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Biology of Reproduction 59, 30-38 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Evolutionary Change in the Endocrinology of Behavioral Receptivity: Divergent Roles for Progesterone and Prolactin within the Genus Phodopus1

H.J. McMillana, , and K.E. Wynne-Edwards2,a

a Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Siberian (Phodopus sungorus) and Djungarian (P. campbelli) hamsters are phenotypically similar and were long considered subspecies. Progesterone (P4) and prolactin (PRL) changes (determined by repeated sampling of individuals) during the behavioral receptivity of both an ovulatory cycle and a postpartum mating, as well as the hormonal requirements for behavioral receptivity, were determined. Changes in P. sungorus were similar to well-described hormonal changes in rats, mice, and golden hamsters, suggesting that previously described differences between P. campbelli and those species had evolved recently. Specifically, 1) P4 facilitated behavioral receptivity at low priming doses of estradiol in P. sungorus but was not needed in P. campbelli; 2) in P. sungorus, P4 increases were synchronous across females and of similar amplitude during each estrus, whereas in P. campbelli, P4 increases were less synchronous across females and were reduced in amplitude postpartum; and 3) PRL profiles were similar (high average PRL levels, few high surges detected) in each species on Day 18, but on proestrus, cyclic P. sungorus had elevated PRL levels and frequent surges while cyclic P. campbelli had lower PRL levels and rare surges. As the endocrinology of P. campbelli also differs from known laboratory rodents in other ways, additional within-genus divergence is predicted.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For the majority of spontaneously ovulating rodent species, a period of estradiol-17ß (E2) priming, followed by a brief exposure to progesterone (P4), induces behavioral receptivity in the ovariectomized female (see [1, 2] for classic reviews). In intact females, elevated levels of peripheral P4 and prolactin (PRL) are also common around the time of receptivity. This highly conserved pattern of increased hormone secretion is involved in proceptive behaviors [3], and the induction of behavioral receptivity [4, 5].

The Djungarian hamster (P. campbelli), like the rat, mouse, and golden hamster, exhibits a 4-day estrous cycle with a spontaneous ovulation [6]. Behavioral receptivity is entrained to the light-dark cycle and occurs around dusk on proestrus with ovulation early the next morning (estrus) [7, 8]. In the absence of coitus, the corpora lutea that differentiate from the ovulated follicles are ephemeral, secreting P4 between 24 and 48 h postovulation (diestrus 1), and completely regress by the morning of the third day (diestrus 2) [7, 9, 10]. As the corpora lutea regress, E2 secretion from the developing follicles increases, culminating in an E2 surge on proestrus [7]. Surges of P4 are also reliably detected on proestrus, although their timing is variable across individuals [9]. Thus, the known steroid endocrinology of the unmated ovulatory cycle of P. campbelli is fundamentally similar to that of other, commonly studied, laboratory rodents [11, 12]. Given that similarity, the demonstration that priming with physiological doses of E2 was sufficient to induce behavioral receptivity in P. campbelli was unexpected [7, 9].

At that time, it was not known whether P. campbelli was representative of a wide range of spontaneously ovulating rodent species not previously investigated, or whether the absence of a role for P4 in the induction of behavioral receptivity in P. campbelli had evolved recently. The present study used a closely related dwarf hamster species as a test of the hypothesis that the role of P4 in proestrus in P. campbelli was a rare example of recent divergence from an ancient, highly conserved pattern of hormone secretion and function.

Siberian hamsters (P. sungorus) do not occur in sympatry with P. campbelli, although they were considered subspecies until recently [13, 14] and viable hybrid offspring can be produced in laboratory crosses [15]. The two species are phenotypically similar and reach the same adult weight (18–35 g), and each has both a 4-day estrous cycle and an 18-day gestation [16–18]. However, they differ in several aspects of their social behavior [19–21]. In particular, successful reproduction in P. campbelli, unlike the majority of small-bodied mammals [22, 23], and unlike P. sungorus, is dependent upon biparental care [24–27].

Although the reproductive endocrinology of male P. sungorus has been extensively studied as a model for seasonal breeding (e.g., [28–30]), and the assay systems for steroid and PRL quantification are established, very little is known about the reproductive endocrinology of P. sungorus females [16]. The hypothesis predicted that their patterns of P4 and PRL secretion, and the function of those hormones during behavioral receptivity, would be similar to those of other spontaneously ovulating rodents and would differ from those of P. campbelli.

Thus, the current study quantified and compared the patterns of P4 and PRL secretion for each species of Phodopus during the behavioral receptivity of both an ovulatory cycle and a postpartum mating. Hormonal changes in serum during these two periods of behavioral receptivity were expected to differ as a result of the endocrine demands of parturition and impending lactation co-occurring with postpartum mating [31–33]. In addition, hormone replacement in ovariectomized female P. sungorus was used to determine the steroid hormone requirements for the induction of behavioral receptivity, and an independent sample of females were used to determine E2 and LH changes in P. sungorus around the time of ovulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Siberian (P. sungorus) and Djungarian (P. campbelli) hamsters were descendants of breeding colonies established in 1981 and most recently outbred with wild animals in 1990 [26]. Ambient temperature was 18 ± 1°C with a 14L:10D photoperiod (the middle of the dark phase = 0000 h). Dim red light provided illumination during the dark phase. All animals were housed in Nalgene (Nalge Nunc International, Rochester, NY) 27 x 21 x 14-cm cages lined with wood shavings, and provided with food (Purina 5001 lab chow; Ralston-Purina, St. Louis, MO) and water ad libitum. All females used were adult virgins (90–140 days). Females were paired with unrelated adult males (> 90 days) that had demonstrated their fertility by siring at least one litter.

