The perforatorium Ф an extension of the nuclear membrane of the rat spermatozoon.
код для вставкиСкачатьT H E PEHE’ORATORIUM - AN EXTENSION O F T H E NUCLEAR MEMBRANE O F THE R A T SPERMATOZOOX Y . CLERMONT, IC, EINBERG, C. 1’. LBBLOKI) AND S. WAGNER Department of A m t o m y , and Eaton Electronics h b o r a t o r y , ,IlcGiZZ U n i v e r s i t y , M o n t r e a l ELEVEN FIGURES The sickle-shaped nucleus of the r a t spermatozoon is prolonged by a refringent structure stainable by iron hematoxylin - the perforatorium. This structure was recognized a s early a s 1887 by Jensen; later it was depicted as a short pointed rod by Niessing (1897), Duesberg (’08), arid Retzius (’09). Blaiidau (’51) o n examination of the freshly fertilized rat ovum observed the perforatorium free of the nucleus and described it a s a rod, one end of which was Y-shaped. Austin and Sapsford (’51) noted that when sodium hydroxide digestion of sperniatozoa was followed under the microscope, the perforatorium remained visible after the otlier components of the head had dissolved. On the whole, littlc is known concerning the structure and significance of the perforatoriurn. Therefore, to extend our meager knowledge of this subject, rat spermatozoa were studied with the light, electron, and phase contrast microscopes. METHOD The light m i c r o s c o p e was used to examine spermatozoa in smears o r sections of testis and epididymis of adult rats. Fixation in Hellp’s fluid was followed by staining with the periodic acid-Schiff (also designated PA-Schiff), Masson trichrome, toluidine blue, or malachite green techniques. The PA-Schiff technique was often combined with one of the 1 T H E 1 V 4 T O V I r 4 1 ~RFCORI) \ O l i JANUARY 1 9 5 5 121, N O 1 nuclrar couiiterstaiiis, lic~iiiwtos;vliii,nialachite j i i w i i , or Feulgcln nuclear stain. In addition, smears and wholc testes W P ~ Y A fixed in Flcrriinirig’s fluid prior to iron lieniatoxylin staiiiirig. Th e u 1t r a - st ruet ure o f sp P rin at o zoa wa s iiire s t iga t ed by cJlectrou ~riic~rosco)iy. The spermatozoa were collected froni the tail portion of tlit. epididyrnis. This organ was cant iiito sriiall pieces wliicli were then suspended in 10 cnii o f salinc a t roo111 temperature. L a r g ~particles wcli-e rcniovecl by filtraof tion through cheese cloth. T o 3 ciii3 of tlie filtrate, I 2% osiiiic acid was addcd, and tlie niisture u7as allowed to stand for 10 minutes. 32icrodrops of this solution W C I Y smcared on grids previously coated with a film of formvai(O.SC/( foimvar dissolved in ethylene dicliloride) arid dried iii a desiccator f o r half an hour. Residual crystals of sodium chloride were removed by careful washing with distilled water. The preparations were then dried in V ( I C U O for two houi.6. Sonir~w(~i’egold s l ? a d o ~ e dbefore clecti.on inicroscopy, otliei-s were not. The isolated perforatoriuni was iiivestigated with the phnso contrast niicroscope. A s suggested by Austin and Sapsford ( ’51), a droplet o f 1N sodium hytlrosidc~was added to a drop of sperm suspension on a glass slide. A cover glass was placed o w 1 * the misturc. The prfoi-atoriuni could then he seen intlcpciident of the nucleus. RESULTS of spcrnzutozoa ( i s S P ordiizc~ry Irzicrosrope ~ Hwith the I n smt’(irs o f epididyrriis or testis, rat spermatozoa tend to lie sideways. The s p r r m head, after ii*oiiheriiatoxyliri staining (fig. I ) , appears t o be composed of the liookrd, lightly stained riucalcus and the pcrfoi-atorium - a darkly stained rod, lying along tlie concave (ventral) and apical portion of the nucleus. This iwd extends beyolid the nuclear apes, curves slightly and tapel-s to R point. IYheii the head of the spermatozoon happened