Placentation and fetal membrane development in the South American coati, Nasua nasua(Mammalia, Carnivora, Procyonidae)

Carnivores are regarded as having zonary, labyrinthine placentas with an endotheliochorial barrier. In addition, specialized areas are common, such as hematomas or hemophagous organs for the uptake of uterine secretion or the phagocytosis of extravasated maternal blood components, serving as mode of iron supply [1‐3]. However, placental research in carnivores has mainly concentrated on domestic dogs and cats [4‐11], rather than other species (e.g., [12‐14]). Within Musteloidea, the mink, ferret and sea otter have been investigated [15‐18], in addition to the raccoon and certain other species, including the coatimundi Nasua narica[19‐21], (Figure 1A). Because more information, especially on wildlife species, is essential to better understanding the evolution of placentation in carnivores, we herein describe this system in the South American coati Nasua nasua (Linnaeus, 1766), which is native to southern South America, for the first time [22]. Coatis are omnivorous, medium-sized animals with a body mass of approximately 6 kg [21, 22]. These animals live in groups organized within a complex social system consisting of up to 70 individuals. Two different groups are usually present; one composed entirely of adult males and a second group consisting of an alpha female (dominant), adult females, and young [23, 24]. Females reach sexual maturity at 2 years of age, and males do so at approximately 3 years. The mating season usually occurs in late spring, from September to November. This is the only time when adult reproductive males meet with female groups [22‐24]. After 10–11 weeks of gestation, 2–6 very altricial young with a body weight of 100 to 180 g are born [21, 22]. Conclusions about the evolutionary course of the characters have been drawn from comparative analyses and analyzed within the relationships of carnivoran groups (Figure 1A), as indicated by recent morphological and molecular phylogenetics [25, 26]. We use the terminology of phylogenetic systematics [27] in differentiating between derived character conditions and ancestral ones, with special reference to ancient conditions or the stem species patterns of Carnivora.

The project was approved by the Bioethics Commission of the School of Veterinary Medicine and Animal Science, University of Sao Paulo, Brazil (Protocol Nr. 1598/2009), on July 1st, 2009, and by the Biodiversity Authorization and Information System – SISBIO (Protocol Nr. 21030–1) on September 17th, 2010. The study was performed from July 2009 to November 2011.

In total, four placentas from mid-gestation (n = 2) to near term (n = 2) were collected from 2 wildlife individuals captured in Mangabeiras Park in Belo Horizonte-MG, Brazil. Hemi-ovariohysterectomy was performed following surgical and anesthetic protocols used for domestic carnivores [28].

Measurements of the occipital-sacral distance of the fetuses’ heads were performed with a stainless-steel caliper, using the nuchal crest at one end and the last sacral vertebra at the opposite end as references (crown-rump, CR), following the methodology proposed by Evans and Sack [29]. The CR distances and the external features of the fetuses were used to estimate the age of each individual. The weight (g) was determined using a digital scale (0.0001 g; BEL Engineering).

After macroscopic examination of the main placental structures (zonary placenta, chorion, allantois, amnion, yolk sac, and hemophagous organs), the tissues were fixed in 4% paraformaldehyde for histology and immunohistochemical staining. The samples were stained for vimentin (1:400, 0.N.602, sc-73259, Santa Cruz Biotechnology, Santa Cruz, CA, USA) to identify mesenchymal cells and endothelium, for cytokeratin (1:300; M0821, Dako, Carpinteria, CA, USA) to identify epithelial and trophoblastic cells, and for proliferating cell nuclear antigen (PCNA; 1:300; PC10, sc-56, Santa Cruz Biotechnology, Santa Cruz, CA, USA) to identify proliferating cells. For this staining, we followed protocols established by our group [30]. The negative control was goat anti-mouse IgG (1:500; AP308F, Chemicon International Temecula, CA, USA). The slides were examined with an Olympus BX40 microscope with a Zeiss KS400 image analysis system. For scanning and transmission electron microscopy, placental samples were fixed in 2.5% glutaraldehyde and processed following established protocols [10, 11].

Extended non-invasive, non-villous, fetomaternal contact zones similar to what is observed in epitheliochorial placentas are widespread in carnivores [4‐21] and are known as the polar zone of the paraplacenta [7]. These zones likely uptake histiotrophe from endometrial glands [8] and represent an ancient character condition of the group Carnivora. The hyena is unique among carnivores because it presents a hemochorial villous placenta with intimate contact between the fetal and the maternal circulation via so-called intraepithelial capillaries in the syncytial trophoblast [13]. In contrast, true epitheliochorial placentas, serving as main fetomaternal contact zones, i.e., the complete reduction of invasive trophoblasts in maternal tissue interactions into strictly noninvasive forms, are regarded as evolutionarily derived conditions that have occurred in several clades of eutherian mammals, such as the ungulates and relatives [2, 31‐34]. Because there is little detailed information on the foundational processes of mammalian trophoblast invasion and its restriction, it is unclear whether the ontogenetic establishment of the carnivore paraplacenta follows similar processes as do the epitheliochorial placentas of close relatives within Laurasiatheria [2, 32]. However, regarding the main fetomaternal exchange region of the placenta, comparative data [4‐21] have confirmed that endotheliochorial, labyrinthine placentation belongs to the ancestral carnivore pattern [2, 32]. Within the group, only hyenas have haemochorial placentas [13, 14], which are hemomonochorial in nature, consisting of a continuous layer of syncytial trophoblasts, a basal lamina, and the fetal capillary endothelium [35].

Regarding the fine structure of the fetomaternal interface, the following characters are widespread among carnivores and seemed to belong to their stem species pattern or ancestral pattern: an endotheliochorial barrier in the zonary or circumferential placenta, in addition to a yolk sac and a large allantoic sac that persist until term. In contrast, the coati and other procyonids were derived from the ancient carnivore condition, maintaining the cellular trophoblast to term [19‐21].

Hematomas (hemophagous organs), as specialized quasi-haemochorial zones in addition to the main endotheliochorial placenta, are widespread in carnivores. These structures are usually temporary, are mainly built by phagocytosing cytotrophoblasts [9, 20] that are associated with uterine glands and extravasated blood, serve as the iron supply, and take up histiotrophe [4‐21]. Thus, these organs represent multifunctional or heterophagous areas [1, 3, 14]. The ancestral pattern of carnivores likely had such areas at the placental margin, occurring in both Caniformia and Feliformia. Nasua and other Musteloidea [19‐21] have large, multilobular organs in a central position. Moreover, Nasua nasua possesses a derived condition in that the organ persists, fully functional, until near term. Areolae are absent in the coati and in other Musteloidae, cats and hyenas [4, 7, 8, 10, 13‐21] and likely are not part of the carnivore stem species pattern [2].