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Biology of Reproduction 64, 1439-1443 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Aromatase Messenger RNA Is Derived from the Proximal Promoter of the Aromatase Gene in Leydig, Sertoli, and Germ Cells of the Rat Testis1

Marilena Lanzino3,,a, Stefania Catalano3,,a, Christelle Genisselb, Sebastiano Andoa, Serge Carreaub, Kent Hamrac, and Michael J. McPhaul2,,d

a Department of Cell Biology, Faculty of Pharmacy, University of Calabria, 87030 Rende (CS), Italy b Department of Biochemistry, University of Caen, UPRES EA 2608, 14032-Caen, France c Green Center for Reproductive Biology and the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857 d Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857

ABSTRACT

It has long been recognized that individual cell types within the testes possess the capacity to synthesize estrogen. A number of studies on different species have demonstrated that the levels of aromatase expression and the patterns of regulation are distinct between the different cell types of the testes. Whereas a variety of promoters have been shown to contribute to the patterns of aromatase expression in different cell lineages, studies using ovarian RNA, testis RNA, and Leydig cell tumor lines have demonstrated that the same promoter (promoter II) was used in each. Recent experiments using potent aromatase inhibitors or analysis of animals in which the genes encoding the estrogen receptor-alpha (ER-{alpha}) or the aromatase, P450, are defective, have confirmed the importance of local estrogen formation in normal testicular function. In order to permit experiments to identify the elements controlling aromatase expression in the individual cell compartments of the testes, we prepared RNA from purified preparations of Leydig, Sertoli, and germ cells. Using specific oligonucleotide primers, the sites of initiation of the aromatase mRNA were determined using rapid amplification of cDNA ends (RACE) and nucleotide sequence analysis of the resulting cDNA fragments. Our results indicate that aromatase mRNA is derived from the proximal promoter (PII) of the aromatase gene in each of the major cell types of the rat testes.

aromatase, gene regulation, Leydig cells, Sertoli cells, spermatid

INTRODUCTION

The principal steroid product of the testis is testosterone, which plays a central role in the control of spermatogenesis. In addition to the production of androgen, the capacity of the testis to synthesize estrogens has been well established [1–3]. Unlike androgens, however, the role that estrogen plays in the physiology of the testis has not been well understood. Estrogens have been shown to play a significant role in regulating testicular steroidogenesis through modulation of microsomal enzymes (17 hydroxylase and 17–20 desmolase) in the adult rat [4, 5]. In addition, it has been suggested that estrogens may have a mitogenic role in both Sertoli and Leydig cell proliferation, and in the control of complex morphological and biochemical changes that occur during spermatogenesis [6].

These descriptive studies have been paralleled by experiments in animals that have suggested the importance of estrogen to normal testicular function. In primates, the long-term use of aromatase inhibitors was found to lead to a selective defect in spermiogenesis [7]. These inferences have been supported by data from the characterization of mice in which the ER{alpha} or aromatase genes have been disrupted. Disruption of the ER{alpha} gene led to a progressive atrophy of the seminiferous epithelium that was traced to an alteration in fluid dynamics within the testis [8–10]. Disruption of the murine aromatase gene yielded a different phenotype. Homozygous mutant mice were initially fertile, but displayed an age-dependent decrease of fertility [11]. In aggregate, these studies have demonstrated that estrogens play distinct and unsuspected roles in regulating the resorption of fluid by the epididymis and in spermatogenesis.

The biosynthesis of estrogens from androgens is catalyzed by the enzyme complex termed, aromatase, which is composed of two polypeptides, a specific form of cytochrome P450 (P450arom, encoded by the CYP19 gene), and a ubiquitous, nonspecific flavoprotein, NADPH-cytochrome P450 reductase [12]. Aromatase is expressed in a number of cells and tissues, including ovarian granulosa cells [13], testicular Leydig and Sertoli cells [14–17], placenta [18, 19], adipose tissue [20, 21], skin, and several sites in the brain (hypothalamus, hippocampus, and amygdala) [22, 23]. This tissue-specific expression appears to be accomplished by a number of different mechanisms. In some cases, the CYP19 gene appears to be regulated by the use of tissue-specific promoters (in humans, cows, pigs, horses, chickens, and rats) [24–28]. A promoter proximal to the translation start site, called promoter II (PII) [29–31], regulates the expression of P450arom in ovaries of human, rat, mouse, and chicken. PII is also dominant in fetal gonads [32] and in two rat Leydig tumor cells (R2C and H540) [33–35].

The localization of P450arom within the testis has been a subject of considerable interest during recent decades. In the rat there is an age-related change in the cellular localization of the aromatization site, primarily in Sertoli cells in immature animals, but located in Leydig and germ cells (pachytene spermatocytes and spermatids) in adults [17, 36–40]. The present study was undertaken in order to identify which promoter is employed in the expression of P450arom transcripts in the different cell types of the rat testis. Analyses of the aromatase cDNA clones isolated from these cells suggest that PII is the principal promoter that is active in each of these cell lineages.

