Deposition of collagen III and alterations in basement membrane integrity as candidate prognostic markers in prostate cancer.

The extracellular matrix surrounding the tumor undergoes changes in its organization during the metastasis process. The present study aims to quantify total collagen, collagen I (Col I) and collagen III (Col III), analyze the alignment of collagen fibers and assess the basement membrane integrity in samples from patients with metastatic and non-metastatic prostate cancer. Tissue samples from 60 patients were classified into groups based on prognostic parameters: better prognosis (n = 20), worse prognosis without metastasis (n = 23) and metastatic (n = 17). Picrosirius red with further analysis under polarizing microscope was used to quantify (with validation using immunohistochemistry) and analyze collagen alignment, and Periodic Acid Schiff staining was used to analyze the basement membrane integrity. The Col I/Col III ratio was found to be higher in the metastatic group than in the groups with better prognosis (p = 0.012) and worse prognosis without metastasis (p = 0.018). Basement membrane integrity constitution in malignant tumor tissue differed from that of adjacent non-tumor tissue (p < 0.001). Moreover, the worsening in the tumor tissue integrity was positively correlated with worse prognostic parameters. All in all, absence of Col III and basement membrane integrity might be indicators of poor prognosis in prostate cancer.

Cellular and molecular antiproliferative effects in 2D monolayer and 3D-cultivated HT-29 cells treated with zerumbone.

Zerumbone (ZER) is a phytochemical isolated from plants of the Zingiberaceae family. Numerous studies have demonstrated its diverse pharmacological properties, particularly its potent antitumorigenic activity. This study aimed to assess the antiproliferative effects of ZER on HT-29 cells cultivated in both two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture systems. The evaluation of growth (size), cell death, and cell cycle arrest in 3D spheroid HT-29 cells was correlated with mRNA expression data. Treatment of 2D cells revealed that ZER exhibited cytotoxicity at concentrations above 30 µM, and an IC50 of 83.54 µM (24-h post-ZER treatment) effectively suppressed cell migration. In the 3D model, ZER induced an increase in spheroid volume over a 72-h period attributed to disaggregation and reconfiguration of characteristic zones. Analysis of cell death demonstrated a significant rise in apoptotic cells after 24 h of ZER treatment, along with cell cycle arrest in the G1 phase. Furthermore, ZER treatment resulted in alterations in mRNA expression, affecting key signaling pathways involved in cell death (BCL2 and BBC3), endoplasmic reticulum stress (ERN1), DNA damage (GADD45A), cell cycle regulation (CDKN1A, NFKB1, MYC, and TP53), and autophagy (BECN1 and SQSTM1). These findings suggested that ZER holds promise as a potential candidate for the development of novel anticancer agents that can modulate crucial cell signaling pathways. Additionally, the use of the 3D culture system proved to be a valuable tool in our investigation.

Placental development in the early stages of red-rumped agouti pregnancy (Dasyprocta leporina Linnaeus, 1758).

BACKGROUND: Hystricomorpha rodents display a similar placentation model to humans. The present study was carried out considering the scarcity of information concerning the placental development in agouti. OBJECTIVE: Describe the microscopy of the placenta, subplacenta and yolk sac of agoutis in early pregnancy and report on the inversion of the yolk sac. METHODS: Fifteen females between the 14th-32nd day of gestation were used following euthanasia. Gestational buttons were collected, fixed, processed, stained to optical microscopy or immunohistochemistry. RESULTS: Chorioallantoic placenta (CP) ranged from conical to a half-sphere, as follows: from the 14th to 17th day, the CP displays an inverted "V" shape, predominantly formed by cytotrophoblasts; from 20 to 22 days, formed almost entirely by cytotrophoblasts; at 28 days, a half sphere, with distinct lobes and interlobular area, numerous maternal gaps delimited by syncytiotrophoblasts and trophoblast giant cells; at 32 days, globose and undergoing the maturation process. Subplacenta, located between decidua and CP, initially presents septa consisting of simple columnar epithelium and after 17 days, comprising stratified epithelium. Visceral yolk sac (VYS) is attached to two CP projections between 14 and 17 days, formed by a simple cubic epithelium and inverted. Between 20 and 22 days, the epithelium displays apical villous projections with cytoplasmic vacuoles and a vascularized mesoderm. After the 24th day, the VYS near the placenta is pleated, very vascularized and villous, with decreased villi sizes further away from the placenta. CONCLUSION: The agouti CP displays similar characteristics to other hystricomorpha, including placenta lobulation, a subplacenta and an inverted vitelline placenta.

