Abstract
In the present work, we emphasize the studies about integrins and their receptors in pig placental interface at different times of gestation. Uterine placental interface (n = 24) of 17, 30, 60 and 70 days of gestation (dg) and non-pregnant uterus (n = 4) of crossbred sows were used. The presence of αvβ3 (ITGAV (integrin subunit alpha V) ITGB3 (integrin subunit beta 3)) and α5β1 (ITGA5 (integrin subunit alpha V) ITGB3 (integrin subunit beta 3)) integrins, and their ligands fibronectin (FN) and osteopontin (OPN/ SPP1), was detected by immunohistochemistry, and the immunolabeled area percentage (IAP) and the optical density (OD) were determined. The integrins and its ligands analyzed have presented peaks of expression in early and mid-gestation, both in IAP and in the OD area, decreasing at 70 dg. These temporal changes showed us that the molecules studied in this work participate in embryo/feto–maternal attachment, variably. Besides, we found a significant correlation both in the intensity and in the extension of immunostaining for trophoblastic FN and endometrial αvβ3, and trophoblastic OPN and endometrial α5β1, throughout the entire pig pregnancy. At late gestation, there is notable placental remodeling with subsequent removal or renewal of folds at the uterine–placental interface that results in the loss of focal adhesions. The decrease of the expression of some integrins and their ligands in late gestation, particularly at 70 dg, would demonstrate that there would be other adhesion molecules and other ligands that could be participating in the establishment of the maternal–fetal interface.
Lay summary
To carry a successful pregnancy, the formation of the placenta in pigs depends on adhesion molecules. Some of these molecules called integrins bind to other molecules such as fibronectin (FN) and osteopontin (OPN/SPP1). The variation in those molecule amounts during gestation would indicate which molecule is participating and what role it plays in pregnancy. We worked with pig placentas of early, mid- and late- gestation and non-pregnant uteruses. αvβ3 (ITGAV (integrin subunit alpha V) ITGB3 (integrin subunit beta 3)) and α5β1 (ITGA5 (integrin subunit alpha 5) ITGB1 (integrin subunit beta 1)) integrins, FN and OPN were found until mid-gestation but not at late gestation, suggesting that other types of molecules have a role in the last period of gestation.
Introduction
In mammals, during gestation, the endometrial luminal epithelium undergoes a series of morphological changes. In pig gestation, there is an elevated rate of reabsorption/loss of the embryos/fetuses, and this is traduced in economic loss in farmers and industry (Bosch 2001, Murphy et al. 2009, Kridli et al. 2016). Some of those authors reported that this reabsorption rate could have a relation with the molecule’s dialogue that takes place at the feto–maternal interface. The endometrial changes allow receptivity to the trophoblast and are related to adhesion molecule modifications on both epithelia. Of these adhesion molecules, there are different integrins and their ligands (Reddy & Mangale 2003, Murphy 2004, Zeiler et al. 2007, Bazer et al. 2009, Srinivasan et al. 2009, Bazer et al. 2012, Jalali et al. 2018, Schäfer-Somi et al. 2020).
Integrins are transmembrane glycoproteins that form dimers and bind to different ligands (Rashev et al. 2005). The main ligands of the integrins include a large number of extracellular matrix (ECM) proteins, such as osteopontin (OPN/ SPP1), collagens, laminins, and fibronectin (FN), among others (Plow et al. 2000). Interestingly, on certain occasions such as implantation and placentation, integrins can not only be expressed in the basal region but also in the apical region, and their ligands, usually located in the ECM, can be secreted into the apical glycoclix. This allows the attachment of the epithelial surfaces that integrate the feto–maternal interface (Rout et al. 2004), the endometrial luminal epithelium and trophoblast. In mammals, integrins represent excellent markers of normal and abnormal stages of uterine receptivity and may be the key in the mechanisms of implantation and regulation of pregnancy (Sutherland et al. 1993, MacLaren & Wildeman 1995, Elnaggar et al. 2017).
In pigs, the role of integrins and their ECM ligands in the period of peri-implantation in early pregnancy (10–15 days of gestation (dg)) has been studied in numerous works (Bowen et al. 1996, Burghardt et al. 1997, Jaeger et al. 2001, García et al. 2004, Rashev et al. 2005, Bowen & Hunt 2008, Bazer et al. 2012, Jalali et al. 2018). The integrins αvβ3 (ITGAV (integrin subunit alpha V ITGB3 (integrin subunit beta 3)) and α5β1 (ITGA5 (integrin subunit alpha 5 ITGB1 (integrin subunit beta 1)) have as ligands the FN and the OPN. These proteins have been studied in early gestation in pigs (Bowen et al. 1996, Tuo & Bazer 1996, García et al. 2004, Rashev et al. 2005, Erikson et al. 2009, Williamson & Koncurat 2009). The OPN, secreted by uterine epithelium and immune cells, may interact with receptors (i.e. integrins) expressed on the conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig (Garlow et al. 2002, White et al. 2005, Johnson et al. 2014).