MATERIALS AND METHODS

Purification of Sertoli Cells

The testes of immature (15- and 21-day-old) Wistar rats were minced and then subjected to enzymatic treatments with collagenase-dispase (0.05%), soybean trypsin inhibitor (0.005%), deoxyribonuclease (0.001%) in Ham F-12/Dulbecco modified Eagle medium (Mediatech, Inc., Herndon, VA) (DMEM; 1:1, v:v) for 15 min at 37°C. Purification of Sertoli cells was carried out as previously described [41]. The purified Sertoli cells were incubated with fresh medium without serum for 24 h, and then treated for additional 24 h in presence or absence of 0.5 mM dibutyryl-cAMP (dbcAMP). Contamination with peritubular cells, evaluated by cytochemical detection of alkaline phosphate activity, was not evident [42]. The proportion of Leydig cells present was evaluated by cytochemical detection of 3ß-hydroxysteroid dehydrogenase activity [43] and was estimated to comprise less than 1% of the cell cultures.

Preparation of Leydig Cells

Testes of mature rats (90 days) were decapsulated and submitted to an enzymatic treatment with collagenase-dispase (0.05%), soybean trypsin inhibitor (0.005%), and deoxyribonuclease (0.001%) in Ham F-12/DMEM (1:1, v:v) for 10 min at 32°C. The Leydig cells were purified on discontinuous Percoll gradients [44] and characterized by histochemical staining for 3ß-HSD; colored cells were identified as Leydig cells (3ß-HSD positives).

The RNA was extracted from freshly purified Leydig cells (>95% pure, the remaining cells were macrophages and fibroblasts).

Isolation of Rat Germ Cells by Sedimentation at Unit Gravity

To isolate purified populations of pachytene spermatocytes and round spermatids, a cellular suspension was generated from the testes of two adult rats and then fractionated over a 2%–4% BSA gradient for ~3 h at 4°C using a Staput Apparatus according to the method of Bellve [45]. The peak pachytene spermatocytes (F #6–10) and spermatid (F #25–32) fractions were identified by Nomarski differential interference contrast microscopy, and determined to be greater than 98% tetraploid and greater than 99% haploid, respectively, following analysis by Feulgen.

RNA Isolation

Total cellular RNA was extracted from cultured Sertoli cells using the Total RNA Isolation System kit (Promega, Madison, WI). The Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX) was used to purify RNA from germ cells. The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis prior to use.

Rapid Amplification of cDNA Ends and Southern Blot Analysis

Rapid amplification of cDNA ends (RACE) was performed using 10 µg of total RNA from Sertoli cells and 5 µg of total RNA from Leydig and germ cells using the Marathon cDNA amplification kit (Clontech Laboratories, Palo Alto, CA). For the first amplification, antisense primer, AS1 (5'-AGCCAGGACCTGGTATGGAAGATGAGCTCT-3', located in exon II) was used in combination with the adapter primer. For the second, nested amplification, antisense primer, AS2 (5'-AATCAGGAGGAGGAGGCCCATGATCAG-3', located in exon II) was combined with the adapter primer. The amplified products were run on 1.2% agarose gel and blotted on ZetaProbe Blotting Membranes (Bio-Rad Laboratories, Hercules, CA). The membrane was hybridized with a specific antisense oligonucleotide, AS3 (5'-ATGGCACTGACAGGCACAGTT-3') for exon II. The oligonucleotide was labeled with [32P]ATP using polynucleotide kinase. The positions of the oligonucleotides relative to the structure of the aromatase gene promoter localized immediately upstream of the translation start site is shown in Figure 1.



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  		FIG. 1. Schematic representation of PII and nucleotide sequences of the isolated RACE clones. Above is represented a schematic map of the proximal promoter (PII) of the aromatase rat gene. The transcription start site is indicated by an asterisk. In this numbering scheme, the initiator, methionine, is located at +97. Arrows show the positions of the oligonucleotide primers (AS1, 2, and 3) used to perform the RACE reactions and analyze the products. Below is shown a schematic representation of 40 individual clones isolated from different testicular cell types. While the majority of clones isolated are consistent with derivation from PII, in some samples (e.g., 15-day-old Sertoli cells), cDNA clones were isolated that suggested contribution from upstream elements

Sequencing

PCR products were subcloned into the pCR 2.1 vector (TA Cloning Kit, Invitrogen) and sequenced using the Thermosequenase kit (U.S. Biochemicals).

Animal Care

All animals were treated according to the "Principles of Laboratory Animal Care" formulated by the U.S. National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals, prepared by the U.S. National Institutes of Health. The Animal Care and Research Advisory committees at the individual institutions approved all animal protocols.

RESULTS

In order to obtain a comprehensive view of the promoters used in the different cell lineages that express aromatase mRNA in the male gonad, the RACE method was used to amplify the 5' ends of the aromatase mRNAs expressed in purified rat Sertoli cells, Leydig cells, and germ cells (pachytene spermatocytes and spermatids). The general structure of the aromatase gene around PII and the positions of the oligonucleotides employed in the studies are shown in Figure 1.

