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Early cytodifferentiation of human prostatic urethra and Leydig cells.

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Early Cytodifferentiation of Human Prostatic Urethra
and Leydig Cells
Department of Anatomy and Laboratory of Electron Microscopy, University of Turku,
SF-20520 Turku 52, Finland
The ultrastructure of the urethral epithelium and mesenchyme of the 6- to 9-week-old human embryos was studied in order to reveal
early morphological signs of prostatic development. The morphological changes
of the urethral wall were correlated with the cytodifferentiation of the Leydig
cells of the same embryos. Throughout the study the urethral epithelium had
two or more layers of cuboidal cells. The ultrastructure of the cells was primitive and they did not achieve characteristics of the secretory prostatic cell. The
surface cells had well developed apical junctions and slender cytoplasmic processes projecting into widened intercellular spaces appeared during the developmental period. The urethral mesenchyme showed the most salient changes. The
mesenchymal cells adjacent to the urethral epithelium differentiated in the
ninth week into fibroblast-like cells with an elongated shape and cytoplasmic
processes. Granular endoplasmic reticulum appeared in the cytoplasm and collagen fibers were seen in the intercellular space. Mesenchymal cell processes
contacting the continuous basal lamina under the epithelium were present. No
direct epithelio-mesenchymal cellular contacts could be seen. The differentiation of the mesenchyme before the epithelial outgrowths suggests that the mesenchyme has an essential role in the glandular development.
Electron microscopic study of the Leydig cells showed that the amount of
agranular endoplasmic reticulum increased considerably in the ninth week.
This agrees with earlier biochemical findings on the capability of Leydig cells
to produce androgens by this time. The temporal relationship between the
cytodifferentiation of Leydig cells and the urethral wall is consistent with the
idea that in the human, fetal androgens induce prostatic development.
The prostate gland is formed from the upper
part of the definitive urogenital sinus in the
region into which the mesonephric and paramesonephric ducts open. The glands constituting the prostate appear as solid branching
cords growing from the epithelium of the
urogenital sinus into the surrounding mesenchyme (Lowsley, '12, '30; Johnson, '20).Animal experiments have shown that fetal androgen-sensitivity of the target tissues is necessary for the prostatic development and that
the development is prevented by the administration of antiandrogens (Jirasek, '71; Jost et
al., '73; Neuman et al., '70). It is likely, that
androgens initiate the prostatic development
acting on the epithelium of the urogenital
sinus. Organ culture experiments with epithelio-mesenchymal recombinations suggest that
ANAT. REC. (1979) 194: 429-444.
the mesenchyme has also an essential role in
the prostatic development (Cunha, '73, '75).
Findings on sex reversed mice heterozygous
for testicular feminization even suggest that
the androgen responsiveness of underlying
mesenchymal cells may be more important
than that of epithelial cells (Ohno, '77; Drews
and Dieterich, '78).
The first light microscopic signs of prostatic
development in man appear in the tenth developmental week. Epithelial outgrowths are
seen in the condensed mesenchyme surrounding the urogenital sinus a t the level of the
mesonephric duct openings. Experimental evidence for the role of androgens in the developReceived July 24, '78 Accepted Feb 8, '79
' Present address Department of Anatomy, Harvard Medical
School, 25 Shattuck Street, Boston, Massachusetts 02115, U S A
ment of human prostate is limited. Human
fetal testis is known to produce androgens
from the eighth week onwards (Niemi et al.,
'67; Huhtaniemi et al., '70; Siiteri and Wilson,
'74) which is well before the onset of the
prostatic development. Furthermore, the male
accessory sex organs are lacking in the
patients with testicular feminization (French
et al., '65, '67; Simmer et al., '65; Southern et
al., '65).
The aim of the present study was t o examine
the ultrastructure of the prostatic development in man with special attention to any evidence of epithelio-mesenchymal interactions.
