- Email: [email protected]
Histological and hormonal studies in rats bearing ovarian implants in the spleen. Effects of portacaval anastomosis
Europ. 3". Cancer Vol. 10, pp. 35-40. Pergamon Press 1974. Printed in Great Britain
Histological and Hormonal Studies in Rats Bearing Ovarian Implants in the Spleen.Effects of Portacaval Anastomosis SUSAN E. SEAGEK 1, A. R. BOYNS 2 and L. H. BLUMGART 3. 1 Tenovus Institute and Department of Surgery; 2 Tenovus Institute and Department of Medical Biochemistry; 3Department of Surgery, Welsh National School of Medicine, Heath, Cardiff, Wales Abstract--Ovaries were implanted into the spleens of ovariectomized rats. Plasma FSH levels increased rapidly after implantation and this was followed by an increase in plasma concentrations of LH. These raised levels were sustainedfor at least 140 days after operation. Plasma prolactin levels showed no change. Portacaval anastomosis prevented the increases in plasma hormone concentrations, blocked the growth of fresh implants and caused atrophy of established implants.
INTRODUCTION BISKIND and Biskind [1] first documented the changes which occur in ovarian implants into the spleen of ovariectomized rats. The implants were found to enlarge considerably and to undergo a sequence of histological changes culminating in the formation of masses of theca cells, "granulosa cell" tumours being found in some of the animals. Similar findings have been reported in rats and rabbits [2-4] in guinea pigs [5] and in mice [6, 7, 8]. T h e mechanism for such growth is believed one of over production of pituitary gonadotrophins following inactivation of ovarian oestrogens in the liver [1]. The availability of radioimmunoassays for rat pituitary hormones now allows precise measurements to be made of serial changes in plasma gonadotrophins following splenic implantation of ovarian tissue. It has also been suggested that failure of some implants to undergo hyperplasia and hypertrophy may be related to the development of adhesions between the spleen and parietal peritoneum giving rise to a portal-systemic collateral circulation [2, 8, 9].
This paper reports an investigation into the histological and hormonal changes following the implantation of ovarian tissue into the spleen of rats. The effect of portacaval anastomosis on the growth of ovarian implants was also examined.
MATERIALS AND METHODS Ten-week-old, female Sprague-Dawley rats were used. They were fed on rat diet 41B and water ad libitum. Operative technique Anaesthesia was induced with ether and, in most cases, continued with a single intraperitoneal injection of veterinary Nembutal (0.07 ml/100 g body weight). Ovariectomy and implantation were carried out through a ventral, mid-abdominal incision. The ovaries were removed after ligation of their vessels, adherent fat and the oviducts discarded and the left ovary halved. The spleen was gently mobilized and an incision made in the capsule of its gastric surface. The capsule was then elevated and one half of the left ovary placed within the "pocket". A flap ofomentum was sutured around implant and spleen to minimise the formation of adhesions between the spleen and parietal peritoneum.
Accepted 26 November 1973. *Present address: Department of Surgery, University of Glasgow, Glasgow, Scotland.
35
36
Susan E. Seager, A. R. Boyns and L. H. Blumgart
End to side portacaval shunting was carried out by a modification of the method of Bismuth et al. [10]. The portal vein was divided high at the hilum of the liver and anastomised (end to side) to the superior vena cava, using 7/0 silk mounted on a 12 m m atraumatic needle. The animals were killed at intervals after operation. A post-mortem examination was carried out and the presence of any adhesions between the spleen and parietal peritoneum noted.
Blood samples Blood was taken from the external jugular vein (with the rats under ether anaesthesia) or by decapitation of the conscious animal. Samples were placed in tubes containing EDTA. The plasma was then separated by centrifugation and stored at --20°C until assay.
Measurement of plasma hormones Immunoreactive prolactin, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured in plasma samples by double antibody radioimmunoassays using kits distributed by the National Institute for Arthritis and Metabolic Diseases (NIAMD), Bethesda, Md., U.S.A. The assay protocols were essentially similar to those used previously (I1).
Splenic implantation of ovarian tissue with simultaneous bilateral ovariectomy Pairs of rats from a group (Ia) of 75 animals were killed at intervals for up to 180 days after operation and tissue taken for histological study. Blood was taken under ether anaesthesia from a group (Ib) of animals before implantation and by decapitation 1 day, 7 days, 9 days, or 20 days after implantation. In another experiment, a group (Ic) of animals were bled under ether anaesthesia before implantation and the same animals bled repeatedly at intervals from 20 to 140 days after implantation.
Splenic implantation of ovarian tissue with delayed bilateral ovariectomy Ovarian implantation was performed in a group (II) of rats but the right ovary was left in situ. Twenty-five days later, the remaining ovary was removed. The animals were killed 25 days after total ovariectomy.
