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1968
Ovarian Function in the Ovoviviparous Elasmobranch Squalus acanthias: A Histological and Histochemical Study
Valentine A. Lance College of William & Mary - Arts & Sciences
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Recommended Citation Lance, Valentine A., "Ovarian Function in the Ovoviviparous Elasmobranch Squalus acanthias: A Histological and Histochemical Study" (1968). Dissertations, Theses, and Masters Projects. Paper 1539624642. https://dx.doi.org/doi:10.21220/s2-xewt-rw73
This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. OVARIAN FUNCTION IN THE OVOVIVIPAROUS ELASMOBRANCH
SQUALUS ACANTHIAS; A HISTOLOGICAL AMD
HISTOCHEMICAL STUDY
A Thesis
Presented to
The Faculty of the Department of Biology
The College of William and Mary in Virginia
In Partial Fulfillment
Of the Requirements for the Degree of
Master of Arts
By
Valentine A. Lance
1968 APPROVAL SHEET
This ■thesis is submitted in partial fulfillment of
the requirements for the degree of
Master of Arts
/-
Valentine A. Lance
Approved, September 1968
lan P. Callard* Ph.D.
L: Robert E . L. Black f Ph.D.
... J * A . Simonsf Ph.D.
Typing Services Agency 913 Jackson Drive Williamsburg, Va. 23185 Phone: 229-8827
435987 ACKNOWLEDGMENTS
The writer wishes to express his appreciation to
Dr. Ian P. Callard# under whose supervision this study was conducted# for his patient guidance and criticism through out the investigation.
The author would also like to thank Dr. Robert
E® L. Black and Dr. Jarid A. Simons for their helpful criticism and encouragement during the writing of the manuscript. TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ...... iii
LIST OF T A B L E S ...... v
LIST OF FIGURES ...... vi
ABSTRACT ...... ix
INTRODUCTION ...... 2
MATERIALS AND METHODS ...... 11
RESULTS ...... 23
DISCUSSION ...... 37
APPENDIX ...... 55
BIBLIOGRAPHY ...... 78 LIST OF TABLES
Table Page 5 1. , 3-beta Hydroxysteroid Dehydrogenase Activity in Ovarian Tissues of Squalus acanthias L ...... ^
2. Steroid Dehydrogenases and Substrates Used in this S t u d y ...... 57 LIST OF FIGURES
Figure Page
1. Mechanism of Histochemical Demonstration of Hydroxysteroid dehydrogenase in Tissue Sections ...... 58
2. Biogenesis of Hormones in the Ovary (from Ryan and Smith (1965))...... 59
3. Stages of Gestation...... 60
4. ABSTRACT
The histology and histochemistry of the ovary of the ovoviviparous elasmobranch, Squalus acanthias were studied and correlated with the stage of the reproductive cycle.
The enzymes NAD and NADP diaphorase, glucose- 6-phosphate dehydrogenase# 3-beta-hydroxysteroid dehydrogenase# and 3-alpha-hydroxysteroid dehydrogenase were shown to be present in various ovarian tissues.
The enzyme localization and distribution were correlated with the histological picture and the findings discussed# in the light of literature concerning ovarian endocrine function in lower vertebrates. OVARIAN FUNCTION IN THE OVOVIVIPAROUS ELASMOBRANCH
SQUALUS ACANTHIAS: A HISTOLOGICAL AND
HISTOCHEMICAL STUDY INTRODUCTION
The phenomenon of viviparity is believed to have evolved independently several times within the vertebrate
sub-phylum. In mammals viviparity is under the control of
a well defined endocrine system, and a considerable amount of experimental data have clearly established the organs
and hormones involved. Of no little interest are the ques
tions of whether viviparity in lower vertebrates is con
trolled by the same glands and hormones, and how the condi
tion of viviparity evolved from the primitive oviparous
state.
There is good evidence that the initial phase of the
female reproductive cycle, follicular development and ovula tion# is physiologically similar throughout the vertebrate
series (cfi tiisaw# 1959# 1961bt. for discussion). Gonadotropic
hormones are released from the anterior lobe of the pituitary
which stimulate the development of gametes# initiate steroid
ogenesis and induce ovulation. In mammals it has been amply
documented (Guillem.anr 1967) that the release of these hor mones, FSK (follicle stimulating hormone) and LB (luteinizing hormone), from the pituitary is under the control of the hypo thalamus, The hypothalamus produces substances termed
’release factors’ (RFs) which reach the anterior pituitary by way of the hypophyseal portal system (Pops and Fielding,
1936, Green and Harris, 1947, 1949), where they stimulate specific cells to produce the specific tropic hormones involved. There is experimental evidence for a similar sys tem in bird3 (Assermtacher, 1958) and anatomical evidence for a hypophyseal portal system in reptiles, amphibians and some fishes (Green, 1966),
The cyclostomes do not possess a distinct hypophyseal portal system, but a thin mantle plexus has been described in both the ammocoete and adult animal (Green, 1951)* The work of Dodd et al. (1960) on the cyclostome, Lampetra fluviatiiis, has shown that even in this primitive vertebrate the pituitary is necessary for the maturation of the gonads and the development of the secondary sex characteristics.
Kypophysectomised animals failed to produce mature gametes and did not undergo the morphological changes that normally occur during the breeding season, whereas the sham-operated controls reproduced normally. The failure of the secondary sex characteristics to appear in the hypophysectomised animals was believed to be due to the absence of steroid hormones normally secreted by the mature gonads.
The elasmobranchs as a group possess many features in common with the higher vertebrates that are not found in teleosts; fertilization is always internal, the ova are 4 large and few in number, they possess paired sex ducts , and the embryological development of the gonads resembles the amniota more than the teleosts (Chieffi, 1967), Within the subclass are oviparous^ovoviviparous and viviparous species.
A well defined hypophyseal portal system has been described
(Meurling, 1960? Mellinger, 1961), but Follenius (1965) has pointed out that the ventral lobe of the pituitary is not under neurovascular or neural control. Seasonal changes in the histology of the adenohypophysis have been observed
(Della Corte, 1961; Della Corte and Chieffi, 1961), but the cells that secrete gonadotropins have yet to be positively identified.
The work of Bisaw and Abr&mowitz (1938, 1939),
Vivien (1940) and Dodd et al. (I960) has shown conclusively that the pituitary is the site of gonadotropin production in the elasmobranchs, and Witsehi (1955) has reported positive bioassays for FSH and LR activity from elasmobranch pitui tary extracts. The elasmobranch pituitary is unique in that it is divided into anatomically discrete lobes. Dodd et al.
(I960) have shown that in Scy1iorl^inus canicuius the ventral lobe alone is the site of gonadotropin synthesis. He states that care must be taken in evaluating the results of early experiments involving hypophysectomy in elasmobranchs since the ventral lobe may not have been completely excised. 5
Removal of the ventral lobe in the oviparous Scyliorhinus
canicmlus (Dodd et al., 1960) resulted in immediate cessa
tion of ovulation and atresia of all follicles larger than
4 mm. The sham-operated controls continued to ovulate nor
mally. However# the oviducts of the hypophysectomised
animals appeared to be unaffected and continued to secrete
horny egg-case material, Vivien (1940) in an incomplete
study noted that the gonads of hypophysectomised
Scyliorhinus canicula resembled those of immature animals
several weeks after the operation. Hisaw and Albert (1937,
1939# unpublished# quoted by Dodd, 1960) found that ovula
tion ceased and follicular atresia occurred in viviparous
R’ustelus canis following hypophysectomy, They also observed
that ovulation could be re-initiated by homoplastic pitui
tary implants. However# hypophysectomy of Hustalus soon
after ovulation did not prevent normal emtrvologic develop
ment for up to 3 months. The normal gestation in this
species is 10-11 months.
This evidence would suggest that normal ovarian
function as far as gamete production is dependant upon the
hypophysis in elasznobr&nchs, but that successful gestation does not require the presence of the pituitary* Either
ovarian steroids are not necessary for the maintenance of
pregnancy in Mustelus canis# or their production by the 6 ovary is not under the control of the pituitary* Experiments involving castration in elasmobranchs have so far been unsuc cessful (Dodd et al*/ 1960), so the role of the ovary during gestation in this vertebrate group has not been directly tested* Howeverf injections or implantations of steroids into immature animals does cause some development of the reproduc tive tract and. secondary sex characteristics* Dodd and
Goddard (1961) implanted pellets of estradiol benzoate into immature female Scyllorhinus canlculus and were able to show some stimulation of the reproductive tract. The oviducts of the treated animals were extremely hyperemic and fully dif ferentiated* and in several cases the oviducts were packed with secretions of the horny egg-case material* The response was greatest in animals that were almost mature and was absent in small immature animals* Mature animals showed no additional stimulation of the oviducts* Hisaw and Abramowltz (1939) reported that pellets of 11 female hormone” implanted into yearling female Mustelus canis produced a maturation of the genital tract equivalent to that seen in a normal three year old adult *
Of central importance in the evolution of viviparity is the corpus luteum* In mammals * after ovulation and fertil ization* gestation is under the control of the corpus luteum which develops from the post-ovulatory follicle* This 7 structure produces the steroid hormone progesterone which is essential for the maintenance of pregnancy. The corpus luteum in turn,, is under the control cf the hypothalarno- pituitary unit for at least the first portion of gestation.
