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Compensatory ovarian hypertrophy occurs by a mechanism distinct from compensatory growth in the regenerating liver
Compensatory ovarian hypertrophy occurs by a mechanism distinct from compensatory growth in the regenerating liver Ronald D. Alvarez, MD, William E. Grizzle, MD, PhD, Lori J. Smith, and Donald M. Miller, MD, PhD Birmingham, Alabama The mechanism by which compensatory ovarian growth occurs is complex and not completely understood. To compare the molecular events in compensatory ovarian growth with those known to occur in other compensatory growth processes such as the regenerating liver, the temporal pattern of proto-oncogene expression and dexoyribonucleic acid synthesis was investigated in rat ovarian tissue after unilateral castration. One hundred fifty female rats were subjected to either a left hemioophorectomy or a sham oophorectomy. Twenty-four rats from each group were put to death at 3 and 14 days after the initial procedure and the ovaries were weighed. There was a mean compensatory weight Increase in the right ovaries of the hemioophorectomy group of 7.9% at 3 days and 22.5% at 14 days. The temporal pattern of proto-oncogene expression was determined by removing the right ovary from SIX rats in each group at 4, 8, 12, 24, 36, and 48 hours after the initial procedure. The ovaries were paired into three samples in each group for each time point and the ribonucleic acid was extracted. Dot blot hybridization was performed on each ribonucleic acid sample with radiolabeled complementary dexoyribonucleic acid probes for the proto-oncogenes c-myc, c-HA-ras, and c-fos. There was no significant increase In proto-oncogene expression in the right ovaries of the hemioophorectomy group when compared with the right ovaries of the sham oophorectomy group. The temporal pattern of dexoyribonucleic aCid synthesis was determined by removing the right ovary from three rats in each group at 8, 12, 24, 36, and 48 hours after the initial procedure. Each rat had been injected intra peritoneally with ["HI thymidine 2 hours before the right oophorectomy. The specific activity of dexoyribonucleic acid extracted from each ovarian sample did not demonstrate a significant increase in ovarian dexoyribonucleic acid synthesis after hemioophorectomy or any significant difference in dexoyribonucleic acid synthesis between the hemioophorectomy and the sham oophorectomy groups. This report concludes that compensatory ovarian growth occurs by a mechanism distinct from compensatory growth in the regenerating liver. (AM J OBSTET GVNECOL 1989;161 :1653-7.)
Key words: Compensatory ovarian growth Compensatory ovarIan growth has been demonstrated to occur in the rat and other animal models after unilateral castration. I. " It has been hypothesized by some investigators that compensatory ovarian growth occurs as a result of an increase in gonadotropin secretion stimulated by declining levels of ovarian steroids or by declining levels of an "inhibin-like" factor.3•7 Others have postulated a functional role of the peripheral autonomic nervous system in compensatory ovarian growth."-IO In other compensatory growth processes, cellular proliferation is preceded by an increase in protooncogene expression. For example, increased expression of the proto-oncogenes c-myc, c-Ha-ras, and c-fos From the Departments of ObstetriCs and GYlecology, Pathology, and Inlernal Medlcme, The Ulllvemty of Alabama at Bmnmgham. Presented at the Thlrt.~-s!xth Annual M eetmg of the SOCiety for Gynecologic InvestlgallOl/, San Diego. Callfonlla. March 15-18, 1989. Reprmt requests: Ronald D. Alva rez, MD, OHB 550, Umvemty StatlOll. UnIVersity of Alabama at Bmnmglwl/I, Bmnl1lghal/1, AL 3529-1
616116507
precedes an increase in dexoyribonucleic acid (DNA) synthesis and cell proliferation in the regenerating liver after partial hepatectomy."-16 No study to date has reported the molecular events in compensatory ovarian growth or compared these events with the molecular events in regenerating liver. It is the purpose of this study to do so by investigating the temporal pattern of proto-oncogene expression and DNA synthesis in rat ovarian tissue after unilateral castration.
