The Influence of Mating and Related Stimuli on Plasma Levels of Luteinizing Hormone, Follicle Stimulating Hormone, Prolactin, and Testosterone in the Male Rat1 FREJA KAMEL, WILLIAM W. WRIGHT, EDWARD J. MOCK, AND ARTHUR I. FRANKEL2 Department of Biological Sciences, State University of New York at Binghamton, Neiv York 13901
killed immediately after mating for varying times up to 1 h, and their levels of LH, FSH, PRL, and T were measured. Both LH and PRL levels peaked 5 min after the start of mating and the declined, while FSH levels remained unchanged. T levels rose slowly and were significantly elevated only after an hour of mating. These results show that levels of LH and PRL, but not FSH, are increased during mating in the male rat, and suggest that the increase in T levels may be due to the prior increase in LH and PRL levels. Furthermore, the rapidity of the rise in LH and PRL levels, coupled with the consistent response of these hormones to unsuccessful attempts to mate, suggests that rather than following mating, the increases in LH and PRL may precede it. (Endocrinology 101: 421, 1977)
ABSTRACT. Male rats were exposed to several stimuli associated with mating, and their resting levels of LH, prolactin (PRL), and testosterone (T) were compared to their hormone levels after stimulation. Sexually experienced rats showed two- to fourfold increases in levels of these hormones after both mating with estrous females and attempting to mate with unreceptive females. Only LH levels were significantly elevated by the odor of estrous females or by non-tactile contact with estrous females. Neither transfer to an empty mating arena nor interaction with another male altered the rats' hormone levels. The LH and PRL levels of sexually naive rats showed a similar pattern of response, but T levels in naive rats were unaffected by all stimuli including mating. In a further study, rats were
T
HE ABILITY of male rats to release hormones during mating remains a subject of controversy. In an early study (1) levels of plasma LH (but not FSH) were increased 5 min after mating. However, a recent report (2) indicated that plasma levels of LH, FSH, and prolactin (PRL) remained unchanged during the hour following mating. These results suggested that gonadotropins might not mediate the matinginduced increase in plasma testosterone (T) levels which had been observed directly (3) as well as inferred from the effect of cohabitation on accessory sex organ weights (4,5). Recently we reported that mating was in fact associated with elevations in plasma Received November 1, 1976. 1 Supported by U.S. Public Health Service Grant HD 04081. A preliminary report of these findings was presented at the 9th Annual Meeting of the Society for the Study of Reproduction, Philadelphia, Pa., 1976 (Abstract #138). 2 Reprint requests to: Dr. Arthur I. Frankel, Department of Biological Sciences, State University of New York at Binghamton, Binghamton, New York 13901.
Binghamton,
levels of LH and PRL as well as T (6). Our study also suggested that successful copulation was not a necessary component of the stimulus resulting in hormone increases. Rats attempting to mate with unreceptive females showed elevations in levels of LH and PRL equivalent to those seen in mating rats. Furthermore, in mating rats, levels of LH and T were as high after one intromission as after ejaculation. These results indicated that ejaculation at least was not always required to elicit hormone release. The observation that related stimuli can elicit hormone release as readily as successful copulation is not unique, since a number of studies have suggested that interrupted mating, unsuccessful attempts to mate, or sensory stimuli associated with mating can elevate hormone levels in males of several other species—rabbit (7,8), hamster (9), vole (10), and mouse (11)—as well as in the rat (3,12), although not in the human male (13). To clarify this issue, we exposed male rats to a variety of stimuli associated with mating and measured their hormonal re-
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ternated with control sessions, in which the male was placed alone in the cage for 35 min. In a further effort to minimize pre-experimental handling, sexual experience in Study 4 was provided by placing an intact female into each male's home cage for at least one week preceding the experiment. Experimental exposure to mating and other stimuli was provided in semicircular plexiglass mating arenas (41 x 77 cm) lined with wood shavings. The shavings were replaced each time an Materials and Methods arena was used. Arenas used for stimuli not Four studies were conducted. Studies 1-3 involving an estrous female had never been exexamined the effect of various stimuli on plasma posed to females. In Studies 1-3, the males were hormone levels, and Study 4 followed the time placed in the arenas for a total of 30 min. course of hormone release during mating. The Stimulus animals were introduced into the arenas 10 min after the experimental animals, so that subjects were male Sprague-Dawley rats either purchased from Russell Miller Farms (Cazenovia, interactions lasted for 20 min. Stimuli used in N.Y.) and received in our laboratory when 50 days these studies were: control—empty arena lined old (Study 1) or raised in our colony from rats with clean shavings, odor—arena which had conoriginally purchased from this supplier (Studies tained 6 estrous females for 30 min immediately 2-4). The rats were housed 2 per cage and had preceding the experiment, non-tactile contact— access to Purina Rat Chow and water ad lib. estrous female on the far side of a double wire The animal room was maintained at constant mesh barrier, castrate female—ovariectomized, temperature (21 ± 1 C) and on an artificial 12 h unreceptive female, estrous female, and another light-12 h dark cycle (lights off 1100-2300 h EST male. In Study 4, the male was placed in a for Studies 1 and 4 and 1000-2200 h EST for mating arena. After 15 min, an estrous female was introduced into the arena, and the male was Studies 2 and 3). removed after varying periods of time. In all studies the sexual behavior of mating rats was Behavioral procedures observed, and the times of the first intromission Sexual experience and experimental mating and ejaculation of each ejaculatory series were sessions were conducted in the animal room dur- noted. ing the dark phase of the light-dark cycle (2 to 6 h after lights off) under dim red light. Females Experimental protocol used as stimulus animals were brought into The 41 rats in Study 1 (December 1974) were behavioral estrus with sc injections of estradiol benzoate (0.01 mg) and progesterone (1.0 mg) in given five weekly sessions of sexual experience sesame oil. Only fully receptive females were beginning when they were 65 days old. During the final two sessions the rats' sexual behavior used. Since we had observed previously that pre- was observed. The rats were assigned to one of experimental exposure to the mating arenas led five experimental groups (control, odor, nonthe rats to associate the arenas with mating, tactile contact, castrate female, estrous female) in the sexual experience sessions in Studies 1 and such a way that pre-experimental sexual be2 were conducted in ordinary stainless steel cages havior was equivalent among groups. The group (26 x 42 cm). The male was placed in the cage for with estrous females also included 6 rats which 5 min, after which an estrous female was intro- did not ejaculate during either of the final two duced into the cage. The first three sessions were experience sessions or during the experimental terminated after an additional 30 min regardless mating session. These constituted a sixth group, of the animals' behavior. The final two sessions designated slow maters; the rest were designated were terminated after 20 min, but if the rat intro- normal maters. There were 6-8 rats per group. mitted the session was continued to 30 min or the One blood sample was collected immediately first post-ejaculatory intromission, whichever after removing the rat from its home cage (restcame first. The experience sessions were al- ing levels), and another was collected after ex-
sponse. Furthermore, since we had observed that hormones appeared to reach maximal levels almost as soon as the rats began to mate, we also studied the time course of hormone release during mating. In this fashion we hoped to specify both the behavioral and the sensory components of the mating situation necessary to elicit hormone release.
