J. Embryol. exp. Morph. 95, 261-277 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
Some histochemical and ultrastructural observations on
the early foetal pig testis
C. J. A . H . V . VAN V O R S T E N B O S C H , C. M . J. E . VAN R O S S U M - K O K ,
B . C O L E N B R A N D E R AND C. J. G. W E N S I N G
Department of Veterinary Anatomy, State University Utrecht, Yalelaan 1, 3508 TD
Utrecht, The Netherlands
SUMMARY
Testes of foetal pigs between 26 to 35 days post coitwn (p.c.) were investigated histochemically
and ultrastructurally.
Diaphorase and A5-3/3-hydroxysteroid dehydrogenase activities were studied using, respect­
ively, NADH and pregnenolone and dihydroxy androsterone as substrates.
Ultrastructurally, attention was focused on the development of mesenchymal cells and on the
sustentacular cells in the primitive sex cords in an attempt to detect the origin of Leydig cells.
Histochemically there is a concentration of activity toward the interstitium with increasing
age. Also the reactions increase in intensity. Ultrastructurally no evidence for Leydig cell
development from Sertoli cells could be observed. Mesenchymal cells between the sex cords
show a development toward Leydig cells. This is absent in mesenchymal cells in the future tunica
albuginea.
Before 30 days p.c. no 'true' Leydig cells can be observed morphologically. The role of the
rough endoplasmic reticulum/mitochondrial complex, which is present in many mesenchymal
and sustentacular cells, is discussed.
INTRODUCTION
Gonadal development in the male pig has been studied biochemically, histo­
chemically as well as morphologically (Moon & Raeside, 1972; Moon, Hardy &
Raeside, 1973; Moon & Hardy, 1973; van Straaten & Wensing, 1978; Raeside
& Middleton, 1979; Ford, Christenson & Maurer, 1980; van Vorstenbosch,
Colenbrander & Wensing, 1982,1984a; van Vorstenbosch, Spek, Colenbrander &
Wensing, 1984e).
A large part of the foetal period has been investigated thoroughly but there is
still a short but physiologically significant period lasting from 28 days until 35 days
post coitwn (p.c.) that has been largely neglected in morphological studies.
During this period the transient testosterone production starts and peaks (Raeside
& Sigman, 1975; Raeside & Middleton, 1979; Ford etal. 1980). Testosterone
production in the male gonad at the onset of this period is occurring before the
Leydig cells appear at day 30p.c. (Pelliniemi, 1975a,b, 1979).
Key words: testis, pig foetus, histochemical, ultrastructural, Leydig cells.
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Table 1. Record of numbers and ages of animals used in experiments
Days post coitum*
Number of foetuses used
26t
3
28
4
30
10
35
6
* Mating date precisely known.
f Sex determined by karyotyping.
Although smooth endoplasmic reticulum (SER) is an important organelle
normally involved in steroid synthesis (Christensen, 1975), no SER is found in the
differentiating cells in the pig gonad up to 27 days p.c.
At 35 days p.c. Leydig cells are well developed and contain appreciable
amounts of SER. These cells also contain peculiar complexes of mitochondria and
long profiles of rough endoplasmic reticulum (RER) which are in close spatial
relationship. These complexes are also found in preSertoli cells of 35 days p.c. and
wane in both cell types between 52 and 60 days p.c., coinciding with the decline of
testosterone production (van Vorstenbosch et ai. 1982, 1984a; Ford etal. 1980).
They are already present in most cell types of male and female gonads of 27 days
p.c. (Pelliniemi, 1975fc).
This study aimed: (1) to determine possible sites of testosterone production
based upon the distribution pattern in the testis of 3^-hydroxysteroid de­
hydrogenase (3/3-HSD) from 26 to 35 days post coitum, and (2) to compare
morphologically the first detectable Leydig cells with those at the peak of
testosterone synthesis at 35 days p.c.
MATERIALS AND METHODS
Foetuses of Yorkshire and Dutch Landrace crossbred pigs have been used. The sampling ages
were 26, 28, 30 and 35 daysp.c. The day of insemination was precisely known and considered as
day 0 post coitum. Numbers and ages of the animals used are recorded in Table 1. The method of
collecting the foetuses has been described earlier (van Vorstenbosch et al. 1982).
Histochemistry
The gonadal ridge area of animals of 26 days p.c. was excised. The entire testes of foetuses of
28 days p.c. were used, while those of animals of 30 or 35 days p.c. were cut into smaller parts.
