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Studies on the Function of the Denervated Rabbit Ovary: Human Chorionic Gonadotropin-Induced Ovulation*†
Vol. 26, No.4, April1975 Printed in U.S A.
FERTILITY AND STERILITY Copyright
0
1975 The American Fertility Society
STUDIES ON THE FUNCTION OF THE DENERVATED RABBIT OVARY: HUMAN CHORIONIC GONADOTROPIN-INDUCED OVULATION*t STUART WEINER, M.D.,* KAREN H. WRIGHT, B.S., AND EDWARD E. WALLACH, M.D.
Department of Obstetrics and Gynecology, Pennsylvania Hospital and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19107
Review of the literature suggests that the innervation of the mammalian ovary may play a role in the selection and early development of follicles, L 2 follicular and hilar4 •5 steroidogenesis, sensory and visceromotor reflexes originating in the ovary, 6 ovarian blood flow, and the mechanism of ovulation. 2 •4 •7 - 10 The forces responsible for extrusion of the follicular contents at the time of ovulation have never been clearly defined. Although several studies indicate that intrafollicular pressure is not increased when ovulation occurs, 11 the presence of perifollicular smooth muscle fibers in most mammalian ovaries, 12 associated with adrenergic ovarian innervation of varying density/ 3 suggests that a neuromuscular mechanism may be important in this process. The significance of the sparse cholinergic innervation of the ovary 13 is also uncertain. In addition, the ability of adrenergic blocking agents to block ovulation, 8 as well as to inhibit ovarian contractility/ further suggests a significant neural function in the control of ovulation. The fact that the endogenous contractile activity of the rabbit and monkey ovary is greatest at the time of ovulation7 Received March 27, 1974. *Supported by The Population Council Grant M73.103. tPresented at The 30th Annual Meeting of The American Fertility Society, April 4 to 6, 1974, Hollywood, Florida. *Reprint requests: Stuart Weiner, M.D., Pennsylvannia Hospital, Eighth and Spruce Streets, Philadelphia, Pennsylvania 19107.
lends further support to this hypothesis. Many of these problems have been recently reviewed. 14 In order to identify more precisely the role, if any, of central neural activity in the control of ovulation, a technique for specific denervation of the in vivo rabbit ovary was developed in a previous study .15 The effect of this method of ovarian denervation on human chorionic gonadotropin (HCG)-induced ovulation in the rabbit is described in the present study. The rabbit ovarian stroma contains several adrenergic and fewer cholinergic nerve fibers. 13 The enhanced activity of mammalian hypothalamic centers, which mediate ovulation after certain external stimuli/ 6 suggests that an efferent neural pathway might be involved in such "reflex ovulation." Ablation of the ovarian nerves should reveal the significance ofthis pathway in the control of ovulation. The differences between the mechanisms of coitus-induced and HCG-induced ovulation will also be discussed in this paper. MATERIALS AND METHODS
Deneruation Technique. The method of ovarian denervation developed in our laboratory 15 was used. Briefly, this involved isolating the ovarian artery at laparotomy and stripping it of its adventitia and any grossly visible nerve bundles, using microsurgical techniques. The excised tissue was stained with methylene blue to assure that neural tissue had been removed. A ligature of
363
364
April1975
WEINERETAL
