- Email: [email protected]
Fertility ratio in male rats
Physiology & Behavior 68 (2000) 611–618
Fertility ratio in male rats: Effects after denervation of two pelvic floor muscles J. Manzoa,*, M.I. Vazqueza, M.R. Cruzb, M.E. Hernandeza, P. Carrilloa, P. Pachecoa,b b
a Instituto de Neuroetología, Universidad Veracruzana, Apartado Postal 566, Xalapa, Ver. 91001, Mexico Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, Ciudad Universitaria, México, D.F. 04510, Mexico Received 18 February 1999; received in revised form 3 June 1999; accepted 21 October 1999
Abstract Fertility ratio is defined here as the proportion of females that a male can impregnate after a constant period of in-polygyny living. This ratio was investigated in male rats after denervation of two pelvic floor muscles, the pubococcygeus and iliococcygeus. Denervation was carried out by transecting the somatomotor branch of the pelvic nerve. The lesion did not modify the sexual behavior of males or their overall fertility, but decreased the weight of the ejaculated seminal plug. Consequently, the number of days living in cohabitation to induce pregnancy was increased in lesioned males (ⵑ13 days) compared with intact and sham animals (ⵑ5 days). These results showed that the fertility ratio was optimal when intact/sham males cohabited with females for two consecutive estrous cycles, but that lesioned males needed up to four cycles to induce most pregnancies. Two hypotheses are raised by our results. The first is that pelvic floor denervation decreases the forceful tension required to expel the semen from the prostatic urethra to the vagina, then an incomplete seminal plug is expelled. The second is that denervation cut afferent fibers that reflexively promote the continence of the semen deposited in the prostatic urethra during seminal emission, allowing some to leak out before ejaculation. The latter hypothesis can also explain the recovery of the fertility ratio in lesioned males. It could be a compensatory mechanism mediated by the pudendal nerve supply to the coccygeus muscle, the other pelvic floor muscle. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ejaculation; Pubococcygeus; Iliococcygeus; Coccygeus; Copulatory behavior; Infertility; Seminal plug; Seminal emission; Pelvic nerve
1. Introduction Fertility is a key event in the reproduction of mammals. Adult males have a high probability to impregnate a female if copulation occurs around her ovulatory period. However, the malfunction of some physiological mechanism in the masculine reproductive system, or the experimental manipulation of the subject, can decrease the reproductive potential even if typical copulatory behavior is achieved. Thus, to quantify the normal or disrupted fertility of males when observational analysis does not detect modifications in behavior, we introduce the fertility ratio concept, defined as the proportion of females that a male can impregnate after a constant period of in-polygyny living. In male rats, there are several factors that affect fertility without altering behavior. One of them is the quality of expulsion and deposition of a seminal plug into the female vagina during ejaculation [1–4]. The seminal plug is a semisolid mass normally present in the urethra of adult male rats * Corresponding author. Tel.: 52 (28) 12-5748; Fax: 52 (28) 12-5748. E-mail address: [email protected]
[5], composed of secretions from the seminal vesicles and coagulating glands and hardened by a complex of accompanying proteins [6–10]. Ablation of these sex glands does not impair sexual behavior of male rats, but they become infertile [7,11–13]. Infertility is due to the absence of a seminal plug, because males deposit sperm in the vagina with the capacity to fertilize eggs if intrauterine insemination is carried out [14]. This means that a seminal plug in the vagina provides a pump-like action, functioning to increase the movement and number of spermatozoa inside the uterus and fallopian tubes [3]. Thus, the absence of the seminal plug reduces the probability of sperm reaching and fertilizing the ovum. Furthermore, the mechanical effect of the seminal plug works together with uterine wall contractions induced chemically after ejaculation by semen substances from the seminal vesicles and coagulating glands [3,4,15]. Alterations also occur within the male reproductive system that influence the expulsion of the seminal plug during ejaculation. Transection of the hypogastric nerve, for example, does not alter the copulatory behavior of males, but they fail to expel semen and seminal plug, and therefore, become infertile [16,17]. This effect is due to the role of the
0031-9384/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0031-9384(99)00 2 1 9 - X
612
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
sympathetic hypogastric nerve in the emission process [18– 21]. On the other hand, the role of striated muscles in ejaculation is also important for the appropriate expulsion and deposition of semen and plug into the vagina. The muscles studied to date are those at the base of the penis that have an intense contractile activity during ejaculation and serve the appropriate expulsion and deposition of the seminal plug [22–24]. Removal of these muscles induces infertility in males because they lose the ability to expel the whole plug [25]. In our laboratory, we have been investigating the role of the iliococcygeus (Icm) and pubococcygeus (Pcm) muscles in the reproductive physiology of the rat. These muscles, together with the coccygeus, form the striated muscular complex known as the pelvic floor [26,27] or levator ani [28]. Icm and Pcm are innervated by the somatomotor branch (Smb) of the pelvic nerve that is composed of afferent and motor axons from the L6 and S1 spinal cord segments [29]. The Smb seems completely somatic, unlike the viscerocutaneous branch of the pelvic nerve that carries afferent fibers from viscera and perigenital skin and the well known autonomic fibers supplying the pelvic plexus [29–35]. Denervation of Icm and Pcm in rats, by transection of the Smb, does not alter reproductive parameters in females [36,37] or in males [32]. However, we observed that this surgery in males reduces the weight of the ejaculated seminal plug. This finding led us to the present study with the aim to further analyze both the effect of Icm and Pcm denervation on the expulsion of semen and the effect of the low weight seminal plug on the fertility ratio of Smb-transected males.
nerve as described elsewhere [29]. To avoid regeneration, a 5-mm portion of the nerve branch was excised (Fig. 1). During sham surgery, the branch was dissected and hooked but not cut. After surgery subjects were placed in a warm chamber (ⵑ24⬚C) until they had recovered from anesthesia, and then returned to the animal room. No health problem was detected in subjects after the lesion of Smb; thus, they did not require special postsurgical care to recover. At the end of the study the animals were deeply anesthetized and the branch transection confirmed before the rats were killed with an overdose of pentobarbital. The examination of all animals with nerve transections revealed no evidence of regeneration. 2.3. Mating tests One copulatory series per animal was observed twice a week before (four tests) and after (seven tests) surgery. Tests were conducted during the last third of the dark period. After 5 min adaptation of the male in a Plexiglas cylindrical arena (50 cm high ⫻ 50 cm diameter), a receptive female was introduced. The introduction of the female indicated the beginning of the test, and the male ejaculatory pattern, the end. The parameters of copulation (see below) were recorded in a PC computer using the SBR software [37]. When an ejaculatory pattern was observed, the female
2. Materials and methods 2.1. Subjects and housing Sexually experienced male Wistar rats, weighing 250– 350 g, were assigned to a group where the pelvic nerve somatomotor branch (Smb) was bilaterally transected (n ⫽ 12) or to a group that was Sham operated (n ⫽ 5). A third group of males was used to obtain the seminal plug from the vagina of mated females (see below). Ovariectomized receptive females were used for mating tests. Receptivity was induced after females were injected subcutaneously with 10 g of estradiol benzoate and 2 mg of progesterone, 48 and 4 h before tests, respectively. Rats were housed in plastic cages (50 ⫻ 30 ⫻ 20 cm) containing wood chip bedding, and kept in a room maintained at 22 ⫾ 2⬚C and under a 12: 12 LD light schedule (lights on at 2200 h). Commercial pelleted rodent chow (Purina, México) and water were available ad lib. 2.2. Surgery Smb transection was carried out under sodium pentobarbital anesthesia (26 mg/kg i.p.). The nerve branch was located in the pelvic area, after a ventral abdominal incision, where it crosses the internal iliac vessel. Smb travels more centrally than the viscerocutaneous branch of the pelvic
Fig. 1. Drawing of the pelvic nerve and its branches, the somatomotor and the viscerocutaneous. The image shows that both the pelvic and pudendal nerves originates from the L6–S1 Trunk, formed in turn from the L6 and S1 spinal nerves. The landmarks to find the pelvic nerve and its branches are the iliac vessels, mainly the internal iliac vein. Asterisks show the two places where the somatomotor branch was cut in the experiments; hence, a portion of the branch was excised.
