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Endocrinology, doi:10.1210/en.2003-1055
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Endocrinology Vol. 145, No. 1 318-329
Copyright © 2004 by The Endocrine Society

Regulation of Sertoli Cell Number and Activity by Follicle-Stimulating Hormone and Androgen during Postnatal Development in the Mouse

Heather Johnston, Paul J. Baker, Margaret Abel, Harry M. Charlton, Gary Jackson, Lynne Fleming, T. Rajendra Kumar and Peter J. O’Shaughnessy

Institute of Comparative Medicine, University of Glasgow Veterinary School (H.J., P.J.B., G.J., L.F., P.J.O.), Glasgow, United Kingdom G61 1QH; Department of Human Anatomy and Genetics, University of Oxford (M.A., H.M.C.), Oxford, United Kingdom OX1 3QX; and Departments of Pathology and Molecular and Cellular Biology, Baylor College of Medicine (T.R.K.), Houston, Texas 77030

Address all correspondence and requests for reprints to: Prof. P. J. O’Shaughnessy, Institute of Comparative Medicine, University of Glasgow Veterinary School, Bearsden Road, Glasgow, United Kingdom G61 1QH. E-mail: p.j.o'shaughnessy{at}vet.gla.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The roles of FSH and androgen in the postnatal development of Sertoli cell number and function have been investigated using mice that lack FSH (FSHßKO), FSH-receptors (FSHRKO), or androgen receptors (Tfm). At birth and d 5, Sertoli cell number was normal in FSHRKO and FSHßKO mice, but was significantly reduced on d 20 and in adulthood. In contrast, Sertoli cell number was reduced at birth in Tfm mice and remained significantly less than normal up to adulthood. Sertoli cell activity was determined through measurement of 11 different mRNA transcript levels. From birth to adulthood, the expression of most transcripts increased, with a significant rise occurring between d 5 and 10. In animals lacking FSH stimulation, mRNA expression (measured per Sertoli cell) was largely normal on d 5, but was reduced in seven transcripts on d 20 and in five transcripts at adulthood. In Tfm mice two transcripts showed reduced expression on d 5, and four were reduced on d 20, although expression in adult Tfm mice did not differ from that in normal cryptorchid controls. The results show that 1) testosterone, but not FSH, is required for Sertoli cell proliferation during fetal and early neonatal life; 2) FSH and testosterone both regulate the late stages of Sertoli cell proliferation; 3) FSH has a general trophic effect on Sertoli cell activity in the pubertal and adult mouse; and 4) androgens are required for specific transcript expression during prepubertal development. Specific effects of androgens were not seen in the adult, although these may be masked by the effects of cryptorchidism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SERTOLI cell acts as the central regulator of testicular development and function. The initial genetic events determining testis differentiation give rise to the Sertoli cells (1, 2), with development of other gonadal somatic cell types likely to depend on Sertoli cell differentiation. In addition, the Sertoli cells act to regulate primordial germ cell proliferation and development during fetal growth (3). Postnatally, the Sertoli cells are essential for development and maintenance of spermatogenesis through direct interactions with the developing germ cells, and in the adult animal the overall germ cell number is dependent upon Sertoli cell number (4). In addition, it is likely that adult Leydig cell activity, function, and survival are dependent on the continued presence of Sertoli cells (5, 6). It is clear, therefore, that regulation of Sertoli cell proliferation and activity during development and in the adult animal is crucial for normal adult fertility.

Studies over a number of years have shown that FSH can act to regulate Sertoli cell function. Initial evidence came from the demonstration that FSH could maintain spermatogenesis in hypophysectomized rats (7), and it was subsequently shown that FSH could stimulate aspects of Sertoli cell function in culture (8, 9). More recently, the use of microarrays has allowed identification of more than 300 genes that are regulated by FSH in cultured Sertoli cells (10). Despite these clear effects of FSH on Sertoli cell function in vitro, however, FSH ß-subunit knockout (FSHßKO) mice and FSH receptor knockout (FSHRKO) mice are fertile, albeit with a reduced germ cell number and sperm quality (11, 12, 13). A number of studies have shown that FSH acts to regulate Sertoli cell number in vivo (14, 15, 16, 17, 18), which would explain in part the reduced germ cell number in the knockout mouse models, but also raises questions about the definitive role of FSH in regulating Sertoli cell function in vivo.

