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Biology of Reproduction 63, 440-447 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular article

Testicular Recrudescence in the Male Black Bear (Ursus americanus): Changes in Testicular Luteinizing Hormone-, Follicle-Stimulating Hormone-, and Prolactin-Receptor Ribonucleic Acid Abundance and Dependency on Prolactin

L.A. Howell-Skallaa, D. Bunickb, R.A. Nelsonc, and J.M. Bahr1,,a

a Department of Animal Sciences, b Department of Veterinary Biosciences, and c Carle Foundation, Department of Internal Medicine, University of Illinois, Urbana, Illinois 61801

ABSTRACT

Testicular recrudescence in male black bears (Ursus americanus) is initiated in January and completed in May. The goals of this study in the black bear were to determine 1) if testicular abundance of LH-receptor (LHr), FSH-receptor (FSHr), and prolactin-receptor (PRLr) mRNA changes during recrudescence; 2) if these changes in mRNA abundance are associated with changes in serum LH, PRL, and testosterone (T) concentrations; and 3) if the spring increase in serum PRL concentrations is required for testicular recrudescence. Serum was obtained monthly from nine male bears for 2 yr, except in July and August. To suppress endogenous PRL, four bears were treated with Parlodel LAR, 50 mg per 70 kg body weight, monthly from January through May, whereas five bears served as controls. Testicular biopsies were obtained in January, March, and May and analyzed for LHr, FSHr, and PRLr mRNA abundance using reverse transcriptase-competitive polymerase chain reaction. The LHr and PRLr mRNA abundance was low in January, increased in March, and remained high in May, whereas the FSHr mRNA abundance remained constant. Serum concentrations of PRL and T increased in March, coincident with the increase in testicular LHr and PRLr mRNA abundance. Suppression of serum PRL concentrations during testicular recrudescence 1) prevented the increase in testicular LHr and PRLr mRNA abundance observed among control bears in March, 2) lowered serum T concentrations in March and April, and 3) resulted in reduced testis size in May. We conclude that testicular LHr and PRLr mRNA are seasonally regulated, and that PRL has a role in testicular recrudescence in the black bear.

FSH, FSH receptor, gene regulation, LH, prolactin, seasonal reproduction, spermatogenesis

INTRODUCTION

Testicular activity of bears in temperate climates is seasonal. Spermatogenesis and steroidogenesis are maximal before and during the breeding season, which extends from May until July, with peak breeding occurring in June [1–3]. Testes then regress from July until December. During this time, spermatogenesis ceases, and steroidogenesis is greatly reduced [4]. Testicular recrudescence is initiated while the bear is still denning in mid-January [3, 4]. Indeed, histological studies have revealed the presence of spermatogonia and spermatocytes and more organized seminiferous tubules in January compared with October. Serum testosterone (T) concentrations do not increase until late February or March [5–7], indicating that steroidogenesis is reinitiated later in recrudescence. Seasonal changes in gonadal activity of the black bear are hypothesized to be controlled by photoperiod [5, 7]; however, the hormonal signals responsible for initiating testicular recrudescence are not known.

Testicular recrudescence is assumed to be controlled predominantly by seasonal changes in LH and FSH. Spermatogenesis appears to be initiated by FSH in most seasonally breeding species, including the hamster, woodchuck, and ram, whereas LH initiates and maintains Leydig cell steroidogenesis [8, 9]. Even though serum FSH concentrations have not yet, to our knowledge, been reported in the black bear, GnRH-stimulated LH secretion was greater during the spring and summer than in the fall and winter [10]. In certain species, seasonal changes in prolactin (PRL) also influence testicular function. In the golden hamster, the increasing serum PRL concentrations observed during longer days up-regulate testicular LH receptors, thus increasing the sensitivity of the testis to stimulation by LH [11]. Recently, our laboratory showed that serum PRL concentrations in three male black bears were lowest in December, during short days, and increased gradually from January until May, coincident with increasing day length [12].

