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Free cell types have been defined in mammal pineal gland parenchyma: pinealocytes or principal cells, supportive or interstial cells and pigmented cells. Numerous terms have been used to designate the second type, including interstitial cells, type II pinealocytes, glial cells and astrocytes. Ultrastructural and immunohistochemical techniques were used to study the second cell type in sheep embryo pineal glands. 32 embryos were studied from day 54 of development through birth. Specimens were arranged in four age-groups, defined in terms of tho most relevant histological features: group 1 (54 to 67 days of prenatal development), group 2 (71 to 92 days), group 3 (98 to 113 days) and group 4 (118 to 150 days). Embryo pineal glands were sliced parasagitally after 1 hour in Carnoy's fluid. One of the two portions thus obtained was fixed in 10% neutral formalin saline and processed by paraffin-embedding methods. Sections 3 urn thick were cut and stained with hematoxylin and eosin (HE) for routine morphological examination, and with phosphotungstic acid hematoxylin (PTAH) for detection of glial-type cells. An avidin-biotin-peroxidase complex (ABPC) was carried out on deparaffinized pineal samples for detection of glial fibrillary acidic protoin (GFAP),the main proteincomponentof intermediateastrocytefilaments. 3 The other half of each pineal gland was used for ultrastructural analysis. Tissue blocks (1 mm) were immersed in ice-cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), and embedded in epoxy resin. Ultrathin sections cut from blocks obtained for electron microscopy were stained with colloidal gold (10 nm) for detection of GFAP posrtive cells. The pineal parenchyma in groups 1 and 2 displayed a undorm morphology; cells were all of one type, identified as pinealoblasts. In groups 3 and 4, in addition to pinealoblasls, a second cell type with different characteristics was detected. Cell morphology was similar in both these groups, and cells were generally located in the perivascular space. Nuclei were small, oval or rounded,and electron-dense, and thus readily distinguishable from the vesicular nuclei of pinealoblasts. Ultrastructural analysis confirmed light microscopic findings: a second cell type was evident in groups 3 and 4, in addition to the pinealoblasts present in all groups. Type II cells were less numerous, less electron-dense and showed a clear preference for perivascular locations. In group 3 these cells had oval nuclei, with a slender rim of chromatin bordering the nuclear envelope. The electron-lucent nucleoplasm, frequently contained a nucleolus of clearly-defined nucleolemma, The most characteristic feature of these cells was the presence of very long processes with numerous microfilaments. The endoplasmic reticulum was mostly granular; cisternae had fairly narrow lumina. Lysosomes wrth clearly-defined limrtingmembrane, diplosomes and ribosomes were observed in perinuclear cytoplasm. Ultrastructurally, type IIcells in group 4 closely resembled those observed in group 3, except that the former exhibited a greater number of filaments. Filaments showed a clear tendency to fuse with pinealoblast processes. A further differential characteristic was that type II cells in group 4 were more electron dense than those of group 3, due in part to the abundance of ribosomes, small electron-dense mitochondria, microfilaments and condensed chromatin. GFAP positive cells were observed in the embryonic pineal parenchyma in groups 3 and 4. In group 3 these were distributed uniformly throughout the gland (Fig. 1). Immunopositive cells (whose appearance resembled that of the CNS astrocytes used as positive control) displayed small, dense, ovoid nuclei, and an intensely-staining rim of cytoplasm bordering negative nuclei. GFAP positive cells displayed a small number of processes, with varying diameters and arrangedboth longitudinallyand transversally Cell processes were interwoven amongst pinealoblasts, and around blood vessels to form a limiting barrier. In group 4 embryos, GFAP positive cells were either oval or elongated in shape. Cytoplasmic processes, which were more numerous than in group 3, varied in both diameterand orientation. Figure 1. Embryo, 98 days. Positive staining cells (GFAP +) scattered throughout the pineal gland. ABPC x 350

The presence of supportive cells in the prenatal development of sheep pineal gland.