Material and methods Animals. Fischer rats (weight range 106 to 177 gm) were housed under controlled conditions of temperature, humidity, and light (lights on 12 hours and lights off 12 hours). All animals were fed water and standard rat chow as desired. Institutional guidelines for the care and experimental use of animals were strictly adhered to. Determination of compensatory growth. Twelve female rats were subjected to a le ft hemioophorectomv and 12 female rats were subjected to a sham oophorectomy both through a ventral midline abdominal in-
1653
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Fig. 1. Mean fold increases in proto-oncogene expres,lOn III the hemioophorectomy (ULO) and sham oophorectomy (SHAM) groups. The right ovaries were excised from each group at various intervals between 4 and 48 hours after the initial procedure. RNA extracted from ovarian samples was analyzed by dot hybridization to radiolabeled cDNA probes for the proto-oncogenes c-myr, c-HA-ras, and cjus.
cis ion with the animals under ketamine anesthesia. Encapsulating fibroadipose tissue was trimmed from the excised ovaries and the ovaries were weighed individually. At 3 and 14 days after the initial procedure, the remaining ovaries were excised from six rats in each group, trimmed of fibroadipose tissue, and individually weighed .. The amount of compensatory ovarian growth was calculated by the formula: Compensatory growth (%)
R - R " , x 100
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where Ro is the mean weight of the right ovaries in the hemioophorectomy group and R, is the mean weight of the right ovaries in the sham oophorectomy group. The experiment was repeated in 24 additional female rats, and the mean compensatory ovarian growth was determined from the results of the two experiments. Determination of proto-oncogene expression.
Thirty-six female rats were subjected to a left hemioophorectomy and 36 were subjected to a sham oophorectomy. At 4, 8, 12, 24, 36, and 48 hours after the initial surgery, six rats from each group were put to death. The right ovaries were excised and paired into three samples in both study groups for each time point and immediately frozen in liquid nitrogen. Total cellular ribonucleic acid (RNA) was isolated from each sample by the method described by Chomczynski and Sacchi. '7 RNA was extracted from pulverized frozen tissue homogenized in a guanidium thiocyanate solution. After phenol-chloroform extraction the RNA was precipitated with isopropanol and quantitated by absorption spectrophotometry. RNA samples from each time point in both the hemioophorectomy group and the sham oophorectomy group were analyzed by dot hybridization. IX Each RNA sample was assayed by hybridization to complementary DNA (cDNA) probes for the c-rnye, c-Ha-ra~, and c-fo.1 protooncogenes. The cDNA insert for each probe was purified by preparative gel electrophoresis and radiolabeled by nick translation. '" Autoradiograms of each filter were analyzed by laser densitomeu'y, and the mean increase in proto-oncogene expression was plotted for both the hemioophorectomy and the sham oophorectomy group. Determination of DNA synthesis. Fifteen female rats were subjected to a left oophorectomy and 15 underwent a sham oophorectomy. At 6, 10, 22, 34, and 46 hours after the initial procedure, three rats from each group were injected intra peritoneally with 200 mCi of ['H) thymidine diluted in 1 ml of saline solution. Two hours later the right ovaries were excised from each rat and immediately frozen in liquid nitrogen. Cellular DNA was isolated from each frozen ovary specimen by proteinase K digestion and phenol extraction. 20 The rate of DNA synthesis in the hemioophorectomy group and in the sham oophorectomy group was determined by measuring the specific activity of each DNA sample and by calculating the mean specific activity for each time point.
