GENERAL
AND
COMPARATIVE
38, 153-161 (1979)
ENDOCRINOLOGY
Steroid-Synthesizing Cells in the Embryonic and Adult Gonads of the Domestic Pigeon, Columba livia (Gmelin) BHAGYASHRI Deparrmeni
V. BHUJLE,’ VINAYAK B. NADKARNI, AND M. APPASWAMY RAO
of Zoology,
Karnaiak University. Karnatak State, India
Dharwad-580
003.
Accepted January 28, 1979 Steroidogenic cells in the developing gonads of Columba livia from Day 9 of incubation till the bird attains maturity have been histochemicahy identified by demonstrating the presence of A5-3/3-hydroxysteroid dehydrogenase (A5-3@HSDH). In the embryonic ovary the medulla, containing A5-3&HSDH-positive cells, gradually seems to migrate into the cortical region with growth of the embryo and later it dwindles in 15 to 20-day-old hatchlings. In later stages, As-3/3-HSDH is found in some cells of theta interna of normal follicles, hypertrophied granulosa cells of atretic follicles, the granulosa and theta interna of postovulatory follicles. and the interstitial stromal cells. On Day 9 of incubation in the male embryo the convoluted solid seminiferous cords show a weak A5-3@HSDH activity. One day prior to hatching, AS-3/3-HSDH activity disappears from the seminiferous cords but appears in the interstitial Leydig cells. In the 4-month-old adult pigeon, A5-3pHSDH activity is seen in the Leydig cells as well as in the seminiferous epithelium.
It has been shown that the embryonic gonads of vertebrates produce secretions which have the same effects as the steroid hormones of adult gonads (reviews by Wolff, 1962a,b; Haffen, 1975). Steroids have been detected in the amniotic and allantoic fluids and blood of chick and quail (St011 and Maraud, 1956; Ozon, 1965, 1969). Avian embryonic gonads cultured in vitro can utilize various radioactive precursors and convert them into steroid sex hormones (Weniger et al., 1967; Akram and Weniger, 1969; Weniger, 1970; Galli and Wassermann, 1972, 1973; Guichard et al., 1972a,b, 1977). There are reports on the identification of steroidogenic cells in the embryonic and adult gonads of birds (Narbaitz and Kolodny, 1?64; Chieffi et al.; 1964; Chieffi and Botte, 1965; Woods and Domm, 1966; ’ Present address: Department of Biology, Chowgule College, Margao-403 691, Goa-India.
Narbaitz and De Robertis, 1968; Scheib and Haffen, 1968, 1969; Woods and Weeks, 1969; Arvy and Hadjiisky, 1970; Scheib, 1973; Tingari, 1973; Bhujle and Nadkarni, 1974). Although, there is sufficient evidence for the steroidogenic potentiality of avian embryonic gonads, there is a difference of opinion about the sites of steroidogenesis in the avian embryonic testis. In the present work, an attempt is made to identify the steroidogenic cells and study the steroidogenic potential of the gonads of the domestic pigeon, Columba livia (Gmelin) from the embryonic stage until they attain maturity. MATERIALS
AND METHODS
Pigeon embryos from Day 9 of incubation were obmined from the pigeon colony maintained by the Zoology Department. Embryos prior to Day 9 of incubation were not studied as it was difficult to cut frozen sections of the gonads of these embryos. The head of the embryo was removed and the torso containing the
S.P. 153
0016-6480/79/060153-09$01.00/O Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
154
BHUJLE,
NADKARNI,
AND
++ ++++
RAO
++ +++t
+t ++ t+++ tttt I:: +ttt tt+t
5=B rg Z$ F
++++ +t+t
++ ++
:: tt++ ++tt tt+t +t t+::
I I I I
I
I
I
tttt ++ :: i-t++ t+ti
++ ++++ ++++ ++4+
tttt t+t+
tt ++
I I I I t-I t-I +++-I t++i
STEROID
DISTRIBUTION
OF
CELLS IN PIGEON
As-3P-HSDH, G-6-PDH, livia
BETWEEN
DAY
AND
9
Enzyme and substrate*
9- to IFdayold embryo
As-3p-HSDH DHA Pregnenolone G-6-PDH NADH-diaphorase A5-3/3-HSDH DHA Pregnenolone G-6-PDH NADH-diaphorase A5-38-HSDH DHA Pregnenolone G-QPDH NADH-diaphorase AS-3p-HSDH DHA Pregnenolone G-6-PDH NADH-diaphorase
I Day prior to hatching to 30 days after hatching 40- to IIO-days old pigeon
I IO- to 120days-old pigeon
TABLE 2 NADH-DIAPHORASE ACTIVITY IN THE TESTIS OF Columbn
OF INCUBATION
Age
155
GONADS
AND
Seminiferous cords
SEXUAL
MATURITY=
Seminiferous tubule epithelium
k + ++ ++
Tubules are not formed
-
tubules are not formed
Leydig cells Leydig cells are not encountered ++ ++ +++ +++
Cords are transformed into tubules
-
fi++ ++++ ++++
Cords are transformed into tubules
+ + ++ ++
+++ +++ +++++ +++++
n Intensity of reaction is graded (-) to (+ + + + +); (-) denotes the absence of reaction and (+ + + + +) a maximal reaction. * All the chemicals are of Sigma grade, obtained from Sigma Chemical Company, St. Louis, MO. vertebral column, adrenals, gonads, and renal tissue was trimmed and rapidly frozen over dry ice for the histochemical study. The torsos from some other embryos of the same age were fixed in Bouin’s fluid for histological studies. In addition, studies were also made of the gonads of pigeons between I and 130 days after hatching (Tables I and 2). The frozen tissues were sectioned at 12 pm in a Pearse-Slee cryostat maintained at -20”. The sections were incubated aerobically at 37” in appropriate media for the localization of A5-3P-hydroxysteroid dehydrogenase (HSDH), G-6-PDH, and NADHdiaphorase activity. For the demonstration of A5-3/3HSDH, dehydroepiandrosterone (DHA) and pregnenolone were used as substrates. The incubation media were prepared following the procedure of Baillie et al. (1966). After incubation, the sections were fixed in 10% neutral formalin for half an hour and were mounted in glycerol jelly. Alternate sections from each tissue were incubated in medium lacking either the substrate or the coenzyme (NAD or NADP) and served as controls.
