Stereological analysis of the New Zealand rabbits (Oryctolagus cuniculus) placenta

Abstract

The onset of gestation is characterized by growth, morphological and functional changes of the placenta. We aim to evaluate the placental compartments in New Zealand rabbits by means of stereological methods. The fetal and maternal portion of placenta (12, 14, 18 and 20 gestational days) was randomly sampled for the stereological analysis. Histological sections were scanned to estimate fetal (labyrinth and junctional) and maternal (decidua) compartment volumes. The total volume of the placenta for the ages of 12, 14, 18 and 20 days was, respectively, 320 mm³, 340 mm³, 940 mm³ and 1300 mm³. The volume of the labyrinth was 56 mm³, 119 mm³, 231 mm³ and 481 mm³, respectively. The volume of junctional zone was 75 mm³, 76 cm³, 238 mm³ and 314 mm³, respectively. The volume of decidua was 174 mm³, 143 mm³, 469 mm³ and 504 mm³, respectively. We concluded that the rabbit´s placenta compartments varied according to the gestational period, increasing continuously over the 20 gestational days. However, on the onset of the development of the placenta the decidua presented faster growth, whereas after the 20 days of development, the labyrinth developed more quickly. This study represents an aid to the understanding of placentation in humans.

Key words
decidua; junctional zone; labyrinth; placentation; stereology

INTRODUCTION

Rabbits represent favorable experimental models for pharmacological and toxicity researches, the placenta is an important organ to understand vascular maternal-fetal components and toxic effects through placental barrier, as well as during the growth of embryo/fetus (Hayashi & Takeshi 2014). Similar with human placenta, rabbits have a discoidal and hemochorial placenta that is why they are chosen to in vitro fertilization, embryological, organogenesis and reproductive pathologies researches (e.g. congenital malformations and intrauterine growth restriction) (Hayashi & Takeshi 2014, Püschel et al. 2010).

The placenta is responsible for maintain the gestation, as well as fetus development, it is composed by a maternal portion (basal plate) and a fetal portion (labyrinth zone), which include large blood vessels responsible for gas exchanges between mother and fetus, with elimination of fetal metabolites during gestation (Flynn et al. 2006). Besides that, placenta has immunological properties, protect the embryo/fetus against xenobiotics and mechanical traumas and release hormones and cytokines (Furukawa et al. 2014).

In the beginning of gestation placenta grows rapidly so that can promote adequate nutritional support contributing to fetus development; in humans, this growth is evidenced mainly in the first weeks with dynamic changes in its structure and function (Knöfler et al. 2019). Therefore, the weight of placenta is of paramount importance do determinate the organ functionality during gestation, its size is obtained from surface dimension (lateral and expansive growth of chorionic plate related with the area of maternal-fetal interaction) and thickness (related with vascularization) (Cardosoa et al. 2012). Any alteration in placental development leads to dysfunction of its structures and functionality, affecting the communication maternal-fetal, consequently impairing nutrition and growth fetal that can be related with chronical diseases such as hypertension and diabetes (Tarrade et al. 2013) and pregnancy complications such as miscarriage, stillbirth, pre-term labour, intrauterine growth restriction and preeclampsia (Knöfler et al. 2019).

Normal and abnormal morphology of placenta can be evaluated with quantitative methods measuring tridimensional data from two-dimensional structures (e.g. size, length, thickness, surface area and volume). Stereology is one of those quantitative techniques that uses specimen fragments to determinate the size of whole organ (Gundersen et al. 1999, Heidari et al. 2015) and this method has been used to estimate surface area, density and absolute volumes of the placenta in some species, such as human (Heidari et al. 2015), rat (Šerman et al. 2015) and cow (Adeyinka et al. 2016), however few reports have been carried out in rabbit’s placenta. As elucidated, rabbits are suitable animal models for reproductive researches and with similarity with human species, the aim of this researcher was evaluated the modifications of placental tissues of New Zealand White rabbits (Oryctolagus cuniculus) using stereological analysis.

MATERIALS AND METHODS

Samples collection

This study was approved by the Ethical Committee of the Faculty of Animal Science and Food Engineering from the University of São Paulo (USP), Pirassununga Campus– SP (N° 13.1.1910.74.9). Placentas of 10 New Zealand rabbits were used in different phases of the gestational period: 12, 14, 18 and 20 days. The gestational age was estimated by Crown-Rump length and by the external fetal characteristics, as described for the species (Evans & Sack 1973).

