| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Testis |
Department of Medicine, Division of Endocrinology, Metabolism and Molecular Medicine, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Leydig cells, male reproductive tract, spermatogenesis, steroid hormone receptors, testis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Targeted disruption of Dax1 in male mice results in infertility, decreased testicular size, and degeneration of germinal epithelium [7]. A thorough histological examination of the male reproductive tract from the Dax1 knockout (Dax1KO) mouse revealed that the rete testis, the passageway for sperm to leave the testis, is obstructed by ectopic clusters of Sertoli cells [8]. Tissue near the rete testis exhibits disruption of the basal lamina and Leydig cell infiltration of the seminiferous tubules, suggesting that deficiency of Dax1 results in alterations of multiple somatic cell types within the male gonad.
Because Dax1 is expressed in both Leydig and Sertoli cells, the relative contributions of Sertoli and Leydig cell dysfunction in the Dax1KO model cannot be ascertained. To directly evaluate the role of Dax1 in Sertoli cells, DAX1 was expressed by the müllerian-inhibiting substance promoter (MIS-DAX1) in the genetic background of the Dax1KO model [9]. Sertoli cell-specific expression of Dax1 was sufficient to rescue fertility and sperm counts but did not substantially reverse the histological abnormalities or testicular weight. This finding implies an important function of Dax1 in other testis cell types. An examination of Leydig cells from the Dax1KO mice revealed up-regulation of Cyp19 expression [10]. Cyp19 encodes aromatase, the enzyme that converts testosterone to estradiol. Estradiol acts as a mitogen to somatic cells of the testis, and overexpression of aromatase results in degeneration of the germinal epithelium and Leydig cell hyperplasia [11]. Therefore, it is possible that the Dax1KO testis phenotype is, in part, caused by steroidogenic dysfunction of the Leydig cells.
To address this hypothesis, we used the LH-receptor promoter to target DAX1 expression (LHR-DAX1) to Leydig cells and bred this transgenic line onto the Dax1KO background. As in the Sertoli cell-specific DAX1 rescue, fertility was improved, but Leydig cell-specific expression of DAX1 was not sufficient to correct the severe testicular dysgenesis seen in Dax1KO mice. Thus, Dax1 appears to play an important functional role in both Sertoli and Leydig cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A 2.1-kilobase (kb) fragment of the LHR upstream promoter (-2082 to +1) was previously reported to drive and maintain expression of ß-galactosidase within the Leydig cells of transgenic mice [12, 13]. The LHR-DAX1 transgene (Fig. 1A) was prepared by ligating the LHR promoter to the human DAX1 cDNA (1.5 kb) and simian virus 40 polyadenylation signal (0.7 kb). A FLAG (DYKDDDDK) epitope was inserted at the amino-terminus of DAX1 to allow documentation of protein expression. To ensure that the FLAG epitope did not affect Dax1 function, we tested the transcriptional activity of the fusion protein in a standard luciferase assay and observed no difference in activity compared to DAX1 (data not shown). Transgenic mice were created by the microinjection of the LHR-DAX1 transgene into pronuclei of 1-cell stage mouse embryos obtained from C57BL/6 females. Six separate mouse lines were created, each originating from a different founder. All experiments were carried out with mice from two lines that had similar levels of transgene expression. The presence of the LHR-DAX1 transgene was confirmed by polymerase chain reaction (PCR) of genomic DNA obtained from tail biopsies (Puregene; Gentra Systems, Minneapolis, MN) using primers corresponding to LHR (5'-agcatactggcctagccaccgga-3') and DAX1 (5'-cgtttgcttcgcgctcataagca-3'). To generate animals of each genotype (wild type [WT], Dax1KO [KO], LHR-DAX1 transgenic [TG], and KO;TG [RS]), TG males were bred to Dax1del/wt females. Each genotype was produced at the expected ratios, housed in a barrier facility, and allowed food and water ad libitum.
|
Measurements of Fertility
All procedures were approved by the Northwestern University Animal Care and Use Committee, Master Protocol 027-07-193. To measure fertility, one animal of each genotype (WT, KO, TG, and RS) at 9 wk of age was housed with a CD1 female (age, 
9–12 wk). The following week (age, 10 wk), another CD1 female was placed with the breeding pair. At the third week of study (age, 11 wk), the female placed with the male at 9 wk was removed and replaced with a new female. Thus, only two females were housed with the male at one time, for a period of 2 wk. This protocol was carried out until the male animals reached 12 wk of age. Eight animals of each genotype were used in the fertility study. After the 2 wk of breeding, females were then killed, their uteri removed, and the embryos counted. Statistically, the presence of any embryo was considered to be a successful pregnancy, whereas the number of embryos present was used to calculate litter size and summed among females from one male to calculate total pups/breeding.
Measurement of Sperm Count and Testicular Weight
At 12 wk of age, the fertility study was concluded, and the males were killed. The right testis was weighed, photographed, fixed in 10% formalin or Bouin fixative, and embedded in paraffin for histological analysis. The right epididymis and ductus deferens were removed and placed in 1 ml of prewarmed Quinn Sperm Washing Medium (Sage BioPharma, Bedminster, NJ) for 15 min at 37°C [9]. After incubation for 15 min, a 1:10 dilution was made into 1 ml of Quinn medium. Twenty microliters were then applied to a hemocytometer. Sperm counts were made separately for both motile and total sperm, and three counts were made for each animal.
Reverse Transcription-PCR
The left testis was dissected and RNA extracted with Trizol (Invitrogen, Carlsbad, CA). The RNA was then treated with DNase (Ambion, Inc., Austin, TX) and quantified spectrophotometrically. Reverse transcription (RT) was performed with 1 µg of RNA, and cDNA was amplified to 34 cycles with the following primers to Cyp19 and RpL19 as a control: Cyp19F, 5'-tggagaacaattcgccctttc-3'; Cyp19R, 5'-ttcgtcaggtctccacgtctct-3'; RpL19F, 5'-ctgaaggtcaagggaatgtg-3'; and RpL19R, 5'-ggacagagtcttgatgatctc-3'. Primer sequences and conditions for Cyp11a, Cyp17, and StAR are the same as those used previously [10].
Measurements of Hormones
Blood was taken from mice at the time of death by cardiac puncture, and serum was collected after centrifugation. Testicular lysates were generated by homogenization of individual testis in 1 ml of phosphate-buffered saline, followed by centrifugation to pellet out insoluble debris. Serum and lysates were frozen and sent to the Ligand Assay and Analysis Core Laboratory of the University of Virginia (Charlottesville, VA) for measurement by RIA.
Histology and Immunohistochemistry
Embedded tissue was sectioned (thickness, 3 µm) on a Jung RM 2025 (Leica, Nussloch, Germany) microtome. Sections were examined on a Zeiss Axioskop (Zeiss, Thornwood, NY), whereas gross tissue was viewed with a Leica MZLFIII (Leica, Heerbrugg, Switzerland) dissecting microscope. For both microscopes, pictures were taken with a Color MagnaFire (Optronics, Goleta, CA) digital camera. Testes were stained with hematoxylin-and-eosin using standard protocols. For immunohistochemistry, sections were deparaffinized by serial washes in xylenes and ethanol, followed by antigen retrieval in sodium citrate buffer (pH 6.0). Autofluorescence was quenched by 15-min incubation in sodium borate (pH 8.5). Sections were then blocked in normal serum, followed by incubation in primary antibody overnight. Fluorescein isothiocyanate or horseradish peroxidase-conjugated-Anti-FLAG (M2, 1:100; Sigma-Aldrich, St. Louis, MO) was used to localize expression of the FLAG-tagged transgene. GATA-4 (C-20, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA) was used to identify Sertoli cells. Secondary antibodies were applied for 2 h at room temperature (Donkey Cy3-anti-Rabbit and Donkey Cy3-anti-Goat from Jackson Immunoresearch, West Grove, PA; DAB from Vector, Burlingame, CA). Sections were washed and mounted with Fluorescent Mounting Media (Vector, Burlingame, CA). Sections were viewed with a Zeiss Axioskop.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mice of all genotypes (WT, TG, KO, and RS) were generated at expected ratios, indicating that the transgene was not deleterious to reproduction. Expression of the LHR-DAX1 transgene was assessed by real-time quantitative RT-PCR and was similar in the two lines analyzed (data not shown). Cellular localization of the epitope-tagged LHR-DAX1 transgene was determined by immunohistochemistry. Leydig cells were strongly positive, which is consistent with the anticipated pattern for LHR-driven expression [12, 13] (Fig. 1B). Fluorescence was also seen in scattered primary spermatogonia and elongated spermatids, as previously documented for this promoter [12]. Analysis by RT-PCR was used to detect DAX1 mRNA and revealed that the LHR promoter did not drive DAX1 expression until after weaning, as observed previously with this 2.1-kb fragment.