RESULTS Ovary
The embryonic
ovary on Day 9 of incuba-
tion is well defined into medulla and cortex. The cortex is occupied by oocytes (Fig. 1) and the medulla consists of rounded cells. A5-3/3-HSDH activity was found in some cells in the medulla but no enzyme activity was found in the cortical region. In embryos between day 9 of incubation and Day 5 after hatching, there was an increase in the thickness of the cortical layer and the medulla gradually degenerated. The A5-3/3-HSDH-positive medullary cells seemed to have migrated to the cortical region (Fig. 2). Ten to fifteen days after hatching, the oocytes from the cortical region were enveloped by follicular cells and formed primary follicles. A5-3p-HSDH activity was found in some interfollicular cells and also in the ooplasm of the follicles. In pigeons 20-25 days after hatching there was a prominent growth of the cortex whereas medullary tissue was found only in traces (Fig. 3). At this stage some primary follicles
156
BHUJLE,
NADKARNI,
AND
RAO
STEROID
CELLS IN PIGEON
undergoing atresia were encountered. A53/3-HSDH activity was found in the cells of the stroma and ooplasm. The ovaries of 40to 60-day-old pigeons showed vitellogenic follicles of different sizes. In follicles of about 500 pm in diameter, the follicular layer could not be differentiated into granulosa and theta. In follicles of about 570 pm in diameter some cells of theta interna showed A5-3/3-HSDH activity. The interstitial stromal cells also showed A53/3-HSDH activity. At the age of 4 months, pigeons start laying eggs and so the ovary of these pigeons is taken as the mature ovary. The mature ovary consists of primary, vitellogenic, postovulatory, and atretic follicles. No enzyme activity was found in the follicular layer of primary follicles. In developing follicles, 500-570 pm in diameter, A53/3-HSDH activity was found in some cells of theta intema and as follicles grew in size the number of theta interna cells showing enzyme activity increased. No enzyme activity was seen in the granulosa cells. However, a weak enzyme activity was seen in the hypertrophied granulosa cells of the atretie follicles. In postovulatory follicles, an intense As-3P-HSDH activity was seen in the granulosa and theta interna cells. The enzyme activity in postovulatory follicles persists for 4-5 days after ovulation and diminishes subsequently. G-6-PDH and NADH-Diaphorase
Both of same cells ity during However,
Activity
these enzymes were found in the that showed As-3/I-HSDH activdifferent stages of development. the activity of G-6-PDH and
GONADS
157
NADH-diaphorase was more intense when compared to that of A5-3/3-HSDH. Further, most of the theta interna cells of large vitellogenic follicles showed activity of both the enzymes (Table 1, Fig. 4). Testis
In histological sections, the testis of a 9-day-old embryo was distinguished from the embryonic ovary by the presence of prominent convoluted seminiferous cords. A weak A5-3/3-HSDH activity was seen inside the seminiferous cords 1 day prior to hatching, (Fig. 5). A5-3P-HSDH activity disappeared from the seminiferous cords but appeared in the Leydig cells that are found between the cords. There were no histological changes in the testis till about 30 days after hatching. A5-3p-HSDH activity was found only in the interstitial Leydig cells. Thirty to forty days after hatching, a lumen appeared in seminiferous cords, thus forming seminiferous tubules, but still the tubules were lined by a single layer of epithelial cells, the stem spermatogonial cells. First spermatogonial divisions were seen in the testis 70-75 days after hatching. In the testes of 90- to loo-day-old pigeons the seminiferous epithelium was many layered and AS-3p-HSDH activity was observed in the Leydig cells. In the testes of 120- to 130-day-old pigeon, different stages of spermatogenesis and sperm bundles were found. An intense As-3@-HSDH reaction was seen in the interstitial cells and a weak activity was observed in the peripheral layer of the germinal epithlium of seminiferous tubules (Fig. 7).