Early studies of the regulation of testicular function also suggested that in addition to FSH, testosterone could act to maintain spermatogenesis in hypophysectomized rats (19). This has subsequently been confirmed by a number of studies and is clearly illustrated by the failure of spermatogenesis in Tfm mutant mice, which lack functional androgen receptors (20), and in hypogonadal (hpg) mice, which show full spermatogenesis after testosterone treatment (18, 21). The experimental chimera studies by Lyon et al. (22) and the fact that germ cells do not express the androgen receptor gene (23) provide strong evidence that androgenic control of spermatogenesis must be mediated by Sertoli cells. Androgens have been shown to affect Sertoli cell function in culture (24), although these effects are generally less marked than the effects of FSH, and in many studies androgens have not had any direct effect in vitro. Thus, although a role for androgens in regulating spermatogenesis through the Sertoli cell is clear, the mechanism by which this is achieved remains to be fully elucidated.

Studies into the roles of FSH and androgen in the regulation of Sertoli cell function have come largely through work with isolated, cultured Sertoli cells in vitro or indirectly through a measurement of spermatogenesis. To examine directly the role of FSH and androgen in the development, regulation, and function of Sertoli cells, we have measured Sertoli cell number and gene expression profiles in FSHßKO and FSHRKO mice, in which FSH activation of the Sertoli cell does not occur, and in Tfm mice, which lack a functional androgen receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The generation of both FSHßKO and FSHRKO mice has been described previously (11, 13). Both colonies were on a mixed C57BL6/129 background and were maintained at the University of Oxford under United Kingdom Home Office regulations. As both FSHRKO and FSHßKO adult males are fertile, the colonies were maintained by breeding homozygous males with heterozygous females. Heterozygous males were used as controls for these mutants in this study. Tfm mice were bred on a C3H/HeH-101/H genetic background from stock animals obtained originally from the Medical Research Council Radiobiology Unit (now the Medical Research Council, Mammalian Genetics Unit, Harwell, UK). The testes of normal mice undergo final descent to the scrotum on about 25 d, whereas in Tfm mice the testes remain intraabdominal into adult life. To control for the failure of testicular descent in Tfm mice, normal animals on the same background were surgically rendered cryptorchid at 21 d and used for experiments when adults (25). Studies of Sertoli cell gene expression in normal mice at different developmental stages used animals on a C3H/HeH-101/H background. In all experiments the day of birth was designated d 1, and animals were killed between d 1 and adulthood (d 90–180) as indicated in the text. The testes from each animal were either frozen in liquid N2 for subsequent study of specific mRNA levels or fixed overnight in Bouin’s fluid and stored in 70% ethanol for subsequent measurement of cell number.

Stereology
For stereology, testes were fixed in Bouin’s fluid and stored in 70% ethanol. Tissue was embedded in Technovit 7100 resin, cut into sections (20 µm thickness), and stained with Harris’ hematoxylin. The total testis volume was estimated using the Cavalieri principle (26), and the slides used to estimate the number of cells were also used to estimate testis volume. The optical disector technique (27) was used to count the number of Sertoli cells in each testis. Sertoli cells were identified by their distinctive nucleus and position on the periphery of the tubule (28). The numerical density of Sertoli cells 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).

Measurement of mRNA levels
For quantification of the content of specific mRNA species in testes during development, a real-time PCR approach was used that employed the TaqMan PCR method after RT of the isolated RNA (29). To allow specific mRNA levels to be expressed per testis and to control for the efficiency of RNA extraction, RNA degradation, and the RT step, an external standard was used (30, 31). 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 residual genomic DNA was removed by deoxyribonuclease treatment (DNA-free, Ambion, Inc., supplied by AMS Biotechnology, Abingdon, UK). The RNA was reverse transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase (Superscript II, Life Technologies, Paisley, UK) as described previously (32, 33).

The sequences of the primers and probes used for real-time PCR were either previously described (34) or are shown in Table 1Go. The membrane-bound kit-ligand (KLm) transcript is generated by alternate splicing, which removes exon 6 of the primary KL transcript. The reverse primer used in the real-time studies spans the KLm-specific exon/exon boundary. The FSHR primer/probe set was designed to hybridize across exons 9 and 10 of the gene and will, therefore, only detect transcripts containing those exons (33).


