49 GOBLET-CELLS-AND-ENTEROCYTES-OF-SOREX-ARANEUS 49 Zoologica Poloniae (2002) 47/3-4: 49-57 NUMBER OF GOBLET CELLS AND ENTEROCYTES IN THE SMALL INTESTINE OF SEXUALLY IMMATURE MALES AND FEMALES OF SOREX ARANEUS L. BOGDANA WILCZYÑSKA AND MARTA JAROSZEWSKA Department of Histology and Embriology of Vertebrates, Nicolas Copernicus University, 87-100 Toruñ, ul. Gagarina 9, Abstract. The number of goblet cells and enterocytes as well as the height of the enterocytes forming the epithelial lamina of the mucosa in the small intestine were determined in young males and females of Sorex araneus L. The material included small intestines of 7 males and 9 females of S. araneus in the first calendar year of their lives. The intestine was divided into three fragments: anterior, mid and posterior. The number of goblet cells per circumference of the villous longitudinal section was 7.11±1.014; 8.01±1.221; 12.90±2.934 (males) and 6.46±1.139; 8.45±1.921; 12.02±3.541 (females) in the consecutive fragments of the small intestine. The number of enterocytes per circumference of the villous longitudinal section for the males was similar in the anterior (151.55±38.144) and mid (l60.l4±30.613) fragments of the intestine and higher than in the posterior fragment (139.17±26.486). Likewise, the corresponding values for the females are identical in all the three fragments, reaching c. 195.79 enterocytes. The ratio of the number of enterocytes to the number of goblet cells in both sexes was the highest in the anterior fragment of the intestine: 20.26±3.572 (males) and 31.15±7.352 (females). The height of enterocytes decreased along the small intestine, with that in males reaching 21.19±2.523 µm (anterior fragment) and 16.77±3.127 µm (posterior fragment); in females, these values ranged from 16.14±1.657 µm to 15.30±2.126 µm, respectively. INTRODUCTION The common shrew S. araneus was subject to a number of studies concerning its biology (DEHNEL, 1949; KUBIK, 1951; BOROWSKI et al., 1952; SERAFIÑSKI, 1955; PUCEK, 1960; SKOCZEÑ, 1970; BUCHALCZYK, 1972). Some studies dealt with seasonal changes in the structure of the organs, the skeletal
50 B.-WILCZYÑSKA-AND-M.-JAROSZEWSKA 50 system in particular (DZIER YKRAJ-ROGALSKA, 1952; BAZAN, 1953; CROWCROFT et al., 1959; BUCHALCZYK et al., 1964; PUCEK, 1965, 1970; PUCEK M. 1965; HYVÄRINEN, 1969, 1968). A number of authors pointed to age-dependent-changes in the structure of the internal organs (FONS et al., 1984; PUCEK, 1970; SIUDA, 1964; BIELAK et al., 1960). There are scarcely any studies on the structure of the alimentary canal in relation to the food taken by the common shrew. The food consists mainly of insects (over 90% of food contents) as well as other invertebrates such as snails, myriapods, young mammals, and small frogs (KOWALSKI, 1964). It is clear that the alimentary canal of the shrew must be adapted to digesting protein-rich food, which additionally contains much chitin per unit volume. The studies of MYRCHA (1967) and WILCZYÑSKA (1996) define a relationship between the histological structure of the esophagus and stomach and the kind of food taken. In order to complete the data confirming the relationship between the food kind and the histological structure of the segments of the alimentary canal, the epithelium from the small intestine of the common shrew was studied in detail. The number of absorptive cells (enterocytes) as well as of goblet cells producing epithelium were assessed. Changes in the structure of the epithelium of the small intestine, which take place throughout the shrew's life, constitute an interesting aspect here. Consequently, the first stage of the studies was conducted on the small intestine of sexually immature males and females with the body mass of 7-8.8 g. The shrews of this mass, caught in the summer months, are the heaviest among the individuals born every year and are during the "stage of relative summer stability" in a physiological sense. Afterwards, the "autumn regress" takes place in relation to the body size and the mass of the inner organs (DEHNEL, 1949; KUBIK, 1951; BOROWSKI et al., 1953; PUCEK, 1965). In the spring, these individuals called wintering ones (DEHNEL, 1949) become sexually mature and start their intense reproduction. MATERIAL AND METHOD The small intestine of the sexually immature males (7) and females (9) of the common shrew (S. araneus) constituted the material. Their body mass ranged from 7.0 to 8.8 g (Table l). The animals were caught between July and August 2000 in Warmiñsko-Mazurskie voievodship. The removed small intestine was kept for 3 days in a mixture of 10% formalin solution with 75% ethanol in the ratio 20:1. Then, they were kept for a month in 4% formalin solution. The intestine was divided into three equal fragments: anterior, mid, and posterior. The length of consecutive fragments was measured on drawings made in stereo microscope equipped with the drawing apparatus. The sum of the lengths of consecutive fragments equaled the total length of the small intestine (Table 1).
