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A histochemical study of ovarian function in the ovoviviparous elasmobranch, Squalus acanthias
GENERAL
AND
COMPARATIVE
Histochemical
ENDOCRINOLOGY
Study
13,
255-267
of Ovarian
Elasmobranch, VALENTINE
(19639)
Squalus
LANCE2
Received
Function
AND
March
in the
acanthiasl IAPt: P. CALLARD
10, 1969
Ovarian tissues from the ovoviviparous elasmobranch, Squu1u.s acanthl,as were examined histochemically for the distribution of NAD and KADP diaphorase: glucose&phosphate dehydrogenase (G+PDH), 3 beta hydroxysteroid dehydrogenase (3,8HSD), 3 alpha hydroxysteroid dehydrogenase (3cxHSD), 17 beta hydrosysteroid dehydrogenase (17pHSD), and 20 beta hydroxysteroid dehydrogenase (20pHSD). The histochemical distribution of the enzymes was correlated with the histological picture obtained from contiguous sections. NAD, NADP diaphorase, and G-6-PDH were distributed throughout the ovary and positive reactions were obtained in all tissues tested. 3/3HSD activity was demonstrated in the granulosa of follicles at various stages of development, increasing in intensity as the follicle matured. After ovulation, 3pHSD was found in the post-ovulatory follicle (L’corpus luteurn”). No 3pHSD activity was observed in atretic follicles (“preovulatorycorpora lutea”). 3aHSD was only observed as a weak reaction in the granulosa of late preovulatory follicles, and no activity of 17/3HSD and BOPPED was observed in any ovarian component tissues.
nzarmorata and Scyliorhirxus stellar6 ‘nave shown that elasmobranch ovarian tissue is capable of synthesizing steroids in,
In recent years there have been a great many studies on the occurrence of steroid hormones in the lower verteb,rates (see reviews by Gottfried, 1964; Ozon, 1966; Chieffi, 1966; Nandi, 1967). The elasmobran&s in particular have been shown to be a rich source of steroid hormones. Progesterone and estrogens have been identified in extracts of ovarian tissue from Squalus sucicleyi (Wotiz et al., 1958, 1960), Scyliorhinus canicula (Simpson et al., 1963) Torpedo rtza~rnoruta (Chieffi and Lupo, 1963)) and Syualus acunthias (Gott#fried, 1964). Changes in the plasma levels of a number of st.eroids during the reproductive cycle of Torpedo marmorata have also been recorded (Buonnanno et al., 1964; Lupo di Prisco et al., 1967). The studies of CMlard and Leathem (1965) on Raia erillacea and Squab a&ant&as, and of Lupo di Prisco et al. (1966) on Torpedo
Baillie et al., cal demonstration
1965,
1956).
The
histochemi-
of a steroid dehydrogerrase is generally taken as evidence that the part,icular tissue is capable of steroid synthesis. To date, however, there has been only one report, on the histochemical dcm-
1 Supported by NSF Grant GB 6917 and the College of William and Mary. ’ Pyesent address: Department of Zoology, University of Hong Kong, Hong Kong. 255
256
LANCE
AND
on&ration of A5,3P-hydroxysteroid dehydrogenase (3,8-HSD) in the elasmobranch ovary (Lupo et al., 1965). These authors were able to demonstrate positive 3P-HSD activity in postovulatory follicles (corpora lutea) in the oviparous Scyliorhinus stellaris, but not in the atretic follicles, and conversely, positive 3/3-HSD activity in atretic follicles but not in postovulatory follicles in the ovoviviparous Torpedo marmorata. They did not report whether any activity in the developing follicles was observed, Chieffi (1967) suggests that the distribution of this enzyme in the elasmobranch ovary is related to mode of reproduction rather than taxonomic differences so that ovoviviparous species develop eneymically active corpora lutea from atretic follicles and oviparous species from post-ovulatory follicles. Squab acanthias is an ovoviviparous elasmobranch with a gestation period of 20-22 months and possesses ovarian corpora lutea-like structures derived from both atretic and postovulatory follicles, the corpora lutea from the postovulatory follicles persisting in the ovary for a greater part of the gestation period (Hisaw and Albert, 1947). In view of the paucity of information on the steroid-producing glands in elasmobranchs, it is of considerable interest to establish what ovarian structures show evidence of steroidogenesis, and whether seasonal variations in activity are apparent. In the present investigation ovaries from S. acanthias were studied by histochemical methods at various phases of the reproductive cycle. MATERIALS
AND
METHODS
Ovaries from S. acanthins collected in the Chesapeake Bay area were obtained from Harburton Marine Laboratory, Harburton, Virginia, during the period December 1967-April 1968, and were shipped to the laboratory on dry ice or in Bouin’s fixative. The frozen tissues were maintained on dry ice until sectioned. The snoutvent lengths and the number and size of the embryos in the oviducts were recorded to estimate the reproductive status of each animal. The snoutvent length of the nonpregnant animals ranged between 33 and 51 cm, while that of the pregnant animals ranged between 42 and 56 cm. Since none
CALLARD
of the ovaries from animals with a snout-vent length of less than 40 cm contained follicles larger than 15 mm in diameter, these were classed as immature. The stage of gestation was determined by the length of the embryos according to Hisaw and Albert (1947) : &age A, recently ovulated (candle stage) ; stage B, embryos from 3.5 to 7.5 cm; stage C, embryos from 12 to 20 cm; stage D, embryos from 23 to 29 cm. No animals in stage B of the gestation period were collected. All of the tissues were examined grossly for the presence of follicles and corpora lutea in all stages of development. A total of 36 ovaries was selected for sectioning; 6 in Bouinb fluid for routine histology and 30 frozen for histochemistry. The frozen tissues were divided into the following groups: (1) immature (n= 4); (2) mature but nonpregnant (n= 10); (3) recently ovulated (n = 9) ; and (4) contained embroys ranging in size from 10-22 cm (n = 7). The frozen tissues were sectioned at 20,~ on an A0 rotary freezing microtome maintained at --25”C, placed on gelatin-coated cover slips and tested for 3,6-HSD, 3a-HSD, 17p-HSD, and 20/?HSD by a modification of the method of Levy et al. (1959). Steroid substrates used in this study were A” pregnen3,&ol-20-one (pregnenolone) and androstan-3,8-ol-17-one (dehydroepiandrosterone, DHEA) for 3/3-HSD ; androstan-3c+ol-17-one (androsterone) for 3a-HSD; A4 androsten-17,G-ola-one (testosterone), and estradiol-17P for 17,@HSD ; and A4 pregnen-20P-ol-3-one (20.&hydroxyprogesterone) for 2O,&HSD (Mann Chemical, Inc.). The steroids were dissolved in propylene glycol or dimethyl formamide (Baillie et al., 1966) at 1 mg/ml. In the case of 17p-HSD, substrate concentrations of 2 mg/ml and 4 mg/ml were also tested. The following buffers were tested in preparing the incubation medium: phosphate (pH 6.8, 7.5, 8.01 and 8.8), Tris (pH 7.5 and 8.0) and elasmobranch Ringer at pH 7.6 (Lockwood, 1961). Nitro-BT (Sigma) was used as the final electron acceptor. Both NAD and NADP (Sigma) were tested as cofactors in each of the enzyme reactions. The histochemical reactions were tested at room temperature (22°C) and at 37°C for periods from l-6 hr, with some sections being tested with an overnight incubation. The effect of rinsing the sections in acetone prior to incubation was also studied. Alternate sections were incubated in a medium lacking the steroid substrate as a control. After incubation the sections were rinsed briefly in phosphate buffer, fixed in 10% formalin for 10 minutes, dehydrated, and mounted in Permount. Alternate frozen sections to those being tested for steroid dehydrogenases were tested for glucose-
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HISTOCHEMISTRY
6-phosphate dehydrogenase (G-6-PDH) by the method of Cohen (1959), NAD- and NADPdiaphorase by the method of Bara (19651, and stained for hpid with fat red 7. Sections were also tested for the presence of cholesterol by a modification of the Schultz method (Lillie, 1965), or stained with hematoxylin and eosin. The material fixed in Bouin’s fluid was embedded in paraffin and stained routinely with hematoxylin and eosin or Mallory’s triple stain. RESULTS
General Microanatomy In X. acanthias the paired ovaries are suspended from the anterior dorsal wall of the body cavity by a broad mesovarium. The ovary is made up of germinal epithehum, follicles in all stages of development, a connective tissue stroma, and a hematopoietic tissue, the epigonal organ which invests the entire structure. The follicular wall of the developing ovum consists of the zona radiata; the vitelline membrane; t’he granulosa, composed of a single layer of columnar epithelium; the theta interna; and the theca externa. The theea. interna appears to be composed mainly of connective tissue cells (see Fig. 1). As the follicle increases in size there is a gradual increase in thickness of the theta interna. The cells of the theca externa are not always distinguishable in the large preovulatory follicles (#-45 mm in diameter). For convenience, postovulatory and atretic follicles have been divided into four stages. The histology of these structures has been described by Hisaw and Hisaw (1959). “Corpus lute~urn’f (Postovulatory Follicle, Fig. 2). Stage 1 is characterized by an extremely thick theta and a folded granulosa which does not completely fill the center of the structure. Stage 2 is essentially similar to stage I, but the granulosa elements now completely fill the area enclosed by the thecal elements. In stage 3 involution has begun, as indicated by vacuolization of the qanulosa elements and connective tissue &filtration. The theca is much reduced in thickness. In stage 4 involution is advanced, the structure is greatly reduced in size wit,11extreme conn&ive tissue infiltra-
IN - &&Us
257
tion and degeneration of the granulosa cells. Atretic Follicle (Preovulatory Corpus Luteum). Stage 1 is characterized by a thin thecal layer, with elements of theta intruding into the follicle along with the highly folded and invaginat,ed granulosa elements. Phagocytosis of yolk has begun. In stage 2, as shown in Fig. 3 ~hago~~-t5sis of the yolk is complete and the folds of hypertrophied follicular elements have begun to anatomose. At stage 3 the yolk granules have disappeared and a thinwalled solid structure, very similar to a stage 3 postovulatory follicle is formed. Stage 4 could not be distinguished from a stage 4 postovulatory follicle. Histochemistry S/3-HXD. Positive reactions were obtained with KAD as a cofactor in ovaries from all stages of t,he reproductive cycle. With NADP as a cofactor a weak reaction was obtained in the granulosa of some of the large follicles (ea. 40 mm diameter), No activity could be demonstrated using NADP as a cofactor in any other tissues. Using NAD as a cofactor 3p-HSD acGvity was observed in the granulosa, and occasionally in the t’heca externa, in developing follicles of all sizes. In foilicles less than 2 mm in diamet,er (Fig. 4j 8 weak reaction was observed in some sections but not in others. It was impossible to determine whether the reaction was of t’hecal or granulosal origin since the tissues have not clearly differentiated at ishisstage. The reaction was seen to increase in intensity with an increase in follicular diameter (see Table 1). flowever, not all follicles in the early stages of development gave positive results. Many sections containing follicles less than 20 mm in diameter were completely negative. All follicles over 20 mm in diameter (Figs. 5, 6) showed enzymic activity in the granuiosa. Activiq was discernible in the cells of theea externa in some of the follicles in all developmental stages (Fig. 5). No activity was observed in the theca interna in any section. Postovulatory follicles, or corpora lutea, showed intense activit’y in the cells derived from
258
LANCE
AND
CALLARD
FIG. 1. Cross section, paraffin, of mature follicle showing granulosa (arrow), Hematoxylin and eosin, X 100. FIG. 2. Cross section, paraffin, of corpus luteum, stage 1, showing thickened elements (arrows). Hematoxylin and eosin, X 100. FIG. 3. Cross section, paraffin, of atretic follicle, showing folded, hypertrophied toxylin and eosin, X 100. FIG. 4. Cross section, frozen, of l- and 5-mm follicles, showing A5, 3 &HSD
theta, theta
and yolk and folded
granulosa activity
(arrow). (arrows).