Periovulatory E2, LH, and Timing of Ovulation in P. sungorus

A total of 30 P. sungorus females were paired with experienced breeder males at dusk on each of four consecutive days to identify the day on which they were in proestrus (behavioral receptivity [6]). They were then assigned to one of six groups destined to be bled from the orbital sinus and immediately killed by cervical dislocation on the subsequent proestrus at 1200 h, 1600 h, 2000 h, or on the subsequent estrus at 0000 h, 0400 h, or 0800 h. Because behavioral receptivity begins before dusk (1900 h), all individuals except those in the earliest (1200 h and 1600 h) groups were given a second behavioral test to confirm the stage of their estrous cycle before they were sampled.

Blood was stored at 4°C overnight. The next day, the clot was removed, and serum (approximately 500 µl/female [8]) was separated by centrifugation and stored at -20°C until assayed for LH, E2, and P4 content. In addition, each ovary for each female was visually inspected for the presence of corpora lutea, cleaned, and weighed (± 0.1 mg). Likewise, the uterus was described and weighed (± 0.1 mg). Oviducts were flushed with 0.1 ml of physiological saline so that their contents could be examined for the presence of tubular ova.

Steroid Requirements for the Induction of Behavioral Receptivity in P. sungorus

Twenty-six P. sungorus females were ovariectomized between 1600 and 1900 h under a mixture of ketamine hydrochloride (100 mg/kg) and acepromazine maleate (1.0 mg/kg) anesthetic. Immediately after surgery, females were given an injection (100 µl s.c.) of estradiol benzoate (EB) in sesame oil. Beginning of EB treatment immediately after surgery provided direct comparison with data for the Djungarian hamster [7, 9]. Although the experimental design did not control for the stage of the female's cycle at the time of ovariectomy (OVX), it had the advantage of minimizing effects resulting from changes in metabolism, receptor number and distribution, and hypothalamic peptide release expected to occur over a period of several weeks following OVX.

The EB was administered in one of five doses: 0.25, 0.625, 1.25, 1.875, or 2.5 µg of EB per female (n = 6, 5, 4, 5, 6 females). On each of the next two days, EB injection followed a behavioral test for receptivity that was terminated after 2 h or when a female showed lordosis and received one intromission [6]. On the third day of behavioral observations (72 h post-OVX), the EB injection was accompanied by 0.1-mg injection of P4 (s.c. in sesame oil). Females were retested for behavioral receptivity at 76 h to determine whether the combination of P4 and EB would facilitate receptivity for those females not receptive under EB priming alone.

To determine levels of E2 resulting from the various doses of EB, an additional three groups of 5 females each were ovariectomized and received injections of EB (0.25, 1.25, or 2.5 µg) at 0 and 24 h post-OVX. At 48 h post-OVX, females were given a test for behavioral receptivity and then bled from the orbital sinus [8]. The resulting serum was assayed for E2 content.

Behavioral Tests

The presence or absence of successful intromission was the sole indicator of behavioral receptivity recorded during behavioral tests. Males were removed from the cage immediately after intromission to prevent ejaculation or pseudopregnancy, and to minimize neuroendocrine responses that might affect subsequent behavioral tests [10]. Lordosis in Phodopus is brief and just allows intromission. There is considerable running between mounts, and a female will roll onto her back if a male attempts to mount when she is not receptive [20]. Latencies were not recorded because indicator males differ in the speed with which they attempt intromission and females differ in the natural timing of first behavioral receptivity in freely interacting pairs (unpublished observations). Thus, interactions between male and female behaviors, the time of day, and the need to separate male-female pairs all precluded additional quantitative measures in this study. A distinctive proceptive vaginal scent mark in Phodopus [20, 21] was seen in the ovariectomized, behaviorally receptive females, but this was not systematically recorded.

Repeated-Sampling Paradigm

Animals were housed in eight experimental cages identical to the home-cage except that the stainless steel hopper was replaced with a clear acrylic plastic lid. A minimum of 24 h was allowed for acclimation to the cage before sampling. Food was available on the floor of the cage. Water was continuously available through the lid. A glass tube entered each cage through the lid and delivered a continuous flow of compressed air near the floor of the cage. Vent holes in the lid allowed return circulation. Air flow was interrupted only at the time of sampling. Experimental cages were surrounded by an opaque curtain to prevent the animals from associating the arrival of the experimenter with handling.

