Schematic representation of the PI3K-Akt signaling pathway model and cellular responses in ovarian follicular cells (FCs) associated with competent oocytes that were able to reach the blastocyst stage (BL) and incompetent oocytes that were unable to initiate embryo development after maturation (NC).

<p>Pentagons indicate whether a given component of the pathway was upregulated (upward arrow) or downregulated (downward arrow) in FCs from the BL and NC groups. Lines ending in arrowheads indicate stimulation, whereas lines ending in bars indicate blockage.</p

Bovine primer sequences used in the RT-PCR amplification.

<p>Bovine primer sequences used in the RT-PCR amplification.</p

Relative expression of mRNAs for genes responsible for the downstream cellular effects of PI3K-Akt signaling on non-cleaved (NC), cleaved non-blastocyst (CNB) and blastocyst (BL) groups.

<p>Bars depict means and error bars depict standard errors of the means. Different superscripts (a and b) indicate significant differences (p < 0.05).</p

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

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 (22–30 dpf), middle (35–40 dpf) and late (45–50 dpf) gestational periods showing the expression of H3K27me3 (green/nuclear) and/or POU5F1 (red/nuclear) and co-localization with POU5F1⁺ (yellow/orange) (Scale bars are 50 μm).</p

Dynamic expression of PGCs during the intermediate and late development periods of the male canine gonadal ridge.

<p>(A-D) Photomicrographs of sections of canine embryos at 35, 40, 45 and 50 dpf. (A1-D1) Histological section of male canine gonadal ridge during the early gestation period (35–45 dpf) (Scale bars are 50 μm). (A2-D2/A3-D3/C4-D4) Testes sections at 35, 40, 45 and 50 dpf immunoassayed for the early germ cell marker (nuclear) POU5F1 (red) and/or the late germ cell markers (cytoplasmic) DDX4, DAZL and (nuclear) DPPA3 (green) (Scale bars are 50 μm).</p

Gene expression correlations determined according to the <i>Pearson</i> coefficient.

<p>The correlation (<i>Pearson</i>) profiles of the pluripotent and germinative genes in the canine PGCs during the early, middle and late periods. Dark blue indicates a positive correlation, and dark red indicates a negative correlation.</p

RT-qPCR analysis of several key pluripotent and germ cell-associated genes in PGCs among the periods.

<p><i>POU5F1</i>, <i>NANOG</i>, <i>DDX4</i>, <i>DPPA3</i> and <i>DAZL</i> were normalized to the <i>GADPH</i> gene (N = 3 biological samples with technical triplicates for each period, p<0.1). Asterisks denote statistically significant differences among the periods.</p

Sequences of the primers used for RT-qPCR.

<p>Sequences of the primers used for RT-qPCR.</p