- Email: [email protected]
Contractility of rat testicular seminiferous tubules in vitro : Prostaglandin F1α and indomethacin
CONTRACTILITY
OF RAT TESTICULAR
PROSTAGLANDIN
SEMINIFEROUS
TUBULES -IN VITRO:
Flcl AND INDOMETHACIN
Louis E. Buhrley3 and LeGrande
132
C. Ellis
Department of Biology Utah State University Logan, Utah 84322
ABSTRACT
The frequency of spontaneous -___ in vitro contractions of seminiferous tubules of the rat appeared to be increased in a dosedependent manner by prostaglandin Fla. PGFla treatment increased the tonus of the smooth muscle cells in the wall of the tubules as indicated by a reduction in the diameter of the tubules. When the tubules were rinsed successively with fresh Tyrode's solution, the contractile frequency was diminished. Returning the original bathing medium to the tubules restored their contractile frequency, as did treatment of the rinsed tubules with PGFla (low7 M). Preinjecting the rats with indomethacin tended to reduce the contractile frequency of the extirpated tubules. Treating the tubules with a solution of indomethacin for 90 min. -in vitro was more effective than pretreatment -__ in viva in reducing contractile frequency, but a combination of these two procedures produced the greatest inhibition. PGFlo restored the contractile frequency of the indomethacin-treated tubules. Our results indicate that PGs modulate the -in vitro contractility of the tubules.
ACKNOWLEDGMENTS
We thank Dr. John E. Pike of the Upjohn Co. for the PGFlcl and Dr. Horace Brown of Merck Sharp and Dohme for the indomethacin.
This work was supported by the U. S. Atomic Energy Commission Grant No. AT(ll-l)-1602 and Utah State University Research Project U-300. Send reprint requests to L. C. Ellis, Department of Biology, Utah State University, Logan, Utah 84322. A preliminary report of this work was presented at the Fourth International Congress on Hormonal Steroids, Mexico City, September 2-7, 1974. Predoctoral fulfillment
Candidate of the Biology Department. Submitted of the requirements for a Ph.D. in Physiology.
as partial
PROSTAGLANDINS JULY 1975
VOL. 10 NO. 1
151
PROSTAGLANDINS
INTRODUCTION
The seminiferous tubules of rat and mouse testes contain a circular layer of tissue that resembles the smooth muscle cells of other organs (1,Z). These cells are responsible for the contractile movements of the tubules (3). Since the contractions appear to move as a wave along the tubule towards the rete testis (4), it has been suggested that they play a role in the release and transport of mature spermatozoa through the tubules to the excurrent ducts (3,4,5). If the contractions are functionally significant, reproductive capabilities could be altered by modifying whatever factors regulate the contractility. Neuronal control seems unlikely since there is a paucity of both cholinergic and adrenergic fibers to the tubules (6). Niemi and Kormano (7) have shown, however, that oxytocin produces an increase in the contractile frequency. Also, Suvanto and Kormano (3) have suggested that other factors such as internal pressure, hormones and glutamic acid may be involved in regulating tubular motility. As substantiation, we know that differentiation of the contractile cells and initiation of contractile activity in the tubules of young mice depend upon an intact pituitary (8). Also, tubules from newborn rats, when grown in organ culture, require androgens for normal maturation and development (9). Prostaglandins (PGs) have pronounced effects on smooth muscle (10). In general, PGs of the F series enhance smooth muscle contractility (11). For example, PGFlo stimulates the contractile activity of the rat uterus (12), rabbit fallopian tubes (13) and the rat testicular capsule (14). The presence of PGs in rat testes (U-17), and the presence of the enzymes for their synthesis (18) and inactivation (19), suggest that these compounds play a role in modulating the contractility of the The present study was undertaken, therefore, to ascertain if tubules. PGFld alters the -__ in vitro contractile activity of the seminiferous tubules of the rat. We also examined the effects on tubular contractility of indomethacin, an inhibitor of PG biosynthesis (20), to further elucidate the possible role of PGs in regulating contractility of the tubules.
