The yolk sac (YS) is the main source of embryonic nutrition during the period when the placenta has not yet formed. It is also responsible for hematopoiesis because the blood cells develop from it as part of the primitive embryonic circulation. The objective of this study was to characterize the transitional area between the YS and primitive gut using the techniques of light microscopy, transmission electron microscopy, and immunohistochemistry to detect populations of pluripotent cells by labeling with Oct4 antibody. In all investigated embryos, serial sections were made to permit the identification of this small, restricted area. We identified the YS connection with the primitive intestine and found that it is composed of many blood islands, which correspond to the vessels covered by vitelline and mesenchymal cells. We identified large numbers of hemangioblasts inside the vessels. The mesenchymal layer was thin and composed of elongated cells, and the vitelline endodermal membrane was composed of large, mono‐ or binucleated cells. The epithelium of the primitive intestine comprised stratified columnar cells and undifferentiated mesenchymal cells. The transitional area between the YS and the primitive intestine was very thin and composed of cells with irregular shapes, which formed a delicate lumen containing hemangioblasts. In the mesenchyme of the transitional area, there were a considerable number of small vessels containing hemangioblasts. Using Oct4 as a primary antibody, we identified positive cells in the metanephros, primordial gonad, and hepatic parenchyma as well as in YS cells, suggesting that these regions contain populations of pluripotent cells. Microsc. Res. Tech. 76:756–766, 2013. © 2013 Wiley Periodicals, Inc.

Male capacity for spreading genes to a great number of descendents and to determine population dynamics depend directly on the genital organs. Morphological studies in pinnipeds are scarce and the functional meaning of some characteristics has never been discussed. We hypothesized that Arctocephalus australis (A. australis) shows morphophysiological adaptations in order to guarantee the perpetuation of the species in the unique annual mating season. Seven males, dead from natural causes, had their genital organs collected and fixed for morphological description. Some features differ from other described mammalian males and are closely related to the biology and reproductive cycle of this species, as the scrotal epidermis, absence of glandular portion in the ductus deferens and spermatogenic epithelium suggest a recrudescent testis period. The corona glandis exhibits a singular arrangement: its erectile border looks like a formation of petals and its association with the os penis gives a "lily-flower" form to this region. We propose the name margo petaliformis to this particular erectile border of the corona glandis because of its similarity to a flower corola. The male genital organs of A. australis show morphological features compatible with adaptation to environment requirements and reproductive efficiency.

The scrotal-testicular biometry was evaluated in goats raised in Piaui state, Brazil, presenting different levels of scrotal division, in rainy and dry periods of the year. For this study, eighteen male goats at mating age were accomplished and arranged into three groups (6 animals each), obeying the classification as goats with no scrotal bipartition (GI), goats showing scrotal bipartition up to 50% of testicular length (GII), and goats with more than 50% of scrotal bipartition (GIII). The biometry of the scrotal-testicular was made evaluating the scrotal length (SL), scrotal circumference (SC), testicular length (TL) and testicular volume (TV). The results were evaluated following the variance analysis (ANOVA) and the SNK test applied on the average comparisons. The analysis of the data demonstrated high values, in dry and rainy periods, of SC (24.63cm/ 26.97cm), SL (16.61cm/ 18.24cm), TL (5.32cm/ 5.93cm), TV (173.81cm³/ 203.01cm³). This supports the hypothesis of the influence of the period of the year and of the scrotal bipartition on the scrotal-testicular biometry in goat.

Low efficiency of somatic cell cloning by nuclear transfer has been associated with alterations of placental vascular architecture. Placental growth and function depend on the growth of blood vessels; VEGF-A and bFGF are the most important factors controlling neovascularization and vascular permeability in the placenta. We hypothesize that the VEGF-A and bFGF systems are disrupted in placentomes from cloned animals, contributing to the placental abnormalities that are common in these clones. We determined mRNA expression and protein tissue localization of VEGF-A, bFGF, and their receptors in placentomes from cloned and non-cloned bovine fetuses at term. Real-time RT-PCR revealed that VEGFR-2 mRNA was increased in cloned male-derived placentomes, while mRNA of bFGF and its receptors were decreased in placentomes of cloned females. VEGF-A system proteins were found to be located in placentomal endothelial, maternal and fetal epithelial and stromal cells; there was a variable pattern of cellular distribution of these proteins in both cloned and non-cloned animals. Alterations in the expression of VEGF-A and bFGF systems suggest that angiogenic factors are involved in abnormal placental development in cloned gestations, contributing to impaired fetal development and poor survival rates.

