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Biology of Reproduction 62, 1571-1578 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Prolactin-Induced Expression of Intercellular Adhesion Molecule-1 and the Accumulation of Monocytes/Macrophages During Regression of the Rat Corpus Luteum1

Kristen K. Olsona, and David H. Townson2,a

a Department of Animal and Nutritional Sciences, University of New Hampshire, Durham, New Hampshire 03824-3590


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intercellular adhesion molecule-1 (ICAM-1) is thought to facilitate the recruitment and migration of monocytes/macrophages to sites of inflammation. Here we investigated whether the luteolytic effect of prolactin in the hypophysectomized rat is associated with the expression of ICAM-1. In addition, we examined the effect of exogenous testosterone (or its potential conversion to estradiol endogenously) on the corpus luteum to address recent speculation that ovarian steroids might augment luteal regression. Immature, 30-day-old rats were ovulated with eCG and hCG and then hypophysectomized; this resulted in a single cohort of persistent corpora lutea. The rats were assigned randomly into four treatment groups: vehicle treatment without or with testosterone (VEH-T4, VEH+T4) and prolactin treatment without or with testosterone (PRL-T4, PRL+T4). Corpora lutea of control rats exhibited minimal ICAM-1 staining and contained relatively few monocytes/macrophages. In contrast, corpora lutea of prolactin-treated rats exhibited prominent ICAM-1 staining and contained numerous monocytes/macrophages. Testosterone did not overtly affect ICAM-1 staining, numbers of monocytes/macrophages, or concentrations of plasma progestins (progesterone and 20{alpha}-dihydroprogesterone) in either VEH or prolactin treatment groups; notwithstanding, luteal weights increased significantly in response to testosterone in VEH+T4 rats compared to VEH-T4 rats and prolactin-treated rats. We conclude that ICAM-1 expression and monocyte/macrophage accumulation are associated with prolactin-induced luteal regression in the rat and that these aspects are not influenced by testosterone.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The immunological characteristics of luteal regression, particularly those pertaining to immune cell trafficking within the corpus luteum, are not well understood. Immune cells accumulate within the corpus luteum of the rat [1–5], guinea pig [6], rabbit [7], pig [8, 9], sheep [10], cow [11, 12], and human [13] and are thought to participate in cell-mediated killing by phagocytosis [14], as well as the release of cytotoxic factors [15, 16] and cytokines [7, 17]. Studies in the rat [2–4], pig [18], sheep [19, 20], cow [12, 20], and human [21] indicate that expression of the proinflammatory chemokine, monocyte chemoattractant protein-1 (MCP-1), is involved in the recruitment of monocytes/macrophages into corpora lutea during luteal regression. However, in conjunction with chemokine expression, the accumulation of monocytes/macrophages in tissues during inflammatory responses is also highly dependent on cellular adhesion [22–24]. Specifically, cellular adhesion facilitates monocyte attachment to the vascular endothelium and enables monocyte/macrophage migration in response to chemokines within the tissue. Three major families of adhesion molecules have been identified and are associated with immune cell attachment and migration. They include the selectins, integrins, and molecules of the immunoglobulin supergene family (e.g., intercellular adhesion molecule-1 [23]).

Intercellular adhesion molecule-1 (ICAM-1) is considered to have a prominent role in monocyte/macrophage adhesion events. ICAM-1 is a 90- to 114-kDa cell surface protein that promotes monocyte/macrophage adhesion through a variety of ligands [25–27] and exists in both membrane-bound and circulating forms [25, 28, 29]. Expression of ICAM-1 is induced by proinflammatory cytokines, including interleukin-1, tumor necrosis factor-{alpha}, and interferon-{gamma} [30–32]. The cell types that commonly express ICAM-1 include endothelial cells, fibroblasts, monocytes, macrophages, and as seen more recently, granulosa cells of the ovary [30, 33, 34].

