INTRODUCTION

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INDIVIDUALS OF MANY VERTEBRATE species undergo seasonal fluctuations in both gonadal size and function that serve to synchronize reproduction with favorable environmental conditions. In nontropical rodents, testicular atrophy, or regression, is reliably induced in the laboratory with exposure to short (<12 h light/day) photoperiods that are typical of autumn and winter at high (>30°) latitudes (4). Short days are transduced into an inhibitory reproductive signal by the pineal indolamine hormone melatonin (11).

In many laboratory studies of small rodents, testicular regression requires 6-12 wk of continuous exposure to short day lengths, during which both spermatogenesis and steroidogenesis stop. After 25-30 wk of regression, the inhibitory effects of melatonin wane and the testes redevelop fully (12). The timing of testicular recrudescence in nature allows male rodents to begin breeding early in the spring before the onset of long day lengths.

Adequate blood supply is crucial for growth, development, and maintenance of all tissues. Growth and formation of new blood vessels from existing capillary beds are achieved via a process termed angiogenesis. This vascular development, stimulated by a variety of growth factors, includes proliferation and migration of endothelial cells to form tubular assemblies, which then assemble into mature blood vessels (10). In healthy adult mammals, physiological angiogenesis occurs almost exclusively in the reproductive organs. In Syrian hamsters (Mesocricetus auratus), for example, testicular recrudescence involves an increase in both densities of testicular blood vessels and vascular permeability (16). These transient processes manifest after 1 wk of exposure to long days in hamsters but disappear with prolonged long-day exposure (16). Angiogenic processes in other adult tissues are primarily associated with pathophysiological conditions, such as wound healing or tumor development (1, 9, 19).

The process of blood vessel growth is regulated by paracrine growth factors (22). Proliferation of endothelial cells is stimulated by the binding of the mitogen VEGF to its receptors, KDR/Flk-1 and Flt-1 (25). Secretion of the endothelium-specific VEGF protein appears to function as a chemoattractant, stimulating endothelial cell migration (22). VEGF also has a potential role in the stimulation of vasodilation and microvascular hyperpermeability and can be inhibited by a variety of angiogenesis inhibitors (22, 25).

In female reproductive tissues, VEGF is expressed continuously throughout the ovarian cycle but is downregulated during periods of vasculature regression (1). Prevention of VEGF-Flt-1 binding, through the injection of soluble, truncated Flt-1 receptors, can inhibit angiogenesis in the corpus lutuea of rats (Rattus norvegicus) without the disruption of mature vasculature (8).

During the gradual atrophy of the testes observed in rodents undergoing seasonal regression, vascular support is withdrawn, and presumably new angiogenesis is inhibited, but this assumption remains undetermined. Individuals regrowing their reproductive organs in the spring undergo a temporary increase in blood vessel growth (15, 16); however, it is not understood how vascularization contributes to regression of the testis.

In white-footed mice, the cellular mechanisms of photoperiod-induced gonadal regression involve apoptosis, rather than necrotic cell death (23). A two- to threefold increase in apoptotic germ cells occurs in the testes of mice during gonadal regression. Likewise, Western analysis indicates an upregulation of the Fas receptor protein, an early transducer of the apoptotic signal, in the testes of mice exposed to short days (23).

A decrease in angiogenic processes may contribute to the increase in apoptotic cell death induced by the onset of testicular regression, in which case the reduction of VEGF protein would be expected to precede gonadal regression and would be detected early in the progression of atrophy. Alternatively, loss of blood vessel maintenance may occur as a result of loss of testicular volume. In this case, VEGF would not be expected to decline until the testes had significantly regressed. In the present study, white-footed mice (Peromyscus leucopus) were maintained in either long (LD 16:8) or short (LD 8:16) photoperiods for up to 10 wk. The angiogenic and blood vessel maintenance protein VEGF was assayed from animals at each of these stages of testicular regression, and the time courses of changes in VEGF protein expression and testicular mass were compared with determine the contribution of VEGF to gonadal regression.

	METHODS

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Animals

Experimental Protocol

Reproductive assessment. To assess the extent of testicular regression, the left testis was weighed and mass was doubled for a paired testis mass measurement. From each animal, (n = 5/group), five seminiferous tubule diameters, a reliable estimate of reproductive activity, were measured using under bright-field illumination (×40) on a Zeiss Axioplan 2 microscope (Thornwood, NY) with Stereoinvestigator software (Microbrightfield, Colchester, VT). In addition, spermatogenic activity was also evaluated in five seminiferous tubules per animal (n = 5/group) to determine reproductive competence using the spermatogenic index developed by Grocock and Clarke (13). This index rates the extent of spermatogenic activity in the seminiferous epithelium. Scores assigned ranged from 1 to 5. A value of "5" was given to large tubules displaying complete spermatogenesis; a score of "1" was assigned to small tubules that contained primarily Sertoli cells, spermatogonia, and few primary spermatocytes.