INFLUENCE OF MATING ON PLASMA HORMONES posure to the appropriate stimulus. The two samples were collected one week apart in a random order. Each group was represented on each day of blood collection. The rats were 97-108 days old at the time of blood collection. The pre-experimental handling procedures in Study 2 (April 1976; 32 rats) were identical to those used in Study 1. The rats were divided into four groups (control, male, odor, estrous female) on the basis of their pre-experimental sexual behavior (7-8 rats per group). Two rats which did not mount the female during either of the final two experience sessions or during the experimental mating session were designated non-maters. Blood was collected as in Study 1; the rats were 93-103 days old at the time of blood collection. Study 3 (August 1975) employed 46 sexually naive rats. Each rat was randomly assigned to one of six experimental groups (control, male, odor, non-tactile contact, castrate female, estrous female; 7-9 rats per group). Blood was collected as in Study 1; the rats were 133-148 days old at the time of blood collection. The 52 rats in Study 4 (January 1975) had minimal sexual experience (see Behavioral procedures). Blood was collected from randomly constituted groups before (0 min) or 5, 15, 30, or 60 min after the introduction of the female into the arena. Resting levels were determined as in Study 1. Blood was also collected from two rats alter 15 min during which they did not mount the female (non-maters). Blood was collected from at least one rat at each time on each day; the order of collection was varied, so that, for example, the 60 min sample was not always collected last. The rats were 112-136 days old at the time of blood collection. Blood collection and hormone assay In Studies 1-3 blood samples (1.5 ml) were collected into heparinized syringes by cardiac puncture of etherized rats. The samples were kept in an ice bath for up to 1 h before centrifugation. In Study 4 blood samples were collected by decapitating etherized rats. The samples were allowed to clot at room temperature before centrifugation. All samples were collected between 3 and 5.5 h after lights off, within 1 min of the time indicated. Plasma or serum was stored at —20 C until assay. Samples were analyzed for T by radioimmunoassay as previously described (14). LH was meas-
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ured using the ovine-ovine rat LH radioimmunoassay (15). Values are expressed in terms of the NIAMDD standard RP-1. The sensitivity of the assay was 2.0 ng. The precision of the assay was estimated using replicate measurements of a pool of serum from male rats. The intraassay coefficient of variation for 200 /x\ samples (67.8% binding) was 9.0%, and the interassay coefficient of variation was 12.0%. Samples were analyzed for PRL using the NIAMDD rat PRL assay kit. Values were expressed in terms of the NIAMDD standard RP-1. The sensitivity of the assay was 0.23 ng. The intraassay coefficient of variation for 50 fi\ samples (44.0% binding) was 11.9%, and the interassay coefficient of variation was 16.0%. Samples were analyzed for FSH using the NIAMDD rat FSH assay kit. Values are expressed in terms of the NIAMDD standard RP-1. The sensitivity of the assay was 20 ng. The intrassay coefficient of variation, estimated by comparing duplicate measurements of each sample, was 15.3%. Samples were analyzed in duplicate, usually at two different volumes. All samples from a single study were analyzed for a particular hormone at the same time. Statistical analysis Statistical analysis (16) was performed using two-way analysis of variance for repeated measures. Comparisons within and among groups were made by analyzing the simple main effects. Further comparisons among groups were made using the Neuman-Keuls test for a posteriori multiple comparisons.
Results
Studies 1 and 2 examined the effect of various stimuli on the plasma hormone levels of sexually experienced rats. Study 1 (Fig. 1) showed that transfer of the rat to a novel mating arena (control) had no significant effect on the levels of any hormone measured. Odor and non-tactile contact produced a significant increase above resting levels only in LH levels (139% and 116%, respectively). The castrate females were unreceptive, and never permitted intromission, although the males mounted them repeatedly. Nevertheless, this stimulus produced a significant elevation in levels
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KAMEL ETAL.
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RESTING LEVEL
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LEVEL IN ARENA
Both of these groups showed a significant rise in LH levels; however, the rise shown by the slow maters (233%) was more substantial than that shown by the normal maters (135%). Only the slow maters showed a significant increase in PRL levels (186%), and only the normal maters showed a significant increase in T levels (93%). The results of Study 2 (Fig. 2) confirmed those of Study 1. Transfer to an arena had no significant effect. Odor produced a signif-
60
i
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•
LEVEL IN ARENA
40
20
***
ISO CONTROL
ODOR
SLOW MATERS
FIG. 1. Mean plasma levels of LH, prolactin, and testosterone in sexually experienced male rats (Study 1). Levels were measured after removing the rat from its home cage (resting level) and after exposing it to the appropriate stimulus (level in arena). Stimuli were: control—empty arena; odor—odor of estrous female; (9)—non-tactile contact with estrous female; -? — ovariectomized, unreceptive female; 9—estrous female (normal maters); slow maters—estrous female (slow maters). Each bar represents the mean value for 6 to 8 animals. In Figs. 1-3 the vertical lines represent the SE and the asterisks indicate the significance of the change in levels within a group (analysis of variance): *P < 0.05; **P < 0.01; ***P < 0.001.
of all three hormones (LH: 347%; PRL: 132%; T: 87%). All the rats exposed to estrous females mated; the normal maters had a median intromission latency (IL) of 2.0 min, a median ejaculation latency (EL) of 9.0 min (first series), and a median ejaculation frequency (EF) of 2 per 20 min. The slow maters had a median IL of 9.0 min; none of the rats in this group ejaculated.
Z
100
50
2.0
1.0
CONTROL
ODOR
FIG. 2. Mean plasma levels of LH, prolactin, and testosterone in sexually experienced male rats (Study 2). Stimuli are as in Fig. 1 with the addition of 6 — male. Each bar represents the mean value for 7 or 8 animals.
