Amylases of the genital tract I. Isoamylases of genital peritoneal fluid G.
SKUDE.
P.
A.
L.
WESTROM,
tract tissue homogenates
and
M.D.
MARDH,
M.D. M.D.
Malmb and Lund, Sweden Homogenates of tissue from the female genitul tract ton&n i.coamyhzseswhich are, to a certain extent, electrophoretically distinguishable ,fromthe pancreatic and salizq isoamylases. In healthy nonpregnant women, high le-c~el~s of activity of genital isoamvlases were found in tissue homogenates of cervical and Fallopian tube mucosa, whereas actiuif~ was weak or absent in homogenates of endorwtrium. The isoam~laws of thecewical mucosa had an electrophoretic migration rat0 toward the anode*i&ntic.al to that of the saliuary main fraction, whereas the isoamylases of the Fallopiatl tube migrated j&ter. SpeciJic genital isoamylase activities were also demonstrable in peritoneal fluid collected from the cul-de-sac. During the menstrual cycle, these acti7jitie.s .showd a midc\;cle peak. In pregnant women, the levels of activity of the genital i.sonmylases in peritoneal puid were lower than in nonpregnant women. In homogrnntr.~ of the mak nccew~~ genital glands, the isoamylases spec$ic for the genital tract were present in minute amount.T. Thr isoamvlases speciJic for the genital tract were not detectable in .serum irl either sex. (AK;. OBSTET. GYNECOL. 126: 652, 1976.) THE AMYLASE ACTIVITY in serum is derived from the pancreatic and salivary glands.’ That there is local production of amylase in the epithelium of the human genital tract is deduced from observations of high levels of amylase activity in the contents of cysts derived from the Miillerian or mesonephric duct.*-’ In human subjects as well as in some animal species, i.e., cow. sheep, and golden Syrian hamster, the concentration of amylase in homogenates of the uterine tubes is high. In some species, i.e., dog, cat, rat, mouse, and guinea pig, no evidence of tubal amylase production is found, at least not during estrus.6’ ’ The present report describes the patterns of the
From the Department of Clinical Chemistry, University of Lund, Ma&5 GeneraiHospital, Malm& ihe Institute of Medical Microbioloev. University of Lund, and the Department of Obst&ics and G$&ology, University Hospital, Lund. Supported by grants from the Swedish Medical Research Council (Projects Nos. B76-13X-581.12B and 876-16X-04509). Received for publication .4ccepted May
Februav
4, 1976.
19, 1976.
Reprint requests: Dr. L. Westrijm, Department of Obstetrics and Gynecologlv, University of Lund, S-221 Lund, Sweden.
85
isoamylases found in tissue homogenates of various parts of the female and male genital tracts as well as the isoamylase pattern in peritoneal fluid obtained from the cul-de-sac of nonpregnant and pregnant women.
Clinical material end spedmen cobution Tissue specimen. FaMupian tubes. One or both Fallopian tubes were obtained from 14 women. Five of them, between 25 and 35 years of age, underwent legal sterilization for nonmedical reasons. With the patient’s consent, the operation was performed as a salpingectomy. Uterine tubes were obtained at hysterectomy from seven women between 35 and 45 years old. These seven women were still menstruating. Tubes from one postmenopausal woman were obtained at ’ hysterectomy. From one pregnant patient, in the tkventieth \veek of gestation, the tubes were removed and the pregnancy was interrupted because of diabetes mcllitus. With the exception of this woman, who was treated with insulin, and three women who were using oral contraceptives, neither hormonal nor radiologic treatment leas given to any of the tvomen bef’orr operation. Malignancy was not found in any of the specimens removed at operation.
Volume Number
Amylases of genital tract. I 653
126 6
E~&W~T~UWL This was obtained from hysterectomy specimens of three of the women of child bearing age. Decidual tissue was obtained from the pregnant diabetic woman. C~rz~icrrl ~U~KU.W. Mucosa was obtained from the above-mentioned three hysterectomy specimens. C’rinary bladder mucosn. prostatic glad, prostatic utriclr, arid scmirral z~iclrs. Specimens from these structures were obtained from one male subject, operated upon because of carcinoma of the urinary bladder. Peritoneal fluid. Peritoneal fluid from the cul-de-sac was collected at laparoscopy from 15 women. between 19 and 3-l years old. The laparoscopy was performed as part of an investigation of infertility in five women and because of acute abdominal pain, which laparoscopy revealed to be ovulatory, in 10 women. All 15 women had normal internal genital organs and were menstruating regularly. The peritoneal fluid was aspirated by means of a cannula which was introduced into the abdominal cavity through a separate incision in the abdominal wall. Peritoneal fluid was also obtained from the cul-de-sac of six pregnant women in the eighth to twelfth gestational weeks M.ho were undergoing legal abortion. These samples were collected by a needle puncture of the cul-de-sac through the posterior vaginal fornix. Serum samples. A blood specimen was drawn at operation from all subjects.