fine black silk was then tied around the ovarian artery, without obstructing its blood flow, to act as a mechanical barrier to delay and disorganize reinnervation of the ovary. Earlier studies 15 indicate that this technique renders the ovary devoid of adrenergic nerve fibers for up to four weeks. HCG-Induced Ovulation. Twenty-two New Zealand White rabbits underwent unilateral ovarian denervation; the contralateral ovaries were left intact to serve as controls. After a three-week isolation period to ensure that the animals would be in anestrus, ovulation was induced in each rabbit by an intravenous injection of 50 IU of HCG (APL, Ayerst Laboratories, New York). Ten to twenty-two hours after HCG administration, each rabbit was subjected to a second laparotomy under sodium pentobarbital anesthesia. Each ovary was carefully inspected, and the numbers of early mature, preovulatory, ruptured, and hemorrhagic follicles present were recorded. Early mature follicles were identified as clear, cystic follicles, which bulged little or not at all above the ovarian surface, and which had diameters ranging from 0.5 to 1.0 mm. Preovulatory follicles were greater than 1.0 mm in diameter, remained clear and homogenous throughout their contents, and bulged above the surface ofthe ovary. These follicles demonstrated no evidence of rupture when the follicular apex or stigma was examined under the dissecting microscope. Perforation of the stigma identified ruptured follicles; collapse of the follicular wall was often noted. The fluid content of ruptured follicles was usually clear, although some intrafollicular hemorrhage was frequently observed. Hemorrhagic follicles were larger than preovulatory follicles, demonstrated sparse or diffuse hemorrhage into the follicular fluid, but lacked evidence of rupture at the stigma. Two rabbits (no. 8 and no. 9) were maintained under anesthesia for six
hours, so that their ovaries could be studied at two-hour intervals from 10 to 16 hours after HCG administration. After each follicle count, peritoneal lavage with physiologic saline solution was performed, and the laparotomy incision was closed with towel clips. After the second laparotomy, the rabbits were killed and their ovaries were bisected longitudinally. One half of each ovary was quickly immersed in isopentane cooled in liquid nitrogen, to be freezedried and stained for adrenergic nerve fibers, using the technique of Falck et alP The other half of each ovary was fixed in Bouin's solution and subjected to routine histologic study. RESULTS
Follicle Counts. As demonstrated in Table 1, no significant differences in follicle counts were noted between denervated and intact ovaries after HCG-induced ovulation. In addition, the numbers and proportions of stimulated follicles which progressed to rupture did not differ significantly between the two groups of ovaries. The individual follicle counts for each rabbit at each time interval after HCG administration are detailed in Table 2. The failure of ovarian denervation to alter the time course of ovulation or TABLE 1. Summary of Follicle Counts• after HCG-Inducedb Ovulation in 22 Rabbits Ovary
Follicles
Early mature Preovulatory Ruptured Hemorrhagic Activated (total) Activated follicles/ovary (mean) Ruptured follicles/ovary (mean)
Denervated Intact (control)
29 17 69 3
19 20 76 8
118
123
5.4
5.6
3.1
3.5
•Observations made from 10 to 22 hours after HCG administration. h50 IU of HCG intravenously.
365
HCG-INDUCED OVULATION
Vol. 26, No.4
TABLE 2. Follicle Counts after HCG-lnduced 8 Ovulation in 22 Rabbits Ovary Intact (control)
Denervated
Rabbit no.
Early mature
8
9
3 3 2 1 1
10 11
12 14 15 16 17 18 19 20 21 23 25 26 27 28 29 30 33 40 8
5 1 2 1 1 1 3 1 1 4 2 2 2
Preovulatory
6 3 2 2 4 3 2 2 1 1 1 1
1 1 3
Ruptured
4 5 6 3 5 5 4 3 3 2 4 6 4 1 4 4 3 3 1 3 3 4 3 3
Hemorrhagic
Early mature
1 1 1 1 2 2 1 2 3
Hours
Preovulatory
2 1 1 1 5 4 2 2 1 1 1 3
2 1 1 1 2 1
4
2
3 2 4 2
2 3
Ruptured
Hemorrhagic after HCG
2 2 2 2 3 3 5 6 2 4 2 1 5 3 4 7 2 6 2 3 4 7 3 5
1 1 2 3
1
1
10 12 14 16 10 12 14 16 16 11
18 17 17 17 17 17 17 17 17 17 20 20 20 20 20 20 22 10
50 1U of HCG intravenously.