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INFLUENCE OF MATING ON PLASMA HORMONES icant elevation only in LH levels (108%). The males exposed to estrous females mated somewhat more.rapidly than those in Study 1; median values for IL, EL (first series), and EF were 0.9 min, 6.7 min, and 2 per 20 min, respectively. Levels of LH, PRL, and T were significantly elevated in this group (LH: 108%; PRL: 401%; T: 139%). The increase in LH levels was comparable to that shown by the normal maters in Study 1, but the increase in PRL levels was much greater. The male-male interactions included fighting and some mounting of each male by the other. However, this stimulus had no significant effect on the hormone levels of the experimental males. Neither of the non-maters in Study 2 attempted to mount the female during the experimental mating session. The hormone levels of these rats are shown in Table 1. Their resting levels of LH were within the normal range for this group, although slightly low, and their PRL levels were normal, but their T levels were extremely low. Only one (#4) showed an increase in LH levels (240%) after exposure to the female. Neither showed much increase in PRL levels (30%, 48%) in comparison to the rest of the group. Both showed an increase in T levels (107%, 207%), but their resting levels were so depressed that these values were still below the range seen in the rest of the group after mating. Study 3 investigated the ability of the stimuli used in Studies 1 and 2 to alter the
•
RESTING LEVEL
•
LEVEL IN ARENA
••I.I
mill CONTROL
o*
ODOR
(O)
ll
&
O
FIG. 3. Mean plasma levels of LH, prolactin, and testosterone in sexually naive male rats (Study 3). Stimuli are as in Figs. 1 and 2. Each bar represents the mean value for 7 to 9 animals.
hormone levels of sexually naive rats (Fig. 3). Once again, neither transfer to an arena nor interaction with another male elevated hormone levels significantly. Odor produced a significant rise only in LH levels (95%), while non-tactile contact had no sigTABLE 1. Plasma hormone levels (ng/ml) of non-mating rats in Study 2 nificant effect. Attempts to mate with castrate females led to a significant increase in Rat levels of LH (189%) and PRL (138%). Al#4 Hormone Sample* #11 though these rats were sexually naive, all the males in the group exposed to estrous LH Resting level 12.0 12.0 females mated. Median values for IL, EL, 15.7 Female 40.8 and EF were 6.1 min, 10.6 min, and 1 per 20 Prolactin 36.8 Resting level 57.7 min, respectively. This stimulus produced 54.4 Female 74.9 a significant elevation in levels of both LH 0.15 0.27 Testosterone Resting level Female 0.83 (265%) and PRL (75%). The rise in LH levels 0.31 was comparable to that seen in the slow * Resting level is level measured immediately after maters in Study 1, but the rise in PRL levels removing rat from home cage. Female is level measured was considerably smaller. All stimuli, inafter 20 min exposure to estrous female.
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»0
T
t
r
»
_
too
I H—+
FlG. 4. Mean serum levels of LH, FSH, prolactin, and testosterone in sexually experienced male rats at various times during mating (Study 4). Each point represents the mean value for 7 to 9 animals. The vertical lines represent the SE, and the asterisks indicate the significance of the difference from resting levels (Neuman-Keuls test). The isolated circles represent the hormone levels of 2 rats which did not mate.
eluding mating, failed to elevate T levels significantly in naive rats. Study 4 examined the time course of hormone release during mating (Fig. 4). During the experimental mating session the sexual behavior of the rats in this study was similar to that of the normal maters in Study 1. Median values for IL and EL (first series) were 3.0 min and 10.0 min, respectively,
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for all groups together. The rats in the 60 min group had a median EF of 4 per 60 min. LH levels were somewhat, although not significantly, elevated before the introduction of the female into the arena (0 min). Since in this study the arenas were used for all groups indiscriminately, this elevation may have been due to odors remaining in the arenas. LH levels were significantly higher than resting levels only after 5 and 30 min of mating (including refractory periods), and had declined to values comparable to resting levels by 60 min. FSH levels remained unchanged during 60 min. PRL levels did not rise before the introduction of the female into the arena. After 5 and 15 min, these levels were significantly higher than both the resting levels and the levels observed at 0 min. Following this, PRL levels declined. T levels rose slowly, so that, although they were elevated after 30 min, they were significantly higher than resting levels and 0 min levels only after 60 min. As in Study 2, neither of the non-maters attempted to mount the female. Their hormone levels (isolated circles, Fig. 4) after 15 min exposure to the female were comparable to the resting levels seen in this study, and did not rise to the levels seen in the 15 min group. Discussion These studies confirm our earlier report (6) that mating is associated with increases in plasma levels of LH and PRL as well as T. Although the increases observed were not particularly large (two- to four-fold), in four studies mating produced a significant elevation in LH and PRL levels. In fact, these hormones appeared to be more responsive than T to mating. On the other hand, the results of Study 4 confirm previous reports that FSH levels are unaffected by mating (1,2). In addition, Studies 1-3 show that successful copulation is not required to elicit hormone release. They extend our previous observation that attempting to mate with an unreceptive female can release LH,
INFLUENCE OF MATING ON PLASMA HORMONES PRL, and T, and also show that the odor of and non-tactile contact with estrous females can both release LH. Finally, the results of Study 4 are consistent with the hypothesis that the increase in T levels is due, at least partially, to the prior elevation in LH and PRL levels. The elevations in LH and PRL levels appeared to be relatively transient. In Study 4, after 60 min of mating, levels of both hormones had declined to values comparable to resting levels. This decline may be due to a negative feedback effect produced by the rising T levels. On the other hand, the apparent transience of the elevations may be a result of the rats' behavior. Rats do not mate continuously, but in a sequence of disconnected episodes (series) separated by refractory periods. Therefore, a rat was considered to be "mating" if no more than 15 min elapsed between intromissions, and "actively mating" during each ejaculatory series (excluding refractory periods). In these studies rats sampled during their refractory periods had lower hormone levels than those sampled while actively mating. In Study 4, the groups sampled at earlier times had a higher proportion of actively mating rats than those sampled at later times. Thus, LH and PRL levels appeared to decline as time progressed. The possibility that hormones are elevated only during active mating may be responsible for the rapid fall in LH levels observed by Taleisnik et al. (1), since these investigators sampled their rats only after mating. It may also be responsible for the recent observation of Balin and Schwartz (2) that LH and PRL levels were not changed by mating, since blood collection in their study was not begun until 15 min following the completion of an ejaculatory series. The tendency for a rat's hormone levels to depend on its behavior at the time of blood collection may also explain certain inconsistencies in the data presented here. In Studies 1-3, rats which mated faster (Studies 1 and 2) had lower LH levels and higher T levels than rats which mated more
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slowly (Study 3 and slow maters, Study 1). It is possible that LH levels had already declined in rapidly mating rats, since many of them were in their refractory periods at the time of blood collection. Conversely, in slowly mating animals, T levels may not have begun to rise. There is, however, no clearcut relation between the rate of mating and PRL levels, and the failure of the normal maters in Study 1 to show an increase in PRL levels remains anomalous. The correspondence between the rate of mating and T levels may also explain the failure of all stimuli to elevate T levels in sexually naive rats, since these animals mated relatively slowly. We previously reported that, in naive rats, levels of LH, PRL, and T all failed to rise significantly after mating (6). However, the present study shows that naive rats are capable of responding to mating with significant increases in LH and PRL levels. Nevertheless, T levels in naive rats showed only a slight increase after 20 min of mating. This may be due to the slow rate at which these rats mated. On the other hand, the testes of naive rats may be less responsive to stimulation than those of experienced ones since Herz et al. (17) have shown that cohabitation increases the testicular concentration of T. In addition to showing that hormone levels increase during mating, these studies also demonstrate that this effect is specific. Although hormone levels rose when rats were placed in an empty arena, these increases were small and non-significant. The increases observed following mating were much larger, suggesting that they were not due to stress alone. However, successful copulation was not necessary to elicit a rise in hormone levels, since several of the other stimuli were also effective. Non-tactile contact with an estrous female was reported to result in increased T levels after times as short as 10 min (3). We have been unable to confirm this report, since after 20 min only LH levels were significantly elevated by odor and non-tactile contact in our studies. Naive rats were somewhat less re-
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KAMELETAL.
