Sites of Steroid Synthesis in the Ovary of the Cyclic Hamster: A Histochemical Study SRINIVAS K. SAIDAPUR ' AND GILBERT S. CREENWALD Departments of Physiology and Gynecology and Obstetrics, Ralph L. Smith Research Center, University ofKansas Medical Center, Kansas City, Kansas 66103, U.S.A.
ABSTRACT The histochemistry of lipids, A5-3P-,20a and 17P-hydroxy-steroid dehydrogenases, glucose-6-phosphate and isocitrate dehydrogenases, NADH and NADPH-diaphorases and acid phosphatase has been studied in the ovary of the cyclic hamster. None of the enzymes studied was present in the preantral follicles on day 1 (day of ovulation) or any day of the 4-day cycle. With the development of antral follicles (on day 3) a few fine lipid droplets appeared in the granulosa cells. The theca interna exhibited moderate amounts of NADH-diaphorase, A5-3P-HSDH,17P-HSDH and slight NADPH-diaphorase, G-6-PDH and ICDH, whereas the granulosa cells of antral follicles showed weak A5-3P-HSDH,NADHdiaphorase and moderate 17P-HSDHbut intense NADPH-diaphorase, G-6-PDH and ICDH activities. All enzyme activities in both theca and granulosa became more pronounced on proestrus (day 4). In the corpora lutea (CL) all the above enzymes were present on day 1and increased on day 2, followed by a rapid decline on days 3 and 4. Lipids were sparsely represented in CL on days 1 and 2 but showed marked accumulations on days 3 and 4. Likewise, acid phosphatase increased on days 3 and 4. The enzyme 2001HSDH was not detectable in the CL. The interstitium showed moderate amounts of lipids, A5-3P-HSDHand NADHdiaphorase while 17P-HSDH,NADPH-diaphorase, G-6-PDH and ICDH were only weakly present. There were no appreciable changes in the lipid and various enzymes in the interstitium throughout the cycle except for A5-3/3-HSDHwhich increased slightly on the afternoon of day 4. Acid phosphatase was not detected in appreciable amounts in the interstitium throughout the cycle. These findings suggest that preantral follicles are unlikely to contribute to steroidogenesis. The antral follicles are the principal source of estrogen, with the granulosa being the major site of synthesis. The interstitium can serve as a major source of progesterone (P) while the theca interna and granulosa cells of antral follicles may form additional sites for P synthesis on the afternoon of day 4. The CL in the hamster are very short-lived and steroidogenesis in them may proceed beyond the synthesis of progesterone. The present findings are correlated with the steroid levels found during the cycle. Appropriate comparisons are also made with the rat ovary. The histochemistry of the mammalian ovary has been studied extensively in several species (references in Baillie e t al., '66). However, for the hamster there are only isolated studies on the ovary describing the distribution of lipids and cholesterol (Knigge and Leathem, '56; Guraya and Greenwald, '66) or A53&hydroxysteroid dehydrogenase (Baillie et al., '66; Blaha and Leavitt, '70, '74) and 17P-hydroxysteroid dehydrogenase (Blaha and Leavitt, AM. J. ANAT. (1978) 151: 71-86.
'74). Other enzymes related directly or indirectly to steroidogenesis, such as glucose-6phosphate dehydrogenase (G8-PDH) and isoAccepted August 11, '77. I Supported by an International Postdoctoral Fellowahip of the Population Council. Biomedical Division, Nen York, U.S.A. Permanent oddrear: Deoartment of Zoolow.KarnatakUnivernitv. - . Dhawar .580 003, India. Send reprint requeatsto Dr. G . 8. Greenwald, Departmentof Physiology, Univenity of Kamas Medical Center. Kanaae City, Kanm 66103.
71
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SRINIVAS K. SAIDAPUR AND GILBERT S. GREENWALD
citrate dehydrogenase (ICDH), acid phosphatase, etc., have not been studied in the hamster ovary. The present paper is a systematic study of the histochemical changes in the ovary of the cyclic hamster dealing with (1)lipids (steroid hormone precursors) ; (2) steroid dehydrogenases, A5-3P-hydroxysteroid dehydrogenase (3P-HSDH) and 17P-hydroxysteroid dehydrogenase (17P-HSDH) (involved in steroid hormone biosynthesis) with reference to both distribution and substrate specificity; (3) G-6PDH and ICDH, which are involved in carbohydrate metabolism but are known to be important sources generating NADPH needed for steroid hydroxylations; and (4) acid phosphatase, a lysosomal enzyme. The distribution was also studied of both NADH and NADPH diaphorases, which are prerequisites for the histochemical localization of NAD- and NADP-linked dehydrogenases. The changes in these enzyme activities are correlated with (1)the development of follicles, (2) the formation and fate of corpora lutea (CL), and (3) patterns of steroids in blood and ovary as reflected in published and unpublished studies from this laboratory. An additional objective of this paper is to compare and contrast these findings with the ovarian histochemistry of the rat and other mammalian species. MATERIALS AND METHODS
Adult female golden h a m s t e r s (Mesocricetus auratus) weighing 80-120 g and maintained on a 14-hour light: 10-hour-dark schedule were used after three to four consecutive 4-day cycles with eight to ten animals in each group. Day 1is defined a s the day of ovulation. Hamsters were killed on days 1to 4 a t 0900 hours and on day 4 animals were also killed a t 1200, 1500 (proestrus), 1800 and 2300 hours (estrus). The animals were killed by decapitation and the ovaries were excised immediately and frozen over dry ice. Sections were cut a t 14 pm thickness in a cryostat maintained a t -2O"C, and processed for different histochemical tests. The lipids were stained using Sudan black B or oil red 0. The procedure for 3P-HSDH and 178-HSDH was according to Baillie et al., ('661, using different substrates (table 2). The details of the method for 3P-HSDH, G-6-PDH and diaphorases is similar to that described previously (Bara, '65; Saidapur and Nadkarni, '72).
The procedure for ICDH and acid phosphatase was as described by Pearse ('72). Appropriate controls were employed for all tests (Pearse, "72). For the hamster, attempts to demons t r a t e 20a-hydroxysteroid dehydrogenase (20a-HSDH) following the method of Balogh ('64) were unsuccessful, whereas the same incubation medium demonstrated enzyme activity in the rat ovaries which served as controls. OBSERVATIONS
The distribution and intensity of lipids and different enzyme activities observed on different days of the cycle are summarized in tables 1 and 2 and therefore only a brief description is given below. 1. Lipids A detailed description of the distribution of lipids and cholesterol has been reported previously for the hamster ovary (Guraya and Greenwald, '65). The present findings are essentially similar. The distribution and intensity-pattern of lipids with both Sudan black B and oil red 0 was the same. Normal preantral follicles did not contain any lipid droplets in theca or in granulosa cells. However, with the formation of antral follicles on days 3 and 4 of the cycle, the granulosa cells contained a few fine lipid droplets, while the thecal cells were negative (fig. 1)to Sudan black B or oil red 0. The newly formed CL (fig. 2) did not contain appreciable amounts of lipid droplets on day 1 of the cycle but they increased progressively on day 2 and reached their maximum (fig. 3) on day 4. The interstitium contained moderate amounts of lipid droplets throughout the cycle and did not show any appreciable changes during the cycle. The granulosa cells of atretic follicles (both preantral and antral) contained large coarse lipid droplets (fig. 4). 2. Diaphorases
(A) NADH-diaphorase showed a very specific distribution (table 1).The diaphorase was absent in both theca and granulosa cells of the preantral follicles while it appeared on the transformation into antral follicles (figs. 5,6). The intensity of diaphorase activity was distinctly greater in the theca interna than in granulosa cells. The CL contained diaphorase activity on all days of the cycle; however, the enzyme was maximal on day 2 and thereafter decreased progressively. Diaphorase activity in the interstitiurn was essentially the
73
STEROIDOGENIC SITES IN HAMSTER OVARY TABLE 1
Distribution of lipids, diaphorases, G-6-PDH,ICDH, and acid phosphatase in the hamster ovary Enzyme/lipid
1. Lipida
(Sudanblack B or oil red 0) 2. NADH-diaphorase
Day of cycle
Preantral follicle
TWG'
+
4 1 2
-
1 2
3 4
1 n L
3 4
5. Acid phosphatase
G
(G)
1
3
4. G-6-PDHIICDH
Th
Atretic follicle
2 3
4
3. NADPH-diaphorase
Antral follicle
1 2
3 4
+
+ ++ + ++ ++++
++ ++ ++ ++ ++ ++ ++ ++ ++ ++
CL'
IT 3
+ + ++ ++++ + +++ ++ + + +++ ++ + + +++ ++ + + +++ ++
+++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + + + ++ -
+ + + -
' Th, Theea interna; G, granuloea. Corpora lutea. Interstitiurn. Intensity of reaction is graded (-) to (+ + + +); (-) denotes the abaence of reaction and ( + + + + ) , a maximal reaction.
