Testosterone and oestradiol in concert protect seminiferous tubule maturation against inhibition by GnRH-antagonist
2011; Wiley; Volume: 34; Issue: 5pt2 Linguagem: Inglês
10.1111/j.1365-2605.2011.01146.x
ISSN2047-2927
AutoresRenata Walczak-Jędrzejowska, Krzysztof Kula, Elżbieta Oszukowska, Katarzyna Marchlewska, Wojciech Kula, Jolanta Słowikowska‐Hilczer,
Tópico(s)Ovarian function and disorders
ResumoInternational Journal of AndrologyVolume 34, Issue 5pt2 p. e378-e385 ORIGINAL ARTICLEFree Access Testosterone and oestradiol in concert protect seminiferous tubule maturation against inhibition by GnRH-antagonist R. Walczak-Jedrzejowska, R. Walczak-Jedrzejowska Department of Andrology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorK. Kula, K. Kula Department of Andrology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorE. Oszukowska, E. Oszukowska Department of Andrology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorK. Marchlewska, K. Marchlewska Department of Reproductive Endocrinology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorW. Kula, W. Kula Chair of Clinical and Experimental Physiology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorJ. Slowikowska-Hilczer, J. Slowikowska-Hilczer Department of Reproductive Endocrinology, Medical University of Lodz, Lodz, PolandSearch for more papers by this author R. Walczak-Jedrzejowska, R. Walczak-Jedrzejowska Department of Andrology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorK. Kula, K. Kula Department of Andrology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorE. Oszukowska, E. Oszukowska Department of Andrology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorK. Marchlewska, K. Marchlewska Department of Reproductive Endocrinology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorW. Kula, W. Kula Chair of Clinical and Experimental Physiology, Medical University of Lodz, Lodz, PolandSearch for more papers by this authorJ. Slowikowska-Hilczer, J. Slowikowska-Hilczer Department of Reproductive Endocrinology, Medical University of Lodz, Lodz, PolandSearch for more papers by this author First published: 28 April 2011 https://doi.org/10.1111/j.1365-2605.2011.01146.xCitations: 9 Krzysztof Kula, Department of Andrology, Chair of Andrology and Reproductive Endocrinology, Medical University of Lodz, PL 91-425, 5 Sterling str, Lodz, Poland. E-mail: krzysztof.kula@umed.lodz.pl AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary Oestradiol enhances follicle stimulating hormone (FSH) action on seminiferous tubule maturation, but the relative involvement of oestradiol and testosterone remains unclear. This study compares the influences of oestrogen and androgen in FSH and testosterone-deficient rats. Animals were injected daily GnRH-antagonist alone (Ant) or combined with 17β-oestradiol benzoate (EB), or testosterone propionate (TP), or both from post-natal day (pnd) 5 to 15. Hormone levels, tubule growth, cell numbers, germ cell apoptosis and proliferation, and Sertoli cell maturation were evaluated on pnd 16. Ant decreased serum FSH and testosterone levels to ∼60% and ∼50% of control values, respectively, and decreased tubule growth, Sertoli cell number and maturation. Germ cell number declined by apoptosis. Co-administration of EB stimulated spermatogonia proliferation and maintained FSH levels (86% of control). Tubule growth, Sertoli cell number and spermatocyte apoptosis remained normal after TP co-administration, but Sertoli cell maturation, germ cell number and spermatogonia survival were reduced. Co-administration of EB with TP prevented all inhibitions. In conclusion, administration of oestradiol with testosterone, but neither one alone, protected seminiferous tubule maturation against inhibition caused by Ant-induced disruption. Oestrogen was involved in stimulating germ cell proliferation and the maintenance of Sertoli cell maturation, whereas androgen affected seminiferous tubule growth and spermatocyte survival. Introduction Follicle stimulating hormone (FSH) and testosterone are important