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Expression and Regulation of ⁵-Desaturase, ⁶-Desaturase, Stearoyl-Coenzyme A (CoA) Desaturase 1, and Stearoyl-CoA Desaturase 2 in Rat Testis

Andrology Laboratory, Department of Gynecology and Obstetrics,² Institute of Clinical Biochemistry and Department of Clinical Chemistry,³ Rikshospitalet University Hospital, N-0027 Oslo, Norway

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and ⁵- and ⁶-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of ⁵- and ⁶-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, ⁵- and ⁶-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. ⁵- and ⁶-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)₂cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.

epididymis, follicle-stimulating hormone, gene regulation, Sertoli cells, testis

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In mammalian cells, the dietary essential fatty acids, linoleic acid (18:2[n-6]) and linolenic acid (18:3[n-3]), are converted to the important fatty acids 20:4(n-6) and 22:5(n-6) [1] and 22:6(n-3) [2] by alternating steps of elongation and desaturation. These modifications include both ⁵-desaturation and ⁶-desaturation, where ⁶-desaturase catalyzes the rate-limiting step in this conversion [3]. The ⁹-desaturases introduce a double bond in position 9 in the nonessential stearic acid, 18:0, to form oleic acid, 18:1(n-9). Oleic acid can go through the same steps of desaturation and elongation as linoleic and linolenic acid, providing the cell with 20:3(n-9) and 22:4(n-9). In this way the ⁹-desaturation provides alternative precursors when the cell suffers from essential fatty acid deficiency (EFAD). In rodents two isoforms of ⁹-desaturase are found: stearoyl-CoA desaturase 1 (SCD1) and stearoyl-CoA desaturase 2 (SCD2).

The rodent SCD1 and SCD2 were cloned already in the 1980s [4, 5]. But it was not until 1999, when Cho and Clarke cloned the human ⁵- and ⁶-desaturase [6, 7], followed by the cloning of the rat ⁵- and ⁶-desaturase [8, 9], that studies of the expression and regulation of all the four genes were made possible. Earlier reports show that SCD1 is expressed in liver and adipose tissue, while SCD2 is expressed in brain, spleen, heart, lymphocytes, and lungs [10]. ⁵- and ⁶-desaturase are expressed in different rodent tissues with the highest level of expression in adrenal gland, liver, brain, and testis and lower in white adipose tissue and kidney [11]. Several reports have been published on the hormonal regulation of desaturase activity, mainly in liver [12–15], while only a few studies have been performed with respect to the regulation of desaturase gene expression. ⁶-desaturase mRNA is reported to increase eightfold in the liver of insulin-treated diabetic rats [16].

The testis is an extraordinary organ regarding the fatty acid metabolism. Although rich in polyunsaturated fatty acids (PUFAs), the testis is continuously drained of these fatty acids as the spermatozoa are transported to the epididymis. This makes a great contrast to other PUFA-rich tissues, like the brain and retina. Testicular cells are rich in PUFAs, with different fatty acids dominating, depending on the species. The rat testis has a high content of 22:5(n-6) [17], whereas 22:6(n-3) is dominating in human [18] and monkey [19] testis. It has earlier been described that germ cells are especially rich in PUFAs, more than the Sertoli cells [20], while the Sertoli cells seem to be more active in converting the dietary essential fatty acids to 22:5(n-6) and 22:6(n-3) than germ cells are [21]. A PUFA transport from the Sertoli cells to the germ cells has therefore been postulated.