same on all days of the cycle. The granulosa of atretic follicles contained appreciable amounts of diaphorase. (B) NADPH-diaphorase.The preantral follicles completely lacked this enzyme. In the antral follicles the distribution and intensity of NADPH diaphorase was inversely related to NADH diaphorase in that the theca interna cells contained only a slight amount of NADPH diaphorase in contrast to the granulosa cells (fig. 7). However, the distribution of the NADPH diaphorase was similar to that of NADH diaphorase in CL, interstitial gland cells and atretic follicles, except that the intensity of formazan deposition was slightly less (table 1). 3. G-6-PDH and ZCDH activities (figs. 8, 9, 10) Both these enzymes are NADP-dependent and showed a similar distribution and intensity-pattern.The intensity and distribution of both G-6-PDH and ICDH resembled that of NADPH diaphorase, especially in the antral follicles and CL (figs. 8, 9) but the activities were markedly lower in the interstitium (table 1) and also in follicles undergoing atresia. The enzyme activities in the granulosa cells of the antral follicles were inten-
sified on day 4, reaching their maximum by 2300 hours (fig. 10). 4. A5-3P-HSDHenzyme activity
In general, the distribution of 3P-HSDH paralleled that of NADH diaphorase, although the intensity of the former reaction was slightly lower. The preantral follicles lacked 3P-HSDH (fig. 11).With the development of antral follicles, enzyme activity appeared in the theca interna and in the granulosa cells (fig. 121, with maximal values (fig. 13) reached on day 4. The enzyme activity in the CL was maximal on day 2 (fig. 14) but decreased rapidly by day 3 and was scarcely represented on day 4 (fig. 15). The interstitial 3pHSDH appeared the same from day 1 to 3 of the cycle and increased slightly on day 4. Atretic antral follicles contained some enzyme activity in both theca interna and granulosa cells, while in the case of preantral atretic follicles the activity was seen only in the granulosa cells. The hamster ovary is able to utilize a variety of substrates for the demonstration of 3P-HSDH (table 2). Of the five substrates used, pregnenolone, 17a-hydroxypregnenolone and 16-dehydropregnenolone produced a formazan deposition of equal magnitude while
74
SRINIVAS K. SAIDAPUR AND GILBERT S. GREENWALD TABLE 2
A53p-HSDH activity in the hamster ouary Substrate
1. Pregnenolone 2. 17a (OH) pregnenolone 3. 16-dehydropregnenolone 4. Dehydroepiandrosterone 5. Epiandrosterone
Day of cycle
Preantral follicle
ThIG
1 2
3 4 1
2 3 4
6. Control
1 2 3
Antral fo11ic1e Th G
-
-
-
-
+ ++
+ ++ +++ +++ + + ++
+
+ ++
Atretic fo11ic1e
(GI
CL
IT
++ +++ +++ +++ ++ +++++++ -
+ ++ ++ -
++ ++ ++
+++ -
4 Intensityofreactioniagraded (-) to ( +
+ + + ) ; (-)denotes theabsenceofenzymeactivity,and ( +i + +),amaximalreaction
dehydroepiandrosterone and epiandrosterone gave a slightly less intense reaction.
5. 17P-HSDH enzyme activity The 17P-HSDH was also NAD-dependent and the cellular sites of its distribution resembled 3P-HSDH. The preantral follicles completely lacked any enzyme activity. With the development of antral follicles on day 3, 17P-HSDH appeared in both theca interna and granulosa cells. On day 4 there was a slight increase in thecal 17P-HSDH but the enzyme activity increased markedly in the granulosa cells (fig. 16).The enzyme was also present in the interstitial gland cells but was poorly represented on all days of the cycle. The activity of 17P-HSDHin the CL resembled 3PHSDH, in that the CL showed 17P-HSDH on the day of their formation (day l),increased slightly on day 2 and declined markedly on subsequent days. Both testosterone and 17Pestradiol were utilized with equal magnitude.
by a slight decrease on day 4. The atretic granulosa cells of preantral as well as antral follicles contained abundant acid phosphatase (fig. 19).
Comparison with the rat A. Antral Follicles. In the rat, granulosa cells of antral follicles do not contain any appreciable amount of lipid, while theca interna cells contain lipid (fig. 20) and cholesterol. In contrast, the theca interna cells of antral follicles in the hamster do not contain lipid or cholesterol. The distribution of NADH-diaphorase, 3P-HSDH and 17P-HSDHare similar in both hamster and rat antral follicles (fig. 21), except that the granulosa cells of the hamster are more reactive than in the rat. However, striking differences exist with regard to the distribution of NADPH-diaphorase, G-6-PDH and ICDH enzymes (figs. 22, 23). Unlike the antral follicles of the rat, in the hamster theca interna cells exhibit markedly low enzyme activity while the granulosa cells of these folli6 . Acid phosphatase cles exhibit the strongest activity. Acid phosAcid phosphatase was not detectable in the phatase is not present in the healthy antral preantral (fig. 17) follicles and was present in follicles of either rat (Lobel et al., '61) or hamonly trace amounts in the granulosa cells of ster. B. Corpora Lutea. The CL of hamster and antral follicles on day 3, with a slight increase on day 4. The theca of antral follicles rat ovary contain diaphorases, 3P-HSDH,17pcompletely lacked acid phosphatase. The in- HSDH, G-6-PDH and ICDH enzymes. Howterstitium contained only trace amounts of ever, the CL of the hamster are very shortacid phosphatase, with no changes observed lived (persisting 4-5 days), unlike those of the throughout the cycle. The newly formed CL on rat which persist for three or more cycles. Furday 1 did not contain any appreciable amount thermore, older and degenerating CL in the of the enzyme. On day 2 the CL contained rat ovary develop 20a-HSDH activity while in some acid phosphatase, with the maximum the hamster degenerating CL do not exhibit reaction developing on day 3 (fig. 18), followed this enzyme activity. The first generation of
STEROIDOGENIC SITES IN HAMSTER OVARY
75
do not. I t was reported previously (Guraya and Greenwald, '65) that injection of PMS into hamsters results in depletion of lipids and cholesterol from the granulosa cells, indicating that the granulosa cells of the antral follicles are capable of responding to gonadotropins by secreting steroid hormones. The Occurrence of A5-3P-HSDHin the theca interna and granulosa of the antral follicles suggests their capacity to oxidize A5-3P-hydroxysteroids to A'-3-ketosteroids, a step that occurs during the biosynthesis of all the biologically active steroid hormones. Generally, 3P-HSDH activity is associated with the production of progesterone in the ovary and therefore its occurrence in the theca interna and granulosa cells of the antral follicles suggests the capacity of both cell types to synthesize the hormone. Incubation in vitro of granulosa and thecal cells of hamster preovulatory follicles confirms their independent ability to produce progesterone (Makris and Ryan, '75). DISCUSSION The occurrence of 17P-HSDHin both theca Preantral follicles interna and granulosa cells of antral follicles Preantral follicles in the hamster ovary suggests the possible interconversions of 17Plack lipids, cholesterol, steroid dehydroge- hydroxysteroids to 17-ketosteroids. The ennases and other enzymes related to steroido- zyme 17P-HSDH is generally associated with genesis both in the granulosa and thecal cells. estrogen synthesis and its occurrence in these This indicates that these follicles do not cells indicates their capacity to synthesize possess any steroidogenic potentiality a t this estrogen(s). Furthermore, it is interesting stage of their growth and development. Thus that this enzyme activity is greater in granthe contribution by the preantral