regulators of spermatogenesis, even though FSH and androgen receptors are not expressed by germ cells. FSH stimulates number, proliferation and differentiation of spermatogonia in rats (Kula, 1988; Boitani et al., 1993; Kula et al., 2001; Walczak-Jedrzejowska et al., 2008), primates (van Alphen et al., 1988) and men (Kula, 1991), whereas androgen seems to be necessary for the progression of meiosis and post-meiotic steps (Kula et al., 1983; De Gendt et al., 2004; Abel et al., 2008). The discovery of oestrogen receptor β (ERβ) in spermatogonia (Saunders et al., 1998) was preceded by the finding that 17β-oestradiol benzoate administration to immature rats increases the number of spermatogonia, whereas testosterone does not (Kula, 1988). This has been confirmed recently in adult rats in vitro, where oestradiol, as well as ERβ-selective agonist 5α-androstane-3β,17β-diol, was able to stimulate DNA synthesis in rat spermatogonia, but the non-aromatizable androgen dihydrotestosterone which binds to the androgen receptor was not (Wahlgren et al., 2008). It has been also shown that oestradiol can act as a male germ cell survival factor in man (Pentikainen et al., 2000) and in rodents (Gancarczyk et al., 2004; D'Souza et al., 2005). In the rat, the onset of spermatogenesis occurs shortly after birth. Up to post-natal day (pnd) 5, the gonocytes differentiate into spermatogonia and on pnd 15, spermatogonia become spermatocytes at the pachytene stage of meiotic prophase (Clermont & Perey, 1957). In this period, the biosynthesis of testosterone and 5α-reduced androgens from pregnenolone by testicular tissue in vitro is minimal, whereas FSH secretion initiates and increases progressively (Kula et al., 1983). To control spermatogonia development, FSH may interact with oestrogen. During the initial 12 days of life, FSH maximally stimulates oestrogen biosynthesis in the rat's testes (Pomerantz, 1980), and oestradiol administration from pnd 5 until 15 was shown to enhance dramatically the stimulatory effect of FSH on seminiferous tubule growth, germ cell numbers and Sertoli cell maturation in rats resulting in precocious initiation of spermatogenesis (Kula et al., 2001). In the presence of FSH and functional androgen receptor qualitatively (but not quantitatively), complete spermatogenesis has been achieved by chronic oestrogen treatment in hypogonadal mice (Ebling et al., 2000; Lim et al., 2008). The aim of the present study was to investigate whether oestrogen and androgen interaction is required during initiation of seminiferous tubule maturation. Endogenous FSH and testosterone deficiencies were produced in immature rats by GnRH antagonist treatment and both oestrogen and androgen were co-administered either individually or together. Materials and methods Animals and hormone treatment Male Wistar rats, born on the same day, were randomly divided into experimental groups of 10 to 12 animals. Each group was kept in a separate cage together with a lactating female. Animals were maintained at a stable temperature (22 °C) and diurnal light–dark cycles (12L : 12D) with free access to food and water. From pnd 5 until 15, male pups were given daily subcutaneous injections with (i) 500 μg/kg b.w. of GnRH-antagonist Cetrorelix alone (Ant) (Merck Serono Europe Ltd, London, UK) or (ii) Ant + 12.5 μg of 17β-oestradiol benzoate (Ant + EB) (Oestradiolum benzoicum; Jelfa, Jelenia Gora, Poland) or (iii) Ant + 2.5 mg of testosterone propionate (Ant + TP) (Testosteronum propionicum; Jelfa) or (iv) Ant + EB + TP or (v) solvents for hormones (control group – C). The dose of Ant was based on a previous study where a dose of highly potent GnRH antagonist, detirelix, administered for 2 weeks to adult rats, suppressed serum concentrations of LH, FSH and testosterone (Chandolia et al., 1991). The dose of EB applied here was shown to inhibit germ cell survival (Walczak-Jedrzejowska et al., 2008) without affecting the secretion of LH and FSH (Kula et al., 2001), and the dose of TP also did not influence FSH secretion in immature rats (Walczak-Jedrzejowska et al., 2009). Experiments were performed in accordance with Polish legal requirements, under the licence given by the Commission of Bioethics at the Medical University of Lodz, Poland. Processing of the tissue On pnd 16, euthanasia was performed. Animals were anaesthetized with methohexital sodium (Brietal; Eli Lilly & Co., Indianapolis, IN, USA) and fentanyl (Fentanyl; Polfa, Warsaw, Poland) and weighed. Blood samples were taken by cardiac puncture. The testes were excised and weighed. Then, after fixation in Bouin's solution for up to 24 h, they were processed through graded alcohols and embedded in paraffin wax blocks. Hormonal assays The clotted blood was centrifuged and serum was collected for hormonal determination. All samples were measured in the same assay. Rat FSH (rFSH) concentrations were determined by double antibody radioimmunoassay (Ortho-Clinical Diagnostic, Amersham, UK) with a sensitivity of 1.0 ng/mL. The concentrations of testosterone and oestradiol were determined by competitive immunoassay (Ortho-Clinical Diagnostic). The sensitivities were 0.009 ng/mL for testosterone and 0.003 ng/mL for oestradiol. Morphometry of the seminiferous tubules Sections 5 μm thick were stained with haematoxylin and eosin. All morphometric analyses were performed using Lxand v3.60HM image analysis software (Logitex, Lodz, Poland). The diameters of 50 seminal tubule cross-sections were measured for each animal. The volume density (Vv) of the seminiferous tubules, expressed as a percentage, was obtained by the point counting method described previously (Kula et al., 2001; Walczak-Jedrzejowska et al., 2009). The absolute volume of the seminiferous tubules (V) was determined by multiplying Vv by testicular weight (Vt), as an equivalent of the fresh testis volume (the specific gravity of testicular tissue is about 1.0). The total length of the seminiferous tubule (L) was calculated using the transformed standard equation for a tube model (L = V/πr2), where V was the seminiferous tubule volume and r, the radius of the tubule (Wing & Christensen, 1982). Germ cell and Sertoli cell number Germ cells and Sertoli cells were identified on the basis of previously reported morphological characteristics and their location in the tubules (Clermont & Perey, 1957). Besides Sertoli cells, the following germ cell types can be distinguished in a 16-day-old rat: type A, intermediate (In) and B spermatogonia, spermatocytes in preleptotene, leptotene, zygotene and pachytene stages. The numbers of Sertoli cells and different types of germ cells were determined in 50 randomly chosen, round cross-sections of the seminiferous tubules for each animal. The diameters of 50 round or oval nuclei were measured for each cell type. For type A spermatogonia and Sertoli cells, the nuclei diameter was estimated as the average of the height and width measurements because of their irregular or oval shapes (Johnson, 1985). All cell counts were corrected for section thickness and nucleus diameter by the Abercombie formula (Abercrombie, 1946). The results were expressed per seminiferous tubule cross-section. The total number of spermatogonia and spermatocytes per testis was calculated as the product of the total length of the seminiferous tubule and the particular cell type number, expressed per tubule cross section (Marshall & Plant, 1996). Morphometric parameters of Sertoli cell maturation To assess Sertoli cell function, the percentage of round-shaped seminiferous tubule cross-sections containing a clear lumen (larger than 100 μm2) was assessed for each animal. The nuclear area of Sertoli cells was measured in 50 longitudinally sectioned Sertoli cells in each rat. All measurements were performed using Lxand v3.60HM image analysis software (Logitex). Germ cell proliferation and apoptosis The proliferative activity of the cells was studied by immunohistochemical labelling of proliferating cell nuclear antigen (PCNA) as described in detail elsewhere (Walczak-Jedrzejowska et al., 2008). An anti-rat PCNA monoclonal mouse