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Amylases of the genital tract
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<,ni7ed. and their isoamylase patterns were determint*d. Amylase determinations. The total amvlase activity was determined with the Phadebas amylase test.* Agarose gel electrophoresis was used to qarate the various isoamylases.Y The isoenzyme patterns of the corresponding serum and peritoneal fluid samples were visuallv (:ompared, and the contribution of the genital tra( t isoamylases was determined. The judgment was hasccl on the staining intensity on the anodal side ol the salivary main fraction of the peritoneal fluid samples, compared with that of serum (Fig. 1). The conrrihutions of’ the individual groups of isoamylascs to the total amylase activity of serum and peritoneal fIllid collected from the cul-de-sac were also deterrni~led bv dcnsitometric scanning. Evidence of amylase identity. 1.0 A\ccrtain the amylase identity of the starch-degrading t.n/vtne produced by the specimens studied. LOO /*I ‘of each homogenate or peritoneal fluid specimen to be tested was mixed with 5 ~1 of rabbit anti-human parotid amylase. After incubation for 30 minrlrcs at room temperature, the mixture was centrifugr~d. and the supernatant was used for electrophoretic separation. Rf5SUltS
Tissue homogenates. Fallopian tubr. As exemplified by Sample 2 (Fig. 1). the specific isoamylases from the Fallopian tube specimens consisted of one main fraction lvhose migration rate toward the anode was faster that1 that of the salivary main fraction. In addition to this genital main fraction, there were two weak fractions--une with a mobility equal to that of the salivery main liaction and one which migrated even faster than the genital main fraction. NJ difference was observed with respect to isoamylase patterns between homogenates from different parts of the tubes. A different Fallopian tube isoamylase pattern is shown as Sample 3 (Fig. 1). In this case, the fraction showing the most pronounced enzyme activity had a still higher migration rate toward the anode. In addition, in this specimen, there was one more enzyme component on the anodal side of the genital main fraction. In homogenates from the uterine tuhes obtained from the pregnant woman. no specific genital isoamvlase activitv coul~l bc demonstrated. Endometrium. In homogenates from the endometrium from the main part of the uterine. c’orpus, tlo *Pharmacia,
Uppsala, Sweden
654
Skude, Mirdh, and Westriim
2 Fig. 1. Isoamylase
3
Fig. 2. Typical
patterns of serum and tissue homogenates from the genital tract. P denotes main pancreatic isoamylase fraction; S, main salivary fraction; G, genital tract isoamylase. The anode is above. Sample 1: Normal serum. Sample 2: Fallopian tube homogenate. Sample 3: Fallopian tube homogenate. Sample 4: Cervical mucosa homogenate. Sample 5: Prostatic gland homogenate. Sample 6: Seminal vesicle homogenate.
isoamylase patterns of serum (left) and peritoneal fluid (right). For abbreviations, see legend to Fig. 1. Sample pair 1: Nonpregnant woman. Two genital tract-specific fractions in peritoneal fluid not detectable in serum. Sample 2: Nonpregnant woman. One genital tract-specific fraction in peritoneai fluid not detectable in serum. Sampie 3: Nonpregnant woman. Additional fast-migrating salivary isoamylase fractions detectable in serum
Table
Table II. Mean values and ranges of the isoamylasc activities (units per liter) in peritoneal fluid from 15 nonpregnant and six pregnant women
I. Mean
activities
values and ranges of the isoamylase per liter) in serum from 15
(units
nonpregnant
and
six pregnant
I
Non/nqnant umwn
1 -Mean
Isoamylase
Pancreatic Salivary Total
Pregiuznt u’*meri
I
1
Range
90 73
1
Mean
40-160 40-l 10
163
obtained
from
80-230
be detected.