Seasonal changes in testicular function are hypothesized to be not only the result of changes in circulating gonadotropins and PRL but also the result of changes in testicular gonadotropin- and PRL receptors (i.e., LH receptor [LHr], FSH receptor [FSHr], and PRL receptor [PRLr]) [13]. Indeed, testicular content of LHr, FSHr, and PRLr increased during testicular recrudescence among several species with seasonal changes in reproduction [13–15]. On the other hand, little information exists regarding the seasonal regulation of testicular gonadotropin-receptor and PRLr gene expression. Based on studies in other species, we hypothesized that testicular LHr, FSHr, and PRLr mRNA would increase during testicular recrudescence, and that suppressing the spring increase in serum PRL concentrations would decrease testicular abundance of LHr mRNA. Therefore, the aims of this study were to determine 1) if testicular abundance of LHr, FSHr, and PRLr mRNA changes during recrudescence; 2) if these changes in mRNA abundance are associated with changes in serum LH, PRL, and T concentrations; and 3) if the spring increase in serum PRL concentrations is required for testicular recrudescence. Regarding the third aim, we suppressed the spring increase in serum PRL with a long-acting form of bromocriptine and measured 1) serum PRL, LH, and T concentrations; 2) testicular size; and 3) testicular LHr, FSHr, and PRLr mRNA abundance.

MATERIALS AND METHODS

Animals

Nine sexually mature American black bears (Ursus americanus) maintained at the Carle Foundation Bear Research Facility in Champaign County, Illinois, were used in this study. Bears were housed individually in covered indoor/outdoor (i.e., open-air) enclosures and exposed to natural environmental conditions. Food and water were available ad libitum throughout the year, except during winter denning (typically October–March), when only water was provided. All experimental protocols were approved by the Laboratory Animal Care Committee of the University of Illinois at Urbana-Champaign and Carle Hospital's Institutional Animal Care and Use Committee. Bears were anesthetized with 5.0 mg/kg body weight of Telazol (Fort Dodge Laboratories, Fort Dodge, IA) administered with a dart gun. Telazol contains tiletamine hydrochloride and zolazepam hydrochloride.

Experimental Design and Sample Collection

This study was conducted during a 2.5-yr period. Serum for hormone analysis (specific aims 2 and 3) was obtained monthly from nine male bears from November 1994 until March 1997. Blood (180 ml) was removed from the femoral vein and centrifuged at 1200 x g for 20 min at 4°C. The resulting serum was stored at -20°C until assayed for LH, PRL, and T. Serum was not obtained during July or August to minimize unnecessary stress to the bears during high ambient temperatures. Specific aims 1 and 3 were investigated from January 1996 through May 1996. The goal was to determine the effect of preventing the long day-induced increase in serum PRL concentrations on various parameters of testicular recrudescence. Serum concentrations of PRL were pharmacologically suppressed with Parlodel LAR, a long-acting form of bromocriptine (batch 001MFD1194, kindly provided by Dr. Iona Lancranjan [Novartis, Basel, Switzerland]). Bromocriptine is a dopamine-receptor agonist that is routinely used to suppress the secretion of PRL. A trial study conducted on two male bears in May 1995 determined that an i.m. injection of Parlodel LAR, 50 mg per 70 kg body weight, suppressed serum PRL concentrations to basal levels 2 and 4 wk postinjection (data not shown). Based on these results, we then treated bears with Parlodel LAR, 50 mg per 70 kg body weight, in January, February, March, and April. Bears were first weighed, and then the appropriate dose (~150 mg) was administered. Four bears were treated with Parlodel LAR, and five bears served as controls (i.e., no injection). Blood and testicular size measurements (i.e., length and width) were obtained monthly. To determine testis size, the scrotal skin was drawn tightly over the testis, and the length and width were then measured using calipers. Data are presented in units of square-centimeters (length x width) and are the average of both the left and right testes. Testicular biopsies were obtained from all bears in January, March, and May. Each testis underwent biopsy, and the samples (size, <50 µg) were placed in a sterile, 1.5-ml, screw-top tube and then immediately frozen in liquid nitrogen and stored on dry ice until transferred to a -80°C freezer. Each month, blood and testicular biopsies were obtained before administration of Parlodel LAR.