granules. granulosa HemaX30.
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HISTOCHEMISTRY
IN
259
&LlClh5
FIG. 3. Cross section, frozen, of 20-mm follicle, showing A$ 3 &HSD activity and gradosa (on right), X 100. FIG. 6. Cross section, frozen, of 30-mm follicle, showing A.5, 3 p-HBD activity isolated pocket of activity in the theea, X30. FIG. ‘2’. Cross section, frozen, of 40-mm follicle, showing A5, 3 fl-HSD activity visible activity in the theca, x 100. FIG. 8. Cross section, frozen, of corpus iuteum stage I, showing AS, 3 @-HSD
(arrows)
in theca
(on !
(arrow)
in granulo;;a
(arrow)
in granulosa
activity
(arrow),
X30.
260
LANCE
TABLE As,3 BETA-HYDROXYSTEROID ACTIVITY
IN OVARIAN
AND
1
DEHYDROGENASE TISSUES OF
Xqualus acanthiaP
Follicle size (mm) Follicular growth
Theta externa
1.0
?
10.0
-t
20.0 40.0 Atretic follicles Stage of
phase
+ + -
A B C D
+ ++ +++ +++t “Luteal
gestation Postovulatory phase
Granulosa
Theta
-I+++ + Not observed f+ -
* Rated from weak to strong reactions on a 4-point system.
the follicular granulosa (“lutein cells”) (Fig. 8). The reaction was not quite so intense as that seen in the granulosa of late preovulatory follicles of 40 mm or more (see Table 1 and Fig. 7). Corpora lutea of stages 2 and 3 showed less activity than did those of stage 1, the reaction apparently decreasing in intensity as gestation proceeded (Fig. 9). No activity was observed in any other ovarian tissue, and atretic follicles gave no reaction at any stage in their development. Omission of the acetone rinse prior to incubation caused large formazan granules to form on the intracellular lipid droplets. When sections were incubated after being rinsed in cold acetone the reaction appeared equally intense and the formazan crystals much finer and more evenly distributed. The reaction was seen to increase in intensity when incubated between 1 and 2 hr, but no significant increase in activity could be observed when the sections were incubated beyond this time. When the reaction was run at pH 8.8 a slight diffuse reaction in the control sections was observed. No detectable differences in activity could be observed between tissues incubated in media at pH 7.5 and 8.0, and control sections were negative. At pH 6.8
CALLARD
the activity was seen to be much weaker. There was no observable difference between the reaction when incubated in an elasmobranch Ringer’s medium or a phosphatebuffered medium. The Tris (pH 8.8) medium also gave a slight reaction in the control section. All tissues that gave a positive reaction with DHA as a substrate also showed a positive reaction with pregnenolone as a substrate. The intensity of the reaction appeared to be slightly weaker when pregnenolone was used. With propylene glyco1 as the steroid solvent some slight activity in control sections was observed. This false reaction was not apparent when dimethyl formamide was used. At room temperature (22°C) the reaction was seen to proceed at a much slower rate than at 37”; i.e., after 2 hr the reaction run at 22°C was much weaker than that run at 3i’“C, but formazan deposits of equal intensity could be achieved if the 22°C incubation was continued 4-6 hr. Running the tissue sections through an alcohol series after fixation caused a slight reduction in the formazan deposits, but it was not considered significant. 3-r-HSD. A weak activity was observed in the granulosa of late preovulatory follicles (ea. 40 mm in diameter, Fig. 10). No other tissue showed any trace of activity, and activity was not observed in follicles less than 40 mm in diameter. 1’7p-HSD and 90,&f-HSD. Neither of the enzymes was demonstrated in any of the ovarian tissues using the techniques described. Increasing the substrate eoncentrations or varying the incubation conditions also failed to produce positive reactions. G-6-PDH. A positive reaction was obtained in all tissues tested. The enzyme appeared to be distributed throughout the ovary. Fine formaean granules were apparent in the connective tissue cells, the cells of the epigonal organ, and in all cellular components of the follicular apparatus. The granulosa of the pre- and postovulatory follicles gave a stronger reaction than did other ovarian tissues and the reaction appeared strongest in postovulatory
OVARIAN
Frc. activity FIG.
Fra. activity FIG.
HISTOCHEMISTRY
~iv Squalus
9. Cross section, frozen, of corpus luteum stage 3, adjacent to developing follicle with AS, 3 8in granulosa and theca (arrows), X30. 10. Cross section, frozen, of 40-mm follicle, 3 U-HRD in granulosa, X 100. Il. Cross section, frozen, of corpus luteum stage I, showing glucose-6-phosphate dehydrogc (arrows) ) X 30. 12. Cross section, frozen, 40-mm follicle, SAD-diaphorase act#ivity (arrows), X100.
follicles of stages 1 and 2 (Fig. 11) . The reaction in atretic follicles and late stage postovulatory follicles was considerably weaker than that of stage 1.
SADand NADP-diaphorase. distribution of bot,h of these enzymes similar t’o that of G-B-PDH. The reac in the granuloaa of both pre- and 1
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ovulatory follicles appeared slightly more intense than in other ovarian tissues. However, no differences could be detected in reaction intensities among follicles in the different stages. The NAD-diaphorase (Fig. 12) reaction appeared to be slightly stronger than the NADP-diaphorase reaction in all tissues tested. Lipid. Bright red lipid droplets were observed in the granulosa cells of large preovulatory follicles (30-40 mm). However, the yolk-lipid granules also stained bright red with this stain and tended to obscure some of the staining reactions in the granulosa of smaller follicles. In follicles in which there was no 3P-HSD reaction, the granulosa failed to stain with fat red 7B. Postovulatory follicles showed intense lipid staining in the cells derived from the granulosa. The lipid appeared to be located in the cytoplasm of the “lutein cells” in discrete round droplets. The theta interna and theta externa were negative. The reaction was present in follicles in all stages of development and involution. Atretic follicles also showed intense bright red lipid staining in the granulosa cells in all stages of atresia. Cholesterol. Using the standard Schultz technique all sections tested failed to show the presence of cholesterol. However, when the mordanting time was increased from 3 to 6 days positive reactions were obtained in some sections of postovulatory follicles from all four stages. Not all sections tested with an extended mordanting period gave positive results. Some sections of stage 1 pos+ovulatory corpora lutea failed to demonstrate a positive Schultz reaction even after 8 days of mordanting, whereas alternate sections from the same block of tissue showed positive lipid staining and 3P-HSD activity. All secstions of atretic follicles tested failed to exhibit positive reactions. No other ovarian structure gave a positive reaction.
