Equine endometrosis pathological features: are they dependent on NF-κB signaling pathway?

Research Areas: Agriculture ; Veterinary Sciences ; ZoologyEndometrosis is an important mares’ disease which considerably decreases their fertility. As classic endometrial classification methods might be insufficient for tissue pathological evaluation, further categorization into active/inactive and destructive/non-destructive types was developed by Hoffmann and others. This study aimed to compare NF-κB pathway genes transcription among histopathological types of endometrosis, following Hoffmann and co-authors’ classification. Endometrial samples, collected postmortem from cyclic mares (n = 100) in estrus or diestrus, were classified histologically and used for gene transcription assessment. Gene transcription of NF-κB subunits (RelA, NF-κB1, NF-κB2), pro-inflammatory molecules (MCP-1, IL-6), and hyaluronan synthases (HAS 1, HAS 2, HAS 3) was compared among endometrosis types (active, non-active, destructive, non-destructive). Most individual mRNA samples showed high expression of RelA, NF-κB1, and MCP-1 gene transcripts and the destructive type of endometrosis, simultaneously. The expression of RelA and NF-κB1 genes was higher in active destructive group than in the other groups only in the follicular phase, as well as being higher in the inactive destructive group than in the others, only in the mid-luteal phase. The increase in gene transcription of the NF-κB canonical activation pathway in destructive endometrosis may suggest the highest changes in extracellular matrix deposition. Moreover, the estrous cycle phase might influence fibrosis pathogenesis.info:eu-repo/semantics/publishedVersio

The dog: nonconformist, not only in maternal recognition signaling

Although similar at the molecular and cellular levels, endocrine mechanisms governing reproductive function in the domestic dog (Canis familiaris) differ markedly at the regulatory level from those known in other domestic animal species. Some of the events, e.g., the lack of luteolysis in the absence of pregnancy, resulting in similar luteal function and, therefore, hormonal profiles in early pregnant and nonpregnant animals, are species-specific. Consequently, no early gestation marker has so far been identified for the dog. Following implantation, relaxin of fetal placental origin can be detected and used for pregnancy diagnosis. Characterized by the lack of an active luteolytic principle from intra- or extra-luteal sources, the canine reproductive cycle appears to represent a “basic” form of mammalian reproductive function with apparently reduced opportunities for facilitating fecundity and hastening reproduction. Nevertheless, in the dog some kind of mechanism for synchronization between blastocyst development and uterine preparation for pregnancy must have evolved in order to support gestation. Driven by this assumption, studies including our recent investigations have been initiated aimed at characterizing some of the embryo-mediated effects of the preimplantation embryo on the canine uterus. Moreover, the lack of a uterine luteolysin and consequently the absence of a need to develop an antiluteolytic strategy make the dog an interesting model for investigating early evolutionary mechanisms involved in the preparation for implantation and ensuring embryo survival. These mechanisms result in an inverse relationship between the duration of pregnancy and of the nonpregnant cycle in the dog, compared with all other domestic animal species

Gene expression profiling of the canine placenta during normal and antigestagen-induced luteolysis

The domestic dog is the only domestic animal species that does not produce steroids in the placenta and instead relies on luteal steroids throughout pregnancy. Nevertheless, the canine placenta is highly responsive to steroids, and withdrawal of progesterone (P4) affects the feto-maternal unit, initializing the parturition cascade. Similar effects can be observed during antigestagen-induced abortion. Here, aiming to provide new insights into mechanisms involved in the termination of canine pregnancy, next generation sequencing (NGS, RNA-seq) was applied. Placental transcriptomes derived from natural prepartum and antigestagen-induced abortions were analyzed and compared with fully developed mid-gestation placentas. The contrast "prepartum luteolysis over mid-gestation" revealed 1973 differentially expressed genes (DEG). Terms associated with apoptosis, impairment of vascular function and activation of signaling of several cytokines (e.g., IL-8, IL-3, TGF-β) were overrepresented at natural luteolysis. When compared with mid-term, antigestagen treatment revealed 135 highly regulated DEG that were involved in the induced luteolysis and showed similar associations with functional terms and expression patterns as during natural luteolysis. The contrast "antigestagen-induced luteolysis over prepartum luteolysis" revealed that, although similar changes occur in both conditions, they are more pronounced during natural prepartum. Among P4-regulated DEG were those related to immune system and cortisol metabolism. It appears that, besides inducing placental PGF2α output, both natural and induced P4 withdrawal is associated with disruption of the feto-maternal interface, leading to impaired vascular functions, apoptosis and controlled modulation of the immune response. The time-related maturation of the feto-maternal interface needs to be considered because it may be clinically relevant