Prolactin-induced luteal regression in the hypophysectomized rat is well documented [35–37], including immunological aspects such as MCP-1 expression, apoptosis, and monocyte/macrophage recruitment [3, 4]. The appeal of this model is that it permits study of the factors that potentially influence luteal regression but limits or eliminates the influence of pituitary, adrenal, and ovarian hormones. Recently, however, it has been suggested that steroids possibly facilitate immunological events within the corpus luteum and thus augment luteal regression [4]. Testosterone and estradiol in particular are known to stimulate the growth and development of the rat corpus luteum [38–41], but might also enhance its regression by influencing chemokine expression and activating immune cells [42, 43]. Estradiol production by the corpus luteum is reduced following hypophysectomy in the rat [35]; however, aromatase activity is retained [44]. Under these circumstances, treatment with exogenous testosterone partially restores estradiol production in vitro [44] and in vivo [38].

In the current study, the primary objective was to determine whether prolactin-induced regression of the rat corpus luteum is associated with increased expression of ICAM-1. In addition, we took advantage of the capacity of rat luteal tissue to aromatize testosterone to estradiol [44] to explore the potential local effects of steroid on inflammatory events associated with luteal regression.


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

The University of New Hampshire Animal Care and Use Committee approved the experiments reported (IACUC #970403). Immature female Sprague-Dawley rats (30 days of age) were ovulated by s.c. injection of gonadotropins (5 IU eCG followed 56 h later by 5 IU hCG). The rats were hypophysectomized at 33 days of age by the vendor (Charles River Laboratories, Portage, MI), and they arrived at our laboratory 5 days later. Previous studies have shown that methods similar to these result in the formation of a physiological number of corpora lutea (16–22 corpora lutea per rat), which persist for months [45] and continue to produce 20{alpha}-dihydroprogesterone (20{alpha}-DHP) and small amounts of progesterone (P4) for at least 40 days post-hypophysectomy [35]. Administration of exogenous prolactin to these rats, however, provokes both a decline in luteal steroidogenesis and the structural demise of the corpus luteum [3, 46].

At 39 days of age, the rats were assigned randomly into four groups: vehicle treatment without or with testosterone (VEH-T4, VEH+T4) and prolactin treatment without or with testosterone (PRL-T4, PRL+T4). Testosterone was administered to examine, simultaneously, the potential effects of testosterone and of estradiol (formed via luteal aromatase) on inflammatory events associated with prolactin-induced luteal regression. The administration of testosterone was based upon the findings of Kim and Greenwald [44], who showed that the corpus luteum of the hypophysectomized rat retains aromatase activity for up to 30 days post-hypophysectomy and is capable of converting exogenous testosterone to estradiol. In the current study, testosterone was administered to rats in vivo through the use of Silastic implants (Dow Corning, Midland, MI; 1.98-mm i.d., 3.18 mm-o.d., 1-cm filled length, 2-cm total length) containing crystalline testosterone. The number of implants available for the study, however, was limited (8 implants were generously provided by P. Landis Keyes, University of Michigan). Therefore, a total of 10–14 rats were assigned randomly to the four treatment groups in each experiment, and the experiment was repeated three times. For the implant/sham operation procedure, rats were anesthetized via inhalation of Metofane (methoxyflurane; Schering-Plough Animal Health; Union, NJ; NDC #0061–5038–01). Wound closure was accomplished using 9-mm sterilized suture clips (Robaz, Rockville, MD). At 43 days of age, sham-operated and testosterone-implanted rats were further subdivided into groups receiving twice-daily injections, i.m., of 0.9% saline or ovine prolactin (Sigma, St. Louis, MO; #L-6520, 312 µg/rat per injection in 250 µl of 0.15 M NaCl, 0.03 M NaHCO3, 0.1% BSA solution). All four groups (VEH+T4, n = 5 rats; VEH-T4, n = 12 rats; PRL+T4, n = 9 rats; PRL-T4, n = 11 rats) were treated daily for 3 days before decapitation at 46 days of age. Trunk blood was obtained from each rat for assay of plasma progestins (P4 and 20{alpha}-DHP). The ovaries from each rat were removed and prepared for immunohistochemical staining of ICAM-1 and monocytes/macrophages or for dissection of individual corpora lutea (see below, Luteal Weight). The sella turcica was inspected for pituitary fragments using a dissecting scope; one rat was excluded from the experiment (PRL-T4 group) for an incomplete hypophysectomy.

Progestin Assays

Progestin assays for 20{alpha}-DHP and P4 were conducted in the Chemistry Core of the Michigan Diabetes Research and Training Center. Plasma concentrations were measured by RIA in petroleum extracts of plasma according to methods described previously [47, 48].

