Original Paper Neuroendocrinology 1992;56:393-396
3 Department of Psychology, Harvard University, Cambridge, Mass.; b Institute of Animal Behavior, Rutgers The State University, Newark, N.J., USA
Key Words Ovulation Caesarian section Pelvic nerve Hypogastric nerve
Reflexive Ovulation in the Rat Induced by Caesarian Section, Is Blocked by Pelvic and/or Hypogastric Nerve Transection
Abstract Reflexive ovulation in the rat, induced by caesarian section performed on day 22 of pregnancy, was blocked by prior bilateral transection of the pelvic and/ or hypogastric nerves, which convey afferent activity from the reproductive tract. Tubal ova and hemorrhagic ovarian follicles were counted 24 h after bilateral nerve transections or sham neurectomy. Whereas the median num bers of ova and hemorrhagic follicles in the sham neurectomy group were 4.5 and 3.5, respectively, these values in each of the neurectomy groups were 0. The present findings indicate that caesarian section activates the pelvic and hypogastric nerves to trigger ovulation. This suggests that normally the rat is a spontaneous ovulator during the estrous cycle (nonpregnancy) phase of its reproductive cycle, and may become a reflexive ovulator at the parturient phase of its reproductive cycle.
Rats and mice are typically regarded as species that ovulate spontaneously, in response to circadian light/ dark cycle-induced hormonal changes [1]. Ferrets, cats, minks, rabbits and voles are typically regarded as reflexive ovulators, in which ovulation is triggered by coitus [2, 3], In rats, reflexive ovulation has been induced experimen tally, e.g. by vomeronasal stimulation [4] or by the sensory stimulation that results from mating, under conditions in which spontaneous ovulation is blocked by exposure to constant light [5], or by barbiturate. In the latter case, the reflexive ovulation was blocked by vaginal deafferentation [6], Ovulation has also been induced by mating stimula tion in mice [7] and immature rats [8] under appropriate honnonal priming conditions [3].
Received: July 10. 1991 Accepted after revision : Januaiy23, 1992
In addition to the above experimentally manipulated conditions, under natural conditions, spontaneous ovula tors may normally become reflex ovulators at a specific phase of their reproductive cycle, i.e. parturition. It has been suggested that postpartum ovulation in the rat may be triggered by the LH release induced by the sensory mechanostimulation of the uterus that occurs during de livery of the fetuses at parturition [9]. This is based on observation of a substantial increase in serum LH starting as early as 2 h (usually 3.5-11 h) after delivery, the major release persisting for 4-6 h [9], Consistent with this are the findings that barbiturate or LH-antiserum administra tion, immediately following parturition, was most effec tive in blocking ovulation. That is, phénobarbital com-
B.R. Komisaruk Institute o f Animat Behavior Rutgers - The State University Newark, NJ 07102 (USA)
Downloaded by: King's College London 137.73.144.138 - 1/18/2019 5:59:54 AM
5. Tiffany Cunninghama Jay S. Rosenblatt Barry R Komisarukh
pletely inhibited postpartum ovulation when adminis tered during the hour following delivery, partially inhib ited the ovulation when administered 1-7 h postpartum, and failed to inhibit ovulation when administered 8-9 h postpartum [10]. LH-antiserum successfully blocked ovu lation only when administered within 1 h postpartum [10]. Furthermore, Johnson [11] found that the time of day when the postpartum LH surge and subsequent ovu lation occurred covaried, on an individual basis, in close temporal relation to the time of day when the individual parturition occurred. The phase of the light cycle interacts with the time of parturition in determining the timing of postpartum ovulation [11, 18]. These findings suggest that there exist both a reflexogenic and a circadian component to the control of postpartum ovulation. As a test of the possible role of uterine afferent activity in stimulating postpartum ovulation, Johnson [11] per formed caesarian section (CS) at 9 a.m. on day 22 of preg nancy and observed that ovulation followed within 18 h. Ligature of the uterine horns at the time of CS blocked the induced ovulation [11], It seems likely that the ovula tion blockage was due to the uterine ligation’s interrup tion of uterine afferent activity generated by the CS proce dure. Since uterine afferent activity is conveyed via the hypogastric and pelvic nerves [ 12, 13], we hypothesized that transection of the hypogastric and/or pelvic nerves prior to CS (on day 22 of pregnancy) would interrupt the ovulation-inducing effect of CS. If reproductive tract (i.e. uterus, cervix and vagina) denervation indeed blocks CSinduced ovulation, this would support the concept that the sensory mechanostimulation generated by the pas sage of the fetuses through the reproductive tract during natural parturition reflexively stimulates ovulation by generating reproductive tract afferent activity. These re sults have been presented in abstract form [14].