to vicwcd from tlie latero-ventral side, it was seen that tlic portion of the perforatoriuiii in contact with the nucleus splits into two “prongs.” These are applied to either side of tile ventral surface of the nucleus ; caudally, they curve dorsally, aiid gradually vanish. In smears staiiied by the PA-Schiff technique with o r without nuclear counterstain, the spermatozoa nuclei were shown to be partly enclosed in a pale purple membrane, the head cap, which dorsally includes a deep purple thickening, the Fig. 1 Two heads of r a t sperinatozoa taken from t h e epididgniis, fixed with osmic acid fumes, and stained with iron hematoxylin. X approx. 1000. The two heads of spermatozoa (upper left and lower right) exhibit a dark, curved structure located 011 their ventral (concave) edge and extending beyond it. I t is the perforstorium. acrosome. The PA-Schiff technique did not stain the perforatorium, neither did H a r r i s hematoxylin aiid other nuclear counterstains. I n sections of testis arid cpididymis, useful information was obtained hy examination of cross sections of sperniatoxoa heads cut a t various lcvcls. Thus, a t a short distance in front of the nucleus, that is, a t level AA’ (fig. a), the PA-Schiff techniqnc showed that the liead cap enclosed a seemingly empty space, the shape of which resembled a three-pointed star (fig. 2, a’). The dorsal point of the star was surmounted by a brightly staining circular spot, the ci’oss section of the acrosonio. Tlic iron Iieniatosylin twhiiiquc sliow.5.eil the cross section of tlicl pcrforatorium to be a dark staining threepointed s t a r (fig. 2, a") ; mid, therefore, tlie perforatorinin was iiot a cyliiiclrical mcl hut r a t h w an cllongxted pyramid. taiiiiiig, this structure was sliowii t o occnpy most of tlie empty space noted iii PA-Schiff preparations (fig. 2, a ) . Whtlxi t l i t l spcrniatoma w c i ~ ecut tlii-oii$i the nntci-ior poi*tion of' tlic nucleus, tliat is, at levcl BY,',the PA-Scliiff techiiiqucl showed the acimoiiie aiid licntl c a p agaiii eiiclosing the Fig. 2 Ulagrain of the head of a rat s p i ~ : i t o z o o i(~a b o \ e ) and cross-sections at \ : i i i o i i s levels (hclowj. I’ERFOBATORIUM OF IlAT SPEILMBTOZOO N 5 other constituents of the head (fig. 2, b’). Iron hematoxylin preparations showed the star-like cross section of the perforatoriuin 11012’ centered by a light area, the cross section of the nuclear a p e s (fig. 2, If’ and b). There was no space between perforatoriuni aiid riucleus (fig. 2, h ) . The dorsal point of tlie star was reduced to n sniall protuberance on top of the iiucleus, wliilc the two ventral points were quite prominent. Tliese wcrc identified as the two prongs of the perf oratorium seen iii snienrs 011 citlier side of the ventral surface of the nucleus. It was felt that the dorsal point of the s t a r was tlie cross section of a third, smaller prong of thc perforatoriuni. Furthermore, the three prongs were joined together by a membrane which stained like the prongs theniselves (fig. 2, bl’). F a r t h e r back, at level CC’, thc head cap arid acrosome again enclosed the other structures (fig. 2, c’). The large cross section of the lightly stained nucleus was eiicapsulatcd by t h e three deeply staining prongs of the perf oratoriuin and the intervening menihrane (fig. 2, c” and c). The dorsal prong was now barely distinguisliable, hut tlie two vcnti*al ones were still prominent. The membrane uriitiny the prongs was conspicuous and tightly fitted the nucleus. Still farther hack, at level DD’, the three prongs of the perforatoriuni (fig. 2, d”) could iio longer be distinguished from the nienibranc covering the nucleus. Similarly, in the posterior third of the