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Cytogenesis of immunoreactive gonadotropic cells in the fetal rat pituitary at light and electron microscope levels
DEVELOPMENTAL
58, 148-163
BIOLOGY
(1977)
Cytogenesis of Immunoreactive Gonadotropic Ceils in the Fetal Rat Pituitary at Light and Electron Microscope Levels C. TOUGARD, Groupe
de Neuroendocrinologie
Received
R. PICART,
AND A. TIXIER-VIDAL
CeEEulaire, Laboratoire de Physiologic Cetlutaire, Marcelin Berthelot, 75231 PARIS 05, France May
13, 3976;
accepted
in revised
form
February
Collige
de France,
11 Place
14,1977
The cytogenesis of immunoreactive gonadotropic cells in the fetal rat pituitary was analyzed at the light and electron microscope levels using the indirect peroxidase-labeled antibody method and antisera against ovine FSH (A-oFSH) and ovine LH (A-oLH), and its two subunits (A-oLHp and A-oLHa). At the light microscope level, the first immunoreactive cells were detected on the seventeenth day postcopulation (dpc) with A-oLHp. Cells immunochemically stained with A-oLHa and A-oLH were generally observed 24 hr later. At the electron microscope level, the first immunoreactive cells were detected on 16 dpc with A-oLH@. These first immunoreactive cells were small, but already displayed some small secretory granules (80-120 nm). On 17 dpc, gonadotropic cells were stained with A-oLH@ as well as with A-oLHar and AoFSH. On 18 dpc, the number and the size of immunoreactive cells began to increase. By 19 dpc, they displayed an important development of ergastoplasmic cisternae and Golgi zone. At term, nevertheless, the ultrastructural features of fetal gonadotropic cells still differed from those of adult gonadotropic cells. INTRODUCTION
pouy and Magre, 1973; Svalander, 1974). Immunocytochemical techniques which have been already successfully applied to adult pituitaries may be used to describe the first immunoreactive cells and to follow their ultrastructural evolution during fetal development. Such immunochemical studies have been performed at the light microscope level in the fetal human pituitary (Dubois and Dubois, 1974; Bugnon et al., 1974; Baker and Jaffe, 1975; Dupouy, 1975). In the fetal rat pituitary, adrenocorticotropic (ACTH) and prolactin (PRL) cells were found on Day 16, thyrotropic (TSH) cells on Day 17, luteinizing hormone (LH)-producing cells on Day 18, and growth hormone (GH)-producing cells on Day 19 (Setalo and Nakane, 1972). According to Dupouy (1975) who used an immunofluorescence method, ACTH cells were found on Day 18 only. So far, no report has been published on the ultrastructural features of fetal immunoreactive cells. Our purpose in the present work was: (i) to detect the first immunoreactive gonado-
The morphological differentiation of the fetal rat pituitary has been extensively studied at the light and electron microscope levels (Jost and Tavernier, 1956; Yoshimura et al., 1970; Svalander, 19741, but, at early stages of development, the identification of the cell types remained difficult. With the light microscope, the histochemical techniques which permit differentiating cell types in the adult pituitary were unsuccessful when applied to the fetal pituitary because of the low stainability of the cells. The first granulated cells which appear under the electron microscope do not display the ultrastructural features of mature adult cells and cannot be identified on such bases. In the fetal rat pituitary, the first granulated cells appear between the sixteenth and seventeenth days of gestation, but morphological analogy with adult pituitary cell types does not occur before the eighteenth and nineteenth days (Yoshimura et al., 1970; Du148 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
TOUGARD,
PICART,
AND TIXIER-VIDAL
tropic cells using four antibodies that we have already applied to adult rat pituitaries (Tougard et aE., 19’73; Tixier-Vidal et al., 197.51, antisera against ovine LH and its two subunits (A-oLH, A-oLH& AoLHa), and antiserum against ovine FSH (A-oFSH); (ii) to describe the ultrastructure of such cells and to follow their evolution during fetal development; and (iii> to compare their ultrastructural features to those of adult mature immunoreactive gonadotropic cells previously described (Tougard et al., 1973; Tixier-Vidal et al., 1975; Moriarty, 1975, 1976). MATERIALS
AND
METHODS
Cytogenesis
of Gonadotropic
Cells
149
pm-thick paraffin sections were treated as previously described by using the indirect peroxidase-labeled antibody method (Tougard et al., 1973; Tixier-Vidal et al., 1975). Serial sections were respectively stained with the four antisera. Control sections were subjected to the following treatments: 3, 3’-diaminobenzidine tetrahydrochloride (DAB; Sigma) reaction without any incubation with antiserum; specific antiserum, then DAB reaction; normal guinea pig serum (NGPS) followed by DAB reaction; anti-guinea pig immunoglobulin bound to peroxidase (A-IgG-per) , then DAB reaction; NGPS followed by AIgG-per, then DAB reaction; specific antiserum, then A-IgG-per and, finally, DAB reaction in medium without H,O,.
Animals. Sprague-Dawley female and male rats were mated for one night. Fertilization was verified by the presence of For the electron microscope study. The sperm in vaginal smears on the following pituitaries from stage 16 to 21 dpc were morning, and that day was designated as fixed in picric acid formaldehyde (PAF) (Zamboni and De Martino, 1967) for 6 hr at the first day postcopulation (1 dpc). Four series of fetuses were studied at different 4°C and then were washed overnight in 0.1 times of the year. Fetal pituitaries were M phosphate buffer, pH 7.3, with 10% sucollected from stage 13 dpc to 21 dpc. Pregcrose. Thick sections (75, 100, or 150 pm) nant rats were decapitated without anes- were cut without freezing with a Sorvall thesia, and the fetuses were removed and TC2 sectioner. These sections as well as killed by decapitation. The base of the half-pituitaries were prepared for immucranium of each fetus was opened under a nochemical staining as previously dedrop of fixative. After a 30-min in situ scribed for adult pituitaries (Tougard et fixation, the pituitaries from stage 16 to 21 al., 1973). Control treatments used were dpc were dissected in fresh fixative. For the same as for the light microscope study. the youngest fetuses, from stage 13 to 15 Ultrathin sections were examined under the electron microscope without further dpc, the whole basal region surrounding the pituitary was removed and immersed staining. The sections were picked up on in fixative. single slot grids coated with parlodion. For the light microscope study. FortyAntisera. Antisera (AS) were the same eight pituitaries were fixed in Herlant as those previously used for adult rat pitui(1960) fixative for 24-48 hr. They were taries (Tougard et al., 1973; Tixier-Vidal et then dehydrated and embedded in paraf- al., 1975). They were obtained by B. Kerfin. In the first series, pituitaries were delhue (Laboratoire des Hormones Polytaken every 2 days from stage 13 to 21 dpc; peptidiques, CNRS) by immunizing in the second and third series, each day guinea pigs with ovine FSH and ovine LH from stage 18 to 21 dpc and, in the fourth and its two subunits (LHP and LHa) preseries, each day from stage 16 to 21 dpc. pared in the same laboratory. The racharacteristics as well For the last series, the sex of each fetus dioimmunological was determined, and males and females as the immunocytochemical specificity of were treated separately. these antisera were described in detail For immunochemical staining, 3- to 5- elsewhere (Kerdelhue et al., 1971, 1972;
150
DEVELOPMENTALBIOLOGY
Tougard et al ., 1973; Tixier-Vidal et al ., 1975). For the light microscope study, the sections were generally incubated for 1.5 hr in one of the four antisera diluted l/50 to l/100. In one experiment, the dilution of each antiserum was correlated with its radioimmunological titer (dilution for each antisera: A-FSH, l/10; A-LH, l/20; A-LHP, l/1000; A-LHa, l/55), and, in that case, the incubation time was 6 or 24 hr. For the electron microscope study, sections were incubated for 3 hr, and three antisera (AFSH, A-LHa, A-LHP) were diluted l/20. Sheep antibodies against guinea pig yglobulin were purified and linked to horseradish peroxidase by Dr. B. Kerdelhue according to Avrameas (1969). RESULTS
Light
Microscope
Study