About late gestation in pigs, we previously determined, using a qualitative evaluation, the expression of integrin αvβ3 (ITGB3) and FN at the fetal–maternal interface during pig placentation; then, we found that this expression was related to the levels of estrogens and progesterone (Velez et al. 2018) and with the expression of the α5β1 (ITGB1) integrin and the OPN (Velez et al. 2019). In the present work, we emphasize the studies about the interaction between the molecules that participate in the development of pig placental interface at different times of gestation to observe which integrin/ligand would participate in the molecular events that allow successful implantation and placentation using morphometric techniques. Thus, for the first time, we determine the optical density (OD) and the immunostaining area percentage (IAP) of the αvβ3 integrin and the α5β1 integrin and its ligands, FN and OPN, in the maternal–fetal interface of pig placenta in different gestation periods. Then, we performed a correlation analysis between the expression of these integrins and their ligands in the endometrial epithelium and trophoblast to observe the relationship between these receptors and their ligands during pig gestation.
Materials and methods
Animals
We used uteri of 24 crossbred sows (Landrace × Large White) between their second and third births. Specimens were divided into five groups according to stages of development: 17 days (n = 4, middle implantation windows, approximately) (reviewed inBazer et al. 2012, Geisert & Spencer 2021), 30 days (n = 4, onset of ossification and immune system development; the uterine–placental interface starts folding) (Butler et al. 2009, Seo et al. 2020, Geisert & Spencer 2021), 60 days (n = 4, exponential growth of placenta completed) (Wooding & Burton 2008) and 70 days (n = 4, placenta development plateaued and fetus growing exponentially) (Cristofolini et al. 2013). Additionally, we used the uteri of non-pregnant sows (NP; n = 4). The organs were obtained from slaughterhouses near General Pico, La Pampa, Argentina. The sows were healthy as certified by the assessment of medical records provided by the farm owner. Additionally, the gestation data of each sow was reported to us. Animals were sacrificed according to the animal welfare manual of the National Agrifood Health and Quality Service (known in Spanish as SENASA) (SENASA 2015).
Sample collection
Placental samples were obtained as follows. Each uterine horn was analyzed along the greater curvature, and approximately 8 g of maternal and fetal porcine placental tissue was extracted from each implantation site. At 17 days of gestation, fetal placental samples were taken by palpating the endometrial mucosal surface, each filament detached easily and was immediately immersed in formol 40% to be fixed. Samples of the uterus were also taken from the same area where the filament was removed. In pregnant sows of 30, 60 and 70 dg, the uterine horns were palpated to detect the location of the embryos or fetuses. Samples were obtained from the feto–maternal interface and were immediately placed in 10% buffered formalin for subsequent histological treatment (determination of integrins and their ligands by immunohistochemistry). Non-pregnant sow uteri were also used as a control. The samples were taken from the greater curvature of both the left horn and the right horn.
Histology
The reproductive tracts were washed promptly with Hanks' saline solution, 10,000 U/mL penicillin, 10 mg/mL streptomycin and 2.5 μg/mL fungizone and stored at 4°C until processing in the laboratory. To maintain tissue integrity at the uterine–placental tissue, the fixation was immediately performed. Briefly, placental 1 cm3 tissue samples, both maternal and fetal, were excised and fixed in 10% phosphate-buffered formalin for 20 h. The samples were stored in 70% ethanol. Subsequently, the tissues were dehydrated using ascending concentrations of ethanol from 70 to 100% and then embedded in paraffin. Sections were cut at 5 μm and mounted on positively charged slides (Genex-brand®, Genex Diagnostics SRL, Buenos Aires, Argentina). The age of the concepti was determined by the crown-rump length of the embryos/fetuses according to Marrable´s table (Marrable 1971). All experimental procedures were revised and approved by a Scientific Committee of Faculty of Veterinary Science of the National University of La Pampa (research code: Res CD 311/2017).
αvβ3 integrin, α5β1 integrin, FN and OPN determination – labeled streptavidin biotin (LSAB) method
After 20 min at 60°C in the oven, the slices were deparaffinized and rehydrated according to standard procedures. Briefly, sections were incubated three times in xylene 100% for 10 min, two times in alcohol at 100°C for 10 min, two times in alcohol at 96°C for 10 min and one time in alcohol at 70°C for 5 min. After washing with phosphate-buffered saline (PBS) three times, we proceeded with the LSAB method. To inhibit the endogenous peroxidase activity, the sections were incubated with hydrogen peroxide 5% for 20 min at room temperature and then were rinsed in PBS two times for 5 min. After this, the sections were heated in citrate buffer solution (pH = 6), for 12 min in a microwave oven and then rinsed again in PBS. The sections were treated with avidin–biotin blocking reagent (Cell Marque, Rocklin, CA, USA) to block biotin endogenous and then washed in PBS. Next, the sections were treated with bovine serum albumin 3% for 15 min at room temperature to block non-specific binding. Subsequently, sections were treated with primary antibodies against αvβ3 integrin, α5β1 integrin, FN and OPN (Table 1) overnight at 4°C. The sections were washed three times with PBS for 5 min and incubated for 20 min at room temperature with biotinylated secondary IgG antibody (K0690; biotinylated link universal (Dako Cytomation, Denmark); this last procedure was not performed for αvβ3 determination as it is a biotinylated primary antibody), washed three times with PBS for 5 min followed by treatment with peroxidase-labeled streptavidin (streptavidin/HRP, Dako Cytomation) for 20 min at room temperature. After being washed three times with PBS for 5 min, the peroxidase activity was developed with a substrate-chromogen system (DAB (3,3′-diaminobenzidine tetrahydrochloride)chromogen and substrate buffer, Dako Cytomation) for 15 min. Subsequently, the sections were rinsed with water followed by hematoxylin staining (Biopur, Rosario, Argentina) for 10 s. The slides were washed with tap water (10 min) and were dehydrated. The samples were mounted and observed under an Axiophot light microscope (Zeiss), and the images were captured by digital camera Canon, PowerShot G6 (Canon, Tokyo, Japan). As negative controls, the primary antibodies were replaced and treated with isotype control antibody. The OD and immunolabeled area percentage (IAP) were quantified by using the Image J software (Ferreira & Rasband 2012).