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
was immediately transferred to another arena. This second arena had a black floor with no bedding material and was already occupied by a male used to dislodge the seminal plug, if any, from the vagina. Plugs are generally dislodged within three to six intromissions [38,39]. To prevent ejaculation, males used for this procedure were only allowed four intromissions, and females were brought to a second male if it was necessary. If no plug was dislodged within eight intromissions, then it was assumed that none was present. Plugs obtained were immediately weighed. 2.4. Copulatory parameters The observational study of sexual behavior in males allowed the identification of three distinctive copulatory movements, i.e., mounts, intromissions, and ejaculation. A male had several mounts and intromissions before ejaculation. The performance of these copulatory movements guided the quantification of copulatory parameters. The number of mounts (NM) and number of intromissions (NI) represent the frequency of each movement before ejaculation. The time elapsed from the introduction of the female with the male to the first mount or intromission was recorded as the latency of mount (LM) or latency of intromission (LI), respectively. If the first copulatory movement executed by the male was an intromission, then LM ⫽ LI. The time elapsed from the first intromission to the ejaculation was recorded as the latency of ejaculation. A proportion of intromissions (hits) was obtaining by computing NI/ (NM⫹NI), the result is known as the hit rate parameter of copulatory behavior, and reflects the capacity of the male to intromit—hence, to have erection of the penis. 2.5. Fertility tests Results from mating tests (see below) showed effects of Smb transection on the weight of the seminal plug expelled after ejaculation. Thus, the present experiment was designed to test whether this modification is reflected in the fertility ratio of the male. In doing this, intact virgin females and sexually experienced males, proved previously to be fertile, were used. Each experimental male (n ⫽ 12) was placed in a cage for reproduction (60 ⫻ 40 ⫻ 22 cm) with three females for 20 days. After that period, or earlier if pregnancy was conspicuous, each female was placed alone in a cage and was observed until parturition or for a maximum of 21 days, when it was assumed that the female had not become pregnant. Gestation was considered to last 21 days; therefore, to calculate the first day of pregnancy a calendar backcount from the day of parturition was made. With this procedure it was possible to obtain the number of days between the formation of a reproduction group and the first day of pregnancy of each female. After being in the reproduction groups, the 12 males were operated as described above, and two groups were obtained, sham (n ⫽ 4) and Smb transected (n ⫽ 8). Then, 5 days after surgery they were
613
placed again in reproduction groups with new females, and the same day counts were made. 2.6. Statistics Results are presented as means ⫾ SEM. Statistics were performed using the GB-STAT Software (V6.0, Dynamic Microsystems, Inc., USA). Behavioral parameters of copulation were analyzed with the analysis of variance (ANOVA) for repeated measures (a post hoc analysis was not necessary in any case). ANOVA tables showed the effect of surgery, tests, and the interaction surgery–test, and the outcomes are presented below only if relevant. Paired comparison of the variance in seminal plug weight between sham and experimental subjects was analyzed by the Cochran’s C (CC) test, and is presented as a box-and-whisker plot as described elsewhere [40]. For the fertility data, the proportions of pregnant females were compared using the Fisher Exact Test, while the number of days to fertilize a female was analyzed by the Kruskal–Wallis ANOVA (KW), followed by paired comparisons with the Mann–Whitney U-test. The continuous 20 days that females were exposed to inpolygyny living with males to become pregnant were divided in four 5-day periods. This procedure was utilized taking into consideration the average number of days that the female estrous cycle lasts. The fertility ratio was calculated for each 5-day period with the following formula: FR = PFp ⁄ TPF
(1)
where FR ⫽ fertility ratio, PFp ⫽ number of pregnant females per period, and TPF ⫽ total number of pregnant females during the whole 20 days of male–female cohabitation. FR was calculated per subject and used as the male value for statistic analysis. Between-groups comparisons for each 5-day period were performed using KW, followed by a paired comparison with the Mann–Whitney U-test. Notice that the formula does not consider females that did not become pregnant. They were excluded for two reasons: (a) every male fertilized at least one female in his group, and (b) there is a chance that some females reflect infertility (or pseudopregnancy) of their own and not because the partner male.
3. Results Male sexual behavior was not modified after bilateral transection of the pelvic nerve somatomotor branch (SmbTx). As shown in Table 1, no copulatory parameter was altered by the surgery when compared with Sham animals. However, although the ANOVA for the weight of the seminal plug did not reveal a reliable change in the surgery–test interaction (Table 1), it showed a significant effect of surgery, F(1, 15) ⫽ 4.24, p ⫽ 0.05, and tests, F(6, 90) ⫽ 2.33, p ⫽ 0.03. The surgery data indicated that Smb transection produced a decrease in the weight of the seminal plug expelled with ejaculation (Fig. 2).
4.2 ⫾ 3.21 2.91 ⫾ 0.57 7.2 ⫾ 0.86 8.58 ⫾ 0.73 3.6 ⫾ 0.6 5.91 ⫾ 0.97 4.8 ⫾ 0.8 10.33 ⫾ 2.46 189 ⫾ 55.86 178.1 ⫾ 17.2 0.74 ⫾ 0.12 0.75 ⫾ 0.04 175 ⫾ 8 130 ⫾ 19 3.8 ⫾ 1.39 3.66 ⫾ 0.97 8.4 ⫾ 0.50 7.58 ⫾ 1.22 6.2 ⫾ 2.24 5.25 ⫾ 1.05 7.6 ⫾ 2.18 15.75 ⫾ 6.97 203.6 ⫾ 34.3 136.1 ⫾ 19.7 0.72 ⫾ 0.09 0.70 ⫾ 0.06 106 ⫾ 31 133 ⫾ 19
3.0 ⫾ 1.22 3.16 ⫾ 0.77 6.0 ⫾ 1.09 7.16 ⫾ 0.68 6.2 ⫾ 2.31 7.75 ⫾ 3.6 16.8 ⫾ 9.84 10.83 ⫾ 3.95 157.2 ⫾ 37.1 152.5 ⫾ 24.6 0.69 ⫾ 0.09 0.70 ⫾ 0.05 167 ⫾ 26 118 ⫾ 13
Test 6, AS Test 5, AS
2.8 ⫾ 1.06 3.33 ⫾ 0.84 7.8 ⫾ 0.73 8.41 ⫾ 0.84 4.8 ⫾ 0.58 7.25 ⫾ 2.19 6.2 ⫾ 1.49 9.58 ⫾ 2.11 161.6 ⫾ 24.6 160.5 ⫾ 23.9 0.75 ⫾ 0.08 0.74 ⫾ 0.05 170 ⫾ 41 148 ⫾ 31
Fig. 2. Overall significant reduction in the weight of the seminal plug expelled by males with transection of the pelvic nerve somatomotor branch (Smb-Tx), as compared to sham-operated subjects (Sh). *p ⬍ 0.05.
Seminal plug weight
Hit rate
Latency of ejaculation
Latency of intromission
Latency of mount
Number of intromissions
Data are Mean ⫾ SEM. BS ⫽ before surgery; AS ⫽ after surgery. Latencies in seconds, seminal plug weight in milligrams. Statistics show the surgery–test interaction from the repeated measures ANOVA.
1.6 ⫾ 0.74 2.83 ⫾ 0.58 10.0 ⫾ 1.22 8.16 ⫾ 0.84 5.0 ⫾ 0.54 5.08 ⫾ 0.73 5.4 ⫾ 0.24 6.91 ⫾ 1.0 184.4 ⫾ 24.5 159.1 ⫾ 27.2 0.86 ⫾ 0.05 0.77 ⫾ 0.04 143 ⫾ 3 102 ⫾ 22 13.0 ⫾ 6.04 5.75 ⫾ 1.78 9.4 ⫾ 0.74 9.83 ⫾ 0.62 6.0 ⫾ 0.89 5.41 ⫾ 0.82 26.8 ⫾ 19.3 8.66 ⫾ 1.58 283 ⫾ 52.88 247.5 ⫾ 53.7 0.52 ⫾ 0.1 0.69 ⫾ 0.05 137 ⫾ 25 116 ⫾ 10
5.8 ⫾ 1.8 4.5 ⫾ 1.17 7.4 ⫾ 0.87 9.16 ⫾ 0.67 8.6 ⫾ 1.63 8.16 ⫾ 1.53 20.4 ⫾ 9.43 13.33 ⫾ 3.41 188 ⫾ 33.92 187.2 ⫾ 26.2 0.59 ⫾ 0.08 0.71 ⫾ 0.05 146 ⫾ 17 89 ⫾ 22
Test 1, AS
4.6 ⫾ 1.43 3.83 ⫾ 0.77 8.6 ⫾ 0.97 8.08 ⫾ 1.32 4.2 ⫾ 0.86 14.08 ⫾ 5.30 11.4 ⫾ 2.31 35.4 ⫾ 18.4 198 ⫾ 37.06 146.9 ⫾ 24.5 0.66 ⫾ 0.07 0.67 ⫾ 0.05 170 ⫾ 20 137 ⫾ 10 Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Number of mounts
Test 4, BS Group Parameter
Table 1 Copulatory parameters of Sham (Sh) and Smb-transected (Smb-Tx) male rats
Test 2, AS
Test 3, AS
Test 4, AS
Test 7, AS
Statistics
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618 F(6, 90) ⫽ 1.60, NS F(6, 90) ⫽ 1.15, NS F(6, 90) ⫽ 0.30, NS F(6, 90) ⫽ 1.27, NS F(6, 90) ⫽ 0.25,NS F(6, 90) ⫽ 0.87, NS F(6, 90) ⫽ 1.65, NS
614
As indicated, the repeated-measures ANOVA yielded a significant difference across tests in the seminal plug weight parameter. This difference was due to the high variability in the weight of the plug that Smb-transected males expelled following surgery. Performing paired comparisons of the homogeneity of variances in this parameter, significant differences were found in the first (CC ⫽ 0.787, p ⬍ 0.05), second (CC ⫽ 0.780, p ⬍ 0.05), third (CC ⫽ 0.991, p ⬍ 0.01), and sixth (CC ⫽ 0.920, p ⬍ 0.01) postsurgery tests (Fig. 3). The fertility potential of males was altered by Smb transection when analyzed per each 5-day period, although the overall data showed that at the end males were capable of inducing pregnancy in most females independently of the surgical group to which they belonged (Table 2). As a consequence, the number of days needed to induce pregnancy was reliably increased in Smb-transected males. Intact and sham-operated males induced pregnancy after living for about 5 days with the females, while Smb-Tx males did so after cohabiting for around 13 days, a difference that was statistically significant in the ANOVA [KW 2(2) ⫽ 14.95, p ⬍ 0.01] and paired comparisons (Fig. 4). The fertility ratio showed that intact and sham males induced 100% of pregnancies during the first and second 5-day periods. Smb-Tx males were unable to induce pregnancy in the first period, but recovered partially in the second and continued to fertilize females up to the fourth period. These results made the Smb-Tx group to be significantly different from intact and sham groups in the first [KW 2(2) ⫽ 9.81, p ⬍ 0.01], third [KW 2(2) ⫽ 8.0, p ⬍ 0.01], and fourth [KW 2(2) ⫽ 6.42, p ⬍ 0.01] periods, but not in the second [KW 2(2) ⫽ 1.36, p ⬎ 0.05], and these differences were reflected in the paired comparisons (Fig. 5).
