Catalase activity in human spermatozoa and seminal plasma

Catalase activity was determined in human semen by measuring the oxygen burst with a Clark electrode, after H₂O₂ addition. Significant catalase activities (mean ± SD) were found in migrated, motile spermatozoa (44 ± 17 nmoles O₂/min/10⁸ cells) and in seminal plasma of normozoospermic men (129 ± 59 nmoles O₂/min/ml). It has been demonstrated that seminal catalase originated from prostate; however, its activity was not correlated with the usual prostatic markers (such as citric acid and zinc). Our data suggest a multiglandular function secreted by this organ. The catalase activities measured in seminal samples from asthenozo-ospermic, infertile men were found lower than those from normozoospermic subjects. The understanding of the relative contribution of the different enzyme systems against O₂ toxicity (superoxide dismutase, catalase, glutathione peroxidase) seem to be a priority area of research to understand disturbances of sperm function.     

Sperm selection capacity of the human zona pellucida.

Previous hemizona assay (HZA) results have illustrated a positive and significant correlation between the percentage of morphologically normal spermatozoa in the semen and the number of spermatozoa tightly bound to the zona pellucida. The present study was designed to evaluate the morphologic features using strict criteria of spermatozoa tightly bound to the zona pellucida. Semen samples of 4 normozoospermic and 11 teratozoospermic men were used to compare the percentage of normal spermatozoa in the semen with that found 1) after swim-up separation and 2) bound to the zona under HZA conditions. The mean (+/- SEM) % normal forms for normozoospermic men in semen, after swim-up and zona-bound spermatozoa were 21.5 +/- 1.6, 27.5 +/- 2.9, and 44.8 +/- 3.4, respectively. A significantly higher % of normal forms were found among zona-bound sperm compared to swim-up forms (p = 0.02) and seminal sperm (p = 0.02). The mean % of normal sperm forms present in semen, after swim-up and zona pellucida-binding for teratozoospermic men, were 3.7 +/- 0.9, 5.8 +/- 1.6 and 15.6 +/- 3.1, respectively. Significant differences existed between the % of normal sperm forms found in the swim-up and zona-bound spermatozoa (P = less than 0.01 and P = less than 0.0003, respectively) compared to the original ejaculates. Results indicate that a selective process against abnormal spermatozoa occurs at the site of the zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the human sperm proacrosin-acrosin system using gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophesis

Proteolytic enzymes in extracts of human sperm have been identified and partially characterized using a technique which incorporates gelatin into a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) system. Initially, semen characteristics from four donors were evaluated. Following this, washed sperm were acid extracted and proacrosin and acrosin activities determined spectrophotometrically. Proteinase activity in unactivated sperm extracts was then extracts was then demonstrated using the gelatin-SDS-PAGE system. Three major (Mr approximately equal to 47,000-54,000) and four faint (Mr approximately equal to 34,000-38,000) bands of digestion were observed. Upon activation of sperm extracts it was observed that maximum esterase activity occurred within 7 min of activation while maximum proteinase activity required approximately 15 min. When gels were washed and incubated in the presence of 50 mM benzamidine, no digestion bands were observed. This indicates that all of the digestion bands were due to trypsin-like proteinases. Finally, upon serial dilution of sperm extracts it was found that this SDS-PAGE system is sensitive enough to detect proteinase activity from as few as 30,000 sperm.     

Phospholipases A2 in the reproductive system of the bull

1. Phospholipase A2 activities were studied in the reproductive organs, seminal plasma and spermatozoa of adult bulls. 2. Phosphatidylethanolamine and phosphatidylcholine with 14C-labelled linoleic (lino-PE, lino-PC) or arachidonic acid (ara-PE, ara-PC) at sn-2 position as well as a fluorescent derivative (4-pyrenylbutyric acid) of phosphatidylcholine (PPC) were used as substrates. 3. The radioactive substrates were hydrolysed most strongly by homogenates of the prostate and Cowper's gland, but also seminal vesicle and its secretory fluid, seminal plasma and ejaculated spermatozoa contained hydrolytic activity. The fluorescence substrate was most strongly hydrolysed by homogenates of ampulla and seminal vesicle as well as its secretory fluid, seminal plasma and ejaculated spermatozoa. 4. Seminal plasma and seminal vesicle fluid contained a Ca2(+)-independent enzyme (enzyme I), which hydrolysed only PPC, while another Ca2(+)-dependent enzyme (enzyme II) hydrolysed only the radioactive substrates. 5. Both enzymes were purified from the seminal vesicle fluid and their biochemical properties were analysed. In SDS-PAGE enzyme I preparation resulted in two major bands with molecular weights of 16,000 and 60,000 in equal quantities and minor band at 15,000. The binding of the enzyme I to Con A-Sepharose indicated that it is a glycoprotein and it had multiple pI-values from 3.75 to 5.0. Enzyme II gave in SDS-PAGE two closely located bands with molecular weights of about 15,000 and 16,000 (major band). Isoelectric focusing showed one band at pI 4.7. Both enzymes appear to bind to spermatozoa at ejaculation but their function remains to be shown.     

