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A histochemical study of the ovaries of women suffering from the Stein-Leventhal syndrome
A histochemical study of the ovaries of women suffering from the Stein-Leventhal syndrome STOYAN I. DOKUMOV, M.D. GEORGI I. DASHEV, M.D. Sofia, Bulgaria
I N 1 9 3 5 Stein and Leventhal reported a syndrome and described its clinical, histo~ logic, and therapeutic characteristics. Since the initial description, much attention has been directed toward this interesting syndrome especially during recent years. However, neither the clinical data nor the bio~ chemical and histologic investigations have been able so far to explain the etiology or pathogenesis of this disturbance. Particularly, it could not be settled whether the disturbed ovarian function was primary or secondary and the relation between the ovarian dysfunction and that of other endocrine glands was unclear. The purpose of this paper is to report the study of the ovaries of women suffering from the Stein-Leventhal syndrome by means of some histochemical tests, since only the histologic findings in the ovaries are typical and constant in this hormonal disturbance.
women, aged 19 to 35, killed in accidents. The ovaries were prepared during the first few hours after death.
Methods Glycogen. Paraffin sections, fixation in 70 per cent alcohol, staining according to Best's method. Ascorbic acid. Paraffin section, fixation in levulose, staining according to J. Leblon. Alkaline and acid phosphatase. Frozen and paraffin sections, fixation in absolute alcohol and acetone, staining according to Gomori' s method. Neutral lipids. Frozen sections, fixation in 10 per cent formol, staining with Sudan black. Cholesterol. Frozen sections, fixation in 10 per cent formol, staining according to Schultze. Neutral and acid mucopolysaccharides. Paraffin sections, fixation in 10 per cent formol, staining with toluidin blue and P.A.S. (periodic acid-Schiff reagents).
Material The ovaries of 25 women, aged 21 to 34 years, suffering from the Stein-Leventhal syndrome (as determined clinically, biochemically, and histologically through the routine histologic tests) were examined. The material obtained by the cuneiform resection of the ovaries was prepared according to several techniques as noted. Controls were the ovaries of 10 healthy
Results The capsule of the ovary. The tunica albuginea was found to be irregularly thickened with a significant proliferation of collagen fibers. No areas of cuboidal epithelium were detectable on the surface of the ovary. The capsule of the ovaries consisted of a dense connective tissue, rich in collagen fibers and P.A.S.-positive substances (Fig. 1) . In some areas, however, where the capsule was looser, there was an accumulation
From the Postgraduate Medical Institute (ISUL), Clinic of Endocrinology and Metabolic Diseases (Direcror: Prof. lw. Pentshev).
183
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Dokumov and Dashev
of the P.A.S.-positive substances. These were related to the reticular invasion of the ovarian cortex. No other enzymatic activity was demonstrable besides the presence of
Fig. l. Deposition of P.A.S.-positive substances in the thickened ovarian capsule. ( P.A.S., hematoxylin . ><: 144. )
Fig. 2. Primordial follicle with Call-Exner bodies and deposition of P.A.S.-positive substances. (P.A.S., hematoxylin. x144.)
Fig. 3. Elevated alkaline phosphatase actJVJty in the theca interna cells. (Gomori. x l44. )
.May l'i, 1963 Am . ] . Obst. & Gyncc.
glycoproteins. A positive glycogen reaction was not found and only an insignificant alkaline phosphatase activity was present. The capsular cells did not contain cholesterol, neutral lipids, or ascorbic acid granules. Primordial and primary follicles. The number of the primordial and primary follicles was greatly reduced and, when present they were, as a rule, found at some distance below the surface of the ovary. The ovum was surrounded by a single layer of flattened granulosa cells and a deposition of P.A.S.-positive substances was distinctly seen in the location of formation of the zona pellucida (Fig. 2) . Cali-Exner bodies were found in the follicles with a well-developed granulosa (i.e., more than 3 layers ) . They possessed a strongly positive P.A.S. reaction, whereas the glycogen reaction remained negative. This P.A.S.-positive reaction was more striking than it was in the normal ovaries. In the granulosa layers ascorbic acid granules were found but the alkaline and acid phosphatase activity remained within normal limits. Only single granules of glycogen were seen and no neutral lipids and cholesterol could be detected. Developing Graafian follicles. Graafian follicles in different stages of development were also found in the ovarian cortex. The principal alterations from the normal were noticeable in the cells of the theca. These cells seemed bigger, vacuolated, with a light cytoplasm and hyperchromic nuclei . The cytoplasm of these cells contained a large number of ascorbic acid granules and an increased alkaline and acid phosphatase activity (Figs. 3 and 4 ) . Compared with the findings in the normal ovaries this alkaline and acid phosphatase activity was significantly higher in this stage of development of the ovarian follicle. Glycogen was not demonstrated. P.A.S.-positive substances were encountered both in the cells of the theca and in the spaces between them. The ovum also showed a positive P.A.S. reaction. The accumulation of the P.A.S.-positive substances in the growing follicle showed no differences when comparison was made with the normal ovary. In contrast to the normal, however, ascorbic
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Fig. 4. Elevated acid phosphatase activity in the theca interna cells. (Gomori. x144.)
