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Alkaline Phosphatase Activity in the Theca of Ovarian Follicles of the Hen Throughout Follicular Development
PHYSIOLOGY AND REPRODUCTION Alkaline Phosphatase Activity in the Theca of Ovarian Follicles of the Hen Throughout Follicular Development C. CHAPEAU, H. ENGELHARDT, G. J. KING, and R. J. ETCHES Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, NIG 2W1 assessment or paraffin for alkaline phosphatase histochemistry. Alkaline phosphatase activity was found to be specific for cells of the theca interna in preovulatory follicles. Activity was first detectable in very small white follicles, the earliest stage in which the theca interna could be distinguished from the theca externa, and highest in the yellow yolky follicles. After ovulation, activity decreased to undetectable levels by the POF4 to 5 stages in POF within 4 to 5 d after ovulation. This study has shown that in the domestic hen, alkaline phosphatase activity is present in cells adjacent to the basal lamina of the theca interna of preovulatory follicles and POF up to 4 to 5 d after ovulation. Identification of the functional significance of this histological reaction will provide new information on ovarian function in the hen.
{Key words: alkaline phosphatase, ovary, theca, hen, follicle) 1996 Poultry Science 75:1536-1545
INTRODUCTION The hen's ovary consists of five to eight large yolkfilled follicles (the F x series with Fi being the largest and F n being the smallest), numerous small yellow follicles (4 to 10 mm in diameter), large white follicles (2 to 4 mm in diameter), small white follicles (less than 2 mm in diameter), and several postovulatory follicles (POF series). The large yolky follicles constitute most of the mass of the ovary and are classified according to their size and proximity to ovulation as Fi (largest) through F5 to F8- A mature preovulatory follicle consists of an oocyte enclosed by the perivitelline layer, which is in turn surrounded by a granulosa layer, basement membrane, theca interna, and theca externa. Production of steroid hormones by these follicles is believed to involve cooperation between three cell types: progesterone precursor provided by granulosa cells is converted to androgen by the theca interna, which is in turn metabolized to estrogen by cells of the theca externa (Porter et ah, 1989; Nitta et al, 1991; Kato et al, 1995).
Received for publication March 8, 1996. Accepted for publication June 19, 1996.
Much less is known about developmental stages in the pool of small follicles. Robinson and Etches (1986) showed that there were maturational differences, in terms of steroid production and response to LH, within the pool of small (< 10 mm) follicles. Due to their large numbers, Robinson and Etches (1986) estimated that small follicles were in fact the principal source of dehydroepiandrosterone and estrogen, and a significant source of androstenedione in the hen's ovary. Several lines of evidence suggest that the theca may be the sole source of steroids in these small follicles (Davidson et ah, 1979; Yoshimura et al, 1989; Kowalski et al, 1991; Tilly et al, 1991a,b; Nitta et al, 1993). When it is first formed, the theca consists of a single layer; differentiation into the theca interna and externa occurs when the follicle reaches approximately 2 mm in size (Hodges, 1974). The smaller classes of follicles have received relatively little attention in morphological studies. In a series of publications investigating the anatomy of the hen's ovary at the electron microscope level, Dahl (1970, 1971) described thecal glands as distinct structures within the theca interna. However, his studies concentrated on ultrastructural detail without reference to stage of follicular development and have not been featured in subsequent studies of ovarian anatomy.
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ABSTRACT Within the hen's ovary, the theca tissue of small follicles that have not yet entered the preovulatory hierarchy is a major contributor to ovarian steroidogenesis. Relatively little is known about developmental stages in the theca layer within this pool of follicles and of the histological properties of this tissue. In pigs and sheep, alkaline phosphatase activity has been identified in the theca of preovulatory follicles and in theca-derived cells in the corpus luteum. The objectives of this study were to document morphological changes in the theca layer through pre- and postovulatory follicular development, and to assess alkaline phosphatase activity at these developmental stages. Ovarian tissue containing small white, large white, small yellow, large yellow, and postovulatory follicles (POF) was obtained from mature White Leghorn hens and embedded in either methacrylate for morphological
ALKALINE PHOSPHATASE ACTIVITY IN THE THECA
MATERIALS AND METHODS Animals Ovaries were obtained from five mature White Leghorn hens (Gallus domesticus) within 10 min after cervical dislocation. The stages of follicular development were identified macroscopically as small white follicles, large white follicles, small yellow follicles, large yellow follicles (F series), and POF.
Tissue Processing for Morphological Assessment Individual pieces of ovarian tissue containing small white, large white, and intact larger follicles were dissected free of most associated stromal tissue and immersed for 1 h in 4% paraformaldehyde, 2% glutaraldehyde in phosphate buffer (pH 7.2, 0.1 M). After initial fixation, follicles of less than 10 mm diameter were cut in half and those greater than 10 mm diameter were cut into several pieces. All materials were fixed for an additional 2 h, washed three times for 15 min each in phosphate buffer, and stored in the same buffer at 4 C until further processing. Follicle halves and segments were dehydrated through an ascending series of ethanols and embedded in JB-4 methacrylate resin, 1 oriented to obtain cross-sections. Sections (2-^m) were placed on uncoated slides, air dried, and stained with toluidine blue O (TBO) in 1% sodium borate.
Tissue Processing for Alkaline Phosphatase Reaction Individual follicles were dissected free and immersed in 80% ethanol overnight. Dehydration of follicles through an ascending series of formalin-free fresh ethanol baths routinely gave the best results for preservation of alkaline phosphatase activity. Follicles were oriented to obtain
iElectron Microscope Services, Fort Washington, PA 19034. Fisher Scientific, Unionville, ON, Canada, L3R 8G6.
2
cross-sections and embedded in paraffin. Five-micron sections were placed on glass slides and heated on a hot plate at 56 C overnight to melt the paraffin. This step ensures coating of the tissue to protect it from the atmosphere. Sections were kept at 4 C until the reaction was performed.
Alkaline
Phosphatase
Reaction
A modified calcium-cobalt Gomori method (Gomori, 1952) was used to localize alkaline phosphatase activity. In this method, sodium glycerophosphate is the substrate and phosphate ions freed by the action of the enzyme combine with calcium ions to form insoluble calcium phosphate. Sections were deparaffinized in xylene, 2 rehydrated through a descending series of ethanols, and incubated for 2 h at room temperature in a freshly c o m b i n e d s o l u t i o n of 50 mL 0.1 M s o d i u m glycerophosphate, 2 95 mL 0.2 M calcium chloride dihydrate, 2 65 mL 0.5 M magnesium sulfate heptahydrate, 2 20 mL 0.1 M sodium diethylbarbiturate, 2 and 20 mL tap water. Sections were then washed for 1 min in water, incubated for 5 min in 0.06 M cobalt chloride hexahydrate, 2 rinsed for 2 min in water, treated for 5 min in 0.04 M sodium sulfide nonahydrate, 2 and washed for 2 min in water. In this reaction, calcium phosphate is converted first into cobalt phosphate and ultimately to cobalt sulfide, an insoluble black compound readily visible under the microscope. Sections were counterstained with Ehrlich's hematoxylin 2 for 20 min, washed under running tap water for 3 min, blued in a bath of ammonia water, and rinsed in water for 3 min. Slides were allowed to air dry overnight and were mounted with Permount. 2 Negative controls consisted of slides processed through the same procedure except that the substrate sodium glycerophosphate was replaced by water.