Since all vertebrates (with the possible exception of birds) develop ovarian structures resembling corpora
lutea following ovulation (Harrisonr 1948) , it is of inter est to determine whether this structure is under the control of the pituitary or plays any part in the hormonal regula
tion of viviparity in lower vertebrates*
In the elasmobranch ovary there are structures resem bling the mammalian corpus luteum derived from both post- ovulatory and atretic follicles* In the ovoviviparous elasmobranch, Squalus acanthias, corpora lutea derived from post-ovulatory follicles are present in the ovary for a greater part of the gestation period (Hisaw and Abrams, 1947]*
Similar corpus luteum-like bodies have been adequately described in several other elasmobranch species (Wallace,
1904? Samuel, 1943, 1945; Chieffi, 1961 Chieffi and Botte,
1961, Hisaw and Hisaw, 1959)*
While there is no evidence that, these structures or any hormones they may produce are involved in the maintenance of pregnancy in elasmobranchsr the oviducts do undergo impres sive modifications during pregnancy, including extreme 8
hyperemia, villus formation and secretory activity (Ranzi,
1935., 1936, quoted by Neeclham, 1942 and Budker, .1958), It
is possible that ovarian hormones of follicular and luteal origin may contribute to this oviduct growth and function.
Hisaw (1963b) has suggested that steroids present in the yolk sacs of developing embryos may act directly on the oviduct to maintain it during pregnancy. Chieffi (1961),
however, has observed that there is an increase in corpora
lutea derived from atretic follicles, with a corresponding
increase in the length of the uterine folds during gestation
in the ovoviviparous Torpedo marmorata. He speculates that
these structures in Torpedo might be the source of steroid
hormones during the gestation period*
In spite of a lack of evidence directly implicating
ovarian hormones in pregnancy maintenance in elasraobranchs ,
there is some evidence for ovarian steroid biosynthesis along pathways similar to those known for mammalian tissue in Raia
erinacea and Squalus acanthias (Callard and Leathern, 1965),
and in Torpedo marmorata and Scyliorhinus stellarls (Lupo di 5 Prisco et a l , 1966). Demonstration of 3-bet a-
hydroxysteroid dehydrogenase in elasmobranch ovarian tissue
(Lupo di Prisco et al,, 1965)* as well as identification of
the products of in vitro ovarian steroid synthesis, indicates
that the follicular apparatus, growing ova and corpora lutea 9 all have steroidogenic potential (Chieffi, 1966)* Ho infor mation is available regarding the steroidogenic potential of isolated luteal tissue of elasmobranch ovaries as compared to whole ovaries* Progesterone and estrogens have been identified in ovarian tissue of Squalus sucklevi (Wotiz et al., 1958, 1960), Sojliorhinus canicula (Simpson et al.,
1963), Torpedo marmorata (Chieffi, 1962; Chieffi and Lupo,
1963) , and Squalus acanthias (Gottfried, 1964). Changes in plasma levels of progesterone and estrogens during the repro ductive cycle of Torpedo maraqrata have also been recorded
(Buonanno et al*, 1964; Lupo di Prisco et al., 1967)*
It is apparent that at least certain ovarian functions in elasmobranchs are under pituitary control. However, the role of the ovary in steroid synthesis during gestation, and particularly the function of the corpus Xuteum is not under stood* In order to gain some insight into the endocrine role of the various ovarian components during the reproductive cycle and gestation, the following histological and histochemical study of the ovoviviparous elasmobranch Squalus ^anthias was undertaken.
The role of the hydroxysteroid dehydrogenases in steroid biosynthesis is well established (Dorfman and Ungar,
1965). Samuels (1951) first identified * 3 beta- hydroxystero1d dehydrogenase in mammalian steroid producing 10 glands,, and a histochemical method for its intracellular localization in frozen tissue sections was developed by
Wattenfoerg (1958)„ The demonstration of the hydroxysteroid dehydrogenase depends on the transfer of hydrogen, removed from the hydroxysteroid, via HAD or MADP to the tetrazolium salt which is reduced and precipitates as insoluble blue crystals at the site of the reaction, As can foe seen in
Appendix, Figure 1, the reaction is dependent on another enzyme, MAD or MADP diaphorase which transfers the hydrogen from MAD or NAJ>P to the tetrazolium salt* The technique has since been modified for the demonstration of several other hydroxysteroid dehydrogenases (Levy et a L , 1959;
Pearson and Grose, 1959* Balogh, 1964? Eaillie et al,, 1965,
1966). The role of the hydroxysteroid dehydrogenases tested in the known mammalian biosynthetic scheme is indi cated in Appendix, Figure 2.
Glucose-6-phosphate dehydrogenase in ovarian tissue has been shown to vary in activity at different stages in the estrous cycle in rats (Deane et al., 1962) and to increase in activity in response to LK (McKerns, 1965).
The localization of these enzymes in Squalua could indicate sites of steroid biosynthesis and differences in steroidogenic activity related to the reproductive cycle * MATERIALS AND METHODS
A . ANIMALS
Ovaries from Squalus acanthias 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 Williamsburg on dry ice or in Bouin's fixative. Tissues from a total of 107 animals were collected during this periods 95 frozen on dry ice and 12 fixed in Bouin*s. The frozen tissues were maintained on dry ice until ready for use. The snout-vent lengths and the number and size of the embryos in the ovi ducts were recorded to estimate the reproductive status of each animal. The snout-vent length of the non~pregnant animals ranged between 33 and 51 cm, while that of the preg nant animals ranged between 42 and 56 cm. Since none 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).
Stage A Recently ovulated (candle stage)
Stage B. Embryos from 3.5 to 7.5 cm. 12
Stage C* Embryos from 12 to 20 cm,
Stage D. Embryos from 2 3 to 29 cm.
(See Appendix Figure 3.) No animals in stage B of the gesta tion 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 were selected for sectioning? 6 were fixed in Bouin * s for routine histology and 30 were frozen for enzyme histochemistry. The frozen tissues were divided into the following groups;
a) immature (n = 4)
b) mature but non-pregnant (n *= 10)
c) recently ovulated (n * 9)
d) contained embryos ranging in size from
10 - 22 cm (n = 7).
B, REAGENTS
1. Steroids. Androsterone (androstane-3-alpha-ol-
17-one)f dehydroepiandrosterone (androstane-3-beta~ol~17- one); testosterone (^, androstene-17~beta-ol-3-one), estradiol-17-beta (estra-1P 3, 5 ,(10)-triene-3, 17-beta- diol), and 20-beta-hvdroxyprogesterone ( ^ , pregnen~20-beta- ol-3-one) were obtained in crystal form from Steraloids,
Inc„t n.Y. Each steroid was dissolved in dimethyl formamide
(img/ml) and stored in 50 ml stoppered bottles at 4°C. Fresh 13 solutions were prepared every 3 months,
2. Cofactors, Nicotinamide adenine dinucleotide
(NAB) and nicotinamide adenine dinucleotide phosphate (NADP) from Sigma Chemical Co, were stored in a dessicator at
-20°C. Solutions of € mg/ml of each in distilled water were prepared fresh for each experiment. The reduced forms
(MADE and HADFH) (Sigma) for the diaphorase reactions were treated similarly,
3. Buffers» All chemicals were of analytical reagent quality, Elasmobranch Ringer's (pH 7,2) was pre pared according to Lockwood (1961)* (See Appendix for formula,) Tris buffer (Sigma) pH 8.8 and phosphate buffers of pH 8.8, 8,0, 7.5 and 8,8 were also prepared (see Appendix).
All buffers were checked on a Beckman Zeromatic pH meter and stored in polyethylene bottles at 4°C. Adjustments of pH were made with concentrated HC1 or NaOH. Fresh solutions were prepared every 4 weeks,
4. Tetrazolium salt. Kitro-blue tetrazolium (Sigma) was stored dessicated in the dark at 4°C, Fresh solutions
(1 mg/ml in distilled water) were prepared weekly and stored in the dark at 40C.
5. Glucose-6-phosphate, The disodium salt of glucose-6-phosphate (Sigma) was stored dessicated at -~20°€, until ready for use. 14
C, HISTOLOGICAL AND HISTOCHEMICAL METHODS
1. Frozen^ Sections
Preparation of coverslips:
To prevent frozen tissue sections from fall
ing off the cover slips during the histochemical procedure
the cover slips were treated as follows; Precleaned cover
slips were first coated with a 0.5% gelatin solution. To
ensure a thin even coating the gelatin was applied by pass-
ing a thin glass rod that had been dipped in the solution
over the cover slip. The cover slips were then dried for
15 minutes on a slide warmer. The dry cover slips were then
passed over a beaker of boiling 40% formaldehyde and returned
to the slide warmer until dry. The treated cover slips were
stored between paper towels until ready to receive the tis
sue sections.
