| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a United Graduate School of Veterinary Science, Gifu University, Gifu 501-1193, Japan
b Laboratory of Theriogenology, Faculty of Agriculture, Gifu University, Gifu 501-1193, Japan
c The Institute of Japanese Black Bear in Ani, Akita 018-4731, Japan
d Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan
e Laboratory of Veterinary Surgery, Faculty of Agriculture, Gifu University, Gifu 501-1193 Japan
ABSTRACT
We examined changes in the concentrations of serum progesterone (P4), estradiol-17ß (E2), FSH, LH, prolactin (PRL), and inhibin to determine their interaction and their effect on the reproductive endocrine controls of pregnant and nonpregnant female Japanese black bears. Fourteen female bears were used in this study over a 2-yr period. In the first year, six of the bears were divided into two groups; a pseudopregnant group and a nonpregnant group. In the second year, the remaining eight bears were also divided into two groups; a pregnant group and a nonpregnant group. Pregnant and pseudopregnant bears had similar P4 trends with both groups exhibiting a significant increase in December, which is the suspected time of implantation in pregnant bears. These trends correlated with an increase in PRL levels, whereas low levels of LH were maintained throughout the year. Nonpregnant bears maintained low concentrations of P4, and compared with pregnant and pseudopregnant bears, they also exhibited a delayed elevation in PRL. Luteinizing hormone activity varied among individual animals, but regardless of reproductive status, fluctuation patterns of E2, FSH, and inhibin did not differ among bears. Our results suggest that PRL may play a luteotropic role in both pregnant and pseudopregnant bears, and is possibly responsible for inducing reactivation of the dormant corpus luteum that precedes implantation in the Japanese black bear.
corpus luteum function, pregnancy, progesterone, prolactin, seasonal reproduction
INTRODUCTION
The Japanese black bear (Ursus thibetanus japonicus) is one of the largest mammalian species to exhibit the reproductive strategy of seasonal breeding, a trait common to other ursids in the Northern hemisphere. Specifically, female bears have intriguing traits such as obligate delayed implantation during the gestation period and parturition during the denning period [1]. The mating season occurs between May and August, and there is some variation due to latitude between Japanese black bears [2] and American black bears [3], as well as Hokkaido brown bears [4]. The fertilized egg undergoes quiescence at the blastocyst stage for about 4–5 mo during the course of its development. In black and brown bears, implantation occurs between late November and early December, followed by parturition between the end of January and the beginning of February [5–8]. Thus, the postimplantation period lasts for about 2 mo in bears.
Although the "delayed implantation" phenomenon is of great interest, its mechanism remains an enigma. Obligate delayed implantation occurs in some carnivore species, in which members of the Mustelidae family, especially minks and western spotted skunks, have been studied extensively for their endocrinological and morphological changes, particularly during the periimplantation period. Delayed implantation in these species is believed to occur due to insufficient pituitary secretion, which results in incomplete differentiation of the corpus luteum (CL) and reduced luteal secretion [9]. The concentration of peripheral progesterone (P4) is therefore maintained at a relatively low level during the delayed implantation period, whereas a dramatic P4 increase occurs in response to CL reactivation at implantation. The delayed implantation characteristic is exhibited by mustelids as well as bears [6, 8–12]. The CL reactivation that precedes implantation is induced by an increase in prolactin (PRL), which in minks and western spotted skunks, is associated with increasing day length. This has been confirmed in several experiments through the administration of PRL or dopamine agonists and antagonists alike [13–17], or by providing an artificial photoperiod or administration of melatonin [18–20]. Although estrogen and progesterone alone will not induce implantation in mustelids, studies on minks and ferrets [9, 21–24] have shown successful induction of the CL in ovariectomized animals. This shows that one or more luteal factors, separately from progesterone, are necessary to induce implantation. In addition, gene expression of leukemia inhibitory factor (LIF), one of the cytokines essential to implantation in mice [25], and its receptor (LIFRß), are increased in the uterus prior to implantation. This suggests that LIF may be involved in preparing the uterus for implantation in minks and western spotted skunks as well. Concentrations of LIFRß mRNA were increased significantly by PRL administration, but these levels were not physiologically sufficient. Thus, some component other than PRL appears to be involved in the gene expression of LIFRß as well as that of LIF, and the function and regulation of these genes in minks and skunks remains obscure [26–28]. On the other hand, Polejaeva et al. [29] reported that PRL appeared to act directly on embryonic cells to terminate diapause in vitro. Consequently, in minks and spotted skunks, PRL plays a role as part of the trigger that induces the dormant CL and embryo reactivation, but it remains to be determined precisely how and where PRL works to reactivate the CL, trigger implantation, or to link luteal proteins or cytokines such as LIF. In bears and European badgers, the pituitary regulation of luteal function and implantation are regarded as being different from those in minks and skunks because implantation occurs in December [15, 30]. It has not yet been determined which hormone acts as a trigger to induce the CL and blastocyst into reactivation at implantation in these species.