At the light microscope level, the immunoreactive cells were easily identifiable. In control sections, the cells were uncolored except for the pseudoperoxidase reaction of red blood cells. Immunoreactive cells were never found before stages 17 or 19 dpc (Table 1). In the four series, the first immunoreactive cells were observed after staining with anti-LHP (Fig. 1). They were localized at the ventral and lateral parts of the fetal pars distalis. At later ages, immunoreactive cells became more numerous and more widely distributed in the pars distalis (Figs. 2 and 3). They were often associated in small clusters of three or four cells. Cells immunoreactive with A-oLH and A-oLHa! were observed only 24 hr after the first A-oLH P-positive cells, that is, respectively between stages 18 and 20 dpc, depending on the series. The same 24hr delay was also observed when the AoLH/3 was diluted more (lo- to 20-fold) in accordance with its radioimmunological titer which was higher than those of A-oLH and A-oLHa (see Methods and Materials). Cells immunoreactive with A-oFSH appeared between stages 20 and 21 dpc of fetal life. Even with a high dilution of A-oLH&
VOLUME 58, 1977
the number of cells immunoreactive for this antiserum was always higher than those for A-oLH and A-oLHa: approximately 15-25% instead of 2-10%. With AoFSH, only a few positive cells were found in all series (see Table 1). Whatever the antiserum, immunoreactive cells displayed a similar ovoid shape, although some were elongated in older fetuses. Surprisingly, some cells of the 19dpc pars intermedia were stained by AoLH/3 (Fig. 5) and were still present on 21 dpc. They were localized in the zone adjacent to the pars distalis or at the periphery of the pituitary cleft. Electron
Microscope
Study
In spite of the small size of the fetal pituitaries, immunoreactive cells were found only in incubated slices and never in incubated half-pituitaries. There are several possible explanations for this difference. The presence of a connective tissue envelope around the pituitary opposes the penetration of the antibody. The gonadoi tropic cells are infrequent in the midsagittal zone of the gland which was directly exposed to the antibody in half-pituitaries. The slight dissociation of the cells which occurred within incubated slices may have favored the penetration of the antisera and other reagents. As previously observed for adult pituitary (Tougard et al., 19731, the periphery of the slices was slightly damaged, but the positive cells were found more centrally (Fig. 4) and could not be confused with damaged cells of the border of the section. Positive cells were found only after treatment with specific antisera; they were recognized by the increased electron density of their secretory granules and ground cytoplasm (Fig. 6). In contrast, after treatment with normal guinea pig serum, the glandular cells displayed a low electron density in their organelles including secretory granules; only the border of the slice displayed an unspecific staining. The poor quality of cell preservation after PAF fixation has been already ob-
TOUCARD,
PICART,
AND TIXIER-VIDAL
Cytogenesis
TABLE DETECTION, Series
I
A-oLH A-oLHP A-oLHa A-oFSH A-oLH A-oLHP A-oLHo A-oFSH A-oLH A-oLHP A-oLHa
III
Days
Antiserum 13-15 -b -
16 nd nd nd nd nd nd nd nd nd nd nd nd
151
Cells
1
WITH THREE ANTISERA, OF IMMUNOREACTIVE G~NADOTROPIC CELIS DURING THE LAST DAYS OF GESTATION: LIGHT MICROSCOPE
A-0FSH
II
of Gonadotropic
17 c -
IN THE FETAL RAT PITUITARY STUDY~
postcopulation 18 nd nd nd nd + ++ +
19 k ++ + -
20
21
nd nd nd nd -
+ ++ +++ ++ + + + t +
+ + +++
ttt
t
++
-
-
-
+ + -
+
IV Male
Female
A-oFSH A-oLH A-oLHP A-oLHa A-oFSH A-oLH A-oLHP A-oLHa
nd nd nd nd nd nd nd nd
-
-
a For each stage two fetal pituitaries were taken from two different 5 pm) were separately incubated with the four antisera. b Significance of symbols: (-) no staining; (k) 1 or 2 immunoreactive strongly stained; (+ t) 5 to 20 immunoreactive cells; (t t +) more done.
served for the adult rat pituitary (Tougard et al., 1973). Considering the well-known difficulties involved in fixation of fetal pituitary, the results obtained here can be considered as optimal for the fixative that we used. This is evidenced by the good preservation of the mitochondria and nuclei. Only glutaraldehyde or osmium tetroxide would permit a better fixation of the cell membrane systems. Unfortunately, in using the same technical approach previously for the adult rat pituitary, we found that gonadotropic cells do not retain their immunoreactivity after glutaraldehyde fixation (unpublished results). Immunoreactive cells were already present, although very few in number, at the first stage that we examined, 16 dpc, but only after staining with A-oLH@ These cells displayed a large nucleus, a small
-
than
+t+
-
t
+ -
mothers.
Serial
+
t+
paraffin
cells slightly stained; 20 immunoreactive
+ t+
?I sections
(3 or
( +) 1 to 5 cells cells; nd, not
cytoplasmic area with numerous mitochondria, and some flattened ergastoplasmic cisternae (Fig. 7). Although the majority of pituitary cells were devoid of secretory granules at this stage, the first immunoreactive cells always contained a few small secretory granules (80-120 nm). The immunochemical staining was restricted to these small granules. On 17 dpc, immunoreactive cells were observed after staining with A-oLH/3 as well as with AoLHa and A-oFSH (Table 2). They all displayed the same ultrastructural features as immunoreactive cells on 16 dpc, but the reaction product was present on the ground cytoplasm in addition to the secretory granules. On 18 dpc, cells immunoreactive with the three antisera became more numerous. They generally displayed an ovoid shape, but some were larger than at the previous stages. The reaction prod-
FIGS. l-4. Staining with A-oLHP of fetal rat pituitaries taken at progressive stages of gestation. FIG. 1. Paraffin section (3 pm) of a 17-dpc fetal pituitary. Some cells (single arrow) are slightly stained. The red blood cells (double arrow) display a pseudoperoxidase reaction. 330 x . FIG. 2. Paraffin section (3 pm) of a 1%dpc fetal pituitary. Immunoreactive cells are more strongly stained and more numerous than those in Fig. 1. They are distributed in all the pars distalis. 330 x . FIG. 3. Paraffin section (3 pm) of a 20-dpc fetal pituitary. Immunoreactive cells are larger and darker than those in Figs. 1 and 2. They are very numerous and associated in small clusters. 330 x . FIG. 4. Araldite section (1 pm) from a slice of a IS-dpc fetal pituitary treated with A-oLH@ before embedding. Immunoreactive cells are easily identifiable in a large peripheral zone of the section, but they are never found in the core of the slice. 330 x . 152
TOUGARD,
PICART,
AND
TIXIER-VIDAL
Cytogenesis
of Gonadotropic
Cells
153
FIG. 5. Serial paraffin sections of a 19-dpc rat fetal pituitary. (a) Section treated with A-oLH/3 diluted l:lOOf! for 24 hr. Immunoreactive cells (single arrow) are seen in the pars distalis (PD). A few cells (double arrow) are also stained in the pars intermedia (PI). PC: pituitary cleft. 330 X. (b) Next section treated with A-oLHa diluted 1:55 for 24 hr. No immunoreactive cells can be seen with this antisera, either in the pars distalis or in the pars intermedia. 330 x
uct was always localized on secretory granules and on the ground cytoplasm (Fig. 8). A few cells displayed a variability in the size of their secretory granules (from 80 to 150 nm), with very few larger ones (250 nm). The ergastoplasmic cisternae were scarce and generally flattened. On 19 dpc, the ultrastructural organization of the immunoreactive cells varied depending on the respective antiserum. Cells which were stained by the three antisera displayed a rounded or ovoid shape and an ultrastructural organization identical to that of cells described at 18 dpc, that is, a few ergastoplasmic cisternae and variability in the diameter of their secretory granules (80-250 nm). Cells which
were stained only by A-oLHa and A-oFSH but not by A-oLH/3 displayed an elongated or polygonal shape. They were characterized by very small secretory granules (50120 nm) generally lining the plasma membrane and by vesicular ergastoplasmic cisternae (Fig. 9). At this stage, some cells immunoreactive with A-oLH@ were also found in the pars inter-media at the periphery of the pituitary cleft. On 20 and 21 dpc, almost all immunoreactive cells displayed an ovoid shape with a large cytoplasmic area, although some small cells with a high nucleus/cytoplasm ratio still remained. The extension of the cytoplasmic area corresponds to an important development of ergastoplasmic
154
DEVELOPMENTAL
FIG. 6. Rat fetal pituitary (21 dpc) treated ble by the increased electron density of their other cells are negative. 7000 X .
