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Changes in levels of plasminogen activator activity in normal and germ-cell-depleted testes during development
259
Molecular and Cellular Endocrinology,
Elsevier/North-Holland
26 (1982) 259-267 Scientific Publishers, Ltd.
CHANGES IN LEVELS OF PLASMINOGEN IN NORMAL AND GERM-CELL-DEPLETED
ACTIVATOR ACTIVITY TESTES DURING DEVELOPMENT
Martial LACROIX, Fannie E. SMITH and Irving B. FRITZ Banting and Best Department (Canada)
of Medical Research,
Received 30 September 198l;accepted
University of Toronto,
Toronto MSG IL6
29 December 1981
Levels of plasminogen activator activity were determined in testes obtained from normal and irradiated rats of various ages. During normal development, plasminogen activator activity per g testis increased rapidly between 40 and 60 days of age, but a comparable rise did not occur in germ-cell depleted testes of irradiated rats. Levels of enzyme in various populations of testicular cells were highest in Sertoli cells (varying between 1800 and 6300 units/mg protein in cells maintained under different culture conditions), and lowest in peritubular myoid cells (about 1 unit/mg protein), with intermediate levels in germinal cells (ranging between 147 and 560 units/mg protein in residual bodies, spermatocytes and spennatids). No protease inhibitor could be detected in germ-cell extracts. The addition to the medium in which Sertoli cells were in culture of particles which can be phagocytosed (autoclaved E. coli) resulted in an increased formation of plasminogen activator activity by Sertoli cells. A synergistic enhancement of enzyme production resulted following the addition of submaximal quantities of dibutyryl cyclic AMP and autoclaved bacteria to Sertoli cells in culture. On the basis of these data, we suggest that the presence of advanced germinal cells during gonadal development may stimulate the synthesis of plasminogen activator by Sertoli cells, mediated in part by the phagocytosis of residual bodies by Sertoli cells which occurs prior to spermiation.
Levels of plasminogen activator activity in testes of normal rats increase during development, with the greatest increment occurring between 40 and 60 days of age (Lacroix et al., 1979). Preliminary data also indicated that Sertoli cells were the primary source of plasminogen activator in testis (Lacroix et al., 1979). In this communication, we report quantitative changes in testicular plasminogen activator activity during development, and we provide additional information on the amounts of plasminogen activator in peritubular myoid cells, spermatocytes, spermatids and Sertoli cells. Data to be presented support the conclusion that most of the plasminogen activator activity in testis arises from Sertoli cells, and that amounts produced by Sertoli cells increase during gonadal maturation.
0303-7207/82/0000-0000/$02.75
0 Elsevier/North-Holland
Scientific Publishers, Ltd.
260
MATERIALS
Martial Lacroix, Fannie E. Smith, Irving B. Fritz
AND METHODS
Germ-cellenriched preparations were obtained by the “Statput” sedimentation procedure described by Go et al. (1971). Germ-cell-depleted animals were obtained by irradiating (125 rad) pregnant rats at their 19th day of gestation (Beaumont, 1960); the male rats from the ensuing litters were raised to the appropriate age required in the experiments. The extent of germ-cell depletion was monitored by histological examination. The testes of ether-anesthetized rats were perfused through the testicular artery with Ca’+-depleted Hank’s buffered saline solution (HBSS-). Anesthetized animals were then killed by cervical dislocation, and testes were decapsulated, weighed and transferred to a glass homogenizer (Kontes No. 24) containing 10 ml 0.1 M TrisHCl (pH 8.1 at 32’) and 0.1% (v/v) Triton X-100 per gram of wet testicular tissue. Testes were homogenized (10 strokes with a motor-driven Teflon pestle), and aliquots of the supension, snap-frozen in liquid nitrogen, were kept at -60 “C until samples from all age groups had been accumulated. Sertoli cells maintained in culture for variable periods of time were washed twice with ice-cold HBSS-. The cells were scraped off the dish with a rubber policeman and centrifuged at 600 X g for 5 min. 10 vol. of cold 0.1 M Tris (pH 8.1 at 32 “C) were added to each volume of cell pellet, and the suspension was transferred to a conical glass homogenizer fitted with a glass pestle. Samples were homogenized, and aliquots were assayed immediately for plasminogen activator activity (Lacroix et al., 1977) or were stored at -60 “C as described above until analyzed. To assay protease inhibitor activity, we measured the possible effects of germcell extracts on the degradation of [ ‘251]fibrin by purified plasmin. To prepare the plasmin, we dissolved rabbit plasminogen, purified by the techniques of Deutsch and Mertz (1970) from plasma, in 75 mM Tris-HCl (pH 8.1 at 32 “C) containing 25% (v/v) glycerol. We incubated this solution for 6 h at 37 “C with 3000 Ploug units urokinase (Calbiochem, lot No. 901315) after which over 96% of the added plasminogen had been converted to plasmin. This plasmin, at a concentration of 0.83 pg/ml, degraded [ 1251]fibrin linearly at a rate of 15%/h for periods up to 3 h. The [ 1251]fibrin used as substrate in these assays had been coated onto the surface of 96-well (Falcon) culture plates as [125 I] fibrinogen (8.3 pg, containing approx. 1 X lo5 cpm per well) by procedures previously described (Strickland and Beers, 1976; Lacroix et al., 1977). On the day of the assay, the coated [1251]fibrinogen in each well was converted to [ ’ 251] librin by the addition of 0.2 unit bovine thrombin (Calbiochem, lot No. 700357) present in 0.2 ml glycerol (0.25%,v/v) in 0.1 M TrisHCl (pH 8.1). The contents of the wells were incubated for 2 h at 32”, after which the solution was removed, and the [125I] fibrin coated wells were washed twice with 0.2 ml sterile 0.1 M Tris-HCl buffer (pH 8.1). The 1251-labeled fibrin prepared in these wells was the substrate immediately employed for the detection of possible inhibitors of the degradation by plasmin, and also for the detection of plasminogen-dependent assays for plasminogen activator activity. Results are expressed
261
Testis plasminogen activator during development
as the percentage of total radioactivity released from the 12’ I-labeled fibrin into the soluble fraction, with 100% being the amount released after incubation with trypsin (0.4% for 2 h). Other materials and procedures were the same as those previously described (Lacroix et al., 1977, 1979; Lacroix and Fritz, 1982).