Anesthetic (isoflurane [1-chloro-2,2,2-trifluoroethyl difluoromethyl ether] vaporized in oxygen) was delivered via the same tubing that provided compressed air, and induced anesthesia in less than 30 sec [34]. This method of repeated anesthesia does not influence PRL levels. Groups sampled 12 h out of phase for the same number of successive samples give the same P4 and PRL results (i.e., whether a sample at 1300 h is the first or the seventh drawn, the hormone titers are the same [9, 34]). A similar "out-of-phase" control was also included in the present study (see postpartum estrus below).

Under anesthetic, 75-µl blood samples were drawn from the orbital sinus into heparinized microcapillary tubes and centrifuged. Plasma was diluted for subsequent RIA and stored at -20°C until assayed for P4 and PRL content. Recovery from anesthesia was immediate upon reestablishment of the compressed air stream. No more than 900 µl of blood (12 successive samples at 2-h intervals) was drawn from any female. This sampling regimen has no adverse effect upon ovulatory cycles or gestation. Hematocrit falls from about 55% to about 45% over the first three samples and then stabilizes without further decrease. Complete hematocrit recovery requires about five days [9, 34].

Proestrus. Before sampling, at least two ovulatory cycles were followed in each female to confirm that females were showing a regular, 4-day cycle. Because vaginal smears are not effective to monitor cycles in Phodopus, a standard behavioral test in which males were introduced to the female cage daily and lordosis with a single intromission was used to recognize proestrus [6, 7] was applied. Seven P. sungorus females were each sampled once every 2 h during the 14-h light phase (0500–1900 h) on the subsequent proestrus. Behavioral receptivity was confirmed during the cycle following the last blood sample. Directly comparable data for P. campbelli (n = 9) were available in the published record [9].

Postpartum estrus. Ten pairs of P. sungorus and 11 pairs of P. campbelli were observed for the day of mating (Day 0) [6]. Pairs remained together throughout the study. Females were sampled between 0100 and 2300 h on Day 18. During that time, both parturition and a postpartum mating typically occur. A second group of P. sungorus (n = 6) were sampled from 1300 h on Day 18 through 1100 h the next day (Day 1 of lactation) and served as an internal control for sampling-order effects on hormone titers.

Hormone Determination

The P4 RIA (antibody #337; G.D. Niswender, Colorado State University, Fort Collins, CO) is in routine use in Phodopus (e.g., [9]). The range of sensitivity of the assay was 13.2–134.3 pg/tube (85–20% binding). Plasma samples were assayed in triplicate at 5 µl, giving a measured range of 2.6–26.9 ng/ml. Determinations outside those limits were rounded to the limiting value before analyses.

The E2 RIA (antibody to E2; Del Collins, Emory University, Atlanta, GA) has also been used extensively in Phodopus [7, 8] and has a range of sensitivity of 3.5–55 pg/tube. Serum samples were assayed in duplicate at 100 µl, giving a measured range of 35–550 pg/ml.

The PRL RIA used anti-hamster PRL (rat; Dr. A.F. Parlow, Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center, Torrance, CA; #AFP-7472988) as the primary antibody, hamster PRL (#AFP-10302-E) as the reference preparation, and goat anti-rat gamma globulin (titer P3, lot #9TA05Y; Antibodies Inc., Davis, CA) as the second antibody. The assay has been used extensively in P. campbelli (e.g., [9, 34]) and yields a validation in P. sungorus (unpublished results) the same as that published for P. campbelli [35]. Heterologous hamster PRL (Dr. F. Talamantes, University of California, Santa Cruz) has also been used to measure P. sungorus plasma [16]. Values are presented as ng/ml although the heterologous assay system may not provide absolute hormone determination in Phodopus. As with the steroid RIA, the range of sensitivity of the assay was truncated at 85% (33 pg/tube) and 20% (1880 pg/tube) binding. All plasma samples were assayed in triplicate at 1 µl, giving a measured range of 33–1880 ng/ml. Values outside those limits were rounded to the appropriate limit before analyses.

Levels of LH in serum were determined with the same assay technique as PRL but using the National Hormone and Pituitary Program ovine LH kit (Dr. A.F. Parlow) for iodination and as the reference preparation (range 0.024 through 6.25 ng/tube), with antiserum GDN15-o-LH (G.D. Niswender, Colorado State University, Fort Collins, CO) diluted in 3% normal rabbit serum at a working dilution of 1:24 000. Secondary antibody was sheep anti-rabbit gamma globulin. This assay had been validated previously for male P. sungorus [36]. Parallelism was confirmed for both P. campbelli and P. sungorus female serum for sample volumes between 25 and 100 µl. Samples were assayed as single determinations at 100 µl.

Assay Variance

Both E2 and LH determinations were completed in single assays so that only the intraassay variance of 3.6% could be calculated for each hormone.

