Iberian red deer need to be conserved for their economic role and for their genetic importance as an important component of the ecosystem. Modifications currently being made to traditional management systems require a better understanding of the structure, function and development of their alimentary system. Here we describe a histomorphometric and immunohistochemical analysis of the stomach of 25 red deer embryos and fetuses from 30 days of gestation until birth (235 days). Differentiation of the reticular compartment from the primitive gastric tube begins at 67 days, forming a three-layered structure: epithelium, pluripotential blastemal tissue and serosa. The primitive reticular cells are initiated as small epithelial evaginations (primary ribs) at 117 days. At 142 days, lateral growths appear from the primary reticular ribs, forming the corneum papillae. The secondary reticular ribs form at 142 days as growths from the primary ribs. The uneven height of primary and secondary reticular ribs leads to the formation of cells of varying size. Growth of the reticular ribs involves the lamina propria but not the submucosa, so clear separation of these layers is maintained during histodifferentiation. Formation of the tunica muscularis from the pluripotential blastemal tissue begins at 67 days of intrauterine life, as two layers of longitudinally and circularly arranged myoblasts. Differentiation of the muscularis from the mucosa occurs at approximately 205 days, as longitudinal projections of the internal bundles of the tunica muscularis form the musculature of the primary ribs. The secretion of neutral and acid mucopolysaccharides by the reticular epithelial layer begins at 67 days, establishing the gradual adaptation of the mucosa to its protective function in postnatal life. Neuroendocrine (non-neuron enolase) and glial cells (glial fibrillary acidic protein and vimentin) were detected by immunohistochemistry, in a similar localization and intensity to that reported in the rumen. The neuropeptides vasoactive intestinal peptide and neuropeptide Y showed a positive immunoreaction in the reticular epithelium from 142 days of prenatal life, again earlier than reported for the rumen. In comparison with domestic ruminants, deer were shown to be less precocious with regard to development of gastric tube, in their capacity to secrete neutral mucopolysaccharides, and in their neuroendocrine nature, as determined by the detection of positive neuroedocrine and/or glial cells.

Morphometric and immunohistochemical study of the rumen of red deer during prenatal development.

This study sought to chart the ontogenesis of the goat rumen by histomorphometric examination, scanning electron microscopy and immunohistochemical analysis. A total of 140 goat embryos and fetuses were used, from the first stage of prenatal life until birth. The appearance of the rumen from the primitive gastric tube was observed at 35 days of prenatal life (CRL 3 cm, 23% gestation). By 38 days (CRL 4.3 cm CRL, 25% gestation) the ruminal wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Ruminal pillars were visible at 46 days (CRL 6 cm, 30% gestation), and by 76 days (CRL 18 cm, 50% gestation) ruminal papillae were starting to appear. Under scanning electron microscopy, by 50 days (CRL 7.7 cm, 33% gestation) small ruminal papillae were observed protruding from the surface. Finally, neuroendocrine cells (synaptophysin, SYP) were detected at 53 days (CRL 9 cm CRL, 35% gestation), while glial cell markers (glial fibrillary acidic protein--GFAP, and vimentin-VIM) were found at 108 days (CRL 31 cm, 72% gestation) and 39 days (CRL 4.4 cm, 26% gestation), respectively. Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) were detected immunohistochemically at 113 days (CRL 33 cm, 75% gestation) and 120 days (CRL 35 cm, 80% gestation), respectively. In conclusion, histomorphogenesis of the rumen in goats was similar to that reported in deer, but rather slower than observed for sheep or cattle.

Histomorphometric and immunohistochemical study of the goat rumen during prenatal development.

The structure of the pineal gland of 32 clinically healthy ovine embryos at different stages of development was studied. Embryos were arranged in four age groups, each containing eight embryos (four males and four females), defined in terms of the most relevant histological features: group 1 (27 to 69 days of prenatal development), group 2 (70 to 97 days), group 3 (98 to 116 days), and group 4 (117 to 150 days). At around 30 days of prenatal life, according to topographic criteria, the pineal outline begins to differentiate into a dorsal evagination of the diencephalic medium line, close to the anterior and posterior commissures. The growth of the pineal is biphasic. The ontogenic-proliferative phase begins at 30 days and includes the invasion of ependymal cells and the proliferation of the pineal parenchyma cells. The hypertrophic-differentiation phase includes the volume increment of the pinealoblasts and their differentiation into pinealocytes; this occurs at around 118 days. At around 98 days, the gland acquires its definitive compact appearance due to 1) glandular growth in constant volume and 2) the obliteration of pineal recess. The glandular structure displays a parenchyma made up of pinealoblasts, interstitial cells, and cells containing pigment. The pineal stroma is structured in pseudolobes formed by reticular and collagen fiber septae, which constitute together the interstitial cell prolongation net, which is the support structure of the whole glandular cytology. Capillaries are detected all over the glandular surface, being more abundant in the medullary zone. At around 98 days of prenatal development, VIP (Vasoactive Intestinal Peptide) positive fibers, distributed around blood vessels and among pinealoblasts were detected.