RESULTS In testes obtained from 30- or 40-day-old non-irradiated rats, the levels of plasminogen activator activity were approx. 3.5 X 10’ units per g testis (Table 1). Between 40 and 60 days of age, the average weight per testis rose 82% while the level of plasminogen activator activity per testis increased 342%, resulting in over a 2fold rise in specific activity (Table 1). During later periods of maturation (up to 80 days of age), the rise in plasminogen activator activity per testis was proportional to the gain in testicular weight (Table 1). In germ-cell-depleted testes obtained from 30- to 40-day-old irradiated rats, the levels of plasminogen activator activity were 4.87 to 6.67 X 10’ units per gram testis (Table 2). These values were higher than those in normal testes obtained from non-irradiated rats of comparable age. In contrast, the weights of germ-cell-depleted testes were considerably lower, along with amounts of plasminogen activator activity per testis (compare data in Tables 1 and 2). In the interval between 40 and 65 days of age, the specific activity remained relatively unchanged, and the rise in plasminogen activator activity was directly proportional to the increase in weight of germ-cell-depleted testes (Table 2). In histological sections of testes from 65-dayTable 1 Levels of plasminogen development
Age (days)
activator
Average weight of testis (g)
activity
in homogenates
of testes
Plasminogen activator activity per testis
from
normal
rats during
Plasminogen activator activity per g testis
(units X lows) 30 40 50 60 70 80
0.36 0.75 1.22 1.36 1.46 1.86
1.31 2.51 7.70 11.1 10.7 15.5
f + f f f f
0.30 0.44 0.44 0.50 0.49 1.12
_ 3.51 3.38 6.35 8.20 7.28 8.34
f f f + + +
0.55 0.61 0.39 0.59 0.12 0.61
The testes from 2 rats of each age group were perfused, homogenized and plasminogen activator activity measured as described in Materials and Methods. The average activity (kS.E.M.) is presented.
Martial Lacroix, Fannie E. Smith, Irving B. Fritz
262
Table 2 Levels of plasminogen activator activity in homogenates testes during development Age (days)
Average weight of testis (g)
of testes from germ-cell-depleted
Plasminogen activator activity per testis
rat
Plasminogen activator activity per g testis
(units X 10e5) 30 40 50 65
0.08 0.18 0.25 0.38
0.37 1.19 1.31 2.41
f 0.04 f 0.15 f 0.73 + 0.14
4.87 6.61 5.38 6.30
f 0.39 + 0.74 t 0.53 f 0.31
Germ-cell-depleted testes were from rats irradiated in utero as described in Materials and Methods. Average plasminogen activator activity (+S.E.M.) for 4 testes from rats of each age specified were determined by methods described in Materials and Methods. Table 3 Levels of plasminogen activator activity in various populations of testicular cells Cell type or preparation
Age of rat (days)
Levels of plasminogen activator activity (units/mg protein)
Whole testis
28 36
2670 t 39 3378 + 171
Peritubular myoid cells
20 30 36 58
0.86 0.66 1.09 0.19
Spermatocytes
28 36
170+37 289 f 54
Spermatids
28 36
560 f 29 122 + 10
69
410 f 53
69
147 f 21
20 20 20
3500 _+ 600 1800 f 900 1800 f 1100
20 20
5900 f 800 6300 5 900
Spermatocytes
plus spermatids
Residual bodies Sertoli cells in culture Control: 1 day Control: 2 days Control: 3 days oFSH (NIH S13,300 ng/ml) 1 day 2 days
+ 0.27 _+0.14 c 0.11 + 0.14
Samples were prepared and assayed for plasminogen activator activity as described in Materials and Methods. The preparations “spermatocytes plus spermatids” correspond to fractions collected from the Staput chamber containing approx. the same number of cells of each type. Results are the average values (+S.D.) calculated from 2 or 3 separate Expts.