Variances in P4 determinations were small, with inter- and intraassay coefficients of variance of 5.3% and 9.7%. Comparable variances for PRL determinations were larger at 9.6% and 12.2%. Because the potential for error when handling samples of 1 µl is large, and different specific activities in different iodinations have modest effects on the standard curve that are magnified in the final blood determinations because of the small volume assayed, universal truncation of the assay sensitivity beyond predetermined 85% and 20% binding levels was applied on the basis of an original sample of assays over four iodinations [34]. As the measured range still spanned almost two orders of magnitude, "low" and "high" PRL levels could be recognized with confidence. Therefore, to err on the side of conservative interpretation of results, PRL data were presented as mean ± SE, but statistical comparisons were restricted to counting the frequency of surges >= 900 ng/ml. Surges were defined as any sample >= 900 ng/ml, on the basis of a statistical analysis detailed in Edwards et al. [34]. That analysis identified significant outliers (more than 2 standard deviations away from the mean value) as peak levels of PRL and then averaged a large number of those peak values (n = 69) to reach the 900 ng/ml definition.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Periovulatory E2, LH, and Timing of Ovulation in P. sungorus

In the 30 females sampled between noon on proestrus and 0800 h on estrus, oviductal ova were found in one female at 2000 h, four of five females at midnight, and all females sampled on estrus. Thus, ovulation and movement of the ovulated ova into the lumen of the oviduct were generally complete before midnight on proestrus. Paired ovarian weight increased from noon on proestrus (7.3 ± 0.7 mg) to more than 10 mg at all subsequent sampling times (except for 2000 h when weight was 9.5 ± 1.3 mg and not significantly different from that at noon). Mean ovarian weights were lower in P. sungorus (9.5–10.8 mg) than in P. campbelli (18 mg declining to 14 mg [8]), as is typical for these two species [37]. Uterine fresh weight was maximal at 1600 h and significantly heavier than at all other times except midnight. This maximal uterine weight coincided with a discrete peak in LH and maximum P4 levels (Fig. 1). E2 levels were maximal at over 200 pg/ml in the first sample at noon on proestrus and had declined to very low levels before the 2000-h sample.



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  		FIG. 1. Mean (± SE) circulating levels of P4 (diamonds), LH (circles), and E2 (histogram) in female P. sungorus at six time points between noon of proestrus and 0800 h on estrus (n = 5 females per time). At 1600 h, both P4 and LH were significantly higher than at all other times. At 2000 h, levels of P4 were still elevated relative to all samples except the 1600-h peak. Maximal E2 levels at noon on proestrus were higher than in all other samples. The shaded background indicates the 10-h dark phase.

Steroid Requirements for the Induction of Behavioral Receptivity

Across the 26 females tested over 76 h (four behavior tests), increasing numbers of females were receptive with increasing dose administered and increasing number of injections and days post-OVX (Fig. 2). Eight females were receptive during the 24-h test (31%), suggesting they had been ovariectomized at a stage of their estrous cycle that already had high E2 [7]. Eight females (7 of which were the same as those receptive on the previous day) were receptive at 48 h (31%). Six of them had received doses high enough to have resulted in E2 levels as high as or higher than peak proestrous surge levels at the time of testing (see below). Twelve of 26 females were receptive during the 72-h test (46%). All twelve remained receptive at 76 h (4 h after the combined EB + P4 dose).



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  		FIG. 2. Summary of the percentage of female P. sungorus (n = 26) receptive during each of the four sequential tests for behavioral receptivity. Results are for all females tested, irrespective of the dose administered. Females that were receptive in one test were not always receptive in successive tests. Responses of individual females, and their doses, are detailed in Figure 3.

Of the remaining 14 females not receptive at 72 h, all but one became receptive by 76 h. Thus, 25 of 26 females (96%), including all females at the lowest E2 doses, were receptive at 76 h after the dual injections (Fig. 3).



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  		FIG. 3. Schematic representation of the experimental design and behavior test results used to determine the steroid requirements for the induction of behavioral receptivity in ovariectomized Phodopus females. Thickened regions of the x-axis locate the dark portion of the 14L:10D photoperiod. Asterisks denote tests for behavioral receptivity, which began before lights-off. Vertical arrows indicate the time and hormone administered as well as the behavioral tests used to quantify the response of females to E2 treatment alone and facilitation of receptivity by P4. Each horizontal row represents the behavior test results for a single female with "-" indicating no behavioral receptivity and "R" indicating lordosis with intromission.

The three injected doses of EB resulted, respectively, in 93 ± 13, 155 ± 4, and 320 ± 30 pg/ml levels of E2 in serum at 48 h (24 h after the last EB injection), and overlapped the range of E2 levels measured in unmanipulated females during the E2 peak on proestrus (Fig. 4). Levels of E2 were significantly higher in P. sungorus than the corresponding levels in P. campbelli as determined in the same assay (p <= 0.005). Of the 15 females bled at 48 h, only one was receptive during the behavior test immediately before the blood was drawn. She had received the intermediate dose of 1.25 µg and was central in the distribution of E2 levels at 155 pg/ml.



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  		FIG. 4. Positive relationship between the dose of EB administered to ovariectomized females (doses administered as µg/female and subsequently converted to µg/g based on body weight at the time of OVX) and the level of E2 measured in serum samples taken 24 h after the second injection. Data are shown for P. sungorus (filled circles) and P. campbelli (open circles) females (n = 15 for each species). Data for P. campbelli have been published in a summarized form in [9]. Methodological details and statistical comparisons are provided in the text.