MATERIALS
AND METHODS
Sexually mature rats (Holtzman) were anesthetized with ether. The animals were placed on their dorsal side and an incision was made through the scrotum. The testes were extirpated, the tunica albuginea was cut and removed, and the tubules placed in a glass water-jacketed muscle warmer that contained oxygenated Tyrode's solution at 35 C. Individual tubules were obtained by placing the mass of tubules in a warmed Petri dish filled with Tyrode's solution, and the tubules were teased from the interstitial tissue with microdissecting forceps under a dissection microscope. Small segments of tubules, approximately one cm in length, were placed in several drops of Tyrode's solution on a microscope slide. The slide was placed inside a double-walled Plexiglass
152
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
chamber that was attached to the microscope stage. Warm water was circulated through the outer chamber to maintain the internal temperature at approximately 35 C. The temperature was monitored by placing small chips of temperature-sensitive liquid crystal sheets (Edmund Scientific Co.) inside the chamber. The bottom of the chamber was covered with Tyrode's solution to maintain a high internal humidity and prevent evaporation of the bathing medium. The tubules were examined in transmitted light with a magnification of 440 X. The contraction frequency was determined by observing the tubules for periods of one minute and counting the contractions at a preselected site on the tubule. The diameter of the tubule as a measure of tonus and the depth of depression of the wall of the tubule during an actual contraction were determined with an ocular micrometer. Eight or more tubules from a number of different rats were observed. The tubules were first examined in Tyrode's solution (control) and then transferred with a disposable capillary pipet to a solution of PGFlo on the same slide. The PGFl, had been dissolved in 95% ethanol and diluted with Tyrode's solution containing 0.1 g glucose/ Observations were initiated within five minutes of the transfer. 100 ml. In some experiments, after the control observations were completed, The tubules were then the Tyrode's solution was drawn off and retained. rinsed with fresh Tyrode's solution and observations were again made. This procedure was repeated a second time. Next, the original solution was returned to the tubules in the muscle warmer and the contractile activity was again ascertained. Some of the tubules were subsequently rinsed several times and then placed in a solution of PGFlo (low7 M). Four different experiments were conducted to determine the effect In the first investigation, of indomethacin on tubular contractions. one group of rats received 2 i.p. injections of indomethacin (10 mg/kg) Control animals received injections in corn oil each day for two days. of corn oil only. The testes were removed from both groups of animals two to three hours after the last injection. After the contractile activity of the tubules had been ascertained, they were placed in a solution of PGFlo (10m7 M) and examined for contractility. In the second investigation, tubules from untreated animals were placed in a solution of indomethacin (10 ug/ml). Oxygen was bubbled through the solution and the temperature was kept at 35 C. The tubules were observed after they had remained in the indomethacin for approximately 1:~ hours. Control tubules from untreated animals were placed After the in a beaker of Tyrode's solution and similarly treated. initial observations, the tubules from the indomethacin bath were treated with PGFlo (low7 M). in vivo and -in vitro experiments Next, the combined effect of these _Tubules from animals pretreated with indomethacin were was determined. placed in the indomethacin solution (10 ug/ml) for 1% hours and then These tubules were subsequently placed in the PGFlo solution examined. The final experiment consisted and the contractile activity observed. of adding a solution of indomethacin (10 ug/ml) directly to untreated The tubules were examined for contractubules on a microscope slide. tile activity within 5 minutes.
JULY 1975
VOL. 10 NO. 1
153
PROSTAGLANDINS
RESULTS
Stimulation of the frequency of the tubular contractions by PGFlc, appeared to be dose-dependent (Fig. 1). The increase was statistically significant at each concentration, and the level of significance rose for each concentration of PG used. This treatment also caused an increase in tonus as reflected by a decrease in the diameter of the tubules (Fig. 2), but the depth of the contractions was not significantly altered (data not shown).
14.0
12.0
10.0 I : . G
6.0
4 t E
6.0
u
4.0
2.0
Prostaglsndin F1, (M)
Figure 1.
154
Stimulation of the contractile frequency of the seminiferous The vertical bars represent the standard tubules by PGFla. error of the mean. * PCO.02; ** pco.01; + p
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
Ll ... r-l
Control
*:*:*: Fl, (II!) *::. Prostaglandin
400
g 3 ;
300
=
200
B H g
100
i
i:
-. :. -::. .. .... . ..... . ..... . ..... . ..... . ..... . ..... ...... ... ::: ......... ...... ...... ...... ...... . .. .. . ...... ...... ...... ...... lo-8
Figure
.
Effect of PGFlo on the tonus (diameter) of the semini* PCO.05. ferous tubules.
Changing the Tyrode's solution that surrounded the tubules (Fig. 3) resulted in a slight decrease in contractile frequency after the first rinse (PcO.1). A second series of rinses significantly decreased the When the tubules were placed in this original frequency (P
JULY 1975
VOL. 10 NO. 1
155
PROSTAGLANDINS
10.0
8.0
-
I4 *
6.0
-
f
T **
L
Control
Figure 3.
156
1st
2nd
Change
Change
Original
P%
Bath
Effect on frequency of tubular contractions produced by: (a) successive changes in the Tyrode's solution, (b) readdition of the original bathing medium, and (c) addition (10m7M) to the rinsed tubules. * PcO.10; ** P
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
8.0
c
8.0 f
Control
Figure 4.
1st
2nd
Original
Change
Change
8ath
W,
Decrease in the depth of the tubular contractions produced by successive changes in the bathing solution and restoration of activity by addition of the original bath and PGFla (10m7M) to the rinsed tubules. * P
JULY 1975
VOL. 10 NO. 1
157
PROSTAGLANDINS
Pretreatment of rats with indomethacin injections caused a slight decrease in the frequency (P0.10) of the contractions (Fig. 5). Bathing the tubules in an indomethacin solution more effectively reduced contractile frequency than did the indomethacin injections. The combination of these two procedures, i.e., placing tubules from indomethacininjected animals in an indomethacin solution, resulted in a still greater reduction of the frequency. In each experiment, PGFlu effectively increased the frequency of the contractions. No effect could be discerned when the tubules were placed in a solution of indomethacin for only five to ten minutes. Placing the tubules in a solution of indomethacin for 90 min. reduced the depth of contractions (PcO.01 - data not shown). Tonus was diminished when indomethacin-pretreated tubules were placed in a solution of indomethacin for 90 min. (PcO.05 - data not shown).
