Changes in the composition of the extracellular matrix accumulated by mesenchymal stem cells during in vitro expansion

One of the approaches to preserve the properties of mesenchymal stem cells (MSCs) during in vitro expansion is to use cell culture substrates. MSCs are known to generate the extracellular matrix (ECM) proper to preserve their proliferative capacity in vitro, but extensive expansion is considered to deprive MSCs of the capacity to prepare such ECM. In order to examine the features of ECM proper that is required to preserve the proliferative capacity of MSCs, we analyzed the changes in the composition of ECM accumulated by MSCs during in vitro expansion. Biochemical and immunological analysis showed that collagen and laminin content decreased during expansion. Immunofluorescence and ultrastructural analyses showed that the ECM structure changed from a dynamic fibrous, porous and steric structure to a static, crammed, and planar one. The results of Western blotting analysis suggested loose intermolecular association in ECM molecules accumulated by extensively proliferated MSCs. The ECM prepared by extensively proliferated MSCs was less effective to recover their proliferative capacity than that prepared by less proliferated cells. Our results suggest that a cell culture substrate to expand MSCs requires abundance in collagen and basement membrane components, and steric, porous and fibrous structure in which ECM molecules are tightly associated.     

Early exercise advances the maturation of glycosaminoglycans and collagen in the extracellular matrix of articular cartilage in the horse

REASON FOR PERFORMING STUDY: Training at a very young age may influence the characteristics of the collagen network of articular cartilage extracellular matrix (ECM) in horses. OBJECTIVES: To investigate whether increasing workload of foals results in significant changes in the biochemical composition of articular cartilage ECM. METHODS: Thoroughbred foals (n = 33) were divided into 2 different exercise groups from age 10 days-18 months. One group (PASTEX; n = 15) was reared at pasture; the other (CONDEX; n = 18) underwent a specific additional training programme that increased workload by 30%. At mean age 18 months, 6 animals from each group were subjected to euthanasia. The proximal articular surface of the proximal phalanx of the right hindlimb was examined for the presence of damage using the cartilage degeneration index (CDI). Samples were taken from 2 sites with known different loading patterns. Slices were analysed for DNA, glycosaminoglycans (GAG), collagen and post translational modifications of collagen (formation of hydroxylysylpyridinoline [HP] and pentosidine crosslinks, and hydroxylysine [Hyl]), and exercise groups and different sites compared. RESULTS: There were no differences in CDI between PASTEX and CONDEX animals, indicating the absence of extra joint damage due to the exercise regimen. There were site-related differences for most biochemical variables, corroborating earlier reports. All biochemical variables showed differences between PASTEX and CONDEX groups at one of the sites, and some at both. GAG and collagen levels were lower in the CONDEX group whereas Hyl, HP crosslinks and pentosidine crosslinks were higher. CONCLUSIONS AND POTENTIAL RELEVANCE: A measurable effect of the conditioning exercise was demonstrated. The margin between too much and too little work when training foals may be narrower than intuitively presumed.     

Characterization of the extracellular matrix in the chorioallantoic membrane of water buffalo (Bubalus bubalis) in early gestation

Placenta is formed by a parenchyma rich in extracellular matrix (ECM), and this structure guarantees the proper development of the embryo and placental functioning. Recently, studies have focused on the characterization of ECM in the placenta and foetal membranes of different species. This work aimed to analyse the composition of the ECM and to quantify the types of collagens in its composition. For this, 33 chorioallantoic membranes were used at different gestational ages, which were grouped into five groups. Subsequently, haematoxylin–eosin staining, Masson trichrome and picrosirius were performed for histological analysis. Through the technique of polarized light, it was possible to quantify the total collagen present in the membranes and finally the immunohistochemical technique was performed to verify the presence of collagens and glycoproteins. It was possible to verify that the chorioallantoic membranes have, in all the gestational periods of the initial third of gestation, the same histological structures, being the most significant difference the membrane thickening that occurs gradually during the gestation. However, we notice the appearance of binucleate cells only from group II. In addition, it was verified that a gradual increase of collagen occurs until the group IV, yet from the group V begins to occur a decrease of this protein. In addition, collagen I, collagen III, fibronectin and laminin were present in all membranes. With this, we concluded that the buffalo chorioallantoic membrane presents ECM in constant remodelling at the beginning of gestation and can be used as biomaterial in works on regenerative biology.     