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  		TABLE 1. Sequences of primers/probes used in this study

 
The real-time PCRs were carried out in a 25-µl volume using a 96-well plate format. Components for real-time PCR were purchased from PE Applied Biosystems (Warrington, UK), apart from primers and probes that were obtained from MWG Biotech (Milton Keynes, UK). Each PCR well contained reaction buffer (with passive reference), 5 mM MgCl2, 200 µM deoxy-NTPs, 300 nM of each primer, 200 nM probe, and 0.02 U/µl enzyme (AmpliTaq Gold, Warrington, UK). Reactions were carried out, and fluorescence was detected on a GeneAmp 5700 system (PE Applied Biosystems). For each sample a replicate was run, omitting the RT step, and a template negative control was run for each primer/probe combination. The quantity of each measured cDNA was then expressed relative to the internal standard luciferase cDNA in the same sample. This method allows direct comparison of expression levels per testis between different samples (30). To determine mRNA levels per Sertoli cell (see Fig. 4GoGo), values were divided by the total Sertoli cell number.



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  		FIG. 4. Sertoli cell mRNA levels during development in normal, FSHßKO, FSHRKO, and Tfm (AR-null) mice. RNA was extracted from testes at different ages, and cDNA was prepared as described in Materials and Methods. Real-time PCR was used to measure cDNA levels relative to an external standard (luciferase) added during RNA extraction. Two-factor ANOVA was used to determine whether there was a significant difference in specific mRNA levels between each of the control groups during development. A, Control groups were not significantly different, and data were analyzed with pooled control data at each age as described in Materials and Methods. Within each age, groups not sharing the same letter superscript were significantly different. B, There was a significant difference between control groups. These control groups have been pooled for illustrative purposes, but data were analyzed without pooling as described in Materials and Methods. Groups with an asterisk were significantly different from the appropriate control group at that age. The mean ± SEM of between three and 23 animals at each age are shown.

 


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  		FIG. 4A. Continued.

 
Statistical analysis
Differences between groups at each age were determined by a single-factor ANOVA, followed by Fisher’s post hoc test. To examine the effects of age on Sertoli cell number, a single-factor ANOVA was carried out within each group, followed by Fisher’s post hoc test. Where there was significant heterogeneity of variance, data were log-transformed before analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Testis morphology
On d 5, seminiferous tubules from normal animals contained mainly Sertoli cells and gonocytes, which were found in the lumen of the tubule and on the basement membrane (Fig. 1AGo). Testis morphology in FSHRKO and Tfm mice on d 5 was similar to that in controls (Fig. 1Go, B and C), although Tfm testes were significantly smaller (1.97 ± 0.1 vs. 1.0 ± 0.17 mm3; P < 0.05). By d 20, the tubules had enlarged in the normal animal and contained mainly Sertoli cells, with spermatogonia and spermatocytes undergoing proliferation (Fig. 1DGo). In the FSHRKO mouse (Fig. 1EGo) at 20 d, overall testis morphology was very similar to normal, although testis size was significantly reduced (24.3 ± 0.4 vs. 7.6 ± 0.9 mm3; P < 0.01). In 20-d-old Tfm mice, overall tubule diameter was similar to normal, but with a higher variation (Fig. 1Go, D vs. F). Tubules in d 20 Tfm mice lacked a lumen and did not show the full range of spermatogenic cell division seen in normal males at this age (Fig. 1FGo). Instead, scattered spermatocytes undergoing division were seen within each tubule. Testes in 20-d-old Tfm mice were significantly smaller than those in controls (24.3 ± 0.4 vs. 4.3 ± 0.3 mm3; P < 0.01). In adult normal and FSHRKO mice, all stages of spermatogenesis were seen as previously described (13), although tubule diameter was reduced in FSHRKO animals (Fig. 1Go, G and H). In adult Tfm mice, tubular morphology was similar to that at 20 d of age, with spermatogenesis not progressing beyond the early spermatocyte stage (Fig. 1IGo). A similar disruption to spermatogenesis was seen in normal mice with experimental cryptorchidism induced before testicular descent (Fig. 1JGo). In about one third of the tubules in adult Tfm mice, clumps of cells with an indistinct morphology were seen in the center of the tubule (arrowed in Fig. 1IGo). At all ages, the morphology of testes from FSHßKO mice was indistinguishable from that of FSHRKO mice (data not shown).



