Partial denervation of the ovaries by transection of the suspensory ligament does not inhibit ovulation in rats treated with pregnant mare serum gonadotropin.код для вставкиСкачать
THE ANATOMICAL RECORD 213:392-395 (1985) Partial Denervation of the Ovaries by Transection of the Suspensory Ligament Does Not Inhibit Ovulation in Rats Treated With Pregnant Mare Serum Gonadotropin GUNNAR SELSTAM, ENS10 NORJAVAARA, TOMAS TEGENFELT, SVEN LUNDBERG, CHRISTINA SANDSTROM, A N D SVEN A K E PERSSON Department o f Physiology, University o f Ume& S-901 87 Ume&(G S , E N , T T , C S ) and Division of Expenmental Medicine, National Defence Research Institute, Department 4, S-901 82 Umec (S L , 5' -A P ) , Sweden ABSTRACT Adrenergic nerves reach the ovary via two routes: along the arteries to the ovary and via the suspensory ligament. Results from earlier investigations suggest that denervation of the nerves along the arteries does not influence the ovulatory process. In the present study we have examined whether denervation by transection of the ovarian suspensory ligament influences the ovulatory process. Partial denervation of the ovary by transection of the ovarian suspensory ligament, sham operation, or only anesthesia were performed on immature 25-day-old rats. To induce ovulation, pregnant mare serum gonadotropin (PMSG) was injected in the morning (0800-09301, when the rats were 26 days old. This PMSG treatment normally induces ovulation around 0200 in the early morning of day 29 with subsequent formation of corpora lutea. Rats were killed 5-8 hr, 3 days, and 5 days after this ovulation time. Ovarian interstitial norepinephrine levels were markedly decreased after transection of the suspensory ligament. Ovulations had occurred in all denervated, as well as sham-operated, and control rats. The various groups did not differ in the number of ovulations per rat. Thus, the adrenergic nerves in the suspensory ligament appear not to be necessary for ovulation. Whether catecholamines themselves play a role in the ovulatory process cannot be elucidated from this experiment, since the norepinephrine content in the ovary was not totally depleted. It seems unlikely that adrenergic nerves reach the corpus luteum via the suspensory ligament, since transection of this structure did not change the luteal content of norepinphrine. Adrenergic nerve terminals have a close relation to the follicular wall in the Graafian follicle of the ovary as first shown for the rabbit (Owman and Sjoberg, 1966) and later for a large number of other mammalian species (for reference see Bahr et al., 1974; Burden, 1978; Stefenson et al., 1981).The theca externa layer contains smooth muscle elements together with adrenergic nerve endings. It has been suggested that contractile activity in this fibromuscular unit could play a role in the ovulatory process (Owman et al., 1979).Recently it has been found that adrenergic nerves pass not only along the ovarian and uterine arteries, but also via the ovarian suspensory ligament and end in the ovary (Lawrence and Burden, 1980).These authors also showed that transection of these nerves caused a n approximately 50% decrease in the ovarian content of norepinephrine as well as disappearance of free nerve endings, whereas nerves along vessels remained intact. In the present investigation we have examined whether the nerves passing through the suspensory ligament are necessary for the ovulatory process. The inci0 1985 ALAN R. LISS, INC. dence of ovulations and ovulatory rates were determined in a well-characterized ovulatory model (pregnant mare serum gonadotropin [PMSG] rat ovulatory model) after denervation by transection of the suspensory ligament. MATERIALS AND METHODS Animals Female Sprague-Dawley rats (Anticimex, Sollentuna, Sweden) were used. The rats were kept under standardized environmental conditions (temperature 22-25°C; light period 0500-1900; humidity 40-60%) and were given free access to standard diet pellets (R3, AstraEwos) and water. Animal care was carried out daily at Received J a n u a r y 28, 1985; accepted J u n e 12, 1985. E. Norjavaara's present address is: Department of Physiology, University of G t e b o r g , S-400 33 Goteborg, Sweden. Address reprint requests to Dr. Gunnar Selstam, Endocrine Division, Department of Physiology, University of Umeb, 901 87 Umeb, Sweden. 