Carbonic anhydrase isoenzymes CAI and CAII in semen, decidua, chorionic villi and various fetal tissues

Early Human Development 51 (1998) 205–211

Carbonic anhydrase isoenzymes CAI and CAII in semen, decidua, chorionic villi and various fetal tissues S. Ali Akbar

a ,b ,1

b a, , K.H. Nicolaides , P.R. Brown *

a

Molecular Biology and Biophysics Section, Biomedical Sciences Division, King’ s College London, Strand, London WC2 2 LS, UK b Harris Birthright Research Centre for Fetal Medicine, Department of Obstetrics and Gynaecology, King’ s College Hospital, Denmark Hill, London, UK Received 7 July 1997; received in revised form 17 November 1997; accepted 18 November 1997

Abstract ELISA methods were used to determine the concentrations of CAI and CAII in spermatozoa, seminal plasma, ovarian follicular fluid, decidual tissues, chorionic villi and various fetal tissues at 8–11 weeks gestation. Both CAI and CAII were expressed in all subjects except ovarian follicular fluid samples. The possible physiological role(s) of carbonic anhydrases in fertilization, implantation and fetal development are discussed.  1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: CAI; CAII; Semen; Decidua; Chorionic villi; Fetal tissues

1. Introduction Carbonic anhydrases (CA) (EC 4.2.1.1) are zinc metalloenzymes that catalyse the interconversion of CO 2 and HCO 32 . The enzyme is thought to regulate a variety of cellular functions such as gas transport, acid / base regulation, secretion of ions, calcification, and has some role in intermediary metabolism [1,2]. The demonstration of early expression of carbonic anhydrase isoenzymes CAI and *Corresponding author. 1 Present address: Department of Biochemistry, Allama Iqbal Medical College, Lahore, Pakistan. 0378-3782 / 98 / $19.00  1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S0378-3782( 97 )00119-9

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CAII in human fetal blood and placental tissues in our laboratory [3–6], and the suggestion of Clark [7] that CA is probably regulated differently in each of the tissues in which it occurs in the adult, prompted investigation of other fetal tissues for the presence and distribution of the isoenzymes. Insight into the biological significance of the individual isoenzymes and information about the factors regulating them might be gained from knowledge of their different distributions in various tissues and the specialized functions of those tissues. Therefore a variety of normal fetal tissues were examined for the expression of these isoenzymes. Decidual tissues which are the transformed stromal part of the gestational endometrium fuse with the chorion and form the maternal part of the materno-fetal interphase (placenta). Decidual tissues not only produce hormones such as prolactin, but are also thought to have specific functions in implantation, development and maintenance of the pregnancy [8] and there is some evidence that endometrial carbonic anhydrase facilitates the implantation of the blastocyst in rabbits [9]. Therefore, decidual tissues were examined for the presence of CAI and CAII. Okamura et al. [10] demonstrated that bicarbonate in seminal plasma stimulated the motility of the mammalian spermatozoa. There has been some evidence that follicular fluid contains chemotactic factors which attract the sperm [11]. Although CA activity was detected histochemically in the granulosa cells of maturing or mature human follicles [12] and CAII was localized in the epithelia of the seminal vesicle, ampulla of the ductus deferens and distal ductus deferens [13], we know of no report of CAI or CAII in seminal plasma or ovarian follicular fluid. Therefore, semen and follicular fluid were investigated for the presence of the isoenzymes.

2. Materials and methods

2.1. Subjects Decidua, chorionic villus and fetal tissues were obtained with consent of the subjects immediately after termination of pregnancy under section 2 of the 1967 Abortion Act at 8–12 weeks of gestation. The tissues were separated, carefully dissected and placed in clean containers. Samples of ovarian follicular fluid, spermatozoa and seminal plasma were kindly provided by Dr. V. Bolton, King’s College Hospital, London. The semen samples were obtained from healthy persons by masturbation and the spermatozoa microscopically ascertained to be normal in terms of motility and morphology. All tissues were thoroughly washed in Krebs-Ringer bicarbonate glucose (1%, w / v) solution (pH 7.4) containing heparin (5000 i.u. / l) and homogenized with nine volumes of PBS-Tween in a small glass homogenizer. After centrifugation (4000 rev. / min, 48C), for 10 min, supernatants were removed and used for assay. Spermatozoa were washed twice with PBS, resuspended in PBS-Tween (about 500 m l), frozen and thawed twice, centrifuged (4000 rev. / min, 48C for 5 min) and the supernatant used for enzyme assay.

