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Regular Article |
a Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
ABSTRACT
We investigated the effects of aging on Sertoli cell-germ cell interactions from Brown Norway rats using the induction of four specific mRNAs as markers. The testes from aging (24 mo old) Brown Norway rats can be normal size or regressed. One marker, a von Ebner's-like protein, is expressed in coculture and "in vivo" in germ cells from normal testes of 6- and 24-mo-old rats but not in germ cells from regressed testes of 24-mo-old rats. A second germ cell marker, the Huntington disease protein, is expressed in all germ cells. Two Sertoli cell markers, a serotonin receptor and a novel gene, are induced in Sertoli cells by meiotic germ cells. The serotonin receptor mRNA is expressed in Sertoli cells from 20-day, 6-mo, and 24-mo normal testes but not in those from 24-mo regressed testes. The novel gene is induced in Sertoli cells from all testes. We conclude that Sertoli cells from aged regressed testes are unable to respond to selective signals from germ cells from young rats, and germ cells from regressed testes show a similar selective loss. Such disruptions in communication between Sertoli cells and germ cells likely contribute to germ cell loss during aging.
aging, gene regulation, Sertoli cells, spermatid, spermatogenesis, testis
INTRODUCTION
Reproductive aging in the male is characterized by dramatic changes in the seminiferous epithelium that eventually lead to decreases in steroidogenesis and spermatogenesis [1–3]. In the testes of aging mammals, total or partial regression of the seminiferous tubules is often seen as germ cell loss occurs. Along with variations in the population of germ cells present in the aging seminiferous tubules, Sertoli cells often show morphological changes ranging from multinuclearization and polymorphic mitochondria to an appearance of a less differentiated state [4, 5].
Brown Norway rats are an excellent model system for studying aging in mammals because this strain of rat does not exhibit many pathologies related to aging. Morphological analyses of the testes of Brown Norway rats reveal decreases in seminiferous tubule diameter, length, and volume that progress with age [2]. In 22-mo-old Brown Norway rats, testes sizes often vary, with one testicle showing regression and the other remaining normal in size [1]. In aging testes, atrophic tubules are often seen adjacent to tubules showing all the stages of spermatogenesis, suggesting that the process of degeneration starts at foci within each tubule [2, 3, 6]. Johnson et al. [7, 8] have shown that testicular atrophy is correlated with decreases in Sertoli cell number and the number of spermatids associated with each Sertoli cell.
During spermatogenesis, germ cells differentiate in close association with Sertoli cells. Distinct and well-defined populations of germ cells interact with Sertoli cells in a cyclic pattern determining the cycle of the seminiferous epithelium for a species. Sertoli cells secrete a large number of transport proteins and regulatory factors that are crucial for germ cell development [9]. Although the need for germ cells to maintain the cyclicity of Sertoli cells has been established [10–12], the specific germ cell factors involved in this process are less well understood. Clearly, interactions between somatic and germ cells cross-regulate gene expression [11, 12].
In contrast to aging testes with complete spermatogenesis, regression in aging testes is accompanied by alterations in the expression of numerous Sertoli cell proteins, including cathepsin, transferrin, clusterin, and inhibin [2, 13]. These declines closely parallel reduction in the number of spermatids associated with Sertoli cells. The germ cell loss during aging could be due to changes in the ability of Sertoli cells to support germ cell development or could be an intrinsic property of germ cell aging, or both.
To determine whether abnormalities in Sertoli cells or germ cells in testes of aging rats may disrupt Sertoli cell and germ cell communication, we have utilized cocultures of Sertoli cells and germ cells to assay markers genes known to be induced by cell-cell interactions [14, 15]. In cocultures and "in vivo", Sertoli cell factors up-regulate mRNAs encoding a von Ebner's-like protein and the Huntington disease protein in male germ cells, whereas a factor from pachytene spermatocytes induces mRNAs encoding a serotonin receptor and a novel gene in Sertoli cells [14, 15]. We utilized both cocultures and in vivo systems to investigate the effect of aging on the gene expression of these specific Sertoli cell and germ cell markers. We focused efforts on comparing the functional capabilities of Sertoli cells and germ cells from regressed and normal size testes of 24-mo-old Brown Norway rats. The Sertoli cells from aged regressed testes were unable to respond to selective signals from germ cells from young rats. Moreover, germ cells from regressed testes did not respond to certain signals from young Sertoli cells, showing a similar selective lose of induction capability.
