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Adult albino rats were injected intravenously with labelled homologous albumin, globulins and fibrinogen. Labelling was done with acid rhodamine B (C.I. No. 45100) and fluorescein isothiocyanate. The amount of fluorescent dye bound to proteins was measured spectrophotometrically. Paper electrophoretic mobility and clotting time of fibrinogen were also checked after labelling. As a control, free fluorescent dye solutions were injected into another lot of rats.Animals were killed at intervals from 10 minutes to 12 days. Blood samples were taken to determine spectrophotometrically the concentration of labelled proteins in the circulation. Paraffin or unfixed fresh frozen sections from all tissues were examined with the fluorescence microscope.Histoimmunological studies were also made using fluorescent antibody against the different rat serum fractions. Coons's technique was applied to tissue sections from normal rats or from animals previously injected with unlabelled serum fractions and killed between 3 and 12 hours later. The following principal observations were made:1) Globulins bind a larger amount of dye than albumin, while fibrinogen bound the least.2) Labelling does not change grossly the electrophoretic mobility of albumin and globulins but slightly modified the clotting time of fibrinogen.3) Time-concentration curves of albumin and globulins in the circulation are very much alike and reveal a fast and a slow component with 2.6 day time half-life for the former and 3.1 half-life for the latter.4) Histologically labelled albumin and globulins are seen first in the lumen and on the walls of all blood vessels, and up to 8 hours they appeared to accumulate widely in connective tissue structures as well as in basement membranes of dermis and the mucosae of digestive, respiratory and urogenital tracts; fluorescence does not cross the basement membranes in most organs, nor does it pass the perichondrium, periosteum, or blood capillaries in the nervous system into surrounding parenchyma. Finally, labelled proteins began to disappear from all these sites after 12 hours, but persist longer in the macrophages of liver, lymph nodes, spleen and bone marrow. Fibrinogen was not seen diffusing out from vessels to the surrounding connective tissue.5) That in animals previously injected with unlabelled serum fractions, histoimmunological techniques also showed the highest specific fluorescence in sections of connective tissue, stained with labelled antialbumin and antiglobulins; labelled antifibrinogen gave only a weak fluorescence in the same structutres.6) These results confirm and extend our previous studies on the role of connective tissue as a transient depot for the extravascular pool of serum proteins.

Rat serum proteins were labelled in vitrowith a fluorescent dye, acid rhodamine B, C.I. No. 45100, using Chadwick's method. Labeled serum was injected intravenously in albino rats weighing 150-200 gm, in doses of 0.8 cc/100 gm body weight. Biochemical and histological findings indicate that there is an equilibrium among serum proteins, connective tissue (as temporary place for storage of these) and reticuloendothelial system, thyroid, ovary and renal tubuli contorti cells (as a place for prolonged deposit of serum proteins).

It is well known that steroids secreted by the Leydig cells inhibit gonadotrophin secretion (Ramirez & McCann, 1965; Bogdanove, 1967). Three decades ago, it was suggested that some cellular component of the seminiferous tubule might regulate the secretion of pituitary gonadotrophins (Vigdoff, Hill, Vehrs & Kubin, 1939; McCullagh & Schaffenburgh, 1952). Although later findings (Allanson & Deanesly, 1962; Katsh & Duncan, 1968) seemed to offer some support to this hypothesis, no conclusive proof has yet been presented. Moreover, testicular extract or a glycoprotein substance related to germinal cells administered to intact and castrated male rats, failed to demonstrate an inhibitory effect on pituitary gonadotrophin concentrations (Debeljuk & Mancini, 1970). The purpose of the present work was to achieve selective damage of spermatogonia in rat testes, temporarily arresting the

Ferrocyanide was detected histologically in adult rat testes by the Prussian blue reaction from 3 min to 3 hr following intravenous injection. Ferrocyanide appeared in the lumen of the testicular blood vessels almost immediately following injection. After 10 min, ferrocyanide was detected in most seminiferous tubule walls and, 20 to 30 min later, Prussian blue deposits were localized in the lumen and epithelium of some seminiferous tubules. The results suggest that the continuity of the extratubular and intratubular ferrocyanide compartments is interrupted in part by tubular wall structures. Possible explanations for the intratubular diffusion of ferrocyanide are discussed.

A highly purified and potent hyaluronidase preparation extracted from bull testis was employed to induce heterologous antibody by sensitizing adult rabbits. Specific anti-enzyme antibodies were demonstrated by Ouchterlony's method, and by passive cutaneous anaphylaxis (pca), and haemagglutination and complement fixation tests. In order to localize the cellular site of the accumulation of the enzyme, direct and indirect immunofluorescent techniques were applied to tissue sections and to isolated germ cells of adult bull testis. Specific fluorescence adequately checked by control reactions appeared in the perinuclear area of spermatids, in the acrosomes of spermatozoa and with less certainty in the cytoplasm of other cells tentatively identified as spermatocytes. Non-specific fluorescence could be seen in the basal cell line of the seminiferous tubules.

Adult outbred guinea pigs were sensitized, without added adjuvants, with a single dose of homogenates prepared from homologous testis previously subjected to thermal injury in vivoor in vitro. It was observed: (A) With exception of positive results obtained with the sperm immobilizing test, no antisperm antibodies of anaphylactic, precipitating, and complement–fixing types were induced. (B) Delayed hypersensitivity, as revealed by skin test and inhibition of macrophage migration, appeared positive in parallel with testicular and epididymal damage similar to the so–called allergic orchitis. (C) Only homogenate prepared with acute thermal damaged testis was able to provoke such a response but not that prepared with testis having thermal chronic lesions. (D) Homogenates from normal adult testis or prepared with kidney having acute thermal nephritis gave negative results.

Experimental allergic orchitis is an organ-specific disorder, which is immunologically mediated, but interpretations of histopathological and immunological studies of the temporal sequence of events leading to aspermatogenesis still remain controversial. Some authors have held that degeneration of germinal cells of the testis is the predominant change which results in aspermatogenesis (Freund, Lipton & Thompson, 1953; Freund, Thompson & Lipton, 1955). On the other hand, destruction of the germinal epithelium has been described following the appearance of early mononuclear infiltrates in the intertubular space with direct invasion of the seminiferous tubules (Waksman, 1959), and the route of sperm passage through the rete testis, the ductulis efferentes and the caput epididymidis (Brown, Glynn & Holborow, 1963; Brown & Glynn, 1969; Johnson, 1970). Tung, Unanue & Dixon (1970) postulated that mononuclear cells can break through the basement membrane and penetrate into the seminiferous tubules. Infarction, oedema and ischaemia have all been causally related

Mancini, Vilar, Alvarez & Seiguer (1965) have demonstrated the passage through the blood—testis barrier of labelled serum proteins injected intravenously into rats. Horseradish peroxidase was earlier introduced as a tracer by Straus (1957a) and applied to the ultrastructural level by Karnovsky (1965) and Graham & Karnovsky (1966).Sedar (1966a, b) reported, in two abstracts, that horseradish peroxidase injected intravenously into the hamster became localized in the epididymis and ductuli efferentes, and in the rete testis. Reddy & Svoboda (1967) observed that horseradish peroxidase injected intravenously into the rat was taken up by the cells of the testis. Recently, Fawcett, Leak & Heidger (1970), studying the permeability of the blood—testis barrier in guinea-pigs, reported the uptake of horseradish peroxidase injected interstitially in the testis.The different results obtained