CALLARD
griseum (Samuel, 1945)) and Scyliorhinus stellaris (Chieffi and Botte, 1961)) the granulosa being composed of a single layer of columnar epithelium. In the ovoviviparous elasmobranchs Rhinobatus granulatus (Samuel, 1943) and Torpedo marmorata (Chieffi, 1961), and in oviparous Raja spp. (Botte, 1963) however, the granulosa is composed of two cell types, a Iarge yolk secreting cell and a smaller columnar cell (Chieffi, 1961). All of the species described as having a two cell-type granulosa belong to the order Batoidei, and all those with a single cell-type granulosa to the SeZachii. It may be, though the number of species studied is small, that a similar situation exists throughout the two orders. Presumably, the two orders evolved a viviparous mode of reproduction independentIy and in parallel (Breder and Rosen, 1966). It is possible that different ovarian endocrine structures evolved in the two groups. To date there have been no histochemical studies of the developing follicles in the elasmobranch ovary. The histochemical data presented in this study show that NAD and NADP-diaphorase are present in all ovarian tissues. The ubiquitous distribution of these enzymes in Squalus ovarian tissue would rule out their absence as a limiting factor in hydroxysteroid dehydrogenase localization (Baillie et al., 1966). S/3-HSD was consistently demonstrated in the granulosa cells, and occasionally in the theta externa of the developing follicle. The activity of the enzyme was seen to increase with an increase in follicular diameter, the most intense reaction being demonstrated in follicles just prior to ovulation (i.e., in the 40-50-mm diameter range). G-6-PDH was also seen to be most active in the granulosa, though differences in intensity of the reaction were impossible to detect in follicles of different size, and weak enzymic activity was observed in all other ovarian tissues. The en7vme 3,8-HSD catalyzes the conDISCUSSION vercion of nregnenolone to progesterone and The histology of the developing follicle dehvdroeniandrosterone to androstenedione, of S+aluus acanthias is essentially similar and it is believed to hold a key position in to that of the ovovivinarous Spinax niger the biosynthesis of the various steroid hormones (Talalay, 1965). It is generally ac(Wallace, 19041, oviparous Chiloscyllium
OVARIAN
HISTOCHEMISTRY
that in all species t,he activity of this enzyme is essential in the early biosynthetic pathways leading to the production of the biologically active steroid hormones (Goldman et al., 1965). Histochemical studies on the ovaries of various mammalian species (Rubin et al., 1963) have shown t’hat the enzyme is particularly concentrated in the theta interna, and present but. less active in the granulosa and interstit,ial tissue of a great many species. A number of other steroid substrates possessing a 3,8-hydroxy group also give positive histochemical results in rat ovarian tissue (Goldberg et al., 1964). Baillie et al. (1966) suggest,that there are separate substrate-specific 3-/3-hydroxysteroid dehydrogenases. Although DNA appeared to give a. slightly better reaction than did pregnenolone in sollIe sections of Squlaus ovarian tissue, no differences in cellular tissue distribution of the formazan crystals with either substrate was observed. It is therefore, unlikely that’ tsvo separate substratespecific 3P-HSDs are present in Squalus ovarian tissue. The slight positive reaction in the granulosa of 4%50-mm follicles with II’ADP as a cofactor and pregnenolone or DWA as substrate might indicate the presence of an XADP-dependent 3,8-HSD. In vertebrates below the Mammalia there is little experimental evidence concerning steroid hormone biosynthesis in ovarian tissue, and histochemical studies on tine locat,ion and distribution of 3pHSD in ovarian follicles have given extremely variable results. Hardisty and Barnes (1968) reported positive 3/3-HSD activity in t’he ovarian granulosa cells of the cyclostome Lampetra j?wiatiiis, the histochemical reaction being most intense during the breeding season. Bara (1965’) showed that in the mackerel 8corYhhersco~aber the S/3-HSD activity was restricted to the thecal cells of the normal follicles, whereas Lambert (1966) found that, in the guplty, Poecilia reticulata, 3/3NSD activity was confined to the cells of the granulosa. Pesonen and Ranola (1962) were unable to detect any steroid dehydrogenase activity in the ovaries of Xenopzds cepted
IN
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laevis and Bufo bufo; Joly (1965) reported positive S/3-HSD activity in the ganulos and weak activity in the theta interns of Salamandra salamandra; and &on ~~96~) was able to demonst’rate 3P-HSD activity in the follicular cells of the newt, Pleurodeles waltii. However; Ferguson (unpublished, cited by Baillie et al.: 1966) showed positive activity in the interstitial tissue of the frog ovary, but not in tbe granulosa or t.heca, whereas Botte and Cottino (1964) were able to demonstrate 3pHSD activity in the granuiosa and t,heca cells, but not in the interstitium of Rar,a esculenta and T,ritur,h5 cristatus. In reptiles 3/3-HSD has been demonstrated I&ochemically in the granulosa and theea ceils of a number of species, including Lace&a sicda (Botte and Delrio, i965) ; Xa’atri:r: sipedon pictivenlris (Callard, 1966) ; and Sceloporus cyanogenys and D ipsosn~z~s dorsalis dorsalis (unpublished observations). In the fowl, as in reptiles, the 3/% HSD reaction has been demonstrated in both the granulosa and thcca of the drveloping follicle (Botte, 1963) ; and ‘Woods and Domm (1966) also reported positive activity in the interstitial cells of the fowl ovary. These histochemical data suggest that both the theta and granulosa of the developing follicle in lower vertebrates arc involved to some extent in steroid biosynthesis. The variable results may reflect differences in enzymic activity in different tissues, differences in distribut’ion of the enzyme, or simply differences in technique. Nevertheless, it appears tha2t, throughout the vertebrate series: the ovarian follicular apparatus has the enzymic ability to convert h”,3/%hyclroxgsteroids to a&,3-ketoeteroids. This hypothesis is supported, to a certain extent, by in vitro incubation experiments with l”Clabeled pregnenolone as substrate when Primarily follicular tissue was incubated (elasmobranchs, Callard and Lcathem. 1965; teleost, Reinbot,h et al., 1966; amphibians, Callard and Leathem, 1966; Ozon, 1967; reptiles, Callard and L&hem. 1964, 1965). In all cases the ovarian incubates were able to synthesize progester-one from the pregncnoione precursor.
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The distribution of G-6-PDH in the developing follicles in Squab showing intense activity in the granulosa, together with the presence of lipid droplets and increasing activity of 3,&HSD concomitant with an increase in follicular diameter, would suggest that the granulosa cells of the developing follicle in this species are probably one site of steroid biosynthesis. The steroid extraction studies of Simpson et a2. (1963) strengthen this view. They showed that ovarian tissue from SquaZus acanthias with mature follicles contained higher concentrations of estradiol-17P per unit weight than did tissue from ovaries with immature follicles (32 ,ug of estradiol/kg of tissue to 11.7 pg of estradiol/kg of tissue). These biochemical and histochemical data suggest that ovarian steroid biosynthesis increases prior to ovulation and that these steroids are responsible for the accompanying changes in the oviduct. The function of “corpora lutea” in the elasmobranch ovary, irrespective of whether they originate from pre- or postovulatory follicles remains obscure. From the present study it is seen that in S. acanthias 3@-HSD activity is present in corpora lutea derived from post,ovulatory follicles, and that this activity is confined to the cells derived from the follicular granulosa, though occasionally weak activity could be detected in the outer theta cells. The reaction was most intense in corpora lutea from ovaries of animals that had recently ovulated (stage A of the gestation period [Hisaw and Albert, 1947; see Material and Methods]) and gradually decreased in intensity as gestation progressed. The corpora lutea derived from atretic follicles, though histologically similar in appearance in the late stages to postovulatory corpora lutea, never at any time showed 3P-HSD activity. G-6-PDH activity was also most intense in the modified granulosa cells of the corpus luteum; however, atretic follicles also showed a weak reaction for this enzyme. The distribution of lipid as demonstrated by staining with fat red 7B could not be correlated with enzyme activity. Both postovulatory and atretic follicles showed considerable lipid with this stain. The results
CALLARD
of the Schultz cholesterol test on S. acanthias ovarian tissues indicate that cholesterol is present at sites of activity of 3/% HSD in postovulatory follicles. There is abundant evidence that cholesterol is one precursor of all the steroid hormones (Talalay, 1965), and it’s location in steroid-producing glands has often been correlated with steroid biosynthesis. In oviparous Xcyliorhinus stellaris a positive 3,8-HSD activity in corpora lutea derived from postovulatory follicles was reported, and no reaction was found in atretic follicles (Lupo et al., 1965). In ovoviviparous Torpedo marmorata the reverse was observed, a positive 3,8-HSD reaction in atretic follicles and no reaction in the postovulatory follicles. In each case in which a posi’ive 3,8-HSD reaction occured a positive Schultz test for cholesterol was obtained. Botte (1963) also reported positive histochemical demonstration of cholesterol in postovulatory follicles of several species of Raja (all oviparous), but did not attempt enzyme histochemistry. In the structures that failed to show enzyme activity (in Torpedo and Xcyliorhinus) the cholesterol test was negative (Lupo et al., 1965)) as was the atretic follicle in Rajn (Botte, 1963). These authors did not state whether the enzyme activity was confined to the cells derived from the granulosa, nor whether any differences in the intensity of the histochemical reaction was observed among the species, or at different stages in the reproductive cycle, Chieffi (1961) observed that corpora lutea from atretic follicles increased in number at the beginning of pregnancy in Torpedo marmorata, and that there was a direct relationship between increasing numbers of such LLcorpora lutea” and the lengthening of the uterine folds as gestation progressed. From these data, plus the fact that these structures show positive 3/3-HSD activity, he suggests that in ovoviviparous Torpedo the atretic follicles are the source of steroid hormones involved in some way with gestation. He also speculates that the histochemical picture of luteogenesis in elasmobranchs may be related to different modes of reproduction rather than to
OVARIAN
HISTOCHEMISTRY
taxonomic distribution, thus ovoviviparous species develop corpora lutea from atretic follicies and oviparous species from postovulatory follicles. The histochemical picture of luteogenesis in ovoviviparous Squalus acanthias presented here resemb’les that’ of the oviparous Scyliorhinus stellaris (corpora lutea develop from postovulatory follicles) and therefore does not correlate with mode of reproduction postulated by Chieffi (1967). If, as the hist.oc~hemicaI data suggest, the granulosa cells of the developing follicle in Squalus acanthias are capable of synthesizing steroids, it would appear that these same cells retain this capacity in the postovulatory follicle, though perhaps to a lesser extent. Whether or not these structures actually secrete steroids, or that steroid hormones are necessary for the maintenance of gestation in ovoviviparous elasmobranchs is not known. The sparse experimental data indicate that the corpus luteum does not function in the maintenance of pregnancy in the few species studied. The fact that Squab acanthias embryos can be maintained in seawater outside the oviduct for periods up to 173 days (Jones and Price, 1967) would inclic,ate that, the development of the embryos in this species is not dependent on maternal steroid hormones. Hisaw and Abrams (1937, 1939 unpublished, cited by Dodd, 1960) found that hypophysectomy in viviparous Mustelus cnnis had no effect on the embryos for the first three months of the ten month gestation period, and that there was an increase in the number of atretie follicles. Hisaw (1959) cites this as evidence that the corpus luteum is not under the control of pituitary hormones as in mammals, and is nonfunctional as far as the maintenance of gestation is concerned. However, as Price (1964) pointed out, the yolk sac placenta of Mustelus does not att’ach to the uterine wall until the fourth month of pregnancy. One would not expect any drastic effect on the embryos as a result of hypophysectomy prior to that time. Until successful ovariectomy is performed on live-bearing elasmobranchs the question must remain unsettled. The presence of a weak 3~HSD activity
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f%$Lahs
265
in the granulosa of mature follicles but not in the corpora lutea in Squalus is difficult to explain. This enzyme catalyzes, reversibly, the reactions converting androsterone to Sn-androstanedione, and etiocholanolone to 5/3-androstanedione (Baillie et al., 1966) ~ Ferguson (unpublished, quoted by Baiilie et al., 1966) was able to demonstrate an intense activity in the corpus luteum of the rat. It is possible that in Squalus the presence of this enzyme in the granulosa cells simply reflects part of the catabolic pathway of s’ceroids, since Callard and Leathem (1965) found significant amounts of etiocholanolone after incubating Squalus ovarian tissue ,in vitro with l”C-testosterone. No 17P-KSD activity was detected in any ovarian sections tested in this study. This is surprising in view of the high levels of estrone and e&radio1 found in elasmobrnnch ovarian tissue (Gottfried, 1964) suggesting significant. 17&HSD activity. The GL uVit’i.0 studies of Callartl and Eeathem (19%) using the same species, and Lupo et aE. (1966), using Torpedo, have shown that elasmobraneh ovarian tissue is capable oi converting testosterone to adrostenedione, thus indicating the presence of t,his enzyme. Recently, Simpson et al. (1968) have demonstrated the in vitro biosynthesis of e&radio1 17p from androstenedione by the follicular membranes of ripe follicles of Xcyltiorhinus caniculus, A. Failure to get a positive 17/X-HSD react,ion in Syz~alu3 ovary could be due to technical problems. However, a section of rat ovarian tissue gave a positive reaction under identical conditions. It is possible t#hat there are species differences in steroid dehydrogenases a,nd. t different, conditions to those used in this study are necessary for the histochemical demonstration of 17p-H$D in Squulus owrisn tissue. REFEREIWES K. C., FERGUSON, 34. M., D. (1965). Histochemicai demonstration of 20,&hydroxysteroid dehydrogenases. J. Eh,zdocrinoZ. 32, 337-339. BAILLIE, A. FL. F~~cusoa, M. M., AND MoKXIART, D. (1966). “Advances in Steroid Ristochemistry?’ Academic Press: New York. BAILLIE, AND
A. M.,
M&.&RT,
CALMAN,
266
LANCE
AND
BALOGH, K. (1964). A histochemical method for demonstrating 2On-hydroxysteroid dehydrogenase activity in rat ovaries. /. Nistochem. C’ytothem. 12, 670-673. BARA, G. (1965). Histochemical localization of 3P-hydroxysteroid dehydrogenase in the ovaries of a teleost fish, Scomber scomber L. Gen. Comp.