Sectioning;
Just prior to sectioning the ovaries were removed from the dry ice container, cut into blocks about
2 cm square, and examined grossly for the presence of fol
licles , corpora lutea and corpora atretica. Blocks con
taining these structures in all stages of development were prepared. The blocks were then fixed to previously chilled microtome chucks with 5.0% gelatin and replaced in the dry
ice container until sectioned. 15
The frozen blocks were sectioned at 20 u on an
A O Spencer cryostat maintained at-30°C, The sections were
placed directly on to warm (room temperature) gelatin -
coated cover slips and placed in Columbia jars. In a num
ber of instances alternate sections from each block were
tested for enzymic activity, cholesterol/ lipids, and
stained with hematoxylin and eosin so that correlations
between structure and enzyme activity., and enzyme activity
and lipid location could be determined.
2, Paraffin Sections
Fixation, dehydration and embedding ^
The ovaries in Bouin's fixative were cut into
pieces about 2 cm square, dehydrated through an alcohol
series, and embedded in paraffin (see Appendix
for procedure).
Sectioning and mounting;
The paraffin blocks were trimmed with a knife
and fixed to microtome chucks by heating the chucks and pressing them firmly against the paraffin blocks. The blocks were then sectioned at 10 u on an AO-Spencer microtome. The
sections were floated on water on glass slides that had been
previously treated with MayerSs egg albumin and heated on a
slide wanner until the sections were flattened, The water was then drained off and the slides were left on the slide 16
warmer overnight to ensure attachment of the sections. Tis
sues were stained in either hematoxylin or Malloryjs triple
stain (see Appendix).
D . ENZYME REACTIONS TESTED
1- G lucose-6-phosphate dehydrogenase
A few parallel sections to those being tested
for hydroxysteroid dehydrogenases were tested for the pres
ence of glucose-6-phosphate dehydrogenase by a modification
of the method of Cohen (1959). The procedure was as fol
lows? Tissue sections were cut at 20 u on a cryostat main
tained at -30° C ? mounted on gelatin-coated cover slips and
placed into two sets of Columbia jars containing the fol
lowing media;
Test Control
phosphate buffer pH 7,5 5.0 ml 5.0 ml NaoHPO4 .7H20 16g/l. in distilled HjO
Nitro-blue tetrazolium 1 mg/ml in distilled water 2.0 ml 2 0 ml
Nicotinamide adenine dinucleotide phosphate (NAD?) 5 rag/ml in distilled water 1.0 ml 1.0 ml
Glucose-6-phosphate added dry directly to medium 15 mg 0
The sections were incubated for 30 minutes at 37°C, rinsed 17 in cold buffer and fixed in 10% formalin for 15 minutes.
After fixation the slides were dehydrated through an alco hol series and mounted in Permount.
2. NADH? and NADPH^ diaphorase
The presence of these enzymes was tested for by a modification of the method of Scarpelli et al. (1958) (in
Lillie, 1965), Frozen tissue sections were mounted on cover slips as described above and incubated for 15 minutes at
37°C in the following media?
Test Control
Phosphate buffer pH 7.5 Na^KPO^, 7 ^ 0 16 g/1. in distilled H^O 4.0 ml 5,0 ml
Hitro-blue tetrazolium 1 mg/ml in distilled water 2,0 ml 2.0 ml
NADH2 or HADPH2
6 mg/ml in distilled water 1.0 ml 0
The sections were then rinsed in buffer, fixed in 10 * for malin for 15 minutes, dehydrated through an alcohol series and mounted in Permount.
3. Hydroxysteroid dehydrogenases
a) To determine optimum conditions for the demon stration of the hydroxysteroid dehydrogenases the following variables were tested;
(1) Incubation Time 18
Incubation times were varied between
1 and 6 hours. In addition some of the tissues were incu bated overnight,
(2) Temperature
The reactions were tried at room temperature and at 37°C.
(3) Buffers
Bach of the reactions was tested using
Elasmobraneh Ringer solutionf Tris (Sigma) and phosphate buffer.
(4) pH
Each of the different enzyme reactions was run at pH 6,8, 7.5, 8.0 and 8.8.
(5) Steroid Solvents
The reactions were tested with the steroid substrates dissolved in both propylene glycol and dimethyl f ormaznide,
(6) Cofactors
Both HAD and HAD? were tested as cofactors in all the reactions.
(7) Substrate Concentrations
In the case of 3-alpha~~ hydroxy steroid dehydrogenase, 17**beta-hydroxysteroid dehydrogenase and
20-beta-hy&roxysteroid dehydrogenase substrate concentrations 19 of 1 mg/ml? 2 mg/ml and 4 mg/ml were tested*
(8) Acetone Rinse
An acetone rinse prior to incubation removes all lipids and endogenous substrates that might give minimal reaction in control tissue sections. Baillie et al. (1966) advises against an acetone rinse as he con siders it might interfere with the enzyme activity* How ever, Rosenbaum (personal communication) states that for- roazan crystals are insoluble in fats and will aggregate in clumps on top of the lipid droplets in the tissue, and, therefore, give a distorted picture of enzyme localisation.
Each of the reaction© was tested with both the inclusion and omission of the acetone rinse prior to incubation.
(9) Mounting Media
Baillie et al. (1966) suggested that dehydrating the tissue sections through an alcohol series removes some of the formazan crystals and recommends an aqueous mounting medium. Both glycerin jelly and Persnount were tested.
b) 3 -beta- hydroxysteroiddehydrogenase
A modified method of Levy et al. (1959) was used for the demonstration of this enzyme. Both pregnenolone and dehydcepiandrosterone v/ere tested as substrates. The 20 procedure was as follows;- Tissue sections were cut at 20 u on a cryostat maintained at -BO G , mounted on gelatin- coated cover slips and rinsed in cold acetone for 5 min utes and cold phosphate buffer for 2 minutes. The sections were divided into two sets of Columbia jars containing the following media;
Test Control
Phosphate buffer pH 7.5 ,7H^0 16 g/1. in distilled water 4.4 ml 4.8 ml
Nitro-blue tetrazoliuir 1 mg/ml in distilled water 2.0 ml 2.0 ml nicotinamide adenine dinucleotide 6 mg/ml in distilled water prepared fresh 0.8 ml 0.8 ml
Dehydroepiandrosterone or pregnenolone 1 mg/ml in dimethyl formamide 0.4 ml 0
Following a 2-hour incubation period at 37°C the sections were rinsed in buffer, fixed in 10% formalin for 15 minutes, dehydrated through an alcohol series and mounted in Permount.
c) 3-alpha-hydroxysteroid dehydrogenase , 17-
beta-hydroxystaroid dehydrogenase, 2 0-beta-
hydroxysteroid dehydrogenase,
The method used for the demonstration of each of the above enzymes was identical to that outlined above for 21
3-beta-hydroxysteroid dehydrogenase, with the exception of the substrate used. For 3 -- alpha* hydroxys tero id dehydro genase androsterone was the substrate tested. For 17-beta** hydroxystercid dehydrogenase both testosterone and estradiol v;ere used as substrates. And for 20-"beta-hydroxysteroid dehy drogenase the substrate was 2Q-beta~hydroxyprogesterone (see
Appendix Table 2). In all hydroxysteroid dehydrogenase tests rat ovarian tissue was incubated as a control for the procedure.
4. Cholesterol
The presence of cholesterol in steroid secreting glands may be demonstrated histochemi ca1ly by the Schultz method as modified by Mallory (Lillie, 1965). This test gives a specific color reaction in frozen tissue sections that contain cholesterol or cholesterol esters. Since the mammalian adrenal gland is particularly rich in cholesterol, control sections of rat adrenal were tested with each sec tion of Sgualus ovarian tissue. The method as follows was modified slightly from the original by increasing the mor danting time from 3 to 6 days.
Tissue sections were mounted on cover slips as for the enzyme reactions then treated as follows
a) Fixed in 10% formalin for 15 minutes.
b) /Iordanted in 2.5% iron alum in tightly stoppered Columbia jars for 6-3 days at 37°C. 22
c) Rinsed in distilled water and blotted dry.
d) A few drops of acetic-sulfuric acid mixture were then dropped on to the tissue section with a glass rod.
e) The cover slip with the tissue section was then placed on a clean dry slide and examined immediately.
The presence of cholesterol or its esters is indicated by a blue-green color that appears within 5 minutes, turning brown within half an hour.
5, Lipids
Sections from each ovarian structure tested for enzymic activity were stained for lipids with Fat Red 7B
(Ciba) and counter-stained with hematoxylin. RESULTS
HISTOLOGY
In Sgualus acanthias the paired ovaries are sus
pended from the anterior dorsal wall of the body cavity by
a broad mesovarium, The ovary is made up of germinal epi
thelium, follicles in all stages of development, a connec
tive tissue stroma, and a hemopoietic tissue, the epigonal
organ which invests the entire structure.
1, Preovulatory Follicle (see Appendix, Figures
4 and 5)
The follicular wall of the developing ovum con
sists of the zona radiata, the vitelline membrane, the gran ulosa, the theea interna and the theca externa. In follicles up to one millimeter in diameter the follicular epithelium, or granulosa, and theca are not clearly differentiated from each other, The cells surrounding the vitelline membrane are round to ovoid in shape with large, round, centrally located nuclei. The cytoplasm is slightly basophilic and the chrom atin material appears to be located at the periphery of the nucleus. As the follicle increases in diameter, the granu
losa and theca become clearly distinguishable, a discrete membrana propria at the base of the granulosa separating the 24 two layers. The granulosa appears to he composed of only one cell type arranged in a single layerr the cells gradually increasing in height with an increase in follicular diameter.