Details of the antibodies used in immunohistochemistry of porcine placenta.
| Antibody | Working dilution/ concentration | Manufacturer |
|---|---|---|
| Monoclonal mouse anti-integrin αvβ3 antibody conjugated with biotin | 1:500 | MAB1976B; Chemicon |
| Monoclonal mouse anti-integrin α5β1 antibody | 1:1500 | MAB1969; Chemicon |
| Polyclonal rabbit anti-fibronectin porcine antibody | 1:200 | ab23751; Abcam |
| Polyclonal rabbit anti-osteopontin porcine antibody | 1:1500 | ab8448; Abcam |
| Biotinylated secondary IgG antibody (biotinylated link universal) | Ready to use | K0690; Dako Cytomation |
IgG, immunoglobulin G; αvβ3, integrin alpha v beta 3; α5β1, integrin alpha 5 beta 1.
Analysis of the results
Qualitative description of the expression of the molecules
A qualitative description of the observed immunostaining of the molecules was performed in the epithelia of the fetal–maternal interface in the different gestation periods studied in the present work.
Quantification of the expression of α5β1 integrin, αvβ3 integrin, FN and OPN
In each microscopic field, taken with a 40× objective, the different morphometric determinations of each structure were performed: endometrial luminal epithelium and trophoblast. Samples immunostained with the previously mentioned antibodies were used. All slides were viewed under a Carl Zeiss light microscope and photographed with a 7.1-megapixel Canon PowerShot G6 camera (Canon) attached to the microscope. The images were processed with the Axiovision software (AxioVision 4.8, Carl Zeiss) and saved in TIFF format. Then, the OD and the IAP of the analyzed molecules were determined. The images were studied with commercial software for digital image analysis based on segmentation processes (ImageJ) (Ferreira & Rasband 2012, http://rsb.info.nih.gov/ij). To analyze the percentage of area that presented immunostaining (IAP), the area to be analyzed was delimited and the ratio between the specifically marked area and the total area of the epithelium of each image was determined. Color separation was performed on each image using the ‘color deconvolution’ tool, which was selected to separate the original image into three monochrome images (red–green–blue: RGB). Of the RGB images, the red images were selected to quantify DAB development. With these 8-bit DAB images, the area to be analyzed was delimited. Subsequently, the segmentation of the image to be quantified was carried out using the ‘threshold’ tool (threshold) to separate the pixels darker than the threshold value.
To determine the OD of the expression of the integrins and the ligands, we proceeded to delimit only the immunolabeled area on each structure to be evaluated from the same 8-bit DAB image. Then with the ‘Analyze/Measure’ tool, we obtained the measurement results of the delimited area (in pixels), the average brown intensity level (mean), minimum brown intensity (minimum), and the maximum brown intensity (maximum). To calculate the OD, the following formula was used: OD = log (maximum/mean), where max = 255 for an image of 8-bit. The results of each structure, of each gestation period, were expressed as the mean ± s.d. of the data obtained from 20 photos analyzed. Also, the median was determined in each group.
Statistical analysis
Statistical analysis was performed using Infostat software (Di Rienzo et al. 2010). ANOVA and Tukey tests were used. Significant differences were defined as those with P < 0.05.
From the repetitions of the OD and IAP values of the molecules measured in the endometrium and the trophoblast of pregnant sows at different gestational stages, the mean and median for each molecule in each tissue were calculated. The Spearman correlation (PROC Corr, SAS 9.4) was estimated between the endometrial and trophoblastic molecules studied (α5β1, αvβ3, FN and OPN). Their expression was analyzed in the epithelia that compose the fetal–maternal interface in pig gestation, both in intensity immunostaining (OD) and in immunostaining extension (IAP). The statistical significance value for the correlation coefficient was defined as 0.05.