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
Fig. 3. Box-and-whisker plot showing the variability in the weight of the seminal plug ejaculated by sham (Sh) and pelvic nerve somatomotor branch transected (Smb-Tx) male rats in a test before surgery (BS) and the seven tests after surgery (AS-n). The histogram-like display shows the box with quartiles ends, the median as a horizontal line inside the box, and the whiskers extending to the farthest data. Significant differences in the variance comparison (Sh vs Smb-Tx per test) were found at p ⬍ 0.05 (*) and p ⬍ 0.01 (**).
4. Discussion The data confirm our preliminary results that denervation of Icm and Pcm produces a decrease in the weight of the ejaculated seminal plug. Although this alteration is just an overall reduction, the effect produced on the fertility ratio is remarkable. The results clearly show that after Smb transection copulatory behavior remains completely normal as previously reported [32], but the expulsion and/or deposition of the seminal plug in the vagina is modified. Every male was fertile by the end of the experiments, but Smb-transected males needed to stay with females around 13 days to induce pregnancy, while intact or sham animals required only 5 days. Thus, living 5 days with a female is the minimum time for a normal male to induce pregnancy, a period that we named the pregnancy induction time (PIT). Each PIT is cor-
615
Fig. 4. Number of days needed by intact (Int), sham (Sh), and pelvic nerve somatomotor branch transected (Smb-Tx) male rats, to induce pregnancy after 20 days of in-polygyny living with three females. The figure shows that Smb-Tx males require significantly (p ⬍ 0.01) more days to induce pregnancy, while Int and Sh males require around 5 days, a period that we named Pregnancy Induction Time.
related with the duration of an estrous cycle of the female rat. In this sense normal males require only one PIT to fertilize females, while Smb-transected males require up to four consecutive PITs. The fertility ratio, then, as calculated by each consecutive PIT, showed that in the first the Smbtransected males were unable to induce pregnancy, but that their capacity to fertilize was restored beginning in the second PIT and continuing until the fourth. This means that infertility in Smb-transected males is transient, i.e., they do not become permanently infertile, but need to cohabit several days (from two to four estrous cycles) to induce pregnancy. Despite their altered fertility, these males showed normal patterns in their copulatory behavior, and results showed that transient infertility was due to effects on the weight of the seminal plug. The proper expulsion and deposition of a normal seminal plug in the rat is crucial for the induction of pregnancy [1–4], and any factor affecting it has a direct consequence for fertility [12,14,25].
Table 2 Percentage of fertilized females by Intact, sham (Sh) and Smb-transected (Smb-Tx) male rats Group
% (0–5 Days period)
% (5–10 Days period)
% (10–15 Days period)
% (15–20 Days period)
% (Total)
Intact (n ⫽ 12) Sh (n ⫽ 4) Smb-Tx (n ⫽ 8)
50 75 (NS) 0 (*)
44.44 25 (NS) 25 (NS)
0 0 (NS) 25 (*)
0 0 (NS) 29.16 (*)
94.44 100 (NS) 79.16 (NS)
Statistics ⫽ Fisher Exact Test of proportions; intact as the comparison group; NS ⫽ not significant; *p ⬍ 0.01.
616
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
Fig. 5. Fertility ratio of intact, sham, and pelvic nerve somatomotor branch transected (Smb-Tx) male rats, after 20 days of in-polygyny living with three females. The whole time was divided in four 5-days periods (0–5, 5– 10, 10–15, and 15–20), and between-groups comparisons were done per period. Intact and sham males fertilized all females in the first and second periods, while Smb-Tx males were significanlty different (p ⬍ 0.01) because did not induce pregnancy in the first period, started in the second, and fertilized most females by extending their matings up to the fourth period.
The main question here is what these pelvic floor muscles do in the ejaculatory process to contribute for the expulsion of the seminal plug, and hence to fertility. Considering that the striated muscles are controlled by the somatic nervous system, it is suggested that they play little part in the autonomic-regulated physiology of seminal emission. The activity of the sex glands and structures having a role in emission is controlled by the sympathetic component of the autonomic nervous system [20,41–43]. Lesion of the hypogastric nerve, the main peripheral sympathetic pathway in the pelvic area, produces infertility by blocking the deposition of sperm and semen material in the prostatic urethra [20,21]. Thus, the ejaculatory pattern of copulatory behavior is normal but no semen is expelled. As alterations in the sympathetic innervation were not induced in our experimental animals, we propose two hypotheses to explain our results. The first suggests that denervation of Icm and Pcm decreases the forceful tension required to expel the emission-accumulated semen from the prostatic urethra to the female vagina. It is known that ejaculation is a two-step process—first seminal emission, and second the powerful mechanical event promoted mainly by the contraction of striated muscles, that is, the ejaculation per se. However, the muscles reported to be involved in the ejection of the semen are the ischiocavernosus and the bulbospongiosus [23,25,42,44,45]. Thus, this proposal is the first indicating a possible role of pelvic floor muscles in producing force for the expulsion of the ejaculate. If true, it means that after ejaculation of Smb-transected males, a quantity of semen is expelled, but some remains in the male urethra due to the lack of a complete ejection force.
The second hypothesis suggests that transection of the Smb cut afferent fibers from the Icm and Pcm that reflexively promote the continence of the semen that is deposited in the prostatic urethra during seminal emission. This idea came from our previous finding that afferent innervation of Pcm reflexively induces urinary continence in male rats by relaxing the bladder wall and allowing the urine to be retained inside the bladder [46]. The role of pelvic floor musculature in the physiology of continence is a topic that has received attention for several years. The activity of pelvic floor muscles alleviates malfunctions such as impotence and incontinence of feces and urine [47–51]. Resulting relief seems to be related to a spinal inhibitory reflex triggered by afferents from these muscles that in turn activate efferent nerves controlling continence [46,52]. The urethra is a common path for the expulsion of semen and urine, thus denervation of pelvic floor muscles could induce both urinary incontinence [53] and seminal incontinence. Therefore, incontinence of the semen could occur as the emission process is developed during the several mounts and intromissions that precede the ejaculation in the copula of the male rat. The semen arriving to the prostatic urethra is not retained completely, and some may leak to the outside. Drops of semen material are then probably removed by the male during the very common genital grooming after intromissions. Thus, after ejaculation the quantity of semen deposited in the female vagina is decreased. The present proposal also explains the high variability in the weight of the seminal plug of denervated animals. This variability is probably the outcome of several factors such as the frequency and quality of genital grooming, the latency of ejaculation, or the number of mounts and intromissions before ejaculation. Grooming could mainly account for the quantity of semen removed before ejaculation, reducing the quantity of semen deposited in the vagina during ejaculation. According to our observations, male rats eat semen material and even seminal plugs that they remove from the vagina if a previous male has ejaculated or after their own postejaculatory interval. Finally, our second hypothesis is also useful in explaining the recovery of the fertility ratio of Smb-transected males after four consecutive PITs. It is known that the pelvic floor is the complex structured by the Icm, Pcm, and coccygeus (Ccm) muscles [27,28]. Transection of Smb denervates Icm and Pcm but not Ccm. This latter muscle is innervated by a branch of the pudendal nerve in both female and male rats [29,54]. Whether the afferent innervation of Ccm also participates in promoting urinary/semen continence is a topic that has not been studied to date. However, it is possible to argue that the three muscles organizing the pelvic floor engage in triggering spinal-mediated inhibitory reflexes to promote continence. Thus, we suggest that alterations produced by denervation of Icm and Pcm are transient effects that are overcome by a plastic compensatory process carried out by neurons projecting to the Ccm. This plasticity could be central, could be peripheral, or could be both.
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
The latter discussion has been focused to the male. However, it is important to consider that the reproductive physiology of the female cannot be completely excluded. The female reproductive system could also be self-adjusted to become fertile if continuously receive a reduced amount of semen material.
[18]
[19] [20]
Acknowledgments This research was supported by the Grant No. 4157P-N from the National Council for Science and Technology (CONACYT-México) to J.M.