Results The mean compensatory weight increase in the right ovaries of the rats who had undergone left hemioophorectomy was 7.9% at 3 days and 22.5% at 14 days. This degree of compensatory ovarian growth is in agreement with that seen by previous investigators.1. 2. >.7 This compensatory growth is less dramatic than that seen in the regenerating liver, which has a 300% to 500% increase in mass within 7 to 14 days after partial hepatectomy. II As demonstrated in Fig. 1, there is no significant difference in the level of expression of the protooncogenes c-rnyr, c-HA-ras, and c-Ios between the right
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ovanes ot the hemioophorectomized rats and the right ovaries of the sham oophorectomized group. This is in clear contradistinction to the observations in regenerating liver. Previous studies conducted in our laboratory have demonstrated a sevenfold to eightfold increase in C-ntyc expression within 3 hours after partial hepatectomy (Campbell VW, Rousell J, Rigsby D, et al. Selective inhibition of c-myc expression by mithramycin prevents hepatocyte proliferation in regenerating livet'. Unpublished data). Othet's have t'eported as much as a fifteenfold increase in c-myc expression during the same time period"e-" The c-myc expression returns to baseline, only to rise again to a less pronounced level of expression between 8 and 24 hours after partial hepatectomy (Campbell VW, Rousell J, Rig~by D, et al. Selective inhibition of c-myc expression by mithramycin prevents hepatocyte proliferation in regenerating liver. Unpublished data). The c-Ha-ras expression has been demonstrated by our previous studies' and by othet's'" I', 'h to inuease 200% to :~()O% within 48 hours after partial hepatectomy. Fig. 2 graphically compares our observations of the pattern of c-1IIyc and c-Ha-rw expression in compensatory ovarian growth and in regenerating liver. In similar studies, c10s levels have been shown to be at least fourfold above normal 30 minutes after partial hepatectomy, only to decline by 2 hours. U A second twofold increase in c-jo.1 expression occurs at 8 hours after partial hepatectomy. As sholl'n 111 FIg. :{, there is no significant II1crease in ovarian DNA synthesis after hemioophorectomy. The level of DNA synthesis in the hemioophorectomy and the sham oophorectomy groups is not significantly dif~ ferent at any time during the first 48 postoperative hours . In contrast, there is a dramatic burst of DNA synthesis in the regenerating liver that occurs 20 to 24 hours after partial hepatectomy." Our previ(lU~ studies (Campbell VW, ROllsell J, Rigsby D. et a!. Selective inhibition of c-myc expres~i()n by mithramyt'in prevents hepatocyte proliferation in regenerating liver. Unpublished data) have demonstrated peak [ ' H]thymidine incorporation into regenerating hepatocytes approximately 20 hours after partial hepatectomy, The pattern of DNA ~ynthesis in the regenerating liver is compared with that in compensatory ovarian growth in Fig, 4,
Comment In this investigation the molecular events sUlTounding compensatory ovanan growth have been compared with those that occur during liver regenet'ation, The results of these experiments indicate that the early events of compensatory ovarian growth are quite dif~ ferent from those of liver regeneration, The distinction between these types of compensatory growth may reHect two ba~ic mechanisms by which different organs
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TIME Fig. 2. Mean 1()ld IIIcre.tse in proto-oncogene expres~lon III regenerating liver and (ompensalOry ovarian growth. In previous studies (Campbell VW. Rousell J. Rigsby D. et al. Selective inhibulOn of c-myc expre,sion by mithramycin prevents hepatocyte proliferation in regenerating liver. Unpublished data) RNA extracted from regenerating liver witlun 24 hours .trter partial hepatectomy wa~ .1Ilalyzed b} dOl hybndization to radiolabeled eDNA prohes lor the proto-oncogenes (-lIIyr and c-HA-rfl.l,
