Differential Effects of Male Stimuli on Follicle-Stimulating Hormone, Luteinizing Hormone, and Prolactin Secretion in Prepubertal Female Mice F. H. BRONSON AND JOEL A. MARUNIAK Department of Zoology, the University of Texas, Austin, Texas 78712 ABSTRACT. Puberty may be induced rapidly in young female mice by exposing them to adult males. The relevant male stimuli include a urinary pheromone and tactile cues, the latter acting in a potentiating capacity. The specific action of the urinary pheromone on pituitary gonadotropins was the subject of the present research. Immature females of a standard size were either paired with adult males, isolated and their bedding sprayed with male urine, or maintained as isolated controls. Exposure to male urine resulted in a rapid and significant release of LH (30 min) which was maintained at an average of 50% higher than levels in isolated controls throughout the 48 h experiment; no immediate changes were observed in either serum FSH or prolactin, but FSH was depressed and prolactin rose progressively during later sampling periods. Cohabitation with an adult male yielded parallel
S
TIMULI associated with adult males are of profound importance in regulating the timing of the pubertal processes in female mice. Young females housed in the absence of these cues show greatly prolonged vaginal and uterine cycles which are anovulatory until a quite advanced age and during which FSH concentrations remain relatively constant (1). Cohabitation of a single female with an adult male, on the other hand, rapidly organizes the pubertal ovulation (2-4). The overall impact of this phenomenon on puberty in female mice can be appreciated by noting that the difference in age at which the first ovulation occurs in the presence or absence of male cues may amount to several weeks (5). The relevant male cues include a urinary pheromone and tactile stimuli, the latter acting primarily in a potentiating capacity (6-8). The immediate responses of the young females to the totality of these cues Supported by U. S. Public Health grant HD-03803 from the National Institute of Child Health and Human Development. Received July 14, 1975.
but decidedly stronger responses in the concentrations of all three hormones. Exogenous estrogen, administered in a second experiment at a dosage previously found to mimic the puberty-inducing action of a male, suppressed serum FSH and LH while elevating serum prolactin. Thus, the present results suggest a) that the male's urinary stimulus exerts its action on immature females via LH release with no immediate and/or direct effects on the other two tropic hormones, and b) that the delayed alterations in serum FSH and prolactin concentrations accompanying male- and/or urine-exposure are secondary consequences of an LH-induced release of estradiol. Attempts to induce the entire pubertal cycle in intact females with subovulating doses of LH, nevertheless, were unsuccessful. (Endocrinology 98: 1101, 1976)
include a rapid release of LH, followed shortly by a dramatic increase in serum estradiol, no immediate changes occurring in either serum FSH or progesterone (9). Normal periovulatory changes in serum levels of the pituitary and ovarian hormones then occur at some later time, the duration of the intervening interval depending upon the stage of development at which maleexposure is initiated (10,11). The present experiments had several objectives, the primary one being to document, if possible, the female's gonadotropic response to urinary odors without contamination by the male's tactile cues. This was accomplished. Exposure to male urine immediately (30 min) and significantly elevated serum LH without initially altering serum concentrations of either FSH or prolactin. Serum levels of the latter two hormones, however, were both altered during later sampling periods. A second experiment, then, examined a possible estradiol basis for these delayed responses. Finally, several attempts were made to induce (trigger) the entire pubertal cycle with subovulatory doses of exogenous LH.