In the progression of germ cell differentiation from spermatogonia to condensing spermatids in mouse, the total amount of 22:5(n-6) increases from 2% to 20% of total fatty acids but is lowered in mature sperm [22]. During sperm maturation in the epididymis, the acrosome membrane undergoes a well-defined series of chemical, physical, and functional changes. These changes include the addition of highly unsaturated phosphatidylcholine. Studies on ram sperm indicate an increase in total 22:6(n-3) content from 14% to 25% [23], which leads to a decrease in general membrane stability. At the same time, addition of cholesterol and an actively maintained asymmetric transmembrane phospholipid distribution modulate these effects to stabilize the membrane of the mature sperm for storage. Furthermore, the tail of the monkey spermatozoa has a higher content of 22:6(n-3) than the head. It has therefore been speculated whether this distribution of PUFAs is important for the membrane fluidity needed for proper motility [24]. These PUFA asymmetries, both the acrosome transmembrane and the head-to-tail asymmetry, are necessary to produce a fertile gamete.

It has further been reported that there is a significant lower level of 22:6(n-3) in the fatty acid composition of phospholipids of spermatozoa collected from patients with impaired sperm motility and patients with low sperm concentration compared to normal patients [25]. The reason for this difference is not known.

In this study, we investigate the testicular expression and regulation of the four desaturases involved in the metabolism of polyunsaturated fatty acids.

	MATERIALS AND METHODS

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Animals

Weaning Sprague-Dawley rats were obtained from M&B (Ry, Denmark). After separation from their mother, the rats were given a standard diet from Grimstone Aldbrough (Hull, UK). Whole testis, epididymis, liver, and kidney were dissected from CO₂-asphyxiated rats, frozen in the vapor phase of liquid nitrogen, and kept at -80°C for later RNA extraction.

Germ Cell Preparation

Germ cells were isolated from decapsulated testes from 32-, 44-, and 58-day-old Sprague-Dawley rats. An enriched germ cell suspension was obtained by consecutive treatment with collagenase, trypsin, and DNase [26, 27]. The cells were then further fractionated by centrifugal elutriation, followed by separation in density gradients of Percoll, into pachytene spermatocytes (PS) and round spermatids (RST) [28]. By this we were able to achieve PS fractions with purity 87–96% and RST fractions with purity 90–95%. The purities were evaluated through flow cytometry with respect to ploidity (data not shown). For each germ cell isolation (PS and RST), testes from five animals were used. Two separate preparations representing all three ages were analyzed.

Sertoli Cell Preparation

Primary cultures of Sertoli cells were obtained from testes of infant (19-day-old) and juvenile (35-day-old) Sprague-Dawley rats, according to the method of Dorrington et al. [29]. These ages were chosen in order to obtain highly purified Sertoli cell preparations (19-day-old rats) and to study more mature Sertoli cells (35-day-old rats). Sertoli cells were plated on 10-cm Nunc Dishes (Nunc, Copenhagen, Denmark) to a final concentration of 10⁶/ml in 12.5 ml Eagles Minimum Essential Medium (MEM, 21090-022; Gibco BRL, Grand Island, NY; nonessential amino acids included). The MEM was supplemented with L-glutamine (2 mM), penicillin (100 IU/ml), streptomycin (0.10 mg/ml), fungizone (2.5 mg/ml), and 10% fetal bovine serum. Cultures were kept at 34°C in a humidified atmosphere of 5% CO₂ in air. On Day 3 after plating, the medium was changed to MEM without fetal bovine serum, and germ cells remnants (<4.0.10⁵/ml) were removed mechanically (19-day-old) or by hypotonic shock treatment (10% MEM in water, 35-day-old). On Day 5 the cells were harvested for RNA extraction (19-day-old: SC > 98%, 35-day-old: SC > 93%). These preparations were used for the localization studies. For the stimulation experiments, cultured Sertoli cells from 19-day-old rats were used.

In addition, freshly isolated preparations, enriched in Sertoli cells from testes of both 19- and 35-day-old rats, were analyzed (SC > 65%). For these preparations the cells were harvested for RNA extraction directly after isolation, without being plated [29].

For both age-groups (19 and 35 days) and isolation steps (cultured and freshly isolated cells), two separate preparations representing 10 animals each were analyzed.