After dissection, materials were frozen in liquid nitrogen and stored at -70°C. Cryostat sections
were used for the histochemical reactions.
The NADH diaphorase reaction to detect Leydig cells was carried out as described by
Dierichs, Wrobel & Schilling (1973) and as modified by van Straaten etal. (1978).
Pregnenolone and dihydroepiandrosterone (DHA) were used as substrates for A5-3j3hydroxysteroid dehydrogenase (3/3-HSD).
The reactions and the estimation of their intensity were carried out as described by van
Straaten etal. (1978).
Electron microscopy
The entire gonad or samples of the gonads were taken at ages 26,28 and 30 days p.c. according
to the method described above. The tissue handling was according to the method described
earlier (van Vorstenbosch etal. 1982).
Observations on the early foetal pig testis
263
Sex determination
From 28 days onward sex can be determined by histological investigation, as sex cord anlagen
are visible in the male foetus. At 26 days p.c. sex is determined by karyotyping. The method
consists of trypsinization of the mesonephros and limb buds, and the subsequent culture of the
isolated cells for one or two days followed by karyotyping (Bosma, Colenbrander & Wensing,
1973).
RESULTS
Histochemistry
The results are shown in Table 2 and Fig. 1.
NADH-diaphorase
At 26 days p.c. NADH showed a weak overall reaction in the gonadal ridge. At
28 days the pattern of distribution of the reaction was still the same, however, the
intensity had increased. At 30 days p.c. the pattern had changed; the activity now
was clearly limited to the interstitium; in the sex cord anlagen there was no activity
at all. At 35 days the pattern was comparable to 30 days p.c. with an increase in the
intensity of the reaction.
A-5'-3ß-hydroxysteroid dehydrogenase
At 26 days p.c. a diffuse 3/2-HSD reaction was present all over the gonad with
DHA as a substrate. With pregnenolone as a substrate the reaction was negative.
At 28 days p.c., both with DHA and pregnenolone as substrates, the reaction
was positive in the area of the cord anlagen. The intensity of the reaction with both
substrates was about the same. The localization of this reaction was not as diffuse
as the reaction at 26 days p. c.
At 30 days p.c. the reaction after using both substrates was much more intense
and located mostly in the interstitium. The reaction with pregnenolone as sub­
strate showed about twice the intensity compared to DHA.
At 35 days p.c. the intensity of the 3/3-HSD reaction became less intense with
pregnenolone as a substrate in comparison to the reaction at 30 days p.c., but
it was clearly located in the interstitium only. Both substrates gave the same
intensity of reaction.
Table 2. Results of histochemistry
Days post coitum
26
28
30
35
Diaphorase
NADH
+
++
+++
+++++
3j0-HSD
Pregnenolone
DHA
+
++
++
++++
++
+++
+++
The reaction intensity scaled from + (very weak) to + + + + + (very intense).
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Fig. 1. (A,B). Semithin sections stained with toluidine blue; (C,F) cryosections;
(C-E) NADH as substrate (diaphorase); (F) Pregnenolone as substrate (A5-3/?-HSD).
(A,C,E) are 28 days p.c. and (B,D,F) 35 daysp.c. of age. (A,B, D-F) Bar, 200 jum; (C)
bar, 800 jum.
At 26 days p.c. the male as well as the female gonadal anläge showed the same
distribution pattern of these reactions. With the method used it is very difficult to
pinpoint the exact position until about 35 days p.c. since there seems to be a
reaction in both interstitium and cords.
Electron microscopy
At 26 and 28 days p.c. no Ley dig cells can be observed. In no cells of the
gonad of either male or female could any SER be detected. In most cells,
Observations on the early foetal pig testis
265
with the exception of germ cells and mesothelial cells, mitochondria/(R)ough
(E)ndoplasmic (R)eticulum complexes can be observed frequently. They consist
of a close spatial relationship between RER and mitochondria.
From 28 days p.c. onwards the testis consists of four compartments.
(1) At the periphery a cover of one or some layers of mesothelial cells. This
layer does not obviously change between 28 and 30 days p.c.
(2) A peripheral mesenchymal cell layer. At 28 days p.c. this layer is not
well developed. At 30 days this compartment is clearly developed and well
recognizable as the future tunica albuginea.
(3) The sponge-like sex cord anläge. At 28 days p.c. this anläge is often in very
close vicinity to the mesothelial cell layer cover of the testis. At 30 days p.c. the
periphery of the anläge has moved centrally.