Emergency cesarean section in dogs: Usefulness of amniotic fluid biochemical parameters and placental morphology as indicators of neonatal viability.

In recent years, special attention has been paid to the analysis of fetal fluids and placental histopathology to identify parameters that can be used as indicators of maternal reproductive quality, embryonic viability, and fetal and neonatal health. Newborn health reflects the functioning of the fetal adnexa and its relationship with maternal tissues. Therefore, evaluating these components is promising for the early detection of newborns at risk. This study aimed to detect the biochemical characteristics of the amniotic fluid (AF) and histopathological characteristics of the placenta for comparison between canine neonates born by elective (EL) and emergency (EM) cesarean sections (CSs) and associate the results with neonatal viability in the first 24 h. A total of 38 neonates born by ELCS (n = 19) and EMCS (n = 19) were selected. AF was collected to analyze the concentration of its biochemical components [alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine, urea, total protein, albumin, total and direct bilirubin, lactate, glucose, potassium, chloride, calcium, and sodium]. Histopathological processing of the placenta was used to describe the lesions and identify the arrangement of collagen fibers using hematoxylin-eosin and picrosirius staining. There was an increase in ALP activity (P = 0.035) and the concentrations of lactate (P < 0.001) and potassium (P = 0.031), and a decrease in chlorides (P < 0.001) in the AF of neonates in the EMCS group. The comparisons between the groups did not show differences between the presence and extent of lesions in the placenta; however, a difference was observed in the arrangement of collagen fibers in the placental structure. A comparison between AF and histopathological findings showed a negative correlation (r = -0.609, P = 0.003) between glucose concentration and the presence of necrosis in the placental labyrinth. It was observed that the composition of the AF changed owing to the influence of the type of cesarean, possibly caused by prolonged hypoxia in cases of dystocia. ALP activity and lactate, potassium, and chloride concentrations in the AF might be explored as markers of neonatal health in EMCS. Under the conditions of this study, no correlations were found between the placenta's histopathological characteristics and the neonates' viability.

Diosgenin increases BBC3 expression in HepG2/C3A cells and alters cell communication in a 3D spheroid model.

Preclinical studies have shown that diosgenin, a steroidal sapogenin, is a promising phytochemical for treating different pathological conditions, such as cancer, diabetes, and cardiovascular diseases. However, the toxicological safety of this molecule for therapeutic use in humans needs to be better understood. Thus, this study aimed to evaluate the mechanisms of action of diosgenin in HepG2/C3A human hepatocellular carcinoma cells. Cytotoxicity, genotoxicity, alterations in the cell cycle, and cell death (apoptosis) were investigated and associated with the gene expression profile of pathways involved in these processes. The effects of diosgenin on the growth of spheroids were also tested. Diosgenin induced a dose-dependent reduction in cell viability and cell cycle arrest in S and G2/M phases and apoptosis in response to DNA damage. Apoptosis was associated with an increase in the expression of BBC3, a participant in the intrinsic apoptosis pathway. Diosgenin also promoted an increase in volume and greater cellular breakdown in spheroids. These results allowed a better understanding of the toxicity of diosgenin in human cells and contributed to the development of treatments based on this phytochemical.

Gonadal differentiation during embryonic and fetal development of male New Zealand rabbits (Oryctolagus cuniculus) / Diferenciación gonadal durante el desarrollo embrionario y fetal de conejos machos neozelandeses (Oryctolagus cuniculus)