Electron Microscopic (Energy Dispersive X-ray Analysis) Study of Human Male Reproductive Organs and Semen

Recently, technological advancement helped to improve our knowledge on trace elements in human male reproductive organs and its secretion, semen. In this study, employing energy dispersive x-ray analysis facilities on electron microscope, presence of different elements in human male reproductive organs-??testis, epididymis, caput, corpus and cauda, prostate gland, seminal vesicle, Cowper??s gland and vas deferens??seminal plasma and spermatozoa pellet was studied. Several elements were observed. Gold was one among them that was present in seminal plasma and spermatozoa. It was also present in epididymis caput. Authors consider epididymis caput as the source of gold in semen.     

Beta-galactosidase in the seminal plasma and reproductive organs of the bull

The distribution of beta-galactosidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of beta-galactosidase was found in testis and in different parts of the epididymis, where the activity seemed to be partly in secretory (cauda secretion) and partly in non-secretory, bound form (caput to cauda epididymidis). Gel filtration on Sepharose 6B at pH 7.0 revealed two beta-galactosidase forms (GF-1, Mr approximately 500,000-600,000 and GF-2, Mr approximately 190,000-220,000) in reproductive organs and seminal plasma. The pH-optimum of both beta-galactosidase forms was about 3.75-4.75. Hg2+ and p-chloromercuribenzoate inhibited strongly these activities. Further, form GF-2 seemed to be slightly more sensitive to the thermal inactivation at 50-70 degrees C than form GF-1. In chromatofocusing beta-galactosidase activities in bull seminal plasma coeluted with those of the cauda epididymidis (pI-values 7.5-6.4). On the contrary, prostate, Cowper's gland, testis, ampulla and seminal vesicles had enzyme activities eluting at lower pI-values (6.3-4.2). Thus, the seminal plasma activity is mainly an indicator for the function of the epididymal cauda.     

Fatty acid composition of human spermatozoa and seminal plasma levels of oxidative stress biomarkers in subfertile males

INTRODUCTION: The lipid composition of the sperm membrane has a significant effect on the functional characteristics of spermatozoa. PATIENTS AND METHODS: In the present study, fatty acid composition of spermatozoa and seminal plasma levels of free 15-F(2t)-Isoprostane and catalase were assayed in men with normozoospermia, asthenozoospermia, asthenoteratozoospermia, and oligoasthenoteratozoospermia. RESULTS: In spermatozoa from asthenozoospermic men only oleic acid levels showed a significant difference from normozoospermic men. In spermatozoa from asthenoteratozoospermic and oligoasthenoteratozoospermic samples all of the tested fatty acids were significantly higher than those from normozoospermic samples. Seminal plasma levels of catalase were significantly lower in all patients while levels of free 15-F(2t)-Isoprostane were significantly higher in all patients compared with normozoospermic men. DISCUSSION: Spermatozoa from pathological samples may have higher levels of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), than spermatozoa from normozoospermic men. Therefore, damage induced by lipid peroxidation would be higher in spermatozoa from pathological samples than those from normozoospermic men.     

Characterization of serine proteinases isolated from rat submaxillary gland: with special reference to the degradation of rat kininogens by these enzymes