Laboratory methods Tissue homogenates. After careful rinsing in 0.9 per cent sodium chloride. the tissue specimens were homogenized with a tissue homogenizer* in 0.025M phosphate buffer, pH 7.0, containing 0.9 per cent sodium chloride. The homogenates then underwent sonication. The clear supernatant obtained after centrifugation at 4,000 g for 15 minutes was used for electrophoretic analysis. Organ cultures. Specimens from the tubal isthmus, ampulla. ancl fimbria of three of the Fallopian tubes removed at operation were maintained as organ cultures.H Minimum essential medium? (MEM) was used without further addition for the transportation of the specimens and as organ culture medium. The organ cultures were set up in Petri dishes. The tissue pieces were nailed to silicone rubber disks. Incubation was made in 10 per cent carbon dioxide at 37” C.* The isoamvlases were studied in the medium used to transport the tubes and in the organ culture medium after one, tlvo, three, and five days of incubation, at which incerlals half of the medium in the Petri dishes *Turnes. tFlox Laboratories
Ltd..
England.
was replaced. At the end of the culture !naintenance period, the tissue samples were homog I;IW Sample 5: Rabbit anriscrum diluted in sodium c-hlctrlrte sc~ltrtiorr. higher values (more than 15) al.oun(l the csrimarc-d time of ovulation. ,4part from this c)tr:lator! peak, there were no differences in t hc p;t!terIb of the isoamylases in peritoneal fluid betwc~~ \\omt’n \%ho used oral contraceptives or intrauterillt tin ic-es ar~tl those who did 1101 use any contraccptivcs In the pregnant women, the pcritonc*al fluidiscrum nonpancreatic isoamylase activitv cluotktlt \xrietl between 0.6 and 2.2 (mean 1X). Evidence of amylase identity. ‘I’he addition ot antiamylase to the tissue homogenates axrrl the specimens from the cul-de-sac completely abolishcti the salivar) and pancreatic isoamylase acrivities as wt:ll as the additional starch hydrolyzing activity fi~r~nrl II> the samples from the female genital tract (Sanil>lc i)atrs 1 10 3, Fig. 3).
Comment The present study confirms earlier reports’~ ’ on a local production of enzymes with xtivity in organs derived from the Miillerian ducts and mesonephron. In health) nonpregnanr wmi611, sl.xGtic genital isoamylases were demonstrable in tissut homogenates of Fallopian tube and cervical mucosa but not endomrtrium. The isoamylases of the cervical m~~cosa had an electrophoretic migration rate identical IO that ol’ the salivary main fraction, whereas the isoamylases of the Fallopian tubes generally migrated f;tstc*r loward the anode. The facrs that different parts of the female genital tract show different specihc genital isoamvlase activities
656
Skude,
Mirdh,
and WestrGm
and patterns and that culture media of Fallopian tube organ cultures show no decrease in isoamylase activity with time, despite the fact that half the volume of the media was changed daily, strongly suggests that production of the specific genital isoamylases is local. The particular cells responsible for this local enzyme production are not yet known. The amount of isoamylase produced in the genital organs is small compared to that produced by the pancreatic and salivary glands. No isoamylase activity corresponding to that found in tissue homogenates of the Fallopian tube could be detected in serum. “Leakage” of enzymes from the Fallopian tubes into the peritoneal cavity is likely, as specific genital isoamylases were identified in peritoneal fluid from the cul-de-sac. The isoamylase pattern of this fluid reflects that
of
the
tissue
homogenates
of
Fallopian
tubes,
although some of the weaker fractions of the specific genital isoamylases found in tissue homogenates were not detectable in peritoneal fluid. Peritoneal fluid ma)
REFERENCES 1. Meites, S., and Rogols, S.: Amylase isoenzymes, CRC Crit. Rev. Clin. Lab. Sci. 2: 103, 1971. 2. Skude, G.: Sources of the serum isoamylases and their normal range of variation with age, Stand. J. Gastroenterol. 10: 577, 1975. 3. Green, C.: Identification of alpha-amylase as a secretion of the human Fallopian tube and “tube-like” epithelium of Miillerian and mesonephric duct origin, AW J. Osszr. GYNECOL. 73: 402. 1957. 4. Hobbs, J. R.. and Aw, S. E.: Urinary isoamylases, it1 Duback, U. C., editor: Enzymes in urine and kidney, Bern, 1968, Hans Huher, p. 281. 5. Vacikova. A.: Assessment of electrophoretic mobilities of some human isoamylases, J. Chromatogr. 69: 349, 197?. 6. McGeachin, R. L., Hargan, L. A., Porter, B. A., and Daus.