Degenerative changes of the cranial cruciate ligament harvested from dogs with cranial cruciate ligament rupture

Degenerative cranial cruciate ligament (CCL) rupture is characterized histologically by degenerating extracellular matrix (ECM) and chondroid metaplasia. Here, we describe the progression of chondroid metaplasia and the changes in the expression of ECM components in canine CCL rupture (CCLR). CCLs from 26 stifle joints with CCLR (CCLR group) and normal CCLs from 12 young beagles (control group) were examined histologically and immunohistochemically for expression of type I (COLI), type II (COLII), type III collagen (COLIII) and Sry-type HMG box 9 (SOX9). Cell density and morphology of CCLs were quantified using hematoxylin–eosin staining. The percentage of round cells was higher in the CCLR group than in controls. COLI-positive areas were seen extensively in the connecting fibers, but weakly represented in the cytoplasm of normal CCLs. In the CCLR group, there were fewer COLI-positive areas, but many COLI-positive cells. The percentages of COLII-, COLIII- and SOX9-positive cells were higher in the CCLR group than in controls. The number of spindle cells with perinuclear halo was high in the CCLR group, and most of these cells were SOX9-positive. Deposition of COLI, the main ECM component of ligaments, decreased with increased COLIII expression in degenerated CCL tissue, which shows that the deposition of the ECM is changed in CCLR. On the contrary, expression of SOX9 increased, which may contribute to the synthesis of cartilage matrix. The expression of COLII and SOX9 in ligamentocytes showed that these cells tend to differentiate into chondrocytes.     

A new three-dimensional glass scaffold increases the in vitro maturation efficiency of buffalo (Bubalus bubalis) oocyte via remodelling the extracellular matrix and cell connection of cumulus cells

At present, many three-dimensional (3D) culture systems have been reported, improving the oocyte quality of in vitro maturation (IVM), yet the mechanism still needs to be further explored. Here we examined the effects of a new self-made 3D glass scaffold on buffalo oocyte maturation; meanwhile, the underlying mechanism on buffalo oocyte maturation was also detected. Compared to the two-dimensional (2D) glass dish culture, results revealed that the 3D culture can improve the first polar body rate of oocytes, subsequent cleavage and blastocysts rate of parthenogenetic activation embryos (p < .05). The extracellular matrix-related proteins COL1A1, COL2A1, COL3A1, FN and cell connection-related proteins N-cadherin, E-cadherin, GJA1 were found higher in cumulus cells of 3D culture. Moreover, in cumulus cells, proteins of the PI3K/AKT pathway reported being regulated by FN and E-cadherin including PI3K P85 and p-AKT were also higher in 3D culture. Furthermore, proapoptosis proteins P53, BAX, caspase-3 were lower in both cumulus cells and oocytes in 3D culture, while proteins PCNA and BCL2 showed the opposite result. Results also showed that the apoptosis was inhibited, and the proliferation was enhanced in cumulus cells of 3D culture. Finally, the cumulus expansion-related genes HAS2, CD44, HMMR, PTX3, PTGS2 were found higher in cumulus cells of 3D culture. Taken together, the 3D culture could promote oocyte maturation by regulating proteins correlated with the ECM, cell connection and PI3K/AKT pathway, inhibiting the apoptosis of cumulus cells and oocytes, enhancing the proliferation of cumulus cells and the cumulus expansion.     

Functional role of type VI collagen during early feather development of the chicken embryo in vitro