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  		FIG. 1. Light micrographs showing normal, FSHRKO, and Tfm testes at d 5, d 20, and adulthood and cryptorchid testes from adult animals. The top row of light micrographs (A–C) shows morphology in d 5 animals, the second row (D–F) in d 20 animals, and the third row (G–I) in adult animals. A section of testis from a normal adult animal, surgically rendered cryptorchid before puberty, is shown in the bottom row (J). The arrow in I points to an intratubular cell mass, which was present in about 30% of tubules from adult Tfm mice. The bar shown represents 50 µm in all micrographs.

 
Sertoli cell number
In normal mice, Sertoli cell number increased 6-fold from d 1 to d 20 and then remained static into adulthood (Fig. 2Go). Sertoli cell number in FSHRKO and FSHßKO animals were normal on d 1 and 5. On d 20, Sertoli cell number was reduced in FSHßKO animals relative to that in control mice and did not differ significantly from that in FSHRKO mice. In the adult animal, Sertoli cell numbers in both FSHRKO and FSHßKO mice were reduced to about 60% of normal. Statistical analysis of the developmental changes in cell numbers in these groups showed that cell number in FSHßKO mice reached a peak on d 5 and did not change thereafter (Fig. 2Go). FSHRKO mice tended to show more variation, but at no time did they differ significantly from FSHßKO mice.



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  		FIG. 2. Sertoli cell number in control, FSHRKO, FSHßKO, Tfm, and cryptorchid mice. Cell numbers in each null group and in appropriate controls were measured as described in Materials and Methods. Two-factor ANOVA showed that there was no difference between different control groups, and control values were, therefore, pooled. Results show the mean ± SEM for between three and 12 animals in each group. Overall, two-factor analysis of the data showed that there were significant age and animal group effects and that there was a significant interaction between the two groups. Further single-factor ANOVA was carried out within each age and within each animal group. The results of analysis within each age are shown in the figure, and columns within each age that do not share a common letter superscript are significantly different. Further analysis within each group was carried out to examine changes in cell number during development, and results are summarized in the table below.

Within each group, ages that do not share a common letter superscript are significantly different.

 
Sertoli cell numbers in Tfm mice were significantly less than control values on d 1 and remained lower than all other groups at each age measured (Fig. 1Go). Cell number peaked on d 20 in Tfm mice and then declined significantly to about 30% of control levels in adulthood (Fig. 2Go). There was no difference in Sertoli cell number between normal control animals and animals rendered cryptorchid before puberty (Fig. 2Go).

Developmental changes in Sertoli cell gene expression in normal mice
Real-time PCR was used to examine changes in Sertoli cell gene expression during postnatal development in normal mice (Fig. 3GoGo). Expression was measured relative to an external standard (luciferase) added to each tissue before RNA extraction. The results, therefore, indicate the relative gene expression per testis. The 11 mRNA species examined in this study were chosen because they have been shown to be expressed in Sertoli cells and because, within the testis, they show little or no expression in any other cell type (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). The mRNA species studied can be broadly divided into four functional groups: 1) tight junction components [zonula occludens 1 (ZO-1) and claudin 11], 2) tissue remodeling factors [cystatin-TE (Cys-TE) and tissue plasminogen activator (tPA)], 3) lipid-binding proteins [epidermal fatty acid-binding protein (FABP) and androgen-binding protein (ABP)], and 4) endocrine and paracrine factors [KLm, platelet-derived growth factor-A (PDGF-A), anti-Mullerian hormone (AMH), desert hedgehog (Dhh), and FSHR]. Of the 11 transcript species measured in this study, 10 showed a general increase in expression from birth to adulthood, with Cys-TE showing the highest overall expression at all ages. The one exception to this pattern was AMH, which showed no change in expression from birth to around d 10, before declining significantly to adulthood. In the 10 transcripts showing an increase in expression after birth, the most consistent change was a significant increase in expression on d 10, which was seen in all cases. After 10 d, expression per testis either remained fairly static (ABP, Dhh, and tPA) or increased further into adulthood (FABP, KLm, PDGF-A, claudin 11, ZO-1, and Cys-TE).