follicles of ulosa cells than in theca interna cells. It any steroid hormones during the estrous cycle seems apparent that granulosa cells are more is not likely. However, on day 1 of the cycle, capable of synthesizing estrogens than the before the appearance of antral follicles, low theca interna cells surrounding them. This levels of estrogen are detected in the blood conclusion is also based on the fact that both (Baranczuk and Greenwald, '73; Saidapur and G-6-PDH and ICDH enzymes are characterisGreenwald, '781, suggesting a n extra-follicu- tically abundant in the granulosa cells compared to the theca interna cells. It is well lar source of estrogen on day 1 of the cycle. known that both ICDH and G-6-PDH are Antral follicles important sources of NADPH needed for hyWith the formation of antral follicles (by droxylations during steroidogenesis (McKerns, the evening of day 2 or the morning of day 3), '69; Wiest and Kidwell, '69). Recent culture important changes take place in the theca in- studies of Makris and Ryan ('75) on isolated terna and granulosa cells. The theca interna granulosa and theca cells of hamster antral does not contain appreciable amounts of lipids follicles indicates a synergism between the and reacts negatively to the Schultz test for two cell types for the production of maximal cholesterol and/or its esters, while the gran- estrogen, while more recent studies by the ulosa cells contain sparse lipids and choles- same authors (Makris and Ryan, '77) show terol and its esters (Guraya and Greenwald, that maximal aromatase activity of the pre'65). This is unlike the situation in many other ovulatory follicle is associated with the granspecies such as the rat, bat, cat, dog and Amer- ulosa cells rather than the theca interna cells. ican opossum (Guraya and Greenwald, '641, Thus, the present histochemical observations wherein the theca interna cells contain lipids are in close agreement with the findings in and cholesterol but the normal granulosa cells vitro. Furthermore, the rise in circulating and
CL in the rat do not contain any appreciable amount of acid phosphatase, while those belonging to the second generation (formed 4-8 days previously) show conspicuous acid phosphatase activity. In the hamster, CL begin to show acid phosphatase activity as early as day 2 with a pronounced increase on day 3. C. Interstitial Gland Cells. The interstitial gland cells of both species contain diaphorases, 3P-HSDH, 17P-HSDH, G-6-PDH and ICDH. However, NADPH-diaphorase and NADP-linked enzymes such as G-6-FDH and ICDH are poorly represented in the interstitium of hamster ovary, unlike the situation in the rat ovary. D. The overall reactivity for 3P-HSDH and 17P-HSDHenzymes was considerably lower in the hamster ovary, so that the intensity of staining in a 14-pm section incubated for two to three hours was weaker than in 14-pm sections of rat ovary incubated for only 30 to 45 minutes.
76
SRINIVAS K. SAIDAPUR AND GILBERT S. GREENWALD
ovarian estrogen concentrations on days 3 and 4 (Shaikh, '72; Baranczuk and Greenwald, '73; Saidapur and Greenwald, '78) correlates with the development of antral follicles (Greenwald, '61) and the appearance of the various enzyme systems described above. The absence of any appreciable amount of acid phosphatase in either theca interna or granulosa cells indicates the apparent lack of any degenerating cells in the healthy antral follicles. Acid phosphatase, a lysosomal enzyme, is generally associated with autolytic processes and therefore is found only in degenerating or atretic follicles, as reported previously for the rat (Lobel et al., '61). In the atretic follicles in the hamster, the degenerative activity is limited to the granulosa cells, as indicated by the abundance of acid phosphatase. The theca interna cells of atretic follicles do not show signs of degeneration and therefore contribute to the formation of interstitial gland cells, a s suggested previously (Guraya and Greenwald, '65). Corpora Zutea
In the CL on days 1 and 2, lipids are represented sparsely but abundant 3P-HSDH, ICDH, G-6-PDH and diaphorases are present, indicating the luteal capacity for progesterone synthesis. Progesterone levels found in the blood on days 1and 2 of the cycle (Leavitt and Blaha, '70;Lukeszwaska and Greenwald, '70; Blaha and Leavitt, '74; Ridley and Greenwald, '75; Saidapur and Greenwald, "78) probably reflect the luteal origin of progesterone. Unlike the CL of rat and other mammalian species studied so far (references in Baillie et al., '66; Pupkin et al., '66) the CL in the hamster ovary are very short-lived. By day 3, characteristic degenerative changes are observed in light and electron microscopic studies (Leavitt et al., '731, including accumulation of large coarse lipid droplets (Guraya and Greenwald, '65) and increased acid phosphatase. There is a decrease in the histochemically demonstrable 3P-HSDH and other enzymes studied. Previous biochemical (Leavitt et al., '73;Chatterjee and Greenwald, '76) and present histochemical studies are therefore in agreement. Moderate amounts of 17P-HSDH are found in the hamster CL on days 1and 2 and there is a considerable decrease thereafter, consistent with the degenerative changes. The CL on different days of the cycle also contain varying amounts of progesterone, testosterone, estra-
diol and estrone (Saidapur and Greenwald, "78). The presence of these steroids as well as 3P-HSDH and 17P-HSDH in the CL suggests not only that they are capable of synthesizing progesterone but also that steroidogenesis can proceed beyond progesterone synthesis. The absence of 20a-HSDH in the degenerating CL in the hamster ovary seems to be a case of species-variation and supports previous biochemical findings (Shaikh, '72; Leavitt et al., '73). Therefore, the mechanisms regulating the regression of CL in the hamster appear to be different from those in the rat and probably involve mainly the elevated levels of acid phosphatase. Interstitial gland celk;
With regard to the interstitial gland there are no appreciable changes in their lipid content and in the various enzymes studied, except for 3P-HSDH which increases slightly on the afternoon of day 4. The occurrence of intense 3P-HSDH activity in the interstitium throughout the cycle suggests its capacity to secrete progesterone. Although the enzyme 17P-HSDH is poorly represented in the interstitium of the hamster ovary, its presence suggests the possibility of testosterone-androstenedione and estradiol-estrone interconversions. The most striking feature of the interstitium of the hamster ovary is the sparsely represented G-6-PDH and ICDH (unlike the situation in the rat ovary) which might in turn further reduce the capacity of the hamster interstitium to synthesize estrogens. The apparent lack of acid phosphatase indicates that there is no appreciable degeneration of the interstitium during the estrous cycle. The Occurrence of coarse lipids, abundant acid phosphatase, some 3P-HSDH and other enzymes in atretic follicles is consistent with that observed by earlier investigators in other mammals (Lobel e t al., '61; Rubin et al., '63; Baillie e t al., '66). However, whether atretic follicles participate in steroid hormone production or not in the hamster is unknown. ACKNOWLEDGMENTS
The research was supported by grants from NIH (HD 00596) and The Ford Foundation. LITERATURE CITED Baillie, A. H., M. M. Fergwon and D. McKhart 1966 In: D%velopments in Steroid Histochemistry. Academic Press, New York. Balogh, K. 1964 A hietochemical method for the demon~
STEROIDOGENIC SITES IN HAMSTER OVARY stration of 2Oa-hydroxysteroid dehydrogenase activity in r a t ovaries. J. Histochem. cytmhem., 12: 670-673. Bara, G. 1966 Glucoee-6-pho~phatedehydrogenase activity in the ovaries of Scomber seomber L. Experientia, 21: 638-640. Baranczuk, R., and G. S. Greenwald Peripheral levels of estrogen in t h e cyclic hamster. Endocrinology, 92: 805-812. Blaha, G. C., and W. W. Leavitt 1970 The distribution of ovarian A63@hydroxysteroid dehydrogenase activity in the golden hamster during the estrous cycle, pregnancy, and lactation. Biol. Reprod., 3: 362-368. 1974 Ovarian steroid dehydrogenase hiatochemistry and circulating progesterone in aged golden hamsters during the estrous cycle and pregnancy. Biol. Reprod., 11: 153-161. Chatterjee, S., and G. S. Greenwald 1976 Biochemical changes in the corpus luteum of the cyclic hamster. J. Endocr., 68: 251-256. Greenwald, G. S. 1961 Quantitative study of follicular development in the ovary of the intact or unilaterally ovariectomized hamster. J. Reprod. Fertil., 2: 351-361. Guraya. S. S., and G. S. Greenwald 1964 A comparative histoehemical study of interstitial tiasue and follicular atresia in the mammalian ovary. Anat. Rec.. 149: 411-434. 1966 A histochemical study of the hamster ovary. Am. J. Anat.. 116: 257-268. Knigge, K. M.,and J. H. Leathem 1956 Growth and atresia of folliclas in the ovary of t h e hamster. Anat. Ree., 124: 679-707. Leavitt, W. W., C. R. Basom, J. N. Bagwell, and G . 5. Blaha 1973 Structure and function of the hamster corpus luteum during the estrous cycle. Am. J. Anat., 136: 236-250. Leavitt, W. W., andG. C. Blaha 1970 Circulatingprogesterone levels in the golden hamster during the estrous cycle, pregnancy, and lactation. Biol. Reprod., 3: 353-361. Lobel, B. L., R. M. Rosenbaum and H.W. Deane 1961 Enzyme correlates of physiological regreasion of follicles and corpora lutea in ovaries of normal rats. Endocrinology, 68: 232-247. Lukaszewska, J. H., and G. S. Greenwald 1970 Progester-
77
one levels in the cyclic and pregnant hamster. Endocrinology, 86: 1-9. Makris, A., and K. Ryan 1975 Progesterone, a n b t e n e dione, testosterone, estrone and estradiol synthesis in hamster ovarian follicle cells. Endocrinology, 96: 694-701. 1977 Aromatase activity of isolated hamster granulosa cells and theca. Steroids, 29: 66-72. McKerns, K. W. 1969 Studies on t h e regulation of ovarian function by gonadotropins.In: The Gonads. K. W. McKerns, ed. Appleton Century Crofts, New York, pp. 137-174. Pearee, A. G. E. 1972 Histochemistry. Theoretical and applied. Vol. 11. Third edition. Churchill Livingatone, London. Pupkin, M., H.Bratt, J. Weisz, C. W. Lloyd and K. Balogh, Jr. 1966 Dehydrogenases in the rat ovary. I. A histochemical study of A5-3p-and 2Ou-hydroxysteroid dehydmgenases and enzymes of carbohydrate oxidation during t h e estrous cycle. Endocrinology, 79: 316-327. Ridley, K., and G. S. Greenwald 1975 Progesterone levels measured every two hours in the cyclic hamster. Proceed. ings of the Society for Experimental Biology and Medicine, 149: 10-12. Rubin, B. L., H. W. Deane and J. A. Hamilton 1963 Biochemical and histochemical studies on A6-3p-hydmxysteroid dehydrogenase activity in the adrenal glands and ovaries of diverse mammals. Endocrinology, 73: 748-763. Saidapur, S. K., and G. S. Greenwald 1978 Peripheral h l d and ovarian levels of sex steroids in the cyclic hamster. Biol. Reprod., in press. Saidapur, S. K., and V. B. Nadkarni 1972 A5-3p-Hydroxysteroid dehydrogenase and glucose-6-phosphatedehydrogenase in t h e testis of toad, Bufo melanostictus (schneider). Indian J. Exp. Biol., 10: 425-427. Shaikh, A. A. 1972 Estrone, estradiol, progesterone and 17a-hydroxyprogesterone in the ovarian venous plasma during the estrous cycle of the hamster. Endocrinology, 91: 1136-1140. Wiest, W. G., and W. R. Kidwell 1969 The regulation of pro. gesterone secretion by ovarian dehydrogenases. In: The Gonads. K. W. McKerns, ed. Appleton Century Crofts, New York, pp. 295-325.
PLATE
1
EXPLANATION OF FIGURES
1 Section through the day3 ovary stained for lipids with oil red 0. A few lipid droplets
are present in the granulosa (G)but not in the theca interna (Th)of an antral follicle. Moderate amounts of lipid droplets are seen in the interstitial gland cells ( I N ) . 2 Day-1 corpus luteum (CL) showing the presence of only sparse lipids (stained with
Sudan black B). 3 Day-3 CL showing the accumulation of lipid droplets. Note the absence of any lipid
droplets in the preantral follicle (PAF).Stained with oil red 0. 4
The accumulation of large, coarse lipid droplets in the granulosa (GIcells of an atretic antral follicle. The theca interna cph) is negative to oil red 0. Scale lines indicate 50 pm.
78
STEROlDOGENlC SITES IN HAMSTER OVARY Srinivae K. Saidapur and Gilbert S. Greenwald
PLATE 1
79
PLATE 2 EXPLANATION OF FIGURES 5 Portion of day-1 ovary showing NADH-diaphorase in the interstitium (IGC) and its
absence in the preantral follicles. 6 NADH-diaphoraae in the antral follicle (day-3 ovary). The diaphorase is more intense in the theca interna (Th) and interstitium (IGC) than in the granulosa (G) cells. 7 NADPH-diaphorase in the antral follicle (day-3ovary) showing an inverse relation with NADH-diaphorase (compare with fig. 6). The enzyme ia characteristically
more abundant in granulosa (G) than in either theca interna (Th) or interstitial gland cells (IGC). 8,9 G-6-PDH and ICDH, respectively, in the day2 CL.Note their poor representation in
the interstitial gland cells (IGC). 10 Strongest G.6-PDH (and also ICDH) activity is seen in the granulosa cells of the antral follicle on the night of estrus (2300h).
Scale lines indicate 50 p m .
80
STEROIDOGENIC SITES IN HAMSTER OVARY Srinivas K. Saidapur and Gilbert S. Greenwald
PLATE 2
81
PLATE 3 EXPLANATION OF FIGURES
11 Day-1 ovary showing A5-3P-HSDHin the interstitial gland cells (IGC). Note the absence of enzyme activity in the preantral follicles (PAF).
12 Day-3 antral follicle. The As-3P-HSDHappears first in theca interna (Th). Slight enzyme activity is also observed in the granulosa cells (G). 13 Portion of a day-4 antral follicle (1600 h) showing an increase in A6-3pHSDHin both theca interna (Th)and granulosa (G) cells (compare with fig. 12). 14 Day-2 CL showing intense A6-3@-HSDHactivity. 15 Note t h e marked decline in luteal A6-3P-HSDH on day 4. Compare with the in. terstitium (IGC). 16 Portion of a n antral follicle (day 4,1500 hours) showing 17P-HSDH activity in both theca interna (Th) and granulosa (GI cells. Scale lines indicate 50 fim.
82
STEROIDOGENlC SITES IN HAMSTER OVARY Srinivan K. Saidapur and Gilbert S. Greenwald
PLATE 3
83
PLATE 4 EXPLANATION OF FIGURES
17 Acid phosphatase is absent in the preantral follicles (day-1 ovary).G , granulosa cells; Th, theca interna. 18 Day-3 CL showing intense acid phosphatase activity. 19 An atretic follicle showing intense acid phosphatase in the granulosa (G) cells. Scale lines indicate 50 pm.
a4
STEROIDOGENIC SITES IN HAMSTER OVARY Srinivas K. Saidapur and Gilbert S. Greenwald
PLATE 4
85
PLATE 5
STEROIDOGENIC SITES IN HAMSTER OVARY Srinivas K. Saidapur and Gilbert S. Greenwald
EXPLANATION O F FIGURES 20 Antral follicle of rat ovary showing lipid droplets [stained with Sudan black B) in the theca interna (Th)and also in the interstitial gland cells ( I N ) but not in granulosa (G)cells (compare with fig. 1).
21 NADH-diaphorasein the antral follicle of rat ovary, showing its distribution mainly in the theca interna (Th) and also in the interstitium (IGC). 22
NADPH-diaphorase in the rat ovary, showing activity mainly in the theca interna (Th) and not in the granulosa cells (G), unlike in the hamster (compare with fig. 7).