In the R2C rat Leydig tumor cell line, initiation of transcription of aromatase mRNA occurs approximately 100 nucleotides upstream of the initiator, methionine [33, 34]. Consistent with these prior results, RACE analysis of R2C RNA identified a single predominant band approximately 250 nucleotides in length (Fig. 2). Using the same method of analysis, bands of similar size were identified using RNA prepared from Sertoli cells isolated at 15 days of age. Similar sized bands were also identified in samples from Sertoli cells isolated from 21-day-old animals and from Leydig cells (90 days of age). Although additional species of aromatase transcripts were detected following hybridization with aromatase-specific probes, analysis of each of these populations by sequence analysis suggested that the majority of transcripts were derived from the promoter immediately upstream of the site of translational initiation. Some small proportion of transcripts appeared to be initiated from a site further upstream (Fig. 2B).



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  		FIG. 2. RACE product amplification of P450arom from rat testicular cells. The amplified products were separated on a 1.2% agarose gel and visualized using ethidium bromide staining (A), and transferred to Zeta-Probe membranes. Aromatase specific fragments were detected by hybridization of the membrane with oligonucleotide AS3 for exon II (B). Lanes 1–2, R2C cells (cDNA diluted 1:10 and 1:50); lanes 3–4, Sertoli cells from 15 days (1:10 and 1:50 cDNA dilution); lane 6, R2C cells; lane 7, Sertoli cells 21 days; lane 8, Sertoli cells 21 days + dbcAMP; lane 10, R2C cells; lane 11, Leydig cells; lane 12, spermatids; lane 13, pachytene spermatocytes. Negative controls (no cDNA added) are shown in lanes 5, 9, and 14

Although aromatase mRNA has been identified as being expressed in cells of the germinal epithelium, the sites of transcription initiation of aromatase mRNA have not been previously identified in these cells. As depicted in Figure 2, whereas some heterogeneity is observed, our results suggest that the majority of transcripts in these cells are derived from PII.

DISCUSSION

The capacity of individual cell types within the testes to synthesize estrogen has long been recognized. Furthermore, it has become evident from such studies that the pattern of aromatase expression differs among the different cell types of the testis, differing substantially when studied at different ages [17, 36–40].

Despite these observations, data that estrogen may serve a functionally important role in the function of the testis has been limited. Shetty et al. [7] demonstrated that the long-term administration of potent aromatase inhibitors to male Bonnet monkeys led to a dramatic reduction of sperm counts. Their studies suggested that these reductions were caused by an inhibition of spermiogenesis [7]. The characterization of mice carrying targeted disruptions of the ER{alpha} and aromatase genes have provided additional insights into the role that estrogen plays in normal testicular physiology. These studies have demonstrated that male mice carrying disruptions of either gene display reduced fertility. In the case of mice carrying the disrupted ER{alpha} gene, the reduction of fertility was traced by changes in fluid absorption that resulted in pressure atrophy of the seminiferous tubules [8–10]. Disruption of the aromatase gene, by contrast, yielded mice that are initially fertile, but they displayed progressive abnormalities of spermatogenesis that appeared in an age-dependent fashion. These studies demonstrated that spermatogenesis primarily was arrested at early spermiogenic stages, and identified a significant reduction in round and elongated spermatids. These findings were believed to be consistent with a requirement for local estrogen synthesis in order to support normal spermatogenesis [11].

A number of different mechanisms have evolved in different species to regulate the expression of aromatase. In some instances, distinctive promoter elements are employed to direct expression of aromatase mRNAs encoding a single aromatase protein derived from a single aromatase gene [24–28]. In other species, distinctive patterns of regulation are achieved by the evolution of distinct aromatase genes [24, 25]. In a minority of instances, distinctive patterns of aromatase expression are derived from the transcription of the aromatase gene from a single promoter element [35].

The results of previous studies have implicated PII (proximal promoter) in controlling the expression of aromatase in the testis [32]. These experiments have examined only a limited number of sample types, and no information was available for the structure of the aromatase mRNAs that are present in the individual cell types. The experiments reported here establish that the promoter located immediately upstream of the site of transcriptional initiation (PII) directs the expression of aromatase mRNA in Sertoli cells, Leydig cells, and in germ cells. Although our results cannot exclude a contribution of a small number of aromatase mRNAs from distinctive promoter elements (e.g., in Sertoli cells from 15-day-old animals), our findings suggest that investigations directed at the study of the control of aromatase mRNA expression in individual cell compartments should be focused on elements contained within the proximal promoter of the aromatase gene (PII).

FOOTNOTES

First decision: 25 October 2000.

1 Supported by grant I-1090 from the Robert A. Welch Foundation, and DK03892 from the National Institutes of Health. Back

2 Correspondence: Michael J. McPhaul, Department of Internal Medicine, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8857. FAX: 214 648 8917; michael.mcphaul{at}utsouthwestern.edu Back

3 These authors contributed equally to this work. Back

Accepted: December 15, 2000.

Received: September 14, 2000.

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