The purpose of the present study was to examine annual changes in several hormones from the pituitary and the ovaries in pregnant and nonpregnant Japanese black bears, and to identify the possible relationship among these hormones. The focus of this relationship is on the regulation of luteal function relative to the maintenance of pregnancy on the basis of P4 changes. Furthermore, we wished to determine which hormone induces CL reactivation, which then provokes termination of delayed implantation.
MATERIALS AND METHODS
Animals
Fourteen captive, sexually mature female Japanese black bears were used in this experiment over a 2-yr period, 1998 and 1999. The bears were managed at Ani Mataginosato Bear Park, Akita, in northeastern Japan (40°N, 140.1°E). All animals were fed primarily cornmeal with some fruit and commercial bear pellets as supplements, and water was provided ad libitum during the active season (from April to November). Animals had access to water but not to food during the denning season (from December to the following March).
Design of the Experiment
In the first year, six female bears were randomly divided into two groups of three animals each. The animals in one group were housed together in isolation in an indoor run (3.47 x 4.88 m) and were given the chance to mate with several different males four or five times during the breeding season (July 6 to August 10). No positive fetal images were recognized by ultrasonographic examination on January 6, and ultimately, no bear gave birth.
In the second group, two of the three had been isolated together from other animals in an indoor run throughout the year. However, the single remaining female had numerous opportunities to meet male bears through a fence during the breeding period when the animals in the first group had been allowed to copulate. She could touch the male bears but she could not mate.
In the second year, six female bears were kept together with male bears in an outdoor run (25 x 50 m) from late April to mid-September (including the mating season) in order to avoid artificial disturbances in their breeding pattern. In mid-September the animals were isolated in indoor runs until early December, when they were transferred to individual delivery rooms (1.8 x 2 m), where they remained until the following April. Each of the six female bears gave birth between January 23 and February 2. As control animals, two female bears were maintained together but were completely isolated from other bears in an indoor run throughout the year.
Sampling
Sampling in the first year started from May 1998 until April 1999 and was conducted between Day 15 and Day 20 of each month, except for the period from November 17 to February 15, when the sampling was performed at 10-day intervals. In the second year, because we wished to minimize any extra stress from sampling attributed to pregnancy, sampling was carried out mid-monthly between September 1999 and February 2000 as well as on parturition day or the day after parturition. Animals were immobilized by blow dart or spear injections with either a combination of ketamine HCl (Ketaral, Sankyo, Japan) and medetomidine HCl (Domitor, Meiji, Japan) at doses of 5 mg/kg and 0.04 mg/kg body weight (BW), respectively, or a mixture of zolazepam and tiletamine HCl (Zoletil, Virbac, France) at a dose of 9 mg/kg BW. Blood samples for hormone assays were collected from the jugular vein into vacuum tubes after immobilization. Collected blood was centrifuged at 1200 x g for 15–20 min and the separated serum was stored at -30°C until assay.
Radioimmunoassay of P4 and E2
Serum concentrations of P4 and E2 were measured by the radioimmunoassay (RIA) method described by Palmer et al. [31]; E2 assay was performed with a minor modification as described previously [32]. The antisera against P4 (HAC-AA63-06RBP84) and E2 (FD121, Medical System Service Teikokuzouki, Kanagawa, Japan) were used at a final dilution of 1:56 000 and 1:420 000, respectively. The radioligands used were [1,2,6,7,16,17-3H(N)]-progesterone and [2,4,6,7,16,17-3H(N)]-estradiol (NET-1112 and NET-517, respectively; both from New England Nuclear Life Science Products). The minimum detectable concentrations of P4 and E2 were 0.04 ng/ml and 5.33 pg/ml, respectively. The intraassay and interassay coefficient variations were 12.62% and 16.11% for P4, and 8.20% and 11.39% for E2, respectively.