Control lndomethnin
10.0 .
Prostaglrndin
2.0
Pretreated
Solution
(80 Figure 5.
158
Min)
Pntmated
Solution
So&ion
(5 Min)
Changes in the contractile frequency of the tubules produced by: (a) indomethacin pretreatments (10 mg/kg); (b) tubules placed in solution of indomethacin (10 ng/ml) for 90 min.; (c) combined effect of treatments (a) and (b); (d) tubules placed in solution of indomethacin (10 yg/ml) for 5 min. Tubules were also treated with PGFlu (lop7 M) following the indomethacin treatments. *P
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
DISCUSSION
In this investigation, -in vitro contractile frequency of rat seminiferous tubules was increased by prostaglandin Flo in what appeared to be a dose-dependent manner (Fig. 1). The decrease in diameter of the tubules showed that there was an increase in the tonus of the smooth muscle cells surrounding the tubules (Fig. 2). These tubular responses resembled the increased frequency of contraction and tonus changes of the testicular capsule produced by PGFla (12). That PGFlo did not increase the depth of the contractions is surprising, since other agents that increase the frequency of tubular contractility usually also increase the depth (unpublished observations). The reason for this lack of effect is unknown at the present time. PGFlo was used in this study as the first in a series of investigations of the effects of various PGs on tubular contraction as has been reported for the rabbit testicular capsule (14,17,24,25). The seminiferous tubules also respond similarly to PGF2, (Farr and Ellis, unpublished data).
from tile sult into
The stimulation of contractile activity by PGFlo probably results an increased availability of intracellular Ca+t for the contracprocesses (21). This mobilzation of extracellular Ca* could refrom a release of bound Ca* or a movement of extracellular Ca++ the cell (22,23).
The reduction in contractile activity (frequency and depth of contractions) after successive changes of the Tyrode's solution bathing the tubules (Fig. 4 and S), plus the resumption of contractility when the tubules were resuspended in the original bathing media, indicate that endogenous factors necessary for contractility had been released by the contracting tubules into the initial bathing solution. In similar studies (24,25), the spontaneous contractions of the rabbit testicular capsule were reduced or eliminated by successive changes of the solution bathing the tissue. Extraction of the original solution yielded a PGlike residue that restored contractile activity. These workers concluded that PG-like compounds appeared to be responsible for modulating the contractile activity of the testicular capsule. Our present data suggest the presence of PGs in the bathing media of actively contracting tubules. That PGs are needed for -in vitro contractility of rat seminiferous tubules is further indicated by the observation that rinsing tubules several times with Tyrode's solution and then treating them with PGFla, increased both the frequency and depth of contractions. Spontaneous contractions of isolated rat uteri were also inhibited by indomethacin (26). Since the release of PGs into the fluid bathing the uteri was simultaneously decreased, these workers concluded that the Their observations are synthesis of PGs was inhibited by indomethacin. consistent with other reports of an inhibition of PG synthesis by indoIn this report, Northover (28) has shown that indomethacin (20,27). methacin inhibits the movement of Ca* into smooth muscle cells and decreases the uptake of this ion by the endoplasmic reticulum. He has
JULY 1975
VOL. 10 NO. 1
159
PROSTAGLANDINS
attributed the inhibitory action of indomethacin to this action. Since PGs also alter the binding of Ca* by cell membranes (21,29), additional work is needed to clarify the mechanisms involved in the inhibition of smooth muscle by indomethacin. Our observation that bathing tubules in a solution of indomethacin in vitro was more effective than pretreatment injections in inhibiting -contractile frequency, could best be explained by a difference in the effective concentration of the drug at the site of action. It has been suggested that indomethacin penetrates the cell membrane very slowly (27). Perhaps the blood-testis barrier further reduces the accumulation of indomethacin within the tubules. In each indomethacin experiment, the reduced frequency of tubular contractions was always increased by the application of a solution of PGFlo. This is consistent with the increased frequency of contractions noted earlier when PGF was added to the rinsed and less active tubules. Our results show that % t e smooth muscle cells of the seminiferous tubules of the rat are responsive to PGFl,. The findings are in accord with the hypothesis that the PGs modulate -in vitro contractions of the tubules and are important for normal testicular function.
REFERENCES
160
1.
Study of the normal and irradiated Lacy, D. and J. Rotblat. boundary tissue of the seminiferous tubules of the rat. Exp. Cell Res. 21:49, (1960).
2.
Ross, M. H. The fine structure and development of the peritubular contractile cell component in the seminiferous tubules of the Am. J. Anat. 121:523, (1967). mouse.