The changes in the epithelium and adjacent
mesenchyme of the urogenital sinus were reviewed and correlated with the cytodifferentiation of Leydig cells. No previous electron
microscopic study has been published on
human prostatic development.
stained with ethanolic uranyl acetate and
lead citrate, and examined with a Jeol Jem T 8
or Jeol Jem 100 C electron microscope.
The fine structure of the prostatic
Seventh and eighth weeks of
The 7-week-old male embryos have a colliculus seminalis in the cranial part of the
urethra and the mesonephric and paramesonephric ducts open into a narrow urethral
lumen. The urethral epithelium is composed of
two to three cell layers. The oval shaped cells
are 20 p m high. On the apical surface of the
epithelial cells there are a few microvilli and
occasional cilia (fig. 1).The zonula occludens,
-zonula adherens and macula adherens complex form the cell junctions a t the luminal
border (fig. 2). The plasma membranes of the
Male embryos a t the age of six to nine weeks luminal cells are straight and closely apposed
were obtained from legal abortions. The ages except basally where the intercellular spaces
were determined from foot-length (FL) rang- are slightly widened. The oval and smoothly
ing from 4 mm to 7 mm or crown-rump length outlined nucleus is located centrally or basally
(CR) ranging from 24 mm to 51 mm according and occupies one-half to two-thirds of the cell
to Streeter ('20) and the sex of the embryos volume. The nucleolus has a pars amorpha and
was identified by the histological examination a pars fibrosa. The chromatin is homogenously
of the gonads.
dispersed. Small and round mitochondria are
located predominantly in the apical part of
Specimen preparation
the cell and are frequently seen adjacent to
The embryos were obtained from the hos- the endoplasmic reticulum; some polysomes
pital in glutaraldehyde fixative. The gonads and Golgi complexes can also be observed in
and the prostatic part of the urethra of the the epithelial cells. The epithelium rests on
male embryos were immediately (within 1 closely attached basal lamina (fig. 3).
hour) removed under a stereomicroscope, cut
The mesenchyme is loose and composed of
transversally into 1-mm thick slices and fixed roundish primitive cells (fig. 4). There is
a t +4"C for three hours.
ample space between the epithelial basal lamThe effects of various fixatives on the epi- ina and the mesenchymal cells. The round,
thelial and mesenchymal cells were examined large nuclei of the mesenchymal cells have
first. The best results were obtained with 0.24 one or two nucleoli. Most of the heterochromol/l glutaraldehyde (Glutaraldehyde EM, matin is attached to the nuclear envelope. The
TAAB Laboratories) and 4.1 mmol/l CaC1, in cells near the basal lamina have small mito0.1 mol/l sodium cacodylate-HC1 buffer, pH chondria, polysomes and some granular endo7.4. After fixation the tissue samples were plasmic reticulum. No Golgi complexes are
washed overnight in 0.23 mol/l sucrose and seen. As a sign of incipient synthetic activity
postfixed in 39 mol/l osmium tetroxide in 0.2 some collagen fibers are found in the intermol/l s-collidin-HC1 buffer, pH 7.4. After de- cellular space (fig. 4 a t arrow heads). The cells
hydration in ethanols the specimens were em- located at a greater distance from the basal
bedded in Epon (Epon 812, Merck). Transverse lamina have fewer mitochondria and poly1-pm sections of the whole area were cut with somes. Myoblastic tissue has not yet developed
an LKB-Huxley ultramicrotome and stained but developing nerve cells are occasionally
with toluidine blue for light microscopy. Thin seen in the periphery of the urethral mesensections were cut with a diamond knife, chyme (fig. 5).
Ninth and the beginning of the tenth
week of development
At the end of the ninth week the epithelium
still contains several cell layers (figs. 8, 9). In
the luminal epithelial cells the most prominent change in comparison with the earlier
stage is the
cytoplasmic processes projecting into a widened intercellular space (figs. 9 and 10 at arrow heads).
At the luminal surface the amount Of microvilli has increased, and junctional complexes
in the
basal layers of the epithelium have a central
large, roundish nucleus and polysomes (fig.