Endocrinol.
5, 284-296.
BOTTE, V. (1963). Osservazioni istologiche ed istochemiche sui follicoli post-ovulatori e atresici de Raja spp. Acta Med. Romana 1, 1-7. Bo-~TE, V. (1963a). La localiezazione della steroideS/3-olo-deidrogznasi nsli’ovaio di pollo. Rend. Ist.
Univ.
Camerino
4, 205-209.
LOTTE, V., .~ND CO~~INO, E. (1964). Richerche istochemiche sulla distribuzione de1 colesterole e di alcuni enzimi della steroidogenesi nei follicoli ovarici e post-ovulatori di Rana esculenta e Triturus elistatus. Boll. 2001. 31, 491-499. BOTTE, V., AND DELRIO, G. (1965). Ricerche istochemiche sulla distribuzione dei 3-dei l’i-chetosteroidi e di alcuni enzimi del!a steroidogenesi nell’ovario di Lacerta si?~uZu. BoEt. Zool. 32, 191-198. BREDER, C. M., AND ROSEN, D. E. (1966). “Modes of Reproduction in Fishes.” Natural History Press, Garden City, New York. BUONNANNO, C., CHIEFFI, G., AND IMPARTO, E. (1964). Variazione de1 tasso plasmatico di deidroepiandrosterone e androsterone nel corso de1 ciclo riproduttivo di Torpedo marmorata (S-lacio ovoviviparo). Publ. Staz. Zool. Napoli 34,
66-74.
CALLARD, I. P., AND LEATHEM, J. H. (1964). In vitro synthesis of progesterone by ovaries and adrenals of snakes. Proc. Sac. Exptl. Biol. Med. 115, 567-569. CALLARD, I. P., AND LEATHE~I, J. H. (1965). In vitro steroid synthesis by the ovaries of elasmobranchs and snakes. Arch. Anat. Microscop. Morphol.
Exptl.
54,
CALLARD, I. P. (1966). synthesis. Excerpta 111, 216. CALLARD, I. P., AND Steroid synthesis by Gen.
Comp.
Endocrinol.
35-48.
Reptilian Med.
Intern.
LEATHEM, amphibian
gonadal Concr.
steroid Ser.
J. H. (1966’). ovarian tissue.
7, 80-84.
CHIIWFI, G. (1961). La luteogenesi nei Selacei ovovivipari. Ricerche istologiche e istochimiche in Torpedo marmorata e Torpedo ocellata. Publ. Staz. Zool. Napoli 32, 145-166. CHIEFFI, G., AND BOTTE, V. (1961). La luteogenesi in Scyliorhinus stellaris. Boll. Zool. ‘28, 203-269. CHIEFFI, G., .~ND Lwo, C. (1963). Identification of sex hormones in the ovarian extracts of Torpedo marmaratn and Bufo vdgaris. Gen. Conp. Endocrinol. 3, 149-152.
CALLARD
CHIEFFI, G. (1966). Occurrence of steroids in gonads of non-mammalian vertebrates and sites of their biosynthesis. Excerpta Med. Intern. Congr. Ser. 132, 1047-1057. CHIEFFI, G. (1966). The reproductive system of elasmobranchs : developmental and endocrinological aspects. In “Sha,rks, Skates and Rays” (Perry W. Gilbert, ed.). Johns Hopkins Press, Baltimore, Maryland. COHEN, R. B. (1959). Histological localization and significance of glucose-6-phosphate dehydrogenase system in adrenal cortex. Proc. Sot. Exptl. Biol. Med. 101, 405-407. DODD, J. M. (1960). Gonadal and gonadotrophic hormones in lower vertebrates. In “Marshall’s Physiology of Reproduction” (A. S. Parkes, ed.) pp. 417-582. Longmans, Green, New York. DOIFMAN. R. I., AND UNGAR, F. (1965). Metabolism of Steroid Hormonrs.” Academic Press. New York. GLICB, D., AND OCHS, M. J. (1955). Studies in histochemistry : quantitative distribution of cholesterol in adrenal glands of the cow, rat and monkey, and effects of stress condit,ions, ACTH, cortisone and desoxycorl-icostciroIir. Endocrinology
56, 285-298.
GOLDBXW, B., JONXS, G. E. S., AND B~RI~owI~., II. 1. (1964). A histochemical study of substr ;tt,c> specificity for the steroid 3p-01 dehydrog(,u:iscs and isomcrase systems in human oval>’ ami testis. J. Histochem. Cytochem. 12, 580-859. GOLDMAN, A. S., YAI~OVAC, TV. C., ANL) BONC;IOVANNI, A. M. (1965). Persistent effects of a sgnthetic androgen derivative on activities of 3phydroxysteroid dehydrogenase and glucose-6phosphate dehydroyenase in rats. B~doc~~irmlog~/ 77,
1105-1115.
GOTT~TRIED,H. (1964). The occurrence and biological significance of steroids in tlrr lomcr vertebrates. Steroids 3, 219-242. H~I~DISTY, M. W., AND BARNES, I<. (196s). Activity of S/3-ol-dehydrogenase in cyclostome gonad. Nature 218, 880. HIS~W, F. L., AND ALBERT, A. (1947). Observat.ions cf the reproduction of the spiny dogfish, Squalus acanthias. Biol. Bull. 92, 187-199. HISAW, F. L. (1959). Endocrine adaptations of the mammalian estrous cycle and gestation. In ‘Comparative Endocrinology” (A. Gorbman, ed.) pp. 533552. Wiley, New York. HISAW, F. L., JR., AND HISAW; F. L. (1959). Corpora lutes of elasmobranch fishes. Anat. Record
135,
269277.
JOLY, J. (1965). Mise en evidence histochimique d’une AS-3-hydroxysteroide deshydrogenase dans l’ovaire de 1’Urodele Salamandra salamandrn (L.) a differents stades du cycle sexuel. Comet. Rend.
2161, 1569-1571.
OVARIAN
HISTOCHEMISTRY
R. T., AND PRICE, K. S. (196’7). A method for maintaining spiny dogfish, SqWdus acarsthias pups artiticially. Copeia 2, 471-472. La~~mw, J. G. D. (1966). Location of hormone production in the ovary of the guppy, Poecilia Teticulata. Experientia 22, 476. JONES,
LEVY,
R.,
DEANE,
H.
IV.,
.4x11 RUBIN,
B. L.
(1959).
Visualization of steroid h’, 3P-ol-dehydrogenase activit+- in tissues of intact and hypophyseetomizcd rats. Er&crklogy 65, 932-943. LILLIE, R. D. (1965). “Histopathologic Technic and Practical Histochemistry.” McGraw-Hill, Xew York LOCKWOOU, A. P. M. (1961). “Ringer” solutions and some notes on the physiological basis of their ionic composition. Comp. Biochem. Physiol. 2, 241-289. LUPO, C., BOWE, V., AND CRIEFFI, CT. (1965). Diffarenze nella capacita di biosintesi degli ormone strroidi tra follicoli l>ost-ovulatori ed atresici in Tcvrpedo maTmorata e Scyliorhinus stellaris. Bdi. zooz. 32, 185-191. LUPO,
C.,
TJoTTE,
I'.,
AXI
G.
CHIEFFI,
(1966).
Steroid biosynthesis in the ovary of elasmobranch fishes, Excelpta Med. Intern. Congy. Ser. 111, Lu~o.
368. C.,
JTELLANO,
6..
.~ND
CHIEFFI,
G.
(1967).
Steroid hormowa in the plasma of the elasmobranch, Torpedo marmorata at various stages of the sexual cycle. Gcn. Comp. Endockzol. 8, 326331. ~NANDI, j. (1967). Comparative endocrinology of steroid hormonw in vertebrates. Am. Zool. 7, 115-133. &ON. R. (1966). Isolement et identification de strroid?s cher; les vrrtebres inferieurs et les okaus. An,%. Biol. Animnle B&him. Biophys. 6, 537-551. Ozov, R. (1967). Synthese, in vitro des hormones steroides dans le testicule et l’ovaire de l’amwaltii Michah. phibiar, urodele, Pleurodeles Gen. Comp. Endocrinol. 8, 214-227. PEARSON, B., AND GROSE, F. (1959). Histochemical demonstration of 178-hydroxysteroid dehydrogcnase by use of tetrazolium salt. Proc. Sac. Expll. Bioi. Med. 100, 636-635. PESOTEN. S., AND Ra~o~a, d. (1962). Observations on the metabolism of adrenal and gonadal steroids in Xenopus Zaezk and B& bufo. Gel&. Camp. Ewlocrinol. 2, 425-432. PRICE. K. S. (1964). Environments during fetal canis Mus tehs development of dogfish, (Mitchell) and Squalvs acanthias Linnaeus, and some comparisons with skates and rays. Ph.D. thesis, TJniversity of Delaware, Newark, Delaware. REINBOTH,
R..
