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We have developed a method to separate and isolate the mesenchymal cells from the epithelial cells in the left Müllerian duct of the developing chick. We then cultured the mesenchymal cells in a serum-free medium. Through an enzyme-linked immunosorbent assay, we detected fibronectin synthesis and release into the medium at stages of Müllerian duct development. Our results demonstrate that the amount of fibronectin secreted by cultured cells gradually decreased in accordance with Müllerian duct differentiation. Similar observations found in the developing embryonic intestine indicate that the highest fibronectin synthesis occurs during early stages of development, when morphogenetic movement and mesenchymal-epithelial interaction are prominent features of embryonic organ differentiation and growth.

The synthesis of cellular and nuclear basic proteins in the first generation of the repopulated pachytene spermatocyte (RPS) and the control pachytene spermatocyte (CPS) was studied by the incorporation of 3H-arginine into proteins. After pretreatment with gossypol for various lengths of time, the synthetic activity for cellular protein in the RPS was slightly inhibited. In comparison to CPS, on the other hand, the inhibition ranged from 12% to 26% (for gossypol pretreatment from 3 to 12 weeks, respectively). However, the synthetic activity for nuclear basic protein in the RPS was drastically reduced in comparison to that of the CPS. The reduction ranged from 46% to 61% in RPS after receiving gossypol pretreatment for 3 to 12 weeks, respectively. Gel electrophoretic separation of the basic protein extracted from the pachytene cell indicated that the major basic proteins are nucleosomal linker and core proteins, i.e., histone H1, H2A,B, H3, and H4, and with a lesser amount of sperm-specific BP and X1 proteins. After gossypol treatment in the RPS, the synthesis of sperm-specific proteins (BP and X1) and core histones (H2A and H4) became drastically reduced. Finally, the effect of gossypol on the ratio of nuclear basic protein to DNA in RPS and the direct correlation of this ratio to nucleosomal spacing and chromatin structure are discussed.

Mature male rats were treated with gossypol for various lengths of time after which the treatment was stopped to allow the arrested spermatogonia to revive. Fifteen days after withdrawal of the drug treatment, the arrested spermatogonia entered into the meiotic division and then reached the pachytene stage. The meiotic cells derived from the arrested spermatogonia were the first generation of the differentiated germ cells after long-term gossypol treatment. Centrifugal elutriation technique was used to isolate the repopulating pachytene spermatocytes (RPS) or the control pachytene spermatocytes (CPS) from the rat testis with or without receiving gossypol pretreatment. In vitro culture condition for RPS and CPS was established for the synthesis of DNA by the measurement of 3H-thymidine incorporation. The activity for DNA synthesis in RPS was studied and compared to that of the CPS. It was concluded that after gossypol treatment for various times, the incorporation of 3H-thymidine into the cellular thymidine pool of RPS was not affected. However, the activity for DNA synthesis in RPS was significantly lower than that in the CPS. The synthetic activity for DNA was reduced by 14%, 26%, 42%, 40%, and 40% in the RPS for the gossypol pretreatment of 3, 4, 6, 7, and 8 weeks, respectively.