11).An occasional Golgi complex is seen in the
cytoplasm. Oval or round mitochondria are
found in the apical parts of these cells and in
the basal cells near the basal lamina. Some
mitochondria are in close association with
granular endoplasmic reticulum.
The cellular density of the mesenchyme has
increased considerably. The previously homogenous mesenchyme has undergone differentiation into three concentric zones around
the epithelium. The inner zone cells have developed into elongated fibroblastic cells with
markedly increased granular endoplasmic reticulum. These changes indicate an increased
synthetic activity of the cells which is confirmed by the accumulation of collagen fibers
in the intercellular space (fig. 13). In the middle zone the mesenchymal cells are still primitive (fig. 14). Besides nerve cells myoblastic
cells are now seen in the outer zone and the
amount of intercellular collagen has increased.
In contrast to the earliest stage, where the
mesenchyme and epithelium were clearly separated, the interface of the epithelium and the
mesenchyme now shows morphological signs
of interaction. The basal lamina is folded but
maintains its continuity. In several places the
mesenchymal cells have cytoplasmic processes in close contact with the basal lamina
(fig. 12). However, no direct contacts between
the epithelial and mesenchymal cells are seen.
The differentiation of the interstitial
cells in the testis
In the seventh week of development early
stages of round to ovoid Leydig cells are seen
among t h e predominant undifferentiated
mesenchymal cells (fig. 6 at arrow heads). The
Leydig cells have a round nucleus with a very
prominent nucleolonema located near the nu-
clear envelope. Many mitochondria, developing agranular endoplasmic reticulum
and Some
dense granules can
be seen in the cytoplasm at this stage (fig. 7).
The testicular Leydig cells mature by the
end of the ninth week, They grow larger and
their number increases considerably (figs. 15
and 16 at
heads). They begin to gather
into small clusters. Their round nuclei exhibit
prominent nucleoli and their cytoplasm is
closely packed with agranular endoplasmic reticulum. Ultrastructurally the Leydig cells
now have the characteristics of steroid-secret.
ing cell (fig. 17).
The differentiation of the mesenchyme adjacent to the epithelium of the urogenital
sinus was the first ultrastructural sign of incipient prostatic development in man. The mesenchymal changes were found to correlate
with the Leydig cell differentiation. The epithelial cells of the urogenital sinus had a
primitive appearance and did not achieve the
morphological characteristics of secretory
prostatic cells during the developmental period covered in this study. The differentiation
of the mesenchyme before the epithelial outgrowth suggests that the mesenchyme has an
important role in the glandular development.
The mesenchymal cells close to the epithelium
differentiated into fibroblastic cells with elongated shape and cellular processes. Granular
endoplasmic reticulum appeared in their cytoplasm and collagen fibers were seen in the intercellular space. A continuous basal lamina
was seen under the epithelium and no epithelio-mesenchymal contacts were observed.
However, the mesenchymal cell processes did
contact the basal lamina and these processes
may be important for the proliferative outgrowth of epithelial cells. Similar processes
have also been in salivary glands developing
in organ culture (Cutler, '77).
The surface epithelial cells had well-developed apical junctions, apparently for sealing
the luminal surface. This agrees with the earlier findings of Hoyes et al. ('72) on the urinary tract. The cytoplasmic processes into intercellular maces further improve cellular
communicatibn and contacts. -They may be
important for coordinated function of epithelial cells in the prostatic development.
Several lines of evidence suggest that androgens are necessary for the development
of the male sex accessory organs (Jost, '73;
Jirasek, '71; Berthezene et al., '76). In several
animal species and man, fetal Leydig cells
have been shown to produce androgens
(Acevedo e t al., '63; Lipsett and Tullner, '65;
Noumura et al., '66; Bloch, '67; Attal, '68;
Huhtaniemi et al., '70; Jirasek, '71; Picon, '76;
Veyssiere et al., '76). Siiteri and Wilson ('74)
have shown that human fetal testis makes
testosterone from appropriate precursors from
the eight week onwards in vitro. The maximal
rates are achieved in the twelfth or thirteenth
week (Siiteri and Wilson, '74). In addition, enzymes of steroid biosynthesis have been demonstrated histochemically in the testis of 8.3week-old human embryos (Niemi et al., '67).