Very
homogenates
of
some
areas
Range 40-120 40-l 10
143
could in
1
72 72
90-260
specific isoamylase activity weak activity was observed endometrium
women
close
to the
uterine
orifices of the Fallopian tubes. In homogenates from decidua obtained in the twentieth gestational week from the patient with diabetes mellitus, no specific isoamylase
activity
was
three
patients
pattern
was
fraction
corresponding
lite
demonstrable.
mucosa. In homogenates
Crruical from all
fraction
of the
corresponding (Sample 4, fraction, Specimrns
characterized
a weak from
zone
investigated,
the
by
prominent
to the tubal
to the Fig. 1).
of cervical one
samples,
of amylase
the male genital
isoamylase
slowest migrating i.e.,
salivary main In front of
main satel-
a migration
rate
isoamylase fraction the cervical main
activity tract.
mucosa
was detectable.
A very weak zone
Pancreatic Nonpancreatic
40 306
10-l 15 115-670
32 121
IO-70 50-250
Total
345
2 1 O-700
152
60-290
of amylase activity, corresponding in mobility to the main specific genital fraction in homogenates of Fallopian tubes, could be identified in the homogenates of prostatic tissue and possibly also in homogenates of the seminal vesicles. In homogenates of the other structures of the male genital tract. i.e., bladder mucosa and prostatic could
utricle,
no specific
genital
isoamylase
activity
be demonstrated (Samples 5 and 6, Fig. 1). Organ culture specimens. The total amylase activity and the specific genital isoamylase patterns Jvere identical in homogenates of Fallopian tube specimens analyzed immediately after removal from the patient and in specimens after five days in organ culture. The corresponding activities and patterns in media collected on different occasions during maintenance of the organ cultures were likewise identical, i.e., show-
Amylases of genital tract. I 655
ing the specific genital isoamylase pattern of the Fallopian tubes. In fresh MEM, no amylase activity was detectable. Serum. The mean values and ranges of the isoamylase activity in serum from the nonpregnant and pregnant women are given in Table I. The specific genital isoamylases seen in homogenates of tissue from rhe Fallopian tubes as well as in the peritoneal fluid were not detectable in serum. Due to genetic variations, some additional fast-migrating salivary fractions were seen in serum from some women (Sample 3, Fig. 2). Peritoneal fluid. The pattern of the level of- isoamylase activity in the peritoneal fluid of healthy nonpregnant women \vas characterized by the pancreatic main fraction, the salivary main component migrating together with one of the genital tract isoamylases, a higher level of enzyme which gave this fraction activity than that of the corresponding fraction in the serum samples, and, in front of this combined salivary and genital isoamylase fraction, two (Sample 1, Fig. 1) or one (Sample 2, Fig. 2) additional broad fraction having rather diffuse margins. Due to genetic variations of the salivary isoamylases, some women demonstrated additional fast-migrating salivary isoamylase fractions in both serum and peritoneal fluid samples (Sample 3, Fig. 2). These fast-migrating salivary isoamylase fractions were always very narrow and distinct and were easily- distinguishable from the genital tract isoamylases. In the peritoneal fluid of healthy nonpregnant women, the total level of amylase activity was more than twice that of serum (Table II). .4s it \\as not possible to distinguish all of the isoamylase fractions of genital origin from those of the salivary glands, the sums of the activities of these Two groups of isoamylases Lvere determined in the peritoneal fluid specimens. The mean ~ZO~~CUItruth isoamylase activity accounted for more than 85 per cent of the total activity in nonpregnant women (Table t I). Visual estimation of the contribution to the peritoneal fluid of the isoamylases from the genital tract revealed their presence in all nonpregnant women studied. In the pregnant women, the specific genital isoamylase activities were considerably reduced or even absent. The quotient between nonpancreatic isoamylase activity in fluid from the cul-de-sac and that in serum, which can be regarded as a measure of the genital isoamylase contribution to the fluid in the cul-de-sac, variecl in healthy nonpregnant women between 1.5 and 16.4 (mean 4.8). The quotients were about two in the early and late stages of the menstrual cycle but reached
Fig. 3. Change in isoamylase panern amylase. For abbreviations, see legend 1: Tubal homogenate befke (left) addition of anti-amylase: Samples 2 cul-de-sac
before
and after
addition
after xtitlition of antito F kg 1. %mplr pair dntl attr‘r fright) rhc alttl “. .I! t’ fro111 thr
trt .~rlti+n,viase.