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  		FIG. 3. Changes in Sertoli cell mRNA levels during development in the normal mouse. RNA was extracted from testes at different ages, and cDNA was prepared as described in Materials and Methods. Real-time PCR was used to measure cDNA levels relative to an external standard (luciferase) added during RNA extraction. The mean ± SEM of between three and six animals at each age are shown. Groups that do not share the same letter superscript are significantly different.

 


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  		FIG. 3A. Continued.

 
Sertoli cell gene expression in FSHßKO, FSHRKO, and Tfm mice
To determine the roles of FSH and androgen in the development of Sertoli cell function, the expression of Sertoli cell-specific genes was measured during postnatal development in normal, FSHßKO, FSHRKO, and Tfm mice. The results were corrected for differences in Sertoli cell number by dividing measured mRNA levels calculated per testis (as described above) by the number of Sertoli cells in each group (Fig. 4GoGo).

In FSHßKO and FSHRKO mice, Sertoli cell RNA transcript levels were largely normal on d 5 after birth. The exceptions were ABP, which was significantly reduced by about 50% in FSHRKO mice, and FSHR, which, as expected, was absent in FSHRKO mice. On d 20, in contrast, there was a significant reduction in ZO-1, PDGF-A, KLm, Cys-TE, FABP, and ABP expression in both FSHßKO and FSHRKO mice. In addition, there was a reduction in claudin 11 expression, although this was only significant in FSHßKO mice. On d 5, FSHR expression was absent in FSHRKO mice. In affected genes, the reduction in expression was between 50 and 80%. No change in Dhh, FSHR (in FSHßKO mice), AMH, or tPA expression was seen. In adult animals, levels of expression of ZO-1, claudin 11, PDGF-A, and Cys-TE remained significantly reduced, and tPA expression was also reduced. In contrast to that in 20-d-old animals, KLm, FABP, and ABP expression was not different from normal in adult FSHR-KO or FSHß-KO animals. The expression of Dhh, AMH, and FSHR (in FSHßKO mice) remained normal throughout postnatal development. Interestingly, FSHR expression was detectable at low levels in adult FSHRKO mice using the real-time primer/probe combination that spans exons 9 and 10 of the cDNA.

In Tfm mice, levels of the mRNA species measured in this study were normal per Sertoli cell on d 5, with the exceptions of tPA and FABP, which were significantly reduced. On d 20, levels of PDGF-A, Cys-TE, tPA, and FABP were reduced significantly in Tfm mice, whereas other mRNA species were unaffected. The effects of the Tfm mutation on Sertoli cell function in the adult animal are more complex because adult Tfm mice are cryptorchid. Induced cryptorchidism per se in normal animals had no effect on the expression of most of the mRNA species measured with the exception of ZO-1, PDGF-A, and FABP, in which expression was significantly reduced, and AMH, in which there was a significant increase in expression. Using the cryptorchid animal as the appropriate control, the Tfm mutation had little effect on gene expression in the adult mouse; the only exception was an increase in KLm mRNA levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sertoli cell number
Sertoli cells proliferate in the mouse through fetal and neonatal development to reach a maximum about 15–20 d postpartum (28, 46). In mice lacking FSH stimulation, the normal Sertoli cell number at birth and during the early postnatal period indicates that FSH is not required for the determination of Sertoli cell number during the fetal and neonatal periods and only becomes necessary during the final establishment of Sertoli cell number. Other studies have reported, in contrast, that late fetal Sertoli cell proliferation in the rat is regulated by FSH (16, 47). This discrepancy may be due to species differences or to differences in techniques used. Earlier studies altered Sertoli cell FSH stimulation through fetal decapitation, use of organ culture, or treatment with FSH antibodies; Sertoli cell labeling, rather than cell number, was measured as an end point. It is possible that in these earlier studies testicular androgen levels may have been affected by the in vivo treatments, although the labeling index may not necessarily reflect final cell numbers. It is worth noting that the results of this study (see below) and others (48) have indicated that fetal Sertoli cell function is largely independent of FSH stimulation. In the adult, the difference in Sertoli cell number between control and FSHßKO animals is similar to that reported previously (17).

