DNA synthesis and cell renewal of mouse intestinal epithelium were studied with radioautography after injection of thymidine-H&#179; to know the difference of
the mode of epithelial cell generation relating to the different frequency of cancer developement in several parts of small and large intestines. Succinic dehydrogensase activity was also observed by histochemical method. Renewal time of the intestinal epithelium of mouse is about three days throughout the intestine with somewhat longer time in rectum and anus, and relatively shorter one in ileum compared to the other parts of the intestine. Daily regenerating rate was low in large intestine, especially in rectum and anus. Strong activity of succinic
dehydrogenase appeared in the bottom of crypt and seems to be correlated to the active cell division. Epithelial cells in large intestine move very slowly upward and few of them seem to move to the opposite side or stay long time
at one place. Intermitotic time is about 27 hours in small intestine and about 40 hours in large intestine. These suggest some relations between the mode of the epthelial cell renewal and cancer development. Because in human the
frequency of cancer development is very high in large intestine, rectum and anus, and the epithelial renewal of these areas is supposed to be delayed similarly as in mice.</p

Kawashima, Takao

Ogata, Takuro

Seito, Takashi

Shimatani, Nobuto

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 20, Issue 4 1966 Article 4AUGUST 1966DNAsynthesis and cell renewal in small andlarge intestines of mouseTakashi Seito∗ Nobuto Shimatani†Takao Kawashima‡ Takuro Ogata∗∗∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.DNAsynthesis and cell renewal in small andlarge intestines of mouse∗Takashi Seito, Nobuto Shimatani, Takao Kawashima, and Takuro OgataAbstractDNA synthesis and cell renewal of mouse intestinal epithelium were studied with radioau-tography after injection of thymidine-H3 to know the difference of the mode of epithelial cellgeneration relating to the different frequency of cancer developement in several parts of small andlarge intestines. Succinic dehydrogensase activity was also observed by histochemical method.Renewal time of the intestinal epithelium of mouse is about three days throughout the intestinewith somewhat longer time in rectum and anus, and relatively shorter one in ileum compared tothe other parts of the intestine. Daily regenerating rate was low in large intestine, especially inrectum and anus. Strong activity of succinic dehydrogenase appeared in the bottom of crypt andseems to be correlated to the active cell division. Epithelial cells in large intestine move veryslowly upward and few of them seem to move to the opposite side or stay long time at one place.Intermitotic time is about 27 hours in small intestine and about 40 hours in large intestine. Thesesuggest some relations between the mode of the epthelial cell renewal and cancer development.Because in human the frequency of cancer development is very high in large intestine, rectum andanus, and the epithelial renewal of these areas is supposed to be delayed similarly as in mice.∗PMID: 4226977 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 20, 167-114 (1966)DNA SYNTHESIS AND CELL RENEWAL IN SMALL ANDLARGE INTESTINES OF MOUSETakashi SEITO, Nobuto SHIMATANI, Takao KAWASHIMAand Takuro OGATADepartment of Surgery, Okayama University Medical School,Okayama, Japan (Director; Prof. S. Tanaka)Received for publication, May 20, 1966As is well known that the small intestinal epithelial cells are incessantlyfading at the top of villi and being renewed by the cells coming from thebottom of villi keeping a "steady state system". This has been established bythe recent works by using radioisotopes of thymidine. QUASTLER and SHERMAN(1959)1 reviewed the cell kinetics of intestinal epithelium of mouse by usingradioisotopes. In 1948 LEBLOND already estimated the renewal time of theepithelium of small intestine of rat to be 1.4.-..1.6 days calculating from themitotic rate of the epithelium. Later in 1958, LEBLOND and his associates2•3 madeprecise observations on the mouse intestine by using thymidine-C14 and -H3,revealing the renewal time in several areas of the mouse intestine; 48 hours induodenum, 72 hours in jejunum, ileum and colon. BERTALANFFY (1960)4 esti-mated the renewal time of rat intestinal epithelium from mitotic rate, whichwas observed by using corchicine and he found that it was 1.3 days in jejunum,10 days in colon, 6.2 days in rectum, and 4.3 days in anal epithelium. In 1961OEHLERT and B0cHNER5 observed the renewal time of the intestinal epitheliumof mouse to be 48 hours in duodenum, less than 48 hours in jejunum, and 72hours in colon. Quite recently, KASAHARA (1966)6 made similar observations onthe mouse epithelium and calculated it to be as 43 hours in duodenum andjejunum, and 46 hours in ileum. In other words, the renewal time of theintestinal epithelium of mouse is found to be 40.