Photomicrographs of the hepatic parenchyma of Wistar rats subjected to ischemia and reperfusion.

<p>Groups (10, 20, and 30 minutes ischemia) and subgroups (15, 30, 60 and 120 minutes reperfusion): I10 and R15 (A), I10 and R30 (B), I10 and R60 (C), I10 and R120 (D), I20 and R15 (E), I20 and R30 (F), I20 and R60 (G), I20 and R120 (H), I30 and R15 (I), I30 and R30 (J), I30 and R60 (K) e I30 and R120 (L). Note the positive immunoreactivity for caspase-3 protein (brown coloration).</p

Photomicrographs of the hepatic parenchyma in control group animals: Negative control (C-) and positive control (C+).

<p>Note the positive immunoreactivity for caspase-3 in the C+ group (brown coloration).</p

Liver parenchyma photomicrographs of Wistar rats subjected to ischemia and reperfusion.

<p>Groups (10, 20, and 30 minutes ischemia) and subgroups (15, 30, 60, and 120 reperfusion): I10 and R15 (A), I10 and R30 (B), I10 and R60 (C), I10 and R120 (D), I20 and R15 (E), I20 and R30 (F), I20 and R60 (G), I20 and R120 (H), I30 and R15 (I), I30 and R30 (J), I30 and R60 (K) e I30 and R120 (L). Note: vascular congestion, microvesicles, hydropic degeneration, necrosis, and pyknotic nuclei. Hematoxylin-eosin staining (HE).</p

Mean values of vascular congestion (A), microvesicles (B), hydropic degeneration (C), necrosis (D), and pyknotic nuclei (E) in the liver parenchyma of the ischemia (I), reperfusion (R), and control (C) group animals.

<p>The data were recorded by optical microscopy after hematoxylin-eosin staining (HE). Ischemic groups: I10–10 minutes of ischemia, I20–20 minutes of ischemia and I30–30 minutes of ischemia. Reperfusion subgroups: R15–15 minutes of reperfusion, R30–30 minutes of reperfusion, R60–60 minutes of reperfusion and R120–120 minutes of reperfusion (*P < 0.05).</p

Photomicrographs of the liver parenchyma of control group animals (C).

<p>Note: central vein, hepatocytes, and sinusoidal capillaries. Hematoxylin-eosin staining (HE).</p

Mean values of positive immunoreactivity for caspase-3 protein in the hepatic parenchyma of ischemia (I), reperfusion (R) and control (C) group animals.

<p>The data were recorded by optical microscopy after immunohistochemical staining. Ischemic groups: I10 –ischemia 10 minutes, I20 –ischemia 20 minutes, and I30 –ischemia 30 minutes. Reperfusion subgroups: R15 –reperfusion 15 minutes, R30 –reperfusion 30 minutes, R60 –reperfusion 60 minutes, and R120 –reperfusion 120 minutes. (P < 0.05).</p

Photomicrographs of the pulmonary parenchyma of control group animals (C).

<p>Note: alveolar septum, alveolar duct, alveolar sac, alveoli, and bronchioles. Hematoxylin-eosin staining (HE).</p

Photomicrographs of the pulmonary parenchyma of Wistar rats subjected to ischemia and reperfusion.

<p>Groups (10, 20, and 30 minutes ischemia) and subgroups (15, 30, 60, and 120 reperfusion): I10 and R15 (A), I10 and R30 (B), I10 and R60 (C), I10 and R120 (D), I20 and R15 (E), I20 and R30 (F), I20 and R60 (G), I20 and R120 (H), I30 and R15 (I), I30 and R30 (J), I30 and R60 (K), I30 and R120 (L). Note: vascular congestion, degeneration of bronchial epithelium, alveolar septal thickening, interstitial edema, and hemorrhage. Hematoxylin-eosin staining (HE).</p

Dynamics of male canine germ cell development

<div><p>Primordial germ cells (PGCs) are precursors of gametes that can generate new individuals throughout life in both males and females. Additionally, PGCs have been shown to differentiate into embryonic germ cells (EGCs) after <i>in vitro</i> culture. Most studies investigating germinative cells have been performed in rodents and humans but not dogs (<i>Canis lupus familiaris</i>). Here, we elucidated the dynamics of the expression of pluripotent (<i>POU5F1</i> and <i>NANOG</i>), germline (<i>DDX4</i>, <i>DAZL</i> and <i>DPPA3</i>), and epigenetic (5mC, 5hmC, H3K27me3 and H3K9me2) markers that are important for the development of male canine germ cells during the early (22–30 days post-fertilization (dpf)), middle (35–40 dpf) and late (45–50 dpf) gestational periods. We performed sex genotype characterization, immunofluorescence, immunohistochemistry, and quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analyses. Furthermore, in a preliminary study, we evaluated the capacity of canine embryo PGCs (30 dpf) to differentiate into EGCs. To confirm the canine EGCs phenotype, we performed alkaline phosphatase detection, immunohistochemistry, electron and transmission scanning microscopy and RT-qPCR analyses. The PGCs were positive for <i>POU5F1</i> and H3K27me3 during all assessed developmental periods, including all periods between the gonadal tissue stage and foetal testes development. The number of <i>NANOG</i>, DDX4, DAZL, DPPA3 and 5mC-positive cells increased along with the developing cords from 35–50 dpf. Moreover, our results demonstrate the feasibility of inducing canine PGCs into putative EGCs that present pluripotent markers, such as POU5F1 and the <i>NANOG</i> gene, and exhibit reduced expression of germinative genes and increased expression of H3K27me3. This study provides new insight into male germ cell development mechanisms in dogs.</p></div

PGCs epigenetic markers during development.

<p>(A-H) Sections of the male canine gonadal ridge and PGCs during the early (25–30 dpf), middle (35–40 dpf) and late (45–50 dpf) gestational periods showing the expression of 5mC (dab staining–brown/nuclear). (E-H) 5mC was clearly expressed in the gonocytes and foetal testes even though the spermatogonial cells were not positive for 5mC (white arrow, E to H). (E1-H1) However, the spermatogonial cells continued to show POU5F1 positivity (red/nuclear). (Scale bars are 50 μm).</p