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The development of adipose tissue lipoprotein lipase (LPL) activity was determined cytochemically in sections from normal and decapitated pig fetuses. Cellular LPL activity was first evident in 60-day-old fetuses. Capillaries were stained for LPL activity in 85-day-old and older fetuses and in young pigs. Cellular synthesis of LPL and capillary staining were not concurrent events. LPL activity developed after adipocyte differentiation and after structural development of capillaries. The cellular and capillary staining of fat cell clusters throughout development and in decapitated fetuses indicated a possible inverse relationship between cellular synthesis of LPL and capability of capillaries to transport LPL to luminal sides. These observations indicate that LPL activity develops in a pattern quite distinct from that observed in vitro. This discrepancy may be due to the involvement of blood vessels in the in vivo developmental pattern.

Perirenal adipose tissue samples were obtained from fetuses removed from pregnant (crossbred) sows at 3 stages of gestation (70, 90 and 110 days). Phosphatase histochemistry, succinate dehydrogenase (SDH) histochemistry and factor VIII antigen immunocytochemistry were conducted on fresh-frozen cryostat sections. Age-associated changes in nucleosidediphosphatase (NDPase) reactions in the arteriolar system were correlated with the morphological development of the medial layer of arterioles and arteries. For instance, a strong NDPase reaction in small arterioles was associated temporally with the assumption of a normal smooth-muscle cell morphology and arrangement in the medial layer. Age-associated changes in blood vessel reactions for factor VIII antigen and alkaline phosphatase activity were not correlated with morphological development. In the youngest fetuses, alkaline phosphatase activity was evident in large and small arterioles, but in the oldest fetuses, alkaline phosphatase activity was restricted to the smallest arterioles and vessels associated with them. Arteriolar differentiation was demonstrable with either adenosine triphosphatase (ATPase) or inosine diphosphatase (IDPase) reactions. Primordial stromal cells around differentiated arterioles were reactive for ATPase but not for IDPase activities. In older fetuses, there were large areas that contained ATPase-reactive stromal cells, no adipocytes, differentiated (ATPase and IDPase) arterioles and few capillaries. Positive reactions for SDH were evident in the ATPase-reactive stromal areas that contained no adipocytes. Differentiated adipocytes were SDH- and ATPase-reactive. These data illustrate the utility of differential phosphatase histochemistry to identify adipose tissue primordia.

Subcutaneous adipose tissue was obtained from fetuses removed from pregnant obese (Ossabaw) and lean (crossbred) sows at three stages of gestation (70, 90, and 110 days). Histochemical analysis for nucleoside phosphatase (NPase), alkaline phosphatase (APase), and NADH tetrazoleum reductase (NADH-TR) was conducted on fresh-frozen cryostat sections. Age- associated changes in NPase and NADH-TR reactions in the arteriolar system were correlated with the morphological development of the medial layer of arterioles and arteries. For instance, a strong NPase reaction in small arterioles was associated temporally with the assumption of a normal smooth muscle cell morphology and arrangement in the medial layer. In the youngest fetuses, strong NADH-TR reactions were only evident in small and presumptive arterioles and venules (associated with fat cells). Little NADH-TR reactivity was evident in larger arterioles and venules in 70-day tissue. Arteries and large arterioles were distinguished from veins and venules (strong reactions vs. weak reactions) with NADH-TR and NPase reactions in the oldest fetuses. In the younger fetuses, the NPase distinction (arterioles vs. veinules) was obvious before NADH-TR distinction. Small adipocyte-associated vessels were APase positive in the youngest fetuses, but APase reactivity was limited to short segments of vessel between arterioles and capillaries in the oldest fetuses. With the following exceptions, all the above observations were independent of fetal strain. In obese fetuses (110 day) small venules and small arterioles were equally reactive for NPase activity. Capillaries in obese fetuses (110 day) were NADH-TR reactive, whereas no activity was evident in capillaries from lean fetuses (110 day). The histology of intimal and medial vessel walls was independent of fetal strain. However, at every fetal age the adventitial layer of large arterioles and the collagenous stroma (around fat cell clusters) was thicker and better organized in obese fetuses. In the present study, differential enzyme histochemistry was utilized to distinguish the differentiation of small adipocyte-associated arterioles from larger arterioles. Furthermore, structural evidence of the arteriolar system clearly precedes metabolic and histological differentiation of tunica media cells. Finally, metabolic and structural traits of the stromal vascular cells of adipose tissue were sensitive to a fetal obese pheno-type.

