Bovine placentome development during early pregnancy

M. B. Aires1, K. Y. Degaki2, V. Dantzer3 and A. T. Yamada2 1 Department of Morphology, Federal University of Sergipe, Sao Cristovao 49100-000, SE, Brazil 2 Laboratory of Development and Reproductive Biology, Institute of Biology, PO Box 6109, University of Campinas,

Campinas 13083-970, SP, Brazil 3 Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, Copenhagen

University, Dyrlægevej 16, 1870 Frederiksberg C, Copenhagen, Denmark

Maternal side of bovine placentome develops from restricted areas of the endometrium known as caruncle (CAR) in response to embryo implantation. CAR mucosa grows in close association with cotyledon, establishing a site of maternal-fetal exchange which systematically modulates this interface during pregnancy. The present study established time-course histological standards in the transition from caruncle to functional placentome and respective correspondence with embryo developmental age. Four well characterized morphological stages (S1-S4), based on CAR endometrial response, were distinguished during early bovine placentation. CAR growth was evidenced by increased epithelial and subepithelial stratum area detected at S2, which continued during S3 and S4. Anti-PCNA immunostaining confirmed intense proliferative activity of CAR epithelial and stromal cells from S1 to S4, contributing to the growth of CAR projections. The continuous growth and development of CAR as a result of cell proliferation and unique endometrial tissue remodeling, increases the surface contact area available for nutrient exchange and mechanical anchorage to effectively support embryo implantation and synepitheliochorial placentation.

Keywords: placentome development; caruncle; embryo; synepitheliochorial placentation

1. Introduction

The bovine embryo undergoes a prolonged implantation process, beginning with apposition of the trophoblast onto the uterine epithelial surface around the 19th gestational day (gd) followed by tenuous adhesion, attachment and interaction between the trophoblastic and uterine epithelial cells by gd 30 [1, 2, 3, 4]. Accordingly, successful bovine synepitheliochorial placentation depends on primary recognition between the trophoblast cells of the allantochorion and the uterine luminal epithelial cells developing the unique heterotypic syncytia at the maternal-fetal interface [5], without trophoblast invasion into the stroma of the caruncle (CAR) or intercaruncle (IC) endometrium [6]. Anchorage of the conceptus in the uterus depends on caruncle endometrial response. There are multiple CAR throughout the bovine endometrium, slightly protruding into the lumen, that together with cotyledons develop into about 100 maternal-fetal placental units called placentomes [7]. The placentome is the structural and functional unit that mediates maternal/fetal gaseous, nutrient and metabolic waste exchanges of the ruminant placenta [8, 9, 10], and demands extensive local tissue remodeling including vascular remodeling for adequate blood supply [11, 12]. Therefore, involvement of the focal and unique response of the CAR mucosa in placentome development is crucial for successful cotyledonary synepitheliochorial placentation [5, 6, 10]. However, the vast majority of knowledge on cow placentome physiopathology is based on histological or biochemical studies using later gestational stage or adopting large intervals of gestational periods [8, 13, 14, 15, 16, 17]. Moreover, despite the importance of placentome formation for embryo implantation, information reviewing the stages of CAR growth and remodeling during this process is so far lacking. Indeed, current cattle breeding technology obtains fertilization rates that can reach up to 90% from successful insemination, with an average calving rate of about 55%, indicating an embryonic-fetal mortality of about 35% [18]. Reasons for most pregnancy losses (70 - 80%) during the first 3 weeks after insemination in the cow [19] and around 13% during 4th to 7th weeks of gestation [20] are largely unknown. Here, we aim to establish time-course histological standards in the transition from CAR to functional placentome and respective correspondence with cow embryo developmental age. Additionally, placentome histoarchitecture as an efficient conceptus anchorage point was reviewed. Morphological evidences suggest a weak interaction between the trophoblast and uterine epithelium at the maternal-fetal interface of synepitheliochorial placentation before placentome formation.

2. Study design

2.1 Uterine samples of artificially inseminated pregnant cows

Paraffin sections from artificially inseminated cow uteri on gestational days (gd) 20, 23, 26, 28, 29, 30, 32, 37 and 40 were processed according to previous study of Pfarrer et al. (2006) [12] and kindly donated by Dr. Henrik Callesen (Aarhus University). The CAR and IC regions were analyzed under light microscopy to establish primer histological

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references for early pregnant endometrial changes. These histological references were used in the prospective study of unknown gestational day (gd) pregnant uteri to check and establish time-course histological standards and respective embryo developmental stage based on crown-rump length (CRL).