Repeated Sampling of Plasma P4 and PRL Levels

The sampling procedure (anesthetizing females and newborn pups, repeated bleeding of females) did not result in infanticide or litter reduction in any P. campbelli females. One P. sungorus female reduced her litter size by one pup during sampling. Two additional P. sungorus litters died during the week after sampling.

Circulating P4 Levels

P. sungorus. The repeated sampling technique identified large surges of P4 in all (n = 7) P. sungorus females sampled during the light phase of proestrus (Fig. 5a). P4 levels did not vary from female to female (no effect of female individual differences on cycle P4 level 0500–1900; (F6,55 = 1.3, p = 0.3) but changed with time (F7,55 = 13.8, p < 0.0001), with levels at 1500 h higher than in all previous samples, and levels at 1700 h and 1900 h significantly higher than at all other times. Each female had her maximum P4 level in either the 1700-h (n = 4) or 1900-h (n = 3) sample. That maximum P4 was 17.1 ± 2.8 ng/ml (range: 9.1–26.9 ng/ml).



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  		FIG. 5. Mean (± SE) levels of plasma P4 obtained from repeated sampling of individual P. sungorus and P. campbelli females throughout proestrus (circles) or postpartum estrus (Day 18; triangles). Data for P. campbelli on proestrus are redrawn from [9]. Shaded areas denote the dark portion of the 14L:10D cycle. After 1300 h on Day 18, sample size for P. sungorus increased with the inclusion of additional females from an overlapping group. Statistical differences are discussed in the text.

All 16 P. sungorus females in the postpartum group gave birth on Day 18. This length of gestation was consistent with that of a large sample of 63 primiparous females paired in the course of a subsequent study (unpublished results), in which 61 births occurred on the 18th day (the remaining two litters were first seen on Day 19), and with that of a previous study, in which 14 of 14 females gave birth on the 18th day [18].

Large increases in P4 level were detected in 14 of 16 P. sungorus females sampled on the day of parturition (Fig. 5a). P4 levels did not differ in females sampled throughout Day 18 and females sampled beginning at 1300 h on Day 18 (F1,81 = 1.2, p = 0.3) and did not yield an interaction term that suggested any difference in the timing of maximal P4 surges (F5,81 = 0.5, p = 0.8). Therefore, there was no evidence that sampling sequence affected P4 results, and samples from the two studies were combined for subsequent analyses.

P4 levels changed with time (F5,92 = 21.3, p <= 0.0001), with values at 1700 h higher than at all other times, and values at 1500 h and 1900 h higher than the low values common to all times before 1500 h and after 1900 h. Of the 14 females with P4 surges, P4 levels were maximal in 2 females at 1500 h, 9 females at 1700 h, and 3 females at 1900 h. Individual P4 levels reached a maximum of 14.3 ± 1.0 ng/ml (range: 8.7–19.4 ng/ml). All females had basal plasma P4 levels by 2300 h on Day 18.

Thus, postpartum P4 surges (Day 18) were similar to those seen on proestrus, with the only differences 1) the absence of surge levels in two females on Day 18 and 2) the higher P4 levels at 1900 h on proestrus than at 1900 h on Day 18 (t16, 7 = 2.3, p <= 0.03). Peak amplitudes within individuals were similar (t23 = 1.2, p = 0.3).

The timing of parturition was strongly linked to the timing (and detection) of P4 increases. All 14 females with P4 increases gave birth well before mid-day (all litters but one were born between 0500 h and 0900 h; the last litter was born between 0900 h and 1100 h). Both of the remaining females, without P4 increases, gave birth between the 1300-h and 1500-h samples. For the females with P4 increases, the average interval between first record of pups and elevated P4 levels was 7.1 ± 0.6 h. Females were not systematically observed for postpartum mating.

P. campbelli. In P. campbelli, P4 surges were detected in all females (n = 9) on proestrus [9]. There was no evidence of individual differences in P4 level (F8,71 = 0.7, p = 0.7) but P4 levels changed with time (F7,71 = 8.6, p <= 0.0001; Fig. 5b). Levels at 1500 h and 1700 h were significantly higher than at all other times except 1900 h. Levels at 1900 h remained significantly higher than those between 0500 h and 1100 h. Timing of female maxima ranged from 1100 h (n = 1) through 1300 h (n = 4) and from 1500 h (n = 3) to 1700 h (n = 1), with maxima of 13.5 ± 1.8 ng/ml (range 8.7–26.4 ng/ml).

All P. campbelli females gave birth on Day 18 (n = 11), and 10 of them had elevated P4. There was no evidence of individual differences in P4 level (F10,118 = 1.2, p = 0.3), but P4 levels changed with time (F11,118 = 8.6, p <= 0.0001; Fig. 5b). Levels at 1500 h and 1700 h were higher than those in all other samples. Levels at 1900 h were higher than all levels before 1300 h and after 1900 h. Timing of female maxima ranged from 1500 h (n = 5) through 1700 h (n = 3) and from 1900 h (n = 1) to 2300 h (n = 1), with maxima of 7.8 ± 0.6 ng/ml (range 5.2–11.8 ng/ml) for those 10 females with surges.