head, at level EE’ (fig. a), the membrane alone was seen, where it appeared to be somewdiat thinner tliaii anteriorly (fig. 2, e” and e). It may be concluded from tliese obscrvatioiis that thc meinbrarious web uniting the prongs of the perforatorium (fig. 2, b”) extends caudally to (’over the whole iiucleus (fig. 2, c”, d”, e”). The question then arose as to whether this was the nuclear niembrane itself. The nuclear membrane of the maturing spermatids was tlien examined a t tlie last few steps of spermiogeiirsis (as defined by Leblond and Clermont, ’,i2a), using sections stained with Masson trichronie after Helly fixation. At step 15 of sperniiogeiicsis the nuclear nienibrane wis thin and faintly basophilic. At step 16, tlie 11ieiii1)riiiic surrounding the nucleus w a s more definite ant1 acidophilic, and was taken to be a soiriewliat modified nuclear niem1)ranc. ,4t step 17 and following, an acidopliilic extension of this nnclea 1- mernhranc protruding apically b c p n d the nucleus made its appearance. This structure was iriilistiiigiiishahle from the perforatoriuin H S observed in preparations stained with iron bcmatoxylin. The perforatoriurn was thus identified as an extension of thc nuclear rnenihrane. 017 niic.rogrtrph.s of t h e opiccil s pel riii n t 0x0a h ea d As already noted hy Randall and Friedlaeiidc~*( ’ S O ) , the eiis of the rat spermatozoon was opaque to the clectro~l hearn a d n o structural detail could be seen. I n contrast, tho hf7ad c a p was fairly transparent, as seen in mishadowed (figs. (5-9) and shadowed (figs. 10 and 11) preparations. Along the dorsal edge, the ncrosome was seen as a rod-like tliickeiiing of the head cap (figs. 8 and 11). TTThethei-the heads w c 1 ~ shadowed in the general direction of the vcntral (fig. 10) 01’ dorsal (fig. 11) surfaces, tlic acrosome gar(’ approximatelp tlie sanio type of shadow. Therefore, this structu1.e must h a r e heen regularly circular or ovoid i n cross section. The acrosome curved smoothly d o n g the dorsal edge of tlic head cap and tapered as it approached the extrcriie tip of the cap. Its caudal region, however., could not he clistingvishd from the rest of the head cap. The perfornforizrvz was seen heponcl tlie nuclear a p e s within tlie head cap in both unsliadowed (figs. 6-9) and slradowPt1 preparations (figs. I0 and 11). The v e n t i d and dorsal reyioiiq of the perforatorium appeared different in preparations shadowed from a ventral (fig.10) or from a dorsal (fiy. 11) direction. The ventral reg<oii of the pcrforatorii-rm was iiidicated hy a sharp line more 01- less continuous with tlic ventral region of the nucleus (figs.8-9 and l l ) , while the dorsal portion of the pcrforatoriuin seernet1 to Inlc~nrlsiiioot11l;v into the head cap (fig. 8). These images werc. in agrwnient with the concept of the perforatorium as an elongated pyramid with a triangular cross section as s l i o ~ ~inn figure 2, a. Rructzrrc of t7Le p w f o m t o r i r r l n as s ~ with m t h e phase microscope Sodicni hydroxide slowly dissolved parts of tlie head of spermatozoa. Tlie initial changp was a complete dissolution Figs. 3-3 Phase contrast photographs of heads of rat spermatozoa treated \\itti I N NaOH f o r either half : i n hour (figs. 3 and 4) o r 1-2 hours (fig. - 5 ) . X approx. 