sponsive to these stimuli; only odor, and not non-tactile contact, resulted in elevated LH levels. This is somewhat surprising, since presumably non-tactile contact includes odor, as well as other sensory stimuli. Apart from mating itself, the most effective stimulus in these studies was provided by unreceptive females. Attempts to mate with these females elicited increases in levels of all three hormones in experienced rats, and in levels of LH and PRL in naive ones. These increases were as great as those seen following mating. This effect has also been observed in the vole (10) and in the mouse (11). Furthermore, Maruniak and Bronson (18) have recently shown that the ability of female mouse urine to elevate LH levels in male mice is independent of the endocrine status of the female. Thus, the sensory cues provided by an estrous female, as well as those involved in intromission and ejaculation, are not necessary to increase hormone levels. Nevertheless, not all social interactions were effective in increasing hormone levels, since exposure to male stimulus animals did not result in increased hormone levels. The non-maters in Studies 2 and 4 provide further evidence that the observed hormone increases are specifically related to mating, since after exposure to a female these rats had lower hormone levels than the other rats. The non-maters in Study 4 may have had chronically low hormone levels, which, after an equivalent proportional increase in the presence of the female, still failed to reach the values attained by the rest of the group. On the other hand, these rats could have had normal resting levels, which did not increase in the presence of the female. The latter seems to be true of the LH and PRL levels of the non-maters in Study 2; after exposure to an estrous female neither of these rats showed much increase in PRL levels (compared to the rest of the group), and only one had elevated LH levels. Both of these rats did have very low T levels, and this may be related to their failure to mate. However,
Endo ' 1977 Vol 101 ( No 2
Harding and Feder (19) have recently shown that male guinea pigs with low levels of sexual activity were similar to high activity males in their resting levels of plasma T, but failed to show an equivalent increase in T levels after mating or non-tactile exposure to an estrous female. It is possible that deficiencies in mating behavior are generally related, not to chronically low hormone levels, but to an inability to show an increase in hormone levels during exposure to a female. In conclusion, these studies show that levels of LH and PRL, as well as T, are increased by mating. These elevations were particularly pronounced when blood was collected from rats which were actively mating. On the other hand, these studies also show that while the increases are specifically related to mating, they do not depend on successful copulation. Thus, it is not correct to say that increases in hormone levels follow mating in the male, as is the case in the female rat (20). In fact, the large elevations consistently observed after totally unsuccessful attempts to mate suggest that the increases may actually precede mating. In this context it is interesting that LHreleasing hormone (LHRH) has been shown to activate mating behavior in castrated, steroid-primed male and female rats (21,22). In the male, the initial result of exposure to a female may be an increase in LHRH secretion. This would increase LH secretion while simultaneously serving to facilitate mating behavior. The increase in LH levels would synergize with the increase in PRL levels to stimulate the secretion of T (23,24). Increased T levels would, in the long run, aid in maintaining the reproductive capacity of the male. However, the mechanism by which PRL levels are increased is not clear. Nor is it clear why FSH levels would be totally unaffected by an increase in LHRH secretion, although FSH levels have been shown to be less responsive to LHRH than LH levels (25). Nevertheless, it is attractive to hypothesize that a single mechanism is involved in the maintenance of both the
INFLUENCE OF MATING ON PLASMA HORMONES physiological and the behavioral aspects of reproduction in the male. Acknowledgments We thank the following individuals and organizations for generous donations of reagents used in radioimmunoassays: Dr. B. V. Caldwell, Yale University School of Medicine, antibody to testosterone, Dr. G. D. Niswender, Colorado State University, antibody to LH, Dr. L. E. Reichert, Jr., Emory University, ovine LH used for radioiodination, and the NIAMDD rat pituitary hormone program for donation of radioimmunoassay kits to measure LH, prolactin and FSH.
References 1. Taleisnik, S., L. Caligaris, and J. J. Astrada, Endocrinology 79: 49, 1966. 2. Balin, M. S., and N. B. Schwartz, Endocrinology 98: 522, 1976. 3. Purvis, K., and N. B. HaynesJ Endocrinol 60: 429, 1974. 4. Folman, Y., and D. DroriJ Reprod Fertil 11: 43, 1966. 5. Thomas, T. R., and C. N. Neiman,Endocrinology 83: 633, 1968. 6. Kamel, F., E. J. Mock, W. W. Wright, and A. I. Frankel, Honn Behav 6: 277, 1975. 7. Endroczi, E., and K. Lissak, Ada Physiol Acad Sci Hung 21: 203, 1962. 8. Saginor, M., and R. Horton,Endocrinology 82: 627, 1968. 9. Macrides, F., A. Bartke, F. Fernandez, and W. D'Angelo, Neuroendocrinology 15: 355, 1974.
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10. Charlton, H. M., F. Naftolin, M. C. Sood, and R. W. Worth,/ Reprod Fertil 42: 167, 1975. 11. Macrides, F., A. Bartke, and S. Dalterio, Science 189: 1104, 1975. 12. Bliss, E. L., A. Frischat, and L. Samuels, Life Sci 11: 231, 1972. 13. Lincoln, G. A., Nature 252: 232, 1974. 14. Frankel, A. I., E. J. Mock, W. W. Wright, and F. Kamel, Steroids 25: 73, 1975. 15. Niswender, G. D., A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr., Proc Soc Exp Biol Med 128: 807, 1968. 16. Keppel, G., Design and Analysis, Prentice-Hall, Englewood Cliffs, New Jersey, 1973. 17. Herz, Z., Y. Folman, and D. Drori,/ Endocrinol 44: 127, 1969. 18. Maruniak, J. A., and F. H. Bronson, Endocrinology 99: 963, 1976. 19. Harding, C. F., and H. H. Feder, Endocrinology 98: 1198, 1976. 20. Moss, R. L., and K. J. Cooper, Endocrinology 92: 1748, 1973. 21. Pfaff, D. W., Science 182: 1148, 1973. 22. Moss, R. L., S. M. McCann, and C. A. Dudley, In Gispen, W. H., Tj. B. van Wimersma Greidanus, B. Bohus, and D. de Wied (eds.), Hormones, Homeostasis and the Brain, Elsevier, New York, 1975, p. 37. 23. Hafiez, A. A., A. Bartke, and C. W. Lloyd, J Endocrinol 52: 327, 1972. 24. Bartke, A., and S. Dalterio, Biol Reprod 15: 90, 1976. 25. Arimura, A., L. Debeljuk, and A. V. Schally, Endocrinology 91: 529, 1972.