51 GOBLET-CELLS-AND-ENTEROCYTES-OF-SOREX-ARANEUS 51 Table 1 Characteristics of the examined shrews Animal males females No Mass Body length Intestine Mass Body length Intestine [g] [mm] length [mm] [g] [mm] length [mm] l 7.0 45.7 9.08 7.2 64.0 8.89 2 7.1 62.0 11.65 7.3 59.7 8.16 3 7.4 52.6 8.26 7.3 56.3 9.06 4 7.5 62.9 11.16 7.5 51.7 11.44 5 7.6 66.7 12.17 7.6 64.0 11.00 6 7.6 62.0 11.68 7.8 56.2 14.03 7 8.0 61.4 10.63 8.0 63.0 8.81 8 8.5 57.5 8.58 9 8.8 61.3 7.39 Mean±SD 7.5±0.34 59.0±7.26 10.66±1.46 7.8±0.56 59.3±4.73 9.71±2.08 Histological processing of the material followed the routine protocol for electron microscopy, using spurr. The blocks were cut perpendicularly to the long axis of the segment into sections 1-2µm thick, stained with methylene blue with azure II on borax. The height of intestinal villi from their base to the top was measured in light microscope with calibrated eye-piece. Along the circumference of the vertical section through the villi, both goblet cells and enterocytes were counted and the ratio of the enterocytes to the goblet cells was estimated. The measurements were taken individually for each intestinal fragment. Both arithmetical mean and standard deviation were calculated. The significance of the differences between the data from the consecutive fragments was estimated, with the test for small, untied samples. RESULTS The villi in all the small intestine were found to be higher in the males compared to females. Their height was 332.30±73.164 µm (males) and 310.85±98.755 µm (females) in the anterior fragment of the small intestine, 364.65±90.665 µm (males) and 316.66±44.269 µm (females) in the mid fragment (Table 2). These differences are statistically insignificant. In the posterior fragment of the intestine the height of the villi was similar for both sexes and amounted to 305.09±71.028 µm (males) and 300.30±18.564 µm (females). The number of enterocytes along the circumference of the vertical section through the villi was smaller in the males than in the females. There were 151.55±38.144 enterocytes (males) in the anterior fragment of the intestine
52 B.-WILCZYÑSKA-AND-M.-JAROSZEWSKA 52 Table 2 Structural proportions of the mucosa membrane of the small intestine of the young males and females of the common shrew Fragment of small intestine Height of villi [µm] Height of enterocytes [µm] No of enterocytes along circumference of vertical section of villi No of goblet cells along circumference of vertical section of villi No of enterocytes per number of goblet cells males anterior 332.30 ±73.164 21.19±2.523 151.55±38.144 7.11±1.014 20.26±3.572 mid 364.65 ±90.665 18.11±1.766 160.14±30.613 8.01±1.221 19.09±4.687 posterior 305.09 ±71.028 16.77±3.127 139.17±26.486 12.90±2.934 10.52±3.015 females anterior 310.85 ±98.755 16.14±1.657 200.92±54.841 6.46±1.139 31.15±7.352 mid 316.66 ±44.269 18.29±3.068 195.99±22.401 8.45±1.921 24.18±5.814 posterior 300.30 ±18.564 15.30±2.126 190.48±47.158 12.02±3.541 17.99±10.902 and 200.92±54.841 (females). The difference was statistically insignificant. In the mid fragment the number of enterocytes amounted to 160.14±30.613 (males) and 195.99±22.401 (females) (p 0.1), whereas in the posterior fragment it was 139.17±24.486 and 190.48±47.158, respectively. In both sexes the number of mucous cells in the epithelial lamina of the intestine increased along the intestine. In the consecutive segments of the intestine, these values were similar for both sexes. In the anterior fragment the number of the mucocytes was 7.11±1.014 (males) and to 6.46±1.139 (females); in the mid fragment 8.01±1.221 (males) and 8.45±1.921 (females); and 1.90±2.934 (males) and 12.02±3.541 (females) - in the posterior. In both sexes the difference in the number of mucous cells between the anterior and posterior fragments was significant (p 0.01). In the males the difference between the mid and posterior fragments was also significant (p 0.05). The cells were randomly scattered among enterocytes in all the fragments of the small intestine. They were of characteristic shape, reflected in their name - goblet cells. Sometimes, their small accumulations could be seen only at the base of the villi. The ratio of the number of enterocytes to the number of goblet cells decreased along the intestine in both sexes, being smaller in the males in all the intestinal fragments. This ratio amounted to 20.26±3.572 (males) and 31.15±7.352 (females) in the anterior fragment. The difference was statistically significant (p 0.05). The corresponding values in the mid fragment of