Portacaval shuntfollowed by splenic implantation of ovarian tissue and bilateral ovariectomy. A portacaval shunt was performed on a group (III) of 24 animals. Three to five days later, half of the left ovary was implanted into the spleen and the rat ovariectomized. Eighty per cent of the animals survived both operations. The animals were killed in pairs daily from I to 14 days and weekly from the 14 to the 77th day after implantation.
Splenic implantation of ovarian tissue with bilateral ovariectomy and a delayedportacaval shunt Ovarian implants were performed in a group (IV) of 10 rats. A portacaval shunt was then performed 15-25 weeks after implantation at which time measurements were made of the exterior length and width of the implant protruding from the spleen. Eighty per cent of the animals survived both procedures. The animals were killed 4 weeks after portacaval shunting and the size of the implants again measured. In four control rats, laparotomy alone was performed at 15-25 weeks and the size of the implant measured. Animals were killed 4 weeks later and the size of the implants again measured. RESULTS
Splenic implantation of ovarian tissue with simultaneous bilateral ovariectomy Histological examination of the implants revealed a characteristic series of changes commencing with a period of necrosis and inflammation in the first days after implantation and progressing to the formation of discrete corpora lutea (Fig. 1). Luteomata or disorganised luteal cells similar to those described by Biskind and Biskind [1] were not observed within the time limits of the experiment (180 days). Plasma levels of FSH rose rapidly after operation and remained elevated throughout the experimental period. Increases in plasma LH occurred more slowly but again the concentrations were sustained at a high level thereafter. Plasma prolactin levels tended to fall after operation but the changes were not statistically significant (Tables 1 and 2).
Splenic implantation of ovarian tissue with delayed bilateral ovariectomy Removal of the contralateral ovary 25 days after hemiovaxiectomy and ovarian implantation produced hyperplasia of the implants after
Fig. i. Splenic implantation of ovarian tissue and simultaneous bilateral ovariectomy (Group Ia). Massed corpora lutea in an implant 49 days after operation./4, & E. x i00
Fig. 2. Portacaval shunt followed by splenic implantation of ovarian tissue with bilateral ovariectomy (Group III). Diffuse luteal cells in an implant 14 days after implantation. H. & E . x I26. (to face p. 36)
Fig. 3. Splenic implantation of ovarian tissue with bilateral ovariectomy and a delayed portacaval shunt. Atrophic changes in an implant 4 weeks after portacavaI shunt. H. & E. x 120.
Histological and Hormonal Studies in Rats Bearing Ovarian Implants in the Spleen
37
Table 1. Concentrations of LH, FSH and prolactin in the plasma of rats, bilaterally ovariectomized and with implants of ovarian tissue in the spleen (Group Ib ) Days after implantation
Number of Rats
0
16
1
4
7
4
9
4
20
4
ng/rnl LH
FSH
89 (69-248) 86 (60-103) "153 (90-175) "155 (90-378) t488 (200-595)
PRL
258 (160-850) t 1570 (775-1420) t1830 (1420->2500) t2100 ( 1470-2130) t > 2500 (1385-> 2500)
11 (1-I07) 49 (18-53) 2 (1-9) 6 (1-72) 1 (I-1)
Blood samples taken under ether anaesthesia from 16 rats before operation and by decapitation of sets of 4 rats at I, 7, 9 or 20 days after operation. LH, luteinizing hormone, (NIAMD Rat LH-RP-1); FSH, follicle stimulating hormone (NIAMD Rat FSH-RP-1); PRL, prolactin (NIAMD Rat prolactin-RP-l). P(vs 0 days); *<0.05; t<0"002; (MannWhitney U test). Median and range are shown for each set of values.
Table 2. Concentrations of LH, FSH and prolaetin in the plasma of 5 rats, bilaterally ovarieetomized and with implants of ovarian tissue in the spleen (Group Ic) Days after implantation
Number of rats
0
5
20
5
35
5
49
5
140
5
ng/ml LH
FSH
PRL
70 (65-75) 7900 (685-1010) ?590
335 (285-510) t > 2500 (>2500->2500) * > 2500
33 (13-37) 19 (16-21) 21
(53o-6o5)
( > 2500- > 2500)
(10-45)
• 783 (745-845) t870 (260-1270)
*2500 ( > 2500- > 2500) > 2500 (210->2500)
24 (20-47) 30 (9-102)
Blood samples taken under ether anaesthesia before operation and at 20, 35, 49 and 140 days after operation. LH, luteinizing hormone (NIAMD Rat LH-RP-1) ; FSH, follicle stimulating hormone (NIAMD Rat FSH-RP-1); PRL prolactin (NIAMD-Rat prolactin-RP-1). P(vs 0 days); *<0.016; "~<0.008; (MannWhitney U test). Median and range are shown for each set of values.
a further 25 days. Plasma hormone levels in unilaterally castrated animals possessing ovarian implants in the spleen were similar to those seen in intact animals (Table 3). Removal of the remaining ovary produced a rapid increase in plasma FSH levels and a slower increase in L H levels. There was a slight fall in plasma concentrations of prolactin.