263
Testis plasminogen activator during development
Table 4 Absence of plasmin inhibitor activity in extracts of germ cells Additions to plasmin
Plasmin activity (% of trypsin)
Tri-HCl
36.9 * 0.7
_
36.7 35.1 38.7 38.2
N.D. N.D. N.D. N.D.
buffer alone
Spermatocytes Spermatids Spermatozoa Residual bodies
?: 0.2 f 0.4 f 1.4 f 1.1
Inhibitor activity
Possible protease inhibitor activity in germ-cell extracts was assayed by procedures described in Materials and Methods. Germ-cell extracts (50 ~.rl),containing between 4 and 50 fig protein, were prepared from each of the indicated germ-cell fractions obtained by the “Staput” sedimentation procedure. These extracts were added to vessels containing 50 ~1 plasmin (3.3 fig/ml) and 100 ~10.1 M Tris-HCI (pH 8.1 at 32°C). The levels of ’ ’ 5I-labelled fibrin degradation products released are reported, expressed as the percentage of total counts obtained after proteolysis by trypsin. Mean values (*SD.), measured in triplicate determinations, are presented. N.D., no inhibitor activity detected.
old irradiated rats, spermatocytes were the most advanced germinal cells evident, whereas spermatids and spermatozoa were abundant in normal testes of 60-day-old non-irradiated rats (data not shown). The specific activity of plasminogen activator in testes from 60-day-old non-irradiated rats was 30% higher than that in germ-celldepleted testes from 65-day-old rats, whereas the levels per testis from normal 60day-old rats were 360% greater than those in testes from 65-day-old irradiated rats (Tables 1 and 2). Levels of plasminogen activator activities in various populations of testicular cells are summarized in Table 3. Levels measured in whole extracts of testes from 28- or 36day-old rats (2670-3378 units/mg protein) overlapped with values in extracts of Sertoli cells prepared from testes of 20-day-old rats and maintained in culture under conditions stated (1800-6300 units/mg protein). The amounts of plasminogen activator activity in peritubular myoid cells were inappreciable (0.66-l .09 units/mg protein). Levels detected in spermatocyte and spermatid preparations ranged from 122 to 560 units/mg protein, with somewhat lower amounts in residual bodies (Table 3). The presence of fragments of Sertoli cells in the testicular germ-cell fractions cannot be ruled out. If the spermatocyte and spermatid fractions were contaminated by as little as 5% Sertoli cells, this would contribute at least 100 units/mg protein. Low levels of detectable plasminogen activator activity in germ-cell fractions could have resulted from the presence of a protease inhibitor. To assess this possibility, we measured effects of germ cell extracts on the activity of known amounts of plasmin. Results obtained indicated the absence of detectable plasmin inhibitor activity in germ cells (Table 4).
Martial Lacroix, Fannie E. Smith, Irving B. Fritz
264
Table 5 Effects of autoclaved bacteria and dibutyryl CAMP on plasminogen activator activity released into the medium by Sertoli cells in culture Additions to basal medium
Expt. No. 1
Expt. No. 2
None dbcAMP (0.04 mM) Autoclaved E. coli
0.07 r 0.42 0.61 f 0.38 0.00 f 0.42
0.00 _+0.22 0.00 f 0.29 10.9 _+0.75
Autoclaved E. coli plus dbcAMP (0.04 mM)
9.74 + 0.67
17.7
+ 1.28
15.8
f 0.97
dbcAMP (0.1 mM)
16.2
* 0.73
Escherichia
coli were washed twice in HBSS and the final pellet resuspended in the same buffer. The bacteria were autoclaved (120°C for 30 min), centrifuged and resuspended in modified Eagle’s minimal essential medium at a concentration of 8 X lo8 particles/ml and stored frozen until used. Fresh medium was added to Sertoli cells seeded the preceding day. The indicated concentrations of dibutyryl CAMP together with approx. 20 bacteria per Sertoli cell in Expt. No. 1, and 40 bacteria per Sertoli cell in Expt. No. 2, were used. On the next day, the medium was harvested and centrifuged. Plasminogen activator activity was assayed as described in Materials and Methods. Each well contained about 7 pg Sertoli cell DNA and 1.0 ml medium. Results are the arithmetic means (tS.D.) of activity measured in samples of tissues culture medium taken from four different cell culture wells treated as indicated.
Levels of plasminogen activator activity in extracts of Sertoli cells, prepared from testes of 20-day-old rats and cultured in basal medium, were approximately 3500 units/mg protein at the end of the first day in culture, and 1800 units/mg protein in cells cultured for 48 h (Table 3). In cells cultured in the presence of FSH for 1 or 2 days, levels of plasminogen activator activity were approx. 6000 units/mg protein (Table 3). Under other conditions, we have detected levels as high as 17 000-40 000 units/mg protein in Sertoli cells cultured in the presence of FSH or dibutyryl CAMP (Lacroix and Fritz, 1982, and unpublished observations). The addition to the culture medium of particles which could be phagocytosed by Sertoli cells resulted in an increased formation of plasminogen activator activity. In experiments with autoclaved E. coli as the phagocytic particle, maximal enzyme activity was observed when’approx. 100 bacteria were present per Sertoli cell. A strongly synergistic effect was obtained when submaximal quantities of dibutyryl CAMP and dead bacteria were added (Table 5). A less strong synergism was observed when submaximal quantities of dibutyryl CAMP and IgG aggregates were added (Lacroix et al., 1979), and on some occasions, only an additive effect was obtained (data not shown).