C.ALI.ARD,
1. P., AWD
LEATHEM,
3. H.
IN
&@US
26’7
(1966). 1,~ vitro steroid synthesis by the ovaries of the teleost fish, Centropl-isfes striaiua. Gen. Camp. Endoerkol. 7, 326-328. KUBIN, B. L., DEANE, H. IV., AND HAMILTOS, J.R. (1963). Biochemical and histochemiea! studies on A’, SP-hydroxysteroid dehydrogenase activity in the adrenal glands and ovaries of diverse mammals. Endocrinology 73, 74~783. SAMUEL. >fl. (1943). Studies on the corpus lutrum in hY&Lobatus granzdutus Cuv. Proc. F,,dia)l Acad. Sci. Sect. B. 18, 133-157. S.ZM M. 1194.5). The corpus luteum in c:i&oacyllium griseum Mull. and Henle. Proc. Ixdi,uj~ Acad. Sci. sect. B. 22, 113-123. S.4MUEL6, L. T., &LMP&ICH, M. L., LAs,4TEI-I, A$. B., AID E.EICI-I, H. (1951). An enzyme in end:)wine tissues which oxidizes A~, 3~-hydrox,vsteroids to (Y, a unsaturakd lietOne.5. Scic~nc~ 113, 490-491. SIMPSOZ, T. H., WRIGHT, R. S., AND HUNT. 8. V. (1963) Sex hormones in fish. II. The oestrogens of Scyliorhi~~us ccozic&ts. J. EndocrirLol. 28, 49S-51)7. SIMPBON, T. H., \~RIGFiT, R. S., AND &,TTFKIED, H. (1963). Steroids m the semen of dogfish (Squalus ncan thias). J. Endocrinol. 26, 48Qc49&. SIMPSON,
T.
H..
WRIGHT,
R.
s.,
IND
&NT.
8. ‘\'.
(1968). Steroid biosynthesis in ,u%fm by the component tissues of the ovary of dogfish, Sc&iorhinus ~a&&.~ i. J. E’ndocl-lnal. 42, 519-527.
P. (1965). Enzymatic mechanisms in steroid biochemistry. Anti. Rev. Biocfrem. 34, 347-350. WALLAm, w. (1904). Observations on ovarian ova and follicles in certain teleostean and elasmobranch fishes. &art. J. Microscop. Xci. 47, X61213.
TAL.*.LLAY,
~'ATTENRFXW,
1~.
w.
(1958).
~iC~OSCO~~iC
i-l&O-
chemical demonstration of steroid-3p-ol-d&ydrogenase in tissue sections. 6. Hihtochem. Cytocitem. 6, 225-232. ~TCIOOS, J. E., AND DOM~I, i. V. (1966). A hkochemical identificat,ion of the androgen producing cells in the gonads on the domestic fowl and albino rat. Gen. Camp. Endocrinol. 7? 559-570. WOTIZ,
H. a., RINGLER,
BOTTICELLI,
C.,
HISAW,
F.
L.,
JR.;
I. Identification of estradiol-.17,8 from dogfish ova (8guaZw swkb~i). d. &ol. Chem. 231, 589-592.
AND
T;l'OTIZ,
H.
N..
BOTTICELLI,
e.
R.,
%.4W,
F. I>., h.,
AKD OLSEE, A. G. (1960). Estradiol-l7/?, e&on-e. and progesterone in the ovaries of do&& (Squalus suckeyi). Proc. Natl. Aead. 8& U. S. 46, 580-583
AND
COMPARATIVE
Histochemical
ENDOCRINOLOGY
Study
13,
255-267
of Ovarian
Elasmobranch, VALENTINE
(19639)
Squalus
LANCE2
Received
Function
AND
March
in the
acanthiasl IAPt: P. CALLARD
10, 1969
Ovarian tissues from the ovoviviparous elasmobranch, Squu1u.s acanthl,as were examined histochemically for the distribution of NAD and KADP diaphorase: glucose&phosphate dehydrogenase (G+PDH), 3 beta hydroxysteroid dehydrogenase (3,8HSD), 3 alpha hydroxysteroid dehydrogenase (3cxHSD), 17 beta hydrosysteroid dehydrogenase (17pHSD), and 20 beta hydroxysteroid dehydrogenase (20pHSD). The histochemical distribution of the enzymes was correlated with the histological picture obtained from contiguous sections. NAD, NADP diaphorase, and G-6-PDH were distributed throughout the ovary and positive reactions were obtained in all tissues tested. 3/3HSD activity was demonstrated in the granulosa of follicles at various stages of development, increasing in intensity as the follicle matured. After ovulation, 3pHSD was found in the post-ovulatory follicle (L’corpus luteurn”). No 3pHSD activity was observed in atretic follicles (“preovulatorycorpora lutea”). 3aHSD was only observed as a weak reaction in the granulosa of late preovulatory follicles, and no activity of 17/3HSD and BOPPED was observed in any ovarian component tissues.
nzarmorata and Scyliorhirxus stellar6 ‘nave shown that elasmobranch ovarian tissue is capable of synthesizing steroids in,
In recent years there have been a great many studies on the occurrence of steroid hormones in the lower verteb,rates (see reviews by Gottfried, 1964; Ozon, 1966; Chieffi, 1966; Nandi, 1967). The elasmobran&s in particular have been shown to be a rich source of steroid hormones. Progesterone and estrogens have been identified in extracts of ovarian tissue from Squalus sucicleyi (Wotiz et al., 1958, 1960), Scyliorhinus canicula (Simpson et al., 1963) Torpedo rtza~rnoruta (Chieffi and Lupo, 1963)) and Syualus acunthias (Gott#fried, 1964). Changes in the plasma levels of a number of st.eroids during the reproductive cycle of Torpedo marmorata have also been recorded (Buonnanno et al., 1964; Lupo di Prisco et al., 1967). The studies of CMlard and Leathem (1965) on Raia erillacea and Squab a&ant&as, and of Lupo di Prisco et al. (1966) on Torpedo
Baillie et al., cal demonstration
1965,
1956).
The
histochemi-
of a steroid dehydrogerrase is generally taken as evidence that the part,icular tissue is capable of steroid synthesis. To date, however, there has been only one report, on the histochemical dcm-
1 Supported by NSF Grant GB 6917 and the College of William and Mary. ’ Pyesent address: Department of Zoology, University of Hong Kong, Hong Kong. 255
256
LANCE
AND
on&ration of A5,3P-hydroxysteroid dehydrogenase (3,8-HSD) in the elasmobranch ovary (Lupo et al., 1965). These authors were able to demonstrate positive 3P-HSD activity in postovulatory follicles (corpora lutea) in the oviparous Scyliorhinus stellaris, but not in the atretic follicles, and conversely, positive 3/3-HSD activity in atretic follicles but not in postovulatory follicles in the ovoviviparous Torpedo marmorata. They did not report whether any activity in the developing follicles was observed, Chieffi (1967) suggests that the distribution of this enzyme in the elasmobranch ovary is related to mode of reproduction rather than taxonomic differences so that ovoviviparous species develop eneymically active corpora lutea from atretic follicles and oviparous species from post-ovulatory follicles. Squab acanthias is an ovoviviparous elasmobranch with a gestation period of 20-22 months and possesses ovarian corpora lutea-like structures derived from both atretic and postovulatory follicles, the corpora lutea from the postovulatory follicles persisting in the ovary for a greater part of the gestation period (Hisaw and Albert, 1947). In view of the paucity of information on the steroid-producing glands in elasmobranchs, it is of considerable interest to establish what ovarian structures show evidence of steroidogenesis, and whether seasonal variations in activity are apparent. In the present investigation ovaries from S. acanthias were studied by histochemical methods at various phases of the reproductive cycle. MATERIALS
AND
METHODS
Ovaries from S. acanthins collected in the Chesapeake Bay area were obtained from Harburton Marine Laboratory, Harburton, Virginia, during the period December 1967-April 1968, and were shipped to the laboratory on dry ice or in Bouin’s fixative. The frozen tissues were maintained on dry ice until sectioned. The snoutvent lengths and the number and size of the embryos in the oviducts were recorded to estimate the reproductive status of each animal. The snoutvent length of the nonpregnant animals ranged between 33 and 51 cm, while that of the pregnant animals ranged between 42 and 56 cm. Since none
CALLARD
of the ovaries from animals with a snout-vent length of less than 40 cm contained follicles larger than 15 mm in diameter, these were classed as immature. The stage of gestation was determined by the length of the embryos according to Hisaw and Albert (1947) : &age A, recently ovulated (candle stage) ; stage B, embryos from 3.5 to 7.5 cm; stage C, embryos from 12 to 20 cm; stage D, embryos from 23 to 29 cm. No animals in stage B of the gestation period were collected. All of the tissues were examined grossly for the presence of follicles and corpora lutea in all stages of development. A total of 36 ovaries was selected for sectioning; 6 in Bouinb fluid for routine histology and 30 frozen for histochemistry. The frozen tissues were divided into the following groups: (1) immature (n= 4); (2) mature but nonpregnant (n= 10); (3) recently ovulated (n = 9) ; and (4) contained embroys ranging in size from 10-22 cm (n = 7). The frozen tissues were sectioned at 20,~ on an A0 rotary freezing microtome maintained at --25”C, placed on gelatin-coated cover slips and tested for 3,6-HSD, 3a-HSD, 17p-HSD, and 20/?HSD by a modification of the method of Levy et al. (1959). Steroid substrates used in this study were A” pregnen3,&ol-20-one (pregnenolone) and androstan-3,8-ol-17-one (dehydroepiandrosterone, DHEA) for 3/3-HSD ; androstan-3c+ol-17-one (androsterone) for 3a-HSD; A4 androsten-17,G-ola-one (testosterone), and estradiol-17P for 17,@HSD ; and A4 pregnen-20P-ol-3-one (20.&hydroxyprogesterone) for 2O,&HSD (Mann Chemical, Inc.). The steroids were dissolved in propylene glycol or dimethyl formamide (Baillie et al., 1966) at 1 mg/ml. In the case of 17p-HSD, substrate concentrations of 2 mg/ml and 4 mg/ml were also tested. The following buffers were tested in preparing the incubation medium: phosphate (pH 6.8, 7.5, 8.01 and 8.8), Tris (pH 7.5 and 8.0) and elasmobranch Ringer at pH 7.6 (Lockwood, 1961). Nitro-BT (Sigma) was used as the final electron acceptor. Both NAD and NADP (Sigma) were tested as cofactors in each of the enzyme reactions. The histochemical reactions were tested at room temperature (22°C) and at 37°C for periods from l-6 hr, with some sections being tested with an overnight incubation. The effect of rinsing the sections in acetone prior to incubation was also studied. Alternate sections were incubated in a medium lacking the steroid substrate as a control. After incubation the sections were rinsed briefly in phosphate buffer, fixed in 10% formalin for 10 minutes, dehydrated, and mounted in Permount. Alternate frozen sections to those being tested for steroid dehydrogenases were tested for glucose-