Hormonal Treatment of Sertoli Cells

Stimulation experiments were started on Day 5 after plating. Cells were treated with dexamethasone (D-2915, Sigma, St. Louis, MO), insulin (Sigma I-6643), ovine follicle-stimulating hormone (FSH; Sigma F-8174), (Bu)₂cAMP (Sigma D-0627), and testosterone (A6950-000, Steraloids Inc., Newport, RI) for 3 or 12 h. The hormones were dissolved in PBS (Sigma D-8662), except for testosterone, which was dissolved in ethanol to a final concentration of 0.001%. In half the controls ethanol was added to a final concentration of 0.001%. Before being harvested, the cell cultures were washed twice with 5 ml MEM without fetal bovine serum. The concentrations and the duration of the different stimulations can be found in the figure legends. For each Sertoli cell stimulation experiment, testes from 22 animals were used, resulting in approximately 20 culture dishes. All treatments were repeated two to six times. The duplicates shown in Figures 4 and 5 represent RNA extracted from two separate cultures.

View larger version (63K): [in this window] [in a new window]   FIG. 4. Regulation of SCD1, SCD2, and 5- and 6-desaturase mRNA in rat Sertoli cells in response to dexamethasone and insulin. Sertoli cells from 19-day-old rats were isolated, and stimulation experiments in the primary cultures started on Day 5 after plating. The cells were treated with dexamethasone (0.1 µM and 1.0 µM) and insulin (0.02 µM and 2.0 µM) for 3 and 12 h. Each lane contains 20 µg of total RNA (A). The duplicates represent two separate cultures, and the results are representative for four to six experiments. The ethidium bromide stained 18S rRNA signal is shown below the blots. SCD1: stearoyl-CoA desaturase 1 (5.9-kb transcript), SCD2: stearoyl-CoA desaturase 2 (5.9-kb transcript), 5: 5-desaturase (4.1-kb transcript), and 6: 6-desaturase (3.8-kb transcript). The SCD1 blot in this figure is result of a 3-wk exposure. The lower graphs represent intensity readings from the scanning of the autoradiograms normalized to the 18S rRNA signals (B). The autoradiograms were imported by an Epson Expression 1680 Pro scanner and the intensities read with Kodak Digital Science 1D Image Analysis Software v. 3.5. The values plotted are relative to the values of their respective 3-h controls

View larger version (77K): [in this window] [in a new window]   FIG. 5. Regulation of SCD1, SCD2, and 5- and 6-desaturase mRNA in rat Sertoli cells in response to testosterone, FSH, and (Bu)2cAMP. Sertoli cells from 19-day-old rats were isolated and the stimulation experiments in the primary cultures started on Day 5 after plating. The cells were treated with testosterone (0.1 µM and 1.0 µM), FSH (0.1 IU/ml and 1.0 IU/ml), and (Bu)2cAMP (500 µM) for 3 and 12 h. All lanes marked with • correspond to cell cultures, which contained a final ethanol concentration of 0.001%, necessary for dissolving testosterone. Each lane contains 20 µg of total RNA (A). The duplicates represent two separate cultures, and the results are representative for two experiments. The ethidium bromide stained 18S rRNA signal is shown in the lower panel. SCD1: stearoyl-CoA desaturase 1 (5.9-kb transcript), SCD2: stearoyl-CoA desaturase 2 (5.9-kb transcript), 5: 5-desaturase (4.1-kb transcript), and 6: 6-desaturase (3.8-kb transcript). The SCD1 blot in this figure is a result of a 2-wk exposure. The lower graphs represent intensity readings from the scanning of the autoradiograms normalized to the 18S rRNA signals (B). The autoradiograms were imported by an Epson Expression 1680 Pro scanner and the intensities read with Kodak Digital Science 1D Image Analysis Software v. 3.5. The values plotted are relative to the values of their respective 3-h controls without ethanol

RNA Extraction and Northern Analysis

Total RNA was isolated by homogenization of tissue or cells in guanidine isothiocyanate, followed by centrifugation through a CsCl cushion and phenol-chloroform extraction [30]. RNA denatured in 50% formamide and 6% formaldehyde was size-fractionated (20 µg/lane) through a 1.5% agarose gel containing 6.7% formaldehyde with circulating sodium phosphate running buffer (20 mM) [31]. The RNA was transferred to a Magna nylon transfer membrane (MSI, Westborough, MA) by capillary blotting. Ethidium bromide staining of the gel was used to verify equal loading, and the 18S rRNA stain for each blot is shown in the figures.