males were moved from their wire bottom cages and placed into tubs with hardwood shavings (Beta-Chip, Warrensburg, N.Y., USA). On day 22, each female received one of the following treatments: (1) CS plus bilateral pelvic and hypogastric neurectomy (n = 5), (2) CS plus bilateral pelvic neurectomy (n = 7), (3) CS plus bilateral hypogastric neurectomy (n = 5), or (4) CS plus sham neurectomy (n = 6). CS was performed on all animals immediately following nerve transections, between 9 a.m. and 6 p.m. To determine whether they had ovulated, the ovaries and fal lopian tubes were removed and examined in all females between 23 and 24 h post-CS. The ovaries were examined microscopically for the number of ruptured follicles, and the fallopian tubes for the number of ova present [10]. For this procedure, females were anes thetized with a mixture of Ketaset and Rompun (0.1 m l/100 g bodyweight). On the day of surgery, animals were anesthetized with a mixture of chloral hydrate, alcohol and water (I g/ml ethanol/1.5 ml dis tilled water; dose 300 mg chloral hydrate/kg body weight). An inci sion was made along the midline of the ventrum, approximately 2 inches rostral to the most caudal nipple. Each uterine horn (fetusfilled) was reflected to one side in order to expose the colon as a landmark; each horn was then covered with saline-soaked gauze. With the colon pulled to one side, the hypogastric nerve was identi fied leaving the mesentericus ganglion and coursing alongside the ureter. A longitudinal incision was made, cutting the nerve in two places 3-5 mm apart, and the segment was removed. The colon was pulled to the opposite side and the procedure was repeated for bi lateral transection. To locate the pelvic nerve, the bifurcation of the vena cava into the common iliac veins was identified as a landmark. Where the internal iliac vein disappears in the dorsal muscle of the rat, the pelvic nerve is visible. Gentle retraction of the muscle exposed the nerve. Here, too, a longitudinal incision (2-4 mm) was made and this procedure was repeated on the opposite side. For sham neurec tomies, the aforementioned procedues were followed for location of both the hypogastric and pelvic nerves. Each animal was observed for intact nerves, the nerves were lifted, but no incisions were made. After completion of the designated neurectomies or sham oper ations, a small incision was made on either uterine horn (above the cervix) and the pups and the placenta were manually expelled from the uterus. Each horn was sutured with 6-0 surgical thread and re placed. The abdominal wall was sutured with 4-0 surgical thread and the incision closed with wound clips.
Materials and Methods Results
394
As summarized in table 1, the incidence of CS-induced ovulation, measured by direct visualization of ova in the fallopian tubes and ovulation points in the ovaries 24 h post-CS, was significantly attenuated or abolished by hypogastric and/or pelvic neurectomy. The median num ber of ova and ovulation points observed in the CS-only group was 4.5 and 3.5, respectively, whereas the median number of ova and ovulation points in each of the three other groups: hypogastric neurectomy only, pelvic neur ectomy only, and combined hypogastric-pelvic neurec-
Cunningham/Rosenblatt/Komisaruk
Neurectomy Blocks Caesarian-Section Induced Ovulation in Rats
Downloaded by: King's College London 137.73.144.138 - 1/18/2019 5:59:54 AM
A total of 23 Sprague-Dawley CD (Charles River, Kingston, N.Y., USA) females (90 days or older), either IAB bred or pur chased from Charles River, were kept socially housed (4-5 rats per cage) in wire bottom cages on a reverse 12-hour light/dark cycle (lights on 7 p.m.). Food and water were available ad libitum for the duration of the experiment. Females were mated during early after noon. Day 1 of gestation was designated as the day when mating was confirmed by the presence of a copulation plug under the wire cage or when sperm were found in the vaginal smear taken early the next morning. Animals were then individually housed on day 2 on the same reverse light cycle. On day 8, females were moved to a normal 12-hour light/dark cycle (lights on 7 a.m.), in order to syn chronize births on day 22. The day before parturition, day 21, fe
tomy, was zero. Only one rat ovulated in any of the neur ectomy groups, and it had received pelvic neurectomy. Each of the neurectomy groups differed significantly from the control CS-only group on each measure. Specifically, the median number of ova in each neurectomy group (all 0) was significantly lower than in the control, CS-only group (4.5) (p < 0.01, Mann-Whitney U test). Further more, the median number of ovulation points in each neurectomy group (0) was significantly lower than in the control CS-only group (3.5) (p < 0.05-0.01, MannWhitney U test).
