Abstract
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.
Highlights
Because Primordial germ cells (PGCs) are precursors of gametes that are capable of generating new individuals and transmit genetic material to future generations [2]
The primary or primitive gonads were located in the medial body region and were designated "undifferentiated" because we could not determine the sex of the embryo through morphological or histological analyses
Mice and swine [19,44,56,57], our results showed that supplementation of the culture conditions with these factors allowed the canine putative embryonic germ cells (EGCs) to proliferate and form colonies in cells that were positive for alkaline phosphatase (AP)
Summary
Because PGCs are precursors of gametes that are capable of generating new individuals and transmit genetic material to future generations [2]. Fertility in humans and other species relies on the successful development of PGCs [5]. PGCs originate from a small population of cells in the extra-embryonic mesoderm in the posterior region in E7.0–7.25 mice [6]. PGCs development begins during the third week of gestation in the posterior region of the yolk sac at the allantois base [7]. As the mammalian embryo develops, the PGCs migrate into the embryo towards the somatic cells of the developing gonadal ridge, where they are reorganized to form future gametes [8,9]
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