antibody (ready to use; DakoCytomation, Glostrup, Denmark) was applied as a primary antibody. As a negative control, the sections were incubated with non-immune serum instead of the primary antibody. To detect nuclei with DNA fragmentation, terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) was performed. Apoptotic cells were visualized using the In Situ Death Detection Kit POD (Roche Molecular Biochemicals, Mannheim, Germany) as described previously (Walczak-Jedrzejowska et al., 2007). The proliferating and apoptotic indices (percentages) of spermatogonia or spermatocytes were examined in 300–500 subsequent cell nuclei at 1000× magnification in the light microscope (Nikon Eclipse E600; Nikon, Kanagawa, Japan). The PCNA-positive and TUNEL-positive spermatogonia or spermatocytes were indentified based on their size, the shape of cell nucleus and their location within the seminiferous tubule cross-section (Clermont & Perey, 1957; Meachem et al., 2005b). Statistics The distribution of the data was analysed using Shapiro–Wilk's test. All data were normally distributed. Statistical analysis of the data was performed using one-way anova followed by a post hoc test (Scheffe test). The results are presented as mean ± SD. Differences were considered significant at p < 0.05. Results Hormone levels The serum FSH level decreased to 63% after Ant-alone, to 52% after Ant + TP and to 66% of C after Ant + EB + TP. After Ant + EB, serum FSH level decreased to only 86% of C and was significantly higher than after Ant alone and the remaining treatments (Fig. 1A). The serum level of testosterone was suppressed after Ant or Ant + EB to 54% or 44% of C, respectively, and was 500-fold higher in groups receiving TP (Fig. 1B). The serum level of oestradiol did not change after Ant alone, increased 6-fold after receiving EB and increased 2-fold after Ant + TP (Fig. 1C). Figure 1Open in figure viewerPowerPoint Changes in serum hormones levels and testicular growth parameters in 16-day-old rats treated from post-natal day (pnd) 5 to 15 with GnRH-antagonist alone (Ant) or in combination with 12.5 μg of 17β-oestradiol benzoate (Ant + EB), 2.5 mg of testosterone propionate (Ant + TP) or both (Ant + EB + TP). (A) serum level of rat-follicle stimulating hormone (r-FSH), (B) serum level of testosterone, (C) serum level of oestradiol, (D) relative testicular weight, (E) seminiferous tubule diameter, (F) seminiferous tubule length. Values shown are mean ± SD. ap < 0.001, bp < 0.01, cp < 0.05 vs. C-control group; dp < 0.001, fp < 0.05 vs. Ant + EB (post hoc Scheffe test). Body and testes weight and seminiferous tubule morphometry Body weight was 33.4 ± 3.5 g in C. It decreased after Ant or Ant + TP to 82%, or 85% of C (27.6 ± 2.9 and 28.7 ± 4.3 g, respectively; p < 0.01), and did not change after Ant + EB or Ant + EB + TP (32.2 ± 5.2 or 31.5 ± 3.7 g, respectively). Relative paired testes weight was halved after Ant and Ant + EB, relatively increased after Ant + TP, reaching 88% of C, and was maintained at C level after Ant + EB + TP (Fig. 1D). Growth of the seminiferous tubules was inhibited after Ant or Ant + EB as seminiferous tubule diameter decreased to 87% and length to 50–65% of C. Both parameters were maintained at C level after Ant + TP or Ant + EB + TP (Fig. 1E,F). Sertoli cell maturation Seminiferous tubule lumen formation was arrested after Ant application, reduced to 12% of C after Ant + EB or Ant + TP (3.41 ± 4.37% or 3.62 ± 1.62%, respectively vs. 29.2 ± 5.29% for C, p < 0.001) and was maintained at C level after Ant + EB + TP (33.3 ± 4.88%). Sertoli cell nuclear area was reduced to 89–90% of C after Ant (25.62 ± 1.40 μm2 vs. 28.73 ± 1.40 μm2 for C, p < 0.05), Ant + EB (25.49 ± 1.56 μm2, p < 0.01) or Ant + TP application (25.84 ± 0.95 μm2, p < 0.05) and was maintained at C level after Ant + EB + TP (30.20 ± 1.51 μm2). Sertoli cell and germ cell number Table 1 shows that Sertoli cell number was reduced to 50% of C after Ant, to 60% after Ant + EB and increased, reaching C value, after Ant + TP or Ant + EB + TP. Ant