Moreover, the development of the male organs
is prevented or seriously disturbed by the
administration of antiandrogens, such as
cyproterone acetate (Neuman et al., '70). In
testicular feminization syndrome, the male
accessory sex organs do not develop because of
the androgen-insensitivity of the target organs (French et al., '65, '67; Southern et
al., '65).
The morphological differentiation of human
fetal Leydig cells can be divided into the following four phases (Pelliniemi and Niemi,
'69) :
(1) predifferentiation phase (at the age of 5-8 weeks!
(2) differentiation phase (8-14 weeks)
(3) maturity phase (14-18 weeks)
(4) involution phase (18-40 weeks)
In the predifferentiation phase Leydig cells
appear in the developing gonads. In the fourteenth week the number of Leydig cells
reaches its maximum, with their relative
volume representing about half of the whole
testis. From the eighteenth week onwards
their number progressively decreases and
they practically disappear until birth. Electron microscopic studies on the fetal Leydig
cells showed that the amount of agranular endoplasmic reticulum increases considerably in
the ninth developmental week. This implies
that there is a functional maturation of the
cells, because the amount of agranular endoplasmic reticulum is known to correlate with
the activity of steroid synthesis (Black, '67;
Narbaitz and Adler, '67). These morphological
findings are consistent with the earlier histochemical and biochemical results (Niemi e t
al., '67; Siiteri and Wilson, '74). The temporal
relationship between the cytodifferentiation
of Leydig cells and mesenchymal cells of the
urethral wall is consistent with the hypothesis that fetal androgens induce the prostatic
development in man.
Differentiation of prostate results from precisely timed epithelio-mesenchymal interactions in rodents. The use of organ culture techniques and of epithelio-mesenchymal recombinations have revealed that androgens act on
the epithelium. However, there is no doubt
that mesenchyme plays an important role in
the morphogenesis of the glandular epithelium, but the nature of these mesenchymal
factors is not known (Cunha, '73, '75; Ohno,
'77; Drews and Dieterich, '78). On the basis of
the present morphological observations we
suggest that the early differentiation of the
mesenchymal cells adjacent to the basal lamina could be induced by testicular androgens
and is a prerequisite for the later differentiation of human prostatic epithelium.
Thanks are due to Mrs. Sirpa From, and
Mrs. Marita Soderstrom, and Mrs. Raija Andersen, and Mr. Mauno Lehtimaki for technical help and Mrs. Iris Dunder for typing the
manuscript. Special thanks to the staff of the
Turku City Hospital and Professor Kalevi
Koski for providing the embryos. Dr. Martin
Dym and Dr. Anita Hoffer are gratefully acknowledged for their valuable comments on
the manuscript.
This work was supported by the Laake Research Foundation and The Finnish Cultural
Foundation (Suomen Kulttuurirahasto).
Acevedo, H. F., L. R. Axelrod, E. Ishikawa and F. Takaki
1963 Studies in fetal metabolism. 11. Metabolism of progesterone-4-C" and pregnenolone-7a-H3 in human fetal
testes. J. Clin. Endocrinol., 23: 885-890.
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Berthezene, F., M. G. Forest, J. A. Grimaud, B. Claustrat
and R. Mornex 1976 Leydig cell agenesis: a cause of male
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Black, V. H. 1967 Differentiation of interstitial cells in
fetal guinea pig testes. Anat. Rec., 157: 214-215.
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1975 Age-dependent loss of sensitivity of female
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1 The ultrastructure of t h e apical part of the urethral epithelium of a 6-week-old embryo. The oval nuclei (N) are basally located in t h e cells and t h e apical part contains
mitochondria (M), polysomes (P), granular endoplasmic reticulum (GR) and Golgi
complex (arrow). The cellular outlines are straight and a few microvilli and solitary
cilia (C) are seen at the apical surface. x 5,000.