1150. hi figuics 3 aud 4, thc iiucleiih ( N ) showv early signs of disintcyyxtion, iiamclly, a loss of refringenee of the apex. The perforatorium ( P ) resists hydrolysis by the alkali :ind displays its free apex as x7ell as the dorsal arid one ventral prong. The prongs a r e continuous with the thin nuclear niemhr:ine ( M ) which is iiow detached froin the nucleus. (Eotll head cap and ncrosoiiie hare heeii complctelr tlissolvcd by this cliemiezd trextment.) I n figure 5, the nucleus is almost completely disintegr:rted. The perforatorium which still resists alkaline hydrolysis is slightly tilted and thus shows the two latero-ventral prongs. Neither the dorsal prong nor the intervening membrane can be clearly distinguished in this photograph. of the acrosonie and head cap. The nucleus a t first showed a swelling of the caudal region of the nucleus (figs. 3 and 4 ) , later followed by a slow dissolution first noticeahle a t its anterior tip and gradually extending to the ~v110lestructure. As this was taking place, the perforatorium which a t first was tightly attached to the surface of the nuclear apex progressively loosened, but kept its refringence, shape, and position relative to the nucleus. Examination u~iclrrt1ic.w coiiclitioiis made i t clear that the perforatoriuin is attachcd to tlie nucleus by three rather than two prongs. Tlio small dorsal prong is visible in figures 3 aiid 4, iii which only oiie of tlic sturdier ventral prongs can be semi. Iii figure ,5,both ventral proiigs may be identified, but tlic dorsal prong is barely visible. The distanccl between the two vcntral proiigs w a s shortcli* tliaii tbwt between eitllcr of tlicril and the dorsal prong. ~’anclallytlic three prongs becaiiie progressively less proniiiiciit. Tlicy were continuous with tlie nuclear membrane which d s o persisted under sodiurri hydroxide treatment. This membrane loosened away from the nucleus a t an early stage (fig. 3). Apparently, tlie nierrilnwie extended over tlic whole nuclear surface, except f o r a small triangular area surrounding thc point of attachnient of the tail piece. (In figure 4, this area appears to be covered by another membrane whicli is apparently wrapped around tlic middle piece of the tail a s well.) Witliin tlic nest two 01’ three hours, as the nuclear material dissolved, the refri~igenceof the nucleus decreased. I n the cwd o n l ~ iits “ghost” persisted (fig. 5). The perforatorium, liowcvei*, retained a rigid shape, while the nuclear membrane became wrinkled and even tuallp broke down in places. DIscus8Ior\: Th c di s t i $1 c t ion, b t ui c ~ 2 .(Ic T O S 0111 c (1 (1 11 11 d p c r f o TOt o rizt m Ti1 the past tlie aci-osonie and the perforatorium h a w often been confused. Thus, Dumberg ( ’OS), in his otlierwisc excellent stndy of rat spermiogenesis, stated that the rod stained hy iron liernatoxylin along the ventral edge and apex of tlir spermatozoon nucleus was the “acrosomc” (altliough he liad proper17 identified this structurc in the earl^ stages of spermioxcnesis). Austin and Sapsfoid ( ’51), by means of tlie pliaw contrast microscope, came t o the conclusion that thc perforatorium was a thickened part of the liead cap. How evtli*, 1 .cbloncl and Clei*nioiit ( ’,i3a), using preparations PERFORATORIUM O F R A T SPEHMATOZOON 9 stained with the periodic acid-Schiff technique established tliat tlie acrosome was embedded within the head cap and that both structures were part of a single structural unit derived from the Golgi zone and referred to a s the “acrosomic system” ; but tlie perforatorium had different tinctorial properties and, in fact, appeared to be completely indepnident of this system. The present work, particularly the electron microscope studies (figs. 6-11), showed definitely that acrosome and perforatorium were distinct entities. The electron microphotographs of shadowed preparations (fig.11)suggested that beyond the nucleus, tlie perforatorium was a n elongated three sided pyramid distinct from the acrosome. A similar conclusion was reached by examination with the ordinary microscope of cross sections of spermatozoa heads, since these showed the apical