant queens and can thereby be considered as an important endocrine organ supporting feline pregnancy.

Are glucocorticoids auto- and/or paracrine factors in early bovine embryo development and implantation?

We determined the transcript content of three genes involved in the metabolism of glucocorticoids (GC) in bovine in vitro fertilized embryos (2-blastomere stage until hatched blastocyst), trophoblast as well as the oviduct (Day 2-4 of the estrous cycle) and endometrium (Day 16 of the cycle and pregnancy). Since mRNA expression of the glucocorticoid receptor and two enzymes responsible for GC production (11ß-HSD1 and 2) was demonstrated in the embryos in all pre-implantation stages as well as in the endometrium and oviduct, it is suggested that GC may serve as auto-/paracrine factors in the development of bovine pre-implantation embryos.

Evidence for Increased Content of PGF2α, PGE2, and 6-keto-PGF1α in Endometrial Tissue Cultures From Heavy Draft Mares in Anestrus With Endometritis.

Endometritis is one of the most important causes of infertility in mares. Mares may suffer from endometritis outside the breeding season; however, pathological condition is often undiagnosed in anestrus. The aim of this study was to examine whether the secretion profiles of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), and a metabolite of prostacyclin I2 (6-keto-PGF1α) differ in endometrial tissue cultures of heavy draft mares in anestrus with endometritis compared to those without endometritis. The endometrial biopsies were collected from 51 heavy draft mares. Inclusion criteria for the control group were absence of endometritis confirmed by histology and no ovarian activity. Inclusion criteria for the experimental group were presence of endometritis showing polymorphonuclear cells and/or lymphocytes infiltration in endometrium and no ovarian activity. Retrospectively, 22 mares were enrolled in this study. The content of PGF2α (P < .05) and PGE2 (P < .001) in the culture medium was distinctly elevated in mares suffering from endometritis, compared to control mares. The relative mRNA abundance responsible for prostaglandins synthesis, that is, PGF2α synthase (PGFS; P < .01), PGE2 synthase (PGES; P < .01), and prostaglandin-endoperoxide synthase (PTGS2; P < .01), were also increased in endometrial tissue of mares with endometritis compared to control mares. The content of 6-keto-PGF1α (P < .0001) in endometrial tissue cultures from mares with endometritis was strikingly elevated compared to those without endometritis; however, plasma concentration of 6-keto-PGF1α was not significantly different between experimental and control groups. This leads to the conclusion that augmented endometrial secretion of PGF2α, PGE2, and 6-keto-PGF1α is associated with endometritis even in mares in anestrus.

Advanced age in mares affects endometrial secretion of arachidonic acid metabolites during equine subclinical endometritis.

Even if mares continue to breed up to an advanced age, in aging mares reproductive failure is quite common. Subclinical endometritis, which occurs more often in aging mares than in younger counterparts, may cause prolongation or shortening of the inter-estrus period or the corpus luteum lifespan. We hypothesized that during subclinical endometritis the secretion of selected arachidonic acid metabolites may differ in aging mares compared to younger females. To verify this thesis, ex vivo organ cultures of endometrium were established with subsequent measurements of concentrations of prostaglandin E2 (PGE2), 6-keto-PGF1α and both leukotrienes (LTs), LTB4 and LTC4 in the culture supernatants. The endometrial biopsies were obtained from 82 mares of known breeding history. This study revealed that the concentrations of the selected arachidonic acid metabolites, which act both as immunological mediators and endocrine modulators in the reproductive organs, depends on the mares' ages. Spontaneous endometrial secretion of PGE2, 6-keto-PGF1α and LTC4 was increased in mares aged 16-23 years that suffered from subclinical endometritis, compared with control counterparts. Moreover, secretion of these metabolites was higher in endometritis-positive mares aged 16-23 years than in younger females. We conclude that advanced age in mares further disturbs the immuno-endocrine balance in endometritis-positive mares.

Cells expressing CD4, CD8, MHCII and endoglin in the canine corpus luteum of pregnancy, and prepartum activation of the luteal TNFα system.