SUMMARY: The rabbit is considered an ideal animal model for studies that describe abnormalities in the testicles due to the similar morphogenetic mechanisms of sexual development and diseases commonly found in humans. The aim of this study was to determine the male sexual differentiation of the New Zealand rabbit (Oryctolagus cuniculus) through development. The gestational age was estimated and classified as 9, 12, 14, 16, 18, 20, 23 and 28 gestational days. The morphological and sexual determination were performed by histological analysis of the reproductive tract in the embryos and fetuses (9-28 days) as well as by immunohistochemistry- Desert hedgehog-Dhh- (testis-specific protein on Y chromosome- 16, 20, 23 days and adult rabbits). Gonads were observed from the 14th day in an undifferentiated stage and with homogeneous aspect. Sexual differentiation was observed from the 16th day with presence of cells forming gonadal cords and Dhh+ cells in the gonadal parenchyma. From the 18th gestational day testicular cords were observed, which evolved into organized seminiferous tubules. The formation of the efferent ducts and ductus deferens and epididymis was observed on the 20th and 23rd days, respectively. The differentiation of the external genitalia occurred from the 23rd days from the anogenital distance and was identified to identify the penile structures. In summary, the features of the sexual differentiation were determined by observation of the Dhh+ protein in embryos from the 16th day to adulthood, and the morphological particularities observed from the 18th gestational day, determined by differentiation of the external genitalia from the 23rd day.

RESUMEN: El conejo se considera un modelo animal ideal para estudios que describen anomalías a nivel testícular debido a que presenta mecanismos morfogenéticos similares al desa- rrollo sexual y enfermedades que se encuentran comúnmente en los seres humanos. El objetivo de este estudio fue determinar la diferenciación sexual masculina del conejo de Nueva Zelanda (Oryctolagus cuniculus) a través del desarrollo. La edad gestacional se estimó y clasificó en 9, 12, 14, 16, 18, 20, 23 y 28 días gestacionales. La determinación morfológica y sexual se realizó mediante análisis histológico del tracto reproductivo en los embriones y fetos (9 - 28 días) así como mediante inmunohistoquímica -Desert hedgehog-Dhh- (proteína testicular específica en el cromosoma Y- 16, 20, 23 días y conejos adultos). Las gónadas se observaron a partir del día 14 en un estadio indiferenciado y con aspecto homogéneo. Se observó diferenciación sexual a partir del día 16 con presencia de células formadoras de cordones gonadales y células Dhh+ en el parénquima gonadal. A partir del día 18 de gestación se observaron cordones testiculares, que evolucionaron a túbulos seminíferos organizados. La formación de los conductos eferentes, deferentes y del epidídimo se observó a los 20 y 23 días, respectivamente. La diferenciación de los genitales externos ocurrió a partir del día 23 desde la distancia anogenital y se utilizó para identificar las estructuras del pene. En conclusión, las características de la diferenciación sexual se determinaron mediante la observación de la proteína Dhh en embriones desde el día 16 hasta la edad adulta, y las particularidades morfológicas observadas a partir del día 18 de gestación, determinadas por diferenciación de los genitales externos a partir del día 23.

Central nervous system development in rabbits (Oryctolagus cuniculus L. 1758).

The present study describes the embryonic and fetal development of the central nervous system in rabbits from the seventh day after conception until the end of the full-term fetal period. A total of 19 embryonic and fetal samples were carefully dissected and microscopically analyzed. Neural tube closure was observed between 7.5 and 8 days of gestation. Primordial encephalic vesicle differentiation and spinal canal delimitation were observed on the 12th day of gestation. Histologically, on the 15th day of gestation, the brain, cerebellum, and brain stem were delimited. On the 18th day of gestation, the cervical and lumbar intumescences of the spinal cord were visible. On the 28th day of gestation, four-cell layers could be distinguished in the cerebral cortex, while the cerebellar cortex was still differentiating. Overall, the morphological aspects of the embryonic and fetal developmental phases in rabbits were highly similar to those in humans. Thus, the present study provides relevant information highlighting rabbits as an excellent candidate animal model for preclinical research on human neurological diseases given the high adaptability of rabbits to bioterium conditions and the similarity of morphological events between rabbits and humans.

The morphology of paca (Cuniculus paca) testis with high dose of letrozole an aromatase inhibitor / La morfología del testículo de paca (Cuniculus paca) con altas dosis de letrozol, un inhibidor de la aromatasa

SUMMARY: The study reported the influence of the high and acute dose of Letrozole on the testis morphology in paca (Cuniculus paca), an aromatase inhibitor that reduces the endogenous estrogen, the essential hormone for spermatogenesis. Morphological changes were observed in seminiferous epithelium with germ cells with apoptotic characteristics and presence of vacuoles and nuclei in pycnose.

RESUMEN: El objetivo de este estudio fue analizar la influencia de una dosis alta de Letrozol en la morfología de los testículos de la paca (Cuniculus paca), un inhibidor de la aromatasa que reduce el estrógeno endógeno, la hormona esencial para la espermatogénesis. Se observaron cambios morfológicos en el epitelio seminífero con células germinales con características apoptóticas y la presencia de vacuolas y núcleos en picnosis.