From the homogenate of rat submaxillary gland, two kinds of serine proteinases, named tentatively proteinases A and B, were isolated and their chemical properties and activities toward rat kininogens were examined, in comparison with those of submaxillary kallikrein. Proteinase A with Mr of 28,200 rapidly cleaved high-molecular-weight (HMW) kininogen into a protein of 67 kDa, which retained thiol-proteinase inhibitory activity, but had lost the correcting activity of HMW kininogen on the prolonged clotting time of Fitzgerald trait plasma. It liberated bradykinin from HMW kininogen but did not liberate kinin from T-kininogen and did not degrade T-kininogen. On the other hand, proteinase B with Mr of 30,400 showed a very weak activity for the liberation of kinin from T-kininogen and the cleavage of T-kininogen at pH 8.0. However, the enzyme extensively degraded T-kininogen at pH 4.5. Proteinase B also degraded HMW kininogen at pH 4.5 and pH 8.0, but liberated bradykinin only at pH 8.0. Thiol-proteinase inhibitory activities of HMW kininogen and T-kininogen were inactivated after the incubation with proteinase B at pH 4.5 but not at pH 8.0, while the correcting activity of HMW kininogen on the Fitzgerald trait plasma was inactivated at pH 4.5 and 8.0. The NH2-terminal amino acid sequences of proteinases A and B were different from each other, and distinguishable with those of serine proteinases in rat submaxillary gland so far reported. These results provide evidence that in addition to the known kallikrein, there exist at least two kinds of serine proteinases in rat submaxillary gland, both of which liberate bradykinin from rat HMW kininogen at pH 8.0 and modulate the functional activities of HMW kininogen and T-kininogen, degrading these proteins at pH 8.0 or 4.5.     

Localization and characterization of glutathione peroxidase (GPx) in boar accessory sex glands, seminal plasma, and spermatozoa and activity of GPx in boar semen

Boar ejaculate owes its characteristic large volume mainly to accessory sex gland (ASG) secretions. These are main contributors to the protective functions of seminal plasma, especially against oxidative damage. Numerous antioxidants have been detected in ASG secretions, and, respectively, in seminal plasma. However, as regards one key antioxidant protector -- the Se-dependent enzyme glutathione peroxidase (GPx) -- there is no agreement yet among researchers as to its presence in boar seminal plasma. Nevertheless, the beneficial effect of dietary Se supplementation on male fertility has been widely recognized. The aim of the present study was to investigate the localization and characterization of GPx in boar ASGs, seminal plasma, and spermatozoa, as well as to evaluate GPx activity in boar semen. Immunohistochemical assays demonstrated GPx presence in the epithelial cells, vacuole membranes, and vascular endothelium of boar seminal vesicle, prostate and bulbourethral glands. Western blot analysis demonstrated the presence of a monomer form of GPx with MW 20 kDa in lysates from seminal vesicle, prostate, bulbourethral glands, and spermatozoa, but not in seminal plasma. Surprisingly, peroxidase activity detected in seminal plasma from normal ejaculates was nearly three times as high as in spermatozoa. Our findings confirmed the presence of immunoreactive GPx in the boar reproductive tract, while further investigation is still warranted to uncover the exact protein forms involved and their function.     

steve bAccumulation of nerve growth factor and its receptors in the uterus and dorsal root ganglia in a mouse model of adenomyosis

Background

The pregnancy hormone human chorionic gonadotropin (hCG) and its free subunits (hCG alpha, hCG beta) are produced in the male reproductive tract and found in high concentrations in seminal fluid, in particular hCG alpha. This study aimed to elucidate changes in peptide hormone profiles in patients showing abnormal semen analyses and to determine the genuineness of the highly abundant hCG alpha.

Methods

Seminal plasma was obtained from 45 male patients undergoing semen analysis during infertility workups. Comprehensive peptide hormone profiles were established by a panel of immunofluorometric assays for hCG, hCG alpha, hCG beta and its metabolite hCG beta core fragment, placental lactogen, growth hormone and prolactin in seminal plasma of patients with abnormal semen analysis results (n = 29) versus normozoospermic men (n = 16). The molecular identity of large hyperglycosylated hCG alpha was analyzed by mass-spectrometry and selective deglycosylation.

Results

hCG alpha levels were found to be significantly lower in men with impaired semen quality (1346 +/- 191 vs. 2753 +/- 533 ng/ml, P = 0.022). Moreover, patients with reduced sperm count had reduced intact hCG levels compared with normozoospermic men (0.097 +/- 0.022 vs. 0.203 +/- 0.040 ng/ml, P = 0.028). Using mass-spectrometry, the biochemical identity of hCG alpha purified from seminal plasma was verified. Under non-reducing conditions in SDS-PAGE, hCG alpha isolated from seminal plasma migrated in a manner comparable with large free hCG alpha with an apparent molecular mass (Mr, app) of 24 kDa, while hCG alpha dissociated from pregnancy-derived holo-hCG migrated at approximately 22 kDa. After deglycosylation with PNGase F under denaturing conditions, all hCG alpha variants showed an Mr, app of 15 kDa, indicating identical amino acid backbones.

Conclusions

The findings indicate a pathophysiological relevance of hCG, particularly its free alpha subunit, in spermatogenesis. The alternative glycosylation pattern on the free large hCG alpha in seminal plasma might reflect a modified function of this subunit in the male reproductive tract.     