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1102
BRONSON AND MARUNIAK
Materials and Methods Animals and general procedures. As detailed elsewhere, previous work in this laboratory has resulted in a series of procedures designed to insure a uniform timing of the male-induced, pubertal response of CF-1 females (9,10). Briefly, the age of the female is ignored and cohabitation is initiated at particular body weights. Cohabiting 17.5-18.4 g females with adult males results in 75-85% of them attaining a proestrous uterus on the afternoon of the third day and ovulating that night. Since many of the hormonal changes underlying this phenomenon are subtle and could be obscured by diluting this "typical" response with the 15-25% fastand/or slow-responders, sera of most of these "aberrant" females can be eliminated before assay on the basis of previously established limits on uterine weight. Thus a combination of these two culling procedures results in a large population of females consistently and uniformly approaching their pubertal ovulation. If the 3-day induction system described above is used with females weighing 15.5-16.4 g, the proestrous uterus occurs on schedule but ovulation does not, the positive feedback of ovarian steroids over LH secretion being immature at this weight (11). The lighter weight range, however, has the advantage of greater homogeneity among isolated control females (10). Finally, regardless of which weight class of female is used, and what end measurement is desired (hormone concentration, uterine weight, or ovulation), the presence of the male is necessary for only the first 48 h of this 3-day induction period (9,12). This paper reports several experiments, all using the 3-day induction system described above. End measures included a) serum levels of gonadotropins as assessed at specific times during the first 48 h, b) uterine weight as determined at 54 h (mid-afternoon of proestrus in the 3-day scheme), or c) ovulation as assessed on day 4. With the exception of those experiments involving ovulation, 15.5-16.4 g females were used routinely in these studies since typically there was no need for a mature positive feedback system and a high degree of uniformity among control females was desirable. The experiments involving ovulation used 17.5-18.4 g females. In all of the present experiments, then, CF-1 females were purchased from Carworth Farms at 10-10.9 g body weight. Females were housed 6 per cage in a male-free room and were weighed
Endo • 1976 Vol 98 • No 5
daily (0800 h) until they reached either 15.516.4 g or 17.5-18.4 g, at which time they were delegated to an experimental treatment. Several animal rooms were used for the various experiments; all had similar ambient conditions (27 ± 1 C; 12 air exchanges per hour, once through air supply; 14:10, light:dark cycle, lights on at 0500 h). All mice were housed in 18 x 29 x 13 cm opaque polyethylene cages and provided with Purina mouse chow. The effect of male urine on serum LH, FSH, and prolactin. Females weighing 15.5-16.4 g were housed one per cage in the same male-free room where they had been maintained since their arrival. At 0900 h of the following day they were delegated to one of three treatment conditions: a) continued isolation in the male-free room; b) exposed to male urine in a second animal room housing no other animals; or c) paired with an adult male in a third animal room otherwise containing only singly-housed adult males. Females were killed and their trunk blood collected individually at 0 h (isolated controls only), or after V2, 1, 3, 6, 24, or 48 h of such treatment. Limits were established during preliminaiy studies for each mean uterine weight under each treatment condition, and for all times at which sera were to be collected during the primary experiment. During later collection, then, individual sera were eliminated, before assay, in the case of any female whose uterus weighed more or less than the established limits. Limits on uterine weight used in the present experiment were:
S
TIMULI associated with adult males are of profound importance in regulating the timing of the pubertal processes in female mice. Young females housed in the absence of these cues show greatly prolonged vaginal and uterine cycles which are anovulatory until a quite advanced age and during which FSH concentrations remain relatively constant (1). Cohabitation of a single female with an adult male, on the other hand, rapidly organizes the pubertal ovulation (2-4). The overall impact of this phenomenon on puberty in female mice can be appreciated by noting that the difference in age at which the first ovulation occurs in the presence or absence of male cues may amount to several weeks (5). The relevant male cues include a urinary pheromone and tactile stimuli, the latter acting primarily in a potentiating capacity (6-8). The immediate responses of the young females to the totality of these cues Supported by U. S. Public Health grant HD-03803 from the National Institute of Child Health and Human Development. Received July 14, 1975.
but decidedly stronger responses in the concentrations of all three hormones. Exogenous estrogen, administered in a second experiment at a dosage previously found to mimic the puberty-inducing action of a male, suppressed serum FSH and LH while elevating serum prolactin. Thus, the present results suggest a) that the male's urinary stimulus exerts its action on immature females via LH release with no immediate and/or direct effects on the other two tropic hormones, and b) that the delayed alterations in serum FSH and prolactin concentrations accompanying male- and/or urine-exposure are secondary consequences of an LH-induced release of estradiol. Attempts to induce the entire pubertal cycle in intact females with subovulating doses of LH, nevertheless, were unsuccessful. (Endocrinology 98: 1101, 1976)
include a rapid release of LH, followed shortly by a dramatic increase in serum estradiol, no immediate changes occurring in either serum FSH or progesterone (9). Normal periovulatory changes in serum levels of the pituitary and ovarian hormones then occur at some later time, the duration of the intervening interval depending upon the stage of development at which maleexposure is initiated (10,11). The present experiments had several objectives, the primary one being to document, if possible, the female's gonadotropic response to urinary odors without contamination by the male's tactile cues. This was accomplished. Exposure to male urine immediately (30 min) and significantly elevated serum LH without initially altering serum concentrations of either FSH or prolactin. Serum levels of the latter two hormones, however, were both altered during later sampling periods. A second experiment, then, examined a possible estradiol basis for these delayed responses. Finally, several attempts were made to induce (trigger) the entire pubertal cycle with subovulatory doses of exogenous LH.