LHR-DAX1 RS Mice Have Increased Fertility and Litter Size
Mutation of Dax1 causes infertility in both humans and mice [7, 14]. Fertility was quantified by comparison of successful pregnancies achieved in the WT, TG, KO, and RS animals. As previously reported [7, 9], fertility was markedly impaired in KO males (28%) (Fig. 2A). Although a slight decrease in fertility was observed in animals transgenic for DAX1 (100% vs. 86%), the selective expression of DAX1 in Leydig cells (RS mice) increased fertility to approximately half of WT levels (53%), which is almost double that of KO animals. The RS males produced an average of 20 pups over 4 wk of mating, whereas KO males produced an average of only 8 pups (Fig. 2B).
|
LHR-DAX1 RS Mice Have Decreased Testicular Weight, Normal Levels of Aromatase, and Increased Sperm Count
The testicular weights of TG animals were similar to those of WT animals. As previously observed [7, 9], the weight of the KO testis is approximately half that of a normal testis (Fig. 3, A and B). Conversely, animals with the DAX1 transgene and deletion of Dax1 (RS mice) had testicular weights that were equivalent to those of the KO mice (Fig. 3, A and B), confirming that Leydig cell expression of Dax1 is not sufficient to rescue the reduced size of the KO testis.
|
To determine if Leydig cell expression of DAX1 could rescue the blockage of sperm passage from the testis, sperm counts were measured in mice of all four genotypes. Although the transgene alone had no significant effect on sperm production in TG animals, a significant increase in sperm was observed in RS animals compared to KO mice (P ≤ 0.01) (Fig. 3C). Sperm counts in RS mice were approximately half those of WT and TG mice, but more than 4-fold higher than those of KO mice, which likely explains the increased fertility in RS animals.
Dax1 transcriptionally represses expression of aromatase (Cyp19), and mutation of Dax1 causes overexpression of aromatase in Leydig cells [10]. Analysis by RT-PCR confirmed a 2-fold elevation of Cyp19 in Dax1-deficient animals. Leydig cell rescue of DAX1 restored aromatase mRNA to WT levels (Fig. 3D) and decreased intratesticular estradiol levels (Fig. 3E). No significant differences were observed in the gonadotropin levels of the four genotypes (Fig. 3E). Examination of mRNAs encoding the steroidogenic enzymes Cyp11a, Cyp17, and StAR by RT-PCR revealed no significant differences in expression levels in the four genotypes (data not shown).
LHR-DAX1 Transgene Does Not Correct Testicular Pathology in KO Mice
Several pathological features are seen in the KO mouse. The diameter of individual seminiferous tubules is prominently increased. In addition, spermatogenesis is almost completely absent, because the seminiferous epithelium is denuded except for residual Sertoli cells that exhibit swirling cytoplasm (Fig. 4C) [7]. Introduction of the LHR-DAX1 transgene minimally altered the testicular histology of RS animals compared to KO mice (Fig. 4, C and D). Although the tubular pathology is not as advanced at 12 wk in RS mice, significant dilation of tubular lumen, vacuolation, and loss of germ cells were still observed. These findings suggest that loss of Dax1 in adult Leydig cells is not the major determinant of the KO testis phenotype.
|
LHR-DAX1 RS Mice Retain Abnormal Rete Testis Pathology
The efferent ductules and rete testis of the KO mouse are obstructed by Sertoli cells that block the outflow of the sperm from the testis [8]. Normally, this area is unobstructed, but a few tubules near the rete testis may display minimal vacuolation (Fig. 5A). In both the KO and RS mice, significant disruption of spermatogenesis is seen, with vacuolation and breakdown of tubule borders (Fig. 5, B and C). Similar to the pathology present throughout the testis, the disorganization of seminiferous tubules in RS mice was not as severe as in KO animals at 12 wk, but the process had been initiated. GATA-4 immunostaining was used to identify Sertoli cells (Fig. 5D) [15]. In KO gonads, the Sertoli cells were scattered throughout the rete testis (Fig. 5E). In the RS animals, a similar distribution of GATA-4 staining cells was found in the rete testis (Fig. 5F).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The increase in successful pregnancies in the RS animals compared to the KO mice suggests that Leydig cell expression of DAX1 is sufficient to restore spermatogenesis to a threshold level that is required for fertility. However, the rate of successful pregnancies in the RS mice was only a little more than half that seen in the WT or TG mice. This finding indicates that Dax1 is required in other temporal or spatial patterns to fully restore fertility. In contrast, mice with Sertoli cell-specific expression of DAX1 had almost complete restoration of fertility [9]. Because Sertoli cells are in direct contact with germ cells, it is not unexpected that expression of Dax1 in these cells more effectively restores fertility. Alternatively, differences in the magnitude and timing of the transgene may also explain the different reproductive phenotypes in these transgenic models. The LHR transgene is reported to be induced at 5 wk postnatal in Leydig cells, whereas the MIS transgene is first expressed embryonically, at 15.5 days postcoitus (dpc) [12, 16]. The pattern of expression driven by the MIS promoter may more closely resemble the natural pattern of Dax1 expression, which is initiated at 9.5 dpc and continues throughout gonadal development in both sexes [1]. The effect of transgene expression in scattered germ cells is consistent with previous reports of this promoter. Its effects on reproduction are unknown but likely are minimal, because the TG mice have normal fertility.