Testis plasminogen activator during development
26.5
DISCUSSION Levels of plasminogen activator activity in testes of normal Wistar rats increased most rapidly during the period of development between 40 and 60 days of age (Table 1). During this interval, the weight gain in normal testis is associated primarily with an increased number of spermatids. While the number of Sertoli cells per testis is reported to increase by less than lo%, the number of spermatids increases by at least 70% (de Jong and Sharpe, 1977). A comparable rise in specific plasminogen activator activity did not occur in germ-cell depleted testes of irradiated rats between 40 and 65 days of age (Table 2). These data suggest that the rise in plasminogen activator activity levels in normal testes during gonadal maturation are associated with the increase in numbers of spermatids. However, the amounts of plasminogen activator activity in spermatids and spermatozoa are relatively low (Table 3) indicating that enzyme levels in these cells could not account for the rise in testicular specific activity observed during normal development. We have also observed very low levels of plasminogen activator in spermatozoa obtained from the epididymides of adult rats (unpublished results). The data are consistent with the possibility that the formation of advanced germinal cells during development stimulates the synthesis of plasminogen activator by a different population of testicular cells. We postulate that the production of plasminogen activator by Sertoli cells is stimulated by the association of Sertoli cells with spermatids. This possibility is supported by the higher levels of plasminogen activator activity in Sertoli cells than in any other cell types in the seminiferous tubule (Table 3). We have observed that the production of plasminogen activator by seminiferous tubules, prepared from normal testes of adult rats and maintained in organ culture, is lo-loo-fold greater by cells in segments at stages VII and VIII of the cycle of the seminiferous epithelium than by cells in segments at any other stage (Lacroix et al., 1981; Fritz et al., 1981). Stages VII and VIII are those stages during which spermatozoa are released into the lumen during the process of spermiation (Leblond and Clermont, 1952; Russell, 1977). Phagocytosis of dead E. coli or IgG aggregates by Sertoli cells results in the increased production of plasminogen activator (Table 5, and Lacroix et al., 1979). Phagocytosis by Sertoli cells of residual bodies shed by late spermatids prior to spermiation may be one of the stimuli which increases the production of plasminogen activator by Sertoli cells at stages VII and VIII of the cycle (Lacroix et al., 1981). It is unlikely that interstitial cells contribute appreciable amounts of plasminogen activator activity to levels present in testis. We base this conclusion upon the absence of detectable quantities of plasminogen activator in Leydig cells, measured by the fibrin-agar overlay technique (Lacroix et al., 1979). In addition, Leydig cells in germ-celldepleted testes of irradiated rats secrete sufficient androgens to maintain normal weights of accessory sex glands in these animals (Lacroix and Fritz, unpublished observations). It therefore appears that the absence of a rise in
266
Martial Lacroix, Fannie E. Smith, Irving B. Fritz
plasminogen activator specific activity levels in testes of irradiated rats during development (Table 2) is not caused by a deficiency of androgen production or by a failure of Leydig cell function. All cell types examined in testis other than Sertoli cells had relatively low levels of plasminogen activity (Table 3). However, we did not investigate enzyme activity in vascular or lymphatic endothelial cells in the interstitium. The relatively low levels of plasminogen activator activity per testis in germcell-depleted rats suggest that these cell types do not contribute appreciably to the rise observed in testes of normal rats during development. If Sertoli cells indeed constitute the primary source of plasminogen activator activity in testis, it follows that the specific activity in Sertoli cells should be considerably higher than that in whole testis. We estimate that levels of plasminogen activator activity/mg protein in whole testis are approx. 3000-3500 in testes from 30 to 40day-old (Tables 1 and 3), and about 8000 in testes from 60 to 80day-old animals (Table 1). This calculation assumes an average protein content of 10% of testis wet weight. Levels of plasminogen activator activity in Sertoli cells prepared from testes of 20day-old rats varied from 1800 to 3500 units/mg protein in cells cultured for l-3 days in basal medium (Table 3). In contrast, levels were considerably elevated in cells cultured in the presence of FSH or dibutyryl CAMP, ranging from approx. 6000 units/mg protein in cells cultured in the presence of FSH (Table 3) to values as high as 17 000-40 000 units/mg protein in cells cultured in the presence of dibutyryl CAMP (Lacroix and Fritz, 1982). These data demonstrate a considerable potential for increased production of plasminogen activator activity by Sertoli cells maintained under varying conditions. Since levels of plasminogen activator produced by cells at stages VII and VIII of the cycle of the seminiferous epithelium are far greater than amounts produced by cells at other stages of the cycle (Lacroix et al., 1981; Fritz et al., 1981), it appears likely that heterogeneous populations of Sertoli cells exist within seminiferous tubules in testes of adult rats. Experiments are in progress to determine factors controlling differing rates of production of plasminogen activator by Sertoli cells located in different stages of the cycle, and to explore mechanisms by which cell association patterns influence plasminogen activator production by Sertoli cells maintained in organ culture.
ACKNOWLEDGEMENTS This study was supported by grants from the Canadian MRC and the Banting Foundation. We are grateful to Drs. P. Tung, M. Shirley and K. Burdzy for their advice during the preparation of the germ-cell fractions. We also thank Donna McCabe for her help in preparing this manuscript.