2.2 Uterine samples of unknown gestational day pregnant cows for histological prospective study

Cow (Bos sp.) fresh uteri obtained at the slaughterhouse were dissected to access the lumen from anti-mesometrial side and examined macroscopically for the presence of embryos and fetuses. The CAR areas were easily recognized macroscopically as round protrusions over the uterine surface compared with the flat IC surfaces. When present, embryos and fetuses were removed for CRL evaluation [21] and the mesometrial side of respective endometrium containing CAR and IC tissues was carefully collected to keep the chorionic membrane attached to the uterine surface. Eight non-pregnant uteri corresponding to the luteal phase (LP) of the estrous cycle and 38 pregnant uteri were further processed for the prospective study. All the animal slaughter was in accordance with humane slaughter methods of the Brazilian Ministry of Agriculture, Livestock and Food Supply. Uterine fragments were immersed and fixed in 4% paraformaldehyde and 0.1M phosphate-buffered saline, pH 7.4 for 24 h at 4oC and processed for conventional paraffin (Histosec, Merck) embedding. Serial 5 μm thick paraffin sections collected onto poly-L-lysine coated glass slides were stained with hematoxilin-eosin (HE) for histological analysis under light microscopy (Eclipse 800, Nikon) and digital images collected with Cool Snap (Nikon) digital camera and Image ProPlus software (Media Cybernetics). The cross-analysis of unknown gd pregnant uteri with artificially inseminated cows, established four time-course histological standards (S1, S2, S3 and S4) of CAR endometrial remodeling, with respective ranges of embryo CRL and estimated gestational days.

2.3 Caruncle growth and remodeling

Three representative samples from each previously established endometrial stages (S1-4) and LP uteri were used to evaluate the proliferation rate of CAR and IC endometrial cells by anti-PCNA (proliferating cell nuclear antigen) immunoperoxidase staining. Briefly, deparaffinized sections were treated with 3% H2O2 in methanol followed by 5% defatted milk in 0.1M PBS pH 7.4. Overnight incubation with rabbit polyclonal anti-PCNA antibody (1 mg/mL, Bethyl Laboratories) diluted 1:300 in 0.1M PBS followed by incubation with biotinylated goat anti-rabbit antibody (1 mg/mL, Dako) secondary antibody diluted 1:600 in 0.1M PBS and then treated with with the LSAB+ system (Dako). After revealed with diaminobenzidine tetrahydrochoride (Sigma) and hydrogen peroxide, the sections were counterstained with methyl green (Merck) pH 4.4, dehydrated and permanent mounted with Entelan resin (Merck). Negative controls were performed by incubation with normal rabbit serum (Dako, USA). PCNA-positive cells were identified as those presenting nuclear positive immunoreactivity. The number of positive cells was counted considering 1000 nuclei using 40x objective distinguishing epithelium (E) and subepithelial stratum (SS) cells for CAR. Three independent counting were performed in non-serial sections of LP and each of S1-S4 stages. The growth rate of CAR area during the implantation stage was quantified by morphometry using HE-stained, sagittal sections from CAR tissues of LP (n=3) and S1-S4 stages (n=3). The entire E and SS regions were digitalized using light microscope previously calibrated with 20X objective and set up with digital camera using ImageProPlus software. The images were merged using Adobe Photoshop to create a single entire image of each CAR section and the total area corresponding to the E and SS was measured with ImageProPlus software. The results were expressed as square millimeter (mm2).

2.4 Placentome histoarchitecture reconstruction

The fully developed placentome histoarchitecutre were revised to better understand the efficiency of chorionic membrane anchorage in the synepitheliochorial placentation. Serial sections of placentomes at S4 stage (two placentomes from 3 different animals) were processed for conventional periodic acid-Schiff (PAS) stain and analyzed under fluorescence microscopy [22]. PAS fluorescence well distinguishes the bright-red fluorescent CAR villi loose connective tissue content from those dark embryonic chorion mesenchyme. The digitalized sequential images were evaluated to analyze the spatial arrangements of bovine placentome.

2.5 Statistical analysis

All data are expressed as means and standard errors of means (SEM). Comparisons between LP endometrium and each of S1-S4 stages were performed by on way ANOVA followed by Tukey test. A value of p < 0.05 was considered significant.