rupture became greater as the interval from the time of HCG injection increased. There was no significant difference between the curves for the denervated and intact ovaries. Histologic Observations. As confirmed by our previous study/ 5 the denervation technique resulted in no apparent ischemic or trophic changes in the routine hematoxylin and eosin stained sections of the ovaries. In addition, the fluorescent stain demonstrated the presence of adrenergic nerve fibers in the intact ovaries, while no such fluorescent structures were found in the denervated specimens (Figs. 2 and 3). Microscopic examination also revealed no difference between the deHours After HCG (50IU lntrtn•nously) nervated and the intact ovaries in the FIG. 1. Graphic representation of the percent of mature follicles which ruptured at increasing inter- distribution of maturing follicles throughvals after HCG administration. Note the nearly out the cortex. In other words, no increase identical curves of denervated and control ovaries. in the numbers of mature subcortical
percentages of stimulated follicles which ruptured after HCG is illustrated graphically in Figure 1. The proportion of stimulated follicles which progressed to
366
WEINERETAL
FIG. 2. Stroma of an intact ovary demonstrating numerous fluorescing adrenergic nerve fibers (arrows) (x 40).
April1975
FIG. 3. Stroma of a denervated ovary devoid of fluorescent adrenergic nerve fibers (x40).
follicles which might not have been ap- ovarian, postganglionic neurons has been parent at the time of laparotomy was suggested, 18 and the endogenous activity noted. Finally, no significant numbers of these fibers may remain intact after of retained ova in apparently ruptured the ovary is decentralized. Indeed, the follicles could be detected. spontaneous contractility of the in vitro rabbit ovary and its responsiveness to DISCUSSION adrenergic agents 7 suggests that this Ovarian denervation did not alter folli- organ possesses endogenous neuromuscular activation, maturation, or ovulation cular mechanisms. Other studies suggest in the rabbit after intravenous injection of ~hat systemically administered adrenHCG. The vascular stripping technique ergic agents which alter ovarian contracused in these studies rendered the ovary tility and ovulation also act on a local devoid of adrenergic nerves. No specific ovarian level, rather than through the histologic stain for cholinergic fibers was central nervous system. 8 Nevertheless, used, but the ovary was probably severed the hypothalamus remains an important from all its connections with the central mediator in the reflex ovulation noted nervous system by this method. Never- in some mammals. 16 While an efferent theless, the presence of entirely intra- hormonal message from the hypothal-
Vol. 26, No. 4
367
HCG-INDUCED OVULATION
amus to the preovulatory follicle has been demonstrated with the identification of the"LH-surge," a comparable efferent neural pathway remains in doubt. The results of this study suggest that such a pathway, if it exists at all, is not essential for HCG-induced ovulation to occur in the rabbit. HCG, however, stimulates ovulation by a local effect on the ovary. We are, therefore, presently investigating the effect of ovarian denervation on coitus-induced ovulation in the rabbit, since this process is mediated by the central nervous system. While the neuromuscular activity of the human ovary appears quite similar to that of the rabbit, 19 the apparent absence of reflex ovulation in the primate makes extrapolation of these results to human ovarian physiology uncertain. The implications are nevertheless suggestive for more completely defining the neurohormonal mechanisms controlling mammalian ovulation. In conclusion, HCG-induced follicular development and rupture are not changed after ovarian denervation in the rabbit. This suggests that these ovarian functions may be independent of central nervous system connections. Since coitus-induced ovulation is mediated by the hypothalamus in those mammals capable of reflex ovulation, the effect of ovarian denervation on this physiologic sequence is currently being investigated. These studies should define whether an efferent neural pathway, in addition to the recognized hormonal mechanisms, is essential for reflex ovulation in the rabbit. SUMMARY
The described method for selective sympathetic denervation of the in vivo rabbit ovary involved stripping the ovarian artery of its nerve bundles and adventitial tissue. The ovary was then entirely free of fluorescent-staining adrenergic nerves.