killed immediately after mating for varying times up to 1 h, and their levels of LH, FSH, PRL, and T were measured. Both LH and PRL levels peaked 5 min after the start of mating and the declined, while FSH levels remained unchanged. T levels rose slowly and were significantly elevated only after an hour of mating. These results show that levels of LH and PRL, but not FSH, are increased during mating in the male rat, and suggest that the increase in T levels may be due to the prior increase in LH and PRL levels. Furthermore, the rapidity of the rise in LH and PRL levels, coupled with the consistent response of these hormones to unsuccessful attempts to mate, suggests that rather than following mating, the increases in LH and PRL may precede it. (Endocrinology 101: 421, 1977)
ABSTRACT. Male rats were exposed to several stimuli associated with mating, and their resting levels of LH, prolactin (PRL), and testosterone (T) were compared to their hormone levels after stimulation. Sexually experienced rats showed two- to fourfold increases in levels of these hormones after both mating with estrous females and attempting to mate with unreceptive females. Only LH levels were significantly elevated by the odor of estrous females or by non-tactile contact with estrous females. Neither transfer to an empty mating arena nor interaction with another male altered the rats' hormone levels. The LH and PRL levels of sexually naive rats showed a similar pattern of response, but T levels in naive rats were unaffected by all stimuli including mating. In a further study, rats were
T
HE ABILITY of male rats to release hormones during mating remains a subject of controversy. In an early study (1) levels of plasma LH (but not FSH) were increased 5 min after mating. However, a recent report (2) indicated that plasma levels of LH, FSH, and prolactin (PRL) remained unchanged during the hour following mating. These results suggested that gonadotropins might not mediate the matinginduced increase in plasma testosterone (T) levels which had been observed directly (3) as well as inferred from the effect of cohabitation on accessory sex organ weights (4,5). Recently we reported that mating was in fact associated with elevations in plasma Received November 1, 1976. 1 Supported by U.S. Public Health Service Grant HD 04081. A preliminary report of these findings was presented at the 9th Annual Meeting of the Society for the Study of Reproduction, Philadelphia, Pa., 1976 (Abstract #138). 2 Reprint requests to: Dr. Arthur I. Frankel, Department of Biological Sciences, State University of New York at Binghamton, Binghamton, New York 13901.
Binghamton,
levels of LH and PRL as well as T (6). Our study also suggested that successful copulation was not a necessary component of the stimulus resulting in hormone increases. Rats attempting to mate with unreceptive females showed elevations in levels of LH and PRL equivalent to those seen in mating rats. Furthermore, in mating rats, levels of LH and T were as high after one intromission as after ejaculation. These results indicated that ejaculation at least was not always required to elicit hormone release. The observation that related stimuli can elicit hormone release as readily as successful copulation is not unique, since a number of studies have suggested that interrupted mating, unsuccessful attempts to mate, or sensory stimuli associated with mating can elevate hormone levels in males of several other species—rabbit (7,8), hamster (9), vole (10), and mouse (11)—as well as in the rat (3,12), although not in the human male (13). To clarify this issue, we exposed male rats to a variety of stimuli associated with mating and measured their hormonal re-
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ternated with control sessions, in which the male was placed alone in the cage for 35 min. In a further effort to minimize pre-experimental handling, sexual experience in Study 4 was provided by placing an intact female into each male's home cage for at least one week preceding the experiment. Experimental exposure to mating and other stimuli was provided in semicircular plexiglass mating arenas (41 x 77 cm) lined with wood shavings. The shavings were replaced each time an Materials and Methods arena was used. Arenas used for stimuli not Four studies were conducted. Studies 1-3 involving an estrous female had never been exexamined the effect of various stimuli on plasma posed to females. In Studies 1-3, the males were hormone levels, and Study 4 followed the time placed in the arenas for a total of 30 min. course of hormone release during mating. The Stimulus animals were introduced into the arenas 10 min after the experimental animals, so that subjects were male Sprague-Dawley rats either purchased from Russell Miller Farms (Cazenovia, interactions lasted for 20 min. Stimuli used in N.Y.) and received in our laboratory when 50 days these studies were: control—empty arena lined old (Study 1) or raised in our colony from rats with clean shavings, odor—arena which had conoriginally purchased from this supplier (Studies tained 6 estrous females for 30 min immediately 2-4). The rats were housed 2 per cage and had preceding the experiment, non-tactile contact— access to Purina Rat Chow and water ad lib. estrous female on the far side of a double wire The animal room was maintained at constant mesh barrier, castrate female—ovariectomized, temperature (21 ± 1 C) and on an artificial 12 h unreceptive female, estrous female, and another light-12 h dark cycle (lights off 1100-2300 h EST male. In Study 4, the male was placed in a for Studies 1 and 4 and 1000-2200 h EST for mating arena. After 15 min, an estrous female was introduced into the arena, and the male was Studies 2 and 3). removed after varying periods of time. In all studies the sexual behavior of mating rats was Behavioral procedures observed, and the times of the first intromission Sexual experience and experimental mating and ejaculation of each ejaculatory series were sessions were conducted in the animal room dur- noted. ing the dark phase of the light-dark cycle (2 to 6 h after lights off) under dim red light. Females Experimental protocol used as stimulus animals were brought into The 41 rats in Study 1 (December 1974) were behavioral estrus with sc injections of estradiol benzoate (0.01 mg) and progesterone (1.0 mg) in given five weekly sessions of sexual experience sesame oil. Only fully receptive females were beginning when they were 65 days old. During the final two sessions the rats' sexual behavior used. Since we had observed previously that pre- was observed. The rats were assigned to one of experimental exposure to the mating arenas led five experimental groups (control, odor, nonthe rats to associate the arenas with mating, tactile contact, castrate female, estrous female) in the sexual experience sessions in Studies 1 and such a way that pre-experimental sexual be2 were conducted in ordinary stainless steel cages havior was equivalent among groups. The group (26 x 42 cm). The male was placed in the cage for with estrous females also included 6 rats which 5 min, after which an estrous female was intro- did not ejaculate during either of the final two duced into the cage. The first three sessions were experience sessions or during the experimental terminated after an additional 30 min regardless mating session. These constituted a sixth group, of the animals' behavior. The final two sessions designated slow maters; the rest were designated were terminated after 20 min, but if the rat intro- normal maters. There were 6-8 rats per group. mitted the session was continued to 30 min or the One blood sample was collected immediately first post-ejaculatory intromission, whichever after removing the rat from its home cage (restcame first. The experience sessions were al- ing levels), and another was collected after ex-
sponse. Furthermore, since we had observed that hormones appeared to reach maximal levels almost as soon as the rats began to mate, we also studied the time course of hormone release during mating. In this fashion we hoped to specify both the behavioral and the sensory components of the mating situation necessary to elicit hormone release.