Radioimmunoassay of LH and PRL
Serum immunoreactive LH and PRL concentrations were determined using heterologous double-antibody canine RIA methods, exactly as previously described [33]. For the LH assay, canine antiserum of LH with a final dilution of 1:350 000 (AFP8311890Rb), purified canine LH for iodination (AFP5214B), and canine LH standard (AFP5216B) were used. For the PRL assay, canine PRL antiserum with a final dilution of 1:350 000 (AFP1062091GP) and canine purified PRL (AFP2451B) for iodination and reference were used. As a second antibody, goat anti-rabbit serum (H-23) and goat anti-guinea pig serum (HAC-GPA2-01GTP80) were employed for LH and PRL assays, respectively. The minimum detectable concentrations were 0.04 ng/ml for the LH assay and 0.24 ng/ml for the PRL assay. Intraassay and interassay coefficients of variation were 12.56% and 15.06% for LH, and 7.23% and 10.06% for PRL, respectively.
Radioimmunoassay of FSH
The immunoreactive FSH measurement procedure was followed by a heterologous double-antibody canine FSH RIA described previously by Nakada et al. [34]. Purified rat FSH (NIDDK-I-5) iodinated by means of the chloramine T method, anti-human FSH rabbit serum (M 91), and canine FSH (LER-1685-3 A) as a reference standard were employed in this assay. Results were expressed in terms of canine FSH. Samples (100 µl), assay buffer (50 µl; 1% BSA in 0.05 M PBS), and FSH antiserum diluted 1:12 000 (100 µl) were incubated for 24 h at 32°C. Following incubation, 50 µl of labeled FSH was added, mixed, and incubated for another 24 h at 32°C. Subsequently, 100 µl of goat anti-rabbit serum diluted 1:200 in 0.05 M PBS containing 5% polyethylene glycol was added, mixed, and incubated for an additional 12 h at 4°C. After the reaction mixture was centrifuged at 1700 x g for 30 min and the supernatant was decanted, radioactivity in the pellets was counted by gamma spectrometry (COBRA 2005; Packard Instrument Co., Meriden, CT). Parallelism between standard canine FSH and immunoreactive FSH in the bear pituitary extract and serum was tested. The pituitary extract was prepared by homogenizing whole pituitary from a male Japanese black bear in 1 ml of 0.05 M PBS on ice. The homogenate was centrifuged at 20 800 x g for 30 min at 4°C. The supernatant was stored at -30°C until assay. The assay sensitivity was 2.69 ng/ml. The intraassay and interassay coefficients of variation were 11.04% and 8.82%, respectively.
Inhibin Radioimmunoassay
Serum immunoreactive inhibin concentrations were measured by the double-antibody RIA method using bovine inhibin described by Hamada et al. [35]. The purified 32-kDa bovine inhibin was iodinated by the chloramine T method. The antiserum against bovine inhibin (TNDH-1) was prepared by immunization with partially purified bovine follicular fluid (bFF) inhibin of a castrated male Japanese white rabbit. Results were expressed in terms of bFF 32-kDa inhibin.
Duplicate or triplicate aliquots (100 µl) of reference standards ranging from 1.95 to 1000 pg/tube or the unknown samples of bear serum and 50 µl of antiserum (TNDH-1) finally diluted 1:800 000 with 0.05 M EDTA-PBS containing 0.4% normal rabbit serum plus 150 µl assay buffer (5% BSA in 0.05 M PBS) were incubated for 24 h at 32°C. The labeled antigen (5000 cpm/50 µl in 5% BSA-PBS) was then added and incubated for a further 24 h at 32°C. After incubation, 50 µl of anti-rabbit serum diluted 1:200 in 0.05 M PBS containing 5% polyethylene glycol was added and incubated for an additional 12 h at 4°C. After the reaction mixture was centrifuged at 1700 x g for 30 min and the supernatant was decanted, radioactivity in the pellets was counted by gamma spectrometry. Parallelism between standard bovine inhibin and immunoreactive inhibin in the bear ovary extract and serum was assessed. The ovarian extract was prepared by homogenizing a piece of ovary (0.36 g) containing middle and small follicles obtained from an adult female Japanese black bear in 2 ml of 0.05% PBS on ice. The homogenate was centrifuged at 20 800 x g for 30 min at 4°C and the supernatant, diluted serially in 5% BSA-PBS, was provided with the assay validation. The assay sensitivity was 0.1 ng/ml. The intraassay and interassay coefficients of variation were 9.01% and 15.67%, respectively.
