Histology and histopathology Vol.30,nº11 (2015)

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Open Access
Effect of boric acid supplementation of ostrich water on the expression of Foxn1 in thymus
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2015) Xiao, Ke; Rahman Ansari, Abdur; ur Rehman, Zia; Khaliq, Haseeb; Song, Hui; Tang, Juan; Wang, Jing; Wang, Wei; Sun, Peng-Peng; Zhong, Juming; Peng, Ke-Mei
Foxn1 is essential for thymus development. The relationship between boric acid and thymus development, optimal dose of boric acid in ostrich diets, and the effects of boric acid on the expression of Foxn1 were investigated in the present study. Thirty healthy ostriches were randomly divided into six groups: Group I, II, III, IV, V, VI, and supplemented with boric acid at the concentration of 0 mg/L, 40 mg/L, 80 mg/L, 160 mg/L, 320 mg/L, 640 mg/L, respectively. The histological changes in thymus were observed by HE staining, and the expression of Foxn1 analyzed by immunohistochemistry and western blot. TUNEL method was used to label the apoptotic cells. Ostrich Foxn1 was sequenced by Race method. The results were as following: Apoptosis in ostrich thymus was closely related with boric acid concentrations. Low boric acid concentration inhibited apoptosis in thymus, but high boric acid concentration promoted apoptosis. Foxn1- positive cells were mainly distributed in thymic medulla and rarely in cortex. Foxn1 is closely related to thymus growth and development. The nucleotide sequence and the encoded protein of Foxn1 were 2736 bases and 654 amino acids in length. It is highly conserved as compared with other species. These results demonstrated that the appropriate boric acid supplementation in water would produce positive effects on the growth development of ostrich thymus by promoting Foxn1 expression, especially at 80mg/L, and the microstructure of the thymus of ostrich fed 80 mg/L boric acid was well developed. The supplementation of high dose boron (>320mg/L) damaged the microstructure of thymus and inhibited the immune function by inhibiting Foxn1 expression, particularly at 640mg/L. The optimal dose of boric acid supplementation in ostrich diets is 80 mg/L boric acid. The genomic full-length of African ostrich Foxn1 was cloned for the first time in the study.
Open Access
Flutamide alters β-catenin expression and distribution, and its interactions with E-cadherin in the porcine corpus luteum of mid- and late pregnancy
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2015) Grzesiak, Malgorzata; Mitan, Agata; Janik, Marcelina E.; Knapczyk-Stwora, Katarzyna; Slomczynska, Maria
This study examined whether flutamideinduced androgen deficiency during mid- and late pregnancy in pigs affected luteal expression of adherens junction protein, β-catenin, and its interactions with Ecadherin. Flutamide (50 mg/kg body weight) was administered into pregnant gilts between days 43-49 (GD50F), 83-89 (GD90F) or 101-107 (GD108F) of gestation. Corpora lutea (CLs) were obtained on day 50, 90 or 108 of pregnancy (n=8-11 per each group). Total β-catenin and E-cadherin expression was examined at mRNA (real-time PCR) and protein (Western blot) level. Moreover, subcellular β-catenin fractions were extracted and immunoblotted. Immunohistochemistry was used for β-catenin localization. To determine whether flutamide disturbs β-catenin/E-cadherin mutual interactions, coimmunoprecipitation using anti-β-catenin antibody was performed. Furthermore, phosphorylation of Ecadherin was assessed. Flutamide exposure led to decreased β-catenin mRNA expression in all examined groups (p<0.001 or p<0.01), but protein level was lower only in the GD90F and GD108F groups (p<0.05). Ecadherin mRNA (p<0.05 or p<0.01) and protein (p<0.05) levels were up-regulated in all flutamidetreated groups when compared to controls. β-catenin was predominantly found in membranes of luteal cells with no significant changes after antiandrogen treatment. βcatenin/E-cadherin complexes were more abundant in the GD90F (p<0.05) and GD108F (p<0.01) groups than in controls due to enhanced E-cadherin phosphorylation at serine 838/840 in those animals (p<0.05). Overall, although androgen deficiency affected β-catenin expression in the CL of pregnancy in pigs, a compensatory mechanism by enhanced interactions with E-cadherin is possible. Thus, androgen signaling via androgen receptors appears to be crucial in the regulation of luteal cells cross-talk.
