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a Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Johns Hopkins School of Public Health, Baltimore, Maryland 21205
b Department of Psychology,
c Department of Neuroscience, Johns Hopkins University, Baltimore, Maryland 21218
ABSTRACT
To determine the extent to which testicular regression involves apoptotic cell death, photosensitive adult starlings were photostimulated for up to 9 wk by exposure to long-day (18 h of light) photoperiods. Apoptotic activity in recrudescing and regressing testes was assessed by in situ TUNEL labeling. Absolute testis mass in male starlings increased after 2 wk of photostimulation and subsequently decreased with continued long-day exposure. Seminiferous tubule diameter also increased after 1–3 wk of photostimulation, then decreased as photorefractoriness developed. Testosterone concentrations increased significantly by Week 2 of photostimulation and declined with further light exposure. TUNEL labeling was significantly elevated in germ cells with 4 wk of photostimulation. An approximate 7-fold increase in the degree of apoptotic cell death was observed over the course of gonadal regression. Incidences of TUNEL labeling in somatic Sertoli cells also increased. Light and electron microscopy examination confirmed that these somatic cells displayed morphological characteristics of apoptotic death. In rodents, Sertoli cells have not been previously reported to die during gonadal regression. These results suggest that seasonal testicular regression in European starlings is mediated by apoptosis.
apoptosis, seasonal reproduction, Sertoli cells, steroid hormones, testis
INTRODUCTION
Transition from short to long day lengths induces the onset of seasonal reproductive function in European starlings (Sturnus vulgaris) ([1–5], reviewed in [6, 7]). In photosensitive starlings, long day lengths (>12 h of light/day) initially induce testicular development in adult males. Continued exposure to long photoperiods results in testicular regression and a loss of the hypothalamo-pituitary-gonadal response to stimulatory long days. The onset of photorefractoriness, a lack of gonadal responsiveness to stimulatory day lengths, is characterized by a marked decline in GnRH and, subsequently, the gonadotropins, LH and FSH, to undetectable concentrations [6, 8]. These endocrine changes, along with elevated prolactin concentrations, evoke testicular regression, a rapid atrophy in both testicular volume and function [2, 8–10]. Endocrine cycles regulated by changes in photoperiod have been well-characterized in starlings. In addition, the endocrine mechanisms underlying photorefractoriness have been studied in the laboratory [e.g., 10, 11]. It is, however, unknown how these photoperiod-induced changes are translated into the cellular signals that mediate testicular atrophy.
In long-day breeding photoperiodic rodents, testicular regression occurs after 
6 wk of short-day exposure and involves apoptotic cell death [12, 13]. In both peripubertal Djungarian hamsters (Phodopus sungorus) and adult white-footed mice (Peromyscus leucopus), increases in apoptotic cell death occur in the testes prior to or concomitant with reductions in plasma testosterone concentrations and testis mass [13, 14]. It is possible that the pattern of testicular cell death observed in rodents might be similar to the process in birds (i.e., high levels of apoptosis with no or little necrotic cell death coinciding with gonadal regression). Although photoperiod serves as the primary predictive cue for individuals in both of these seasonal-breeding animals, several differences in gonadal regression are apparent between birds and rodents. For example, the environmental stimuli that induce gonadal regression differ between rodents that breed seasonally and most temperate zone birds. Importantly, the degree to which testis size is reduced after the breeding season is much more extensive (90%–95% compared with 50%–70% regression) in most birds than it is in the majority of rodents. Finally, the timing of gonadal regression is generally more rapid in most birds than in rodents. The present experiment was conducted to determine the contribution of apoptotic cell death to gonadal regression in starlings.
Apoptosis, or programmed cell death, is a regulated form of cell death defined by a number of cellular biochemical and morphological changes [15–17]. Activation of the apoptotic protein cascade results in nuclear and cytoplasmic condensation, changes in mitochondrial membrane potential (
), internucleosomal cleavage of DNA, and disintegration or "blebbing" of the cell membrane as a result of cytoskeletal element destruction [17]. Disintegration of the membrane ultimately leads to formation of apoptotic bodies that are phagocytosed by macrophages or neighboring cells [15, 17–19]. In contrast, necrotic cell death is characterized by nuclear swelling, membrane eruption, and subsequent inflammation due to a cell-mediated immune response [20, 21]. Necrotic cell death does not require the complex web of gene activation and protein expression typical of apoptosis.