be used for the analysis of variation in specific genital isoamylase activities. Such variations were noted during the menstrual cycle, the specific genital isoarnylase activities
showing
a peak
activity
around
the
estimateci
time of ovulation. It was also noted that the activities were low or absent during an intrauterine pregnancy. Studies of isoamylase activities in peritoneal fluid have also been made in patients with infections of the Fallopian tubes” and in those with ectopic pregnant-1 In the male subject, genital isoamylase activity was found in the tissue homogenates of the prostate and seminal vesicles. However, the isoamylases were produced in amounts so minute that they could not bc detected in seminal plasma.” The biological significance of the isoamylases in the female genital tract is not known. The demonstrated variations of the specific genital isoamylases during the menstrual physiologic
cycle
suggest
processes
a role
of these
enzymes
in the
of reproduction.
A. T.: Amylase in Fallopian tubes, Proc. Sot. Exp. Biol. Med. 99: 130, 1958. 7. Skude. G., and Mirdh, P.-A.: Isoamylases in blood, urine, and tissue homogenates from some experimental animals, Stand. J. Gastroenterol. 1976. In press. 8. Mirdh, P.-A., W’estriim, L., von Mecklenburg, C., and Hammar. E.: Studies on ciliated epithelia of the human genital tract. I. Swelling of the cilia of Fallopian tube epithelium in organ cultures infected with M~coplamo homirzir, Br. J. Vener. Dis. 52: 52, 1976. 9. Skude, G.: Electrophoretic separation, detection, and variation of amylase isoenzymes, Stand. J. Clin. Lab. Invest. 35: 41, 1975. 10. Westriim, L., Skude, G., and M%-dh, P.-A.: Amylases ot the genital tract. II. Peritoneal fluid isoamylases in acute salpingitis, AM. J. OBSTET. GYNECOL. 136: 657, 1976.
SKUDE.
P.
A.
L.
WESTROM,
tract tissue homogenates
and
M.D.
MARDH,
M.D. M.D.
Malmb and Lund, Sweden Homogenates of tissue from the female genitul tract ton&n i.coamyhzseswhich are, to a certain extent, electrophoretically distinguishable ,fromthe pancreatic and salizq isoamylases. In healthy nonpregnant women, high le-c~el~s of activity of genital isoamvlases were found in tissue homogenates of cervical and Fallopian tube mucosa, whereas actiuif~ was weak or absent in homogenates of endorwtrium. The isoam~laws of thecewical mucosa had an electrophoretic migration rat0 toward the anode*i&ntic.al to that of the saliuary main fraction, whereas the isoamylases of the Fallopiatl tube migrated j&ter. SpeciJic genital isoamylase activities were also demonstrable in peritoneal fluid collected from the cul-de-sac. During the menstrual cycle, these acti7jitie.s .showd a midc\;cle peak. In pregnant women, the levels of activity of the genital i.sonmylases in peritoneal puid were lower than in nonpregnant women. In homogrnntr.~ of the mak nccew~~ genital glands, the isoamylases spec$ic for the genital tract were present in minute amount.T. Thr isoamvlases speciJic for the genital tract were not detectable in .serum irl either sex. (AK;. OBSTET. GYNECOL. 126: 652, 1976.) THE AMYLASE ACTIVITY in serum is derived from the pancreatic and salivary glands.’ That there is local production of amylase in the epithelium of the human genital tract is deduced from observations of high levels of amylase activity in the contents of cysts derived from the Miillerian or mesonephric duct.*-’ In human subjects as well as in some animal species, i.e., cow. sheep, and golden Syrian hamster, the concentration of amylase in homogenates of the uterine tubes is high. In some species, i.e., dog, cat, rat, mouse, and guinea pig, no evidence of tubal amylase production is found, at least not during estrus.6’ ’ The present report describes the patterns of the
From the Department of Clinical Chemistry, University of Lund, Ma&5 GeneraiHospital, Malm& ihe Institute of Medical Microbioloev. University of Lund, and the Department of Obst&ics and G$&ology, University Hospital, Lund. Supported by grants from the Swedish Medical Research Council (Projects Nos. B76-13X-581.12B and 876-16X-04509). Received for publication .4ccepted May
Februav
4, 1976.