BIOLOGY
VOLUME
58, 1977
with A-OLHW Two immunoreactive secretory granules and cytoplasm.
cells are easily identifiaThe nucleus (N) and the
TOUGARD,
PICART,
AND TIXIER-VIDAL
Cytogenesis
of Gonadotropic
Cells
FIG. 7. Rat fetal pituitary (16 dpc) treated with A-oLHP. Two immunoreactive gonadotropic cells (G) display a large nucleus and a small cytoplasmic area. The reaction product is localized on the secretory granules. A few linear ergastoplasmic cisternae are underlined with reaction product (arrow). The nucleus and the numerous mitochondria Cm) are negative. 9000 x
cisternae which are flattened or slightly dilated (Figs. 10 and 11). At the same time the Golgi was enlarged. These moditkations mainly concerned the cells which were immunoreactive with the three antisera, including A-oLHP. These cells were
far more numerous than on 19 dpc and of their secretory granules displayed the same size range (from 80 to 150 nm), again with a few larger ones (300 nm) in some cells. In all these cells, the reaction product
156
DEVELOPMENTAL
BIOLOGY TABLE
DETECTION, Series
WITH THREE ANTISERA, OF IMMUNOREACTIVE DURING THE LAST DAYS OF GESTATION: Antiserum
VOLUME
58, 1977
2 GONAWTROPIC CELLS IN THE FETAL RAT PITUITARY ELECTRON MICROSCOPE STUDY” Days
postcopulation
16
17
18
19
20
21
III
A-oFSH A-oLHP A-oLHa
nd* nd nd
nd nd nd
+ +++ k
nd nd nd
+++ +++ +++
+++ +++ +++
IV
A-oFSH A-oLHP A-oLHo
+ -
+ + f
nd nd nd
+++ +++ +++
nd nd nd
nd nd nd
4 At each stage, a pool of 10 fetal pituitaries were with each of the three antisera. Immunoreactive cells sections. b Significance of symbols: (-1 no staining; (2) one to five cells strongly stained; (+ + +) more than five
collected, and TC2 sections were detected on l-pm-thick or two immunoreactive immunoreactive cells;
were incubated separately sections or on ultrathin
cells slightly nd, not done.
FIG. 8. Bat fetal pituitary (18 dpc) treated with A-oFSH. This immunoreactive secretory granules (80-200 nm in diameter). The reaction product is found on both ground cytoplasm. The ergastoplasmic cisternae are very scarce. 14,000 x
stained,
(+)
one
cell contains a few secretory granules and
TOUCARD,
PICART,
AND TIXIER-VIDAL
Cytogenesis
of Gonadotropic
Cells
FIG. 9. Rat fetal pituitary treated with A-oLHa. This elongated immunoreactive cell contains a few very small secretory granules (50-120 nm), mainly located at the periphery of the cell, and vesicular ergastoplasmic cisternae (arrow). 11,500 X.
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DEVELOPMENTAL
BIOLOGY
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FIG. 10. Rat fetal pituitary (21 dpc) treated with A-oLH& This immunoreactive elongated and parallel ergastoplasmic cisternae and small secretory granules product is found on secretory granules and on some cytoplasmic areas (arrow). plasmic cisternae is always negative. 17,000 X .
cell contains numerous (80-150 nm). The reaction The content of the ergasto-
FIG. 11. Rat fetal pituitary (21 dpc) treated with A-oLHp. This immunoreactive cell displays an ovoid shape with a large cytoplasmic area. It contains numerous secretory granules (80-150 nm) and flattened (single arrow) or dilated (double arrow) ergastoplasmic cisternae. The reaction product is found on the secretory granules and on the ground cytoplasm. 15,000 x 159
160
DEVEJAJPMENTAL
BIOLOGY
was localized on secretory granules and on the ground cytoplasm. The content of the ergastoplasmic cisternae was always negative (Figs. 10 and 11). DISCUSSION
Specificity
of Antisera
The specificity of the immunochemical staining has been previously tested by radioimmunological study (Kerdelhue et al., 1971, 1972), as well as by analysis of the effects on the staining of previous adsorption with several other pituitary hormones (Tougard et al., 1973; Tixier-Vidal et al., 1975). On the basis of such studies, it can be assumed that staining with A-oLH and/ or A-oLH/3 identifies rat LH molecules but not FSH or TSH. In contrast, staining with A-oLHa and/or A-oFSH does not discriminate LH from FSH and TSH. Chronology of the Immunocytochemical Differentiation of Gonadotropic Cells during Fetal Life
At the light microscope level, the first immunoreactive cells observed were always those stained with A-oLH@ Cells immunoreactive with A-oLH and A-OLHCX only appeared 24 hr later. The same result was obtained when A-oLHP was diluted 20-fold more than the other antisera. These differences cannot result from individual variations between fetuses, since the four antisera were respectively applied to serial sections for each fetal pituitary examined. Nevertheless, we cannot conclude from this result that LHP is the first subunit which is synthesized in the fetal since we used antisera rat pituitary, against ovine hormone to localize rat gonadotropic hormone. Indeed, Vaitukaitis et al. (1972) have shown that the interspeties cross-reactivity resides in the p subunits of pituitary glycoprotein hormones, whereas the zoological specificity resides in the (Y subunits. This may explain our detection of the /? subunit before the a subunit or the native hormone. This can also explain why, at later stages, we found
VOLUME
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a larger number of immunoreactive cells with A-oLH/3 than with the other antisera. The more precocious detection of AoLH/3-positive cells might be also related to technical conditions which affected the antigenicity against A-oLHa and A-oFSH more than that against A-oLHp. Consistent with this possibility is the fact that, in the fourth experimental series (Tables 1 and 2) in which fetal pituitaries were taken from the same mothers for both light (LM) and electron (EM) microscope studies, positive cells were detected for the three antisera very much sooner with EM than with LM techniques. This could suggest that fixation and/or embedding alter, to varying degrees, the antigenicity of the hormones. This is particularly true for FSH which seems selectively denatured or extracted after histological treatments. A similar observation was already discussed for adult rat pituitary cells (Tixier-Vidal et al., 1975). The chronology of the appearance of immunochemical staining with the four antisera also varied from one series to another (Table 1). This cannot be attributed to differences in the quality of fixation or to individual variations between the mothers, since we used fetuses of several mothers for each series. At present we have no explanation for such variations. When male and female pituitaries were collected separately (fourth experimental series), the positive cells in the female were found 24 hr later than those in the male. The only report we found on the differentiation of immunoreactive gonadotropic cells in the fetal rat pituitary mentioned the appearance of LH-positive cells on Day 18 (Setalo and Nakane, 1972), using picric acid formaldehyde for fixation and polyethylene glycol for embedding. The human fetal pituitary has been more extensively studied with immunocytochemical techniques (Dubois and Dubois, 1974; Dubois et al., 1975; Bugnon et al., 1974; Pasteels et al., 1974; Baker and Jaffe, 1975). All these
TOUGARD,
PICART,
AND
TIXIER-VIDAL
authors found cells immunoreactive for an anti-HCG (Bugnon et al., 1974; Baker and Jaffe, 1975) or for an anti-ovine LH (Dubois and Dubois, 1974; Dubois et al., 1975; Pasteels et al., 1974) at approximately 10 weeks. Cells immunoreactive for an antiporcine LHp were detected later, at 23 weeks (Baker and Jaffe, 1975) or between 14 and 20 weeks, depending on the sex (Dubois and Dubois, 1974; Dubois et al., 1975). It is of interest to compare these cytological results with the radioimmunoassay data of Hagen and McNeilly (1975) who found (Ysubunit and native LH before p subunit during development of the fetal human pituitary. Whatever the technique (LM or EM), the presence of immunoreactive cells with A-oLHP in the pars intermedia is a surprising fact. We have previously verified (Tougard et al., 1973) that our A-oLHp did not cross-react with ACTH. In adult rat pituitary, moreover, the same antisera did not stain any cells in the pars intermedia. Ultrastructural Features of the First Immunoreactive CeZZs
The ultrastructure of the first detected immunoreactive cells, on 16 and 17 dpc, was the same with the three AS. They were small and rounded with a high nucleus/cytoplasm ratio and a few small secretory granules. They look like the socalled ambiguous cells which are the first granulated cells to appear on Day 16, as described by several authors using conventional electron microscope techniques (Yoshimura et al., 1970; Svalander, 1974). Since our observations at the EM level started on Day 16, we do not know whether immunoreactive gonadotropic cells exist at earlier stages. Nevertheless, this seems most doubtful because of the extreme paucity and primitive ultrastructural organization of the positive cells on Day 16. Their ultrastructure does not differ from that of the neighboring granular cells. Therefore, it is probable that the immunochemical differentiation precedes the morphological
Cytogenesis of Gonadotropic
Cells
161