Structure of the ovine pineal gland during prenatal development

The aim of this study is to describe differences in the ontogenesis of the rumen in the sheep (domestic ruminant) and deer (wild ruminant). A total of 50 embryos and fetuses of Merino sheep and 50 of Iberian deer were used, from the first stages of prenatal life until birth. For the study, the animals were divided into five experimental groups according to the most relevant histological characteristics. The appearance of the rumen from the primitive gastric tube was earlier in the sheep (22% gestation, 33 days) than in the deer (25% gestation, 66 days). In both cases it displayed a primitive epithelium of a stratified, cylindrical, non-ciliary type. At around 28% gestation in the sheep (42 days) and 26% (67 days) in the deer, the rumen was configured of three clearly-differentiated layers: internal or mucosal, middle or muscular and external or serosal. In both species the stratification of the epithelial layer was accompanied by modifications in its structure with the appearance of the ruminal pillars and papillae. The pillars appeared before the papillae and the appearance of both structures was always earlier in the deer (pillars: 70 days, 27% gestation; papillae: 97 days, 36% gestation) than in the sheep (pillars: 42 days, 28% gestation; papillae: 57 days, 38% gestation). The outlines of the ruminal papillae appeared as evaginations of the basal zone toward the ruminal lumen, dragging in their formation the basal membrane, the lamina propria and the submucosa. The tegumentary mucosa of the rumen was without secretion capability in the first embryonic phases. From 67 days (26% gestation) the neutral mucopolysaccharides appeared in the deer and at 46 days (30% gestation) in the sheep. In both cases they continued to decrease until birth, this diminution being more pronounced in the deer. Finally, the presence of neuroendocrine and glial cells was detected in the deer at earlier stages than in the sheep.

Ontogenesis of the rumen: a comparative analysis of the Merino sheep and Iberian red deer.

Light and electron microscopic immunocytochemical observations were made to demonstrate central pinealopetal fibers immunoreactive for γ-aminobutyric acid (GABA) and synapses between their terminals and pinealocytes in the pineal gland of four rodent (Wistar-King rat; mouse; Syrian hamster, Mesocricetus auratus; Hartley strain guinea pig) and one nonrodent (tree shrew, Tupaia glis) species. GABA-immunoreactive myelinated and unmyelinated fibers and endings were found in the parenchyma of the pineal gland of all the animals examined. In the rodent species, GABAergic fibers were mainly found in the intermediate and proximal portions of the pineal gland and were nearly or entirely absent in the distal portion of the gland. Abundant GABAergic fibers were evenly distributed throughout the gland of the tree shrew. In all the animals, the habenular and posterior commissures contained abundant GABA-positive fibers, and some of them were followed to the pineal gland. GABA-positive endings made synaptic contact with pinealocytes, occasionally in mice and guinea pigs, and frequently in tree shrews; no synapses were observed in Syrian hamsters and rats. In the pineal gland of all the animals, GABA-immunoreactive cell bodies were not detected, and sympathetic fibers were not immunoreactive for GABA. These data indicate that GABAergic fibers are main pinealopetal projections from the brain. In view of the difference in the distribution of these fibers, central GABAergic innervation may play a more significant role in nonrodents than in rodents. The frequent occurrence of GABAergic synapses on pinealocytes in the tree shrew suggests that GABA released at these synapses directly controls activity of pinealocytes of this animal. J. Comp. Neurol. 430:72–84, 2001. © 2000 Wiley-Liss, Inc.

Central GABAergic innervation of the mammalian pineal gland: a light and electron microscopic immunocytochemical investigation in rodent and nonrodent species.

Ultrastructural and immunohistochemical techniques were used to study the second cell type in sheep embryo pineal glands. Thirty-two embryos were studied from day 54 of development through birth. Specimens were arranged in four age groups, defined in terms of the most relevant histological features: Group 1 (54-67 days of prenatal development), Group 2 (71-92 days), Group 3 (98-113 days), and Group 4 (118-150 days). At 98 days, a second cell type was observed which differed from pinealoblasts and showed uniform ultrastructural characteristics similar to those of astrocytes in the central nervous system. Ultrastructural homogeneity was not matched by the results of histochemical and immunohistochemical analysis: while all Type II cells stained positive to phosphotungstic acid hematoxylin, only 50% expressed glial fibrillary acidic protein. In the course of ovine intrauterine development, the vascular affinity of this second cell population, composed of glial-like or astrocytic cells at varying stages of maturity, leads to the formation of a limiting pineal barrier. This barrier may constitute the morphological expression of a hypothetical functional involvement in the exchange of substances between blood and pineal parenchyma.

Antonio J. Masot

Papers

A combined immunohistochemical and electron microscopic study of the second cell type in the developing sheep pineal gland