53 GOBLET-CELLS-AND-ENTEROCYTES-OF-SOREX-ARANEUS 53 the intestine were 19.09±4.687 (males) and 24.18±5.814 (females), whereas in the posterior fragment they were 10.52±3.015 and 17.99±10.902 respectively. In these fragments the difference between males and females was statistically insignificant. By contrast, the differences in the values of this ratio between the fragments of the intestine were significant in the following cases: between the anterior and posterior fragments (p 0.01) and between the mid and posterior fragments (p 0.05) in males, and between anterior and posterior fragments (p 0.05) in females. In both sexes, the height of enterocytes gradually decreased along the intestine, with the enterocytes being higher in the anterior intestine fragment in males compared to females. Their height amounted to: 21.19±2.523 µm and to 16.14±1.657 µm, respectively. The difference was statistically significant (p 0.01). In the next fragments of the intestine the height of enterocytes was similar for males and females, amounting to 18.11±1.766 µm (males) and 18.29±3.068 µm (females) in the mid fragment, and to 16.77±3.127 µm and 15.30±2.126 µm in the posterior fragment respectively. Only in the males the height of enterocytes varied between the anterior and mid fragments (p 0.01) as well as between the anterior and posterior fragments (p 0,05). DISCUSSION The results of the studies showed differences in the structure of the epithelial lamina and the height of the villi between the consecutive fragments of the small intestine of sexually immature males and females of the common shrew. In both sexes the villi were higher in the first two fragments of the intestine (anterior and mid ones) than in the posterior fragment. The differences are of 16.3% (males) and of 5.4% (females). This fact conforms to the rule stating that in the parts of the small intestine where food absorption is the most intense, the villi are highest, i.e. with the biggest absorptive area (KONTUREK, 1985). In these fragments of the small intestine in both sexes the number of absorptive cells (along the circumference of the vertical section through the villi) was also higher. There were differences between males and females. The number of enterocytes was considerably smaller in the males. In the females the number of enterocytes in the initial fragment was by 49.37 higer than in males; in the mid fragment - by 35.85 more; and by 51.31 more in the posterior fragment. It results from the fact that enterocytes in the epithelial lamina of the small intestine of females are considerably narrower and smaller than those in males. The height of the villi is identical for both sexes, therefore, the entire absorptive area of the villi (formed by the top part of enterocytes) is the same for both sexes. The number of enterocytes along the circumference of vertical section of the villi was found to change directly in proportion to their heights.
54 B.-WILCZYÑSKA-AND-M.-JAROSZEWSKA 54 The number of the goblet cells in both sexes increases almost twofold from the anterior to the posterior fragment of the intestine (1.8 times). The further the intestinal fragment, the more intense the secretion of mucus covering its internal surface. The presence of the mucus facilitates transport of food items as well as indigestible substances towards the large intestine. The mucus of the small intestine, especially in its posterior fragment, enters in contact with a great amount of solid food. The presence of the increased number of mucus-producing cells maximally decreases the possibility of damaging the mucous membrane by the chitin present in the food of the common shrew. The animals were caught according to permission of Ministry of the Environment no. DLOPiKog.