(4) Spaces within the cord anläge. They will give rise to the future interstitium.
It is in this area that at 30 days p.c. Ley dig cells can be detected next to the
mesenchymal cells and only there. They occur patchwise and still large areas are
devoid of them.
The four compartments will be described below at 30 days p.c. If there are
differences to 28 days p.c. and 35 days p.c. they will be mentioned explicitly
(Fig- 2).
(1) Mesothelial layer
The testis is covered with slightly elongated cuboidal epithelial cells, which are
interconnected by tight junction-like structures. Further cell contacts are very
loose. Except for a few small projections on the coelomic side, the cell membrane
is smooth. Coated pits or signs of pinocytosis are rarely observed. The cells mostly
rest upon a well-defined basal lamina. Their nuclei are almost always located in the
apical part of the cells. Except for numerous polysomes and free ribosomes their
cell organelles are not extensively developed. Among these cells there are many
containing lipid-like droplets of various sizes. Mitochondria are slender and long
with lamellar cristae. The nuclei are somewhat irregular and do not possess
prominent nucleoli.
Especially before 35 days p.c. this layer seems to be very dynamic and frequently
cells can be observed that are leaving the layer to migrate downward into the
peripheral layer of loose mesenchymal cells. Amongst these migrating cells are not
only cells containing lipid-like droplets, but also cells without 'lipid' droplets. We
have no clear idea about their fate. After 35 days p\c. the droplet-containing cells
are no longer present. (Figs 3A,B, 4A.)
(2) Peripheral mesenchymal layer
The bulk of the mesenchymal cells are slender and have long processes. They
often contact each other with short blunt projections showing tight-junction-like
connections. The cells often possess a well-developed Golgi apparatus. The
cytoplasmic matrix is very lucent. A few long RER profiles, incompletely covered
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with ribosomes, can be observed (so-called transitional ER). Mitochondria are
mostly elongated and have lamellar cristae. They are not very often in close
contact with the RER. (Fig. 4B.)
In this compartment other cell types can be observed, firstly lipid-containing
mesothelial-like cells which apparently have migrated from the mesothelial layer.
They can be found deep in the mesenchymal layer. Especially before 35 days p.c. it
is often difficult to interpret the spatial configuration of the basal lamina of the
mesothelial layer. In a number of cases this structure cannot only be found
underneath the mesothelial layer, but also underneath mesenchymal cells just
Fig. 2. 28 days p.c. Spaces as shown in the sex cord anläge are the future interstitium.
m, mesothelial layer; p, peripheral mesenchymal layer; s, sex cord anläge. x2500.
Bar, 10 ^m.
Observations on the early foetal pig testis
Fig. 3. (A) 28 days p.c. Between normal mesothelial cells (n), cells can be observed
containing lysosome-like bodies and lipid droplets. Ribosome-rich cells (r) positioned
below the lipid containing cells, can also be found between the mesothelial cells. Basal
lamina (arrowheads). X8000. (B) 28 days p.c. enlargement from Fig. 1A. Note the
well-developed Golgi apparatus and the thick bundle of actin-like material (arrow).
Note also the discontinuity of the basal lamina under the cell marked (b) (asterisk).
Arrowheads under basal lamina. xl5 000. Bars, 1 /-tm.
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below this layer. A second cell type can be distinguished which is also mesen­
chymal but differs from the bulk of the mesenchymal cells in possessing a huge
amount of free or clustered ribosomes and short cell projections. (Fig. 3A,B)
Fig. 4. (A) 28 days p.c. Mesothelial cells. Note the lipid droplet in a mesothelial cell
and a cell containing many lipid droplets which is positioned below the normal cell
layer. X8000. (B) 28 days p.c. Normal mesenchymal cells in the peripheral layer. Tight
junction-like cell connections (arrowheads), x 15 000. Bars, 1/an.
Observations on the early foetal pig testis
269
(3) The sex cord anläge
The sex cord anläge comprises an anastomosing system of cell plates or cords.
At 28 days p.c. they are still in close proximity to the mesothelial cell layer, but at
30 days their periphery has clearly moved more centrally. This anläge consists of
two cell types: Sertoli cells and germ cells. The latter are spread diffusely through
the cord. We did not study their morphology.
The cords are surrounded by a basal lamina, which is sometimes weakly
developed. The Sertoli cells (sustentacular cells), are wedge shaped. They are
interconnected by loose cell contacts and the width of their intercellular spaces
varies, but frequently their cell membranes run more or less parallel. Coated pits
are rare. The Golgi apparatus is well developed. The most characteristic feature is
the very large number of mitochondria in close vicinity with long RER profiles,
which have few ribosomes. The cisterns are always filled with flocculent material.