RNA Isolation and cDNA Preparation

To obtain sufficient amounts of RNA, individual biopsy samples from two or three bears had to be pooled. This resulted in four January control groups, two March control and two March Parlodel LAR groups, and two May control and two May Parlodel LAR groups. Total RNA was isolated from testicular biopsy samples using TRIZOL Reagent (Life Technologies, Gaithersburg, MD), quantified spectrophotometrically (A260/A280), aliquoted into smaller volumes, and stored at -80°C. Isolated RNA was reverse transcribed into cDNA using the RETROscript First-Strand Synthesis Kit (Ambion Inc., Austin, TX) according to manufacturer's instructions. Briefly, 2 µg of total RNA were combined with 4 µl deoxyribonucleoside triphosphates (dNTPs) (2.5 mM of each dATP, dGTP, dCTP, and dTTP), 2 µl of random decamers (50 µM), and 12 µl of nuclease-free water. Contents were centrifuged briefly, heated for 3 min at 75°C, and then placed on ice. Subsequently, 2 µl of 10x RT buffer (500 mM Tris-HCl [pH 8.3], 750 mM KCl, 30 mM MgCl2, and 50 mM dithiothreitol), 1 µl of placental RNase inhibitor (1 U), and 1 µl of Moloney-Murine Leukemia Virus reverse transcriptase (100 U) were added to the reaction and incubated at 42°C for 1 h. To inactivate the reverse transcriptase, the reaction was incubated at 92°C for 10 min and then placed on ice.

Reverse Transcriptase-Competitive Polymerase Chain Reaction

Reverse transcriptase-competitive polymerase chain reaction (RT-cPCR) was used to semiquantitatively determine changes in the relative abundance of LHr, FSHr, and PRLr mRNA in both control and Parlodel LAR-treated bears. Internal DNA standards (i.e., fragments of neutral DNA with the same primer-binding sequences as the gene target) of known concentration were coamplified with target cDNA. DNA standards that could be amplified with LHr, FSHr, or PRLr primers were made with the PCR MIMIC Construction Kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions. For each receptor RT-cPCR assay, two sets of primers were designed: 1) a set of gene-specific primers, and 2) a set of composite primers for construction of cDNA standards. The gene-specific primers were based on the ursine sequence obtained previously in our laboratory (Genbank Accession numbers: LHr, AF169790; FSHr, AF169791; PRLr, AF169792) [16]. Primer sequences and expected product sizes are provided in Table 1. Primers for the housekeeping gene cyclophilin were obtained from Ambion (mouse cyclophilin primers) and yielded a 116-base pair (bp) product.


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  		TABLE 1. Details of RT-cPCR primers, targets, and standards