OVARIAN
HISTOCHEMISTRY
6-phosphate dehydrogenase (G-6-PDH) by the method of Cohen (1959), NAD- and NADPdiaphorase by the method of Bara (19651, and stained for hpid with fat red 7. Sections were also tested for the presence of cholesterol by a modification of the Schultz method (Lillie, 1965), or stained with hematoxylin and eosin. The material fixed in Bouin’s fluid was embedded in paraffin and stained routinely with hematoxylin and eosin or Mallory’s triple stain. RESULTS
General Microanatomy In X. acanthias the paired ovaries are suspended from the anterior dorsal wall of the body cavity by a broad mesovarium. The ovary is made up of germinal epithehum, follicles in all stages of development, a connective tissue stroma, and a hematopoietic tissue, the epigonal organ which invests the entire structure. The follicular wall of the developing ovum consists of the zona radiata; the vitelline membrane; t’he granulosa, composed of a single layer of columnar epithelium; the theta interna; and the theca externa. The theea. interna appears to be composed mainly of connective tissue cells (see Fig. 1). As the follicle increases in size there is a gradual increase in thickness of the theta interna. The cells of the theca externa are not always distinguishable in the large preovulatory follicles (#-45 mm in diameter). For convenience, postovulatory and atretic follicles have been divided into four stages. The histology of these structures has been described by Hisaw and Hisaw (1959). “Corpus lute~urn’f (Postovulatory Follicle, Fig. 2). Stage 1 is characterized by an extremely thick theta and a folded granulosa which does not completely fill the center of the structure. Stage 2 is essentially similar to stage I, but the granulosa elements now completely fill the area enclosed by the thecal elements. In stage 3 involution has begun, as indicated by vacuolization of the qanulosa elements and connective tissue &filtration. The theca is much reduced in thickness. In stage 4 involution is advanced, the structure is greatly reduced in size wit,11extreme conn&ive tissue infiltra-
IN - &&Us
257
tion and degeneration of the granulosa cells. Atretic Follicle (Preovulatory Corpus Luteum). Stage 1 is characterized by a thin thecal layer, with elements of theta intruding into the follicle along with the highly folded and invaginat,ed granulosa elements. Phagocytosis of yolk has begun. In stage 2, as shown in Fig. 3 ~hago~~-t5sis of the yolk is complete and the folds of hypertrophied follicular elements have begun to anatomose. At stage 3 the yolk granules have disappeared and a thinwalled solid structure, very similar to a stage 3 postovulatory follicle is formed. Stage 4 could not be distinguished from a stage 4 postovulatory follicle. Histochemistry S/3-HXD. Positive reactions were obtained with KAD as a cofactor in ovaries from all stages of t,he reproductive cycle. With NADP as a cofactor a weak reaction was obtained in the granulosa of some of the large follicles (ea. 40 mm diameter), No activity could be demonstrated using NADP as a cofactor in any other tissues. Using NAD as a cofactor 3p-HSD acGvity was observed in the granulosa, and occasionally in the t’heca externa, in developing follicles of all sizes. In foilicles less than 2 mm in diamet,er (Fig. 4j 8 weak reaction was observed in some sections but not in others. It was impossible to determine whether the reaction was of t’hecal or granulosal origin since the tissues have not clearly differentiated at ishisstage. The reaction was seen to increase in intensity with an increase in follicular diameter (see Table 1). flowever, not all follicles in the early stages of development gave positive results. Many sections containing follicles less than 20 mm in diameter were completely negative. All follicles over 20 mm in diameter (Figs. 5, 6) showed enzymic activity in the granuiosa. Activiq was discernible in the cells of theea externa in some of the follicles in all developmental stages (Fig. 5). No activity was observed in the theca interna in any section. Postovulatory follicles, or corpora lutea, showed intense activit’y in the cells derived from
258
LANCE
AND
CALLARD
FIG. 1. Cross section, paraffin, of mature follicle showing granulosa (arrow), Hematoxylin and eosin, X 100. FIG. 2. Cross section, paraffin, of corpus luteum, stage 1, showing thickened elements (arrows). Hematoxylin and eosin, X 100. FIG. 3. Cross section, paraffin, of atretic follicle, showing folded, hypertrophied toxylin and eosin, X 100. FIG. 4. Cross section, frozen, of l- and 5-mm follicles, showing A5, 3 &HSD
theta, theta
and yolk and folded
granulosa activity
(arrow). (arrows).
granules. granulosa HemaX30.
OVARIAX
HISTOCHEMISTRY
IN
259
&LlClh5
FIG. 3. Cross section, frozen, of 20-mm follicle, showing A$ 3 &HSD activity and gradosa (on right), X 100. FIG. 6. Cross section, frozen, of 30-mm follicle, showing A.5, 3 p-HBD activity isolated pocket of activity in the theea, X30. FIG. ‘2’. Cross section, frozen, of 40-mm follicle, showing A5, 3 fl-HSD activity visible activity in the theca, x 100. FIG. 8. Cross section, frozen, of corpus iuteum stage I, showing AS, 3 @-HSD
(arrows)
in theca
(on !
(arrow)
in granulo;;a
(arrow)
in granulosa
activity
(arrow),
X30.
260
LANCE
TABLE As,3 BETA-HYDROXYSTEROID ACTIVITY
IN OVARIAN
AND
1
DEHYDROGENASE TISSUES OF
Xqualus acanthiaP
Follicle size (mm) Follicular growth
Theta externa
1.0
?
10.0
-t
20.0 40.0 Atretic follicles Stage of
phase
+ + -
A B C D
+ ++ +++ +++t “Luteal
gestation Postovulatory phase
Granulosa
Theta
-I+++ + Not observed f+ -
* Rated from weak to strong reactions on a 4-point system.
the follicular granulosa (“lutein cells”) (Fig. 8). The reaction was not quite so intense as that seen in the granulosa of late preovulatory follicles of 40 mm or more (see Table 1 and Fig. 7). Corpora lutea of stages 2 and 3 showed less activity than did those of stage 1, the reaction apparently decreasing in intensity as gestation proceeded (Fig. 9). No activity was observed in any other ovarian tissue, and atretic follicles gave no reaction at any stage in their development. Omission of the acetone rinse prior to incubation caused large formazan granules to form on the intracellular lipid droplets. When sections were incubated after being rinsed in cold acetone the reaction appeared equally intense and the formazan crystals much finer and more evenly distributed. The reaction was seen to increase in intensity when incubated between 1 and 2 hr, but no significant increase in activity could be observed when the sections were incubated beyond this time. When the reaction was run at pH 8.8 a slight diffuse reaction in the control sections was observed. No detectable differences in activity could be observed between tissues incubated in media at pH 7.5 and 8.0, and control sections were negative. At pH 6.8
CALLARD
the activity was seen to be much weaker. There was no observable difference between the reaction when incubated in an elasmobranch Ringer’s medium or a phosphatebuffered medium. The Tris (pH 8.8) medium also gave a slight reaction in the control section. All tissues that gave a positive reaction with DHA as a substrate also showed a positive reaction with pregnenolone as a substrate. The intensity of the reaction appeared to be slightly weaker when pregnenolone was used. With propylene glyco1 as the steroid solvent some slight activity in control sections was observed. This false reaction was not apparent when dimethyl formamide was used. At room temperature (22°C) the reaction was seen to proceed at a much slower rate than at 37”; i.e., after 2 hr the reaction run at 22°C was much weaker than that run at 3i’“C, but formazan deposits of equal intensity could be achieved if the 22°C incubation was continued 4-6 hr. Running the tissue sections through an alcohol series after fixation caused a slight reduction in the formazan deposits, but it was not considered significant. 3-r-HSD. A weak activity was observed in the granulosa of late preovulatory follicles (ea. 40 mm in diameter, Fig. 10). No other tissue showed any trace of activity, and activity was not observed in follicles less than 40 mm in diameter. 1’7p-HSD and 90,&f-HSD. Neither of the enzymes was demonstrated in any of the ovarian tissues using the techniques described. Increasing the substrate eoncentrations or varying the incubation conditions also failed to produce positive reactions. G-6-PDH. A positive reaction was obtained in all tissues tested. The enzyme appeared to be distributed throughout the ovary. Fine formaean granules were apparent in the connective tissue cells, the cells of the epigonal organ, and in all cellular components of the follicular apparatus. The granulosa of the pre- and postovulatory follicles gave a stronger reaction than did other ovarian tissues and the reaction appeared strongest in postovulatory
OVARIAN
Frc. activity FIG.
Fra. activity FIG.
HISTOCHEMISTRY
~iv Squalus
9. Cross section, frozen, of corpus luteum stage 3, adjacent to developing follicle with AS, 3 8in granulosa and theca (arrows), X30. 10. Cross section, frozen, of 40-mm follicle, 3 U-HRD in granulosa, X 100. Il. Cross section, frozen, of corpus luteum stage I, showing glucose-6-phosphate dehydrogc (arrows) ) X 30. 12. Cross section, frozen, 40-mm follicle, SAD-diaphorase act#ivity (arrows), X100.
follicles of stages 1 and 2 (Fig. 11) . The reaction in atretic follicles and late stage postovulatory follicles was considerably weaker than that of stage 1.