RESULTS Morphological features used to estimate stage of follicular development included follicle size, presence of clearly defined theca interna and externa layers, and the appearance of the granulosa layer. Ovaries contained follicles at all stages of development up to and including the Fi, which is the largest and nearest to ovulation. Postovulatory follicles were also present, ranging from POF1 (most recently ovulated) to POF7. For some ovaries, it was difficult to judge the sequence of follicles past POF5 because of size similarity. Developmental changes and alkaline phosphatase activity of the follicles throughout follicular maturation and senescence are summarized in Table 1 and selected examples of the micrographs that provided this information are presented below. Even among the very small follicles (less than 1 mm in diameter and clearly visible only by microscope), several developmental stages were discernible. The smallest follicles examined possessed a very thin theca
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Alkaline phosphatase activity has been demonstrated in theca cells in the pig (Corner, 1948) and sheep (O'Shea et ah, 1980) and has been used to identify thecaderived cells for up to 48 h after ovulation in the ewe (O'Shea et ah, 1980) and up to the 6th wk of pregnancy in the sow (Corner, 1948). The objectives of this study were to document morphological changes in the theca layer through pre- and postovulatory follicular development at the light microscope level, and to assess alkaline phosphatase activity at these developmental stages. If this enzyme activity is confined to the theca interna at all stages of follicular development, alkaline phosphatase may prove useful in histological studies of ovarian function in the domestic hen.
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CHAPEAU ET AL. TABLE 1. Summary of alkaline phosphatase histochemistry in developing hen follicles Developmental stage
Alkaline phosphatase reaction 1
Salient features
Very small follicle
thin, undifferentiated T; cuboidal G layer distinguishable Ti; pseudostratified G layer first visible without microscope; brush border on G clearly defined Ti and Te; 1 to 2 mm diameter 2 to 4 m m diameter thick Te, thin Ti; 4 to 10 mm diameter
Very small follicle
Intensity
Distribution 3
-/+
intermittent in Ti
+ to +++
F8 F7 F6
G layer intact G layer begins to dissociate less cell-cell contact between G
+++ to ++++ +++ to ++++ +++ to ++++
F5
less cell-cell contact between G
+++ to ++++
F4 F3 F2
stratified G layer stratified G layer lower columnar G layer; breakdown of brush border compressed cuboidal G layer; no brush border G layer partially detached from T; folding of basement membrane G layer partially detached from T; folding of basement membrane lumen filling with G basement membrane lost; T and G no longer distinguishable basement membrane lost; T and G no longer distinguishable
++++ ++++ ++++
intermittent in Ti but strong in regions intermittent in Ti but aggregated near capillaries intermittent in Ti thicker than in LWF; continuous around periphery of the follicle intermittent in Ti intermittent in Ti intermittent in Ti but thicker than in F7_8 intermittent in Ti but thicker than in F7_g present throughout the Ti present throughout the Ti present throughout the Ti
++++
present throughout the Ti
+++ to ++++
aggregated near capillaries within Ti
+++
aggregated near capillaries within Ti
+ -/+
sporadic in Ti sporadic in Ti
Very small white follicle Small white follicle Large white follicle Small yellow follicle
Fl POF1 POF2 POF3 POF4 POF5 to 7
+ to +++ + to +++ ++ to ++++
-
iProminent morphological features highlighted; see text for further details. T = theca, Ti : theca interna; Te = theca externa, G = granulosa. intensity scoring: - = no reaction, + = very weak but definite, ++ = moderate, +++ = strong, ++++ = very strong. 3 See results for detailed description of distribution.
layer composed of connective tissue but without distinctive theca interna (Figure 1, left). The granulosa layer, which consisted of a single layer of cuboidal cells with dark nuclei, completely surrounded the oocyte. A prominent pronucleus was visible in the vacuolated oocyte. Alkaline phosphatase activity could not be demonstrated in any follicles at this stage (Table 1). The next stage of development was characterized by a theca externa layer comprised of a typical connective tissue matrix with spindle-shaped fibroblasts, and in some regions an identifiable theca interna lying between the theca externa and granulosa layers. In the earliest stages of its differentiation, the theca was a thinly stratified layer containing large elongated polyhedral cells with large nuclei and frothy vacuolated cytoplasm occupying intermediate or basal positions within the layer (Figure 1, right). The region contained a mixture of vacuolated cells and those with dense cytoplasm interspersed with spindle-shaped fibroblasts in a collagen matrix. Capillaries were found in the theca interna at this stage. The granulosa layer was now pseudostratified. The vacuolated oocyte had no affinity for TBO
stain. Granulosa cell apices had a fuzzy appearance in methacrylate sections, suggesting the presence of a brush border. Although alkaline phosphatase activity could not be demonstrated in most follicles at this stage, very weak activity could be detected in cells of the theca interna of a few follicles (data not shown). Enzyme activity was localized to small groups of cells, giving an uneven, patchy appearance to the reaction. The very small white follicles were the first stage that could be distinguished without a microscope. The main differences between this stage and the previous one was that the fuzzy appearance at the apex of the granulosa cells could now be resolved as a brush border in methacrylate sections. This feature persisted until near the final stages of follicular maturation (F2 stage). Mitotic figures were present but not common. Lipid droplets with an affinity for TBO stain could now be observed at the periphery of the white yolk; the inner part of the yolk was vacuolated as noted for previous stages. In the largest follicles of this category, lipid droplets were more numerous and distributed evenly in the white yolk in paraffin sections.
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Ovarian structure
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In methacrylate sections of small white follicles (Figure 2A), lipids appeared to accumulate into larger droplets but had not yet fused together. The prominent theca externa had differentiated into a more fibroblastic layer and was considerably thicker than the theca interna. The theca interna layer of these follicles (1 to 2 mm) exhibited moderate alkaline phosphatase activity in a zone just outside the granulosa layer (Table 1). No morphological distinction could be made between alkaline phosphatase-positive and -negative cells. In the largest follicles of this category, the reaction was much more intense in the vicinity of capillaries but was still restricted to patches. At this stage as well as at previous stages, the reaction tended to be greatest near the basement membrane and gradually decreased in intensity toward the stromal interface. The alkaline phosphatase reaction of large white follicles was of similar intensity and distribution as that of small white follicles (Table 1). As follicles matured into the small yellow category, the theca became thicker and both the theca externa and a thin theca interna contained cells that stained less intensely than those in very small follicles (compare Figure 1 and Figure 2B), probably due to the accumulation of lipid-like material within their cytoplasm. Both the size and staining intensity of lipid droplets in the yellow yolk were increased due to the fusion of previously coalescing smaller droplets. The region exhibiting alkaline phosphatase activity in the theca interna increased in thickness and now completely surrounded the follicle (Figure 2C). The intensity of the
reaction ranged from weak to strong or very strong (Table 1). Alkaline phosphatase activity in the theca interna was similar in intensity for stages Fg through F5, ranging from strong to very strong. A few mitotic figures could occasionally be seen in the theca interna. By the F2 stage, the granulosa cells possessed a low columnar morphology (Figure 3A). In the Fi follicle (Figure 3B), granulosa cells had assumed a compressed cuboidal profile. Lipidlike accumulations were again present in both granulosa and theca interna cells. The perivitelline layer was now evident as a homogeneous band between the apical membrane of the granulosa cells and the yolk. Between the F4 and F1 stages, the intensity of the alkaline phosphatase reaction was maximal and very uniform, extending throughout the entire theca interna (Table 1). The most recent POF (POF1) was a flattened cupshaped structure with a wide fissure. The lumen was bounded on each side by the granulosa layer that persisted after discharge of the oocyte (Figure 4A). The granulosa layer was partially detached from the theca layer; granulosa cells were vacuolated and had assumed a plump, rounded shape. The basement membrane was folded together with the granulosa cells resulting in a wrinkled appearance. The arrangement of both theca layers was relaxed and undulating with fibroblasts assuming irregular shapes, suggesting a decrease in tension within the wall. In an older POF1, the granulosa layer was completely detached from the basement membrane and had escaped into the lumen of the evacuated follicle (not shown). Developing capillaries
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FIGURE 1. A very small follicle (< 1 mm diameter; left) and follicle wall segment from a slightly larger follicle. The smallest follicle had a well defined cuboidal granulosa layer (G) separated from an undifferentiated theca (T) by a distinct basement membrane. There was a prominent pronucleus (P) within the vacuolated oocyte. The granulosa layer of the larger follicle (right) was pseudostratified and the theca was beginning to differentiate. The theca contained large elongated polyhedral cells (arrow), (methacrylate, TBO, bar = 50 (im).