[21] [22]
[23]
References [24] [1] Blandau RJ. On factors involved in sperm transport through the cervix uteri of the albino rat. Am J Anat 1945;77:253–72. [2] Hart BL. Role of testosterone secretion and penile reflexes in sexual behavior and sperm competition in male rats: a theoretical contribution. Physiol Behav 1983;31:823–7. [3] Matthews M, Adler NT. Facilitative and inhibitory influences of reproductive behavior on sperm transport in rats. J Comp Physiol Psychol 1977;91:727–41. [4] Matthews M, Adler NT. Systematic interrelationship of mating, vaginal plug position, and sperm transport in the rat. Physiol Behav 1978; 20:303–9. [5] Kunstyr I, Kupper W, Weisser H, Naumann S, Messow C. Urethral plug—a new secondary male sex characteristic in rat and other rodents. Lab Anim 1982;16:151–5. [6] Carballada R, Esponda P. Role of fluid from seminal vesicles and coagulating glands in sperm transport into the uterus and fertility in rats. J Reprod Fertil 1992;95:639–48. [7] Cukierski MA, Sina JL, Prahalada S, Robertson RT. Effects of seminal vesicle and coagulating gland ablation on fertility in rats. Reprod Toxicol 1991;5:347–52. [8] Fawell SE, Higgins SJ. Formation of rat copulatory plug: purified seminal vesicle secretory proteins serve as transglutaminase substrates. Mol Cell Endocrinol 1987;53:149–52. [9] Koren E, Schon E, Lukac J. The coagulation of insoluble and basic proteins from rat seminal vesicle secretion with vesiculase: influence of collagenase-like peptidase from rat testis. J Reprod Fertil 1975;42: 491–5. [10] Sofikitis N, Takahashi C, Kadowaki H, Okazaki T, Shimamoto T, Nakamura I, Miyagawa I. The role of the seminal vesicles and coagulating glands in fertilization in the rat. Int J Androl 1992;15:54–61. [11] Beach FA, Wilson JR. Mating behavior in male rats after removal of the seminal vesicles. Proc Natl Acad Sci USA 1963;49:624–6. [12] Carballada R, Esponda P. Structure of the vaginal plugs generated by normal rats and by rats with partially removed seminal vesicles. J Exp Zool 1993;265:61–8. [13] Tisell LE, Larsson K. Unimpaired sexual behavior of male rats after complete removal of the prostate and seminal vesicles. Invest Urol 1979;16:274–5. [14] Cukierski MA, Sina JL, Prahalada S, Wise LD, Antonello JM, MacDonald JS, Robertson RT. Decreased fertility in male rats administered the 5 alpha-reductase inhibitor, finasteride, is due to deficits in copulatory plug formation. Reprod Toxicol 1991;5:353–62. [15] Crane LH, Martin L. Postcopulatory myometrial activity in the rat as seen by video- laparoscopy. Reprod Fertil Dev 1991;3:685–98. [16] Larsson K, Swedin G. The sexual behavior of male rats after bilateral section of the hypogastric nerve and removal of the accessory genital glands. Physiol Behav 1971;6:251–3. [17] Lucio RA, Manzo J, Pacheco P. Pelvic and hypogastric nerves co-
[25]
[26]
[27] [28] [29]
[30] [31] [32]
[33] [34] [35]
[36]
[37]
[38]
[39]
[40] [41] [42]
617
participation in the mediation of male rat sexual behavior. Soc Neurosci Abstr 1991;17:1059. Billups KL, Tillman S, Chang TS. Ablation of the inferior mesenteric plexus in the rat: alteration of sperm storage in the epididymis and vas deferens. J Urol 1990;143:625–9. Kimura Y, Adachi K, Kisaki N, Ise K. On the transportation of spermatozoa in the vas deferens. Andrologia 1975;7:55–61. Kimura Y, Miyata K, Adachi K, Kisaki N. Peripheral nerves controlling the closure of internal urethral orifice during ejaculation. Urol Int 1975;30:218–27. Kolbeck SC, Steers WD. Neural regulation of the vas deferens in the rat: an electrophysiological analysis. Am J Physiol 1992;263:R331–8. Holmes GM, Chapple WD, Leipheimer RE, Sachs BD. Electromyographic analysis of male rat perineal muscles during copulation and reflexive erections. Physiol Behav 1991;49:1235–46. Holmes GM, Sachs BD. The ejaculatory reflex in copulating rats: normal bulbospongiosus activity without apparent urethral stimulation. Neurosci Lett 1991;125:195–7. Wallach SJR, Hart BL. The role of the striated penile muscles of the male rat in seminal plug dislodgement and deposition. Physiol Behav 1983;31:815–21. Sachs BD. Role of striated penile muscles in penile reflexes, copulation, and induction of pregnancy in the rat. J Reprod Fertil 1982;66: 433–43. Brink EE, Pfaff DW. Vertebral muscles of the back and tail of the albino rat (Rattus norvegicus albinus). Brain Behav Evol 1980;17:1– 47. McKenna KE, Nadelhaft I. The organization of the pudendal nerve in the male and female rat. J Comp Neurol 1986;248:532–49. Schröder HD. Organization of the motoneurons innervating the pelvic muscles of the male rat. J Comp Neurol 1980;192:567–87. Pacheco P, Martinez–Gomez M, Whipple B, Beyer C. Komisaruk BR. Somato-motor components of the pelvic and pudendal nerves of the female rat. Brain Res 1989;490:85–94. Hulsebosch CE, Coggeshall RE. An analysis of the axon populations in the nerves to the pelvic viscera in the rat. J Comp Neurol 1982;211:1–10. Langworthy OR. Innervation of the pelvic organs of the rat. Invest Urol 1965;2:491–511. Lucio RA, Manzo J. Martinez–Gomez M, Sachs BD, Pacheco P. Participation of pelvic nerve branches in male rat copulatory behavior. Physiol Behav 1994;55:241–6. Mallory B, Steers WD, De Groat WC. Electrophysiological study of micturition reflexes in rats. Am J Physiol 1989;257:R410–21. Purinton PT, Fletcher TF, Bradley WE. Gross and light microscopic features of the pelvic plexus in the rat. Anat Rec 1973;175:697–705. Steers WD, Mallory B, De Groat WC. Electrophysiological study of neural activity in penile nerve of the rat. Am J Physiol 1988;254: R989–1000. Burden HW, Price GT, Renegar RH, Hodson CA. Effects of peripheral nerve lesions during pregnancy on parturition in rats. Anat Embryol (Berl) 1990;182:499–501. Martinez–Gomez M, Cruz Y, Pacheco P, Aguilar–Roblero R, Hudson R. The sensory but not muscular pelvic nerve branch is necessary for parturition in the rat. Physiol Behav 1998;63:929–32. Claro F, Del Abril A, Segovia S, Guillamon A. SBR: a computer program to record and analyse sexual behavior in rodents. Physiol Behav 1990;48:489–93. McClintock MK, Anisko J, Adler NT. Group mating among norway rats II. The social dynamics of copulation: competition, cooperation, and mate choice. Anim Behav 1982;30:410–25. Tukey JW. Explanatory Data Analisis. Reading, MA: Addison–Wesley, 1977. Bacq ZM. Impotence of the male rodent after sympathetic denervation of the genital organs. Am J Physiol 1931;96:321–30. Benson GS. Male sexual function: erection, emission, and ejaculation. In: Knobil E, Neil JD, editors. The Physiology of Reproduction. New York: Raven Press, Ltd., 1994. pp. 1489–1506.
618
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
[43] Ricker DD, Chang TSK. Neuronal input from the inferior mesenteric ganglion (IMG) affects sperm transport within the rat cauda epididymis. Int J Androl 1996;19:371–6. [44] De Groat WC, Booth AM. Physiology of male sexual function. Ann Intern Med 1980;92:329–31. [45] Sachs BD. Potency and fertility: hormonal and mechanical causes and effects of penile actions in rats. In: Balthazart J, Prove E, Gillis R, editors. Hormones and behavior in higher vertebrates. Berlin: Springer Verlag, 1983. pp. 86–110. [46] Manzo J, Esquivel A, Hernandez ME, Carrillo P, Martinez–Gomez M, Pacheco P. The role of pubococcygeus muscle in urinary continence in the male rat. J Urol 1997;157:2402–6. [47] Claes H, Baert L. Pelvic floor exercise versus surgery in the treatment of impotence.Br J Urol 1993;71:52–7. [48] Dubrovsky B, Filipini D. Neurobiological aspects of the pelvic floor muscles involved in defecation. Neurosci Biobehav Rev 1990;14: 157–68. [49] Ishigooka M, Hashimoto T, Sasagawa I, Nakada T. Reduction in
[50] [51] [52]
[53]
[54]
norepinephrine content of the rabbit urinary bladder by alpha-2 adrenergic antagonist after electrical pelvic floor stimulation. J Urol 1994;151:774–5. Merril DC, Conway C, DeWolf W. Urinary incontinence. Treatment with electrical stimulation of the pelvic floor. Urology 1975;5:67–72. Teague CT, Merril DC. Electric pelvic floor stimulation. Mechanism of action. Invest Urol 1977;15:65–9. Sundin T, Carlsson C-A, Kock NG. Detrusor inhibition induced from mechanical stimulation of the anal region and from electrical stimulation of pudendal nerve afferents. An experimental study in cats. Invest Urol 1974;11:374–8. Gilpin SA, Goslin JA, Smith ARB, Warrel DW. The pathogenesis of genitourinary prolapse and stress incontinence of urine. A histological and histochemical study. Br J Obstet Gynaecol 1989;96:15–23. Pacheco P, Camacho MA, Garcia LI, Hernandez ME, Carrillo P, Manzo J. Electrophysiological evidence for the nomenclature of the pudendal nerve and sacral plexus in the male rat. Brain Res 1997;763: 202–8.
