The perforatorium Ф an extension of the nuclear membrane of the rat spermatozoon.

T 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