All but one P. campbelli female gave birth between 0300 h and 0700 h on Day 18 and had a postpartum P4 increase. The female that gave birth after mid-day (first pup seen at 1500 h) was the female with no P4 change.

Therefore, P. campbelli P4 differed between proestrus and Day 18 in several ways: 1) Day 18 P4 maxima were significantly lower than proestrous maxima (t17 = 3.2, p <= 0.005; and they were largely nonoverlapping since only 2 proestrous females fell within the range of Day 18 values and only three Day 18 females fell within the range of proestrous females); 2) levels of P4 at 1500 h were significantly higher during a cycle; and 3) one Day 18 female did not have an increase in P4 during sampling.

Species comparison. When the two species were compared over time, there was no difference in P4 maxima on proestrus (t14 = 1.1, p = 0.3), but the maxima on Day 18 were almost twice as high in P. sungorus (t23 = 5.3, p <= 0.0001), with higher average P4 levels between 1700 h and 2100 h. In other words, the decreased and low peak levels of P4 during the postpartum estrus of P. campbelli were the distinctive result (Fig. 5).

Circulating PRL Levels

As was typical in previous studies of PRL levels in P. campbelli females [9, 34], levels of PRL varied rapidly from minimum to maximum detectable levels within single females. Thus, statistical comparisons focused on the presence or absence of surge levels of PRL rather than on the mean (± SE) values shown in Figure 6. Surges were defined as any sample >= 900 ng/ml ([34]; see Materials and Methods for more details).



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  		FIG. 6. Mean (± SE) levels of plasma PRL obtained from repeated sampling of individual P. sungorus and P. campbelli females throughout proestrus (circles) or postpartum estrus (Day 18; triangles). Data for P. campbelli on proestrus are redrawn from [9]. Shaded areas represent the dark portion of the 14L:10D cycle. After 1300 h on Day 18, sample size for P. sungorus increased with the inclusion of additional females from an overlapping group. Samples for P. campbelli at 1100 h on Day 18 were damaged before assay. Statistical differences are discussed in the text.

P. sungorus. On proestrus, there was an effect of time of sample on the level of PRL in P. sungorus plasma, with high PRL levels at dawn and in the late afternoon (Fig. 6a). When sampling began at 0500 h, 5 of the 7 females were at surge levels. Between 0700 h and 1100 h, only 4 of 21 samples were at surge levels (3 females). At 1300 h and 1700 h, 6 of 7 and 7 of 7 females, respectively, were at surge levels. Overall, 30 of the 56 PRL levels (54%) were above 900 ng/ml, and each female had at least two samples at surge levels (range 2–6 per female).

Between 1300 h and 2300 h on Day 18, PRL levels in Day 18 P. sungorus females that had been sampled since 0100 h were higher than PRL levels in the second group, which been sampled since 1300 h (F1,108 = 4.09, p = 0.05). However, that difference was not significant at any single time, nor was there any evidence that the magnitude of the difference changed over time. As a result, both sets of PRL determinations were combined for use in subsequent analyses. Because eight samples (from 6 females) were damaged before analyses for PRL, and 6 females from the second group were added to the 1300 h onward samples, the resulting sample sizes ranged from 8 to 16 females.

Unlike the results for proestrus, there was no evidence of a systematic change in PRL levels over time in P. sungorus on Day 18. At each sample time, at least two females had PRL levels over 900 ng/ml. However, across the 148 samples, only 39 samples (26%, which included 9 of 10 females sampled all day and 4 of 6 females sampled just 6 times) were at surge levels.

For P. sungorus, this represented a significantly lower proportion of samples at surge levels on Day 18 compared to proestrus ({chi}2(dof=1) = 12.3, p < 0.0005). Therefore, within P. sungorus, the PRL profile on Day 18 differed from proestrus in the absence of temporal pattern (low synchrony between females) and in the low overall frequency of surge levels.

There was no clear temporal relationship between parturition and PRL surges. Surge levels of PRL were not reliably associated with parturition, remating (when seen), or increases in P4 for individual females.

P. campbelli. Like P. sungorus, P. campbelli had high PRL levels in the late afternoon, but P. campbelli did not have a dawn surge of PRL (Fig. 6b). Only 4 of the 72 PRL determinations in P. campbelli on proestrus were at surge levels. Those five samples from four females all occurred between 1300 h and 1500 h.

In P. campbelli, PRL levels on Day 18 were higher than on proestrus, with no evidence of temporal patterning. Likewise, surges were rarer on proestrus than on Day 18 in P. campbelli ({chi}2(dof=1) = 30.7, p < 0.0001).

Unlike the case with P4, there was no clear temporal relationship between parturition and PRL surges. Surge levels of PRL were not reliably associated with parturition, remating (when seen), or increases in P4 for individual females.