Primers, Probes, and Hybridization

Single-stranded probes were synthesized and labeled with [-³²P]dCTP (Amersham Biosciences, Buckinghamshire, UK) by PCR, using a short synthetic DNA template [32]. For the ⁵-desaturase probe, we used a 20-mer antisense primer, 5'-GGGCACCAAGGAAGTTCCAA-3', providing a 108-mer probe covering the area between base pair 321 and 428 open reading frame (ORF) of the ⁵-desaturase cDNA (PubMed accession no. AF320509). For ⁶-desaturase, we used a 20-mer antisense primer, 5'-GGGAGGTAGCAAGGACAAAG-3', providing a 100-mer probe covering the area between base pair 415 and 514 (ORF) of the ⁶-desaturase cDNA (PubMed accession number AB021980). For the SCD1 probe, we used a 24-mer antisense primer, 5'-TATGCATTTATCATGTATGCTTAG-3', providing a 106-mer probe covering the area between base pair 1202 and 1307 (ORF) of the SCD1 cDNA (PubMed accession no. J02585). Finally, for SCD2, we used an 18-mer antisense primer, 5'-CACAGCCCGAGTGTTGAA-3', providing a 106-mer probe covering the area between base pair 1828 and 1933 (ORF) of the rat SCD2 cDNA (PubMed accession no. NM_031841). All primers and templates were obtained from MedProbe (Oslo, Norway). The probes were hybridized to the filters in 50% formamide, 5x Denhart's, 5x SSC, 0.1% SDS, 0.25 mg/ml nonhomological salmon DNA (Sigma, D-9156), 50 mM sodium phosphate (pH 6.5) at 42°C. The filters were washed in 2x SSC/0.1% SDS at room temperature for 4 x 10 min and 0.1x SSC/0.1% SDS at 50°C for 2 x 30 min. Autoradiography was carried out at -80°C with Hyperfilm ECL (Amersham Biosciences).

	RESULTS

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Localization

In this study, we show that in rat, ⁵- and ⁶-desaturase are highly expressed in testis and epididymis (Fig. 1). Liver and kidney were included as examples of tissues expressing high and low levels of ⁵- and ⁶-desaturase, respectively (Fig. 1). ⁵- and ⁶-desaturase follow the same expression pattern in these four tissues. Interestingly, the ⁵- and ⁶-desaturase expression in the epididymis seems to be as high, or even higher, than in liver.

View larger version (35K): [in this window] [in a new window]   FIG. 1. Expression of SCD1, SCD2, and 5- and 6-desaturase in different tissues from adult rat. Total RNA (20 µg/lane) was extracted from whole liver, kidney, epididymis, and testis tissue from 91-day-old rats. The ethidium bromide stained 18S rRNA signal is shown in the lower panel. SCD1: stearoyl-CoA desaturase 1 (5.9-kb transcript), SCD2: stearoyl-CoA desaturase 2 (5.9-kb transcript), 5: 5-desaturase (4.1-kb transcript), and 6: 6-desaturase (3.8-kb transcript). All lanes are from same blot. Each blot is representative for three individual experiments

SCD1 seems not to be expressed in testis like in kidney while expressed in epididymis at the same level as in liver (Fig. 1). In whole testis tissue from adult rats (91-day-old), SCD1 was not detectable with Northern blot. On the other hand, in testicular cell preparations, SCD1 can be observed in Sertoli cells (Fig. 2). SCD2 is expressed in testis and epididymis (Fig. 1), whereas in kidney and liver the expression is very low or absent.