Immunohistochemistry. After exposure to methoxyflurane vapors for rapid anesthesia (Metofane; Schering-Plough, Union, NJ), animals were perfused through the heart with 50 ml 0.9% saline followed by 150 ml Bouin's solution as a fixative. After fixation, the right testis was removed, weighed, and postfixed in Bouin's solution for 24 h. Tissue was then dehydrated in a series of ethanol solutions (50, 70, and 100%) before paraffin embedding.

VEGF protein electrophoresis. VEGF activity was assessed in testes collected after 2, 4, 6, 8, or 10 wk of exposure to the experimental photoperiod. Before perfusion, animals were anesthetized under methoxyflurane anesthesia (Metofane; Schering-Plough) and the left testis was removed, immediately frozen, and stored at 80°C before processing for protein extraction. Minced tissue (~0.1 g) was sonicated in two volumes of homogenization buffer (1 M Tris · HCl, pH 7.4; 0.5 M EDTA, pH 8.0; 10% SDS) containing proteolytic inhibitors 4-(2-aminoethyl)-benezensulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin. Samples were incubated at 0°C for 30 min and centrifuged for 30 min at 14,000 rpm. The supernatant was extracted, boiled for 5 min at 95°C with an equal volume of Laemmli buffer (containing 10% -mercaptoethanol), and protein concentration was determined by Bradford assay. Proteins were loaded 20 µl/lane at 25 µg/ml into SDS-PAGE gel (12% solution) and electrophoresed and then transferred onto nitrocellulose (Hybond ECL, Amersham Life Sciences, Aylesbury, UK). Benchmark prestained protein ladder (GIBCO-BRL, Gaithersburg, MD) was used to determine transfer efficiency and to estimate protein size. After a 1-h blocking period, blots were incubated for 1 h with VEGF antibody diluted to 1:800 (sc716, Santa Cruz Biotechnology), washed with TBS-Tween buffer, and incubated for 1 h in peroxidase-conjugated goat anti-rabbit IgG (1:20,000, Jackson ImmunoResearch Laboratories, West Grove, PA). Primary antibodies were detected by an enhanced chemiluminescence detection kit (Amersham Life Sciences). For comparison between gels, protein isolated from a single control animal was loaded onto each gel alongside experimental samples in 10-, 20-, and 40-µg concentrations. VEGF protein levels were quantified by densitometric analysis of blots using the MCID imaging system (Imaging Research, St. Catherine's, Ontario, Canada). The calculated optical density value (area × optical density) for each experimental animal was compared with the internal standard curve created from the control samples. Quantification from optical density readings is presented as micrograms of protein equivalent to the internal standard of each blot.

Statistical Analysis

	RESULTS

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Reproductive Measures

Testis mass. Paired testis mass was significantly decreased in short-day compared with long-day housed animals (P < 0.05, n = 5/group) (Fig. 1). In males housed for 10 wk in short days, testis mass was significantly reduced compared with all males housed in long photoperiods and males housed in short days for 2, 4, 6, and 8 wk (P < 0.05 for each comparison) (Fig. 1). Among males housed in long days, there was not a significant difference in paired testis mass (P > 0.05) (Fig. 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Paired testes mass (mg) in Peromyscus leucopus housed for up to 10 wk in either long (16L:8D) or short (8L:16D) days [F = 3.70, degrees of freedom (df) = 9, P = 0.01]. * Significant difference compared with all other groups (P < 0.05).

Seminiferous tubule diameters. Chronic exposure to short- compared with long-day photoperiods significantly reduced seminiferous tubule diameters in white-footed mice (P < 0.05, n = 5/group) (Table 1). After 8 wk, the average diameter for males housed in short days was reduced 27% compared with males housed in long days (P < 0.05). Average tubule diameters of males housed in short days at 8 wk was significantly reduced compared with males housed in long days for 2, 6, 8, and 10 wk and males housed in short days for 2, 4, and 6 wk (P < 0.05) (Table 1). After 10 wk of photoperiod exposure, average seminiferous tubule diameters were reduced 29% in short- compared with long-day housed males; diameters after 10 wk of short-day exposure were significantly reduced compared with all long-day housed males and males housed in short days for 2, 4, and 6 wk (P < 0.05 for each comparison) (Table 1).

Spermatogenic index. Spermatogenic index, a measure of reproductive competence, was reduced with long-term exposure to short photoperiods (P < 0.05, n = 5/group) (Table 1). After 8 and 10 wk of short-photoperiod exposure, spermatogenic activity was significantly reduced compared with all males housed in long days and males housed in short days for 2, 4, and 6 wk (P < 0.05 in all cases) (Table 1).

