Cellular changes in the gonads, livers and adrenal glands of Japanese quail as affected by the insecticide Kepone

TOXICOLOGY

AND

APPLIED

Cellular

PHARMACOLOGY

Changes

of Japanese

Quail

31,491-504(1975)

in the Gonads, as Affected

Livers by the

and Adrenal Insecticide

Glands Kepone

VICTOR P. EROSCHENKO~ AND WILBOR 0. WILSON Department

of Anatomy, School of Veterinary Medicine, University of California, Davis, California 95616 and Department of Aaian Sciences, University of California, Davis, California 95616 Received

May

8, 1974;

accepted

September

IO, 1974

Cellular Changes in the Gonads, Livers and Adrenal Glands of Japanese Quail as Affected by the Insecticide Kepone. EROSCHENKO, V. P. AND WILSON, W. 0. (1975). Toxicol. Appl. Phamacol. 31,491-504. A histological study of normal reproductive organs, livers, and adrenal glands from immature and adult Japanese quail of both sexes and of quail fed 200 ppm of the insecticide Kepone under long and short photoperiods was carried out. Kepone increased cellular proliferation, cytodifferentiation and tubular gland formation in the oviducts of immature quail when kept under 6 hr of light and 18 hr of darkness (6L: 18D) and 16 hr of light and 8 hr of darkness (16L: 8D). No cellular changes were noted in the oviducts from laying quail. Ovarian tissue from Kepone-treated quail contained more primary oocytes and smaller follicles when compared to the control birds. Liver cells from all experimental birds were filled with large, lipidlike inclusions. Hypertrophy of adrenal cortical and medullary cells was noted in all Kepone-treated birds. A detrimental effect of Kepone was recorded in the testes of immature and adult quail under the 16L: 8D light regimen. Highly dilated seminiferous tubules, reduced germinal epithelium and spermatozoa, and abundant intraluminal cellular debris were common. The results indicate that the insecticide Kepone has an estrogenlike effect on the oviducts of immature females and on the testes of both immature and mature males. The toxicity of various pesticides is of general importance because of their wide use in agriculture. While these pesticides destroy insects, they may have a direct action on the reproduction of birds and mammals confined to the treated fields. Diminished reproduction of various species following consumption of diets containing residues of chlorinated hydrocarbons have been suggested by several reports. DDT in particular has been most often implicated as it has been most widely used and studied. KeponeZ (decachlorotetracyclodecanone) is a complex polycyclic chlorinated insecticide that has been useful in controlling agricultural leaf-eating pests, insects, roaches, ants and other pests (Brown, 1966; Epstein and Legator, 1971). However, Kepone also interferes with the reproduction of birds and mammals and affects various organs of the body (Dewitt et al., 1962; Huber, 1965; McFarland and Lacy, 1969; Eroschenko and Wilson, 1974). Various concentrations of Kepone inhibited reproduction of bobwhite quail and ’ Present address: Department of Biological Sciences, University of Idaho, Moscow, Idaho 83843. z Supplied by the Agricultural Division of Allied Chemicals. N.Y. Copyright ( ) 1975 by Academic Press, Inc. All rights ofreproduction I” any form reserved. Printed in Great Britain

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altered feather pigmentation of male pheasants, quail and ducks to resemble the plumage of adult females (Dewitt et al., 1962). Kepone reduced reproduction in mice (Huber, f965) and decreasedegg production in laying chickens (Naber and Ware, 1964). Kepone hasalso been shownto exert estrogenicactivity in the reproductive organs of the Japanese quail. Administration of Kepone to quail that were immature, hypophysectomized, ovariectomized or kept under 6 hr of light and 18 hr of darkness(6L: 18D) produced greatly enlarged oviducts. Feeding Kepone to maturing quail resulted in enlarged ovaries containing greater number of developing follicles (McFarland and Lacy, 1969; Eroschenko and Wilson, 1974). Feeding Kepone to immature and mature quail also produced enlarged edematous testes. Furthermore, livers and adrenal glands were significantly heavier in Keponetreated quail of different agesand of both sexes(Eroschenko and Wilson, 1974). The purpose of this investigation was to carry out a histological comparison of the reproductive organs, livers, and adrenal glands from normal quail of both sexesand different ages with those of similar quail that were maintained under long and short photoperiods and fed 200 ppm of the insecticide Kepone. METHODS

