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Seasonal changes of the blood-testis barrier in viscacha Lagostomus maximus maximusA freeze-fracture and lanthanum tracer study.

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THE ANATOMICAL RECORD 236:459-464 (1993)
Seasonal Changes of the Blood-Testis Barrier in Viscacha
(Lagostornus maximus maximus): A Freeze-Fracture and
Lanthanum Tracer Study
ALFONSINA MORALES AND JUAN C. CAVICCHIA
Instituto de Histologia y Embriologia, Facultad de Ciencias Mkdicas, Uniuersidad
Nacional de Cuyo, Mendoza, Argentina
ABSTRACT
Adult male viscachas (Lagostomus maximus maximus)
were gathered from their natural habitat during the period of complete
spermatogenesis (June) and during the month of maximum testicular regression (August). The testes were processed by conventional electron microscopic technique using lanthanum nitrate (electron-denseintercellular
tracer) to define the intercellular spaces below the inter-Sertoli tight junctions and by freeze-fracture techniques.
During complete spermatogenesis the tracer surrounds spermatogonia,
preleptotene, and leptotene spermatocytes and stops at the level of the
inter-Sertoli tight junctions below all germ cells displaying synaptonemal
complexes (zygotene-pachytene spermatocytes) and germ cells in more
advanced stages of differentiation. Conversely, during testicular regression the tracer percolates all intercellular spaces between Sertoli cells and
the remaining germ cells (spermatogonia and few preleptotene and leptotene spermatocytes). During complete spermatogenesis, freeze-fracture
replicas exhibit numerous inter-Sertoli tight junction strands parallel to
each other and to the basal lamina. During spermatogenesis decay, the
inter-Sertoli tight junctions are found to be short, tortuous, frequently interrupted, and often associated with extented membranous areas of gap
junctions. 0 1993 Wiley-Liss, Inc.
Key words: Blood-testis barrier, Spermatogenesis, Sertoli cell, Testis, Seasonal breeders, Lagostornus maximus maximus, Intercellular
communications
The concept of the blood-testis barrier (BTB) emerges
from the physiological studies of Setchell and coworkers (Setchell et al., 1969; Setchell and Waites, 1975),
who demonstrated significant differences in electrolytes, carbohydrates, amino acids, steroids, and proteins between the adluminal seminiferous compartment and the extratubular fluids.
The morphologic correlate of this permeability barrier was later found by Fawcett et al. (19701, who described inter-Sertoli tight junctions that were judged to
be the stopping device for electron-opaque intercellular
tracers. In more recent works, the BTB was found t o be
a widespread phenomenon. It has been observed first in
mammals and later in other vertebrates as reptiles,
birds, amphibians, fishes, and also invertebrates as
nematodes, snails, and insects (Cavicchia and Moviglia, 1983; Bergmann et al., 1984; Miranda and Cavicchia, 1986, 1988; Bergmann and Schindelmeiser,
1987; Cavicchia and Miranda, 1988).
Increasing evidence indicates that the dynamic architecture of the seminiferous tubules and the development and synchronized differentiation of the distinct
germ cell types are modulated by local control mechanisms. The classic concept of hypophysial regulation of
0 1993 WILEY-LISS. INC.
the seminiferous epithelium through LH and FSH has
been drastically modified by recent findings that demonstrate that the cellular components of the seminiferous tubules are closely interrelated by a paracrine control (Sharpe, 1984; Le Margueresse and Jegou, 1988).It
has been established that the germ cells close to the
basal lamina of the seminiferous epithelium (spermatogonia, preleptotene, and leptotene spermatocytes)
reside in a basal compartment below the inter-Sertoli
tight junctions in free communication with extratubular fluids and that the zygotene spermatocytes (identified by the presence of synaptonemal complexes) and
other germ cells in a more advanced degree of differentiation are located in an isolated intratubular environment termed the adluminal compartment (Cavicchia and Dym, 1977, 1978; Dym and Cavicchia, 1977,
1978).