This technique was used to study the effects ot' ovarian denervation on HCGinduced ovulation. After HCG was administered to 22 rabbits which had previously undergone unilateral ovarian denervation, the ovaries were observed for follicular maturation and rupture. Control ovaries demonstrated a mean of 5.6 stimulated follicles/ovary; denervated ovaries had a mean of 5.4. An average of 3.5 follicles/control ovary ruptured; an average of 3.1 follicles/denervated ovary ruptured. Furthermore, the time course of ovulation after HCG did not differ between denervated and intact ovaries. These results indicate that HCG-induced ovulation in the rabbit is not interrupted by ovarian denervation. Acknowledgments. The authors gratefully acknowledge the assistance of Thomas Henry and Susan Pisano. We also wish to thank Pramuan Virutamasen, M.D., for his invaluable advice during the early parts of this study. Human chorionic gonadotropin (APL) was generously supplied by Garfield Davies, Ayerst Laboratories, New York, New York.
REFERENCES 1. Fink G, Schofield GC: Experimental studies on
2.
3.
4.
5.
6.
the innervation of the ovary in cats. J Anat 109:115, 1971 Brink CE, Grob HS: Response of the denervated mouse ovary to exogenous gonadotropins. Presented at the Sixth Annual Meeting of the Society for the Study of Reproduction, Athens, Georgia, August 13-16, 1973 Spies HG, Niswender GD: Levels of prolactin, LH and FSH in the serum of intact and pelvicneuroectomized rats. Endocrinology 88:937, 1971 Hill RT: Paradoxical effects of ovarian secretions. In The Ovary. Vol. II. Edited by S Zuckerman. New York, Academic Press, 1962, p 231 Neilson D, Jones GS, Woodruff JD, et al: The innervation of the ovary. Obstet Gynecol Surv 25:889, 1970 Labate JS, Reynolds SRM: Sensory pathways of the ovarian plexus. Am J Obstet Gynecol 34:1, 1937
368
WEINERETAL
April1975
7. Virutamasen P, Wright KH, Wallach EE: 14. Bahr J, Kao L, Nalbandov AV: The role of Effects of catecholamines on ovarian contraccatecholamines and nerves in ovulation. Bioi tility in the rabbit. Obstet Gynecol 39:225, Reprod 10:273, 1974 1972 15. Weiner S, Wright KH, Wallach EE: Selective 8. Ferrando G, Nalbandov AV: Direct effect on ovarian sympathectomy in the rabbit. Fertil the ovary of the adrenergic blocking drug Steril 26:353, 1975 dibenzyline. Endocrinology 85:38, 1969 16. Law OT, Sackett GP: Hypothalamic potentials in 9. Marshall JM: Adrenergic innervation of the the female rat evoked by hormones and female reproductive tract: Anatomy, physiology vaginal stimulation. Neuroendocrinology 1:31, and pharmacology. Rev Physiol 62:6, 1970 1965 10. Marshall JM: Effects of catecholamines on the 17. Falck B, Hillarp NA, Thiene G, et al: Fluoressmooth muscle of the female reproductive tract. cence of catecholamines and related compounds Ann Rev Pharmacal 13:19, 1973 condensed with formaldehyde. J Histochem 11. Espey LL, Lipner H: Measurement of intraCytochem 10:348, 1962 follicular pressure in the rabbit ovary. Am J 18. Owman C, Sjostrand NO: Short adrenergic Physiol 205:1067, 1963 neurons and catecholamine-containing cells in 12. Okamura H, Virutamasen P, Wright KH, et vas deferens and accessory male genital glands al: Ovarian smooth muscle in the human being, of different mammals. Z Zellforsch Abt rabbit, and cat: Histochemical and electronHistochem 66:300, 1965 microscopic study. Am J Obstet Gynecol 112: 19. Diaz-Infante Jr. A, Virutamasen P, Connaugh183, 1972 ton JF, et al: In vitro studies of human 13. Jacobowitz D, Wallach EE: Histochemical and ovarian contractility. Obstet Gynecol 44:830, chemical studies of the autonomic innervation 1974 of the ovary. Endocrinology 81:1132, 1967
FERTILITY AND STERILITY Copyright
0
1975 The American Fertility Society
STUDIES ON THE FUNCTION OF THE DENERVATED RABBIT OVARY: HUMAN CHORIONIC GONADOTROPIN-INDUCED OVULATION*t STUART WEINER, M.D.,* KAREN H. WRIGHT, B.S., AND EDWARD E. WALLACH, M.D.