MATERIALS AND METHODS
Isolation of Sertoli Cells
Sertoli cells were isolated from testes of 20-day-old, 6-mo-old, and 24-mo-old Brown Norway rats (Charles River, Kingston, MA and NIA, Bethesda, MD), using the method of Le Magueresse and Jegou [16] as previously described [17, 18]. To maximize the purity of Sertoli cells from adult rats, following trypsin incubation the testicular cell suspensions were filtered through glass wool to remove spermatozoa, the incubation time of the cells with collagenase was increased to 15–16 min (from 10 min) with continued monitoring by phase contrast microscopy until optimal cellular dissociations were seen, and hypotonic treatments to remove contaminating germ cells were carried out for 5–6 min instead of the usual 2–3 min. The Sertoli cells were cultured at 32°C in a humidified atmosphere of 5% CO2-95% air in Ham F-12, Dulbecco minimal essential medium (v/v) containing gentamycin (4 µg/ml), insulin (10 µg/ml), and transferrin (5 µg/ml) at a constant density of 2 x 106 cells/cm2 in polystyrene 
-irradiated Petri dishes (Falcon, Oxnard, CA). After 2 days of culture, the Sertoli cells were hypotonically shocked in 20 mM Tris HCl, pH 7.4, to remove nearly all germ cells. Twenty-four hours later, Sertoli cells were cocultured with germ cells for 24 h. The absence of significant numbers of contaminating cells was demonstrated by the absence of germ cell markers in the Sertoli cell RNA preparations following hybridization.
Isolation of Germ Cells
Testes from 6- and 24-mo-old Brown Norway rats were decapsulated and incubated with collagenase (1 mg/ml) for 15 min at 37°C. After the seminiferous tubules settled, interstitial cells were removed by decanting the supernatants. The seminiferous tubules were incubated with trypsin (1 mg/ml) for 20 min at 37°C, pipetted several times to obtain a single cell suspension, and filtered through 20-mm nylon mesh. After counting, the germ cells were used as described below. The absence of substantial numbers of contaminating Sertoli cells was demonstrated by the absence of Sertoli cell markers in the germ cell RNA preparations following hybridization.
Cocultures of Sertoli Cells and Germ Cells
Sertoli cells (2 x 106) from 20-day-old, 6-mo-old, and 24-mo-old rats were cocultured with germ cells (8 x 106) from 6- or 24-mo-old rats for 24 h. Germ cells were cultured in medium supplemented with 2 mM sodium pyruvate and 6 mM DL-lactate at a density of 8 x 106 cells/ml. At the end of culture, cells were scraped from the plates and RNA was purified [14, 15].
Northern Blot Hybridization
Complementary DNA probes were radiolabeled by random priming and denatured at 100°C for 5 min immediately before hybridization. Northern blot analysis was performed with GeneScreen membranes (DuPont/NEN, Boston, MA) as described previously [17, 18].
RESULTS
We have previously demonstrated that RNAs encoding a von Ebner's-like protein and the Huntington disease protein are up-regulated in germ cells cocultured with Sertoli cells [14], and mRNAs encoding a serotonin receptor and a novel gene are up-regulated in Sertoli cells cocultured with pachytene spermatocytes [15]. In this study, we used these genes as markers to evaluate functional expression in Sertoli cells and germ cells derived from the testes of young, sexually mature, and aged Brown Norway rats. At 24 mo of age, Brown Norway rats have testes of either normal or regressed sizes [3]. We describe below the differences in mRNA induction capabilities in Sertoli cells and germ cells isolated from regressed and normal testes. We define regressed testes as those that are smaller than normal-size testes in 24-mo-old rats of similar size.