INFLUENCE OF MATING ON PLASMA HORMONES posure to the appropriate stimulus. The two samples were collected one week apart in a random order. Each group was represented on each day of blood collection. The rats were 97-108 days old at the time of blood collection. The pre-experimental handling procedures in Study 2 (April 1976; 32 rats) were identical to those used in Study 1. The rats were divided into four groups (control, male, odor, estrous female) on the basis of their pre-experimental sexual behavior (7-8 rats per group). Two rats which did not mount the female during either of the final two experience sessions or during the experimental mating session were designated non-maters. Blood was collected as in Study 1; the rats were 93-103 days old at the time of blood collection. Study 3 (August 1975) employed 46 sexually naive rats. Each rat was randomly assigned to one of six experimental groups (control, male, odor, non-tactile contact, castrate female, estrous female; 7-9 rats per group). Blood was collected as in Study 1; the rats were 133-148 days old at the time of blood collection. The 52 rats in Study 4 (January 1975) had minimal sexual experience (see Behavioral procedures). Blood was collected from randomly constituted groups before (0 min) or 5, 15, 30, or 60 min after the introduction of the female into the arena. Resting levels were determined as in Study 1. Blood was also collected from two rats alter 15 min during which they did not mount the female (non-maters). Blood was collected from at least one rat at each time on each day; the order of collection was varied, so that, for example, the 60 min sample was not always collected last. The rats were 112-136 days old at the time of blood collection. Blood collection and hormone assay In Studies 1-3 blood samples (1.5 ml) were collected into heparinized syringes by cardiac puncture of etherized rats. The samples were kept in an ice bath for up to 1 h before centrifugation. In Study 4 blood samples were collected by decapitating etherized rats. The samples were allowed to clot at room temperature before centrifugation. All samples were collected between 3 and 5.5 h after lights off, within 1 min of the time indicated. Plasma or serum was stored at —20 C until assay. Samples were analyzed for T by radioimmunoassay as previously described (14). LH was meas-
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ured using the ovine-ovine rat LH radioimmunoassay (15). Values are expressed in terms of the NIAMDD standard RP-1. The sensitivity of the assay was 2.0 ng. The precision of the assay was estimated using replicate measurements of a pool of serum from male rats. The intraassay coefficient of variation for 200 /x\ samples (67.8% binding) was 9.0%, and the interassay coefficient of variation was 12.0%. Samples were analyzed for PRL using the NIAMDD rat PRL assay kit. Values were expressed in terms of the NIAMDD standard RP-1. The sensitivity of the assay was 0.23 ng. The intraassay coefficient of variation for 50 fi\ samples (44.0% binding) was 11.9%, and the interassay coefficient of variation was 16.0%. Samples were analyzed for FSH using the NIAMDD rat FSH assay kit. Values are expressed in terms of the NIAMDD standard RP-1. The sensitivity of the assay was 20 ng. The intrassay coefficient of variation, estimated by comparing duplicate measurements of each sample, was 15.3%. Samples were analyzed in duplicate, usually at two different volumes. All samples from a single study were analyzed for a particular hormone at the same time. Statistical analysis Statistical analysis (16) was performed using two-way analysis of variance for repeated measures. Comparisons within and among groups were made by analyzing the simple main effects. Further comparisons among groups were made using the Neuman-Keuls test for a posteriori multiple comparisons.
Results
Studies 1 and 2 examined the effect of various stimuli on the plasma hormone levels of sexually experienced rats. Study 1 (Fig. 1) showed that transfer of the rat to a novel mating arena (control) had no significant effect on the levels of any hormone measured. Odor and non-tactile contact produced a significant increase above resting levels only in LH levels (139% and 116%, respectively). The castrate females were unreceptive, and never permitted intromission, although the males mounted them repeatedly. Nevertheless, this stimulus produced a significant elevation in levels
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KAMEL ETAL.
•
RESTING LEVEL
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LEVEL IN ARENA
Both of these groups showed a significant rise in LH levels; however, the rise shown by the slow maters (233%) was more substantial than that shown by the normal maters (135%). Only the slow maters showed a significant increase in PRL levels (186%), and only the normal maters showed a significant increase in T levels (93%). The results of Study 2 (Fig. 2) confirmed those of Study 1. Transfer to an arena had no significant effect. Odor produced a signif-
60
i
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RESTING LEVEL
•
LEVEL IN ARENA
40
20
***
ISO CONTROL
ODOR
SLOW MATERS
FIG. 1. Mean plasma levels of LH, prolactin, and testosterone in sexually experienced male rats (Study 1). Levels were measured after removing the rat from its home cage (resting level) and after exposing it to the appropriate stimulus (level in arena). Stimuli were: control—empty arena; odor—odor of estrous female; (9)—non-tactile contact with estrous female; -? — ovariectomized, unreceptive female; 9—estrous female (normal maters); slow maters—estrous female (slow maters). Each bar represents the mean value for 6 to 8 animals. In Figs. 1-3 the vertical lines represent the SE and the asterisks indicate the significance of the change in levels within a group (analysis of variance): *P < 0.05; **P < 0.01; ***P < 0.001.
of all three hormones (LH: 347%; PRL: 132%; T: 87%). All the rats exposed to estrous females mated; the normal maters had a median intromission latency (IL) of 2.0 min, a median ejaculation latency (EL) of 9.0 min (first series), and a median ejaculation frequency (EF) of 2 per 20 min. The slow maters had a median IL of 9.0 min; none of the rats in this group ejaculated.
Z
100
50
2.0
1.0
CONTROL
ODOR
FIG. 2. Mean plasma levels of LH, prolactin, and testosterone in sexually experienced male rats (Study 2). Stimuli are as in Fig. 1 with the addition of 6 — male. Each bar represents the mean value for 7 or 8 animals.