The umbilical cord blood (UCB) is an important source of pluripotent stem cells, which motivated researches on ontogeny and transplantation. The morphological characterization of umbilical cord cells is the first step to establish subsequent experiments on these areas. Although some information on humans can be found, no data on UCB is available for bovines. Therefore, this work is the first attempt to conduct an ultrastructural characterization of bovine umbilical cord blood. Blood was collected from the umbilical cord of twenty fetuses by punction of the umbilical vein. Samples were processed for whole leucocytes observation by centrifugation and the buffy coat was collected. Cells were washed and pelleted and prepared according to the standard protocol of the transmission electron microscopy. The presence of cells with morphologic characteristics compatible with the precursors from the erythrocytic, neutrophilic, eosinophilic, basophilic, and lymphocytic lineages was observed. Atypical cells with peculiar morphological features, strongly similar to apoptotic cells, were seen. Bovine neutrophils with three types of cytoplasmic granules were also found in the blood. The ultrastructural characteristics of observed bovine UCB cells where similar to those found in other species, suggesting that bovines could possibly constitute an experimental model for approaches on UCB cells research.

The functions of placental oestrogens during equine pregnancy are still unclear. Yet, they may act predominantly as local regulators of growth and differentiation in the microplacentomes. Thus, expression patterns of oestrogen receptors (ERs) alpha and beta were investigated in the microcotyledonary placenta from pregnant mares at 110, 121, 179, 199 and 309 days of gestation by immunohistochemistry. In microplacentomes, both the ER isoforms were detected in trophoblast (T) cells, chorionic villous stroma (FS), microcaruncular epithelium (ME) and microcaruncular stroma (MS). Proportions of positive cells were 38-91% (T), 11-41% (FS), 55-89% (ME), 17-51% (MS) for ERalpha and 66-76% (T), 21-37% (FS), 41-68% (ME) and 24-55% (MS) for ERbeta. Between days 110 and 199, proportions of cells positive for progesterone receptor (PR) varied between 19% and 62% (T), 3% and 50% (CS), 15% and 46% (ME), and 4% and 33% (MS). At day 309, PR was virtually absent in T, CS and ME (percentages < 0.1), whereas in MS 14.3% of cells were still positive. The expression of ERs and PR in equine microplacentomes gives evidence for a role of placental steroids as regulators of placental growth, differentiation and function. The detection of ERalpha, ERbeta and PR in foetal and maternal vascular tissue suggests that placental steroids are also involved in the control of placental angiogenesis and /or vascular functions. The co-localization of ERs with aromatase in T suggests auto- or intracrine functions of oestrogens in this cell type.

The study is based on 141 pregnant Bos indicus cows, from days 20 to 70 post-insemination. First, special attention was given to the macroscopically observable phenomena of attachment of the conceptus to the uterus, i.e. the implantation, from about days 20 to 30 post-insemination up to day 70, and placentome development by growth, vascularization and increase in the number of cotyledons opposite to the endometrial caruncles. Secondly, as for the conceptuses, semiquantitative, statistical analyses were performed of the lengths of chorio-allantois, amnion and yolk sac; and the different parts of the centre and two extremes of the yolk sacs were also analysed. Thirdly, the embryos/foetuses corresponding to their membranes were measured by their greatest length and by weight, and described by the appearance of external developmental phenomena during the investigated period like neurulation, somites, branchial arcs, brain vesicles, limb buds, C-form, pigmented eye and facial grooves. In conclusion, all the data collected in this study from days 20 to 70 of bovine pregnancy were compared extensively with corresponding data of the literature. This resulted in an 'embryo/foetal age-scale', which has extended the data in the literature by covering the first 8 to 70 days of pregnancy. This age-scale of early bovine intrauterine development provides model for studies, even when using slaughtered cows without distinct knowledge of insemination or fertilization time, through macroscopic techniques. This distinctly facilitates research into the cow, which is now being widely used as 'an experimental animal' for testing new techniques of reproduction like in vitro fertilization, embryo transfer and cloning.