Portacaval shuntfollowed by splenic implantation of ovarian tissue and bilateral ovariectomy. In this group (III) it was noted that the implants rapidly atrophied and little more than a scar was visible on the surface of the spleen by the 77th day after implantation. Histological examination of the implants showed that an initial period of necrosis and
38
Susan E. Seager, A. R. Boyns and L. H. Blumgart Table 3. Effect of removal of remaining ovary on the concentrations of LH, FSH and prolactin in the plasma of unilaterally ovariectomized rats bearing implants of ovarian tissue in the spleen (Group II) Days after total ovariectomy
Number of rats
0
9
43 (15-120)
25
4
t438 (160-720)
ng/ml LH
FSH
PRL
390 (320->2500) t2290 (1690->2500)
7 (4-26) *4 (2-6)
Blood samples taken under ether anaesthesia immediately before and 25 days after total ovariectomy. LH, Luteinizing hormone (NIAMD Rat LH-RP-1); FSH, follicle stimulating hormone (NIAMD Rat FSH-RP-1); PRL, prolactin (NIAMD Rat prolactin-RP-1). P (vs 0 days); * < 0.05; ~ < 0.02; (Mann-Whitney U test). Median and range are shown for each set of values.
Table 4. Histological changes in ovarian implants in the spleen of bilaterally ovarieaomized rats. Effect of portaeaval anastomosis performed at time of ovarian implantation in the spleen Histological findings Age of implant at autopsy (days) 1-4
5-7
8-14 21-35
42-77
Bilateral ovariectomy and portacaval anastomosis
Bilateral ovarieetomy alone Necrosis in follicles and corpora lutea. Acute inflammatory reaction. Occasional surviving follicles. Some necrosis: inflammatory reaction resolving. Development of new follicles. Luteinization beginning. New follicles. Luteinization, some large corpora lutea becoming cystic, haemorrhagic or fibrotic. Few new follicles. Large corpora lutea with cystic, haemorrhagic or fibrotic reaction. Some compression of corpora lutea. Large corpora lutea undergoing cystic haemorrhagic or fibrotic reaction.
Necrosis: inflammation.
Necrosis: inflammation resolving: new follicles. Necrosis: inflammation. Some isolated patches of luteal cells. Necrosis: follicles: patches of luteal cells. Follicles.
Table 5. Effect of portacaval shunt (or laparotomy alone) on the size of ovarian implants in the spleen of bilaterally ovariectomized animals (Group IV)
Operation
Number of rats
P-C shunt
I0
Laparotomy (Controls)
4
Weeks after implant 20 (15-25) 18
(15-18)
Width (mm)
Length (mm) Before
After
6.0 (3.7-10.0)
3.5 (1.9-6.3)
4.6
(3.4-5.5)
5.7
(4.5-5.9)
Before 5.0 (1.9-10.0)
After 2.0 (1"2-4-5)
3.5
4.0
(0-5-4.0)
(2.0-4.4)
Animals killed 4 weeks after operation. P (shunt vs controls). < 0.02 (before/after for length) ; P (shunt vs controls). < 0.002 (before/after for width). (Mann-Whitney U test with correction for varying ages of implants). Median and range are shown for each set of values.
Histological and Hormonal Studies in Rats Bearing Ovarian Implants in the Spleen associated inflammatory reaction occurred, similar to that seen in Group I, but the inflammatory change appeared to last somewhat longer (Table 4). Although follicular development was evident, the follicles did not enlarge nor did they progress to luteal formation. In some animals, isolated groups of luteal cells were noted within the stroma from the 14th day onwards (Fig. 2). There was no further development within the implant up to the 77th post-operative day. Single blood samples taken from individual animals at 2-13 days after shunt and implant revealed no increase in plasma level of LH and FSH.