Serine proteinase inhibitors of seminal plasma of teleost fish: distribution of activity, electrophoretic profiles and relation to proteinase inhibitors of blood

Anti-proteinase activity has been found in seminal plasma of eight teleost fish species: brown trout, rainbow trout, brook trout, lake whitefish, bream, northern pike, Danube salmon and burbot. This activity correlated with seminal plasma protein and sperm concentrations. Using a mammalian (bovine) trypsin for detecting proteinase inhibitors it was found for the first time that there are species-specific electrophoretic profiles of anti-proteinase activity. One to three bands could be identified by this method. However, additional proteinase inhibitors could be identified by using fish (cod) trypsin. These inhibitors were detected in seminal plasma of salmonids and coregonids and have a slow migration rate. Fast-migrating proteinase inhibitors were present in rainbow, brown and brook trout, northern pike, whitefish and burbot. These inhibitors could be detected in brook and brown trout by using either trypsins. However, they were detected only with bovine trypsin in rainbow trout, northern pike, whitefish and burbot. These results suggest that multiple forms of serine proteinase inhibitors exist in seminal plasma of teleost fish and they differ in their affinity toward serine proteinases. Seminal plasma serine proteinase inhibitors of rainbow trout migrated during electrophoresis similarly to blood plasma proteinase inhibitors, and suggests that the two inhibitors may be similar or the same. Anti-proteinase specific activity was similar in blood and seminal plasma. Proteinase inhibitors of fish seminal plasma seem to be an important part of sperm physiology, possibly related to protection of spermatozoa. Staining for detection of serine proteinase inhibitors also allowed detection of presence of nonspecific esterase in seminal plasma of most species.     

Metalloproteinase activities expressed during development and maturation of the rat prostatic complex and seminal vesicles.

The objective of this study was to characterize proteinase activities expressed during development and maturation of the prostate gland and seminal vesicles of the rat by using gelatin-and casein-containing SDS polyacrylamide gel zymography. The prostatic complexes of 2- and 10-day-old animals and the individual lobes of the prostate (ventral, dorsolateral, and anterior [coagulating gland]) and the seminal vesicles of 15-day-old animals expressed prominent gelatinolytic activities of approximately 64, 71, and 76 kDa. These activities had properties of metalloproteinases; i.e., they were stimulated by Ca2+ and inhibited by EDTA and EGTA. They were greatly diminished by 52 days of age (immediately postpuberty) and were not detected in the dorsal lobe of the adult. Less active gelatinolytic proteinases with molecular masses of approximately 34 and 43 kDa were expressed in the developing prostatic complexes and individual lobes and seminal vesicles, but they were not detected in postpubertal animals. Weak gelatinolytic activities of 82, 85, and 89 kDa were found in the prostatic complexes; these activities were greatly diminished in all prostate lobes with sexual maturation but were expressed in the seminal vesicles at all ages. A large-molecular-mass Ca(2+)-independent proteinase of 130 kDa or greater was first detected in the dorsolateral prostate at 21 days of age. This activity was expressed in both the lateral and dorsal lobes of the adult but was greater in the lateral lobe. Proteinase activities of about 22 and 26 kDa that were not stimulated by Ca2+ were detected in the ventral prostate at 15 days of age by means of both gelatin and casein gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of isolated acrosomal matrices from hamster spermatozoa

The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5.2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70-80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin-SDS-PAGE. The major activity corresponded to bands of relative molecular masses (Mr) of 56,000, 51,000 and 48,000 with two minor bands of Mr 30,000 and 28,000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC-PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of Mr 60,000 and 72,000.     

Fertilization-promoting peptide in reproductive tissues and semen of the male marmoset (Callithrix jacchus)