Uterine responses to early pre-attachment embryos in the domestic dog and comparisons with other domestic animal species

In the dog, there is no luteolysis in the absence of pregnancy. Thus, this species lacks any anti-luteolytic endocrine signal as found in other species that modulate uterine function during the critical period of pregnancy establishment. Nevertheless, in the dog an embryo-maternal communication must occur in order to prevent rejection of embryos. Based on this hypothesis, we performed microarray analysis of canine uterine samples collected during pre-attachment phase (days 10-12) and in corresponding non-pregnant controls, in order to elucidate the embryo attachment signal. An additional goal was to identify differences in uterine responses to pre-attachment embryos between dogs and other mammalian species exhibiting different reproductive patterns with regard to luteolysis, implantation, and preparation for placentation. Therefore, the canine microarray data were compared with gene sets from pigs, cattle, horses, and humans. We found 412 genes differentially regulated between the two experimental groups. The functional terms most strongly enriched in response to pre-attachment embryos related to extracellular matrix function and remodeling, and to immune and inflammatory responses. Several candidate genes were validated by semi-quantitative PCR. When compared with other species, best matches were found with human and equine counterparts. Especially for the pig, the majority of overlapping genes showed opposite expression patterns. Interestingly, 1926 genes did not pair with any of the other gene sets. Using a microarray approach, we report the uterine changes in the dog driven by the presence of embryos and compare these results with datasets from other mammalian species, finding common-, contrary-, and exclusively canine-regulated genes

Interplacental uterine expression of genes involved in prostaglandin synthesis during canine pregnancy and at induced prepartum luteolysis/abortion

BACKGROUND: In the non-pregnant dog, ovarian cyclicity is independent of a uterine luteolysin. This is in contrast to pregnant animals where a prepartum increase of luteolytic PGF2alpha occurs, apparently originating in the pregnant uterus. Recently, the placenta as a source of prepartum prostaglandins (PGs) was investigated, indicating fetal trophoblast cells as the likely main source. However, the possible contribution of uterine interplacental tissues to the production of these hormones has not yet been thoroughly examined in the dog. METHODS: Several key factors involved in the production and/or actions of PGs were studied: cyclooxygenase 2 (COX2, PTGS2), PGF2alpha-synthase (PGFS/AKR1C3), PGE2-synthase (PGES), and the respective receptors FP (PTGFR), EP2 (PTGER2) and EP4 (PGTER4), 15-hydroxyprostaglandin dehydrogenase (HPGD), PG-transporter (PGT, SLCO2A1) and progesterone receptor. Their expression and localization patterns were assessed by Real Time PCR and immunohistology in the interplacental uterine sites from pregnant dogs during the pre-implantation period (days 8-12), post-implantation (days 18-25), mid-gestation (days 35-40) and during antigestagen-induced luteolysis/abortion. RESULTS: Whereas only low COX2 expression was observed in uterine samples at all the selected time points, expression of PGFS/AKR1C3 strongly increased post-implantation. A gradual increase in PGES-mRNA expression was noted towards mid-gestation. FP-mRNA expression decreased significantly with the progression of pregnancy until mid-gestation. This was associated with clearly detectable expression of HPGD, which did not change significantly over time. The expression of FP and EP2-mRNA decreased significantly over time while EP4-mRNA expression remained unaffected. The antigestagen-treatment led to a significant increase in expression of COX2, PGES, EP2 and PGT (SLCO2A1) mRNA. COX2 was localized predominantly in the myometrium. The expression of PGFS/AKR1C3, which was unchanged, was localized mostly to the surface luminal epithelium. The expression of EP4, PGT and HPGH did not change during treatment, they were co-localized with PGES and EP2 in all uterine compartments. CONCLUSIONS: The data clearly demonstrate the basic capability of the canine pregnant uterus to produce and respond to PGs and suggests their functions both as local regulatory factors involved in the establishment and maintenance of pregnancy, as well as potential contributors to the process of parturition, supporting the myometrial contractility associated with fetal expulsion