Immature and mature coturnix of each sex, obtained from the University of California, Davis, were used in these experiments. At three weeks of age the chicks were separatedinto control and experimental groups according to sex. placed into individual wire floor cagesand subjected to different photoperiods. In the tirst experiment, three-week old control birds of each sex under 16 hr of light and 8 hr of darkness (16L: SD) were given turkey starter rations and water ad libittm until 6 weeks of age. Another group of the sameage received a similar diet to which 200 ppm of the insecticide Kepone was added. Kepone was first dissolved in acetone and thoroughly mixed into the feed. At 4 weeks of age six to eight experimental and control birds were randomly selectedfrom each sex and sacrificed weekly until 6 weeks of age. In the secondexperiment. 6-week-old birds of both sexes.previously kept under 6 hr of light and 18 hr of darkness (6L: I8D) for 3 weeks, were fed a ration containing 200 ppm Kepone for three weeks.The control birds remained on the uncontaminated turkey starter. Six to eight birds of each sex from the experimental and control groups were randomly selectedeach week and sacrificed from the 6th to 9th week inclusive. In the third experiment, 24 week old birds of both sexesunder 16L: 8D light regimen were kept in individual cagesand were fed turkey starter and water ad libiturv. Another group of birds of the sameage received food containing 200 ppm Kepone for exactly 21 days. At the end of the Kepone feeding, six to eight birds of both sexesfrom the control and experimental group were randomly selectedand terminated. At the time of termination in each experiment, ovaries, oviducts, testes. livers and adrenal glands from both the control and experimental group were excised and fixed in Tellyesniczky’s fixative solution. Samplesof each tissuewere embeddedin paraffin, sectioned at 7 1)and stained with Alcian blue-periodic acid Schiff (AB-PAS) and Masson’s trichrome stains(Lillie, 1954).The slideswereexamined with the microscope and photographed.