Sertoli Cells from Regressed Testes but Not Normal Testes from Aging Brown Norway Rats Cannot Induce the von Ebner's-Like Protein mRNA in Germ Cells
Sertoli cells from prepubertal (20 day old), sexually mature (6 mo old), and aged (24 mo old, normal-size testes) Brown Norway rats can induce the 1.2-kilobase (kb) mRNA of the von Ebner's-like protein in cocultures with germ cells from prepubertal and 6-mo-old rats (Fig. 1A, lanes 2 and 5; Fig. 3B, lane 2). However, Sertoli cells derived from regressed testes of 24-mo-old rats fail to induce the von Ebner's-like protein mRNA in germ cells (Fig. 1A, lanes 8 and 9). As previously demonstrated for Sprague Dawley rats [14], control germ cells cultured in the absence of Sertoli cells do not express the von Ebner's-like protein mRNA (Fig. 1A, lanes 10 and 11; Fig. 3B, lane 3). This failure is not due to germ cell death after 24 h in culture because identical differential display patterns are obtained from germ cell RNA preparations before and after 24 h in culture [14]. The inability of Sertoli cells from 24-mo-old [fprats with regressed testes to induce the von Ebner's-like protein mRNA in cocultures is also seen in vivo, where 24-mo-old rats with regressed testes do not express the mRNA encoding von Ebner's-like protein (Fig. 1A, lane 14). However, testes from 24-mo-old rats with normal-size testes express the von Ebner's-like protein mRNA (Fig. 3A, lane 8).
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The Huntington disease protein mRNA is a second germ cell marker induced by a Sertoli cell factor [15]. It is up-regulated in germ cells by Sertoli cells from 20-day-old (Fig. 1A, lanes 2 and 3), 6-mo-old (Fig. 1A, lanes 5 and 6), and 24-mo-old animals with regressed testes (Fig. 1A, lanes 8 and 9) and by Sertoli cells from 24-mo-old animals with normal-size testes (Fig. 3B, lane 2). The Huntington disease protein mRNA is also detected in vivo in RNA prepared from 20-day, 6-mo, and 24-mo regressed (Fig. 1A, lanes 12–14) and 24-mo normal testes (Fig. 3A, lane 8). We conclude that Sertoli cells from regressed but not normal testes of 24-mo-old Brown Norway rats have lost the ability to induce the von Ebner's-like protein mRNA but not the Huntington disease protein mRNA in germ cells.
Germ Cells from Regressed Testes from Aging Brown Norway Rats Express Meiotic and Postmeiotic Markers
In contrast to the inability to express the von Ebner's-like protein mRNA in germ cells from regressed testes of 24-mo-old rats, three other germ cell markers are expressed (Fig. 1, A and B; Fig. 2). Lactate dehydrogenase (LDH) C4, a germ cell-specific isoprotein first expressed during meiosis [19], is detected in testis RNA isolated from 20-day old, 6-mo-old, and 24-mo-old animals with regressed testes (Fig. 1B, lanes 1–3). Hemiferrin, a second germ cell-specific protein first expressed in postmeiotic male germ cells [20], is also expressed in the regressed testes of 24-mo-old rats (Fig. 2B, lane 4). The Huntington disease protein mRNA is induced in coculture and expressed in vivo in 24-mo-old rats with regressed testes (Figs. 1 and 3). The presence of LDH C4 and hemiferrin mRNAs in regressed testes (Figs. 1–3) indicates the presence of meiotic and postmeiotic germ cells in sufficient numbers to be detectable by the relatively insensitive technique of Northern blotting, rather than reverse transcription-polymerase chain reaction, suggesting that a reduced but substantial number of germ cells remain in the regressed testes we have analyzed. Thus, although germ cells from regressed testes cannot express the von Ebner's-like mRNA, other mRNAs encoding the Huntington disease protein, LDH C, and transferrin continue to be expressed.
