Buffalo Placenta Research Articles

Comprehensive transcriptional profiling and functional study of lymphangiogenic growth factor in placentome of early-stage pregnant water buffalo.

The aim of the present study was to evaluate the expression and localization of lymphangiogenic factors (VEGF-C and VEGF-D), their receptor (VEGFR3) and lymphatic endothelial marker (LYVE1) in buffalo placenta during early pregnancy [EP], and to investigate the functional role of lymphangiogenic growth factors in placental lymphangiogenesis. The mRNA and protein expression of VEGF-C, VEGF-D, their receptor VEGFR3 and LYVE1 showed significant expression in EP1 (29-42 days) and EP2 stages (51-82 days) both in caruncle (maternal part) and cotyledon (foetal part) of the buffalo placenta. Immunoreactivity of VEGF-C, VEGF-D and LYVE1 was observed around the endometrial gland, in lymphatics and trophoblast cells, whereas VEGFR3 mainly localized in lymphatics of the caruncle and cotyledons. Cultured trophoblast cells were treated with VEGF-C/VEGF-D (50, 100 and 150 ng/ml) and combined doses of VEGF-C and VEGF-D (150 ng/ml) each for different time durations (24, 48 and 72 h). The mRNA expression of LYVE1 and PCNA was significantly (p < .001) upregulated with VEGF-C and VEGF-D and combined treatment (@150 ng/ml), as well as significantly downregulating Caspase-3 at 48 and 72 h. Thus, the present study provides evidence that lymphangiogenic factors are expressed in buffalo placental compartments and they may play a significant role in the regulation of placental function in water buffaloes.

Pregnancy-associated glycoproteins (PAGs) concentrations in water buffaloes (Bubalus bubalis) during gestation and the postpartum period

For the first time in literature this study describes the pregnancy-associated glycoprotein (PAG) profile of buffalo cows during gestation and the post-partum period using antiserum raised against PAG-molecules purified from buffalo placenta (AS#860).Ninety-eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and then biweekly until the end of pregnancy. Pregnancy was confirmed by ultrasonography on days 28 and 45, and by rectal palpation from day 60 onwards. Blood samples were suspended for the non-pregnant cows on day 45, while the blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 post-calving. A cut-off value of 1.0 ng/mL was used in order to discriminate between pregnant and non-pregnant buffaloes. We used Linear Mixed models after Log(x+1) transformation to analyse the PAG concentrations.Fifty-two buffalo cows had become pregnant out of 98 synchronized (53%) and 46 remained non-pregnant (47%) as shown by ultrasonography and the PAG analysis. Significant differences (P < 0.001) in PAG concentrations were observed between the pregnant and non-pregnant buffaloes from day 23 as the PAG of the non-pregnant cows was always close to zero. Conversely, the PAG of the pregnant cows increased progressively from day AI until day 105 post-insemination and then stabilized until the end of pregnancy. Regarding pregnancy diagnosis, the sensitivity of PAG-RIA 860 system (ability of the test to correctly identify pregnant buffalo) ranged from 23% on day 23–98% on day 28 post AI; the specificity (ability to correctly identify non-pregnant buffaloes) was 100% throughout the sampling period. PAG progressively decreased from parturition to day 25 post-partum; from day 30 post-partum, the concentrations fell below 1 ng/mL and were close to 0 on the last day of observation (50 d post-partum).In conclusion, our results showed that RIA-860 is highly accurate for diagnosing pregnancy in buffaloes starting from day 28 of gestation. Furthermore, the rapid disappearance of PAG concentration after calving means that a cut-off limit in post-partum for detecting a new pregnancy is not required.

Characterization and distribution of the caveolins-1 and -2 in water buffalo placenta

Characterization and distribution of the caveolins-1 and -2 in water buffalo placenta