Results
Determination of integrin and their ligands expression using image quantification: OD and the IAP at the placental interface
αvβ3 integrin
αvβ3 integrin was analyzed in the endometrial luminal epithelium and feto–maternal interface from non-pregnant and pregnant pigs, respectively (Fig. 1; Table 2). The integrin immunostaining was different according to each gestation period and tissue analyzed. OD values are given in detail in Figs 1K and L, and the staining can be observed in Fig. 1A, B, C, D, E, F, G, H and I images. No immunostaining for αvβ3 was observed in the non-pregnant uterus (Fig. 1A; OD: 0.03; NP). Concerning gestation, two immunostaining peaks of αvβ3 (strong cytoplasmic expression) at 17 dg and 60 dg were observed in the endometrial epithelium (OD: 0.39 and OD: 0.6, respectively). In the trophoblast (Fig. 1L; OD: 0.35) at 60 dg, a strong expression of the αvβ3 integrin was observed (OD: 0.49; Fig. 1G). The immunostaining was expressed continuously in the medial–apical portion of the trophoblast cells. At 70 dg, the expression of the αvβ3 integrin decreased significantly in the endometrial cells (Fig. 1H; OD: 0.23; Fig. 1N; P < 0.0001). In this period, the immunostaining of the αvβ3 integrin showed us a slight patchy pattern in the apical membrane of the trophoblast cells (Fig. 1I; OD: 0.24; Fig. 1N). The percentage of immunolabeled area analysis of αvβ3 integrin during pig gestation is in detail in Figs 1M and N. The greatest expression area of αvβ3 integrin in the endometrial epithelium at 60 dg was observed (Fig. 1M). However, the αvβ3 integrin in the trophoblast expanded more cell area in early/mid-gestation than in late gestation (Fig. 1N).
αvβ3 integrin expression at different development stages (17, 30, 60 and 70 dg) and non-pregnant sow (NP) uteri. (A) Representative images of αvβ3 immunohistochemical staining in the non-pregnant uterus (A) and placental tissue at 17 (B, C), 30 (D, E), 60 (F, G) and 70 (H, I) dg (400×). A different brown color scale indicates positive staining. Note negative immunostaining in negative control (J). Arrowheads indicate the luminal endometrial epithelium and arrows show the trophoblast. (K) Optical density (OD) in the endometrial tissue. (L) OD in the trophoblast. (M) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (N) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
αvβ3 integrin expression at different development stages (17, 30, 60 and 70 dg) and non-pregnant sow (NP) uteri. (A) Representative images of αvβ3 immunohistochemical staining in the non-pregnant uterus (A) and placental tissue at 17 (B, C), 30 (D, E), 60 (F, G) and 70 (H, I) dg (400×). A different brown color scale indicates positive staining. Note negative immunostaining in negative control (J). Arrowheads indicate the luminal endometrial epithelium and arrows show the trophoblast. (K) Optical density (OD) in the endometrial tissue. (L) OD in the trophoblast. (M) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (N) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
αvβ3 integrin expression at different development stages (17, 30, 60 and 70 dg) and non-pregnant sow (NP) uteri. (A) Representative images of αvβ3 immunohistochemical staining in the non-pregnant uterus (A) and placental tissue at 17 (B, C), 30 (D, E), 60 (F, G) and 70 (H, I) dg (400×). A different brown color scale indicates positive staining. Note negative immunostaining in negative control (J). Arrowheads indicate the luminal endometrial epithelium and arrows show the trophoblast. (K) Optical density (OD) in the endometrial tissue. (L) OD in the trophoblast. (M) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (N) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
Qualitative description of the expression of the molecules – αvβ3/α5β1/FN/OPN.
| Luminal endometrial epithelium | Trophoblast | |||||||
|---|---|---|---|---|---|---|---|---|
| FN | αvβ3 | OPN | α5β1 | FN | αvβ3 | OPN | α5β1 | |
| Non pregnant uteri | (–) | (–) | (++) | (–) | N/A | N/A | N/A | N/A |
| Gestation day | ||||||||
| 17 | (+) | (++) | (+) | (+++) | (++) | (++) | (+++) | (++) |
| 30 | (+) | (+) | (+++) | (++) | (++) | (++) | (+++) | (++) |
| 60 | (++) | (+++) | (+++) | (+) | (+++) | (+++) | (+++) | (+) |
| 70 | (+–) | (+) | (++) | (–) | (+) | (+) | (++) | (–) |
(–) = negative, (+) = weak positivity, (++) = moderate positivity, (+++) = strong positivity.
FN, fibronectin;. N/A, not applicable; OPN, osteopontin.
α5β1 integrin
α5β1 integrin was analyzed in the endometrial luminal epithelium and feto–maternal interface from non-pregnant and pregnant pigs (Fig. 2; Table 2). The integrin immunostaining was different according to each gestation period and tissue was analyzed. OD values are given in detail in Figs 2I and J. The α5β1 integrin was not observed in the endometrial luminal epithelium of the non-pregnant uterus (Fig. 2A; OD: 0.07; NP). Regarding gestation, strong immunostaining of α5β1 integrin was found in the endometrial luminal epithelium only at 17 dg (Fig. 2B; Fig. 2I; OD: 0.39). Then, its immunostaining decreased significantly (P < 0.0001). With respect to the trophoblast, α5β1 integrin was most expressed at 17 and 30 dg (OD: 0.22 and 0.19, respectively; Fig. 2C and D, arrow; Fig. 2J). In late gestation (70 dg), there was no immunostaining of α5β1 integrin (Fig. 2F and G; Fig. 2I and J). The percentage of immunolabeled area analysis of α5β1 integrin during pig gestation is presented in Figs 2K and L. The IAP of α5β1 integrin showed significant differences between the different periods of gestation studied in this work (P < 0.0001). The greatest expression area of α5β1 integrin in the endometrial epithelium (Fig 2K) and trophoblast at 30 dg was observed (Fig 2L).