-..48 hours in duodenum, 43.-..72 hours in iejunum, 46.-..72 hours in ileum and 72 hours in colon, showingthat the shorter the renewal time is in duodenum, the longer is it in jeiunum,ileum and colon.It is generally known that in human beings the rate of cancer developmentin the intestine is higher in rectum and colon and very low in small intestine.Concerning this, ONOE (1964)7 suggested a close correlation between the renewaltime of intestinal epithelium or regenerating process and the cancer formation.With his view as an incentive a series of study was conducted on the renewal1671Seito et al.: DNAsynthesis and cell renewal in small and large intestines ofProduced by The Berkeley Electronic Press, 1966168 T. SEITO, N. SHIMATANI, T. KAWASHIMA and T. OGATAtime of mouse intestinal epithelium by injecting thyminide-Hs, and the resultsof the study revealed the delayed renewal time of the epithelium of rectum andanus and low regenerating rates in large intestine where the cancer develops athigher rates, suggesting some relations between the modes of the epithelial cellrenewal and cancer development.MATERIALS AND METHODSAdult Strong-A mice weighing about 30 g were injected intravenously with1 p.c per 19m of thymidine-Hs (specific activity 14.8 c/mM, Japanese Radio-chemical Center). The animals were killed 1/2, 2, 4, 8, 24, 32, 40, 48, 72,96 and 120 hours after injection. Specimens were obtained from duodenumnear pylorus, jejunum near Treiz's ligament, ileum near coecum, coecum, trans-verse colon, rectum and anus near transitional part to squamous epithelium.Tissues were fixed in 10 per cent neutral formalin for one day and embedded inparaffin and then sectioned 4/l thick. Autoradiographs were prepared by thestripping film technique with Kodak AR-10 stripping film for three weeks at5°C, thereafter stained with hematoxilin and eosin for observation (D·19-Bsolution was used for development).Specimens for succinic dehydrogenase staining were prepared as follows.Small and large intestines two hours after the injection were cut at 151-' in acryostat kept at - 20°C without fixation, then incubatcd with the followingmedium for 30 minutes at 37°C; M/5 sodium succinate 5 ml, M/lO phosphatebuffer pH 7.6, 5 ml, nitro BT 5 mg/3 ml, 6 ml, aq. dist. 10 ml.RESULTSTable 1 shows labeled index, situation, and density of labeled cells ateach sacrifice time after injection calculated from neighboring five crypts. Thelabeling index is the number of labeled cell per all epithelial cells of a crypt andvilli.At 30 minutes after injection radioautographic reactions were observed onlyin crypts near the bottom. In the epithelial cells of villi or on the surface oflarge intestine such reactions were absent. Labeled cells were more abunduntin the crypt cells of small intestine than in those of large intestine. Grain countswere 15--30 per nucleus in the majority.At two hours after the injection situation and labeled index hardly differedfrom that at 30 minutes but at four hours labeled cells moved a little towardthe luminal side in the crypt.At eight hours these labeled nuclei disseminated in the crypt and stronglylabeled cells reached the upper end of the crypt in small intestine. On the other2Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/4DNA Synthesis and Cell RenewalTable 1 Position and Per Cent of Labeled Cells169IleumColonY2hr 2hrs 4 hrs 8hrs 24hrs 32hrs 40hrs 2days 3days 4dllYSDuodenum J~ 9.1 f~ 9.5 IN 12.0 J~ 22.2 J~ 28.1 m32.2 m572 ~ 82.3 ~ 61.3 m50.2W ~ ~ ~ ~ 00 ~ ~ ~ ill. rn 9.0 rn 13.2 fn 10.5 fn1 20.2 m45.6 ill 44.9 m55.9 m75.6 m58.0 m' 25.9Jejunum IH~ Il~ J!\¥J 1JtwJ JH~ JJ~ JJl Wr IJl V! JJI~J JJl w~13 ~25 \95 ~6.1 ~52 rt3.J ~68 \ 50S \~S ~~Goecum 1»17.21JJ 11.1 \ lUlu 10.1 1u 18.1)57.7)41.11» 56.1 1»SUM 429l 7.1 1u 5.41u lO.1lJ 7.1l! 141 111 54S 1u3Bn 19~J) 19.1)34SI!: Anusband, in large intestine the dissemination was not so marked but several labeled-cells were found far from the base of crypt.At 24 hours the migration of labeled cells were prominent. In small inte-stine, especially in ileum, strongly labeled cells reached about two thirds fromthe top of the villi. Duodenum and jejunum showed a similar tendency. Inlarge intestine the migration of labeled cells upward luminal side was observedbut not so marked as seen in small intestine. As a whole, grain counts in the-crypt cells of small intestine decreased. Occasionally strongly