-4201/262/00 from 2000 year. LICZBA KOMÓREK KUBKOWYCH I ENTEROCYTÓW W JELICIE CIENKIM NIEDOJRZA YCH P CIOWO SAMCÓW I SAMIC RYJÓWKI AKSAMITNEJ SOREX ARANEUS L. STRESZCZENIE Przeprowadzone badania mia³y na celu okreœlenie liczby komórek kubkowych i enterocytów oraz wysokoœci enterocytów buduj¹cych blaszkê nab³onkow¹ b³ony œluzowej jelita cienkiego m³odych samców i samic ryjówki aksamitnej. W tym celu pobrano jelito cienkie od 7 samców i 9 samic ryjówki aksamitnej w pierwszym kalendarzowym roku ich ycia. Jelito podzielono na trzy fragmenty: pocz¹tkowy, œrodkowy i koñcowy. Stwierdzono, e liczba komórek kubkowych, przypadaj¹ca na obwód pod³u nego przekroju przez kosmki, wzrasta wraz z d³ugoœci¹ jelita i w trzech jego czêœciach jest jednakowa dla obu p³ci. Wynosi ona w pocz¹tkowym fragmencie 7,11±1,014 (samce) i 6,46±1,139 (samice), w œrodkowym 8,01±8,1,221 (samce) i 8,5±1,921 (samice) i odpowiednio 12,90±2,934 i 12,02±3,541 w koñcowym fragmencie. Zarówno u samców jak i u samic ró nica w liczbie komórek miêdzy pocz¹tkowym i koñcowym fragmentem jelita jest istotna statystycznie (p 0,01). U samców liczba enterocytów na obwodzie pod³u nego przekroju przez kosmki jelita cienkiego jest wiêksza i zbli ona w pocz¹tkowym (151,55±38,144) i œrodkowym (160,14±30,613) fragmencie jelita i mniejsza w koñcowym (139,17±26,486). Analagiczne dane dla samic s¹ jednakowe we wszystkich trzech fragmentach i wynosz¹ œrednio: 195,79 enterocytów. Stosunek liczby enterocytów do liczby komórek kubkowych zarówno u samców jak i u samic jest najwy szy w pocz¹tkowej czêœci jelita i wynosi: 20,26±3,572 u samców i 31,15±7,352 u samic. Ró nica w wartoœci tego wspó³czynnika miêdzy samcami i samicami jest istotna statystycznie tylko w pocz¹tkowym fragmencie jelita (p 0,05). Wysokoœæ enterocytów maleje wzd³u jelita cienkiego i wynosi u samców w pocz¹tkowym fragmencie jelita 21,19±2,523 µm, w œrodkowym 18,11±1,766 µm i w koñcowym 16,77±3,127 µm. Ró nica
55 GOBLET-CELLS-AND-ENTEROCYTES-OF-SOREX-ARANEUS 55 w wysokoœci enterocytów miêdzy pocz¹tkowym i koñcowym fragmentem jelita jest istotna statystycznie (p 0,05) jedynie u samców. U samic wartoœæ ta wynosi odpowiednio 16,14±1,657 µm, 18,29±3,068 µm 15,30±2,126 µm. Miêdzy samcami i samicami ró nica w wysokoœci enterocytów jest istotna statystycznie tylko w pocz¹tkowym fragmencie jelita (p 0,01). REFERENCES BIELAK, T., PUCEK, Z., 1960: Seasonal changes in the brain weight of the common shrew (Sorex araneus araneus L., 1978). Acta Theriol., 13: 297-300. BOROWSKI, ST., DEHNEL, A., 1953: Materia³ do biologii Soricidae. AUMCS, 6: 305-448. BUCHALCZYK, A., 1972: Seasonal variation in the activity of shrews. Acta Theriol., 17: 221-243. BUCHALCZYK, A., KORYBSKA, Z., 1964: Variation in the weight of the brown adipose tissue of Sorex araneus L., 1978. Acta Theriol., 14: 193-215. BAZAN, I., 1953: Die morpho-histologischen Veranderungen des Thymus in Lebenszyklus von Sorex araneus L. AUMCS, 5: 253-304; CROWCROFT, P., INGLES, J.M., 1959: Seasonal changes in the brain-case of the common shrew (Sorex araneus L). Nature, 183: 907-908 ; DEHNEL, A.,1949: Badania nad rodzajem Sorex L. AUMCS, 17: 17-102. DEHNEL, A., 1952: Biologia rozmna ania ryjówki Sorex araneus L. w warunkach laboratoryjnych. AUMCS, 11: 359-373. DZIER YKRAJ - ROGALSKA, I., 1953: Zmiany histomorfologiczne tarczycy S. a. araneus L. AUMCS VII, 4: 213-252. FONS, R., STEPHAN, H., BARON, G., 1984: Brains of Soricidae. Z. f. zool. Systematik u. Evolutionforschung, 2: 145-158. HYVÄRINEN, H., 1968: On the seasonal variation of the activity of alkaline phosphatase in the kidney of the bank vole (C. glareolus SCHR.) and the common shrew (S. araneus L.). Aquilo Ser.Zool., 5: 7-11. HYVÄRINEN, H., 1969: On he seasonal changes in the skeleton of the common shrews (Sorex araneus L.) and their physiological background. Aquilo Ser. Zool., 7: 1-32. KONTUREK, S., 1985: Fizjologia uk³adu trawiennego. Fizjologiczne podstawy gastroenterologii. PZWL, Warszawa. KOWALSKI, K., 1964: Klucze do oznaczania krêgowców Polski. Ssaki, Mammalia. PWN, Warszawa. KUBIK, J., 1951: Analiza pu³awskiej populacji Sorex araneus araneus L. i Sorex minutus minutus. AUMCS, 2: 335-372. MYRCHA, A., 1967: Comparative studies on the morphology of the stomach in the Insectivora. Acta Theriol., 14: 223-244. PUCEK, M., 1965: Water contents and seasonal changes of the brain-weight in shrews. Acta Theriol., 24: 353-367. PUCEK, Z., 1960: Sexual maturition and variability of the reproductive system in young shrews (Sorex L.) in the first calendar life. Acta Theriol., 12: 269-296. PUCEK, Z., 1965: Seasonal and age changes in the weight of internal organs of shrews. Acta Theriol., 26: 369-438. PUCEK, Z., 1970: Seasonal and age changes in shrews as an adaptive process. Symp. Zool. Soc. London, 26: 189-207.
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