FIG. 1. Section of the ovary of 9-day-old embryo of pigeon showing the cortical (C) and medullary (M) regions. Arrows point to oocytes. Scale line indicates 50 pm. FIG. 2. A5-3PHSDH activity in the interstitial cells (I) of the cortical region (arrow) of the ovary of the 2-day-old pigeon. M indicates degenerating medulla. Scale line indicates 100 pm. FIG. 3. Section of the ovary of 25day-old pigeon showing developing follicles (F). Note the degenerating medulla (M). Scale line indicates 100 pm. FIG. 4. G-6-PDH activity in the ovary of the adult pigeon. The activity is found in the theta interna cells (TH) of growing follicles and in the granulosa cells (G) and theta intema of postovulatory follicles. G-6-PDH activity is also found in the interstitial cells (I). Scale line indicates 100 pm.
158
BHUJLE,
FIG. 5. line indicates FIG. 6. line indicates FIG. 7. the activity
NADKARNI,
AND
RAO
A5-3P-HSDH activity in the seminiferous cords (arrows) of an 1 l-day-old embryo. Scale 125 pm. NADH-diaphorase activity in the Leydig cells of the testis of a 45-day-old pigeon. Scale 125 pm. A5-3P-HSDH activity in the testis of an adult pigeon. I = Leydig cells. Arrows indicate in the seminiferous epithelium. Scale line indicates 200 +m.
STEROID
CELLS
IN
PIGEON
GONADS
159
hatching and thereafter the interstitial cells form the principal site of steroidogenesis. Both of these enzyme activities occurred In the embryonic ovary as well as the in the same cells that showed A5-3/3-HSDH testis, pregnenolone is preferentially utiactivity during different stages of developlized during the demonstration of A5ment. However, the intensity of reaction of 3P-HSDH activity suggesting that A4both of the enzymes was more pronounced pathway of steroidogenesis is more preva(Fig. 6, Table 2). Of the two substrates, pregnenolone and lent than A5-pathway in the embryonic DHA, used for the demonstration of A5- gonads of the pigeon. Similar observations have been made by Boucek et al. (1966) for 3@HSDH activity, the intensity of reaction the embryonic gonads of the chick. In vitro with pregnenolone was greater than that studies on quail testis by Scheib er al. with DHA in the embryonic gonads. How(1974) reveal that DHA is a less efficient ever, in the gonads of the posthatching precursor than pregnenolone. birds, A5-3@HSDH activity was of similar Soon after hatching, in the ovary of C. magnitude, with both substrates. livia, A5-3/3-HSDH-positive cells appear in the cortical region and the medulla shows DISCUSSION degeneration suggesting a shift of steThe presence of A5-3P-HSDH activity in roidogenesis from the medulla to the corthe islets of cells in the medulla and its tex. Similar observations have been made absence in the cortex of the embryonic by Narbaitz and De Robertis (1968) on the ovary of the pigeon suggests the ability of chick ovary. A weak A5-3&HSDH activity A5-3p-HSDH-positive medullary cells to in the ooplasm of the primary follicles oxidize A5-3@-hydroxysteroids to A4-3- suggests its capacity to convert A5-3/3ketosteroids. This observation is in agree- hydroxysteriods to A4-3-ketosteroids. It is ment with those of Narbaitz and Kolodny interesting to note that the amount of inter( 1964) and Scheib and Haffen ( 1968, 1969) stitial tissue showing A5-3P-HSDH activity in their studies on chick embryos. Howis greater in a young ovary (before the apever, Woods and Weeks (1969) have ob- pearance of many vitellogenic follicles) served a diffused A5-3j3-HSDH activity in than in the ovary of an adult pigeon where some cells of the cortex, in addition to the the theta interna cells of vitellogenic follipositive A5-3p-HSDH activity in some cles are involved in steroidogenesis. An inmedullary cells. crease in number of A5-3p-HSDH-positive Chieffi et al. (1964) have stated that A5- theta interna cells with the increase in the 3P-HSDH is diffused throughout the chick size of follicles may be associated with intestis from Day 8 throughout embryonic creased synthesis of estrogens as estrogen synthesis appears to be a consistent condevelopment. But according to Narbaitz (Lofts and Kolodny (1964) the embryonic testis of comitant of follicular development the chick is completely devoid of any spe- and Bern, 1972). cific enzyme activity. Scheib and Haffen It is reported that the postovulatory folli(1968, 1969) have observed a weak enzyme cles found in birds are transient and of unsignificance (Dodd, activity in the seminiferous cords of the certain physiological 6.5day-old chick which becomes more in- 1977). However, the presence of A5-3/3HSDH activity in the granulosa and theta tense by 7-8 days and remains throughout interna cells of the postovulatory follicle in the embryonic development. Our observaC. livia persisting for 3-4 days after ovulations on the embryonic testis of pigeon tion suggests that they may have a stesuggests that the seminiferous cords form the site of steroidogenesis till 1 day prior to roidogenic role. AS-3p-HSDH has also been G-6-PDH
and NADH-Diaphorase
Activity
160
BHUJLE,
NADKARNI,
reported in the postovulatory follicles of fowl and quail (Chieffi and Botte, 1965; Woods and Domm, 1966; Sayler rt al., 1970). In the testis, after hatching, A5-3p-HSDH activity is found in the Leydig cells but no enzyme activity is seen inside the seminiferous epithelium until 120- 130 days, suggesting that the Leydig cells are involved in steroid secretion. The seminiferous epithelium shows steroidogenic potentiality only in the adult pigeon. Similar observations have been reported by Tingari (1973) for the testis of the adult fowl. The presence of G-6-PDH in the same cells that showed A5-3/3-HSDH activity in the developing gonads, provides additional albeit indirect, of the steevidence, roidogenic potentiality of these cells, as G-6-PDH is known to generate the NADPH required for the hydroxylations of steroids during steroidogenesis (Weist and Kidwell, 1969). ACKNOWLEDGMENTS The authors are grateful to the Karnatak University, Dharwad, India, for providing laboratory facilities. The work was supported by a Senior Fellowship from the C.S.I.R. of India to the first author. The free gift of chemicals by W.H.O., Geneva, is gratefully acknowledged.