portion of the perforatorium as a three-pointed star (fig. 2, a”). f%trpe noid ncrtiire of t h e perforatoritcwa Observations with the phase contrast microscope on suspensions of spermatozoa treated with sodium hydroxide revealed that tlie perforatorium was resistant to mild alkaline hydrolysis, while the acrosomic system was rapidly, and the nucleus slowly, hydrolyzed. The perforatorium which was thus r~ioi’eo r less isolated was a rigid, forked structure with tlirce prongs, one dorsal and two latero-ventral. These prongs were shown to be thickened portions of the nuclear membrane (fig.2, b a ~ i dca). The nucleus of the r a t spermatozoon was surrounded by a nienibrane wliicli was tentatively considered to be the nuclear riienibrane itself. This membrane, like the perforatorium, eshihited two unusual properties : a moderate degree of acidopliilia and H remarkable resistance to alkaline digestion. The acidopliilia appeared toward the end of spermiogenesis (stage If;), that is, just before the perforatorium emerged as a small protrusion of the nuclear apex, which gradually became more 10 Y. C L E E M O K T A N D O T H E R S aiid more proniiiicnt ( TJebloncland C'lerniont, '52a) .l It would seem that cliatiges in tlie nuclear xiieriibraxie associated with the appearance of acidophilia iiiduced the apical portion of this riicinbranc to protrude beyond the nuclear apes and thus give rise to the perforatoriurn. The mrchanisni by which tlie perforatorium develops froni the nuclear niembi.aiie might bc : (1) shrinkage of the iiucleus without ( ~ o r r e s p i i d i n gshrinkage of the niernbranc ; the nucleus would liavc to recede from the apical region of the head, whereas tlir menihraiic would persist t o become the perforat orium ; ( 2 ) uii cspaiisioti and lengthening of tlie nuclear niernliranc which beyolid the riuclcar apex would forni the perforatoi-iuin (possibly due t o addition of an acidophilic material witliin the substance of the riic~nil)ranc~).llcasnrcrnents of lieads of sperniatitls at tlie last few steps of sperniiogenesis revealed that the length of tlicl nucleus tlecreasecl slightly, namely, fr*oiii 11.1 p at step 15 to 10.6 p a t steps 18 arid 19 011 the avei*agc. This slight slir-iiikage of the riucleiis was too stnall t o accouiit for tlie formation of the peiforatoriurii (which measiu'eb 3.2 p beyond tlle tip of the nucleus). Thus, t!iv data favor the altcrnative hypothesis that ail expaiisioii of the nnclear nit~iiibi*anegives rise t o the pcrfoi*;itor iinn . SI'MA[ARY ()hervations with tlic phase contrast niicroscope, the clectron microscope, as well as with tlie ordinary light microscope, revealed tlie following details of the strncturc of the liead of r a t s p c r n dl ~t ozoa : Tilt. fo1wa1d (lisplnceinmt of the h ( ~ a ( 1cap previously tlebciihrcl I J Lebloncl ~ a i d C'lc~rn~oiit( '5%) at stel) 1 7 of spcriiiiogenrsis coulrl be oxplainccl 1 ) thc ~ t,ict that the c.inerging prrforatoriuni piislhcs tlir lieail rap forward. * Wlirtlle~f acto r s euterii:il to tlie cell influenw the hy~)otlretical e ~ ~ ) n n s i o of ii tlir modified nuclear meinbrane c:iii only be surmised. In this regaid, arguments hnxr been prewnted b? ~~elJ~OllC1 :ind ('lerniont ( '321,) t o indicate that tlir Scitoli rlernents a n d prcsiiiii:ihl,v tlie otlicr crlls of the srrniniferous rpithrlinm :IS wrll :ire subjected t o an incrcnsiiig lateral pressure :it stages IV-VI of the cyclc, t h a t is, a t the time n h e n the older yriieiation of spermatids reaches stel) 17. Surli n pressure might inrlntc the clonffntioii of :I meniburine enilowrd with PERFOHATORIUM O F BAT SPERMATOZOON 11 1. Tlie acrosome and head cap, which together form the * ‘acrosomic system,” a r e distinct from the perforatorium. 