3.
The relation between -__ in vitro conSuvanto, 0. and M. Kormano. tractions of the rat seminiferous tubules and the cyclic stage J. Reprod. Fertil. 21:227, (1970). of the seminiferous epithelium.
4.
Clermont, Y. Contractile elements in the limiting membrane of the seminiferous tubules of the rat. Exp. Cell Res. 15:438, (1958).
5.
Ross, M. H. and I. R. Long. Science 153:1271, tubules.
6.
In Hodson. N. The nerves of the testis, epididymus and scrotum. "The Testis" (eds. A. D. Johnson, W. R. Gomes and N. L. Vandemark) Vol. I, pp. 47-99, Academic Press, N. Y., (1970.
7.
Contractility of the seminiferous tubule Niemi, M. and M. Kormano. Ann. Med. of the postnatal rat testis and its response to oxytocin. Exp. Biol. Fenn. 43:40, (1965).
Contractile (1966).
cells in human seminiferous
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
8.
Bressler, R. S. and M. H. Ross. Differentiation of peritubular myoid cells of the testis: Effects of intratesticular implantation of newborn mouse testes into normal and hypophysectomized adults. Biol. Reprod. 6:148, (1972).
9.
Hovatta, 0. Effect of androgens and antiandrogens on the development of the myoid cells of the rat seminiferous tubules (organ culture). Zellforsch. 131:299, (1972).
10.
Weeks, J. R. (1972).
11.
Horton, E. W. Hypotheses on physiological Physiol. Rev. 49:122, (1969).
12.
Eliasson, R. Studies on prostaglandin occurrence, formation and biological actions. Acta Physiol. Stand. 46, Suppl. 158:1, (1959).
13.
Horton, E. W. and I. H. M. Main. A comparison of the biological activities of four prostaglandins. Br. J. Pharmacol. 21:182, (1963).
14.
Johnson, J. M., J. L. Hargrove and L. C. Ellis. Prostaglandin Flcl induced stimulation of rabbit testicular contractions -in vitro. Proc. Sot. Exp. Biol. Med. 138:378, (1971).
15.
Prostaglandins Carpenter, M. P. and B. Wiseman. Fed. Proc. 29:248 (Abstr.), (1970).
16.
Ellis, I.. C. Rat testicular prostaglandin synthesis and its Fed. Proc. 31:295, (1972). relationship to androgen synthesis.
17.
Seeley, R. R. Regulation of rabbit testicular capsular motility: Ph. D. prostaglandins, sex steroids, and sympathomimetic agents. Dissertation. Utah State University, Logan, Utah, (1973).
18.
Comparative aspects of prostaChrist, E. J. and D. A. van Dorp. Biochim. Biophys. Acta glandin biosynthesis in animal tissues. 270:537, (1972).
19.
The distribution of1 3 Anggard, E., C. Larsson and B. Samuelsson. 15-hydroxy prostaglandin dehydrogenase and prostaglandin-delta ’ reductase. Acta Physiol. Stand. 81:396, (1971).
20.
Ferreira, S. H., S. Moncada and J. R. Vane. Indomethacin and Nature aspirin abolish prostaglandin release from the spleen. New Biol., 231:237, (1971).
21.
Carsten, M. E. Prostaglandin's part in regulating uterine contraction by transport of calcium. In "The Prostaglandins: Clinical Applications in Human Reproduction" (ed. E. M. Southern) pp. 59-66, Futura Publishing Co., Mount Kisco, N. Y., (1972).
JULY 1975
The prostaglandins.
VOL. 10 NO. 1
Annu. Rev. Pharmacol.
12:317,
roles of prostaglandins.
of rat testis.
161
PROSTAGLANDINS
162
22.
Ramwell, P. W. and J. E. Shaw. Biological significance of the prostaglandins. Recent Prog. Horm. Res. 26:139, (1970).
23.
Eagling, E. M., H. Love11 and V. R. Pickels. Prostaglandin, myometrial 'enhancement' and calcium. J. Physiol. (Lond.) 213:53p, (1971).
24.
Hargrove, J. L., R. R. Seeley and L. C. Ellis. Contractions of rabbit testes -in vitro: Permissive role of prostaglandins for the actions of calcium and some smooth-muscle stimulating agents. Prostaglandins 3:469, (1973).
25.
Hargrove, J. L., R. R. Seeley and L. C. Ellis. Prostaglandin-like substances: Initiation and maintenance of rabbit testicular contraction -in vitro. Proc. Sot. Exp. Biol. Med. 142:205, (1973).
26.
The contribution of prostaglandin Vane, J. R. and K. I. Williams. production to contractions of the isolated rat uterus. Br. J. Pharmacol. 48:629, (1973).
27.
Indomethacin and aspirin Raz, A., H. Stern and R. Kenig-Wakshal. inhibition of prostaglandin E synthesis by sheep vesicles microsome powder and seminal vesicles s 1ices. Prostaglandins 3:337, (1973).
28.
