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Centre for Interdisciplinary Research in Animal Health

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Metallopeptidades 2 and 9 genes epigenetically modulate equine endometrial fibrosis
Publication . Alpoim-Moreira, Joana; Fernandes, Carina; Pimenta, Jorge; Bliebernicht, Miguel; Rebordão, Maria Rosa; Castelo-Branco, Pedro; Szóstek-Mioduchowska, Anna; Skarzynski, Dariusz J.; Ferreira-Dias, Graça
Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA methylation commonly mediates gene repression. Thus, this study aimed to evaluate if the transcription of some genes involved in equine endometrosis was altered with endometrial fibrosis, and if the observed changes were epigenetically modulated, through DNA methylation analysis. Endometrial biopsies collected from cyclic mares were histologically classified (Kenney and Doig category I, n = 6; category IIA, n = 6; category IIB, n = 6 and category III, n = 6). Transcription of COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1, and TIMP2 genes and DNA methylation pattern by pyrosequencing of COL1A1, MMP2, MMP9, TIMP1 genes were evaluated. Both MMP2 and MMP9 transcripts decreased with fibrosis, when compared with healthy endometrium (category I) (P < 0.05). TIMP1 transcripts were higher in category III, when compared to category I endometrium (P < 0.05). No differences were found for COL1A1, COL1A2, COL3A1 and TIMP2 transcripts between endometrial categories. There were higher methylation levels of (i) COL1A1 in category IIB (P < 0.05) and III (P < 0.01), when compared to category I; (ii) MMP2 in category III, when compared to category I (P < 0.001) and IIA (P < 0.05); and (iii) MMP9 in category III, when compared to category I and IIA (P < 0.05). No differences in TIMP1 methylation levels were observed between endometrial categories. The hypermethylation of MMP2 and MMP9, but not of COL1A1 genes, occurred simultaneously with a decrease in their mRNA levels, with endometrial fibrosis, suggesting that this hypermethylation is responsible for repressing their transcription. Our results show that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation and therefore, might be promising targets for therapeutic use.
Ultrasonographic detected adrenomegaly in Clinically Ill Cats: a retrospective study
Publication . Oliveira, João; Dias, Maria Joana; Fontes, Ana Paula; Englar, Ryane E.; Vicente, Gonçalo; Ferreira, Rui Lemos; Galac, Sara; Leal, Rodolfo Oliveira
This retrospective study aimed to assess the prevalence of ultrasonographic detected adrenomegaly in clinically ill cats, evaluating the final established diagnosis, describe adrenal ultrasound findings and if the adrenomegaly was suspected or incidental. Abdominal ultrasonography reports of cats presenting to a veterinary teaching hospital between October 2018 and February 2021 were retrospectively reviewed. Cats showing adrenomegaly (one or both glands having a dorsoventral axis >4.8 mm) were selected and medical records respectively evaluated. Nine-hundred and eighty-three ultrasonographical reports were selected, of which, 68 (7%) disclosed adrenomegaly. European/Domestic Short-Hair (62/68; 91%) male (44/68; 65%) castrated (35/44; 80%) cats were overrepresented. Adrenomegaly was an incidental finding in 62/68 (91%) cats while in 6/68 (9%) it was identified in the context of investigating a potential adrenal disease. Concerning established diagnosis, chronic kidney disease was overrepresented (25/68; 37%), followed by endocrinopathies (20/68; 29%). Adrenomegaly was bilateral in 53% (36/68) of cases. In unilateral cases (32/68; 47%), it was more prevalent on the left side (23/32; 72%), with a normal-sized contralateral adrenal gland. Left adrenal demonstrated a larger size and a tendency to oval shape. This study assesses the prevalence of adrenomegaly in clinically ill cats, reinforcing it can be an incidental ultrasound finding.
Post-transcriptional silencing of Bos taurus prion family genes and its impact on granulosa cell steroidogenesis
Publication . Pimenta, Jorge M.B.G.A.; Pires, Virgínia M.R.; Nolasco, Sofia; Castelo-Branco, Pedro; Marques, Carla C.; Apolónio, Joana; Azevedo, Rita; Fernandes, Mónica T.; Lopes-da-Costa, Luís; Prates, José; Pereira, Rosa M.L.N.
Prion proteins constitute a major public health concern, which has partly overshadowed their physiological roles in several scenarios. Indeed, these proteins were implicated in male fertility but their role in female fertility is relatively less explored. This study was designed to evaluate the role of SPRN and PRNP prion family genes in bovine follicular steroidogenesis pathways. Post-transcriptional SPRN and PRNP silencing with siRNAs was established in bovine granulosa cell (GC) in vitro culture, and gene expression and progesterone and estradiol concentrations were evaluated. SPRN knockdown, led to a down regulation of CYP11A1 mRNA levels (2.1-fold), and PRNP knockdown led to an upregulation of SPRN mRNA levels (2.3-fold). CYP19A1 expression and estradiol synthesis was not detected in any experimental group. Finally, SPRN knockdown led to a mild reduction in progesterone production in GCs and this was the only experimental group that did not exhibit an increment in progesterone levels after 48 h of culture. As a conclusion, it was possible to detect the expression of the SPRN gene in bovine GCs, a potential interaction between SPRN and PRNP regulation, and the impact of SPRN expression on CYP11A1 and progesterone levels. These findings bring new insights into the role of these genes in ovarian steroidogenesis and female reproductive physiology. (c) 2022 Elsevier Inc. All rights reserved.

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Funding agency

Fundação para a Ciência e a Tecnologia

Funding programme

6817 - DCRRNI ID

Funding Award Number

UIDB/00276/2020

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