Immunohistochemistry. Photoperiod had a marked effect on the expression of VEGF in the testes (Fig. 2). Qualitative analysis of VEGF in testicular cross sections (n = 6 sections/animal, 4 animals/group) revealed that labeling was abundant in both the seminiferous tubules and in interstitial regions in males housed in long days. Figure 2A is representative of cross sections from long-day housed males. VEGF staining was observed in vascular epithelial cells in long-day housed males, however, staining was most prominent in the interstitial Leydig cells and in the intratubular Sertoli cells (Fig. 2, A and C). Little immunoreactivity was noted in germ cells. The extent of staining throughout cross sections did not differ among males housed in long days. In contrast, short-day exposure induced a dramatic reduction in VEGF labeling; Fig. 2B shows a representative male housed for 10 wk in short photoperiods. Comparatively few endothelial cells were labeled in short-day housed males, and staining in Sertoli cells was severely reduced after 10 wk of short-day exposure. The general decrease in the extent of labeling is depicted in Fig. 2B. Figure 2D illustrates the reduced staining in endothelial and Sertoli cells. VEGF labeling was still noted in Leydig cells in the interstitium of short-day housed males, however, the extent of staining was greatly reduced.

View larger version (167K): [in this window] [in a new window]   Fig. 2.   Extent of vascular endothelial growth factor (VEGF) labeling and localization of VEGF in seminiferous epithelium of white-footed mice. Light micrographs shown are representative examples of mice housed for 10 wk in either long (A, C) or short (B, D) days. Arrows identify stained Sertoli cells in the seminiferous tubule, interstitial Leydig cells are indicated by *. A, B: magnification ×200, scale bar 100 µm. C, D: magnification ×1,000, scale bar 25 µm.

VEGF protein electrophoresis. Testicular VEGF expression was detected after protein electrophoresis by Western blot analysis (n = 5/group). The pattern of total testis VEGF expression did not differ among males housed in long days. In contrast, VEGF protein levels in the testis were significantly reduced after exposure to short days (P < 0.05) (Fig. 3).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Representative Western blot analysis of testicular VEGF protein expression in white-footed mice housed for 2, 4, 8, or 10 wk in long) or short days (n = 5/group). Lanes 1, 3, 5, and 7 represent long-day housed animals after 2, 4, 8, or 10 wk, respectively. Lanes 2, 4, 6, and 8 represent animals housed in short days (LD 8:16) for 2, 4, 8, or 10 wk, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Mean (±SE) VEGF protein expression in the testes of white-footed mice housed for 2, 4, 8, or 10 wk in long or short days (n = 5/group) (F = 2.87, df = 9, P = 0.002). Quantification from optical density readings is presented as micrograms of protein equivalent to the internal standard of each blot. No significant differences were observed among males housed in long days (P > 0.05). * Significant difference (Fisher's least-significant difference, P < 0.05) in mean VEGF expression compared with males at 2, 4, 8, and 10 wk of long-day exposure.

	DISCUSSION

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The present study demonstrates that VEGF protein levels in the testes are reduced after white-footed mice are exposed to short (LD 8:16) compared with long (LD 16:8) photoperiods. The decline in VEGF expression occurred within 2 wk after males were transferred to short days. Testicular expression of VEGF remained reduced from week 2 to 10 of short-day exposure, compared with males housed in long days through 10 wk. In the same animals, reproductive function, as assessed by measurements of seminiferous tubule diameter and spermatogenic activity, was not reduced until 8 wk of short-day exposure. In addition, testicular regression did not occur until 10 wk after initial exposure to short days. Therefore, the data are inconsistent with the hypothesis that changes in VEGF are a consequence of gonadal regression.

VEGF immunoreactivity indicates that this protein accumulates in Leydig and Sertoli cells in the testes of white-footed mice, with low amounts of staining in germ cells and vascular endothelial cells. This is consistent with the pattern of VEGF expression observed in the testes of rats, humans, and chickens (4, 5, 16). Transfer to short days resulted in reduced VEGF expression in both of these somatic cell types in white-footed mice; however, the reduction was most evident in Sertoli cells of short-day housed males. The distribution of VEGF in the testis supports previous hypotheses of a paracrine mitogenic and support function for this growth factor, which maintains functional vessels within the seminiferous epithelium (5, 16).