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RESULTS Eflects qf’ Depone

on the Ordrcts

Histological assessment of the magnum, isthmusand shell gland from the 4 week old control bird oviducts under I6L :8D light regimen indicated that development had not occurred. The surface epithelium was pseudostratified columnar with attenuated cytoplasm occupied largely by the nucleus.No indication of secretory activity or ciliogenesis was noted. This lack of cellular differentiation in the surface epithelium did not permit histological recognition of the various parts of the oviduct. No tubular gland formation or secretory activity was noted at this time (Fig. 1). The oviduct of five week old control birds increased in size, and mucosal folds were pronounced. Initial formation of the tubular glands from the surface epithelium was evident in all parts of the oviduct. The epithelial cells were taller, with the nuclei located either apically or basally. Cilia covered the surface of someepithelial cells and different stagesof mitosis were observed in both the surface and in the tubular gland cells in all regions of the oviduct. Small amounts of secretory material, as revealed by the AB-PAS positive reaction, were demonstrated in the secretory epithelial and in the tubular gland cellsof both the magnum and isthmus regions of the oviduct (Fig. 3). In the uterus AB-PAS positive material was noted only at the most apical portions of someof the surface epithelial cells, whereas,in the tubular gland cells no such secretion was recorded. One week’s consumption of the ration with insecticide Kepone by the birds under the 16L: 8D light regimen produced marked histological changesin the entire oviduct. Some of the surface epithelial cells were profusely ciliated, others greatly enlarged, and still others were undergoing various stagesof mitosis. Highly branched tubular glands were likewise prominent in all parts of the oviduct (Fig. 2). Secretory product, shown by the AB-PAS positive reaction, was present in the secretory epithelial and tubular gland cells of the magnum and isthmus. In the uterus only the surface epithelial cells indicated a faint outline of AB-PAS positive secretory material. Two weeksof the Kepone ration produced further cytodifferentiation and development of the oviduct. Preparations with AB-PAS stain showed that the secretory epithelial cells and the highly branched tubular glands of both the magnum and isthmus wereengorged with secretion. Tubular gland cells of the isthmus were filled with smaller secretory granules, those in the magnum were engorged with larger coalescedgranules (Fig. 4). Indication of secretory material was noted only in the surface epithelial cells of the uterus. Histological comparisonsof the magnum, isthmus and uterus of the oviduct from the 6 and 27 week old laying control quails and from the Kepone-treated 6 and 27 week old laying quail under the 16L:8D light regimen revealed that the groups were identical. Samplesfrom theseoviducts were taken at the time when no egg waspresent in the oviduct orjust as the calcified egg was in the processof pigmentation. Secretory cells in the magnum and isthmus epithelium were engorged with various amounts of secretion and distorted the cytoplasm of the adjacent ciliated cells. The tubular gland cells and their lumina in the magnum and isthmus were similarly engorged with different sized secretory granules causing a basaldisplacement of their nuclei. In the uterus, only the surface epithelial cells demonstrated some AB-PAS positive secretory granules. The reproductive organs of control female quail kept under 6L: 18D light regimen from the third to ninth week of ageremained in a complete dormant state. Histology of

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the oviduct showed no evidence of either mitoses, cellular differentiation, gland formation or secretory activity. However, Kepone ration for three weeks caused cellular development, hypertrophy, and cytodifferentiation in the oviduct. The morphology of

FIG. 1. Magnum section of oviduct from a 4 week old control quail. Surface epithelial cells (arrows) did not indicate cytodifferentiation or secretory activity. No tubular gland formation was noted and the stroma was prominent. Massons’s Trichrome x 1400. FIG. 7. Section Cytodifferentiation, tion and presence x800.

of the magnum from a 4 week old quail’s oviduct that was fed Kepone for one week. mitotic activity (arrows), prominent cilia (arrow A), complex tubular gland formaof secretory granules in all the cells were characteristic features. Masson’s Trichrome

the entire oviduct closely resembled that illustrated in Figs. 2 and 4, namely, a highly developed and differentiated surface epithelium, tubular glands. and secretory activity.

KEPONE

EFFECTS

IN

JAPANESE

QUAIL

FK. 3. Section from the magnum of a 5 week old control quail oviduct. Cytodifferentiation and tubular gland formation were in its initial stages. Appearance of cilia (arrow A) and mitotic activity in surface epithelial and tubular gland cells (arrows) was also apparent. Masson’s Trichrome x1400. FIG. 4. Section from the magnum of a 5 week old quail oviduct that was fed Kepone for two weeks. Surface epithelial and tubular gland cells (TG) were filled with secretory products which appeared either granular or vacuolated as result of staining. Cilia on surface cells were also prominent (arrow). Masson’s Trichrome x1400.

Eject of Kepone on the Otlaries Histology of the 6 week old control ovaries from birds under 16L: 8D light showed a hierarchy of primary oocytes and larger, developing follicles containing accumulations of lipidlike inclusions or yolk droplets. Interstitial cells contained granulated cytoplasm and lipid vacuoles (Fig. 5). The histology of the ovaries from the Kepone treated 6 week old quail were especially characterized by an abundance of primary oocytes and small

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WILSON

fol liclies. However, a distinct follicular hierarchy was less evident than in the coSntrol ov ariles. Larger developing follicles and the interstitial cells had the same fea tur .es as thexe observed in the controls (Fig. 6). The histology of 27 week old control ova rie s and thexe from Kepone-fed adult birds under 16L: 8D light appeared identical. These were

section of the 6 week old control quail ovary. A hierarchy of primary follicles (Df) was apparent. Masson’s Trichrome x150.

ooc

w s (00)

F and

5. Low power -ger developing

F The feat

6. Low power section of an ovary from a 6 week old quail that received three weeks of KC :pone. esence of multiple primary oocytes (00) and less evident follicular hierarchy was a char ‘act eristic I. Masson’s Trichrome x 150.