Fertility ratio in male rats: Effects after denervation of two pelvic floor muscles J. Manzoa,*, M.I. Vazqueza, M.R. Cruzb, M.E. Hernandeza, P. Carrilloa, P. Pachecoa,b b
a Instituto de Neuroetología, Universidad Veracruzana, Apartado Postal 566, Xalapa, Ver. 91001, Mexico Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apartado Postal 70228, Ciudad Universitaria, México, D.F. 04510, Mexico Received 18 February 1999; received in revised form 3 June 1999; accepted 21 October 1999
Abstract Fertility ratio is defined here as the proportion of females that a male can impregnate after a constant period of in-polygyny living. This ratio was investigated in male rats after denervation of two pelvic floor muscles, the pubococcygeus and iliococcygeus. Denervation was carried out by transecting the somatomotor branch of the pelvic nerve. The lesion did not modify the sexual behavior of males or their overall fertility, but decreased the weight of the ejaculated seminal plug. Consequently, the number of days living in cohabitation to induce pregnancy was increased in lesioned males (ⵑ13 days) compared with intact and sham animals (ⵑ5 days). These results showed that the fertility ratio was optimal when intact/sham males cohabited with females for two consecutive estrous cycles, but that lesioned males needed up to four cycles to induce most pregnancies. Two hypotheses are raised by our results. The first is that pelvic floor denervation decreases the forceful tension required to expel the semen from the prostatic urethra to the vagina, then an incomplete seminal plug is expelled. The second is that denervation cut afferent fibers that reflexively promote the continence of the semen deposited in the prostatic urethra during seminal emission, allowing some to leak out before ejaculation. The latter hypothesis can also explain the recovery of the fertility ratio in lesioned males. It could be a compensatory mechanism mediated by the pudendal nerve supply to the coccygeus muscle, the other pelvic floor muscle. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ejaculation; Pubococcygeus; Iliococcygeus; Coccygeus; Copulatory behavior; Infertility; Seminal plug; Seminal emission; Pelvic nerve
1. Introduction Fertility is a key event in the reproduction of mammals. Adult males have a high probability to impregnate a female if copulation occurs around her ovulatory period. However, the malfunction of some physiological mechanism in the masculine reproductive system, or the experimental manipulation of the subject, can decrease the reproductive potential even if typical copulatory behavior is achieved. Thus, to quantify the normal or disrupted fertility of males when observational analysis does not detect modifications in behavior, we introduce the fertility ratio concept, defined as the proportion of females that a male can impregnate after a constant period of in-polygyny living. In male rats, there are several factors that affect fertility without altering behavior. One of them is the quality of expulsion and deposition of a seminal plug into the female vagina during ejaculation [1–4]. The seminal plug is a semisolid mass normally present in the urethra of adult male rats * Corresponding author. Tel.: 52 (28) 12-5748; Fax: 52 (28) 12-5748. E-mail address: [email protected]
[5], composed of secretions from the seminal vesicles and coagulating glands and hardened by a complex of accompanying proteins [6–10]. Ablation of these sex glands does not impair sexual behavior of male rats, but they become infertile [7,11–13]. Infertility is due to the absence of a seminal plug, because males deposit sperm in the vagina with the capacity to fertilize eggs if intrauterine insemination is carried out [14]. This means that a seminal plug in the vagina provides a pump-like action, functioning to increase the movement and number of spermatozoa inside the uterus and fallopian tubes [3]. Thus, the absence of the seminal plug reduces the probability of sperm reaching and fertilizing the ovum. Furthermore, the mechanical effect of the seminal plug works together with uterine wall contractions induced chemically after ejaculation by semen substances from the seminal vesicles and coagulating glands [3,4,15]. Alterations also occur within the male reproductive system that influence the expulsion of the seminal plug during ejaculation. Transection of the hypogastric nerve, for example, does not alter the copulatory behavior of males, but they fail to expel semen and seminal plug, and therefore, become infertile [16,17]. This effect is due to the role of the
0031-9384/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S0031-9384(99)00 2 1 9 - X
612
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
sympathetic hypogastric nerve in the emission process [18– 21]. On the other hand, the role of striated muscles in ejaculation is also important for the appropriate expulsion and deposition of semen and plug into the vagina. The muscles studied to date are those at the base of the penis that have an intense contractile activity during ejaculation and serve the appropriate expulsion and deposition of the seminal plug [22–24]. Removal of these muscles induces infertility in males because they lose the ability to expel the whole plug [25]. In our laboratory, we have been investigating the role of the iliococcygeus (Icm) and pubococcygeus (Pcm) muscles in the reproductive physiology of the rat. These muscles, together with the coccygeus, form the striated muscular complex known as the pelvic floor [26,27] or levator ani [28]. Icm and Pcm are innervated by the somatomotor branch (Smb) of the pelvic nerve that is composed of afferent and motor axons from the L6 and S1 spinal cord segments [29]. The Smb seems completely somatic, unlike the viscerocutaneous branch of the pelvic nerve that carries afferent fibers from viscera and perigenital skin and the well known autonomic fibers supplying the pelvic plexus [29–35]. Denervation of Icm and Pcm in rats, by transection of the Smb, does not alter reproductive parameters in females [36,37] or in males [32]. However, we observed that this surgery in males reduces the weight of the ejaculated seminal plug. This finding led us to the present study with the aim to further analyze both the effect of Icm and Pcm denervation on the expulsion of semen and the effect of the low weight seminal plug on the fertility ratio of Smb-transected males.
nerve as described elsewhere [29]. To avoid regeneration, a 5-mm portion of the nerve branch was excised (Fig. 1). During sham surgery, the branch was dissected and hooked but not cut. After surgery subjects were placed in a warm chamber (ⵑ24⬚C) until they had recovered from anesthesia, and then returned to the animal room. No health problem was detected in subjects after the lesion of Smb; thus, they did not require special postsurgical care to recover. At the end of the study the animals were deeply anesthetized and the branch transection confirmed before the rats were killed with an overdose of pentobarbital. The examination of all animals with nerve transections revealed no evidence of regeneration. 2.3. Mating tests One copulatory series per animal was observed twice a week before (four tests) and after (seven tests) surgery. Tests were conducted during the last third of the dark period. After 5 min adaptation of the male in a Plexiglas cylindrical arena (50 cm high ⫻ 50 cm diameter), a receptive female was introduced. The introduction of the female indicated the beginning of the test, and the male ejaculatory pattern, the end. The parameters of copulation (see below) were recorded in a PC computer using the SBR software [37]. When an ejaculatory pattern was observed, the female
2. Materials and methods 2.1. Subjects and housing Sexually experienced male Wistar rats, weighing 250– 350 g, were assigned to a group where the pelvic nerve somatomotor branch (Smb) was bilaterally transected (n ⫽ 12) or to a group that was Sham operated (n ⫽ 5). A third group of males was used to obtain the seminal plug from the vagina of mated females (see below). Ovariectomized receptive females were used for mating tests. Receptivity was induced after females were injected subcutaneously with 10 g of estradiol benzoate and 2 mg of progesterone, 48 and 4 h before tests, respectively. Rats were housed in plastic cages (50 ⫻ 30 ⫻ 20 cm) containing wood chip bedding, and kept in a room maintained at 22 ⫾ 2⬚C and under a 12: 12 LD light schedule (lights on at 2200 h). Commercial pelleted rodent chow (Purina, México) and water were available ad lib. 2.2. Surgery Smb transection was carried out under sodium pentobarbital anesthesia (26 mg/kg i.p.). The nerve branch was located in the pelvic area, after a ventral abdominal incision, where it crosses the internal iliac vessel. Smb travels more centrally than the viscerocutaneous branch of the pelvic
Fig. 1. Drawing of the pelvic nerve and its branches, the somatomotor and the viscerocutaneous. The image shows that both the pelvic and pudendal nerves originates from the L6–S1 Trunk, formed in turn from the L6 and S1 spinal nerves. The landmarks to find the pelvic nerve and its branches are the iliac vessels, mainly the internal iliac vein. Asterisks show the two places where the somatomotor branch was cut in the experiments; hence, a portion of the branch was excised.
