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Endocrinology Vol. 138, No. 1 41-48
Copyright © 1997 by The Endocrine Society

ARTICLES

Estrogens Potentiate the Stimulatory Effects of Follicle-Stimulating Hormone on N-Cadherin Messenger Ribonucleic Acid Levels in Cultured Mouse Sertoli Cells1

Colin D. MacCalman, Spiro Getsios, Riaz Farookhi and Orest W. Blaschuk

Division of Urology, Department of Surgery, McGill University, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1A1; and the Department of Physiology, McGill University (R.F.), Montreal, Quebec, Canada H3G 1Y6

Address all correspondence and requests for reprints to: Dr. Orest W. Blaschuk, Urology Research Laboratories, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. E-mail: MDOB{at}MUSICA.McGILL.CA


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gonadal steroids and FSH are key regulators of Sertoli cell function. N-Cadherin (N-cad) is a calcium-dependent cell adhesion molecule that mediates Sertoli cell-germ cell interactions. We recently demonstrated that steroids, in particular estradiol, are potent regulators of testicular N-cad messenger RNA (mRNA) levels in vivo. In view of the cooperative effects of steroids and FSH on Sertoli cell-germ cell interactions, we examined the combined effects of these hormones on N-cad mRNA levels in cultured mouse Sertoli cells. FSH was capable of increasing N-cad mRNA levels 2-fold in these cells. The effects of FSH on N-cad mRNA levels in cultured Sertoli cells were mimicked by cAMP-inducing agents. Treatment of the Sertoli cell cultures with FSH and estradiol stimulated N-cad mRNA levels 3-fold, whereas steroids alone had no effect on N-cad mRNA levels. These studies demonstrate that FSH and estradiol in combination are required to achieve maximal N-cad mRNA levels in cultured Sertoli cells. The results obtained from these studies substantiate the hypothesis that estrogens play a pivotal role in regulating spermatogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SERTOLI CELLS of the mammalian seminiferous epithelium regulate germ cell development (1). The structural framework for spermatogenesis results from specific junctional interactions that occur between Sertoli cells and germ cells of the testes. FSH and gonadal steroids are both capable of mediating Sertoli cell function (2, 3). FSH receptors have been localized to the Sertoli cell membrane (4). These receptors are coupled to a cAMP-dependent intracellular signaling pathway that is activated upon ligand binding (5, 6). Several studies have indicated that FSH contributes to the regulation of spermatogenesis in the testis (2, 7). For example, FSH has pronounced effects on the organization of the Sertoli cell cytoskeleton (8, 9), facilitating Sertoli cell-germ cell interactions in vitro (10). In addition, the exposure of neonatal rats to FSH has been shown to increase Sertoli cell and germ cell numbers in the mature animal (11). However, the ability of FSH to maintain spermatogenesis in the adult remains unclear.

The administration of either FSH or testosterone (Tt) to hypophysectomized rats can maintain approximately 70% of normal spermatogenesis (12, 13, 14). Tt treatment has distinct effects on Sertoli cells when examined in vitro and in vivo. Sertoli cell morphology is essentially unaffected by Tt treatment in vitro. In contrast, Tt is essential for maintaining Sertoli cell-germ cell junctional complexes in vivo (9). Recent studies indicate that Tt and FSH act in concert to establish Sertoli cell-germ cell interactions and, consequently, regulate germ cell development (8, 9, 15). Omission of either hormone results in a reduction in the number of junctional interactions between Sertoli cells and round spermatids in vitro and in vivo (8, 9). Furthermore, the maximum number of specific junctional interactions between Sertoli cells and round spermatids is observed in the presence of both FSH and Tt (8).

The role of estrogens in spermatogenesis remains unclear. Studies have demonstrated the presence of estrogen receptors in the Sertoli cells and Leydig cells of the testis (16). Sertoli cells, germ cells, and Leydig cells are each capable of synthesizing estrogens from Tt at various stages of testicular development (17, 18). Furthermore, a reduction in testicular estrogen levels inhibits spermatid maturation (19, 20). Normal spermatogenesis has been reported in hypophysectomized rats receiving FSH and estradiol (E2) (21). Recently, mice lacking a functional estrogen receptor (designated ERKO mice) have been shown to have a significantly reduced rate of spermatogenesis (22). Although a detailed developmental analysis of these mice has not been completed, preliminary observations reveal that the seminiferous tubules are collapsed and contain few germ cells in the testes of adult ERKO mice (23). These studies suggest that E2 plays a key role in maintaining spermatogenesis.

