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Endocrinology, doi:10.1210/en.2008-0086
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Endocrinology Vol. 149, No. 7 3279-3285
Copyright © 2008 by The Endocrine Society

Spermatogenesis and Sertoli Cell Activity in Mice Lacking Sertoli Cell Receptors for Follicle-Stimulating Hormone and Androgen

M. H. Abel, P. J. Baker, H. M. Charlton, A. Monteiro, G. Verhoeven, K. De Gendt, F. Guillou and P. J. O'Shaughnessy

Department of Human Anatomy and Genetics (M.H.A., H.M.C.), University of Oxford, Oxford OX1 3QX, United Kingdom; Division of Cell Sciences (P.J.B., A.M., P.J.O.), University of Glasgow Veterinary School, Glasgow G61 1QH, United Kingdom; Laboratory for Experimental Medicine and Endocrinology (G.V., K.D.G.), Catholic University of Leuven, B-3000 Leuven, Belgium; Unité Mixte de Recherche 6175 (F.G.), Institut National de la Recherche Agronomique, Centre National de Recherche Scientifique, Université de Tours, 37380 Nouzilly, France

Address all correspondence and requests for reprints to: P. J. O'Shaughnessy, Division of Cell Sciences, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, United Kingdom. E-mail: p.j.oshaughnessy{at}vet.gla.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Spermatogenesis in the adult male depends on the action of FSH and androgen. Ablation of either hormone has deleterious effects on Sertoli cell function and the progression of germ cells through spermatogenesis. In this study we generated mice lacking both FSH receptors (FSHRKO) and androgen receptors on the Sertoli cell (SCARKO) to examine how FSH and androgen combine to regulate Sertoli cell function and spermatogenesis. Sertoli cell number in FSHRKO-SCARKO mice was reduced by about 50% but was not significantly different from FSHRKO mice. In contrast, total germ cell number in FSHRKO-SCARKO mice was reduced to 2% of control mice (and 20% of SCARKO mice) due to a failure to progress beyond early meiosis. Measurement of Sertoli cell-specific transcript levels showed that about a third were independent of hormonal action on the Sertoli cell, whereas others were predominantly androgen dependent or showed redundant control by FSH and androgen. Results show that FSH and androgen act through redundant, additive, and synergistic regulation of spermatogenesis and Sertoli cell activity. In addition, the Sertoli cell retains a significant capacity for activity, which is independent of direct hormonal regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE ADULT animal, Sertoli cells act primarily to promote and maintain germ cell development. This is achieved by generation of a unique microenvironment within the seminiferous tubules through the formation of a Sertoli cell barrier and regulation of solute movement and secretion into the tubules (1, 2). In addition, Sertoli cells provide cytoarchitectural support and stimulation to the developing germ cells as they undergo proliferation and differentiation. Previous studies have shown that spermatogenesis is regulated by FSH and testosterone and that these hormones act through specific receptors on the Sertoli cell (reviewed in Ref. 3). The role that each of these hormones plays in the regulation of Sertoli cell function and spermatogenesis has become clearer through study of mice lacking specific hormones or hormone receptors. In animals lacking FSH or the FSH receptor (FSHRKO), there is a reduction in Sertoli cell number and germ cell number, but the animals remain fertile (4, 5, 6, 7). In contrast, absence of the androgen receptor (AR), either ubiquitously through a mutation in the receptor (Tfm) or specifically in the Sertoli cells (SCARKO) will cause arrest of spermatogenesis in early meiosis (8, 9, 10, 11). Thus, FSH appears to act to induce proliferation of the Sertoli cells and germ cells and optimize the progress of spermatogenesis, whereas testosterone action on the Sertoli cell is critical for progression through meiosis.

Although it is clear that both FSH and androgen are essential for normal spermatogenesis, what remains unknown is the nature and importance of interaction between the hormones. For example, the degree of overlap, or redundancy, is uncertain, and also, therefore, the extent to which ablation of one hormone or its receptor may be compensated by the presence of the other hormone. Similarly, the degree to which Sertoli cell function and germ cell development is independent of direct hormonal stimulation remains unclear. To answer these questions directly we generated mice lacking both FSH receptor (FSHR) and AR on the Sertoli cell (FSHRKO-SCARKO) and examined the subsequent effects on Sertoli cell function and spermatogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
All mice were bred and all procedures carried out under U.K. Home Office License and with the approval of a local ethical review committee. Mice with a specific Sertoli cell knockout of the AR have been previously generated by crossing male mice [expressing an anti-Müllerian hormone (AMH)-Cre recombinase (Cre) transgene) (12)] with female mice carrying an AR with a floxed exon 2 (ARfl) (9). To produce male mice lacking both FSHR and AR within the Sertoli cell, mice carrying the AMH-Cre transgene (C57-BL6/SJL) and mice carrying the ARfl allele (Swiss-Webster/129) were crossed with FSHRKO mice (C57-BL6/129) (6) and interbred. The groups used for comparison with the double-knockout FSHRKO-SCARKO mice were: 1) hemizygous FSHRKO/+ males expressing AMH-Cre or ARfl, which were considered as control animals (the two groups were initially analyzed separately, but no significant difference between the AMH-Cre and ARfl groups was seen, and the data were combined); 2) FSHRKO-Cre and FSHRKO-ARfl, which were considered FSHRKO animals; and 3) hemizygous FSHRKO/+ SCARKO animals, which were considered SCARKO animals for this study. PCR genotyping was carried out as previously described (9, 13).

Mice were killed at 8 wk and testes snap frozen in liquid nitrogen or fixed overnight. Fixation was either in Bouin’s for subsequent morphometric analysis or 4% paraformaldehyde/1% glutaraldehyde in phosphate buffer [0.1 M (pH 7.2)] for preparation of semithin sections.

Hormone measurements
Blood was collected by cardiac puncture under anesthesia and the serum separated and stored at –20 C until assayed. Serum levels of FSH and LH were measured commercially using imunofluorimetric assays (Delfia; Wallac OY, Turku, Finland) as previously described (14, 15). Serum levels of testosterone were measured by RIA after ether extraction (16).

Measurement of specific mRNA transcript levels
To quantify the content of specific mRNA species in testes from each group, a real-time PCR approach was used after reverse transcription of the isolated RNA. To allow specific mRNA levels to be expressed per Sertoli cell and control for the efficiency of RNA extraction, RNA degradation, and the reverse transcription step, an external standard was used (7, 17, 18). The external standard was luciferase mRNA (Promega UK, Southampton, UK), and 5 ng were added to each testis at the start of the RNA extraction procedure. Testis RNA was extracted using TRIzol (Life Technologies, Paisley, UK), and the RNA was reverse transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase (Superscript II; Life Technologies) as described previously (19). For real-time PCR, the SYBR green method was used in a 96-well plate format using a MX3000 cycler (Stratagene, Amsterdam, The Netherlands). Reactions contained 5 µl 2x SYBR master mix (Stratagene), primer (100 nM), and template in a total volume of 10 µl. At the end of the amplification phase, a melting curve analysis was carried out on the products formed. All primers were designed using Primer Express 2.0 (Applied Biosystems, Warrington, UK) with parameters previously described (20) and were designed so that the amplicon would cross at least one intron. The primers used have been described previously (21, 22). To correct for Sertoli cell number, data from the real-time PCR studies were divided by the Sertoli cell number of each group as measured below.

Histology and stereology
To prepare semithin (1 µm) sections, testes were embedded in araldite and sections stained with toluidine blue. For stereological analysis, testes were embedded in Technovit 7100 resin, cut into sections (20 µm), and stained with Harris’ hematoxylin. The total testis volume was estimated using the Cavalieri principle (23). The optical dissector technique (24) was used to count the number of Sertoli cells and germ cells in each testis. Each cell type was identified by previously described criteria (25, 26). The numerical density of each cell type was estimated using an Olympus BX50 microscope fitted with a motorized stage (Prior Scientific Instruments, Cambridge, UK) and Stereologer software (Systems Planning Analysis, Alexandria, VA).

Statistical analysis
Data were analyzed using two-factor ANOVA with each gene knockout as one of the factors. Where the interaction between the factors was significant, this means that the effect of the double knockout was not simply an additive effect of each gene knockout alone. To show whether differences between individual groups were significant, t tests were used using the pooled variance from the ANOVA. Data were log transformed where appropriate to avoid heterogeneity of variance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotype
Adult FSHRKO-SCARKO mice were normally masculinized, although testes from the double knockouts were significantly smaller than FSHRKO, SCARKO, or control mice (Table 1Go). Seminal vesicle weights were also slightly smaller in FSHRKO-SCARKO mice (Table 1Go). Seminiferous tubule diameter decreased across the groups in the order control>FSHRKO>SCARKO>FSHRKO-SCARKO with an associated increase in the relative abundance of interstitial tissue (Fig. 1Go). All stages of spermatogenesis were present in FSHRKO mice, whereas in SCARKO mice, there was apparent loss of pachytene spermatocytes with a marked reduction in the number of secondary spermatocytes and few round spermatids present (Fig. 1Go). In FSHRKO-SCARKO mice, germ cells entered meiosis, but development of most cells stopped at early pachytene with no secondary spermatocytes or round spermatids apparent (Fig. 1Go).


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  		TABLE 1. Organ weights in 8-wk-old mice

 

Figure 1
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  		FIG. 1. Semithin sections from testes of 8-wk-old normal, FSHRKO, SCARKO, and FSHRKO-SCARKO mice. The FSHRKO mice contained all stages of spermatogenesis, although germ cell number was reduced. In SCARKO mice, spermatogenesis progressed through meiosis, but there was progressive loss of pachytene spermatocytes, and few secondary spermatocytes or round spermatids were observed. In FSHRKO-SCARKO mice, the tubules were of a smaller diameter with large numbers of Sertoli cells (black arrowheads) and smaller numbers of spermatogonia (red arrowheads). Spermatogonia entered meiosis, but development stopped at early pachytene in most cells (yellow arrowhead). Bar, 20 µm.

 
Morphometric analysis showed that Sertoli cell number in 8-wk-old FSHRKO-SCARKO mice was similar to FSHRKO mice and significantly less than control or SCARKO mice (Fig. 2Go). Sertoli cells in SCARKO and FSHRKO-SCARKO mice contained large lipid droplets (not shown) that were not seen in other groups. The total germ cell number was reduced in FSHRKO and SCARKO mice, compared with control, and there was a further marked reduction in FSHRKO-SCARKO mice (Fig. 2Go). The germ cell to Sertoli cell ratio was reduced, although not markedly, in FSHRKO mice, compared with control. In contrast, in SCARKO mice the germ cell to Sertoli cell ratio was reduced to 10% of control, and in the FSHRKO-SCARKO, it was further reduced to 2% of control (Fig. 2Go). Analysis of germ cell types in each group showed that spermatogonial numbers were reduced by about 60% in the FSHRKO and FSHRKO-SCARKO mice (Fig. 3Go). Spermatocyte number was also reduced by about 50% in FSHRKO and SCARKO mice, but in the combined FSHRKO-SCARKO mouse, there was very limited successful progression into meiosis with spermatocyte numbers reduced to 4% of control (Fig 3Go). The number of round spermatids was reduced to about 40% in the FSHRKO mouse with very few (<0.5% of control) postmeiotic cells present in the SCARKO mouse and none in the FSHRKO-SCARKO mouse.


Figure 2
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  		FIG. 2. Morphometric analysis Sertoli and germ cell numbers in 8-wk-old testes from control, FSHRKO, SCARKO, and FSHRKO-SCARKO mice. Cell number was measured using the optical dissector method. Results show the mean ± SEM of four animals per group. Groups with different letter superscripts are significantly different. Where there was a significant interaction between the effects of the two gene knockouts, this is indicated on the figure.

 

Figure 3
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  		FIG. 3. Morphometric analysis of germ cell types in 8-wk-old testes from control, FSHRKO, SCARKO, and FSHRKO-SCARKO mice. Cell number were measured using the optical dissector method. Results show the mean ± SEM of four animals per group. Groups with different letter superscripts are significantly different.