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Serotonin Receptor Induced in Sertoli Cells from Normal-Size Testes from Aged Brown Norway Rats Cannot Be Induced in Sertoli Cells from Regressed Testes
Two transcripts of about 7 and 0.8 kb, encoding a novel serotonin receptor, are expressed in Sprague Dawley rat Sertoli cells cocultured for 24 h with germ cells [15]. A similar induction is seen in Brown Norway rat Sertoli cells from 20-day-old, 6-mo-old, and 24-mo-old rats with normal testes cocultured with germ cells prepared from sexually mature (6 mo old) rats (Fig. 4, lanes 2 and 4; Fig. 5A, lane 2). However, the same germ cells fail to induce the serotonin receptor mRNAs in Sertoli cells derived from regressed testes of 24-mo-old rats (Fig. 4, lane 6). As seen in Sprague Dawley rats [15], induction of the serotonin receptor mRNA requires coculture of germ cells and Sertoli cells and is not detected when Sertoli cells from 20-day-old, 6-mo-old, or 24-mo-old rats are cultured alone (Fig. 4, lanes 1, 3, and 5; Fig. 5A, lane 1).
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The inability of aging Sertoli cells from regressed testes to express the serotonin receptor mRNA is not restricted to coculture. Although the serotonin receptor mRNAs are detected in total RNA from 20-day-old (Fig. 4, lane 8), 6-mo-old (Fig. 4, lane 9), and 24-mo-old normal testes (Fig. 5B, lane 3), the serotonin receptor mRNAs are not present in RNA from regressed testes (Fig. 4, lane 10).
The inability of aged Sertoli cells from regressed testes to express the serotonin receptor mRNAs is selective and does not reflect a general shutdown of Sertoli cell gene expression in regressed testes. A second novel gene, clone 1, is induced in Sertoli cells from 20-day-old and 6-mo-old rats and from 24-mo-old rats with both regressed and normal testes (Figs. 4 and 5A). It is also strongly expressed in vivo in 20-day, 6-mo, and 24-mo normal testes (Fig. 5B), and in 24 mo regressed testes (Fig. 4, lane 10). This suggests the inability of the Sertoli cell to express the serotonin receptor mRNAs is a selective loss of function.
DISCUSSION
In men, reproductive aging is associated with regressed seminiferous tubules containing Sertoli cells of altered appearance and an arrest of germ cell differentiation [3, 4]. In aging Brown Norway rats, similar changes are seen in the seminiferous epithelium. A recent morphological study revealed that at 24 mo of age a breakdown of the blood-testes barrier and striking changes in the structural appearance of Sertoli cells occurs [6]. These changes occur despite testosterone and circulating FSH levels sufficient to maintain spermatogenesis [1, 2]. The cause of these dramatic changes in the aging seminiferous epithelium is unknown, and speculation has centered on a limitation of stem cell renewal or an immunological cause of germ cell degeneration.
Here we propose that a breakdown in germ cell-Sertoli cell communication contributes to testicular regression. The breakdown may be caused by a loss of specific factors or an inability to respond to the factors leading to selective reduction in gene expression. In regressed testes of 24-mo-old Brown Norway rats, changes in Sertoli cell-germ cell interactions in both cocultures and in vivo produce selective losses of gene expression in both germ cells and Sertoli cells. In sexually mature Sprague Dawley rats, genes encoding a von Ebner's-like protein and the Huntington disease protein are induced in germ cells by Sertoli cell factors [14], and genes encoding a serotonin receptor and a novel unknown protein are induced in Sertoli cells by germ cell factors [15]. These genes are induced in 6- and 24-mo-old Brown Norway rats with normal testes, but a dramatic loss of expression is seen in both Sertoli cells and germ cells in regressed testes from 24-mo-old rats. The inability of germ cells from young animals to express the mRNA encoding von Ebner's-like protein but not the Huntington disease protein argues for a specific loss, likely a soluble factor secreted by Sertoli cells [14]. The inability of the Sertoli cells from 24-mo regressed testes to induce von Ebner's-like protein mRNA in germ cells from 6-mo-old animals (Fig. 1, lane 8) argues for a similar loss of inducing factor from the aging Sertoli cells. Moreover, the inability of the germ cells from regressed testes to respond to Sertoli cells from 20-day-old, 6-mo-old, and 24-mo-old rats suggests that although the soluble protein-like Sertoli cell inducing factor for the serotonin receptor [15] is available to the germ cells they are not capable of responding to it. The absence of the von Ebner's-like protein mRNA from the intact 24-mo regressed testes indicates that this deficiency is not a coculture artifact or is lost in cell isolations.