Fig. 5. Ascorbic acid granules in the granulosa cells. (Leblon and methylene blue. x270.)
acid granules were seen in the granulosa cells (Fig. 5). No neutral lipids or cholesterol was detected. Mature Graafian follicles. Mature Graafian follicles were very rarely present in our cases. Follicles in this stage revealed a well-developed granulosa, consisting of 5 to 7 layers. The membrana limitans externa was well defined. The granulosa cells had a positive glycogen reaction and ascorbic acid granules were found in the cytoplasm also. Liquor folliculi reacted positively for the glycoproteins. These P.A.S.-positive substances, however, gave a negative reaction to glycogen, ascorbic acid, neutral lipids, and cholesterol. Alkaline phosphatase activity was found in the intima of the capillaries and also in the theca interna cells as a peripheral rim. The findings in the mature Graafian follicle resembled closely those seen in the normal ovary.
Follicles in atresia. Multiple follicular cysts, which presented follicles in a different stage of cystic atresia, were found beneath the thickened tunica aibuginea. These atretic follicles contained all elements of the growing follicle, but in different stages of degeneration. The cysts varied in size from 0.2 to 0.4 mm. and were full of a light pink jelly giving a positive P.A.S. reaction (Fig. 6). This substance, however, gave a negative glycogen reaction, did not contain ascorbic acid granules, and possessed no alkaline and acid phosphatase activity. It did not contain any neutral lipids or cholesterol. Probably it contained glycoproteins. Compared with the atretic follicles of the normal ovary, greater degeneration was evident. The wall of the cystically degenerated follicles contained some remnants of granulosa. Its cells were atrophied with distinct dystrophic alterations. They were
Fig. 6. Intensive deposition of P.A.S.positive substances in the lumen of a cystic dilated atretic follicle. (P.A.S., hematoxylin. x144.)
186
Dokumov and Doshev
Am.
J.
May 15, 1963 Obst. & Gynec.
Fig. 10. Alkaline phosphatase activity in the theca interna cells and in a capillary wall. ( Gomori . x l44.) Fig. 7. Infiltration with P.A.S.-positive substances of the thickened wall of a capillary around a cystic dilated follicle. (P.A.S., hematoxylin. x l44. )
Fig. 8. Intensive P.A.S.-Positive reaction in the hypertrophied and hyperplastic theca interna cells. (P.A.S., hematoxylin. x144.)
Fig. 9. Positive glycogen reaction in the areas of the hypertrophied theca interna cells. (Best. xl44.)
flattened, had a vacuolated cytoplasm and pyknotic nuclei. The glycogen reaction was less evident in the cytoplasm and the ascorbic acid granules were scantier. No alkaline and acid phosphatase activity was demonstrable. P.A.S.-positive substances and cholesterol were absent. Only single cells had an accumulation of neutral lipids in their vacuolated cytoplasm. Multiple capillaries were seen around the cystic structures. Compared with the capillaries around the normal follicles their wall was more thickened and was strongly infiltrated by P.A.S.-positive substances (Fig. 7). In some areas, however, a diminution of these substances was noticed. This was accompanied by a hyalination and sclerosis of the capillary wall. Probably the alterations in the capillary wall (hyalination and sclerosis) were preceded by an intensive infiltration of P.A.S.-positive substances. The theca interna cells were distinctly hypertrophied and hyperplastic. They stained relatively well, especially the cytoplasm, which was vacuolated in some areas and displayed a clear basophilia. The nuclei were well stained. Occasionally mitoses were found. Some theca interna cells showed evidence of P.A.S.-positive substances (Fig. 8). A slight glycogen reaction and an increased alkaline and acid phosphatase activity were noticed in the cytoplasm of the cells (Figs. 9 and 10) . The ascorbic acid granules were well defined. Some cells, situated in well-circumscribed groups, had a positive reaction for cholesterol and neutral lipids (Fig. 11 I . This
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Fig. 11. Neutral lipids in the cytoplasm of some hypertrophied theca interna cells. (Sudan black. xl44.)