respond to il~iury. In spite of an increase in mass 01 the remaining ovary, there appears to be no increase in the expression of the c-myc, c-Ha-ras, or c-li)s protooncogenes and no increase in DNA synthesis during compematory ovarian growth. The apparent dissimilarities in compensatory growth between these two organ systems raise several key issues, First, does the magnitude of compensatory growth explain the differences in the molecular events during compensatory growth in these two systems? The regenerating liver approaches its original weight by 7 days after partial hepatectomy." However. by 14 days after hemioophorectomy, compensatory growth in the contralateral ovary is only slightly > 207<. Increased proto-oncogene expression may have evolved as a mechanism to initiate more rapid growth in more vital organ systems, The magnitude of compensatory ovarian growth may explain the differences in this process when compared with that in regenerating liver, although the complete absence of even a proportional increase in proto-oncogene expre~sion and in DNA ~yn thesis in compensatory ovarian growth argues against this, Second, although there appears to be no increase in expression of the proto-oncogenes c-m,W. c-Ha-rw, and c-/m. our results do not exclude the pos~ibility that other
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proto-oncogenes may be involved in the compensatory ovarian growth process, Many of the known protooncogenes have been documented to have increased expression in both normal and abnormal (neoplastic) cell transformation processes."~·e:. Although increased c-myc expression has provided an accurate marker for cellular proliferation in other growth processes, the results of this experiment do not exclude the possibility of a more ovary-specific proto-oncogene that may ini-
tiate a slower growth mechani~m in the compensatory ovarian growth process. Last, are hypertrophic, rather than hyperplastic, cell processes involved in the earl\, events of compensatory ovarian growth? If hyperplasia plays a major role in compensatory growth, cell divison a nd DNA svnrhesis should occur. Conrrary to the events in the regenerating liver, there appears to he no measureable increase in Dl\'A synthesis in the remammg ovary within the
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fi,'st 48 hours after hemioophorectomy according to the results of this study. The molecular distinction between hypertrophic and hyperplastic changes is unclear, but it would appear from our data that should hypertrophic changes occur in compensatory ovarian growth, they do so by a pathway different from the hyperpla'tic changes in regenerating liver. REFERENCES I. Arai H. On the cause of the hypertrophy of the surviving ovary after semi-spaying (albino rat) and on the number of ova on it. AmJ Anat 1920;28 :59-79. 2, Hatai S. The effect of castration. spaying or semispaying on the weight of the central nervous system and of the hypophysis of the albl\1o rat; also the effect of ~emi spaying on the remaining o vary. J Exp Zool 1913; 15:297314. 3, Ramirez YD. Sawyer CH. A sex difference in the rat pituitary FSH response to unilateral gonadectomy as revealed by plasma RIA. Endocrinology 1974;94:475-82. 4. Welschen R, Dullaart J . de Jong FH. Interrelationship~ between circulating levels of estradiol-17j3 progesterone. FSH, and LH immediately after unilateral ovariectomy in the cyclic rat. BioI Reprod 1978; 18:42 1-7. 5. Benson B. Sorrentino S. Evans JS. Increase in serum FSH following unilateral ovariectomy in the rat. Endocrinology 1969;84:369-74. 6. Butcher RL. Changes 1\1 gonadotropins and steroids associated with unilateral ovariectomy of the rat. Endocrinology 1977; 10 I: 830-40 7. Edgren RA. Parlow AF. Peterson DL. Jones Re, On the mechanism of ovarian hypertrophy following hemicastration in rats. Endocrinology 1965; 76:97-102. 8. Burden HW, Lawrence IE. Smith CP, et al. The effects of vagotomy on compensatory ovarian hypertroph y and follicular activation a fter unilateral ovariectomy, Anat Rec 1986;2 14:6 1-6. 9, Burden HW. Lawrence IE. The effect of denervation on com pensatory O\'arian hypertrophy. Neuroendocnnology 1977;23::~68-78.
10, Curry TE, Lawrence IE. Burden HW, Effect ot ovanan sy mpathectomy Oil folli cul ar development during compensatory ovarian hypertrophy in the guinea-pig . .J Reprod Ferti! 1984;71:39..H. II. Bucher NLR. Malt RA. Regeneratio n of liver and kidney, Boston: Little. Brown. 1971. 12. Makino R. Hayo;hl K. Sugimura T. C-myc transcnpt is induced in rat liver at a \'ery early stage of regeneration or b\ cvcloheximide treatment. Nature 1984;310:697-8. 13. Tho~pson NL, Mead .J E, Brown L. Goyette 1\[, Shank PRo Fau;to N , Sequential protooncogene expression during rdt liver regeneration. Cancer Res 1986 :46:3111-7. 14. Goyette M, Petropoulos Cj , Shank PRo Fausto N. Regulated transcription ot c-Ki-ras and c-mvc during compensatorv growth of rat liver. Mol Cell BiolJ 984;4: 1493-8. 15, Goyette M. Petropoulos C,I. Shank PR, Famto N, Expression of a cellular oncogene during hver regeneration Science 1983;219:510-2, 16. Fausto N. Shank PRo Oncogene expres,ion in hver rege neration dnd hepatocarcinogenesis. Hepatologv 1983; 3: 1016-23. 