In contrast to animals lacking FSH stimulation, androgens appear to be essential for Sertoli cell proliferation during fetal life and throughout the postnatal, prepubertal period. Interestingly, Sertoli cell number in adult Tfm mice was significantly lower than on d 20, suggesting that androgens may be required to maintain Sertoli cell number in the adult animal. In this study Sertoli cells are defined in part by their position on the periphery of the seminiferous tubules. We have noted that in sections of adult Tfm testes some tubules contained clumps of cells within the center of the tubule, similar to those is seen in KL-deficient and claudin 11-KO mice (49, 50). These have been reported to contain Sertoli cells that have become detached from the basement membrane (6, 50), and as these cells were not included in the Sertoli cell counts reported here, they may account for the apparent decrease in cell number in the adult. If these cell clumps contain functional Sertoli cells, then gene expression levels, measured per Sertoli cell in the adult Tfm mouse, will have been slightly overestimated.

The hpg mouse is an alternative and useful model for the study of Sertoli cell development because it lacks GnRH through a natural deletion leading to a loss of circulating LH and FSH (51). Comparison between hpg mice and those lacking FSH stimulation or androgen receptors can be complex, because adult hpg mice have severely depleted levels of both FSH and testosterone, although neither hormone is completely absent. Sertoli cell number is normal during fetal development in hpg mice (28), which would support the hypothesis that FSH does not regulate fetal Sertoli cell number, as androgen levels are normal during this stage of hpg development (52). After birth, Sertoli cell number is reduced during the early postnatal period in the hpg mouse (28) with numbers similar to those seen in Tfm mice in this study. A significant reduction in androgen levels occurs around birth in hpg mice (52), suggesting that the early postnatal reduction in Sertoli cell number is caused by androgen withdrawal. Treatment of hpg mice with FSH or androgen will increase adult Sertoli cell number (18), which is consistent with the results reported here.

Sertoli cell gene expression during normal development
Sertoli cell activity in this study has been assessed by detailed measurement of a limited number of mRNA species that provides a restricted picture of gene expression in the Sertoli cell during development, but will indicate, nevertheless, general trends in cell activity.

With the exception of AMH, the clearest developmental change in normal Sertoli cell gene expression (per testis) occurred between d 5 and 10. Sertoli cell number increases during this period, but in most cases the increase in cell number would not account for the increase in gene expression, suggesting an overall increase in Sertoli cell activity. This is also the period during which changes in the expression of at least some other Sertoli cell genes is first seen (53), suggesting that there is an overall change in Sertoli cell activity at this time. The interval between postnatal d 5 and 10 is crucial in mouse testis development. During this period spermatagonial proliferation and meiosis begin (54), adult Leydig cells first start to differentiate (55, 56), and the blood-testis barrier begins to form (57). The Sertoli cell is known to play a central role in regulating testis function (6), and these early postnatal developments in the testis may all be related to changes in Sertoli cell function. Between d 5 and 10 there is little change in circulating levels of FSH (58, 59) (also confirmed in our own studies; data not shown) and a drop in intratesticular androgen (60). This suggests that Sertoli cell sensitivity to hormone stimulation increases or another, as yet unknown, factor is involved in Sertoli cell regulation at this time.

Sertoli cell gene expression in FSHRKO, FSHßKO, and Tfm mice
Tight junction components.
Tight junctions between Sertoli cells are the main functional component of the blood-testis barrier. Both ZO1 and claudin-11 are integral to the structure of these tight junctions (61), and claudin-11-KO mice lack tight junctions between Sertoli cells, leading to infertility (50). The significant reduction in levels of both ZO1 and claudin-11 transcripts in mice lacking FSH stimulation on d 20 indicates that FSH is involved in the regulation of these genes. Sertoli cell tight junctions may, therefore, be compromised in these animals, and this may provide a partial explanation of the reported reduction in sperm quality in these mutants. In the Tfm mutants, there was little difference in claudin 11 expression at any age during postnatal development. Similarly, levels of ZO-1 were normal up to 20 d, although there was a dramatic decline in expression in both adult normal cryptorchid mice and adult Tfm mice. As adult Tfm mice are cryptorchid, it appears that androgen resistance does not affect ZO-1 expression beyond the changes induced by cryptorchidism itself. Other studies have shown that tight junctions are abnormal in the adult Tfm mouse (62), and cryptorchidism has been reported to be associated with defective development of the blood-testis barrier (63).