1101
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 15:56 For personal use only. No other uses without permission. . All rights reserved.
1102
BRONSON AND MARUNIAK
Materials and Methods Animals and general procedures. As detailed elsewhere, previous work in this laboratory has resulted in a series of procedures designed to insure a uniform timing of the male-induced, pubertal response of CF-1 females (9,10). Briefly, the age of the female is ignored and cohabitation is initiated at particular body weights. Cohabiting 17.5-18.4 g females with adult males results in 75-85% of them attaining a proestrous uterus on the afternoon of the third day and ovulating that night. Since many of the hormonal changes underlying this phenomenon are subtle and could be obscured by diluting this "typical" response with the 15-25% fastand/or slow-responders, sera of most of these "aberrant" females can be eliminated before assay on the basis of previously established limits on uterine weight. Thus a combination of these two culling procedures results in a large population of females consistently and uniformly approaching their pubertal ovulation. If the 3-day induction system described above is used with females weighing 15.5-16.4 g, the proestrous uterus occurs on schedule but ovulation does not, the positive feedback of ovarian steroids over LH secretion being immature at this weight (11). The lighter weight range, however, has the advantage of greater homogeneity among isolated control females (10). Finally, regardless of which weight class of female is used, and what end measurement is desired (hormone concentration, uterine weight, or ovulation), the presence of the male is necessary for only the first 48 h of this 3-day induction period (9,12). This paper reports several experiments, all using the 3-day induction system described above. End measures included a) serum levels of gonadotropins as assessed at specific times during the first 48 h, b) uterine weight as determined at 54 h (mid-afternoon of proestrus in the 3-day scheme), or c) ovulation as assessed on day 4. With the exception of those experiments involving ovulation, 15.5-16.4 g females were used routinely in these studies since typically there was no need for a mature positive feedback system and a high degree of uniformity among control females was desirable. The experiments involving ovulation used 17.5-18.4 g females. In all of the present experiments, then, CF-1 females were purchased from Carworth Farms at 10-10.9 g body weight. Females were housed 6 per cage in a male-free room and were weighed
Endo • 1976 Vol 98 • No 5
daily (0800 h) until they reached either 15.516.4 g or 17.5-18.4 g, at which time they were delegated to an experimental treatment. Several animal rooms were used for the various experiments; all had similar ambient conditions (27 ± 1 C; 12 air exchanges per hour, once through air supply; 14:10, light:dark cycle, lights on at 0500 h). All mice were housed in 18 x 29 x 13 cm opaque polyethylene cages and provided with Purina mouse chow. The effect of male urine on serum LH, FSH, and prolactin. Females weighing 15.5-16.4 g were housed one per cage in the same male-free room where they had been maintained since their arrival. At 0900 h of the following day they were delegated to one of three treatment conditions: a) continued isolation in the male-free room; b) exposed to male urine in a second animal room housing no other animals; or c) paired with an adult male in a third animal room otherwise containing only singly-housed adult males. Females were killed and their trunk blood collected individually at 0 h (isolated controls only), or after V2, 1, 3, 6, 24, or 48 h of such treatment. Limits were established during preliminaiy studies for each mean uterine weight under each treatment condition, and for all times at which sera were to be collected during the primary experiment. During later collection, then, individual sera were eliminated, before assay, in the case of any female whose uterus weighed more or less than the established limits. Limits on uterine weight used in the present experiment were:
