KERATIN STRUCTURES OF CERATOTHERIUM Jseph C. Daniel Jr. Michael Adam Ann C. Van Orden Departments f Bilgical Sciences and Mechanical Engineering Old Dminin University Nrflk, VA 23508 INTRODUCTION Keratin, in dense mlecular frms, is the prevalent cmpnent f many anatmical features f mammals (extensively reviewed by Marshall et. al. 1991). In additin t being a key ingredient in the cmplex mrphlgy f skin, it is the primary structural material f hair, fur, quills, vibrissae, scales, cmbs, hfs, nails, claws, sheaths f sme head hrns, nse hrns, balleen and the bill f the Platypus. The rhincers has fur such anatmical structures, namely tail hair, ear hair, hf and hrn. Given the interest in this labratry f develping substitutes fr rhin hrn, pssibly frm the hair r hf trimmings f the animal, t be used in prescriptins f traditinal riental medicine, it became imprtant t cmpare these structures in an effrt t ascertain and expse their similarities and differences. The reprt f Butter et. al. (1990) presents the mlecular distinctins between the three main types f mammalian keratin (hair, hf, hrn) and cncludes that "Keratin is nt a single substance but a cmplex mixture f prteins and the sulfur cntaining diamin acid cystine." The gal f the study reprted here was t explre the micrscpic differences in keratin structures f the rhincers. Numerus studies have been cnducted n the cmpsitin f rhin hrn (eg. Butler, et. al. 1990; Lynch, et. al. 1973; Grves, 1971; Ryder, 1972), but the literature is almst bereft f reprts abut rhin hf r hair, pssibly the nly ne being fund in Van Orden and Daniel (1993). Hrn, at all structural levels, differs smewhat between the species f rhincers (Butler, et. al. 1990, Grves, 1971, Earland et. al. 1962), but t evade any cnfusin frm these differences, all f the samples used were frm the white rhincers (Cerattherium simum), specifically frm ne animal, the male at the Virginia Zlgical Park - "Rufus." METHODS AND MATERIALS In 1990 and 1992, respectively, Rufus brke the tip f his large hrn and a prtin f the hf f his right freleg. The pieces were recvered by the keepers and given t the senir authr. The keepers als cut hair samples frm bth the tail and the ear fr this study. All samples were stred dry between the time f cllectin and their preparatin fr study. Bth types f hair were prepared in tw ways. T facilitate viewing them micrscpically in lngitudinal array, hairs were psitined in parallel n an aluminum stub, care being given t evade verlapping, and cated with 100 A f AulPd. They were phtgraphed at different magnificatins with a scanning electrn micrscpe (Cambridge Sterscan 100). T see the internal rganizatin, revealed by crss sectins, hairs were imbedded in epxy resin using standard histlgical techniques, cut in sectin and plished t a smth surface. Standard metallurgical techniques fr hard cmpsite materials were used t btain the smth surface, namely thrugh a series f prgressively finer grit sand-paper and final plish with diamnd slurry and 0.3 micrn alumina as described by Van Orden & Daniel (1993). Individual hairs were measured and apprpriate fields were phtgraphed thrugh ptical light micrscpes. The denser structures, hf and hrn, were cut with a fine-tthed saw bth with and acrss the grain striatins and plished as described. The plished surfaces were studied, measured and phtgraphed micrscpicall y. 1995 AZA REGIONAL CONFERENCE PROCEEDINGS 537
RESULTS Plates A-D in Figure 1 shw all structures in lngitudinal psitin (sectin) and Plates E-H in Figure 2 are phtgraphs f the crss sectins. The sectins f hm cnfirm the cmpacted filamentus structure already well knwn frm the papers referenced in the intrductin. The filaments are cmpsed f tw layers surrunding an inner cre r medulla, the middle ne (crtex) being cntained by a denser bunding layer, the cuticle. The utside diameter f the filaments ranges frm 300 ~m t 500 ~m, averaging 420 ~m; n average the cuticle is 30 ~m thick, the crtex 160 ~m and the medulla abut 35 ~m. The filaments frequently are in direct cntact with each ther, smetimes blending their crtex inside f a cmmn cuticle, but are mstly separated up t abut 150 ~m apart and the intervening space filled with a matrix netwrk f tiny fibers. Van Orden and Daniel (1992) used energy dispersive X-ray spectrscpy and X-ray diffractin analysis t shw that bth the filaments and the matrix fibers were cmpsed f keratin. The paper f Lynch et. al. (1973) shws that the filaments are built f cncentric laminae f flat crtical cells, pssible f tw different types. The lngitudinal sectins als cnfirmed the preliminary bservatin f Van Orden and Daniel (1992) that the filaments f hm are nt cntinuus structures that grw unimpeded frm fllicles t hm tip. Rather they are interrupted, elngated spindle-like filaments that appear t interdigitate and expse their bare, runded ends at pints f breakage. This fact supprts the speculatin that the fllicles prducing the filaments