At this stage the staining properties of the granulosa are similar to those of the primordial granulosa, the cytoplasm is lightly staining and only the periphery of the nuclei stains, As the cells assume a columnar shape large vacuoles appear in the cytoplasm. The nuclei are now situated close to the basement membrane and occupy about one third to one half of the cell volume.
The theca interna is composed largely of connective tissue that stains an intense blue with Mallory‘s triple stain» Interspersed between the fibrous stroma are small flattened cells with densely staining, basophilic nuclei.
In the smaller follicles, one to five millimeters in dia meter, the theca externa is not clearly defined? but in sec tions where it is evident it is composed of one or two layers of medium-sized, irregularly-shaped cells with round to oval nuclei that are weakly basophilic. Occasionally one or two nucleoli are visible. The cytoplasm stains lightly. These cells are not evident in all preparations, and where they do appear they are not always in a continuous layer.
This is essentially the picture that is met with in 25 all follicles prior to ovulation, the only changes being a gradual increase in thickness in the connective tissue stroma of the theca and a decrease in size of the vacuoles in the granulosa cells as the diameter of the follicles increases.
2. Postovulatory Follicles
a) Stage I (see Appendix, Figures 6 and 7).
Immediately after ovulation the collapsed follicle is thrown into complex folds with prominent, finger-like extensions of follicular epithelium, along with the basement membrane and blood cells projecting into the lumen* The cells of the follicular epithelium have under gone considerable histologic differentiation. The indivi dual cells have hypertrophied and the nuclei are now moder ately basophilic,: the cytoplasm is still lightly stained and the coll boundaries are difficult to make out. The folli cular epithelium is nov; separated from the theca. interna and numerous blood cells appear in the spaces between the two layers. This layer is now many cells in thickness. It is possible that the granulosa is pushed into this many layered appearance by the rapid contraction of the follicle follow ing ovulation. The preovulatory follicle of 40-50 milli meters in diameter contracts to a structure of about 15 milli meters at its greatest diameter. 26
The outstanding feature of the postovulatory follicle at this stage is the extreme thickness of the con-” nective tissue layer of the theca interna (100-200 u from
20-50 u in the preovulatory follicle) . This sheath is corn-' posed mainly of connective tissue fibers that stain deep blue with Mallory's triple stain- The nuclei of the cells interspersed between the fibers are still strongly baso philic but are now irregularly-shaped f possibly due to the forces of contraction as the egg is expelled. The theca externa, when visible, retains the same histologic appear ance and is clearly differentiated from the theca interna.
Sometimes these cells appear as isolated pockets adjacent to the connective tissue sheath of the theca interna.
b) Stage II (see Appendix, Figures S and 9).
A cross section of the postovulatory folli cle at this stage reveals little change in the staining p r o perties of the different components. The granulosa cells now completely fill the lumen of the follicle and the cells retain their hypertrophied appearance. The nuclei are a little more compact and seem to have a slightly increased affinity for hematoxylin. Large vacuoles are apparent in the lightly staining cytoplasm. A few strands of connective tissue fibers are interspersed between the columns of gran ulosa cells, but the theca interna is still separate and 27
peripheral. The theca interna is now .much reduced in thick ness (ca. 50 u). As in the previous stages, cells of the
theca externa are not always apparent.
c) Stage III (see Appendix,, Figures 10 and 11).
This stage is marked by the appearance of extremely large vacuoles in the cytoplasm of the granulosa cells. The staining properties remain essentially the same
though the nuclei are now less compacted. Some connective
tissue strands from the theca interna, accompanied by blood vessels, project into the mass of granulosa cells. The
theca interna is now barely visible, a few connective tis
sue fibers and small connective tissue cells surround the
luteal- like body. The cells of the theca externa were extremely difficult to locate in the sections examined and when observed, had undergone no apparent change. The overall diameter of the follicle is reduced and is gener ally compressed laterally by the new crop of developing ova.
d) Stage IV (see Appendix, Figures 12 and 13).
At this stage the process of involution is well under way. The structure is greatly reduced in size with many more connective tissue elements and blood vessels pushing into the body of granulosa cells. The granulosa cells show definite signs of disintegration, the nuclei are pycnotic and the cell boundaries indistinct, Many cells are clearly ruptured. Gradually the connective tissue fibers, blood vessels and hemopoietic tissue of the epigonal organ come to occupy most of the area of the degenerating struc ture .
3 * Atre tic_Pollicles
Follicles in all stages of development are seen to undergo atresia. In the smaller follicles, up to 2 mm in diameter the cells simply atrophy and are resorbed. In the larger follicles the mechanism is somewhat more complex and may be divided into the following stages:
a) Stage X (see Appendix, Figures 14 and 15),
The first sign of atresia is the breakdown of the vitelline membrane, next,- strands of granulosa cells along with blood vessels and connective tissue elements from the theca interna migrate into the yolk mass. The follicle still maintains its spherical shape. The cells of the gran ulosa are enormously hypertrophied, columnar, with large clear vacuoles in the cytoplasm. The nuclei are ovoid to round and moderately basophilic. The major difference between the folds of connective tissue and granulosa that invaginate the follicle in this structure and those in the postovulatory follicle is the presence of elements of the theca interna. In the postovulatory follicle the theca 29 interna remains separate from the folds of granulosa and blood vessels.
b) Stage II (see Appendix, Figures 16 and 17).
.Most of the yolk has now disappeared and the follicle is no longer spherical. The folds of follicular epithelium and connective tissue begin to anastomose. The granulosa ceils are still very large but no longer contain the large round vacuoles in the cytoplasm. The nuclei are spherical and moderately basophilic and are now situated close to the free end of the cell. The cytoplasm is lightly stained and exhibits a fine reticular structure. Increasing amounts of connective tissue and connective tissue cells are visible in the invagiaatad folds of granulosa.
c) Stage III (see Appendix, Figures 18 and 19).
All the yolk granules have now disappeared and there is no longer any space in the lumen of the folli cle. The structure is much reduced in size and begins to resemble the postovulatory follicle of Stage III.. The gran ulosa cells show signs of early involution. The nuclei are still fairly compact but the outlines of the cells are no longer clear and the cytoplasmic reticulum is no longer vis ible. h great many connective tissue cells and blood cells accompanied by fibrous material are visible between the invag inations.. The only difference between this structure and the postovulatory follicle is the greater number of connective tissue elements and blood cells present,
d) Stage IV.
It was impossible to distinguish between the atretic and postovulatory follicles at this stage.
SNZY/iE HISTOCKS?;ISTAY
1, Glu co s e ~ 6 ~ pho s pha t e ciehydrogenase (see Appendix.
Figure 20) .
A positive reaction we3 obtained in all tissues tested, The enzyme appeared to be distributed throughout the ovary„ Fine formazan granules were apparent in the con nective tissue cells, the cells of the epigonal organ, and
•in all cellular components of the follicular apparatus. The granulosa of the pre- and postovulatory follicle gave a stronger reaction than the theca interna and other ovarian tissues, but it was difficult to detect differences in inten sity among the different stages. The reaction in the granu losa of late postovulatory follicle (Stage III) however, wa considerably veaher than that of Stage I.
2. ico tjlnamide adenine dinucleotide and nicotin
amide adenine dinucleotide phosphate diaphorase
(see Appendix, Figure 21).
The distribution of both of these enzyme was sim ilar to that of glucose-6 -phosphate dehydrogenase. As was 31
seen for that enzyme the reaction in the granulosa was more intense than in the theca interna, but not, however, than
the other ovarian tissues. No differences could be detected between reaction intensities from follicles in the different stages. The NADHp reaction appeared to be slightly stronger than the NADPH2 in all tissues tested.
3. Hydroxysteroid dehydrogenases
a) 3-'beta-'hydroxys teroid dehydrogenase (see
Appendix, Figures 22 through 32)«
Positive reactions were obtained with NAD as a cofactor in ovaries from all stages of the reproductive cycle. With NADP as a cofactor a weak reaction was obtained only in the granulosa of large preovulatory follicles (ca,
40 mm diameter). Ho activity could be demonstrated using
NADP as a cofactor in any other tissues.
Using NAD as a cofactor 3-beta-hydroxysteroid dehydrogenase activity was observed in the granulosa, and occasionally in the theca externa, in developing follicles of all sizes. In follicles less than 2 mm in diameter a weak reaction was observed in some sections but not in others. It was impossible to determine*whether the reaction was of thecal or granulosal origin since the tissues had not clearly differentiated at this stage. The reaction in the granulosa was seen to increase in intensity with an increase 32 in follicular diameter (see Appendix, Table 1). However, 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 fol licles over 20 mm in diameter showed enzymic activity in the granulosa. Activity was discernible in the cells of the theca externa in some of the follicles in all developmental stages. Postovulatory follicles, or corpora lutea, showed intense activity in the cells derived from the fol licular granulosa ('lutein cells*). The reaction was not quite as intense as that seen in the granulosa of late pre ovulatory follicles of 40 mm or more (see Appendix, Table
1 ). Corpora lutea of Stages II and III shox^ed less activ ity than Stage I, the reaction apparently decreasing in intensity as gestation proceeded.