KEPONE

EFFECTS

IN JAPANESE

QUAIL

497

characterized by a hierarchy of primary follicles, larger developing follicles and large, yolk filled follicles. Histologic examination of the ovaries from both the experimental and control birds kept under 6L : 18D light regimen did not reveal any obvious differences in their cellular composition or alterations. Both types of ovaries were characterized by primary oocytes and larger developing follicles. There was no apparent difference in the numbers of primary oocytes or larger follicles.

FIG. 7. Adrenal gland t?om a 6 week old control quail was characterized strands (C) and by clumps of polygonal medullary cells (M). This feature glands from all control birds of either sex. Masson’s Trichrome x1400.

by columnatcortical cell was common in the adrenal

FIG. 8. Adrenal gland from a 6 week old quail that was fed Kepone for three weeks. Hypertrophy of cortical (C) and medullary cells (M) was characteristic of all Kepone-treated birds. The cortical cells appeared taller and the medullary cells more polygonal. The presence of granulation and the enlarged amount of cytoplasm could indicate increased secretory activity. Masson’s Trichrome x1400.

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Efikcts of Kepone on the Lioer The histology of the liver cells from control birds indicated a granular cytoplasm and small lipidlike inclusions. Microscopic examination of liver cells from all Kepone-fed

FIG. 9. Low magnification section of a functional testis from a control quail 6 weeks old. The seminiferous tubules (S) and the lumina were prominent. Masson’s Trichrome x90. FIG. 10. Section of an enlarged, edematous testis from a 6 weeks old quail fed Kepone for 3 weeks. The characteristic feature was the great distention and dilation of seminiferous tubules (S) by a waterlike fluid. Masson’s Trichrome x90.

birds showed that the majority of the hepatic cells had plurivacuolar lipid accumulations that severely displaced and distorted the cytoplasm and the nuclei. These lipid accumulations in the liver cells created a “Swiss cheese-like” appearance, since in the routine tissue preparations large empty spaceswere noted within theseliver cells.

KEPONE

EFFECTS

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Effects of Kepone on the Adrenal Glands The adrenal glands of the nontreated birds contained an intermingling of cortical and medullary cells. The cortical cells were arranged in strands with a double row of columnar cells containing a granular cytoplasm. The nuclei were spherical and basally located. The polygonal-shaped medullary cells and their centrally situated nuclei were larger than the cortical cells. Their cytoplasm was granular with small, lipidlike vacuoles (Fig. 7). The adrenal glands from all Kepone-fed birds of both sexes showed cortical and medullary cell hypertrophy. The cortical cells were taller, more columnar with larger and more granular cytoplasm. As a result, the cortical strands appeared more prominent. Similar effects of hypertrophy were evident in the medullary cells, which appeared more polygonal with a larger amount of granular cytoplasm (Fig. 8). Effects of Kepone on the Testes Histological examination of the 6 week old control testes from birds kept under 16L: 8D light revealed full spermatogenic activity. The lumina of the seminiferous tubules were lined by clumps of.tightly packed, maturing spermatozoa oriented with their heads toward the germinal epithelium (Figs. 9 and 11). Histology of the 27 week

FIG. 11. Higher magnification of testis shown in Fig. 9. Multilayered germinal epithelium, activity of spermatogonia and spermatocytes, and presence of spermatozoa clumps (arrows) large number of sperms was a common feature. Masson’s Trichrome x1400.

mitotic bearing

FIG. 12. Higher magnification of testis shown in Fig. 10. Reduction and disruption of germinal epithelium apparently as a result of the great distention and dilation of the seminiferous tubules (S) by fiuid.Spermclumpswerelessprominentandcontainedfewerspermsthat appeared disoriented (arrows). Masson’s Trichrome x 1400.