Tissue remodeling factors.
Cys-TE is a member of the cystatin superfamily of cysteine proteinases, and it may have a number of functions in the testis, including germ cell-Sertoli cell interactions (43). Overall testicular expression of Cys-TE was more than 10 times greater than that of any other transcript measured in this study, suggesting an important role in Sertoli cell function. Reduced levels of Cys-TE in d 20 and adult mice lacking FSH stimulation indicate a role for FSH in regulating this gene. In contrast, the other tissue remodeling factor, tPA, was not significantly affected by lack of FSH stimulation at any age. In Tfm mice, levels of both Cys-TE and tPA were significantly reduced on d 20, but were normal in the adult animal. This suggests that there is a transient period around puberty during which these transcripts require androgen stimulation, but that they become refractory to the effects of androgens in the adult animal.

Lipid-binding proteins.
Both FABP and ABP were reduced on d 20 in FSHßKO and FSHRKO mice, but were normal in adult animals, showing that FSH is required during the pubertal period for normal expression, but that expression becomes independent in the adult animal. FABP is involved in the uptake and transport of fatty acids essential for the nourishment of the surrounding cell types (42) and may play a role in germ cell maturation. It is possible, therefore, that in the adult animal control of FABP expression becomes dependent primarily on Sertoli cell-germ cell interaction as spermatogenesis becomes established. This would be consistent with continued reduced levels of expression of FABP in normal cryptorchid and Tfm adult mice. The role of ABP in the testis is uncertain, but it may be involved in regulating germ cell apoptosis during the prepubertal period (64). Reduced levels of ABP during this period may, therefore, also contribute to reduced germ cell number in the adult FSHRKO and FSHßKO mice.

Endocrine and paracrine factors.
The growth factors Dhh and PDGF-A are required during testicular development for normal Leydig cell differentiation (41, 65). The expression of Dhh was unaffected by the mutations used in this study, whereas, in contrast, PDGF-A transcript levels were reduced in animals lacking FSH stimulation. This contrasts with earlier studies in the rat that suggested that PDGF-A expression is inhibited by FSH (66). It is likely that this apparent discrepancy is due to species differences in the expression and control of PDGF-A (67). Interestingly, cryptorchidism (and the Tfm mutation) caused a marked reduction in PDGF-A expression, suggesting that adult expression is also dependent on germ cell maturation. This would be consistent with demonstrated stage-dependent expression in the mouse testis (41).

The membrane-bound form of Kit ligand has been shown to be necessary for the maintenance of differentiated germ cells and for their entry into and/or completion of meiosis (68). The significantly lower levels of KLm transcripts on d 20, when meiosis is beginning, in mice lacking FSH stimulation indicates that FSH regulates the expression of this gene in prepubertal mice. Normal expression of KLm in the adult animal shows, however, that this regulation is transient and is not required once spermatogenesis is established.

The FSHR is known to be down-regulated in the testis by exposure to high circulating levels of FSH (69, 70). In the results reported here, however, the lack of circulating FSH did not affect the expression of FSHR, suggesting that within the normal physiological range, FSHR expression is not regulated by FSH. In adult mice lacking androgen receptors, there was a reduction in FSHR expression, indicating that androgens can play a role in regulating FSHR in the adult animal. It is possible, therefore, that one of the trophic effects of androgen on the Sertoli cell is to increase sensitivity to FSH.

AMH is produced by Sertoli cells from early fetal life to puberty (71). At puberty, synthesis is down-regulated, possibly through the synergistic action of intratesticular testosterone and meiotic germ cells (71). In adult mice, there was a small increase in AMH mRNA levels in cryptorchid and Tfm mice compared with normal controls, which would be consistent with this hypothesis, although it is clear that the overall drop in AMH expression and AMH serum levels (72) is not prevented by androgen insensitivity. Normal levels of AMH expression in mice lacking FSH indicate that FSH is not required for AMH expression.