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INFLUENCE OF MATING ON PLASMA HORMONES icant elevation only in LH levels (108%). The males exposed to estrous females mated somewhat more.rapidly than those in Study 1; median values for IL, EL (first series), and EF were 0.9 min, 6.7 min, and 2 per 20 min, respectively. Levels of LH, PRL, and T were significantly elevated in this group (LH: 108%; PRL: 401%; T: 139%). The increase in LH levels was comparable to that shown by the normal maters in Study 1, but the increase in PRL levels was much greater. The male-male interactions included fighting and some mounting of each male by the other. However, this stimulus had no significant effect on the hormone levels of the experimental males. Neither of the non-maters in Study 2 attempted to mount the female during the experimental mating session. The hormone levels of these rats are shown in Table 1. Their resting levels of LH were within the normal range for this group, although slightly low, and their PRL levels were normal, but their T levels were extremely low. Only one (#4) showed an increase in LH levels (240%) after exposure to the female. Neither showed much increase in PRL levels (30%, 48%) in comparison to the rest of the group. Both showed an increase in T levels (107%, 207%), but their resting levels were so depressed that these values were still below the range seen in the rest of the group after mating. Study 3 investigated the ability of the stimuli used in Studies 1 and 2 to alter the
•
RESTING LEVEL
•
LEVEL IN ARENA
••I.I
mill CONTROL
o*
ODOR
(O)
ll
&
O
FIG. 3. Mean plasma levels of LH, prolactin, and testosterone in sexually naive male rats (Study 3). Stimuli are as in Figs. 1 and 2. Each bar represents the mean value for 7 to 9 animals.
hormone levels of sexually naive rats (Fig. 3). Once again, neither transfer to an arena nor interaction with another male elevated hormone levels significantly. Odor produced a significant rise only in LH levels (95%), while non-tactile contact had no sigTABLE 1. Plasma hormone levels (ng/ml) of non-mating rats in Study 2 nificant effect. Attempts to mate with castrate females led to a significant increase in Rat levels of LH (189%) and PRL (138%). Al#4 Hormone Sample* #11 though these rats were sexually naive, all the males in the group exposed to estrous LH Resting level 12.0 12.0 females mated. Median values for IL, EL, 15.7 Female 40.8 and EF were 6.1 min, 10.6 min, and 1 per 20 Prolactin 36.8 Resting level 57.7 min, respectively. This stimulus produced 54.4 Female 74.9 a significant elevation in levels of both LH 0.15 0.27 Testosterone Resting level Female 0.83 (265%) and PRL (75%). The rise in LH levels 0.31 was comparable to that seen in the slow * Resting level is level measured immediately after maters in Study 1, but the rise in PRL levels removing rat from home cage. Female is level measured was considerably smaller. All stimuli, inafter 20 min exposure to estrous female.
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»0
T
t
r
»
_
too
I H—+
FlG. 4. Mean serum levels of LH, FSH, prolactin, and testosterone in sexually experienced male rats at various times during mating (Study 4). Each point represents the mean value for 7 to 9 animals. The vertical lines represent the SE, and the asterisks indicate the significance of the difference from resting levels (Neuman-Keuls test). The isolated circles represent the hormone levels of 2 rats which did not mate.
eluding mating, failed to elevate T levels significantly in naive rats. Study 4 examined the time course of hormone release during mating (Fig. 4). During the experimental mating session the sexual behavior of the rats in this study was similar to that of the normal maters in Study 1. Median values for IL and EL (first series) were 3.0 min and 10.0 min, respectively,
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for all groups together. The rats in the 60 min group had a median EF of 4 per 60 min. LH levels were somewhat, although not significantly, elevated before the introduction of the female into the arena (0 min). Since in this study the arenas were used for all groups indiscriminately, this elevation may have been due to odors remaining in the arenas. LH levels were significantly higher than resting levels only after 5 and 30 min of mating (including refractory periods), and had declined to values comparable to resting levels by 60 min. FSH levels remained unchanged during 60 min. PRL levels did not rise before the introduction of the female into the arena. After 5 and 15 min, these levels were significantly higher than both the resting levels and the levels observed at 0 min. Following this, PRL levels declined. T levels rose slowly, so that, although they were elevated after 30 min, they were significantly higher than resting levels and 0 min levels only after 60 min. As in Study 2, neither of the non-maters attempted to mount the female. Their hormone levels (isolated circles, Fig. 4) after 15 min exposure to the female were comparable to the resting levels seen in this study, and did not rise to the levels seen in the 15 min group. Discussion These studies confirm our earlier report (6) that mating is associated with increases in plasma levels of LH and PRL as well as T. Although the increases observed were not particularly large (two- to four-fold), in four studies mating produced a significant elevation in LH and PRL levels. In fact, these hormones appeared to be more responsive than T to mating. On the other hand, the results of Study 4 confirm previous reports that FSH levels are unaffected by mating (1,2). In addition, Studies 1-3 show that successful copulation is not required to elicit hormone release. They extend our previous observation that attempting to mate with an unreceptive female can release LH,
INFLUENCE OF MATING ON PLASMA HORMONES PRL, and T, and also show that the odor of and non-tactile contact with estrous females can both release LH. Finally, the results of Study 4 are consistent with the hypothesis that the increase in T levels is due, at least partially, to the prior elevation in LH and PRL levels. The elevations in LH and PRL levels appeared to be relatively transient. In Study 4, after 60 min of mating, levels of both hormones had declined to values comparable to resting levels. This decline may be due to a negative feedback effect produced by the rising T levels. On the other hand, the apparent transience of the elevations may be a result of the rats' behavior. Rats do not mate continuously, but in a sequence of disconnected episodes (series) separated by refractory periods. Therefore, a rat was considered to be "mating" if no more than 15 min elapsed between intromissions, and "actively mating" during each ejaculatory series (excluding refractory periods). In these studies rats sampled during their refractory periods had lower hormone levels than those sampled while actively mating. In Study 4, the groups sampled at earlier times had a higher proportion of actively mating rats than those sampled at later times. Thus, LH and PRL levels appeared to decline as time progressed. The possibility that hormones are elevated only during active mating may be responsible for the rapid fall in LH levels observed by Taleisnik et al. (1), since these investigators sampled their rats only after mating. It may also be responsible for the recent observation of Balin and Schwartz (2) that LH and PRL levels were not changed by mating, since blood collection in their study was not begun until 15 min following the completion of an ejaculatory series. The tendency for a rat's hormone levels to depend on its behavior at the time of blood collection may also explain certain inconsistencies in the data presented here. In Studies 1-3, rats which mated faster (Studies 1 and 2) had lower LH levels and higher T levels than rats which mated more
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slowly (Study 3 and slow maters, Study 1). It is possible that LH levels had already declined in rapidly mating rats, since many of them were in their refractory periods at the time of blood collection. Conversely, in slowly mating animals, T levels may not have begun to rise. There is, however, no clearcut relation between the rate of mating and PRL levels, and the failure of the normal maters in Study 1 to show an increase in PRL levels remains anomalous. The correspondence between the rate of mating and T levels may also explain the failure of all stimuli to elevate T levels in sexually naive rats, since these animals mated relatively slowly. We previously reported that, in naive rats, levels of LH, PRL, and T all failed to rise significantly after mating (6). However, the present study shows that naive rats are capable of responding to mating with significant increases in LH and PRL levels. Nevertheless, T levels in naive rats showed only a slight increase after 20 min of mating. This may be due to the slow rate at which these rats mated. On the other hand, the testes of naive rats may be less responsive to stimulation than those of experienced ones since Herz et al. (17) have shown that cohabitation increases the testicular concentration of T. In addition to showing that hormone levels increase during mating, these studies also demonstrate that this effect is specific. Although hormone levels rose when rats were placed in an empty arena, these increases were small and non-significant. The increases observed following mating were much larger, suggesting that they were not due to stress alone. However, successful copulation was not necessary to elicit a rise in hormone levels, since several of the other stimuli were also effective. Non-tactile contact with an estrous female was reported to result in increased T levels after times as short as 10 min (3). We have been unable to confirm this report, since after 20 min only LH levels were significantly elevated by odor and non-tactile contact in our studies. Naive rats were somewhat less re-
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KAMELETAL.