23 ICDH activity in the antral follicle of rat ovary is restricted to theca interna (Th) cells rather than to granulosa (G) cells (compare with fig. 10).
Scale lines indicate 50 pm.
86
ABSTRACT The histochemistry of lipids, A5-3P-,20a and 17P-hydroxy-steroid dehydrogenases, glucose-6-phosphate and isocitrate dehydrogenases, NADH and NADPH-diaphorases and acid phosphatase has been studied in the ovary of the cyclic hamster. None of the enzymes studied was present in the preantral follicles on day 1 (day of ovulation) or any day of the 4-day cycle. With the development of antral follicles (on day 3) a few fine lipid droplets appeared in the granulosa cells. The theca interna exhibited moderate amounts of NADH-diaphorase, A5-3P-HSDH,17P-HSDH and slight NADPH-diaphorase, G-6-PDH and ICDH, whereas the granulosa cells of antral follicles showed weak A5-3P-HSDH,NADHdiaphorase and moderate 17P-HSDHbut intense NADPH-diaphorase, G-6-PDH and ICDH activities. All enzyme activities in both theca and granulosa became more pronounced on proestrus (day 4). In the corpora lutea (CL) all the above enzymes were present on day 1and increased on day 2, followed by a rapid decline on days 3 and 4. Lipids were sparsely represented in CL on days 1 and 2 but showed marked accumulations on days 3 and 4. Likewise, acid phosphatase increased on days 3 and 4. The enzyme 2001HSDH was not detectable in the CL. The interstitium showed moderate amounts of lipids, A5-3P-HSDHand NADHdiaphorase while 17P-HSDH,NADPH-diaphorase, G-6-PDH and ICDH were only weakly present. There were no appreciable changes in the lipid and various enzymes in the interstitium throughout the cycle except for A5-3/3-HSDHwhich increased slightly on the afternoon of day 4. Acid phosphatase was not detected in appreciable amounts in the interstitium throughout the cycle. These findings suggest that preantral follicles are unlikely to contribute to steroidogenesis. The antral follicles are the principal source of estrogen, with the granulosa being the major site of synthesis. The interstitium can serve as a major source of progesterone (P) while the theca interna and granulosa cells of antral follicles may form additional sites for P synthesis on the afternoon of day 4. The CL in the hamster are very short-lived and steroidogenesis in them may proceed beyond the synthesis of progesterone. The present findings are correlated with the steroid levels found during the cycle. Appropriate comparisons are also made with the rat ovary. The histochemistry of the mammalian ovary has been studied extensively in several species (references in Baillie e t al., '66). However, for the hamster there are only isolated studies on the ovary describing the distribution of lipids and cholesterol (Knigge and Leathem, '56; Guraya and Greenwald, '66) or A53&hydroxysteroid dehydrogenase (Baillie et al., '66; Blaha and Leavitt, '70, '74) and 17P-hydroxysteroid dehydrogenase (Blaha and Leavitt, AM. J. ANAT. (1978) 151: 71-86.
'74). Other enzymes related directly or indirectly to steroidogenesis, such as glucose-6phosphate dehydrogenase (G8-PDH) and isoAccepted August 11, '77. I Supported by an International Postdoctoral Fellowahip of the Population Council. Biomedical Division, Nen York, U.S.A. Permanent oddrear: Deoartment of Zoolow.KarnatakUnivernitv. - . Dhawar .580 003, India. Send reprint requeatsto Dr. G . 8. Greenwald, Departmentof Physiology, Univenity of Kamas Medical Center. Kanaae City, Kanm 66103.
71
72
SRINIVAS K. SAIDAPUR AND GILBERT S. GREENWALD
citrate dehydrogenase (ICDH), acid phosphatase, etc., have not been studied in the hamster ovary. The present paper is a systematic study of the histochemical changes in the ovary of the cyclic hamster dealing with (1)lipids (steroid hormone precursors) ; (2) steroid dehydrogenases, A5-3P-hydroxysteroid dehydrogenase (3P-HSDH) and 17P-hydroxysteroid dehydrogenase (17P-HSDH) (involved in steroid hormone biosynthesis) with reference to both distribution and substrate specificity; (3) G-6PDH and ICDH, which are involved in carbohydrate metabolism but are known to be important sources generating NADPH needed for steroid hydroxylations; and (4) acid phosphatase, a lysosomal enzyme. The distribution was also studied of both NADH and NADPH diaphorases, which are prerequisites for the histochemical localization of NAD- and NADP-linked dehydrogenases. The changes in these enzyme activities are correlated with (1)the development of follicles, (2) the formation and fate of corpora lutea (CL), and (3) patterns of steroids in blood and ovary as reflected in published and unpublished studies from this laboratory. An additional objective of this paper is to compare and contrast these findings with the ovarian histochemistry of the rat and other mammalian species. MATERIALS AND METHODS
Adult female golden h a m s t e r s (Mesocricetus auratus) weighing 80-120 g and maintained on a 14-hour light: 10-hour-dark schedule were used after three to four consecutive 4-day cycles with eight to ten animals in each group. Day 1is defined a s the day of ovulation. Hamsters were killed on days 1to 4 a t 0900 hours and on day 4 animals were also killed a t 1200, 1500 (proestrus), 1800 and 2300 hours (estrus). The animals were killed by decapitation and the ovaries were excised immediately and frozen over dry ice. Sections were cut a t 14 pm thickness in a cryostat maintained a t -2O"C, and processed for different histochemical tests. The lipids were stained using Sudan black B or oil red 0. The procedure for 3P-HSDH and 178-HSDH was according to Baillie et al., ('661, using different substrates (table 2). The details of the method for 3P-HSDH, G-6-PDH and diaphorases is similar to that described previously (Bara, '65; Saidapur and Nadkarni, '72).
The procedure for ICDH and acid phosphatase was as described by Pearse ('72). Appropriate controls were employed for all tests (Pearse, "72). For the hamster, attempts to demons t r a t e 20a-hydroxysteroid dehydrogenase (20a-HSDH) following the method of Balogh ('64) were unsuccessful, whereas the same incubation medium demonstrated enzyme activity in the rat ovaries which served as controls. OBSERVATIONS
The distribution and intensity of lipids and different enzyme activities observed on different days of the cycle are summarized in tables 1 and 2 and therefore only a brief description is given below. 1. Lipids A detailed description of the distribution of lipids and cholesterol has been reported previously for the hamster ovary (Guraya and Greenwald, '65). The present findings are essentially similar. The distribution and intensity-pattern of lipids with both Sudan black B and oil red 0 was the same. Normal preantral follicles did not contain any lipid droplets in theca or in granulosa cells. However, with the formation of antral follicles on days 3 and 4 of the cycle, the granulosa cells contained a few fine lipid droplets, while the thecal cells were negative (fig. 1)to Sudan black B or oil red 0. The newly formed CL (fig. 2) did not contain appreciable amounts of lipid droplets on day 1 of the cycle but they increased progressively on day 2 and reached their maximum (fig. 3) on day 4. The interstitium contained moderate amounts of lipid droplets throughout the cycle and did not show any appreciable changes during the cycle. The granulosa cells of atretic follicles (both preantral and antral) contained large coarse lipid droplets (fig. 4). 2. Diaphorases
(A) NADH-diaphorase showed a very specific distribution (table 1).The diaphorase was absent in both theca and granulosa cells of the preantral follicles while it appeared on the transformation into antral follicles (figs. 5,6). The intensity of diaphorase activity was distinctly greater in the theca interna than in granulosa cells. The CL contained diaphorase activity on all days of the cycle; however, the enzyme was maximal on day 2 and thereafter decreased progressively. Diaphorase activity in the interstitiurn was essentially the
73
STEROIDOGENIC SITES IN HAMSTER OVARY TABLE 1
Distribution of lipids, diaphorases, G-6-PDH,ICDH, and acid phosphatase in the hamster ovary Enzyme/lipid
1. Lipida
(Sudanblack B or oil red 0) 2. NADH-diaphorase
Day of cycle
Preantral follicle
TWG'
+
4 1 2
-
1 2
3 4
1 n L
3 4
5. Acid phosphatase
G
(G)
1
3
4. G-6-PDHIICDH
Th
Atretic follicle
2 3
4
3. NADPH-diaphorase
Antral follicle
1 2
3 4
+
+ ++ + ++ ++++
++ ++ ++ ++ ++ ++ ++ ++ ++ ++
CL'
IT 3
+ + ++ ++++ + +++ ++ + + +++ ++ + + +++ ++ + + +++ ++
+++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ + + + ++ -
+ + + -
' Th, Theea interna; G, granuloea. Corpora lutea. Interstitiurn. Intensity of reaction is graded (-) to (+ + + +); (-) denotes the abaence of reaction and ( + + + + ) , a maximal reaction.