2. The perforatoriurn is a rigid and refringent structure which fits tightly onto the nuclear apex. It is continued over the nucleus by three prongs, which a r e thickenings of the nuclear membrane. Tlie perforatorium appears to be an outgrowth of this membrane, arising a s a result of the changes taking place in the head of the spermatid during the latter part of sperniiogenesis. ACKNOWLEDGMENT This work was supported by a grant from the National Research C‘oiincil of Canada. LITKRATURE CITED 1931 The development of the r a t spermatid. AUSTIN, C”. K., AND C. SAPSFORD J. Roy. Micr. Sac., 7 2 : 397-406. BIANDAIJ,.T. 1951 Observations on the morphology of r a t spermatozoa mounted J I ~ media of different refractive mdices and examined with phase microscope. Aiiat. Rec., 1 0 9 : (Suppl.), 11. UIWSBERG,J. 1908 La, spermiog6nAse chez le rat. Arch. f . Zellforschung, 2 : 137-180. JENSEN, 0. S. 1887 IJntersucliungen uibei die Samenkorpcr der Saugetiere, r o g e l und Amphibien. Arch. f . mikr. Anat., 30: 379-425. I,EBLOND,C P., A N D Y. CLERl t ONT 1952a Spermiogenesis of rat, mousp, hamster and guinea pig xs revealed by the ‘ ‘periodic arid-fuchsin sulfurous acid” technique. Am. J. Anat., 9 0 : 167-215. - ______ 19521s Dcfiiitioii of the stages of the cycle of t h e seminiferous epithelium in the rat. Ann. New York Acad. Sci., 5 5 : 518-573. SIESSING, G. 1897 Die Reteiligung von Centralknrper niid Fiphkre ain Aufban des Samcnfadens bri Saugetieren. Arch. f . micr. Anat.. 49 : 111-142. K A N D ~ L LJ. , T., A N D M. TI. G. FRIEDLAENDER 1950 The microstructure of rain zpermatoma. Exp. Cell Resrarch, 1 : 1 4 6 . RETZIUS, G. 1909 Die Spermieii dcr Nagetiere. Hiol. Untersuchungen, Neue Folgr XIX, Stockholm. PLATE 1 G-11 Electron microphotographs of lieads of rat sperniatozo:i fixed i n osnric acid. The preparations esnniinod under the c,lcctron niicrostolie were either niisliatlowed (figs. 6-9) or shadowecl with gold (figs. 10 a n d 11). G and 7 \Vhole he:& of spermatozoa. x npprox. 6,000 (fig. ti) and 4,500 (fig. 7 ) . The nuclciis :nid thc perforatorium ( P ) :ire opaque t o tlic clectron beam and appear dark, wliilo tlie t r a n s p r c n t head cap is the gray structure surrounding the apex of the hcad. Caud:illy the head cap Is closely attached t o the nucleus and cannot be distinguished rxccpt f o r the cand:il edges (C, C', fig. 6 ; see also fig. 7 ) . The point of a t t x h m e n t of the tail piece to tl!c nucleus, that is, the ventral angle of the nucleus, is shown a t T (fig. 6 ) . Tlie :xpproxiinate apical limit of the iiuclriis i s intlicated liy tlic tip of the arrow at S (fig. 6 ) . 8 and 9 Apical rc>gion of Iie:ids of sporni:itozo:i. X approx. 10,000 (fig. 8) and 11,000 (fig. 9 ) . T h e tlors:il (cnnvcx! edge of the liead cap sliows ;I rod-likc tliiclwning, tlie ncriisonie ( A ) . These picturcs clearly show t1i:it the pointed, opaque pcrforatoriiim has a sharli rcntral edge nirrl n iiinre diffuse dorsal limit. 1 0 TI!(, a p e s of the hciad of ;I sperixitozooii sliatlowed with gold froiii tlic vtwtr:il direction. x :ipprox. 10,500. I n this 1Irep:ir:itioii the ventral edgc of the Iicad cap is visible a s 1 ~ 1 1as the :icrosonic on tlic, dorsal edge. Tlie 1ironounrerl thic.kness of the perforatoriuni m:iy he seen. 11 The upex of the hrad of a spcririatozooii s1i:idowed with gold f r o m the dorsal dircetion. x :lppro". 10,500. Tho ;icrosonie appears like a t>ajwringrod on the dorsal edge of tlie head call. Kote thc sharp line m:iilc by the sliadow along the ventr:tl side of the perforatoriiun. Tlle struct,ures in t,hc wntr:il wiic>avity of t,lie Iic~n(1arc not visible. 12
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