Immunohistochemistry (ICAM-1, Monocytes/Macrophages [ED1], and Activated Macrophages [ED2])

Ovaries were frozen in OCT compound (Miles Laboratories, Elkhart, IN) and prepared as frozen sections (6 µm). Methods used to detect and evaluate ICAM-1 expression, monocytes/macrophages, and activated macrophages were similar to those described previously [2–4]. Sections of ovaries from all treatment groups were stained simultaneously throughout all immunostaining procedures (10–12 ovarian sections per rat per antibody). The following monoclonal antibodies were used: mouse anti-rat monocyte/macrophage antibody (ED1 clone; 1:200; Chemicon, Temecula, CA), mouse anti-rat activated macrophage antibody (ED2 clone; 1:200; Accurate Chemical and Scientific Corp., Westbury, NY), and mouse anti-rat ICAM-1 (IA29 clone; 1:1000; R&D Systems, Minneapolis, MN). For simplicity, monocytes/macrophages will be herein referred to as ED1-positive cells, and activated macrophages as ED2-positive cells. Frozen sections were air dried, fixed in 95% ethanol at 4°C for 10 min, and transferred to 0.3% H2O2 in methanol at 4°C for 15 min to quench endogenous peroxidase activity. Sections were rinsed 3 times (5 min each) in PBS containing 1% BSA (PBS-1% BSA) before blocking with 10% normal horse serum for 15 min at 37°C. Sections were rinsed again with PBS-1% BSA solution and incubated with antibody for 1 h (ICAM-1) or 30 min (ED1 and ED2) at 37°C. Sections were then rinsed 3 times (5 min each) with PBS-0.1% BSA before incubation at 37°C for 30 min with biotinylated horse anti-mouse immunoglobulin (1:200 dilution). Amplification of the antigen-antibody complex was achieved using avidin-biotin-peroxidase (ABC kit; Vector/Novocastra, Burlingame, CA) for 30 min at 37°C. The color reaction was precipitated for all antibodies using 3-amino-9-ethylcarbazole (AEC kit; Vector/Novocastra) for 10 min at room temperature. Tissue sections were then rinsed, counterstained with hematoxylin, rinsed, and mounted using aqueous mounting medium (Dako, Carpinteria, CA). Nonspecific staining was assessed by omission of the primary antibody and was undetectable in all instances.

Immunohistochemical expression of ICAM-1 was assessed qualitatively in blind fashion by two independent observers. Slides of the ovarian sections from all treatment groups were coded prior to evaluation. Observations of immunodetectable ICAM-1 staining ranged from little to no staining in the corpora lutea to prominent peripheral and parenchymal staining. There was 95% agreement between the two observers in the characterization of ICAM-1 staining.

Numbers of ED1- and ED2-positive cells in the corpora lutea were estimated by counting positively stained cells within 2–8 nonoverlapping fields in each of 2–7 corpora lutea per rat. A red precipitate surrounding a darkly stained nucleus signified a positively stained cell. Positively stained cells were counted using a microscope equipped with a x40 objective. Cell numbers are expressed as the average number of positively stained cells per high-power field. The mean number of cells per high-power field obtained from the corpora lutea of one rat represents n = 1.

Luteal Weight

Freshly excised ovaries from each rat were rinsed in PBS (pH 7.0), and the corpora lutea from one ovary were dissected with magnification provided by a dissecting microscope. The corpora lutea were kept hydrated on Whatman (Clifton, NJ) no. 4 filter paper saturated with PBS during collection. For each rat, the average wet weight of the corpora lutea was calculated by dividing the total weight of corpora lutea by the total number of corpora lutea per ovary. The average wet weight of the corpora lutea from one rat represents n = 1.