In the dog, knowledge about involvement of the immune system in controlling luteal function is restricted to observations showing a time-dependent invasion of immune cells into the corpus luteum (CL) of non-pregnant bitches. Therefore, this study investigated the presence of CD4-, CD8-, MHCII- and endoglin-expressing cells in CL collected throughout pregnancy from pre-implantation until prepartum luteolysis. Immunohistochemistry and semi-quantitative RT-PCR were applied. The time-dependent expression of CD4, CD8 and endoglin was more strongly related to formation of the CL, whereas MHCII was induced during luteolysis. Next, the luteal expression of TNFα and its receptors, TNFR1 and TNFR2, was analyzed in non-pregnant dogs between days 5-65 after ovulation and during pregnancy. Moreover, the effects of progesterone withdrawal were investigated in mid-pregnant dogs treated with an antigestagen aglepristone. The TNFα system was induced in the early CL of non-pregnant dogs. In pregnant dogs, expression of TNFα did not vary much, contrasting with increased expression of both receptors in the post-implantation period and significantly decreased expression at mid-gestation; prepartum luteolysis was characterized by increased TNFR2 expression. Apart from the downregulated expression of TNFR1, the changes observed following antigestagen treatment resembled those observed during normal prepartum luteolysis. A modulatory function of the TNFα system during formation of the canine CL is suggested, possibly related to the strong accompanying vascularization and luteal infiltration with activated macrophages. Contrasting with the slow luteal regression in non-pregnant dogs, in pregnant animals the upregulation of TNFR2 expression during prepartum luteolysis implies functional involvement of the TNFα system during that time.

Identifying diagnostic endocrine markers and changes in endometrial gene expressions during pyometra in cats.

Pyometra is a significant reproductive problem in cats. The aims of this study were to evaluate (i) the immunological profile of queens by studying plasma concentrations of metabolites of prostacyclin I2 (6-keto-PGF1α), leukotriene B4 (LTB4) and leukotriene C4 (LTC4); and (ii) the gene transcription profiles of Toll-like receptors (TLRs) 2 and 4 (TLR2/4), PGE2-synthase (PGES), PGF2α-synthase (PGFS) and prostaglandin-endoperoxide synthase 2 (PTGS2) in the feline endometrium throughout the estrous cycle, after medroxyprogesterone acetate (MPA) treatment and during pyometra. The concentration of plasma 6-keto-PGF1α in pyometra was increased in comparison to other groups studied (p<0.01). Endometrial mRNA coding for TLR2 was up-regulated in cats suffering from pyometra compared to other groups (p<0.001). Expression of mRNA for TLR4 was up-regulated in endometria originating from MPA-treated cats, pyometra and late diestrus cats, compared with tissues from cats during estrus and anestrus (p<0.05). As expected, endometrial mRNA for PTGS2 was up-regulated only in pyometra, compared with other groups (p<0.001). Similarly, endometrial mRNA for PGFS was up-regulated in pyometra, compared with endometria from anestrus, late diestrus and from MPA-treated cats (p<0.05), or from cats during estrus (p<0.01). Overall, these results indicate that plasma concentrations of LTB4 and LTC4 are not useful diagnostic markers since they were not increased in queens with pyometra, in contrast to 6-keto-PGF1α. In addition, treatment with MPA evoked neither endocrine nor molecular changes in endometria of cats.

Type of Inflammation Differentially Affects Expression of Interleukin 1ß and 6, Tumor Necrosis Factor-α and Toll-Like Receptors in Subclinical Endometritis in Mares.

Mares that fail to conceive or lose their embryos, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). In this study, the question was addressed: does SE fully activate selected mechanisms of innate immunity in mares? For this aim, expression of mRNAs for Toll-like Receptor 2 and 4 (TLR 2/4), interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor α (TNF) was examined in control mares versus either mares suffering from chronic endometritis (ChE) or subacute suppurative endometritis (SSE). The concentrations of IL-1ß, IL-6 and TNF-α in supernatants from endometrial tissue cultures after 4 h incubation were measured using the enzyme immunoassay (EIA) method. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. Based on histopathological assessment, mares were classified as suffering from ChE, SSE or as being healthy. In addition, immuno-localization of both TLR2 and TLR4 as well as TNF-α was investigated in the equine endometria. The mRNA expression of TLR2 (P < 0.01), IL-1ß (P < 0.0001), IL-6 (P < 0.0001) and TLR4 and TNF (P < 0.05) was up-regulated in endometria of mares suffering from SSE compared with unaffected mares. Concentrations of IL-6 and TNF-α were increased only in mares exhibiting SSE, compared with unaffected (P < 0.01 for both) and ChE mares (P < 0.05 for both). Immuno-localization of TNF-α and TLRs was confirmed, both in unaffected and SE-affected endometria, and was present in the luminal and glandular epithelia and stromal cells. The severity of inflammation impacts the immune response and fosters activation of innate immunity mechanisms, as observed in the endometria of mares. The intracellular localization of TLRs and TNF-α in the endometria indicates a key role of endometrial epithelial and stromal cells in the immune response and inflammation.