Northover, B. J. Effect of anti-inflammatory drugs on the binding of calcium to cellular membranes in various human and guinea-pig Br. J. Pharmacol. 48:496, (1973). tissues.
29.
Carsten, M. E. Prostaglandins and oxytocin: Prostaglandins 5:33, uterine smooth muscle.
Their effects on (1974).
JULY 1975
VOL. 10 NO. 1
OF RAT TESTICULAR
PROSTAGLANDIN
SEMINIFEROUS
TUBULES -IN VITRO:
Flcl AND INDOMETHACIN
Louis E. Buhrley3 and LeGrande
132
C. Ellis
Department of Biology Utah State University Logan, Utah 84322
ABSTRACT
The frequency of spontaneous -___ in vitro contractions of seminiferous tubules of the rat appeared to be increased in a dosedependent manner by prostaglandin Fla. PGFla treatment increased the tonus of the smooth muscle cells in the wall of the tubules as indicated by a reduction in the diameter of the tubules. When the tubules were rinsed successively with fresh Tyrode's solution, the contractile frequency was diminished. Returning the original bathing medium to the tubules restored their contractile frequency, as did treatment of the rinsed tubules with PGFla (low7 M). Preinjecting the rats with indomethacin tended to reduce the contractile frequency of the extirpated tubules. Treating the tubules with a solution of indomethacin for 90 min. -in vitro was more effective than pretreatment -__ in viva in reducing contractile frequency, but a combination of these two procedures produced the greatest inhibition. PGFlo restored the contractile frequency of the indomethacin-treated tubules. Our results indicate that PGs modulate the -in vitro contractility of the tubules.
ACKNOWLEDGMENTS
We thank Dr. John E. Pike of the Upjohn Co. for the PGFlcl and Dr. Horace Brown of Merck Sharp and Dohme for the indomethacin.
This work was supported by the U. S. Atomic Energy Commission Grant No. AT(ll-l)-1602 and Utah State University Research Project U-300. Send reprint requests to L. C. Ellis, Department of Biology, Utah State University, Logan, Utah 84322. A preliminary report of this work was presented at the Fourth International Congress on Hormonal Steroids, Mexico City, September 2-7, 1974. Predoctoral fulfillment
Candidate of the Biology Department. Submitted of the requirements for a Ph.D. in Physiology.
as partial
PROSTAGLANDINS JULY 1975
VOL. 10 NO. 1
151
PROSTAGLANDINS
INTRODUCTION
The seminiferous tubules of rat and mouse testes contain a circular layer of tissue that resembles the smooth muscle cells of other organs (1,Z). These cells are responsible for the contractile movements of the tubules (3). Since the contractions appear to move as a wave along the tubule towards the rete testis (4), it has been suggested that they play a role in the release and transport of mature spermatozoa through the tubules to the excurrent ducts (3,4,5). If the contractions are functionally significant, reproductive capabilities could be altered by modifying whatever factors regulate the contractility. Neuronal control seems unlikely since there is a paucity of both cholinergic and adrenergic fibers to the tubules (6). Niemi and Kormano (7) have shown, however, that oxytocin produces an increase in the contractile frequency. Also, Suvanto and Kormano (3) have suggested that other factors such as internal pressure, hormones and glutamic acid may be involved in regulating tubular motility. As substantiation, we know that differentiation of the contractile cells and initiation of contractile activity in the tubules of young mice depend upon an intact pituitary (8). Also, tubules from newborn rats, when grown in organ culture, require androgens for normal maturation and development (9). Prostaglandins (PGs) have pronounced effects on smooth muscle (10). In general, PGs of the F series enhance smooth muscle contractility (11). For example, PGFlo stimulates the contractile activity of the rat uterus (12), rabbit fallopian tubes (13) and the rat testicular capsule (14). The presence of PGs in rat testes (U-17), and the presence of the enzymes for their synthesis (18) and inactivation (19), suggest that these compounds play a role in modulating the contractility of the The present study was undertaken, therefore, to ascertain if tubules. PGFld alters the -__ in vitro contractile activity of the seminiferous tubules of the rat. We also examined the effects on tubular contractility of indomethacin, an inhibitor of PG biosynthesis (20), to further elucidate the possible role of PGs in regulating contractility of the tubules.