Species comparison. In P. campbelli, the highest PRL average on proestrus (527 ng/ml at 1300 h) was similar to the lowest PRL average in P. sungorus (474 ng/ml at 0700 h), and PRL surges on proestrus were significantly rarer in P. campbelli than in P. sungorus ({chi}2(dof=1) = 34.8, p < 0.0001). In contrast, PRL levels were higher and surges were more frequent in P. campbelli on Day 18 ({chi}2(dof=1) = 8.9, p < 0.005). Thus, the differences between proestrus and Day 18 were in opposite directions in P. sungorus and P. campbelli (Fig. 6).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In many respects, physiological changes in P. sungorus and P. campbelli over the time of behavioral estrus were similar. Like P. campbelli [8], P. sungorus mated before dusk on proestrus of an ovulatory cycle and ovulated before midnight. Circulating E2 levels reached almost 200 pg/ml in the early afternoon of proestrus and declined soon after dark [7]. Maximal levels of P4 were found in each species in the mid-afternoon, peaking after E2 and, at least in P. sungorus, at the same time as LH (although samples were only assayed at 4-h intervals, 1600 h is the LH maximum in golden hamsters and Sprague-Dawley rats on the same photoperiod [12, 38]).

As far as could be determined from the behavioral observations, which were restricted to the presence or absence of successful intromission, steroid hormone requirements for the induction of behavioral receptivity in P. sungorus were similar to those found in the classic studies in the golden hamster [39]. At high doses, a substantial proportion of females became behaviorally receptive after E2 treatment alone. There was also clear evidence of facilitation of behavioral receptivity by P4, and that facilitation was effective in eliciting receptive behavior even against a background of low priming doses of E2.

Thus, as predicted by the hypothesis that the absence of a need for P4 facilitation had evolved recently in P. campbelli, P. sungorus were similar to other spontaneously ovulating small mammals [1, 2], but not to P. campbelli. In P. campbelli, like many species with induced ovulation [3, 40], all EB treatments that produced E2 levels near those seen on proestrus (physiological levels) resulted in behavioral receptivity [7, 9]. Furthermore, no ovariectomized P. campbelli female that became receptive under EB treatment shifted back out of receptive behavior without prolonged exposure to P4 ([7, 9], unpublished data), whereas P. sungorus females that showed behavioral receptivity on one day would not always show receptivity the next. As additional confirmation of the sufficiency of E2 in eliciting behavioral receptivity in P. campbelli, ovariectomized P. campbelli with E2 implants (silicone elastomer capsules, s.c.) will mate daily for several weeks [10]. Therefore, while these data could not rule out a shared role for P4 in subtler measures of behavioral receptivity than successful intromission, they were clear evidence of a species difference within the genus Phodopus.

Levels of E2 in the two species were similar, although direct comparisons were limited because the available samples were all at least 4 h apart, and not synchronized. At noon on proestrus, the level of E2 in peripheral P. sungorus serum was around 200 pg/ml, while interpolation of earlier results for P. campbelli suggested that those females were at about 150 pg/ml, rising to a maximum of almost 200 pg/ml at 1430 h [7]. There were, however, indications that the E2 surge ended sooner in P. sungorus. Levels of E2 in P. campbelli females were still about 150 pg/ml at 1930 h, by which time levels of E2 in P. sungorus had returned to baseline. Aspects of E2 metabolism may also differ in the two species of Phodopus, since EB injections given from the same stock solutions, and measured in the same assay, resulted in higher E2 levels in ovariectomized P. sungorus. Thus, evolutionary changes in sensitivity to E2 may have accompanied changing roles for P4 in the facilitation of behavioral receptivity in the genus Phodopus.

In addition to the facilitation of behavioral receptivity, there are other known functions for P4 surges on proestrus. These include elicitation of proceptive behaviors and suppression of agonistic behaviors [41], control of the precise timing of behavioral receptivity [42, 43], and the eventual inhibition of behavioral receptivity [44, 45]. Neither proceptivity, nor aggression, nor the inhibition of behavioral receptivity was quantified in the present study. However, because P4 surges in P. campbelli were asynchronous relative to the synchronous P4 surges in P. sungorus, a role in timing receptivity was unlikely for P. campbelli. Other functional roles for P4 on proestrus may be similar in the two species.

There is, however, an alternative interpretation of these results which would be consistent with the hypothesis that the absence of a role for P4 in the behavioral receptivity of P. campbelli had evolved recently. Rather than seek a functional role for P4 increases on proestrus, the increases seen in P. campbelli may be considered nonfunctional relics of a recent time in the phylogenetic history of P. campbelli when the P4 surges synchronized and facilitated behavioral receptivity. Such an interpretation would also be consistent with the patterns of P4 secretion in each species during the postpartum estrus.

