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The blood-testis barrier and its formation relative to spermatocyte maturation in the adult ratA lanthanum tracer study.

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The Blood-Testis Barrier and Its Formation Relative to
Spermatocyte Maturation in the Adult Rat:
A Lanthanum Tracer Study
LONNIE D. RUSSELL
Department ofAnatomy, University of Miami, School ofMedicine, Miami, Florida 33152
ABSTRACT
An electron-opaque substance, lanthanum, was utilized to determine when germ cells of the rat first cross the blood-testis barrier in adult spermatogenesis. Intravascularly perfused lanthanum was shown to surround all
spermatogonia, preleptotene spermatocytes and early leptotene spermatocytes of
Stage IX in the adult rat testis. Lanthanum was excluded from the spaces around
more mature cells by newly-formed tight junctions between adjoining Sertoli
processes. These processes had previously intervened between the leptotene cells
and the basal lamina. The results are in close agreement with those of a previous
study (Russell, '77a) which indicated that leptotene cells are the first cells of
adult spermatogenesis to enter the intermediate compartment and to reside
beyond a permeability barrier.
During the course of spermatogenesis, germ testis. It was indicated in that study that the
cells are seen a t various levels within the leptotene spermatocytes of Stage IX (classifiseminiferous tubule. The most primitive cells cation of Leblond and Clermont, '52) are the
(i.e., spermatogonia, newly-formed sperma- first cells in spermatogenesis to enter an
tocytes) are observed a t the periphery (base) environment which possesses the major, if not
of the tubule lying on or near the basal lami- the only, characteristic, i.e., permeability barna. As these cells become committed to the rier, of a blood-testis barrier. In addition, the
maturation process, they are generally found existence of a transient intermediate comin positions much closer to the tubular lumen. partment was postulated; one which would
A t some point in their movement toward the contain the leptotene spermatocytes, and
lumen of the tubule, they eventually come to would assure that the continuity of the bloodreside on the luminal aspect of the Sertoli-Ser- testis barrier be maintained.
A method of determining precisely when
toli occluding junctions (Dym and Fawcett,
'70; Fawcett, '74). These junctions, present spermatogenic cells are no longer exposed to
above the spermatogonia and newly formed blood-borne substances, and thus have passed
spermatocytes, have been shown to be an ef- a permeability barrier, would be to use a high
fective barrier preventing the centripetal molecular weight tracer substance. Tracers
passage of large molecular weight substances have been used by several investigators in the
such as lanthanum nitrate (Dym and Fawcett, past to demonstrate that these molecules
'70). Once cells of the basal compartment have readily enter the basal compartment of the
traversed this blood-testis barrier, they are testis (Dym and Fawcett, '70; Dym, '73;
believed to reside in a separate compartment Neaves, '73; and others). By introducing the
nearer to the lumen, termed the adluminal tracer into the vascular system, it could be
compartment (Dym and Fawcett, '70). The al- determined after studying specific developtered milieu around cells of the adluminal mental stages, exactly when during spercompartment (Setchell, '67; Tuck et al., '70) is
Received May 17. '77. Accepted Aug. 5, '77.
thought to be favorable for the continued de' Present address to which all correspondence should be sent: Development of these cells (Dym, '73).
partment of Physiology, Southern Illinois Universlty, Carbondale,
Illinois
62901.
Russell ('77a) has studied the mechanism
"Above" and "below" when used to describe relative positions
by which spermatocytes are moved from the within the seminiferous tubule indicate respectlvely positions either
basal to the adluminal compartment of the rat nearer the lumen or nearer the base of the tubule.
ANAT. REC.
(1978)190: 99-112.
99
100
LONNIE D. RUSSELL
matogenesis t h a t the substance no longer
surrounds these cells. The absence of extracellular tracer around a cell type which had previously been surrounded by this substance
would indicate t h a t a permeability barrier
had formed, restricting t h e entry of t h e tracer.