same on all days of the cycle. The granulosa of atretic follicles contained appreciable amounts of diaphorase. (B) NADPH-diaphorase.The preantral follicles completely lacked this enzyme. In the antral follicles the distribution and intensity of NADPH diaphorase was inversely related to NADH diaphorase in that the theca interna cells contained only a slight amount of NADPH diaphorase in contrast to the granulosa cells (fig. 7). However, the distribution of the NADPH diaphorase was similar to that of NADH diaphorase in CL, interstitial gland cells and atretic follicles, except that the intensity of formazan deposition was slightly less (table 1). 3. G-6-PDH and ZCDH activities (figs. 8, 9, 10) Both these enzymes are NADP-dependent and showed a similar distribution and intensity-pattern.The intensity and distribution of both G-6-PDH and ICDH resembled that of NADPH diaphorase, especially in the antral follicles and CL (figs. 8, 9) but the activities were markedly lower in the interstitium (table 1) and also in follicles undergoing atresia. The enzyme activities in the granulosa cells of the antral follicles were inten-
sified on day 4, reaching their maximum by 2300 hours (fig. 10). 4. A5-3P-HSDHenzyme activity
In general, the distribution of 3P-HSDH paralleled that of NADH diaphorase, although the intensity of the former reaction was slightly lower. The preantral follicles lacked 3P-HSDH (fig. 11).With the development of antral follicles, enzyme activity appeared in the theca interna and in the granulosa cells (fig. 121, with maximal values (fig. 13) reached on day 4. The enzyme activity in the CL was maximal on day 2 (fig. 14) but decreased rapidly by day 3 and was scarcely represented on day 4 (fig. 15). The interstitial 3pHSDH appeared the same from day 1 to 3 of the cycle and increased slightly on day 4. Atretic antral follicles contained some enzyme activity in both theca interna and granulosa cells, while in the case of preantral atretic follicles the activity was seen only in the granulosa cells. The hamster ovary is able to utilize a variety of substrates for the demonstration of 3P-HSDH (table 2). Of the five substrates used, pregnenolone, 17a-hydroxypregnenolone and 16-dehydropregnenolone produced a formazan deposition of equal magnitude while
74
SRINIVAS K. SAIDAPUR AND GILBERT S. GREENWALD TABLE 2
A53p-HSDH activity in the hamster ouary Substrate
1. Pregnenolone 2. 17a (OH) pregnenolone 3. 16-dehydropregnenolone 4. Dehydroepiandrosterone 5. Epiandrosterone
Day of cycle
Preantral follicle
ThIG
1 2
3 4 1
2 3 4
6. Control
1 2 3
Antral fo11ic1e Th G
-
-
-
-
+ ++
+ ++ +++ +++ + + ++
+
+ ++
Atretic fo11ic1e
(GI
CL
IT
++ +++ +++ +++ ++ +++++++ -
+ ++ ++ -
++ ++ ++
+++ -
4 Intensityofreactioniagraded (-) to ( +
+ + + ) ; (-)denotes theabsenceofenzymeactivity,and ( +i + +),amaximalreaction
dehydroepiandrosterone and epiandrosterone gave a slightly less intense reaction.
5. 17P-HSDH enzyme activity The 17P-HSDH was also NAD-dependent and the cellular sites of its distribution resembled 3P-HSDH. The preantral follicles completely lacked any enzyme activity. With the development of antral follicles on day 3, 17P-HSDH appeared in both theca interna and granulosa cells. On day 4 there was a slight increase in thecal 17P-HSDH but the enzyme activity increased markedly in the granulosa cells (fig. 16).The enzyme was also present in the interstitial gland cells but was poorly represented on all days of the cycle. The activity of 17P-HSDHin the CL resembled 3PHSDH, in that the CL showed 17P-HSDH on the day of their formation (day l),increased slightly on day 2 and declined markedly on subsequent days. Both testosterone and 17Pestradiol were utilized with equal magnitude.
by a slight decrease on day 4. The atretic granulosa cells of preantral as well as antral follicles contained abundant acid phosphatase (fig. 19).
Comparison with the rat A. Antral Follicles. In the rat, granulosa cells of antral follicles do not contain any appreciable amount of lipid, while theca interna cells contain lipid (fig. 20) and cholesterol. In contrast, the theca interna cells of antral follicles in the hamster do not contain lipid or cholesterol. The distribution of NADH-diaphorase, 3P-HSDH and 17P-HSDHare similar in both hamster and rat antral follicles (fig. 21), except that the granulosa cells of the hamster are more reactive than in the rat. However, striking differences exist with regard to the distribution of NADPH-diaphorase, G-6-PDH and ICDH enzymes (figs. 22, 23). Unlike the antral follicles of the rat, in the hamster theca interna cells exhibit markedly low enzyme activity while the granulosa cells of these folli6 . Acid phosphatase cles exhibit the strongest activity. Acid phosAcid phosphatase was not detectable in the phatase is not present in the healthy antral preantral (fig. 17) follicles and was present in follicles of either rat (Lobel et al., '61) or hamonly trace amounts in the granulosa cells of ster. B. Corpora Lutea. The CL of hamster and antral follicles on day 3, with a slight increase on day 4. The theca of antral follicles rat ovary contain diaphorases, 3P-HSDH,17pcompletely lacked acid phosphatase. The in- HSDH, G-6-PDH and ICDH enzymes. Howterstitium contained only trace amounts of ever, the CL of the hamster are very shortacid phosphatase, with no changes observed lived (persisting 4-5 days), unlike those of the throughout the cycle. The newly formed CL on rat which persist for three or more cycles. Furday 1 did not contain any appreciable amount thermore, older and degenerating CL in the of the enzyme. On day 2 the CL contained rat ovary develop 20a-HSDH activity while in some acid phosphatase, with the maximum the hamster degenerating CL do not exhibit reaction developing on day 3 (fig. 18), followed this enzyme activity. The first generation of
STEROIDOGENIC SITES IN HAMSTER OVARY
75
do not. I t was reported previously (Guraya and Greenwald, '65) that injection of PMS into hamsters results in depletion of lipids and cholesterol from the granulosa cells, indicating that the granulosa cells of the antral follicles are capable of responding to gonadotropins by secreting steroid hormones. The Occurrence of A5-3P-HSDHin the theca interna and granulosa of the antral follicles suggests their capacity to oxidize A5-3P-hydroxysteroids to A'-3-ketosteroids, a step that occurs during the biosynthesis of all the biologically active steroid hormones. Generally, 3P-HSDH activity is associated with the production of progesterone in the ovary and therefore its occurrence in the theca interna and granulosa cells of the antral follicles suggests the capacity of both cell types to synthesize the hormone. Incubation in vitro of granulosa and thecal cells of hamster preovulatory follicles confirms their independent ability to produce progesterone (Makris and Ryan, '75). DISCUSSION The occurrence of 17P-HSDHin both theca Preantral follicles interna and granulosa cells of antral follicles Preantral follicles in the hamster ovary suggests the possible interconversions of 17Plack lipids, cholesterol, steroid dehydroge- hydroxysteroids to 17-ketosteroids. The ennases and other enzymes related to steroido- zyme 17P-HSDH is generally associated with genesis both in the granulosa and thecal cells. estrogen synthesis and its occurrence in these This indicates that these follicles do not cells indicates their capacity to synthesize possess any steroidogenic potentiality a t this estrogen(s). Furthermore, it is interesting stage of their growth and development. Thus that this enzyme activity is greater in granthe contribution by the preantral follicles of ulosa cells than in theca interna cells. It any steroid hormones during the estrous cycle seems apparent that granulosa cells