Statistical Analyses

Cell counts, plasma progestins, and luteal weight data were analyzed using a two-way ANOVA with the following factors: without/with prolactin and without/with testosterone. Residual plots revealed heterogeneity of variance in the initial statistical analysis of the plasma progestin data. Therefore, these data were log transformed to minimize variance and then reanalyzed. Tukey's multiple comparison procedures were used to compare means among treatment groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical Expression of ICAM-1 and ED1- and ED2-Positive Cells

Prominent peripheral and parenchymal staining of ICAM-1 was evident in the corpora lutea of 15 of 19 (80%) prolactin-treated rats (Fig. 1, A–D). The corpora lutea of the remaining 4 rats also contained peripheral/parenchymal ICAM-1 staining, but the intensity of the immunostaining was somewhat diminished. A "wreath-like" pattern of staining surrounding the corpora lutea was the most distinct indication of ICAM-1 expression (arrowheads, Fig. 1, A and B), but staining was also observed in areas within the luteal tissue (arrows, Fig. 1, C and D). In contrast, relatively little to no ICAM-1 staining was evident in corpora lutea of 13 of 16 (81%) control rats (Fig. 1, E and F). Staining was limited to the peripheral edges of the corpora lutea in these instances and was localized to the larger blood vessels (arrowheads, Fig. 1F). In the remaining 3 rats, relatively moderate ICAM-1 expression, including parenchymal staining, was observed. Immunodetectable ICAM-1 was also evident in grossly atretic follicles, but not in other follicles within the same region of the ovary (Fig. 1G). Nonspecific staining was absent in all sections in which the primary antibody was omitted (Fig. 1H). No overt changes in ovarian ICAM-1 staining due to testosterone exposure were observed in either prolactin-treated or control rats.



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  		FIG. 1. Immunohistochemical expression of ICAM-1 (red precipitate) in ovarian sections of hypophysectomized rats after 72-h treatment with prolactin (A–C, D, G) or vehicle (E, F, H). A–C) Staining of ICAM-1 along the periphery (arrowheads) and within the parenchyma (arrows) of corpora lutea of prolactin-treated rats. x80. D) Higher magnification of C: note staining in parenchyma (arrow). x100. E, F) Limited ICAM-1 staining along edges between two corpora lutea of a control rat. Original magnification, x80. F) Higher magnification of E: note intense staining associated with large blood vessel (arrowheads). Original magnification, x100. G) Staining of ICAM-1 within a grossly atretic follicle (arrow): note absence of ICAM-1 staining in adjacent follicles and ovarian stroma. Original magnification, x80. H) Same section as in F: omission of mouse anti-rat ICAM-1 antibody (nonspecific staining). Original magnification, x100

Corpora lutea from prolactin-treated rats contained numerous ED1- and ED2-positive cells (Fig. 2, A and B; also Fig. 3). In contrast, significantly fewer ED1- and ED2-positive cells (P < 0.01) were observed in the corpora lutea of control rats (Fig. 2, C and D; also Fig. 3). Exposure to testosterone did not alter the numbers of ED1- and ED2-positive cells detected in corpora lutea of either prolactin-treated or control rats (P > 0.05; Fig. 3).



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  		FIG. 2. Immunohistochemical localization of monocytes/macrophages (ED1-positive cells) after 72-h treatment with prolactin (A, B) or vehicle (C, D). A) Same section as in Figure 1C: numerous monocytes/macrophages were present (reddish-stained cells). Original magnification, x80. B) Higher magnification of A: note numerous monocytes/macrophages in parenchyma (arrows). Original magnification, x100. C) Same section as in Figure 1E: few monocytes/macrophages detectable. Original magnification, x80. D) Higher magnification of C: note that most monocytes/macrophages were adjacent to large blood vessel (arrows). Original magnification, x100.



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  		FIG. 3. Numbers of ED1- and ED2-positive cells per high power field in corpora lutea of rats exposed or not exposed to testosterone prior to treatment with prolactin or vehicle (PRL-T4, n = 11 rats; PRL+T4, n = 9; VEH-T4, n = 11; VEH+T4, n = 5). For a given cell type (ED1-positive, ED2-positive), letters indicate differences among groups (P < 0.01)

Luteal Weights and Plasma Progestins

The interaction of treatment (VEH vs. PRL) and testosterone (with vs. without) for luteal weight was significant (P < 0.01; Fig. 4). Luteal weights of VEH-treated rats were heavier than those of prolactin-treated rats, and the corpora lutea increased in mass in response to exogenous testosterone (P < 0.05; Fig. 4). In contrast, prolactin treatment caused a significant decline in luteal weight (P < 0.01; Fig. 4) that was unaffected by the presence of testosterone. For plasma progestins, there was a significant treatment effect (P < 0.01), but no testosterone effect or treatment-by-testosterone interaction (P > 0.01; Figs. 5 and 6).