Pluripotent stem cells proliferation is associated with placentation in dogs.

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.

Term placenta of the southern elephant seal (Mirounga leonina).

INTRODUCTION: The pinnipeds' placenta has been described as zonary, annular, labyrinthic and endotheliochorial, like that of the terrestrial carnivores. This article describes the placenta of Mirounga leonina, a phocid pinniped, focusing on some morphological features related to fetal nutrition. METHODS: Placental samples from three elephant seals were collected and conditioned after natural delivery at the Antarctic Specially Protected Area 132. Histological and ultrastructural studies were conducted; cytokeratins, vimentin, α-smooth muscle actin, and desmin proteins were detected using immunohistochemistry. RESULTS: The placentas were zonary, lobed, belt-shaped, and showed multiple vivid orange areas, which corresponded to bilirubin crystalline pigment found among chorionic villi and inside trophoblast cells. In the labyrinth, cytotrophoblast cells were isolated and there was a scant syncytium interposed between maternal and fetal vessels. Fetal vessels were small, round, and frequently intratrophoblastic, while maternal vessels were large, irregular, sinuous, and thin-walled. Vimentin and actin were detected in some scattered non-vascular cells throughout the labyrinth. Broad areas of degenerated and necrotic maternal components were also observed. DISCUSSION: The placentas of pinniped and fissiped carnivores share several traits. However, some remarkable features might maximize respiratory efficiency, collaborating to endure deep-diving hypoxia. Some of them, as the notably large sinuous maternal capillaries and fetal capillary indentation into the syncytium, are shared, e.g., by Phocidae and Mustelidae. Besides hemotropic nutrition taking place through an extremely narrow barrier, the abundant necrotic material and hematic products might allow substantial endocytosis of detritus even in term placentas, in this species giving birth to precocious offspring.

Skin development in 14 to 60-day-old Guinea pig fetus / Desarrollo de la piel del feto de conejillo de indias de 14 a 60 días

To contribute knowledge of an important experimental model for studies on skin embryology, a study was carried out to describe the morphological events of the skin during the intrauterine life of Cavia porcellus from the 10th to the 60th day of gestation. Embryos and fetuses were dissected, and the skin of the nasal, cranial, lumbar and anal regions was processed byoptical microscopy. At 30 days the first hairs, called lanugos, were observed in the cranial region. The morphological description showed that a few days can make a great difference in development.

Con el objetivo de contribuir al conocimiento de un importante modelo experimental para estudios sobre embriología de la piel, se llevó a cabo un estudio para describir los eventos morfológicos de la piel durante la vida intrauterina de Cavia porcellus desde el día 10 hasta el día 60 de gestación. Los embriones y los fetos se disecaron y se procesó la piel de las regiones nasal, craneal, lumbar y anal, mediante microscopía óptica. A los 30 días se observaron los primeros vellos, llamados lanugos, en la región craneal. La descripción morfológica mostró que unos pocos días pueden marcar una gran diferencia en el desarrollo.

Metabolites and Lipids Associated with Fetal Swine Anatomy via Desorption Electrospray Ionization - Mass Spectrometry Imaging.

Chemical imaging by mass spectrometry (MS) has been largely used to study diseases in animals and humans, especially cancer; however, this technology has been minimally explored to study the complex chemical changes associated with fetal development. In this work, we report the histologically-compatible chemical imaging of small molecules by desorption electrospray ionization (DESI) - MS of a complete swine fetus at 50 days of gestation. Tissue morphology was unperturbed by morphologically-friendly DESI-MS analysis while allowing detection of a wide range of small molecules. We observed organ-dependent localization of lipids, e.g. a large diversity of phosphatidylserine lipids in brain compared to other organs, as well as metabolites such as N-acetyl-aspartic acid in the developing nervous system and N-acetyl-L-glutamine in the heart. Some lipids abundant in the lungs, such as PC(32:0) and PS(40:6), were  similar to surfactant composition reported previously. Sulfatides were highly concentrated in the fetus liver, while hexoses were barely detected at this organ but were abundant in lung and heart. The chemical information on small molecules recorded via DESI-MS imaging coupled with traditional anatomical evaluation is a powerful source of bioanalytical information which reveals the chemical changes associated with embryonic and fetal development that, when disturbed, causes congenital diseases such as spina bifida and cleft palate.