Development of the subplacenta in Galea spixii (Rodentia, Caviidae)

s / Placenta 35 (2014) A1eA112 A23 molecules in the fetal epithelium, and in the placental angiogenesis, despite the large distribution of the CAVs -1 and -2 in water buffalo placenta. Funding: FAPESP P1.40. DEVELOPMENT OF THE SUBPLACENTA IN GALEA SPIXII (RODENTIA, CAVIIDAE) Ferdinando Bezerra , Phelipe Favaron , Andrea Mess , Maria Angelica Miglino , Moacir de Oliveira a Department of Animal Science, Universidade Federal Rural do SemiArido, Mossoro, Rio Grande do Norte, Brazil; b Surgery Department, University of Sao Paulo, Sao Paulo, SP, Brazil The subplacenta of caviomorph rodents is considered to be the developmental origin for trophoblast invasion, which is relatively similar to the process in human beings. Objectives: We described subplacental development in a cavy species, Galea spixii. Methods: Tissues were collected from 12 females, ranging from day 15 to 55 of gestation. Samples were processed by means of histology, immunohistochemistry, cytochemistry, and electron microscopy. Results: On day 15 to 23, the subplacenta consisted of a single cytotrophoblast layer and a syncytium lining the maternal lacunae. Fetal circulation was present. On day 30, the subplacenta was more compact with well-defined border. Then it showed intense vascularisation. The lobules were composed by syncytiotrophoblast in the central part, and mesenchyme surrounded by cytotrophoblast at the borders of the organ. On day 45, degeneration of tissues was evident and syncytiotrophoblast was more abundant than cytotrophoblast. The syncytium was vacuolized and showed signs of apoptosis. Near term (days 53-55), more evident signs of cell death and reduction of subplacental tissue occurred. Proliferative activity of the subplacenta, evaluated by PCNA and AgNOR, was high early in early gestation and then continously decreased. Conclusion: In conclusion, the development of the subplacenta in Galea started early on reached a maximum around day 30 and became necrotic towards the end of gestation. Financial support: INCTC and NETCEM. P1.41-N. MORPHOLOGICAL AND VASCULAR CHANGES IN THE CORPUS LUTEUM DURING EARLY GESTATION OF BOVINE SCNT-DERIVED PREGNANCIES F abio Pinaffi, Izabelle Jardim, Juliano Sangalli, Rafael Sampaio, Flavio Meirelles, Luciano Silva University of Sao Paulo, Pirassununga, Sao Paulo,

Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females

BackgroundPregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species.ResultsAmino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n = 33) and non-pregnant (n = 26) water buffalo females.ConclusionsOur results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards.

Tissue-specific promoter methylation coincides with Cyp19 gene expression in buffalo ( Bubalus bubalis) placenta of different stages of gestation

Aromatase is the key enzyme for estrogen biosynthesis and is encoded by Cyp19 gene. Placental cotyledons are the main site of Cyp19 gene expression during pregnancy. The present study was aimed to investigate if DNA methylation and thus epigenetic mechanisms play a potential role in stage-specific regulation of Cyp19 expression in placental cotyledons of pregnant water buffaloes ( Bubalus bubalis). Significantly higher expression of Cyp19 gene ( p < 0.05) in placental cotyledons of early gestation period and post parturition period was found in comparison to mid-gestation placenta. Tissue-specific promoter driven transcript analyses showed that the change in expression was mainly due to change in the relative abundance of transcripts from exon I.1 while the transcripts from exon II showed comparatively less variation. Methylation analysis of 5 CpG dinucleotides of placenta-specific promoter I.1 and proximal promoter, PII showed hypo-methylation of PI.1 in early and term placenta while hyper-methylation in mid-placenta. However, PII was found to be hypomethylated in all the three tissues. In conclusion, result of the present study demonstrated that stage-specific methylation status of PI.1, the major promoter responsible for aromatase expression in buffalo placental cotyledons, coincides with the change in expression of Cyp19 gene in different stages of pregnancy.

Transplacental Transfer of Iron in the Water Buffalo (Bubalus bubalis): Uteroferrin and Erythrophagocytosis

The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental-maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl's reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal-placental tissues in buffalo throughout pregnancy.