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  		FIG. 4. Luteal wet weights from rats exposed or not exposed to testosterone prior to treatment with prolactin or vehicle (PRL-T4, n = 11 rats; PRL+T4, n = 9; VEH-T4, n = 11; VEH+T4, n = 5). Different letters indicate differences between treatment groups (P < 0.01). An asterisk (*) indicates a difference within group (P < 0.05).



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  		FIG. 5. Plasma progesterone concentrations for rats exposed or not exposed to testosterone prior to treatment with prolactin or vehicle (PRL-T4, n = 11 rats; PRL+T4, n = 9; VEH-T4, n = 11; VEH+T4, n = 5). Different letters indicate differences between treatment groups (P < 0.01)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have shown that prolactin-induced regression of the rat corpus luteum is associated with immune response events including the expression of MCP-1, apoptosis, and the recruitment of monocytes/macrophages [3, 4]. It has also been suggested that the onset of the prolactin surge during proestrus in normal cycling rats initiates these events and that steroids might influence this process [4]. In most inflammatory responses, both the coordinated expression of cellular adhesion molecules (e.g., ICAM-1) and the local release of chemokines (e.g., MCP-1) facilitate recruitment of immune cells [23, 24]. Whether steroids impact the inflammatory response within the corpus luteum has not been examined. These issues led us to consider in the current study whether 1) ICAM-1 is expressed in the rat corpus luteum during prolactin-induced luteal regression and 2) whether prolactin-induced luteal regression is influenced by steroids.

The results of this study indicate that prolactin stimulates ICAM-1 expression in the rat corpus luteum. Specifically, prominent staining for ICAM-1 and the numerous populations of ED1- and ED2-positive cells in regressing corpora lutea of prolactin-treated rats contrasted with the limited staining of ICAM-1 and the relatively sparse numbers of ED1- and ED2-positive cells detected in corpora lutea of control rats. Luteal regression was confirmed by the decline of luteal weight and by the decrease in plasma progestins. Whether these effects were the result of direct or indirect actions of prolactin is uncertain. However, prolactin is known to regulate transcriptional factors in rat luteal cells [49] and to activate macrophages within gonadal tissue [50]. The findings of the present study are therefore supportive of the luteolytic actions of prolactin in the rat as documented by others [3, 4, 35–37] and extend our current knowledge of inflammatory responses that occur during luteal regression.

In a typical inflammatory response, adhesion molecules mediate the attachment of circulating monocytes to the endothelium of capillaries and their migration into surrounding tissue. Expression of ICAM-1 is considered to be a pivotal component of this process [23, 24]. A variety of cell types are known to express ICAM-1 (e.g., fibroblasts, smooth muscle cells, and endothelial cells; [30]), and some of these cell types are components of the corpus luteum. Cytokines such as tumor necrosis factor-{alpha} are known to induce ICAM-1 expression [24], and tumor necrosis factor-{alpha} expression increases within the corpus luteum during luteal regression [51]. In the current study, there was evidence of vascular ICAM-1 staining. In corpora lutea of control rats, ICAM-1 staining was greater than that in negative control sections, but was restricted to the largest blood vessels. In corpora lutea of prolactin-treated rats, prominent staining was observed within the parenchyma, but was also associated spatially with the capillary network known to encompass each structure. These findings indicate that there is constitutive and induced expression of ICAM-1 associated with the vasculature of the rat corpus luteum, similar to that described previously for other organs of the rat [52].

Expression of adhesion molecules and of chemokines is associated with immune cell-mediated tissue growth and repair [23]. Consistent with this idea, immune cells may mediate tissue remodeling during luteal regression and ultimately dispose of the corpus luteum. We speculate that the events that facilitate this process include the increased expression of ICAM-1 and chemokines such as MCP-1 in response to luteolytic stimuli, as well as the marked accumulation of phagocytic cells (including monocytes, macrophages, and eosinophils), all of which contribute to the selective and expeditious removal of luteal tissue.