The present study used in situ TUNEL labeling and morphological assessment to determine whether gonadal regression in European starlings is mediated by apoptosis. We further sought to compare the timing of endocrine events with apoptosis over the course of the gonadal cycle, as has recently been described in rodents [12, 13].
MATERIALS AND METHODS
Animals
Adult male starlings (Sturnus vulgaris; n = 56) were trapped in the wild in November–December 1998 with permission on the U.S. Department of Agriculture (USDA) dairy farms in Beltsville, Maryland. Males were housed in cages (49 cm x 95 cm x 51 cm; n = 8 birds per cage) and supplied with food (Turkey Starter Crumbs, Purina Mills, Inc., St. Louis, MO) and water ad libitum. Temperature remained constant at 20 ± 2°C. All experiments were conducted in our facilities, which have been approved by the Association for Assessment and Accreditation of Laboratory Animal Care. All starlings were housed until they were photosensitive in a short (8 h of light per day) photoperiod, and were then transferred to a stimulatory photoperiod of 18 h of light per day (18L). At 1, 2, 3, 4, 5, 6, and 9 wk of 18L exposure, testes were removed and assayed (n = 8 males per group) for evidence of cell death.
For morphology studies, 15 adult male starlings were trapped and caught in November 1999 at the USDA dairy farms. These males were housed as just described, and maintained in an 18L photoperiod for 1, 2, 4, 6, and 9 wk (n = 3 per group).
Experimental Protocol
Body and testis mass were determined at autopsy. Seminiferous tubule diameter, a reliable measure of reproductive competence [22], was assessed by taking the average measure of 10 nearly round tubules per animal. Round tubules were randomly selected by an experimenter who was blind to group assignment, and measured using Stereoinvestigator software (Microbrightfield, Colchester, VT).
Testosterone Radioimmunoassay
At the end of each photoperiod exposure terminal blood samples were collected by puncture of the alar wing vein into heparinized tubes and centrifuged for 15 min at 2500 x g at 4°C. After separation, plasma was stored at -80°C until plasma testosterone values were determined via 125I double antibody radioimmunoassay (RIA; ICN Biomedicals, Costa Mesa, CA). The ICN testosterone RIA is highly specific; sensitivity is to 25 pg and cross-reactions with other steroids are <0.1% to 7.8%. This assay has been validated for use in starlings [23].
Tissue Processing
Twenty to 24 h following collection of the terminal blood sample, starlings were anesthetized with an overdose of secobarbital (0.09 ml, 50 mg/ml i.m.). The left testis and epididymis were removed through an incision on the ventral surface of the abdomen, weighed, snap-frozen, and stored at -80°C for future experiments. Animals were then perfused through the left ventricle with 50 ml of 0.9% saline followed by 500 ml of 10% neutral buffered formalin (Electron Microscopy Sciences, Fort Washington, PA) as a fixative. After fixation, the right testis and epididymis were removed, weighed, and postfixed in 10% formalin for 96 h. Tissue was then washed in PBS and dehydrated in 70% ethanol prior to paraffin embedding.
For TUNEL labeling, 6-µm sections were collected from every 50 µm of tissue and stained for apoptotic activity. Briefly, tissue sections were deparaffinized, endogenous peroxidases were quenched with incubation in 3% H2O2, and sections were incubated in a 1:1500 dilution of terminal deoxynucleotidyl transferase (1 unit TdT: 3.3 µl labeling buffer) (Trevigen TACS 2TdT, Gaithersburg, MD) to label fragmented 3' OH termini. Twelve testis cross-sections were counted per animal. Cells that incorporated the labeled biotinylated nucleotides were considered TUNEL-positive (apoptotic) and were counted under brightfield illumination (40x) on an Axioplan 2 microscope (Zeiss, Thornwood, NY) using Stereoinvestigator software (Microbrightfield). Negative control sections, processed without TdT, did not show positive labeling. Control slides, provided by Trevigen and processed alongside experimental sections, labeled TUNEL-positive. Biotinylated nucleotides indicating DNA fragmentation were visualized using streptavidin-horseradish peroxidase, and colorimetrically detected with Trevigen TACS Blue Label. Apoptotic activity was quantified by counting the number of cells that were positive for TUNEL staining within each testis cross-section. To control for reduction of testis size, the number of TUNEL-positive cells was expressed as the number of apoptotic cells per total number of seminiferous tubules within each testis cross-section.