Programmed necrosis - a new mechanism of steroidogenic luteal cell death and elimination during luteolysis in cows.

Programmed necrosis (necroptosis) is an alternative form of programmed cell death that is regulated by receptor-interacting protein kinase (RIPK) 1 and 3-dependent, but is a caspase (CASP)-independent pathway. In the present study, to determine if necroptosis participates in bovine structural luteolysis, we investigated RIPK1 and RIPK3 expression throughout the estrous cycle, during prostaglandin F2α (PGF)-induced luteolysis in the bovine corpus luteum (CL), and in cultured luteal steroidogenic cells (LSCs) after treatment with selected luteolytic factors. In addition, effects of a RIPK1 inhibitor (necrostatin-1, Nec-1; 50 µM) on cell viability, progesterone secretion, apoptosis related factors and RIPKs expression, were evaluated. Expression of RIPK1 and RIPK3 increased in the CL tissue during both spontaneous and PGF-induced luteolysis (P < 0.05). In cultured LSCs, tumor necrosis factor α (TNF; 2.3 nM) in combination with interferon γ (IFNG; 2.5 nM) up-regulated RIPK1 mRNA and protein expression (P < 0.05). TNF + IFNG also up-regulated RIPK3 mRNA expression (P < 0.05), but not RIPK3 protein. Although Nec-1 prevented TNF + IFNG-induced cell death (P < 0.05), it did not affect CASP3 and CASP8 expression. Nec-1 decreased both RIPK1 and RIPK3 protein expression (P < 0.05). These findings suggest that RIPKs-dependent necroptosis is a potent mechanism responsible for bovine structural luteolysis induced by pro-inflammatory cytokines.

Altered secretion of selected arachidonic acid metabolites during subclinical endometritis relative to estrous cycle stage and grade of fibrosis in mares.

Mares that fail to become pregnant after repeated breeding, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). Contact with infectious agents results in altered synthesis and secretion of inflammatory mediators, including cytokines and arachidonic acid metabolites, and disturbs endometrial functional balance. To address the hypothesis that SE affects the immune endocrine status of the equine endometrium, spontaneous secretion of prostaglandin E(2) (PGE(2)), prostaglandin F(2α) (PGF(2α)), 6-keto-PGF(1α )(a metabolite of prostacyclin I(2)), leukotriene B(4) (LTB(4)), and leukotriene C(4) (LTC(4)) was examined. In addition, secretion of these factors was examined relative to the grade of inflammation, fibrosis, and estrous cycle stage. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. On the basis of histopathologic assessment, mares were classified as suffering from first-grade SE, second-grade SE, or being healthy. The grade of fibrosis and the infiltration of endometrial tissue with polymorphonuclear leukocytes were examined by routine hematoxylin-eosin staining. In mares suffering from SE, the secretion profiles of PGE(2), 6-keto-PGF(1α), LTB(4), and LTC(4) were changed compared to mares that did not suffer from endometritis. The secretion of PGE(2) and 6-keto-PGF1α was increased, whereas that of LTB(4) and LTC(4) was decreased. Secretion of 6-keto-PGF(1α) was increased in first- and second-grade SE (P < 0.01). The concentration of PGI(2) metabolite was increased only in inflamed endometrium, independently of the inflammation grade, but was not affected by fibrosis. Prostaglandin E(2) secretion was increased in second-grade SE (P < 0.05). The secretion of LTB(4) decreased in both first- and second-grade SE (P < 0.05), whereas secretion of LTC(4) was decreased only in second-grade SE (P < 0.05). Fibrosis did not change the secretion profile of PGE(2), PGF(2α), and 6-keto-PGF(1α) during the course of SE. However, the secretion profile of LTB(4) was affected during the course of fibrosis. Evident divergences between PGE(2) and PGF(2α) profiles and in PGE2:PGF(2α) ratios in the control versus SE mares observed during the course of diestrus contribute to shortened or prolonged interestrous intervals observed clinically in SE mares.

Validation of reference genes in the feline endometrium.

The aim of the study was to find the most stable reference genes from: ACTB, GAPDH, RPL30, CYC, RPL17, RPS7 and YWHAZ in the feline endometrium. Three free software packages, geNorm, NormFinder and BestKeeper were used. In geNorm analysis, the most stable gene was RPS7 (at a primer concentration 1000 nM) or YWHAZ (500 and 250 nM). According to NormFinder and BestKeeper, ACTB (at all examined primer concentrations) followed by RPS7 and CYC were the most stable genes. Based on geNorm results at least two genes from among RPS7, RPL30, ACTB or YWHAZ should be chosen for Real Time-PCR result normalization.