In each RT-cPCR assay, 2 µl of experimental cDNA was titrated against 10-fold serial dilutions of the DNA standard (10-1, 10-2, 10-3, 10-4, 10-5, and 10-6 attomol/µl). Each reaction contained 2 µl of cDNA sample, 2 µl of DNA standard, and 46 µl PCR master mix containing 5 µl 10x buffer (3.5 mM [LHr], 3.0 mM [FSHr], or 1.5 mM [PRLr] MgCl2), 31 µl of H2O, 8 µl of dNTPs (200 µm of each dNTP), 0.5 µl of forward primer, 0.5 µl of reverse primer, and one TaqBead Hot Start Polymerase (1.25 U; Promega, Madison, WI), for a total volume of 50 µl. The PCR program consisted of an initial 5-min denaturation at 94°C, followed by 30 cycles of denaturing (1 min at 94°C), annealing (2 min at 69°C for LHr, at 50°C for FSHr, and at 53°C for PRLr), and extending (3 min at 72°C). After the final cycle, the reaction was extended for 7 min and then held at 4°C. Cyclophilin cDNA was amplified in all samples with standard Taq buffer using the following 30-cycle PCR program as recommended by Ambion: 94°C for 3 min; five cycles of 94°C for 20 sec (denaturation), 45°C for 30 sec (annealing), and 72°C for 40 sec (extension); 25 cycles of 94°C for 20 sec (denaturation), 55°C for 30 sec (annealing), and 72°C for 40 sec (extension); and a final extension at 72°C for 5 min. The number of PCR cycles was optimized for all primer sets to ensure that cDNA products were amplified during the exponential phase of the PCR reaction. Negative controls included both sterile water and cDNA RT product, from which the reverse transcriptase had been omitted. The PCR products (5 µl) were electrophoresed on a 2% agarose gel and stained with ethidium bromide. The gel image was captured with a CCD camera imaging system (FOTO/Analyst Image Analysis System; Fotodyne Inc., Hartland, WI), and band densities analyzed densitometrically with Collage Image Analysis Software (Fotodyne). For each assay, a test titration was set up to determine three or four standard concentrations that yielded band intensities in the range of the gene target. The ratio of band intensities for the gene target to the DNA standard was obtained and then compared against other samples to determine relative changes in gene expression among multiple samples. Cyclophilin was amplified in each sample to ensure relatively equal starting concentrations of RNA.

Hormone Assays

The heterologous RIA used to determine LH concentrations in bear serum was previously established and validated in our laboratory [10]. A monoclonal anti-bovine LH antibody (lot 2, 518B7) that detects LH from many different mammalian species [17] was kindly provided by Dr. Jan Roser at the University of California-Davis. Purified bovine LH (USDA-bLH-B-6) was kindly provided by Dr. John Proudman (U.S. Department of Agriculture Animal Hormone Program). Intraassay and interassay coefficients of variation were 3.9% (n = 6) and 6.3% (n = 7), respectively. Sensitivity of the LH assay was 80 pg. Bear serum PRL concentrations were determined by a heterologous RIA previously established and validated in our laboratory [12]. Purified porcine prolactin (USDA-pPRL-B-1) used for iodination, and standards and primary antiserum (goat anti-porcine PRL) were kindly provided by Dr. D.J. Bolt (U.S. Department of Agriculture Animal Hormone Program). Intraassay and interassay coefficients of variation were 7.1% (n = 6), and 9.2% (n = 7), respectively. Sensitivity of the PRL assay was 30 pg. Serum T concentrations were determined in double-extracted bear serum with an RIA previously validated in our laboratory [5]. The cross-reactivity of the antiserum was reported by Bahr et al. [18]. Sensitivity of the T assay was 8 pg. Recovery of labeled T (1000 cpm) added to serum before extraction was at least 70%. Intraassay and interassay coefficients of variation were 3.5% (n = 4) and 10.2% (n = 16), respectively. Hormone concentrations were determined using the RIAEIA Parallelism Program with Hot Recovery, written by Dr. Ming-Che J. Wu (Taiwan Livestock Research Institute, Hsinhua, Tainan, Taiwan).

Statistical Analysis

Statistical analyses were performed using SAS Software (SAS Institute Inc., Cary, NC). Differences in serum concentrations of LH, PRL, and T were analyzed with a two-way ANOVA with repeated measures for one factor (month) with SAS PROC MIXED. Values for serum LH, PRL, and T concentrations were log transformed to minimize heterogeneity of variance indicated by a preliminary analysis conducted using PROC GLM. Classes were Parlodel LAR treatment, bear, and month. Data were first analyzed for year effect; when none was found, data from multiple years were combined. To assess the effects of Parlodel LAR treatment on hormone concentrations, the linear model incorporated a nested effect, with bear being nested within treatment. Data were analyzed for treatment, month, and treatment x month interactions. To maximize statistical power, bears treated with Parlodel LAR were compared with all other control bears (2.5-yr period). Terms were included in the model to account for control bears not being in the same time period. When a significant interaction was found, means were then analyzed with the test of least significant differences. Data are presented as mean ± SEM.