sponsive to these stimuli; only odor, and not non-tactile contact, resulted in elevated LH levels. This is somewhat surprising, since presumably non-tactile contact includes odor, as well as other sensory stimuli. Apart from mating itself, the most effective stimulus in these studies was provided by unreceptive females. Attempts to mate with these females elicited increases in levels of all three hormones in experienced rats, and in levels of LH and PRL in naive ones. These increases were as great as those seen following mating. This effect has also been observed in the vole (10) and in the mouse (11). Furthermore, Maruniak and Bronson (18) have recently shown that the ability of female mouse urine to elevate LH levels in male mice is independent of the endocrine status of the female. Thus, the sensory cues provided by an estrous female, as well as those involved in intromission and ejaculation, are not necessary to increase hormone levels. Nevertheless, not all social interactions were effective in increasing hormone levels, since exposure to male stimulus animals did not result in increased hormone levels. The non-maters in Studies 2 and 4 provide further evidence that the observed hormone increases are specifically related to mating, since after exposure to a female these rats had lower hormone levels than the other rats. The non-maters in Study 4 may have had chronically low hormone levels, which, after an equivalent proportional increase in the presence of the female, still failed to reach the values attained by the rest of the group. On the other hand, these rats could have had normal resting levels, which did not increase in the presence of the female. The latter seems to be true of the LH and PRL levels of the non-maters in Study 2; after exposure to an estrous female neither of these rats showed much increase in PRL levels (compared to the rest of the group), and only one had elevated LH levels. Both of these rats did have very low T levels, and this may be related to their failure to mate. However,
Endo ' 1977 Vol 101 ( No 2
Harding and Feder (19) have recently shown that male guinea pigs with low levels of sexual activity were similar to high activity males in their resting levels of plasma T, but failed to show an equivalent increase in T levels after mating or non-tactile exposure to an estrous female. It is possible that deficiencies in mating behavior are generally related, not to chronically low hormone levels, but to an inability to show an increase in hormone levels during exposure to a female. In conclusion, these studies show that levels of LH and PRL, as well as T, are increased by mating. These elevations were particularly pronounced when blood was collected from rats which were actively mating. On the other hand, these studies also show that while the increases are specifically related to mating, they do not depend on successful copulation. Thus, it is not correct to say that increases in hormone levels follow mating in the male, as is the case in the female rat (20). In fact, the large elevations consistently observed after totally unsuccessful attempts to mate suggest that the increases may actually precede mating. In this context it is interesting that LHreleasing hormone (LHRH) has been shown to activate mating behavior in castrated, steroid-primed male and female rats (21,22). In the male, the initial result of exposure to a female may be an increase in LHRH secretion. This would increase LH secretion while simultaneously serving to facilitate mating behavior. The increase in LH levels would synergize with the increase in PRL levels to stimulate the secretion of T (23,24). Increased T levels would, in the long run, aid in maintaining the reproductive capacity of the male. However, the mechanism by which PRL levels are increased is not clear. Nor is it clear why FSH levels would be totally unaffected by an increase in LHRH secretion, although FSH levels have been shown to be less responsive to LHRH than LH levels (25). Nevertheless, it is attractive to hypothesize that a single mechanism is involved in the maintenance of both the
INFLUENCE OF MATING ON PLASMA HORMONES physiological and the behavioral aspects of reproduction in the male. Acknowledgments We thank the following individuals and organizations for generous donations of reagents used in radioimmunoassays: Dr. B. V. Caldwell, Yale University School of Medicine, antibody to testosterone, Dr. G. D. Niswender, Colorado State University, antibody to LH, Dr. L. E. Reichert, Jr., Emory University, ovine LH used for radioiodination, and the NIAMDD rat pituitary hormone program for donation of radioimmunoassay kits to measure LH, prolactin and FSH.
References 1. Taleisnik, S., L. Caligaris, and J. J. Astrada, Endocrinology 79: 49, 1966. 2. Balin, M. S., and N. B. Schwartz, Endocrinology 98: 522, 1976. 3. Purvis, K., and N. B. HaynesJ Endocrinol 60: 429, 1974. 4. Folman, Y., and D. DroriJ Reprod Fertil 11: 43, 1966. 5. Thomas, T. R., and C. N. Neiman,Endocrinology 83: 633, 1968. 6. Kamel, F., E. J. Mock, W. W. Wright, and A. I. Frankel, Honn Behav 6: 277, 1975. 7. Endroczi, E., and K. Lissak, Ada Physiol Acad Sci Hung 21: 203, 1962. 8. Saginor, M., and R. Horton,Endocrinology 82: 627, 1968. 9. Macrides, F., A. Bartke, F. Fernandez, and W. D'Angelo, Neuroendocrinology 15: 355, 1974.
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10. Charlton, H. M., F. Naftolin, M. C. Sood, and R. W. Worth,/ Reprod Fertil 42: 167, 1975. 11. Macrides, F., A. Bartke, and S. Dalterio, Science 189: 1104, 1975. 12. Bliss, E. L., A. Frischat, and L. Samuels, Life Sci 11: 231, 1972. 13. Lincoln, G. A., Nature 252: 232, 1974. 14. Frankel, A. I., E. J. Mock, W. W. Wright, and F. Kamel, Steroids 25: 73, 1975. 15. Niswender, G. D., A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr., Proc Soc Exp Biol Med 128: 807, 1968. 16. Keppel, G., Design and Analysis, Prentice-Hall, Englewood Cliffs, New Jersey, 1973. 17. Herz, Z., Y. Folman, and D. Drori,/ Endocrinol 44: 127, 1969. 18. Maruniak, J. A., and F. H. Bronson, Endocrinology 99: 963, 1976. 19. Harding, C. F., and H. H. Feder, Endocrinology 98: 1198, 1976. 20. Moss, R. L., and K. J. Cooper, Endocrinology 92: 1748, 1973. 21. Pfaff, D. W., Science 182: 1148, 1973. 22. Moss, R. L., S. M. McCann, and C. A. Dudley, In Gispen, W. H., Tj. B. van Wimersma Greidanus, B. Bohus, and D. de Wied (eds.), Hormones, Homeostasis and the Brain, Elsevier, New York, 1975, p. 37. 23. Hafiez, A. A., A. Bartke, and C. W. Lloyd, J Endocrinol 52: 327, 1972. 24. Bartke, A., and S. Dalterio, Biol Reprod 15: 90, 1976. 25. Arimura, A., L. Debeljuk, and A. V. Schally, Endocrinology 91: 529, 1972.