~~
Received September 11, 1992; accepted January 6, 1993.
Address reprint requests to Dr. J. C. Cavicchia, Instituto de Histologia y Embriologia, Casilla de Correo 56, 5500 Mendoza, Argentina.
460
A. MORALES AND J.C. CAVICCHIA
Figs. 1-4.
461
BLOOD-TESTIS BARRIER IN VISCACHA
In the few studies on seasonal breeders with cyclic
annual spermatogenesis, the inter-Sertoli tight junctions exhibit a cyclic formation and disassembly of the
BTB (mink, Pelletier, 1986, 1988; lizards, Cavicchia
and Miranda, 1988; duck, Pelletier, 1990; toad, Cavicchia and Moviglia, 1983). Since we believe that in these
models with cyclic spermatogenesis the unusual feature of formation and decay of inter-Sertoli junctions
and the apparent close correlation with neighboring
germ cells are of real interest, we have studied another
mammal belonging to this group, the viscacha (Lagostomus maximus maximus).
The viscacha presents an annual photoperiod. During the winter months the pineal gland stimulated by
the long darkness inhibits gonadal development. Conversely, spermatogenesis displays complete activity
during spring and summer (Fuentes et al., 1991;
Reiter, 1980).
MATERIALS AND METHODS
Ten adult male viscachas were gathered from their
natural habitat in a desert area located at 33"20' south
latitude, during June and August. In June, testicular
weight was 5.37 i 1.75 gm (mean 2 standard deviation) and histological examinations (see below) indicate complete spermatogenic activity. During August,
testicular weight was 1.38 ? 0.69 gm and histological
observations demonstrate maximal testicular involution. The animals were killed by shot near their burrows and the testes were dissected immediately and
fixed by immersion in 0.1 M cacodylate buffer (pH
7.4)-5% glutaraldehyde, with 2% lanthanum hydroxide as final concentration (Revel and Karnovsky, 1967;
Neaves, 1977; Ross, 1977; Camatini et al., 1982). Small
blocks were postfixed in 1%osmium-tetroxide in the
same buffer, dehydrated in graded series of acetone,
and embedded in Epon 812 (Luft, 1961).
Sections of 1 Fm were stained with toluidine bluesodium borate for light microscopy. Ultrathin sections
were stained with uranyl acetate and lead citrate (Reynolds, 1963).
For freeze-fracture, small samples of tissue were
fixed in 5% glutaraldehyde in 0.1 M cacodylate buffer
Fig. 1 . Seminiferous tubule with complete spermatogenesis in June.
The tracer percolates the basal lamina (BL) and surrounds leptotene
spermatocytes (L). Pachytene spermatocytes (PIand spermatids (Sd)
are not surrounded by the tracer. S: Sertoli cell. 3,500 x .
pH 7.4 and immersed in 25% glycerol used as cryoprotectant for 24 h and frozen in Freon 22 and liquid nitrogen. Blocks were freeze-fractured in a 301 BAF
Balzer, PA apparatus at -105" C, and shadowed with
carbon-platinum. Replicas were cleaned in sodium hypochlorite and mounted on 200# copper grids. Membrane faces were labeled as either E (exoplasmic) face
or P (protoplasmic) face.
RESULTS
Viscachas in June
The average weights of the body and testis coincide
with those already reported by Fuentes et al. (1991) for
viscacha (5.2 k 1.1 kg and 5.37 t 1.75 gm, respectively). The seminiferous epithelium is made up of Sertoli cells and germ cells, which develops a complete
maturation wave along the tubule and presents different cell associations (see inset, Fig. 2). We found a wellorganized blood-testis barrier, determined by interSertoli tight junctions. The two compartments are
delineated in the following way: the basal one, occupied
by spermatogonia, preleptotene, and leptotene spermatocytes (Figs. 1,3), and the adluminal one, containing the more mature cells. The tracer was unable to
surround zygotene-pachytene spermatocytes, which
are easily identified by the presence of synaptonemic
complexes (Solari, 1969) and also all the cells at more
advanced maturation stages.
