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ABSTRACT: Active immunization of boars against gonadotropin-releasing hormone (GnRH) inhibits luteinizing hormone (LH) and testicular steroids, so that mitosis of spermatogonia is reduced and apoptosis increased. To clarify whether high amounts of estrogens which are synthesized in the boar testis support spermatogenesis, a group of 6 boars was immunized against GnRH and then infused for 7 weeks with estradiol (E2-17β). For comparison, intact boars and immunized boars were infused with saline only. Testicular tissue was then analyzed by immunocytochemistry for apoptosis (TUNEL, EM), mitosis (Ki67), and estrogen receptor α (ERα). The specificity of ERα staining was confirmed by RT-PCR and Western blot. Immunization decreased LH and testosterone to minimal concentrations in immunized and E217β-infused immunized boars, whereas follicle-stimulating hormone (FSH) was not significantly altered. Estradiol decreased to base levels after immunization. Infusion increased E2-17β in peripheral blood plasma of the immunized boars to physiological levels. Except for A-spermatogonia, all spermatogenic cells decreased after immunization by about 60%. After estradiol infusion, cell counts increased again and were intermediate between control and immunized boars. Mitosis of spermatogonia was reduced by nearly 50% due to immunization but was partly restored by E2-17β infusion. Expression of ERα was localized in spermatogonia, suggesting stimulation of mitosis, which was further confirmed due to its predominant occurrence in stage I of the seminiferous epithelial cycle (main stage of cell division). Apoptosis was minimal in boars but elevated in the other 2 groups. Data showed that estrogens in physiological concentrations supported mitosis but were not sufficient to normalize sperm production because apoptosis was still high.
Testicular androgens are responsible for the maintenance of male reproductive functions, including spermatogenesis. In addition, small amounts of estrogens are measurable in peripheral plasma of males. Such trace amounts may result from steroidogenesis in the testes but also in cells of the adrenal cortex (Conley et al, 1996), as shown for many species, leading to peripheral concentrations on the order of 5–20 pg/mL (Eiler and Graves, 1977; Overpeck et al, 1978; Melnyk et al, 1992; Bujan et al, 1993). Several cell types in the testes were shown to express aromatase activity, such as Sertoli cells, Leydig cells, germ cells, and peritubular cells, depending on the species (Brodie and Inkster, 1993; Tsubota et al, 1993; Almadhidi et al, 1995; Levallet et al, 1998; Carreau et al, 2001; Fra˛cek et al, 2001; for review: O'Donnell et al, 2001). Testicular synthesis also explains the occurrence of small amounts of estrogens in semen as reported, eg, for the rat (Free and Jaffe, 1979), the bull (Ganjam and Amann, 1976; Eiler and Graves, 1977), and man (Bujan et al, 1993; Luboshitzky et al, 2002). Concentrations are on the order of 150 pg/mL (Reiffsteck et al, 1982). Studies in rats demonstrated the occurrence of estrogen receptors α and β (ERα, ERβ) both in Sertoli cells and in several types of spermatogenic cells, so that a role of estrogens for spermatid adhesion to Sertoli cells was suggested (Miura et al, 1999; Ebling et al, 2000; Pelletier et al, 2000; Hess, 2003).
In a few species, high amounts of estrogens are synthesized in the Leydig cells and are measurable in high concentrations in peripheral blood plasma, as shown for the stallion (Raeside, 1969; Setchell and Cox, 1986; Claus et al, 1992; Lemazurier et al, 2001) and the boar (Velle, 1958). In the latter species, peripheral concentrations may reach nearly 280 pg/mL blood plasma (Claus and Hoffmann, 1980). In the mature boar, aromatase activity occurs exclusively in the Leydig cells (Fra˛cek et al, 2001; Mutembei et al, 2005). Sertoli cell expression of aromatase is limited to a narrow period of fetal development (Claus et al, unpublished data). Substitution experiments with castrated boars demonstrated that the synergistic action of androgens and estrogens is necessary to ensure normal function of accessory sex glands, male libido, and sexual behavior (Joshi and Raeside, 1973; Parrot and Booth, 1984; Booth, 1988), and also the establishment of the anabolic potential (van Weerden and Grandadam, 1976).
In the boar, high amounts of total estrogens also occur in the fluid of spermatogenic tubules. They are primarily represented by 17β-estradiol (E2-17β), which may reach concentrations of 41 ng/mL in this fluid (Claus et al, 1985). A high proportion of E2-17β is transferred into the semen, so that the total amounts per ejaculate may reach 110 ng. These amounts influence female reproductive functions such as sperm transport and ovulation (Claus, 1989, 1990). The high tubular concentrations of estrogens additionally suggest an effect on spermatogenesis. An individual function of estrogens and a possible synergism with androgens remain to be clarified. Two types of estrogen receptors are well described, estrogen receptors α and β (ERα, ERβ). In the boar the presence of the receptor types in different cells within the testes was investigated by immunocytochemistry and the ERβ demonstrated in Sertoli cells (Mutembei et al, 2005), germ cells (Rago et al, 2004; Mutembei et al, 2005), and Leydig cells (Mutembei et al, 2005). ERα immunoreactivity was shown in germ cells (Rago et al, 2004; Mutembei et al, 2005) and Leydig cells (Rago et al, 2004; Mutembei et al, 2005). A more detailed study, however, using in situ hybridization combined with laser-assisted cell picking revealed a clear separation between expression of ERα and ERβ between cell types, so that ERα expression was found to be limited to spermatogonia and spermatocytes, whereas ERβ expression occurred only in Sertoli cells (Lekhkota et al, 2005). It appears, therefore, that E2-17β may be directly involved in the regulation of mitotic activity of early stages of spermatogenesis, but a possible effect cannot be separated from an effect of androgens so far. Active immunization of boars with potent antigens against gonadotropin-releasing hormone (GnRH) leads to an inhibition of luteinizing hormone (LH), so that androgen and estrogen formation in the testes drops (Metz et al, 2002). Follicle-stimulating hormone (FSH) concentrations are not influenced by active immunization, so that blood plasma concentrations do not differ significantly between normal and immunized boars (Awoniyi et al, 1988; Wagner and Claus, 2004). In consequence, immunization allows us to study spermatogenesis in the absence of steroids and additionally to perform substitution experiments to clarify individual or combined effects of androgens and estrogens.