MATERIALS
AND METHODS
Sexually mature rats (Holtzman) were anesthetized with ether. The animals were placed on their dorsal side and an incision was made through the scrotum. The testes were extirpated, the tunica albuginea was cut and removed, and the tubules placed in a glass water-jacketed muscle warmer that contained oxygenated Tyrode's solution at 35 C. Individual tubules were obtained by placing the mass of tubules in a warmed Petri dish filled with Tyrode's solution, and the tubules were teased from the interstitial tissue with microdissecting forceps under a dissection microscope. Small segments of tubules, approximately one cm in length, were placed in several drops of Tyrode's solution on a microscope slide. The slide was placed inside a double-walled Plexiglass
152
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
chamber that was attached to the microscope stage. Warm water was circulated through the outer chamber to maintain the internal temperature at approximately 35 C. The temperature was monitored by placing small chips of temperature-sensitive liquid crystal sheets (Edmund Scientific Co.) inside the chamber. The bottom of the chamber was covered with Tyrode's solution to maintain a high internal humidity and prevent evaporation of the bathing medium. The tubules were examined in transmitted light with a magnification of 440 X. The contraction frequency was determined by observing the tubules for periods of one minute and counting the contractions at a preselected site on the tubule. The diameter of the tubule as a measure of tonus and the depth of depression of the wall of the tubule during an actual contraction were determined with an ocular micrometer. Eight or more tubules from a number of different rats were observed. The tubules were first examined in Tyrode's solution (control) and then transferred with a disposable capillary pipet to a solution of PGFlo on the same slide. The PGFl, had been dissolved in 95% ethanol and diluted with Tyrode's solution containing 0.1 g glucose/ Observations were initiated within five minutes of the transfer. 100 ml. In some experiments, after the control observations were completed, The tubules were then the Tyrode's solution was drawn off and retained. rinsed with fresh Tyrode's solution and observations were again made. This procedure was repeated a second time. Next, the original solution was returned to the tubules in the muscle warmer and the contractile activity was again ascertained. Some of the tubules were subsequently rinsed several times and then placed in a solution of PGFlo (low7 M). Four different experiments were conducted to determine the effect In the first investigation, of indomethacin on tubular contractions. one group of rats received 2 i.p. injections of indomethacin (10 mg/kg) Control animals received injections in corn oil each day for two days. of corn oil only. The testes were removed from both groups of animals two to three hours after the last injection. After the contractile activity of the tubules had been ascertained, they were placed in a solution of PGFlo (10m7 M) and examined for contractility. In the second investigation, tubules from untreated animals were placed in a solution of indomethacin (10 ug/ml). Oxygen was bubbled through the solution and the temperature was kept at 35 C. The tubules were observed after they had remained in the indomethacin for approximately 1:~ hours. Control tubules from untreated animals were placed After the in a beaker of Tyrode's solution and similarly treated. initial observations, the tubules from the indomethacin bath were treated with PGFlo (low7 M). in vivo and -in vitro experiments Next, the combined effect of these _Tubules from animals pretreated with indomethacin were was determined. placed in the indomethacin solution (10 ug/ml) for 1% hours and then These tubules were subsequently placed in the PGFlo solution examined. The final experiment consisted and the contractile activity observed. of adding a solution of indomethacin (10 ug/ml) directly to untreated The tubules were examined for contractubules on a microscope slide. tile activity within 5 minutes.
JULY 1975
VOL. 10 NO. 1
153
PROSTAGLANDINS
RESULTS
Stimulation of the frequency of the tubular contractions by PGFlc, appeared to be dose-dependent (Fig. 1). The increase was statistically significant at each concentration, and the level of significance rose for each concentration of PG used. This treatment also caused an increase in tonus as reflected by a decrease in the diameter of the tubules (Fig. 2), but the depth of the contractions was not significantly altered (data not shown).
14.0
12.0
10.0 I : . G
6.0
4 t E
6.0
u
4.0
2.0
Prostaglsndin F1, (M)
Figure 1.
154
Stimulation of the contractile frequency of the seminiferous The vertical bars represent the standard tubules by PGFla. error of the mean. * PCO.02; ** pco.01; + p
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
Ll ... r-l
Control
*:*:*: Fl, (II!) *::. Prostaglandin
400
g 3 ;
300
=
200
B H g
100
i
i:
-. :. -::. .. .... . ..... . ..... . ..... . ..... . ..... . ..... ...... ... ::: ......... ...... ...... ...... ...... . .. .. . ...... ...... ...... ...... lo-8
Figure
.
Effect of PGFlo on the tonus (diameter) of the semini* PCO.05. ferous tubules.
Changing the Tyrode's solution that surrounded the tubules (Fig. 3) resulted in a slight decrease in contractile frequency after the first rinse (PcO.1). A second series of rinses significantly decreased the When the tubules were placed in this original frequency (P
JULY 1975
VOL. 10 NO. 1
155
PROSTAGLANDINS
10.0
8.0
-
I4 *
6.0
-
f
T **
L
Control
Figure 3.
156
1st
2nd
Change
Change
Original
P%
Bath
Effect on frequency of tubular contractions produced by: (a) successive changes in the Tyrode's solution, (b) readdition of the original bathing medium, and (c) addition (10m7M) to the rinsed tubules. * PcO.10; ** P
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
8.0
c
8.0 f
Control
Figure 4.
1st
2nd
Original
Change
Change
8ath
W,
Decrease in the depth of the tubular contractions produced by successive changes in the bathing solution and restoration of activity by addition of the original bath and PGFla (10m7M) to the rinsed tubules. * P
JULY 1975
VOL. 10 NO. 1
157
PROSTAGLANDINS
Pretreatment of rats with indomethacin injections caused a slight decrease in the frequency (P
Control lndomethnin
10.0 .