 
Hormone profile
Serum levels of LH were significantly elevated in FSHRKO mice but were not significantly affected in SCARKO mice. In FSHRKO-SCARKO mice, LH levels were significantly greater than all other groups (Table 2Go). There were no significant differences in serum FSH or testosterone levels between groups.


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  		TABLE 2. Serum hormone levels in 8-wk-old animals

 
Sertoli cell mRNA levels
To examine Sertoli cell function in the four groups, real-time PCR was used to measure the levels of 14 mRNA transcripts that are specifically or predominantly expressed in the Sertoli cells. Transcript levels were measured relative to an external control and data normalized for Sertoli cell number (7) (Table 3Go and Fig. 4Go). Five of the transcripts tested (Sox9, Itgb1, Wt1, Trf, and Dhh) showed no significant difference in abundance per Sertoli cell among the four groups, although between-animal variation was relatively high for some transcripts (Table 3Go). Of the remaining nine transcripts, four showed a significant alteration in levels related only to the presence of the AR (Rhox5, Aqp8, Tjp1, and Gata1), one transcript responded to the loss of either FSHR or AR (Slc38a5), one responded only to the loss of FSHR (Cts12), and three transcripts (Espn, Msi1, and Slc7a4) showed a significantly more marked response to the loss of both receptors than to either FSHR or AR alone (Fig. 4Go).


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  		TABLE 3. mRNA transcript levels in 8-wk-old mice: transcripts unaffected in FSHRKO, SCARKO, or FSHRKO-SCARKO mice

 

Figure 4
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  		FIG. 4. Levels of Sertoli cell-specific mRNA transcripts in testes from 8-wk-old control, FSHRKO, SCARKO, and FSHRKO-SCARKO mice. Transcripts levels were measured relative to an external standard by real-time PCR and corrected for Sertoli cell number as described in Materials and Methods. Results show the mean ± SEM of five or six animals per group. Groups with different letter superscripts are significantly different. Where there was a significant interaction between the effects of the two gene knockouts, this is indicated on the figure.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Generation of the FSHRKO-SCARKO mouse provides us with a base from which to examine how FSH and androgen act and interact through the Sertoli cell to regulate testicular function. In addition, it allows us to determine what aspects of Sertoli cell function are independent of direct hormonal input. The phenotype shows there is additive, synergistic, and redundant regulation of spermatogenesis and the Sertoli cell by FSH and androgen but that regulation of a significant number of Sertoli cell transcripts (and, by implication, Sertoli cell function) is independent of direct hormonal control.

Interactions between the effects of FSH and androgens on testicular function have been studied previously using models such as the hypophysectomized rat or the hypogonadal (hpg) mouse, which lacks GnRH with consequent loss of circulating gonadotrophins (27). The advantage of the FSHRKO-SCARKO mouse, in contrast to these models, is the specificity and/or totality of the knockout effects. All current data suggest that the FSHR is confined to the Sertoli cells in the male (28, 29), whereas SCARKO mice have been generated to lack ARs only in the Sertoli cells (9). Thus, the targeted ablations affect only the Sertoli cells in this double knockout, and although there may be knock-on effects on gonadotrophin levels or androgen production, these are not complicating factors because the Sertoli cells are unresponsive to hormonal stimulation.

Effectively, the FSHRKO-SCARKO mouse provides a baseline control from which the direct effects of FSH and androgen on Sertoli cell function can be assessed. Thus, in the SCARKO mouse, the Sertoli cells are stimulated by FSH and not androgen; in the FSHRKO mouse, they are stimulated by androgen and not FSH, whereas in the normal mouse, they are exposed to both hormones. From this perspective, it can be seen that the action of FSH is to increase Sertoli cell number, total germ cell number, and the number of germ cells associated with each Sertoli cell. This effect is achieved by increasing the number of spermatogonia and enhancing the entry of these cells into meiosis. In contrast, androgens have no direct effect on Sertoli cell number but cause a marked increase in total germ cell number and thus the number of germ cells per Sertoli cell. The principal effect appears to be through increased entry into meiosis and, crucially, by enabling completion of meiosis. Together, the hormones have an additive effect on entry into meiosis but act synergistically to stimulate completion of meiosis and entry into spermiogenesis. In addition, it is clear from the double knockout that germ cells can initiate meiosis without direct hormonal stimulation through the Sertoli cell, supporting the conclusions of early in vitro studies (30). The general description of hormone action on spermatogenesis is also consistent with earlier hormone replacement studies, which have shown, for example, that FSH and androgen will increase or restore spermatocyte number (31, 32, 33), whereas androgens will increase the number of postmeiotic germ cells in the hpg mouse (31, 34).