Placentas were fixed in paraformaldehyde 4% (PFA 4%) for 48 hours. The samples were then stored in ethanol 70% until stereological analysis.

Quantitative study

Placenta sampling

The placental lobes were chosen randomly. Thus, the same probability of choice between right and left lobe was maintained.

Stereological analysis

Placentas were weighed (g) and the total volume of placenta (Vref) was calculated dividing the weight by the density (1.05g/cm³) (Mayhew 2006). They were then sectioned, perpendicular to the horizontal plane to generate vertical sections, approximately 1.5 mm thick. These sections were sampled following the systematic uniform randomly (Sur) sampling (Gundersen et al. 1999), resulting in a total of 8 to 12 sections. From these sections were generated 15 to 20 histological sections SUR sampled and used to estimate the volume fraction and the total volume of each placental zones.

Histological procedures

The samples were fixed in paraformaldehyde (PFA) 4% for 48 hours and histological standard procedures were done to embedded the samples in paraffin. The paraffin blocks were exhaustively cut into 5µm sections and stained with hematoxylin and eosin (H&E). The slides were scanned by Pannoramic Scan System (3D Histec) to quantitative and qualitative placental zones analysis.

Volume fractions of the placental zones (Vv)

Volume fractions of the placental zones were estimated by point-counting method. The point spacing was 0.4mm. The points hitting the labyrinth zone were counted and divided by the total points hitting the entire placenta: Vv (labyrinth)= ∑p (labyrinth)/ ∑p (placenta). The same procedure was done to decidua and junctional zones: Vv (junctional zone)= ∑p (junctional zone)/ ∑p (placenta) and Vv (decidua)= ∑p (decidua)/ ∑p (placenta).

Total volume of the placental zones (V)

The total volume of each zone was estimated multiplying the volume fraction of each zone by the total volume of placenta (Vref): V (labyrinth)= Vv (labyrinth) x Vref; V (junctional zone)= Vv (junctional zone) x Vref; V (decidua)= Vv (decidua) x Vref. The quantitative results were shown as mean value followed by the coefficient of variation (CV).

RESULTS

Qualitative study

In the macroscopic analysis, the New Zealand rabbit placenta was characterized as discoidal and bilobular, maintaining the morphology during the all the gestational period analyzed (Figure 1a). In addition, the placenta of this species is characterized, in terms of maternal-fetal interaction, in hemodicorial, choroalanoid and labyrinth, allowing the nutrient exchanges.

Figure 1
a) Fetal and maternal portion of discoidal and bilobular placenta of New Zealand rabbit on the 16^th day of gestation (Bar scale: 1 cm). b) Photomicrography of the placental compartments (labyrinth, junctional and decidua zones on the 16^th day; scale bar: 1mm). c) Trophoblastic cells of the labyrinth (Lab) and spongiotrophoblasts (asterisk) of the junctional zone (JZ) (20^th day; Scale bar: 500 μm). d) Trophoblasts (black arrow) in contact with maternal blood (yellow arrow, 20^th day; Scale bar: 50 μm). Dec: decidua; JZ: junctional zone; Lab: labyrinth. Hematoxylin & Eosin staining.

The placental disc was characterized histologically by the labyrinth zone composed by the trophoblastic cells, separating the maternal-fetal circulations, distinguishing itself from the rest of the cellular components due to the large size of the nucleus and the globose format (Figure 1c, d). The junctional zone, composed of spongiotrophoblasts and glycogen-rich cells, was visualized between the labyrinth and decidua (Figure 1b, c). The decidua was characterized by the presence of deciduous cells, originating from stromal cells of the endometrium, and maternal arteries in close contact with the endometrium (Figure 1b).

Total volume of placenta (Vref)

The mean total volume of the placenta (fetal and maternal portions) presented a gradual increase according to the development of gestation: 12 days (320 mm³) 14 days (340 mm³), 18 days (940 mm³) and 20 days (1300 mm³) (Table I; Figure 2).

Figure 2
Total volume of the rabbit placenta (mm³) from the 12 days to the 20 days of gestation.

Table I
Mean value of total placental volume and placental compartments (mm³) followed by coefficient of variation (CV) for 12 to 20 days of gestation in New Zealand rabbits.