diversification of the anterior pituitary cell types. At that stage of development, the most numerous pituitary cells are devoid of secretory granules. Such agranular cells were never found immunoreactive for any of the three antisera. It seems that, at least for gonadotropic cells, the immunochemical differentiation coincides with the achievement of the secretory pathway with the formation of secretory granules. Ultrastructural Evolution oflmmunoreactive Gonadotropic Cells during Fetal Life
On 18 dpc, the immunoreactive cells begin to develop in both their number and their size. The cell growth corresponds mainly to development of the ergastoplasmic cisternae and the Golgi zone. At the same time, the secretory granules show a slight increase in their number and diameter. These changes take place at an increasing rate from 18 to 21 dpc. In addition, a slight diversification in the ultrastructural organization of the immunoreactive cells appears on 19 dpc, depending on the antisera. The cells which are stained with A-oLH/3 and, therefore, can be considered as LH cells contain small secretory granules, the diameter of which varies from 80 to 150 nm, with a few larger ones (250-300 nm). Their ergastoplasmic cisternae are generally flattened or slightly dilated. The other cell type is stained by A-oLHa and A-oFSH only. It differs from the latter by the very small size of the secretory granules (50-120 nm) and the vesicular aspect of their ergastoplasmic cisternae. On the basis of their immunochemical staining, these cells may contain either FSH or TSH. They become very scarce on 21 dpc. At these stages, the LH cells become more numerous, but do not display further diversification in their ultrastructural organization. At all stages and in all cell types the subcellular distribution of the reaction product was identical to that already described for adult rat pituitary (Tougard et
162
DEVELOPMENTAL
BIOLOGY
al., 1973), that is, it was located on the secretory granules and the ground cytoplasm with the content of the ergastoplasmic cisternae always negative. The significance of such subcellular localizations has been already discussed for the adult rat pituitary (Tougard et al., 1973). The absence of a positive reaction in the ergastoplasmic cisternae may be the result of lack of penetration of antisera into the cisternae, dilution of the hormone, or masking of its antigenic sites during the hormonal transit into the endoplasmic reticulum. The same situation was observed in gonadotropic cells of the normal male rat, but, after stimulation by castration, the content of the ergastoplasmic cisternae became strongly positive in some cells (Tougard et al., 1973). Comparison Twes
with Adult
Gonadotropic
Cell
Previous electron microscope studies (Kurosumi and Oota, 1968) of the adult rat pituitary using conventional techniques distinguished two types among gonadotropic cells, considered, respectively, as FSH cells and LH cells. These two cell types as well as numerous intermediary forms were later found similarly immunoreactive with antisera specific for rat LH (A-oLH, A-oLH@ Tougard et al., 1973; AbLHp: Moriarty, 1975). The same cell types were also stained with A-oFSH (Tixier-Vidal et al., 1975) as well as with an ArFSHp specific for rat FSH (Moriarty, 1976). This led to the conclusion that both LH and FSH are contained within the same cell, at least in some gonadotropes. The ultrastructural duality of the gonadotropic cells need not reflect an immunochemical duality. In contrast to the adult gonadotropic cells, the LH-positive cells of the rat fetal pituitary display a single type of ultrastructural organization which, at the end of gestation, appears similar to the socalled “LH cell type” of Kurosumi and Oota (1968). At the same time, neverthe-
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58,
1977
less, both LH and FSH were measurable by radioimmunoassay in pituitaries taken from fetuses of the same mothers used for the immunocytochemical study. From 18 to 19 dpc, the FSH and LH pituitary contents sharply increased, and FSH was 30 times more abundant than LH (Kerdelhue, personal communication). The secretion of FSH, therefore, does not necessarily imply ultrastructural heterogeneity of the gonadotropic cells as observed in the adult. This raises a question concerning the mechanisms involved in such a differentiation. It is clear from our study that this process occurs only after birth. We are very grateful to Dr. B. Kerdelhue and Dr. M. Jutisz for the generous gift of all antisera used in this study as well as for helpful advice. We also wish to acknowledge the excellent technical assistance of 0. Locquet and C. Pennarun. This work was supported by grants from the D.G.R.S.T. (Contract No. 72 7 0 100, France) and from the C.N.R.S (E.R. No. 89, France). REFERENCES S. (19691. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Zmmunochemistry 6, 43-52. BAKER, B. L., and JAFFE, R. B. (1975). The genesis of cell types in the adenohypophysis of the human fetus as observed with immunochemistry. Amer. J. Anat. 143, 137-161. BUGNON, C., LENYS, D., BLOCH, B., and FELLMANN, D. (1974). Etude cytoimmunologique des phenomenes de differentiation cellulaire precoce dans l’adenohypophyse foetale humaine. C. R SPances Sot. Biol. 168, 460-466. DUBOIS, P. M., and DUBOIS, M. P. (1974). Mise en evidence par immunofluorescence de l’activite gonadotrope LH dans l’antehypophyse foetale humaine. In “International Symposium on Sexual Endocrinology of the Perinatal Period. May 30-31, 1974” ( M. G. Forest and J. Bernard, eds.1, Vol. 32, pp. 37-62. INSERM Paris. DUBOIS, P. M., BEGEOT, M., and DUBOIS, M. P. (19751. Dissociation entre les sous-unitis (Y et p dans l’antihypophyse foetale humaine. Ann. Endocrinol. 36, l-2. DUPOUY, J. P. (1975). Fonction corticostimulante de l’hypophyse chez le foetus et chez la mere, en fin de gestation. J. Physiol. 71, 133A. DUPOUY, J. P., and MAGRE, S. (1973). Ultrastructure des cellules granulees de l’hypophyse foetale du rat. Identification des cellules corticotropes et AVRAMEAS,
TOUGARD,
PICART,
AND TIXIER-VIDAL
thyrbotropes. Arch. Anat. Microsc. 62, 185-205. HAGEN, C., and MCNEILLY, A. S. (1975). Identification of human luteinizing hormone, follicle-stimulating hormone, luteinizing hormone P-subunit and gonadotrophin (Y subunit in foetal and adult pituitary glands. J. Endocrinol. 67, 49-57. HERLANT, M. (1960). Etude de deux techniques nouvelles destinkes & mettre en Bvidence les diffkentes catigories cellulaires pksentes dans la glande pituitaire. Bull. Microsc. Appl. 10, 37-44. JOST, A., and TAVERNIER, R. (1956). Application de la mkthode $ l’acide pkriodique Schiff (Mac Manus) & l’ktude de l’hypophyse foetale du Rat. C.R. Acad. Sci. (Paris) 243, 1353-1356. KERDELHUE, B., PITOULIS, S., and JUTISZ, M. (1971). Etude par radioimmunologie de la spkificiti des immunk-ums de l’hormone lutkinisante (LH) ovine et de ses sous-unit.&. C.R. Acad. Sci. (Paris) 273, 511-514. KERDELHUE, B., KANN, G., and JUTISZ, M. (1972). Dosage radioimmunologique de la FSH chez le mouton et le rat. In “Colloque sur les Hormones Glycoprotkiques Hypophysaires” (M. Jutisz, ed.), pp. 177-192. INSERM, Paris. KUROSUMI, K., and OOTA, Y. (1968). Electron microscopy of two types of gonadotrophs in the anterior pituitary glands of persistent estrous and diestrous rats. 2. Zellforsch. 85, 34-46. MORIARTY, G. C. (1975). Electron microscopic immunocytochemical studies of rat pituitary gonadotrophs. A sex difference in morphology and cytochemistry of LH cells. Endocrinology 97, 12151225. MORIARTY, G. C. (1976). Immunocytochemistry of the pituitary glycoprotein hormones. J. Histothem. Cytochem. 24, 846-863. PASTEELS, J. L., G~USSET, P., DANGUY, A., and EcTORS, F. (1974). Gonadotropin secretion by human fetal and infant pituitaries. Zn “International
Cytogenesis
of Gonadotropic
Cells
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Symposium on Sexual Endocrinology of the Perinatal Period, May 30-31, 1974” (M. G. Forest and J. Bertrand, eds.), Vol. 32, pp. 13-36. INSERM, Paris. SETALO, G., and NAKANE, P. K. (1972). Studies on the functional differentiation of cells in fetal anterior pituitary glands of rats with peroxidase-labeled antibody method. Anat. Rec. 172, 403. SVALANDER, C. (1974). Ultrastructure ofthe fetal rat adenohypophysis. Acta Endocrinol. 76 [Suppl. 1881, 1-114. TIXIER-VIDAL, A., TOUGARD, C., KERDELHUE, B., and JUTISZ, M. (1975). Light and electron microscopic studies on immunocytochemical localization of gonadotropic hormones in rat pituitary using antisera against ovine FSH, ovine LH and its two subunits. Ann. N.Y. Acad. Sci. 254, 433461. TOUGARD, C., KERDELHUE, B., TIXIER-VIDAL, A., and JUTISZ, M. (1973). Light and electron microscope localization of binding sites of antibodies against ovine luteinizing hormone and its two subunits in rat adenohypophysis using peroxidase-labeled antibody technique. J. Cell Biol. 58, 503-521. VAITUKAITIS, J. L., Ross, G. T., REICHERT, L. E., JR., and WARD, D. N. (1972). Immunologic basis for within and between species cross-reactivity of luteinizing hormone. Endocrinology 91, 13371342. YOSHIMURA, F., HARUMIYA, K., and HIYAMA, H. (1970). Light and electron microscopic studies of the cytogenesis of anterior pituitary cells in perinatal rats in reference to the development of target organs. Arch. Histol. Japon. 31, 333-369. ZAMBONI, L., and DE MARTINO, C. (1967). Buffered picric acid formaldehyde: A new rapid fixative for electron microscopy. J. Cell Biol. 35, 148a.