REFERENCES Akram, H., and Weniger, J. P. (1969). Secretion d’ oestrone et d’oestradiol par les gonades embryonnaires d’oiseaux. Gen. Comp. Endocrinol. 12, 568-573.
Univ.
J. (Natur.
Sri.)
13, 241-246.
Bhujle, B. V., and Nadkami, V. B. (1977). Steroid synthesizing cellular sites in the developing gonads of the domestic pigeon, Columba livia. In “Proc. First International Symposium on Avian
RAO
Endocrinology, Calcutta Jan. 1977.” (B. K. Follett, ed.). Abstract 77. Boucek, R. J.. Gyori, E., and Alvarez, R. (1966). Steroid dehydrogenase reactions in developing chick adrenal and gonadal tissues. Gen. Comp. Endocrinol.
7, 292-303.
Chieffi, G., and Botte, V. (1965). The distribution of some enzymes involved in the steroidogenesis of hen’s ovary. Experientia, 21, 16-17. Chieffi, G., Manelii, H., Botte, V., and Mastrolia, L. ( 1964). 11differenziamento istochimico dell’ interrenale e dei tessuti somatici della gonade embrionale di polloi: Comportamento della 3/3-olodeidrogenasi. Acta. Emhryol. Morphol. Exp. 7, 89-91. Dodd, J. M. (1977). The structure of the ovary of nonmammalian vertebrates. In “The Ovary” (S. Zuckerman, ed.), Vol. I, pp. 219-255. Academic Press, New York. Galien L., and Le Foulgoc, M. Th. (1957). Dectection par Auorimttrie et colorimetrie de steroides sexuels dan les gonades embryonnaires de poulet. C. R. Sot. Rio/. 151, 1088-1089. Gahi, F., and Wasserman, G. F. (1972). Steroid biosynthesis by testes and ovaries of 15day-old chick embryos. Gen. Comp. Endocrinol. 19, 509-514. Galli, F., and Wasserman, G. F. (1973). Steroid biosynthesis by gonads of 7- and IO-day-old chick embryos. Gen. Comp. Endocrinol. 21, 77-83. Guichard, A., Cedard, L., and Haffen, K. (1973a). Aspect comparatif de la synthtse de steroides sexuels par les gonads embryonnaires de poulet a differents stades du developpement (etude en culture organotypique a partir de precurseurs radioactifs). Gen. Comp. Endocrinol. 20, 16-28. Guichard, A., Cedard, L., Haffen, K., and Scheib, D. (1973b). Metabolisme de la pregnenolone et de la progesterone radioactives par les gonades embryonnaires de caille (Coturnix coturnixjapanica) en culture organotypique. Gen. Comp. Endocrinol.
Arvy, H., and Hadjiisky, P. (1970). Histoenzymologie des activites deshydrogenasiques dans I’ovaire de Coturnix coturnix L. et de Callus gallus L. (Phasianides, Galliformes). C. R. Ass. Anat. 54, 50-62. Baillie, A. H., Ferguson, M. M., and Hart, D. Mck. (1966). “Developments in Steroid Histochemistry.” Academic Press, New York. Bhujle, V. B., and Nadkami, V. B. (1974). Steroid synthesizing cellular sites in the gonads and the interrenal cells of pigeon, Columba livia. Marthawada
AND
21, 474-478.
Guichard, A., Cedard, L., Mignot, Th. M., Scheib, D., and Haffen, K. (1977). Radioimmunoassay of steroids produced by cultured chick embryonic gonads: Differences according to age, sex and size. Gen.
Comp.
Endocrinol.
32, 255-265.