SADand NADP-diaphorase. distribution of bot,h of these enzymes similar t’o that of G-B-PDH. The reac in the granuloaa of both pre- and 1
262
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ovulatory follicles appeared slightly more intense than in other ovarian tissues. However, no differences could be detected in reaction intensities among follicles in the different stages. The NAD-diaphorase (Fig. 12) reaction appeared to be slightly stronger than the NADP-diaphorase reaction in all tissues tested. Lipid. Bright red lipid droplets were observed in the granulosa cells of large preovulatory follicles (30-40 mm). However, the yolk-lipid granules also stained bright red with this stain and tended to obscure some of the staining reactions in the granulosa of smaller follicles. In follicles in which there was no 3P-HSD reaction, the granulosa failed to stain with fat red 7B. Postovulatory follicles showed intense lipid staining in the cells derived from the granulosa. The lipid appeared to be located in the cytoplasm of the “lutein cells” in discrete round droplets. The theta interna and theta externa were negative. The reaction was present in follicles in all stages of development and involution. Atretic follicles also showed intense bright red lipid staining in the granulosa cells in all stages of atresia. Cholesterol. Using the standard Schultz technique all sections tested failed to show the presence of cholesterol. However, when the mordanting time was increased from 3 to 6 days positive reactions were obtained in some sections of postovulatory follicles from all four stages. Not all sections tested with an extended mordanting period gave positive results. Some sections of stage 1 pos+ovulatory corpora lutea failed to demonstrate a positive Schultz reaction even after 8 days of mordanting, whereas alternate sections from the same block of tissue showed positive lipid staining and 3P-HSD activity. All secstions of atretic follicles tested failed to exhibit positive reactions. No other ovarian structure gave a positive reaction.
CALLARD
griseum (Samuel, 1945)) and Scyliorhinus stellaris (Chieffi and Botte, 1961)) the granulosa being composed of a single layer of columnar epithelium. In the ovoviviparous elasmobranchs Rhinobatus granulatus (Samuel, 1943) and Torpedo marmorata (Chieffi, 1961), and in oviparous Raja spp. (Botte, 1963) however, the granulosa is composed of two cell types, a Iarge yolk secreting cell and a smaller columnar cell (Chieffi, 1961). All of the species described as having a two cell-type granulosa belong to the order Batoidei, and all those with a single cell-type granulosa to the SeZachii. It may be, though the number of species studied is small, that a similar situation exists throughout the two orders. Presumably, the two orders evolved a viviparous mode of reproduction independentIy and in parallel (Breder and Rosen, 1966). It is possible that different ovarian endocrine structures evolved in the two groups. To date there have been no histochemical studies of the developing follicles in the elasmobranch ovary. The histochemical data presented in this study show that NAD and NADP-diaphorase are present in all ovarian tissues. The ubiquitous distribution of these enzymes in Squalus ovarian tissue would rule out their absence as a limiting factor in hydroxysteroid dehydrogenase localization (Baillie et al., 1966). S/3-HSD was consistently demonstrated in the granulosa cells, and occasionally in the theta externa of the developing follicle. The activity of the enzyme was seen to increase with an increase in follicular diameter, the most intense reaction being demonstrated in follicles just prior to ovulation (i.e., in the 40-50-mm diameter range). G-6-PDH was also seen to be most active in the granulosa, though differences in intensity of the reaction were impossible to detect in follicles of different size, and weak enzymic activity was observed in all other ovarian tissues. The en7vme 3,8-HSD catalyzes the conDISCUSSION vercion of nregnenolone to progesterone and The histology of the developing follicle dehvdroeniandrosterone to androstenedione, of S+aluus acanthias is essentially similar and it is believed to hold a key position in to that of the ovovivinarous Spinax niger the biosynthesis of the various steroid hormones (Talalay, 1965). It is generally ac(Wallace, 19041, oviparous Chiloscyllium
OVARIAN
HISTOCHEMISTRY
that in all species t,he activity of this enzyme is essential in the early biosynthetic pathways leading to the production of the biologically active steroid hormones (Goldman et al., 1965). Histochemical studies on the ovaries of various mammalian species (Rubin et al., 1963) have shown t’hat the enzyme is particularly concentrated in the theta interna, and present but. less active in the granulosa and interstit,ial tissue of a great many species. A number of other steroid substrates possessing a 3,8-hydroxy group also give positive histochemical results in rat ovarian tissue (Goldberg et al., 1964). Baillie et al. (1966) suggest,that there are separate substrate-specific 3-/3-hydroxysteroid dehydrogenases. Although DNA appeared to give a. slightly better reaction than did pregnenolone in sollIe sections of Squlaus ovarian tissue, no differences in cellular tissue distribution of the formazan crystals with either substrate was observed. It is therefore, unlikely that’ tsvo separate substratespecific 3P-HSDs are present in Squalus ovarian tissue. The slight positive reaction in the granulosa of 4%50-mm follicles with II’ADP as a cofactor and pregnenolone or DWA as substrate might indicate the presence of an XADP-dependent 3,8-HSD. In vertebrates below the Mammalia there is little experimental evidence concerning steroid hormone biosynthesis in ovarian tissue, and histochemical studies on tine locat,ion and distribution of 3pHSD in ovarian follicles have given extremely variable results. Hardisty and Barnes (1968) reported positive 3/3-HSD activity in t’he ovarian granulosa cells of the cyclostome Lampetra j?wiatiiis, the histochemical reaction being most intense during the breeding season. Bara (1965’) showed that in the mackerel 8corYhhersco~aber the S/3-HSD activity was restricted to the thecal cells of the normal follicles, whereas Lambert (1966) found that, in the guplty, Poecilia reticulata, 3/3NSD activity was confined to the cells of the granulosa. Pesonen and Ranola (1962) were unable to detect any steroid dehydrogenase activity in the ovaries of Xenopzds cepted
IN
~yZd&S
263
laevis and Bufo bufo; Joly (1965) reported positive S/3-HSD activity in the ganulos and weak activity in the theta interns of Salamandra salamandra; and &on ~~96~) was able to demonst’rate 3P-HSD activity in the follicular cells of the newt, Pleurodeles waltii. However; Ferguson (unpublished, cited by Baillie et al.: 1966) showed positive activity in the interstitial tissue of the frog ovary, but not in tbe granulosa or t.heca, whereas Botte and Cottino (1964) were able to demonstrate 3pHSD activity in the granuiosa and t,heca cells, but not in the interstitium of Rar,a esculenta and T,ritur,h5 cristatus. In reptiles 3/3-HSD has been demonstrated I&ochemically in the granulosa and theea ceils of a number of species, including Lace&a sicda (Botte and Delrio, i965) ; Xa’atri:r: sipedon pictivenlris (Callard, 1966) ; and Sceloporus cyanogenys and D ipsosn~z~s dorsalis dorsalis (unpublished observations). In the fowl, as in reptiles, the 3/% HSD reaction has been demonstrated in both the granulosa and thcca of the drveloping follicle (Botte, 1963) ; and ‘Woods and Domm (1966) also reported positive activity in the interstitial cells of the fowl ovary. These histochemical data suggest that both the theta and granulosa of the developing follicle in lower vertebrates arc involved to some extent in steroid biosynthesis. The variable results may reflect differences in enzymic activity in different tissues, differences in distribut’ion of the enzyme, or simply differences in technique. Nevertheless, it appears tha2t, throughout the vertebrate series: the ovarian follicular apparatus has the enzymic ability to convert h”,3/%hyclroxgsteroids to a&,3-ketoeteroids. This hypothesis is supported, to a certain extent, by in vitro incubation experiments with l”Clabeled pregnenolone as substrate when Primarily follicular tissue was incubated (elasmobranchs, Callard and Lcathem. 1965; teleost, Reinbot,h et al., 1966; amphibians, Callard and Leathem, 1966; Ozon, 1967; reptiles, Callard and L&hem. 1964, 1965). In all cases the ovarian incubates were able to synthesize progester-one from the pregncnoione precursor.
