GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
27,271-275
(1975)
NOTE
Histochemical Observations on the Corpus of the House Sparrow (Passer domesticus)
Luteum Ovary
A histochemical study has been made of the postovulatory follicle (or corpus luteum) in The luteal cell mass is gradually the ovary of the house sparrow (Passer domesticus). formed by hypertrophy of the granulosa and theta intema cells which develop diffuse lipoproteins, and lipid droplets consisting Iirst of phospholipids and some triglycerides, and later of triglycerides, cholesterol and/or its esters and some phospholipids. The granulosa luteal cells develop lipids earlier, and in larger amount, than the theta luteal cells. The functional significance of the morphological and histochemical features of the sparrow corpus luteum is briefly discussed in relation to steroid hormone synthesis.
In Mammalia, Reptilia, and Amphibia, the hypertrophy and “luteinization” of granulosa cells after ovulation is closely accompanied by the development of diffusely distributed lipoproteins and lipid droplets which have been presumed to be related to steroidogenesis (Guraya, 1971, 1973, 1975). In view of these histochemical findings, correlated with electron microscope studies, it was of interest to make similar observations on the corpus luteum of house sparrow ovary. MATERIAL
(see Table 1). The granulosa luteal cells also develop some deeply sudanophilic lipid droplets (Fig. l), consisting of phospholipids and triglycerides (see Table 1).
AND METHODS
Ovarian material from sexually mature sparrows (Passer domesticus) was used. Female specimens were shot during the breeding periods (April-July and September-October). The recovery of an egg from the oviduct was the criterion for recent ovulation. The ovaries were immediately removed and placed in physiological saline (Baker, 1944). Fixing fluids and histochemical techniques employed for the study of lipids were the same as previously reported (Guraya, 1968).
Observations The evolution and involution of the postovulatory follicle or corpus luteum in the house sparrow can conveniently be divided into a series of stages: Stuge 1. Immediately after ovulation, the granulosa cells hypertrophy and develop a diffusely distributed sudanophilic substance (Fig. 1), which appears to be lipoprotein, judged from its histochemical reactions
FIGS. l-5. Photomicrographs of successive stages of formation of the sparrow corpus luteum fixed in formaldehyde-calcium, postchromed in dichromate-calcium and frozen sections coloured with Sudan black B. Fig. 1. Portion of newly ruptured follicle showing hypertrophied granulosa cells (GC) and thecal layers (TL). x 109.07.
271 Copyright @I 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
0 0 p++ 0 +++ +++ 0 0 0 0
++ + 0 0 p+++ p+to++ ++to+++ ++to+++ 0 0 0 0
++to+++ +to++ 0 0 p++++ p++ +to++ ++ 0 0 +to++ 0
+++ ++
Stage 3
Lipid droplets
A 0 +++ ++
0 0 p++++ p++ +to++
++++ ++to+++
Stage 4
TABLE 1 CORPUS LUTEUM
IN THE
Stage 2
OF LIPIDS
; 0 +++
0 0 p+++ -
+++-t
Stage 5
SPARROW
Remarks
OVARY~
in the lipid droplets Confirming the presence of phospholipids in the lipid droplets Revealing the presence of cholesterol and/or its esters
Revealing phospholipids
Revealing neutral lipids in the lipid droplets
Showing the presence of diffuse and discrete lipid droplets in both granulosa and theta luteal cells at different stages of corpus luteum Contirming the presence of lipids
OF THE HOUSE
D Key to abbreviations: *, After treatment with; FCA + PC, formaldehyde-calcium and postchromed in dichromate-calcium; WB + PE, weak Bouin’s followed by pyridine extraction; P, Pink; GC, Granulosa luteal cells; TC, Theta luteal cells; -, Granulosa luteal cells not seen; f, weak reaction; + +, moderate reaction; + + +, strong reaction; + + + + , very strong reaction; 0, negative.