finding suggested that perhaps the hypertrophied and hyperplastic cells, situated around the cystic, atretic follicles, possessed an elevated vital activity or that they produced certain well-defined substances. The interstitium around the follicular structures was infiltrated by a dense connective tissue which in some areas had undergone hyalination and sclerosis. In other areas the connective tissue grew looser and showed P.A.S.positive substances. A particular enzymatic activity was not observed. Corpora lutea and corpora albicantia. These were not found in any of our cases. Comment
Although the scope and the range of the histochemical methods used in this study are limited and incomplete, some characteristics in the ovaries of women suffering from the Stein-Leventhal syndrome have been demonstrated. These were found both in the structures encountered in the normal ovary (primordial, primary, growing follicle, follicles undergoing the different degrees of atresia) and in the formations absent in a normal ovary (the hypertrophied and hyper-
REFERENCES I. McKay, D . G., Pinkerton, J. M., Hertig, A. T., and Danziger, S. : Obst. & Gynec. 18: 13, 1961. 2. Pinkerton, J. M., McKay, D. G., Adams, E. C., and Hertig, A. T.: Obst. & Gynec. 18: 152, 1961.
plastic theca interna) . Regardless of the kind of structures affected, however, the alterations were located mainly in the cells producing the steroid hormones. It is the histochemical characteristics of these cells that reveal the differences in cellular metabolism, when comparison is made with the same cells of a normal ovary. These histochemical findings are probably the sequence of a changed vital activity of the cell or of an abnormal production of definite substances, probably steroid hormones, their precursors or metabolic products. Conclusions 1. The ovaries of women suffering from the Stein-Leventhal syndrome disclose distinct histochemical differences from normal ovanes. 2. These histochemical differences are located both in the structures physiologically found in a normal ovary and in structures absent in a normal ovary. 3. The main histochemical differences are localized in the cells producing the steroid hormones.
Clinic of Endocrinology Postgraduate Medical Inst itute (ISUL) Belo Mo re str. 8 Sofia, Bulgaria
I N 1 9 3 5 Stein and Leventhal reported a syndrome and described its clinical, histo~ logic, and therapeutic characteristics. Since the initial description, much attention has been directed toward this interesting syndrome especially during recent years. However, neither the clinical data nor the bio~ chemical and histologic investigations have been able so far to explain the etiology or pathogenesis of this disturbance. Particularly, it could not be settled whether the disturbed ovarian function was primary or secondary and the relation between the ovarian dysfunction and that of other endocrine glands was unclear. The purpose of this paper is to report the study of the ovaries of women suffering from the Stein-Leventhal syndrome by means of some histochemical tests, since only the histologic findings in the ovaries are typical and constant in this hormonal disturbance.
women, aged 19 to 35, killed in accidents. The ovaries were prepared during the first few hours after death.
Methods Glycogen. Paraffin sections, fixation in 70 per cent alcohol, staining according to Best's method. Ascorbic acid. Paraffin section, fixation in levulose, staining according to J. Leblon. Alkaline and acid phosphatase. Frozen and paraffin sections, fixation in absolute alcohol and acetone, staining according to Gomori' s method. Neutral lipids. Frozen sections, fixation in 10 per cent formol, staining with Sudan black. Cholesterol. Frozen sections, fixation in 10 per cent formol, staining according to Schultze. Neutral and acid mucopolysaccharides. Paraffin sections, fixation in 10 per cent formol, staining with toluidin blue and P.A.S. (periodic acid-Schiff reagents).
Material The ovaries of 25 women, aged 21 to 34 years, suffering from the Stein-Leventhal syndrome (as determined clinically, biochemically, and histologically through the routine histologic tests) were examined. The material obtained by the cuneiform resection of the ovaries was prepared according to several techniques as noted. Controls were the ovaries of 10 healthy
Results The capsule of the ovary. The tunica albuginea was found to be irregularly thickened with a significant proliferation of collagen fibers. No areas of cuboidal epithelium were detectable on the surface of the ovary. The capsule of the ovaries consisted of a dense connective tissue, rich in collagen fibers and P.A.S.-positive substances (Fig. 1) . In some areas, however, where the capsule was looser, there was an accumulation
From the Postgraduate Medical Institute (ISUL), Clinic of Endocrinology and Metabolic Diseases (Direcror: Prof. lw. Pentshev).