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
was immediately transferred to another arena. This second arena had a black floor with no bedding material and was already occupied by a male used to dislodge the seminal plug, if any, from the vagina. Plugs are generally dislodged within three to six intromissions [38,39]. To prevent ejaculation, males used for this procedure were only allowed four intromissions, and females were brought to a second male if it was necessary. If no plug was dislodged within eight intromissions, then it was assumed that none was present. Plugs obtained were immediately weighed. 2.4. Copulatory parameters The observational study of sexual behavior in males allowed the identification of three distinctive copulatory movements, i.e., mounts, intromissions, and ejaculation. A male had several mounts and intromissions before ejaculation. The performance of these copulatory movements guided the quantification of copulatory parameters. The number of mounts (NM) and number of intromissions (NI) represent the frequency of each movement before ejaculation. The time elapsed from the introduction of the female with the male to the first mount or intromission was recorded as the latency of mount (LM) or latency of intromission (LI), respectively. If the first copulatory movement executed by the male was an intromission, then LM ⫽ LI. The time elapsed from the first intromission to the ejaculation was recorded as the latency of ejaculation. A proportion of intromissions (hits) was obtaining by computing NI/ (NM⫹NI), the result is known as the hit rate parameter of copulatory behavior, and reflects the capacity of the male to intromit—hence, to have erection of the penis. 2.5. Fertility tests Results from mating tests (see below) showed effects of Smb transection on the weight of the seminal plug expelled after ejaculation. Thus, the present experiment was designed to test whether this modification is reflected in the fertility ratio of the male. In doing this, intact virgin females and sexually experienced males, proved previously to be fertile, were used. Each experimental male (n ⫽ 12) was placed in a cage for reproduction (60 ⫻ 40 ⫻ 22 cm) with three females for 20 days. After that period, or earlier if pregnancy was conspicuous, each female was placed alone in a cage and was observed until parturition or for a maximum of 21 days, when it was assumed that the female had not become pregnant. Gestation was considered to last 21 days; therefore, to calculate the first day of pregnancy a calendar backcount from the day of parturition was made. With this procedure it was possible to obtain the number of days between the formation of a reproduction group and the first day of pregnancy of each female. After being in the reproduction groups, the 12 males were operated as described above, and two groups were obtained, sham (n ⫽ 4) and Smb transected (n ⫽ 8). Then, 5 days after surgery they were
613
placed again in reproduction groups with new females, and the same day counts were made. 2.6. Statistics Results are presented as means ⫾ SEM. Statistics were performed using the GB-STAT Software (V6.0, Dynamic Microsystems, Inc., USA). Behavioral parameters of copulation were analyzed with the analysis of variance (ANOVA) for repeated measures (a post hoc analysis was not necessary in any case). ANOVA tables showed the effect of surgery, tests, and the interaction surgery–test, and the outcomes are presented below only if relevant. Paired comparison of the variance in seminal plug weight between sham and experimental subjects was analyzed by the Cochran’s C (CC) test, and is presented as a box-and-whisker plot as described elsewhere [40]. For the fertility data, the proportions of pregnant females were compared using the Fisher Exact Test, while the number of days to fertilize a female was analyzed by the Kruskal–Wallis ANOVA (KW), followed by paired comparisons with the Mann–Whitney U-test. The continuous 20 days that females were exposed to inpolygyny living with males to become pregnant were divided in four 5-day periods. This procedure was utilized taking into consideration the average number of days that the female estrous cycle lasts. The fertility ratio was calculated for each 5-day period with the following formula: FR = PFp ⁄ TPF
(1)
where FR ⫽ fertility ratio, PFp ⫽ number of pregnant females per period, and TPF ⫽ total number of pregnant females during the whole 20 days of male–female cohabitation. FR was calculated per subject and used as the male value for statistic analysis. Between-groups comparisons for each 5-day period were performed using KW, followed by a paired comparison with the Mann–Whitney U-test. Notice that the formula does not consider females that did not become pregnant. They were excluded for two reasons: (a) every male fertilized at least one female in his group, and (b) there is a chance that some females reflect infertility (or pseudopregnancy) of their own and not because the partner male.
3. Results Male sexual behavior was not modified after bilateral transection of the pelvic nerve somatomotor branch (SmbTx). As shown in Table 1, no copulatory parameter was altered by the surgery when compared with Sham animals. However, although the ANOVA for the weight of the seminal plug did not reveal a reliable change in the surgery–test interaction (Table 1), it showed a significant effect of surgery, F(1, 15) ⫽ 4.24, p ⫽ 0.05, and tests, F(6, 90) ⫽ 2.33, p ⫽ 0.03. The surgery data indicated that Smb transection produced a decrease in the weight of the seminal plug expelled with ejaculation (Fig. 2).
4.2 ⫾ 3.21 2.91 ⫾ 0.57 7.2 ⫾ 0.86 8.58 ⫾ 0.73 3.6 ⫾ 0.6 5.91 ⫾ 0.97 4.8 ⫾ 0.8 10.33 ⫾ 2.46 189 ⫾ 55.86 178.1 ⫾ 17.2 0.74 ⫾ 0.12 0.75 ⫾ 0.04 175 ⫾ 8 130 ⫾ 19 3.8 ⫾ 1.39 3.66 ⫾ 0.97 8.4 ⫾ 0.50 7.58 ⫾ 1.22 6.2 ⫾ 2.24 5.25 ⫾ 1.05 7.6 ⫾ 2.18 15.75 ⫾ 6.97 203.6 ⫾ 34.3 136.1 ⫾ 19.7 0.72 ⫾ 0.09 0.70 ⫾ 0.06 106 ⫾ 31 133 ⫾ 19
3.0 ⫾ 1.22 3.16 ⫾ 0.77 6.0 ⫾ 1.09 7.16 ⫾ 0.68 6.2 ⫾ 2.31 7.75 ⫾ 3.6 16.8 ⫾ 9.84 10.83 ⫾ 3.95 157.2 ⫾ 37.1 152.5 ⫾ 24.6 0.69 ⫾ 0.09 0.70 ⫾ 0.05 167 ⫾ 26 118 ⫾ 13
Test 6, AS Test 5, AS
2.8 ⫾ 1.06 3.33 ⫾ 0.84 7.8 ⫾ 0.73 8.41 ⫾ 0.84 4.8 ⫾ 0.58 7.25 ⫾ 2.19 6.2 ⫾ 1.49 9.58 ⫾ 2.11 161.6 ⫾ 24.6 160.5 ⫾ 23.9 0.75 ⫾ 0.08 0.74 ⫾ 0.05 170 ⫾ 41 148 ⫾ 31
Fig. 2. Overall significant reduction in the weight of the seminal plug expelled by males with transection of the pelvic nerve somatomotor branch (Smb-Tx), as compared to sham-operated subjects (Sh). *p ⬍ 0.05.
Seminal plug weight
Hit rate
Latency of ejaculation
Latency of intromission
Latency of mount
Number of intromissions
Data are Mean ⫾ SEM. BS ⫽ before surgery; AS ⫽ after surgery. Latencies in seconds, seminal plug weight in milligrams. Statistics show the surgery–test interaction from the repeated measures ANOVA.
1.6 ⫾ 0.74 2.83 ⫾ 0.58 10.0 ⫾ 1.22 8.16 ⫾ 0.84 5.0 ⫾ 0.54 5.08 ⫾ 0.73 5.4 ⫾ 0.24 6.91 ⫾ 1.0 184.4 ⫾ 24.5 159.1 ⫾ 27.2 0.86 ⫾ 0.05 0.77 ⫾ 0.04 143 ⫾ 3 102 ⫾ 22 13.0 ⫾ 6.04 5.75 ⫾ 1.78 9.4 ⫾ 0.74 9.83 ⫾ 0.62 6.0 ⫾ 0.89 5.41 ⫾ 0.82 26.8 ⫾ 19.3 8.66 ⫾ 1.58 283 ⫾ 52.88 247.5 ⫾ 53.7 0.52 ⫾ 0.1 0.69 ⫾ 0.05 137 ⫾ 25 116 ⫾ 10
5.8 ⫾ 1.8 4.5 ⫾ 1.17 7.4 ⫾ 0.87 9.16 ⫾ 0.67 8.6 ⫾ 1.63 8.16 ⫾ 1.53 20.4 ⫾ 9.43 13.33 ⫾ 3.41 188 ⫾ 33.92 187.2 ⫾ 26.2 0.59 ⫾ 0.08 0.71 ⫾ 0.05 146 ⫾ 17 89 ⫾ 22
Test 1, AS
4.6 ⫾ 1.43 3.83 ⫾ 0.77 8.6 ⫾ 0.97 8.08 ⫾ 1.32 4.2 ⫾ 0.86 14.08 ⫾ 5.30 11.4 ⫾ 2.31 35.4 ⫾ 18.4 198 ⫾ 37.06 146.9 ⫾ 24.5 0.66 ⫾ 0.07 0.67 ⫾ 0.05 170 ⫾ 20 137 ⫾ 10 Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Sh Smb-Tx Number of mounts
Test 4, BS Group Parameter
Table 1 Copulatory parameters of Sham (Sh) and Smb-transected (Smb-Tx) male rats
Test 2, AS
Test 3, AS
Test 4, AS
Test 7, AS
Statistics
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618 F(6, 90) ⫽ 1.60, NS F(6, 90) ⫽ 1.15, NS F(6, 90) ⫽ 0.30, NS F(6, 90) ⫽ 1.27, NS F(6, 90) ⫽ 0.25,NS F(6, 90) ⫽ 0.87, NS F(6, 90) ⫽ 1.65, NS
614
As indicated, the repeated-measures ANOVA yielded a significant difference across tests in the seminal plug weight parameter. This difference was due to the high variability in the weight of the plug that Smb-transected males expelled following surgery. Performing paired comparisons of the homogeneity of variances in this parameter, significant differences were found in the first (CC ⫽ 0.787, p ⬍ 0.05), second (CC ⫽ 0.780, p ⬍ 0.05), third (CC ⫽ 0.991, p ⬍ 0.01), and sixth (CC ⫽ 0.920, p ⬍ 0.01) postsurgery tests (Fig. 3). The fertility potential of males was altered by Smb transection when analyzed per each 5-day period, although the overall data showed that at the end males were capable of inducing pregnancy in most females independently of the surgical group to which they belonged (Table 2). As a consequence, the number of days needed to induce pregnancy was reliably increased in Smb-transected males. Intact and sham-operated males induced pregnancy after living for about 5 days with the females, while Smb-Tx males did so after cohabiting for around 13 days, a difference that was statistically significant in the ANOVA [KW 2(2) ⫽ 14.95, p ⬍ 0.01] and paired comparisons (Fig. 4). The fertility ratio showed that intact and sham males induced 100% of pregnancies during the first and second 5-day periods. Smb-Tx males were unable to induce pregnancy in the first period, but recovered partially in the second and continued to fertilize females up to the fourth period. These results made the Smb-Tx group to be significantly different from intact and sham groups in the first [KW 2(2) ⫽ 9.81, p ⬍ 0.01], third [KW 2(2) ⫽ 8.0, p ⬍ 0.01], and fourth [KW 2(2) ⫽ 6.42, p ⬍ 0.01] periods, but not in the second [KW 2(2) ⫽ 1.36, p ⬎ 0.05], and these differences were reflected in the paired comparisons (Fig. 5).