Mo activity was observed in any other ovarian tissue, and atretic follicles gave no reaction at any stage in their development,
b) Influence of temperature, pF and other vari
ables on the histochemical demonstration of
3-beta-hydroxysteroid dehydrogenase„
Omission of the acetone rinse prior to incuba tion caused large formasan granules to form on the lipid drop lets. When sections were incubated after being rinsed in cold 33 acetone the reaction was equally intense, and the formazan
crystals appeared much finer and more evenly distributed.
The reaction was seen to increase in intensity when incubated between 1 and 2 hours, but no significant
increase in activity could be observed when the sections were
incubated beyond this time.
When the reaction was run at pH 8.3 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, the activity was seen
to be much weaker, There was no observable difference between the reaction when incubated in an elasmobranch
Ringer5s medium or a phosphate buffered medium. The Tris (pH 8.8) medium gave a diffuse reaction in the control sec tion .
All tissues that gave a positive reaction with dehydroepiandrosterone 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.
Kith propylene glycol as the steroid solvent
some activity in control sections was observed, but not when dimethyl formamide was used. 34
At room temperature (22°C) the reaction was seen to proceed at a much slower rate than at 37°C} i.e.., after two hours the reaction run at 22°C was much weaker than that run at 37°C, but activities of equal intensities could be achieved if the 22 0 incubation was continued 4*6 hours.
Running the tissue sections through an alco hol series after fixation did cause a slight reduction in the forma2 an deposits/ 'but it was not considered significant.
c) 3 - alpha •• hydroxyste roid dehydrogenase (see
Appendix, Figure 33).
A weak activity was demonstrated only.in the granulosa of late preovulatory follicles (40 mra). No other tissue showed, any trace of acrivity.
d) .17 - be t a •*■■ hydroxy steroid dehydrogenase;, 20-beta-
hydroxysteroid dehydrogenase.
neither of the above enz\nes was demonstrated in any of the ovarian tissues using the techniques described.
Increasing the substrate concentrations or varying the incu bation conditions also failed to produce positive reactions.
The absence of activity did not appear to be due to inadequa cies in the technique since positive reactions for these enzymes were obtained in rat ovarian tissue sections,
4. Fat Red 7B (see Appendix, Figures 34 through 36).
Bright red lipid droplets were seen in the granulosa cells of large preovulatory follicles (30-40 rm) .. However the yolk*lipid granules also stained a bright red with this stain and tended to obscure some of the reactions in the granulosa of smaller follicles. In follicles where there was no "r > 3 beta hydroxysteroid dehydrogenase 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 granulosa cells in discrete round droplets. The theca interna and the theca externa were negative. The reaction was present in the 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
5 Cholesterol
Using the standard Schultz technique all sec tions tested failed to show the presence of cholesterol, whereas the control sections of rat adrenal gave an intense positive reaction. However, when the mordanting time was increased from 3 to 6 days, positive reactions were obtained in some postovulatory follicles of all stages.
Not all sections tested gave positive results. Some sections of Stage I corpora Lutea failed to demonstrate a positive 36
Schultz reaction even after 8 days of mordanting. Only two sections containing atretic follicles (Stages II and III) were tested with a 6-day mordanting period and both failed to react positively. No other ovarian structures gave a positive reaction. DISCUSSION
The histology of the developing follicle of Sgualujf acanthias is essentially similar to that of ovoviviparous
Spinax niger (Wallace,- 1904) , and oviparous ChiloscyIlium griseum (Samuel, 1945), and Scyliojrhinus stellaris (Chieffi and Botte, 1961), i.e., the granulosa is composed of a sin gle layer of uniform columnar cells. In the ovoviviparous elasmobranchs Rhinobatus grajnulatus (Samuel, 194 3) and
Torpedo marmorata (Chieffi, 1961), and in oviparous Raja spp. (Botte, 1963), however, the granulosa is composed of two col1 types, a large yolk secreting cell with a reti cular nucleus and abundant cytoplasm, and a small columnar cell with a densely staining nucleus and lightly staining cytoplasm (Chieffi, 1961). All of the species described as having a two cell-type granulosa belong to the order
Batoidei, and all of those with a single cell type granu losa, Selachii. One can speculate that a similar situation may exist throughout the two orders. Presumably, the two orders evolved a viviparous mode of reproduction independently and in parallel (Rreder and Rosen, 1966). It is possible that different ovarian endocrine structures evolved in the two groups.
The theca interna in all species described appears to be largely composed of connective tissue fibers with a few small connective tissue calls interspersed. The theca interna of Squalus showed a similar histologic picture.
The outer thecal layer ("theca externa ) described in this study differs markedly from the theca externa as described in Rhinobatus granulatus (Samuel, 1943). In that species the theca externa is composed of concentrically arranged parsnchymatic , elongatef connective tissue cells.
In Squalus on the other hand the cells do not appear to be typically of connective tissue origin. Further, these cells were not always present a© a discrete layer in the developing follicle • It is possible that this 'theca externa is in fact interstitial tissue. However, its origin at this time is unknown, and the cells were not observed in any other location in the ovary.
To date there have been no histochemical studies of the developing follicles in the elasraobraxich ovary. The histochemical data presented in this study show that NAD and NADP diaphorase are present in all ovarian tissues,.
These enzymes function in transferring the hydrogen ion from the hydroxvsteroid molecule via a pyridine nucleotide to the tetrazolium salt, and must be present in order to demonstrate the histochemical localization of hydroxysteroid dehydrogenases (see Appendix, Figure 1). The ubiquitous 39 distribution of >?AD and HA.DP diaphorase in Squalus ovarian tissue would rule out the absence of these enzymes as a lim iting factor in hydroxysteroid dehydrogenase localization* 5 * 3--beta-hydroxysteroid dehydrogenase was consis tently demonstrated in the granulosa cells, and occasionally observed in the outer thecal cells (theca externa5) 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),
Glucose-6-phosphate dehydrogenase was also seen to be most active in the granulosa, though differences in intensity of the reaction were impossible to detect in follicles of dif ferent size, and weak enzymic activity was observed in all other ovarian tissues, 5 The enzyme , 3-beta-hydroxysteroid dehydrogenase catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androsteneaione, and it is believed to hold a key position in the biosynthesis of the various steroid hormones (Talalay, 1965), It is generally accepted that in all species the activity of this enzyme is essen tial in the early biosynthetic pathways leading to the produc tion of almost all the biologically active steroid hormones
(Goldberg et al,, 1965). Histocheniical studies have shown 5 that ' , 3-beta-hyd.roxysteroid dehydrogenase is present in all the steroid secreting glands of mammals (Wattenberg,
1958; Levy et al., 1959; Baillie et al., 1966). Studies on the ovaries of various maxusialian species (hubin et al.,
1963) have shown that the enzyme is particularly conspicuou in the theca interna,, and present hut shows less activity in the granulosa and interstitial tissue of a great many species.
A number of other steroid substrates possessing a
3-beta hydroxy group also give positive histochemical results in rat ovarian tissue (Goldberg et al., 1964).
Baillie et al. (1966) suggest that there are separate sub strate specific 3“beta--hydroxysteroid dehydrogenases.
Although dehydroepiandrosterone appeared to give a slightly better reaction than pregnenolone in some sections of
Sgualus ovarian tissue, no differences in intracellular or tissue distribution of the formazan crystals with either substrate were observed. It is, therefore, unlikely that two separate substrate specific 3'* bet a “hydroxy steroid dehy. drcgenases are present in Sgualus ovarian tissue.
The slight positive reaction in the granulosa of
40-50 run follicles with hADP as a cofactor and pregnenolone or dehydroepiandrosterone as substrate might indicate the presence of an KADP-dependent , 3 d;eta-hydroxysteroid 41 dehydrogenase However the reaction could b e due to V.P..D
impurities in the NADP, thus demonstrating the normal NAD- dependent enzyme.
In vertebrates below the mammalia there is little experimental evidence concerning steroid hormone biosyn thesis in ovarian tissue, and histochemical studies on the location and distribution of hydroxysteroid dehydrogenases
in ovarian follicles have given extremely variable results.