Small RER profiles are seldom seen. Clusters of ribosomes are present. The cell
matrix of the majority of the cells is dark. There are no signs of the lipid droplets in
the basal compartment of the cell, which are so characteristic of later stages of
development. A modest number of membrane-bound electron-dense bodies are
present. The nucleus is somewhat irregular and shows no prominent nucleoli.
Between 28 and 35 days the organization of these cells does not markedly change.
(Fig. 5A,B.)
(4) The interstitium between the developing cords
Inside this interstitial area mesenchymal cells show an electron-dense matrix.
The cell membrane is smooth except for some small protrusions. There are two
cell types that differ from the cells in the peripheral layer. The first type can be
followed morphologically until they have developed into young Ley dig cells.
These young Leydig cells are intermingled between undifferentiated interstitial
cells. The RER profiles are small and transitional but no true SER can be
found. Mitochondria are elongated or ovoid. Some tubular cristae can be found.
Mitochondrial/RER complexes are frequently present. The nucleus is often ovoid
and shows a prominent nucleolus. Gap junctions or epitheloid cell connections are
sometimes seen. Inside the cell a well-developed Golgi apparatus can be seen. The
second type of mesenchymal cells shows some very long RER profiles in intimate
relationship with mitochondria. The cell matrix is rather dense. The cellular
organization does not fit the type mentioned before. (Figs 6B, 7.)
In the interstitium differentiated single or clustered Leydig cells are present in
patches from 30 days onwards. The clustered cells are epitheloidally connected
with some interdigitations and gap junctions. SER in tubular form is abundantly
present. Mitochondria have mixed lamellar and tubular cristae, but sometimes
only tubular cristae. The cisterns of the SER can be slightly swollen, giving the
cells a rather characteristic appearance. The ER is distributed all over the cell.
RER/mitochondrial complexes are present but are far less obvious than in most
other mesenchymal cells in this compartment. RER long profiles are rather sparse,
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Fig. 5. (A) 28 days p.c. Sertoli cells (s) and part of a germ cell (g). Note the filled
cisternae of the RER. x 15 000. (B) 28 days p.c. Sertoli cell (s) and parts of germ cell
(g). Note the intimate relationship between mitochondria and transitional RER with
well-filled cisternae. x27000. (Insert) 28 days p.c. Conspicuous spatial coupling of
RER and mitochondria. X35000. Bars, ljum.
but small profiles are common and display mostly the transitional form. Lipid
droplets can be observed occasionally. The nucleus is mostly ovoid and possesses
a well-developed nucleolus. The cells are mostly surrounded by a basal lamina.
(Figs 8, 9.)
Observations on the early foetal pig testis
Fig. 6. (A) 28 days p.c. Mesenchymal cell from the interstitial compartment be­
tween sex cord anlagen. Note the intimate RER mitochondrial relationship, x 18 000.
(B) 28 days p.c. Mesenchymal cell from the interstitial compartment between the sex
cord anlagen; from this form of mesenchymal cells in the interstitium a line can be
followed towards Leydig cells, x 18 000. Bars, ljum.
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C . J . A . H . V . VAN VORSTENBOSCH AND OTHERS
Fig. 7. 28 days p.c. This cell in the interstitium shows already some characteristics of a
young Leydig cell such as a gap junction; transitional endoplasmic reticulum, x 18 000.
Bar, ljum.
The main difference between Leydig cells of 30 days p.c. and of 35 days p.c. of
age is the increase in number, and an increase in branched tubular SER.
(Fig. 10A,B,C.)
Obvious morphological relationships cannot be determined between the Sertoli
cells and the Leydig cells. Both cell types show a totally different morphology and
intermediate forms are never observed. Indications of Sertoli cells migrating into
the interstitium were never observed.
DISCUSSION
Androgen production in the early foetal gonads has been reported in different
species such as sheep (Attal, 1969), rabbit (Lipsett & Tullner, 1965; Catt etal.
1975; George, Catt, Neaves & Wilson, 1978), rat (Noumura, Weisz & Lloyd,
1966), pig (Stewart & Raeside, 1976). In the pig, androgen production starts
before the appearance of Leydig cells (Moon, Hardy & Raeside, 1973; Ford et al.
1980; Pelliniemi, 1975).