Sexual determination and differentiation during embryonic and fetal development of New Zealand rabbit females / Determinación y diferenciación sexual durante el desarrollo embrionario y fetal de conejos hembras de Nueva Zelanda

The aim of this study was to know the embryonic and fetal development of the female rabbit genital system (Oryctolagus cuniculus), describing its main phases and the moment of sexual differentiation. Eleven pregnant New Zealand female rabbits were used in different gestational phases. The day of coitus was determined as day 0. For each stage a minimum of two animals was considered. The samples were obtained every two days from the ninth day post-coitus (dpc) until the 28th dpc. The gestational period was divided in two: animals with undifferentiated sex (group 1) and animals with differentiated sex (group 2). The ages of embryos and fetuses were estimated through the crown-rump method. Subsequently, embryos and fetuses were dissected, fixed and processed to be embedded in paraffin (Histosec). The histological analysis was performed on sections stained with hematoxylin and eosin. Immunohistochemical analysis to determine sexual differentiation was performed on samples from the 16th, 18th and 28th dpc. Desert Hedgehog (Dhh) and Indian Hedgehog (Ihh) primary antibodies, respectively, were used to identify cells of the male and female germinal epithelium. The immunohistochemical results showed that at the 16th dpc, female sexual differentiation was evident, since positive expression of the Ihh protein was observed. Sexual differentiation was obtained through histological analysis on the 18th dpc and through anatomical observation of the external genitalia on the 24th dpc. Knowing the characteristics of the embryonic and fetal development of the female rabbit genital system as well as the moment of sexual differentiation make it possible to establish bases for future research that address the physiology and pathology of these organs. Thus, any alteration in the chain of events of sexual determination and differentiation must search for an explanation from the knowledge of the possible normal mechanisms affected.

El objetivo de esta investigación fue conocer el desarrollo embrionario y fetal del sistema genital femenino de conejo (Oryctolagus cuniculus), describiendo sus principales fases y el momento de la diferenciación sexual. Se utilizaron 11 conejos hembras gestantes neozelandesas, en diferentes fases gestacionales. El día del coito se determinó como día 0. Para cada etapa fue considerado un mínimos de dos animales. Las muestras fueron obtenidas cada dos días, a partir del noveno día post-coito (dpc) hasta el 28 dpc. El periodo gestacional fue dividido en dos: animales con sexo indiferenciado (grupo 1) y, animales con sexo diferenciado (grupo 2). Las edades de los embriones y los fetos fueron estimadas a través del método de crown-rump. Posteriormente, embriones y fetos fueron disecados, fijados y procesados para su inclusión en parafina (Histosec). El análisis histológico se realizó en secciones teñidas con Hematoxilina y Eosina. El análisis inmunohistoquímico para determinar la diferenciación sexual fue realizado en muestras de 16, 18 y 28 dpc. Para identificar células del epitelio germinativo masculino y feminino se utilizaron los anticuerpos primarios Desert Hedgehog (Dhh) e Indian Hedgehog (Ihh), respectivamente. Los resultados inmunohistoquímicos mostraron que a los 16 dpc se evidenció diferenciación sexual femenina, ya que se observó expresión positiva de la proteína Ihh. La diferenciación sexual, a través del análisis histológico fue obtenida a los 18 dpc y a través de la observación anatómica de los genitales externos a los 24 dpc. Conocer las características del desarrollo embrionario y fetal del sistema genital femenino de conejo, así como, el momento de la diferenciación sexual, permiten sentar bases para futuras investigaciones que aborden la fisiología y patología de estos órganos. Así, cualquier alteración en la cadena de eventos de la determinación y diferenciación sexual deberá buscar una explicación a partir del conocimiento de los posibles mecanismos normales afectados.

Mid-pregnancy ewe shearing and the effects on fetus liver and muscle glycoprotein deposits.