Department of Obstetrics and Gynecology, Pennsylvania Hospital and University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19107
Review of the literature suggests that the innervation of the mammalian ovary may play a role in the selection and early development of follicles, L 2 follicular and hilar4 •5 steroidogenesis, sensory and visceromotor reflexes originating in the ovary, 6 ovarian blood flow, and the mechanism of ovulation. 2 •4 •7 - 10 The forces responsible for extrusion of the follicular contents at the time of ovulation have never been clearly defined. Although several studies indicate that intrafollicular pressure is not increased when ovulation occurs, 11 the presence of perifollicular smooth muscle fibers in most mammalian ovaries, 12 associated with adrenergic ovarian innervation of varying density/ 3 suggests that a neuromuscular mechanism may be important in this process. The significance of the sparse cholinergic innervation of the ovary 13 is also uncertain. In addition, the ability of adrenergic blocking agents to block ovulation, 8 as well as to inhibit ovarian contractility/ further suggests a significant neural function in the control of ovulation. The fact that the endogenous contractile activity of the rabbit and monkey ovary is greatest at the time of ovulation7 Received March 27, 1974. *Supported by The Population Council Grant M73.103. tPresented at The 30th Annual Meeting of The American Fertility Society, April 4 to 6, 1974, Hollywood, Florida. *Reprint requests: Stuart Weiner, M.D., Pennsylvannia Hospital, Eighth and Spruce Streets, Philadelphia, Pennsylvania 19107.
lends further support to this hypothesis. Many of these problems have been recently reviewed. 14 In order to identify more precisely the role, if any, of central neural activity in the control of ovulation, a technique for specific denervation of the in vivo rabbit ovary was developed in a previous study .15 The effect of this method of ovarian denervation on human chorionic gonadotropin (HCG)-induced ovulation in the rabbit is described in the present study. The rabbit ovarian stroma contains several adrenergic and fewer cholinergic nerve fibers. 13 The enhanced activity of mammalian hypothalamic centers, which mediate ovulation after certain external stimuli/ 6 suggests that an efferent neural pathway might be involved in such "reflex ovulation." Ablation of the ovarian nerves should reveal the significance ofthis pathway in the control of ovulation. The differences between the mechanisms of coitus-induced and HCG-induced ovulation will also be discussed in this paper. MATERIALS AND METHODS
Deneruation Technique. The method of ovarian denervation developed in our laboratory 15 was used. Briefly, this involved isolating the ovarian artery at laparotomy and stripping it of its adventitia and any grossly visible nerve bundles, using microsurgical techniques. The excised tissue was stained with methylene blue to assure that neural tissue had been removed. A ligature of
363
364
April1975
WEINERETAL
fine black silk was then tied around the ovarian artery, without obstructing its blood flow, to act as a mechanical barrier to delay and disorganize reinnervation of the ovary. Earlier studies 15 indicate that this technique renders the ovary devoid of adrenergic nerve fibers for up to four weeks. HCG-Induced Ovulation. Twenty-two New Zealand White rabbits underwent unilateral ovarian denervation; the contralateral ovaries were left intact to serve as controls. After a three-week isolation period to ensure that the animals would be in anestrus, ovulation