393 OVULATION AFTER PARTIAL OVARIAN DENERVATION the end of the day. No other rats, male or female, were kept in this animal room. Denervation Immature 25-day-old rats were denervated. The rats were anesthetized with pentobarbital and the abdomen was opened with bilateral dorsal incisions. The ovarian suspensory ligament was localized and two silk threads were tied a few millimeters apart. The part of the ligament between the ligatures was cut away. The muscle layer and skin were sutured separately with ligatures. This procedure is similar to that described by Lawrence and Burden (1980), except that the ligament was surgically transected and not cauterized. In sham-operated rats, the ligament was exposed and silk threads were introduced, but not tied. Control rats were anesthetized only. Induction of Ovulation A well-characterized rat ovulatory model was used in which follicular maturation and ovulations are induced shortly before normal puberty. A single dose of 6 IU or 8 IU of pregnant mare serum gonadotropin (Sigma) was injected subcutaneously into the neck skin in the morning (0800-0900) when rats were 26-days-old,i.e., the day after denervation. This treatment is known to induce follicular maturation, and in the afternoon of day 28 a preovulatory endogenous surge of gonadotropins occurs that results in ovulation early around 0200 of day 29. On day 29, ovulations are easily recognized in a stereomicroscope by the presence of rupture points on newly formed corpora lutea and the appearance of oocytes in the ampullar region. The length of the luteal phase is approximately 10-12 days after which these rats start regular estrous cycles. For details and references about the model see, for example, Norjavaara (1984) or Norjavaara et al. (1984). That ovulations had occurred a t 5-8 h r after the ovulation time was checked as described above. Occurrence of ovulations at 3 and 5 days after the ovulation time was checked in a stereomicroscope by counting of corpora lutea. In experiments performed 5-8 hr after the ovulation time, oocytes were always found in the ampullar region of the oviduct. and 0.5 M Tris-HC1 buffer (pH 8.4-8.6). The tubes were gently agitated and then centrifuged. The catecholamines were eluated with 200 pl 1 M perchloric acid, and 10-20 p1 of the eluate was injected into a highperformance liquid chromatography (HPLC) system. This system consisted of the solvent delivery system 6000A and the U6K loop injector (Waters Assoc., Millford, MA) equipped with a n LC-4 electronic controller, a glassy carbon electrode, and an Ag/AgCl reference electrode (Bioanalytical Systems, West Lafayette, IN). The working potential was 700 mV. Norepinephrine was separated on a n analytical column (Nucleosil 5 SA) of dimensions 20 mm x 4.6 mm without a precolumn. The mobile phase was a sodium acetatekitrate buffer (5.75 g citric acid, 6.80 g sodium acetate and 2.40 g sodium hydroxide per liter; pH adjusted to 5.2 with acetic acid). It was degassed and filtered (Millipore 0.2 pm) and delivered at ambient room temperature at a rate of 2 ml/min. Norepinephrine was quantitated by comparing peak height ratio between a sample and internal standard, DHBA, with that of a n authentic standard of norepinephrine. The method used is essentially a modification of that described by Keller et al. (1976). Statistical Analyses Values are given as mean f SEM. Comparisons between groups were made by the Mann-Whitney nonparametric U test (Siegel, 1956). A P value of less than 0.05 was considered significant. RESULTS The effects of transection of the ovarian suspensory ligament on ovulation and norepinephrine content in the whole ovary 5-8 h r and 3 days after ovulation are shown in Table 1. Transection of the