Because identification criteria for the successive
germ cell types have been sufficiently described (Solari, 1969; Russell and Frank, 1978; Cavicchia and Sacerdote, 19881, they are not repeated here. Cross sections of seminiferous tubules in Figures 2 and 3 show
the association of spermatogonia, leptotene and
pachytene spermatocytes, and early round spermatids.
The tracer surrounded cells up to leptotenes, showing
that the Sertoli cells have established the blood-testis
barrier below the pachytenes. In the association of
spermatogonia, zygotene, diplotene and secondary
spermatocytes, and oblong spermatids, the tracer
stopped below the zygotenes (Figs. 2, 4). Freeze-fracture replicas display numerous strands of particles parallel to each other and to the basal lamina at the basal
portion of the lateral plasma membrane of Sertoli cells
(see Fig. 7). This arrangement coincides with BTB impermeability to the tracer. The intramembrane particles remain mostly a t the E face of cryofracture replicas. Sertoli cells show the characteristic irregular
shape and infolded nuclei with an even distribution of
the chromatin (Figs. 1, 2).
Viscachas in Augusf
Fig. 2.Seminiferous tubule with complete spermatogenesis in June.
The tracer surrounds spermatogonia (G).Zygotene spermatocytes (Z),
and round or elongated spermatids (Sd) are not surrounded by the
tracer. BL: basal lamina. L: lumen. 3,500 x . Inset: Light microscopy
of seminiferous tubules with complete spermatogenesis in June.
600 x .
Fig. 3. Higher magnification of leptotene spermatocytes (L) surrounded by the tracer. BL: basal lamina. 9,000 X .
Fig. 4.Higher magnification of zygotene spermatocytes (Z) in the
adluminal compartment. Arrows indicate synaptonemal complexes.
The tracer surrounds some spermatogonia (G). B L basal lamina.
7,000 x .
The testis weight is 0.7-0.8 gm. This average
weight, which indicates gonadal regression, agrees
with the result of Fuentes et al. (1991). The seminiferous epithelium is made up only by Sertoli cells, spermatogonia, and few preleptotene and leptotene spermatocytes. The Sertoli cell nuclei are located at
different heights in the epithelium, containing abundant heterochromatin and a prominent nucleolus.
These cells display a pleiomorphic profile and the typical nuclei turn into an oval regular profile (Fig. 5).
Using lanthanum, an electron-opaque tracer, the intercellular spaces between Sertoli cells of the seminiferous cords, which were now, during the inactive pe-
462
A. MORALES AND J.C. CAVICCHIA
Figs. 5-9.
463
BLOOD-TESTIS BARRIER IN VISCACHA
riod, devoid of a lumen (inset Fig. 51, were completely
permeated. The tracer percolated from the basal lamina up to the center of the core (Figs. 5, 6). Scarce,
tortuous, and frequently interrupted tight junctions between Sertoli cells were observed in freeze-fracture
replicas (Figs. 8,9). They were usually associated with
extensive gap junctions (Fig. 8).
DISCUSSION
Since marked involution of the germinal line takes
place in the seminiferous epithelium of viscacha during
a certain period of the year, this seems to be a suitable
model for the study of seasonal changes of spermatogenesis and their relation to inter-Sertoli junction variations. Moreover, according to Lincoln’s classification
(19811,this rodent belongs to the group of animals that
suffer maximal seasonal regression, decreasing testis
size to a mere 10%of the size reached during complete
spermatogenic activity.
The maximum testicular weight corresponds to the
autumn months (from April to June) and undergoes an
abrupt decay during August. The other parameters related to involution, e.g., low levels of plasmatic testosterone and intense decrease of the number of interstitial cells, which display hyperchromatic nuclei, were
also described (Fuentes et al., 1991). In viscacha, the
inter-Sertoli tight junctions have patterns similar to
the one described in the guinea pig (Pelletier and
Friend, 1983) and the rat (Cavicchia and Sacerdote,
1988). Also, the intramembrane particles of inter-Sertoli tight junctions are located preferentially in the E
face, as is the case for all the species previously studied
except the mink (Pelletier, 1988). In this species, during testicular regression, intramembrane particles of
the inter-Sertoli tight junctions are mostly in the P face
of the membranes.