The molecular mechanisms by which estrogens regulate spermatogenesis remain to be determined. We have recently shown that E2, but not Tt, dihydrotestosterone (DHT), or progesterone, is capable of regulating N-cadherin (N-cad) messenger RNA (mRNA) levels in the immature mouse testis (24). N-cad is a calcium-dependent cell adhesion molecule that mediates cellular interactions in a homotypic manner (25, 26). N-cad is present in the mouse and rat testes (27, 28, 29, 30). Immunolocalization studies revealed that N-cad is present at the interface between Sertoli cells and spermatocytes (28, 29, 31). Furthermore, antibodies directed against N-cad inhibit Sertoli cell-germ cell interactions in vitro (32). This cell adhesion molecule is, therefore, directly involved in maintaining the collective organization of the testis (31).

FSH has been shown to regulate aromatase levels in the testis of the prepubertal mouse and rat (33, 34). It has been suggested that there is a cooperative effect between steroids and FSH in maintaining spermatogenesis (35). In the present study, we have examined the ability of FSH and gonadal steroids, alone or in combination, to regulate N-cad mRNA levels in cultured Sertoli cells. In addition, as the action of FSH in Sertoli cells involves stimulation of cAMP production (5, 6, 36), we have investigated the combined effects of steroids and this intracellular second messenger on N-cad mRNA levels in these cells. We report that E2 potentiates the effects of FSH on N-cad mRNA levels in cultured Sertoli cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of Sertoli cells
Sertoli cells were isolated from the testes of 17-day-old Swiss-Webster mice using a method adapted from Dorrington et al. (5) and O’Brien et al. (37). The mice were killed by cervical dislocation, and the testes were removed, decapsulated, and finely chopped. Forty testes were digested at 32 C in 20 ml Hanks’ balanced salt solution (HBSS; Life Technologies, Burlington, Canada) supplemented with 1 mg/ml trypsin (type 3; Sigma Chemical Co., St. Louis, MO) and 5 µg/ml deoxyribonuclease (type 1; Sigma Chemical Co.). After incubation for 30 min with shaking (60 oscillations/min), the tubules were transferred to a 50-ml plastic tube and allowed to sediment at unit gravity for 2 min. The supernatant was removed, and the tubules were incubated with 1 mg/ml soybean trypsin inhibitor (type 1-s; Sigma Chemical Co.) for 2 min. The tubules were washed three times with HBSS (Life Technologies), and then transferred to a 125-ml Erlenmeyer flask containing 1 mg/ml collagenase (type 1; Sigma Chemical Co.) in 20 ml HBSS for 30 min. The digest was centrifuged at 100 x g for 2 min, and the Sertoli cell pellet was washed three times with Ca- and Mg-free HBSS (Life Technologies) containing 1 µg/ml deoxyribonuclease (type 1; Sigma Chemical Co.). The cells were resuspended at a ratio of 1:10 (vol/vol) in Eagle’s MEM (Life Technologies) supplemented with fungizone (625 µg/liter), streptomycin (100 mg/liter), penicillin (105 IU/liter), nonessential amino acids (1%), and L-glutamine (1%; Life Technologies). Aliquots of 1 ml were transferred to Primaria culture plates (100 x 20 mm; Fisher Scientific, Montreal, Canada) containing 9 ml supplemented MEM. The plates were incubated at 32 C for 48 h under a water-saturated atmosphere of 5% CO2 in air. Contaminating germ cells were then removed by hypoosmotic lysis (38). The Sertoli cells were cultured for an additional 3 days in fresh medium before being used in the experiments. The primary cultures were composed of at least 98% Sertoli cells, as judged by the criteria of Tung and Fritz (39), such as determining the number of vimentin (a Sertoli cell marker)-positive cells relative to the number of vimentin-negative cells using immunocytochemical methods.

Hormone treatments
Sertoli cells were cultured under the following conditions. To determine the effects of human FSH (8466 IU/mg; lot AFP-570D, National Pituitary Program, NIDDK) alone on N-cad mRNA levels, cultures were exposed to either increasing doses of FSH (0–100 ng/ml) for 12 h or a fixed dose of FSH (30 ng/ml) for 3, 6, 12, or 24 h. Steroid effects were examined by culturing the Sertoli cells with increasing doses (0, 50 nM, 100 nM, or 1 µM) of E2, Tt, or DHT for 12 h in the absence or presence of FSH (30 ng/ml).