Histochemical and ultrastructural aspects of adipocyte differentiation in subcutaneous tissue of fetal pigs were analyzed in a longitudinal study. A matrix of collagen fibers surrounding adipocytes developed after the establishment of a distinct and continuous PAS-positive basement membrane. The degree of plasma membrane invagination and specialization was positively correlated with the extent of basement membrane and collagen matrix formation. Close spatial relationships between narrow, smooth endoplasmic reticulum, plasma membrane invaginations, the surface of lipid droplets and mitochondria were observed in differentiating adipocytes. Histochemical and ultrastructural criteria for the identification of preadipocytes are: (1) perivascular location; (2) mitochondria localized in the Golgi zone; (3) cytosolic glycogen; (4) rough endoplasmic reticulum with cisternae uniformly and approximately 600 Å wide; (5) free ribosomes and few polysomes, and (6) lipid droplets encased by microfilaments. These criteria permitted clear distinction from obvious fibroblasts and macrophages. Other stromal cells were morphologically abnormal. Occasionally, adipocytes and perivascular cells exhibited close intercellular contacts that were morphologically distinct from intercellular contacts between contiguous endothelial cells.

The relationship between mammary gland development and adipocyte formation in rat subcutaneous tissue was studied. Tissue samples from newborn rats and young rats were prepared for histological and histochemical analysis. The development of subcutaneous adipocytes in the young rat is associated temporally and spatially with mammary gland development. The growing mammary gland ducts and the blood vessels and nerves that serve the ducts may physically interact with connective tissue cells, resulting in a population of undifferentiated cells. Adipocytes and a wide variety of cell types may be derived from this pool of undifferentiated cells. Histochemical characteristics of cells of immature ends of mammary gland ducts indicated hypoxic conditions. These conditions may have directed a vascularization process that resulted in a network of adipocytes and capillaries around mammary gland ducts. Thus, under normal conditions, adipocyte formation is an integral phase of mammary gland development in the fetal and yount rat.

Connective tissue cells that are histochemically and morphologically distinct from ‘fibroblasts’ are localized around developing hair follicles in the pig and rat. Immature adipose tissue is limited to small areas immediately around fully descended hair follicles in the rat hypodermis. In the present study, connective tissue around large nerves and blood vessels in fetal pig subcutaneous tissue was examined for the presence of enzymes typical of adipocytes. Samples from decapitated pig fetuses were studied so that the effects of an altered hormonal profile could be examined. Samples of dorsal subcutaneous adipose tissue were obtained from fetuses at 65, 70, 85, 90, 110, and 112 days of gestation. Fetuses were decapitated in utero at 45 days of gestation, and adipose tissue samples were obtained from these fetuses at 110 days of gestation. A close spatial relationship was observed between the growth of large blood vessels and nerves and fat cell cluster development in the older ( > 70 days) fetuses. Connective tissue cells that were contiguous with fat cell clusters were histochemically identical to adipocytes. The lipid histochemistry of the reactive connective tissue cells (histochemically identical to adipocyte) was variable in young fetuses. In all 110- and 112-day-old fetuses, the reactive cells contained lipid, whereas the reactive cells in decapitated fetuses were devoid of lipid. The reactive connective tissue cells were not associated with capillaries and did not contain basement membranes. The histochemistry of these cells suggests that they respond to a particular hormonal or metabolic profile as do adipocytes. Connective tissue cells contiguous with fat cell clusters may represent a distinct stage of adipocyte differentiation between the undifferentiated state and the mature adipocyte.