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old control testes revealed similar testicular morphology as seen in the 6 week old control males. On the other hand. all testes from the 6 and 27 week old Kepone-fed quail revealed greatly distended seminiferous tubules and interstitial spaces occupied by

Fro. 13. Desquamated cellular debris in the seminiferous tubules that was prevalent in Kepone-treated 6 week and 27 week old quail. This type of desquamation contained dividing spermatocytes, maturing spermatids and deformed sperm heads (arrows). Masson’s Trichrome x1400. FIG. 14. Edematous testes from a 27 week old quail fed Kepone for three weeks. Disruption ofgerminal epithelium in the form of erosion was seen (arrows). Disorientation and reduction of sperms in the sperm clumps was also a common feature. Masson’s Trichrome x 1400.

fluid of undetermined origin (Fig. IO). Seminiferous tubules of theseedematous testes contained flattened. reduced and generally disrupted germinal epithelium (Figs. 12 and 14). Many of the lumina in these tubules contained desquamated cellular debris consisting of germ cells in various stages of maturation (Fig. 13). Curled, abnormal

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sperm as well as maturing spermatids appeared distorted within the germinal epithelium and in the desquamated debris of the lumina. The sperm bundles were now less compact, due possibly to the decreased number of sperms and to a reduced number of maturing spermatids (Figs. 12 and 14). Histology of the testes from the Kepone-fed birds under 6L.: 18D light regimen did not indicate any cellular alterations from those of the control. The seminiferous tubules were characterized by a single layer of stem spermatogonia and Sertoli cells. No distention of seminiferous tubules or disruption of the germinal epithelium by fluid was noted in any of the testes. DISCUSSION The oviduct of immature avian species differentiates rapidly when the birds are exposed to a proper stimulatory photoperiod (Tanaka et al., 196.5)as well as a proper hormonal treatment. Administration of exogenous estrogen to immature chicks stimulated the growth of the surface epithelium and caused a 3- to 4-fold increase in mitotic activity. In the continuous presenceof estrogen, these cells continued to divide and differentiate forming tubular glands, secretory (goblet) and ciliated cells of the oviduct. Estrogen also induced secretory activity in the surface epithelial cells of the magnum, uterus and in the tubular gland cells of the magnum which contained lysozyme and ovalbumin (Ljungkvist, 1967; Kohler et al., 1969; Oka and Schimke, 1969a; 1969b; Palmiter and Wrenn, 1971; Jackson et al., 1971; Socher and O’Malley, 1973). Thus, cellular proliferation, cytodifferentiation and an increase in size of the oviduct can be physiologically influenced by estrogen. Histology of the enlarged oviducts from the quail fed Kepone exhibited profuse cilia, increasedtubular gland formation and secretion. Enlargement of the entire oviduct was histologically determined to be due not only to a generalized hypertrophy and hyperplasia of all regions, but also to a secretory activity in all the regions of magnum and isthmus. Even when the birds were kept on an “inhibitory” light (6L: 18D) regimen administration of Kepone produced a similar enlargement of the oviduct characterized by high cellular proliferation, ciliogenesis,cytodifferentiation, tubular gland formation, and secretion. Generally. a decreasein day-length from 18 to 6 hr causedan inhibition of sexual maturation and was nonstimulatory to either the testesor ovaries (Mather and Wilson, 1964: Follett and Farner, 1966). Histology. of ovaries from Kepone-fed birds kept under inhibitory light (6L: 18D) as well asfrom laying adult birds did not indicate any cellular alterations or abnormalities different from the controls. On the other hand, maturing experimental birds under 16L: 8D light regimen had significantly heavier ovaries (Eroschenko and Wilson, 1974) characterized by lessevident follicular hierarchy and by a greater number of primary oocytes and developing follicles. McFarland and Lacy (1969) reported that fewer number of eggswere also found in the uterus of Kepone-fed quail, thus implying decreasedovulation or egg laying. In mice, Kepone causedprolonged releaseof FSH and estrogen but apparently blocked the releaseof LH, thus preventing ovulation and corpus luteum formation (Huber, 1965). Experimentation with Kepone on ovariectomized and hypophysectomized quail indicated a dual action for Kepone. McFarland and Lacy (1969) postulated that Kepone