Isolation of new pregnancy-associated glycoproteins from water buffalo ( Bubalus bubalis) placenta by Vicia villosa affinity chromatography

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo ( Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8 kDa and from 57.8 to 73.3 kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

Fator de crescimento fibroblástico básico e seus receptores em relação à atividade proliferativa na placenta bubalina em diferentes fases da gestação

Estudou-se a distribuição espaço-temporal do fator de crescimento fibroblástico básico (bFGF), do receptor 1 do fator de crescimento fibroblástico (FGFR1) e do receptor 2 do fator de crescimento fibroblástico (FGFR2) na placenta bubalina, correlacionando-a à proliferação celular. Para a detecção do bFGF, FGFR1, FGFR2 e antígeno Ki-67, colheram-se 12 placentas de búfalas nos terços inicial, médio e final da gestação, em abatedouros, e realizaram-se testes de imunoistoquímica. Detectou-se e avaliou-se a expressão do bFGF, do FGFR1, do FGFR2 e do antígeno Ki-67 ao longo da gestação. No compartimento fetal da placenta, observaram-se correlações positivas entre a expressão do bFGF e Ki-67, entre FGFR1 e Ki-67 e entre FGFR2 com Ki-67 (r=0,313, 0,358 e 0,384, respectivamente). No epitélio e estroma maternos observaram-se altas correlações entre FGFR1 e Ki-67 (r=0,739 e r=0,511, respectivamente). Os resultados sugerem envolvimento do bFGF, FGFR1 e FGFR2 na proliferação do trofoblasto enquanto no compartimento materno da placenta bubalina apenas o FGFR1 atuaria como modulador dessa atividade.

COMPARATIVE MORPHOLOGICAL FEATURES OF THE PLACENTAL BARRIER AND LOCALIZATION OF ESTROGEN RECEPTORS α IN THE PLACENTA OF COWS AND BUFFALOES

The present study was conducted to identify the morphological characteristics of the placental barrier and localization of estrogen receptors α in the cow and buffalo placentas. Placentomes were collected from 3 pregnant cows and 3 pregnant buffaloes at the 5th and 6th months of gestation respectively. The placentomes were fixed by using 10% neutral buffered formalin for immunohistochimestry and were post fixed in 3% glutaraldehyde for light microscopy. Indirect immunoperoxidase staining methods using monoclonal antibody against bovine ERα as primary antibody was applied. The morphometric data were obtained by using Q500 MC Image processing and analysis program. Histological examination revealed that there is a great similarity in the structure of the placental barrier of cows and buffaloes, whereas typical six slayers of tissue were seen separating the fetal blood from the maternal one. These layers were maternal endothelium, thin layer of maternal connective tissue, maternal uterine epithelial cells, fetal trophoblast cells, fetal connective tissue and fetal endothelium. Nuclear immunoreactivities occurred mainly in the maternal crypt epithelium and maternal connective tissue cells. In about 1000 maternal uterine cryptal cells, 87.6% were positive cells for ERα in the cow placenta and about 67.8% were positive cells in the buffalo placenta. The present study concluded that although there is a great similarity in the structure of the placental barrier and expression of ERα in the placentas of cows and buffaloes interbreeding between the two species is impossible due to the different number of chromosomes set. The placenta of cows seems to be less responsive to estrogen in comparison to that of buffaloes.

Isolation of pregnancy-associated glycoproteins (PAG) from water buffalo (Bubalus bubalis) placenta by use of Vicia villosa bound agarose affinity chromatography

The present study describes the isolation and characterisation of new PAG molecules extracted from mid- and late-pregnancy placentas in the water buffalo (Bubalisbubalis). After extraction, acid and ammonium sulphate precipitation and DEAE chromatography water buffalo PAG (wbPAG) were enriched by Vicia villosa agarose (VVA) affininity chromatography. As determined by Western blotting with anti-PAG-sera, apparent molecular masses of immunoreactive bands from VVA peaks ranged from 59.5 to 75.8 kDa and from 57.8 to 80.9 kDa in the mid- and late- pregnancy placenta respectively. Aminoterminal microsequencing of proteins allowed the identification of three distinct wbPAG sequences wich have ben deposed in the SwissProt database: RGSXLTIHPLRNIRDFFYUG (Acc. n. P85048), RGSXLTILPLRNIID (P85049) and RGSXLTHLPLRNI (P85050). Their comparison to those previously identified revealed that two of them were new since they have not been described yet. Our results confirm the suitability of VVA chromatography in enrichment of multiple PAG molecules expressed in buffalo placenta. Productions of specific antisera can be very useful in immonoistochemical and immunocyitochemical studies of PAG expression in fetomaternal interfaces. Purified native PAG are also required for development on specific immoassays (RIA/ELISA) currently used for pregnancy diagnosis and physiological investigation in farm animal.