α5β1 integrin expression at different development stages (17, 30, 60 and 70 dg) and non-pregnant sow (NP) uteri. Representative images of α5β1 immunohistochemical staining in NP (A), placental tissue at 17 (B, C), 30 (D), 60 (E) and 70 (F, G) dg (400×). A different brown color scale indicates positive staining. Negative control (H). Arrowheads indicate the luminal endometrial epithelium and arrows show the trophoblast. (I) Optical density (OD) in the luminal endometrial epithelium. (J) OD in the trophoblast. (K) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (L) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
α5β1 integrin expression at different development stages (17, 30, 60 and 70 dg) and non-pregnant sow (NP) uteri. Representative images of α5β1 immunohistochemical staining in NP (A), placental tissue at 17 (B, C), 30 (D), 60 (E) and 70 (F, G) dg (400×). A different brown color scale indicates positive staining. Negative control (H). Arrowheads indicate the luminal endometrial epithelium and arrows show the trophoblast. (I) Optical density (OD) in the luminal endometrial epithelium. (J) OD in the trophoblast. (K) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (L) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
α5β1 integrin expression at different development stages (17, 30, 60 and 70 dg) and non-pregnant sow (NP) uteri. Representative images of α5β1 immunohistochemical staining in NP (A), placental tissue at 17 (B, C), 30 (D), 60 (E) and 70 (F, G) dg (400×). A different brown color scale indicates positive staining. Negative control (H). Arrowheads indicate the luminal endometrial epithelium and arrows show the trophoblast. (I) Optical density (OD) in the luminal endometrial epithelium. (J) OD in the trophoblast. (K) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (L) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
Fibronectin
FN was immunodetected in the endometrial luminal epithelium and feto–maternal interface from non-pregnant and pregnant pigs, respectively (Fig. 3; Table 2). The FN immunostaining was different according to each gestation period and tissue analyzed. OD values are presented in Figs 3I and J. Immunostaining of the FN was not observed in the endometrial luminal epithelium of the non-pregnant uterus (Fig 3A; OD: 0.08; Fig. 3I). Regarding gestation, in the endometrium, FN was expressed mostly at 60 dg (Fig 3E; OD: 0.26; Fig. 3I), and then its immunostaining decreased significantly at 70 dg (OD: 0.16; P < 0.0001; Fig. 3G; Fig. 3I). In the trophoblast, FN immunostaining was observed strong at 60 dg (OD: 0.54; P < 0.0001; Fig 3F; Fig. 3J). The IAP of FN during pig gestation is presented in Figs 3K and L. The IAP of FN showed significant differences between the different periods of gestation studied in this work (P < 0.0001). In the endometrium, a greater immunolabeled area at 30 dg (33.02%) and 60 dg (36.43%; P < 0.0001; Fig. 3K) was observed, compared with the rest of the periods analyzed. In trophoblast, FN covered a greater area at 17 and 60 dg (46.05 and 40.07%, respectively) and decreased significantly at 70 dg (9.17%; P < 0.0001; Fig 3L).
FN expression at different development stages (17, 30, 60remo and 70 dg) and non-pregnant sow (NP) uteri. Representative images of fibronectin immunohistochemical staining in NP uterus (A), placental tissue at 17 (B-C), 30 (D), 60 (E-F) and 70 (G-H) dg (400×). A different brown color scale indicates positive staining. Arrowheads indicate the luminal endometrial epithelium and arrows show the trophoblast (I) Optical density (OD) in the luminal endometrial epithelium. (J) OD in the trophoblast. (K) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (L) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
FN expression at different development stages (17, 30, 60remo and 70 dg) and non-pregnant sow (NP) uteri. Representative images of fibronectin immunohistochemical staining in NP uterus (A), placental tissue at 17 (B-C), 30 (D), 60 (E-F) and 70 (G-H) dg (400×). A different brown color scale indicates positive staining. Arrowheads indicate the luminal endometrial epithelium and arrows show the trophoblast (I) Optical density (OD) in the luminal endometrial epithelium. (J) OD in the trophoblast. (K) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (L) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
FN expression at different development stages (17, 30, 60remo and 70 dg) and non-pregnant sow (NP) uteri. Representative images of fibronectin immunohistochemical staining in NP uterus (A), placental tissue at 17 (B-C), 30 (D), 60 (E-F) and 70 (G-H) dg (400×). A different brown color scale indicates positive staining. Arrowheads indicate the luminal endometrial epithelium and arrows show the trophoblast (I) Optical density (OD) in the luminal endometrial epithelium. (J) OD in the trophoblast. (K) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (L) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
Osteopontin
OPN was immunodetected in the endometrial luminal epithelium and feto–maternal interface from non-pregnant and pregnant pigs (Fig 4; Table 2). Significant differences were found among the stages of gestation analyzed in this work. OD values are presented in Figs 4I and J. Low immunostaining of the OPN was observed in the endometrial luminal epithelium of the non-pregnant uterus (Fig. 4A; OD: 0.3; Fig. 4I). However, during gestation, in endometrium, OPN was most expressed at 30 dg and 60 dg (OD: 0.64 and 0.58, respectively; P < 0.0001; Fig. 4D and E). In trophoblast, the OD of OPN was significantly higher at 30 dg (OD: 0.71; Fig. 4D) and then it decreased significantly in late gestation, 60 dg (OD: 0.42; Fig. 4E) and 70 dg (OD: 0.27; Fig. 4G; P < 0.0001; Fig. 4J). The percentage of the immunolabeled area of OPN is presented in Figs 4K and L. Significant differences were found among the stages of pig gestation analyzed in the present work (P < 0.0001). At 30 dg (48.29%) and 60 dg (53.41%), OPN covered a larger area in the endometrial luminal epithelium compared to the rest of the gestation periods. In the fetal placenta, the OPN covered a greater area at 30 dg (59.53%; P < 0.0001; Fig. 4L). At 70 dg, its expression area decreased significantly (22.75%; Fig. 4L).