183
184
Dokumov and Dashev
of the P.A.S.-positive substances. These were related to the reticular invasion of the ovarian cortex. No other enzymatic activity was demonstrable besides the presence of
Fig. l. Deposition of P.A.S.-positive substances in the thickened ovarian capsule. ( P.A.S., hematoxylin . ><: 144. )
Fig. 2. Primordial follicle with Call-Exner bodies and deposition of P.A.S.-positive substances. (P.A.S., hematoxylin. x144.)
Fig. 3. Elevated alkaline phosphatase actJVJty in the theca interna cells. (Gomori. x l44. )
.May l'i, 1963 Am . ] . Obst. & Gyncc.
glycoproteins. A positive glycogen reaction was not found and only an insignificant alkaline phosphatase activity was present. The capsular cells did not contain cholesterol, neutral lipids, or ascorbic acid granules. Primordial and primary follicles. The number of the primordial and primary follicles was greatly reduced and, when present they were, as a rule, found at some distance below the surface of the ovary. The ovum was surrounded by a single layer of flattened granulosa cells and a deposition of P.A.S.-positive substances was distinctly seen in the location of formation of the zona pellucida (Fig. 2) . Cali-Exner bodies were found in the follicles with a well-developed granulosa (i.e., more than 3 layers ) . They possessed a strongly positive P.A.S. reaction, whereas the glycogen reaction remained negative. This P.A.S.-positive reaction was more striking than it was in the normal ovaries. In the granulosa layers ascorbic acid granules were found but the alkaline and acid phosphatase activity remained within normal limits. Only single granules of glycogen were seen and no neutral lipids and cholesterol could be detected. Developing Graafian follicles. Graafian follicles in different stages of development were also found in the ovarian cortex. The principal alterations from the normal were noticeable in the cells of the theca. These cells seemed bigger, vacuolated, with a light cytoplasm and hyperchromic nuclei . The cytoplasm of these cells contained a large number of ascorbic acid granules and an increased alkaline and acid phosphatase activity (Figs. 3 and 4 ) . Compared with the findings in the normal ovaries this alkaline and acid phosphatase activity was significantly higher in this stage of development of the ovarian follicle. Glycogen was not demonstrated. P.A.S.-positive substances were encountered both in the cells of the theca and in the spaces between them. The ovum also showed a positive P.A.S. reaction. The accumulation of the P.A.S.-positive substances in the growing follicle showed no differences when comparison was made with the normal ovary. In contrast to the normal, however, ascorbic
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Fig. 4. Elevated acid phosphatase activity in the theca interna cells. (Gomori. x144.)
Fig. 5. Ascorbic acid granules in the granulosa cells. (Leblon and methylene blue. x270.)
acid granules were seen in the granulosa cells (Fig. 5). No neutral lipids or cholesterol was detected. Mature Graafian follicles. Mature Graafian follicles were very rarely present in our cases. Follicles in this stage revealed a well-developed granulosa, consisting of 5 to 7 layers. The membrana limitans externa was well defined. The granulosa cells had a positive glycogen reaction and ascorbic acid granules were found in the cytoplasm also. Liquor folliculi reacted positively for the glycoproteins. These P.A.S.-positive substances, however, gave a negative reaction to glycogen, ascorbic acid, neutral lipids, and cholesterol. Alkaline phosphatase activity was found in the intima of the capillaries and also in the theca interna cells as a peripheral rim. The findings in the mature Graafian follicle resembled closely those seen in the normal ovary.
Follicles in atresia. Multiple follicular cysts, which presented follicles in a different stage of cystic atresia, were found beneath the thickened tunica aibuginea. These atretic follicles contained all elements of the growing follicle, but in different stages of degeneration. The cysts varied in size from 0.2 to 0.4 mm. and were full of a light pink jelly giving a positive P.A.S. reaction (Fig. 6). This substance, however, gave a negative glycogen reaction, did not contain ascorbic acid granules, and possessed no alkaline and acid phosphatase activity. It did not contain any neutral lipids or cholesterol. Probably it contained glycoproteins. Compared with the atretic follicles of the normal ovary, greater degeneration was evident. The wall of the cystically degenerated follicles contained some remnants of granulosa. Its cells were atrophied with distinct dystrophic alterations. They were
Fig. 6. Intensive deposition of P.A.S.positive substances in the lumen of a cystic dilated atretic follicle. (P.A.S., hematoxylin. x144.)