Statistics
Mean (± SEM) values were determined for serum P4, E2, FSH, LH, PRL, and inhibin concentrations in three mated bears in the first year and in six parturient bears in the second year. Differences among time periods were evaluated by one-way ANOVA and individual differences were further analyzed using the Tukey-Kramer test when significant F values were indicated. In order to evaluate the relationship between CL activity and hormone changes based on serum P4 concentrations, correlation coefficients were calculated between mean values of P4 and the other hormone concentrations. This was analyzed for data from the mated bears in the first year and the parturient bears in the second year from September to January, which corresponds with the luteal phase. A value of P < 0.05 was considered significant.
RESULTS
FSH and Inhibin Assay Validation
The displacement curves for serial dilutions of serum samples from two bears and the pituitary extract were paralleled with the canine FSH standard curve (Fig. 1a). For the inhibin assay, good parallelism was also obtained between the bovine inhibin standard and serial dilutions of sera from two bears and the ovarian extract (Fig. 1b).
|
Hormonal Changes in Mated and Nonmated Bears
Hormone concentrations for mated and nonmated bears in the first year are presented in Figure 2. In mated bears that did not give birth, mean values of serum P4 concentrations showed low levels from May to July (0.47 ± 0.04 ng/ml) and began to increase gradually in August (2.04 ± 0.04 ng/ml). Subsequently, a marked elevation was observed on December 17 or 27 (9.55 ± 1.10 ng/ml), and high levels were maintained until January 6 or 16, when the concentrations began to decrease, and returned to basal levels by February 5 (1.19 ± 0.13 ng/ml). Mean values between November 27 and January 6 were significantly higher compared with those in other months (P < 0.05). Mean values for serum PRL concentrations on September 17, October 17, and November 17 were significantly lower (2.67 ± 1.62, 2.28 ± 0.53, and 1.77 ± 0.47 ng/ml, respectively; P < 0.05) in contrast to high values from May to August (8.67 ± 1.62 ng/ml on average), followed by an elevation of the concentrations on November 27 (8.69 ± 4.39 ng/ml). Serum LH concentrations were at constant, low levels throughout the year (0.06 ± 0.04 ng/ml on average) and no significant difference was revealed among the months. Serum E2 concentrations varied among individuals but were relatively high in May to August, tended to decline toward November, and then became high again in December and January, although there was no apparent statistical difference in these concentrations among individual months. Serum inhibin concentrations ranged between 0.70 ng/ml and 1.47 ng/ml, and no distinct change was observed over the year. Serum FSH concentrations increased in late November and early December and maximal levels were observed on December 27 (114.12 ± 97.74 ng/ml, P < 0.05). The results of correlation coefficients analyzed between changes of P4 and the other hormones from September and January 26 (n = 10) revealed a positive correlation between P4 and PRL changes (r = 0.65, P < 0.05).
|
In nonmated animals, bear 33, which had met male bears through a fence, had a marked elevation in P4 and PRL concentrations on December 7 (12.11 ng/ml and 7.39 ng/ml, respectively), which was consistent with the results in the mated bears. On the other hand, no remarkable P4 increase was observed throughout the year (0.24–2.82 ng/ml) in the remaining nonmated bears, except for a transient elevation in January (7.64 ng/ml) in bear 59, and there were two peaks in serum PRL concentrations in December (7.96–12.06 ng/ml) and January (8.14 ng/ml). However, serum LH concentrations fluctuated, with erratically high levels among individuals between September and January. Changes in serum E2, inhibin, and FSH concentrations were similar to those in mated bears.