Histological Analysis
Following photostimulation for 1, 2, 4, 6, or 9 wk, starlings were perfused through the left ventricle with 0.9% saline followed by 200 ml of 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). After fixation, testes were removed, weighed, cut into 1- to 2-mm slices, and reimmersed in 5% glutaraldehyde for 1 wk. Tissues were then washed in 0.1 M cacodylate buffer and postfixed in a 1% osmium tetroxide, and then dehydrated in a series of ethanols and embedded in an araldite/polybed A12 resin (Electron Microscopy Sciences).
Analysis of dying cells with light microscopy was done with 1-µm-thick sections stained with toluidine blue and analyzed with a Nikon Eclipse E800 microscope (Nikon, Inc., Melville, NY). Digital images were captured with a 5 mHz cooled CCD (Princeton Instruments, Roper Scientific, Trenton, NJ) camera using IP lab software (Scanalytics, Fairfax, VA). For electron microscopy, 80-nm sections were cut on an MT 2 ultramicrotome (Sorvall, Newtown, CT), mounted on 200-mesh copper grids, and stained with uranyl acetate and lead citrate. Grids were analyzed and micrographed on a Hitachi HU 12A electron microscope (Hitachi Ltd., Tokyo, Japan).
Statistical Analysis
Statistical evaluation of differences between experimental groups was performed by ANOVA, and for measurements lacking equal variance, by a Kruskal-Wallis ANOVA on ranks using the Sigma Stat software package (Jandel Scientific, Chicago, IL). To isolate significant differences between groups, the Student-Newman-Keuls test was used for the pairwise multiple comparisons. In all cases, mean differences were considered statistically significant if P < 0.05.
RESULTS
Body Mass, Testis Mass, and Seminiferous Tubule Diameter
No changes were observed in body mass with photoperiod stimulation; average body mass for all groups was 76.3 ± 0.4 g (P > 0.05). As expected, paired testis mass in male starlings increased after 2 and 3 wk of 18L photostimulation and subsequently decreased with continued long-day exposure (P < 0.05 where indicated; Fig. 1A). Seminiferous tubule diameter also increased with 2–3 wk of photostimulation and decreased with further exposure to 18L (P < 0.05 where indicated; Fig. 1B). Compared with measurements taken at Week 1 of photostimulation, seminiferous tubule diameter increased 0.7- and 1.8-fold in males housed in 18L for 2 and 3 wk, respectively (P < 0.05 in both cases; Fig. 1B).
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Testosterone Concentrations
Plasma testosterone concentrations were increased more than 7-fold at Week 2 compared with Week 1 of photostimulation (P < 0.05; Fig. 1C). The transient increase in plasma testosterone peaked after 2 wk of photostimulation; after 3 and 4 wk of photostimulation, testosterone concentrations were only 2.8- and 1.3-fold greater than Week 1 baseline values, respectively (P > 0.05). After 5 wk of photostimulation, testosterone concentrations were below those of Week 1, and remained low for the duration of the experiment (Fig. 1C, P > 0.05).
Apoptosis Detection During the Testicular Cycle
Identification of testicular apoptosis during the reproductive cycle of starlings was performed using in situTUNEL analysis on testis cross-sections. Apoptotic labeling of germ cells was observed in relatively low amounts during testicular development at 1, 2, and 3 wk of photostimulation. By Weeks 3 and 4 of photostimulation, the concentration of interstitial pigment was considerably reduced, and the majority of tubules in all animals contained a full complement of germ cells, including mature, clustered spermatozoa (Fig. 2A). Abundance of spermatozoa and later stage germ cells declined rapidly after 5, 6, and 9 wk of 18L photoperiod exposure, and the concentration of both TUNEL labeling and interstitial pigment increased in the regressing testes of these animals (Fig. 2B). Based on their position within the seminiferous epithelium, stained germ cells were predominantly spermatocytes. After 9 wk of 18L exposure, degeneration of the testes was obvious, and males were no longer reproductively competent.
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The number of germ cells that labeled for apoptosis was quantified by counting the number of TUNEL-positive germ cells per seminiferous tubule within each cross-section (Fig. 3). Similar low levels of apoptotic staining were detected in males exposed to 1, 2, and 3 wk of 18L photoperiod (P > 0.05; Fig. 3). A significant increase in the number of apoptotic germ cells was detected after 4 and 5 wk of 18L exposure (P < 0.05; Fig. 3A). Germ cell apoptotic activity was approximately 7-fold higher at Weeks 4 and 5 compared with Week 1 baseline counts. At 6 and 9 wk, the number of TUNEL-stained germ cells decreased and, by Week 9, the number of apoptotic germ cells per seminiferous tubule was reduced compared with all other time points sampled (P < 0.05; Fig. 3A).