Table 1. Effect of transection of the pelvic and/or hypogastric nerves on ovulation induced by CS
Group
n
Ova
Ovulation points
CS only CS + pelvic neurectomy + hypogastric neurectomy CS + pelvic neurectomy CS + hypogastric neurectomy
6
4.5 (1-11)
3.5 (1-5)
5 7 5
0 (0) 0 (0-2) 0 (0)
0 (0) 0 (0-8) 0 (0)
Values indicate median with the range given in parentheses. The number of ovulation points observed did not always equal the num ber of ova observed.
Discussion
lease that stimulates ovulation. Although it seems un likely that the neurectomy procedure in the present study would interfere with the action of released gonadotropin, we cannot rule out that possibility. The rat is commonly characterized as a spontaneous ovulator, i.e. ovulation occurs during the estrous cycle in the absence of specific mating stimulation but determined by the timing of the light cycle. The present findings sug gest that in addition, the rat may normally manifest re flexive ovulation at a specific phase of its reproductive cycle, i.e. at parturition, when the timing of ovulation is determined by the time of parturition as well as by the timing of the light cycle. There is evidence [18] that these two types of ovulation are different, on the basis that pen tobarbital is more potent in blocking cyclical than post partum ovulation. LH release begins earlier and persists longer when it occurs postpartum than when it occurs during the normal 4-day cycle [ 18], With regard to innervation of the reproductive tract, the hypogastric nerve provides sensory innervation pre dominantly of the proximal two thirds of the uterus and the cervix [12, 13], whereas the pelvic nerve provides sen sory innervation predominantly of the cervix and vagina [19] with minimal innervation of the caudal body of the uterus [ 13]. The firing activity of the hypogastric nerve is increased when fetuses are moved through the cervix [12]. Significant reduction of the incidence of CS-induced ‘postpartum’ ovulation resulted from transection of either or both of these pairs of nerves, and the region innervated in common by both nerves is the cervix. Therefore, it is likely that afferent activity from the cervix via both the hypogastric and pelvic nerves mediates the full expression of this reflexive ovulation. Thus, in the case ofligature of the uterine horns, cervical stimulation by the fetuses was
395
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In the present study, we make the assumption that the sensory stimulation generated by CS is partially com parable to that generated during parturition, because all fetuses are pushed individually toward the cervical end of the uterus where they are expelled via a small incision. Therefore in this CS procedure, it is likely that uterine and possibly cervical, although not vaginal, afferent activity is generated. The median number of ova observed in the present study (4.5) was lower than that reported postpar tum; i.e. it was found that as a result of postpartum mat ing, 12.7 ± 2.3 (mean ± SD) pups were born [15]. Hence, at least this number of ova had been shed postpar tum. (It was also reported in that study that there was no significant difference between the number of pups born as a result of postpartum mating versus mating during the estrous cycle.) We conclude that if the afferent activity re sulting from parturition is the stimulus for postpartum ovulation, then CS is a suboptimal stimulus in that regard. In order to study the effects of neurectomy, natural parturition is the obvious stimulus of choice. However, this is technically difficult, because pelvic neurectomy in terferes with natural parturition by blocking abdominal striated muscle contractions that are coordinated with uterine contractions [16, 17], We are currently engaged in studies that utilize neurectomized females undergoing normal parturition with manual assistance. The present findings, that deafferentation of the repro ductive tract on day 22 of gestation in the rat blocks CSinduced ovulation, imply that under normal conditions afferent activity emanating from the reproductive tract stimulates postpartum ovulation. The mechanostimulation normally generated by the fetuses as they pass through the birth canal at the time of parturition very likely provides the afferent signal for gonadotorpin re-
prevented and ovulation did not occur [11]. A contribu tion by uterine and/or vaginal afferent activity cannot be ruled out. These nerves also play a role in other effects of repro ductive tract mechanostimulation during the reproductive cycle in the rat. The pelvic nerves mediate the induction of pregnancy [ 17], pseudopregnancy [20] and reflexive ovu lation in response to vaginal stimulation [6]. Based on deafferentation studies, pelvic neurectomy, but not hypo gastric neurectomy, disrupts the micturition reflex and parturition [ 16]. The hypogastric nerves mediate analgesia during pregnancy [21 ]. Analgesia produced by vaginocervical mechanostimulation is mediated by both nerves [22 ].