Molecular Mechanism of Equine Endometrosis: The NF-κB-Dependent Pathway Underlies the Ovarian Steroid Receptors’ Dysfunction

Endometrosis is a frequently occurring disease decreasing mares’ fertility. Thus, it is an important disease of the endometrium associated with epithelial and stromal cell alterations, endometrium gland degeneration and periglandular fibrosis. Multiple degenerative changes are found in uterine mucosa, the endometrium. However, their pathogenesis is not well known. It is thought that nuclear factor-κB (NF-κB), a cell metabolism regulator, and its activation pathways take part in it. The transcription of the profibrotic pathway genes of the NF-κB in fibrotic endometria differed between the follicular (FLP) and mid-luteal (MLP) phases of the estrous cycle, as well as with fibrosis progression. This study aimed to investigate the transcription of genes of estrogen (ESR1, ESR2) and progesterone receptors (PGR) in equine endometria to find relationships between the endocrine environment, NF-κB-pathway, and fibrosis. Endometrial samples (n = 100), collected in FLP or MLP, were classified histologically, and examined using quantitative PCR. The phase of the cycle was determined through the evaluation of ovarian structures and hormone levels (estradiol, progesterone) in serum. The transcription of ESR1, ESR2, and PGR decreased with the severity of endometrial fibrosis and degeneration of the endometrium. Moreover, differences in the transcription of ESR1, ESR2, and PGR were noted between FLP and MLP in the specific categories and histopathological type of equine endometrosis. In FLP and MLP, specific moderate and strong correlations between ESR1, ESR2, PGR and genes of the NF-κB pathway were evidenced. The transcription of endometrial steroid receptors can be subjected to dysregulation with the degree of equine endometrosis, especially in both destructive types of endometrosis, and mediated by the canonical NF-κB pathway depending on the estrous cycle phase

The NF-kappa B-signalling pathway in mare's endometrium infiltrated with the inflammatory cells

Research Areas: Agriculture ; Reproductive Biology ; Veterinary SciencesABSTRACT: Endometritis is an important issue decreasing mares' fertility. In the case of endo-metritis, both inflammatory cells infiltration and proinflammatory molecules produc-tion are regulated by various cellular and gene regulatory mechanisms, including the nuclear factor- κB (NF- κB)- dependent pathway. NF- κB- signalling pathway has been recently studied in the equine endometrium in the context of endometrosis. Thus, this study aimed to determine gene transcription of NF- κB subunits (RelA; NF- κB1; NF- κB2), proinflammatory molecules (M C P - 1 ; I L- 6) and hyaluronan synthases (HAS 1; HAS 2; HAS 3) in endometritis and compare them with the intensity and type of inflamma-tory cell infiltration. Endometrial samples, collected post- mortem from cyclic mares in oestrus or dioestrus, were classified histologically and examined using quantitative PCR. Transcription NF- κB subunits genes did not differ with either inflammatory in-tensity or type of inflammatory cell infiltration. Transcription of M C P - 1 and I L- 6 genes increased with the severity of inflammation, with the involvement of HAS 3 and HAS 2 genes, as opposed to HAS 1 genes. These proinflammatory molecules and hyaluronan synthases in the equine inflamed endometrium do not seem to be regulated by the NF- κB pathway. Hence, separate signalling pathways for the development and pro-gression of equine endometritis and endometrosis may be suggestedinfo:eu-repo/semantics/publishedVersio

In vitro decidualisation of canine uterine stromal cells

BACKGROUND: The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology. METHODS: Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n = 6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated. RESULTS: The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ERα were observed in decidualised cells; PRL and ERβ remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle α actin (αSMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable. CONCLUSION: An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12958-015-0066-4) contains supplementary material, which is available to authorized users