are more is not likely. However, on day 1 of the cycle, capable of synthesizing estrogens than the before the appearance of antral follicles, low theca interna cells surrounding them. This levels of estrogen are detected in the blood conclusion is also based on the fact that both (Baranczuk and Greenwald, '73; Saidapur and G-6-PDH and ICDH enzymes are characterisGreenwald, '781, suggesting a n extra-follicu- tically abundant in the granulosa cells compared to the theca interna cells. It is well lar source of estrogen on day 1 of the cycle. known that both ICDH and G-6-PDH are Antral follicles important sources of NADPH needed for hyWith the formation of antral follicles (by droxylations during steroidogenesis (McKerns, the evening of day 2 or the morning of day 3), '69; Wiest and Kidwell, '69). Recent culture important changes take place in the theca in- studies of Makris and Ryan ('75) on isolated terna and granulosa cells. The theca interna granulosa and theca cells of hamster antral does not contain appreciable amounts of lipids follicles indicates a synergism between the and reacts negatively to the Schultz test for two cell types for the production of maximal cholesterol and/or its esters, while the gran- estrogen, while more recent studies by the ulosa cells contain sparse lipids and choles- same authors (Makris and Ryan, '77) show terol and its esters (Guraya and Greenwald, that maximal aromatase activity of the pre'65). This is unlike the situation in many other ovulatory follicle is associated with the granspecies such as the rat, bat, cat, dog and Amer- ulosa cells rather than the theca interna cells. ican opossum (Guraya and Greenwald, '641, Thus, the present histochemical observations wherein the theca interna cells contain lipids are in close agreement with the findings in and cholesterol but the normal granulosa cells vitro. Furthermore, the rise in circulating and
CL in the rat do not contain any appreciable amount of acid phosphatase, while those belonging to the second generation (formed 4-8 days previously) show conspicuous acid phosphatase activity. In the hamster, CL begin to show acid phosphatase activity as early as day 2 with a pronounced increase on day 3. C. Interstitial Gland Cells. The interstitial gland cells of both species contain diaphorases, 3P-HSDH, 17P-HSDH, G-6-PDH and ICDH. However, NADPH-diaphorase and NADP-linked enzymes such as G-6-FDH and ICDH are poorly represented in the interstitium of hamster ovary, unlike the situation in the rat ovary. D. The overall reactivity for 3P-HSDH and 17P-HSDHenzymes was considerably lower in the hamster ovary, so that the intensity of staining in a 14-pm section incubated for two to three hours was weaker than in 14-pm sections of rat ovary incubated for only 30 to 45 minutes.
76
SRINIVAS K. SAIDAPUR AND GILBERT S. GREENWALD
ovarian estrogen concentrations on days 3 and 4 (Shaikh, '72; Baranczuk and Greenwald, '73; Saidapur and Greenwald, '78) correlates with the development of antral follicles (Greenwald, '61) and the appearance of the various enzyme systems described above. The absence of any appreciable amount of acid phosphatase in either theca interna or granulosa cells indicates the apparent lack of any degenerating cells in the healthy antral follicles. Acid phosphatase, a lysosomal enzyme, is generally associated with autolytic processes and therefore is found only in degenerating or atretic follicles, as reported previously for the rat (Lobel et al., '61). In the atretic follicles in the hamster, the degenerative activity is limited to the granulosa cells, as indicated by the abundance of acid phosphatase. The theca interna cells of atretic follicles do not show signs of degeneration and therefore contribute to the formation of interstitial gland cells, a s suggested previously (Guraya and Greenwald, '65). Corpora Zutea
In the CL on days 1 and 2, lipids are represented sparsely but abundant 3P-HSDH, ICDH, G-6-PDH and diaphorases are present, indicating the luteal capacity for progesterone synthesis. Progesterone levels found in the blood on days 1and 2 of the cycle (Leavitt and Blaha, '70;Lukeszwaska and Greenwald, '70; Blaha and Leavitt, '74; Ridley and Greenwald, '75; Saidapur and Greenwald, "78) probably reflect the luteal origin of progesterone. Unlike the CL of rat and other mammalian species studied so far (references in Baillie et al., '66; Pupkin et al., '66) the CL in the hamster ovary are very short-lived. By day 3, characteristic degenerative changes are observed in light and electron microscopic studies (Leavitt et al., '731, including accumulation of large coarse lipid droplets (Guraya and Greenwald, '65) and increased acid phosphatase. There is a decrease in the histochemically demonstrable 3P-HSDH and other enzymes studied. Previous biochemical (Leavitt et al., '73;Chatterjee and Greenwald, '76) and present histochemical studies are therefore in agreement. Moderate amounts of 17P-HSDH are found in the hamster CL on days 1and 2 and there is a considerable decrease thereafter, consistent with the degenerative changes. The CL on different days of the cycle also contain varying amounts of progesterone, testosterone, estra-
diol and estrone (Saidapur and Greenwald, "78). The presence of these steroids as well as 3P-HSDH and 17P-HSDH in the CL suggests not only that they are capable of synthesizing progesterone but also that steroidogenesis can proceed beyond progesterone synthesis. The absence of 20a-HSDH in the degenerating CL in the hamster ovary seems to be a case of species-variation and supports previous biochemical findings (Shaikh, '72; Leavitt et al., '73). Therefore, the mechanisms regulating the regression of CL in the hamster appear to be different from those in the rat and probably involve mainly the elevated levels of acid phosphatase. Interstitial gland celk;
With regard to the interstitial gland there are no appreciable changes in their lipid content and in the various enzymes studied, except for 3P-HSDH which increases slightly on the afternoon of day 4. The occurrence of intense 3P-HSDH activity in the interstitium throughout the cycle suggests its capacity to secrete progesterone. Although the enzyme 17P-HSDH is poorly represented in the interstitium of the hamster ovary, its presence suggests the possibility of testosterone-androstenedione and estradiol-estrone interconversions. The most striking feature of the interstitium of the hamster ovary is the sparsely represented G-6-PDH and ICDH (unlike the situation in the rat ovary) which might in turn further reduce the capacity of the hamster interstitium to synthesize estrogens. The apparent lack of acid phosphatase indicates that there is no appreciable degeneration of the interstitium during the estrous cycle. The Occurrence of coarse lipids, abundant acid phosphatase, some 3P-HSDH and other enzymes in atretic follicles is consistent with that observed by earlier investigators in other mammals (Lobel e t al., '61; Rubin et al., '63; Baillie e t al., '66). However, whether atretic follicles participate in steroid hormone production or not in the hamster is unknown. ACKNOWLEDGMENTS
The research was supported by grants from NIH (HD 00596) and The Ford Foundation. LITERATURE CITED Baillie, A. H., M. M. Fergwon and D. McKhart 1966 In: D%velopments in Steroid Histochemistry. Academic Press, New York. Balogh, K. 1964 A hietochemical method for the demon~