View larger version (83K): [in this window] [in a new window]   FIG. 2. Expression of SCD1, SCD2, and 5- and 6-desaturase in separated testicular cells and whole testis tissue from rats of different ages. Total RNA was extracted from whole testis tissue (10-, 25-, 50-, and 80-day-old rats), enriched germ cell suspension (PS—pachytene spermatocytes and RST—round spermatids; 32-, 44-, and 58-day-old rats), freshly isolated preparations enriched in Sertoli cells, and Sertoli cells cultured for 5 days (19- and 35-day-old rats). Each lane contains 20 µg of total RNA. The ethidium bromide stained 18S rRNA signal is shown in the lower panel. SCD1: stearoyl-CoA desaturase 1 (5.9-kb transcript), SCD2: stearoyl-CoA desaturase 2 (5.9-kb transcript), 5: 5-desaturase (4.1-kb transcript), and 6: 6-desaturase (3.8-kb transcript). Each blot is representative for two individual experiments

In Figure 2, the cellular localization of SCD1, SCD2, ⁵- and ⁶-desaturase in the testis can be seen. Once again, ⁵- and ⁶-desaturase follow the same expression pattern. The highest levels of expression can be observed in freshly isolated preparations enriched in Sertoli cells. Variations in the desaturase expression among the Sertoli cell preparations may reflect the varying amount of germ cell impurities and the activity of the Sertoli cells in the various preparations. ⁵- and ⁶-desaturase are far lower expressed in all the different germ cell fractions than in the Sertoli cells. The age-dependent decrease in the desaturase signals seen in whole testis tissue confirms this cellular localization (Fig. 2). It is unknown whether the very weak signals observed in the germ cells fractions represent desaturase mRNA from germ cells or from contaminating cell types (e.g., Sertoli cells).

A more detailed age curve is presented in Figure 3. Here it can be seen that the signal intensity of both ⁵- and ⁶-desaturase correlates with the relative proportion of Sertoli cells in the developing testis [33–35]. The highest level of ⁵- and ⁶-desaturase mRNA is seen between Day 15 and Day 20, which coincides with the onset of spermatogenesis. At this age the Sertoli cells constitute the major cell fraction (>65%) [33–35]. On Day 25 the first haploid cells appear [34], and as they become more abundant, the desaturase signals, and the relative amount of Sertoli cells decrease proportionally, until leveling off between Day 40 and Day 50.

View larger version (89K): [in this window] [in a new window]   FIG. 3. Expression of SCD1, SCD2, and 5- and 6-desaturase in whole testis tissue from rats of different ages. Total RNA (20 µg/lane) was extracted from whole testis tissue of rats from 6 to 91 days of age. The ethidium bromide stained 18S rRNA is shown in the lower panel. SCD1: stearoyl-CoA desaturase 1 (5.9-kb transcript), SCD2: stearoyl-CoA desaturase 2 (5.9-kb transcript), 5: 5-desaturase (4.1-kb transcript), and 6: 6-desaturase (3.8-kb transcript). Each blot is representative for four individual experiments

SCD1 is expressed in testis, but at very low levels. The SCD1-blots in Figures 2 and 3 are results of more than 3 wk exposure, compared to approximately 1 wk for the other genes. SCD1 is expressed in Sertoli cells, whereas in germ cells the expression is close to absent. This finding is confirmed through repeated experiments (n = 4). As for ⁵- and ⁶-desaturase, the SCD1 expression pattern is confirmed by the age curve (Fig. 2). SCD2 is much higher expressed in testis than SCD1 (Fig. 1) and follows essentially the same distribution as the other desaturases (Fig. 2), with a slightly different age-dependent expression (Fig. 3).