The reason why shearing ewes in mid-pregnancy does increase the lamb birth weight is not completely clears. Therefore, we focused on the analyses of the deposition of glycogen in different fetal tissues to investigate this issue. Thirteen pregnant Australian Merino ewes, raised in native pasture, were separated in two groups. One group (n = 7) was shorn (SE) at 70 days of pregnancy, whereas another group (n = 6) remained unshorn (NSE). Cesarean section was conducted in all the ewes at near parturition, when placenta and fetuses sampling were collected. Placenta, liver and muscle samples were fixed and stained with glycoprotein-reactive acid-Schiff acid for analysis under light microscopy. The quantification of these glycoproteins was performed with the support of a program that analyzes the measurement of the intensity of staining by field. Five random fields from each sample were used, where statistical analyzes was used as normal test T. Among the analyzed regions, the deposition of glycoprotein between SE and NSE groups was statistically different in the hepatic portal vein (54,499.23 µm 2 in SE and 34,830.73 µm2 in NSE) and in the total muscle area of the sample fragment (41,128, 7 µm2 and 31,942.7 µm2 , respectively; P < 0.05). We conclude that shearing ewes at the 70th day of gestation lead to accumulation of glycoproteins in the liver and muscle of fetuses, which may be responsible for the increase in birth weights in that group.

Fetal Membranes-Derived Stem Cells Microenvironment.

Recently, the regenerative medicine has been trying to congregate different areas such as tissue engineering and cellular therapy, in order to offer effective treatments to overcome several human and veterinary medical problems. In this regard, fetal membranes have been proposed as a powerful source for obtainment of multipotent stem cells with low immunogenicity, anti-inflammatory properties and nontumorigenicity properties for the treatment of several diseases, including replacing cells lost due to tissue injuries or degenerative diseases. Morpho-physiological data have shown that fetal membranes, especially the yolk sac and amnion play different functions according to the gestational period, which are direct related to the features of the microenvironment that their cells are subject. The characteristics of the microenvironment affect or controls important cellular events involved with proliferation, division and maintenance of the undifferentiated stage or differentiation, especially acting on the extracellular matrix components. Considering the importance of the microenvironment and the diversity of embryonic and fetal membrane-derived stem cells, this chapter will addressed advances in the isolation, phenotyping, characteristics of the microenvironment, and applications of yolk sac and amniotic membrane-derived stem cells for human and veterinary regenerative medicine.

Phenotype and multipotency of rabbit (Oryctolagus cuniculus) amniotic stem cells.

BACKGROUND: Stem cells are capable of unlimited self-renewal and are able to remain undifferentiated for extended periods of time prior to their differentiation into specific cell lineages. Because of the issues (ethical and religious) involved in the use of embryonic stem cells and the limited plasticity of adult stem cells, an alternative cell source could be foetal stem cells derived from extra-embryonic tissue, which are highly proliferative, grow in vitro and possess interesting immunogenic characteristics. As a result, the amniotic membrane of several species has been studied as an important new source of stem cells. METHODS: Here, we cultured and characterized mesenchymal progenitor cells derived from the rabbit amniotic membrane, and investigated their differentiation potential. In total, amniotic membranes were collected from eight rabbit foetuses and were isolated by the explant technique. The obtained cells were cultured in DMEM-HIGH glucose and incubated at 37 °C in a humidified atmosphere with 5% CO2. RESULTS: The cells adhered to the culture plates and showed a high proliferative capacity with fibroblast-like morphologies. The cells showed a positive response for markers for the cytoskeleton, mesenchymal stem cells and proliferation, pluripotency and haematopoietic precursor stem cells. However, the cells were negative for CD45, a marker of haematopoietic cells. Furthermore, the cells had the capacity to be induced to differentiate into osteogenic, adipogenic and chondrogenic lineages. In addition, when the cells were injected into nude mice, we did not observe the formation of tumours. CONCLUSIONS: In summary, our results demonstrate that multipotent mesenchymal stem cells can be obtained from the rabbit amniotic membrane for possible use in future cell therapy applications.

Vascularization and VEGF expression altered in bovine yolk sacs from IVF and NT technologies.