OPN expression at different development stages (17, 30, 60 and 70 dg) and non-pregnant sow (NP) uteri. Representative images of OPN immunohistochemical staining in NP (A), placental tissue at 17 (B-C), 30 (D), 60 (E) and 70 (F-G) dg (400×). Different brown color scale indicates positive staining. Negative control (H). Arrowheads indicate the luminal endometrial epithelium and arrows indicate the trophoblast. (I) Optical density (OD) in the luminal endometrial epithelium. (J) OD in trophoblast. (K) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (L) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
OPN expression at different development stages (17, 30, 60 and 70 dg) and non-pregnant sow (NP) uteri. Representative images of OPN immunohistochemical staining in NP (A), placental tissue at 17 (B-C), 30 (D), 60 (E) and 70 (F-G) dg (400×). Different brown color scale indicates positive staining. Negative control (H). Arrowheads indicate the luminal endometrial epithelium and arrows indicate the trophoblast. (I) Optical density (OD) in the luminal endometrial epithelium. (J) OD in trophoblast. (K) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (L) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
OPN expression at different development stages (17, 30, 60 and 70 dg) and non-pregnant sow (NP) uteri. Representative images of OPN immunohistochemical staining in NP (A), placental tissue at 17 (B-C), 30 (D), 60 (E) and 70 (F-G) dg (400×). Different brown color scale indicates positive staining. Negative control (H). Arrowheads indicate the luminal endometrial epithelium and arrows indicate the trophoblast. (I) Optical density (OD) in the luminal endometrial epithelium. (J) OD in trophoblast. (K) Immunostaining area percentage (IAP) of the luminal endometrial epithelium. (L) IAP of the trophoblast. Values with different letters indicate significant differences (P < 0.05).
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
Correlation between the αvβ3 and α5β1 integrins and their ligands, FN and OPN
The OD and IAP of αvβ3 and α5β1 integrins and their ligands, FN and OPN, were measured. Those values were measured in the luminal epithelium of the endometrium and trophoblast of pregnant pigs at different gestational stages. The median for each receptor/ligand in each tissue was calculated. The correlation between the medians of the studied molecules in the endometrium and trophoblast was estimated (see results detailed in Table 3). The behavior of these molecules in the epithelia that constitute the maternal–fetal interface in pig gestation, both in immunostaining intensity (OD) and in immunostaining extension (IAP), was analyzed.
Assessment of the correlation between FN and OPN and their receptors (αvβ3 and α5β1) in the endometrium (E) or trophoblast (T) based on different stages of gestation using the Spearman correlation coefficient (SCC). The analysis was performed according to the OD and IAP values determined from the immunolabeling of the molecules through pregnancy.
| FN_T | OPN_T | αvβ3_T | α5β1_T | |||||
|---|---|---|---|---|---|---|---|---|
| r | P | r | P | r | P | r | P | |
| OD | ||||||||
| α5β1_E | 0.43235 | 0.0944 | 0.57059 | 0.021 | ||||
| αvβ3_E | 0.60294 | 0.0134 | 0.07647 | 0.7783 | ||||
| FN_E | 0.28529 | 0.2841 | 0.23235 | 0.3865 | ||||
| OPN_E | 0.32059 | 0.226 | –0.07353 | 0.7867 | ||||
| IAP | ||||||||
| α5β1_E | 0.32941 | 0.2128 | 0.75294 | 0.0008 | ||||
| αvβ3_E | 0.67941 | 0.0038 | 0.37059 | 0.1577 | ||||
| FN_E | 0.35 | 0.1839 | 0.63235 | 0.0086 | ||||
| OPN_E | –0.12353 | 0.6485 | 0.31765 | 0.2306 | ||||
r = SCC; P < 0.05 are statistically significant and presented in bold.