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
Fig. 3. Box-and-whisker plot showing the variability in the weight of the seminal plug ejaculated by sham (Sh) and pelvic nerve somatomotor branch transected (Smb-Tx) male rats in a test before surgery (BS) and the seven tests after surgery (AS-n). The histogram-like display shows the box with quartiles ends, the median as a horizontal line inside the box, and the whiskers extending to the farthest data. Significant differences in the variance comparison (Sh vs Smb-Tx per test) were found at p ⬍ 0.05 (*) and p ⬍ 0.01 (**).
4. Discussion The data confirm our preliminary results that denervation of Icm and Pcm produces a decrease in the weight of the ejaculated seminal plug. Although this alteration is just an overall reduction, the effect produced on the fertility ratio is remarkable. The results clearly show that after Smb transection copulatory behavior remains completely normal as previously reported [32], but the expulsion and/or deposition of the seminal plug in the vagina is modified. Every male was fertile by the end of the experiments, but Smb-transected males needed to stay with females around 13 days to induce pregnancy, while intact or sham animals required only 5 days. Thus, living 5 days with a female is the minimum time for a normal male to induce pregnancy, a period that we named the pregnancy induction time (PIT). Each PIT is cor-
615
Fig. 4. Number of days needed by intact (Int), sham (Sh), and pelvic nerve somatomotor branch transected (Smb-Tx) male rats, to induce pregnancy after 20 days of in-polygyny living with three females. The figure shows that Smb-Tx males require significantly (p ⬍ 0.01) more days to induce pregnancy, while Int and Sh males require around 5 days, a period that we named Pregnancy Induction Time.
related with the duration of an estrous cycle of the female rat. In this sense normal males require only one PIT to fertilize females, while Smb-transected males require up to four consecutive PITs. The fertility ratio, then, as calculated by each consecutive PIT, showed that in the first the Smbtransected males were unable to induce pregnancy, but that their capacity to fertilize was restored beginning in the second PIT and continuing until the fourth. This means that infertility in Smb-transected males is transient, i.e., they do not become permanently infertile, but need to cohabit several days (from two to four estrous cycles) to induce pregnancy. Despite their altered fertility, these males showed normal patterns in their copulatory behavior, and results showed that transient infertility was due to effects on the weight of the seminal plug. The proper expulsion and deposition of a normal seminal plug in the rat is crucial for the induction of pregnancy [1–4], and any factor affecting it has a direct consequence for fertility [12,14,25].
Table 2 Percentage of fertilized females by Intact, sham (Sh) and Smb-transected (Smb-Tx) male rats Group
% (0–5 Days period)
% (5–10 Days period)
% (10–15 Days period)
% (15–20 Days period)
% (Total)
Intact (n ⫽ 12) Sh (n ⫽ 4) Smb-Tx (n ⫽ 8)
50 75 (NS) 0 (*)
44.44 25 (NS) 25 (NS)
0 0 (NS) 25 (*)
0 0 (NS) 29.16 (*)
94.44 100 (NS) 79.16 (NS)
Statistics ⫽ Fisher Exact Test of proportions; intact as the comparison group; NS ⫽ not significant; *p ⬍ 0.01.
616
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
Fig. 5. Fertility ratio of intact, sham, and pelvic nerve somatomotor branch transected (Smb-Tx) male rats, after 20 days of in-polygyny living with three females. The whole time was divided in four 5-days periods (0–5, 5– 10, 10–15, and 15–20), and between-groups comparisons were done per period. Intact and sham males fertilized all females in the first and second periods, while Smb-Tx males were significanlty different (p ⬍ 0.01) because did not induce pregnancy in the first period, started in the second, and fertilized most females by extending their matings up to the fourth period.
The main question here is what these pelvic floor muscles do in the ejaculatory process to contribute for the expulsion of the seminal plug, and hence to fertility. Considering that the striated muscles are controlled by the somatic nervous system, it is suggested that they play little part in the autonomic-regulated physiology of seminal emission. The activity of the sex glands and structures having a role in emission is controlled by the sympathetic component of the autonomic nervous system [20,41–43]. Lesion of the hypogastric nerve, the main peripheral sympathetic pathway in the pelvic area, produces infertility by blocking the deposition of sperm and semen material in the prostatic urethra [20,21]. Thus, the ejaculatory pattern of copulatory behavior is normal but no semen is expelled. As alterations in the sympathetic innervation were not induced in our experimental animals, we propose two hypotheses to explain our results. The first suggests that denervation of Icm and Pcm decreases the forceful tension required to expel the emission-accumulated semen from the prostatic urethra to the female vagina. It is known that ejaculation is a two-step process—first seminal emission, and second the powerful mechanical event promoted mainly by the contraction of striated muscles, that is, the ejaculation per se. However, the muscles reported to be involved in the ejection of the semen are the ischiocavernosus and the bulbospongiosus [23,25,42,44,45]. Thus, this proposal is the first indicating a possible role of pelvic floor muscles in producing force for the expulsion of the ejaculate. If true, it means that after ejaculation of Smb-transected males, a quantity of semen is expelled, but some remains in the male urethra due to the lack of a complete ejection force.
The second hypothesis suggests that transection of the Smb cut afferent fibers from the Icm and Pcm that reflexively promote the continence of the semen that is deposited in the prostatic urethra during seminal emission. This idea came from our previous finding that afferent innervation of Pcm reflexively induces urinary continence in male rats by relaxing the bladder wall and allowing the urine to be retained inside the bladder [46]. The role of pelvic floor musculature in the physiology of continence is a topic that has received attention for several years. The activity of pelvic floor muscles alleviates malfunctions such as impotence and incontinence of feces and urine [47–51]. Resulting relief seems to be related to a spinal inhibitory reflex triggered by afferents from these muscles that in turn activate efferent nerves controlling continence [46,52]. The urethra is a common path for the expulsion of semen and urine, thus denervation of pelvic floor muscles could induce both urinary incontinence [53] and seminal incontinence. Therefore, incontinence of the semen could occur as the emission process is developed during the several mounts and intromissions that precede the ejaculation in the copula of the male rat. The semen arriving to the prostatic urethra is not retained completely, and some may leak to the outside. Drops of semen material are then probably removed by the male during the very common genital grooming after intromissions. Thus, after ejaculation the quantity of semen deposited in the female vagina is decreased. The present proposal also explains the high variability in the weight of the seminal plug of denervated animals. This variability is probably the outcome of several factors such as the frequency and quality of genital grooming, the latency of ejaculation, or the number of mounts and intromissions before ejaculation. Grooming could mainly account for the quantity of semen removed before ejaculation, reducing the quantity of semen deposited in the vagina during ejaculation. According to our observations, male rats eat semen material and even seminal plugs that they remove from the vagina if a previous male has ejaculated or after their own postejaculatory interval. Finally, our second hypothesis is also useful in explaining the recovery of the fertility ratio of Smb-transected males after four consecutive PITs. It is known that the pelvic floor is the complex structured by the Icm, Pcm, and coccygeus (Ccm) muscles [27,28]. Transection of Smb denervates Icm and Pcm but not Ccm. This latter muscle is innervated by a branch of the pudendal nerve in both female and male rats [29,54]. Whether the afferent innervation of Ccm also participates in promoting urinary/semen continence is a topic that has not been studied to date. However, it is possible to argue that the three muscles organizing the pelvic floor engage in triggering spinal-mediated inhibitory reflexes to promote continence. Thus, we suggest that alterations produced by denervation of Icm and Pcm are transient effects that are overcome by a plastic compensatory process carried out by neurons projecting to the Ccm. This plasticity could be central, could be peripheral, or could be both.
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
The latter discussion has been focused to the male. However, it is important to consider that the reproductive physiology of the female cannot be completely excluded. The female reproductive system could also be self-adjusted to become fertile if continuously receive a reduced amount of semen material.
[18]
[19] [20]
Acknowledgments This research was supported by the Grant No. 4157P-N from the National Council for Science and Technology (CONACYT-México) to J.M.