Schultz
AH*PE
Acid haematein
Nile blue
GC++ TC +
Stage 1
REACTIONS
GCO TC 0 FCA + PC GC + blue TC + blue FCA + PC GCO TC 0 WB + PE GC 0 TC 0 FCA+PC GCO TC 0
FCA+PC
Sudan black B (SBB) in 70% ethanol SBB* PE
WB + PE
Fixation
Technique
Diffuse lipids
HIST~CHEMICAL
273
NOTES
FIG. 2. Showing an increasing amount of sudanophilic lipids in the granulosa luteal cells (GC), which are being invaded by the less sudanophilic theta luteal cells (XI). X 109.07. The thecal derivatives show a similar though smaher development of diffusely distributed lipids and lipid droplets (Fig. 1). Stage 2. The thecal cells start invading the granulosa. The lipid droplets of the latter form dense sudanophilic masses which also contain abundant neutral fats (triglycerides) (Table 1). Theta intema cells surrounding the granulosa luteal cells, hypertrophy to form theta luteal cells (Fig. 2), which also develop some diffusely distributed lipoproteins and lipid droplets similar to those of the granulosa luteal cells in stage 1 (Fig. 2). Stage 3. At this stage there occurs a complete intermixing of granulosa and theta luteal cells (Fig. 3), though these can still be distinguished from one another due to differences in their lipid contents (see Table 1); the granulosa luteal cells show relatively more lipid droplets rich in triglycerides and cholesterol and/or its esters. Srage 4. The corpus luteum at this stage is greatly decreased in size due to the progressive degeneration of granulosa luteal cells (Fig. 4). Some regressing granulosa luteal cells filkd with coarse lipid droplets, are still seen among the theta luteal cells, but ultimately, they also disappear from view. Stage 5. The theta luteal cells, which still persist at this stage, accumulate more sudanophilic lipids (Fig. 5), but their number is gradually reduced to form very
FIG. 3. Showing the complete intermixing of highly sudanophilic granulosa luteal cells (GC) and relatively fewer sudanophilic theta luteal cells (TC). x 109.07. small nodules in the stroma, which persist for a considerable time.
DISCUSSION
The most striking and significant chemical change which occurs during the hypertrophy or “luteinization” of granulosa cells after ovulation in the sparrow ovary is the development of diffusely distributed lipoproteins throughout their cytoplasm, which are not seen in the cytoplasm of granulosa cells of developing follicles. The transformation of theta intema cells into theta luteal cells is also closely accompanied by the development of similar lipoproteins but in lesser amount. The diffuse lipoproteins of luteal cells in the sparrow corpus luteum might derive from the membranes of smooth reticulum similar to those described in the granulosa
274
NOTES
FIG. 4. Regressing corpus luteum showing some highly sudanophilic, degenerating granulosa luteal cells (GC). Note development of more sudanophilic lipids in theta luteal cells (TC). x 150.
luteal cells of the hen corpus luteum (Wyburn et al., 1966). The present author (Guraya, 1971, 1973, 1975) has also made a similar suggestion for the luteal cells in Reptilia and Amphibia.
FIG.
x 150.
Correlated morphological, histochemical, and biochemical studies have also suggested that the enzyme activities indicative of steroid hormone synthesis parallel the presence and development of diffuse
5. Corpus luteum in its last stages of involution, showing heavy accumulation of sudanophilic
lipids.
275
NOTES
lipids and membranes of smooth reticulum in steroid-producing cells, indicating a functional association between them (Guraya, 1971, 1975). A similar enzyme activity has also been reported in the granulosa luteal cell mass of birds (Lofts and Bern, 1972; Guraya, 1975). In addition to acting as a site for the synthetic enzymes involved in the biosynthesis of steroid hormones, the membranes of smooth reticulum (or diffuse lipoproteins) may also possibly serve to some extent as a reservoir for the storage of hormone precursor (cholesterol). The granulosa luteal cells of the sparrow corpus luteum show some lipid droplets dbnsisting mainly of phospholipids and some triglycerides to begin with. Similar lipid droplets also develop in the corpora lutea of Mammalia, Reptilia, and Amphibia (see Guraya, 1971, 1975). Wyburn et al. (1%6) have also reported the development of lipid droplets in the luteal cells of the hen’s ovary. It is believed that when lipid droplets are present in abundance, storage is taking place in steroid gland cells and when the amount is less, the hormone is being released (Guraya, 1971, 1975). According to this concept, which evolved from many cytological and histochemical studies, the granulosa luteal cells of the sparrow corpus luteum might be functioning in the secretion of some steroid hormone in stages 1 and 2 and in the storage of hormone precursor (cholesterol) in stages 3 and 4 when the theta luteal cells seem to be active in steroidogenesis as judged by the presence of a few lipid droplets in their cytoplasm. From this discussion, it can be concluded that the luteinization process in the sparrow follicular epithelium and thecal layers after ovulation is cytologically and histochemically the same as that described for mammals. But very divergent opinions have been expressed in regard to the secretory role of the avian corpus luteum (see Lofts and Bern, 1972; Guraya, 1975).
With the involution of the corpus luteum, lipids, consisting of cholesterol and/or its esters, triglycerides, pigments and some phospholipids accumulate in the luteal cells. Simultaneously the various cell organelles are also greatly altered in their fine structure (Wyburn et al., 1966). The enzyme activity indicative of steroidogenesis also disappears concomitantly (Chieffi and Botte, 1965), suggesting the disappearance of the steroidogenic activity in the degenerating granulosa luteal cells. REFERENCES Baker, J. R. (1944). The structure and chemical composition of the golgi elements. Quart. J. Microsc. Sci. 85, 1-71. Chieffi, G., and Botte, V. (1965). The distribution of some enzymes involved in steroidogenesis of hen’s ovary. Experientia 21, 16-17. Guraya, S. S. (1968). Histochemical study of granulosa (follicular) cells in the preovulatory and post-ovulatory follicles of amphibian ovary. Gen. Comp. Endocrinol. 10, 138-146. -.....-(1971). Morphology, Histochemistry and Biochemistry of Human ovarian compartments and steroid hormone synthesis. Physiol. Rev. 51, 785-807. -(1973). Morphological and histochemical observations on the corpora lutea of the snake (Lycodon aulicus) ovary. Acta Morphol. Acad. Sci. Hung.
21, 23!%247.
-(1975). Recent advances in the morphology, histochemistry and biochemistry of steroidsynthesizing cellular sites in non-mammalian vertebrate ovary. Inter. Rev. Cytol., in press. Lofts, B. and Bern, H. A. (1972). The functional morphology of steroidogenic tissue. In “Steroids in Nonmammalian Vertebrates” (David R. Idler, Ed.), pp. 37-117. Academic Press, New York. Wyburn, G. M.,‘J&nston, H. S. and Aitken, R. N. C. (1966). Fate of granulosa cells in the hen’s follicle. Z. Zellforsch. 72, 53-65. SARDUL S. GURAYA RANJIT K. CHALANA Department of Zoology College of Basic Sciences and Humanities Punjab Agricultural University Ludhiana, Punjab, India Accepted
May
26, 1975
AND
COMPARATIVE
ENDOCRINOLOGY
27,271-275
(1975)
NOTE
Histochemical Observations on the Corpus of the House Sparrow (Passer domesticus)
Luteum Ovary
A histochemical study has been made of the postovulatory follicle (or corpus luteum) in The luteal cell mass is gradually the ovary of the house sparrow (Passer domesticus). formed by hypertrophy of the granulosa and theta intema cells which develop diffuse lipoproteins, and lipid droplets consisting Iirst of phospholipids and some triglycerides, and later of triglycerides, cholesterol and/or its esters and some phospholipids. The granulosa luteal cells develop lipids earlier, and in larger amount, than the theta luteal cells. The functional significance of the morphological and histochemical features of the sparrow corpus luteum is briefly discussed in relation to steroid hormone synthesis.
In Mammalia, Reptilia, and Amphibia, the hypertrophy and “luteinization” of granulosa cells after ovulation is closely accompanied by the development of diffusely distributed lipoproteins and lipid droplets which have been presumed to be related to steroidogenesis (Guraya, 1971, 1973, 1975). In view of these histochemical findings, correlated with electron microscope studies, it was of interest to make similar observations on the corpus luteum of house sparrow ovary. MATERIAL
(see Table 1). The granulosa luteal cells also develop some deeply sudanophilic lipid droplets (Fig. l), consisting of phospholipids and triglycerides (see Table 1).
AND METHODS
Ovarian material from sexually mature sparrows (Passer domesticus) was used. Female specimens were shot during the breeding periods (April-July and September-October). The recovery of an egg from the oviduct was the criterion for recent ovulation. The ovaries were immediately removed and placed in physiological saline (Baker, 1944). Fixing fluids and histochemical techniques employed for the study of lipids were the same as previously reported (Guraya, 1968).
Observations The evolution and involution of the postovulatory follicle or corpus luteum in the house sparrow can conveniently be divided into a series of stages: Stuge 1. Immediately after ovulation, the granulosa cells hypertrophy and develop a diffusely distributed sudanophilic substance (Fig. 1), which appears to be lipoprotein, judged from its histochemical reactions
FIGS. l-5. Photomicrographs of successive stages of formation of the sparrow corpus luteum fixed in formaldehyde-calcium, postchromed in dichromate-calcium and frozen sections coloured with Sudan black B. Fig. 1. Portion of newly ruptured follicle showing hypertrophied granulosa cells (GC) and thecal layers (TL). x 109.07.