After parturition, the rat normally mates within 9 h [23] and ovulates within 24 h [11]. The sensory stimula tion generated by parturition could trigger these events, serving an adaptive function of minimizing the latency for the initiation of the rat’s subsquent pregnancy.
Acknowledgments We gratefully acknowledge the technical assistance of Dr. Anne D. Mayer, and the advice of Dr. Carlos Beyer. Supported by NiH grants NLS-25R01NS22948 and GRS 5 S06 RR08223, and the Charles and Johanna Busch Foundation (B.R.K.) and by the Rutgers-CINVESTAV International Exchange Program. Contribution number 537 from the Institute of Animal Behavior.
1 Komisaruk BR, Steinman JL: Genital stimu lation as a trigger for neuroendocrine and be havioral control of reproduction: in Repro duction: A Behavioral and Neuroendocrine Perspective. Ann NY Acad Sei 1986;64—75. 2 Feder HH: Estrous cyclicity in mammals; in Adler NT (ed): Neuroendocrinology of Re production. New York, Plenum Publishing, 1981, pp 279-348. 3 Komisaruk BR, Terasawa E, Rodriguez-Sierra JF: How the brain mediates ovarian response to environmental stimuli; in Adler NT (ed): Neuroendocrinology of Reproduction. New York. Plenum Publishing. 1981, pp 352-355. 4 Johns MA. Feder HH, Komisaruk BR. Mayer AD: Urine-induced reflex ovulation in anovu latory rats may be a vomeronasal effect. Na ture 1978;272:446-448. 5 Brown-Grant K, Davidson JM, Greig F: In duced ovulation in albino rats exposed to con stant light. Horm Behav 1977;8:62-76. 6 Harrington FE. Eggert RG. Wilbur RD: In duction of ovulation in chlorpromazineblocked rats. Endocrinology' 1967:81:877881. 7 Aron C, Asch G, Roos J: Triggering of ovula tion by coitus in the rat. Int Rev Cytol 1966;20:139. 8 Zarrow MX, Clark JH: Ovulation following vaginal stimulation in a spontaneous ovulator and its implications. J Endocrinol 1968: 40:343-352.
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9 Rebar RW, Nakane PK, Midgley AR Jr: Post partum release of luteinizing hormone (LH) in the rat as determined by radioimmunoas say. Endocrinology 1969;84:1352-1358. 10 Ying S, Gove S, Fang VS, Greep RO: Ovula tion in postpartum rats. Endocrinology 1973;92:108-116. 11 Johnson N: Postpartum Ovulation in the Rat; thesis proposal Purdue University, 1972. 12 Peters LC. Kristal MB, Komisaruk BR: Sen sory innervation of the external and internal genitalia of the female rat. Brain Res 1987; 408:199-203. 13 Berkley KJ, Hotta H. Robbins A, Sato Y: Functional properties of afferent fibers sup plying reproductive and other pelvic organs in pelvic nerve of female rat. J Neurophysiol 1990;63:256-272. 14 Cunningham ST, Rosenblatt JS, Komisaruk BR: Evidence that caesarian-section-induced ovulation in the rat is mediated by the pelvic and hypogastric nerves. Abstr Soc Neurosci 1990; 16:396. 15 Gilbert AN, Burgoon DA, Sullivan KA, Adler NT: Mother-weanling interactions in Norway rats in the presence of a successive litter pro duced by postpartum mating. Physiol Behav 1983;30:267-271.