Regarding OD, a direct and statistically significant correlation was found between fetal FN and endometrial αvβ3 integrin in all gestation (Spearman correlation coefficient (SCC): r = 0.79706; P < 0.0002; Fig. 5A). No correlation was found between the fetal FN and the other receptor studied – the α5β1 integrin expressed in the endometrial epithelium (SCC: r = 0.34706; P = 0.1878; Table 3). If we refer to the OPN ligand expressed in the trophoblast, a positive and statistically significant correlation was found with the α5β1 integrin receptor expressed in the endometrial epithelium throughout gestation (SCC: r = 0.57059; P < 0.021; Fig. 5B). We found no correlation between the FN and OPN expressed in the endometrial luminal epithelium with its receptors expressed in the trophoblast during pig gestation.
Positive correlation between FN and OPN and their receptors expressed at the fetal–maternal interface (A, B, C, D and E) during pregnancy, according to optical density (OD) and immunostaining area percentage (IAP) values. (E: endometrium; T: trophoblast). Values of P < 0.05 are considered statistically significant.
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
Positive correlation between FN and OPN and their receptors expressed at the fetal–maternal interface (A, B, C, D and E) during pregnancy, according to optical density (OD) and immunostaining area percentage (IAP) values. (E: endometrium; T: trophoblast). Values of P < 0.05 are considered statistically significant.
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
Positive correlation between FN and OPN and their receptors expressed at the fetal–maternal interface (A, B, C, D and E) during pregnancy, according to optical density (OD) and immunostaining area percentage (IAP) values. (E: endometrium; T: trophoblast). Values of P < 0.05 are considered statistically significant.
Citation: Reproduction and Fertility 4, 1; 10.1530/RAF-22-0079
About the IAP, a direct and statistically significant correlation was observed between the fetal FN and the endometrial αvβ3 integrin in the entire study group (CCS: r = 0.67941; P = 0.0038; Fig. 5C); this relation was not observed with the α5β1 integrin (CCS: r = 0.32941; P = 0.2128). However, a correlation was found between the endometrial FN and its α5β1 integrin receptor expressed in the trophoblast (CCS: r = 0.63235; P = 0.0086; Fig. 5D). Regarding fetal OPN, similar to what happened with OD, a positive correlation was found between the IAP of this molecule and its receptor, the integrin α5β1, expressed in the endometrium (CCS: r = 0.75294; P = 0.0008; Fig. 5E). There was no correlation in IAP between endometrial αvβ3 integrin and fetal OPN. A correlation was not found between OPN expressed in the endometrial luminal epithelium and its receptors expressed in the trophoblast during pig gestation.
Discussion
ln pigs, implantation is central and placentation is diffuse and epitheliochorial. After the adhesion of both epithelia, the trophoblast does not penetrate the endometrium (Bidarimath & Tayade 2017, da Anunciação et al. 2017), and it only adheres to the uterine epithelium (Johnson et al. 2021). In the present work, we determine the OD and the immunolabeled area of two integrins (αvβ3 and α5β1) and their ligands (FN and OPN) to perform a correlation analysis between the values found for each molecule at the placental interface. The analysis was implemented at different times of the swine gestation ranging from the peri-implantation period to advanced periods in which the increment of placental surface ceases (Biensen et al. 1998).
Several authors demonstrate that integrins (αvβ3, αvβ6, α5β1 and α2β1 ) and its ligands (FN, OPN, vitronectin and collagen) expressed in the uterine epithelium would participate in the adhesion of the trophoblast to the endometrium generating the epitheliochorial placenta in pigs (Bowen et al. 1996, 1997, Erikson et al. 2009, Bazer et al. 2012, Geisert & Spencer 2021). At the implantation period, αvβ3 and α5β1 integrins have different ligands such as FN and OPN that allow a contact between trophoblast and endometrial epithelium (Kaneko et al. 2013, Kang et al. 2014, Berneau et al. 2019, Kadry & Calderwood 2020). In the present study, we performed immunohistochemical techniques, and we have quantified the immunostaining obtained from all samples analyzed to evaluate the expression of two integrins (αvβ3 and α5β1) and its ligands (FN and OPN). Our results confirm the results reported by these authors who postulated that integrins and their ligands act like ‘a molecular bridge’ attaching maternal and fetal epithelia during implantation.
In early gestation, in agreement withRashev et al. (2005), we found high expression levels for the α5β1 integrin and FN, between days 15 and 35 of pig gestation. It is important to highlight that despite the strong expression of α5β1 integrin at the implantation period in pigs, it decreased in middle and late gestation. This event reveals that it would participate actively at the implantation process, and its importance decreases in the periods after implantation. Since both pig trophoblast and uterine luminal epithelium expressed α5β1 integrin and FN in early gestation, it can be postulated that both epithelia can bind FN as well as accumulate and assemble FN molecules on the maternal–fetal interface. However, in our study, αvβ3 integrin and OPN were found elevated too. Erikson et al. (2009) demonstrated, at 20 dg, that specific integrin receptors bind directly to OPN expressed in the conceptus and uterus. They reported that OPN binds αvβ3 integrin in pig luminal endometrial cells. In our laboratory, a similar expression of αvβ3 integrin and OPN was found in the endometrium and trophoblast at 17 and 35 dg, demonstrating its participation of their binding in both epithelia. But, despite the fact that the four molecules studied here were expressed at the fetal–maternal interface during early gestation, a positive correlation has only been found particularly in avb3 integrin with FN and in a5b1 integrin with OPN. Thus, we found that ‘trophoblastic OPN’ and ‘endometrial epithelial α5β1 integrin’ were correlated in pig early gestation. A significant correlation both in the intensity (by OD) and in the extension of immunostaining (by IAP) for trophoblastic FN and endometrial αvβ3 integrin at 17 and 30 dg was observed too. This molecule’s expression pattern observed at the fetal–maternal interface in the implantation period shows that they could be participating in a molecular network that would allow a blocking or restriction blastocyst invasion, as was described in human implantation byDamsky et al. (1994), in sheep by Johnson et al. (2001) and in mare byWilsher and Allen (2009).