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acted either directly as an estrogenic agent on the oviduct or indirectly via the hypothalamo-pituitary axis to increase FSH release and thereby ovarian estrogen secretion. However, the greatest “estrogenic effect” of Kepone was recorded when the pituitary was intact. Kepone produced an “estrogen-like” effect on the undeveloped oviducts of sexually immature birds exposed to 16L: 8D light regimen by causing significant weight changes (Eroschenko and Wilson, 1974) cytodifferentiation, ciliogenesis, tubular gland formation and secretory activity. However, this accelerated development and maturation of the oviduct did not produce any evidence of cellular abnormalities or degeneration, Thus, under these conditions Kepone can affect the oviduct by its dual action, as previously postulated. However, its most direct estrogenic action was recorded from the oviducts of birds exposed to reduced light (6L : I8D). The increased weight change of the oviducts, cytodifferentiation, cellular proliferation and maturation could only come about as result of estrogenic action. Similar results were reported in chickens since immature chicks that received Kepone ration had enlarged oviducts (Sherman and Ross, 1961). In laying quail, the amount of estrogen secreted by the ovary was probably maximal for the stimulation of the oviducts since Kepone failed to exert any changes in its weight (Eroschenko and Wilson, 1974). Likewise, the histology of the oviduct from the Keponefed laying birds did not indicate any cellular abnormalities or changes. The morphology of the fully functional quail oviduct was similar to those reported for quail (Tamura and Fujii, 1966a, 1966b; Fertuck and Newstead, 1970) and for the domestic hen (Richardson 1935; Fujii et al., 1965; Wyburn et al., 1970; Aitken, 1971). Fatty infiltration was associated with hepatic enlargement, and the histology of liver cells indicated heavy lipid accumulations. However, no mitotic activity nor areas of pathologic changes were observed in any liver cells. Similar findings were also reported by McFarland and Lacy (1969). Histologic examination of livers from Kepone-fed mice, however, revealed focal necrosis, cellular hypertrophy, hyperplasia and congestion (Huber, 1965). On the other hand, Kepone-fed chicks exhibited no liver enlargement (Sherman and Ross, 1961). Kepone administration to both sexes of the quail produced heavier adrenal glands (Eroschenko and Wilson, 1974) and resulted in hypertrophy of medullary and cortical cells. However, no indication of cellular degeneration or mitosis was evident. Hypertrophy of these cells could indicate their hyperfunction with possible increase of corticosterone secretion into general circulation. At any rate, the morphological alterations of the quail adrenal gland cells apparently constitute the adaptive responses to the systemic presence of the insecticide Kepone. The testes from maturing and fully grown Kepone-fed quail under 16L:gD light regimen were severely affected. Distended seminiferous tubules contained watery fluid and this accumulation of fluid was apparently responsible for the significant increase in testes weight (Eroschenko and Wilson, 1974). Generally, there was a reduction of the germinal epithelium thickness and of the spermatozoa within the sperm clumps. In addition, more of the seminiferous tubules from the testes of Kepone-fed birds contained desquamated and abnormal cellular elements. Thus, part of this detrimental effect of Kepone on the histology of the testes could be due to its “estrogenic action.” It has been shown that estrogen and estradiol benzoate reduced testes size, depressed spermato-