Lofts, B., and Barn, H. A. (1972). The functional morphology of steroidogenic tissue. In “Steroids in Non-Mammalian Vertebrates” (D. R. Idles, ed.), pp. 37-126. Academic Press, New York. Narbaitz, R., and De Robertis, Jr., E. M. (1968). Postnatal evolution of steroidogenic cells in the chick ovary. Histochemie 15, 187-193. Narbaitz, R., and Kolodny, L. (1964). As-38 hydroxysteroid dehydrogenase in differentiating chick gonads. Z. Zellforsch. 63, 612-617. Ozon, R. (1965). Mise en evidence d’hormones ste-
STEROID
CELLS
IN
roides oestrogenes dans le sang de la poule adulte et chez 1’ embryon de poulet. C. R. Acad. Sci. (Paris)
261, 5664-5666.
Ozon, R. (1969). Steroid biosynthesis in larval and embryonic gonads of lower vertebrates. Gen. Comp. Endocrinol. Suppl. 2, 135-140. Scheib, D. (1973). Les cellules secretrices Testiculairis du poussin de la japonase: Differentiation de leur ultrastructure et rapports avec leurs potentiates steroidogenes. Develop. Growth Differentiation, 15, 3 15-328.
Scheib. D., and Haffen, K. (1%8). Sur la localisation histoenzymologique de la 3P-hydroxysteroides deshydrogenase dans les gonades de l’embryon de poulet, apparition et spicificite de I’activitie enzymatique. Ann. Embryol. Morphog. 1,61-72. Sheib, D., and Haffen, K. (1969). Apparition et localisation des hydroxysteroide dishydrogenase (A5-3P-et 17p) dans les gonades de I’embryon et du poussin chez la caille (Coturnix coturnix japonica). Etude histoenzymologique et comparison avec le poulet (Gallus gallus domesticus). Gen.
Comp.
Encodrinol.
12, 586-597.
Scheib, D., Haffen, K., Guichard, A., and Cedard, L. Transformation de la dihydro(1974). epiandrosterone-414C par les gonades embryonnaires de la caille japanaise (Coturnix coturnix juponica) explantees in vitro. Gen. Comp. Endocrinol.
23, 453-459.
PIGEON
161
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Stoll, R., and Maraud, R. (1956). Existence d’ hormones steroidtique dans I’organisme de I’embryon de poulet au tours de sa differentiation sexuelle. C. R. Acad. Sci. (Paris) 242, 1235-1237. Tingari, M. D. (1973). Histochemical localization of 3p- and 17P-hydroxysteroid dehydrogenases in the male reproductive tract of the domestic fowl (Gallus domesticus)
Histochem.
J. 5, 57-65.
Weist, W. G., and Kidwell, W. R. (1969). The regulation of progesterone secretion by ovarian dehydrogenases. In “The Gonads” (K. W. McKems, ed.) pp. 295-325. North-Holland Publishing Company, Amsterdam. Weniger, J. P. (1970). Le testicule embryonnaire de poulet secrete t-h de la testosterone? Arch. Anat. Histol.
Embryol.
Morphog.
53, 99-105.
Weniger, J. P.. Ehrhardt, J. P., and Fritig, B. (1967). Sur la formation d’oestrone et d’oestradiol par les gonades d’embryon de poulet femelles c&iv&es in vitro. C. R. Acad. Sci. (Paris) 264, 838-841. Woods, J. E., and Domm, L. V. (1%6). A histochemical identification of the androgen producing cells in the gonads of the domestic fowl and albino rat. Gen.
Comp.
Endocrinol.
7, 559-570.
Woods, J. E., and Weeks, R. L. (1969). Ontogenesis of the pituitary gonadal axis in the chick embryo. Gen.
Comp.
Endocrinol.
13, 242-254.
AND
COMPARATIVE
38, 153-161 (1979)
ENDOCRINOLOGY
Steroid-Synthesizing Cells in the Embryonic and Adult Gonads of the Domestic Pigeon, Columba livia (Gmelin) BHAGYASHRI Deparrmeni
V. BHUJLE,’ VINAYAK B. NADKARNI, AND M. APPASWAMY RAO
of Zoology,
Karnaiak University. Karnatak State, India
Dharwad-580
003.
Accepted January 28, 1979 Steroidogenic cells in the developing gonads of Columba livia from Day 9 of incubation till the bird attains maturity have been histochemicahy identified by demonstrating the presence of A5-3/3-hydroxysteroid dehydrogenase (A5-3@HSDH). In the embryonic ovary the medulla, containing A5-3&HSDH-positive cells, gradually seems to migrate into the cortical region with growth of the embryo and later it dwindles in 15 to 20-day-old hatchlings. In later stages, As-3/3-HSDH is found in some cells of theta interna of normal follicles, hypertrophied granulosa cells of atretic follicles, the granulosa and theta interna of postovulatory follicles. and the interstitial stromal cells. On Day 9 of incubation in the male embryo the convoluted solid seminiferous cords show a weak A5-3@HSDH activity. One day prior to hatching, AS-3/3-HSDH activity disappears from the seminiferous cords but appears in the interstitial Leydig cells. In the 4-month-old adult pigeon, A5-3pHSDH activity is seen in the Leydig cells as well as in the seminiferous epithelium.
It has been shown that the embryonic gonads of vertebrates produce secretions which have the same effects as the steroid hormones of adult gonads (reviews by Wolff, 1962a,b; Haffen, 1975). Steroids have been detected in the amniotic and allantoic fluids and blood of chick and quail (St011 and Maraud, 1956; Ozon, 1965, 1969). Avian embryonic gonads cultured in vitro can utilize various radioactive precursors and convert them into steroid sex hormones (Weniger et al., 1967; Akram and Weniger, 1969; Weniger, 1970; Galli and Wassermann, 1972, 1973; Guichard et al., 1972a,b, 1977). There are reports on the identification of steroidogenic cells in the embryonic and adult gonads of birds (Narbaitz and Kolodny, 1?64; Chieffi et al.; 1964; Chieffi and Botte, 1965; Woods and Domm, 1966; ’ Present address: Department of Biology, Chowgule College, Margao-403 691, Goa-India.
Narbaitz and De Robertis, 1968; Scheib and Haffen, 1968, 1969; Woods and Weeks, 1969; Arvy and Hadjiisky, 1970; Scheib, 1973; Tingari, 1973; Bhujle and Nadkarni, 1974). Although, there is sufficient evidence for the steroidogenic potentiality of avian embryonic gonads, there is a difference of opinion about the sites of steroidogenesis in the avian embryonic testis. In the present work, an attempt is made to identify the steroidogenic cells and study the steroidogenic potential of the gonads of the domestic pigeon, Columba livia (Gmelin) from the embryonic stage until they attain maturity. MATERIALS
AND METHODS
Pigeon embryos from Day 9 of incubation were obmined from the pigeon colony maintained by the Zoology Department. Embryos prior to Day 9 of incubation were not studied as it was difficult to cut frozen sections of the gonads of these embryos. The head of the embryo was removed and the torso containing the
S.P. 153
0016-6480/79/060153-09$01.00/O Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.
154
BHUJLE,
NADKARNI,
AND
++ ++++
RAO
++ +++t
+t ++ t+++ tttt I:: +ttt tt+t
5=B rg Z$ F
++++ +t+t
++ ++
:: tt++ ++tt tt+t +t t+::
I I I I
I
I
I
tttt ++ :: i-t++ t+ti
++ ++++ ++++ ++4+
tttt t+t+
tt ++
I I I I t-I t-I +++-I t++i
STEROID
DISTRIBUTION
OF
CELLS IN PIGEON
As-3P-HSDH, G-6-PDH, livia
BETWEEN
DAY
AND
9
Enzyme and substrate*
9- to IFdayold embryo
As-3p-HSDH DHA Pregnenolone G-6-PDH NADH-diaphorase A5-3/3-HSDH DHA Pregnenolone G-6-PDH NADH-diaphorase A5-38-HSDH DHA Pregnenolone G-QPDH NADH-diaphorase AS-3p-HSDH DHA Pregnenolone G-6-PDH NADH-diaphorase
I Day prior to hatching to 30 days after hatching 40- to IIO-days old pigeon
I IO- to 120days-old pigeon
TABLE 2 NADH-DIAPHORASE ACTIVITY IN THE TESTIS OF Columbn
OF INCUBATION
Age
155
GONADS
AND
Seminiferous cords
SEXUAL
MATURITY=
Seminiferous tubule epithelium
k + ++ ++
Tubules are not formed
-
tubules are not formed
Leydig cells Leydig cells are not encountered ++ ++ +++ +++
Cords are transformed into tubules
-
fi++ ++++ ++++
Cords are transformed into tubules
+ + ++ ++
+++ +++ +++++ +++++
n Intensity of reaction is graded (-) to (+ + + + +); (-) denotes the absence of reaction and (+ + + + +) a maximal reaction. * All the chemicals are of Sigma grade, obtained from Sigma Chemical Company, St. Louis, MO. vertebral column, adrenals, gonads, and renal tissue was trimmed and rapidly frozen over dry ice for the histochemical study. The torsos from some other embryos of the same age were fixed in Bouin’s fluid for histological studies. In addition, studies were also made of the gonads of pigeons between I and 130 days after hatching (Tables I and 2). The frozen tissues were sectioned at 12 pm in a Pearse-Slee cryostat maintained at -20”. The sections were incubated aerobically at 37” in appropriate media for the localization of A5-3P-hydroxysteroid dehydrogenase (HSDH), G-6-PDH, and NADHdiaphorase activity. For the demonstration of A5-3/3HSDH, dehydroepiandrosterone (DHA) and pregnenolone were used as substrates. The incubation media were prepared following the procedure of Baillie et al. (1966). After incubation, the sections were fixed in 10% neutral formalin for half an hour and were mounted in glycerol jelly. Alternate sections from each tissue were incubated in medium lacking either the substrate or the coenzyme (NAD or NADP) and served as controls.