58, 148-163
BIOLOGY
(1977)
Cytogenesis of Immunoreactive Gonadotropic Ceils in the Fetal Rat Pituitary at Light and Electron Microscope Levels C. TOUGARD, Groupe
de Neuroendocrinologie
Received
R. PICART,
AND A. TIXIER-VIDAL
CeEEulaire, Laboratoire de Physiologic Cetlutaire, Marcelin Berthelot, 75231 PARIS 05, France May
13, 3976;
accepted
in revised
form
February
Collige
de France,
11 Place
14,1977
The cytogenesis of immunoreactive gonadotropic cells in the fetal rat pituitary was analyzed at the light and electron microscope levels using the indirect peroxidase-labeled antibody method and antisera against ovine FSH (A-oFSH) and ovine LH (A-oLH), and its two subunits (A-oLHp and A-oLHa). At the light microscope level, the first immunoreactive cells were detected on the seventeenth day postcopulation (dpc) with A-oLHp. Cells immunochemically stained with A-oLHa and A-oLH were generally observed 24 hr later. At the electron microscope level, the first immunoreactive cells were detected on 16 dpc with A-oLH@. These first immunoreactive cells were small, but already displayed some small secretory granules (80-120 nm). On 17 dpc, gonadotropic cells were stained with A-oLH@ as well as with A-oLHar and AoFSH. On 18 dpc, the number and the size of immunoreactive cells began to increase. By 19 dpc, they displayed an important development of ergastoplasmic cisternae and Golgi zone. At term, nevertheless, the ultrastructural features of fetal gonadotropic cells still differed from those of adult gonadotropic cells. INTRODUCTION
pouy and Magre, 1973; Svalander, 1974). Immunocytochemical techniques which have been already successfully applied to adult pituitaries may be used to describe the first immunoreactive cells and to follow their ultrastructural evolution during fetal development. Such immunochemical studies have been performed at the light microscope level in the fetal human pituitary (Dubois and Dubois, 1974; Bugnon et al., 1974; Baker and Jaffe, 1975; Dupouy, 1975). In the fetal rat pituitary, adrenocorticotropic (ACTH) and prolactin (PRL) cells were found on Day 16, thyrotropic (TSH) cells on Day 17, luteinizing hormone (LH)-producing cells on Day 18, and growth hormone (GH)-producing cells on Day 19 (Setalo and Nakane, 1972). According to Dupouy (1975) who used an immunofluorescence method, ACTH cells were found on Day 18 only. So far, no report has been published on the ultrastructural features of fetal immunoreactive cells. Our purpose in the present work was: (i) to detect the first immunoreactive gonado-
The morphological differentiation of the fetal rat pituitary has been extensively studied at the light and electron microscope levels (Jost and Tavernier, 1956; Yoshimura et al., 1970; Svalander, 19741, but, at early stages of development, the identification of the cell types remained difficult. With the light microscope, the histochemical techniques which permit differentiating cell types in the adult pituitary were unsuccessful when applied to the fetal pituitary because of the low stainability of the cells. The first granulated cells which appear under the electron microscope do not display the ultrastructural features of mature adult cells and cannot be identified on such bases. In the fetal rat pituitary, the first granulated cells appear between the sixteenth and seventeenth days of gestation, but morphological analogy with adult pituitary cell types does not occur before the eighteenth and nineteenth days (Yoshimura et al., 1970; Du148 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0012-1606
TOUGARD,
PICART,
AND TIXIER-VIDAL
tropic cells using four antibodies that we have already applied to adult rat pituitaries (Tougard et aE., 19’73; Tixier-Vidal et al., 197.51, antisera against ovine LH and its two subunits (A-oLH, A-oLH& AoLHa), and antiserum against ovine FSH (A-oFSH); (ii) to describe the ultrastructure of such cells and to follow their evolution during fetal development; and (iii> to compare their ultrastructural features to those of adult mature immunoreactive gonadotropic cells previously described (Tougard et al., 1973; Tixier-Vidal et al., 1975; Moriarty, 1975, 1976). MATERIALS
AND
METHODS
Cytogenesis
of Gonadotropic
Cells
149
pm-thick paraffin sections were treated as previously described by using the indirect peroxidase-labeled antibody method (Tougard et al., 1973; Tixier-Vidal et al., 1975). Serial sections were respectively stained with the four antisera. Control sections were subjected to the following treatments: 3, 3’-diaminobenzidine tetrahydrochloride (DAB; Sigma) reaction without any incubation with antiserum; specific antiserum, then DAB reaction; normal guinea pig serum (NGPS) followed by DAB reaction; anti-guinea pig immunoglobulin bound to peroxidase (A-IgG-per) , then DAB reaction; NGPS followed by AIgG-per, then DAB reaction; specific antiserum, then A-IgG-per and, finally, DAB reaction in medium without H,O,.