alone inhibited total germ cell number. Addition of EB or TP increased it to some extent, whereas Ant + EB + TP treatment maintained the total germ cell number at the C level. The incidence of PCNA-positive spermatogonia increased to 112% of C after Ant + EB, and did not change by other treatments (Table 1). The incidence of PCNA-positive spermatocytes remained unchanged in all experimental groups (data not shown). Table 1. Mean ± SD of Sertoli and total germ cell number per testis expressed as absolute value, and as the percentage of changes vs. control group, and the incidence of spermatogonia proliferation (PCNA-positive Sg) in rats receiving vehicles (C), GnRH-antagonist alone (Ant), Ant with 12.5 μg of 17β-oestradiol benzoate (Ant + EB), Ant with 2.5 mg of testosterone propionate (Ant + TP) and Ant + EB + TP Group (no. animals) Sertoli cell number (106/testis) Total germ cell number PCNA-positive Sg (%) 106/testis % changes vs. C C (12) 28.7 ± 3.3 17.5 ± 0.9 100 75.0 ± 4.0 Ant (10) 14.0 ± 1.0a 5.1 ± 0.4a 29 75.6 ± 4.0 Ant + EB (11) 17.6 ± 2.8a 9.3 ± 1.3a 53 84.0 ± 1.4c Ant + TP (11) 26.3 ± 3.8 13.0 ± 0.6b 74 76.4 ± 3.5 Ant + EB + TP (10) 25.2 ± 0.5 16.2 ± 1.2 92 81.5 ± 1.9 Ant, antagonist; C, control; EB, oestradiol benzoate; PCNA, proliferating cell nuclear antigen; TP, testosterone propionate. a p < 0.001, bp < 0.01, cp < 0.05 vs. C (post hoc Scheffe test). Quantitative changes in germ cell subtypes are shown in Fig. 2. While the number of spermatogonia decreased to 49% and spermatocytes to 20% of C after Ant, a relative increase of spermatogonia number to about 70% of C was seen after both Ant + EB or Ant + TP application. Spermatocyte number relative increase was more evident after Ant + TP (74% of C) than after Ant + EB (41% of C). After Ant + EB + TP application, the numbers of spermatogonia and spermatocytes were maintained on C values (90–95% of C). In relation to Ant-alone-treated group, the number of spermatogonia increased 1.5-fold after Ant + EB or Ant + TP, whereas the number of spermatocytes increased 2-fold and 3.7-fold after Ant + EB and Ant + TP, respectively (Fig. 2A,B). Figure 2Open in figure viewerPowerPoint Changes in germ cell number and the incidence of their apoptosis in 16-day-old rats treated from post-natal day (pnd) 5 to 15 with GnRH-antagonist alone (Ant) or in combination with 12.5 μg of 17β-oestradiol benzoate (Ant + EB), 2.5 mg of testosterone propionate (Ant + TP) or both (Ant + EB + TP). (A) number of spermatogonia, (B) number of spermatocytes, (C) the percentage of TUNEL-positive spermatogonia, (D) the percentage of TUNEL-positive spermatocytes. Values shown are mean ± SD. ap < 0.001, bp < 0.01, cp < 0.05 vs. C-control group; dp < 0.001, ep < 0.01, fp < 0.05 vs. Ant (post hoc Scheffe test). Germ cell apoptosis The incidence of TUNEL-positive spermatogonia increased 2.5-fold that of C after Ant, 2.3-fold after Ant + EB, 1.6-fold after Ant + TP and decreased to the C value after Ant + EB + TP (Fig. 2C). The incidence of TUNEL-positive spermatocytes increased to 3.2 times that of C after Ant, 2.7-fold after Ant + EB and decreased to C values after Ant + TP or Ant + EB + TP (Fig. 2D). Figure 3 presents representative photomicrographs of seminiferous tubule cross-sections showing cellular apoptosis detected by TUNEL. Figure 3Open in figure viewerPowerPoint Representative photomicrographs (A and B) of seminiferous tubule cross-sections presenting cellular apoptosis detected by TUNEL method in 16-day-old rat (control group). Scale bars represent 10 μm. In A, arrows indicate unlabelled spermatogonia (Sg) and Sertoli cell nuclei (S); arrowhead indicates nucleus of labelled spermatogonia. In B, arrows indicate unlabelled Sertoli cell (S), spermatogonia (Sg) and spermatocyte nuclei (Sc); arrowhead indicates nuclei of labelled spermatocytes. Discussion In this study, a decrease in FSH and testosterone secretions was achieved by GnRH-antagonist administration with testosterone deficiency resulting presumably from decreased secretion of LH (Halmos et al., 1996). High