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genesis and partially inhibited gonadal growth in avian species (van Tienhoven, 1961; Parker, 1968 ; Lofts and Murton, 1973). Testicular abnormality and malformed spermatozoa were also recorded in Kepone-fed pheasants (Dewitt et at., 1963). On the other hand, normal spermatogenesis and interstitial cell content were recorded in testes from Kepone-fed mice (Huber, 1965). Kepone can possibly influence the integrity of testicular morphology by other means. Injection of corticosteroids increased testicular size and weight in young cockerels (Conner, 1959). Also, treatment of chicks with desoxycorticosterone produced testes with highly distended seminiferous tubules and flattened germinal epithelium as result of increased intraluminal fluid accumulation (Boas, 1958). Feeding high salt diet to immature chicks produced hypertrophy of the seminiferous tubules and later transformed them into thin walled cysts containing water-clear fluid. (Siller et al., 1972.) In conclusion, a sublethal level of Kepone (200 ppm) affects the histology of organs such as liver and adrenal glands, as well as enhances the development and maturation of the female reproductive organs. None of these organs indicated cellular damage or abnormality as result of three weeks of Kepone diet. However, its effect on the male reproductive system can be considered as detrimental since disrupted germinal epithelium, desquamated germinal cells and decreased spermatogenesis can mean reproductive failure, which can result from the lack of sperm mobility, sperm volume, as well as from the apparent sperm abnormalities. ACKNOWLEDGMENTS

This study was supported

by a U.S. Public Health

Service Grant ES 00054 for the Toxi-

cology Center and by Dr. R. A. Freedland’slaboratory. This investigationwaspresentedin a more extended form as a thesis by the senior author in partial fulfilment for the Ph.D. degree at the University of California, Davis, Calif.

of the requirements

REFERENCES R. N. C. (1971). The oviduct. In Physiology and Biochemistry of the Domestic Fowl (D. J. Bell and B. M. Freeman, eds.), Vol. 3, pp. 1237-1289. Academic Press, New York. BOAS, N. R. (19.58). The effects of desoxycorticosterone acetate on testes size and function in the cockerel. Endocrir1ology 63, 323-328. BROWN, R. L. (1966). Pesticides in clinical practice. C. C. Thomas, Springfield, III. CONNER, M. H. (1959). Effects of various hormone preparations and nutritional stresses on chicks. Poultry Sci. 38, 1340-1343. DEWITT, J. B., CRABTREE, D. G., FINLEY, R. B. AND GEORGE, J. L. (1962). Effects of pesticides on fish and wildlife: A review of investigations during 1960. U.S. Dept. of Interior, Bur. of Fish and Wildlife Serv., Cir. 143. DEWITT, J. B.. STICKEL, W. H. AND SPRINGER, P. F. (1963). Wildlife studies during 1961-62. Patuxent Wildlife Research Center, A review of Fish and Wildlife Service, U.S. Dept. of Interior, Fish and Wildlife Service Circ. 167. EPSTEIN. S. E. AND LEGATOR, M. S. (1971). MutagerzicityofPesticides. M.I.T. Press, Cambridge, Mass. EROSCHENKO, V. P. AND WILSON, W. 0. (1974). Photoperiods and age as factors modifying the effects of Kepone in Japanese quail. Toxicol. Appl. Pharmacol. 29, 329-339. FERTUCK. H. D. AND NEWSTEAD, J. D. (1970). Fine structural observations on magnum mucosa in quail and hen oviducts. Z. Zellforsch. 103, 447459. FOLLETT, B. K. AND FARNER, D. S. (1966). The effects of the daily photoperiod on gonadal growth, neurohypophysial hormone content, and neurosecretion in the hypothalamushypophyseal system of the Japanese quail (Coturni.u coturnix juponica). Cm. Compar. Etrdocrinol. 7. 11 I- 124.

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