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3. Results

3.1 Time-course stages of caruncle (CAR) and intercaruncle (IC) histological changes

Histological analysis of CAR and IC samples of thirty-eight unknown gd pregnant uteri and nine artificially inseminated cow uteri (gd 20, 23, 26, 28, 29, 30, 32, 37 and 40) showed time-course continuous and extensive endometrial changes that affected the CAR and IC regions differently. Taking the non-pregnant CAR and IC endometrium histological features as a reference, the dynamics of CAR remodeling (Fig. 1) was arbitrated into four sequential stages (S1, S2, S3 and S4) and respective correspondence to estimate gd was based on the crown-rump length (CRL) (Table 1). Table 1 Caruncle developmental stages (S1-S4) and corresponding CRL based gestational day (gd) intervals. N – number of animals, CRL – Crown-rump-length (cm).

Stages CRL(cm) gd N S1 0.5 - 0.7 20-26 8 S2 1.0 - 1.3 28-33 10 S3 1.5 - 2.5 35-40 10 S4 5.0 - 8.5 50-60 8

The histology of non-pregnant cow endometrium showed easily distinguished CAR and IC regions (Fig. 1A-C). A similar high columnar epithelial cell sheet was covering both regions. The CAR stroma (Fig. 1A, B) was arranged into three layers; a thin, less cellular sub-epithelial stratum (SS), a thick highly cellular stratum (CS) of loose connective tissue, and a deep vascular stratum (VS) of dense connective tissue containing large and thick walled arterial and venous blood vessels, while the IC stroma was restricted to two layers, lacks the cellular stratum (CS) and contains tubular glands opened to the IC surface (Fig. 1C). The earliest embryo with CRL 0.5 cm showed close apposition and attachment of chorionic membrane on the uterine mucosa. Spots of mono- and bi-nucleated trophoblast cells attached and fused with epithelial cells forming a continuous sheet over the CAR and IC surface, supported by connective stroma similar to those found in non-pregnant endometrium (Fig. 1D-F). These CAR and IC histological characteristics were seen consistently until reaching an embryo size of CRL 0.7 cm, corresponding to gd 20 - 26 (Table 1). These histological parameters were arbitrated as stage 1 (S1). Embryo CRL ranging from 1.0 to 1.3 cm, corresponding to gd 28-33, were arbitrated as stage 2 (S2) (Table 1). Several small and regular endometrial projections were seen exclusively on the CAR surface that gradually increased in size as finger-like villous projections in to the lumen (Fig. 1G - I). Cuboidal epithelial cells with an axis of SS loose connective tissue (Fig. 1H) cover these CAR projections, and deep in the endometrium a large area of CS stroma is dominant in the CAR region. The IC region did not change significantly and similar trophoblast-uterine epithelial cells cover the IC surface, except at the glandular opening site (Fig. 1I). Interestingly, extensive embryo chorionic membrane detached from both CAR (Fig. 1H) and IC (Fig. 1I insert) by disruption of the trophoblast-epithelial interface. This suggests the fragility of the cell-cell interface of synepitheliochorial placenta for mechanical support during the early implantation stage. Stage 3 (S3) related to embryo CRL ranging from 1.5 to 2.5 cm and corresponded with estimated gd 35-40 (Table 1). Compared with S2, the CAR projections showed increased width and length (Fig. 1J) and developed lateral branches that gradually anastomosed with neighboring projections (Fig. 1J, K). The stroma of CAR projection axis was similar to those seen in early SS connective tissue and the large CS region showed rows of dense connective tissue containing thick walled blood vessels that continued into the deeper vascular stratum of dense connective tissue (Fig. 1J). Chorionic membrane deeply intruded into these CAR projections with close interaction of trophoblast cells and uterine epithelial cells seen throughout the entire CAR (Fig. 1K). In contrast to S2, no chorion-epithelial cell disruption or discontinuities were observed at the CAR surface and were matched to gd 37 - 40 of artificially inseminated samples. The more complex CAR projections seem to better support chorion anchorage to sustain the synepitheliochorial placenta. In the IC regions, the trophoblast-uterine epithelium syncytia were restricted to some spots and the chorionic membrane freely floats in the uterine lumen (Fig. 1L). Further prospective analysis of unknown gd cow uterine samples elected eight embryos with CRL ranging from 5.0 to 8.5 cm corresponding with gd 50 - 60 as stage 4 (S4) to configure the early placentome structure. The CAR endometrial projection areas increased to a large net of thick trabecules not resembling the earlier (S2) finger-like villous projections (Fig. 1M, N). The roof of the CAR trabecules was mostly continuous with some open areas from where the chorionic membranes intrude into the CAR trabecular projections (Fig. 1M). Endometrial epithelial cells were in close attachment to mono- or bi-nucleate trophoblast cells at CAR projections. When compared with S2 and S3 stages, the CS area (Fig. 1M) in the CAR stroma was reduced and VS dense connective tissue with large blood vessels

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was close to the CAR projections. IC mucosa showed highly pseudostratified columnar epithelium covering the luminal surface without apposition or attachment of chorionic membranes (Fig. 1O).