[21] [22]
[23]
References [24] [1] Blandau RJ. On factors involved in sperm transport through the cervix uteri of the albino rat. Am J Anat 1945;77:253–72. [2] Hart BL. Role of testosterone secretion and penile reflexes in sexual behavior and sperm competition in male rats: a theoretical contribution. Physiol Behav 1983;31:823–7. [3] Matthews M, Adler NT. Facilitative and inhibitory influences of reproductive behavior on sperm transport in rats. J Comp Physiol Psychol 1977;91:727–41. [4] Matthews M, Adler NT. Systematic interrelationship of mating, vaginal plug position, and sperm transport in the rat. Physiol Behav 1978; 20:303–9. [5] Kunstyr I, Kupper W, Weisser H, Naumann S, Messow C. Urethral plug—a new secondary male sex characteristic in rat and other rodents. Lab Anim 1982;16:151–5. [6] Carballada R, Esponda P. Role of fluid from seminal vesicles and coagulating glands in sperm transport into the uterus and fertility in rats. J Reprod Fertil 1992;95:639–48. [7] Cukierski MA, Sina JL, Prahalada S, Robertson RT. Effects of seminal vesicle and coagulating gland ablation on fertility in rats. Reprod Toxicol 1991;5:347–52. [8] Fawell SE, Higgins SJ. Formation of rat copulatory plug: purified seminal vesicle secretory proteins serve as transglutaminase substrates. Mol Cell Endocrinol 1987;53:149–52. [9] Koren E, Schon E, Lukac J. The coagulation of insoluble and basic proteins from rat seminal vesicle secretion with vesiculase: influence of collagenase-like peptidase from rat testis. J Reprod Fertil 1975;42: 491–5. [10] Sofikitis N, Takahashi C, Kadowaki H, Okazaki T, Shimamoto T, Nakamura I, Miyagawa I. The role of the seminal vesicles and coagulating glands in fertilization in the rat. Int J Androl 1992;15:54–61. [11] Beach FA, Wilson JR. Mating behavior in male rats after removal of the seminal vesicles. Proc Natl Acad Sci USA 1963;49:624–6. [12] Carballada R, Esponda P. Structure of the vaginal plugs generated by normal rats and by rats with partially removed seminal vesicles. J Exp Zool 1993;265:61–8. [13] Tisell LE, Larsson K. Unimpaired sexual behavior of male rats after complete removal of the prostate and seminal vesicles. Invest Urol 1979;16:274–5. [14] Cukierski MA, Sina JL, Prahalada S, Wise LD, Antonello JM, MacDonald JS, Robertson RT. Decreased fertility in male rats administered the 5 alpha-reductase inhibitor, finasteride, is due to deficits in copulatory plug formation. Reprod Toxicol 1991;5:353–62. [15] Crane LH, Martin L. Postcopulatory myometrial activity in the rat as seen by video- laparoscopy. Reprod Fertil Dev 1991;3:685–98. [16] Larsson K, Swedin G. The sexual behavior of male rats after bilateral section of the hypogastric nerve and removal of the accessory genital glands. Physiol Behav 1971;6:251–3. [17] Lucio RA, Manzo J, Pacheco P. Pelvic and hypogastric nerves co-
[25]
[26]
[27] [28] [29]
[30] [31] [32]
[33] [34] [35]
[36]
[37]
[38]
[39]
[40] [41] [42]
617
participation in the mediation of male rat sexual behavior. Soc Neurosci Abstr 1991;17:1059. Billups KL, Tillman S, Chang TS. Ablation of the inferior mesenteric plexus in the rat: alteration of sperm storage in the epididymis and vas deferens. J Urol 1990;143:625–9. Kimura Y, Adachi K, Kisaki N, Ise K. On the transportation of spermatozoa in the vas deferens. Andrologia 1975;7:55–61. Kimura Y, Miyata K, Adachi K, Kisaki N. Peripheral nerves controlling the closure of internal urethral orifice during ejaculation. Urol Int 1975;30:218–27. Kolbeck SC, Steers WD. Neural regulation of the vas deferens in the rat: an electrophysiological analysis. Am J Physiol 1992;263:R331–8. Holmes GM, Chapple WD, Leipheimer RE, Sachs BD. Electromyographic analysis of male rat perineal muscles during copulation and reflexive erections. Physiol Behav 1991;49:1235–46. Holmes GM, Sachs BD. The ejaculatory reflex in copulating rats: normal bulbospongiosus activity without apparent urethral stimulation. Neurosci Lett 1991;125:195–7. Wallach SJR, Hart BL. The role of the striated penile muscles of the male rat in seminal plug dislodgement and deposition. Physiol Behav 1983;31:815–21. Sachs BD. Role of striated penile muscles in penile reflexes, copulation, and induction of pregnancy in the rat. J Reprod Fertil 1982;66: 433–43. Brink EE, Pfaff DW. Vertebral muscles of the back and tail of the albino rat (Rattus norvegicus albinus). Brain Behav Evol 1980;17:1– 47. McKenna KE, Nadelhaft I. The organization of the pudendal nerve in the male and female rat. J Comp Neurol 1986;248:532–49. Schröder HD. Organization of the motoneurons innervating the pelvic muscles of the male rat. J Comp Neurol 1980;192:567–87. Pacheco P, Martinez–Gomez M, Whipple B, Beyer C. Komisaruk BR. Somato-motor components of the pelvic and pudendal nerves of the female rat. Brain Res 1989;490:85–94. Hulsebosch CE, Coggeshall RE. An analysis of the axon populations in the nerves to the pelvic viscera in the rat. J Comp Neurol 1982;211:1–10. Langworthy OR. Innervation of the pelvic organs of the rat. Invest Urol 1965;2:491–511. Lucio RA, Manzo J. Martinez–Gomez M, Sachs BD, Pacheco P. Participation of pelvic nerve branches in male rat copulatory behavior. Physiol Behav 1994;55:241–6. Mallory B, Steers WD, De Groat WC. Electrophysiological study of micturition reflexes in rats. Am J Physiol 1989;257:R410–21. Purinton PT, Fletcher TF, Bradley WE. Gross and light microscopic features of the pelvic plexus in the rat. Anat Rec 1973;175:697–705. Steers WD, Mallory B, De Groat WC. Electrophysiological study of neural activity in penile nerve of the rat. Am J Physiol 1988;254: R989–1000. Burden HW, Price GT, Renegar RH, Hodson CA. Effects of peripheral nerve lesions during pregnancy on parturition in rats. Anat Embryol (Berl) 1990;182:499–501. Martinez–Gomez M, Cruz Y, Pacheco P, Aguilar–Roblero R, Hudson R. The sensory but not muscular pelvic nerve branch is necessary for parturition in the rat. Physiol Behav 1998;63:929–32. Claro F, Del Abril A, Segovia S, Guillamon A. SBR: a computer program to record and analyse sexual behavior in rodents. Physiol Behav 1990;48:489–93. McClintock MK, Anisko J, Adler NT. Group mating among norway rats II. The social dynamics of copulation: competition, cooperation, and mate choice. Anim Behav 1982;30:410–25. Tukey JW. Explanatory Data Analisis. Reading, MA: Addison–Wesley, 1977. Bacq ZM. Impotence of the male rodent after sympathetic denervation of the genital organs. Am J Physiol 1931;96:321–30. Benson GS. Male sexual function: erection, emission, and ejaculation. In: Knobil E, Neil JD, editors. The Physiology of Reproduction. New York: Raven Press, Ltd., 1994. pp. 1489–1506.
618
J. Manzo et al. / Physiology & Behavior 68 (2000) 611–618
[43] Ricker DD, Chang TSK. Neuronal input from the inferior mesenteric ganglion (IMG) affects sperm transport within the rat cauda epididymis. Int J Androl 1996;19:371–6. [44] De Groat WC, Booth AM. Physiology of male sexual function. Ann Intern Med 1980;92:329–31. [45] Sachs BD. Potency and fertility: hormonal and mechanical causes and effects of penile actions in rats. In: Balthazart J, Prove E, Gillis R, editors. Hormones and behavior in higher vertebrates. Berlin: Springer Verlag, 1983. pp. 86–110. [46] Manzo J, Esquivel A, Hernandez ME, Carrillo P, Martinez–Gomez M, Pacheco P. The role of pubococcygeus muscle in urinary continence in the male rat. J Urol 1997;157:2402–6. [47] Claes H, Baert L. Pelvic floor exercise versus surgery in the treatment of impotence.Br J Urol 1993;71:52–7. [48] Dubrovsky B, Filipini D. Neurobiological aspects of the pelvic floor muscles involved in defecation. Neurosci Biobehav Rev 1990;14: 157–68. [49] Ishigooka M, Hashimoto T, Sasagawa I, Nakada T. Reduction in
[50] [51] [52]
[53]
[54]
norepinephrine content of the rabbit urinary bladder by alpha-2 adrenergic antagonist after electrical pelvic floor stimulation. J Urol 1994;151:774–5. Merril DC, Conway C, DeWolf W. Urinary incontinence. Treatment with electrical stimulation of the pelvic floor. Urology 1975;5:67–72. Teague CT, Merril DC. Electric pelvic floor stimulation. Mechanism of action. Invest Urol 1977;15:65–9. Sundin T, Carlsson C-A, Kock NG. Detrusor inhibition induced from mechanical stimulation of the anal region and from electrical stimulation of pudendal nerve afferents. An experimental study in cats. Invest Urol 1974;11:374–8. Gilpin SA, Goslin JA, Smith ARB, Warrel DW. The pathogenesis of genitourinary prolapse and stress incontinence of urine. A histological and histochemical study. Br J Obstet Gynaecol 1989;96:15–23. Pacheco P, Camacho MA, Garcia LI, Hernandez ME, Carrillo P, Manzo J. Electrophysiological evidence for the nomenclature of the pudendal nerve and sacral plexus in the male rat. Brain Res 1997;763: 202–8.