Histo-morphology of the Uterus and Early Placenta of the African Buffalo ( Syncerus caffer) and Comparative Placentome Morphology of the African Buffalo and Cattle ( Bos taurus)

Differences exist in reproductive physiology between African buffalo (Syncerus caffer), cattle (Bos taurus) and water buffalo (Bubalus bubalis). The aim of this study was to histo-morphologically compare the anatomy of non-pregnant and pregnant uteri of buffalo and cattle. Two non-pregnant uteri and placentae of six pregnant African buffalo were used. Early placentome formation (fetal crown rump length (CRL): 2-17.5 cm) in S. caffer and B. taurus was compared. The endometrium of buffalo uteri comprises round to ovoid, dome-shaped and gland-free caruncles. A predominantly simple columnar epithelium of non-ciliated cells covers caruncular tissue, while, additionally, ciliated cells occur in the epithelium of the intercaruncular areas and within the simple columnar or pseudostratified epithelium of the endometrial glands. During early gestation, multiple placentomes develop. Unlike the placentomes in cattle at similar CRL, buffalo placentomes do not develop a caruncular stalk. The sessile, dome-shaped buffalo placentome has simple, slightly conical villi branching less than in cattle, thus indicating different and less complex feto-maternal interdigitation than seen in the latter. A synepitheliochorial interhaemal barrier can be expected in the buffalo placenta, as the occurrence and ultrastructure of trophoblast giant cells resemble those described in cattle.

118 TRANSFER OF IRON FROM MOTHER TO FETUS IN WATER BUFFALO: ERYTROPHAGOCYTOSIS AND UTEROFERRIN

The transplacental transport of iron by uteroferrin (Uf), and the hemophagous areas in the water buffalo placenta were analyzed to clarify the mechanism of blood extravasation in the materno-fetal interface with consequent transfer of iron to the fetus through the trophoblastic erythrophagocytosis (Murai and Yamauchi 1986 Nippon Juigaki Zasshi 48, 75–88) and in the endometrial glands (Bazer et al. 1991 Exp. Hematol. 19 910–19 915). In the water buffalo placenta this mechanism remains unclear; uteroferrin is very important in this process because it is both an iron transporter and a progesterone-induced hematopoietic growth factor. Our objective was to characterize these hemophagous areas and the endometrium of the water buffalo, focusing in the materno-fetal transfer of iron. Small pieces of the placentomes and interplacentomal region of water buffaloes (Bubalus bubalis bubalis, n = 51) in all of the gestation periods were fixed in 4% paraformoldehyde, and 2.5% glutaraldehyde in PBS, processed, and stained for light and transmission electron microscopy to characterize the hemophagous areas and endometrial glands morphologically, histochemically (Perls, acid phosphatase reaction), and immunohistochemically with rabbit anti-pig uteroferrin antibody to confirm the iron transfer. The hemophagous areas were present in the placentome from 4–10-months-pregnant placentae. The Perls reaction for ferric iron staining was negative in the placentome and positive in the endometrial glands, and the acid phosphatase reaction to detect phagocytic activity was positive in the placentome as well as in the interplacentomal region. The uteroferrin immunohistochemical reaction was positive in the trophoblast, mainly in determined regions of the materno-fetal interface and in other points deep in the placentome, and the endometrial glands showed a strong reaction in the epithelium and in the lumina. The ultrastructure of the hemophagous areas revealed ingested erythrocytes inside the epithelial cells of trophoblasts, endocytic vesicles, and caveolae. The endometrial gland epithelium is of the columnar type with microvilli and basal nuclei. The results obtained mainly by histochemistry and immunohistochemistry indicated that the hemophagous areas and endometrial glands are very important sites for iron transfer in water buffalo, and are thus involved in the regulation of fetal hematopoiesis. This work was funded by FAPESP.