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Apoptotic labeling was not limited to germ cells in photostimulated European starlings. Sertoli cells, which are somatic support cells within the seminiferous tubules, were also observed to label TUNEL-positive in males housed for 4, 5, and 6 wk in 18L (Fig. 2, C and D). Sertoli cells were considered TUNEL-positive if the blue apoptotic label was cell-specific and extended from the basement membrane to the lumen of the seminiferous tubule. Figure 2, C and D illustrates typical TUNEL-positive Sertoli cells in males housed for 4 wk in 18L. The pattern of staining observed was consistent and specific; similar labeling was not detected in earlier weeks of photostimulation, nor in adjacent sections of epididymis also processed for TUNEL labeling. As observed with germ cell labeling, the number of TUNEL-positive Sertoli cells was relatively low at 1, 2, and 3 wk of 18L photostimulation, without obvious differences among these groups. At 4 and 5 wk of 18L photoperiod exposure, the number of TUNEL-positive Sertoli cells was significantly increased compared with staining detected after 1, 2, and 3 wk of exposure.
Apoptotic Sertoli cells were quantified by counting the number of TUNEL-positive cells per seminiferous tubule within each cross-section (Fig. 3B). The overall number of TUNEL-positive Sertoli cells was reduced compared with the number of labeled germ cells counted; however, the pattern of apoptotic activity across weeks of photostimulation was similar in both Sertoli and germ cells (note scale, y axis, Fig. 3). Low levels of apoptosis were observed in males exposed to 1, 2, and 3 wk of 18L (P > 0.05; Fig. 3B). As with germ cell labeling, a significant increase in number of TUNEL-positive Sertoli cells was detected after 4, 5, and 6 wk of photoperiod exposure (P < 0.05; Fig. 3B). At 4 and 5 wk the number of TUNEL-positive Sertoli cells was increased approximately 45- and 43-fold, respectively, over Week 1 baseline values (P < 0.05; Fig. 3B). After 9 wk the number of TUNEL-positive Sertoli cells was significantly reduced compared with all other weeks sampled (P < 0.05; Fig. 3B).
Morphological Confirmation of Sertoli Cell Death
To confirm the Sertoli cell death indicated by TUNEL-positive labeling of these somatic cells, morphological characteristics of cell death were examined in plastic-embedded sections at the light and electron microscopy levels. Evidence that spermatocytes, spermatogonia, and Sertoli cells were dying was initially observed at the light level in 1-µm sections stained with toluidine blue from males after 4 wk of photostimulation. Dying cells displayed condensed, darkly staining cytoplasm with irregular nuclei, morphology that is characteristic of apoptosis. Germ cell apoptosis was common in males undergoing testicular regression during Weeks 4–6 of photostimulation. Apoptotic bodies surrounding round or oval degrading spermatocytes were common (not shown). Whereas the majority of cell deaths were noted in the round or ovoid germ cells, darkly staining condensed cells with long, attenuated cytoplasmic processes were also noted in males after 3 wk of photostimulation (Fig. 4A). Identity of these dying cells was confirmed with electron microscopy as darkly stained cells with extended cytoplasmic processes, and were observed to have condensed cytoplasm, irregular nuclei, and marginated chromatin; these are all indications of apoptotic cell death (Fig. 5, B and C). The characteristic morphology of these cells along with these signs of cell death confirmed that Sertoli cells in the starling seminiferous epithelium were dying. Similar to the TUNEL assay results, the overall incidence of dying Sertoli cells was infrequent, and most Sertoli cells appeared healthy (Fig. 4, B and C). At 9 wk, when testicular regression was complete, the normally elongated cytoplasm of the Sertoli cells was retracted, and many tubules contained detached Sertoli cells with irregular nuclei and marginated chromatin in the lumenal space (Fig. 4, B and C).
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DISCUSSION
In European starlings, testicular regression induced by sustained 18L exposure was associated with apoptotic cell death. Photosensitive starlings responded to stimulatory day lengths with an initial increase, and then a decrease in paired testes mass. Relatively low levels of apoptotic cell death characterized the period of testicular regrowth (Weeks 1–3 of photostimulation). Testicular cell death increased significantly after 4, 5, and 6 wk of photostimulation, with both germ and somatic cells labeling positive with the TUNEL assay for apoptosis. Cell death was primarily observed in spermatocytes, spermatogonia, and Sertoli cells; labeling of spermatids and Leydig cells was not observed. Cellular swelling, cell lysis, and macrophage infiltration, all hallmarks of necrotic cell death, were infrequently observed, implying that necrosis does not mediate testicular regression in European starlings.