Development of a normal-sized cohort of Sertoli cells is of importance for male fertility because each Sertoli cell can support only a finite number of germ cells. Previous studies have shown that both FSH and androgen can act to regulate the final Sertoli cell number in the adult animal (7, 35), although initial studies on the SCARKO mouse showed that androgen-dependent regulation of Sertoli cell number does not appear to be through direct effects of androgen on the Sertoli cell (7, 9, 36). In FSHRKO-SCARKO mice, the number of Sertoli cells was identical with the FSHRKO mouse, showing that only FSH is of importance in terms of direct effects on Sertoli cell proliferation with no synergistic or additive effect of androgen apparent. The nuclei of the Sertoli cells in the FSHRKO-SCARKO mice appeared to take up more of a pseudostratified appearance, probably due to the reduced diameter of the tubules. This marked reduction in tubular diameter is unlikely to be due entirely to loss of germ cells in the FSHRKO-SCARKO because tubules in the germ cell-free W/Wv mouse are less markedly affected and the Sertoli cells maintain a simple epithelium (Refs. 37 and 38 and our unpublished observation). This suggests that FSH and androgen regulate the diameter of the seminiferous tubules and maintain the normal morphology of the epithelium.

The significant increase in LH levels in FSHRKO mice has been reported previously (39) and attributed to reduced testosterone production by the testis. In FSHRKO-SCARKO mice, the increase in LH was more marked and may be indicative of further dysfunction in the Leydig cells. Both SCARKO and FSHRKO mice have reduced Leydig cell number (39, 40), and it is possible that the effects are additive in the FSHRKO-SCARKO mouse, although this will require further study. Circulating testosterone levels were unchanged in both FSHRKO and FSHRKO-SCARKO, but there was high variability between animals, and a more reliable indicator of androgen levels may be the reduction in weight of the seminal vesicles in FSHRKO-SCARKO mice. Reduced Leydig cell number or function in the SCARKO, FSHRKO, and FSHRKO-SCARKO mice is further evidence for a hormone-dependent Sertoli cell factor that regulates Leydig cell function (41, 42).

Despite our enhanced understanding of the role played by FSH and androgens in the regulation of spermatogenesis, the cellular mechanisms involved are still in some doubt. It is clear that FSH can regulate levels of a large number of different mRNA transcripts (43), and the overall effect of FSH may be to increase general Sertoli cell activity and thereby enhance germ cell progression and survival. In contrast, only a relatively small number of Sertoli cell genes have been shown unequivocally by array studies to be androgen dependent in a number of different mouse models (22, 44, 45, 46, 47). Somewhat confusingly, there is little overlap of identified androgen-dependent genes between these studies. It is likely that this is due to differences in the age of the animals used and the endocrine environment or previous hormonal exposure of the animals. Nevertheless, there is some accumulating evidence to suggest that androgens are required for functional generation of the Sertoli cell barrier and for development of the specialized tubular environment required for germ cell development (22, 48). Thus, in contrast to FSH, androgen action may be mediated through a relatively small number of changes in Sertoli cell gene expression.