Testis plasminogen activator during development
261
REFERENCES Beaumont, H.M. (1960) Int. .I. Radiat. Biol. 2, 247-256. de Jong, F.H., and Sharpe, R.M. (1977), J. Endocrinol. 75, 197-207. Deutsch, D.G., and Mertz, E.T. (1970) Science 170, 1095-1096. Fritz, LB., Parvinen, M., Karmally, K., and Lacroix, M. (1981) Ann. N.Y. Acad. Sci., in press. Go, V.L.W., Vernon, R.G., and Fritz, LB. (1971) Can. J. Biochem. 49, 753-760. Lacroix, M., and Fritz, LB. (1982) Mol. Cell. Endocrinol. 26, 247-258. Lacroix, M., Smith, F.E., and Fritz, I.B. (1977) Mol. Cell. Endocrinol. 9, 227-236. Lacroix, M., Smith, F., and Fritz, I.B. (1979) Cold Spring Harbor Conferences on Cell Proliferation, Vol. 6, Eds.: G.H. Sato and R. Ross (Cold Spring Harbor Laboratory, Cold Spring Harbor) pp. 239-248. Lacroix, M., Parvinen, M., and Fritz, LB. (1981) Biol. Reprod. 25, 143-146. Leblond, C.P., and Clermont, Y. (1952) Ann. N.Y. Acad. Sci. 55,548-577. Russell, L. (1977) Am. J. Anat. 148,313-328. Strickland, S., and Beers, W.H. (1976) J. Biol. Chem. 251, 5694-5702.
Molecular and Cellular Endocrinology,
Elsevier/North-Holland
26 (1982) 259-267 Scientific Publishers, Ltd.
CHANGES IN LEVELS OF PLASMINOGEN IN NORMAL AND GERM-CELL-DEPLETED
ACTIVATOR ACTIVITY TESTES DURING DEVELOPMENT
Martial LACROIX, Fannie E. SMITH and Irving B. FRITZ Banting and Best Department (Canada)
of Medical Research,
Received 30 September 198l;accepted
University of Toronto,
Toronto MSG IL6
29 December 1981
Levels of plasminogen activator activity were determined in testes obtained from normal and irradiated rats of various ages. During normal development, plasminogen activator activity per g testis increased rapidly between 40 and 60 days of age, but a comparable rise did not occur in germ-cell depleted testes of irradiated rats. Levels of enzyme in various populations of testicular cells were highest in Sertoli cells (varying between 1800 and 6300 units/mg protein in cells maintained under different culture conditions), and lowest in peritubular myoid cells (about 1 unit/mg protein), with intermediate levels in germinal cells (ranging between 147 and 560 units/mg protein in residual bodies, spermatocytes and spennatids). No protease inhibitor could be detected in germ-cell extracts. The addition to the medium in which Sertoli cells were in culture of particles which can be phagocytosed (autoclaved E. coli) resulted in an increased formation of plasminogen activator activity by Sertoli cells. A synergistic enhancement of enzyme production resulted following the addition of submaximal quantities of dibutyryl cyclic AMP and autoclaved bacteria to Sertoli cells in culture. On the basis of these data, we suggest that the presence of advanced germinal cells during gonadal development may stimulate the synthesis of plasminogen activator by Sertoli cells, mediated in part by the phagocytosis of residual bodies by Sertoli cells which occurs prior to spermiation.
Levels of plasminogen activator activity in testes of normal rats increase during development, with the greatest increment occurring between 40 and 60 days of age (Lacroix et al., 1979). Preliminary data also indicated that Sertoli cells were the primary source of plasminogen activator in testis (Lacroix et al., 1979). In this communication, we report quantitative changes in testicular plasminogen activator activity during development, and we provide additional information on the amounts of plasminogen activator in peritubular myoid cells, spermatocytes, spermatids and Sertoli cells. Data to be presented support the conclusion that most of the plasminogen activator activity in testis arises from Sertoli cells, and that amounts produced by Sertoli cells increase during gonadal maturation.
0303-7207/82/0000-0000/$02.75
0 Elsevier/North-Holland
Scientific Publishers, Ltd.
260
MATERIALS
Martial Lacroix, Fannie E. Smith, Irving B. Fritz
AND METHODS
Germ-cellenriched preparations were obtained by the “Statput” sedimentation procedure described by Go et al. (1971). Germ-cell-depleted animals were obtained by irradiating (125 rad) pregnant rats at their 19th day of gestation (Beaumont, 1960); the male rats from the ensuing litters were raised to the appropriate age required in the experiments. The extent of germ-cell depletion was monitored by histological examination. The testes of ether-anesthetized rats were perfused through the testicular artery with Ca’+-depleted Hank’s buffered saline solution (HBSS-). Anesthetized animals were then killed by cervical dislocation, and testes were decapsulated, weighed and transferred to a glass homogenizer (Kontes No. 24) containing 10 ml 0.1 M TrisHCl (pH 8.1 at 32’) and 0.1% (v/v) Triton X-100 per gram of wet testicular tissue. Testes were homogenized (10 strokes with a motor-driven Teflon pestle), and aliquots of the supension, snap-frozen in liquid nitrogen, were kept at -60 “C until samples from all age groups had been accumulated. Sertoli cells maintained in culture for variable periods of time were washed twice with ice-cold HBSS-. The cells were scraped off the dish with a rubber policeman and centrifuged at 600 X g for 5 min. 10 vol. of cold 0.1 M Tris (pH 8.1 at 32 “C) were added to each volume of cell pellet, and the suspension was transferred to a conical glass homogenizer fitted with a glass pestle. Samples were homogenized, and aliquots were assayed immediately for plasminogen activator activity (Lacroix et al., 1977) or were stored at -60 “C as described above until analyzed. To assay protease inhibitor activity, we measured the possible effects of germcell extracts on the degradation of [ ‘251]fibrin by purified plasmin. To prepare the plasmin, we dissolved rabbit plasminogen, purified by the techniques of Deutsch and Mertz (1970) from plasma, in 75 mM Tris-HCl (pH 8.1 at 32 “C) containing 25% (v/v) glycerol. We incubated this solution for 6 h at 37 “C with 3000 Ploug units urokinase (Calbiochem, lot No. 901315) after which over 96% of the added plasminogen had been converted to plasmin. This plasmin, at a concentration of 0.83 pg/ml, degraded [ 1251]fibrin linearly at a rate of 15%/h for periods up to 3 h. The [ 1251]fibrin used as substrate in these assays had been coated onto the surface of 96-well (Falcon) culture plates as [125 I] fibrinogen (8.3 pg, containing approx. 