Reproductive technologies are widely used in cattle, although many are associated with high-embryonic mortality, especially during early gestation, when the yolk sac undergoes macroscopic changes in structure. We hypothesized that vasculogenesis and angiogenesis are affected, thereby affecting embryonic and placental differentiation. To test this, we studied yolk sac development and gene expression of the vascular endothelial growth factor system (VEGF-A, VEGFR-1/Flt-1, VEGFR-2/KDR). Samples from Days 25 to 40/41 of pregnancy from control cattle (n = 8) and from pregnancies established with IVF, (n = 7) or somatic cell nuclear transfer/clones (n = 5) were examined by histology, immunohistochemistry, and quantitative reverse transcriptase PCR. Yolk sacs in IVF- and nuclear transfer-derived pregnancies were immature. Development of villi was sparse in IVF yolk sacs, whereas vascularization was barely formed in clones and was associated, in part, with thin or interrupted endothelium. Transcript levels of the genes characterized exceed minimum detection limits for all groups, except in the mentioned clone with interrupted endothelium. Levels of mRNA for VEGF-A and VEGFR-2 were significantly higher in IVF yolk sacs. Clones had substantial individual variation in gene expression (both upregulation and downregulation). Our data confirmed the broad range in expression of VEGF genes. Furthermore, overexpression in IVF yolk sacs may compensate for an immature yolk sac structure, whereas in clones, patchy expression may cause structural alterations of blood vessels. In conclusion, we inferred that disturbances of yolk sac vasculature contributed to increased early embryonic mortality of bovine pregnancies established with reproductive technologies.

Trend Toward Individualization of the Endocrine and Exocrine Portions of the Giant Anteater Pancreas (Myrmecophaga Tridactyla, Xenarthra).

Considering the physiological importance of the pancreas as an endocrine and exocrine organ, this study described the characteristics of the gross and microscopic morphology of this organ using 16 Myrmecophaga tridactyla individuals. The pancreas was located in the left antimere of the body, was pale in colour and exhibited an elongated shape with a central body and lobulated surface. It was positioned in the abdomen, following the curvatura ventriculi major of the stomach, and was attached to the initial portion of the duodenum. The corpus pancreatis was elongated and showed a caudal curvature of 45°. The pancreas exhibited a facies dorsalis (related to the spleen and stomach) and a facies ventralis (related to the renal capsule and intestine). Macroscopically, a craniodorsal, medial, and caudoventral regions were identified, in addition to the left lobe. Structurally, the organ exhibited two distinct parts: the first had exocrine characteristics, consisting of acini and ducts; the second, which was the endocrine portion, consisted of the pancreatic islets, which were located in the medial, caudoventral and left lobe regions. Ultrastructural analysis identified secretory vesicles containing zymogen granules, mitochondria, Golgi apparatus and rough endoplasmic reticulum in pancreatic centroacinar cells. Morphological data on the anatomy of members of the Xenarthra have revealed important peculiarities of several organs and systems, adding great biological value to the representatives of this group. In addition, these studies significantly contribute not only to knowledge of the biology, taxonomy and, consequently, preservation of these animals but also to the discovery of new experimental models. Anat Rec, 300:1104-1113, 2017. © 2016 Wiley Periodicals, Inc.

Development and maturation of the lung in fetuses of Galea spixii and expression of markers / Desenvolvimento e maturação do pulmão em fetos de Galea spixii e expressão de marcadores

ABSTRACT: The aim of this research was to study the development of the lung in Galea spixii , by gross anatomy, histological analysis and immunohistochemical techniques. Totally, 8 fetuses were used and allocated into three groups of age: Group I (33-35 days), Group II (38-40 days), and Group III (43-45 days) with given crown-rump - CR lenghts. According to the gross morphology, there were no differences in relation to the lung morphology among groups. In relation to lung maturation, the Group I showed early formation of the bronchi and bronchioles, which were richly surrounded by mesenchyme and small blood vessels, typical features of the pseudoglandular stage. Individuals from Group II showed higher amounts of tubular formations in the lung parenchyma and reduced mesenchyme, reaching the canalicular stage. The lung from individuals of the Group III was completely formed, reaching the alveolar phase. In the immunohistochemical analysis, the lung of individuals from both Groups I and II were positive for Pcna, Oct-4, and VEGF. In contrast, there was not labbeling on samples from Group III. Thus, the G. spixii lung anatomy resembles the agouti ( Dasyprocta sp.) lung, in relation to the number of lobes and fissures. In regard to the lung development, G. spixii showed more similarity with the human fetal lung, since both are born in the stage of the alveolar phase, different to other rodent species.