RESULTS

Serum LH, PRL, and T Concentrations

Serum LH concentrations were very low (0.08 to 0.2 ng/ml) throughout the year, and they did not differ between months or between control and Parlodel LAR-treated bears (data not shown). Serum LH concentrations in most of the bears sampled were either at or below the sensitivity threshold of the assay (0.08 ng). Serum PRL concentrations (mean ± SEM) in control bears were low from September until February, with concentrations being lowest in October and November (Fig. 1A). Serum PRL concentrations increased significantly in March (P < 0.05), continued increasing in April, and were highest in May and June. Serum PRL levels were significantly lower (P < 0.05) in Parlodel LAR-treated bears than in control bears in March, April, and May; therefore, Parlodel LAR effectively maintained serum PRL concentrations at basal concentrations throughout the period of testicular recrudescence. Serum T concentrations in control bears were lowest in September, low from October until February, and increased significantly (P < 0.05) in March (Fig. 1B). Serum T concentrations were low again in April and May and highest in June. Serum T concentrations were not different between Parlodel LAR-treated and control bears in February or May, but they were significantly lower in Parlodel LAR-treated bears in March and April (P < 0.05).



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  		FIG. 1. Seasonal changes in serum PRL and T concentrations (mean ± SEM) in untreated (control; open bars) and Parlodel LAR-treated (solid bars) bears. Bears were samples once each month for two consecutive years, except in July and August because of high ambient temperatures. During the first year, all nine bears were controls. During the spring of the second year, four bears were treated with Parlodel LAR, whereas five bears served as controls. June serum samples were obtained only during the first year. No effect of year on hormone concentrations was observed; therefore, data from untreated bears from multiple years were combined. Different letters indicate significant differences (P < 0.05) among control bears. An asterisk indicates a significant difference (P < 0.05) between control and Parlodel LAR-treated bears. A) Serum PRL concentrations. B) Serum T concentrations

Testis Size

In control bears, testes increased in size steadily from January until April, and testes remained large in May (Fig. 2). Testes of Parlodel LAR-treated bears increased in size from January until March but remained the same in April and May. Testes of Parlodel LAR-treated bears were significantly smaller than those of control bears in May (P < 0.05).



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  		FIG. 2. Changes in testis size (mean ± SEM) during testicular recrudescence in control (n = 5; open bars) and Parlodel LAR-treated (n = 4; solid bars) bears. Different letters indicate significant differences (P < 0.05) among control (lower case) or Parlodel LAR-treated (upper case) bears. An asterisk indicates a significant difference (P < 0.05) between control and Parlodel LAR-treated bears

Testicular LHr, FSHr, and PRLr mRNA Abundance

The abundance of LHr mRNA was low in January, during the early stage of recrudescence; increased threefold in March, during the middle stage of recrudescence; and remained elevated during May, the mating season (Fig. 3, A and B). In March, the abundance of LHr mRNA was twofold less in bears with suppressed serum PRL concentrations (i.e., Parlodel LAR-treated bears) than in control bears. In May, no consensus effect of Parlodel LAR on LHr mRNA abundance was observed. Testicular abundance of FSHr mRNA remained constant throughout recrudescence (Fig. 4, A and B). No effect of low serum PRL concentrations (i.e., Parlodel LAR-treated bears) on testicular abundance of FSHr mRNA was observed. Testicular abundance of PRLr mRNA changed during recrudescence: PRLr mRNA levels were low in January, during the early stage of recrudescence; increased twofold in March, during the middle stage of recrudescence; and remained elevated in May, during the mating season (Fig. 5, A and B). In March, the abundance of testicular PRLr mRNA was 1.4-fold less in bears with suppressed serum PRL concentrations (i.e., Parlodel LAR-treated bears) than in control bears. In May, the abundance of PRLr mRNA in Parlodel LAR-treated bears was similar to that in controls. Cyclophilin was amplified in each sample to confirm that equal amounts of RNA were present in each sample (Fig. 6).