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FIGURE 2. A) Follicle wall segment from a small white follicle. Granulosa cells (G) presented a brush border (b) at their apex. The well-defined theca interna (Ti) layer contained large vacuolated cells. The theca externa (Te) was very prominent. Note the change in staining properties and aggregation of lipid droplets (L) in the yolk (Y) in comparison with Figure 1 B). Small yellow follicle. The theca interna layer now contained some larger vacuolated cells. The previously coalescing lipid droplets (L) have fused together into larger lipid spheres, (methacrylate, TBO, bar = 50 pm). C. Alkaline phosphatase reaction in a small yellow follicle. Enzyme activity, indicated by an intense dark reaction, was located throughout the theca interna layer. The granulosa and theca externa layers were not reactive, (paraffin, hematoxylin counterstain, bar = 100 jttm).
approached the basement membrane but were never seen to cross it. The alkaline phosphatase reaction (Figure 4B) was very intense and localized to the entire thickness of the theca interna. In POF2 (Figure 4C), the
enzyme reaction was strong and localized perpendicular to the delta-shaped regions
somewhat less intense but still to the vicinity of capillaries surface of the lumen, forming of very dense black deposits.
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Between these regions, the reaction gradually decreased in intensity but was somewhat stronger toward the basement membrane. In POF3, the reaction was very weak and patchy, whereas the lumen became increasingly difficult to distinguish due to filling with granulosa cells. In POF4 (Figure 5A), discrimination between granulosa and theca cells was difficult due to the disappearance of the basement membrane. By this stage, the enzyme reaction was either undetectable or very weak and patchy. In POF 5 to 7, activity was consistently undetectable. In the smallest postovulatory follicle (Figure 5B), large accumulations of lipids were observed in the remaining granulosa cells.
DISCUSSION As first demonstrated in the pig (Corner, 1948), alkaline phosphatase activity in the hen ovary was specific for the theca interna layer in preovulatory follicles. A positive reaction first appeared in very small white follicles less than 1 mm in diameter, just as theca interna differentiated into a clearly recognizable layer. As folliculogenesis progressed, the staining reaction remained confined to the theca interna. Although alkaline phosphatase activity was initially weak and found in patches within the theca interna, it became
apparent in all cells adjacent to the basal lamina separating the theca and granulosa and increased in intensity as follicles developed to the Fi stage (less than 24 h prior to ovulation). Within the postovulatory follicles, alkaline phosphatase activity decreased to undetectable levels by the POF4 to 5 stages (4 to 5 d postovulation). In mammals, Corner (1948) found alkaline phosphatase activity to be a reliable marker for the identification of the contribution of theca cells to the corpus luteum in the pig but not the rabbit, guinea pig, dog, monkey, or human. This variability between species makes it difficult to speculate on the relevance of this enzyme to ovarian structure and function. In a preliminary report of his studies, Corner (1944) suggested that in tissues in which it was not associated with the storage of inorganic phosphate as in bone, the enzyme may be involved in lipid metabolism. The same suggestion was made by Emmel (1945) and Hard (1946) in their studies in small intestine and placenta, respectively. Our observations of differentiation of the theca interna in very small (< 1 mm) white follicles was in contrast to those of Hodges (1974), who reported that the theca had differentiated into two layers in 2-mm but not in 1-mm follicles. In slightly larger follicles, glandular cells were reported to be grouped
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FIGURE 3. A) F 2 preovulatory follicle. Granulosa cells (G) possessed a low columnar morphology with a fluffy appearance at their apex indicating the presence of a brush border (*). Cells of the theca interna (Ti) and externa (Te) were flat and elongated. One cell (arrow) was in telophase in the theca interna layer. B) The F : preovulatory follicle. Granulosa (G) cells had assumed a compressed cuboidal profile and were beginning to lose their cell-cell attachment, giving them a more rounded appearance. Accumulations of lipid-like material were present in both granulosa and theca interna cells. The arrow indicates the perivitelline layer, (methacrylate, TBO, bar = 50 (im).
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FIGURE 4. A) Follicular wall from a POFl. Collapsed follicle walls were bounded by the granulosa (G) layer, theca interna (Ti) and theca externa (Te). Granulosa cells had assumed a rounded, plump shape and were vacuolated. Theca interna and externa appeared to be more relaxed compared to preovulatory follicles, allowing fibroblasts to assume irregular shapes, (methacrylate, TBO, bar = 100 ^m) Inset: Basement membrane (arrow) folded together with granulosa cells. B) Alkaline phosphatase reaction in a POFl follicle. The reaction ranged from intense to very intense and was still limited to the theca interna layer, (paraffin, hematoxylin counterstain, bar = 200 jim). C) Alkaline phosphatase reaction in a POF2 follicle. At this stage the reaction intensity was much stronger in theca interna cells located in the vicinity of capillaries. Between capillaries the reaction was of moderate intensity, becoming slightly stronger toward the basement membrane, (paraffin, hematoxylin counterstain; bar = 100 /mi).
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FIGURE 5. A) Alkaline phosphatase reaction in a POF4 follicle. By this stage the reaction was very weak and patchy, (paraffin, hematoxylin counterstain, bar = 200 (im). B) Follicle wall from a POF8 follicle. Granulosa cells were filled with lipid-like material. Cellular organization of both granulosa and theca interna layers was loose and "relaxed", (methacrylate, TBO; bar = 50 ^m).
into well-defined islands that were evenly spaced around the circumference of the follicle (Dahl, 1971), whereas in older follicles, these cells were reported to be elongated and arranged in irregular, poorly defined patches (Perry et ah, 1978). In the present study, although resolution with the paraffin-embedded tissue did not permit identification of specific cell types, the continuous band of alkaline phosphatase positive cells argues against a correlation between them and the putative glandular cells.
These glandular theca cells have been identified as steroidogenic cells by both morphological criteria (Dahl, 1970) and the demonstration of steroidogenic enzyme activity (Chieffi and Botte, 1965; Woods and Domm, 1966; Wyburn and Bailie, 1966; Narbaitz and de Robertis, 1968; Boucek and Savard, 1970). Indeed, studies based on histochemical detection of A53j3-hydroxysteroid dehydrogenase activity (Davidson et ah, 1979), steroidogenic capacity in vitro (Tilly et ah, 1991a) and immunolocalization of A5-3/3-hydroxysteroid
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1544
CHAPEAU ET AL.