Animals. Sprague-Dawley female and male rats were mated for one night. Fertilization was verified by the presence of For the electron microscope study. The sperm in vaginal smears on the following pituitaries from stage 16 to 21 dpc were morning, and that day was designated as fixed in picric acid formaldehyde (PAF) (Zamboni and De Martino, 1967) for 6 hr at the first day postcopulation (1 dpc). Four series of fetuses were studied at different 4°C and then were washed overnight in 0.1 times of the year. Fetal pituitaries were M phosphate buffer, pH 7.3, with 10% sucollected from stage 13 dpc to 21 dpc. Pregcrose. Thick sections (75, 100, or 150 pm) nant rats were decapitated without anes- were cut without freezing with a Sorvall thesia, and the fetuses were removed and TC2 sectioner. These sections as well as killed by decapitation. The base of the half-pituitaries were prepared for immucranium of each fetus was opened under a nochemical staining as previously dedrop of fixative. After a 30-min in situ scribed for adult pituitaries (Tougard et fixation, the pituitaries from stage 16 to 21 al., 1973). Control treatments used were dpc were dissected in fresh fixative. For the same as for the light microscope study. the youngest fetuses, from stage 13 to 15 Ultrathin sections were examined under the electron microscope without further dpc, the whole basal region surrounding the pituitary was removed and immersed staining. The sections were picked up on in fixative. single slot grids coated with parlodion. For the light microscope study. FortyAntisera. Antisera (AS) were the same eight pituitaries were fixed in Herlant as those previously used for adult rat pitui(1960) fixative for 24-48 hr. They were taries (Tougard et al., 1973; Tixier-Vidal et then dehydrated and embedded in paraf- al., 1975). They were obtained by B. Kerfin. In the first series, pituitaries were delhue (Laboratoire des Hormones Polytaken every 2 days from stage 13 to 21 dpc; peptidiques, CNRS) by immunizing in the second and third series, each day guinea pigs with ovine FSH and ovine LH from stage 18 to 21 dpc and, in the fourth and its two subunits (LHP and LHa) preseries, each day from stage 16 to 21 dpc. pared in the same laboratory. The racharacteristics as well For the last series, the sex of each fetus dioimmunological was determined, and males and females as the immunocytochemical specificity of were treated separately. these antisera were described in detail For immunochemical staining, 3- to 5- elsewhere (Kerdelhue et al., 1971, 1972;
150
DEVELOPMENTALBIOLOGY
Tougard et al ., 1973; Tixier-Vidal et al ., 1975). For the light microscope study, the sections were generally incubated for 1.5 hr in one of the four antisera diluted l/50 to l/100. In one experiment, the dilution of each antiserum was correlated with its radioimmunological titer (dilution for each antisera: A-FSH, l/10; A-LH, l/20; A-LHP, l/1000; A-LHa, l/55), and, in that case, the incubation time was 6 or 24 hr. For the electron microscope study, sections were incubated for 3 hr, and three antisera (AFSH, A-LHa, A-LHP) were diluted l/20. Sheep antibodies against guinea pig yglobulin were purified and linked to horseradish peroxidase by Dr. B. Kerdelhue according to Avrameas (1969). RESULTS
Light
Microscope
Study
At the light microscope level, the immunoreactive cells were easily identifiable. In control sections, the cells were uncolored except for the pseudoperoxidase reaction of red blood cells. Immunoreactive cells were never found before stages 17 or 19 dpc (Table 1). In the four series, the first immunoreactive cells were observed after staining with anti-LHP (Fig. 1). They were localized at the ventral and lateral parts of the fetal pars distalis. At later ages, immunoreactive cells became more numerous and more widely distributed in the pars distalis (Figs. 2 and 3). They were often associated in small clusters of three or four cells. Cells immunoreactive with A-oLH and A-oLHa! were observed only 24 hr after the first A-oLH P-positive cells, that is, respectively between stages 18 and 20 dpc, depending on the series. The same 24hr delay was also observed when the AoLH/3 was diluted more (lo- to 20-fold) in accordance with its radioimmunological titer which was higher than those of A-oLH and A-oLHa (see Methods and Materials). Cells immunoreactive with A-oFSH appeared between stages 20 and 21 dpc of fetal life. Even with a high dilution of A-oLH&
VOLUME 58, 1977
the number of cells immunoreactive for this antiserum was always higher than those for A-oLH and A-oLHa: approximately 15-25% instead of 2-10%. With AoFSH, only a few positive cells were found in all series (see Table 1). Whatever the antiserum, immunoreactive cells displayed a similar ovoid shape, although some were elongated in older fetuses. Surprisingly, some cells of the 19dpc pars intermedia were stained by AoLH/3 (Fig. 5) and were still present on 21 dpc. They were localized in the zone adjacent to the pars distalis or at the periphery of the pituitary cleft. Electron
Microscope
Study
In spite of the small size of the fetal pituitaries, immunoreactive cells were found only in incubated slices and never in incubated half-pituitaries. There are several possible explanations for this difference. The presence of a connective tissue envelope around the pituitary opposes the penetration of the antibody. The gonadoi tropic cells are infrequent in the midsagittal zone of the gland which was directly exposed to the antibody in half-pituitaries. The slight dissociation of the cells which occurred within incubated slices may have favored the penetration of the antisera and other reagents. As previously observed for adult pituitary (Tougard et al., 19731, the periphery of the slices was slightly damaged, but the positive cells were found more centrally (Fig. 4) and could not be confused with damaged cells of the border of the section. Positive cells were found only after treatment with specific antisera; they were recognized by the increased electron density of their secretory granules and ground cytoplasm (Fig. 6). In contrast, after treatment with normal guinea pig serum, the glandular cells displayed a low electron density in their organelles including secretory granules; only the border of the slice displayed an unspecific staining. The poor quality of cell preservation after PAF fixation has been already ob-
TOUCARD,
PICART,
AND TIXIER-VIDAL
Cytogenesis
TABLE DETECTION, Series
I
A-oLH A-oLHP A-oLHa A-oFSH A-oLH A-oLHP A-oLHo A-oFSH A-oLH A-oLHP A-oLHa
III
Days
Antiserum 13-15 -b -
16 nd nd nd nd nd nd nd nd nd nd nd nd
151
Cells
1
WITH THREE ANTISERA, OF IMMUNOREACTIVE G~NADOTROPIC CELIS DURING THE LAST DAYS OF GESTATION: LIGHT MICROSCOPE
A-0FSH
II
of Gonadotropic
17 c -
IN THE FETAL RAT PITUITARY STUDY~
postcopulation 18 nd nd nd nd + ++ +
19 k ++ + -
20
21
nd nd nd nd -
+ ++ +++ ++ + + + t +
+ + +++
ttt
t
++
-
-
-
+ + -
+
IV Male
Female
A-oFSH A-oLH A-oLHP A-oLHa A-oFSH A-oLH A-oLHP A-oLHa
nd nd nd nd nd nd nd nd
-
-
a For each stage two fetal pituitaries were taken from two different 5 pm) were separately incubated with the four antisera. b Significance of symbols: (-) no staining; (k) 1 or 2 immunoreactive strongly stained; (+ t) 5 to 20 immunoreactive cells; (t t +) more done.
served for the adult rat pituitary (Tougard et al., 1973). Considering the well-known difficulties involved in fixation of fetal pituitary, the results obtained here can be considered as optimal for the fixative that we used. This is evidenced by the good preservation of the mitochondria and nuclei. Only glutaraldehyde or osmium tetroxide would permit a better fixation of the cell membrane systems. Unfortunately, in using the same technical approach previously for the adult rat pituitary, we found that gonadotropic cells do not retain their immunoreactivity after glutaraldehyde fixation (unpublished results). Immunoreactive cells were already present, although very few in number, at the first stage that we examined, 16 dpc, but only after staining with A-oLH@ These cells displayed a large nucleus, a small
-
than
+t+
-
t
+ -
mothers.