Testicular weight was not increased in RS mice compared to KO animals. Testicular volume has a direct relationship to fertility, because germ cells are responsible for much of the testicular weight (60%) [17]. Because the germinal epithelium degenerates in the KO and RS mice, the decreased cell numbers likely result in reduced testicular weight. Nevertheless, we did observe a significant increase in sperm counts in the epididymides of RS mice compared to KO animals. Although the sperm counts were approximately half those of WT and TG mice, the increased number of sperm provides a plausible basis for the increased fertility in RS animals, because only a threshold level of spermatogenesis is required for fertility. Sertoli cell-specific rescue of DAX1 also did not increase testicular volume, and sperm counts were also raised to approximately half the WT values [9]. These results indicate that DAX1 expression in either cell type is not sufficient to increase the size and full spermatogenic capacity of the testis.
Aromatase was restored to WT levels in RS mice. Although aromatase activity has been detected in germ cells and immature Sertoli cells, the majority of aromatase expression is localized to Leydig cells in adult mice [18]. Previous analyses of isolated Leydig cells from the Dax1KO testis revealed overexpression of Cyp19 [10]. Aromatase transcription is directly controlled by the orphan receptor steroidogenic factor 1 (Sf1) [10, 19], and Dax1 represses transcription of Cyp19 by inhibiting Sf1. Dax1 also inhibits the activity of the estrogen receptor, raising the potential for increased estrogen receptor action in the background of Dax1 deficiency [20]. Administration of the selective estrogen receptor modulator, tamoxifen, was sufficient to rescue fertility in the KO mouse, suggesting that excess estrogen contributes to the infertility in this model. Whereas further work is required to explore the relationship between Dax1 and estradiol within the testis, Dax1 may have multiple roles in the regulation of estrogen action.
Testicular pathology was still present in RS animals compared to KO mice, but the dilation, vacuolation, and degeneration of the seminiferous epithelium was less severe in RS animals at 12 wk. Obstruction of the rete testis likely is the proximal cause of infertility in the KO mice [8]. Whereas the area around the rete is normally subject to some pathological change, these findings are usually limited to one or two tubules that become slightly vacuolated. In 12-wk-old KO animals, we observe significant changes in many tubules in proximity to the rete testis. As in other areas of the testis, only slight improvements of the histology near the rete were seen in the RS mice, indicating that the pathogenesis of the rete testis obstruction is not solely caused by deficiency of Dax1 in Leydig cells. It also appears that the expansion of the Sertoli cell population observed in KO mice is not improved in the RS animals, because large aggregates of GATA-4 immunostaining cells may be found in or around the rete testis. It is important to note that Dax1 is expressed embryonically in the area of the mesonephric tubules, which develop into the rete testis. Therefore, Dax1 may play a key role in development of this structure. Further study is required to determine the role of Dax1 in gonadal development.
In conclusion, we evaluated the relative importance of Dax1 function in Leydig cells by transgenically expressing DAX1 in the genetic background of the KO mouse. Although sperm counts increased to a level that was sufficient to partially restore fertility, most of the severe pathology associated with mutation of Dax1 remained unchanged. These findings suggest that Dax1 function is required by multiple cell types in the male gonad.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: J. Larry Jameson, 303 E. Chicago Ave., Tarry 15, Chicago IL 60611. FAX: 312 908 9032; ljameson{at}northwestern.edu
3 Current address: Department of Physiology, University of Turku, Turku, Finland
Received: 18 September 2002.
First decision: 12 October 2002.
Accepted: 5 February 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  G. Mantovani, E. De Menis, G. Borretta, G. Radetti, S. Bondioni, A. Spada, L. Persani, and P. Beck-Peccoz DAX1 and X-linked adrenal hypoplasia congenita: clinical and molecular analysis in five patients. Eur. J. Endocrinol., May 1, 2006; 154(5): 685 - 689. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Naciff, K. A. Hess, G. J. Overmann, S. M. Torontali, G. J. Carr, J. P. Tiesman, L. M. Foertsch, B. D. Richardson, J. E. Martinez, and G. P. Daston Gene Expression Changes Induced in the Testis by Transplacental Exposure to High and Low Doses of 17{alpha}-Ethynyl Estradiol, Genistein, or Bisphenol A Toxicol. Sci., August 1, 2005; 86(2): 396 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Alam, E. T. Maizels, Y. Park, S. Ghaey, Z. J. Feiger, N. S. Chandel, and M. Hunzicker-Dunn Follicle-stimulating Hormone Activation of Hypoxia-inducible Factor-1 by the Phosphatidylinositol 3-Kinase/AKT/Ras Homolog Enriched in Brain (Rheb)/Mammalian Target of Rapamycin (mTOR) Pathway Is Necessary for Induction of Select Protein Markers of Follicular Differentiation J. Biol. Chem., May 7, 2004; 279(19): 19431 - 19440. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