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2.2. Measurement of CAI, CAII and total protein The concentrations of CAI and CAII were measured in all tissue and fluid samples after appropriate dilution with PBS-Tween, using modified double-antibody sandwich enzyme-linked immunosorbent assays (ELISA) described elsewhere [5]. The methods were sensitive enough to detect nanogram amounts of the isoenzymes. The coefficient of variation for CAI assay was 5.9% and for CAII was 5.1% [5]. The total protein concentration was estimated in all samples, using BSA as a standard by the method of Lowry et al. [14].

2.3. Estimation of blood-derived isoenzymes in tissue samples Adult haemoglobin (HbA) was measured in decidual tissues, chorionic villous and ovarian fluid samples while fetal haemoglobin (HbF) was determined in all samples of chorionic villous and fetal tissues, using Double Antibody Sandwich ELISA methods described elsewhere [4]. The measured HbA and HbF values and normal fetal and adult blood data (adult, 13.73 mg CAI / g Hb and 1.45 mg CAII / g Hb; fetuses at 20 weeks of gestation, 39 m g CAI / g Hb and 53 m g CAII / g Hb, [5]) were used to calculate the amounts of isoenzymes derived from fetal and maternal blood. These values were then subtracted from the total amount of the enzyme to calculate the actual level of that enzyme originating in the tissue sample.

3. Results Both CAI and CAII were detected in all except ovarian follicular fluid samples (n 5 5). The concentrations of isoenzymes are shown in Table 1 and Table 2.

4. Discussion This study demonstrated the presence of both CAI and CAII in spermatozoa, seminal fluid, decidua and in a wide variety of fetal tissues. Bicarbonate has been reported to stimulate the motility of the sperm via a bicarbonate-sensitive adenylate cyclase present in the sperm plasma membrane [10]. The suggested mechanisms are alkalinization of the sperm cytosol, increased metabolic activity and increased Ca 21 uptake by the spermatozoa [15]. Seminal plasma which is made up of the secretions of the seminal vesicles (46–80%), the prostate (13–33%), the testes (5%), the bulbourethral and urethral glands, the epididymis and the spermatozoa themselves, contains high concentrations of bicarbonate [16]. Furthermore, spermatozoa themselves are capable of producing bicarbonate [15]. The detection of both CAI and CAII in human spermatozoa and in seminal plasma, the presence of CAII in multiple sites of the male reproductive tract [13], and in mammalian spermatozoa [17] may indicate a physiological role for carbonic anhydrase in the process of fertilization by controlling the concentration of bicarbon-

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Table 1 CAI and CAII in various human tissues. Concentration of CAI and CAII in various normal fetal and maternal tissue extracts Material

Isoenzyme levels ( m g / g TP)

Gestation (weeks)

CAI

CAII

Fetal tissue Heart Adrenal Tongue Gut Brain Brain Liver Lung Pancreas Chorionic villus

8 8 8 9 9 12 12 12 12 9

578 84 144 101 191 134 144 101 17 76

280 469 47 128 32 50 701 43 14 64

Maternal tissue Decidua Decidua

9 12

852 393

148 86

TP, total protein.

ate ions in the seminal plasma and spermatozoa. Bicarbonate ions not only maintain the motility of the spermatozoa but also protect them in the acid environment of the vagina at mid-cycle (seminal plasma can maintain its pH (7.2–7.8) even in the presence of relatively large volumes of powerful acids [16]). The sources of CAII and CAI in seminal plasma are not known. However, it is possible that various parts of the male reproductive tract (e.g. seminal vesicle) which are known sites of CAII [13] also contain CAI and deliver both isoenzymes to the seminal plasma along with other cytosolic components, possibly by apocrine-type secretion (human seminal plasma contains granules and vesicles originating from the prostate as shown by Ronquist and Brody [18]). Although CAI and CAII were not detected in human ovarian follicular fluid in this study, the isoenzymes may be expressed in the oocyte itself; in some mammals, such as the rabbit, CA activity has been detected in the oocyte [12]. Decidual tissues are maternal in origin and functionally important not only for implantation but also for the maintenance of pregnancy. The uterine endometrium in humans is a known source of CA and the activity of the endometrial CA doubles Table 2 CAI and CAII in human semen. Mean concentrations with S.D. of CAI and CAII in spermatozoa and seminal plasma from normal healthy volunteers Material

N

CAI (ng / g TP)

CAII ( m g / g TP)

Spermatozoa Seminal plasma

4 5

2136139 102627

60624 26615

TP, total protein; N, number of samples.