The POF has also been implicated in the control of oviposition through its ability to synthesize prostaglandins (Day and Nalbandov, 1977; Shimada and Saito, 1989). It seems unlikely, however, that alkaline phosphatase activity is related to prostaglandin production, because the former is present in small white, large white, small yellow, and yolk-filled follicle, whereas prostaglandin production is restricted to the two largest
pre- and postovulatory follicles (Shimada and Saito, 1989). In summary, the alkaline phosphatase staining reaction was restricted to cells within the theca interna layer of preovulatory follicles adjacent to the basal lamina separating the granulosa and theca. Alkaline phosphatase activity was detectable as soon as the theca interna could be distinguished and was highest in the yolk-filled follicles. Use of this histochemical marker in combination with other markers of biosynthetic activity in either tissue sections or isolated cell preparations may facilitate functional studies of ovarian theca cells and reveal an as yet unidentified function of theca cells in the avian ovary.
ACKNOWLEDGMENTS The technical assistance p r o v i d e d by Cheryl Anderson-Langmuir is acknowledged. Research funds were provided by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs.
REFERENCES Aitken, R.N.C., 1966. Post-ovulatory development of ovarian follicles in the domestic fowl. Res. Vet. Sci. 7:138-142. Armstrong, D. G., M. F. Davidson, A. B. Gilbert, and J. W. Wells, 1977. Activity of 3/3-hydroxysteroid dehydrogenase in the postovulatory follicle of the domestic fowl (Gallus domesticus). J. Reprod. Fertil. 49:253-259. Bahr, J. M., S.-C. Wang, M. Y. Huang, and F. O. Calvo, 1983. Steroid concentrations in isolated theca and granulosa layers of preovulatory follicles during the ovulatory cycle of the domestic hen. Biol. Reprod. 29:326-334. Boucek, R. J., and K. Savard, 1970. Steroid formation by the avian ovary in vitro {Gallus domesticus). Gen. Comp. Endocrinol. 15:6-11. Chieffi, G., and V. Botte, 1965. The distribution of some enzymes involved in the steroidogenesis of hen's ovary. Experientia 21:16-20. Corner, G. W., 1944. Alkaline phosphatase in the ovarian follicles and corpora lutea. Sci. 100:270-271. Corner, G. W., 1948. Alkaline phosphatase activity in the ovarian follicle and in the corpus luteum. Contrib. Embryol. 32:3-8. Dahl, E., 1970. Studies of the fine structure of ovarian interstitial tissue. 2. The ultrastructure of the thecal gland of the domestic fowl. Z. Zellforsch. Mikrosk. Anat. 109: 195-211. Dahl, E., 1971. Studies of the fine structure of ovarian interstitial tissue. 1. A comparative study of the fine structure of the ovarian interstitial tissue of the rat and the domestic fowl. J. Anat. 108:275-290. Davidson, M. F., A. B. Gilbert, and J. W. Wells, 1979. Activity of ovarian A5-3/3-hydroxysteroid dehydrogenase in the domestic fowl (Gallus domesticus) with respect to age. J. Reprod. Fertil. 57:61-64. Day, S. L., and A. V. Nalbandov, 1977. Presence of prostaglandin F (PGF) in hen follicles and its physiological role in ovulation and oviposition. Biol. Reprod. 16:486-494.
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dehydrogenase in paraffin sections (Nitta et al., 1993) indicate that the theca is likely the sole source of steroids until follicles enter the yolk-filled hierarchy. Two lines of reasoning suggest that alkaline phosphatase activity in the theca was not related to steroidogenic activity. Firstly, enzyme activity increased in intensity as follicles matured from the small white stage to the yolk-filled hierarchy, reaching a maximum in the Fi to F3 follicles, whereas steroidogenesis by the theca decreases as follicles precede from the F5 to Fi stages (Bahr et ah, 1983; Etches and Duke, 1984). Furthermore, the steroidogenic capacity of granulosa cells increases as follicles develop through the F-series hierarchy (Etches, 1984; Nitta et al., 1993), whereas alkaline phosphatase activity was never detected in the granulosa layer at any stage. The distribution of alkaline phosphatase activity in POF in the present study supported the view that both granulosa and theca cells contributed to the avian POF (Aitken, 1966; Fujii and Tamura, 1968; Guraya and Chalana, 1976). Our findings agreed with those of Guraya and Chalana (1976) in the house sparrow, who reported that cells of the theca interna were arranged radially around the granulosa cell mass in early POF and invaded the granulosa cell mass in more advanced POF such that the different cell types became intermixed. Although Guraya and Chalana (1976) believed granulosa and theca cells were distinguishable by differences in staining properties, morphology, and lipid content, the use of alkaline phosphatase as a histochemical marker in the present study provided a more obvious and objective means of identification of thecaderived cells and confirmed that theca and granulosa cells remain anatomically distinct in the POF. Alkaline phosphatase activity decreased with time and was no longer detectable by the POF4 to 5 stage. This decrease may be linked to either a specific decline in functional activity of theca cells or structural regression of the POF. Activity of the A5-3/3-hydroxysteroid dehydrogenase enzyme in the theca of the POF declines rapidly during the first 15 h after ovulation and then falls gradually, becoming undetectable by 50 h (Armstrong et al., 1977). By contrast, A5-3/3-hydroxysteroid dehydrogenase activity in granulosa cells was stable for up to 35 h after ovulation. As the most dramatic decrease in alkaline phosphatase activity in the present study occurred between POF2 and POF3, activity of this enzyme could indeed be related to steroidogenic activity of the POF. In preovulatory follicles, however, the correlation between alkaline phosphatase activity and steroidogenesis was poor.
ALKALINE PHOSPHATASE ACTIVITY IN THE THECA
follicle shifts from the theca layer to granulosa layer with follicular maturation. Biol. Reprod. 48:110-116. O'Shea, J. D., D. G. Cran, and M. F. Hay, 1980. Fate of the theca interna following ovulation in the ewe. Cell Tissue Res. 210:305-319. Perry, M. M., A. B. Gilbert, and A. F. Evans, 1978. Electron microscope observations on the ovarian follicle of the domestic fowl during the rapid growth phase. J. Anat. 125: 481^97. Porter, T. E., B. M. Hargis, J. L. Silsby, and M. E. El Halawani, 1989. Differential steroid production between theca interna and theca externa cells: a three-cell model for follicular steroidogenesis in avian species. Endocrinology 125: 109-116. Robinson, F. E., and R. J. Etches, 1986. Ovarian steroidogenesis during follicular maturation in the domestic fowl (Gallus domesticus). Biol. Reprod. 35:1096-1105. Shimada, K., and N. Saito, 1989. Control of oviposition in poultry. Crit. Rev. Poult. Biol. 2:235-253. Tilly, J. L., K. I. Kowalski, and A. L. Johnson, 1991a. Stage of ovarian follicular development associated with steroidogenic competence in avian granulosa cells. Biol. Reprod. 44:305-314. Tilly, J. L., K. I. Kowalski, and A. L. Johnson, 1991b. Cytochrome P 450 side-chain cleavage (P450SCC) m m e hen ovary. I. P450SCC messenger RNA, immunoactive protein, and enzyme activity in developing granulosa cells. Biol. Reprod. 45:967-974. Woods, J. E., and L. V. Domm, 1966. A histochemical identification of the androgen-producing cells of the domestic fowl and albino rat. Gen. Comp. Endocrinol. 7: 559-570. Wybum, G. M., and A. H. Bailie, 1966. Some observations on the fine structure and histochemistry of the ovarian follicle of the fowl. Pages 30-38 in: Physiology of the Domestic Fowl. C. Horton-Smith, and E. C. Amoroso, ed. Oliver and Boyd, Edinburgh, UK. Yoshimura, Y., T. Okamoto, and T. Tamura, 1989. Enzyme histochemical observations on the ovarian small follicles in relation to the mechanism of atresia in laying hens (Gallus domesticus). Jpn. Poult. Sci. 26:10-16.