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  		FIG. 3. Changes in testicular abundance of LHr mRNA during testicular recrudescence (January, March, and May) in control (open bars) and Parlodel LAR-treated (solid bars) bears as detected by semi-quantitative RT-cPCR. A) Representative ethidium bromide-stained agarose gel showing coamplification of LHr standard (191 bp) and ursine LHr target (364 bp). Each inset contains four PCR reactions run in parallel with a dilution series of standards and a constant amount of sample cDNA. B) Mean LHr abundance expressed as the ratio of LHr target abundance (At) to LHr standard abundance (As). Each bar represents the relative LHr mRNA abundance in a pool of testicular RNA obtained from two or three bears



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  		FIG. 4. Changes in testicular abundance of FSHr mRNA during testicular recrudescence (January, March, and May) in control (open bars) and Parlodel LAR-treated (solid bars) bears as detected by semiquantitative RT-cPCR. A) Representative ethidium bromide-stained agarose gel showing coamplification of FSHr cDNA standard (440 bp) and ursine FSHr target (348 bp). Each inset contains three PCR reactions run in parallel with a dilution series of standards and a constant amount of sample cDNA. B) Mean FSHr abundance expressed as the ratio of FSHr target abundance (At) to FSHr standard abundance (As). Each bar represents the relative FSHr mRNA abundance in a pool of testicular RNA obtained from two or three bears



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  		FIG. 5. Changes in testicular abundance of PRLr mRNA during testicular recrudescence (January, March, and May) in control (open bars) and Parlodel LAR-treated (solid bars) bears as detected by RT-cPCR. A) Representative ethidium bromide-stained agarose gel showing coamplification of PRLr cDNA standard (340 bp) and ursine PRLr target (264 bp). Each inset contains three PCR reactions run in parallel with a dilution series of standards and a constant amount of sample cDNA. B) Mean PRLr abundance expressed as the ratio of PRLr target abundance (At) to PRLr standard abundance (As). Each bar represents the relative PRLr mRNA abundance in a pool of testicular RNA obtained from two or three bears



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  		FIG. 6. RT-cPCR detection of cyclophilin mRNA in each of the cDNA samples previously analyzed for LHr, FSHr, and PRLr mRNA. Amplified cyclophilin was used as a control to demonstrate RNA integrity and to approximate equal starting concentrations. JC, January control; MrC, March control; MrP, March Parlodel; MyC, May control; MyP, May Parlodel

DISCUSSION

We examined the expression of testicular LHr, FSHr, and PRLr mRNA during the early, mid, and late stages of recrudescence (January, March, and May, respectively) in the black bear using RT-PCR. The abundance of testicular LHr mRNA was low in January, increased greatly in March, and remained high during May. These findings concur with previous studies that demonstrated a greater concentration of testicular membrane-bound LHr during the mating season than during the nonmating season in hamsters [19] and in sheep [20]. The small amount of testicular samples available precluded determining changes in receptor protein levels or in receptor binding; however, studies in other species have demonstrated a positive correlation between LHr mRNA expression and protein synthesis [21]. The seasonal pattern of LHr mRNA expression in the bear testis correlated positively with other observed functional parameters, thus supporting the relevance of increased testicular LHr mRNA during recrudescence. In this study, serum T concentrations first increased in March, coincident with increased testicular LHr mRNA. Likewise, immunocytochemical staining for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and P450c17 in the bear testis increased dramatically between January and June [4]. We were not able to detect seasonal changes in basal serum LH concentrations because of very low serum LH concentrations and our inability to take blood samples more frequently than once a month. Even so, our laboratory has demonstrated that GnRH-stimulated LH secretion was more robust among bears in March than in October, December, and June [10].