Xlardiety and Barnes (1968) reported positive 5 , 3--beta--hydroxysteroid dehydrogenase activity in the ovarian granulosa cells of the cyclostoxne, Lampetra fluviatills, the histochemical reaction being most intense during the breeding season. Bara (1966) showed that in the 5 mackerel, Scomber scomber, the , 3-beta -hydroxysteroid dehydrogenase activity vias restricted to the theca cells of the normal follicles, whereas Lambert (1966) found that in 5 the guppy, Poecilia reticulata, , 3- beta hydroxys teroid dehydrogenase activity was confined to the cells of the granulosa. Pesonen and Rapola (1362) were unable to detect any steroid dehydrogenase activity in the ovaries of Xenopus laevis and Bufo bufo; Ferguson (unpublished, quoted by
Baillie et al., 1366) was able to demonstrate 3-beta- hydroxysteroid dehydrogenase activity in the interstitial tissue of the frog ovary, but not in the granulosa or theca; g; however, 02on (1967) showed positive ~, 3-beta- hydroxysteroid dehydrogenase activity in the follicular cells of the newt Pleurodales waltii, and Botte and Cottino (1964) 5 were able to demonstrate , 3 -beta-hydroxysteroid dehy drogenase activity in the granulosa and theca calls., but not in the inters tit ium. of l.ana esculanta and Triturns cristatus, 5 In reptiles r 3-be t a ~hydroxysteroid dehydrogenase activity has bean demonstrated histochemically in the granulosa and theca cells of: the developing follicle of a number of species including Lacerta sicuia (Botte and Delrio, 1965); Natrix sipedon pictivantr1s (Callard, 1966): Sceloporus cyanogenys and Bipsosaurus dorsalis dorsalis (personal observations- and 5 Callard, unpublished) In fov/l, as in reptiles,, the , 3- toeta-hydroxy steroid dehydrogenase reaction has been demon-™ strated in both the granulosa and theca of the developing follicle (Botte, 1963; Ferguson, unpublished, quoted by
Baillie et al., 1966),
These histochemical data suggest that both the theca
■and granulosa of the developing follicle in lower vertebrates are involved, to some extent, in steroid biosynthesis. The variable results may reflect differences in enzymic activity in different tissues,, differences in. distribution of the enzyme, or simply, differences in technique* Nevertheless, it appears that throughout the vertebrate series the ovarian 43 follicular apparatus has the enzymic ability to convert 5 4 5. 3-beta-hydroxys teroids to , 3--k etc steroidsThis is supported to a certain extent by in vitro incubation expert- 14 ments with C -labelled pregnenolone as substrate when pri marily follicular tissue was incubated (elasmobranchs,
Callard and Leathern, 1965; teleost, Reinboth et al„ f 1966, amphibians( Callard and Leathern, 1966; Ozon, 1967; reptiles ,
Callard and Leathern, 1964)* In all cases the ovarian incu bates were able to synthesize progesterone from the pregnen olone precursor.
The NADP-dependent enzyme, glucose-6-phosphate dehy drogenase generates a NADPK which is essential in many of the reactions involved in steroid biosynthesis (Talalay,
1965). Deane et al. (1962) have demonstrated the presence of this enzyme in rat ovarian tissues that show positive 5 , 3~beta~hydroxysteroid dehydrogenase activity. The activity of glucose-6-phosphate dehydrogenase appeared to reflect the secretory activity of the steroid producing cells. However, Taki et al* (1967) could find no correla tion between the [email protected] distribution of this enzyme 5 and , 3 -beta *hydroxysteroid dehydrogenase in human ovaries,, McKerns (1965) showed that in rat ovarian tissue the activity of glucose-6-phosphate dehydrogenase could be enhanced in vitro by luteinizing hormone r which is known to 44 stimulate steroid biosynthesis.
The distribution of glucose*-6-phosphate dehydro genase in the dewloping follicles in Squalus , (i.e., intense activity in the granulosa), together with the pr©~ 5 sence of lipid droplets and increasing activity of ' , 3-beta- hydroxysteroid dehydrogenase 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 a1. (1963) strengthen this view. They showed that ovarian tissue from jSgualus acanjthias with mature follicles contained higher concentrations of estradiol•17- beta per unit weight than tissues from ovaries with immature folli cles (32 ug of estradiol/kg of tissue to 11>7 ug of estradiol/ kg of tissue)* The in vitro studies of Callard and Leathern
(1965) also suggest that the Squalus acanthias ovary is active in steroid biosynthesis, It is possible., as the histcchemical and biochemical data suggest, that ovarian steroid biosynthesis increases prior to ovulation to prepare the oviduct for the reception of the embryos»
The histology and histochemistry of the corpus luteum in the elasmobranch ovary presents a somewhat confus ing picture. In Squalus acanthias the postovulatory follicle develops into a solid gland-like structure consisting of 45 hypertrophied granulosa cells surrounded by a thick connec tive tissue sheath. The connective tissue theca cells remain distinctly separate for at least the first two stages of its development. Samuel (1943) describes the corpus luteurn of
Rhinobatus granulatus as being composed of both granulosa cells and cells from the theca interna. Since the material of Samuel1s study consisted of a single ovary, the reproduc tive status of the animal was not indicated, and no atretic follicles were described, it is possible that the structure described may have been an atretic follicle. The atretic follicle in Sgualgis shows a similar histologic picture to the structure described by Samuel.
In Squalus the :corpus luteurn* formed from an atretic follicle is distinctly different from the corpus luteum formed from a postovulatory follicle (at least in the early stages of development), In the atretic structure.cells from the theca interna migrate into the follicle in the early stages of its development, whereas in the postovulatory corpus luteum the cells of the theca intern® remain separate until the process of involution begins. In Torpedo marmorata and T.. ocollata the reverse is true, the small granulosa cells in an atretic follicle develop into a solid glandular struc ture (the large cells are ‘expelled ) while the postovulatory follicle undergoes an early sclerosis (Chieffi, 1961). The 46 theca interna in this preovulatory corpus luteum remains separate as in the postovulatorv corpus luteum in Squalus.
From the present study it is seen that in S, 5 acanthias . 3-beta-hydroxysteroid dehydrogenase activity is present only in the postovulatory corpora lutea, and that this activity is confined to the cells derived from the follicular granulosar though occasionally weak activity could be detected in the outer theca cells, The reaction was most intense in corpora lutea from ovaries of animals that had recently ovulated (stage A of the gestation period) and gradually decreased in intensity as gestation progressed.
The structures derived from atretic follicles, though histo logically similar in appearance in the late stages to post- ovulatory corpora lutea.. never at any time showed ~ , 3-~heta- hydroxysteroid dehydrogenase activity. Glucose-6-phosphate dehydrogenase also showed intense activity in the modified granulosa cells of the corpus luteumf but as in the preovul atory follicle, no differences in intensity could be detected between the structures at different stages in the cycle. It did appear, however, that the corpora lutea from stage A in the gestation period were slightly more active than corpora lutea from later stages.. The atretic follicles showed a weak glucose-6-'phosphate dehydrogenase activity,
The distribution of lipid as demonstrated by staining 47 with fat red IB could, not be correlated with enzyme activity.
Both corpora lutea and atretic follicles showed considerable staining.. The results of the Schultz cholesterol test on S, m e a n t hias ovarian tissues indicate that cholesterol is pres- 5 ant at sit©© of activity of , 3‘-beta-hydroxystoroid dehy drogenase in postovulatory follicles and is absent in all other ovarian structures. There is abundant evidence that cholesterol is the sole precursor of all the steroid hor mones {Talal&y, 1965) and its location in steroid produc ing glands has often been correlated with steroid biosyn. thesis {Click and Ochs, 1955). The presence of cholesterol in the corpus luteum of Sgrualus^ supported, by the enzyme histochenica 1 data,- suggest© that this structure is active in steroid biosynthesis.
In oviparous Scylicrhinus stellaris a positive , 3-beta^hydroxysteroid dehydrogenase activity in corpora lutea derived from postovulatory follicles was reported, and no reaction was found in atretic follicles (Lupo at al.,
1965),. In ovoviviperous Torpedo raarraorataas with the 5 histological data., the reverse was true# a positive 3 -beta- hydroxysterold dehydrogenase reaction in atretic follicles and no reaction in the postovulatory follicles. In each 5 case where a positive f S-beta-hydroxysteroid dehydro genase reaction occurred. a positive Schultz test for 48 cholesterol was obtained. Botte (1963) also reported posi tive hiss toe hemical demonstration of cholesterol in post- ovulatory follicles of several species of Raja (all ovipar ous) , but did not attempt histochemistry. In the struc tures that failed to show enzyme activity (in Torpedo and
the cholesterol test was negative (Lupo et a h f 196 5) , as was the atretic follicle in Haja (Botte,
1963). These authors did not state whether the enzyme activity was confined to the cells derived from the gran ulosa., nor whether any difference in the intensity of the histochemical reaction was observed between the species, or at different stages in the reproductive cycle,
Chieffi (1961) observed that 'corpora luteaf; 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''corpora lutea and the lengthening of the uterine folds as gestation progressed* From this data, plus the fact that these struc- tures show positive 5 , 3-beta-hydroxysteroid dehydrogenase 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 luteogenesi s in elasmo- branchs may be related to different modes of reproduction 49
rather than to taxonomic distribution,- i.e., ovoviviparous
species develop corpora lutea from atretic follicles and
oviparous species from postovulatory follicles. The histo-
chemical picture of luteogenesis in ovoviviparous Squalus
acanthia3 presented here resembles that of the oviparous
Scy1iorhinus stellaris (corpora lutea develop from post
ovulatory follicles) and, therefore,- does not correlate
with mode of reproduction as postulated by Chieffi (1967).
The presence of a weak 3-aIpha-hydroxysteroid dehy
drogenase activity in the granulosa of mature follicles
(40-50 nun) but not in the corpora lutea in Squalus is dif
ficult to explain. This enzyme catalyzes, reversifoly, the
reactions converting androsterone to 5~alpha-androstan-
edione, and etiocholanolone to 5-beta-androstanedione
(Baillie e t .a1., 1966).Ferguson (unpublished, quoted by
Baillie 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 gran
ulosa cells simply reflects part of the catabolic pathway
of steroids,since Callard. and Leathern (1965) found signifi
cant amounts of etiocholanolone after incubating Squalus
ovarian tissue in vitro with labelled testosterone.