Samplr
4: Normal human strum without alIti-am> 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<,ni7ed. and their isoamylase patterns were determint*d. Amylase determinations. The total amvlase activity was determined with the Phadebas amylase test.* Agarose gel electrophoresis was used to qarate the various isoamylases.Y The isoenzyme patterns of the corresponding serum and peritoneal fluid samples were visuallv (:ompared, and the contribution of the genital tra( t isoamylases was determined. The judgment was hasccl on the staining intensity on the anodal side ol the salivary main fraction of the peritoneal fluid samples, compared with that of serum (Fig. 1). The conrrihutions of’ the individual groups of isoamylascs to the total amylase activity of serum and peritoneal fIllid collected from the cul-de-sac were also deterrni~led bv dcnsitometric scanning. Evidence of amylase identity. 1.0 A\ccrtain the amylase identity of the starch-degrading t.n/vtne produced by the specimens studied. LOO /*I ‘of each homogenate or peritoneal fluid specimen to be tested was mixed with 5 ~1 of rabbit anti-human parotid amylase. After incubation for 30 minrlrcs at room temperature, the mixture was centrifugr~d. and the supernatant was used for electrophoretic separation. Rf5SUltS
Tissue homogenates. Fallopian tubr. As exemplified by Sample 2 (Fig. 1). the specific isoamylases from the Fallopian tube specimens consisted of one main fraction lvhose migration rate toward the anode was faster that1 that of the salivary main fraction. In addition to this genital main fraction, there were two weak fractions--une with a mobility equal to that of the salivery main liaction and one which migrated even faster than the genital main fraction. NJ difference was observed with respect to isoamylase patterns between homogenates from different parts of the tubes. A different Fallopian tube isoamylase pattern is shown as Sample 3 (Fig. 1). In this case, the fraction showing the most pronounced enzyme activity had a still higher migration rate toward the anode. In addition, in this specimen, there was one more enzyme component on the anodal side of the genital main fraction. In homogenates from the uterine tuhes obtained from the pregnant woman. no specific genital isoamvlase activitv coul~l bc demonstrated. Endometrium. In homogenates from the endometrium from the main part of the uterine. c’orpus, tlo *Pharmacia,
Uppsala, Sweden
654
Skude, Mirdh, and Westriim
2 Fig. 1. Isoamylase
3
Fig. 2. Typical
patterns of serum and tissue homogenates from the genital tract. P denotes main pancreatic isoamylase fraction; S, main salivary fraction; G, genital tract isoamylase. The anode is above. Sample 1: Normal serum. Sample 2: Fallopian tube homogenate. Sample 3: Fallopian tube homogenate. Sample 4: Cervical mucosa homogenate. Sample 5: Prostatic gland homogenate. Sample 6: Seminal vesicle homogenate.
isoamylase patterns of serum (left) and peritoneal fluid (right). For abbreviations, see legend to Fig. 1. Sample pair 1: Nonpregnant woman. Two genital tract-specific fractions in peritoneal fluid not detectable in serum. Sample 2: Nonpregnant woman. One genital tract-specific fraction in peritoneai fluid not detectable in serum. Sampie 3: Nonpregnant woman. Additional fast-migrating salivary isoamylase fractions detectable in serum
Table
Table II. Mean values and ranges of the isoamylasc activities (units per liter) in peritoneal fluid from 15 nonpregnant and six pregnant women
I. Mean
activities
values and ranges of the isoamylase per liter) in serum from 15
(units
nonpregnant
and
six pregnant
I
Non/nqnant umwn
1 -Mean
Isoamylase
Pancreatic Salivary Total
Pregiuznt u’*meri
I
1
Range
90 73
1
Mean
40-160 40-l 10
163
obtained
from
80-230
be detected.