Hormonal Changes in Parturient and Nonmated Bears
Hormone concentrations in parturient and nonmated bears in the second year are presented in Figure 3. In the parturient bears, mean values for serum P4 concentrations were high in September (4.29 ± 2.30 ng/ml) compared with those in October (2.54 ± 0.68 ng/ml) and November (3.54 ± 0.81 ng/ml), and were then elevated significantly in December (10.86 ± 1.44 ng/ml, P < 0.05). P4 concentrations in January decreased to half the values of December (6.00 ± 2.07 ng/ml) and dropped to basal levels by the day following parturition (1.66 ± 0.57 ng/ml). Serum PRL concentrations were somewhat higher in September (3.84 ± 1.75 ng/ml) than in October (1.21 ± 0.17 ng/ml) and November (1.15 ± 0.09 ng/ml), were elevated significantly in December (4.42 ± 1.52 ng/ml, P < 0.05), and then decreased slightly in January (4.18 ± 1.08 ng/ml) but began to increase again after parturition. Serum concentrations of LH were somewhat high in September and December but there was no significant differences. Serum E2 concentrations were low between October (14.93 ± 2.79 pg/ml) and November (18.52 ± 10.12 pg/ml) compared with September (34.34 ± 15.10 pg/ml) and December (37.46 ± 16.19 pg/ml), and mean values were significantly different between October and December (P < 0.05). Serum inhibin concentrations varied between individual animals over the period of this experiment and no significant difference was revealed among individual months. FSH levels were low in November (11.58 ± 10.92 ng/ml) but tended to increase in December (54.02 ± 21.71 ng/ml) and January (76.51 ± 51.8 ng/ml), however, the difference was not significant. Meanwhile, results of an analysis of correlation coefficients between P4 and the other hormone changes from September and January (n = 5) revealed no correlation.
|
In the two nonmated bears, serum concentrations of P4 remained at low levels (0.10–1.84 ng/ml) except for a marked rise in January (12.09 ng/ml) in bear 61, and PRL increased gradually from December (1.95–3.08 ng/ml) to February (4.12–6.34 ng/ml). Serum LH concentrations were slightly high in September (0.02–0.21 ng/ml) and January (0.11–0.28 ng/ml). Serum E2 concentrations became higher in December (42.42 pg/ml) and February (38.96 pg/ml) in one bear, but low concentrations (10.01–16.77 pg/ml) were maintained in the other bear, except in February (30.71 pg/ml). Serum inhibin concentrations declined gradually from October (1.36–2.15 ng/ml) to February (1.12–1.33 ng/ml), whereas FSH became high in January (100.88 ng/ml) in one bear, and in February (97.25 ng/ml) in the other bear.
DISCUSSION
Maintenance of pregnancy requires P4, which is primarily secreted from the CL throughout gestation in most carnivores. This is supported by evidence that the placenta does not secrete P4 in dogs [36] and minks [37], and is further supported by findings that indistinguishable patterns of P4 changes between pregnant and pseudopregnant or hysterectomized animals were seen in minks [11], ferrets [38–40], skunks [41], and foxes [42]. In bears, the profiles of P4 during pregnancy have been reported in American black bears [6, 43], polar bears [31], and Hokkaido brown bears [7, 8] as well as in Japanese black bears [32]. The findings are for the most part consistent with the results of the present study. Serum P4 concentrations undergo a slow increase of 2–5 ng/ml during the delayed implantation period, although in the present study, somewhat higher levels were observed in some animals in September. At the suspected time of implantation, a dramatic increase in P4 concentrations occurs consistently and is reflected by CL reactivation, which is characterized by a morphological change in which luteal volume increases 2-fold to 4.5-fold, as described by Wimsatt [5]. Serum P4 concentrations are thereafter maintained at relatively high levels for 30 days and decline steadily to basal levels by parturition. In accordance with some other carnivore species, nonpregnant animals sometimes have similar changes in P4 concentrations as reported in Hokkaido brown bears, a condition defined as pseudopregnancy [7, 8]. On the other hand, nonpregnant animals with consistently low P4 concentrations throughout the year have also been reported in American black bears and polar bears [6, 31]. In the present study, we found both of these nonpregnant findings in Japanese black bears and labeled them with etiological explanations. First, nonpregnant bears located in complete isolation had consistently low P4 concentrations, suggesting that ovulation did not occur in these animals. This can also be supported by findings in the literature indicating that no corpora lutea but numerous atretic follicles were recognized in the ovaries of one unmated bear that had shown low P4 levels until January 6, when she died by accident (data not shown). Although a temporal P4 increase was observed in January in some bears, this may be due to luteinization of atretic follicles in response to a rise in pituitary hormone secretion as seen in minks [44]. Second, in pseudopregnant animals, mated bears that did not give birth and the nonmated bear that was able to see males through a fence in the first year had similar P4 trends as those in pregnant animals. This would suggest that the CL would definitely form after ovulation and function in a manner similar to pregnancy. We cannot rule out the possibility that these mated bears may be pregnant, but knowing that either implantation failure or fetal death at the early stage might occur, it is impossible to diagnose these incidents during the early gestation period. Consequently, this led us to speculate that once ovulation occurs, the CL is formed normally and its fate may surely follow the same course until its regression, regardless of whether or not an embryo existed in the uterine lumen.