Prostaglrndin
2.0
Pretreated
Solution
(80 Figure 5.
158
Min)
Pntmated
Solution
So&ion
(5 Min)
Changes in the contractile frequency of the tubules produced by: (a) indomethacin pretreatments (10 mg/kg); (b) tubules placed in solution of indomethacin (10 ng/ml) for 90 min.; (c) combined effect of treatments (a) and (b); (d) tubules placed in solution of indomethacin (10 yg/ml) for 5 min. Tubules were also treated with PGFlu (lop7 M) following the indomethacin treatments. *P
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
DISCUSSION
In this investigation, -in vitro contractile frequency of rat seminiferous tubules was increased by prostaglandin Flo in what appeared to be a dose-dependent manner (Fig. 1). The decrease in diameter of the tubules showed that there was an increase in the tonus of the smooth muscle cells surrounding the tubules (Fig. 2). These tubular responses resembled the increased frequency of contraction and tonus changes of the testicular capsule produced by PGFla (12). That PGFlo did not increase the depth of the contractions is surprising, since other agents that increase the frequency of tubular contractility usually also increase the depth (unpublished observations). The reason for this lack of effect is unknown at the present time. PGFlo was used in this study as the first in a series of investigations of the effects of various PGs on tubular contraction as has been reported for the rabbit testicular capsule (14,17,24,25). The seminiferous tubules also respond similarly to PGF2, (Farr and Ellis, unpublished data).
from tile sult into
The stimulation of contractile activity by PGFlo probably results an increased availability of intracellular Ca+t for the contracprocesses (21). This mobilzation of extracellular Ca* could refrom a release of bound Ca* or a movement of extracellular Ca++ the cell (22,23).
The reduction in contractile activity (frequency and depth of contractions) after successive changes of the Tyrode's solution bathing the tubules (Fig. 4 and S), plus the resumption of contractility when the tubules were resuspended in the original bathing media, indicate that endogenous factors necessary for contractility had been released by the contracting tubules into the initial bathing solution. In similar studies (24,25), the spontaneous contractions of the rabbit testicular capsule were reduced or eliminated by successive changes of the solution bathing the tissue. Extraction of the original solution yielded a PGlike residue that restored contractile activity. These workers concluded that PG-like compounds appeared to be responsible for modulating the contractile activity of the testicular capsule. Our present data suggest the presence of PGs in the bathing media of actively contracting tubules. That PGs are needed for -in vitro contractility of rat seminiferous tubules is further indicated by the observation that rinsing tubules several times with Tyrode's solution and then treating them with PGFla, increased both the frequency and depth of contractions. Spontaneous contractions of isolated rat uteri were also inhibited by indomethacin (26). Since the release of PGs into the fluid bathing the uteri was simultaneously decreased, these workers concluded that the Their observations are synthesis of PGs was inhibited by indomethacin. consistent with other reports of an inhibition of PG synthesis by indoIn this report, Northover (28) has shown that indomethacin (20,27). methacin inhibits the movement of Ca* into smooth muscle cells and decreases the uptake of this ion by the endoplasmic reticulum. He has
JULY 1975
VOL. 10 NO. 1
159
PROSTAGLANDINS
attributed the inhibitory action of indomethacin to this action. Since PGs also alter the binding of Ca* by cell membranes (21,29), additional work is needed to clarify the mechanisms involved in the inhibition of smooth muscle by indomethacin. Our observation that bathing tubules in a solution of indomethacin in vitro was more effective than pretreatment injections in inhibiting -contractile frequency, could best be explained by a difference in the effective concentration of the drug at the site of action. It has been suggested that indomethacin penetrates the cell membrane very slowly (27). Perhaps the blood-testis barrier further reduces the accumulation of indomethacin within the tubules. In each indomethacin experiment, the reduced frequency of tubular contractions was always increased by the application of a solution of PGFlo. This is consistent with the increased frequency of contractions noted earlier when PGF was added to the rinsed and less active tubules. Our results show that % t e smooth muscle cells of the seminiferous tubules of the rat are responsive to PGFl,. The findings are in accord with the hypothesis that the PGs modulate -in vitro contractions of the tubules and are important for normal testicular function.
REFERENCES
160
1.
Study of the normal and irradiated Lacy, D. and J. Rotblat. boundary tissue of the seminiferous tubules of the rat. Exp. Cell Res. 21:49, (1960).
2.
Ross, M. H. The fine structure and development of the peritubular contractile cell component in the seminiferous tubules of the Am. J. Anat. 121:523, (1967). mouse.
3.
The relation between -__ in vitro conSuvanto, 0. and M. Kormano. tractions of the rat seminiferous tubules and the cyclic stage J. Reprod. Fertil. 21:227, (1970). of the seminiferous epithelium.
4.
Clermont, Y. Contractile elements in the limiting membrane of the seminiferous tubules of the rat. Exp. Cell Res. 15:438, (1958).
5.