1 X lo5 cpm per well) by procedures previously described (Strickland and Beers, 1976; Lacroix et al., 1977). On the day of the assay, the coated [1251]fibrinogen in each well was converted to [ ’ 251] librin by the addition of 0.2 unit bovine thrombin (Calbiochem, lot No. 700357) present in 0.2 ml glycerol (0.25%,v/v) in 0.1 M TrisHCl (pH 8.1). The contents of the wells were incubated for 2 h at 32”, after which the solution was removed, and the [125I] fibrin coated wells were washed twice with 0.2 ml sterile 0.1 M Tris-HCl buffer (pH 8.1). The 1251-labeled fibrin prepared in these wells was the substrate immediately employed for the detection of possible inhibitors of the degradation by plasmin, and also for the detection of plasminogen-dependent assays for plasminogen activator activity. Results are expressed
261
Testis plasminogen activator during development
as the percentage of total radioactivity released from the 12’ I-labeled fibrin into the soluble fraction, with 100% being the amount released after incubation with trypsin (0.4% for 2 h). Other materials and procedures were the same as those previously described (Lacroix et al., 1977, 1979; Lacroix and Fritz, 1982).
RESULTS In testes obtained from 30- or 40-day-old non-irradiated rats, the levels of plasminogen activator activity were approx. 3.5 X 10’ units per g testis (Table 1). Between 40 and 60 days of age, the average weight per testis rose 82% while the level of plasminogen activator activity per testis increased 342%, resulting in over a 2fold rise in specific activity (Table 1). During later periods of maturation (up to 80 days of age), the rise in plasminogen activator activity per testis was proportional to the gain in testicular weight (Table 1). In germ-cell-depleted testes obtained from 30- to 40-day-old irradiated rats, the levels of plasminogen activator activity were 4.87 to 6.67 X 10’ units per gram testis (Table 2). These values were higher than those in normal testes obtained from non-irradiated rats of comparable age. In contrast, the weights of germ-cell-depleted testes were considerably lower, along with amounts of plasminogen activator activity per testis (compare data in Tables 1 and 2). In the interval between 40 and 65 days of age, the specific activity remained relatively unchanged, and the rise in plasminogen activator activity was directly proportional to the increase in weight of germ-cell-depleted testes (Table 2). In histological sections of testes from 65-dayTable 1 Levels of plasminogen development
Age (days)
activator
Average weight of testis (g)
activity
in homogenates
of testes
Plasminogen activator activity per testis
from
normal
rats during
Plasminogen activator activity per g testis
(units X lows) 30 40 50 60 70 80
0.36 0.75 1.22 1.36 1.46 1.86
1.31 2.51 7.70 11.1 10.7 15.5
f + f f f f
0.30 0.44 0.44 0.50 0.49 1.12
_ 3.51 3.38 6.35 8.20 7.28 8.34
f f f + + +
0.55 0.61 0.39 0.59 0.12 0.61
The testes from 2 rats of each age group were perfused, homogenized and plasminogen activator activity measured as described in Materials and Methods. The average activity (kS.E.M.) is presented.
Martial Lacroix, Fannie E. Smith, Irving B. Fritz
262
Table 2 Levels of plasminogen activator activity in homogenates testes during development Age (days)
Average weight of testis (g)
of testes from germ-cell-depleted
Plasminogen activator activity per testis
rat
Plasminogen activator activity per g testis
(units X 10e5) 30 40 50 65
0.08 0.18 0.25 0.38
0.37 1.19 1.31 2.41
f 0.04 f 0.15 f 0.73 + 0.14
4.87 6.61 5.38 6.30
f 0.39 + 0.74 t 0.53 f 0.31
Germ-cell-depleted testes were from rats irradiated in utero as described in Materials and Methods. Average plasminogen activator activity (+S.E.M.) for 4 testes from rats of each age specified were determined by methods described in Materials and Methods. Table 3 Levels of plasminogen activator activity in various populations of testicular cells Cell type or preparation
Age of rat (days)
Levels of plasminogen activator activity (units/mg protein)
Whole testis
28 36
2670 t 39 3378 + 171
Peritubular myoid cells
20 30 36 58
0.86 0.66 1.09 0.19
Spermatocytes
28 36
170+37 289 f 54
Spermatids
28 36
560 f 29 122 + 10
69
410 f 53
69
147 f 21
20 20 20
3500 _+ 600 1800 f 900 1800 f 1100
20 20
5900 f 800 6300 5 900
Spermatocytes
plus spermatids
Residual bodies Sertoli cells in culture Control: 1 day Control: 2 days Control: 3 days oFSH (NIH S13,300 ng/ml) 1 day 2 days
+ 0.27 _+0.14 c 0.11 + 0.14
Samples were prepared and assayed for plasminogen activator activity as described in Materials and Methods. The preparations “spermatocytes plus spermatids” correspond to fractions collected from the Staput chamber containing approx. the same number of cells of each type. Results are the average values (+S.D.) calculated from 2 or 3 separate Expts.
