Microscopy images from: A physiological and histological atlas of reproduction in the North American deer mouse (Peromyscus maniculatus)

The North American deer mouse (Peromyscus maniculatus) exhibits extensive diversity in morphology, physiology, and life history across its broad range. These traits have propelled the deer mouse to model system status across several fields within the biological sciences. Nonetheless, we still lack basic knowledge about some important aspects of this species’ biology. For example, limited information about the deer mouse’s reproductive physiology remains a significant barrier to developing genetic tools for the species and for advancing our current understanding of how this species has been so evolutionarily successful. In this manuscript, we aim to fill this knowledge gap by (1) characterizing body temperature profiles across reproductive stages and (2) generating a detailed histological atlas of placental development. We show that body temperature can be used to diagnose copulation and pregnancy in deer mice, however body temperature cannot be used to predict fertility (likelihood to breed) prior to pairing individuals. Our placental atlas represents the first day-by-day developmental timeline of the placenta in a Peromyscus species, and from it we are able to describe unique organization and behaviors of trophoblast cells, especially at the maternal-fetal interface in the deer mouse. Together, these descriptive datasets provide substantial new comparative data on reproductive physiology in Cricetids, and they provide a foundation for further functional work in this important model species.

Methods
We employed three separate double-labeling protocols to describe specific features of placentation and development. To examine vascular development and remodeling in the implantation site, we used two sets of sections to label two markers of vascular remodeling and endothelial cell organization: the basement membrane protein laminin and the tight-junction protein ZO-1 (26,27). To examine interactions between uterine natural killer cells (uNK cells) and trophoblasts in the implantation site, we also labeled one set of sections for the uNK cell marker perforin.

For all immunohistochemistry protocols, sections were fixed with ice-cold 4% paraformaldehyde, permeabilized using methanol, and blocked using 10% normal goat serum in PBS (Vector Laboratories S-1000). Sections were incubated overnight with one of three primary antibodies: rabbit anti-laminin (1:1000, Thermo Scientific PA116730), or rabbit anti-ZO-1 (1:500, Invitrogen 617300), or rabbit anti-perforin (1:400, Amsbio TP251). After PBS washes the next day, sections were incubated for 1 hour in Alexafluor 568 (1:300, goat-anti-rabbit, Thermo Scientific A32731TR). All sections were then labeled for cytokeratin (a trophoblast cell marker) using a 2 hour incubation with pan-cytokeratin antibody conjugated to FITC (1:300, Sigma-Aldrich F3418, lot: 088M4797V) followed by DAPI to visualize nuclei. Immunostained sections were cover slipped with Fluoromount-G, dried overnight at room temperature, and stored at 4°C until imaging on an Evident Scientific APX100. Three sections per implantation site were imaged and quantified for each protocol at one of three objectives (4x, 10x, or 40x).