Increases in testicular apoptosis occurred after a decrease in plasma testosterone. Plasma concentrations of testosterone peaked after 2 wk of 18L photostimulation, whereas significant increases in apoptotic cell death were noted after 4 wk of 18L. Testosterone is a testicular cell-survival factor; withdrawal of testosterone is associated with apoptotic germ cell death, and exogenous testosterone ameliorates apoptotic germ cell death induced after hypophysectomy [24–26]. In starlings, photoperiod-induced changes in testosterone are a result of prior decreases in synthesis and secretion of GnRH, and subsequently LH and FSH [8–10, 27]. The increases in testicular apoptosis observed in the present experiment are presumably consequences of these endocrine changes, suggesting that withdrawal of gonadotropins and testosterone may influence the cellular events of testicular regression in starlings.
In contrast to the timing and pattern of testicular atrophy in starlings, increases in cell death occur prior to or concomitant with decreases in testosterone in both Djungarian hamsters (Phodopus sungorus) and white-footed mice (Peromyscus leucopus) undergoing photoperiod-induced regression [12, 13]. In P. leucopus, decreases in testosterone occur 2 wk after increases are first observed in expression of apoptotic proteins, TUNEL labeling, and DNA laddering. In Djungarian hamsters, declines in serum FSH concentrations occur prior to increases in testicular apoptosis [12]. Indeed, a decline in FSH is hypothesized to initiate testicular apoptosis in rodents; reduction in testosterone concentrations may amplify the apoptotic signal to the seminiferous epithelium in these rodent species [12, 13].
The progression of testicular regression varied considerably among individual males in the present experiment. At Week 4 of photostimulation starlings could be divided into two distinct groupings based on testicular morphology. Testes of some Week 4 males could be classified as reproductively active, with full cohorts of germ cells and low levels of TUNEL, morphology that is typical of Week 3 males. Testes of other males at Week 4 resembled reproductively inactive males at Week 5. Testes of these males had lacked full generations of germ cells and contained high levels of TUNEL labeling. As a result, variability in TUNEL quantification was high at Week 4. Whether this variability was a result of difference in the timing of testicular regression onset or rate of atrophy is unknown. Despite the variability in atrophy progression, TUNEL labeling was significantly increased after 4, 5, and 6 wk of photostimulation.
Once testicular regression is complete, the seminiferous tubules contain primarily spermatogonia and Sertoli cells [28–29]. In adult testes, Sertoli cells are conventionally considered to exist as a stable population and are notable for their resistance to stimuli that induce death in other testis cell types [26, 30–33]. Changes in Sertoli cell populations associated with breeding condition have been suggested in select seasonal breeding mammalian species (e.g., stallions, red deer, and Corriedale rams); however, in other seasonal species and in hypophysectomized rodents and fowl, changes in Sertoli cell numbers have not been reported [32, 34–38]. When testicular morphology is compared between breeding and nonbreeding seasons in photoperiodic species, the most commonly described changes in Sertoli cells are cell volume and shape [34, 39–41]. Confirmation of Sertoli cell death in seasonally regressing testes, as provided in the present experiment, had not be previously documented.
In the present experiment, starling Sertoli cells labeled TUNEL-positive during testicular regression. TUNEL labeling for apoptosis was cell specific, and cell death was confirmed by assessing morphological changes that are typical of apoptosis at the light and electron microscopic level. Degenerating cells were easily identified by irregularly shaped nuclei, marginated chromatin, attenuating cell membranes, and general structural distortion [40]. In addition, dying cells were characterized by darkly staining condensed cytoplasm. These characteristics of cell death were observed in cells identified by light and electron microscopy as Sertoli cells based on their position within the seminiferous tubule and characteristic elongated cytoplasmic branches.
Targeting Sertoli cells for apoptotic cell death could serve as a mechanism to achieve rapid testicular regression in starling testes. Quick dismantling of the seminiferous epithelium may be achieved through targeted cell death of Sertoli cells as these somatic cells control survival, differentiation, and death for distinct cohorts of germ cells [42]. Desmosome or desmosome-like channels form between germ cells and the cytoplasmic processes of Sertoli cells for this communication [30, 31, 42]. In mammalian testes undergoing induced regression, Sertoli cells secrete the soluble ligand for the Fas receptor, FasL [43]. Germ cells expressing the death receptor, Fas, receive the local signal for apoptosis initiation from Sertoli cells [44, 45]. Exposure of Sertoli cells to toxins has been shown to up-regulate the expression of both Fas ligand on Sertoli cells as well as Fas in germ cells [44–46]. Apoptotic Sertoli death could therefore enhance subsequent induction of apoptosis for local clusters of germ cells. This model of comprehensive programmed cell death would result in the rapid atrophy of the seminiferous epithelium observed in starlings compared with species in which germ cells are exclusively targeted.