To determine whether Tt was having a direct effect on N-cad mRNA levels, Sertoli cells were cultured with either 1 µM Tt plus 30 ng/ml FSH or 1 µM E2 plus 30 ng/ml FSH in the presence or absence of the aromatase inhibitor, 4-androsten-4-ol-3,17-dione acetate (4-ATD; 1 mM; Sigma Chemical Co.). The concentration of 4-ATD used in this study was chosen on the basis of previous studies (40, 41).

The ability of cAMP to stimulate N-cad mRNA levels in Sertoli cells was tested with several different effectors. Sertoli cells were cultured for 6 h in the presence or absence of 1 µM E2 and (Bu)2cAMP (1 mM), forskolin (10 µM), or cholera toxin (1 µg/ml; Sigma Chemical Co.). The concentrations of the agents used in this study were chosen on the basis of previous studies (3, 6).

Northern blot procedures
Total RNA was prepared from the cultured Sertoli cells by the phenol-chloroform method of Chomczynski and Sacchi (42). Approximately 40 µg total RNA were obtained from 1 million cells.

Total RNA was separated by electrophoresis in 1% agarose-formaldehyde gels and transferred onto a charged nylon membrane, as described by MacCalman et al. (43). Approximately 15 µg total RNA were loaded into each well of the gels. The Northern blots were incubated in a solution composed of 3% BSA (Sigma Chemical Co.) dissolved in 5 x SSPE (20 x SSPE consists of 0.2 M sodium phosphate monobasic, pH 7.4, containing 25 mM EDTA and 3 M NaCl) at 37 C for 30 min. They were then transferred to a prehybridization solution of 5 x SSPE containing 50% deionized formamide, 5 x Denhardt’s solution (purchased from 5 Prime, 3 Prime, Boulder, CO), 5% dextran sulfate (obtained from Pharmacia, Piscataway, NJ), 1% SDS, 50 mM sodium phosphate dibasic, and 5 mM sodium phosphate monobasic. The blots were incubated in this solution at 37 C for 60 min. Heat-denatured salmon sperm DNA (final concentration, 0.2 mg/ml; purchased from 5 Prime, 3 Prime) and the radiolabeled N-cad complementary DNA (cDNA) probe were then added to the prehybridization solution. The cDNA probe was described in detail by Chen et al. (44). The probe was radiolabeled by the random primer method of Feinberg and Vogelstein (45) and heat-denatured before being added to the prehybridization solution. The blots were incubated in the presence of the radiolabeled probe for 24 h at 37 C, then washed twice with 2 x SSPE at room temperature (5 min/wash), twice with 2 x SSPE containing 1% SDS at 55 C (30 min/wash), and twice with 0.2 x SSPE at room temperature (30 min/wash). Finally, the blots were subjected to radioautography to detect the hybridization of the radiolabeled probe to the mRNA species. The radioautograms were scanned using an LKB laser densitometer (LKB, Rockville, MD). The Northern blots were then reprobed with a radiolabeled synthetic oligonucleotide specific for 18S ribosomal RNA (rRNA), according to the protocols described by Chen et al. (44). The blots were again subjected to radioautography to detect the hybridization of the radiolabeled probe to the 18S rRNA. The radioautograms were then scanned with the laser densitometer. The absorbance values obtained with respect to each N-cadherin mRNA species were normalized relative to the 18S rRNA absorbance value.

Statistical analysis
The results are presented as the mean relative absorbance (±SE) for three independent experiments. Statistical differences between the treated and untreated groups were assessed by ANOVA. In the presence of a significant F value, individual groups were compared using the least significant difference test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Three N-cad mRNA species of 5.6, 4.7, and 3.8 kilobases were present in all of the total RNA extracts prepared from cultured Sertoli cells. Initially, we examined the effects of gonadal steroids on N-cad mRNA levels in cultured Sertoli cells. The cells were cultured in the presence of varying concentrations (50 nM to 1 µM) of E2, Tt, or DHT for 12 h. These steroids were incapable of stimulating any of the N-cad mRNA species above control levels in the Sertoli cells (Figs. 1Go and 2Go).