RESULTS Ovary
The embryonic
ovary on Day 9 of incuba-
tion is well defined into medulla and cortex. The cortex is occupied by oocytes (Fig. 1) and the medulla consists of rounded cells. A5-3/3-HSDH activity was found in some cells in the medulla but no enzyme activity was found in the cortical region. In embryos between day 9 of incubation and Day 5 after hatching, there was an increase in the thickness of the cortical layer and the medulla gradually degenerated. The A5-3/3-HSDH-positive medullary cells seemed to have migrated to the cortical region (Fig. 2). Ten to fifteen days after hatching, the oocytes from the cortical region were enveloped by follicular cells and formed primary follicles. A5-3p-HSDH activity was found in some interfollicular cells and also in the ooplasm of the follicles. In pigeons 20-25 days after hatching there was a prominent growth of the cortex whereas medullary tissue was found only in traces (Fig. 3). At this stage some primary follicles
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CELLS IN PIGEON
undergoing atresia were encountered. A53/3-HSDH activity was found in the cells of the stroma and ooplasm. The ovaries of 40to 60-day-old pigeons showed vitellogenic follicles of different sizes. In follicles of about 500 pm in diameter, the follicular layer could not be differentiated into granulosa and theta. In follicles of about 570 pm in diameter some cells of theta interna showed A5-3/3-HSDH activity. The interstitial stromal cells also showed A53/3-HSDH activity. At the age of 4 months, pigeons start laying eggs and so the ovary of these pigeons is taken as the mature ovary. The mature ovary consists of primary, vitellogenic, postovulatory, and atretic follicles. No enzyme activity was found in the follicular layer of primary follicles. In developing follicles, 500-570 pm in diameter, A53/3-HSDH activity was found in some cells of theta intema and as follicles grew in size the number of theta interna cells showing enzyme activity increased. No enzyme activity was seen in the granulosa cells. However, a weak enzyme activity was seen in the hypertrophied granulosa cells of the atretie follicles. In postovulatory follicles, an intense As-3P-HSDH activity was seen in the granulosa and theta interna cells. The enzyme activity in postovulatory follicles persists for 4-5 days after ovulation and diminishes subsequently. G-6-PDH and NADH-Diaphorase
Both of same cells ity during However,
Activity
these enzymes were found in the that showed As-3/I-HSDH activdifferent stages of development. the activity of G-6-PDH and
GONADS
157
NADH-diaphorase was more intense when compared to that of A5-3/3-HSDH. Further, most of the theta interna cells of large vitellogenic follicles showed activity of both the enzymes (Table 1, Fig. 4). Testis
In histological sections, the testis of a 9-day-old embryo was distinguished from the embryonic ovary by the presence of prominent convoluted seminiferous cords. A weak A5-3/3-HSDH activity was seen inside the seminiferous cords 1 day prior to hatching, (Fig. 5). A5-3P-HSDH activity disappeared from the seminiferous cords but appeared in the Leydig cells that are found between the cords. There were no histological changes in the testis till about 30 days after hatching. A5-3p-HSDH activity was found only in the interstitial Leydig cells. Thirty to forty days after hatching, a lumen appeared in seminiferous cords, thus forming seminiferous tubules, but still the tubules were lined by a single layer of epithelial cells, the stem spermatogonial cells. First spermatogonial divisions were seen in the testis 70-75 days after hatching. In the testes of 90- to loo-day-old pigeons the seminiferous epithelium was many layered and AS-3p-HSDH activity was observed in the Leydig cells. In the testes of 120- to 130-day-old pigeon, different stages of spermatogenesis and sperm bundles were found. An intense As-3@-HSDH reaction was seen in the interstitial cells and a weak activity was observed in the peripheral layer of the germinal epithlium of seminiferous tubules (Fig. 7).
FIG. 1. Section of the ovary of 9-day-old embryo of pigeon showing the cortical (C) and medullary (M) regions. Arrows point to oocytes. Scale line indicates 50 pm. FIG. 2. A5-3PHSDH activity in the interstitial cells (I) of the cortical region (arrow) of the ovary of the 2-day-old pigeon. M indicates degenerating medulla. Scale line indicates 100 pm. FIG. 3. Section of the ovary of 25day-old pigeon showing developing follicles (F). Note the degenerating medulla (M). Scale line indicates 100 pm. FIG. 4. G-6-PDH activity in the ovary of the adult pigeon. The activity is found in the theta interna cells (TH) of growing follicles and in the granulosa cells (G) and theta intema of postovulatory follicles. G-6-PDH activity is also found in the interstitial cells (I). Scale line indicates 100 pm.
158
BHUJLE,
FIG. 5. line indicates FIG. 6. line indicates FIG. 7. the activity
NADKARNI,
AND
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A5-3P-HSDH activity in the seminiferous cords (arrows) of an 1 l-day-old embryo. Scale 125 pm. NADH-diaphorase activity in the Leydig cells of the testis of a 45-day-old pigeon. Scale 125 pm. A5-3P-HSDH activity in the testis of an adult pigeon. I = Leydig cells. Arrows indicate in the seminiferous epithelium. Scale line indicates 200 +m.