Serial
+
t+
paraffin
cells slightly stained; 20 immunoreactive
+ t+
?I sections
(3 or
( +) 1 to 5 cells cells; nd, not
cytoplasmic area with numerous mitochondria, and some flattened ergastoplasmic cisternae (Fig. 7). Although the majority of pituitary cells were devoid of secretory granules at this stage, the first immunoreactive cells always contained a few small secretory granules (80-120 nm). The immunochemical staining was restricted to these small granules. On 17 dpc, immunoreactive cells were observed after staining with A-oLH/3 as well as with AoLHa and A-oFSH (Table 2). They all displayed the same ultrastructural features as immunoreactive cells on 16 dpc, but the reaction product was present on the ground cytoplasm in addition to the secretory granules. On 18 dpc, cells immunoreactive with the three antisera became more numerous. They generally displayed an ovoid shape, but some were larger than at the previous stages. The reaction prod-
FIGS. l-4. Staining with A-oLHP of fetal rat pituitaries taken at progressive stages of gestation. FIG. 1. Paraffin section (3 pm) of a 17-dpc fetal pituitary. Some cells (single arrow) are slightly stained. The red blood cells (double arrow) display a pseudoperoxidase reaction. 330 x . FIG. 2. Paraffin section (3 pm) of a 1%dpc fetal pituitary. Immunoreactive cells are more strongly stained and more numerous than those in Fig. 1. They are distributed in all the pars distalis. 330 x . FIG. 3. Paraffin section (3 pm) of a 20-dpc fetal pituitary. Immunoreactive cells are larger and darker than those in Figs. 1 and 2. They are very numerous and associated in small clusters. 330 x . FIG. 4. Araldite section (1 pm) from a slice of a IS-dpc fetal pituitary treated with A-oLH@ before embedding. Immunoreactive cells are easily identifiable in a large peripheral zone of the section, but they are never found in the core of the slice. 330 x . 152
TOUGARD,
PICART,
AND
TIXIER-VIDAL
Cytogenesis
of Gonadotropic
Cells
153
FIG. 5. Serial paraffin sections of a 19-dpc rat fetal pituitary. (a) Section treated with A-oLH/3 diluted l:lOOf! for 24 hr. Immunoreactive cells (single arrow) are seen in the pars distalis (PD). A few cells (double arrow) are also stained in the pars intermedia (PI). PC: pituitary cleft. 330 X. (b) Next section treated with A-oLHa diluted 1:55 for 24 hr. No immunoreactive cells can be seen with this antisera, either in the pars distalis or in the pars intermedia. 330 x
uct was always localized on secretory granules and on the ground cytoplasm (Fig. 8). A few cells displayed a variability in the size of their secretory granules (from 80 to 150 nm), with very few larger ones (250 nm). The ergastoplasmic cisternae were scarce and generally flattened. On 19 dpc, the ultrastructural organization of the immunoreactive cells varied depending on the respective antiserum. Cells which were stained by the three antisera displayed a rounded or ovoid shape and an ultrastructural organization identical to that of cells described at 18 dpc, that is, a few ergastoplasmic cisternae and variability in the diameter of their secretory granules (80-250 nm). Cells which
were stained only by A-oLHa and A-oFSH but not by A-oLH/3 displayed an elongated or polygonal shape. They were characterized by very small secretory granules (50120 nm) generally lining the plasma membrane and by vesicular ergastoplasmic cisternae (Fig. 9). At this stage, some cells immunoreactive with A-oLH@ were also found in the pars inter-media at the periphery of the pituitary cleft. On 20 and 21 dpc, almost all immunoreactive cells displayed an ovoid shape with a large cytoplasmic area, although some small cells with a high nucleus/cytoplasm ratio still remained. The extension of the cytoplasmic area corresponds to an important development of ergastoplasmic
154
DEVELOPMENTAL
FIG. 6. Rat fetal pituitary (21 dpc) treated ble by the increased electron density of their other cells are negative. 7000 X .
BIOLOGY
VOLUME
58, 1977
with A-OLHW Two immunoreactive secretory granules and cytoplasm.
cells are easily identifiaThe nucleus (N) and the
TOUGARD,
PICART,
AND TIXIER-VIDAL
Cytogenesis
of Gonadotropic
Cells
FIG. 7. Rat fetal pituitary (16 dpc) treated with A-oLHP. Two immunoreactive gonadotropic cells (G) display a large nucleus and a small cytoplasmic area. The reaction product is localized on the secretory granules. A few linear ergastoplasmic cisternae are underlined with reaction product (arrow). The nucleus and the numerous mitochondria Cm) are negative. 9000 x
cisternae which are flattened or slightly dilated (Figs. 10 and 11). At the same time the Golgi was enlarged. These moditkations mainly concerned the cells which were immunoreactive with the three antisera, including A-oLHP. These cells were
far more numerous than on 19 dpc and of their secretory granules displayed the same size range (from 80 to 150 nm), again with a few larger ones (300 nm) in some cells. In all these cells, the reaction product
156
DEVELOPMENTAL
BIOLOGY TABLE
DETECTION, Series
WITH THREE ANTISERA, OF IMMUNOREACTIVE DURING THE LAST DAYS OF GESTATION: Antiserum
VOLUME
58, 1977
2 GONAWTROPIC CELLS IN THE FETAL RAT PITUITARY ELECTRON MICROSCOPE STUDY” Days
postcopulation
16
17
18
19
20
21
III
A-oFSH A-oLHP A-oLHa
nd* nd nd
nd nd nd
+ +++ k
nd nd nd
+++ +++ +++
+++ +++ +++
IV
A-oFSH A-oLHP A-oLHo
+ -
+ + f
nd nd nd
+++ +++ +++
nd nd nd
nd nd nd
4 At each stage, a pool of 10 fetal pituitaries were with each of the three antisera. Immunoreactive cells sections. b Significance of symbols: (-1 no staining; (2) one to five cells strongly stained; (+ + +) more than five
collected, and TC2 sections were detected on l-pm-thick or two immunoreactive immunoreactive cells;
were incubated separately sections or on ultrathin
cells slightly nd, not done.
FIG. 8. Bat fetal pituitary (18 dpc) treated with A-oFSH. This immunoreactive secretory granules (80-200 nm in diameter). The reaction product is found on both ground cytoplasm. The ergastoplasmic cisternae are very scarce. 14,000 x
stained,
(+)
one
cell contains a few secretory granules and
TOUCARD,
PICART,
AND TIXIER-VIDAL
Cytogenesis
of Gonadotropic
Cells
FIG. 9. Rat fetal pituitary treated with A-oLHa. This elongated immunoreactive cell contains a few very small secretory granules (50-120 nm), mainly located at the periphery of the cell, and vesicular ergastoplasmic cisternae (arrow). 11,500 X.
158
DEVELOPMENTAL
BIOLOGY
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FIG. 10. Rat fetal pituitary (21 dpc) treated with A-oLH& This immunoreactive elongated and parallel ergastoplasmic cisternae and small secretory granules product is found on secretory granules and on some cytoplasmic areas (arrow). plasmic cisternae is always negative. 17,000 X .
cell contains numerous (80-150 nm). The reaction The content of the ergasto-
FIG. 11. Rat fetal pituitary (21 dpc) treated with A-oLHp. This immunoreactive cell displays an ovoid shape with a large cytoplasmic area. It contains numerous secretory granules (80-150 nm) and flattened (single arrow) or dilated (double arrow) ergastoplasmic cisternae. The reaction product is found on the secretory granules and on the ground cytoplasm. 15,000 x 159
160
DEVEJAJPMENTAL
BIOLOGY
was localized on secretory granules and on the ground cytoplasm. The content of the ergastoplasmic cisternae was always negative (Figs. 10 and 11). DISCUSSION
Specificity
of Antisera
The specificity of the immunochemical staining has been previously tested by radioimmunological study (Kerdelhue et al., 1971, 1972), as well as by analysis of the effects on the staining of previous adsorption with several other pituitary hormones (Tougard et al., 1973; Tixier-Vidal et al., 1975). On the basis of such studies, it can be assumed that staining with A-oLH and/ or A-oLH/3 identifies rat LH molecules but not FSH or TSH. In contrast, staining with A-oLHa and/or A-oFSH does not discriminate LH from FSH and TSH. Chronology of the Immunocytochemical Differentiation of Gonadotropic Cells during Fetal Life
At the light microscope level, the first immunoreactive cells observed were always those stained with A-oLH@ Cells immunoreactive with A-oLH and A-OLHCX only appeared 24 hr later. The same result was obtained when A-oLHP was diluted 20-fold more than the other antisera. These differences cannot result from individual variations between fetuses, since the four antisera were respectively applied to serial sections for each fetal pituitary examined. Nevertheless, we cannot conclude from this result that LHP is the first subunit which is synthesized in the fetal since we used antisera rat pituitary, against ovine hormone to localize rat gonadotropic hormone. Indeed, Vaitukaitis et al. (1972) have shown that the interspeties cross-reactivity resides in the p subunits of pituitary glycoprotein hormones, whereas the zoological specificity resides in the (Y subunits. This may explain our detection of the /? subunit before the a subunit or the native hormone. This can also explain why, at later stages, we found
VOLUME
58, 1977