Splenic implantation of ovarian tissue with bilateral ovariectomy and a delayedportacaval shunt No adhesions were observed between the spleen and parietal peritoneum in any of the animals at the time of laparotomy, portacaval shunting or at autopsy. The dimensions of the splenic implants (measured at autopsy) were significantly smaller than those of the same implants measured at the time of portacaval anastomosis (Table 5). Histologically, the implants from those animals which had undergone portacaval shunts were found to be greatly different from animals in Group I. In general they consisted of patches of ovarian stroma and the remaining follicles were found to be atrophic and cystic. The corpora lutea were few in number, small and centrally scarred (Fig. 3). In one animal, luteal cells appeared to be diffused throughout the ovarian stroma. Control animals which had undergone laparotomy (without portacaval shunting) bore implants which had a histological appearance similar to that observed in Group I. DISCUSSION In our experiments, ovarian implants in the spleen underwent a series of histological changes culminating in follicle formation and intense luteinization. Similar observations were made by Biskind and Biskind [1] although they
observed the development of luteomata in implants after 150 days. Biological assays have demonstrated an increase in the content of gonadotrophins in the pituitary and blood after ovarian implantation in the spleen [12-14]. In addition, earlier work also provided evidence that FSH levels in the blood rise more rapidly than those of L H [15]. Our results confirm these reports but we were unable to substantiate the suggestion that raised prolacfin levels may also contribute to the luteinization that occurs [ 16]. Ovarian implants did not grow in unilaterally oveHectomized animals until the remaining ovary was removed. This finding was related to an increase in plasma levels of L H and FSH. O n the other hand splenic implants failed to grow in animals with a portacaval shunt and there was no change in plasma levels of L H and FSH. Therefore there is an excellent correlation between growth of an implant and plasma concentrations of FSH and LH. It is of interest in this respect that injections of pregnant mare serum gonadotrophin (which possesses L H and FSH activity) accelerate the growth of ovarian implants whereas the administration of ovine prolactin has no effect [9]. T u m o u r development was not observed in our studies and may depend on increases in prolactin secretion. Injection of ovine prolactin does not stimulate luteoma formation in rats [9], but this procedure may induce the formation of antibodies to prolactin which would block its biological action. It remains to be determined whether induction of raised plasma concentrations will enhance the formation of tumours in implanted ovaries in an analogous manner to that of m a m m a r y tumours induced by polycyclic hydrocarbons. Acknowledgements--We thank Dr. J. G. Leopold for help in the histological interpretation and Mr. R. G. Newcombe for statistical advice. We are very grateful to the National Institute for Arthritis and Metabolic Diseases (USA) for providing the radioimmunoassay kits, to Professor K. Griffiths for his interest and to the Tenovus Organisation for generous financial assistance. This work was carried out with aid of a Grant from the Welsh Regional Hospital Board to L. H. B.
REFERENCES 1. 2. 3.
39
M . S . BISKINDand G. R. BISKIND. Development of tumours in the rat ovary after transplantation into the spleen. Proc. Soc. exp. Biol. (N. Y.) 55, 176 (1944). B.M. PECKHAM,R. R. GREENEand M. E. JEFFRIES. Granulosa cell tumours in female rats and rabbits. Science107~ 319 (1948). B.M. PECKHAMand R. R. GREENE. Experimentally produced granulosa cell tumours in rats. CancerRes. 12~ 25 (1952).
40
Susan E. Seager, A. R. Boyns and L. H. Blumgart 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
B . M . PECKHAMand R. R. GREENE. Experimentally produced granulosa cell tumours in rabbits. CancerRes. 129 654 (1952). A. LXPSCHUTZ,H. PONC~ DE LEON, E. WOYWOODand O. GAY. Intrasplenic ovarian graft in the guinea pig and the problem of neoplastic reactions of the graft. Rev. Canad. Biol. 5, 181 (1946). M . H . LI and W. U. GARDNER.Tumours in intrasplertic ovarian transplants in castrated mice. Science 1059 13 (1947). M . H . L1 and W. U. GARDNER.Further studies on the pathogenesis of ovarian tumours in mice. Cancer Res. 99 35 (1949). W . U . GARDNER.Development and growth of tumours in ovaries transplanted into the spleen. Cancer Res. 159 109 (1955). G . R . BISKIND, D. E. BERNST~IN and S. M. GosPE. The effect of exogenous gonadotrophins on the development of experimental ovarian turnouts in rats. Cancer Res. 139 216 (1953). H. BISMUTH,J. P. BENHAMOUand J. LASTASTE. Technique of portacaval anastomoses in the rat. Presse med. 719 1859 (1963). V. DA~JT~, M. E. HARPER, A. R. BO'ZNs, E. N. COLE, B. G. BROWNSEYand K. GRIFFITHS.The effect of certain stilboestrol analogues on plasma prolactin and testosterone in the rat. J. Endocr. 579 207 (1973). E . C . JuNGcK, C. G. HELI.ER and W. O. NELSON. Regulation of pituitary gonadotrophic secretion. Inhibition by oestrogen or inactivation by the ovaries. Proc. Soc. exp. Biol (N.Y.) 65, 148 (1947). O . J . MILL~.Rand C. A. PF~.IFFER. Demonstration of increased gonadotrophic hormone production in castrated mice with intrasplenic ovarian grafts. Proc. Soc. exp. Biol (N.Y.) 759 178 (1950). W . E . ACHIT.LESand S. H. STURGIS. The effect of the intrasplenic ovarian graft on pituitary gonadotrophins. Endocrinology 49, 720 (1951). C. BIDDUr.PH and R. K. MEYER. Gonadotrophic hormone secretion in immature hypophysectomised parabiotic rats. Proc. Soc. exp. Biol. (N.Y.) 63, 42 (1946). E. B. ASTWOOD. Regulation of corpus luteum function by hypophyseal luteotrophin. Endocrinology 289 309 (1941).