Open Access
Expression of myostatin in early postnatal mouse masseter and rectus femoris muscles
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2015) Takada, Hiroshi; Miwa, Yoko; Sato, Iwao
Aims: Myostatin (Mstn) is a member of the transforming growth factor-β (TGF-β) family that inhibits muscle differentiation. In this study, we aimed to identify the relationships between Mstn, thyroid hormone receptor alpha (TRα), and myosin heavy chain (MyHC) isoform expression during early postnatal development. Methods: We investigated the expression of Mstn, TRα, and MyHCs (embryonic, slow, IIa, IIb, and IIx) using quantitative real-time RT-PCR and ELISA (Mstn) in postnatal mouse muscles between day 0 and day 10. We also examined the correlations between Mstn, TR and MyHCs during the early development of mouse masseter muscle (MM) and rectus femoris muscle (RFM). Results: Distinct Mstn mRNA expression patterns were observed in the two muscles despite nearly non-significant changes in the Mstn protein abundance in MM. The expression pattern of the TRα mRNA in the MM differed from that observed in the RFM. The expression of MyHC IIa, IIb and IIx mRNAs increased in the MM and decreased in the RFM from day 0 to day 10, whereas embryonic fiber MyHC mRNA expression was similar in both muscle types. Principal component analysis showed the existence of a correlation between: (1) TRα and MyHC, (2) Mstn and MyHC, and (3) TRα and Mstn in MM. The correlations were different in RFM and MM. Cluster analyses identified the distinct clusters: cluster 1, days 0-4 for the MM and day 0 for the RFM;cluster 2, day 6 for the MM and day 2 for the RFM; and cluster 3, days 8-10 for the MM and days 4-10 for the RFM. Conclusions: These data suggest that TRα influences MyHC expression in both muscle types. In addition, Mstn has a limited effect in the MM related to the expression of individual MyHCs, as opposed to its role in the RFM, at early postnatal developmental stages. TRα could be involved in regulating both the temporal expression of MyHCs and Mstn at the early postnatal stages in the MM and RFM.
Open Access
Cell viability evaluation of transdifferentiated endothelial-like cells by quantitative electron-probe X-ray microanalysis for tissue engineering
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2015) Vico, Manuel; Rodríguez-Morata, Alejandro; Garzón, Ingrid; Campos, Fernando; Jaimes Parra, Boris; Pérez-Köhle, Barbara; Buján, Julia; Alaminos, Miguel; Sánchez-Quevedo, Mª Carmen
Development of an efficient vascular substitute by tissue engineering is strongly dependent on endothelial cell viability. The aim of this study was to evaluate cell viability of transdifferentiated endotheliallike cells (Tr-ELC) by using for the first time electron probe X-ray microanalysis (EPXMA), not only to accurately analyze cell viability by quantifying the intracellular ionic concentrations, but also to establish their possible use in vascular tissue engineering protocols. Human umbilical cord Wharton’s jelly stem cells (HWJSC) and endothelial cells from the human umbilical vein (HUVEC) were isolated and cultured. Transdifferentiation from HWJSC to the endothelial phenotype was induced. EPXMA was carried out to analyze HUVEC, HWJSC and Tr-ELC cells by using a scanning electron microscope equipped with an EDAX DX-4 microanalytical system and a solid-state backscattered electron detector. To determine total ion content, the peak-to-local-background (P/B) ratio method was used with reference to standards composed of dextran containing known amounts of inorganic salts. Our results revealed a high K/Na ratio in Tr-ELC (9.41), in association with the maintenance of the intracellular levels of chlorine, phosphorous and magnesium and an increase of calcium (p=0.031) and sulfur (p=0.022) as compared to HWJSC. Calcium levels were similar for HUVEC and Tr-ELC. These results ensure that transdifferentiated cells are highly viable and resemble the phenotypic and microanalytical profile of endothelial cells. Tr-ELC induced from HWJSC may fulfill the requirements for use in tissue engineering protocols applied to the vascular system at the viability and microanalytical levels.
Open Access
Ex vivo and in vivo modulatory effects of umbilical cord Wharton’s jelly stem cells on human oral mucosa stroma substitutes
(Universidad de Murcia. Departamento de Biología Celular e Histología, 2015) Alfonso-Rodríguez, C.A.; González-Andrades, E.; Jaimes-Parra, B.D.; Fernández-Valadé, R. s; Campos, A.; Sánchez-Quevedo, M. C.; Alaminos, M.; Garzon, I.
Novel oral mucosa substitutes have been developed in the laboratory using human umbilical cord Wharton’s jelly stem cells -HWJSC- as an alternative cell source. In the present work, we have generated human oral mucosa substitutes with oral mucosa keratinocytes and HWJSC to determine the influence of these cell sources on stromal differentiation. First, acellular and cellular stroma substitutes and bilayered oral mucosa substitutes with an epithelial layer consisting of oral mucosa keratinocytes -OM samplesor HWJSC -hOM- were generated. Then, tissues were analyzed by light and electron microscopy, histochemistry and immunohistochemistry to quantify all major extracellular matrix components after 1, 2 and 3 weeks of ex vivo development, and OM and hOM were also analyzed after in vivo grafting. The results showed that bioengineered oral mucosa stromas displayed an adequate fibrillar mesh. Synthesis of abundant collagen fibers was detected in OM and hOM after 3 weeks, and in vivo grafting resulted in an increased collagen synthesis. No elastic or reticular fibers were found. Glycoprotein synthesis was found at the epithelialstromal layer when samples were grafted in vivo. Finally, proteoglycans, decorin, versican and aggrecan were strongly dependent on the in vivo environment and the presence of a well-structured epithelium on top. The use of HWJSC was associated to an increased synthesis of versican. These results confirm the usefulness of fibrinagarose biomaterials for the generation of an efficient human oral mucosa stroma substitute and the importance of the in vivo environment and the epithelialmesenchymal interaction for the adequate differentiation of the bioengineered stroma.

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