In white-footed mice, circulating testosterone concentrations decrease after 6-8 wk of short-day exposure (23, 24). The decrease in testicular expression of VEGF occurs after 2 wk of short-day exposure, well in advance of the average time to plasma testosterone reduction in white-footed mice. VEGF expression is regulated by sex steroid hormones in other androgen-dependent tissues (20). Administering physiological concentrations of dihydrotestosterone to adult Sprague-Dawley rats increases the expression of VEGF mRNA in the prostate (20). In addition, plasma testosterone concentrations are highly correlated with testicular capillary blood flow in the seasonally breeding red fox (15). The association between plasma and intratesticular concentrations of testosterone has not been carefully examined in white-footed mice. It is possible that a threshold intratesticular concentration of testosterone is required for VEGF expression typical of long-day housed males. Transfer to short days could induce a rapid reduction of testosterone within the testis. This decrease may not be sufficiently large enough to result in detectable changes in circulating androgen concentrations; however, the intratesticular reduction might be sufficient to prevent long-day levels of VEGF expression.

Alternatively, VEGF expression in the testis, unlike in the prostate, may occur independently of changes in testosterone concentrations. During reproductive recrudescence in the red fox, increases in testicular capillary blood flow precede increases in testis mass and correlate with the onset of spermatogenic activity (15). In the adult golden hamster, increases in angiogenic sprouting occur after 12 wk of short-day exposure before the gonadotropin surge that occurs during spontaneous recrudescence (15). This suggests that testicular vessiculogenesis or angiogenesis must occur before increases in testosterone and implies pituitary control of vessel birth and expansion processes (15). Consistent with this, plasma luteinizing hormone correlates with the decline in testis activity during regression in deer mice (Peromyscus maniculatus), and short photoperiod induces a rapid decrease in serum folicle-stimulating hormone that precedes gonadal regression in Siberian hamsters (Phodopus sungorus) (2, 10). Seasonal remodeling of testicular vasculature via endothelial growth factors may therefore be under gonadotropic or hypothalamic control or both. In addition, VEGF expression may be regulated through the pineal indolamine hormone melatonin. Short day lengths result in an extended duration of nocturnal melatonin secretion that occurs immediately after transfer to short photoperiod (3, 12). Exposure to long durations of melatonin results in reduced gonadotropin-releasing hormone (GnRH) synthesis and secretion, leading to reproductive regression (12). It is possible that increased melatonin could result in decreased VEGF expression in the testes either indirectly, through the GnRH system, or directly, mediated through melatonin receptors in the testes. Additional studies are necessary to specify the endocrine cues and transduction pathways for VEGF during testicular regression.

In addition to its function as an angiogenic growth factor, VEGF is known to function as a key regulator of blood vessel maintenance (7, 14, 18, 21). The involvement of VEGF in vasculature maintenance may result in structural repercussions in the testis that are not immediately observable after short day reductions in protein expression. The rapid decline in VEGF expression after transfer of animals to short days suggests an early loss of vessel support during testicular regression. During testicular regression in adult golden hamsters, decreases in testicular blood vessel volume are significantly and strongly correlated with the decline in testis mass (16). The abrupt reduction in VEGF expression observed in white-footed mice housed in short days in the present study suggests that loss of blood vessel maintenance may be an initial step in the cascade of events that leads to testicular regression.

Inhibition of blood vessel support signals would likely result in a slow degradation of vasculature within the testis. Such degeneration might reduce oxygen and nutrient supply to the seminiferous epithelium, potentially resulting in an increase of apoptotic cell death. In addition, VEGF expression in severe combined immunodeficient mice is associated with increased expression of cell survival factor Bcl-2 and decreased expression of a cysteine protease involved in apoptosis, caspase-3 (18). A reduction of VEGF expression in the testis is therefore anticipated to be correlated with increases in apoptotic cell death. Indeed, increases in germ cell apoptosis are observed after 4 and 6 wk of short-day exposure in white-footed mice (23, 24). Determining whether this cell death is a direct result of blood vessel degeneration or the product of reduced gonadotropin support requires further study. The results of the present study suggest that signals leading to blood vessel degradation occur early in regression; reduction in VEGF occurs before increases in apoptotic germ cell death, reduction of spermatogenic activity, decreases in testis mass, and reductions in plasma testosterone in white-footed mice (23).

In summary, our correlational data suggest that VEGF expression may be involved with regulation of healthy, functioning vasculature in the testicular parenchyma of white-footed mice. Expression levels do not differ among long-day housed males, but transfer to short days results in a rapid decrease in the protein. Decreases in testicular VEGF precede decreases in reproductive competence by ~6 wk and testis mass by ~8 wk, suggesting that changes in VEGF are not a consequence of gonadal regression. Future studies examining the expression of this protein over the time course of reproductive recrudescence should be conducted to further understand the regulatory role that VEGF plays during the testicular cycle of seasonally breeding rodents.