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library and Center for Knowledge Management on March 22, 2015
Emmel, V. E., 1945. Alkaline phosphatase in the Golgi zone of absorbing cells of the small intestine. Anat. Rec. 91:39^8. Etches, R. J., 1984. Maturation of ovarian follicles. Pages 51-73 in: Reproductive Biology of Poultry. F. J. Cunningham, P. E. Lake, and D. Hewitt, ed. British Poultry Science Ltd, Longman Group, Harlow, UK. Etches, R. J., and C. E. Duke, 1984. Progesterone, androstenedione and oestradiol content of the theca and granulosa tissues of the four largest ovarian follicles during the ovulatory cycle of the hen. J. Endocrinol. 103:71-76. Fujii, S., and T. Tamura, 1968. Histological observations on post-ovulatory follicles in domestic fowl. J. Fac. Fish. Anim. Husb. Hiroshima Univ. 7:203-233. Gomori, G., 1952. Microscopic Histochemistry. University of Chicago Press, Chicago, IL. Guraya, S. S., and R. K. Chalana, 1976. Morphology of the post-ovulatory follicle of house sparrow. Acta Biol. Acad. Scientarum Hungaricae 27:261-267. Hard, W. L., 1946. A histochemical and quantitative study of phosphatase in the placenta and fetal membranes of the guinea pig. Amer. J. Anat. 78:47-77. Hodges, R. D., 1974. The Histology of the Fowl. Academic Press, New York, NY. Kato, M., K. Shimada, N. Saito, K. Noda, and M. Ohta, 1995. Expression of P450 17a . hydroxylase and P450 aromatase genes in isolated granulosa, theca interna, and theca externa layers of chicken ovarian follicles during follicular growth. Biol. Reprod. 52:405-410. Kowalski, K. I., J. L. Tilly, and A. L. Johnson, 1991. Cytochrome P450 side-chain cleavage (P450 scc ) in the hen ovary. I. Regulation of P450 s c c messenger RNA levels and steroidogenesis in theca cells of developing follicles. Biol. Reprod. 45:955-966. Narbaitz, R., and E. M. de Robertis, 1968. Postnatal evolution of steroidogenic cells in the chick ovary. Histochemie 15: 187-193. Nitta, H., Osawa, Y., and J. M. Bahr, 1991. Multiple steroidogenic cell populations in the thecal layer of preovulatory follicles of the chicken ovary. Endocrinology 129:2033-2040. Nitta, H., Mason, J. I., and J. M. Bahr, 1993. Localization of 3|S-hydroxysteroid dehydrogenase in the chicken ovarian
1545
{Key words: alkaline phosphatase, ovary, theca, hen, follicle) 1996 Poultry Science 75:1536-1545
INTRODUCTION The hen's ovary consists of five to eight large yolkfilled follicles (the F x series with Fi being the largest and F n being the smallest), numerous small yellow follicles (4 to 10 mm in diameter), large white follicles (2 to 4 mm in diameter), small white follicles (less than 2 mm in diameter), and several postovulatory follicles (POF series). The large yolky follicles constitute most of the mass of the ovary and are classified according to their size and proximity to ovulation as Fi (largest) through F5 to F8- A mature preovulatory follicle consists of an oocyte enclosed by the perivitelline layer, which is in turn surrounded by a granulosa layer, basement membrane, theca interna, and theca externa. Production of steroid hormones by these follicles is believed to involve cooperation between three cell types: progesterone precursor provided by granulosa cells is converted to androgen by the theca interna, which is in turn metabolized to estrogen by cells of the theca externa (Porter et ah, 1989; Nitta et al, 1991; Kato et al, 1995).
Received for publication March 8, 1996. Accepted for publication June 19, 1996.
Much less is known about developmental stages in the pool of small follicles. Robinson and Etches (1986) showed that there were maturational differences, in terms of steroid production and response to LH, within the pool of small (< 10 mm) follicles. Due to their large numbers, Robinson and Etches (1986) estimated that small follicles were in fact the principal source of dehydroepiandrosterone and estrogen, and a significant source of androstenedione in the hen's ovary. Several lines of evidence suggest that the theca may be the sole source of steroids in these small follicles (Davidson et ah, 1979; Yoshimura et al, 1989; Kowalski et al, 1991; Tilly et al, 1991a,b; Nitta et al, 1993). When it is first formed, the theca consists of a single layer; differentiation into the theca interna and externa occurs when the follicle reaches approximately 2 mm in size (Hodges, 1974). The smaller classes of follicles have received relatively little attention in morphological studies. In a series of publications investigating the anatomy of the hen's ovary at the electron microscope level, Dahl (1970, 1971) described thecal glands as distinct structures within the theca interna. However, his studies concentrated on ultrastructural detail without reference to stage of follicular development and have not been featured in subsequent studies of ovarian anatomy.
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ABSTRACT Within the hen's ovary, the theca tissue of small follicles that have not yet entered the preovulatory hierarchy is a major contributor to ovarian steroidogenesis. Relatively little is known about developmental stages in the theca layer within this pool of follicles and of the histological properties of this tissue. In pigs and sheep, alkaline phosphatase activity has been identified in the theca of preovulatory follicles and in theca-derived cells in the corpus luteum. The objectives of this study were to document morphological changes in the theca layer through pre- and postovulatory follicular development, and to assess alkaline phosphatase activity at these developmental stages. Ovarian tissue containing small white, large white, small yellow, large yellow, and postovulatory follicles (POF) was obtained from mature White Leghorn hens and embedded in either methacrylate for morphological
ALKALINE PHOSPHATASE ACTIVITY IN THE THECA
MATERIALS AND METHODS Animals Ovaries were obtained from five mature White Leghorn hens (Gallus domesticus) within 10 min after cervical dislocation. The stages of follicular development were identified macroscopically as small white follicles, large white follicles, small yellow follicles, large yellow follicles (F series), and POF.
Tissue Processing for Morphological Assessment Individual pieces of ovarian tissue containing small white, large white, and intact larger follicles were dissected free of most associated stromal tissue and immersed for 1 h in 4% paraformaldehyde, 2% glutaraldehyde in phosphate buffer (pH 7.2, 0.1 M). After initial fixation, follicles of less than 10 mm diameter were cut in half and those greater than 10 mm diameter were cut into several pieces. All materials were fixed for an additional 2 h, washed three times for 15 min each in phosphate buffer, and stored in the same buffer at 4 C until further processing. Follicle halves and segments were dehydrated through an ascending series of ethanols and embedded in JB-4 methacrylate resin, 1 oriented to obtain cross-sections. Sections (2-^m) were placed on uncoated slides, air dried, and stained with toluidine blue O (TBO) in 1% sodium borate.
Tissue Processing for Alkaline Phosphatase Reaction Individual follicles were dissected free and immersed in 80% ethanol overnight. Dehydration of follicles through an ascending series of formalin-free fresh ethanol baths routinely gave the best results for preservation of alkaline phosphatase activity. Follicles were oriented to obtain
iElectron Microscope Services, Fort Washington, PA 19034. Fisher Scientific, Unionville, ON, Canada, L3R 8G6.