The CL requires endocrine support for its survival and function, but luteotropin secretion from placenta, as with lactogen in rodents and chorionic gonadotropin in primates and horses, has not been reported in terrestrial carnivores. Thus, the CL in carnivore species is controlled by pituitary hormones such as LH and PRL, but not by the products of conception [37]. An appearance of the CL control by such hormones, however, differs even among carnivore species. In dogs, the CL is dependent neither on LH nor PRL during the early gestation period [45], whereas these hormones are required for maintenance of the CL from the mid-luteal phase [46, 47]. The essential role of LH, however, has been bought into question; Onclin et al. [48] have suggested that canine CL function was more sensitive to LH support from mid-gestation but that LH did not appear to be directly luteotropic. In ferrets, both LH and PRL are required for luteal maintenance throughout gestation [49, 50]. As for mustelids exhibiting delayed implantation, the CL may not require pituitary support during early diapause in minks [14], but thereafter PRL undergoes a proximal stimulus to activate the dormant CL at implantation in minks and western spotted skunks. On the other hand, although administration of LH and GnRH failed to activate the dormant CL in minks, skunks, and badgers, anti-GnRH treatment served to reduce P4 concentrations during the postimplantation period, suggesting that LH is necessary for maintenance of late gestation in minks [51]. Luteinizing hormone and its second messenger, cAMP, stimulated steroidogenesis related to P4 synthesis in luteal cells during both preimplantation and postimplantation periods in vitro in minks [52]. In addition, mRNA of both PRL and LH receptors were expressed in a different manner over the course of CL reactivation and early postimplantation, and in minks, PRL up-regulates its own receptor and maintains the LH receptor [53]. Consequently, although the definite regulation of the CL by these hormones remains unclear, it is assumed that LH and PRL regulate the CL sometimes specifically and sometimes synchronously in minks [52]. No information about CL control in bears has been reported, except for a study in American black bears that suggested a possibility that LH may be a luteotropin [54]. In the present study, a new aspect for the possible role of PRL in the regulation of P4 secretion in both pregnant and pseudopregnant bears was discussed. A significant increase in serum PRL concentrations appeared to coincide with a significant increase in serum P4 concentrations in November and December, at the presumed time of implantation, suggesting that PRL may function as a luteotropin in the Japanese black bear. Pseudopregnant bears, moreover, exhibited a significant positive correlation between PRL and P4 concentrations from September to January, strongly suggesting the role of PRL as a luteotropin. Because there was no finding that LH is a luteotropin in the present study, it is assumed that the CL function in bears is regulated by PRL primarily during gestation and that PRL is also possibly involved in inducing implantation.
The proximal stimulus of implantation in minks and western spotted skunks is an elevation in PRL secretion from the pituitary in accordance with increasing day length after the vernal equinox [13–20]. Although such PRL function can be applicable in bears, there appears to be a definite discrepancy between bears and minks or western spotted skunks. In each species, the CL and embryo diapause are terminated at different times, which in turn, affects when implantation occurs. In bears, implantation occurs during the short-day period between late November and early December, whereas it occurs during the long-day period between March and April in minks [55, 56] and between April and May in the western spotted skunk [57]. In most seasonal breeders, changes in PRL secretion undergo seasonal cycles that are driven by the photoperiod, when exposure to long days causes high concentrations of PRL in blood, whereas short days cause low concentrations. In our study, serum PRL concentrations were high from May to August and thus consistent with this theory, whereas the marked rise between November and the following February went against it. Consequently, we conclude that the female Japanese black bear exhibits two increasing phases in the annual pattern of serum PRL concentrations. We call them the "photoperiod-induced state" and "diapause termination-involved state," respectively. However, what induces hyperprolacticemia in the latter state remains to be determined.