was induced in each rabbit by an intravenous injection of 50 IU of HCG (APL, Ayerst Laboratories, New York). Ten to twenty-two hours after HCG administration, each rabbit was subjected to a second laparotomy under sodium pentobarbital anesthesia. Each ovary was carefully inspected, and the numbers of early mature, preovulatory, ruptured, and hemorrhagic follicles present were recorded. Early mature follicles were identified as clear, cystic follicles, which bulged little or not at all above the ovarian surface, and which had diameters ranging from 0.5 to 1.0 mm. Preovulatory follicles were greater than 1.0 mm in diameter, remained clear and homogenous throughout their contents, and bulged above the surface ofthe ovary. These follicles demonstrated no evidence of rupture when the follicular apex or stigma was examined under the dissecting microscope. Perforation of the stigma identified ruptured follicles; collapse of the follicular wall was often noted. The fluid content of ruptured follicles was usually clear, although some intrafollicular hemorrhage was frequently observed. Hemorrhagic follicles were larger than preovulatory follicles, demonstrated sparse or diffuse hemorrhage into the follicular fluid, but lacked evidence of rupture at the stigma. Two rabbits (no. 8 and no. 9) were maintained under anesthesia for six
hours, so that their ovaries could be studied at two-hour intervals from 10 to 16 hours after HCG administration. After each follicle count, peritoneal lavage with physiologic saline solution was performed, and the laparotomy incision was closed with towel clips. After the second laparotomy, the rabbits were killed and their ovaries were bisected longitudinally. One half of each ovary was quickly immersed in isopentane cooled in liquid nitrogen, to be freezedried and stained for adrenergic nerve fibers, using the technique of Falck et alP The other half of each ovary was fixed in Bouin's solution and subjected to routine histologic study. RESULTS
Follicle Counts. As demonstrated in Table 1, no significant differences in follicle counts were noted between denervated and intact ovaries after HCG-induced ovulation. In addition, the numbers and proportions of stimulated follicles which progressed to rupture did not differ significantly between the two groups of ovaries. The individual follicle counts for each rabbit at each time interval after HCG administration are detailed in Table 2. The failure of ovarian denervation to alter the time course of ovulation or TABLE 1. Summary of Follicle Counts• after HCG-Inducedb Ovulation in 22 Rabbits Ovary
Follicles
Early mature Preovulatory Ruptured Hemorrhagic Activated (total) Activated follicles/ovary (mean) Ruptured follicles/ovary (mean)
Denervated Intact (control)
29 17 69 3
19 20 76 8
118
123
5.4
5.6
3.1
3.5
•Observations made from 10 to 22 hours after HCG administration. h50 IU of HCG intravenously.
365
HCG-INDUCED OVULATION
Vol. 26, No.4
TABLE 2. Follicle Counts after HCG-lnduced 8 Ovulation in 22 Rabbits Ovary Intact (control)
Denervated
Rabbit no.
Early mature
8
9
3 3 2 1 1
10 11
12 14 15 16 17 18 19 20 21 23 25 26 27 28 29 30 33 40 8
5 1 2 1 1 1 3 1 1 4 2 2 2
Preovulatory
6 3 2 2 4 3 2 2 1 1 1 1
1 1 3
Ruptured
4 5 6 3 5 5 4 3 3 2 4 6 4 1 4 4 3 3 1 3 3 4 3 3
Hemorrhagic
Early mature
1 1 1 1 2 2 1 2 3
Hours
Preovulatory
2 1 1 1 5 4 2 2 1 1 1 3
2 1 1 1 2 1
4
2
3 2 4 2
2 3
Ruptured
Hemorrhagic after HCG
2 2 2 2 3 3 5 6 2 4 2 1 5 3 4 7 2 6 2 3 4 7 3 5
1 1 2 3
1
1
10 12 14 16 10 12 14 16 16 11
18 17 17 17 17 17 17 17 17 17 20 20 20 20 20 20 22 10
50 1U of HCG intravenously.