Ross, M. H. and I. R. Long. Science 153:1271, tubules.
6.
In Hodson. N. The nerves of the testis, epididymus and scrotum. "The Testis" (eds. A. D. Johnson, W. R. Gomes and N. L. Vandemark) Vol. I, pp. 47-99, Academic Press, N. Y., (1970.
7.
Contractility of the seminiferous tubule Niemi, M. and M. Kormano. Ann. Med. of the postnatal rat testis and its response to oxytocin. Exp. Biol. Fenn. 43:40, (1965).
Contractile (1966).
cells in human seminiferous
JULY 1975
VOL. 10 NO. 1
PROSTAGLANDINS
8.
Bressler, R. S. and M. H. Ross. Differentiation of peritubular myoid cells of the testis: Effects of intratesticular implantation of newborn mouse testes into normal and hypophysectomized adults. Biol. Reprod. 6:148, (1972).
9.
Hovatta, 0. Effect of androgens and antiandrogens on the development of the myoid cells of the rat seminiferous tubules (organ culture). Zellforsch. 131:299, (1972).
10.
Weeks, J. R. (1972).
11.
Horton, E. W. Hypotheses on physiological Physiol. Rev. 49:122, (1969).
12.
Eliasson, R. Studies on prostaglandin occurrence, formation and biological actions. Acta Physiol. Stand. 46, Suppl. 158:1, (1959).
13.
Horton, E. W. and I. H. M. Main. A comparison of the biological activities of four prostaglandins. Br. J. Pharmacol. 21:182, (1963).
14.
Johnson, J. M., J. L. Hargrove and L. C. Ellis. Prostaglandin Flcl induced stimulation of rabbit testicular contractions -in vitro. Proc. Sot. Exp. Biol. Med. 138:378, (1971).
15.
Prostaglandins Carpenter, M. P. and B. Wiseman. Fed. Proc. 29:248 (Abstr.), (1970).
16.
Ellis, I.. C. Rat testicular prostaglandin synthesis and its Fed. Proc. 31:295, (1972). relationship to androgen synthesis.
17.
Seeley, R. R. Regulation of rabbit testicular capsular motility: Ph. D. prostaglandins, sex steroids, and sympathomimetic agents. Dissertation. Utah State University, Logan, Utah, (1973).
18.
Comparative aspects of prostaChrist, E. J. and D. A. van Dorp. Biochim. Biophys. Acta glandin biosynthesis in animal tissues. 270:537, (1972).
19.
The distribution of1 3 Anggard, E., C. Larsson and B. Samuelsson. 15-hydroxy prostaglandin dehydrogenase and prostaglandin-delta ’ reductase. Acta Physiol. Stand. 81:396, (1971).
20.
Ferreira, S. H., S. Moncada and J. R. Vane. Indomethacin and Nature aspirin abolish prostaglandin release from the spleen. New Biol., 231:237, (1971).
21.
Carsten, M. E. Prostaglandin's part in regulating uterine contraction by transport of calcium. In "The Prostaglandins: Clinical Applications in Human Reproduction" (ed. E. M. Southern) pp. 59-66, Futura Publishing Co., Mount Kisco, N. Y., (1972).
JULY 1975
The prostaglandins.
VOL. 10 NO. 1
Annu. Rev. Pharmacol.
12:317,
roles of prostaglandins.
of rat testis.
161
PROSTAGLANDINS
162
22.
Ramwell, P. W. and J. E. Shaw. Biological significance of the prostaglandins. Recent Prog. Horm. Res. 26:139, (1970).
23.
Eagling, E. M., H. Love11 and V. R. Pickels. Prostaglandin, myometrial 'enhancement' and calcium. J. Physiol. (Lond.) 213:53p, (1971).
24.
Hargrove, J. L., R. R. Seeley and L. C. Ellis. Contractions of rabbit testes -in vitro: Permissive role of prostaglandins for the actions of calcium and some smooth-muscle stimulating agents. Prostaglandins 3:469, (1973).
25.
Hargrove, J. L., R. R. Seeley and L. C. Ellis. Prostaglandin-like substances: Initiation and maintenance of rabbit testicular contraction -in vitro. Proc. Sot. Exp. Biol. Med. 142:205, (1973).
26.
The contribution of prostaglandin Vane, J. R. and K. I. Williams. production to contractions of the isolated rat uterus. Br. J. Pharmacol. 48:629, (1973).
27.
Indomethacin and aspirin Raz, A., H. Stern and R. Kenig-Wakshal. inhibition of prostaglandin E synthesis by sheep vesicles microsome powder and seminal vesicles s 1ices. Prostaglandins 3:337, (1973).
28.
Northover, B. J. Effect of anti-inflammatory drugs on the binding of calcium to cellular membranes in various human and guinea-pig Br. J. Pharmacol. 48:496, (1973). tissues.
29.
Carsten, M. E. Prostaglandins and oxytocin: Prostaglandins 5:33, uterine smooth muscle.
Their effects on (1974).
JULY 1975
VOL. 10 NO. 1