263
Testis plasminogen activator during development
Table 4 Absence of plasmin inhibitor activity in extracts of germ cells Additions to plasmin
Plasmin activity (% of trypsin)
Tri-HCl
36.9 * 0.7
_
36.7 35.1 38.7 38.2
N.D. N.D. N.D. N.D.
buffer alone
Spermatocytes Spermatids Spermatozoa Residual bodies
?: 0.2 f 0.4 f 1.4 f 1.1
Inhibitor activity
Possible protease inhibitor activity in germ-cell extracts was assayed by procedures described in Materials and Methods. Germ-cell extracts (50 ~.rl),containing between 4 and 50 fig protein, were prepared from each of the indicated germ-cell fractions obtained by the “Staput” sedimentation procedure. These extracts were added to vessels containing 50 ~1 plasmin (3.3 fig/ml) and 100 ~10.1 M Tris-HCI (pH 8.1 at 32°C). The levels of ’ ’ 5I-labelled fibrin degradation products released are reported, expressed as the percentage of total counts obtained after proteolysis by trypsin. Mean values (*SD.), measured in triplicate determinations, are presented. N.D., no inhibitor activity detected.
old irradiated rats, spermatocytes were the most advanced germinal cells evident, whereas spermatids and spermatozoa were abundant in normal testes of 60-day-old non-irradiated rats (data not shown). The specific activity of plasminogen activator in testes from 60-day-old non-irradiated rats was 30% higher than that in germ-celldepleted testes from 65-day-old rats, whereas the levels per testis from normal 60day-old rats were 360% greater than those in testes from 65-day-old irradiated rats (Tables 1 and 2). Levels of plasminogen activator activities in various populations of testicular cells are summarized in Table 3. Levels measured in whole extracts of testes from 28- or 36day-old rats (2670-3378 units/mg protein) overlapped with values in extracts of Sertoli cells prepared from testes of 20-day-old rats and maintained in culture under conditions stated (1800-6300 units/mg protein). The amounts of plasminogen activator activity in peritubular myoid cells were inappreciable (0.66-l .09 units/mg protein). Levels detected in spermatocyte and spermatid preparations ranged from 122 to 560 units/mg protein, with somewhat lower amounts in residual bodies (Table 3). The presence of fragments of Sertoli cells in the testicular germ-cell fractions cannot be ruled out. If the spermatocyte and spermatid fractions were contaminated by as little as 5% Sertoli cells, this would contribute at least 100 units/mg protein. Low levels of detectable plasminogen activator activity in germ-cell fractions could have resulted from the presence of a protease inhibitor. To assess this possibility, we measured effects of germ cell extracts on the activity of known amounts of plasmin. Results obtained indicated the absence of detectable plasmin inhibitor activity in germ cells (Table 4).
Martial Lacroix, Fannie E. Smith, Irving B. Fritz
264
Table 5 Effects of autoclaved bacteria and dibutyryl CAMP on plasminogen activator activity released into the medium by Sertoli cells in culture Additions to basal medium
Expt. No. 1
Expt. No. 2
None dbcAMP (0.04 mM) Autoclaved E. coli
0.07 r 0.42 0.61 f 0.38 0.00 f 0.42
0.00 _+0.22 0.00 f 0.29 10.9 _+0.75
Autoclaved E. coli plus dbcAMP (0.04 mM)
9.74 + 0.67
17.7
+ 1.28
15.8
f 0.97
dbcAMP (0.1 mM)
16.2
* 0.73
Escherichia
coli were washed twice in HBSS and the final pellet resuspended in the same buffer. The bacteria were autoclaved (120°C for 30 min), centrifuged and resuspended in modified Eagle’s minimal essential medium at a concentration of 8 X lo8 particles/ml and stored frozen until used. Fresh medium was added to Sertoli cells seeded the preceding day. The indicated concentrations of dibutyryl CAMP together with approx. 20 bacteria per Sertoli cell in Expt. No. 1, and 40 bacteria per Sertoli cell in Expt. No. 2, were used. On the next day, the medium was harvested and centrifuged. Plasminogen activator activity was assayed as described in Materials and Methods. Each well contained about 7 pg Sertoli cell DNA and 1.0 ml medium. Results are the arithmetic means (tS.D.) of activity measured in samples of tissues culture medium taken from four different cell culture wells treated as indicated.
Levels of plasminogen activator activity in extracts of Sertoli cells, prepared from testes of 20-day-old rats and cultured in basal medium, were approximately 3500 units/mg protein at the end of the first day in culture, and 1800 units/mg protein in cells cultured for 48 h (Table 3). In cells cultured in the presence of FSH for 1 or 2 days, levels of plasminogen activator activity were approx. 6000 units/mg protein (Table 3). Under other conditions, we have detected levels as high as 17 000-40 000 units/mg protein in Sertoli cells cultured in the presence of FSH or dibutyryl CAMP (Lacroix and Fritz, 1982, and unpublished observations). The addition to the culture medium of particles which could be phagocytosed by Sertoli cells resulted in an increased formation of plasminogen activator activity. In experiments with autoclaved E. coli as the phagocytic particle, maximal enzyme activity was observed when’approx. 100 bacteria were present per Sertoli cell. A strongly synergistic effect was obtained when submaximal quantities of dibutyryl CAMP and dead bacteria were added (Table 5). A less strong synergism was observed when submaximal quantities of dibutyryl CAMP and IgG aggregates were added (Lacroix et al., 1979), and on some occasions, only an additive effect was obtained (data not shown).
