Degradation of phagocytosed spermatozoa by mouse peritoneal macrophages in vitro

Abstract

Summary. Mouse peritoneal macrophages were cultured in vitro in the presence of autologous spermatozoa. The spermatozoa were found to be phagocytosed and degraded, with accumulation of pigment in the cytoplasm, during the first 5 days.Phagocytosis of spermatozoa by macrophages is seen in two lesions of the epididymis: sperm granulomata, in which the spermatozoa have escaped from a ruptured ductule (Glassy & Mostofi, 1956), and 'brown patches', in which the phagocytosis occurs in the lumen of an obstructed ductule (Mitchinson, Sherman & Stainer-Smith, 1975). In both conditions the phagocytosed spermatozoa appear to be converted to cytoplasmic pigment granules resembling lipofuscin.This mechanism might be important in the origin of autoantibodies to spermatozoa. The present study was an attempt to produce sperm phagocytosis in vitro, by culturing mouse peritoneal macro¬ phages with autologous spermatozoa, as a first step in the closer examination of the phenomenon.Eight male mice (strain B10.D2 from the Laboratory Animals Centre, Carshalton, Surrey, U.K.), 3 months of age, were decapitated and injected i.p. with 3 ml sterile Hank's balanced salt solution (HBSS : Flow Laboratories, Irvine, Scotland). After gentle massage, the peritoneum was opened and the fluid was recovered with a syringe, diluted 1:9 with HBSS and dispensed in 1 ml aliquots to each culture dish (35 10 mm: Falcon Plastics, Oxnard, California). Ten dishes, 5 'experimental' and 5 'control' for each mouse, were allowed to stand for 20 min while the macrophages adhered to the bottom and were then washed twice with 1 ml HBSS.The epididymides of each mouse were sliced in 1 ml sterile culture medium so that spermatozoa could be collected without contamination by other cells. The culture medium contained the following constituents in 100 ml: 32-93 ml Eagles modified MEM + Hank's salts + 0-35 g NaHC03/l; 32-93 ml Hank's lactalbumin; 32-93 ml heat-inactivated bovine fetal serum; 1-1 ml 200 mM-L-glutamine solution; and 0-11 ml of a solution of 50 mg gentamicin/ml (all materials from Flow Laboratories).'Control' dishes received 1 ml culture medium and 'experimental' dishes received 0-9 ml culture medium +0-1 ml sperm suspension from the same mouse.The dishes were then gassed with 95 % air + 5 % C02 and incubated for up to 5 days at 37°C. At 24-h intervals the medium was replaced with fresh medium without spermatozoa. Every day 2 dishes, one 'experimental' and one 'control' for each mouse, were withdrawn. The cells were fixed with 10% formol saline, stained with haematoxylin and eosin, and cleared and mounted in epon resin. The percentage of cells containing sperm heads and yellowish pigment was then counted, using xlOOO magnification. It was difficult to decide whether or not there was any pigment in some cells; these were counted as positive in 'control' dishes and negative in 'experimental' dishes. Results and discussionSome of the sperm suspensions showed immediate head-to-head agglutination, the aggregates ranging from several spermatozoa to about 50. Suc...

The passage of spermatozoa to regional lymph nodes in testicular lymph following vasectomy in rams and boars

“…Spermatozoa were recognized in lymph vessels adjacent to such lesions in these experiments and a similar observation has been made with human specimens (Friedman & Garske, 1949; King, 1955;Glassy & Mostofi, 1956 (Ball & Mitchinson, 1977).…”

“…The spermatozoa appear to be degraded with the production of a ceroid pigment (Pearce, 1972) and the presence of small clusters of macrophages containing such material in the paracortex implies migration of sinus macrophages into the nodal pulp. Such a migration may be important in the development of the immune response (Hoefsmit, Kamperdijk & Balfour, 1980 (Ball & Mitchinson, 1977).…”

Frequency and structure of macrophages and abnormal sperm cells in guinea fowl semen

“…The ultrastructure of the nucleus in guinea fowl macrophages also was different from the macrophages in the semen and epididymis of humans and monkeys (Alexander, 1972 ;Phadke, 1975). The phagocytic process appeared to be the same as reported for the phagocytosis of spermatozoa by mouse peritoneal macrophages (Ball and Mitchinson, 1977), which bind the head region first, form phagocytic vesicles, and accumulate residual bodies and undigested microtubules and nuclear material . However, the phatocytic activity was not directed toward the abnormal spermatids.…”

Further observations on spermiophagy by murine peritoneal macrophages in vitro

“…The suspensions were incubated at 37°C for 45-60 min to allow the macrophages to adhere, then vigorously washed twice with HBSS to remove non-adherent cells. The HBSS was replaced by 1 ml samples of culture medium (Ball & Mitchinson, 1977) and the cells were incubated overnight at 37°C in an atmosphere of 5% C02 in air.…”

“…Alternatively, the macrophage may process and present phagocytosed sperm antigens so that they are more likely to induce autosensitization, as is known in many experimental systems (Humphrey, 1982). Ball & Mitchinson (1977) have previously shown that mouse spermatozoa are degraded by macrophages to ceroid pigment within a few days, but the relevance of sperm degradation to antigenicity prompted a study of the earlier events in this process.…”