Days of Gestation	Placenta Total volume	Labyrinth volume	Junctional volume	Decidua volume
12	320 (0,14)	56 (0,30)	75 (0,17)	174 (0,16)
14	340 (0,42)	119 (0,48)	76 (0,14)	143 (0,51)
18	940 (0,07)	231 (0,06)	238 (0,05)	469 (0,01)
20	1300 (0,07)	481 (0,15)	314 (0,22)	504 (0,18)

Total volume of placental compartments

The total volume of labyrinth ranged from 320 mm³ on the 12th day of gestation to 1300 mm³ on the 20th day of gestation. The total volume of the junctional zone ranged from 75 mm³ at 12th to 314 mm³ at the 20th day of gestation and the total volume of the decidua ranged from 174 mm³ on the 12th day to 504 mm³ at the 20th day of gestation (Figure 3).

Figure 3
Total volume of the rabbit placental zones from day 12 to day 20 of gestation.

DISCUSSION

The rabbits present a placenta with hemodichorial structure, presenting two layers of chorion between the maternal and fetal blood, similar to the structure of human placenta comparing to other rodents that present hemotrichorial placenta. Furthermore, the rabbit’s placenta is more similar about the hemodynamic changes and development of diseases during placentation, comparable to the humans (Skoda et al. 2017).

In this study the total volume of New Zealand rabbit placenta was evaluated, as well as the volume of the placental compartments, using the stereology technique, which estimates the three-dimensional structure of an organ from two-dimensional data obtained from smaller fragments of this tissue, improving the interpretation of results regarding the growth, morphogenesis and functionality of an organ as a whole (Shemer et al. 2012).

In general, there was an increase in the total volume of the placenta and the placental compartments (labyrinth, junctional zone/JZ and decidua) during the 20 days of gestation analyzed, mainly between the 14th and the 18th days. In rodent species, such as Necromys lasiurus, in which gestation lasts on average 23 days, there was an increase in the total volume of placenta and placental compartments from the beginning to the middle of the gestational period (10 to 16 days), followed by a near-birth reduction (Favaron et al. 2013); similar results were also described for mice (Coan et al. 2004) and rat (Šerman et al. 2015); while in humans, the placenta shows continuous growth throughout the gestation (Higgins et al. 2011).

In the labyrinth zone, composed of two layers of trophoblasts (syncytiotrophoblast and cytotrophoblast) separating the space of maternal and fetal blood, the duplication of trophoblast layer was observed in all the phases of gestation analyzed, possibly due to a greater requirement of blood and nutrient supply during fetal growth; thus, proliferation and trophoblastic differentiation contributes to the diffusion of O₂ into the labyrinth space (Mayhew 2006). In humans, the fetal part of the placenta (chorionic villi) represents the greater part of the placental compartment (Heidari et al. 2015). In mice, the volume of the labyrinth zone was similar to the junctional zone during late gestation in this species (Coan et al. 2004). Similar data were found in this study in the evaluation of rabbit placenta, which on the 18^th day of pregnancy presented the volume of the labyrinth zone similar to that of JZ; presenting an increase of the labyrinth, in relation to the JZ, in the 20^th day of development.

The junctional zone is located at the maternal-fetal interface and is composed of different cell types: spongiotrophoblasts, glycogen rich cells and giant trophoblastic cells (Furukawa et al. 2014); these cells originate from trophoblasts on the 10^th day of development of the rabbit placenta, but are not present in the human placenta (Fischer et al. 2012); in mice, giant trophoblastic cells are responsible for the invasion of the blastocyst in the maternal uterus, as well as modulate the biological activity of hormones and growth factors (Coan et al. 2004). In this study, the volume of the JZ was increased during gestational days, mainly between the 14^th and 18^th days, but less significant in relation to the labyrinth and decidua zone.

The decidua originates from stromal cells of the endometrium, which is composed of endometrial glands that synthesize and secrete substances essential for embryo/ fetal development (Santos et al. 2012). In this study, we observed a slight reduction in decidua volume on the 14^th day, which increased again in the subsequent days.

Although the stereological study provides quantitative information on a structure and functionality of tissues and organs, other methods can also be applied for the better comprehension about cellular interactions between placental compartments, which directly influence fetal development and growth.

CONCLUSIONS

The three-dimensional quantification, based on stereological methods, allowed to estimate the volume of the placental compartments during the 20 days of gestation in New Zealand rabbits, allowing a better understanding of the modifications that occur during the development of the placenta in this specie, which interfere directly in maternal-fetal exchanges and, consequently, on the growth of the fetus, serving as a subsidy for understanding the functioning of placentation in humans.

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Publication Dates

• Publication in this collection
  26 Feb 2021
• Date of issue
  2021

History

• Received
  3 Mar 2019
• Accepted
  17 June 2019