supraphysiological serum levels of oestradiol or testosterone were achieved by co-administration of EB or TP to Ant-treated rats. EB co-administration maintained higher FSH levels during Ant treatment. Increased secretion of FSH after oestrogen administration to immature or adult rodents has been observed earlier (Atanassova et al., 1999; Ebling et al., 2000; D'Souza et al., 2005; Lim et al., 2008). Ant-alone treatment did not influence the serum level of oestradiol. An increase of oestradiol level after Ant + TP might have resulted from the increased availability of testosterone, the substrate for oestrogen synthesis (Dorrington et al., 1978). The administration of different doses of testosterone or oestradiol to hypogonadal mice or adult rats produced increased intra-testicular levels of the administered steroids when their blood levels rose significantly above normal (Singh et al., 1995; Meachem et al., 1998; D'Souza et al., 2005). In infant male rats, the same daily doses of sex steroids as those in the present study produced 3-fold and 5-fold increases in the intra-testicular concentrations of oestradiol and testosterone, respectively (in preparation). In contrast, a decrease in oestradiol intra-testicular concentration was observed after oestradiol administration to hypogonadal mice, despite an increase in its serum level (Lim et al., 2008). However, these mice exhibited a physiological range of serum oestradiol, far lower than those achieved in the present study. A decrease in the testes weight and Sertoli cell number after Ant alone treatment may be attributed to diminished FSH and testosterone secretions, as both hormones regulate these parameters (Atanassova et al., 2005). However, serum FSH levels remained between 52 and 86% of control value in treated groups and it is not clear if these modest reductions represent a significant loss in a functional FSH activity with respect to Sertoli cell numbers. Moreover, co-administration of TP with Ant maintained testes weight and Sertoli cell number at normal levels suggesting the importance of androgen action. Reduced number and increased apoptosis of germ cells after treatment with Ant alone could be attributed to FSH deficiency. FSH enables spermatogonial survival in adult and immature rats (Shetty et al., 1996; Meachem et al., 1999, 2005a,b). After Ant + TP, the meiotic cell spermatocyte (but not spermatogonia) survival was normalized despite reduced FSH level, confirming the importance of androgen for meiotic progression (Kula et al., 1983; De Gendt et al., 2004). Ant + TP treatment was not, however, sufficient to maintain a normal total germ cell number. It has been demonstrated previously that administration of testosterone reduces spermatogonia differentiation in adult rats (Yang et al., 2004) and eliminates the FSH-induced increase in spermatogonia differentiation in immature rats (Kula, 1988). The combination of Ant + EB stimulated spermatogonia proliferation. This could be attributed either to an indirect effect, mediated by the relative increase in FSH secretion (Ebling et al., 2000; Lim et al., 2008) or to the direct influence of oestrogen on germ cells (Wagner et al., 2006; Wahlgren et al., 2008; Porter et al., 2009). Oestradiol administration stimulated proliferation of spermatogonia in artificially cryptorchid maturating mice in a dose-related manner, not associated with proportional changes in FSH secretion, indicating a direct effect of oestrogen (Li et al., 2007). In addition, during recovery of rat spermatogenesis after irradiation, decreases in the levels of serum FSH and intra-testicular testosterone, induced by Ant treatment, were beneficial for the recovery of spermatogenesis by oestrogen administration. In these rats, oestradiol was a considerably more effective stimulator of spermatogonia recovery than androgen (Shetty et al., 2004). Despite FSH deficiency, all the measured parameters were maintained at quantitatively normal levels after Ant + EB + TP treatment. Maintenance of germ cell number could result from the