Regulation

Dexamethasone and insulin up-regulate both ⁵- and ⁶-desaturase in Sertoli cells (Fig. 4), with a more rapid induction with dexamethasone than with insulin. The ⁵- and ⁶-desaturase signals are slightly higher when the cells are stimulated with 1.0 µM dexamethasone than with 0.1 µM dexamethasone. When looking at the insulin-stimulated Sertoli cells, the most marked up-regulation of ⁶-desaturase is seen with the lowest concentration (0.02 µM) for 12 h. With ⁵-desaturase, the mRNA expression is strongest at the highest concentration of insulin. Furthermore, the induction of ⁵-desaturase by dexamethasone and insulin seem to be weaker than for ⁶-desaturase (Fig. 4).

SCD1 mRNA level is down-regulated both by dexamethasone and by insulin, as readily observed after 3 h. For both hormones at both concentrations, a small up-regulation of SCD1 can be observed from 3 to 12 h, bringing the SCD1 gene expression toward the control levels. The regulation of SCD2 in response to dexamethasone and insulin is less pronounced than for the other desaturases (Fig. 4). Through repeated experiments (n = 6), we have been able to confirm a weak up-regulation of SCD2 mRNA following both dexamethasone and insulin treatment.

The last blot shows the Sertoli cell response to testosterone, FSH, and (Bu)₂cAMP with respect to ⁵-desaturase, ⁶-desaturase, SCD1, and SCD2 (Fig. 5). All four desaturases are up-regulated by FSH. This effect is confirmed through (Bu)₂cAMP stimulation, which propagates an even more pronounced up-regulation. The most dramatic effect is seen with SCD1. SCD1 mRNA turns from almost nondetectable to a high expression when the Sertoli cells are stimulated with FSH (1.0 IU/ml). The ⁵-desaturase, ⁶-desaturase, and SCD2 mRNA FSH response is most pronounced with 0.1 IU/ml FSH. By comparing the testosterone-treated Sertoli cell cultures with the control cultures with added ethanol, it seems that none of the desaturases is regulated by testosterone. On the other hand, ethanol (0.001%) seems to up-regulate all the four desaturases.

	DISCUSSION

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Localization

In this paper we show for the first time, to our knowledge, that SCD1 and SCD2 are expressed in testis. When comparing liver and testis from adult rats, the testis expresses far higher levels of SCD2 mRNA than liver, while the opposite distribution is seen for SCD1. Earlier reports reviewed by Ntambi [36] describe large differences in expression and regulation between the SCDs. SCD1 and SCD2 are both expressed in a tissue-specific manner and regulated differently in various tissues in response to fat free diets, PUFA supplements, and hormones. Some tissues also express these desaturases constitutively. Previously published papers on sperm lipids point in the direction that a functional sperm is dependent on a balanced composition of n-3, n-6, and n-9 fatty acids [24, 25, 37]. Spermatozoa enriched in n-9 fatty acids may turn out to be nonfunctional, for example, unable to undergo the acrosome reaction. As shown in this paper, SCD2 is responsive to in vitro hormonal treatment of testicular cells, indicating that there is a need for regulating the amount of n-9 acids in testis.

Furthermore, we have shown that all four desaturases are highly expressed in epididymis from sexually mature rats. To our knowledge, this has not been described for other tissues. Surprisingly, the epididymal expression of all the four desaturases is as high, or higher, than the hepatic expression. Two possible explanations could be given for this pronounced desaturase expression. Either the PUFAs are desaturated in epididymis as a step in the maturation process of the spermatozoa or the high-order PUFAs are synthesized to secure vital epididymal cells. Studies on ram sperm indicate an increase in total 22:6(n-3) content from 14% to 25% in the spermatozoa when passing through the epididymis [23]. However, this 22:6(n-3) increase is correlated with a selective loss of sperm phospholipids through the epididymis, resulting in an elevated proportion of choline plasmalogen-bound 22:6(n-3) [38].