Although germ cells from 24-mo regressed testes do not synthesize the von Ebner's-like protein mRNA, they do respond to the Sertoli cell factor that induces the Huntington disease protein (Fig. 1A), and they express LDH C4 mRNA (Fig. 1B) and hemiferrin mRNA (Fig. 2). These two proteins are germ cell specific and are initially expressed in meiotic [19] and postmeiotic [20] germ cells, respectively, demonstrating the presence of a population of germ cells from both intervals of spermatogenesis in the regressed testes we have examined. Our results showing a decline in the expression of LDH C4 and hemiferrin genes in 24-mo regressed testes are in agreement with published reports showing that regressed testes contain a variety of tubules, ranging from normal to those virtually devoid of germ cells [2, 6]. The percentage of normal tubules decreases from 95% to 20% with age [2, 3, 6]. The expression pattern of germ cell-specific genes we have detected indicates that the regressed testes from 24-mo-old rats contain meiotic and postmeiotic germ cells (Figs. 1 and 2), and the inability of the germ cells to express the von Ebner's-like mRNA or to induce the serotonin receptor mRNA cannot be explained by the absence of germ cells. Moreover, no induction or expression of either mRNA was seen when up to fourfold (32 x 106) more germ cells were cocultured with Sertoli cells (data not shown). These differences in expression of the von Ebner's-like protein or serotonin receptor mRNAs are also seen in vivo, which suggests a selective loss of a cell's ability to express certain genes with aging. It is generally believed from reductions of inhibin, cyclic protein 2/cathepsin, transferrin, and clusterin expression [3, 13, 21, 22] that decreases in Sertoli cell function with age disrupt the support provided to germ cells, leading to decreases in spermatogenesis. The large reduction of clusterin and transferrin mRNA in the regressed testes (Fig. 2) confirms this hypothesis. We propose that germ cells from aging rats also show reduced function. This loss of function is selective because aging germ cells fail to induce the serotonin receptor mRNA but can induce clone 1 in Sertoli cells. Thus, aging germ cells appear to directly contribute to spermatozoa decreases as males age and perhaps to the increases in preimplantation loss, decreases in average fetal weight, and increases in neonatal death associated with increasing paternal age [23].
How can we interpret these selective decreases in signaling between germ cells and Sertoli cells? The von Ebner's-like protein is a member of the lipocalin protein, a large family of proteins that transport small hydrophobic molecules, including retinoids, steroids, and lipids [14]. The inability of aging Sertoli cells to induce the expression of such a protein in germ cells could easily disrupt cellular transport and cause a decrease in fertility. Similarly, the loss of a putative serotonin receptor in Sertoli cells by a failure of inducing factor from aging germ cells could adversely affect spermatogenesis through a nitric oxide pathway or may be a contributory factor to decreases in testosterone production [15]. Clearly, both the von Ebner's-like protein in germ cells and the putative serotonin receptor of Sertoli cells are likely to play essential but undefined roles in spermatogenesis, and we can only speculate on possible functions.
In summary, two cell type-specific markers of Sertoli cell-germ cell communication, a von Ebner's-like protein and a serotonin receptor, are selectively down-regulated in regressed testes of aging rats, and two others show no change. The selective loss of function in both Sertoli cells and germ cells raises the possibility of aging in both somatic and germinal cells of the mammalian testis and demonstrates the neccesity of two-way cellular communication between differentiating germ cells and their supporting cells during spermatogenesis.
ACKNOWLEDGMENTS
We thank Dr. E. Goldberg, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, for providing the LDH C4 cDNA. We also thank the anonymous reviewers, especially reviewer II, for their insightful comments and suggestions.
FOOTNOTES
1 This work was supported by NIH grants HD-11878 and T32 HD-07305. 
2 Correspondence: Norman B. Hecht, Center for Research on Reproduction and Women's Health, 421 Curie Blvd., 1310 BRBII/III (6142), University of Pennsylvania, Philadelphia, PA 19104. FAX: 215 573 5408;nhecht{at}mail.med.upenn.edu
3 Current address: Department of Surgery, Division of Urology, University of Massachusetts Medical School, Worcester, MA 01655.
Accepted: August 17, 2000.
Received: June 16, 2000.
REFERENCES
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