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  		Figure 1. Radioautograms of a Northern blot probed with radiolabeled N-cad cDNA (upper panel) and then reprobed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA (middle panel). The blot contains RNA extracted from Sertoli cells cultured for 12 h in either the absence of E2 (lane a) or in the presence of 50 nM, 500 nM, or 1 µM E2 (lanes b–d, respectively). The positions of the 28S and 18S rRNA species are shown on the left side of the upper panel. The two radioautograms were scanned using a laser densitometer. The values obtained for the three N-cad mRNA transcripts were then normalized relative to the absorbance values obtained for the 18S rRNA. The results derived from this analysis as well as those from two other studies (radioautograms not shown) are represented (mean ± SEM; n = 3) in the bar graphs.

 


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  		Figure 2. Radioautograms of a Northern blot probed with radiolabeled N-cad cDNA (upper panel) and then reprobed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA (middle panel). The blot contains RNA extracted from Sertoli cells cultured for 12 h in the absence of steroids (lane a) or in the presence of 1 µM E2 (lane b), 1 µM Tt (lane c), or 1 µM DHT (lane d). The positions of the 28S and 18S rRNA species are shown on the left side of the upper panel. The two radioautograms were scanned using a laser densitometer. The values obtained for the three N-cad mRNA transcripts were then normalized relative to the absorbance values obtained for the 18S rRNA. The results derived from this analysis as well as those from two other studies (radioautograms not shown) are represented (mean ± SEM; n = 3) in the bar graphs.

 
In contrast, FSH alone (30 ng/ml) was capable of causing a significant increase in Sertoli cell N-cad mRNA levels within 6–12 h of culture compared to the N-cad mRNA levels in cells that were not exposed to this hormone (Fig. 3Go). The effect of FSH on the Sertoli cell N-cad mRNA levels was also dose dependent, with maximal stimulation occurring at FSH concentrations of 30 and 100 ng/ml (Fig. 4Go).



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  		Figure 3. Radioautograms of a Northern blot probed with radiolabeled N-cad cDNA (upper panel) and then reprobed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA (middle panel). The blot contains RNA extracted from Sertoli cells cultured in the presence of FSH (30 ng/ml) for 0, 3, 6, 12, or 24 h. The positions of the 28S and 18S rRNA species are shown on the left side of the upper panel. The two radioautograms were scanned using a laser densitometer. The values obtained for the three N-cad mRNA transcripts were then normalized relative to the absorbance values obtained for the 18S rRNA. The results derived from this analysis as well as those from two other studies (radioautograms not shown) are represented (mean ± SEM; n = 3) in the bar graphs. Values indicated by asterisks were significantly greater than control values (P < 0.05). All other groups were not significantly greater from each other.

 


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  		Figure 4. Radioautograms of a Northern blot probed with radiolabeled N-cad cDNA (upper panel) and then reprobed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA (middle panel). The blot contains RNA extracted from Sertoli cells cultured for 12 h in either the absence or presence of varying concentrations of FSH. The positions of the 28S and 18S rRNA species are shown on the left side of the upper panel. The two radioautograms were scanned using a laser densitometer. The values obtained for the three N-cad mRNA transcripts were then normalized relative to the absorbance values obtained for the 18S rRNA. The results derived from this analysis as well as those from two other studies (radioautograms not shown) are represented (mean ± SEM; n = 3) in the bar graphs. Values indicated by asterisks were significantly greater than control values (P < 0.05). All other groups were not significantly greater from each other.

 
To determine whether FSH altered the ability of Sertoli cells to respond to the steroids, Sertoli cell cultures were incubated for 12 h in the presence of FSH and varying concentrations of E2 (50 nM to 1 µM; Fig. 5Go). Increasing concentrations of E2 progressively enhanced the effects of FSH on N-cad mRNA levels. A maximal effect was observed at 500 nM E2. There was no further enhancement when the concentration of E2 was increased to 1 µM. Similar results were obtained when the Sertoli cells were cultured in the presence of FSH and Tt (Fig. 6Go). The stimulatory effects of FSH and Tt on the N-cad mRNA species appear to be specific for this aromatizable androgen, as DHT had no effect on N-cad mRNA levels (data not shown).