When we analyzed mid-late gestation, we demonstrated that the protein expression of αvβ3 integrin, FN and OPN remains elevated until 60 dg in the same locations, like endometrial luminal epithelium and trophoblast. However, in that period of gestation, the immunostaining of α5β1 integrin at the feto–maternal interface was low. This is partially in concordance with Frank et al. (2017), who reported the expression of αv and β3 integrins subunits and their ligand OPN in the uterine luminal epithelium and conceptus trophoblast between 9 and 50 dg. We have not observed a decrease of αvβ3 integrin, FN and OPN immunostaining until 70 dg. These findings indicate us that these molecules could be relevant for the adhesion of both epithelia at 60 dg. Focusing on the correlation analysis in this period, it was found that the FN ligand and its receptor, αvβ3 integrin, were positively correlated. Additionally, a peak expression of fetal FN and αvβ3 integrin was found at 60 days. This period coincides with a placental tissue remodeling moment in pigs, with essential modifications in the expression of molecules related to vascular and placental establishment (Cristofolini et al. 2013, Sanchis et al. 2017, Fiorimanti et al. 2018, Stenhouse et al. 2019b ).
The absence of correlation between some of these receptors and their ligands in some pregnancy periods could demonstrate that there would be other adhesion molecules and other ligands that could participate in that adhesion. Thus vitronectin (αvβ3 integrin ligand) (García et al. 2004) or αvβ6 (OPN receptor) (Erikson et al. 2009), both found at the preimplantation stage, could be the molecules participating in those structures and in the periods of gestation analyzed here. Future studies are necessary to corroborate this hypothesis.
It should be highlighted that the integrins and ligands analyzed here decreased their expression significantly at 70 dg, a period of remodeling of placenta with an increase of vascular apoptosis (Sanchis et al. 2017). In human, the trophoblast expression of α5 integrin is associated with hypoxia (Arimoto-Ishida et al. 2009), and it is necessary to design experiments that could explore the relationship between hypoxia and integrin expression in pig, taking into account the importance of this condition in embryo loss and abortus. The importance of changes in integrins and their ligands during pathological processes has been demonstrated in humans with implantation defects (revised inMerviel et al. 2009, Zhou et al. 2021). However, the alterations in integrins are found in some women with fetal loss (Quenby et al. 2007), and trophoblastic integrins are modified in women with early pregnancy loss associated with thyroid disturbs (Vissenberg et al. 2015). In pigs,Stenhouse et al. (2019a) demonstrated that variations of mRNA integrin expression at gestation impacts both the fetal size and sex. However, this unique report found in pig analyzes mRNA in all the tissues and does not discriminate the localization of the molecules. The results of our work establishes a normal pattern of integrin and ligand expression that could be the basis to analyze the changes that occur during embryo/fetus loss. More studies need to be performed in placentas that have undergone reabsorption to clarify if these molecules would be modified in the feto–maternal interface in these situations.
To conclude, we observed that the interactions between integrins αvβ3 and α5β1 and their ligands, FN and OPN, showed changes throughout the placentation. The present work shows that the intensity of the expression of adhesion molecules and their ligands analyzed is not constant throughout gestation. However, the correlations established between particular molecules would allow processes of remodeling and placental growth during gestation in the pig. In this way, they would follow the changes in the placenta that occur during pregnancy.
A better understanding of the molecules that participate in the establishment of the feto–maternal interface will provide us new strategies to reduce the high embryonic/fetal losses that occur in this species.
Declaration of interest
None of the authors have any conflicts of interest to declare.
Funding
This study was partially funded by the National Agency for the Promotion of Science and Technology of Argentina (ANPCYT, PICTO 2011 0242) and the Science and Technology Program of the National University of La Pampa (UNLPam, grant N° 360/11). CV is a postdoctoral fellow, and MC and CB are researchers from the National Scientific and Technical Research Council of Argentina (CONICET). DW and MK are researchers from UNLPam.
Author contribution statement
CV, MC, DW and CB performed the literature review and wrote the article. CB and MK contributed to the research design and provided critical revisions to the article. CV, MG and DW performed experiments and analyzed data. All authors have read and approved the final version of the article.
Acknowledgements
The authors are grateful to Faculty of Veterinary Sciences of National University of La Pampa and ANPCYT which funded this study through grants. PhD Carolina Velez thanks CONICET for fellowship support.
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