Corpora lutea of pregnant and pseudopregnant domestic cats reveal similar steroidogenic capacities during the luteal life span.

In domestic cats, luteal phases of pregnancy and pseudopregnancy (non-pregnant luteal phase) differ in the course and level of plasma progesterone (P4). Therefore, we assumed differences in luteal steroidogenic capacities. Here we present a comprehensive analysis of intraluteal steroid biogenesis in the domestic cat. We quantitatively measured relative mRNA levels of steroidogenic acute regulatory protein (STAR), cytochrome P450 oxidases (CYP), hydroxysteroid dehydrogenases (HSD), steroid reductase (SRD) and enzymes involved in sulfoconjugation of steroids, i.e. sulfotransferase (SULT) and sulfatase (STS). Protein expression was analysed by Western Blot for HSD3B. Additionally, intraluteal steroid contents were determined. During the pseudopregnant luteal phase, expression of STAR (p=0.005), HSD3B1 (p<0.0001), CYP19A1 (p<0.0001) and HSD17B7 (p=0.008) decreased from formation of the corpus luteum (CL) onwards. HSD3B protein expression was highest in the development/maintenance stage of CL and declined during the subsequent luteal phase of pregnancy and pseudopregnancy. This was in accordance with decreasing intraluteal levels of P4, oestrogens and androgens. In contrast, expression of SRD5A1 (p<0.001) increased with progression through stages of the pseudopregnant CL, being indicative of P4 metabolism via an alternate pathway to dihydrotestosterone (DHT). Compared to the formation stage, expression of SULT1E1 was higher in all other luteal stages of pseudopregnancy (p=0.004), implying a potential sulfoconjugation of oestrogens. Expression of CYP11A1 and CYP17A1 was unaffected by the luteal stage (p>0.05), suggesting a permanent capacity of cat CL to convert progestogens via androgen and oestrogen pathways. In general, mRNA expression profiles of steroidogenic enzymes during the pregnant luteal phase reflected the pseudopregnancy profiles. Intraluteal oestrogen (p<0.0001) and androgen (p=0.008) levels were higher in the formation stage compared to the following luteal stages of pseudopregnancy. Concentrations of P4 were higher in the development/maintenance compared to the regression stages (p=0.01). We conclude that cat CL of the same histomorphological stage are characterised by identical steroidogenic capacities independently of an on-going pregnancy.

Possible involvement of oxytocin and its receptor in the local regulation of prostaglandin secretion in the cat endometrium.

Ovarian originated oxytocin (OT) is involved in several reproductive process, amongst them its role in the regulation/modulation of the estrous cycle in several species has been demonstrated. Although the systemic role of endometrial originated prostaglandins (PGs), especially prostaglandin F(2α) (PGF(2α)), is equivocal in cats, their possible involvement in the local regulation of uterine events during the estrous cycle is uncertain. We examined the spontaneous and LH-stimulated OT production in cultured luteal cells, the spatial and temporal arrangement of OT receptors (OTR) in a cat endometrium and, finally the effects of OT on PG secretion and prostaglandin-endoperoxide synthase (PTGS2) expression in the feline cultured endometrial cells. Uteri together with ovaries were collected from adult domestic cats (n=27) at different stages of the estrous cycle, after routine ovariohysterectomy procedures. The endometrial and luteal cells were separated enzymatically. Luteinizing hormone (LH) augmented OT secretion in cultured luteal cells 2-fold compared with control (P<0.05). Oxytocin receptor was abundantly expressed in different ovarian structure, as well as in uterine tissues collected at early/developing and mid-luteal phase. The secretion of PGF(2α) by endometrial epithelial cells was increased by OT at a dose 10(-7)M (P<0.001). Atosiban (specific OTR blocker) alone did not affect PG secretion but atosiban in combination with OT abolished the stimulating effect of OT on PGF(2α) secretion. Oxytocin augmented PGE(2) secretion at a dose 10(-7)M and 10(-6)M in the endometrial stromal cells (P<0.001). The treatment with atosiban did not abrogated positive effect of OT on PGE(2) production in the stromal cells. Effect of OT on PTGS2 mRNA expression, the rate-limiting enzyme in PG production, was examined by Real Time-PCR and PTGS2 mRNA expression was significantly affected by OT in both epithelial and stromal cell cultures (P<0.01). The present observations have shown that OT is locally produced by the early/developing corpora lutea and that corpora lutea delivered OT may regulate PG secretion in a cat endometrium especially at early- and mid-diestrus, by affecting PTGS2 mRNA expression.