Ho 17-beta-hydroxysteroid dehydrogenase activity was detected, in any ovarian sections tested in this study. 50
This is surprising in view of the high levels of estrone and estradiol found in elasmobranch ovarian tissue (Gottfried,
1964) suggesting significant 17 beta--hydroxy steroid dehydro genase activity. The in vitro studies of Callard and
Leathern (1965) using the same species,, and Lupo et al. (1966) using Torpedo have shown that elasmobranch ovarian tissue is capable of converting testosterone to androstenedione, thus indicating the presence of this enzyme. Failure to get a positive 17“beta-hydroxysteroid dehydrogenase reac tion in Sgualus ovary could be due to technical problems.
However, the control rat ovarian section gave positive results under identical conditions. It is possible that there are species differences in steroid dehydrogenases and that different conditions to those used in this study are necessary for the histochemical demonstration in Scjualus ovarian tissue.
The function of corpora lutea in the elasmobranch ovary, irrespective of whether they originate from pre- or postovulatory follicles, remains obscure. Chieffi (1967) suggests that in Torpedo ’''corpora lutea" derived from pre ovulatory follicles function in steroid biosynthesis during pregnancy. If, as the histochemical data presented in this study suggest, the granulosa cells of the developing follicle in Sgualus 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 elasmohranchs is not known. The A sparse experimental data, however, indicate that the corpus
luteum does not function in the maintenance of pregnancy in
the few species studied. Hisaw and Abrams (1937, 193$) unpub
lished. quoted by Dodd, 1960) found that hypophysectomy in viviparous Hustelus cants had no effect on embryos for the first three months of the ten month gestation period, and that there was an increase in the number of atretic folli cles. Hisaw (1959) cites this as evidence that the elasmo branch corpus luteum is not under the control of pituitary hormones as in the mammals, and is non-functional as far as the maintenance cf gestation Is concerned. The fact that
Squalus acanthias- embryos can be maintained in sea water outside the oviduct for periods up to 173 days (Jones and
Price, 1967) would also indicate that the development of the eir,bryos in this species is not dependent on maternal steroid hormones. However, as Price (1964) has pointed out, the yolk sac placenta of Mustelus does not attach to the uterine wall until the fourth month of pregnancy. One would not expect any drastic effect on the embryos as a result of 52 hypophysesfcomy prior to that time. It is obvious that until ovariectomy is performed on live-bearing elasmobranchs the question will remain unsettled.
Some studies of steroids in the blood of elasmo branch s have been performed. Buonanno et al. (1964) and
Lupo do Frisco et al. (196?) analysed steroids in the plasma of Torpedo raarmorata at various stages in the sexual cycle and found higher levels of dehydroepiandrosterone and andro- starone during the gestation period, but were unable to detect progesterone during pregnancy. However, they were able to detect significant amounts of progesterone in the period prior to gestation. These results would seem to indicate that in Torpedo progesterone is not associated with gestation., and that an increase in synthesis of 17- ketosteroids may in some way be related to pregnancy in this species, The origin of these steroids in the plasma remains unknown. It is possible that they may originate in the adrenal gland„
Callard and Leathern (1965) in an in vitro study of
Squalus acanthias ovarian tissue showed that it is capable of converting pregnenolone to progesterone, progesterone to testcsterona# and testosterone to androstenedione, epiandro starone and eticbolanolone. They also showed that the percent conversion of pregnenolone to progesterone in 53 ovarian tissue from pregnant animals was double that of tissue from non-pregnant animals. Whether this increased biosynthetic activity was due to the presence of corpora lutea was not established* The persistence of the corpus luteum in Sc^ualus for a greater part of the gestation period (Hisaw and Albert/ 1947) suggests a correlation between the presence of this structure in the ovary and ges tation *
The histochemical demonstration of an enzyme in a given tissue merely suggests the presence of a particular metabolic pathway,, but gives no clue as to function. In order to deduce functional properties some correlation must be found between reaction intensity and steroid biosynthesis
(Boucek et al./ 1966). Such information is lacking in the elasmobranchs. The histochemical data presented here sug gest that the granulosa cells of the developing follicle and the modified granulosa cells of the corpus luteum are capable of synthesizing steroid hormones. It appears likely that the granulosa cells of developing follicles are the source of the high levels of estrogen found in the ova of this species (Simpson et al., 1963), and that the corpus luteum retains a steroid synthesising capacity for the first few months of gestation, though perhaps to a lesser degree*
Until definitive experiments involving hypophysectorcy, 54 ovariectomy, and in vitro incubations of isolated luteal tissue are performed/ the function of the corpus luteum in elasmobranchs must remain an enigma.
56
APPENDIX A
Table 1.
5 , 3~Beta~Hydroxysteroid Dehydrogenase Activity In Ovarian Tissues of Squalus Acanthias L.
Follicle Size (in mni) Theca Granulosa
Follicular Growth Phase 1.0 ? + 10.0 + ++ 20.0 + +++ 40.0 + +++++
Atretic Follicles —
Stage of ” Luteal Gestation Theca tissue”
Posxiovulatory Phase A + ++++ B a C - ++ D +
aNot observed Steroid Dehydrogenases and Substrates Used in This Study M !3 ISj EH CO Gt W t£ >■* w © P CQ us JD E G © o o d £ d o o © Da CD03 G D U d m G G E h h i I 1 •H •GP s d r-w 00 d rH rH 43© d d © d P £ P 43 d I d G G d co rH <| r- 43p P P P p © © © a)
© *- © - d d © XT'* •iH P '—' P £ P o CO r->CO n© £ © tn p 43 CO d P £ © © P P US © us £ £ £ © ! \ } I j © - ©
o P p •p o cs> *-* CM d O ^ P 43 © 43 Cn © d © d d 1 P G P p P P P P © P © © p © US P P P © © 0 © © © © O O 43 £ P P © © X p £ O C^ P © £ ! O £ © G — tn © G O JP £ O © © p © 0 >i © © O © O tTP >i >1 X—S •rH d o d CO fN O C £ © © G i C %, appendix b 58 MECHANISM OF HISTOCHEMICAL DEMONSTRATION OF HYDROXYSTEROID DEHYDROGENASE IN TISSUE SECTIONS ' NAD Formazan crystals (insoluble colored) HO I Hydroxysteroid Diaphorase dehydrogenase [ i NAOH Tetrazolium salt (soluble colorless) 0 FIGURE 1 59 BIOGENESIS OF HORMONES IN THE OVARY CH3COOH A cetate HO B Cholesterol HO' Pregnenolone Progesterone CH, =0 HO 17- Hydroxypregnenolone 1.7-Hydroxyprogesterone HO' Androstenedione Testosterone HO HO Estradiol Legend: Reactions marked with a C are catalyzed by 3-beta hydroxysteroid dehydrogenase. Reactions marked with a G are catalyzed by 17-beta- hydroxysteroid dehydrogenase. FIGURE 2. From Ryan and Smith (19 65). OVUM CORPUS EMBRYO LUTEUM LENGTH < ffi O o 60 FIGURE 3, Stages of Gestation KEY PAGE FOR INSERTS 61 - 68 Pages to be inserted with photographs Figures 4, 5, 6, 7 61 8,9,10,11 62 12, 13, 14, 15 63 16, 17, 18, 19 64 20, 21, 22, 23 65 24, <£• f 26 , 27 66 28, 29, 30, 31 67 32, 33 68 Page 61 Fig. k. Paraffin Stctloa of a saall follicle showing laaatur* granulosa csllo H.& I,f 1001 Fig. 5. Paraffin section of a nature follicle showing the different lagers of the follicle mil* 8. & 1. ^00X« H. (k I. Heaatoxylin and Bosln / v . > * - t , Page 6la* Fig* 6* Paraffin section of corpus latum shortly after eeulatlon. 8. & E. 3 0 1 . Fig. 7* Sane aa fig. 6. showing detail of theca and granulosa 100X. Page 62* Fig. 8. Corpus lytsws stags XI* Mete solid aass of soils Frocsn section stained with B* & £. 