271 Copyright @I 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
0 0 p++ 0 +++ +++ 0 0 0 0
++ + 0 0 p+++ p+to++ ++to+++ ++to+++ 0 0 0 0
++to+++ +to++ 0 0 p++++ p++ +to++ ++ 0 0 +to++ 0
+++ ++
Stage 3
Lipid droplets
A 0 +++ ++
0 0 p++++ p++ +to++
++++ ++to+++
Stage 4
TABLE 1 CORPUS LUTEUM
IN THE
Stage 2
OF LIPIDS
; 0 +++
0 0 p+++ -
+++-t
Stage 5
SPARROW
Remarks
OVARY~
in the lipid droplets Confirming the presence of phospholipids in the lipid droplets Revealing the presence of cholesterol and/or its esters
Revealing phospholipids
Revealing neutral lipids in the lipid droplets
Showing the presence of diffuse and discrete lipid droplets in both granulosa and theta luteal cells at different stages of corpus luteum Contirming the presence of lipids
OF THE HOUSE
D Key to abbreviations: *, After treatment with; FCA + PC, formaldehyde-calcium and postchromed in dichromate-calcium; WB + PE, weak Bouin’s followed by pyridine extraction; P, Pink; GC, Granulosa luteal cells; TC, Theta luteal cells; -, Granulosa luteal cells not seen; f, weak reaction; + +, moderate reaction; + + +, strong reaction; + + + + , very strong reaction; 0, negative.
Schultz
AH*PE
Acid haematein
Nile blue
GC++ TC +
Stage 1
REACTIONS
GCO TC 0 FCA + PC GC + blue TC + blue FCA + PC GCO TC 0 WB + PE GC 0 TC 0 FCA+PC GCO TC 0
FCA+PC
Sudan black B (SBB) in 70% ethanol SBB* PE
WB + PE
Fixation
Technique
Diffuse lipids
HIST~CHEMICAL
273
NOTES
FIG. 2. Showing an increasing amount of sudanophilic lipids in the granulosa luteal cells (GC), which are being invaded by the less sudanophilic theta luteal cells (XI). X 109.07. The thecal derivatives show a similar though smaher development of diffusely distributed lipids and lipid droplets (Fig. 1). Stage 2. The thecal cells start invading the granulosa. The lipid droplets of the latter form dense sudanophilic masses which also contain abundant neutral fats (triglycerides) (Table 1). Theta intema cells surrounding the granulosa luteal cells, hypertrophy to form theta luteal cells (Fig. 2), which also develop some diffusely distributed lipoproteins and lipid droplets similar to those of the granulosa luteal cells in stage 1 (Fig. 2). Stage 3. At this stage there occurs a complete intermixing of granulosa and theta luteal cells (Fig. 3), though these can still be distinguished from one another due to differences in their lipid contents (see Table 1); the granulosa luteal cells show relatively more lipid droplets rich in triglycerides and cholesterol and/or its esters. Srage 4. The corpus luteum at this stage is greatly decreased in size due to the progressive degeneration of granulosa luteal cells (Fig. 4). Some regressing granulosa luteal cells filkd with coarse lipid droplets, are still seen among the theta luteal cells, but ultimately, they also disappear from view. Stage 5. The theta luteal cells, which still persist at this stage, accumulate more sudanophilic lipids (Fig. 5), but their number is gradually reduced to form very
FIG. 3. Showing the complete intermixing of highly sudanophilic granulosa luteal cells (GC) and relatively fewer sudanophilic theta luteal cells (TC). x 109.07. small nodules in the stroma, which persist for a considerable time.
DISCUSSION
The most striking and significant chemical change which occurs during the hypertrophy or “luteinization” of granulosa cells after ovulation in the sparrow ovary is the development of diffusely distributed lipoproteins throughout their cytoplasm, which are not seen in the cytoplasm of granulosa cells of developing follicles. The transformation of theta intema cells into theta luteal cells is also closely accompanied by the development of similar lipoproteins but in lesser amount. The diffuse lipoproteins of luteal cells in the sparrow corpus luteum might derive from the membranes of smooth reticulum similar to those described in the granulosa
274
NOTES
FIG. 4. Regressing corpus luteum showing some highly sudanophilic, degenerating granulosa luteal cells (GC). Note development of more sudanophilic lipids in theta luteal cells (TC). x 150.
luteal cells of the hen corpus luteum (Wyburn et al., 1966). The present author (Guraya, 1971, 1973, 1975) has also made a similar suggestion for the luteal cells in Reptilia and Amphibia.