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  		Figure 5. Radioautograms of a Northern blot probed with radiolabeled N-cad cDNA (upper panel) and then reprobed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA (middle panel). The blot contains RNA extracted from Sertoli cells cultured for 12 h in the absence of hormone (lane a) or in the presence of 1 µM E2 (lane b), 30 ng/ml FSH (lane c), 30 ng/ml FSH plus 50 nM E2 (lane d), 30 ng/ml FSH plus 500 nM E2 (lane e), or 30 ng/ml FSH plus 1 µM E2 (lane f). The positions of the 28S and 18S rRNA species are shown on the left side of the upper panel. The two radioautograms were scanned using a laser densitometer. The values obtained for the three N-cad mRNA transcripts were then normalized relative to the absorbance values obtained for the 18S rRNA. The results derived from this analysis as well as those from two other studies (radioautograms not shown) are represented (mean ± SEM; n = 3) in the bar graphs. Values indicated by asterisks were significantly greater than control values (P < 0.05). All other groups were not significantly greater from each other.

 


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  		Figure 6. Radioautograms of a Northern blot probed with radiolabeled N-cad cDNA (upper panel) and then reprobed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA (middle panel). The blot contains RNA extracted from Sertoli cells cultured for 12 h in the presence of 1 µM E2 (lane a), 1 mM 4-ATD (lane b), 30 ng/ml FSH (lane c), 30 ng/ml FSH plus 1 mM 4-ATD (lane d), 1 µM Tt plus 30 ng/ml FSH (lane e), 1 µM Tt plus 30 mg/ml FSH plus 1 mM 4-ATD (lane f), 1 µM E2 plus 30 mg/ml FSH (lane g), or 1 µM E2 plus 30 ng/ml FSH plus 1 mM 4-ATD (lane h). The positions of the 28S and 18S rRNA species are shown on the left side of the upper panel. The two radioautograms were scanned using a laser densitometer. The values obtained for the three N-cad mRNA transcripts were then normalized relative to the absorbance values obtained for the 18S rRNA. The results derived from this analysis as well as those from two other studies (radioautograms not shown) are represented (mean ± SEM; n = 3) in the bar graphs. Values indicated by asterisks were significantly greater than control values (P < 0.05). All other groups were not significantly greater from each other.

 
To determine whether Tt was having a direct effect on N-cad mRNA levels, or whether the effect was due to its conversion to E2, Sertoli cells were cultured in the presence of FSH and either Tt or E2 and in the presence or absence of the aromatase inhibitor, 4-ATD (40, 41). In the presence of 4-ATD, there was no enhancement of the FSH effect with Tt (Fig. 6Go). E2 and FSH continued to have an additive effect in the presence of 4-ATD.

Finally, to elucidate the mechanism by which FSH regulates N-cad mRNA levels, Sertoli cells were cultured with (Bu)2cAMP, cholera toxin, or forskolin in the presence or absence of E2. A significant increase in the levels of all three N-cad mRNA transcripts was observed in Sertoli cells cultured with either the cAMP analog or the cAMP-inducing agents (Fig. 7Go). E2 enhanced the effects of these agonists on N-cad mRNA levels.



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  		Figure 7. Radioautograms of a Northern blot probed with radiolabeled N-cad cDNA (upper panel) and then reprobed with a radiolabeled synthetic oligonucleotide specific for 18S rRNA (middle panel). The blot contains RNA extracted from Sertoli cells cultured for 12 h in the in the presence of 1 µM E2 (lane a), 1 mM (Bu)2cAMP (lane b), 10 µM forskolin (lane c), 10 µg cholera toxin (lane d), 1 µM E2 plus 10 µM forskolin (lane e), 1 µM E2 plus 1 mM dibutyrl cAMP (lane f), or 1 µM E2 plus 10 µg cholera toxin (lane g). The positions of the 28S and 18S rRNA species are shown on the left side of the upper panel. The two radioautograms were scanned using a laser densitometer. The values obtained for the three N-cad mRNA transcripts were then normalized relative to the absorbance values obtained for the 18S rRNA. The results derived from this analysis as well as those from two other studies (radioautograms not shown) are represented (mean ± SEM; n = 3) in the bar graphs. Values indicated by asterisks were significantly greater than control values (P < 0.05). All other groups were not significantly greater from each other.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Three N-cad mRNA species of 5.6, 4.7, and 3.8 kb were detected in all extracts prepared from the cultured Sertoli cells. These N-cad mRNA transcripts have been previously detected in mouse heart, brain, ovary, testes, and isolated Sertoli cells (29, 46, 47, 48). The present studies demonstrate that FSH can regulate testicular N-cad mRNA levels in vitro. This FSH stimulation of N-cad mRNA levels can be further enhanced by the addition of E2. All FSH effects could be mimicked by cAMP or the cAMP-inducing agents under our experimental conditions.