16 Higuchi T. Uchidc K. Honda K, Negoro H: Pelvic neurectomy abolishes the fetus-expul sion reflex and induces dystocia in the rat. Exp Neurol 1987;96:443-455. 17 Kollar EJ: Reproduction in the female rat after pelvic nerve neurectomy. Anat Rec 1953;115:641-658. 18 Hoffmann JC, Schwartz NB: Timing of postportum ovulation in the rat. Endocrinology 1965;76:620-625. 19 Komisaruk BR. Adler NT. Hutchison: Genital sensory field: Enlargement by estrogen treat ment in female rats. Science 1972:178: 1295-1298. 20 Carlson RR, De Feo VJ: Role of the pelvic nerve vs. the abdominal sympathetic nerves in the reproductive function of the female rat. Endocrinology 1965;77:1014-1022. 21 Gintzler AR, Peters LC, Komisaruk BR: Atte nuation of pregnancy-induced analgesia by hypcgastric neurectomv in rats. Brain Res 1983;277:186-188. 22 Cunningham ST, Steinman JL, Whipple B, Mayer AD, Komisaruk BR: Differential roles of hypogastric and pelvic nerves in the analge sic and motoric effects of vaginocervical stim ulation in rats. Brain Res 1991;559:337-343. 23 Connor JR, Davis HN: Postpartum estrus in Norway rats. Part I. Behav Biol Reprod 1980;23:994-999.
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Neurectomy Blocks Caesarian-Section Induced Ovulation in Rats
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References
3 Department of Psychology, Harvard University, Cambridge, Mass.; b Institute of Animal Behavior, Rutgers The State University, Newark, N.J., USA
Key Words Ovulation Caesarian section Pelvic nerve Hypogastric nerve
Reflexive Ovulation in the Rat Induced by Caesarian Section, Is Blocked by Pelvic and/or Hypogastric Nerve Transection
Abstract Reflexive ovulation in the rat, induced by caesarian section performed on day 22 of pregnancy, was blocked by prior bilateral transection of the pelvic and/ or hypogastric nerves, which convey afferent activity from the reproductive tract. Tubal ova and hemorrhagic ovarian follicles were counted 24 h after bilateral nerve transections or sham neurectomy. Whereas the median num bers of ova and hemorrhagic follicles in the sham neurectomy group were 4.5 and 3.5, respectively, these values in each of the neurectomy groups were 0. The present findings indicate that caesarian section activates the pelvic and hypogastric nerves to trigger ovulation. This suggests that normally the rat is a spontaneous ovulator during the estrous cycle (nonpregnancy) phase of its reproductive cycle, and may become a reflexive ovulator at the parturient phase of its reproductive cycle.
Rats and mice are typically regarded as species that ovulate spontaneously, in response to circadian light/ dark cycle-induced hormonal changes [1]. Ferrets, cats, minks, rabbits and voles are typically regarded as reflexive ovulators, in which ovulation is triggered by coitus [2, 3], In rats, reflexive ovulation has been induced experimen tally, e.g. by vomeronasal stimulation [4] or by the sensory stimulation that results from mating, under conditions in which spontaneous ovulation is blocked by exposure to constant light [5], or by barbiturate. In the latter case, the reflexive ovulation was blocked by vaginal deafferentation [6], Ovulation has also been induced by mating stimula tion in mice [7] and immature rats [8] under appropriate honnonal priming conditions [3].
