Date Published: February 28, 2018
Publisher: Public Library of Science
Author(s): Aline F. de Souza, Naira C. Godoy Pieri, Kelly C. S. Roballo, Fabiana F. Bressan, Juliana B. Casals, Carlos E. Ambrósio, Felipe Perecin, Daniele S. Martins, Jae Yong Han.
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 in vitro culture. Most studies investigating germinative cells have been performed in rodents and humans but not dogs (Canis lupus familiaris). Here, we elucidated the dynamics of the expression of pluripotent (POU5F1 and NANOG), germline (DDX4, DAZL and DPPA3), 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 POU5F1 and H3K27me3 during all assessed developmental periods, including all periods between the gonadal tissue stage and foetal testes development. The number of NANOG, 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 NANOG 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.
During embryonic development, extensive reprogramming occurs via gene regulation, resulting in changes in cell dynamics and the generation of new cells, such as primordial germ cells (PGCs) . Because PGCs are precursors of gametes that are capable of generating new individuals and transmit genetic material to future generations . Studies investigating PGCs have revealed interesting findings regarding their migration route, in vitro cultivation and differentiation [3,4]. Furthermore, fertility in humans and other species relies on the successful development of PGCs .
This study provides new and important findings regarding the morphology and expression of pluripotent- and epigenetic-related factors in germ cells during canine embryonic and foetal development. Additionally, the derivation and in vitro culture of canine EGCs was possible. We observed heterogeneous germline PGCs and EGCs populations that had specific and development-dependent molecular signatures of the markers POU5F1, NANOG, DDX4, DAZL, and DPPA3. The epigenetic profiles of these cell populations were determined to include the histone repressive markers H3K27me3 and H3K9me2, and changes in the DNA methylation (5mC) and hydroxymethylation of the DNA (5hmC) status were detected. These results not only open new avenues for studying canine reproductive physiology but also serve as models for comparative reproductive biology.