The regulatory function of type VI collagen during early feather development in embryonic chickens was investigated at the cellular and organ levels. Immunohistochemical studies of embryonic chicken skin showed that type VI collagen was distributed in spatial‐specific and temporal‐specific manners related to early feather development. To clarify the role of type VI collagen, we studied the feather development in intact, reconstituted and reconstituted gel skin cultures. When ethyl‐3,4‐dihydroxybenzoate (EDHB) was added to the medium of intact skin as an inhibitor of type VI collagen synthesis, the feather buds did not elongate and the number of neural cell adhesion molecule (NCAM)‐positive cells was reduced. However, the magnitudes of both suppressive effects of EDHB were reduced by the addition of liquid type VI collagen. Similar improvement was also observed in the reconstituted skin with liquid type VI collagen and in the reconstituted gel skin with solid type VI collagen at a low concentration. Moreover, type VI collagen promoted feather bud development in the absence of EDHB. However, a high concentration of solid type VI collagen in the reconstituted gel skin arrested the feather bud elongation, and antitype VI collagen antibodies caused feather buds to become longer and smaller in the reconstituted skin. At the cellular level, type VI collagen affected the proliferation, migration and NCAM expression of mesenchymal cells. These results suggest that type VI collagen regulates early feather development by controlling mesenchymal cell behavior.     

Immunohistochemistry of the extracellular matrix of the normal equine lamina cribrosa

Purpose To use immunohistochemical techniques to identify and localize the structural macromolecules of the extracellular matrix (ECM) of the normal adult equine lamina cribrosa in order to make comparisons to the extracellular matrix of the lamina cribrosa of horses with glaucoma. METHODS: Normal eyes of five adult horses between 5 and 10 years of age were fixed in 10% neutral buffered formalin and embedded in paraffin. Polyclonal rabbit-derived antibodies against human elastin, laminin, fibrillin-1, and collagen types I, III and IV, and polyclonal goat-derived antibodies against collagen type VI were used as primary antibodies. Transverse and longitudinal histologic sections of the optic nerve head and lamina cribrosa were stained using several dilutions of the primary antibodies, biotinylated link antibody, horseradish peroxidase-labeled streptavidin, and 3,3'-diaminobenzidine as a chromogen. The immunohistochemical staining patterns were qualitatively interpreted. RESULTS: The normal adult horse lamina cribrosa labeled positively for collagen types I, III and VI, laminin, elastin and fibrillin. Collagen type VI staining of the laminar ECM was most intense, followed by labeling for collagen types III and I, respectively. Laminar blood vessels were weakly positive for laminin and slightly positive for type IV collagen. The scleral ECM of the laminar insertion zone had more intense labeling for collagen types I and VI than did the laminar plates. CONCLUSIONS: The extracellular matrix of the laminar plates of the adult equine lamina cribrosa is similar to the dog as it consists of elastic and collagen fibers (with collagen types VI, III and I). Both the normal dog and horse lamina display more intense staining of collagen type VI than is found in the ECM of the normal human lamina cribrosa. The macromolecular structure of the equine lamina cribrosa suggests that it is a very resilient structure that may provide some protection to the optic nerve axons during episodes of elevated intraocular pressure.     

Immunohistochemical characterization of the extracellular matrix in normal mitral valves and in chronic valve disease (endocardiosis) in dogs

This study aimed to characterize the composition and distribution of the extracellular matrix (ECM) components in normal canine mitral valves (MV) and in chronic heart valve disease (CVD).MV of 50 dogs (normal (n = 9), mild (n = 13), moderate (n = 17), severe (n = 11) CVD) were investigated macroscopically, histologically (H.-E., picrosirius red) and immunohistochemically (collagen I, III, IV, V, VI, elastin, laminin, fibronectin, heparan sulphate).In normal MV, ECM components were expressed in a typical layered pattern. In mild CVD, basement membrane components (laminin, collagen IV, fibronectin) were increased. Advanced CVD was characterized by myxomatous nodular lesions displaying a marginal and a central region comprised mainly of collagen I, VI and fibronectin in the former and collagen I and III in the latter. Collagen IV and laminin appeared multifocally in marked CVD.In conclusion, not only an accumulation of proteoglycans, but also a distinctly altered expression of basement membrane components, and collagens characterizes CVD.     