2 A higher magnification of the apical part of the epithelial cells of the same embryo.
The arrow points at a well developed cell junction. P, polysomes; M, mitochondrion;
GR, granular endoplasmic reticulum; GR
M, apposition of granular endoplasmic
reticulum and mitochondria. x 16,000.
3 The basal epithelial cells and the basal lamina (BL) of the same embryo. N, nucleus;
NU, nucleolus. x 5,000.
P. Kellokumpu-Lehtinen, R. Santti and L J. Pelliniemi
4 A lower power micrograph of the basal epithelium (E) and t h e adjacent mesenchyme (a 6-week-old embryo). BL, basal lamina; MC, mesenchymal cell; N, nucleus;
arrow, collagen fibers. x 4,000.
5 The peripheral mesenchyme (a 6-week-old embryo). Developing nervous tissue (NT)
and round or oval mesenchymal cells. N, nucleus. x 5,000.
6 A light micrograph of the testis in the seventh week. Well-developed testicular
cords (TC) are seen. The interstitium contains mostly primitive mesenchymal cells
but a few developing Leydig cells (arrow) can also be seen. x 800.
7 A primitive Leydig cell (a 6-week-old embryo) lying close to the basal lamina (BL) of
a testicular cord (TO. MC, mesenchymal cell; N, nucleus. x 5,000.
P Kellokumpu-Lehtinen, R Santti and L J Pelliniemi
8 A light micrograph of the urethral epithelium and mesenchyme (ME)of a 9-weekold embryo. This transverse section shows a colliculus seminalis and mesonephric
ducts (MD), in fusion with the urethral walls. UE, urethral epithelium; PD,
paramesonephric duct. x 150.
9 Ultrastructure of t h e epithelium of a 9-week-old fetus. The superficial cells have a
round or an oval nucleus (N). The arrow points to cellular processes in widened intercellular spaces. HC, heterochromatin; NU, nucleolus; BL, basal lamina.
X 5,000.
10 A higher magnification of the apical part of the epithelium of a 9-week-old fetus.
G, Golgi complex; M, mitochondrion; N, nucleus; MV, microvilli, arrow, cellular
processes in a widened intercellular space. x 16,500.
11 A typical epithelial cell of t h e middle layer (a 9-week-old fetus). NU, nucleolus;
GR, granular endoplasmic reticulum. x 10,000.
P . Kellokumpu-Lehtinen, R. Santti and L. J. Pelliniemi
12 Epithelio-mesenchymal interface (a 9-week-old fetus). Cytoplasmic processes
(arrow) of mesenchymal cells are in contact with t h e epithelial basal lamina (BL)
and opposed by similar processes of epithelial cells (E). x 19,500.
13 Elongated mesenchymal cells (a 9-week-old fetus) with abundant granular endoplasmic reticulum (GR) and more intercellular fibers (CF), N, nucleus. x 5,000.
14 Round mesenchymal cells in the middle layer (a 9-week-old fetus); only a few collagen fibers (CF) are present, N, nucleus. x 5,000.
P Kellokumpu-Lehtinen, R Santti and L. J Pelliniemi
44 1
15 Part of a testis of a 8-week-old fetus. TC, testicular cord; MC, mesenchymal cell;
arrow, Leydig cell. x 800.
16 Part of a testis of a 9-week-old fetus. The amount of Leydig cells (arrow) has
markedly increased. TC, testicular cord. x 800.
17 Ultrastructure of a Leydig cell of a 9-week-old fetus. The amount of tubular
agranular endoplasmic reticulum (AR) has markedly increased. N, nucleus; M, mitochondrion; G , electron dense granule. x 5,000.
P. Kellokumpu.Lehtinen, R. Santti and L. J. Pelliniemi
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cytodifferentiation, leydig, prostatita, urethral, human, early, cells
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