Received: July 10. 1991 Accepted after revision : Januaiy23, 1992
In addition to the above experimentally manipulated conditions, under natural conditions, spontaneous ovula tors may normally become reflex ovulators at a specific phase of their reproductive cycle, i.e. parturition. It has been suggested that postpartum ovulation in the rat may be triggered by the LH release induced by the sensory mechanostimulation of the uterus that occurs during de livery of the fetuses at parturition [9]. This is based on observation of a substantial increase in serum LH starting as early as 2 h (usually 3.5-11 h) after delivery, the major release persisting for 4-6 h [9], Consistent with this are the findings that barbiturate or LH-antiserum administra tion, immediately following parturition, was most effec tive in blocking ovulation. That is, phénobarbital com-
B.R. Komisaruk Institute o f Animat Behavior Rutgers - The State University Newark, NJ 07102 (USA)
Downloaded by: King's College London 137.73.144.138 - 1/18/2019 5:59:54 AM
5. Tiffany Cunninghama Jay S. Rosenblatt Barry R Komisarukh
pletely inhibited postpartum ovulation when adminis tered during the hour following delivery, partially inhib ited the ovulation when administered 1-7 h postpartum, and failed to inhibit ovulation when administered 8-9 h postpartum [10]. LH-antiserum successfully blocked ovu lation only when administered within 1 h postpartum [10]. Furthermore, Johnson [11] found that the time of day when the postpartum LH surge and subsequent ovu lation occurred covaried, on an individual basis, in close temporal relation to the time of day when the individual parturition occurred. The phase of the light cycle interacts with the time of parturition in determining the timing of postpartum ovulation [11, 18]. These findings suggest that there exist both a reflexogenic and a circadian component to the control of postpartum ovulation. As a test of the possible role of uterine afferent activity in stimulating postpartum ovulation, Johnson [11] per formed caesarian section (CS) at 9 a.m. on day 22 of preg nancy and observed that ovulation followed within 18 h. Ligature of the uterine horns at the time of CS blocked the induced ovulation [11], It seems likely that the ovula tion blockage was due to the uterine ligation’s interrup tion of uterine afferent activity generated by the CS proce dure. Since uterine afferent activity is conveyed via the hypogastric and pelvic nerves [ 12, 13], we hypothesized that transection of the hypogastric and/or pelvic nerves prior to CS (on day 22 of pregnancy) would interrupt the ovulation-inducing effect of CS. If reproductive tract (i.e. uterus, cervix and vagina) denervation indeed blocks CSinduced ovulation, this would support the concept that the sensory mechanostimulation generated by the pas sage of the fetuses through the reproductive tract during natural parturition reflexively stimulates ovulation by generating reproductive tract afferent activity. These re sults have been presented in abstract form [14].
males were moved from their wire bottom cages and placed into tubs with hardwood shavings (Beta-Chip, Warrensburg, N.Y., USA). On day 22, each female received one of the following treatments: (1) CS plus bilateral pelvic and hypogastric neurectomy (n = 5), (2) CS plus bilateral pelvic neurectomy (n = 7), (3) CS plus bilateral hypogastric neurectomy (n = 5), or (4) CS plus sham neurectomy (n = 6). CS was performed on all animals immediately following nerve transections, between 9 a.m. and 6 p.m. To determine whether they had ovulated, the ovaries and fal lopian tubes were removed and examined in all females between 23 and 24 h post-CS. The ovaries were examined microscopically for the number of ruptured follicles, and the fallopian tubes for the number of ova present [10]. For this procedure, females were anes thetized with a mixture of Ketaset and Rompun (0.1 m l/100 g bodyweight). On the day of surgery, animals were anesthetized with a mixture of chloral hydrate, alcohol and water (I g/ml ethanol/1.5 ml dis tilled water; dose 300 mg chloral hydrate/kg body weight). An inci sion was made along the midline of the ventrum, approximately 2 inches rostral to the most caudal nipple. Each uterine horn (fetusfilled) was reflected to one side in order to expose the colon as a landmark; each horn was then covered with saline-soaked gauze. With the colon pulled to one side, the hypogastric nerve was identi fied leaving the mesentericus ganglion and coursing alongside the ureter. A longitudinal incision was made, cutting the nerve in two places 3-5 mm apart, and the segment was removed. The colon was pulled to the opposite side and the procedure was repeated for bi lateral transection. To locate the pelvic nerve, the bifurcation of the vena cava into the common iliac veins was identified as a landmark. Where the internal iliac vein disappears in the dorsal muscle of the rat, the pelvic nerve is visible. Gentle retraction of the muscle exposed the nerve. Here, too, a longitudinal incision (2-4 mm) was made and this procedure was repeated on the opposite side. For sham neurec tomies, the aforementioned procedues were followed for location of both the hypogastric and pelvic nerves. Each animal was observed for intact nerves, the nerves were lifted, but no incisions were made. After completion of the designated neurectomies or sham oper ations, a small incision was made on either uterine horn (above the cervix) and the pups and the placenta were manually expelled from the uterus. Each horn was sutured with 6-0 surgical thread and re placed. The abdominal wall was sutured with 4-0 surgical thread and the incision closed with wound clips.