17. Chomczynski P. Sacchi N. Single-step method of RNA isolation bv acid g uanidlum throcyanate-phenolchlorofor m extraction. Anal Biochem 1987: 162: 156-9. 18. T ho mas PS . Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc N"t1 Acad Sci USA 19RO;77 :520 1-:), 19. Rigby PW, Dreckmann M, Rhodes C. Berg P. Labehng deoxyribonucleic acid to hig h specific actl\'lty in vitro by nick translation with DNA pol~mera~e l. .I Mol Bioi 1977;113:237-51. 20. Blin N. Stafford DW. Isolati on of high molecular-weight DNA.. Nucleic Acids R e ~ 1976;3:2303-10. 21. Gre5ham JW. l\IorphologlC st ud~' of dexoyribonudelC aCid synthesis and cell proliferatio n in regenerating rat liver: autoradiography with l 'H]-thymidine. Cancer Res 1962; 22:842-9. 22. Bi;hop JM, Cellular oncogene, and retroviruses. Ann Rev Biochem 1983;52:301-54. 23. Cooper GM. Cellular transforming genes. Science 1982 ; 217:ROI-II. 24. Bishop .1M. Oncogenes, Sci Am 1982;246:80-92. 25. Weinberg RA. A molecular basis of cancer. Sci Am 1983 ,249: 126-43,
Key words: Compensatory ovarian growth Compensatory ovarIan growth has been demonstrated to occur in the rat and other animal models after unilateral castration. I. " It has been hypothesized by some investigators that compensatory ovarian growth occurs as a result of an increase in gonadotropin secretion stimulated by declining levels of ovarian steroids or by declining levels of an "inhibin-like" factor.3•7 Others have postulated a functional role of the peripheral autonomic nervous system in compensatory ovarian growth."-IO In other compensatory growth processes, cellular proliferation is preceded by an increase in protooncogene expression. For example, increased expression of the proto-oncogenes c-myc, c-Ha-ras, and c-fos From the Departments of ObstetriCs and GYlecology, Pathology, and Inlernal Medlcme, The Ulllvemty of Alabama at Bmnmgham. Presented at the Thlrt.~-s!xth Annual M eetmg of the SOCiety for Gynecologic InvestlgallOl/, San Diego. Callfonlla. March 15-18, 1989. Reprmt requests: Ronald D. Alva rez, MD, OHB 550, Umvemty StatlOll. UnIVersity of Alabama at Bmnmglwl/I, Bmnl1lghal/1, AL 3529-1
616116507
precedes an increase in dexoyribonucleic acid (DNA) synthesis and cell proliferation in the regenerating liver after partial hepatectomy."-16 No study to date has reported the molecular events in compensatory ovarian growth or compared these events with the molecular events in regenerating liver. It is the purpose of this study to do so by investigating the temporal pattern of proto-oncogene expression and DNA synthesis in rat ovarian tissue after unilateral castration.
Material and methods Animals. Fischer rats (weight range 106 to 177 gm) were housed under controlled conditions of temperature, humidity, and light (lights on 12 hours and lights off 12 hours). All animals were fed water and standard rat chow as desired. Institutional guidelines for the care and experimental use of animals were strictly adhered to. Determination of compensatory growth. Twelve female rats were subjected to a le ft hemioophorectomv and 12 female rats were subjected to a sham oophorectomy both through a ventral midline abdominal in-
1653
1654 Alvarez et al.
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Fig. 1. Mean fold increases in proto-oncogene expres,lOn III the hemioophorectomy (ULO) and sham oophorectomy (SHAM) groups. The right ovaries were excised from each group at various intervals between 4 and 48 hours after the initial procedure. RNA extracted from ovarian samples was analyzed by dot hybridization to radiolabeled cDNA probes for the proto-oncogenes c-myr, c-HA-ras, and cjus.
cis ion with the animals under ketamine anesthesia. Encapsulating fibroadipose tissue was trimmed from the excised ovaries and the ovaries were weighed individually. At 3 and 14 days after the initial procedure, the remaining ovaries were excised from six rats in each group, trimmed of fibroadipose tissue, and individually weighed .. The amount of compensatory ovarian growth was calculated by the formula: Compensatory growth (%)
R - R " , x 100
R,
where Ro is the mean weight of the right ovaries in the hemioophorectomy group and R, is the mean weight of the right ovaries in the sham oophorectomy group. The experiment was repeated in 24 additional female rats, and the mean compensatory ovarian growth was determined from the results of the two experiments. Determination of proto-oncogene expression.