In addition to a membrane form of ICAM-1, a soluble form of ICAM-1 (sICAM-1) also exists. The origin of sICAM-1 and its interaction with the membrane form of ICAM-1 are unclear. Proposed functions of sICAM-1 include mediation of adhesion of immune cells during transendothelial migration, competition with membrane-bound ICAM-1 to prevent cellular attachment, and the promotion of transmembrane signaling in immune cells [28]. A soluble form of ICAM-1 has been found recently in the ovary [34], but its role in ovarian function is not known. Immunodetectable sICAM-1 is present in follicular aspirates, and granulosa-luteal cells secrete sICAM-1 in vitro following stimulation with lipopolysaccharide [34].

Treatment of rats with testosterone beginning 72 h in advance of prolactin injections and continuing throughout prolactin treatment did not overtly alter ICAM-1 expression, the accumulation of monocytes/macrophages within corpora lutea, or plasma progestin concentrations. Previous studies have shown that corpora lutea of hypophysectomized rats retain the capacity to aromatize testosterone to estradiol [38, 44]. Silastic implants containing crystallized testosterone, and placed s.c., maintained serum androgen and progesterone concentrations, estradiol concentrations within the corpora lutea, and luteal growth at levels comparable to those in pregnant, sham-operated rats [38]. Our goal in the current study was to determine whether testosterone administered systemically, or its potential conversion to estradiol within the corpora lutea (via luteal aromatase; [44]), influences prolactin-induced luteal regression. Considering that monocytes/macrophages express steroid receptors and respond to steroid [42, 53, 54], the possibility that steroid influences monocyte/macrophage populations within the rat ovary is plausible. However, we found no evidence that the administration of testosterone alters the total number or ratio of ED1- and ED2-positive cells. The administration of steroid did have an effect in the corpora lutea, however, as evidenced by the significant increase in luteal weight, but without an accompanying increase in plasma progestin. These observations contrast with findings in the study by Gibori and Keyes [38], indicating that testosterone augmented both luteal growth and serum progestin production in adult pregnant rats in which the corpora lutea had been exposed to pituitary/placental hormones and were secreting progesterone at the time of hypophysectomy. In the current study, immature rats were hypophysectomized immediately following gonadotropin-induced ovulation, and these corpora lutea produce predominantly 20{alpha}-DHP (see above; also [35]). Steroid receptor expression within rat luteal tissue is dependent upon prolactin/placental lactogen [55, 56]. Hence, the diminished response of the corpora lutea to steroid might have been due to the lack of adequate exposure to circulating pituitary and/or placental hormones.

In conclusion, this study is the first to report expression of ICAM-1 in the corpus luteum during luteal regression. Expression of ICAM-1 is most prevalent in the peripheral, ostensibly highly vascular, regions of the corpus luteum and is accompanied by an accumulation of large numbers of monocytes and macrophages. Expression of ICAM-1 and the differentiation of monocytes into activated macrophages were not influenced by steroid. Further, these findings indicate that prolactin stimulates, directly or indirectly, the expression of ICAM-1 in the rat corpus luteum, and that expression of ICAM-1 is consistent with its proposed role in recruitment and activation of monocytes/macrophages.



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  		FIG. 6. Plasma concentrations of 20{alpha}-DHP for rats exposed or not exposed to testosterone prior to treatment with prolactin or vehicle (PRL-T4, n = 11 rats; PRL+T4, n = 9; VEH-T4, n = 11; VEH+T4, n = 5). Different letters indicate differences between treatment groups (P < 0.01)


   ACKNOWLEDGMENTS
 
The authors wish to thank Upjohn Pharmaceuticals for the gift of Lutalyse. We would like to also thank Dr. P. Landis Keyes (University of Michigan Medical School, Ann Arbor, MI) for the Silastic-filled testosterone capsules and suggestions and S. Kitzsteiner of the Chemistry Core Facility of the Michigan Diabetes Research and Training Center (University of Michigan Medical School, Ann Arbor, MI) for conducting the plasma steroid assays.


   FOOTNOTES
 
First decision: 17 August 1999.

1 Supported by USDA grant 98–35208-6654 and by funds from the University of New Hampshire Vice President for Research. Back

2 Correspondence: D.H. Townson, Department of Animal and Nutritional Sciences, Kendall Hall, 128 Main St., University of New Hampshire, Durham, NH 03824–3590. FAX: 603 862 3758;dave.townson{at}unh.edu Back

Accepted: January 11, 2000.

Received: July 1, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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