Our previous studies on ⁶-desaturase activity indicated that the enzyme activity was much higher in Sertoli cells than in germ cells [21]. In this study we confirm these findings by demonstrating far greater ⁶-desaturase mRNA levels in Sertoli cells than in germ cell. Not surprisingly, ⁵-desaturase follows the same expression pattern as ⁶-desaturase. These findings, combined with the knowledge that germ cells are especially rich in PUFAs, more than the Sertoli cells [20], suggest that the PUFAs are modified in the Sertoli cells before transported to the germ cells. A lipid transport between the Sertoli and the germ cell was postulated as early as 1888 by Von Ebner, who was the first to confirm the presence of lipid droplets in the Sertoli cells [39]. The hypothesis has been refined and tested several times, but until today, it has not been supported by data from experiments designed for such studies [20, 21, 40].

Regulation

In this paper we show that dexamethasone and insulin up-regulate ⁵- and ⁶-desaturase gene expression in Sertoli cells, while the picture is more complex concerning the stearoyl-CoA desaturases. SCD1 mRNA is down-regulated by dexamethasone and insulin, while SCD2 is up-regulated by both hormones. The fact that SCD2 is several-fold higher expressed than SCD1 in testis suggests that SCD2 is responsible for the ⁹-desaturase activity in this organ.

Interestingly, the regulation of the desaturases in testicular cells is opposite of what is found in liver. De Gómez Dumm et al. reported as early as 1979 that dexamethasone depresses the ⁵- and ⁶-desaturase activity in liver microsomes while inducing the ⁹-desaturase activity [41]. However, this was an in vivo study on female rat liver microsomes measuring desaturase activity. Disregarding the experimental conditions, the different regulation observed may reflect the roles that liver and testis play in the fatty acid metabolism; the liver provides various tissues with fatty acids, while the testis has a high need of PUFAs. This fact could, from a physiological point of view, explain differences seen in liver and testis with respect to the regulation of ⁵- and ⁶-desaturases.

When looking at regulation of the stearoyl-CoA desaturase expression in dexamethasone-treated testicular cells, both de Gómez Dumm [41] and Marra et al. [42] have described similar trends in liver. At the transcriptional level we observe a weak up-regulation of SCD2 in Sertoli cells, while the two other groups found an increased ⁹-desaturase activity in liver microsomes [41, 42]. Marra et al. further suggested that a dexamethasone-induced protein was responsible for this activation [42].

Concerning the insulin effect on the desaturases, the presented results are in accordance with earlier results published on liver. We find that insulin up-regulates ⁵-desaturase, ⁶-desaturase, and the net stearoyl-CoA desaturase gene expression in rat Sertoli cells. Brenner and coworkers have studied diabetic rats and found sevenfold lower ⁶-desaturase mRNA in liver, whereas after 24-h insulin administration, the ⁶-desaturase mRNA level increased eightfold [16]. In an earlier study they found impaired hepatic ⁵- and ⁹-desaturase activities in addition to impaired ⁶-desaturase activity in diabetic rats [15]. These activities could be restored by insulin treatment.

⁶-desaturase is generally accepted as rate limiting in the formation of arachidonic acid, 20:4(n-6), and of eicosapentaenoic acid, 20:5(n-3), from linoleic acid, 18:2(n-6), and linolenic acid, 18:3(n-3), respectively [3]. It is therefore interesting that the level of ⁶-desturase mRNA is more strongly up-regulated than ⁵-desaturase mRNA by both insulin and dexamethasone.