Starlings survive 3–5 yr in the wild, and seasonal regrowth of testes presumably occurs each spring [1–6]. Development of the germ cell population occurs during those episodes of photostimulation. Although our results suggest that a low number of Sertoli cells undergo cell death during regression, it is unknown if remaining Sertoli cells also undergo mitosis to replace cells lost at the termination of the previous breeding season. If such increases in mitosis occur, Sertoli cells would presumably show some degree of mitotic activity with seasonal photostimulation. Alternatively, Sertoli cells may not be regenerated and, therefore, Sertoli cell death could be involved with mediation of reproductive senescence. Fertility in older birds could be reduced due to yearly Sertoli cell loss, as germ cell numbers are inherently linked to number of Sertoli cells [41].
Sertoli-Sertoli cell tight junctions form the blood testis barrier, creating a privileged adluminal compartment for germ cell development [41]. In hamsters undergoing testicular regression, the permeability of the blood-testis barrier is maintained through regression and is subsequently lost after atrophy is complete [47]. Presumably, selective Sertoli cell death may induce early loss of the integrity of the blood testis barrier in starlings undergoing testicular regression. Alternatively, as low numbers of Sertoli cells were observed to undergo apoptosis, the blood-testis barrier may not have been affected and remained intact during regression. In seasonally breeding mink, alterations in blood-testis barrier permeability during the final seasonal wave of spermatogenesis did not result in an immune response [46]. The present study did not measure immune parameters during early testicular atrophy; however, future studies examining immune function and epithelial barrier permeability during testicular atrophy would address questions concerning loss of barrier integrity, and possible autoimmune response.
Sertoli cell death observed in starlings exposed to an 18L photoperiod for 4, 5, and 6 wk is unlike the pattern of apoptosis observed in white-footed mice in which only germ cells appear to undergo apoptosis during testicular regression [13]. The extent of germ cell death in the starling seminiferous epithelium is also noteworthy: at the peak of apoptosis, nearly five germ cells per seminiferous tubule counted were TUNEL-positive. This far exceeds the extent of germ cell labeling in white-footed mice, in which rarely more than one germ cell per seminiferous tubule counted was labeled TUNEL-positive [13]. The extensive cell death observed in starlings corroborates with the rapid period of testicular regression observed in these birds after exposure to 18L [10].
These results suggest that photoperiod-induced testicular regression in starlings is mediated primarily by apoptosis. Regression in starlings is unique among vertebrate species thus far examined as both germ cells and Sertoli cells undergo cell death. Taken together, the data presented illustrate a difference in testicular regression between songbirds and rodents, suggesting that distinct mechanisms have evolved to address parallel issues of seasonal gonadal function.
ACKNOWLEDGMENTS
We thank Deb Duffy for her instrumental role with experimental planning; Janet Folmer for expert electron microcopy and patient training sessions; Dr. Philip Vanek (Trevigen), for labeling advice; and Brian Spar, Era Hanspel, Jana Kuo, Yvonne Li, Andrew Shapiro, Jen Sartor, and Uade de la Silva for their aid with data collection. We also thank Dr. Brian Prendergast and Deb Duffy for reading the manuscript and providing helpful comments.
FOOTNOTES
First decision: 8 August 2000.
1 This research was supported by NIMH grant 57535 to R.J.N., NICHHD training grant T32-HD-07276 to K.A.Y., NSF grant IBN 9905401 to G.F.B., and NIH grant NS 35467 to G.F.B. 
2 Correspondence and current address: Kelly A. Young, Oregon Regional Primate Center, Division of Reproductive Sciences, Oregon Health Sciences University, 505 NW 185th Ave., Beaverton, OR 97006. FAX: 503 690 5563; youngk{at}ohsu.edu
Accepted: October 4, 2000.
Received: June 23, 2000.
REFERENCES
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indicate a significant difference compared with Week 1 baseline values; ** indicates a significant difference compared with all other weeks (P < 0.05). B) Average seminiferous tubule diameter measurements (mm) ± SEM in European starlings housed in 18L photoperiods for up to 9 wk. 