STEROID
CELLS
IN
PIGEON
GONADS
159
hatching and thereafter the interstitial cells form the principal site of steroidogenesis. Both of these enzyme activities occurred In the embryonic ovary as well as the in the same cells that showed A5-3/3-HSDH testis, pregnenolone is preferentially utiactivity during different stages of developlized during the demonstration of A5ment. However, the intensity of reaction of 3P-HSDH activity suggesting that A4both of the enzymes was more pronounced pathway of steroidogenesis is more preva(Fig. 6, Table 2). Of the two substrates, pregnenolone and lent than A5-pathway in the embryonic DHA, used for the demonstration of A5- gonads of the pigeon. Similar observations have been made by Boucek et al. (1966) for 3@HSDH activity, the intensity of reaction the embryonic gonads of the chick. In vitro with pregnenolone was greater than that studies on quail testis by Scheib er al. with DHA in the embryonic gonads. How(1974) reveal that DHA is a less efficient ever, in the gonads of the posthatching precursor than pregnenolone. birds, A5-3@HSDH activity was of similar Soon after hatching, in the ovary of C. magnitude, with both substrates. livia, A5-3/3-HSDH-positive cells appear in the cortical region and the medulla shows DISCUSSION degeneration suggesting a shift of steThe presence of A5-3P-HSDH activity in roidogenesis from the medulla to the corthe islets of cells in the medulla and its tex. Similar observations have been made absence in the cortex of the embryonic by Narbaitz and De Robertis (1968) on the ovary of the pigeon suggests the ability of chick ovary. A weak A5-3&HSDH activity A5-3p-HSDH-positive medullary cells to in the ooplasm of the primary follicles oxidize A5-3@-hydroxysteroids to A4-3- suggests its capacity to convert A5-3/3ketosteroids. This observation is in agree- hydroxysteriods to A4-3-ketosteroids. It is ment with those of Narbaitz and Kolodny interesting to note that the amount of inter( 1964) and Scheib and Haffen ( 1968, 1969) stitial tissue showing A5-3P-HSDH activity in their studies on chick embryos. Howis greater in a young ovary (before the apever, Woods and Weeks (1969) have ob- pearance of many vitellogenic follicles) served a diffused A5-3j3-HSDH activity in than in the ovary of an adult pigeon where some cells of the cortex, in addition to the the theta interna cells of vitellogenic follipositive A5-3p-HSDH activity in some cles are involved in steroidogenesis. An inmedullary cells. crease in number of A5-3p-HSDH-positive Chieffi et al. (1964) have stated that A5- theta interna cells with the increase in the 3P-HSDH is diffused throughout the chick size of follicles may be associated with intestis from Day 8 throughout embryonic creased synthesis of estrogens as estrogen synthesis appears to be a consistent condevelopment. But according to Narbaitz (Lofts and Kolodny (1964) the embryonic testis of comitant of follicular development the chick is completely devoid of any spe- and Bern, 1972). cific enzyme activity. Scheib and Haffen It is reported that the postovulatory folli(1968, 1969) have observed a weak enzyme cles found in birds are transient and of unsignificance (Dodd, activity in the seminiferous cords of the certain physiological 6.5day-old chick which becomes more in- 1977). However, the presence of A5-3/3HSDH activity in the granulosa and theta tense by 7-8 days and remains throughout interna cells of the postovulatory follicle in the embryonic development. Our observaC. livia persisting for 3-4 days after ovulations on the embryonic testis of pigeon tion suggests that they may have a stesuggests that the seminiferous cords form the site of steroidogenesis till 1 day prior to roidogenic role. AS-3p-HSDH has also been G-6-PDH
and NADH-Diaphorase
Activity
160
BHUJLE,
NADKARNI,
reported in the postovulatory follicles of fowl and quail (Chieffi and Botte, 1965; Woods and Domm, 1966; Sayler rt al., 1970). In the testis, after hatching, A5-3p-HSDH activity is found in the Leydig cells but no enzyme activity is seen inside the seminiferous epithelium until 120- 130 days, suggesting that the Leydig cells are involved in steroid secretion. The seminiferous epithelium shows steroidogenic potentiality only in the adult pigeon. Similar observations have been reported by Tingari (1973) for the testis of the adult fowl. The presence of G-6-PDH in the same cells that showed A5-3/3-HSDH activity in the developing gonads, provides additional albeit indirect, of the steevidence, roidogenic potentiality of these cells, as G-6-PDH is known to generate the NADPH required for the hydroxylations of steroids during steroidogenesis (Weist and Kidwell, 1969). ACKNOWLEDGMENTS The authors are grateful to the Karnatak University, Dharwad, India, for providing laboratory facilities. The work was supported by a Senior Fellowship from the C.S.I.R. of India to the first author. The free gift of chemicals by W.H.O., Geneva, is gratefully acknowledged.
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