rupture became greater as the interval from the time of HCG injection increased. There was no significant difference between the curves for the denervated and intact ovaries. Histologic Observations. As confirmed by our previous study/ 5 the denervation technique resulted in no apparent ischemic or trophic changes in the routine hematoxylin and eosin stained sections of the ovaries. In addition, the fluorescent stain demonstrated the presence of adrenergic nerve fibers in the intact ovaries, while no such fluorescent structures were found in the denervated specimens (Figs. 2 and 3). Microscopic examination also revealed no difference between the deHours After HCG (50IU lntrtn•nously) nervated and the intact ovaries in the FIG. 1. Graphic representation of the percent of mature follicles which ruptured at increasing inter- distribution of maturing follicles throughvals after HCG administration. Note the nearly out the cortex. In other words, no increase identical curves of denervated and control ovaries. in the numbers of mature subcortical
percentages of stimulated follicles which ruptured after HCG is illustrated graphically in Figure 1. The proportion of stimulated follicles which progressed to
366
WEINERETAL
FIG. 2. Stroma of an intact ovary demonstrating numerous fluorescing adrenergic nerve fibers (arrows) (x 40).
April1975
FIG. 3. Stroma of a denervated ovary devoid of fluorescent adrenergic nerve fibers (x40).
follicles which might not have been ap- ovarian, postganglionic neurons has been parent at the time of laparotomy was suggested, 18 and the endogenous activity noted. Finally, no significant numbers of these fibers may remain intact after of retained ova in apparently ruptured the ovary is decentralized. Indeed, the follicles could be detected. spontaneous contractility of the in vitro rabbit ovary and its responsiveness to DISCUSSION adrenergic agents 7 suggests that this Ovarian denervation did not alter folli- organ possesses endogenous neuromuscular activation, maturation, or ovulation cular mechanisms. Other studies suggest in the rabbit after intravenous injection of ~hat systemically administered adrenHCG. The vascular stripping technique ergic agents which alter ovarian contracused in these studies rendered the ovary tility and ovulation also act on a local devoid of adrenergic nerves. No specific ovarian level, rather than through the histologic stain for cholinergic fibers was central nervous system. 8 Nevertheless, used, but the ovary was probably severed the hypothalamus remains an important from all its connections with the central mediator in the reflex ovulation noted nervous system by this method. Never- in some mammals. 16 While an efferent theless, the presence of entirely intra- hormonal message from the hypothal-
Vol. 26, No. 4
367
HCG-INDUCED OVULATION
amus to the preovulatory follicle has been demonstrated with the identification of the"LH-surge," a comparable efferent neural pathway remains in doubt. The results of this study suggest that such a pathway, if it exists at all, is not essential for HCG-induced ovulation to occur in the rabbit. HCG, however, stimulates ovulation by a local effect on the ovary. We are, therefore, presently investigating the effect of ovarian denervation on coitus-induced ovulation in the rabbit, since this process is mediated by the central nervous system. While the neuromuscular activity of the human ovary appears quite similar to that of the rabbit, 19 the apparent absence of reflex ovulation in the primate makes extrapolation of these results to human ovarian physiology uncertain. The implications are nevertheless suggestive for more completely defining the neurohormonal mechanisms controlling mammalian ovulation. In conclusion, HCG-induced follicular development and rupture are not changed after ovarian denervation in the rabbit. This suggests that these ovarian functions may be independent of central nervous system connections. Since coitus-induced ovulation is mediated by the hypothalamus in those mammals capable of reflex ovulation, the effect of ovarian denervation on this physiologic sequence is currently being investigated. These studies should define whether an efferent neural pathway, in addition to the recognized hormonal mechanisms, is essential for reflex ovulation in the rabbit. SUMMARY
The described method for selective sympathetic denervation of the in vivo rabbit ovary involved stripping the ovarian artery of its nerve bundles and adventitial tissue. The ovary was then entirely free of fluorescent-staining adrenergic nerves.