301. Fig. 9. Corpus Inteas stags II Paraffin section H.S. 1001 Fhgt 62m Fig* 10* Corpus lutsu* stag* III Parafftii saction H. & S. 100X Fig* IXm Carpus luteua stags XXX Paraffin section 400X ■V "V v* - ' w j ' i • v «v » y*r,' v V * Y . W ‘ '*■ ■ • *•, ^ . / Y v * S r f O ^ ,J(^: ; V - '4-*. ^ w »r <■ * r A - *- - ■■ fegt ft Fig* 12* Corpus 1st son stags II paraffla S* 1 E« 30* Fig. 13. Sana as Fig. 12. 1001 ' VIZ < v £ ',•W E* A S * B. \ R ? ; f A iA - F S Z m . V i ’-' i'\r Cti Fags 63m Fig. 14 Atretic follicle stage II paraffin H. & E. 10OX Fig. 1 5 . Atretic follicle stage X. Note thecal cells accompanying the invading granulosa. Frozen section H. 8- E. 4O0X f> ; '-tt* ; i i ■ ; :: >;. : *: *•• i - i ,.\> :; ■ •*. • .* •. •; ’;!■ :< & \ fc' $?'-*&&&&'# - V ' ^ V i..-., -r-V.* " « / ' -.v *.. ..- .v • * - «*-. - t V. . - *■ C - 'K. ’ '-*-•*&-:?!*. 1 • • •' v **- *• **< *’ *•'*' 'V- Page 64* Fig* 16* Atretic follicle stage II Hote large lipid vacuoles in granulosa cells. Paraf fin section H E. 1 0 0 X Fig. 17. Detail of an atretic follicle shoeing hypertrophied granulosa cells* Paraffin section H. E. 400X Page 64a Fig* 18. Atretic follicle stage 111 paraffin 8* 1. 301 Fig. 19. Detail of Fig .18 showing folds of granulosa , 200X 31ucose-6»phosphate dehydrogenase activity in corpus luteum stage XI* Mote absence of activity in the theca. 301 KAD-diaphorase activity in follicle wall and interstitial tissue 1 0 0 X Page 65a Fig. 22. 3b~H£D activity in small follicle® 30X Fig. 23* 3&-HSB activity is granulosa and theca externa of 10 mm follicle* 3 0 X 3"b-HSl> - ^-keta-hydroxysteroid dehydrogenase Page 66* Fig. 2b Detail of activity la granulosa theca externa of 2 D m m follicle 2 5 0 X Fig* 25* 3 h-HSD activity in granulosa but not la theca of 30 mm follicle 100X Page 66a Fig. 26. 3£-HH D activity in granulosa and in isolated group of theca externa cells. 100X Fig. 27• activity in granulosa of 40 mm follicle Note intensity of the reaction. ftigt #7* Its* 28* activity it corpoa lataas tltft I % m x Fig. 29* 3b-*HSO aetivitjr la ttriw lata** %OOX Pag* 67a Pig. 3 0 . 3£~Hf-D activity ia corpaa luteu* stag* XXX 100X Fig. 3 1 . Detail of fig. 3 0 . Caspara lctancity iltk that m m ia fig. 2 9 * *oox Fage 08 Fig* 32* 3 -BSD* activity la granulosa of largo folllelo IOOX Fig* 35* 3&-HS© activity in corpus lutoun stage XXI and la granulosa and thoca txttru of adjacent 300 an folllelo* Coapare intensities in the two loeatlons 30X 3-alpha-hydroxysterold dehydrogenase m ^ M - ♦ . •*. SPj.Tr* +* %A gj# * ' Page 63m Fig* 32t* Lipid droplets in the granuloma of 30 mm follicle stained with fat red **002 Fig. 35* Corpus luteurn stage I stained with Fat Bed 7JI showing large lipid droplets* 1002 APPENDIX C FORMULAE AND STAINING PROCEDURES Boilin's fixative Saturated aqueous solution of picric acid 75 ml 40% formaldehyde 25 ml Glacial Acetic acid 5 ml Meyer's egg albumin Fresh egg white 50 ml Glycerin 50 ml Sodium salicylate 1 g Erlich's acid hematoxylin Distilled water 30 ml 95% ethanol 30 ml Glycerin 30 ml Glacial acetic acid 3 ml Hematoxylin 0.7 g Ammonium alum excess Harris hematoxylin Distilled water 200 ml 100% ethanol 10 ml Ammonium alum 20 g Hematoxylin 1 g Mercuric oxide 0.5 c Two drops of glacial acetic acid to 20 ml of stock solution when ready to use. 70 5. Mallory I 0.5% acid fuchsin solution 6. Mallory II Aniline blue, water soluble 0.5 g Orange G 2.0 g Phosphotungstic acid 1.0 g Distilled water 100 ml 7. Eosin Eosin Y, water and alcohol-soluble 5.0 g Distilled water 25 ml 95% ethanol 940 ml 8. Fat Red 7B (Ciba) The dye is made up as a saturated solution in 70% ethanol and filtered before use, 9. Removal of Bouin1s from tissue 70% ethanol 500 ml Lithium carbonate 10 g 3 - 4 changes 10. Acid alcohol 70% ethanol 1 drop concentrated HC1/100ml 70% ethanol 11. Alkaline alcohol 70% ethanol 2 - 3 drops ammonium hydroxide/100ml 70% ethanol 71 12. Scott * s Solution NaHC03 2.0 g MgS04 20 g Distilled water 100 ml 13. Acetic acid - sulfuric acid mixture Place 2 - 5 cc of glacial acetic acid in a small test tube and immerse in ice water. Then, gradually add the same volume of con centrate sulfuric acid while the tube is still in the ice water. 14. Iron alum Ferric ammonium sulfate 2,5 g Distilled water 100 ml 15. Elasmobranch Ringer’s NaCl 16,36 g KC1 0.89 g CaCl26H20 2.19 g NaHCCU 0.38 g HaH Urea 21.6 g Distilled water 1000.0 ml 16. Phosphate Buffer,. pH 8»8 26.8 g £?a 2^ iiPO 4, Distilled water 1000.0 ml Phosphate Buffer, pH 8« 0 20.0 g Ua-HPO,2 4 Distilled water 1000.0 ml 72 Adjust pH with concentrated ECL added drop at a time. Phosphate Buffer, pH 7.5 Na2HP04 16.0 1 g Distilled water 1000.0i ml Adjust with concentrated HC1. Phosphate Buffer, pH 6.8 Na2HP04 10.01 g Distilled water 1000.0i ml Adjust pH with concentrated HC1. 17. Tris (Hydroxymethy1) Aminomethane 12.i g Distilled water 1000. 0 ml 73 PROCEDURE FOR STAINING PARAFFIN SECTIONS WITH MALLORY1S TRIPLE STAIN Place slides ini Xylene 5 min. Xylene 5 min. 95% ethanol 2 min. 80% ethanol 2 min. 70% ethanol 2 min. 50% ethanol 2 min. Distilled water 2 min. Mallory I 12 min. Running tap water 1.5 min. Distilled water 1 min. 5% phosphomolybdic acid 2 min. Mallory II 15 min. Running tap water 5 min. Distilled water 1 min. 50% ethanol 1 min. 95% ethanol 1.5 min. 100% ethanol 3 min. Xylene 5 min. Xylene 5 min. Mount in Permount and heat on a slide warmer for 12 hours. 74 PROCEDURE FOR EMBEDDING TISSUE IN PARAFFIN 1* Cut tissue in blocks about 2 cm square. 2. Fix in Bouin's for 2 - 4 days. 3. 30% ethanol for 30 minutes. 4. Place in 50% ethanol for 5-16 hours. 5. Place in 70% ethanol-lithium carbonate for 3- 5 days. Change every day. 6. Place in 80% ethanol for 12 - 24 hours. 7. Place in 95% ethanol for 5 - 7 hours. 8. Place in 100% ethanol for 1 hour. 9. Place in 100% ethanol-xylene (50?50) for 8 hours. 10. Place in xylene for 24 hours - 3 weeks. 11. Run through three changes of paraffin (+56°C) for 1.5 hours each. 12. Embed in paraffin. PROCEDURE FOR STAINING PARAFFIN SECTIONS WITH HEMATOXYLIN AND EOSIN Place slides in? Xylene 5 m m . Xylene 5 min. 95% ethanol 2 min. 80% ethanol 2 min. 70% ethanol 2 min. 50% ethanol 2 min. Distilled water 2 min. Ehrlich hematoxylin 10 min. Running tap water 2 min. Acid 70% ethanol 1/2 ~ 1 min. Alkaline 70% ethanol 1-2 min. 70% ethanol 2 min. 0.5% alcoholic eosin 1 min. 95 % ethanol 1 min. 100% ethanol 3 min. Xylene 5 min. Xylene 5 min. Mount in Permount and heat on a slide warmer for 12 hours. 76 PROCEDURE FOR STAINING FROZEN SECTIONS WITH HEMATOXYLIN AND EOSIN L Cut tissue sections at 15 u on cryostat at -35°C. 2. Mount on gelatin-coated cover slips* 3 e Place in absolute methanol for 20 - 30 seconds, 4. Remove and add directly several drops of Harris hematoxylin and stain for 60 seconds. 5. Pour off the excess hematoxylin and decolorize briefly in acid tap water (1 drop cone, HC1 in 10 ml H^O). 6. Transfer to alkaline tap water for 10 - 20 seconds (1 drop cone. NaOH in 10 ml ^0) . 7. Drain briefly and allow one or two drops of 0.5% alcoholic eosin to run over the tissue. 8. Rinse for 5 seconds in 95% ethanol. 9. Transfer to 100% ethanol and agitate for several seconds. 10. Transfer to second jar of 100% ethanol and repeat the above step. 11. 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Graduated from Long Island University, New York, with B, S. in biology cum laude in 1966. M. A. candidate, the College of William and Mary, Virginia, 1966-1968, with a concentration in biology. Title of thesis; Ovarian Function in the Ovoviviparous elasmobranch, Squalus acanthias: A histological and histochemicai s tudy. The author currently holds the position of demonstrator in zoology at the University of Hong Kong