Materials and Methods Results
394
As summarized in table 1, the incidence of CS-induced ovulation, measured by direct visualization of ova in the fallopian tubes and ovulation points in the ovaries 24 h post-CS, was significantly attenuated or abolished by hypogastric and/or pelvic neurectomy. The median num ber of ova and ovulation points observed in the CS-only group was 4.5 and 3.5, respectively, whereas the median number of ova and ovulation points in each of the three other groups: hypogastric neurectomy only, pelvic neur ectomy only, and combined hypogastric-pelvic neurec-
Cunningham/Rosenblatt/Komisaruk
Neurectomy Blocks Caesarian-Section Induced Ovulation in Rats
Downloaded by: King's College London 137.73.144.138 - 1/18/2019 5:59:54 AM
A total of 23 Sprague-Dawley CD (Charles River, Kingston, N.Y., USA) females (90 days or older), either IAB bred or pur chased from Charles River, were kept socially housed (4-5 rats per cage) in wire bottom cages on a reverse 12-hour light/dark cycle (lights on 7 p.m.). Food and water were available ad libitum for the duration of the experiment. Females were mated during early after noon. Day 1 of gestation was designated as the day when mating was confirmed by the presence of a copulation plug under the wire cage or when sperm were found in the vaginal smear taken early the next morning. Animals were then individually housed on day 2 on the same reverse light cycle. On day 8, females were moved to a normal 12-hour light/dark cycle (lights on 7 a.m.), in order to syn chronize births on day 22. The day before parturition, day 21, fe
tomy, was zero. Only one rat ovulated in any of the neur ectomy groups, and it had received pelvic neurectomy. Each of the neurectomy groups differed significantly from the control CS-only group on each measure. Specifically, the median number of ova in each neurectomy group (all 0) was significantly lower than in the control, CS-only group (4.5) (p < 0.01, Mann-Whitney U test). Further more, the median number of ovulation points in each neurectomy group (0) was significantly lower than in the control CS-only group (3.5) (p < 0.05-0.01, MannWhitney U test).
Table 1. Effect of transection of the pelvic and/or hypogastric nerves on ovulation induced by CS
Group
n
Ova
Ovulation points
CS only CS + pelvic neurectomy + hypogastric neurectomy CS + pelvic neurectomy CS + hypogastric neurectomy
6
4.5 (1-11)
3.5 (1-5)
5 7 5
0 (0) 0 (0-2) 0 (0)
0 (0) 0 (0-8) 0 (0)
Values indicate median with the range given in parentheses. The number of ovulation points observed did not always equal the num ber of ova observed.
Discussion
lease that stimulates ovulation. Although it seems un likely that the neurectomy procedure in the present study would interfere with the action of released gonadotropin, we cannot rule out that possibility. The rat is commonly characterized as a spontaneous ovulator, i.e. ovulation occurs during the estrous cycle in the absence of specific mating stimulation but determined by the timing of the light cycle. The present findings sug gest that in addition, the rat may normally manifest re flexive ovulation at a specific phase of its reproductive cycle, i.e. at parturition, when the timing of ovulation is determined by the time of parturition as well as by the timing of the light cycle. There is evidence [18] that these two types of ovulation are different, on the basis that pen tobarbital is more potent in blocking cyclical than post partum ovulation. LH release begins earlier and persists longer when it occurs postpartum than when it occurs during the normal 4-day cycle [ 18], With regard to innervation of the reproductive tract, the hypogastric nerve provides sensory innervation pre dominantly of the proximal two thirds of the uterus and the cervix [12, 13], whereas the pelvic nerve provides sen sory innervation predominantly of the cervix and vagina [19] with minimal innervation of the caudal body of the uterus [ 13]. The firing activity of the hypogastric nerve is increased when fetuses are moved through the cervix [12]. Significant reduction of the incidence of CS-induced ‘postpartum’ ovulation resulted from transection of either or both of these pairs of nerves, and the region innervated in common by both nerves is the cervix. Therefore, it is likely that afferent activity from the cervix via both the hypogastric and pelvic nerves mediates the full expression of this reflexive ovulation. Thus, in the case ofligature of the uterine horns, cervical stimulation by the fetuses was
395