This technique was used to study the effects ot' ovarian denervation on HCGinduced ovulation. After HCG was administered to 22 rabbits which had previously undergone unilateral ovarian denervation, the ovaries were observed for follicular maturation and rupture. Control ovaries demonstrated a mean of 5.6 stimulated follicles/ovary; denervated ovaries had a mean of 5.4. An average of 3.5 follicles/control ovary ruptured; an average of 3.1 follicles/denervated ovary ruptured. Furthermore, the time course of ovulation after HCG did not differ between denervated and intact ovaries. These results indicate that HCG-induced ovulation in the rabbit is not interrupted by ovarian denervation. Acknowledgments. The authors gratefully acknowledge the assistance of Thomas Henry and Susan Pisano. We also wish to thank Pramuan Virutamasen, M.D., for his invaluable advice during the early parts of this study. Human chorionic gonadotropin (APL) was generously supplied by Garfield Davies, Ayerst Laboratories, New York, New York.
REFERENCES 1. Fink G, Schofield GC: Experimental studies on
2.
3.
4.
5.
6.
the innervation of the ovary in cats. J Anat 109:115, 1971 Brink CE, Grob HS: Response of the denervated mouse ovary to exogenous gonadotropins. Presented at the Sixth Annual Meeting of the Society for the Study of Reproduction, Athens, Georgia, August 13-16, 1973 Spies HG, Niswender GD: Levels of prolactin, LH and FSH in the serum of intact and pelvicneuroectomized rats. Endocrinology 88:937, 1971 Hill RT: Paradoxical effects of ovarian secretions. In The Ovary. Vol. II. Edited by S Zuckerman. New York, Academic Press, 1962, p 231 Neilson D, Jones GS, Woodruff JD, et al: The innervation of the ovary. Obstet Gynecol Surv 25:889, 1970 Labate JS, Reynolds SRM: Sensory pathways of the ovarian plexus. Am J Obstet Gynecol 34:1, 1937
368
WEINERETAL
April1975
7. Virutamasen P, Wright KH, Wallach EE: 14. Bahr J, Kao L, Nalbandov AV: The role of Effects of catecholamines on ovarian contraccatecholamines and nerves in ovulation. Bioi tility in the rabbit. Obstet Gynecol 39:225, Reprod 10:273, 1974 1972 15. Weiner S, Wright KH, Wallach EE: Selective 8. Ferrando G, Nalbandov AV: Direct effect on ovarian sympathectomy in the rabbit. Fertil the ovary of the adrenergic blocking drug Steril 26:353, 1975 dibenzyline. Endocrinology 85:38, 1969 16. Law OT, Sackett GP: Hypothalamic potentials in 9. Marshall JM: Adrenergic innervation of the the female rat evoked by hormones and female reproductive tract: Anatomy, physiology vaginal stimulation. Neuroendocrinology 1:31, and pharmacology. Rev Physiol 62:6, 1970 1965 10. Marshall JM: Effects of catecholamines on the 17. Falck B, Hillarp NA, Thiene G, et al: Fluoressmooth muscle of the female reproductive tract. cence of catecholamines and related compounds Ann Rev Pharmacal 13:19, 1973 condensed with formaldehyde. J Histochem 11. Espey LL, Lipner H: Measurement of intraCytochem 10:348, 1962 follicular pressure in the rabbit ovary. Am J 18. Owman C, Sjostrand NO: Short adrenergic Physiol 205:1067, 1963 neurons and catecholamine-containing cells in 12. Okamura H, Virutamasen P, Wright KH, et vas deferens and accessory male genital glands al: Ovarian smooth muscle in the human being, of different mammals. Z Zellforsch Abt rabbit, and cat: Histochemical and electronHistochem 66:300, 1965 microscopic study. Am J Obstet Gynecol 112: 19. Diaz-Infante Jr. A, Virutamasen P, Connaugh183, 1972 ton JF, et al: In vitro studies of human 13. Jacobowitz D, Wallach EE: Histochemical and ovarian contractility. Obstet Gynecol 44:830, chemical studies of the autonomic innervation 1974 of the ovary. Endocrinology 81:1132, 1967