In contrast to LHr mRNA expression, FSHr mRNA was equally abundant in January, March, and May. It would be interesting to determine if the testicular abundance of FSHr mRNA changes before the reinitiation of recrudescence in the bear. Supporting a possible role for FSH in reinitiating spermatogenesis, immunocytochemical studies have demonstrated that P450 aromatase, which is regulated by FSH in rat prepubertal Sertoli cells, first appears in the Sertoli cells of the bear during January [4].

Testicular abundance of PRLr mRNA increased in March from that in January, as expected based on PRL-binding studies in the hamster [14] and coincident with a significant increase in serum PRL concentrations. This finding is consistent with other studies that demonstrated a positive relationship between PRLr mRNA, receptor binding, and serum PRL concentrations, and it suggests that PRL up-regulates its own receptor [22, 23].

The role of PRL in testicular function varies with the species. In photoinhibited hamsters, PRL stimulated testicular growth and T secretion via increased and/or maintained Leydig cell LHr [19, 24]. With this in mind, we hypothesized that the spring increase in serum PRL concentrations previously observed in the bear was necessary for testicular recrudescence. Treatment of four male bears with monthly injections of Parlodel LAR from January until May suppressed the serum PRL concentrations throughout the period of testicular recrudescence, from February through May, compared with control bears not treated with Parlodel LAR. Low concentrations of serum PRL during the period of testicular recrudescence prevented the increase in testicular LHr mRNA observed in March and maintained the abundance of LHr mRNA either at or slightly above the January levels. This observation is consistent with numerous studies that demonstrated the ability of PRL to increase LHr mRNA and to up-regulate LH receptors in the ovary of the rat and mink [22, 25] and the testis of the golden hamster [14]. Suppressed serum PRL concentrations during testicular recrudescence in the bear had no effect on FSHr mRNA levels in the testis. This finding agrees with previous studies that showed FSHr is not regulated by PRL [26]. Finally, suppressing the spring increase in serum PRL concentrations reduced the increase in testicular PRLr mRNA abundance observed in control bears during March. This is in accordance with the known ability of PRL to autoregulate its own receptor [27]. Abundance of PRLr mRNA increased modestly from January to March in Parlodel LAR-treated bears, supporting a previous study that showed testicular PRLr is regulated by LH and FSH in addition to PRL [23].

Bears treated with Parlodel LAR exhibited not only suppressed abundance of testicular LHr and PRLr mRNA but also suppressed serum T concentrations in March and April. These results agree with a study that showed treatment of boars with bromocriptine decreased both testicular LHr and serum T concentrations [28]. Indeed, many studies have found a positive relationship between PRL and T production [29, 30]. The PRL stimulates steroidogenesis not only by up-regulating LHr but also by increasing stores of esterfied cholesterol [31] and increasing 3ß-HSD and 17ß-HSD activity in the mouse [32, 33].

Suppression of serum PRL concentrations during testicular recrudescence also resulted in decreased testis size in May, thus suggesting a role for PRL during testicular growth in the bear. Studies have shown PRL to be positively correlated with testis growth in many species, including rams and photoinhibited golden hamsters [11, 34]. Reduced testis size in PRL-deficient bears could result indirectly from low serum T concentrations in March and April, because T is known to maintain normal spermatogenesis in the rat [35]. An alternate explanation is that PRL directly affects spermatogenesis. The PRLr mRNA has been localized in Sertoli cells, spermatogonia, and spermatocytes, with PRL binding being identified in spermatogonia, spermatocytes, elongated spermatids, and spermatozoa [36]. Unfortunately, we were unable to examine the effect of suppressed PRL on spermatogenesis in this study, because we were limited in the amount of available testicular tissue.