Histological and Hormonal Studies in Rats Bearing Ovarian Implants in the Spleen.Effects of Portacaval Anastomosis SUSAN E. SEAGEK 1, A. R. BOYNS 2 and L. H. BLUMGART 3. 1 Tenovus Institute and Department of Surgery; 2 Tenovus Institute and Department of Medical Biochemistry; 3Department of Surgery, Welsh National School of Medicine, Heath, Cardiff, Wales Abstract--Ovaries were implanted into the spleens of ovariectomized rats. Plasma FSH levels increased rapidly after implantation and this was followed by an increase in plasma concentrations of LH. These raised levels were sustainedfor at least 140 days after operation. Plasma prolactin levels showed no change. Portacaval anastomosis prevented the increases in plasma hormone concentrations, blocked the growth of fresh implants and caused atrophy of established implants.
INTRODUCTION BISKIND and Biskind [1] first documented the changes which occur in ovarian implants into the spleen of ovariectomized rats. The implants were found to enlarge considerably and to undergo a sequence of histological changes culminating in the formation of masses of theca cells, "granulosa cell" tumours being found in some of the animals. Similar findings have been reported in rats and rabbits [2-4] in guinea pigs [5] and in mice [6, 7, 8]. T h e mechanism for such growth is believed one of over production of pituitary gonadotrophins following inactivation of ovarian oestrogens in the liver [1]. The availability of radioimmunoassays for rat pituitary hormones now allows precise measurements to be made of serial changes in plasma gonadotrophins following splenic implantation of ovarian tissue. It has also been suggested that failure of some implants to undergo hyperplasia and hypertrophy may be related to the development of adhesions between the spleen and parietal peritoneum giving rise to a portal-systemic collateral circulation [2, 8, 9].
This paper reports an investigation into the histological and hormonal changes following the implantation of ovarian tissue into the spleen of rats. The effect of portacaval anastomosis on the growth of ovarian implants was also examined.
MATERIALS AND METHODS Ten-week-old, female Sprague-Dawley rats were used. They were fed on rat diet 41B and water ad libitum. Operative technique Anaesthesia was induced with ether and, in most cases, continued with a single intraperitoneal injection of veterinary Nembutal (0.07 ml/100 g body weight). Ovariectomy and implantation were carried out through a ventral, mid-abdominal incision. The ovaries were removed after ligation of their vessels, adherent fat and the oviducts discarded and the left ovary halved. The spleen was gently mobilized and an incision made in the capsule of its gastric surface. The capsule was then elevated and one half of the left ovary placed within the "pocket". A flap ofomentum was sutured around implant and spleen to minimise the formation of adhesions between the spleen and parietal peritoneum.
Accepted 26 November 1973. *Present address: Department of Surgery, University of Glasgow, Glasgow, Scotland.
35
36
Susan E. Seager, A. R. Boyns and L. H. Blumgart
End to side portacaval shunting was carried out by a modification of the method of Bismuth et al. [10]. The portal vein was divided high at the hilum of the liver and anastomised (end to side) to the superior vena cava, using 7/0 silk mounted on a 12 m m atraumatic needle. The animals were killed at intervals after operation. A post-mortem examination was carried out and the presence of any adhesions between the spleen and parietal peritoneum noted.
Blood samples Blood was taken from the external jugular vein (with the rats under ether anaesthesia) or by decapitation of the conscious animal. Samples were placed in tubes containing EDTA. The plasma was then separated by centrifugation and stored at --20°C until assay.
Measurement of plasma hormones Immunoreactive prolactin, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured in plasma samples by double antibody radioimmunoassays using kits distributed by the National Institute for Arthritis and Metabolic Diseases (NIAMD), Bethesda, Md., U.S.A. The assay protocols were essentially similar to those used previously (I1).
Splenic implantation of ovarian tissue with simultaneous bilateral ovariectomy Pairs of rats from a group (Ia) of 75 animals were killed at intervals for up to 180 days after operation and tissue taken for histological study. Blood was taken under ether anaesthesia from a group (Ib) of animals before implantation and by decapitation 1 day, 7 days, 9 days, or 20 days after implantation. In another experiment, a group (Ic) of animals were bled under ether anaesthesia before implantation and the same animals bled repeatedly at intervals from 20 to 140 days after implantation.
Splenic implantation of ovarian tissue with delayed bilateral ovariectomy Ovarian implantation was performed in a group (II) of rats but the right ovary was left in situ. Twenty-five days later, the remaining ovary was removed. The animals were killed 25 days after total ovariectomy.
Portacaval shuntfollowed by splenic implantation of ovarian tissue and bilateral ovariectomy. A portacaval shunt was performed on a group (III) of 24 animals. Three to five days later, half of the left ovary was implanted into the spleen and the rat ovariectomized. Eighty per cent of the animals survived both operations. The animals were killed in pairs daily from I to 14 days and weekly from the 14 to the 77th day after implantation.