19, 1976.
Reprint requests: Dr. L. Westrijm, Department of Obstetrics and Gynecologlv, University of Lund, S-221 Lund, Sweden.
85
isoamylases found in tissue homogenates of various parts of the female and male genital tracts as well as the isoamylase pattern in peritoneal fluid obtained from the cul-de-sac of nonpregnant and pregnant women.
Clinical material end spedmen cobution Tissue specimen. FaMupian tubes. One or both Fallopian tubes were obtained from 14 women. Five of them, between 25 and 35 years of age, underwent legal sterilization for nonmedical reasons. With the patient’s consent, the operation was performed as a salpingectomy. Uterine tubes were obtained at hysterectomy from seven women between 35 and 45 years old. These seven women were still menstruating. Tubes from one postmenopausal woman were obtained at ’ hysterectomy. From one pregnant patient, in the tkventieth \veek of gestation, the tubes were removed and the pregnancy was interrupted because of diabetes mcllitus. With the exception of this woman, who was treated with insulin, and three women who were using oral contraceptives, neither hormonal nor radiologic treatment leas given to any of the tvomen bef’orr operation. Malignancy was not found in any of the specimens removed at operation.
Volume Number
Amylases of genital tract. I 653
126 6
E~&W~T~UWL This was obtained from hysterectomy specimens of three of the women of child bearing age. Decidual tissue was obtained from the pregnant diabetic woman. C~rz~icrrl ~U~KU.W. Mucosa was obtained from the above-mentioned three hysterectomy specimens. C’rinary bladder mucosn. prostatic glad, prostatic utriclr, arid scmirral z~iclrs. Specimens from these structures were obtained from one male subject, operated upon because of carcinoma of the urinary bladder. Peritoneal fluid. Peritoneal fluid from the cul-de-sac was collected at laparoscopy from 15 women. between 19 and 3-l years old. The laparoscopy was performed as part of an investigation of infertility in five women and because of acute abdominal pain, which laparoscopy revealed to be ovulatory, in 10 women. All 15 women had normal internal genital organs and were menstruating regularly. The peritoneal fluid was aspirated by means of a cannula which was introduced into the abdominal cavity through a separate incision in the abdominal wall. Peritoneal fluid was also obtained from the cul-de-sac of six pregnant women in the eighth to twelfth gestational weeks M.ho were undergoing legal abortion. These samples were collected by a needle puncture of the cul-de-sac through the posterior vaginal fornix. Serum samples. A blood specimen was drawn at operation from all subjects.
Laboratory methods Tissue homogenates. After careful rinsing in 0.9 per cent sodium chloride. the tissue specimens were homogenized with a tissue homogenizer* in 0.025M phosphate buffer, pH 7.0, containing 0.9 per cent sodium chloride. The homogenates then underwent sonication. The clear supernatant obtained after centrifugation at 4,000 g for 15 minutes was used for electrophoretic analysis. Organ cultures. Specimens from the tubal isthmus, ampulla. ancl fimbria of three of the Fallopian tubes removed at operation were maintained as organ cultures.H Minimum essential medium? (MEM) was used without further addition for the transportation of the specimens and as organ culture medium. The organ cultures were set up in Petri dishes. The tissue pieces were nailed to silicone rubber disks. Incubation was made in 10 per cent carbon dioxide at 37” C.* The isoamvlases were studied in the medium used to transport the tubes and in the organ culture medium after one, tlvo, three, and five days of incubation, at which incerlals half of the medium in the Petri dishes *Turnes. tFlox Laboratories
Ltd..
England.
was replaced. At the end of the culture !naintenance period, the tissue samples were homog I;IW Sample 5: Rabbit anriscrum diluted in sodium c-hlctrlrte sc~ltrtiorr. higher values (more than 15) al.oun(l the csrimarc-d time of ovulation. ,4part from this c)tr:lator! peak, there were no differences in t hc p;t!terIb of the isoamylases in peritoneal fluid betwc~~ \\omt’n \%ho used oral contraceptives or intrauterillt tin ic-es ar~tl those who did 1101 use any contraccptivcs In the pregnant women, the pcritonc*al fluidiscrum nonpancreatic isoamylase activitv cluotktlt \xrietl between 0.6 and 2.2 (mean 1X). Evidence of amylase identity. ‘I’he addition ot antiamylase to the tissue homogenates axrrl the specimens from the cul-de-sac completely abolishcti the salivar) and pancreatic isoamylase acrivities as wt:ll as the additional starch hydrolyzing activity fi~r~nrl II> the samples from the female genital tract (Sanil>lc i)atrs 1 10 3, Fig. 3).