Thirty-six female rats were subjected to a left hemioophorectomy and 36 were subjected to a sham oophorectomy. At 4, 8, 12, 24, 36, and 48 hours after the initial surgery, six rats from each group were put to death. The right ovaries were excised and paired into three samples in both study groups for each time point and immediately frozen in liquid nitrogen. Total cellular ribonucleic acid (RNA) was isolated from each sample by the method described by Chomczynski and Sacchi. '7 RNA was extracted from pulverized frozen tissue homogenized in a guanidium thiocyanate solution. After phenol-chloroform extraction the RNA was precipitated with isopropanol and quantitated by absorption spectrophotometry. RNA samples from each time point in both the hemioophorectomy group and the sham oophorectomy group were analyzed by dot hybridization. IX Each RNA sample was assayed by hybridization to complementary DNA (cDNA) probes for the c-rnye, c-Ha-ra~, and c-fo.1 protooncogenes. The cDNA insert for each probe was purified by preparative gel electrophoresis and radiolabeled by nick translation. '" Autoradiograms of each filter were analyzed by laser densitomeu'y, and the mean increase in proto-oncogene expression was plotted for both the hemioophorectomy and the sham oophorectomy group. Determination of DNA synthesis. Fifteen female rats were subjected to a left oophorectomy and 15 underwent a sham oophorectomy. At 6, 10, 22, 34, and 46 hours after the initial procedure, three rats from each group were injected intra peritoneally with 200 mCi of ['H) thymidine diluted in 1 ml of saline solution. Two hours later the right ovaries were excised from each rat and immediately frozen in liquid nitrogen. Cellular DNA was isolated from each frozen ovary specimen by proteinase K digestion and phenol extraction. 20 The rate of DNA synthesis in the hemioophorectomy group and in the sham oophorectomy group was determined by measuring the specific activity of each DNA sample and by calculating the mean specific activity for each time point.
Results The mean compensatory weight increase in the right ovaries of the rats who had undergone left hemioophorectomy was 7.9% at 3 days and 22.5% at 14 days. This degree of compensatory ovarian growth is in agreement with that seen by previous investigators.1. 2. >.7 This compensatory growth is less dramatic than that seen in the regenerating liver, which has a 300% to 500% increase in mass within 7 to 14 days after partial hepatectomy. II As demonstrated in Fig. 1, there is no significant difference in the level of expression of the protooncogenes c-rnyr, c-HA-ras, and c-Ios between the right
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ovanes ot the hemioophorectomized rats and the right ovaries of the sham oophorectomized group. This is in clear contradistinction to the observations in regenerating liver. Previous studies conducted in our laboratory have demonstrated a sevenfold to eightfold increase in C-ntyc expression within 3 hours after partial hepatectomy (Campbell VW, Rousell J, Rigsby D, et al. Selective inhibition of c-myc expression by mithramycin prevents hepatocyte proliferation in regenerating livet'. Unpublished data). Othet's have t'eported as much as a fifteenfold increase in c-myc expression during the same time period"e-" The c-myc expression returns to baseline, only to rise again to a less pronounced level of expression between 8 and 24 hours after partial hepatectomy (Campbell VW, Rousell J, Rig~by D, et al. Selective inhibition of c-myc expression by mithramycin prevents hepatocyte proliferation in regenerating liver. Unpublished data). The c-Ha-ras expression has been demonstrated by our previous studies' and by othet's'" I', 'h to inuease 200% to :~()O% within 48 hours after partial hepatectomy. Fig. 2 graphically compares our observations of the pattern of c-1IIyc and c-Ha-rw expression in compensatory ovarian growth and in regenerating liver. In similar studies, c10s levels have been shown to be at least fourfold above normal 30 minutes after partial hepatectomy, only to decline by 2 hours. U A second twofold increase in c-jo.1 expression