Morphometric and histochemical study of involution in the rat uterus after parturition

Morphological changes to and collagen loss from the rat uterus during postpartum involution were investigated. The expression patterns of collagen type III and myeloperoxidase (MPO) were also determined. Morphological changes were studied on days 1, 3, 5, 10, 15, 20, 22 and 25 postpartum. As a control, diestrus rats’ uterine were used. Specimens from the uterine horn were embedded in paraffin, cut into 8 µm coronal sections, and stained with hematoxylin‐eosin. The thickness of the longitudinal and circular smooth muscle layers and of the endometrium were measured. The collagen content was determined using hydroxyproline analysis. Immunostaining was used to examine the expression of collagen type III on days 1, 3, 5, 10, 15 and 20 postpartum; and MPO on days 1, 3, 5, 10 and 22 postpartum. The thickness of the smooth muscle layers was found to decrease rapidly postpartum: the circular smooth muscle layer returned to that of a non‐pregnant, control uterus by day 5 postpartum and the longitudinal smooth muscle layer by day 15 postpartum. Eosinophilic cells were observed in the endometrial stroma adjacent to the myometrium on days 10, 15 and 20 postpartum, and were confirmed as collagenous cells. Immunostaining identified collagen type III positive cells in the vessel‐rich layer adjacent to the placental site on days 1, 3, 5 and 10 postpartum, and these cells were confirmed to be phagocytic. Postpartum reduction in the weight of the uterus was accompanied by decreases in both the collagen content and the thickness of the longitudinal and circular smooth muscle layers. Furthermore, the phagocytic cells were shown to express MPO during postpartum involution of the uterus.     

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta.     

Collagen,glycosaminoglycans and matrix metalloproteinase‐2 and metalloproteinase‐9 in the cervix of the ewe during prepubertal development

The tortuous nature of the ovine cervix restricts the transcervical passage of the cannula, and many studies have aimed to understand the endocrine mechanism of the remodelling of cervical tissue in adult ewe. However, little is known about the remodelling of the cervical tissue during the prepubertal development of the lambs. To obtain histochemical and biochemical evidence about the nature of the prepubertal development of the cervix of the ewe, cervices of Corriedale lambs obtained at 0, 1, 2, 4, 6 and 8 months of age (n = 5 to 6 in each) were processed. Neutral and acidic glycosaminoglycans (by PAS‐Alcian stain) were weakly in the cervical stroma and not shown change during the development, whereas the percentage volume of fibrillar collagen (by van Gieson stain) increases throughout the experimental period in the superficial fold stroma and deep wall stroma (p < 0.05). The relative cervical weight (g/kg of body weight) and the collagen concentration (by spectrophotometry, mg/mg wet tissue) showed an early decreasing phase from months 0 to 4 and a later increasing phase from months 4 to 8 (p < 0.05). The latent form of matrix metalloproteinase‐2 (MMP‐2) detected by gelatin zymography (ng/mg protein) decreased from months 0 to 2 and increased from months 4 to 8, whereas the activated form decreased from months 0 to 2, remained low until month 6 and then recovered on month 8 (p < 0.0001). Data suggest that the relative cervical weight biphasic pattern during the development is related to MMP‐2‐dependent changes in the collagen content.     

The valves of the cardiac outflow tract of the starry ray,Raja asterias (Chondrichthyes; Rajiformes): Anatomical,histological and evolutionary aspects

The cardiac outflow tract of chondrichthyans is composed of the myocardial conus arteriosus, equipped with valves at its luminal side, and the bulbus arteriosus devoid of myocardium. Knowledge of the histomorphology of the conal valves is scarce despite their importance in preventing blood backflow to the heart. Current information on the subject refers to a single shark species. The present report is the first to describe the structure of the conal valves of a batoid species, namely, Raja asterias. Hearts from seven starry rays were examined using scanning electron microscopy and histochemical techniques for light microscopy. In all hearts, the conus showed four transverse rows of three pocket‐like valves each. Each valve was composed of a leaflet and its supporting sinus. The leaflet had a stout central body, rich in glycosaminoglycans, which contained fibroblasts, collagen and elastin. The central body was surrounded by two thin fibrous layers, outer and inner, formed mainly by collagen. The valves of the anterior row, which were the largest of the valvular system, were attached proximally to the conus arteriosus and distally to the bulbus arteriosus, and not to the ventral aorta as previously reported for chondrichthyans. The arrangement of the anterior valves in the starry ray is an anatomical pattern that apparently has been preserved throughout the evolution of vertebrates.     