Comment The present study confirms earlier reports’~ ’ on a local production of enzymes with xtivity in organs derived from the Miillerian ducts and mesonephron. In health) nonpregnanr wmi611, sl.xGtic genital isoamylases were demonstrable in tissut homogenates of Fallopian tube and cervical mucosa but not endomrtrium. The isoamylases of the cervical m~~cosa had an electrophoretic migration rate identical IO that ol’ the salivary main fraction, whereas the isoamylases of the Fallopian tubes generally migrated f;tstc*r loward the anode. The facrs that different parts of the female genital tract show different specihc genital isoamvlase activities
656
Skude,
Mirdh,
and WestrGm
and patterns and that culture media of Fallopian tube organ cultures show no decrease in isoamylase activity with time, despite the fact that half the volume of the media was changed daily, strongly suggests that production of the specific genital isoamylases is local. The particular cells responsible for this local enzyme production are not yet known. The amount of isoamylase produced in the genital organs is small compared to that produced by the pancreatic and salivary glands. No isoamylase activity corresponding to that found in tissue homogenates of the Fallopian tube could be detected in serum. “Leakage” of enzymes from the Fallopian tubes into the peritoneal cavity is likely, as specific genital isoamylases were identified in peritoneal fluid from the cul-de-sac. The isoamylase pattern of this fluid reflects that
of
the
tissue
homogenates
of
Fallopian
tubes,
although some of the weaker fractions of the specific genital isoamylases found in tissue homogenates were not detectable in peritoneal fluid. Peritoneal fluid ma)
REFERENCES 1. Meites, S., and Rogols, S.: Amylase isoenzymes, CRC Crit. Rev. Clin. Lab. Sci. 2: 103, 1971. 2. Skude, G.: Sources of the serum isoamylases and their normal range of variation with age, Stand. J. Gastroenterol. 10: 577, 1975. 3. Green, C.: Identification of alpha-amylase as a secretion of the human Fallopian tube and “tube-like” epithelium of Miillerian and mesonephric duct origin, AW J. Osszr. GYNECOL. 73: 402. 1957. 4. Hobbs, J. R.. and Aw, S. E.: Urinary isoamylases, it1 Duback, U. C., editor: Enzymes in urine and kidney, Bern, 1968, Hans Huher, p. 281. 5. Vacikova. A.: Assessment of electrophoretic mobilities of some human isoamylases, J. Chromatogr. 69: 349, 197?. 6. McGeachin, R. L., Hargan, L. A., Porter, B. A., and Daus.
be used for the analysis of variation in specific genital isoamylase activities. Such variations were noted during the menstrual cycle, the specific genital isoarnylase activities
showing
a peak
activity
around
the
estimateci
time of ovulation. It was also noted that the activities were low or absent during an intrauterine pregnancy. Studies of isoamylase activities in peritoneal fluid have also been made in patients with infections of the Fallopian tubes” and in those with ectopic pregnant-1 In the male subject, genital isoamylase activity was found in the tissue homogenates of the prostate and seminal vesicles. However, the isoamylases were produced in amounts so minute that they could not bc detected in seminal plasma.” The biological significance of the isoamylases in the female genital tract is not known. The demonstrated variations of the specific genital isoamylases during the menstrual physiologic
cycle
suggest
processes
a role
of these
enzymes
in the
of reproduction.
A. T.: Amylase in Fallopian tubes, Proc. Sot. Exp. Biol. Med. 99: 130, 1958. 7. Skude. G., and Mirdh, P.-A.: Isoamylases in blood, urine, and tissue homogenates from some experimental animals, Stand. J. Gastroenterol. 1976. In press. 8. Mirdh, P.-A., W’estriim, L., von Mecklenburg, C., and Hammar. E.: Studies on ciliated epithelia of the human genital tract. I. Swelling of the cilia of Fallopian tube epithelium in organ cultures infected with M~coplamo homirzir, Br. J. Vener. Dis. 52: 52, 1976. 9. Skude, G.: Electrophoretic separation, detection, and variation of amylase isoenzymes, Stand. J. Clin. Lab. Invest. 35: 41, 1975. 10. Westriim, L., Skude, G., and M%-dh, P.-A.: Amylases ot the genital tract. II. Peritoneal fluid isoamylases in acute salpingitis, AM. J. OBSTET. GYNECOL. 136: 657, 1976.