STEROIDOGENIC SITES IN HAMSTER OVARY stration of 2Oa-hydroxysteroid dehydrogenase activity in r a t ovaries. J. Histochem. cytmhem., 12: 670-673. Bara, G. 1966 Glucoee-6-pho~phatedehydrogenase activity in the ovaries of Scomber seomber L. Experientia, 21: 638-640. Baranczuk, R., and G. S. Greenwald Peripheral levels of estrogen in t h e cyclic hamster. Endocrinology, 92: 805-812. Blaha, G. C., and W. W. Leavitt 1970 The distribution of ovarian A63@hydroxysteroid dehydrogenase activity in the golden hamster during the estrous cycle, pregnancy, and lactation. Biol. Reprod., 3: 362-368. 1974 Ovarian steroid dehydrogenase hiatochemistry and circulating progesterone in aged golden hamsters during the estrous cycle and pregnancy. Biol. Reprod., 11: 153-161. Chatterjee, S., and G. S. Greenwald 1976 Biochemical changes in the corpus luteum of the cyclic hamster. J. Endocr., 68: 251-256. Greenwald, G. S. 1961 Quantitative study of follicular development in the ovary of the intact or unilaterally ovariectomized hamster. J. Reprod. Fertil., 2: 351-361. Guraya. S. S., and G. S. Greenwald 1964 A comparative histoehemical study of interstitial tiasue and follicular atresia in the mammalian ovary. Anat. Rec.. 149: 411-434. 1966 A histochemical study of the hamster ovary. Am. J. Anat.. 116: 257-268. Knigge, K. M.,and J. H. Leathem 1956 Growth and atresia of folliclas in the ovary of t h e hamster. Anat. Ree., 124: 679-707. Leavitt, W. W., C. R. Basom, J. N. Bagwell, and G . 5. Blaha 1973 Structure and function of the hamster corpus luteum during the estrous cycle. Am. J. Anat., 136: 236-250. Leavitt, W. W., andG. C. Blaha 1970 Circulatingprogesterone levels in the golden hamster during the estrous cycle, pregnancy, and lactation. Biol. Reprod., 3: 353-361. Lobel, B. L., R. M. Rosenbaum and H.W. Deane 1961 Enzyme correlates of physiological regreasion of follicles and corpora lutea in ovaries of normal rats. Endocrinology, 68: 232-247. Lukaszewska, J. H., and G. S. Greenwald 1970 Progester-
77
one levels in the cyclic and pregnant hamster. Endocrinology, 86: 1-9. Makris, A., and K. Ryan 1975 Progesterone, a n b t e n e dione, testosterone, estrone and estradiol synthesis in hamster ovarian follicle cells. Endocrinology, 96: 694-701. 1977 Aromatase activity of isolated hamster granulosa cells and theca. Steroids, 29: 66-72. McKerns, K. W. 1969 Studies on t h e regulation of ovarian function by gonadotropins.In: The Gonads. K. W. McKerns, ed. Appleton Century Crofts, New York, pp. 137-174. Pearee, A. G. E. 1972 Histochemistry. Theoretical and applied. Vol. 11. Third edition. Churchill Livingatone, London. Pupkin, M., H.Bratt, J. Weisz, C. W. Lloyd and K. Balogh, Jr. 1966 Dehydrogenases in the rat ovary. I. A histochemical study of A5-3p-and 2Ou-hydroxysteroid dehydmgenases and enzymes of carbohydrate oxidation during t h e estrous cycle. Endocrinology, 79: 316-327. Ridley, K., and G. S. Greenwald 1975 Progesterone levels measured every two hours in the cyclic hamster. Proceed. ings of the Society for Experimental Biology and Medicine, 149: 10-12. Rubin, B. L., H. W. Deane and J. A. Hamilton 1963 Biochemical and histochemical studies on A6-3p-hydmxysteroid dehydrogenase activity in the adrenal glands and ovaries of diverse mammals. Endocrinology, 73: 748-763. Saidapur, S. K., and G. S. Greenwald 1978 Peripheral h l d and ovarian levels of sex steroids in the cyclic hamster. Biol. Reprod., in press. Saidapur, S. K., and V. B. Nadkarni 1972 A5-3p-Hydroxysteroid dehydrogenase and glucose-6-phosphatedehydrogenase in t h e testis of toad, Bufo melanostictus (schneider). Indian J. Exp. Biol., 10: 425-427. Shaikh, A. A. 1972 Estrone, estradiol, progesterone and 17a-hydroxyprogesterone in the ovarian venous plasma during the estrous cycle of the hamster. Endocrinology, 91: 1136-1140. Wiest, W. G., and W. R. Kidwell 1969 The regulation of pro. gesterone secretion by ovarian dehydrogenases. In: The Gonads. K. W. McKerns, ed. Appleton Century Crofts, New York, pp. 295-325.
PLATE
1
EXPLANATION OF FIGURES
1 Section through the day3 ovary stained for lipids with oil red 0. A few lipid droplets
are present in the granulosa (G)but not in the theca interna (Th)of an antral follicle. Moderate amounts of lipid droplets are seen in the interstitial gland cells ( I N ) . 2 Day-1 corpus luteum (CL) showing the presence of only sparse lipids (stained with
Sudan black B). 3 Day-3 CL showing the accumulation of lipid droplets. Note the absence of any lipid
droplets in the preantral follicle (PAF).Stained with oil red 0. 4
The accumulation of large, coarse lipid droplets in the granulosa (GIcells of an atretic antral follicle. The theca interna cph) is negative to oil red 0. Scale lines indicate 50 pm.
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STEROlDOGENlC SITES IN HAMSTER OVARY Srinivae K. Saidapur and Gilbert S. Greenwald
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES 5 Portion of day-1 ovary showing NADH-diaphorase in the interstitium (IGC) and its
absence in the preantral follicles. 6 NADH-diaphoraae in the antral follicle (day-3 ovary). The diaphorase is more intense in the theca interna (Th) and interstitium (IGC) than in the granulosa (G) cells. 7 NADPH-diaphorase in the antral follicle (day-3ovary) showing an inverse relation with NADH-diaphorase (compare with fig. 6). The enzyme ia characteristically
more abundant in granulosa (G) than in either theca interna (Th) or interstitial gland cells (IGC). 8,9 G-6-PDH and ICDH, respectively, in the day2 CL.Note their poor representation in
the interstitial gland cells (IGC). 10 Strongest G.6-PDH (and also ICDH) activity is seen in the granulosa cells of the antral follicle on the night of estrus (2300h).
Scale lines indicate 50 p m .
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STEROIDOGENIC SITES IN HAMSTER OVARY Srinivas K. Saidapur and Gilbert S. Greenwald
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
11 Day-1 ovary showing A5-3P-HSDHin the interstitial gland cells (IGC). Note the absence of enzyme activity in the preantral follicles (PAF).
12 Day-3 antral follicle. The As-3P-HSDHappears first in theca interna (Th). Slight enzyme activity is also observed in the granulosa cells (G). 13 Portion of a day-4 antral follicle (1600 h) showing an increase in A6-3pHSDHin both theca interna (Th)and granulosa (G) cells (compare with fig. 12). 14 Day-2 CL showing intense A6-3@-HSDHactivity. 15 Note t h e marked decline in luteal A6-3P-HSDH on day 4. Compare with the in. terstitium (IGC). 16 Portion of a n antral follicle (day 4,1500 hours) showing 17P-HSDH activity in both theca interna (Th) and granulosa (GI cells. Scale lines indicate 50 fim.
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STEROIDOGENlC SITES IN HAMSTER OVARY Srinivan K. Saidapur and Gilbert S. Greenwald
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
17 Acid phosphatase is absent in the preantral follicles (day-1 ovary).G , granulosa cells; Th, theca interna. 18 Day-3 CL showing intense acid phosphatase activity. 19 An atretic follicle showing intense acid phosphatase in the granulosa (G) cells. Scale lines indicate 50 pm.
a4
STEROIDOGENIC SITES IN HAMSTER OVARY Srinivas K. Saidapur and Gilbert S. Greenwald
PLATE 4
85
PLATE 5
STEROIDOGENIC SITES IN HAMSTER OVARY Srinivas K. Saidapur and Gilbert S. Greenwald
EXPLANATION O F FIGURES 20 Antral follicle of rat ovary showing lipid droplets [stained with Sudan black B) in the theca interna (Th)and also in the interstitial gland cells ( I N ) but not in granulosa (G)cells (compare with fig. 1).
21 NADH-diaphorasein the antral follicle of rat ovary, showing its distribution mainly in the theca interna (Th) and also in the interstitium (IGC). 22
NADPH-diaphorase in the rat ovary, showing activity mainly in the theca interna (Th) and not in the granulosa cells (G), unlike in the hamster (compare with fig. 7).
23 ICDH activity in the antral follicle of rat ovary is restricted to theca interna (Th) cells rather than to granulosa (G) cells (compare with fig. 10).
Scale lines indicate 50 pm.
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