The influence of the scrotal bipartition and of the year period on the scrotal-testicular thermal regulation was evaluated in male goats raised in Piaui State, Brazil. Eighteen male goats at mating age were accomplished in this study and arranged into three Groups (6 animals each) obeying the classification as goats presenting no scrotal bipartition (Group I), goats showing scrotal bipartition at 50% of the testicular length (Group II), and goats with more than 50% of scrotal bipartition (Group III). The scrotal, testicular and spermatic funiculi temperatures were evaluated invasively with the aid of a digital thermometer and non-invasive with a pyrometer in the proximal, medial and distal portion. The data were acquired during the dry (October-November) and rainy (February-March) period of the year, measured in two shifts: morning (6h00-7h00) and afternoon (14h00-15h00). The results were submitted to variance analysis (ANOVA) following the SNK test for average comparison (p<0.05). The year period interfered on the scrotal-testicular thermal regulation, due to increased temperatures of the scrotal, testicular and spermatic funiculi during the dry period in comparison with the rainy period. The bipartition level was also a factor which contributed to the influence of scrotal-testicular temperature regulation, due to lower average scrotal-testicular temperature rates observed during both periods in the goats with higher levels of scrotal bipartition (>50%). It is possible to conclude that with the experimental conditions applied on this study, the level of scrotal bipartition and the climatic conditions interfere with the scrotal-testicular thermal regulation in goats.

In the bovine synepitheliochorial placenta, restricted trophoblast invasion requires complex interactions of integrin receptors with proteins of the extracellular matrix (ECM) and integrin receptors of neighboring cells. Activated integrins assemble to focal adhesions and are linked to the actin cytoskeleton via signaling molecules including alpha-actinin (ACTN), focal adhesion kinase (PTK2 or FAK), phosphotyrosine, and talin (TLN1). Aims of this study were to assess integrin activation and focal adhesion assembly within epithelial cells of bovine placentomes and low-passage (not transformed) placentomal caruncular epithelial cells cultured on dishes coated with ECM proteins. Immunofluorescence analysis was performed to colocalize the signaling molecules ACTN, PTK2, phosphotyrosine, and TLN1 with each other and with beta(1)-integrin (ITGB1) in placentomal cryosections throughout pregnancy and in caruncular epithelial cells in vitro. Antibody specificity was confirmed by Western blot. Cells were cultured on uncoated dishes, and the dishes were coated with fibronectin (FN), laminin (LAMA), and collagen type IV (COL4), thereby statistically assessing cell number and qualitatively assessing the expression pattern of ITGB1, phosphotyrosine, and TLN1. Results demonstrated integrin activation and focal adhesion assembly in the placentome and that low-passage caruncular epithelial cells maintain integrin-associated properties observed in vivo. Expression and/or colocalization of signaling molecules with ITGB1 confirmed, for the first time, integrin activation and participation in "outside-in" and "inside-out" signaling pathways. The prominent role of ECM, and FN in particular, in integrin signaling is supported by the in vitro enhancement of proliferation and focal adhesion expression. Thus, this in vitro model provides excellent potential for further mechanistic studies designed to elucidate feto-maternal interactions in the bovine placentome.

OBJECTIVE To compare the content of substance P, vasoactive intestinal polypeptide, and neurofilament 200 in biopsy specimens taken from the abomasal wall of healthy cows of 2 breeds. SAMPLE POPULATION Biopsy specimens taken from different sites of the abomasal wall from 20 German Holstein cows and 20 German Fleckvieh cows. PROCEDURES Biopsy specimens were examined immunohistochemically, and the content of substance P, vasoactive intestinal polypeptide, and neurofilament 200 was determined by measuring the immunoreactive areas. RESULTS Significant differences between the breeds were detected. Substance P-immuno-reactive area in the corpus abomasi was significantly smaller in the German Holsteins (geometric mean +/- geometric SD, 679 +/- 1.83 microm2) than in the German Fleckvieh cows (1,020 +/- 1.65 microm2). Concerning vasoactive intestinal polypeptide, differences between breeds were not significant. Overall nerve density in the antral abomasal wall was significantly greater in German Holsteins than in German Fleckvieh cows (immunoreactive areas for neurofilament 200 in German Holsteins was 4,842 +/- 1.29 microm2 and in German Fleckvieh cows was 3,333 +/- 1.63 microm2). Conclusions and Clinical Relevance-The significantly lower content of substance P in the corpus abomasi could explain why German Holstein cows are predisposed to abomasal displacement, compared with German Fleckvieh cows, in which this disease is a rare finding.