In this study we show that all four desaturases are up-regulated by FSH in Sertoli cells, while testosterone does not seem to influence the desaturase gene expression. These results are in accordance with observations done in older animals by Marzouki and Coniglio in the 1980s [43, 44]. Marzouki et al. [43] showed that the testis of hypophysectomized sexually mature male rats converted less [1-¹⁴C] 20:3(n-6) into [1-¹⁴C] 20:4(n-6) and [1-¹⁴C] 22:4(n-6), concluding that hypophysectomy, hence loss of gonadotropin action, inhibited the ⁵-desaturase activity in testis. They further showed that treatment with testosterone did not affect the metabolism of any of the [¹⁴C] substrates in testis [43]. To our knowledge no such studies were performed with respect to ⁶- or ⁹-desaturase activity. Coniglio et al. [44] also showed that the elongation of PUFAs in testis from sexually mature rats was impaired by hypophysectomy and that the elongation activity was restored with FSH treatment but not with testosterone. Furthermore, the induction of the gene expression obtained with (Bu)₂cAMP in this study indicates that increased cAMP level is involved in the FSH-dependent up-regulation of all the desaturases.

When comparing the ⁵-desaturase, ⁶-desaturase, and SCD2 signals in freshly isolated preparations enriched in Sertoli cells with the signals in cultured Sertoli cells (Fig. 2), there is a marked difference. The highest desaturase mRNA levels are observed in the fresh Sertoli cell preparations, even though more germ cells are present in these preparations than in cultured Sertoli cells. Obviously, there must be factors, paracrine or endocrine, or metabolites present in the cell preparations that are lost on culturing.

FSH seems to be a much more important hormone for the Sertoli cells with respect to the desaturation/elongation apparatus than testosterone at this age of the rat. Although the level of androgen receptor (AR) is age dependent with highest level in the adult rats [45], expression of the receptor has been demonstrated in cultured Sertoli cells from 19-day-old animals [46]. In the male, FSH receptors are known to be localized only to the Sertoli cell. In this way FSH acts as an organ-specific regulator. When combining the results from this paper and the previously mentioned papers of Mazouki et al. [43] and Coniglio et al. [44], one can speculate whether FSH, under normal conditions, acts as a key hormone for the whole desaturation/elongation apparatus in the developing testis. It is well known that many, if not most, of the parameters of Sertoli cell function are increased as a result of FSH stimulation [47]. In our opinion, this links the desaturation/elongation apparatus to the Sertoli cell functions.

In 1979, Clausen et al. [34] published a paper where the onset of spermatogenesis is correlated with plasma FSH levels. Plasma FSH level increases approximately five times from Day 19 to Day 35 in the male rat. At the same time this interval covers both the onset of the spermatogenesis and the appearance of the first haploid cells. When comparing the age-dependent desaturase blot (Fig. 3) with the cellular composition of the rat testis [33–35], there seems to be a discrepancy from Day 15 to Day 40. In this interval the desaturase signals in whole testis tissue are higher than expected from the cellular composition with respect to Sertoli cells. It cannot be excluded that the desaturases are present in other somatic cell types of the testis. However, when taking into account the increase in plasma FSH levels occurring around this age, and the fact that FSH up-regulates the gene expression of the desaturases, these differences get a plausible explanation: In the developing testis, FSH enhances the desaturase expression in the Sertoli cells to meet the needs of a functional spermatogenesis.

In conclusion, all four desaturases are expressed testis and epididymis. The high expression in Sertoli cells and low to absent expression in germ cells strengthen the hypothesis of a lipid transport from the Sertoli cells to the germ cells. FSH strongly induces all four desaturases. Thus, we hypothesize that FSH is a key regulator of the desaturase expression in the Sertoli cells.

	ACKNOWLEDGMENTS

 
We want to thank Trine Henrichsen and Cathrine Bjørsvik at Andrology Laboratory, Department of Gynecology and Obstetrics, Rikshospitalet University Hospital, Oslo, for excellent technical work.

	FOOTNOTES

 
¹ Correspondence: Trine B. Haugen, Andrology Laboratory, Department of Gynecology and Obstetrics, Rikshospitalet University Hospital, N-0027 Oslo, Norway. FAX: 47 23 07 29 40; t.b.haugen{at}rh.uio.no

Received: 6 December 2002.

First decision: 6 January 2003.

Accepted: 11 February 2003.

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