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In the present study, we make the assumption that the sensory stimulation generated by CS is partially com parable to that generated during parturition, because all fetuses are pushed individually toward the cervical end of the uterus where they are expelled via a small incision. Therefore in this CS procedure, it is likely that uterine and possibly cervical, although not vaginal, afferent activity is generated. The median number of ova observed in the present study (4.5) was lower than that reported postpar tum; i.e. it was found that as a result of postpartum mat ing, 12.7 ± 2.3 (mean ± SD) pups were born [15]. Hence, at least this number of ova had been shed postpar tum. (It was also reported in that study that there was no significant difference between the number of pups born as a result of postpartum mating versus mating during the estrous cycle.) We conclude that if the afferent activity re sulting from parturition is the stimulus for postpartum ovulation, then CS is a suboptimal stimulus in that regard. In order to study the effects of neurectomy, natural parturition is the obvious stimulus of choice. However, this is technically difficult, because pelvic neurectomy in terferes with natural parturition by blocking abdominal striated muscle contractions that are coordinated with uterine contractions [16, 17], We are currently engaged in studies that utilize neurectomized females undergoing normal parturition with manual assistance. The present findings, that deafferentation of the repro ductive tract on day 22 of gestation in the rat blocks CSinduced ovulation, imply that under normal conditions afferent activity emanating from the reproductive tract stimulates postpartum ovulation. The mechanostimulation normally generated by the fetuses as they pass through the birth canal at the time of parturition very likely provides the afferent signal for gonadotorpin re-
prevented and ovulation did not occur [11]. A contribu tion by uterine and/or vaginal afferent activity cannot be ruled out. These nerves also play a role in other effects of repro ductive tract mechanostimulation during the reproductive cycle in the rat. The pelvic nerves mediate the induction of pregnancy [ 17], pseudopregnancy [20] and reflexive ovu lation in response to vaginal stimulation [6]. Based on deafferentation studies, pelvic neurectomy, but not hypo gastric neurectomy, disrupts the micturition reflex and parturition [ 16]. The hypogastric nerves mediate analgesia during pregnancy [21 ]. Analgesia produced by vaginocervical mechanostimulation is mediated by both nerves [22 ].
After parturition, the rat normally mates within 9 h [23] and ovulates within 24 h [11]. The sensory stimula tion generated by parturition could trigger these events, serving an adaptive function of minimizing the latency for the initiation of the rat’s subsquent pregnancy.
Acknowledgments We gratefully acknowledge the technical assistance of Dr. Anne D. Mayer, and the advice of Dr. Carlos Beyer. Supported by NiH grants NLS-25R01NS22948 and GRS 5 S06 RR08223, and the Charles and Johanna Busch Foundation (B.R.K.) and by the Rutgers-CINVESTAV International Exchange Program. Contribution number 537 from the Institute of Animal Behavior.
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16 Higuchi T. Uchidc K. Honda K, Negoro H: Pelvic neurectomy abolishes the fetus-expul sion reflex and induces dystocia in the rat. Exp Neurol 1987;96:443-455. 17 Kollar EJ: Reproduction in the female rat after pelvic nerve neurectomy. Anat Rec 1953;115:641-658. 18 Hoffmann JC, Schwartz NB: Timing of postportum ovulation in the rat. Endocrinology 1965;76:620-625. 19 Komisaruk BR. Adler NT. Hutchison: Genital sensory field: Enlargement by estrogen treat ment in female rats. Science 1972:178: 1295-1298. 20 Carlson RR, De Feo VJ: Role of the pelvic nerve vs. the abdominal sympathetic nerves in the reproductive function of the female rat. Endocrinology 1965;77:1014-1022. 21 Gintzler AR, Peters LC, Komisaruk BR: Atte nuation of pregnancy-induced analgesia by hypcgastric neurectomv in rats. Brain Res 1983;277:186-188. 22 Cunningham ST, Steinman JL, Whipple B, Mayer AD, Komisaruk BR: Differential roles of hypogastric and pelvic nerves in the analge sic and motoric effects of vaginocervical stim ulation in rats. Brain Res 1991;559:337-343. 23 Connor JR, Davis HN: Postpartum estrus in Norway rats. Part I. Behav Biol Reprod 1980;23:994-999.
Cunningham/Rosenblatt/Komisaruk
Neurectomy Blocks Caesarian-Section Induced Ovulation in Rats
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