a larger number of immunoreactive cells with A-oLH/3 than with the other antisera. The more precocious detection of AoLH/3-positive cells might be also related to technical conditions which affected the antigenicity against A-oLHa and A-oFSH more than that against A-oLHp. Consistent with this possibility is the fact that, in the fourth experimental series (Tables 1 and 2) in which fetal pituitaries were taken from the same mothers for both light (LM) and electron (EM) microscope studies, positive cells were detected for the three antisera very much sooner with EM than with LM techniques. This could suggest that fixation and/or embedding alter, to varying degrees, the antigenicity of the hormones. This is particularly true for FSH which seems selectively denatured or extracted after histological treatments. A similar observation was already discussed for adult rat pituitary cells (Tixier-Vidal et al., 1975). The chronology of the appearance of immunochemical staining with the four antisera also varied from one series to another (Table 1). This cannot be attributed to differences in the quality of fixation or to individual variations between the mothers, since we used fetuses of several mothers for each series. At present we have no explanation for such variations. When male and female pituitaries were collected separately (fourth experimental series), the positive cells in the female were found 24 hr later than those in the male. The only report we found on the differentiation of immunoreactive gonadotropic cells in the fetal rat pituitary mentioned the appearance of LH-positive cells on Day 18 (Setalo and Nakane, 1972), using picric acid formaldehyde for fixation and polyethylene glycol for embedding. The human fetal pituitary has been more extensively studied with immunocytochemical techniques (Dubois and Dubois, 1974; Dubois et al., 1975; Bugnon et al., 1974; Pasteels et al., 1974; Baker and Jaffe, 1975). All these
TOUGARD,
PICART,
AND
TIXIER-VIDAL
authors found cells immunoreactive for an anti-HCG (Bugnon et al., 1974; Baker and Jaffe, 1975) or for an anti-ovine LH (Dubois and Dubois, 1974; Dubois et al., 1975; Pasteels et al., 1974) at approximately 10 weeks. Cells immunoreactive for an antiporcine LHp were detected later, at 23 weeks (Baker and Jaffe, 1975) or between 14 and 20 weeks, depending on the sex (Dubois and Dubois, 1974; Dubois et al., 1975). It is of interest to compare these cytological results with the radioimmunoassay data of Hagen and McNeilly (1975) who found (Ysubunit and native LH before p subunit during development of the fetal human pituitary. Whatever the technique (LM or EM), the presence of immunoreactive cells with A-oLHP in the pars intermedia is a surprising fact. We have previously verified (Tougard et al., 1973) that our A-oLHp did not cross-react with ACTH. In adult rat pituitary, moreover, the same antisera did not stain any cells in the pars intermedia. Ultrastructural Features of the First Immunoreactive CeZZs
The ultrastructure of the first detected immunoreactive cells, on 16 and 17 dpc, was the same with the three AS. They were small and rounded with a high nucleus/cytoplasm ratio and a few small secretory granules. They look like the socalled ambiguous cells which are the first granulated cells to appear on Day 16, as described by several authors using conventional electron microscope techniques (Yoshimura et al., 1970; Svalander, 1974). Since our observations at the EM level started on Day 16, we do not know whether immunoreactive gonadotropic cells exist at earlier stages. Nevertheless, this seems most doubtful because of the extreme paucity and primitive ultrastructural organization of the positive cells on Day 16. Their ultrastructure does not differ from that of the neighboring granular cells. Therefore, it is probable that the immunochemical differentiation precedes the morphological
Cytogenesis of Gonadotropic
Cells
161
diversification of the anterior pituitary cell types. At that stage of development, the most numerous pituitary cells are devoid of secretory granules. Such agranular cells were never found immunoreactive for any of the three antisera. It seems that, at least for gonadotropic cells, the immunochemical differentiation coincides with the achievement of the secretory pathway with the formation of secretory granules. Ultrastructural Evolution oflmmunoreactive Gonadotropic Cells during Fetal Life
On 18 dpc, the immunoreactive cells begin to develop in both their number and their size. The cell growth corresponds mainly to development of the ergastoplasmic cisternae and the Golgi zone. At the same time, the secretory granules show a slight increase in their number and diameter. These changes take place at an increasing rate from 18 to 21 dpc. In addition, a slight diversification in the ultrastructural organization of the immunoreactive cells appears on 19 dpc, depending on the antisera. The cells which are stained with A-oLH/3 and, therefore, can be considered as LH cells contain small secretory granules, the diameter of which varies from 80 to 150 nm, with a few larger ones (250-300 nm). Their ergastoplasmic cisternae are generally flattened or slightly dilated. The other cell type is stained by A-oLHa and A-oFSH only. It differs from the latter by the very small size of the secretory granules (50-120 nm) and the vesicular aspect of their ergastoplasmic cisternae. On the basis of their immunochemical staining, these cells may contain either FSH or TSH. They become very scarce on 21 dpc. At these stages, the LH cells become more numerous, but do not display further diversification in their ultrastructural organization. At all stages and in all cell types the subcellular distribution of the reaction product was identical to that already described for adult rat pituitary (Tougard et
162
DEVELOPMENTAL
BIOLOGY
al., 1973), that is, it was located on the secretory granules and the ground cytoplasm with the content of the ergastoplasmic cisternae always negative. The significance of such subcellular localizations has been already discussed for the adult rat pituitary (Tougard et al., 1973). The absence of a positive reaction in the ergastoplasmic cisternae may be the result of lack of penetration of antisera into the cisternae, dilution of the hormone, or masking of its antigenic sites during the hormonal transit into the endoplasmic reticulum. The same situation was observed in gonadotropic cells of the normal male rat, but, after stimulation by castration, the content of the ergastoplasmic cisternae became strongly positive in some cells (Tougard et al., 1973). Comparison Twes
with Adult
Gonadotropic
Cell
Previous electron microscope studies (Kurosumi and Oota, 1968) of the adult rat pituitary using conventional techniques distinguished two types among gonadotropic cells, considered, respectively, as FSH cells and LH cells. These two cell types as well as numerous intermediary forms were later found similarly immunoreactive with antisera specific for rat LH (A-oLH, A-oLH@ Tougard et al., 1973; AbLHp: Moriarty, 1975). The same cell types were also stained with A-oFSH (Tixier-Vidal et al., 1975) as well as with an ArFSHp specific for rat FSH (Moriarty, 1976). This led to the conclusion that both LH and FSH are contained within the same cell, at least in some gonadotropes. The ultrastructural duality of the gonadotropic cells need not reflect an immunochemical duality. In contrast to the adult gonadotropic cells, the LH-positive cells of the rat fetal pituitary display a single type of ultrastructural organization which, at the end of gestation, appears similar to the socalled “LH cell type” of Kurosumi and Oota (1968). At the same time, neverthe-
VOLUME
58,
1977
less, both LH and FSH were measurable by radioimmunoassay in pituitaries taken from fetuses of the same mothers used for the immunocytochemical study. From 18 to 19 dpc, the FSH and LH pituitary contents sharply increased, and FSH was 30 times more abundant than LH (Kerdelhue, personal communication). The secretion of FSH, therefore, does not necessarily imply ultrastructural heterogeneity of the gonadotropic cells as observed in the adult. This raises a question concerning the mechanisms involved in such a differentiation. It is clear from our study that this process occurs only after birth. We are very grateful to Dr. B. Kerdelhue and Dr. M. Jutisz for the generous gift of all antisera used in this study as well as for helpful advice. We also wish to acknowledge the excellent technical assistance of 0. Locquet and C. Pennarun. This work was supported by grants from the D.G.R.S.T. (Contract No. 72 7 0 100, France) and from the C.N.R.S (E.R. No. 89, France). REFERENCES S. (19691. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Zmmunochemistry 6, 43-52. BAKER, B. L., and JAFFE, R. B. (1975). The genesis of cell types in the adenohypophysis of the human fetus as observed with immunochemistry. Amer. J. Anat. 143, 137-161. BUGNON, C., LENYS, D., BLOCH, B., and FELLMANN, D. (1974). Etude cytoimmunologique des phenomenes de differentiation cellulaire precoce dans l’adenohypophyse foetale humaine. C. R SPances Sot. Biol. 168, 460-466. DUBOIS, P. M., and DUBOIS, M. P. (1974). Mise en evidence par immunofluorescence de l’activite gonadotrope LH dans l’antehypophyse foetale humaine. In “International Symposium on Sexual Endocrinology of the Perinatal Period. May 30-31, 1974” ( M. G. Forest and J. Bernard, eds.1, Vol. 32, pp. 37-62. INSERM Paris. DUBOIS, P. M., BEGEOT, M., and DUBOIS, M. P. (19751. Dissociation entre les sous-unitis (Y et p dans l’antihypophyse foetale humaine. Ann. Endocrinol. 36, l-2. DUPOUY, J. P. (1975). Fonction corticostimulante de l’hypophyse chez le foetus et chez la mere, en fin de gestation. J. Physiol. 71, 133A. DUPOUY, J. P., and MAGRE, S. (1973). Ultrastructure des cellules granulees de l’hypophyse foetale du rat. Identification des cellules corticotropes et AVRAMEAS,
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