Splenic implantation of ovarian tissue with bilateral ovariectomy and a delayedportacaval shunt Ovarian implants were performed in a group (IV) of 10 rats. A portacaval shunt was then performed 15-25 weeks after implantation at which time measurements were made of the exterior length and width of the implant protruding from the spleen. Eighty per cent of the animals survived both procedures. The animals were killed 4 weeks after portacaval shunting and the size of the implants again measured. In four control rats, laparotomy alone was performed at 15-25 weeks and the size of the implant measured. Animals were killed 4 weeks later and the size of the implants again measured. RESULTS
Splenic implantation of ovarian tissue with simultaneous bilateral ovariectomy Histological examination of the implants revealed a characteristic series of changes commencing with a period of necrosis and inflammation in the first days after implantation and progressing to the formation of discrete corpora lutea (Fig. 1). Luteomata or disorganised luteal cells similar to those described by Biskind and Biskind [1] were not observed within the time limits of the experiment (180 days). Plasma levels of FSH rose rapidly after operation and remained elevated throughout the experimental period. Increases in plasma LH occurred more slowly but again the concentrations were sustained at a high level thereafter. Plasma prolactin levels tended to fall after operation but the changes were not statistically significant (Tables 1 and 2).
Splenic implantation of ovarian tissue with delayed bilateral ovariectomy Removal of the contralateral ovary 25 days after hemiovaxiectomy and ovarian implantation produced hyperplasia of the implants after
Fig. i. Splenic implantation of ovarian tissue and simultaneous bilateral ovariectomy (Group Ia). Massed corpora lutea in an implant 49 days after operation./4, & E. x i00
Fig. 2. Portacaval shunt followed by splenic implantation of ovarian tissue with bilateral ovariectomy (Group III). Diffuse luteal cells in an implant 14 days after implantation. H. & E . x I26. (to face p. 36)
Fig. 3. Splenic implantation of ovarian tissue with bilateral ovariectomy and a delayed portacaval shunt. Atrophic changes in an implant 4 weeks after portacavaI shunt. H. & E. x 120.
Histological and Hormonal Studies in Rats Bearing Ovarian Implants in the Spleen
37
Table 1. Concentrations of LH, FSH and prolactin in the plasma of rats, bilaterally ovariectomized and with implants of ovarian tissue in the spleen (Group Ib ) Days after implantation
Number of Rats
0
16
1
4
7
4
9
4
20
4
ng/rnl LH
FSH
89 (69-248) 86 (60-103) "153 (90-175) "155 (90-378) t488 (200-595)
PRL
258 (160-850) t 1570 (775-1420) t1830 (1420->2500) t2100 ( 1470-2130) t > 2500 (1385-> 2500)
11 (1-I07) 49 (18-53) 2 (1-9) 6 (1-72) 1 (I-1)
Blood samples taken under ether anaesthesia from 16 rats before operation and by decapitation of sets of 4 rats at I, 7, 9 or 20 days after operation. LH, luteinizing hormone, (NIAMD Rat LH-RP-1); FSH, follicle stimulating hormone (NIAMD Rat FSH-RP-1); PRL, prolactin (NIAMD Rat prolactin-RP-l). P(vs 0 days); *<0.05; t<0"002; (MannWhitney U test). Median and range are shown for each set of values.
Table 2. Concentrations of LH, FSH and prolaetin in the plasma of 5 rats, bilaterally ovarieetomized and with implants of ovarian tissue in the spleen (Group Ic) Days after implantation
Number of rats
0
5
20
5
35
5
49
5
140
5
ng/ml LH
FSH
PRL
70 (65-75) 7900 (685-1010) ?590
335 (285-510) t > 2500 (>2500->2500) * > 2500
33 (13-37) 19 (16-21) 21
(53o-6o5)
( > 2500- > 2500)
(10-45)
• 783 (745-845) t870 (260-1270)
*2500 ( > 2500- > 2500) > 2500 (210->2500)
24 (20-47) 30 (9-102)
Blood samples taken under ether anaesthesia before operation and at 20, 35, 49 and 140 days after operation. LH, luteinizing hormone (NIAMD Rat LH-RP-1) ; FSH, follicle stimulating hormone (NIAMD Rat FSH-RP-1); PRL prolactin (NIAMD-Rat prolactin-RP-1). P(vs 0 days); *<0.016; "~<0.008; (MannWhitney U test). Median and range are shown for each set of values.
a further 25 days. Plasma hormone levels in unilaterally castrated animals possessing ovarian implants in the spleen were similar to those seen in intact animals (Table 3). Removal of the remaining ovary produced a rapid increase in plasma FSH levels and a slower increase in L H levels. There was a slight fall in plasma concentrations of prolactin.