During complete spermatogenesis, the numerous
strands of the inter-Sertoli junctions, parallel to each
other and to the basal lamina as observed by freezefracture, coincided with tracer impermeability. It
seems clear that the number of strands is closely related to BTB competence.This is in agreement with the
observation of Gilula et al. (1976) in the rat, Nagano
and Suzuki (1976) in the mouse, and Pelletier and
Friend (1983) in the guinea pig. Then, these junctions
belong, on the basis of the number of strands, to the
tight-tight junctions classification of Claude and Good-
Fig. 5. Seminiferous cord during seasonal involution (August). The
tracer percolates the intercellular spaces between Sertoli cells (S) and
remaining spermatogonia(GI. BL: basal lamina. 3,500 x . Inset: Light
microscopy of seminiferous cords in August. 600 X .
Fig. 6. Higher magnification of the same area of previous figure
showing the tracer in the inter-Sertoli spaces. 9,000 X .
Fig. 7. Cryofracture of seminiferous tubules showing numerous inter-Sertoli tight junction strands (arrows) in June. N: spermatocyte
nucleus. 15,000 x .
Figs. 8, 9. Cryofracture of seminiferous cord showing tortuous,
short, and interrupted tight junction strands in August. In Figure 8
an extense gap junction area is indicated (arrowheads).30,000 x .
enough (1973). However, this does not seem to be a
general phenomenon, since it has been reported that
the competence of the BTB is not related to the number
of inter-Sertoli junctional strands (mink, Pelletier,
1986-1988; human, Camatini et al., 1979; duck, Pelletier, 1990). In another model, the biliar canaliculi, the
same situation occurs as described by Martinez Palomo
and Erlij (19751, who suggested that the nature of this
junction may be different.
The most significant finding in this study is the remarkable coincidence between assembling of the BTB
and the presence of germ cells with synaptonemal complexes. This is in good agreement with previous reports
on numerous species, both in vertebrates and invertebrates (rat, Bergman and Dierichs, 1983; dog, Connell,
1980; rabbit, Sun and Gondos, 1986; insect, Miranda
and Cavicchia, 1986, 1988; lizard, Cavicchia and Miranda, 1988; toad, Cavicchia and Moviglia, 1982, 1983;
rat, Cavicchia and Sacerdote, 1991). In Galea rnusteloides, a local wild rodent of the Andes piedmont, the
BTB disassembles during testicular regression (Cavicchia, et al., 1992). However, since this event coincides
with the noticeable seasonal involutionary changes in
germ cells, all possible germ cell-Sertoli cell intercommunication mechanisms may be altered.
According to Pelletier (1986-1988), using horseradish peroxidase as an intercellular tracer, the mink has
a different behaviour regarding the BTB. He found
that in the first and the last spermatogenic wave in the
active testicular phase, the establishment of the BTB is
not coincident with the presence of a particular type of
germ cell. During these periods there is a percolation of
the tracer in the presence of zygotene or pachytene
spermatocytes.
The mink clearly represents an exceptional case,
since 90% of the males show high antibody levels
against spermatozoa during testicular regression
(Tung et al., 1981,1983,1984).In the viscacha, a mammal that is an unambiguously seasonal breeder, we
have found a general pattern of an annual modulation
of the BTB, which reminds one of the features reported
in other seasonal breeders.
ACKNOWLEDGMENTS
The technical help of Mr. Osvaldo Arango and Mr.
Daniel Bari is highly appreciated. The authors also
thank Dr. Fabio Sacerdote for linguistic assistance
with the manuscript. This investigation was supported
by grants from CONICET and CIUNC of Argentina.
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