FIG.
x 150.
Correlated morphological, histochemical, and biochemical studies have also suggested that the enzyme activities indicative of steroid hormone synthesis parallel the presence and development of diffuse
5. Corpus luteum in its last stages of involution, showing heavy accumulation of sudanophilic
lipids.
275
NOTES
lipids and membranes of smooth reticulum in steroid-producing cells, indicating a functional association between them (Guraya, 1971, 1975). A similar enzyme activity has also been reported in the granulosa luteal cell mass of birds (Lofts and Bern, 1972; Guraya, 1975). In addition to acting as a site for the synthetic enzymes involved in the biosynthesis of steroid hormones, the membranes of smooth reticulum (or diffuse lipoproteins) may also possibly serve to some extent as a reservoir for the storage of hormone precursor (cholesterol). The granulosa luteal cells of the sparrow corpus luteum show some lipid droplets dbnsisting mainly of phospholipids and some triglycerides to begin with. Similar lipid droplets also develop in the corpora lutea of Mammalia, Reptilia, and Amphibia (see Guraya, 1971, 1975). Wyburn et al. (1%6) have also reported the development of lipid droplets in the luteal cells of the hen’s ovary. It is believed that when lipid droplets are present in abundance, storage is taking place in steroid gland cells and when the amount is less, the hormone is being released (Guraya, 1971, 1975). According to this concept, which evolved from many cytological and histochemical studies, the granulosa luteal cells of the sparrow corpus luteum might be functioning in the secretion of some steroid hormone in stages 1 and 2 and in the storage of hormone precursor (cholesterol) in stages 3 and 4 when the theta luteal cells seem to be active in steroidogenesis as judged by the presence of a few lipid droplets in their cytoplasm. From this discussion, it can be concluded that the luteinization process in the sparrow follicular epithelium and thecal layers after ovulation is cytologically and histochemically the same as that described for mammals. But very divergent opinions have been expressed in regard to the secretory role of the avian corpus luteum (see Lofts and Bern, 1972; Guraya, 1975).
With the involution of the corpus luteum, lipids, consisting of cholesterol and/or its esters, triglycerides, pigments and some phospholipids accumulate in the luteal cells. Simultaneously the various cell organelles are also greatly altered in their fine structure (Wyburn et al., 1966). The enzyme activity indicative of steroidogenesis also disappears concomitantly (Chieffi and Botte, 1965), suggesting the disappearance of the steroidogenic activity in the degenerating granulosa luteal cells. REFERENCES Baker, J. R. (1944). The structure and chemical composition of the golgi elements. Quart. J. Microsc. Sci. 85, 1-71. Chieffi, G., and Botte, V. (1965). The distribution of some enzymes involved in steroidogenesis of hen’s ovary. Experientia 21, 16-17. Guraya, S. S. (1968). Histochemical study of granulosa (follicular) cells in the preovulatory and post-ovulatory follicles of amphibian ovary. Gen. Comp. Endocrinol. 10, 138-146. -.....-(1971). Morphology, Histochemistry and Biochemistry of Human ovarian compartments and steroid hormone synthesis. Physiol. Rev. 51, 785-807. -(1973). Morphological and histochemical observations on the corpora lutea of the snake (Lycodon aulicus) ovary. Acta Morphol. Acad. Sci. Hung.
21, 23!%247.
-(1975). Recent advances in the morphology, histochemistry and biochemistry of steroidsynthesizing cellular sites in non-mammalian vertebrate ovary. Inter. Rev. Cytol., in press. Lofts, B. and Bern, H. A. (1972). The functional morphology of steroidogenic tissue. In “Steroids in Nonmammalian Vertebrates” (David R. Idler, Ed.), pp. 37-117. Academic Press, New York. Wyburn, G. M.,‘J&nston, H. S. and Aitken, R. N. C. (1966). Fate of granulosa cells in the hen’s follicle. Z. Zellforsch. 72, 53-65. SARDUL S. GURAYA RANJIT K. CHALANA Department of Zoology College of Basic Sciences and Humanities Punjab Agricultural University Ludhiana, Punjab, India Accepted
May
26, 1975