Fig. 1 A-O Photomicrographs of HE stained sections of bovine caruncle (CAR) and intercaruncle (IC) endometrium from non-pregnant luteal phase (LP) (A-C) and pregnant S1 (D-F), S2 (G-I), S3 (J-L) and -S4 (M-O) stages. (A) LP-CAR region with high cellular stroma (CS) and large blood vessels in the vascular stratum (VS) flanked by IC region; (B) LP-CAR showing high columnar epithelium (E) apposed on loose connective tissue of the subepithelial stratum (SS) with blood vessel plexus (arrow heads) and the cellular stratrum (CS); (C) LP-IC with a tubular gland (arrow) in the stroma (ST); (D) S1-CAR showing the chorion (Ch) attached on CAR surface; (E, F) chorionic mono-(MT) and binuclear (BT) trophoblast tightly attached on or forming syncitium (insert) with the endometrial epithelia of CAR and IC regions; (G) small projections (arrow heads) on S2-CAR; (H) CAR finger-like projections covered by cuboidal epithelial (E) cell often disrupted (arrowhead). Chorion (Ch) detachment with epithelial cell disruption was also noted in the IC region (I); (J) increase of number and length of CAR projections (arrow heads); (K) lateral branches that anastomosed transversally (arrowhead) with neighbor villous projections and Ch intrude into the crypt-like space formed between

CAR projections; (L) IC region with detached Ch; (M) Typical placentome with complex arrangements of CAR projections; (N) anastomosis of CAR projections lateral branches at the apical portion (arrowheads); (O) IC region with detached Ch. Scale bars: (A)=5 mm, (D, G, J, M, I, L, O)= 300μm, (B, C, E, F, H) =30μm, (K, N) = 50μm.

3.2 Caruncle growth and remodeling

Development of the CAR endometrium projections encompassed uterine epithelial (E) and subepithelial stratum (SS) expansion. CAR growth owing to hyperplasia evaluated by PCNA immunostaining showed continuous presence of PCNA+ cells in the E and SS of non-pregnant (LP) and pregnant CAR endometrium (Fig. 2A). CAR epithelial cell proliferation gradually increased from S1 to S4 (S1: 315.1 ± 9.2, S2: 466.2 ± 7.0, S3: 477.4 ± 20.0, S4: 559.9 ± 10.0) compared with LP (69.4 ± 6.1), while the increased number of PCNA+ SS cells was restricted to S1 (LP: 230.0 ± 14.1, S1: 344.7 ± 7.0, S2: 233.3 ± 10.0, S3: 185.7 ± 7.0, S4: 176.1 ± 4.9) (Fig. 2B). The morphometry of CAR showed the growth of E and SS areas started at S2 (0.83 ± 0.04 mm2) and continued in S3 (1.13 ± 0.07 mm2) and S4 (5.57 ± 0.74 mm2). The CAR area at S4 was statistically different to that of LP (0.45 ± 0.12 mm2, *p<0.05), S1 (0.47 ± 0.04 mm2, *p<0.05) and S2 (0.83 ± 0.04 mm2, *p<0.05) (Fig. 2C).

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Fig.2 A. Photomicrograph of anti-PCNA immunoperoxidase reaction showing positive nuclei (dark brown) of epithelial (arrows heads) and subepithelial stratum (arrows) cells at CAR area in non-pregnant (LP) and pregnant (S1-S4) uteri. Scale bars: 30μm. B. Histogram showing PCNA positive cells in epithelial (E) and subepithelial stratum (SS) of pregnant (S1-S4) and non-pregnant (LP) endometrium. Values are SEM, n=3/stage,*p < 0.05. C. Histogram of CAR area showing the extensive growth of E and SS area at S4. Values are SEM, n=3/stage,*p < 0.05

3.3 CAR projections of S4 placentome

PAS stained fluorescence microscopy analysis of S4 placentome sections distinguished the bright red fluorescent connective tissue axis of CAR projections in contrast to dark non-fluorescent chorion membrane (Fig. 3A, B). The evaluation of serial sections showed that CAR projections form a trabeculae network with spaces where the chorion villi expanded. The trabecule wall parallel to the major axis of placentome projections was thicker compared with the secondary transversal projections that anastomose with neighboring secondary projections forming longitudinal trabecules. The embryo chorionic membrane deeply intrudes and form branches in the CAR trabecular labyrinth. Interestingly, the top portion of the placentome is covered by thick transversal trabecules with narrow openings in which the chorionic membranes intrude and then expand for tight anchorage.