Ovarian steroids regulate prostaglandin secretion in the feline endometrium.

Sex steroids, i.e. progesterone (P(4)) and 17beta-estradiol (E(2)), fluctuate during the feline estrous cycle and their alterations correspond to many events in cat reproduction. In order to investigate possible effects of sex steroids on prostaglandin (PG) secretion in the cultured endometrial cells, mRNA expressions coding for PG-endoperoxide synthase (PTGS2) in the epithelial and stromal cells harvested with sex steroids were studied by RT-PCR. The effects of ovarian steroids on PG secretion in the epithelial and stromal cells were also investigated. E(2) at a dose 10(-7)M significantly increased prostaglandin F2alpha (PGF(2alpha)) secretion in the epithelial cells (P<0.01). PGF(2alpha) production was intensified by the treatment in combination with both steroids (P<0.001). P(4) at any dose alone had no effect on PGF(2alpha) secretion in the epithelial cells, whereas at a dose 10(-5)M enhanced prostaglandin E2 (PGE(2)) production (P<0.05). The ovarian steroids stimulated both PGF(2alpha) and PGE(2) in the epithelial cells of the feline endometrium via an E(2) receptor (ESR1)- and P(4) receptor (PGR)-dependent genomic-pathway. In contrast to the epithelial cells, neither P(4) nor E(2) affected PG secretion in the stromal cells. PTGS2 mRNA expression was not affected by ovarian steroids in either cell types. The overall results suggest that PG secretion is regulated by P(4) and E(2) and this effect is not due to changes in PTGS2 mRNA expression.

Aspergillosis of a dog genital tract-Case report.

The information about aspergillosis locations in the reproductive organ is scarce. This short paper deals with aspergillosis in the dog genital tract with hyphae present in semen. There are two therapy schemes used in visceral mycoses, non-invasive treatment and surgical intervention. Considering the future reproductive career of the dog, we decided on antifungal drugs administration. Based on the microbiological results, we administered amoxycillin with clavulonate (Synulox 500mg, twice daily) orally. Itraconazole was used as an antimycological agent (Orungal, 100mg, twice daily) every other week. In 8th week of therapy no Aspergillus spp. growth was noted, yet slight Penicillium growth was observed. After 12 weeks of treatment, no fungus growth was present. Neither spores or hyphae were seen in the microscopic examination. Three months after the termination of the therapy, the dog mated with two females. In one case, unifetal pregnancy was diagnosed by ultrasound examination on day 42 after mating. Due to purulent discharge on day 45 after mating, the owner decided to terminate the pregnancy. In the other case, severe pyometra appeared 12 days after the second mating and the owner decided to put the female to sleep. The pathogen eradication from the ejaculates may be treated as a serious success, yet the lack of litters after mating calls for an explanation and consequences of Aspergillus spp. infection need to be considered.

Effects of tumor necrosis factor-alpha and nitric oxide on prostaglandins secretion by the bovine oviduct differ in the isthmus and ampulla and depend on the phase of the estrous cycle.

To determine the possible roles of tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO) in the bovine oviduct, ampulla and isthmus collected during the estrous cycle were exposed for 18 h to TNFalpha, NO donor (NONOate), NO synthase inhibitors (L-NOARG, L-NAME and AMT) and oxytocin (OT) as a positive control. Prostaglandins (PGs) and NO(2)/NO(3) in conditioned media were measured. TNFalpha stimulated PGF(2alpha) secretion on Day 0 (onset of estrus = Day 0) and Days 2-3, in both the ampulla and isthmus, but on Days 18-20 only in ampulla. TNFalpha increased PGE(2) secretion in both fragments in each phase. NONOate did not affect PGF(2alpha) secretion on Days 18-20, whereas this NO donor stimulated PGF(2alpha) secretion in both fragments on Day 0 and Days 2-3. TNFalpha increased NO(2)/NO(3) production in every examined phase in the ampulla and on Days 2-3 in the isthmus. L-NAME lowered NO(2)/NO(3) production regardless of phase or fragment. L-NOARG and AMT lowered NO(2)/NO(3) production in both fragments on Day 0 and Days 2-3. The possible role of TNFalpha, NO or PGs on the oviductal contractility during the early-luteal phase was also examined. Neither TNFalpha nor NONOate influenced contractility in either fragment. Although PGF(2alpha) stimulated the contraction in both fragments, PGE(2) decreased it. When taken together, TNFalpha seems to play some role as a modulator of PGF(2alpha) and PGE(2) production and for transferring the embryo from the oviduct to the uterus by stimulating NO production in the bovine oviduct.