Changes in E2, inhibin, and FSH, the hormones that control follicular development, were also examined in this study. These hormones appeared to change in the presence or absence of the CL and, in particular, changes in inhibin varied throughout gestation in parturient bears. This suggests that some follicles coexist with the CL, which may continue through the series of developmental stages. The origin of inhibin secretion has not been reported in bears to date. Serum E2 concentrations tended to decrease in October and November and to increase from December to January, mainly in parallel with the data in our previous study [32]. However, in the present study, the E2 change appeared to reflect follicular synthesis rather than a change related to pregnancy, because no distinct differences between pregnant and nonpregnant animals were found in E2 trends. Nevertheless, considering the capability of estrogen synthesis in bear placenta [58], the role of estrogen in pregnancy remains to be elucidated. Further investigations with more frequent samplings are necessary in order to determine interactions among these hormones and the roles these hormones play in folliculogenesis and pregnancy in bears.
The manner of ovulation in bears has not been defined, but many scientists tend to speculate that bears are induced ovulators [59]. In the present study, if ovulation had not occurred in all nonmated bears except the nonmated bear that had been exposed to males, it could be speculated that ovulation in bears may require copulation or some other stimulus such as female exposure to a male. Incidents have been reported of spontaneous ovulation even in cats, which are defined as copulation-induced ovulators, implying that the age and interaction between animals could be critical factors in inducing spontaneous ovulation [60, 61]. Bears are thus believed to be induced ovulators but to occasionally exhibit spontaneous ovulation, as seen in other carnivores that are presumed to be induced ovulators [62].
In summary, this is the first evidence describing pituitary-gonadal axis control for female bears based on changes in P4, E2, FSH, LH, PRL, and inhibin concentrations. The results suggest that PRL must be a luteotropic factor that activates or maintains the CL in this species on the basis of a relationship between P4 and PRL changes, although no distinctive association between P4 and LH was observed in the present study. Serum E2, inhibin, and FSH concentrations changed without a correlation to the P4 change over the course of a year, indicating that the changes of these hormones may be associated with follicular development rather than CL activity in this species.
ACKNOWLEDGMENTS
We thank Dr. K. Wakabayashi, the Institute of Endocrinology, Gunma University, Maebashi, Japan, for providing H-23, HAC-AA63-06RBP84, and HAC-GPA2-01GTP80; Dr. A.F. Parlow, National Hormone and Pituitary Program, Harbor-UCLA Medical Center, California, for providing AFP8311890Rb, AFP5214B, AFP5216B, AFP1062091GP, and AFP2451B; the National Hormone and Pituitary Program, Rockville, Maryland, for providing NIDDK-I-5; Dr. S. Lynch, Endocrine Services Limited, Warwickshire, U.K., for providing M91; and Dr. L.E. Reichert, Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, New York, for providing LER-1685-3 A. We are grateful to the staffs of Ani Mataginosato Bear Park and the institute of the Japanese black bear, Akita, Japan, for handling assistance and animal management. We also thank Dr. J.M. Bahr for a critical review of the manuscript.
FOOTNOTES
First decision: 12 December 2000.
1 This work was supported in part by the Sasakawa Scientific Research Grant from The Japan Science Society; and by grant-in-aid 10306020 for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. 
2 Correspondence. FAX: 81 58 293 2955; tsubota{at}cc.gifu-u.ac.jp
Accepted: May 8, 2001.
Received: October 24, 2000.
REFERENCES
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), bear pituitary extract (
), and two female bear sera (
, 
) in RIA using 125I-labeled rat FSH as a tracer. Each value represents the mean of triplicate determinations in both assays. b) Dose-response curves for bovine inhibin as a reference standard (