Very
homogenates
of
some
areas
Range 40-120 40-l 10
143
could in
1
72 72
90-260
specific isoamylase activity weak activity was observed endometrium
women
close
to the
uterine
orifices of the Fallopian tubes. In homogenates from decidua obtained in the twentieth gestational week from the patient with diabetes mellitus, no specific isoamylase
activity
was
three
patients
pattern
was
fraction
corresponding
lite
demonstrable.
mucosa. In homogenates
Crruical from all
fraction
of the
corresponding (Sample 4, fraction, Specimrns
characterized
a weak from
zone
investigated,
the
by
prominent
to the tubal
to the Fig. 1).
of cervical one
samples,
of amylase
the male genital
isoamylase
slowest migrating i.e.,
salivary main In front of
main satel-
a migration
rate
isoamylase fraction the cervical main
activity tract.
mucosa
was detectable.
A very weak zone
Pancreatic Nonpancreatic
40 306
10-l 15 115-670
32 121
IO-70 50-250
Total
345
2 1 O-700
152
60-290
of amylase activity, corresponding in mobility to the main specific genital fraction in homogenates of Fallopian tubes, could be identified in the homogenates of prostatic tissue and possibly also in homogenates of the seminal vesicles. In homogenates of the other structures of the male genital tract. i.e., bladder mucosa and prostatic could
utricle,
no specific
genital
isoamylase
activity
be demonstrated (Samples 5 and 6, Fig. 1). Organ culture specimens. The total amylase activity and the specific genital isoamylase patterns Jvere identical in homogenates of Fallopian tube specimens analyzed immediately after removal from the patient and in specimens after five days in organ culture. The corresponding activities and patterns in media collected on different occasions during maintenance of the organ cultures were likewise identical, i.e., show-
Amylases of genital tract. I 655
ing the specific genital isoamylase pattern of the Fallopian tubes. In fresh MEM, no amylase activity was detectable. Serum. The mean values and ranges of the isoamylase activity in serum from the nonpregnant and pregnant women are given in Table I. The specific genital isoamylases seen in homogenates of tissue from rhe Fallopian tubes as well as in the peritoneal fluid were not detectable in serum. Due to genetic variations, some additional fast-migrating salivary fractions were seen in serum from some women (Sample 3, Fig. 2). Peritoneal fluid. The pattern of the level of- isoamylase activity in the peritoneal fluid of healthy nonpregnant women \vas characterized by the pancreatic main fraction, the salivary main component migrating together with one of the genital tract isoamylases, a higher level of enzyme which gave this fraction activity than that of the corresponding fraction in the serum samples, and, in front of this combined salivary and genital isoamylase fraction, two (Sample 1, Fig. 1) or one (Sample 2, Fig. 2) additional broad fraction having rather diffuse margins. Due to genetic variations of the salivary isoamylases, some women demonstrated additional fast-migrating salivary isoamylase fractions in both serum and peritoneal fluid samples (Sample 3, Fig. 2). These fast-migrating salivary isoamylase fractions were always very narrow and distinct and were easily- distinguishable from the genital tract isoamylases. In the peritoneal fluid of healthy nonpregnant women, the total level of amylase activity was more than twice that of serum (Table II). .4s it \\as not possible to distinguish all of the isoamylase fractions of genital origin from those of the salivary glands, the sums of the activities of these Two groups of isoamylases Lvere determined in the peritoneal fluid specimens. The mean ~ZO~~CUItruth isoamylase activity accounted for more than 85 per cent of the total activity in nonpregnant women (Table t I). Visual estimation of the contribution to the peritoneal fluid of the isoamylases from the genital tract revealed their presence in all nonpregnant women studied. In the pregnant women, the specific genital isoamylase activities were considerably reduced or even absent. The quotient between nonpancreatic isoamylase activity in fluid from the cul-de-sac and that in serum, which can be regarded as a measure of the genital isoamylase contribution to the fluid in the cul-de-sac, variecl in healthy nonpregnant women between 1.5 and 16.4 (mean 4.8). The quotients were about two in the early and late stages of the menstrual cycle but reached
Fig. 3. Change in isoamylase panern amylase. For abbreviations, see legend 1: Tubal homogenate befke (left) addition of anti-amylase: Samples 2 cul-de-sac
before
and after
addition
after xtitlition of antito F kg 1. %mplr pair dntl attr‘r fright) rhc alttl “. .I! t’ fro111 thr
trt .~rlti+n,viase.
Samplr
4: Normal human strum without alIti-am> 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.