RESUMO: Buscou-se estudar o desenvolvimento do pulmão do preá ( Galea spixii ) com ênfase nas características macroscópicas, histológicas e imunohistoquimicas, bem como acompanhar a proliferação celular. Para tanto, foram utilizados 8 fetos, os quais, de acordo com o tamanho (crown-rump - CR), foram divididos em 3 grupos: Grupo I (33-35 dias), Grupo II (38-40 dias) e Grupo III (43-45 dias). Macroscopicamente, não houve diferenças quanto à morfologia do pulmão entre os grupos. Em relação à maturação pulmonar, no grupo I, observou-se o início da formação dos brônquios e bronquíolos, os quais estavam rodeados por grande quantidade de mesênquima e vasos sanguíneos de pequeno calibre, caracterizando o estágio pseudoglandular. Os indivíduos do Grupo II apresentaram maior quantidade de formações tubulares no parênquima pulmonar, assim como redução da quantidade de mesênquima, atingindo, dessa forma, a fase canalicular. No Grupo III, os pulmões apresentaram-se completamente formados, atingindo a fase alveolar. Nas análises de imunohistoquimica, os pulmões dos indivíduos dos Grupos I e II apresentaram marcação para Pcna, Oct-4 e VEGF. Por outro lado, não houve marcação no Grupo III. A anatomia do pulmão do preá se assemelha ao pulmão da cutia ( Dasyprocta sp.) quanto ao número de lobos e fissuras. No que se refere ao desenvolvimento pulmonar, o pulmão do preá apresentou maior similaridade com o pulmão de fetos humanos, visto que ambos, ao nascer, encontram-se na fase alveolar, contrário a outras espécies de roedores.

Development of the central nervous system in guinea pig (Cavia porcellus, Rodentia, Caviidae) / Desenvolvimento do sistema nervoso central em guinea pig (Cavia porcellus, Rodentia, Caviidae)

This study describes the development of the central nervous system in guinea pigs from 12th day post conception (dpc) until birth. Totally, 41 embryos and fetuses were analyzed macroscopically and by means of light and electron microscopy. The neural tube closure was observed at day 14 and the development of the spinal cord and differentiation of the primitive central nervous system vesicles was on 20th dpc. Histologically, undifferentiated brain tissue was observed as a mass of mesenchymal tissue between 18th and 20th dpc, and at 25th dpc the tissue within the medullary canal had higher density. On day 30 the brain tissue was differentiated on day 30 and the spinal cord filling throughout the spinal canal, period from which it was possible to observe cerebral and cerebellar stratums. At day 45 intumescences were visualized and cerebral hemispheres were divided, with a clear division between white and gray matter in brain and cerebellum. Median sulcus of the dorsal spinal cord and the cauda equina were only evident on day 50. There were no significant structural differences in fetuses of 50 and 60 dpc, and animals at term were all lissencephalic. In conclusion, morphological studies of the nervous system in guinea pig can provide important information for clinical studies in humans, due to its high degree of neurological maturity in relation to its short gestation period, what can provide a good tool for neurological studies.(AU)

Este estudo descreve o desenvolvimento do sistema nervoso central em guinea pig do 12º dia pós-concepção (dpc) até ao nascimento. No total, 41 embriões e fetos foram analisados macroscopicamente e por microscopia de luz e eletrônica. O fechamento do tubo neural foi observado no dia 14 e o desenvolvimento da medula espinhal e diferenciação das vesículas primitivas do sistema nervoso central foram observados no dia 20. Histologicamente, o tecido cerebral indiferenciado foi observado como uma massa de tecido mesenquimal entre os dias 18 e 20 e no 25º dia o tecido no interior do canal medular apresentou maior densidade. No dia 30 o tecido cerebral apresentou-se diferenciado, período no qual a medula espinhal preenchia todo o canal vertebral e foi possível observar os estratos cerebral e cerebelar. No dia 45 as intumescências cervical e lombar foram visualizadas e os hemisférios cerebrais estavam divididos, com uma clara distinção entre substância branca e cinzenta no cérebro e cerebelo. O sulco mediano dorsal da medula espinhal e a cauda equina foram evidentes apenas no dia 50. Não houve diferenças estruturais significativas em fetos de 50 e 60 dpc e animais a termo eram todos lisencefálicos. Estudos morfológicos do sistema nervoso em guinea pig podem fornecer informações importantes para estudos clínicos em seres humanos devido ao alto grau de maturidade neurológica em relação ao seu período de gestação curto, fato que servir como excelente ferramenta em estudos neurológicos.(AU)