186
Dokumov and Doshev
Am.
J.
May 15, 1963 Obst. & Gynec.
Fig. 10. Alkaline phosphatase activity in the theca interna cells and in a capillary wall. ( Gomori . x l44.) Fig. 7. Infiltration with P.A.S.-positive substances of the thickened wall of a capillary around a cystic dilated follicle. (P.A.S., hematoxylin. x l44. )
Fig. 8. Intensive P.A.S.-Positive reaction in the hypertrophied and hyperplastic theca interna cells. (P.A.S., hematoxylin. x144.)
Fig. 9. Positive glycogen reaction in the areas of the hypertrophied theca interna cells. (Best. xl44.)
flattened, had a vacuolated cytoplasm and pyknotic nuclei. The glycogen reaction was less evident in the cytoplasm and the ascorbic acid granules were scantier. No alkaline and acid phosphatase activity was demonstrable. P.A.S.-positive substances and cholesterol were absent. Only single cells had an accumulation of neutral lipids in their vacuolated cytoplasm. Multiple capillaries were seen around the cystic structures. Compared with the capillaries around the normal follicles their wall was more thickened and was strongly infiltrated by P.A.S.-positive substances (Fig. 7). In some areas, however, a diminution of these substances was noticed. This was accompanied by a hyalination and sclerosis of the capillary wall. Probably the alterations in the capillary wall (hyalination and sclerosis) were preceded by an intensive infiltration of P.A.S.-positive substances. The theca interna cells were distinctly hypertrophied and hyperplastic. They stained relatively well, especially the cytoplasm, which was vacuolated in some areas and displayed a clear basophilia. The nuclei were well stained. Occasionally mitoses were found. Some theca interna cells showed evidence of P.A.S.-positive substances (Fig. 8). A slight glycogen reaction and an increased alkaline and acid phosphatase activity were noticed in the cytoplasm of the cells (Figs. 9 and 10) . The ascorbic acid granules were well defined. Some cells, situated in well-circumscribed groups, had a positive reaction for cholesterol and neutral lipids (Fig. 11 I . This
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Stein-Leventhal syndrome 187
Fig. 11. Neutral lipids in the cytoplasm of some hypertrophied theca interna cells. (Sudan black. xl44.)
finding suggested that perhaps the hypertrophied and hyperplastic cells, situated around the cystic, atretic follicles, possessed an elevated vital activity or that they produced certain well-defined substances. The interstitium around the follicular structures was infiltrated by a dense connective tissue which in some areas had undergone hyalination and sclerosis. In other areas the connective tissue grew looser and showed P.A.S.positive substances. A particular enzymatic activity was not observed. Corpora lutea and corpora albicantia. These were not found in any of our cases. Comment
Although the scope and the range of the histochemical methods used in this study are limited and incomplete, some characteristics in the ovaries of women suffering from the Stein-Leventhal syndrome have been demonstrated. These were found both in the structures encountered in the normal ovary (primordial, primary, growing follicle, follicles undergoing the different degrees of atresia) and in the formations absent in a normal ovary (the hypertrophied and hyper-
REFERENCES I. McKay, D . G., Pinkerton, J. M., Hertig, A. T., and Danziger, S. : Obst. & Gynec. 18: 13, 1961. 2. Pinkerton, J. M., McKay, D. G., Adams, E. C., and Hertig, A. T.: Obst. & Gynec. 18: 152, 1961.
plastic theca interna) . Regardless of the kind of structures affected, however, the alterations were located mainly in the cells producing the steroid hormones. It is the histochemical characteristics of these cells that reveal the differences in cellular metabolism, when comparison is made with the same cells of a normal ovary. These histochemical findings are probably the sequence of a changed vital activity of the cell or of an abnormal production of definite substances, probably steroid hormones, their precursors or metabolic products. Conclusions 1. The ovaries of women suffering from the Stein-Leventhal syndrome disclose distinct histochemical differences from normal ovanes. 2. These histochemical differences are located both in the structures physiologically found in a normal ovary and in structures absent in a normal ovary. 3. The main histochemical differences are localized in the cells producing the steroid hormones.
Clinic of Endocrinology Postgraduate Medical Inst itute (ISUL) Belo Mo re str. 8 Sofia, Bulgaria