Histochemical investigations of the distribution pattern of 3/3-HSD in the early
foetal pig gonads (crown-rump-length: 1-5-2 cm) showed a positive reaction in
the cord anlagen and a negative reaction in the interstitium for both DHA and
pregnenolone as substrates, with a maximum intensity of the reaction even before
Observations on the early foetal pig testis
273
the appearance of Leydig cells (Moon & Raeside, 1972). The results of the
histochemical study reported in this paper are only partly in agreement with those
of Moon & Raeside (1972). 3ß-HSD with DHA as a substrate showed a steady
increase in intensity, but with pregnenolone as a substrate the reaction peaks at 30
days p.c. Thus before the appearance of Leydig cells, the cord anläge and the
interstitium between this anläge shows a positive 3/3-HSD reaction. In the sections
the precise location of the positive cells could not be identified accurately, but it is
remarkable that the RER-mitochondrial complexes which are so evident in both
these compartments are hardly present in the cells of the peripheral mesenchymal
layer, while in this layer no 3/3-HSD activity was noticed.
Fig. 8. 30 days p.c. Young Leydig cells showing an epitheloid arrangement, mito­
chondria show tubular cristae and small vesicular SER. Note the round nucleus with its
prominent nucleolus, x 18 000. Bar, 1/an.
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C . J . A . H . V . VAN VORSTENBOSCH AND OTHERS
Fig. 9. 30 days p.c. A later stage: young Leydig cell clearly showing well developed
SER of the tubular form. The mitochondria contain tubular cristae and a well developed
Golgi apparatus can be seen. Circular gap junction (arrowhead), x 18000. Bar, 1 jum.
The RER-mitochondrial complexes are still present in both Leydig and Sertoli
cells at 35 days p.c. Thereafter these complexes vanish in Leydig and Sertoli cells,
coinciding with the decrease in testosterone production (van Vorstenbosch et al.
1982,1984a,b; Ford et al. 1980). It is tempting to suggest that these complexes are
the initial sites of testosterone production and that they maintain testosterone
secretory capabilities for some time after the appearance of fully differentiated
Leydig cells. This could explain the overall reaction in cords and interstitium in the
early stage.
Dierichs et al. (1973) and van Straaten et al (1978) used the NADH-diaphorase
reaction as a marker for late foetal and postnatal Leydig cells. We used this
reaction to localize Leydig cells at a much earlier age. We did not expect to find
Observations on the early foetal pig testis
275
Fig. 10. (A) 35 days p.c. Leydig cell: abundant SER and many mitochondria
with tubular cristae. x 15 000. (B) 35 days p.c. Leydig cell: tubular SER. x 18000.
(C) 35 daysp.c. Leydig cell: note the mitochondrial/RER complex, x 18 000. Bars, 1 [im.
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C . J . A . H . V . VAN VORSTENBOSCH AND OTHERS
any activity before the appearance of Ley dig cells. Surprisingly, a positive reaction
was already seen at 26 days.
From 28 days Leydig-like cells begin to appear in between the cords and after 30
days fully developed Leydig cells are present. Between 30 and 35 days the increase
in the number of Leydig cells coincides with the increasing plasma testosterone
concentration (Ford etal. 1980). There is also an increase in SER and a decrease
in transitional E R (van Vorstenbosch etal. 1982). We did not find convincing
evidence that Leydig cells derive from Sertoli cells as has been suggested by
Pelliniemi (1975). The Sertoli cells are already clearly differentiated at 28 days and
have a characteristic morphology. They are confined within a basal lamina. Cells
'breaking through' this lamina into the interstitium were not observed, making
Pelliniemi's suggestion improbable.
The clear difference in structure between the mesenchymal cells of the peri­
pheral layer and the cord anläge suggests an influence of this anläge on the
mesenchymal cells of the latter compartment. This influence might be responsible
for the initiation of Leydig cell differentiation.
The lipid-like droplets present in the cells of the mesothelial layer are quite
remarkable and apparently are characteristic for the period between 28 and 35
days, as they are not observed earlier (Pelliniemi, 1975) or later.
The authors own a debt of gratitude to the following persons for helpful discussions, skilful
technical assistance and personal involvement. Mr H. van Dijk, Mrs L. Michielsen, Mr H. H.
Otter, Mr I. Pel, Mr E. Spek, Mr O. v.d. Veen, Mrs A. J. E. P. Verbruggen, Professor
Dr W. A. de Voogd van der Straaten.
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{Accepted
4 February
1986)