Control of Sertoli cell activity
Overall, little difference in Sertoli cell activity was seen on d 5 between animals lacking FSH stimulation and normal mice of the same age. Receptors for FSH are expressed early in fetal testis development (73), but our results indicate that fetal and early neonatal Sertoli cell development is largely independent of FSH control. This supports earlier studies using cultured rat Sertoli cells (48) and is consistent with normal Sertoli cell numbers in animals lacking FSH stimulation. By d 20, the expression of seven genes was reduced in FSHRKO and FSHßKO animals. This suggests that the rise in Sertoli cell activity seen around d 10 is at least partly driven by FSH. In the adult animal dependence on FSH was largely retained, with five of the 11 genes investigated being significantly reduced compared with normal mice. Despite these clear changes in gene expression in mice lacking FSH stimulation, it should be noted that the spermatogenic process is essentially normal in these animals, although there is an overall reduction in spermatid number per Sertoli cell (17). Thus, although FSH clearly regulates the expression of a number of genes in the Sertoli cell, the reduction in expression in the absence of FSH stimulation is not sufficient to compromise, fatally, the spermatogenic process. The reduced spermatid number in these animals may, however, be a reflection of reduced Sertoli cell function.

In contrast to the relatively normal spermatogenesis in mice lacking FSH stimulation, mice lacking functional androgen receptors show severe disruption to spermatogenesis both before puberty and in the adult animal. The failure of normal germ cell proliferation and differentiation in prepubertal, 20-d-old Tfm mice shows that androgens are essential for this process and, as androgen receptors are present in Sertoli cells but not germ cells (22, 23), this indicates that Sertoli cell function is disrupted during the prepubertal period. Gene expression levels per Sertoli cell on d 5 in Tfm mice were similar to those in normal mice with the exceptions of FABP and tPA. This would indicate that, unlike cell proliferation, Sertoli cell activity is largely independent of androgen action up to d 5. By d 20, the expression levels of four mRNA transcripts were reduced in the Tfm testis, showing that androgens as well as FSH have specific trophic effects on the Sertoli cells at this age. Although both hormones can thus alter Sertoli cell activity around puberty, the considerably greater level of spermatogenic disruption in the androgen-insensitive mice suggests that androgens must regulate an essential subset of Sertoli cell genes critical for the spermatogenic process.

Analysis of Sertoli cell function in adult Tfm mice is complicated by the failure of normal testicular descent in these animals. In normal animals rendered surgically cryptorchid at puberty, gene expression was altered in four of the 11 transcripts measured in this study. The mechanism involved in this effect is likely to be linked to increased temperature in the cryptorchid testis or, possibly, to germ cell loss (74). In adult Tfm mice, only one transcript was significantly different from the cryptorchid controls. It is possible that some of those genes that are sensitive to cryptorchidism are also dependent on androgen for full expression, but our studies suggest, nevertheless, that androgens have only a limited effect on Sertoli cell gene expression in the adult animal.

Overall, our results using FSHßKO, FSHRKO, and Tfm mice show that both FSH and androgen are required for development of the full complement of Sertoli cells in the adult male, although only androgens are crucial during fetal and neonatal development. Sertoli cell activity increases around d 10 in the mouse, possibly reflecting an increase in sensitivity to FSH at this time, although some genes appear to function independently of FSH action through development. Androgens also appear to be important in maintaining Sertoli cell function in the pubertal period, although requirements for androgen are less clear in the adult, and the effects of androgen may be mediated through a limited set of genes not measured here.


   Footnotes
 
This work was supported by funding from the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and The Moran Foundation (Department of Pathology, Baylor College of Medicine).

Abbreviations: ABP, Androgen-binding protein; AMH, anti-Mullerian hormone; Cys-TE, cystatin-TE; Dhh, desert hedgehog; FABP, fatty acid-binding protein; FSHR, FSH receptor; KO, knockout; PDGF-A, platelet-derived growth factor-A; tPA, tissue plasminogen activator; ZO-1, zonula occludens 1.

Received August 14, 2003.

Accepted for publication September 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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R. S. McRae, H. M. Johnston, M. Mihm, and P. J. O'Shaughnessy
Changes in Mouse Granulosa Cell Gene Expression during Early Luteinization
Endocrinology, January 1, 2005; 146(1): 309 - 317.
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K. De Gendt, J. V. Swinnen, P. T. K. Saunders, L. Schoonjans, M. Dewerchin, A. Devos, K. Tan, N. Atanassova, F. Claessens, C. Lecureuil, et al.
A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis
PNAS, February 3, 2004; 101(5): 1327 - 1332.
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