stimulatory effect of oestradiol on the proliferation of spermatogonia and the protective effect of testosterone against spermatocyte apoptosis. In addition, only combined co-treatment maintained normal Sertoli cell maturation. This indicates that Sertoli cell is the platform for oestrogen-androgen interaction. The interaction of oestrogen with diminished availability of FSH might also be of importance as acceleration of Sertoli cell maturation associated with precocious initiation of spermatogenesis has been achieved by simultaneous administration of EB and FSH, but not by either preparation administered individually (Kula et al., 2001). Inter-cellular connections might be a target for the combined effect of oestrogen and androgen. Androgens stimulate the formation of the tight junction between Sertoli cells (Kaitu'u-Lino et al., 2007), which strongly correlates with a decrease of spermatocyte apoptosis (Morales et al., 2007). Oestrogen may regulate N-cadherin mRNA in immature rat testes (Mac Calman & Blaschuk, 1994; Mac Calman et al., 1997), a protein which is the main component of adherens junctions. Furthermore, both oestradiol and testosterone may increase the level of connexin 43 transcripts, the most abundant protein forming gap junctions in the testes of Rana esculenta (Izzo et al., 2009). It was shown that connexin 43-based gap junctions between Sertoli cells and spermatogonia may be involved in an increase in spermatogonia number by controlling their survival in immature rats (Gilleron et al., 2009). During testicular maturation in rats, the blood concentration of oestradiol is several times higher on pnd 5 than during further development (Walczak-Jedrzejowska et al., 2009). This coincides with differentiation of gonocytes into the first spermatogonia, and in fact, it has been shown that oestradiol stimulates proliferation of gonocytes in mice in vitro (Li et al., 1997; Thuillier et al., 2010). Initiation, progression and completion of all pre-meiotic (mitotic) steps take place from birth until pnd 15 (Clermont & Perey, 1957). The activity of aromatase, an enzyme catalysing oestrogen formation from testosterone, is localized in newborn rats exclusively within seminiferous tubules and undergoes sharp dislocation on pnd 15 into Leydig cells for the rest of life (Tsai-Morris et al., 1985). All these data suggest that pre-meiotic steps of spermatogenesis may need oestrogen action. Our study indicates that despite the FSH level being reduced, the combined administration of oestradiol and testosterone, but neither one of the sex steroids alone, eliminates all inhibitions of seminiferous tubule maturation exerted by Ant treatment. The oestrogen component was involved in the stimulation of spermatogonia proliferation and presumably in the maintenance of ongoing Sertoli cell maturation, whereas the androgen component protected Sertoli cell number, seminiferous tubule growth and spermatocyte survival. Hence, the interaction of oestrogen with androgen may facilitate seminiferous tubule maturation in the rat. Acknowledgements We thank Merck Serono Europe Ltd for kindly providing a GnRH-antagonist (Cetrorelix) for our study, and a technician, Boguslawa Cyniak, for histological processing of gonads. This study was financially supported by the Medical University of Lodz grants no. 502-11-711, 502-11-427 and 503-1089-2/3. References Abel MH, Baker PJ, Charlton HM, Monteiro A, Verhoeven G, De Gendt K, Guillou F & O'Shaughnessy PJ. (2008) Spermatogenesis and Sertoli cell activity in mice lacking Sertoli cell receptors for follicle-stimulating hormone and androgen. Endocrinology 149, 3279– 3285. CrossrefCASPubMedWeb of Science®Google Scholar Abercrombie M. (1946) Estimation of nuclear population from microtome sections. Anat Rec 94, 239– 247. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar van Alphen MM, van de Kant HJ & de Rooij DG. (1988) Follicle-stimulating hormone stimulates spermatogenesis in the adult monkey. Endocrinology 123, 1449– 1455. 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