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The influence of the extracellular matrix (ECM) and ECM components on preadipocyte development was examined in primary cultures of adipose tissue stromal-vascular (S-V) cells. Extracellular matrix derived from Engelbreth-Holm-Swarm (EHS) cells or tumors enhanced several aspects of adipogenesis in vitro. In comparison to uncoated and fibronectin substrata, EHS-ECM substratum markedly increased attachment, spreading, and hypertrophy of preadipocytes while antagonizing spreading of non-preadipocytes. In addition, adipocyte number increased (P < .05) on these substrata despite no increase in total cell number: this resulted in a greater (P < .05) proportion of preadipocytes. These effects of EHS-ECM were also observed with laminin substrata per se, whereas types I and IV collagen and fibronectin had no influence. In contrast to all other substrata, adipocyte number decreased and total cell number increased 2.5-fold on ECM derived from corneal endothelial cells; this resulted in the lowest proportion of preadipocytes. Challenging cultures with adipogenic media (+serum) did not counter the inhibitory influence of corneal endothelial ECM, whereas dexamethasone partially neutralized the inhibitory influence of this ECM. These studies clearly show that source or type of the ECM dictated the influence of ECM substrata on preadipocyte development in primary S-V cultures. However, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development. Key Words: Adipocytes, Extracellular Matrix, Differentiation, Tissue Culture

Fetal hypophysectomy (hypox) slightly enhances lipid accretion in adipose tissue and markedly increases fat cell size and de novo lipogenesis. Thyroxine ([T.sub.4]) treatment (14 to 20 d) had no influence on fat cell size but markedly increased lipogenesis, lipid accretion, and apparent fat cell number in hypox fetuses. Although vascular development is intimately associated with preadipocyte development in the fetus, little is known concerning the regulation of vascular development per se by [T.sub.4] and other adipogenic hormones. Therefore, the purpose of the present study was to evaluate the development of qualitative traits of the adipocyte-associated vasculature after fetal hypox with and without [T.sub.4] treatment. Type IV collagen immunoreactivity of and lectin binding by capillaries in large fat cell clusters was reduced by hypox, and binding of the soybean agglutinin lectin was completely lost. Reactivity for cytosolic markers in capillaries was also reduced by fetal hypox. These blood vessel traits were not influenced by [T.sub.4] treatment. The quantitative aspects of capillary development in large fat cell clusters were not influenced by hypox or [T.sub.4] treatment. On a qualitative basis, blood vessels in the skin and in small fat cell clusters were not affected by hypox. Lectin binding by adipocytes was enhanced by hypox regardless of the size of clusters, and [T.sub.4] augmented this response to hypox. These studies demonstrate that endocrine regulation of the differentiation of the adipocyte-associated vasculature is more complex than the endocrine regulation of adipocyte differentiation per se. Key Words: Fetus, Hypophysectomy, Capillaries, Adipocytes, Cytochemistry

The objective of this study was to determine whether AIMAX (a dithiocarbamoylhydrazine derivative) is suitable for determining the effects of norepinephrine (NE) depletion on reproduction in domestic animals. Therefore, the effect of AIMAX (n = 6) on concentrations of biogenic amines in the medial basal hypothalamus (MBH) and anterior hypothalamic area (AHA) of ovariectomized (OVX) rats primed with ovarian steroids was compared to that of diethyldithiocarbamate (DDC; n = 5), a potent dopamine-beta-hydroxylase (DBH) inhibitor, which is chemically similar to AIMAX. Rats that received only ovarian steroids and saline injections served as controls (n = 6). Treatment with DDC resulted in sedation and reduced body temperature. In contrast, rats behaved normally after AIMAX treatment. AIMAX reduced (P < .05) NE but increased (P < .05) dopamine (DA) concentrations in MBH and AHA compared with controls. Similar changes in NE and DA concentrations were observed in DDC-treated rats. However, elevated epinephrine (EPI) levels were measured in MBH and AHA of only DDC-treated rats. Serum LH concentrations were suppressed (P < .005) in both AIMAX- and DDC-treated rats compared with control animals. Because AIMAX, like DDC, suppressed hypothalamic NE content and LH secretion, AIMAX should be useful in studying effects of NE depletion on gonadotropin secretion in domestic animals. Key Words: Hypothalamus, Catecholamines, AIMAX, Rats

In the present study, a technique is described for measuring proliferation of primary porcine preadipocytes. Cultures of stromal-vascular (S-V) cells from dorsal subcutaneous adipose tissue were maintained under several growth conditions. Cells were isolated from cultures at various times and DNA was labeled with propidium iodide (PI) and analyzed by flow cytometry. Treatment of cultures with colchicine allowed identification of S-V cells in mitosis. The relative proportion of replicating cells was dependent on several factors, including serum concentration and cell density at the time of harvest. Using the AD-1 anti-preadipocyte/adipocyte monoclonal antibody, the preadipocyte subpopulation within the S-V cells was identified. Furthermore, by labeling cells with PI concomitant with AD-1, the proportion of replicating preadipocytes was quantified. It is now possible to identify and quantify replicating preadipocytes rapidly under various experimental conditions.

: Hypophysectomy of the pig fetus alters the number of fat cells and fat cell clusters as well as lipogenic enzyme activities in tissues compared with littermate controls. The objective of the present study was to determine whether the altered endocrine environment in vivo affected subsequent development and differentiation of preadipocytes in vitro. Hypophysectomy was performed in utero at 70 d of gestation. At 105 d of gestation, stromal-vascular cells from dorsal subcutaneous adipose tissues were cultured in several differentiation media. Overall, stromal-vascular cultures from hypophysectomized fetuses contained fewer fat cell clusters and concomitantly lower glycerol-3-phosphate dehydrogenase specific enzyme activity than did cultures from littermate controls. Fluorescence-activated flow cytometry using a monoclonal anti-adipocyte/preadipocyte antibody designated AD-1 revealed that hypophysectomy also reduced the proportion of cells expressing the AD-1 cell surface antigen. These results demonstrated that effects of fetal hypophysectomy on cells from adipose tissues are maintained and measurable in culture. In addition, one of the consequences of fetal hypophysectomy may be diminution in the relative proportion of adipocyte precursors. Key Words: Adipocytes, Hypophysectomy, Differentiation, Pigs, Fetus, Flow Cytometry