Portacaval shuntfollowed by splenic implantation of ovarian tissue and bilateral ovariectomy. In this group (III) it was noted that the implants rapidly atrophied and little more than a scar was visible on the surface of the spleen by the 77th day after implantation. Histological examination of the implants showed that an initial period of necrosis and
38
Susan E. Seager, A. R. Boyns and L. H. Blumgart Table 3. Effect of removal of remaining ovary on the concentrations of LH, FSH and prolactin in the plasma of unilaterally ovariectomized rats bearing implants of ovarian tissue in the spleen (Group II) Days after total ovariectomy
Number of rats
0
9
43 (15-120)
25
4
t438 (160-720)
ng/ml LH
FSH
PRL
390 (320->2500) t2290 (1690->2500)
7 (4-26) *4 (2-6)
Blood samples taken under ether anaesthesia immediately before and 25 days after total ovariectomy. LH, Luteinizing hormone (NIAMD Rat LH-RP-1); FSH, follicle stimulating hormone (NIAMD Rat FSH-RP-1); PRL, prolactin (NIAMD Rat prolactin-RP-1). P (vs 0 days); * < 0.05; ~ < 0.02; (Mann-Whitney U test). Median and range are shown for each set of values.
Table 4. Histological changes in ovarian implants in the spleen of bilaterally ovarieaomized rats. Effect of portaeaval anastomosis performed at time of ovarian implantation in the spleen Histological findings Age of implant at autopsy (days) 1-4
5-7
8-14 21-35
42-77
Bilateral ovariectomy and portacaval anastomosis
Bilateral ovarieetomy alone Necrosis in follicles and corpora lutea. Acute inflammatory reaction. Occasional surviving follicles. Some necrosis: inflammatory reaction resolving. Development of new follicles. Luteinization beginning. New follicles. Luteinization, some large corpora lutea becoming cystic, haemorrhagic or fibrotic. Few new follicles. Large corpora lutea with cystic, haemorrhagic or fibrotic reaction. Some compression of corpora lutea. Large corpora lutea undergoing cystic haemorrhagic or fibrotic reaction.
Necrosis: inflammation.
Necrosis: inflammation resolving: new follicles. Necrosis: inflammation. Some isolated patches of luteal cells. Necrosis: follicles: patches of luteal cells. Follicles.
Table 5. Effect of portacaval shunt (or laparotomy alone) on the size of ovarian implants in the spleen of bilaterally ovariectomized animals (Group IV)
Operation
Number of rats
P-C shunt
I0
Laparotomy (Controls)
4
Weeks after implant 20 (15-25) 18
(15-18)
Width (mm)
Length (mm) Before
After
6.0 (3.7-10.0)
3.5 (1.9-6.3)
4.6
(3.4-5.5)
5.7
(4.5-5.9)
Before 5.0 (1.9-10.0)
After 2.0 (1"2-4-5)
3.5
4.0
(0-5-4.0)
(2.0-4.4)
Animals killed 4 weeks after operation. P (shunt vs controls). < 0.02 (before/after for length) ; P (shunt vs controls). < 0.002 (before/after for width). (Mann-Whitney U test with correction for varying ages of implants). Median and range are shown for each set of values.
Histological and Hormonal Studies in Rats Bearing Ovarian Implants in the Spleen associated inflammatory reaction occurred, similar to that seen in Group I, but the inflammatory change appeared to last somewhat longer (Table 4). Although follicular development was evident, the follicles did not enlarge nor did they progress to luteal formation. In some animals, isolated groups of luteal cells were noted within the stroma from the 14th day onwards (Fig. 2). There was no further development within the implant up to the 77th post-operative day. Single blood samples taken from individual animals at 2-13 days after shunt and implant revealed no increase in plasma level of LH and FSH.