occurs at 8 hours after partial hepatectomy. As sholl'n 111 FIg. :{, there is no significant II1crease in ovarian DNA synthesis after hemioophorectomy. The level of DNA synthesis in the hemioophorectomy and the sham oophorectomy groups is not significantly dif~ ferent at any time during the first 48 postoperative hours . In contrast, there is a dramatic burst of DNA synthesis in the regenerating liver that occurs 20 to 24 hours after partial hepatectomy." Our previ(lU~ studies (Campbell VW, ROllsell J, Rigsby D. et a!. Selective inhibition of c-myc expres~i()n by mithramyt'in prevents hepatocyte proliferation in regenerating liver. Unpublished data) have demonstrated peak [ ' H]thymidine incorporation into regenerating hepatocytes approximately 20 hours after partial hepatectomy, The pattern of DNA ~ynthesis in the regenerating liver is compared with that in compensatory ovarian growth in Fig, 4,
Comment In this investigation the molecular events sUlTounding compensatory ovanan growth have been compared with those that occur during liver regenet'ation, The results of these experiments indicate that the early events of compensatory ovarian growth are quite dif~ ferent from those of liver regeneration, The distinction between these types of compensatory growth may reHect two ba~ic mechanisms by which different organs
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TIME Fig. 2. Mean 1()ld IIIcre.tse in proto-oncogene expres~lon III regenerating liver and (ompensalOry ovarian growth. In previous studies (Campbell VW. Rousell J. Rigsby D. et al. Selective inhibulOn of c-myc expre,sion by mithramycin prevents hepatocyte proliferation in regenerating liver. Unpublished data) RNA extracted from regenerating liver witlun 24 hours .trter partial hepatectomy wa~ .1Ilalyzed b} dOl hybndization to radiolabeled eDNA prohes lor the proto-oncogenes (-lIIyr and c-HA-rfl.l,
respond to il~iury. In spite of an increase in mass 01 the remaining ovary, there appears to be no increase in the expression of the c-myc, c-Ha-ras, or c-li)s protooncogenes and no increase in DNA synthesis during compematory ovarian growth. The apparent dissimilarities in compensatory growth between these two organ systems raise several key issues, First, does the magnitude of compensatory growth explain the differences in the molecular events during compensatory growth in these two systems? The regenerating liver approaches its original weight by 7 days after partial hepatectomy." However. by 14 days after hemioophorectomy, compensatory growth in the contralateral ovary is only slightly > 207<. Increased proto-oncogene expression may have evolved as a mechanism to initiate more rapid growth in more vital organ systems, The magnitude of compensatory ovarian growth may explain the differences in this process when compared with that in regenerating liver, although the complete absence of even a proportional increase in proto-oncogene expre~sion and in DNA ~yn thesis in compensatory ovarian growth argues against this, Second, although there appears to be no increase in expression of the proto-oncogenes c-m,W. c-Ha-rw, and c-/m. our results do not exclude the pos~ibility that other
1656 Alvarez et al.
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TIME (hrs) Fig. 3. DNA synthesis In the hemioophorectomy (L'LO) and ;ham ovariectomy (SHAM) g-roups. Each rat was injected intraperitoneally with 200 mCi of [' Hlthymidine at \,Irious interv,lb between 6 and 46 hours after th e initial procedure. The right oV
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TIME (hours) Fig. 4. DNA synthesIs in regenerating liver and compensaton O\'Jl'ldn growth. In PI'C\IOll> studie; (Campbell VW, RouseliJ. Rig~b)' D, et al. Selective inhibitio n of c-myc expression III mithramycin prevents hepatocyte proliferation in regenerating liver. L'npublished data) DNA was extracted hom regenerating liver I hour a fter intraperitoneal injection of[.IHlthYlIlidine at \ariOll~ mtervals between 6 and 30 hours after partial hepatectom) . The Ie\ cl of D:\A sy nthe'i' \\'a, determined b} measuring the specific activity of each sample al each time POlllt.