peridically cease prductin (pssibly during drughts r times f starvatin) and reinstate it when cnditins imprve r upn receipt f sme unknwn signal. Hwever, if all fllicles paused in their filament prductin at the same times, then the discntinuity wuld cause the hm t have ne r mre weak spts especially prne t breakage. T the best f the authrs' knwledge this has nt been bserved. Furthermre, captive rhins are well maintained and therefre nt generally subject t perids f malnutritin, excess thirst r any ther envirnmental stress: As the hm sample used here came frm a captive rhin, it is therefre, mst unlikely that the filament peridicity can be assigned t such events. Apptsis, prgressive cell death, f cells in the fllicles fllwed by renewal is anther pssibility, but nly speculative at best. The questin merits further investigatin. Rhincers' hf des nt have the heavy filamentus structure f hm, but has sparsely separated filaments In a cmpactin f much smaller fibers, partially aligned prxim-distally alng the tip and frntal sides f each digit. These few filaments are smaller than the hm filaments, have less well defined cuticle and crtex and have n dense cre as a medulla, but rather a diffuse amrphus central regin abut 1 00 ~m in diameter. It is impssible t determine whether the few filaments in hf are interrupted alng their length and the entire structure is far less defined than hm Rhin hf resembles hrse hf (Bertram and Gsline 1987), but with fewer and larger filaments. Ryder (1962) gives 25-50 ~m as the diameter f hrse hf filaments separated by 50-100 ~m f matrix material: in the rhin hf the filaments are 100-200 ~m in diameter interspersed by 200-400 ~m f matrix. Tail hair pssesses the same three layers as In the classical structure fund in all hair (eg. Wake, 1979; Pugh et. al. 1989; Marshall et. al. 1991), but aligned differently. Obviusly, because each hair is a separate self-defined structure (as cntrasted t the cmpacted frm fund in hm and hf) the cuticle is, and must be, thicker and supprtive. The crtex is evenly distributed thrughut the central prtin f the hair and running thrugh it, seemingly at randm, are dense cres (medullae) which vary in sectin frm rund t val t semicircular and in number frm ne t six. Ear hair has n medulla, the crtex being the nly central feature. The tall hair is the heaviest, being abut half t tw-thirds the diameter f hm filaments and averaging abut 200 ~m. (Measurements f 15 randmly selected samples ranged frm 154 t 308 ~m with 226 ~m average.) The ear hair is smaller, less straight and averages 66 ~m in diameter frm 15 sample measurements. 538
CONCLUSIONS Rhincers hm is nt just cmpacted hair but a specialized structure f lng, heavy keratin filaments bund tgether in a fibrus netwrk matrix. Hf is mre amrphus with an extensive matrix infiltrated by a few filaments, far less cncentrated than in hrn and substitut ing a less cmpact central area fr the small, dense cre fund in hrn filaments. Ear hair has n medulla, but tail hair is structured in the typical mammalian frmat. Tail hair is abut three t fur times the size (diameter) f ear hair and abut hah the size f hrn filaments; the internal structure f bth types f hair is significantly different than that f the filaments. REFERENCES Bertram, J.E.A. and J.M. Gsline. 1987. Functinal design f hrse hf keratin: The mdulatin f mechanical prperties thrugh hydratin effects. J. Exp. BiI. 130: 121-136. Butler, D.J., P.R. DeFrest, and L. Kbilinsky. 1990. The use f iselectric fcusing t identify rhincers kemtins. J. Frensic Sci. 35: 336-344. Earland, C., P.R. Blakey and J.G.P. Stell. 1962. Mlecular rientatin f sme keratins. Nature 196: 1287-1291. Grves, C.P. 1971. Species chamcters in rhincers hrn. Z. Saugetierkunde 36: 238-252. Lynch, L.J., V. Rbinsn, and CA Andersn. 1973. A scanning electrn micrscpe study f the mrphlgy f rhincers hrn. Aust. J. BiI. Sci. 26: 395-399. Marshall, R.C., D.F.G. Orwin, and J.M. Gillespie. 1991. Structure and bichemistry f mammalian hard keratin. Electrn. Micrsc. Rev. 4: 47-83. Pugh, Pugh, F.H., J.B. Heiser, and W.N. McFarland. 1989. Vertebmte Life-Third Editin. Macmillan Publishing Cmpany, N.V. 899 ppg. Ryder, M.L. 1962. Structure f rhincers hrn. Nature 193: 1199-1201. Van Order, A.C. and J.C. Daniel. 1993. Structure and cmpsitin f rhincers hrn. Mat. Res. Sc. Symp. Prc. 292: 45-56. Wake, M.H. 1979. Hyman's Cmparative Vertebrate Anatmy-Third Editin. Univ. f Chicag Press. 769 ppg. ACKNOWLEDGMENTS The authrs are grateful t Rbert Edahl and Catherine Wilsn fr technical assistance, t the persnnel f the Virginia Zlgical Park fr supplying the keratin samples and t Diane St. Clair fr financial supprt. 539
~. ~ r"" Figure 1. Lngitudinal views f: A. Hrn sectin at 10x. B. Hf sectin at SOx. C. Tail hair intact at 36x. D. Ear hair intact at SOx. A and B are light micrgraphs; C and 0 are scanning electrn micrgraphs.
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