labeled cellsremained in the base of crypt.At 32 hours the labeled cells reached higher place of villi than at 24 hoursin small intestine and at this time labeled cells markedly increased in largeintestine and strongly reacting nuclei were seen at about three fourths of crypt'sheight in coecum and colon but in rectum and anus the upper half of the crypt·cells was not yet labeled. At 40 hours, these migrations of labeled cells towardthe lumen and diminution of grain counts in the crypt cells became more pro-minent.At 48 hours the strongly labeled cells reached the top of villous and surface-epithelium both in small and large intestines. In small intestine these stronglylabeled cells were disseminated in the upper two thirds of the villi. Also inlarge intestine the labeled cells reached the top of the surface but certain cellswith strongly labeled nucleus were sometimes in the crypt far from the luminal3Seito et al.: DNAsynthesis and cell renewal in small and large intestines ofProduced by The Berkeley Electronic Press, 1966170 T. SEITO, N. SHIMATANI, T. KAWASHIMA and T. OGATAside.At 72 hours these strongly radioactive nuclei were observed in the upperone third of the villi or less than in one third of duodenum and jejunum. Inileum the grain counts in the upper side of villous epithelium were less thanFig. 1: Jejunum, after 2 hours >< 400, Fig. 2: Colon, after 30 minutes>< 400, Fig. 3: Ileum, after 32 hours >< 400, Fig. 4: Colon, after 48 hours><4004Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/4DNA Synthesis and Cell Renewal 171those in the former two. Grain counts were decreased gradiently down to thecrypt in all small intestine and no nucleus in the crypt showed grain counts of·over ten. In large· intestine the decrease in the number of labeled cells wasmarked at this time, but occasionally strongly radioactive cells were observed.around the base of crypt.At 96 hours labeled cells were clearly decreased and the grain counts inthese nuclei were very small in most region of the crypt of small and large-intestines excepting occasional nuclei with many grain counts in large intestine.At 120 hours unlabeled cells increased and the majority of labeled nucleihad only one or two grain counts.In addition, succinic dehydrogenase in the intestine of the specimen twohours after the injection was estimated histochemically. The enzyme activitywas intense on the surface of villi and the basal side of the crypt (Fig. 5).Fig. 5 Ileum, succinic dehydrogenase staining X 100DISCUSSIONThymidine-H3 is readily incorporated into DNA in chromosomes soon afterthe injection where DNA synthesis is taking place. There is no transfer oftritium once chromosomes have been labeled. Therefore, labeled thymidine isan adequate tracer for newly formed deoxyribonucleic acid in radioautographs8•9•Radioactive nuclei at 30 minutes and two hours were observed in 20 cellsat the bottom of the crypt both in small and large intestines except in severalcells far away from the bottom selectively in the crypt. These findings indicatethat the DNA synthesis is achieved in these regions. Furthermore, strongsuccinic dehydrogenase activity seems to be intimately associated with theseregions. QUASTLER and SHERMAN1 reported that the majority of labeled cells5Seito et al.: DNAsynthesis and cell renewal in small and large intestines ofProduced by The Berkeley Electronic Press, 1966172 T. SEITO, N. SHIMATANI, T. KAWASHIMA and T. OGATAbelonged to columnar and goblet cells and did not include the Paneth cells andthe silver staining cells, though it was not clear in the present study. Newlyformed cells mainly run up along the side of crypt and villi and are protrudinginto the lumen. However, few cells seem to take the opposite course and arelost from the base of crypt because over eight hours after the injection, whenlabeled cells migrated upward, strongly reacted cells were occasionally observedin the bottom of crypt especially in large intestine.As indicated in Table 2, grain counts in the bottom cells of the crypts wereTable 2 Mean of Grain Counts of 20 Crypt Cells from the Bottom----------_.Times after injection1/2 2 4 8 24 32 40 48 72 96 (hrs)Duodenum 17.5 11.9 17.2 11.6 15.8 6.7 4.8 4.2 3.2 2.7Jejunum 15.9 17.4 16.2 12.5 9.7 7.8 5.9 3.8 2.9 2.2Ileum 15.8 16.2 18.8 12.5 13.2 9.7 4.9 3.2 2.7 2.3Coecum 13.4 13.9 12.8 8.7 12.5 9.5 11.3 4.3 4.4 3.3Colon 17.7 16.9 13.6 12.5 10.7 11.1 7.6 4.9 5.7 2.4Rectum 12.7 14.5 15'1 14.4 15.1 6.1 7.4 4.1 5.9 2.8Anus 13.8 15.0 13.4 14.5 14.5 5.9 10.1 4.7 4.9 2.6II5 ~------------;------------6'----III21 24 32 40 48* 0 Duodenum x Jejunum "Ileum: g5 - - - - - - - - - - - - - - - - - - r-------- -- -~ ----- ------12 961224 8 24 28 32 40 48 n.5· 72 II* 0Coecum x Colon" Rectum D AnusFig. 