2
cross-sections and embedded in paraffin. Five-micron sections were placed on glass slides and heated on a hot plate at 56 C overnight to melt the paraffin. This step ensures coating of the tissue to protect it from the atmosphere. Sections were kept at 4 C until the reaction was performed.
Alkaline
Phosphatase
Reaction
A modified calcium-cobalt Gomori method (Gomori, 1952) was used to localize alkaline phosphatase activity. In this method, sodium glycerophosphate is the substrate and phosphate ions freed by the action of the enzyme combine with calcium ions to form insoluble calcium phosphate. Sections were deparaffinized in xylene, 2 rehydrated through a descending series of ethanols, and incubated for 2 h at room temperature in a freshly c o m b i n e d s o l u t i o n of 50 mL 0.1 M s o d i u m glycerophosphate, 2 95 mL 0.2 M calcium chloride dihydrate, 2 65 mL 0.5 M magnesium sulfate heptahydrate, 2 20 mL 0.1 M sodium diethylbarbiturate, 2 and 20 mL tap water. Sections were then washed for 1 min in water, incubated for 5 min in 0.06 M cobalt chloride hexahydrate, 2 rinsed for 2 min in water, treated for 5 min in 0.04 M sodium sulfide nonahydrate, 2 and washed for 2 min in water. In this reaction, calcium phosphate is converted first into cobalt phosphate and ultimately to cobalt sulfide, an insoluble black compound readily visible under the microscope. Sections were counterstained with Ehrlich's hematoxylin 2 for 20 min, washed under running tap water for 3 min, blued in a bath of ammonia water, and rinsed in water for 3 min. Slides were allowed to air dry overnight and were mounted with Permount. 2 Negative controls consisted of slides processed through the same procedure except that the substrate sodium glycerophosphate was replaced by water.
RESULTS Morphological features used to estimate stage of follicular development included follicle size, presence of clearly defined theca interna and externa layers, and the appearance of the granulosa layer. Ovaries contained follicles at all stages of development up to and including the Fi, which is the largest and nearest to ovulation. Postovulatory follicles were also present, ranging from POF1 (most recently ovulated) to POF7. For some ovaries, it was difficult to judge the sequence of follicles past POF5 because of size similarity. Developmental changes and alkaline phosphatase activity of the follicles throughout follicular maturation and senescence are summarized in Table 1 and selected examples of the micrographs that provided this information are presented below. Even among the very small follicles (less than 1 mm in diameter and clearly visible only by microscope), several developmental stages were discernible. The smallest follicles examined possessed a very thin theca
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Alkaline phosphatase activity has been demonstrated in theca cells in the pig (Corner, 1948) and sheep (O'Shea et ah, 1980) and has been used to identify thecaderived cells for up to 48 h after ovulation in the ewe (O'Shea et ah, 1980) and up to the 6th wk of pregnancy in the sow (Corner, 1948). The objectives of this study were to document morphological changes in the theca layer through pre- and postovulatory follicular development at the light microscope level, and to assess alkaline phosphatase activity at these developmental stages. If this enzyme activity is confined to the theca interna at all stages of follicular development, alkaline phosphatase may prove useful in histological studies of ovarian function in the domestic hen.
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CHAPEAU ET AL. TABLE 1. Summary of alkaline phosphatase histochemistry in developing hen follicles Developmental stage
Alkaline phosphatase reaction 1
Salient features
Very small follicle
thin, undifferentiated T; cuboidal G layer distinguishable Ti; pseudostratified G layer first visible without microscope; brush border on G clearly defined Ti and Te; 1 to 2 mm diameter 2 to 4 m m diameter thick Te, thin Ti; 4 to 10 mm diameter
Very small follicle
Intensity
Distribution 3
-/+
intermittent in Ti
+ to +++
F8 F7 F6
G layer intact G layer begins to dissociate less cell-cell contact between G
+++ to ++++ +++ to ++++ +++ to ++++
F5
less cell-cell contact between G
+++ to ++++
F4 F3 F2
stratified G layer stratified G layer lower columnar G layer; breakdown of brush border compressed cuboidal G layer; no brush border G layer partially detached from T; folding of basement membrane G layer partially detached from T; folding of basement membrane lumen filling with G basement membrane lost; T and G no longer distinguishable basement membrane lost; T and G no longer distinguishable
++++ ++++ ++++
intermittent in Ti but strong in regions intermittent in Ti but aggregated near capillaries intermittent in Ti thicker than in LWF; continuous around periphery of the follicle intermittent in Ti intermittent in Ti intermittent in Ti but thicker than in F7_8 intermittent in Ti but thicker than in F7_g present throughout the Ti present throughout the Ti present throughout the Ti
++++
present throughout the Ti
+++ to ++++
aggregated near capillaries within Ti
+++
aggregated near capillaries within Ti
+ -/+
sporadic in Ti sporadic in Ti
Very small white follicle Small white follicle Large white follicle Small yellow follicle
Fl POF1 POF2 POF3 POF4 POF5 to 7
+ to +++ + to +++ ++ to ++++
-
iProminent morphological features highlighted; see text for further details. T = theca, Ti : theca interna; Te = theca externa, G = granulosa. intensity scoring: - = no reaction, + = very weak but definite, ++ = moderate, +++ = strong, ++++ = very strong. 3 See results for detailed description of distribution.
layer composed of connective tissue but without distinctive theca interna (Figure 1, left). The granulosa layer, which consisted of a single layer of cuboidal cells with dark nuclei, completely surrounded the oocyte. A prominent pronucleus was visible in the vacuolated oocyte. Alkaline phosphatase activity could not be demonstrated in any follicles at this stage (Table 1). The next stage of development was characterized by a theca externa layer comprised of a typical connective tissue matrix with spindle-shaped fibroblasts, and in some regions an identifiable theca interna lying between the theca externa and granulosa layers. In the earliest stages of its differentiation, the theca was a thinly stratified layer containing large elongated polyhedral cells with large nuclei and frothy vacuolated cytoplasm occupying intermediate or basal positions within the layer (Figure 1, right). The region contained a mixture of vacuolated cells and those with dense cytoplasm interspersed with spindle-shaped fibroblasts in a collagen matrix. Capillaries were found in the theca interna at this stage. The granulosa layer was now pseudostratified. The vacuolated oocyte had no affinity for TBO
stain. Granulosa cell apices had a fuzzy appearance in methacrylate sections, suggesting the presence of a brush border. Although alkaline phosphatase activity could not be demonstrated in most follicles at this stage, very weak activity could be detected in cells of the theca interna of a few follicles (data not shown). Enzyme activity was localized to small groups of cells, giving an uneven, patchy appearance to the reaction. The very small white follicles were the first stage that could be distinguished without a microscope. The main differences between this stage and the previous one was that the fuzzy appearance at the apex of the granulosa cells could now be resolved as a brush border in methacrylate sections. This feature persisted until near the final stages of follicular maturation (F2 stage). Mitotic figures were present but not common. Lipid droplets with an affinity for TBO stain could now be observed at the periphery of the white yolk; the inner part of the yolk was vacuolated as noted for previous stages. In the largest follicles of this category, lipid droplets were more numerous and distributed evenly in the white yolk in paraffin sections.