proto-oncogenes may be involved in the compensatory ovarian growth process, Many of the known protooncogenes have been documented to have increased expression in both normal and abnormal (neoplastic) cell transformation processes."~·e:. Although increased c-myc expression has provided an accurate marker for cellular proliferation in other growth processes, the results of this experiment do not exclude the possibility of a more ovary-specific proto-oncogene that may ini-
tiate a slower growth mechani~m in the compensatory ovarian growth process. Last, are hypertrophic, rather than hyperplastic, cell processes involved in the earl\, events of compensatory ovarian growth? If hyperplasia plays a major role in compensatory growth, cell divison a nd DNA svnrhesis should occur. Conrrary to the events in the regenerating liver, there appears to he no measureable increase in Dl\'A synthesis in the remammg ovary within the
Compensatory ovarian hypertrophy
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1657
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fi,'st 48 hours after hemioophorectomy according to the results of this study. The molecular distinction between hypertrophic and hyperplastic changes is unclear, but it would appear from our data that should hypertrophic changes occur in compensatory ovarian growth, they do so by a pathway different from the hyperpla'tic changes in regenerating liver. REFERENCES I. Arai H. On the cause of the hypertrophy of the surviving ovary after semi-spaying (albino rat) and on the number of ova on it. AmJ Anat 1920;28 :59-79. 2, Hatai S. The effect of castration. spaying or semispaying on the weight of the central nervous system and of the hypophysis of the albl\1o rat; also the effect of ~emi spaying on the remaining o vary. J Exp Zool 1913; 15:297314. 3, Ramirez YD. Sawyer CH. A sex difference in the rat pituitary FSH response to unilateral gonadectomy as revealed by plasma RIA. Endocrinology 1974;94:475-82. 4. Welschen R, Dullaart J . de Jong FH. Interrelationship~ between circulating levels of estradiol-17j3 progesterone. FSH, and LH immediately after unilateral ovariectomy in the cyclic rat. BioI Reprod 1978; 18:42 1-7. 5. Benson B. Sorrentino S. Evans JS. Increase in serum FSH following unilateral ovariectomy in the rat. Endocrinology 1969;84:369-74. 6. Butcher RL. Changes 1\1 gonadotropins and steroids associated with unilateral ovariectomy of the rat. Endocrinology 1977; 10 I: 830-40 7. Edgren RA. Parlow AF. Peterson DL. Jones Re, On the mechanism of ovarian hypertrophy following hemicastration in rats. Endocrinology 1965; 76:97-102. 8. Burden HW, Lawrence IE. Smith CP, et al. The effects of vagotomy on compensatory ovarian hypertroph y and follicular activation a fter unilateral ovariectomy, Anat Rec 1986;2 14:6 1-6. 9, Burden HW. Lawrence IE. The effect of denervation on com pensatory O\'arian hypertrophy. Neuroendocnnology 1977;23::~68-78.
10, Curry TE, Lawrence IE. Burden HW, Effect ot ovanan sy mpathectomy Oil folli cul ar development during compensatory ovarian hypertrophy in the guinea-pig . .J Reprod Ferti! 1984;71:39..H. II. Bucher NLR. Malt RA. Regeneratio n of liver and kidney, Boston: Little. Brown. 1971. 12. Makino R. Hayo;hl K. Sugimura T. C-myc transcnpt is induced in rat liver at a \'ery early stage of regeneration or b\ cvcloheximide treatment. Nature 1984;310:697-8. 13. Tho~pson NL, Mead .J E, Brown L. Goyette 1\[, Shank PRo Fau;to N , Sequential protooncogene expression during rdt liver regeneration. Cancer Res 1986 :46:3111-7. 14. Goyette M, Petropoulos Cj , Shank PRo Fausto N. Regulated transcription ot c-Ki-ras and c-mvc during compensatorv growth of rat liver. Mol Cell BiolJ 984;4: 1493-8. 15, Goyette M. Petropoulos C,I. Shank PR, Famto N, Expression of a cellular oncogene during hver regeneration Science 1983;219:510-2, 16. Fausto N. Shank PRo Oncogene expres,ion in hver rege neration dnd hepatocarcinogenesis. Hepatologv 1983; 3: 1016-23. 17. Chomczynski P. Sacchi N. Single-step method of RNA isolation bv acid g uanidlum throcyanate-phenolchlorofor m extraction. Anal Biochem 1987: 162: 156-9. 18. T ho mas PS . Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc N"t1 Acad Sci USA 19RO;77 :520 1-:), 19. Rigby PW, Dreckmann M, Rhodes C. Berg P. Labehng deoxyribonucleic acid to hig h specific actl\'lty in vitro by nick translation with DNA pol~mera~e l. .I Mol Bioi 1977;113:237-51. 20. Blin N. Stafford DW. Isolati on of high molecular-weight DNA.. Nucleic Acids R e ~ 1976;3:2303-10. 21. Gre5ham JW. l\IorphologlC st ud~' of dexoyribonudelC aCid synthesis and cell proliferatio n in regenerating rat liver: autoradiography with l 'H]-thymidine. Cancer Res 1962; 22:842-9. 22. Bi;hop JM, Cellular oncogene, and retroviruses. Ann Rev Biochem 1983;52:301-54. 23. Cooper GM. Cellular transforming genes. Science 1982 ; 217:ROI-II. 24. Bishop .1M. Oncogenes, Sci Am 1982;246:80-92. 25. Weinberg RA. A molecular basis of cancer. Sci Am 1983 ,249: 126-43,