Testis plasminogen activator during development
26.5
DISCUSSION Levels of plasminogen activator activity in testes of normal Wistar rats increased most rapidly during the period of development between 40 and 60 days of age (Table 1). During this interval, the weight gain in normal testis is associated primarily with an increased number of spermatids. While the number of Sertoli cells per testis is reported to increase by less than lo%, the number of spermatids increases by at least 70% (de Jong and Sharpe, 1977). A comparable rise in specific plasminogen activator activity did not occur in germ-cell depleted testes of irradiated rats between 40 and 65 days of age (Table 2). These data suggest that the rise in plasminogen activator activity levels in normal testes during gonadal maturation are associated with the increase in numbers of spermatids. However, the amounts of plasminogen activator activity in spermatids and spermatozoa are relatively low (Table 3) indicating that enzyme levels in these cells could not account for the rise in testicular specific activity observed during normal development. We have also observed very low levels of plasminogen activator in spermatozoa obtained from the epididymides of adult rats (unpublished results). The data are consistent with the possibility that the formation of advanced germinal cells during development stimulates the synthesis of plasminogen activator by a different population of testicular cells. We postulate that the production of plasminogen activator by Sertoli cells is stimulated by the association of Sertoli cells with spermatids. This possibility is supported by the higher levels of plasminogen activator activity in Sertoli cells than in any other cell types in the seminiferous tubule (Table 3). We have observed that the production of plasminogen activator by seminiferous tubules, prepared from normal testes of adult rats and maintained in organ culture, is lo-loo-fold greater by cells in segments at stages VII and VIII of the cycle of the seminiferous epithelium than by cells in segments at any other stage (Lacroix et al., 1981; Fritz et al., 1981). Stages VII and VIII are those stages during which spermatozoa are released into the lumen during the process of spermiation (Leblond and Clermont, 1952; Russell, 1977). Phagocytosis of dead E. coli or IgG aggregates by Sertoli cells results in the increased production of plasminogen activator (Table 5, and Lacroix et al., 1979). Phagocytosis by Sertoli cells of residual bodies shed by late spermatids prior to spermiation may be one of the stimuli which increases the production of plasminogen activator by Sertoli cells at stages VII and VIII of the cycle (Lacroix et al., 1981). It is unlikely that interstitial cells contribute appreciable amounts of plasminogen activator activity to levels present in testis. We base this conclusion upon the absence of detectable quantities of plasminogen activator in Leydig cells, measured by the fibrin-agar overlay technique (Lacroix et al., 1979). In addition, Leydig cells in germ-celldepleted testes of irradiated rats secrete sufficient androgens to maintain normal weights of accessory sex glands in these animals (Lacroix and Fritz, unpublished observations). It therefore appears that the absence of a rise in
266
Martial Lacroix, Fannie E. Smith, Irving B. Fritz
plasminogen activator specific activity levels in testes of irradiated rats during development (Table 2) is not caused by a deficiency of androgen production or by a failure of Leydig cell function. All cell types examined in testis other than Sertoli cells had relatively low levels of plasminogen activity (Table 3). However, we did not investigate enzyme activity in vascular or lymphatic endothelial cells in the interstitium. The relatively low levels of plasminogen activator activity per testis in germcell-depleted rats suggest that these cell types do not contribute appreciably to the rise observed in testes of normal rats during development. If Sertoli cells indeed constitute the primary source of plasminogen activator activity in testis, it follows that the specific activity in Sertoli cells should be considerably higher than that in whole testis. We estimate that levels of plasminogen activator activity/mg protein in whole testis are approx. 3000-3500 in testes from 30 to 40day-old (Tables 1 and 3), and about 8000 in testes from 60 to 80day-old animals (Table 1). This calculation assumes an average protein content of 10% of testis wet weight. Levels of plasminogen activator activity in Sertoli cells prepared from testes of 20day-old rats varied from 1800 to 3500 units/mg protein in cells cultured for l-3 days in basal medium (Table 3). In contrast, levels were considerably elevated in cells cultured in the presence of FSH or dibutyryl CAMP, ranging from approx. 6000 units/mg protein in cells cultured in the presence of FSH (Table 3) to values as high as 17 000-40 000 units/mg protein in cells cultured in the presence of dibutyryl CAMP (Lacroix and Fritz, 1982). These data demonstrate a considerable potential for increased production of plasminogen activator activity by Sertoli cells maintained under varying conditions. Since levels of plasminogen activator produced by cells at stages VII and VIII of the cycle of the seminiferous epithelium are far greater than amounts produced by cells at other stages of the cycle (Lacroix et al., 1981; Fritz et al., 1981), it appears likely that heterogeneous populations of Sertoli cells exist within seminiferous tubules in testes of adult rats. Experiments are in progress to determine factors controlling differing rates of production of plasminogen activator by Sertoli cells located in different stages of the cycle, and to explore mechanisms by which cell association patterns influence plasminogen activator production by Sertoli cells maintained in organ culture.
ACKNOWLEDGEMENTS This study was supported by grants from the Canadian MRC and the Banting Foundation. We are grateful to Drs. P. Tung, M. Shirley and K. Burdzy for their advice during the preparation of the germ-cell fractions. We also thank Donna McCabe for her help in preparing this manuscript.
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