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Ovarian structure
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In methacrylate sections of small white follicles (Figure 2A), lipids appeared to accumulate into larger droplets but had not yet fused together. The prominent theca externa had differentiated into a more fibroblastic layer and was considerably thicker than the theca interna. The theca interna layer of these follicles (1 to 2 mm) exhibited moderate alkaline phosphatase activity in a zone just outside the granulosa layer (Table 1). No morphological distinction could be made between alkaline phosphatase-positive and -negative cells. In the largest follicles of this category, the reaction was much more intense in the vicinity of capillaries but was still restricted to patches. At this stage as well as at previous stages, the reaction tended to be greatest near the basement membrane and gradually decreased in intensity toward the stromal interface. The alkaline phosphatase reaction of large white follicles was of similar intensity and distribution as that of small white follicles (Table 1). As follicles matured into the small yellow category, the theca became thicker and both the theca externa and a thin theca interna contained cells that stained less intensely than those in very small follicles (compare Figure 1 and Figure 2B), probably due to the accumulation of lipid-like material within their cytoplasm. Both the size and staining intensity of lipid droplets in the yellow yolk were increased due to the fusion of previously coalescing smaller droplets. The region exhibiting alkaline phosphatase activity in the theca interna increased in thickness and now completely surrounded the follicle (Figure 2C). The intensity of the
reaction ranged from weak to strong or very strong (Table 1). Alkaline phosphatase activity in the theca interna was similar in intensity for stages Fg through F5, ranging from strong to very strong. A few mitotic figures could occasionally be seen in the theca interna. By the F2 stage, the granulosa cells possessed a low columnar morphology (Figure 3A). In the Fi follicle (Figure 3B), granulosa cells had assumed a compressed cuboidal profile. Lipidlike accumulations were again present in both granulosa and theca interna cells. The perivitelline layer was now evident as a homogeneous band between the apical membrane of the granulosa cells and the yolk. Between the F4 and F1 stages, the intensity of the alkaline phosphatase reaction was maximal and very uniform, extending throughout the entire theca interna (Table 1). The most recent POF (POF1) was a flattened cupshaped structure with a wide fissure. The lumen was bounded on each side by the granulosa layer that persisted after discharge of the oocyte (Figure 4A). The granulosa layer was partially detached from the theca layer; granulosa cells were vacuolated and had assumed a plump, rounded shape. The basement membrane was folded together with the granulosa cells resulting in a wrinkled appearance. The arrangement of both theca layers was relaxed and undulating with fibroblasts assuming irregular shapes, suggesting a decrease in tension within the wall. In an older POF1, the granulosa layer was completely detached from the basement membrane and had escaped into the lumen of the evacuated follicle (not shown). Developing capillaries
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FIGURE 1. A very small follicle (< 1 mm diameter; left) and follicle wall segment from a slightly larger follicle. The smallest follicle had a well defined cuboidal granulosa layer (G) separated from an undifferentiated theca (T) by a distinct basement membrane. There was a prominent pronucleus (P) within the vacuolated oocyte. The granulosa layer of the larger follicle (right) was pseudostratified and the theca was beginning to differentiate. The theca contained large elongated polyhedral cells (arrow), (methacrylate, TBO, bar = 50 (im).
1540
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FIGURE 2. A) Follicle wall segment from a small white follicle. Granulosa cells (G) presented a brush border (b) at their apex. The well-defined theca interna (Ti) layer contained large vacuolated cells. The theca externa (Te) was very prominent. Note the change in staining properties and aggregation of lipid droplets (L) in the yolk (Y) in comparison with Figure 1 B). Small yellow follicle. The theca interna layer now contained some larger vacuolated cells. The previously coalescing lipid droplets (L) have fused together into larger lipid spheres, (methacrylate, TBO, bar = 50 pm). C. Alkaline phosphatase reaction in a small yellow follicle. Enzyme activity, indicated by an intense dark reaction, was located throughout the theca interna layer. The granulosa and theca externa layers were not reactive, (paraffin, hematoxylin counterstain, bar = 100 jttm).
approached the basement membrane but were never seen to cross it. The alkaline phosphatase reaction (Figure 4B) was very intense and localized to the entire thickness of the theca interna. In POF2 (Figure 4C), the
enzyme reaction was strong and localized perpendicular to the delta-shaped regions
somewhat less intense but still to the vicinity of capillaries surface of the lumen, forming of very dense black deposits.
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Between these regions, the reaction gradually decreased in intensity but was somewhat stronger toward the basement membrane. In POF3, the reaction was very weak and patchy, whereas the lumen became increasingly difficult to distinguish due to filling with granulosa cells. In POF4 (Figure 5A), discrimination between granulosa and theca cells was difficult due to the disappearance of the basement membrane. By this stage, the enzyme reaction was either undetectable or very weak and patchy. In POF 5 to 7, activity was consistently undetectable. In the smallest postovulatory follicle (Figure 5B), large accumulations of lipids were observed in the remaining granulosa cells.
DISCUSSION As first demonstrated in the pig (Corner, 1948), alkaline phosphatase activity in the hen ovary was specific for the theca interna layer in preovulatory follicles. A positive reaction first appeared in very small white follicles less than 1 mm in diameter, just as theca interna differentiated into a clearly recognizable layer. As folliculogenesis progressed, the staining reaction remained confined to the theca interna. Although alkaline phosphatase activity was initially weak and found in patches within the theca interna, it became
apparent in all cells adjacent to the basal lamina separating the theca and granulosa and increased in intensity as follicles developed to the Fi stage (less than 24 h prior to ovulation). Within the postovulatory follicles, alkaline phosphatase activity decreased to undetectable levels by the POF4 to 5 stages (4 to 5 d postovulation). In mammals, Corner (1948) found alkaline phosphatase activity to be a reliable marker for the identification of the contribution of theca cells to the corpus luteum in the pig but not the rabbit, guinea pig, dog, monkey, or human. This variability between species makes it difficult to speculate on the relevance of this enzyme to ovarian structure and function. In a preliminary report of his studies, Corner (1944) suggested that in tissues in which it was not associated with the storage of inorganic phosphate as in bone, the enzyme may be involved in lipid metabolism. The same suggestion was made by Emmel (1945) and Hard (1946) in their studies in small intestine and placenta, respectively. Our observations of differentiation of the theca interna in very small (< 1 mm) white follicles was in contrast to those of Hodges (1974), who reported that the theca had differentiated into two layers in 2-mm but not in 1-mm follicles. In slightly larger follicles, glandular cells were reported to be grouped
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FIGURE 3. A) F 2 preovulatory follicle. Granulosa cells (G) possessed a low columnar morphology with a fluffy appearance at their apex indicating the presence of a brush border (*). Cells of the theca interna (Ti) and externa (Te) were flat and elongated. One cell (arrow) was in telophase in the theca interna layer. B) The F : preovulatory follicle. Granulosa (G) cells had assumed a compressed cuboidal profile and were beginning to lose their cell-cell attachment, giving them a more rounded appearance. Accumulations of lipid-like material were present in both granulosa and theca interna cells. The arrow indicates the perivitelline layer, (methacrylate, TBO, bar = 50 (im).
1542
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FIGURE 4. A) Follicular wall from a POFl. Collapsed follicle walls were bounded by the granulosa (G) layer, theca interna (Ti) and theca externa (Te). Granulosa cells had assumed a rounded, plump shape and were vacuolated. Theca interna and externa appeared to be more relaxed compared to preovulatory follicles, allowing fibroblasts to assume irregular shapes, (methacrylate, TBO, bar = 100 ^m) Inset: Basement membrane (arrow) folded together with granulosa cells. B) Alkaline phosphatase reaction in a POFl follicle. The reaction ranged from intense to very intense and was still limited to the theca interna layer, (paraffin, hematoxylin counterstain, bar = 200 jim). C) Alkaline phosphatase reaction in a POF2 follicle. At this stage the reaction intensity was much stronger in theca interna cells located in the vicinity of capillaries. Between capillaries the reaction was of moderate intensity, becoming slightly stronger toward the basement membrane, (paraffin, hematoxylin counterstain; bar = 100 /mi).