6, 7 Mean of Grain Counts of 20 Crypt Cells from the BottomPlotted Logarithmically Against Time6Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/4DNA Synthesis and Cell Renewal 173appearently diminished as the sacrifice time became long after injection. Ifthe daughter cells accept half of DNA with thymidine-H8 and they part it equallyto daughter cells, mean of grain counts in the crypt cells against time shoulddraw a strait line logarithmically (Figs. 6 and 7). Intermitotic time is givenfrom these figures as the time grain counts become a half, it is 27 hours in thesmall intestine and 39.5 hours in the large intestine.Renewal time of epithelial cells and daily regenerating rate are shown inTable 3. SHERMAN and QUASTLER10 reported that the movement of cells alongTable 3 Renewal Time and Daily Regenerating Rate*Renewal time Regenerating rateDuodenum 72 hours 33Jejunum 72 31Ileum 48-72 18-27Coeeum 72 9Colon 72 11Rectum 72-96 7-10Anus 72-96 6-8* Daily regenerating rate shows the number of daily faded epithelial cells.the villi is not caused by the thrust of the cells coming up from the crypt. Inthe present study there are observed hardly any great differences of renewaltime among these regions of the intestine examined since labeling indicesdiffered.SUMMARYDNA synthesis and cell renewal of mouse intestinal epithelium were studiedwith radioautography after injection of thymidine-H8 to know the difference ofthe mode of epithelial cell generation relating to the different frequency of cancerdevelopement in several parts of small and large intestines. Succinic dehydro-gensase activity was also observed by histochemical method. Renewal time ofthe intestinal epithelium of mouse is about three days throughout the intestinewith somewhat longer time in rectum and anus, and relatively shorter one inileum compared to the other parts of the intestine. Daily regenerating rate waslow in large intestine, especially in rectum and anus. Strong activity of suc-cinic dehydrogenase appeared in the bottom of crypt and seems to be correlatedto the active cell division. Epithelial cells in large intestine move very slowlyupward and few of them seem to move to the opposite side or stay long timeat one place. Intermitotic time is about 27 hours in small intestine and about40 hours in large intestine. These suggest some relations between the mode of7Seito et al.: DNAsynthesis and cell renewal in small and large intestines ofProduced by The Berkeley Electronic Press, 1966174 T. SEITO, N. SHIMAtANI, T. KAWASHIMA and T. OGAtAthe epthelial cell renewal and cancer development. Because in human thefrequency of cancer development is very high in large intestine, rectum andanus, and the epithelial renewal of these areas is supposed to be delayedsimilarly as in mice.ACKNOWLEDGEMENTThe authors gratefully acknowledge Prof. S. TANAKA for his advices and encouragementthroughout this work.REFERENCES1. QUASTLER, H. and SHERMAN, F. G.: Cell population kinetics in the intestinal epitheliumof the mouse. Exp. Cell Res. 17, 420, 19592. WALKER, B. E. and LEBLOND, C. P.: Sites of nucleic acid synthesis in the mouse visualizedby radioautography after administration of C-labelled adenine and thymidine. Exp. Cell Res.14, 510, 19583. LEBLOND, C. P. and MESSIER, B.: Renewal of chief cells and goblet cells in the smallintestine as shown by radioautography after injection of thymidine-H3 into mice. Anat.Record 132, 247, 19584. BERTALANFFY, F. D.: Mitotic rates and renewal times of the digestive tract epithelia in therat. Acta Anat. 40, 130, 19605. OEHLERT, W. and BijcHNER, Th.: Mechanismus unci zeitlicher Ablauf der physiologischenRegeneration im mehrschichitigen Plattenepithel und in der Schleimhaut des Magen-Darm-traktes der weissen Maus. Beit. Path. Anat. 125, 374, 19616. KASAHARA, K.: Kinetic studies on mucosal cells of the gastrointestinal tract in normal mice.japan. j. Gastroentero/. 63, 88, 1966 (in Japanese)7. ONOE, T.: The reason why cancer often occurs in large intestine than in small intestine.I gaku no ayumi 48, 695, 1964 (in Japanese)8. AMANO, M., MESSIER, B. and LEBLOND, C. P.: Specificity of labelled thymidine as adeoxyribouncleic acid precursor in radioautography. j. Histochem. Cyiochem. 7, 153, 19599. FITZGERALD, P.]., EIDINOFF, M. L., KNOLL, J. E. and SIMMEL, E. B : Tritium in radio-autography. Science 114, 494, 195110. SHERMAN, F. C. and QUASTLER, H.: DNA synthesis in irradiated intestinal epithelium.Exp. Cell. Res. 19, 343, 19608Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 4http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/4

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