Splenic implantation of ovarian tissue with bilateral ovariectomy and a delayedportacaval shunt No adhesions were observed between the spleen and parietal peritoneum in any of the animals at the time of laparotomy, portacaval shunting or at autopsy. The dimensions of the splenic implants (measured at autopsy) were significantly smaller than those of the same implants measured at the time of portacaval anastomosis (Table 5). Histologically, the implants from those animals which had undergone portacaval shunts were found to be greatly different from animals in Group I. In general they consisted of patches of ovarian stroma and the remaining follicles were found to be atrophic and cystic. The corpora lutea were few in number, small and centrally scarred (Fig. 3). In one animal, luteal cells appeared to be diffused throughout the ovarian stroma. Control animals which had undergone laparotomy (without portacaval shunting) bore implants which had a histological appearance similar to that observed in Group I. DISCUSSION In our experiments, ovarian implants in the spleen underwent a series of histological changes culminating in follicle formation and intense luteinization. Similar observations were made by Biskind and Biskind [1] although they
observed the development of luteomata in implants after 150 days. Biological assays have demonstrated an increase in the content of gonadotrophins in the pituitary and blood after ovarian implantation in the spleen [12-14]. In addition, earlier work also provided evidence that FSH levels in the blood rise more rapidly than those of L H [15]. Our results confirm these reports but we were unable to substantiate the suggestion that raised prolacfin levels may also contribute to the luteinization that occurs [ 16]. Ovarian implants did not grow in unilaterally oveHectomized animals until the remaining ovary was removed. This finding was related to an increase in plasma levels of L H and FSH. O n the other hand splenic implants failed to grow in animals with a portacaval shunt and there was no change in plasma levels of L H and FSH. Therefore there is an excellent correlation between growth of an implant and plasma concentrations of FSH and LH. It is of interest in this respect that injections of pregnant mare serum gonadotrophin (which possesses L H and FSH activity) accelerate the growth of ovarian implants whereas the administration of ovine prolactin has no effect [9]. T u m o u r development was not observed in our studies and may depend on increases in prolactin secretion. Injection of ovine prolactin does not stimulate luteoma formation in rats [9], but this procedure may induce the formation of antibodies to prolactin which would block its biological action. It remains to be determined whether induction of raised plasma concentrations will enhance the formation of tumours in implanted ovaries in an analogous manner to that of m a m m a r y tumours induced by polycyclic hydrocarbons. Acknowledgements--We thank Dr. J. G. Leopold for help in the histological interpretation and Mr. R. G. Newcombe for statistical advice. We are very grateful to the National Institute for Arthritis and Metabolic Diseases (USA) for providing the radioimmunoassay kits, to Professor K. Griffiths for his interest and to the Tenovus Organisation for generous financial assistance. This work was carried out with aid of a Grant from the Welsh Regional Hospital Board to L. H. B.
REFERENCES 1. 2. 3.
39
M . S . BISKINDand G. R. BISKIND. Development of tumours in the rat ovary after transplantation into the spleen. Proc. Soc. exp. Biol. (N. Y.) 55, 176 (1944). B.M. PECKHAM,R. R. GREENEand M. E. JEFFRIES. Granulosa cell tumours in female rats and rabbits. Science107~ 319 (1948). B.M. PECKHAMand R. R. GREENE. Experimentally produced granulosa cell tumours in rats. CancerRes. 12~ 25 (1952).
40
Susan E. Seager, A. R. Boyns and L. H. Blumgart 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
B . M . PECKHAMand R. R. GREENE. Experimentally produced granulosa cell tumours in rabbits. CancerRes. 129 654 (1952). A. LXPSCHUTZ,H. PONC~ DE LEON, E. WOYWOODand O. GAY. Intrasplenic ovarian graft in the guinea pig and the problem of neoplastic reactions of the graft. Rev. Canad. Biol. 5, 181 (1946). M . H . LI and W. U. GARDNER.Tumours in intrasplertic ovarian transplants in castrated mice. Science 1059 13 (1947). M . H . L1 and W. U. GARDNER.Further studies on the pathogenesis of ovarian tumours in mice. Cancer Res. 99 35 (1949). W . U . GARDNER.Development and growth of tumours in ovaries transplanted into the spleen. Cancer Res. 159 109 (1955). G . R . BISKIND, D. E. BERNST~IN and S. M. GosPE. The effect of exogenous gonadotrophins on the development of experimental ovarian turnouts in rats. Cancer Res. 139 216 (1953). H. BISMUTH,J. P. BENHAMOUand J. LASTASTE. Technique of portacaval anastomoses in the rat. Presse med. 719 1859 (1963). V. DA~JT~, M. E. HARPER, A. R. BO'ZNs, E. N. COLE, B. G. BROWNSEYand K. GRIFFITHS.The effect of certain stilboestrol analogues on plasma prolactin and testosterone in the rat. J. Endocr. 579 207 (1973). E . C . JuNGcK, C. G. HELI.ER and W. O. NELSON. Regulation of pituitary gonadotrophic secretion. Inhibition by oestrogen or inactivation by the ovaries. Proc. Soc. exp. Biol (N.Y.) 65, 148 (1947). O . J . MILL~.Rand C. A. PF~.IFFER. Demonstration of increased gonadotrophic hormone production in castrated mice with intrasplenic ovarian grafts. Proc. Soc. exp. Biol (N.Y.) 759 178 (1950). W . E . ACHIT.LESand S. H. STURGIS. The effect of the intrasplenic ovarian graft on pituitary gonadotrophins. Endocrinology 49, 720 (1951). C. BIDDUr.PH and R. K. MEYER. Gonadotrophic hormone secretion in immature hypophysectomised parabiotic rats. Proc. Soc. exp. Biol. (N.Y.) 63, 42 (1946). E. B. ASTWOOD. Regulation of corpus luteum function by hypophyseal luteotrophin. Endocrinology 289 309 (1941).