So far the effects of immunization on spermatogenesis have been compared with normal control boars (Wagner and Claus, 2004). It was found that steroid withdrawal leads to a reduction of the mitotic rate of spermatogonia. In addition, the expression of the glucocorticoid receptor in spermatogonia was increased so that apoptosis was elevated in spermatogonia and spermatocytes. In consequence, reduced mitosis and increased apoptosis led to a decrease of spermatogenic activity in the absence of testicular steroids (Wagner and Claus, 2004).
Based on these effects, the present paper reports the consequences of a continuous infusion with E2-17β on ERα-mediated effects on spermatogenesis in immunized boars.
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As reported earlier, immunization against GnRH led to a nearly complete inhibition of LH but not FSH, so that changes in spermatogenesis can be specifically attributed to the lack of testicular steroids (Metz et al, 2002; Wagner and Claus, 2004). Remaining concentrations of E2-17β were on the order of 12 pg/mL and thus represent the limit of sensitivity of the assay (11.5 pg/mL). Infusion of E2-17β did not alter the minimal testosterone concentrations but increased circulating E2-17β concentrations up to physiological levels. The concentrations obtained were higher compared to control boars, but were of an order which regularly can be determined in older boars (Claus et al, 1983). Thus the experimental design allowed the study of a selective effect of estradiol on spermatogenesis in the boar.
An alternative approach had been chosen by knockout experiments in mice inhibiting either the aromatase activity, and thus aromatization of androgens to estrogens (Fisher et al, 1998; Robertson et al, 1999), or alternatively the estrogen receptor (Lubahn et al, 1993; Krege et al, 1998; for review see O'Donnell et al, 2001). In both cases possible effects of estrogens on different traits of male reproductive physiology had to be studied in the presence of androgens. More recently E2-17β formation was inhibited with an aromatase inhibitor, but effects on spermatogenesis were not investigated (At-Taras et al, 2005).
In our study the expression of ERα in boar testis was additionally confirmed by RT-PCR and by Western blot analysis in all 3 groups. In Western blot the preparations from the nonimmunized control boars obviously led to additional bands which may be attributed to a different endocrine background (eg, due to the presence of androgens). The underlying substances were not further identified. In addition, we could demonstrate previously that ERα expression is limited to early stages of germ cell development (Lekhota et al, 2005). ERα expression in the noninfused immunocastrates shows that it is partly independent from the presence of the ligand. A tendency for elevated receptor density in E2-17β-infused boars may be due to an additional ligand-dependent expression. The ERα could be exclusively localized in A- and B-spermatogonia. Staining, however, was focused on spermatogonia in stage I of the seminiferous epithelial cycle, which represents the main stage of spermatogonial division (Frankenhuis et al, 1982; Garcia-Gil et al, 2002). Stages VI/VII and VIII also belong to the mitotic stages, but mainly represent the G1 and S-phase, whereas actual division (M-phase) occurs in stage I (Guraya, 1987) where also ERα was detected in the present study. A mitosis-supporting effect of estrogens for spermatogenesis is not surprising, because their promotion of cell division is well known for a variety of other tissues, including estrogen-dependent tumors (Altucci et al, 1997; Zhang et al, 1998). In such tissues it was also found that estrogens promote cells to finish the G1-phase and to enter the M-phase (Leung and Potter, 1987; Zhang et al, 1998). A role of estrogens for mitosis of spermatogonia was also assumed for the stallion (Sipahutar et al, 2003) and for rodents and the eel (O'Donnell et al, 2001; Miura et al, 1999).
By the use of subcutaneous E2-17β Silastic implants it was shown in GnRH-deficient mice that the treatment led to an increase of the tubular volume (Ebling et al, 2000), as also shown in the hamster (Pak et al, 2002). In the latter study the effect was attributed to a spermatogenesis-initiating effect of estrogens.
Quantitative analysis revealed that counts of the individual spermatogenic cells other than A-spermatogonia were intermediate in the E2-17β-infused immunized boars, compared to both boars with significantly higher and immunized boars with significantly lower cell counts.
These data thus additionally support a role of E2-17β in spermatogenesis in the boar. This does not mean necessarily that overall production of sperm is improved in E2-17β-infused immunized boars compared to immunized boars, because counteracting apoptosis in the following spermatogenic cells was not significantly different between these 2 groups. In control boars, however, apoptosis was markedly reduced, suggesting an additional role of androgens. It was not possible to compare sperm counts in ejaculates, because semen collection attempts were unsuccessful both in immunized and E2-17β-infused immunized boars. Taken together, the data show that in the boar ERα is localized only in spermatogonia. Infusion of E2-17β led to an increase of mitosis in the tubules of GnRH-immunized boars, indicating that estrogens are necessary for germ cell renewal in the testis of boars.