Finally, an unexpected finding in this study was the decrease in serum T concentrations among control bears in April and May, during the middle to end stages of testicular recrudescence. Serum T concentrations typically begin to increase in February or March, and they continue increasing until May or June [4–7, 10]. A possible explanation lies in the unique denning physiology of the black bear. Bears were denning and, therefore, fasting from November until March. After the bears were sampled in March, denning was ended with the resumption of feeding. This sudden initiation of eating could, at least theoretically, trigger drastic changes in the enterohepatic recirculation of steroids. Steroids are metabolized to inactive forms in the liver via hydroxylation and conjugation and then are released into the bile. Conjugated steroids are not permeable to intestinal epithelial cells, and they are excreted in the feces. However, because of the presence of bacteria in the gut, a considerable amount of conjugated steroid is deconjugated and then returned to the liver via enterohepatic circulation [37]. The functional significance of enterohepatic recirculation is not well understood, but its manipulation can have significant effects on circulating steroid concentrations. In women, high-fiber, vegetarian diets increase the passage of food through the digestive tract and result in decreased reabsorption of estrogens and, therefore, decreased concentrations of serum estrogen [38]. A similar scenario could be occurring in bears. The sudden movement of food through the bear's digestive tract after termination of denning would allow less time for steroid metabolites to be deconjugated and reabsorbed, the result of which might be increased excretion of conjugated steroids and apparent decreased peripheral steroid concentrations. Future studies that both determine the amount of excreted conjugated T and trace the metabolic pathway of injected [14C]-T in the denning and nondenning bear could shed light on this interesting phenomenon.

Certain experimental limitations exist when studying animals seldom used in research. In this investigation of the black bear, we were limited to very small testicular tissue samples and, therefore, were able to measure only changes in receptor mRNA and not in receptor binding. Another limitation was the use of Parlodel LAR to suppress PRL secretion. Bromocriptine is a dopamine agonist that blocks not only PRL secretion but acts directly on the hypothalamus to suppress GnRH and, subsequently, to affect LH and FSH secretion [39]. On the other hand, bromocriptine did not appear to affect LH secretion in boars or rams [28, 40] or FSH secretion in hamsters or rams [19, 30]. We were not able to control for the potential nonspecificity of bromocriptine by including a bromocriptine plus PRL control group because of the large amount of purified PRL required. Studies have been performed in the hamster, however, in which bromocriptine treatment reduced the number of testicular LHr and the simultaneous administration of ovine PRL reversed the effect [14]. Taken together, most studies conclude that bromocriptine can be used as a specific inhibitor of PRL secretion, but caution must be used in drawing definitive conclusions.

To our knowledge, this is the first investigation concerning the role of PRL and testicular LHr, FSHr, and PRLr in testicular recrudescence of the black bear. We hypothesized that increasing serum concentrations of PRL in March increase the testicular content of LHr and PRLr. An increase in LHr would enhance testicular responsiveness to LH, thus stimulating T production. Increased serum T concentrations observed during the premating season support both spermatogenesis and social behaviors necessary for successful mating. Data presented in this study support this hypothesis, because preventing the spring increase in serum PRL concentrations with Parlodel LAR during testicular recrudescence decreased the abundance of testicular LHr and PRLr mRNA, serum T concentrations, and testis size. Future studies should investigate the role of FSH in stimulating the reinitiation of spermatogenesis during the early stages of testicular recrudescence and explore the possibility of using Parlodel LAR as a means of contraception for male bears in zoos.

ACKNOWLEDGMENTS

The authors thank Alicia Howell and Corey Pelz for their statistical expertise and Hong Wang for her technical assistance.

FOOTNOTES

First decision: 30 November 1999.

1 Correspondence: Janice Bahr, Department of Animal Sciences, University of Illinois, 326 Animal Sciences Lab, 1207 W. Gregory Dr., Urbana, IL 61801. FAX: 217 333 8286; j-bahr{at}uiuc.edu Back

Accepted: March 14, 2000.

Received: October 21, 1999.

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