Downloaded from http://ps.oxfordjournals.org/ at UCSF Library and Center for Knowledge Management on March 22, 2015
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FIGURE 5. A) Alkaline phosphatase reaction in a POF4 follicle. By this stage the reaction was very weak and patchy, (paraffin, hematoxylin counterstain, bar = 200 (im). B) Follicle wall from a POF8 follicle. Granulosa cells were filled with lipid-like material. Cellular organization of both granulosa and theca interna layers was loose and "relaxed", (methacrylate, TBO; bar = 50 ^m).
into well-defined islands that were evenly spaced around the circumference of the follicle (Dahl, 1971), whereas in older follicles, these cells were reported to be elongated and arranged in irregular, poorly defined patches (Perry et ah, 1978). In the present study, although resolution with the paraffin-embedded tissue did not permit identification of specific cell types, the continuous band of alkaline phosphatase positive cells argues against a correlation between them and the putative glandular cells.
These glandular theca cells have been identified as steroidogenic cells by both morphological criteria (Dahl, 1970) and the demonstration of steroidogenic enzyme activity (Chieffi and Botte, 1965; Woods and Domm, 1966; Wyburn and Bailie, 1966; Narbaitz and de Robertis, 1968; Boucek and Savard, 1970). Indeed, studies based on histochemical detection of A53j3-hydroxysteroid dehydrogenase activity (Davidson et ah, 1979), steroidogenic capacity in vitro (Tilly et ah, 1991a) and immunolocalization of A5-3/3-hydroxysteroid
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B
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CHAPEAU ET AL.
The POF has also been implicated in the control of oviposition through its ability to synthesize prostaglandins (Day and Nalbandov, 1977; Shimada and Saito, 1989). It seems unlikely, however, that alkaline phosphatase activity is related to prostaglandin production, because the former is present in small white, large white, small yellow, and yolk-filled follicle, whereas prostaglandin production is restricted to the two largest
pre- and postovulatory follicles (Shimada and Saito, 1989). In summary, the alkaline phosphatase staining reaction was restricted to cells within the theca interna layer of preovulatory follicles adjacent to the basal lamina separating the granulosa and theca. Alkaline phosphatase activity was detectable as soon as the theca interna could be distinguished and was highest in the yolk-filled follicles. Use of this histochemical marker in combination with other markers of biosynthetic activity in either tissue sections or isolated cell preparations may facilitate functional studies of ovarian theca cells and reveal an as yet unidentified function of theca cells in the avian ovary.
ACKNOWLEDGMENTS The technical assistance p r o v i d e d by Cheryl Anderson-Langmuir is acknowledged. Research funds were provided by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs.
REFERENCES Aitken, R.N.C., 1966. Post-ovulatory development of ovarian follicles in the domestic fowl. Res. Vet. Sci. 7:138-142. Armstrong, D. G., M. F. Davidson, A. B. Gilbert, and J. W. Wells, 1977. Activity of 3/3-hydroxysteroid dehydrogenase in the postovulatory follicle of the domestic fowl (Gallus domesticus). J. Reprod. Fertil. 49:253-259. Bahr, J. M., S.-C. Wang, M. Y. Huang, and F. O. Calvo, 1983. Steroid concentrations in isolated theca and granulosa layers of preovulatory follicles during the ovulatory cycle of the domestic hen. Biol. Reprod. 29:326-334. Boucek, R. J., and K. Savard, 1970. Steroid formation by the avian ovary in vitro {Gallus domesticus). Gen. Comp. Endocrinol. 15:6-11. Chieffi, G., and V. Botte, 1965. The distribution of some enzymes involved in the steroidogenesis of hen's ovary. Experientia 21:16-20. Corner, G. W., 1944. Alkaline phosphatase in the ovarian follicles and corpora lutea. Sci. 100:270-271. Corner, G. W., 1948. Alkaline phosphatase activity in the ovarian follicle and in the corpus luteum. Contrib. Embryol. 32:3-8. Dahl, E., 1970. Studies of the fine structure of ovarian interstitial tissue. 2. The ultrastructure of the thecal gland of the domestic fowl. Z. Zellforsch. Mikrosk. Anat. 109: 195-211. Dahl, E., 1971. Studies of the fine structure of ovarian interstitial tissue. 1. A comparative study of the fine structure of the ovarian interstitial tissue of the rat and the domestic fowl. J. Anat. 108:275-290. Davidson, M. F., A. B. Gilbert, and J. W. Wells, 1979. Activity of ovarian A5-3/3-hydroxysteroid dehydrogenase in the domestic fowl (Gallus domesticus) with respect to age. J. Reprod. Fertil. 57:61-64. Day, S. L., and A. V. Nalbandov, 1977. Presence of prostaglandin F (PGF) in hen follicles and its physiological role in ovulation and oviposition. Biol. Reprod. 16:486-494.
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dehydrogenase in paraffin sections (Nitta et al., 1993) indicate that the theca is likely the sole source of steroids until follicles enter the yolk-filled hierarchy. Two lines of reasoning suggest that alkaline phosphatase activity in the theca was not related to steroidogenic activity. Firstly, enzyme activity increased in intensity as follicles matured from the small white stage to the yolk-filled hierarchy, reaching a maximum in the Fi to F3 follicles, whereas steroidogenesis by the theca decreases as follicles precede from the F5 to Fi stages (Bahr et ah, 1983; Etches and Duke, 1984). Furthermore, the steroidogenic capacity of granulosa cells increases as follicles develop through the F-series hierarchy (Etches, 1984; Nitta et al., 1993), whereas alkaline phosphatase activity was never detected in the granulosa layer at any stage. The distribution of alkaline phosphatase activity in POF in the present study supported the view that both granulosa and theca cells contributed to the avian POF (Aitken, 1966; Fujii and Tamura, 1968; Guraya and Chalana, 1976). Our findings agreed with those of Guraya and Chalana (1976) in the house sparrow, who reported that cells of the theca interna were arranged radially around the granulosa cell mass in early POF and invaded the granulosa cell mass in more advanced POF such that the different cell types became intermixed. Although Guraya and Chalana (1976) believed granulosa and theca cells were distinguishable by differences in staining properties, morphology, and lipid content, the use of alkaline phosphatase as a histochemical marker in the present study provided a more obvious and objective means of identification of thecaderived cells and confirmed that theca and granulosa cells remain anatomically distinct in the POF. Alkaline phosphatase activity decreased with time and was no longer detectable by the POF4 to 5 stage. This decrease may be linked to either a specific decline in functional activity of theca cells or structural regression of the POF. Activity of the A5-3/3-hydroxysteroid dehydrogenase enzyme in the theca of the POF declines rapidly during the first 15 h after ovulation and then falls gradually, becoming undetectable by 50 h (Armstrong et al., 1977). By contrast, A5-3/3-hydroxysteroid dehydrogenase activity in granulosa cells was stable for up to 35 h after ovulation. As the most dramatic decrease in alkaline phosphatase activity in the present study occurred between POF2 and POF3, activity of this enzyme could indeed be related to steroidogenic activity of the POF. In preovulatory follicles, however, the correlation between alkaline phosphatase activity and steroidogenesis was poor.
ALKALINE PHOSPHATASE ACTIVITY IN THE THECA
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