Trabecular and subchondral bone development of the talus and distal tibia from foal to adult in the warmblood horse

Horses are precocial animals and able to stand and walk within hours after birth. To cope with associated loading, intrauterine bone development has shown to be anticipative. This study provides further insight into the post‐natal development of structurally important features of trabecular and subchondral bone of the talus and sagittal ridge of the tibia of warm‐blooded horses. In all areas studied, the average bone volume fraction showed a gradual increase over time, which was the result of a significant increase in trabecular thickness, without significant changes in the degree of anisotropy. Similar to the mineralised part of the bone, collagen content, measured as average retardation using polarised light microscopy, increased significantly, but the degree of anisotropy of the collagen type I network did not. At birth, the subchondral bone layer had a more trabecular aspect, gradually changing to an even surface with only a few vascular canals at an age of 2 months. Presented results indicate the necessity for a stronger structure, but not for a different structural design after birth, providing further evidence for anticipatory bone development in the horse. More knowledge about the strategies used to cope with mechanical loading after birth might be helpful in understanding the developmental bone and joint diseases.     

Type‐I interferon regulates matrix metalloproteinases clearance of the bovine endometrial spheroid

This study investigated the effect of type‐I interferon (IFN) on the expression of matrix metalloproteinases (MMPs) of the bovine endometrial stromal cells (BES) and epithelial cells (BEE). The cells were separated and purified from the caruncles and cultured in DMEM/F‐12 containing 10% fetal bovine serum. Spheroids were generated by using ascorbate. Zymograms of the supernatant showed that BEE predominantly expressed MMP‐9, whereas MMP‐2 was expressed in BES and homo‐spheroids. While MMPs expression was not detected in hetero‐spheroids. Real‐time quantitative PCR revealed that type‐I IFN and P4 suppressed the gene expression of MMP‐2 and MMP‐9 in hetero‐spheroids, respectively. On the other hand, gelatin zymography analysis of the supernatant showed that type‐I IFN strongly promote the clearance of MMPs. While zymograms of the MMPs stocked in the hetero‐spheroids were significantly reduced by type‐I IFN. Phenylmethanesulfonyl fluoride and leupeptin (both are serine proteinase inhibitors) significantly repressed the clearance of MMP‐2 and MMP‐9 induced by type‐I IFN. Moreover, collagen fibers in hetero‐spheroids significantly decreased after the treatment with type‐I IFN. In conclusion, it was suggested that type‐I IFN participate in the tissue remodeling by regulation the clearance of MMPs.     