The lower P4 level on Day 18 in P. campbelli suggests that active selection has reduced the serum levels of P4 relative to proestrus. That selection may have arisen from the potential for conflict between the need for P4 to facilitate behavioral receptivity and the need to reduce P4 to facilitate parturition. Most mammalian species have low levels of P4 before and during parturition (e.g., mouse [46], cow [47], rat [48], golden hamster [49]). Of those species, only the rat and mouse have a postpartum estrus [31], and temporally structured P4 data for each on the day of parturition are not available. However, the majority of mouse births occur between 1000 h and 1400 h [46], and the same is true for rats, with the peak of births at 1400 h [50, 51]. Even when photoperiod is manipulated during gestation, a large majority of golden hamster births occur during the light phase, with a peak in the early afternoon [52]. Likewise, a previous study of P. sungorus noted that litters are commonly born in the early morning (before 0500 h), in mid-morning (0900–1000 h), and in late afternoon (1600–1700 h) [16]. In the present study, 88% of P. sungorus litters and 91% of P. campbelli litters were born before 0900 h (lights-on at 0500 h). In both species, the detection of a P4 increase on the day of parturition was strongly linked to early delivery, and failure to detect a P4 increase was linked with late delivery. Other studies have shown that 22% of P. campbelli females do not mate until the day after parturition [53], and that P. sungorus females can have proestrous vaginal smears 24–48 h postpartum [16]. Thus, early delivery in Phodopus may be a prerequisite for behavioral receptivity on the same day.

Species differences in the frequency and temporal pattern of PRL surges on proestrus and Day 18 also supported the hypothesis that P. campbelli had not only diverged recently from a common ancestor with a "typical," or ancestral, pattern of PRL secretion, but that P. campbelli might still be undergoing dynamic change (on an evolutionary time scale) with respect to the role of PRL in female reproduction.

In P. sungorus, all females had several proestrous samples that met the definition of a surge, and females were synchronized so that distinct peaks in mean PRL level were recognized at dawn and 1700 h. In P. campbelli, instead of 54% of samples at surge levels, only 6% of samples were over 900 ng/ml. Those surges were also restricted to the late afternoon, making the shape of the PRL curves on proestrus similar in the two species. Proestrous PRL surges are a typical feature of small rodents that have been studied [12, 38, 54–56], and they are known to be involved in the facilitation of behavioral receptivity [57, 58] and successful ovulation [59], although their role has not yet been determined for any other hamster species. Therefore, the presence of PRL surges in P. sungorus and the comparative absence of surges in P. campbelli support, rather than undermine, the hypothesis that the role of PRL in P. campbelli is anomalous and has probably evolved recently.

Several functional roles for PRL surges have been suggested for the dramatic PRL increase on the afternoon before parturition in the rat [48] and mouse [60]. Elevating P4 delays the antepartum PRL surge and delays parturition by the same amount of time, suggesting that the antepartum PRL surge is involved in the initiation of labor [61]. In addition, antepartum PRL is clearly implicated in the stimulation of maternal behavior [62–64]. In contrast, PRL surges on the day of parturition are generally assumed to be suckling-induced [65], and the absence of PRL on the day of parturition is likewise considered evidence that suckling has not begun at that time [60]. In contrast with the very different PRL surge frequencies on proestrus, both species of Phodopus had PRL surges throughout the day on Day 18. That similarity in the two species suggests that PRL has a common function in each species. However, neither P. sungorus nor P. campbelli had a clear relationship between PRL surges and either birth or P4 increases, so that neither a link between pup suckling (which could not be measured directly because pups and females were anesthetized before the experimenter saw them) and PRL surges, nor a link between a PRL surge and behavioral receptivity was evident.

In summary, the facilitation of behavioral receptivity by P4 in P. sungorus, the highly synchronous P4 surges on proestrus and postpartum in P. sungorus, and the frequent, synchronous PRL surges on proestrus in P. sungorus all had strong parallels to available data for other rodent species; and all differed from P. campbelli. The most parsimonious explanation for this result, given the close relatedness of these two species (P. sungorus is closer to the ancestral form: [66]), is that the behavioral and periovulatory endocrinology of P. campbelli reproduction has evolved recently.

Previous studies have noted that other patterns of secretion, and functional roles, of both P4 and PRL during P. campbelli reproduction could not be explained or predicted by comparison with the rat, mouse, and golden hamster [8, 9, 34, 35, 67]. The evolution of biparental care in P. campbelli has recently been explained as an adaptation to overcome conflicts between adaptations for survival in a cold, arid, seasonal habitat, and adaptations for extremely rapid reproduction [27]. If the novel endocrinology of P. campbelli has also evolved recently in response to extreme selection pressure in its ecological niche, then other aspects of P. sungorus reproductive endocrinology may also be more similar to those of rats, mice and golden hamsters than to their congener P. campbelli. Thus, Phodopus may represent a rare example of ongoing evolution in mammalian reproductive endocrinology.


   FOOTNOTES
 
1 Financial support for this research was provided by an NSERC Research Grant and the Advisory Research Council of Queen's University (K.E.W.-E.), the Ontario Graduate Scholarship Program, and a Queen's University Graduate Fellowship (H.J.M.). Induction of behavioral receptivity studies were begun while K.E.W.-E. was a Lalor Foundation Postdoctoral Fellow at the University of Kansas Medical Center and received support from Dr. P.F. Terranova and NSF. Back

2 Correspondence. FAX: (613) 545–6617; wynneedw{at}biology.queensu.ca Back

Accepted: February 17, 1998.

Received: October 17, 1997.


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