The present study reports t h e results of such
a n experiment.
MATERIALS AND METHODS
Testicular tissue of normal, adult male
(225-300g) Sprague-Dawley rats was fixed by
vascular perfusion (Vitale e t al., '73) with a
4% buffered (cacodylate) glutaraldehyde solution containing 2% lanthanum nitrate. The
mixture was prepared according to the methods outlined by Dym and Fawcett ('70). After
20 minutes of fixation, t h e testis was removed
and diced into 1 mm blocks, whereupon these
blocks were fixed by immersion in the perfusate solution for a n additional hour. The
blocks were subsequently post-treated in osmium tetroxide containing 2% lanthanum
nitrate for one hour. A similar concentration
of lanthanum was added to all dehydration
solutions. After dehydration, infiltration, and
embedding, thin sections exhibiting silvergold to gold interference colors were cut
with Porter-Blum MT-2 ultramicrotome. Unstained sections were viewed on a Hitachi HU11C electron microscope. Stages of the cycle
were identified by t h e classification scheme
devised by Leblond and Clermont ('52). Selected areas of longitudinal sections of seminiferous tubules were utilized to study a developmental sequence occurring at the base of
the tubule in Stages VIII and IX.
RESULTS
The penetration characteristics of lanthanum as they relate to t h e testis have been described previously by several investigators
(Dym and Fawcett, '70; Dym, '73; Vitale e t al.,
'73; Connell, '75) and need not be reviewed in
detail in this report. I n general i t has been
shown t h a t l a n t h a n u m readily fills t h e
lymphatic and interstitial spaces within the
testis and gains entrance into t h e basal compartment. Here it surrounds all germ cells of
this compartment but is prevented from
reaching t h e tubular lumen by Sertoli-Sertoli
occluding junctions.
As mentioned in t h e introduction, this
author (Russell, '77a) has investigated t h e
movement of spermatocytes from t h e basal to
the adluminal compartment of t h e rat testis.
I t was shown t h a t one of t h e earliest signs of
this translocation of spermatocytes was their
actual physical separation from the basal lamina. At the beginning of this process, Sertoli
cells undermined t h e newly formed leptotene
cells by extending appendages between them
and the basal lamina or in some cases between
them and Type A spermatogonia. This was
seen to occur in late Stage VIII and in early
Stage IX. I t was also shown that when basal
processes of Sertoli cells met below the young
leptotene cells, azonula adherens and, shortly
thereafter, a Sertoli ectoplasmic ("junctional") specialization (composed of subsurface
bundles of filaments and more deeply placed
saccules of endoplasmic reticulum [Flickinger
and Fawcett, '67; Russell, '77b; and othersl)
formed at the surfaces of these invading processes. In t h e present study, late Stage VIII
and Stage IX were examined in lanthanum
perfused animals. Lanthanum was capable of
passing between Sertoli processes to clearly
outline preleptotene spermatocytes (fig. 2)
and leptotene spermatocytes of Stage VIII.
Also leptotene spermatocytes in tubular segments displaying Stage IX which adjoined
those of Stage VIII (in longitudinal section)
were outlined (figs. 2, 3).4 In many cases,
lanthanum was seen to pass through the space
between Sertoli-Sertoli ectoplasmic ("junctional") specializations to reach these cells
(fig. 2).
At a distance slightly further into the Stage
IX tubule, t h e pattern of lanthanum distribution had changed. Although lanthanum was
always seen around spermatogonia of this
stage, i t never penetrated the spaces around
leptotene cells (fig. 5). However, i t was observed to penetrate a considerable distance
into t h e intercellular cleft between Sertoli cell
processes (fig. 5). E n face profiles of the intercellular space between the two Sertoli cells at
t h e level of these processes indicated t h a t several rows of tight junctions had formed between these cells. This was evidenced by the
electron translucent linearities which were
outlined by lanthanum (figs. 5-71. The failure
' Leptotene spermatocytes were identified by the presence of axial
elements within the nucleus.
a The reader is referred to Perey e t al. ('61) for a detaded descriptlon
of the appearance of longitudinal sections of seminiferous tubules, In
a previous study (Russell, '77a) and in the present study, a logical
step-by-stepsequence of events was observed in longitudinal sections
through Stages VIII and IX. Those portions of tubules displaying
Stage IX which adjoined Stage VlII were always found to be less
advanced than these adjoining Stage X.
GERM CELL ENTRY INTO INTERMEDIATE COMPARTMENT
of lanthanum to surround leptotene cells
could always be correlated with the presence
of these tight junctions.
In the most advanced areas of Stage IX
(those adjoining Stage X and also Stages XXIV), the penetration of lanthanum was
limited to the spaces around spermatogonia
and into the clefts between adjacent Sertoli
cells. However, the extent of its penetration
between Sertoli cells was always minimal (fig.
4). Here i t was only rarely seen to outline a
single tight junction. The pattern of lanthanum penetration just described was apparently repeated during each subsequent cycle as
spermatocytes matured and were moved from
the basal Compartment.
DISCUSSION
The determination of the extracellular milieu of germ cells is no doubt important in understanding the progress of their maturation.
For several years i t has been generally assumed t h a t the environment of the adluminal
compartment is necessary for the meiotic
processes as well as the complex transformation involved in spermiogenesis. Three examples indicating t h a t this environment is
important in influencing germ cell maturation have been shown in recent years. O’Rand
and Romrell (’77) and Millette and Bellve
(’77) presented evidence that pachytene spermatocytes (adluminally positioned cells) develop surface molecules of an antigenic nature
which are specific to these germ cells and to
their successors, yet are not present on spermatogonia of the basal compartment. That
these cells are isolated from the systemic immune system by the blood-testis barrier no
doubt favors the development of these cell surface antigens. In addition, Russell and Clermont (’77b) have shown that cells of the
adluminal compartment are the ones primarily affected by hypophysectomy or hormone replacement (LH and FSH). That study suggested t h a t a special hormonal environment
provided in the adluminal compartment and
controlled by the Sertoli cell was necessary for
continued development of these cells. It has
also been demonstrated that cells will not develop beyond the pachytene phase of meiosis if
placed in an in vitro environment (Steinberger and Steinberger, ’65). Presumably such
a n environment would not be similar to that
established in vivo, since culture media (even
if supplied with the necessary hormones)
j
101
would be equally accessible to cells of both the
basal and adluminal compartments and would
favor the development of cells in one or the
other of the compartments.
In previous publications several investigators (Dym and Fawcett, ’70; Fawcett, ’74, ’75;
Ross and Dobler, ’75) have assumed that preleptotene spermatocytes or spermatogonia
(O’Rand and Romrell, ’77) are the cells which
cross the blood-testis barrier. Russell (’77a)
has shown that although loosening of cells
from the basal lamina and movement of cells
away from this structure occurred during the
preleptotene phase of meiosis, as well as the
preleptotene-leptotene transition phase, the
establishment of a permeability barrier did
not occur until the cells had reached the leptotene phase of development. An indication
that a permeability barrier had formed was
given by the failure of young leptotene cells
to shrink (as did other basal compartment
cells) after hypertonic fixatives were perfused
through the vascular system. The precise timing of the formation of a permeability barrier
in spermatogenesis, as shown in the present
study, is in partial although not complete
agreement with that demonstrated by Russell
(‘77a). In the latter study it was shown that,
with the establishment of a zonula adherens
between the Sertoli processes, the shrinkage
artifact was eliminated or reduced around leptotene cells. The present study demonstrated
that lanthanum penetrated the spaces around
leptotene cells even a t this time. However, a t
a time shortly thereafter (estimated to be a
few hours) and subsequent to the development
of Sertoli ectoplasmic (“junctional”) specializations (Flickinger and Fawcett, ’67; Russell,
’77 and others), the penetration of lanthanum
was blocked. Once tight junctions formed between the two Sertoli cells, the lanthanum
was incapable of reaching the leptotene spermatocyte. The tight junctions between the
two cells appearing as “pale lines in lakes of
electron opaque tracer” “eaves, ’73) indicated that these structures were responsible
for the failure of continued ‘penetration of
lanthanum. During early Stage IX, the lanthanum was capable of outlining several of
these rows of tight junctions before its luminal penetration was halted. This indicates
that as tight junctions were formed below the
Sertoli cell there were, a t least for a short
time, some imperfections in their makeup
which would cause them to be “leaky.” With
102
LONNIE D. RUSSELL
time, lanthanum was only rarely seen to pass
even a single tight junctional profile.
For a short period of time prior to the development of tight junctions, lanthanum was
seen in the spaces between opposing Sertoli
ectoplasmic specializations and was capable
of gaining entrance to the spaces around
leptotene cells. Sertoli ectoplasmic specializations termed junctional specializations by
Flickinger and Fawcett ('671, were characterized (Brokelmann, '61; Nicander, '63; Flickinger and Fawcett, '67) as being composed of
subsurface bundles of filaments and more
deeply placed saccules of endoplasmic reticulum. They are seen both a t the base of the
tubule in the region where two Sertoli cells
meet as well as at the surfaces of Sertoli cells
which oppose germ cells (Ross and Dobler, '75;
Ross, '76, '77; Russell, '77b). The observations
presented in the present study support Russell's contention ('77b) that these specializations are not a junctional component but are
separate entities which may or may not be associated with either the tight, gap, or desmosome variety of junctions. Whether or not
they are involved in cell-to-cell adhesion as
suggested by reports from Ross' laboratory
(Ross and Dobler, '75; Ross, '76, '77) has yet to
be determined.
The present report has emphasized the timing of the formation of a permeability barrier
which restricted the movement of lanthanum
in an upward direction. It might be assumed
that once this barrier had formed, the leptotene spermatocyte had entered the adluminal compartment. This does not, however,
appear to be the case. This author has previously ('77a) indicated t h a t although a new
permeability barrier had formed below the
leptotene spermatocytes of Stage IX, the preexisting ones above these cells were maintained until Stage XI, or in some cases much
later. In a morphological sense, such a compartment sealed by tight junctions above and
below could neither be described as basal nor
adluminal. The simultaneous presence of two
barriers led Russell to postulate the existence
of an intermediate compartment of the testis.
One function of this compartment would be to
act as a transit chamber that wouldassure the
continuity of the blood-testis barrier as the
spermatocytes pass from the basal to the
adluminal compartment. The initial formation of a permeability barrier below the spermatocyte would signal the entry of the lep-
totene cell into the intermediate compartment, not t h e adluminal compartment.
Whether or not the intermediate compartment provides a physiologic environment different from t h a t of the adluminal compartment has yet to be demonstrated.
ACKNOWLEDGMENTS
The technical assistance of Ms. Beryn
Frank and the help of Ms. Yelyena Kowalewicz in preparing and reading the manuscript
is greatly appreciated.
This work was supported by Grant NIH10266 from the National Institute of Child
Health and Human Development.
LITERATURE CITED
Brokelmann, J. 1963 Fine structure of germ cells and
Sertoli cells during the cycle of the seminiferous epithelium in t h e rat. 2. Zellforsch., pp. 820-850.
Connell, C. J. 1975 A freeze-fractured and lanthanum
tracer study of the junction between the Sertoli cells of
the dog. Anat. Rec., 181: 336-337.
Dym, M. 1973 The fine structure of the monkey (Macaca) Sertoli cell and its role in maintaining the bloodtestis barrier. Anat. Rec., 175: 639.656.
Dym, M., and D. W. Fawcett 1970 The blood-testis barrier
in the r a t and the physiological compartmentation of the
seminiferous epithelium. Biol. Reprod., 3: 308-326.
Fawcett, D. W. 1974 Observations on the organization of
the interstitial tissue of the testis and on the occluding
cell junctions in the seminiferous epithelium. Advances
in Biosciences, 10: 83-99.
1975 The Ultrastructure and function of the
Sertoli cell. Handbook of Physiology. Vol. 5, Section 7.
Physiological Society, Washington, D.C., pp. 21-55.
Flickinger, C., and D. W.Fawcett 1967 The junctional specializations of Sertoli cells in the seminiferous epithelium. Anat. Rec., 158: 207-222.
Leblond, C. P., and Y. Clermont 1952 Definition of the
stages of the cycle of the seminiferous epithelium in the
rat. Ann. N. Y. Acad. Sci., 55: 548-573.
Millette, C. F., and A. R. Bellve 1977 Temporal expression
of cell surface antigens during mouse spermatogenesis.
Program of the American Society of Andrology, p. 37.
Neaves, W.B. 1973 Permeability of Sertoli tight junctions to lanthanum after ligation of ductus deferens and
ductuli efferentes, J. Cell Biol., 59: 559-572.
ORand, M. G., and L. J. Romrell 1977 Appearance of cell
surface auto and isoantigens during spermatogenesis in
the rabbit. Develop. Biol., 55: 347-358.
Perey, B., Y.Clermont and C. P. Leblond 1961 The wave of
t h e seminiferous epithelium of the rat. Am. J. Anat., 108;
47-78.
Ross, M. H. 1976 The Sertoli cell junctional specializations during spermiogenesis and a t spermiation. Anat.
Rec., 186: 79-103.
1977 Sertoli-Sertoli junctions and the Sertolispermatid junctions after efferent ductule ligation and
lanthanum treatment. Am. J. Anat., 148: 49-56.
Ross, M. H., and J. Dobler 1975 The Sertoli cell junctional
specializations and their relationship to the germinal epithelium as observed after efferent duct ligation. Anat.
Rec., 183: 267-292.
GERM CELL ENTRY INTO IN TERMEDIATE COMPARTMENT
Russell, L. 1977a Movement of spermatocytes from the
basal to the adluminal compartment of the rat testis. Am.
J. Anat., 148: 313-328.
1977b Observations on rat Sertoli ectoplasmic
(“junctional”) specialization in their association with
germ cells in the r a t testis. Tissue and Cell, 9: 475-498.
Russell, L., and Y. Clermont 1977 Degeneration of germ
cells in normal, hypophysectomized, and hypophysectomized-hormone treated rats. Anat. Rec., 187: 347-366.
Setchell, B. P. 1967 The blood-testicular fluid barrier in
sheep. J. Physiol., 189: 63-65.
103
Steinberger, A., and E. Steinberger 1975 Differentiation of
r a t seminiferous epithelium in organ culture. J. Reprod.
Fert., 9: 243-248.
Tuck, R. R., B. P. Setchell, M. H. Waites and J. A. Young
1970 The composition of fluid collected by micropuncture
and catheterization from the seminiferous tubules and
rete testis of rats. Pflugers Arch., 318: 225-243.
Vitale, R., D. W. Fawcett and M. Dym 1973 The normal development of the blood-testis barrier and the effects of
clomiphene and estrogen treatment. Anat. Rec., 176:
333-344.
Note added in proof: The findings presented in a recent paper by Dym and Cavicchia (Biol.
Reprod., 17: 390-403) are in substantial agreement with those of this study and those
of an earlier study from this laboratory (Russell, ’77b). It should be noted however
that Dym and Cavicchia ascertained in monkeys that late leptotene or zygotene spermatocytes ascend from one compartment to another. The two studies from this laboratory, utilizing the rat, indicate that it is the early leptotene cell which leaves the
basal compartment. This slight discrepancy in the two reports appears to represent a
true species difference. Dym and Cavicchia chose t o comment on a n implicit function
of the intermediate compartment; whereas the stated function “allows the upward
mobility of germ cells without disrupting the continuity of the blood-testis barrier”
was ignored. All parties agree that the junctional complexes reform prior to the dissolution of existing junctions above the cell in question, and that this provides the
means by which the barrier is sealed during germ cell migration. Whether or not a n
additional function of the intermediate compartment will be elucidated in the futuFe
is unclear. Surely, we must first establish a function for the blood-testis barrier (i.e.,
basal vs. adluminal compartment).
which meet above this spermatocyte. The furthermost upward penetration of lanthanum is indicated by
opposing arrows. One surface of the preleptotene spermatocyte is flattened and lies in contact with the limiting membrane. Lanthanum is not seen around a nearby pachytene spermatocyte.
1 Lanthanum is seen around a preleptotene spermatocyte of Stage VIII and between adjacent Sertoli cells (S)
EXPLANATION OF FIGURE
PLATE 1
Figs. 1-7 Unstained sections showing the basal aspect of seminiferous tubules after lanthanum administration. The limiting membrane or basal aspect of the tubule is always seen a t the bottom of the figure when the
page is oriented horizontally.
<:ERM CELL ENTRY 1::rO
Lonnie D Russell
INTERMEDIATE CDMPARTMENT
PLATE 1
3 Early Stage IX tubule showing a Type A spermatogonium and a leptotene spermatocyte outlined by
lanthanum. Basal processes (arrows) from two adjoining Sertoli cells (S)have extended under the leptotene
spermatocyte and met below this cell (arrows). The furthermost extent of lanthanum penetration is indicated by arrowheads.
2 Early Stage IX tubule showing a leptotene spermatocyte removed from the basal lamina. Lanthanum is seen
in the spaces between basal Sertoli cell (S) processes (p) and also surrounds the leptotene cell. The extent of
upward penetration of lanthanum is indicated by arrowheads. Also shown are the ectoplasmic specializations
(paired arrows) which are seen both above and below the spermatocyte.
EXPLANATION OF FIGURES
PLATE 2
GERM CELL ENTRY INTO INTERMEDIATE COMPARTMENT
Lonnie D. Russell
PLATE 2
A Stage X tubule showing Type A spermatogonium, a leptotene spermatocyte and two adjoining Sertoli cells
6).
Tracer substance lies external to the spermatogonia and is seen to extend only a short distance into the
cleft between Sertoli cells (arrowhead). Ectoplasmic specialization (arrows) is observed below the leptotene
spermatocyte.
5 An early Stage IX tubule showing a Type A spermatogonium, leptotene spermatocyte and neighboring Sertali cells (S). Lanthanum is seen in the spaces around a Type A spermatogonium and in the spaces between
adjoining Sertoli (S) processes; however, this substance does not extend to surround the leptotene spermatocyte. Arrowheads indicate the highest level of lanthanum passage.
4
EXPLANATION OF FIGURES
PLATE 3
m
iGI
4a
109
6, 7 High magnification of two late Stage IX tubules showing adjoining Sertoli cells and a Type A spermatogonium (fig. 6 only). These Sertoli cells diverge to surround a leptotene spermatocyte a t a higher level
(not shown; direction indicated by bold arrows). Lanthanum is seen in the interface between a spermatogonium and an adjoining Sertoli cell and also between Sertoli cells. Electron translucent linearities
(arrowheads) in the area of lanthanum deposition indicate newly formed tight junctions. The latter are
well visualized when the plane of section through the intercellular space is oblique. The furthermost
upward penetration of lanthanum is indicated by paired arrows.
EXPLANATION OF FIGURES
PLATE 4
GERM CELL ENTRY INTO INTERMEDIATE COMPARTMENT
Lonnie 0. Russell
PLATE 4
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