Sertoli cells were prepared from 17-day-old mice. Animals of this age were chosen because the yields of Sertoli cells are greater than those obtained from younger animals. Sertoli cells isolated from these animals are highly responsive to FSH (49). FSH action on N-cad mRNA expression was time dependent. FSH caused an increase in the levels of all three N-cad mRNA transcripts after 6 h. N-cad mRNA levels decreased after 24 h of treatment with FSH. This time-dependent effect may be a result of the cells becoming refractory to the hormone. Prolonged exposure to FSH results in desensititzation, an increased rate of cAMP catabolism (50, 51), and receptor down-regulation (36).

Steroids alone were incapable of stimulating N-cad mRNA levels in vitro. In contrast, these hormones are potent regulators of testicular N-cad mRNA levels in vivo (24). Others have previously shown that steroids are often incapable of stimulating Sertoli cell functions in vitro. For example, androgens have little effect on the morphology of Sertoli cells and fail to enhance Sertoli cell-germ cell interactions in vitro (8), whereas Tt is essential for Sertoli cell-germ cell interactions in vivo (14). Androgen and estrogen effects on cultured Sertoli cells may be difficult to elicit due to a reduction in the number of steroid receptors expressed by these cells in vitro (16) or the lack of other factors essential for steroid action (52).

The greatest N-cad mRNA levels were observed in Sertoli cells that were cultured in the presence of FSH and either Tt and E2. DHT did not significantly enhance the effects of FSH on the levels of the three N-cad mRNA species. These observations suggest that Tt may act directly on the N-cad mRNA levels of the Sertoli cells or that it is first metabolized to E2. The ability of the aromatase inhibitor, 4-ATD, to block the additional effect of Tt confirms that E2 is the key steroidal regulator of N-cad mRNA levels in Sertoli cells.

Recently, Cameron and Muffly (8) reported that FSH and Tt are essential for maximal Sertoli cell-germ cell binding in coculture. Unfortunately, the ability of E2 to regulate Sertoli cell-germ cell interactions was not determined in these studies. E2 has been shown to influence Sertoli cell function and germ cell development. For example, spermatid maturation in rats is reduced after the injection of an aromatase inhibitor (19, 20, 53). Nitta et al. (18) reported that the germ cells of the mouse seminiferous epithelium are capable of producing E2, and that this hormone may be involved in germ cell development. Furthermore, mice lacking a functional estrogen receptor have a reduced rate of spermatogenesis (22). Preliminary histological examination of these ERKO mice has revealed abnormalities in the testis, including compromised seminiferous tubules and reduced germ cell numbers (23). Finally, administration of the estrogen antagonist, tamoxifen, in male rats also compromises the structural integrity of the seminiferous epithelium and blocks spermatogenesis (54). Collectively, these observations as well as those presented herein suggest that E2 is involved in regulating spermatogenesis.

The actions of FSH are mediated by specific receptors that are functionally coupled via membrane-associated G proteins to the adenyl cyclase cAMP-generating pathway (6, 36). To better define the mechanism by which E2 and FSH regulate N-cad mRNA levels in cultured Sertoli cells, we examined the ability of E2 and cAMP to regulate N-cad mRNA levels. The cAMP analog or the cAMP-inducing agents examined were capable of increasing N-cad mRNA expression. E2 enhanced the stimulatory effects of cAMP. In this context, it should be noted that Brabant et al. (55) and Coutifaris et al. (56) demonstrated that E-cadherin mRNA levels are regulated by cAMP in thyrocytes and choriocarcinoma cells, respectively.

In conclusion, these studies demonstrate that FSH alone is incapable of maximally stimulating N-cad mRNA levels in cultured Sertoli cells. E2 in conjunction with FSH appears to be necessary to achieve maximal N-cad mRNA expression in these cells. We speculate that the previously reported ability of FSH and E2 to coregulate Sertoli cell-germ cell interactions is due at least in part to their ability to comodulate the testicular levels of the cell adhesion molecule, N-cad. The results presented herein support the hypothesis that estrogens in addition to androgens are important gonadal steroid regulators of spermatogenesis.


   Footnotes
 
1 Dedicated to the memory of Irving B. Fritz. This work was supported by funds from the Medical Research Council of Canada. Back

Received April 26, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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