ovarian suspensory ligament resulted in a marked decrease in the norepinephrine content of the ovary that was seen both 5-8 hr and 3 days after ovulation. Despite the decrease in norepinephrine content neither the incidence nor the number of ovulations was inhibited (Table 1). The catecholamines dopamine and epinephrine could not be detected in the ovary (data not shown). To determine in which compartment in the ovary the decrease in norepinephrine content occurs after suspenDetermination of Norepinephrine sory ligament transection, the content of norepinephrine Rats were decapitated with a guillotine and ovaries was determined in extirpated corpora lutea and in the were rapidly excised and placed in ice-cold saline. Sur- remaining ovary after all corpora lutea had been extirrounding tissue was dissected away and the ovaries pated (this compartment is referred to as interstitium). were inspected for signs of ovulation. Whole ovaries, all The decrease of ovarian norepinephrine content after corpora lutea from one ovary, or the interstitial tissue this type of denervation was registered only in the inter(the remaining ovary after extirpation of all corpora stitial compartment, since the corpus luteal levels of lutea) were placed in preweighed tubes containing a norepinephrine was unaltered (Table 2). Also in this standard solution. This solution contained 0.3-0.5 ml experiment no changes in number of ovulations ocice-cold 0.1 M perchloric acid with 0.4 mM sodium bi- curred. sulphite and the internal standard 3,4-dihydroxybenzylDISCUSSION amine (DHBA, Sigma). The tubes were weighed and deep-frozen (-80°C) until analysis. The frozen tissue The present study demonstrates that interruptions of samples were thawed and homogenized by pulsed soni- nerves in the ovarian suspensory ligament, with subsecation for 1-1.5 min with a Branson sonifier (model B15, quent reduction in ovarian levels of norepinephrine, does output level a t setting 7) equipped with a microtip. The not influence the number of ovulations. Therefore, these duty cycle was set a t 60% and the tubes were cooled in nerves seem not to play an obligatory role in ovulation. ice during sonication. After centrifugation at 9,000 g for The participation of catecholamines in the ovulatory 5 min the supernatant was transferred to a test tube process cannot, however, be ruled out, since the ovary containing acid-washed A1203 (Anton and Sayre, 1962) was not totally depleted of norepinephrine. The remain- 394 G. SELSTAM ET AL. TABLE 1. Norepinephrine content of the ovary and ovulatory rates after partial denervation by transection of the suspensory ligament' Time after ovulation 5-8 hr 3 days Group N Control 4 Sham-operated3 Denervated 6 Sham-operated4 Denervated 6 Ovulation Number of ovulations Incidence per rat' 414 10.2 k 0.9 313 6/6 414 616 Norepinephrine content 9.0 f 0.9 10.2 f 0.8 (NS) 13.3 i 0.9 14.3 f 2.2 (NS) Ovarian weight (mgY (nglg ovarian wet weight) 41.8 f 5.3 44.5 f 4.0 45.1 f 1.8(NS) 41.3 f 3.4 48.7 f 3.7 (NS) 114 f 28 94 f 12 27 f 3* 184 f 26 29 f 10* 'Denervation was performed by transection of the ovarian suspensory ligament with its nerves in immature 25-day-old rats. In shamoperated rats the abdomen was opened and ligatures were put in place but not tied. Control rats were anasthetized only. The rats were given 6 IU of PMSG on day 26 of age. This treatment is known to induce ovulation around 0200 of day 29. Rats were killed 5-8 hr and 3 days after the ovulation time. Ovulations were examined and norepinephrine content measured as described in Materials and Methods. Values are presented as mean f SEM. Differences between groups were tested by the Mann-Whitney U test. 2NS = not significant. *P < 0.05 versus control or sham-operated rats. TABLE 2. Norepinephrine content of ovarian interstitiurn and corpus luteum and ovulatory rates after partial denervation by transection of the suspensory ligament' Grouu N Sham-operated 8 Denervated 8 Incidence 818 818 Ovulation Number of ovulations in left ovaryz 9.4 f 2.2 8.0 1.5 (NS) Norepinephrine content (ngig wet wt.) Interstitium Coruus luteurn' 271 f 20 146 f 16* 35 f 6 37 + 6 (NS) 'Ovarian suspensory ligament with its nerves were transected surgically in immature 25-day-old rats. The rats were given 8 IU of PMSG on day 26. This treatment is known to induce ovulation around 0200 of day 29 with subsequent formation of corpora lutea. The rats were killed when 33 days of age, bearing 5-day-old corpora lutea. For estimation of ovulatory rates and norepinephrine content see Materials and Methods. Values are presented as mean k SEM. Differences between groups were tested with Mann-Whitney U test. 'NS = not significant. *P < 0.05 compared to sham-operated rats. ing norepinephrine content may have been sufficient to support ovulation; alternatively, a n increased sensitivity to norepinephrine may have been elicited. This later possibility was pointed out by Owman and co-workers (Owman et al., 1979), who suggested that contractility of the follicular wall promoted the ovulatory process and that contractile sensitivity to catecholamines increased after denervation of sympathetic nerves following the ovarian arteries. Increased sensitivity to catecholamines may also occur in the ovary after transection of the suspensory ligament in the rat, since the ovarian Padrenergic receptor content has been shown to increase after this type of denervation (Aguado and Ojeda, 1984a). That catecholamines can participate in ovulation is also supported by several in vivo and in vitro studies in which adrenergic antagonists have been shown to inhibit the ovulation (for references see Owman et al., 1979 and Moudgal and Razdan, 1981) and by the fact that adrenergic agonist can enhance ovulation (Kobayashi et al., 1983). Furthermore, the fact that ovarian tissue autografted to the subcutis demonstrates that ovulation is inhibited although follicles develop and luteinize with entrapped ova (Chichal et al., 1976). However, further studies are needed to elucidate the mechanism and the role of catecholamines and other amines in the ovulatory process. Lawrence and Burden (1980)reported that transection of the suspensory ligament resulted in disappearance of free nerve endings throughout the interstitium. Interestingly, in the present study transection of the suspensory ligament decreased norepinephrine content in the interstitial compartment of the ovary, but not in the corpus luteum. If the luteal norepinephrine is localized in nerve endings, this finding may indicate that the sparse innvervation of the rat corpus luteum (Burden, 1978)is mainly of vascular origin and does not reach the corpus luteum via the suspensory ligament. We conclude that redundant activity of nerves in the suspensory ligament is not necessary for ovulation. However, activities in adrenergic nerves of the suspensory ligament andor along arteries to the ovary may influence other ovarian processes such as steroidogenesis (Burden and Lawrence, 1977; Capps et al., 1978; Weiss et al., 1982; Aguado and Ojeda, 1984b). ACKNOWLEDGMENTS This study was supported by the Swedish Medical Research Council (04X-05653), Stiftelsen Lars Hiertas OVULATION AFTER PARTIAL OVARIAN DENERVATION Minne, A ke Wibergs Stiftelse, and the Swedish Medical Association. LITERATURE CITED Aguado, L.I., and S.R. Ojeda (1984b) Ovarian adrenergic nerves play a role in maintaining preovulatory steroid secretion. Endocrinology, 114t1944-1946. Aguado, L.I., and S.R. Ojeda (1984a) Prepubertal ovarian function is finely regulated by direct adrenergic influences. Role of noradrenergic innervation. Endocrinology, 114: 1845-1853. Anton, A.H., and D.F. Sayre (1962) A study of the factors affecting the aluminum oxide trihydroxyindole procedure for the analysis of catecholamines. J.Pharmacol. Exp. Ther., 138:360-375. Bahr, J., L. Kao, and A.W. Nalbandov (1974) The role of catecholamines and nerves in ovulation. Biol. Reprod. 10273-290. Burden, H.W. (1978) Ovarian innervation. In: The Vertebrate Ovary. R.E. Jones, ed. 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