264
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The distribution of G-6-PDH in the developing follicles in Squab showing intense activity in the granulosa, together with the presence of lipid droplets and increasing activity of 3,&HSD concomitant with an increase in follicular diameter, would suggest that the granulosa cells of the developing follicle in this species are probably one site of steroid biosynthesis. The steroid extraction studies of Simpson et a2. (1963) strengthen this view. They showed that ovarian tissue from SquaZus acanthias with mature follicles contained higher concentrations of estradiol-17P per unit weight than did tissue from ovaries with immature follicles (32 ,ug of estradiol/kg of tissue to 11.7 pg of estradiol/kg of tissue). These biochemical and histochemical data suggest that ovarian steroid biosynthesis increases prior to ovulation and that these steroids are responsible for the accompanying changes in the oviduct. The function of “corpora lutea” in the elasmobranch ovary, irrespective of whether they originate from pre- or postovulatory follicles remains obscure. From the present study it is seen that in S. acanthias 3@-HSD activity is present in corpora lutea derived from post,ovulatory follicles, and that this activity is confined to the cells derived from the follicular granulosa, though occasionally weak activity could be detected in the outer theta cells. The reaction was most intense in corpora lutea from ovaries of animals that had recently ovulated (stage A of the gestation period [Hisaw and Albert, 1947; see Material and Methods]) and gradually decreased in intensity as gestation progressed. The corpora lutea derived from atretic follicles, though histologically similar in appearance in the late stages to postovulatory corpora lutea, never at any time showed 3P-HSD activity. G-6-PDH activity was also most intense in the modified granulosa cells of the corpus luteum; however, atretic follicles also showed a weak reaction for this enzyme. The distribution of lipid as demonstrated by staining with fat red 7B could not be correlated with enzyme activity. Both postovulatory and atretic follicles showed considerable lipid with this stain. The results
CALLARD
of the Schultz cholesterol test on S. acanthias ovarian tissues indicate that cholesterol is present at sites of activity of 3/% HSD in postovulatory follicles. There is abundant evidence that cholesterol is one precursor of all the steroid hormones (Talalay, 1965), and it’s location in steroid-producing glands has often been correlated with steroid biosynthesis. In oviparous Xcyliorhinus stellaris a positive 3,8-HSD activity in corpora lutea derived from postovulatory follicles was reported, and no reaction was found in atretic follicles (Lupo et al., 1965). In ovoviviparous Torpedo marmorata the reverse was observed, a positive 3,8-HSD reaction in atretic follicles and no reaction in the postovulatory follicles. In each case in which a posi’ive 3,8-HSD reaction occured a positive Schultz test for cholesterol was obtained. Botte (1963) also reported positive histochemical demonstration of cholesterol in postovulatory follicles of several species of Raja (all oviparous), but did not attempt enzyme histochemistry. In the structures that failed to show enzyme activity (in Torpedo and Xcyliorhinus) the cholesterol test was negative (Lupo et al., 1965)) as was the atretic follicle in Rajn (Botte, 1963). These authors did not state whether the enzyme activity was confined to the cells derived from the granulosa, nor whether any differences in the intensity of the histochemical reaction was observed among the species, or at different stages in the reproductive cycle, Chieffi (1961) observed that corpora lutea from atretic follicles increased in number at the beginning of pregnancy in Torpedo marmorata, and that there was a direct relationship between increasing numbers of such LLcorpora lutea” and the lengthening of the uterine folds as gestation progressed. From these data, plus the fact that these structures show positive 3/3-HSD activity, he suggests that in ovoviviparous Torpedo the atretic follicles are the source of steroid hormones involved in some way with gestation. He also speculates that the histochemical picture of luteogenesis in elasmobranchs may be related to different modes of reproduction rather than to
OVARIAN
HISTOCHEMISTRY
taxonomic distribution, thus ovoviviparous species develop corpora lutea from atretic follicies and oviparous species from postovulatory follicles. The histochemical picture of luteogenesis in ovoviviparous Squalus acanthias presented here resemb’les that’ of the oviparous Scyliorhinus stellaris (corpora lutea develop from postovulatory follicles) and therefore does not correlate with mode of reproduction postulated by Chieffi (1967). If, as the hist.oc~hemicaI data suggest, the granulosa cells of the developing follicle in Squalus acanthias are capable of synthesizing steroids, it would appear that these same cells retain this capacity in the postovulatory follicle, though perhaps to a lesser extent. Whether or not these structures actually secrete steroids, or that steroid hormones are necessary for the maintenance of gestation in ovoviviparous elasmobranchs is not known. The sparse experimental data indicate that the corpus luteum does not function in the maintenance of pregnancy in the few species studied. The fact that Squab acanthias embryos can be maintained in seawater outside the oviduct for periods up to 173 days (Jones and Price, 1967) would inclic,ate that, the development of the embryos in this species is not dependent on maternal steroid hormones. Hisaw and Abrams (1937, 1939 unpublished, cited by Dodd, 1960) found that hypophysectomy in viviparous Mustelus cnnis had no effect on the embryos for the first three months of the ten month gestation period, and that there was an increase in the number of atretie follicles. Hisaw (1959) cites this as evidence that the corpus luteum is not under the control of pituitary hormones as in mammals, and is nonfunctional as far as the maintenance of gestation is concerned. However, as Price (1964) pointed out, the yolk sac placenta of Mustelus does not att’ach to the uterine wall until the fourth month of pregnancy. One would not expect any drastic effect on the embryos as a result of hypophysectomy prior to that time. Until successful ovariectomy is performed on live-bearing elasmobranchs the question must remain unsettled. The presence of a weak 3~HSD activity
IN
f%$Lahs
265
in the granulosa of mature follicles but not in the corpora lutea in Squalus is difficult to explain. This enzyme catalyzes, reversibly, the reactions converting androsterone to Sn-androstanedione, and etiocholanolone to 5/3-androstanedione (Baillie et al., 1966) ~ Ferguson (unpublished, quoted by Baiilie et al., 1966) was able to demonstrate an intense activity in the corpus luteum of the rat. It is possible that in Squalus the presence of this enzyme in the granulosa cells simply reflects part of the catabolic pathway of s’ceroids, since Callard and Leathem (1965) found significant amounts of etiocholanolone after incubating Squalus ovarian tissue ,in vitro with l”C-testosterone. No 17P-KSD activity was detected in any ovarian sections tested in this study. This is surprising in view of the high levels of estrone and e&radio1 found in elasmobrnnch ovarian tissue (Gottfried, 1964) suggesting significant. 17&HSD activity. The GL uVit’i.0 studies of Callartl and Eeathem (19%) using the same species, and Lupo et aE. (1966), using Torpedo, have shown that elasmobraneh ovarian tissue is capable oi converting testosterone to adrostenedione, thus indicating the presence of t,his enzyme. Recently, Simpson et al. (1968) have demonstrated the in vitro biosynthesis of e&radio1 17p from androstenedione by the follicular membranes of ripe follicles of Xcyltiorhinus caniculus, A. Failure to get a positive 17/X-HSD react,ion in Syz~alu3 ovary could be due to technical problems. However, a section of rat ovarian tissue gave a positive reaction under identical conditions. It is possible t#hat there are species differences in steroid dehydrogenases a,nd. t different, conditions to those used in this study are necessary for the histochemical demonstration of 17p-H$D in Squulus owrisn tissue. REFEREIWES K. C., FERGUSON, 34. M., D. (1965). Histochemicai demonstration of 20,&hydroxysteroid dehydrogenases. J. Eh,zdocrinoZ. 32, 337-339. BAILLIE, A. FL. F~~cusoa, M. M., AND MoKXIART, D. (1966). “Advances in Steroid Ristochemistry?’ Academic Press: New York. BAILLIE, AND
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BALOGH, K. (1964). A histochemical method for demonstrating 2On-hydroxysteroid dehydrogenase activity in rat ovaries. /. Nistochem. C’ytothem. 12, 670-673. BARA, G. (1965). Histochemical localization of 3P-hydroxysteroid dehydrogenase in the ovaries of a teleost fish, Scomber scomber L. Gen. Comp.
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BOTTE, V. (1963). Osservazioni istologiche ed istochemiche sui follicoli post-ovulatori e atresici de Raja spp. Acta Med. Romana 1, 1-7. Bo-~TE, V. (1963a). La localiezazione della steroideS/3-olo-deidrogznasi nsli’ovaio di pollo. Rend. Ist.
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LOTTE, V., .~ND CO~~INO, E. (1964). Richerche istochemiche sulla distribuzione de1 colesterole e di alcuni enzimi della steroidogenesi nei follicoli ovarici e post-ovulatori di Rana esculenta e Triturus elistatus. Boll. 2001. 31, 491-499. BOTTE, V., AND DELRIO, G. (1965). Ricerche istochemiche sulla distribuzione dei 3-dei l’i-chetosteroidi e di alcuni enzimi del!a steroidogenesi nell’ovario di Lacerta si?~uZu. BoEt. Zool. 32, 191-198. BREDER, C. M., AND ROSEN, D. E. (1966). “Modes of Reproduction in Fishes.” Natural History Press, Garden City, New York. BUONNANNO, C., CHIEFFI, G., AND IMPARTO, E. (1964). Variazione de1 tasso plasmatico di deidroepiandrosterone e androsterone nel corso de1 ciclo riproduttivo di Torpedo marmorata (S-lacio ovoviviparo). Publ. Staz. Zool. Napoli 34,
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CHIEFFI, G. (1966). Occurrence of steroids in gonads of non-mammalian vertebrates and sites of their biosynthesis. Excerpta Med. Intern. Congr. Ser. 132, 1047-1057. CHIEFFI, G. (1966). The reproductive system of elasmobranchs : developmental and endocrinological aspects. In “Sha,rks, Skates and Rays” (Perry W. Gilbert, ed.). Johns Hopkins Press, Baltimore, Maryland. COHEN, R. B. (1959). Histological localization and significance of glucose-6-phosphate dehydrogenase system in adrenal cortex. Proc. Sot. Exptl. Biol. Med. 101, 405-407. DODD, J. M. (1960). Gonadal and gonadotrophic hormones in lower vertebrates. In “Marshall’s Physiology of Reproduction” (A. S. Parkes, ed.) pp. 417-582. Longmans, Green, New York. DOIFMAN. R. I., AND UNGAR, F. (1965). Metabolism of Steroid Hormonrs.” Academic Press. New York. GLICB, D., AND OCHS, M. J. (1955). Studies in histochemistry : quantitative distribution of cholesterol in adrenal glands of the cow, rat and monkey, and effects of stress condit,ions, ACTH, cortisone and desoxycorl-icostciroIir. Endocrinology
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GOTT~TRIED,H. (1964). The occurrence and biological significance of steroids in tlrr lomcr vertebrates. Steroids 3, 219-242. H~I~DISTY, M. W., AND BARNES, I<. (196s). Activity of S/3-ol-dehydrogenase in cyclostome gonad. Nature 218, 880. HIS~W, F. L., AND ALBERT, A. (1947). Observat.ions cf the reproduction of the spiny dogfish, Squalus acanthias. Biol. Bull. 92, 187-199. HISAW, F. L. (1959). Endocrine adaptations of the mammalian estrous cycle and gestation. In ‘Comparative Endocrinology” (A. Gorbman, ed.) pp. 533552. Wiley, New York. HISAW, F. L., JR., AND HISAW; F. L. (1959). Corpora lutes of elasmobranch fishes. Anat. Record
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(1966). 1,~ vitro steroid synthesis by the ovaries of the teleost fish, Centropl-isfes striaiua. Gen. Camp. Endoerkol. 7, 326-328. KUBIN, B. L., DEANE, H. IV., AND HAMILTOS, J.R. (1963). Biochemical and histochemiea! studies on A’, SP-hydroxysteroid dehydrogenase activity in the adrenal glands and ovaries of diverse mammals. Endocrinology 73, 74~783. SAMUEL. >fl. (1943). Studies on the corpus lutrum in hY&Lobatus granzdutus Cuv. Proc. F,,dia)l Acad. Sci. Sect. B. 18, 133-157. S.ZM
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P. (1965). Enzymatic mechanisms in steroid biochemistry. Anti. Rev. Biocfrem. 34, 347-350. WALLAm, w. (1904). Observations on ovarian ova and follicles in certain teleostean and elasmobranch fishes. &art. J. Microscop. Xci. 47, X61213.
TAL.*.LLAY,
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