Nasua nasua, coati, is a mammal of the Carnivora order and Procyonidae family. It lives in bands composed of females and young males. The pineal gland or epiphysis of brain is endocrine, producing the melatonin. Its function is the control of the cycle of light environment, characteristic of day and night. For this research, five adult coatis were used, originating from CECRIMPAS-UNIfeob (Proc. IBAMA 02027.003731/04-76), Brazil. The animals were killed and perfusion-fixed in 10% formaldehyde. Pineals were measured and a medium size was found to be 2.3-mm-long and 1.3-mm-wide. Pineal gland was located in the habenular commissure in the most caudal portion of the third ventricular roof, lying in a dorso-caudal position from the base to the apex. Pinealocytes were predominantly found in the glandular parenchyma. Distinct and heterogeneous arrangements of these cells throughout the three pineal portions were observed as follows: linear cords at the apex, circular cords at the base of the gland, whereas at the body a transition arrangement was found. Calcareous concretions could be observed in the apex. The pineal gland was classified as subcallosal type [Rec. Méd. Vét.1, 36 (1956)] and as AB type [Prog. Brain Res. 42, 25 (1979); The Pineal Organ, Berlin/Heidelberg: Springer-Verlag (1981)].

This study was conducted to investigate endometrial and placental structural changes that occurred in response to mid-gestational termination of pregnancy in queens using aglepristone, a progesterone receptor antagonist. Thirteen European Shorthair pregnant queens were either treated with aglepristone (10 mg/kg body weight; subcutaneously) twice on days 25 and 26 after first mating (am; group I; n = 9), or remained untreated and served as control (group II; n = 4). Queens of group I were ovariohysterectomized between days 30 and 41 am, either at the onset (n = 3) or during (n = 1) abortion and 12 h (n = 1), 24 h (n = 3) or 10 days after abortion (n = 1). Queens of group II were ovariohysterectomized on day 30 am. Tissue was collected from the cervix, and the interplacental zone as well as the paraplacenta/placental girdle of the uterus, subjected to standard histological procedures and evaluated using light microscopy. During abortion, gaps appeared within the paraplacenta and the placental girdle which were filled with blood, leading to an embryo-maternal disconnection. Blood was also observed within the uterine lumen as well as the interstitial mucosal stroma of the cervix and the placental girdle zone and probably originated from damaged venules, whilst arterioles remained intact. As the interval between abortion and surgery increased, the interstitial and luminal haemorrhages became less pronounced and completely disappeared except interstitial remnants 10 days after abortion. The endometrial regeneration was not fully completed on day 10 after abortion and a few cystically dilated glands were evident. In conclusion, abortion of queens through aglepristone given during mid-gestation is assumed to be the result of damage of uterine venules. This leads to an interstitial haemorrhages and bleeding into the uterine lumen, subsequently resulting in utero-placental detachment.

Background/Aims: Interaction of trophoblastic integrins with the extracellular matrix plays a role in embryo implantation and trophoblast invasion. The phenomenon of restricted trophoblast invasion, observed in the bovine epitheliochorial placenta offers intriguing conditions to study invasive processes. The migration of bovine trophoblast giant cells is accompanied by the expression of specific integrins and corresponding extracellular matrix ligands. Methods: Primary cultures of different cell populations from cow placentomes were established and characterized, and in vitro phenotypes were compared with in vivo conditions by immunofluorescence. Results: Propagated epithelial cells were positive for cytokeratin and vimentin, while fibroblasts contained α-smooth muscle actin, desmin and vimentin. Epithelial cells coexpressed integrin subunits α6 and β1 with laminin, and fibroblast cells were positive for αv, β3, fibronectin and laminin. In contrast to cells in vivo, cultured epithelial cells secreted fibronectin, while collagen IV was not detected. The occurrence of integrin subunits was confirmed at mRNA level by RT-PCR. Conclusion: We have established cell cultures isolated from maternal and fetal components of bovine placentomes expressing typical cytoskeletal filaments and integrin receptors also present in their in vivo counterparts. These bovine placentomal cells provide a suitable in vitro model for the study of cell-cell interactions.

Objectives: In bovine placentomes migrating trophoblast giant cells have been shown to express various growth factors. To relate these findings to placentomal function and growth, the expression of vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2, and total VEGF protein were quantitatively analysed in the course of gestation.