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during the secretory (luteal) phase of the menstrual cycle [19]. The presence of CA in the endometrium and maternal part of the placenta of other mammals e.g. rabbit, sheep and guinea-pig is also well established [20,21]. In the pregnant rabbit in the pre-implantation stage 6–7 days after mating, the blastocyst fluid is remarkably rich in bicarbonate and the endometrium in CA [22]. On the basis of experiments in rabbit with carbonic anhydrase inhibitor (ethoxzoleamide), Boving [9] concluded that implantation of the blastocyst is facilitated by the presence of increased carbonic anhydrase activity. He suggested that increased levels of endometrial CA facilitate both the production and removal of carbon dioxide in the endometrial cell. As carbon dioxide is carried off by the maternal circulation, alkaline products are left behind and the local rise in pH causes disaggregation of the epithelial cells. The trophoblast, being syncytial, remains intact and replaces the epithelium. Boving [9] further suggested that increased alkalinity may also influence the attachment of the blastocyst by making it more sticky. Pincus and Bialy [23] showed that Diamox, a highly specific inhibitor of CA, terminated pregnancy in rats when administered directly into the uterine lumen but Edgren et al. [24], failed to repeat these results. In this study both CAI and CAII were found in fetal heart, adrenal, tongue, gut, brain, liver, lung and pancreas very early in embryonic life. The findings support previous reports documenting the presence of CA activity in fetal liver, lung, kidney, stomach and medulla oblongata from 8 weeks of gestation [25,26]. Lonnerholm and Wistrand [27] detected CA activity in lung tissues from fetuses of gestational ages ranging 14–26 weeks and in fetal kidney between 12–26 weeks of gestation [28]. In pancreas, CA activity was first detected at about 20 weeks of gestation [26]. Although CA is thought to be involved in the secretion of pancreatic bicarbonate in adults [29], the low level of CA isoenzymes in the fetal pancreas reported here might be related to the fact that during the fetal life the intestinal-juice-producing activity of this gland is very small, whereas the heart which has important functions to fulfil from a very early embryonic age, has higher levels. The level of CAII was found to be higher than that of CAI in human fetal liver at 12 weeks of gestation (Table 1) and a similar relationship is observed in adult mammalian liver [29,30]. It was observed that the level of CAII was higher than CAI in human fetal gut (Table 1), confirming the results of Lonnerholm and Wistrand [31] who showed that CAII was consistently higher than CAI in jejunum, ileum and colon. It has been suggested [31] that CA in the intestinal mucosa facilitates electrolyte transfer e.g. reabsorption of sodium chloride and excretion of bicarbonate. Adamson and Waxman [32] who demonstrated that administration of acetazolamide in fetal lambs lowered the rate of lung liquid secretion by about 65% and significantly decreased the chloride concentration in this secreted fluid, suggested that CA was involved in lung liquid secretion in fetal lambs. Lonnerholm and Wistrand [27] suggested a similar role for the enzyme in the lung of the human fetus on the basis of the difference in cellular distribution of CA in fetal lung compared to adult lung. A high concentration of CAII in the fetal adrenal glands compared to other fetal organs (except liver) at 8 weeks of gestation was also observed in this study.

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Although exogenous corticosteroids increased the fetal erythrocyte CA activity in rabbits at 24–30 days of gestation [33], the significance of high levels of CAII in adrenal gland observed here is difficult to explain. In summary, expression of CA isoenzymes in spermatozoa, seminal plasma, decidual tissues, placental tissues and in a variety of other fetal tissues very early in intrauterine life suggests that the enzyme has an important role in the processes of fertilization, implantation and feto-placental development.

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