To examine the effect of ablating both FSHR and AR on Sertoli cell activity, we measured the abundance of 14 mRNA species known to be expressed specifically or predominantly within the Sertoli cell population in the adult testis (21, 22, 49). The 14 transcripts divided into three groups, those showing no significant response to ablation of either or both receptors, those sensitive to loss of the AR, and those sensitive to loss of both receptors. The relatively large number of transcripts (~35%), which were unaffected by loss of hormone responsiveness, indicates that a significant proportion of Sertoli cell activity may be independent of hormone action. In addition, 20% of transcripts (Espn, Msi1, and Slc7a4) showed a degree of redundancy between the effects of FSH and androgen. Overall, therefore, extrapolating from these data, it appears likely that a significant number of Sertoli cell genes are either hormone independent or can be stimulated by either hormone, which may act to ensure an adequate baseline of cell activity irrespective of fluctuations in hormone levels. When considering changes in Sertoli cell activity after alterations in hormone or hormone receptor levels, a possible confounding factor is the effect of germ cell loss on Sertoli cell mRNA transcript levels (50, 51). All of the transcripts reported in this study, however, have been shown to be unaffected by germ cell ablation (21).

The transcripts shown in Fig. 4Go were selected for study largely because they are expressed specifically or predominantly in the Sertoli cells and illustrate different aspects of hormonal regulation of the cell. Nevertheless, the altered expression of some of these transcripts, particularly in the SCARKO and FSHRKO-SCARKO mice, offers some further clues to the specific mechanism of hormonal action on the Sertoli cell. ESPN, for example, is an actin-bundling protein (52) and an integral part of the ectoplasmic specializations that are specific to the Sertoli cell and contribute to the Sertoli cell (blood testis) barrier (53). Similarly, TJP1 is integral to the structure of tight junctions that are a major component of the Sertoli cell barrier (54). The reduction in Espn and Tjp1 levels in the SCARKO and FSHRKO-SCARKO mouse would therefore be likely to contribute to the overall disruption of the Sertoli cell barrier seen in the absence of the AR (48).

The marked loss of Aqp8 transcripts in the SCARKO was surprising because previous array studies have not identified Aqp8 as androgen dependent (22, 44, 47). This discrepancy may have come about, however, because earlier array studies used immature testes (10d and 20d) and expression of Aqp8 begins only around d 16 in the rat testis (55). The aquaporins are a family of water-transporting proteins that act to allow osmotically driven movement of water across cell membranes or epithelial layers. Interestingly, in mice lacking AQP8, there is a significant enlargement of the testis (56), although whether this is due to changes in fluid accumulation or cell numbers is unknown. Loss of Aqp8 in the SCARKO mouse and the pattern of expression of the solute carriers (Slc7a4 and Slc38a5) in the different groups are further evidence that both FSH and androgens act directly or indirectly to regulate the internal environment of the seminiferous tubules and thereby facilitate germ cell development (22).

This study shows that spermatogenesis is dependent on the action of FSH and androgen on the Sertoli cell with only the initial onset of meiosis apparently independent of direct hormonal regulation. The two hormones can both act to maintain the meiotic germ cell population, but there is an absolute need for androgen to complete meiosis. Spermiogenesis does not appear to require FSH because the ratio of round spermatids to mature sperm in the FSHRKO is similar to control (not shown), although the models are not informative on the role of androgen in this process. Because other cell types in the testis express the AR, it is also possible that androgens may have additional indirect effects on spermatogenesis, and this will require further study of FSHRKO-ARKO mice.


   Acknowledgments
 
We thank Professor D. G. de Rooij for assistance with identification of germ cell morphology, Biomedical Services (Oxford) for care of the animals and Mohan Masih for histology.


   Footnotes
 
This work was supported by the Wellcome Trust.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 10, 2008

Abbreviations: AMH, Anti-Müllerian hormone; Aqp8, aquaporin 8; AR, androgen receptor; Cre, Cre recombinase; Cst12, cystatin 12; Dhh, desert hedgehog; Espn, espin; FSHR, FSH receptor; FSHRKO, animals lacking FSHR; FSHRKO-SCARKO, mice lacking both FSHR and AR on the Sertoli cell; Gata1, GATA binding protein 1; Itgb1, β1 integrin; Msi 1, Musashi homolog 1; Rhox5, reproductive homeobox 5; SCARKO, Sertoli cell-selective AR knockout; Slc7a4, solute carrier family 7a4; Slc38a5, solute carrier family 38a5; Sox9, SRY-box containing gene 9; Tjp1, tight junction protein 1; Trf, transferrin; Wt1, Wilms tumor homolog.

Received January 22, 2008.

Accepted for publication April 1, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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