Models in vivo of wound healing in the horse and the role of growth factors

Abstract Wound models attempt to simulate the natural healing processes in wounds. However, all models have significant limitations due to the complexity of the tissue repair process. Much can be learned from wound models in vitro by the use of cell culture techniques. The horse can provide a suitable naturally occurring model of chronic wound healing because it has many similarities to wound healing encountered in human medicine. The tissue architecture was investigated with regard to extracellular matrix and growth factor distribution during wound healing and growth factors were consistently present in the wound area. Biochemical investigations revealed increased levels of hydroxyproline, collagen, and TGFβ1 in exuberant granulation tissue. Equine wound models were established in vitro using cell culture techniques and growth factors had significant effects on the growth of the cells and their ability to synthesize collagen. Two gelatinases (MMP-2 and MMP-9) were detected in the tissues and wound fluid samples investigated. Zusammenfassung Wundmodelle haben zum Ziel, die natürlichen Heilungsprozesse in Wunden zu simulieren. Allen Modellen sind durch den komplexen Gewebsheilungsprozess deutliche Grenzen gesetzt. Durch die Verwendung von Zellkulturtechniken kann von Wundmodellen in vitro viel abgeleitet werden. Das Pferd stellt wegen der vielen Ähnlichkeiten zur humanmedizinischen Wundheilung ein geeignetes Modell für chronische Wundheilung dar. Die Gewebsarchitektur wurde bezüglich der Verteilung von extrazellulärer Matrix und von Wachtumsfaktoren während der Wundheilung untersucht; Wachtsumsfaktoren waren ständig in der Wunde vorhanden. Biochemische Untersuchungen ergaben erhöhte Hydroxyprolin-, Kollagenund TGFβ1-Spiegel. Wundmodelle beim Pferd in vitro und Zellkulturtechniken wurden entwickelt; Wachstumsfaktoren hatten deutliche Wirkung auf die Zellen und ihre Fahigkeit zur Kollagensynthese. Zwei Gelatinasen (MMP-2 und MMP-9) wurden im Gewebe identifiziert und Wundflüssigkeitsproben untersucht. [Cochrane, C.A. Models in vivo of wound healing in the horse and the role of growth factors. (Wundheilungsmodelle in vivo beim Pferd und die Rolle von Wachstunisfaktoren). Veterinary Dermatology 1997; 8 : 259–272] Resumen Los modelos de heridas intentan estimular el proceso natural de curación de heridas. Sin embargo, todos 10s modelos presentan limitaciones considerables debido a la complejidad del proceso de curación de heridas. Se puede aprender mucho de modelos de heridas in vitro mediante el uso de técnicas de cultivo celular. El caballo puede suponer un modelo natural adecuado de curación crónica de heridas ya que se asemeja a la curación de heridas en medicina humana. Se investigó la arquitectura tisular en referencia a la matriz extracelular y la distribución de factores de crecimento viendo que los factores de crecimiento se encontraban presentes de forma constante en el área de la herida. Las investigaciones bioquímicas revelaron incremento en los niveles de hidroxiprolina, colágeno y TGFbl en tejido de granulación exhuberante. Se establecieron modelos in vitro de heridas equinas utilizando técnicas de cultivo celular y los factores de crecimiento tuvieron efectos significativos en el crecimiento de las células y su capacidad de sintetizar colágeno. Se detectaron dos gelatinasas (MMP-2 y MMP-9) en 10s tejidos y muestras de fluidos de las heridas investigadas. [Cochrane, C.A. Models in vivo of wound healing in the horse and the role of growth factors. (Modelos de curacion de heridas in vivo en el caballo y el papel de 10s factores de crecimiento). Veterinary Dermatology 1997; 8 : 259–272] Résumé Les modèles de plaies tentent de simuler les processus naturels de cicatrisation. Cependant tous les modèles ont des limitations significatives dues à la complexité des processus de réparation des tissus. Beaucoup peut être étudié sur des modèles de plaies in vitro suite à l'utilisation des techniques de culture cellulaire. Le cheval peut constituer un modèle nature1 fiable de cicatrisation chronique de plaie de par les nombreuses similarités qu'il partage avec la cicatrisation des plaies en médecine humaine. L'architecture tissulaire a étéétudiée concernant la matrice extracellulaire et la distribution des facteurs de croissance pendant la cicatrisation, et les facteurs de croissance se sont montrés présents de façon consistante dans la zone de la plaie. Des investigations biochimiques ont révélé une élévation des taux d'hydroxyproline, collagène, et TGFβ1 dans la granulation tissulaire exubérante. Des modèles équins de plaies ont étéétablis in vivo en utilisant des techniques de culture cellulaire et les facteurs de croissance ont montré des effets significatifs sur la croissance des cellules et leur capacité de synthétiser le collagène. Deux gélatinases (MMP-2 et MMP-9) ont été détectées dans les tissus et les échantillons de fluide des plaies ont été analysés. [Cochrane, C.A. Models in vivo of wound healing in the horse and the role of growth factors. (Modeles in vivo de cicatrisation des plaies chez le cheval et role des facteurs de croissance). Veterinary Dermatology 1997; 8 : 259–272]     

The expression of VEGF,myoglobin and CRP2 proteins regulating endometrial remodeling in the porcine endometrial tissues during follicular and luteal phase

Endometrial remodeling is important for successful embryo development and implantation in pigs. Therefore, this study investigated change of proteins regulating endometrial remodeling on follicular and luteal phase in porcine endometrial tissues. The endometrial tissue samples were collected from porcine uterus during follicular and luteal phase, vascular endothelial growth factor (VEGF), myoglobin and cysteine‐rich protein 2 (CRP2) proteins were expressed by immnofluorescence, immunoblotting, and determined by 2‐DE and MALDI‐TOF/MS. We found that VEGF, myoglobin and CRP2 were strongly localized in endometrial tissues during luteal phase, but not follicular phase. The protein levels of VEGF, myoglobin and CRP2 in endometrial tissues were higher than luteal phase (P < 0.05). These results may provide understanding of intrauterine environment during estrous cycle in pigs, and will be used in animal reproduction for developing specific biomarkers in the future.