127 VILLOUS ARCHITECTURE AND FETO-MATERNAL INTERDIGITATION IN THE AFRICAN BUFFALO (SYNCERUS CAFFER) DURING DIFFERENT GESTATION STAGES

Anchorage of the ruminant placenta is achieved by complementary indentation of chorionic villi into endometrial crypts. Geometrical patterns of interdigitation vary greatly among ruminants. Fetal villosity has been studied in other bovid species (Bos taurus, Bubalus bubalis) closely related to the African buffalo. The present study provides first information on villous architecture and feto-maternal anchorage in the African buffalo. Tissue samples from six pregnant cows (crown/rump length in cm: 2, 3, 14, 17, full-term) were collected during disease eradication programs in the Hluhluwe Game Reserve and from a buffalo breeding centre at Phalaborwa (afterbirths, n = 3). Tissue was fixed with 2.4% glutaraldehyde in Millonig's phosphate buffer by immersion or, if possible, via vascular perfusion through uterine and umbilical arteries prior to separation of feto-maternal tissue for scanning electron microscopy (SEM) preparation. Samples were further prepared for light microscopy and SEM using standard techniques. SEM samples were examined in a Philips XL 20 Microscope operated at 7 kV. Fetal villi consisted of a vascularized mesenchymal core covered by trophoblast epithelium. At a crown/rump length of 2 and 3 cm, the cotyledons comprised numerous short finger-like villi (length = 0.15–0.46 mm) originating from the chorionic plate. Some of these primary (stem) villi divided longitudinally a short distance from their origin. These relative simple villi were accommodated within corresponding caruncular crypts. At later gestation (crown/rump length: 14 and 17 cm), fetal villi had lengthened (length = 2–7.5 mm) and lateral (secondary) branching had started. The latter appeared in the form of bulbous or elongated structures projecting at approximately right angles from the stem villus. During the last month of gestation and in post-partum samples, primary (8–12 mm) and secondary villi had developed considerably in length. Secondary villi displayed a complex array of tertiary (terminal) villi of variable shape and size. A rich capillary network was observed within the connective tissue core of the tertiary villi as well as in the corresponding endometrial septae forming the walls of the caruncular crypts. Fully developed, each primary villus with its attendant secondary and tertiary villi formed a slender conical-shaped unit, also referred to as a cotyledonary villous tree. This paper provides the first description of villous architecture and placentome development in the African buffalo. The specific branching pattern of cotyledonary villi and complementary caruncular crypts in the African buffalo placenta, as described in this study, appears less complex when compared to that in the placenta of related Bos and Bubalus species.

Microvascular architecture of the fetal cotyledons in water buffaloes ( Bubalus bubalis) during different stages of pregnancy

To elucidate the morphological background of physiological differences between bovine and buffalo gestation forty-two placentae ranging from the 3rd to 10th month of pregnancy were used to study the microvascular architecture of the fetal cotyledons in the buffalo. The tissues were prepared for light and scanning electron microscopy by paraformaldehyde fixation and corrosion casting of the fetal cotyledonary vascular system. Histology and vascular casts revealed the buffalo fetal cotyledons to consist of a series of conical villous trees changing from a wide to slender shape during pregnancy, and with a base strictly facing the fetal side. The branches of these trees, intermediate and terminal villi, projected radially from the stem, thus representing a rough type of villous tree. A second type of tree lacked these branches and was therefore termed smooth villus. The rough type was most prevalent, and the intermediate and terminal villi showed capillary complexes arranged in stories by the 4th to 5th month of gestation. The stories became broader and denser with the progress of pregnancy (6th to 7th month of gestation), due to extensive growth of new capillaries and simultaneous development of convolutions causing the vascular ridges of the terminal villi to appear bushy. Near term (9th to 10th month) the capillary system became very dense, particularly at the margin of the vascular ridges, leaving only narrow spaces for the corresponding maternal septal tissue. In detail, at its base the trunk of each villous tree contained a single central stem artery which originated from the allantochorionic arterial system, and 1-3 parallel peripheral stem veins. When approaching the cone tip, these vessels branched into new stem arteries and veins, each giving rise to arterioles and venules according to the principle vascularization of the stem villus first, followed by intermediate and terminal villi. The capillary complex of the terminal villi consisted of arterial capillary limbs, capillary loops with sinusoidal dilatations and anastomoses, and venous capillary limbs. The latter converged into venules of terminal and intermediate villi which joined stem veins leading into allantochorionic veins. In conclusion, the fetal vasculature of the buffalo placentome was greatly increased from early pregnancy to near term. This was denoted by the general development of stem villous trees and an increase in the volume and density of the capillary system of the terminal villi, reflecting the increasing need of materno-fetal substance exchange in the buffalo placenta, particularly near term.