Fertilization-promoting peptide (FPP) is present in the prostate gland and semen of some mammals, and has been shown to enhance the fertilizing ability of both epididymal mouse and ejaculated human spermatozoa. The novel peptide may prove of importance for the treatment of some cases of male infertility, and a suitable animal model would be useful to test this hypothesis. To this end, we examined reproductive tissues and semen of the male marmoset for the presence of FPP. Peptides were extracted from seminal plasma, testes, prostate, and bulbourethral glands of intact and castrated male marmosets. The peptides were identified by ion-exchange chromatography followed by radioimmunoassay. The mean concentration of FPP immunoreactivity in semen from intact males was 58.7 nM (SE ± 9.9 nM, n = 10), and anion-exchange chromatography revealed FPP as the only immunoreactive peptide present. Analysis of tissues revealed that FPP in semen was likely to be derived from the prostate gland, which contained this peptide as the major source of immunoreactivity (10.86 pmol/gland; SE ± 4.39 pmol/gland, n = 4). Only low concentrations of FPP were detectable in the bulbourethral glands, and the peptide was undetectable in the testis. Surprisingly, FPP was readily detectable in the seminal plasma from one castrated marmoset and was present in the prostate gland from 3 castrates at levels which did not differ significantly from those in intact animals (5.47 pmol/gland, SE ± 1.64 pmol/gland, n = 3). Plasma testosterone measurements indicated that residual circulatory androgens remained after castration, which may be consistent both with the maintenance of mating behavior and the presence of prostatic FPP. We conclude that FPP is present within the prostate gland and seminal plasma of the marmoset at concentrations consistent with a role in male fertility in this species. Mol. Reprod. Dev. 47:113–119, 1997. © 1997 Wiley-Liss, Inc.     

Selenium,Copper and Zinc in Seminal Plasma of Men with Varicocele,Relationship with Seminal Parameters

Varicocele has been associated with decrease in seminal parameters. Selenium (Se), copper (Cu), and zinc (Zn) are trace elements essential for normal spermatogenesis of mammals and play a critical role as antioxidant defense system enzymes. Se, Cu, and Zn are associated with sperm quality in fertile and infertile men. However, there is little information about Se, Cu, and Zn concentrations in semen in patients with varicocele and its association with seminal parameters. The purpose of this study was to determine the concentrations of Se, Cu, and Zn in semen of patients with varicocele and the relationship with seminal parameters. Total Reflection X-Ray Fluorescence was used for the fist time in the seminal fluid analysis. The concentration of selenium in men with varicocele was smaller than the normozoospermic group, while no differences were observed for both concentrations of zinc and copper. A significant positive correlation between zinc and selenium concentration was observed. Selenium in seminal plasma correlates with a good spermatozoa concentrations, motility, and morphology. Additionally, a significant positive correlation was observed between zinc levels and sperm count. In conclusion, a decrease in selenium concentration was associated with detriment of seminal parameters. A study should be conducted to evaluate the benefits of both zinc and selenium supplementation to improve seminal parameters in patients with varicocele.     

Carnitine, acetylcarnitine and the activity of carnitine acyltransferases in seminal plasma and spermatozoa of men, rams and rats.

The concentration of total carnitine (i.e. carnitine plus acetylcarnitine) was measured in seminal plasma and spermatozoa of men and rams. In ram semen, there was a close correlation between the concentration of spermatozoa and that of total carnitine in the seminal plasma, indicating that the epididymal secretion was the sole source of seminal carnitine. The percentage of total carnitine present as acetylcarnitine was 40% in seminal plasma and 70-80% in spermatozoa. The acetylation state of carnitine in seminal plasma was apparently not influenced by the metabolic activity of spermatozoa in ejaculated ram semen as no change was found in the plasma concentration of carnitine or acetylcarnitine up to 45 min after ejaculation. In spermatozoa, the activity of carnitine acetyltransferase (EC 2.3.1.7) was approximately equivalent to that of carnitine palmitoyltransferase (EC 2.3.1.21); and the activity of these enzymes was similar in ram and human spermatozoa but greater in rat spermatozoa. It is concluded that there is no correlation between the content of either total carnitine or the carnitine acyltransferases and the respiratory capacity of spermatozoa.     

猕猴精浆纤溶酶原激活因子的来源及在精子获能中的作用

我们的前期工作表明,不育症人精液中纤溶酶原激活因子（plasminogen activator;PA）活性明显升高;给成年办和猕猴注射长效睾酮诱发无精过程中,精液PA含量也伴随上升,为进一步查明PA的来源和对精子的作用,原位杂交检测组织型PA（tPA）,尿激酶型PA（uPA）及PA抑制因子－1（PAI-1)泊mRNAs在成年健康猕附睾、前列腺和精囊中的表达。体外培养猕猴精子,培液中加入uPA、tPA及其底物纤溶酶原（plasminogen）,测试PA对精子活力、顶体反应及激活卵子的影响。结果表明,猕猴附睾、前列腺和精囊均表达tPA、uPA和PAI-1 mRNAs。加入uPA能维持精子的活力,使精子产生超激活运动,诱导顶体反应的发生,并使精子获得激活卵子的能力,这说明猕猴精浆PA除来源于睾丸外,可能主要来源于附睾及附性腺;在体外,uPA,而不是tPA,可能诱导精子获能。     

Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma

The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids. This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.