Fig. 3 Illustrative images of bright field (A) and corresponding (B) fluorescence microscopy of PAS stained S4 placentome sections. Bright red fluorescence of extra-cellular matrix contents of caruncle subepithelial stroma (SS) in contrast to deep non fluorescence of chorion mesenchyme (Ch). The anastomosis of transversal braches in the apical portion of projections closed a large surface of CAR (arrowhead) limiting the chorion insertion in narrow foramens (arrow).

4. Discussion and conclusion

We systematically evaluated CAR and IC histological changes and distinguished four sequential time-course stages (S1-S4) considering the CAR area growth and cell proliferation in response to conceptus implantation. The classic model of placentome development describes the formation of villi (fetal component) and crypts (maternal component) at the CAR surface [9, 23, 24]. The process is described with fetal villi invading or pushing the CAR endometrium forming crypts where chorion membrane could grow. Nevertheless, according to our data, placentome development begins with the formation of CAR projections during S2 from gd 28 onward. At S1, both CAR and IC surface responses are similar and demonstrate an association between the continuous chorionic membrane and endometrial epithelium to form the maternal-fetal interface. The rupture of the chorion-uterine epithelial cell layer through the CAR and IC surface is consistently seen at S2, and coincides with the second highest period of pregnancy loss in cattle [20]. The tight chorion-uterine epithelia unit formed

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at this stage may only weakly support the quickly growing embryo mass and any mechanical stretching in the uterine environment could provoke rupture of this unit and, as consequence, cause implantation failure. The growth of CAR projections involves extensive hypertrophy of the endometrium [25] as evidenced by the increase of E and SS area detected at S2, which continued during S3 and increased considerably at S4. This is a consequence of high proliferative activity of both CAR luminal epithelial cells and stromal cells starting at S1 and should trigger the projection growth during S2 at the CAR surface. However, it is interesting to note the intensive proliferation of epithelial cells supporting CAR projections growth from S2 onward is not proportional to stromal cell proliferation. The less prominent proliferation indicated by PCNA expression on stromal cells predicts that the CAR stroma remodeling and hypertrophy is owed to the increased synthesis and secretion of extracellular matrix components by stromal cells rather than hyperplasia of these cells, as suggested by Kaidi et al. (1995) [26]. Increased collagen I, III, IV and laminin contents at the same locations of CAR villi during the first trimester of pregnancy provides the tensile strength resistance necessary to support mechanical stretching of chorion attachment onto the endometrial projections [16, 24, 27]. The formation of lateral branches of CAR projections and their anastomosis with neighboring projections results in an intricate trabeculae network starting at S3. The evaluation of CAR projections at S4 with PAS fluorescence confirmed several transverse plaques that anastomose to neighboring projections from their roots to apices. Furthermore, the CAR histoarchitecture at the last stage examined also showed the massive lateral growth of the apical portion of projections, restricting the chorion insertion area in narrow openings over the placentome surface. Similar findings were reported by Schmidt et al. (2006) [28] in buffalo, with dome-shaped placentomes exhibiting few apical holes where finger-like chorionic villi intruded deeply and expanded into them. Another interesting observation made during histological revision of pregnant cow endometrium, was the gradual increase of detached chorionic membrane from the IC epithelium as the placentome developed. After S4, the IC region did not show extensive chorion attachment to the epithelium, which was reorganized by high pseudostratified columnar cells. Tight attachment, adhesion and syncytial formation between chorionic trophoblast and epithelial cells through the CAR and IC surface during S1 and S2, are crucial and well-known phenomena for successful embryo implantation [5, 29]. Development of the CAR endometrium to form functional placentomes synchronized with embryo implantation is a complex biological process of tissue remodeling accomplished by well-orchestrated cellular proliferation and differentiation. Further efforts to understand the precise role of morphoregulatory genes could reveal new insights into the mechanistic participation of each cell population at the maternal-fetal interface during a successful pregnancy. Here, we have reviewed and established time-course morphological stages to easily recognize the CAR endometrial response during early bovine placentation. The continuous growth and development of CAR as a result of cell proliferation and unique endometrial tissue remodeling, increases the surface contact area available for nutrient exchange and mechanical anchorage to effectively support embryo implantation and synepitheliochorial placentation.

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