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BOR - Papers in Press, published online ahead of print November 12, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.023135
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BIOLOGY OF REPRODUCTION 70, 687–693 (2004)
DOI: 10.1095/biolreprod.103.023135
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Retinoic Acid Inhibits Rat XY Gonad Development by Blocking Mesonephric Cell Migration and Decreasing the Number of Gonocytes

Hui Li, and Kwan Hee Kim1

School of Molecular Biosciences, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-4234


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitamin A (also called retinol) and its derivatives, retinoic acids (RAs), are required for postnatal testicular function. Abnormal spermatogenesis is observed in rodents on vitamin A-deficient diets and in retinoic acid receptor {alpha} (RAR{alpha}) knockout mice. In contrast, RA has an inhibitory effect on the XY gonad development in embryos. To characterize this inhibitory effect of RA, we investigated the cellular events that are required for the XY gonad development, including cell migration from the adjacent mesonephros into the gonad, fetal Sertoli cell differentiation, and survival of gonocytes. In organ cultures of Embryonic Day 13 (E13) XY gonads from rats, all-trans-retinoic acid (tRA) inhibited mesonephric cell migration into the gonad. Moreover, treatment with tRA decreased the expression of Müllerian-inhibiting substance in Sertoli cells and dramatically reduced the number of gonocytes. Increased apoptosis was detected in the XY gonads cultured with tRA, suggesting that the loss of gonocytes could be due to increased apoptosis. In addition, Am580, a synthetic compound that exhibits RAR{alpha}-specific agonistic properties, mimicked the inhibitory effects of tRA on the XY gonad development including mesonephric cell migration and gonocyte survival. Conversely, a RAR{alpha}-selective antagonist, Ro 41-5253, suppressed the inhibitory ability of tRA on the XY gonad development. These results suggest that retinoic acid acting through RAR{alpha} negatively affects fetal Sertoli cell differentiation and gonocyte survival and blocks the migration of mesonephric cells, thereby leading to inhibition of the XY gonad development.

embryo, gamete biology, Sertoli cells, steroid hormone receptors, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The initial event in gonadal development in both males and females is the formation of a morphologically identical primordium. In the presence of Sry (sex-determining region of the Y chromosome), the bipotential gonad differentiates and develops along a male-specific pathway by Embryonic Day 13.5 (E13.5) in rats [1, 2]. The mesenchymal cell migration into the XY gonad from the adjacent mesonephros is required for testicular cord formation such that, if migration is blocked, testicular cords fail to form [3]. This cell migration contributes to the peritubular myoid cells and vascular endothelial cells [4]. In a similar time frame, fetal Sertoli cells begin to differentiate and aggregate with primordial germ cells (PGCs), as flattened peritubular myoid cells form the outer cell layer of the seminiferous cords, encompassing both Sertoli and germ cells. Consequently, the highly organized testicular cords become separated from the interstitial region [5–7]. Once the PGCs are enclosed inside the testicular cords, they change morphologically and differentiate into gonocytes. Meanwhile, peritubular myoid and Sertoli cells secrete extracellular matrix components for a basal lamina, which forms a structural platform for testicular cords.

By Embryonic Day 14 (E14) in rats, the mesonephros contains both Müllerian and Wolffian ducts, which are the precursors of female and male reproductive tracts, respectively [8, 9]. During male sexual differentiation, fetal Sertoli cells secrete Müllerian-inhibiting substance (MIS), which induces regression of the Müllerian duct by apoptosis [10, 11]. This event occurs between E14 and E17 in rats and is required for normal male reproductive development [12, 13]. Subsequent to the action of MIS on the Müllerian duct, Leydig cells produce testosterone, which induces differentiation of the Wolffian ducts into male reproductive tracts. In females, the Wolffian ducts passively regress, resulting in development of Müllerian ducts and, thereby, female reproductive tracts.

Vitamin A (also called retinol) and its metabolic derivatives, retinoic acids (RAs), are required for various fundamentally physiological processes, including embryonic development, cellular proliferation and differentiation, vision, and reproduction [14–16]. Retinol and RA deprivation or exposure to excess retinol can result in major abnormalities in embryonic development and adult physiology [17–19].

The action of RA is mediated through two families of nuclear receptors, the retinoic acid receptors (RARs), which recognize both all-trans-retinoic acid (tRA) and 9-cis-retinoic acid, and the retinoid X receptors (RXRs), which recognize only 9-cis-retinoic acid [20]. Each family consists of three subtypes: {alpha}, ß, and {gamma}. The RXRs can homodimerize or heterodimerize with RARs and other nuclear receptors, including thyroid hormone receptor, vitamin D receptor, and peroxisome proliferator-activated receptors [21], whereas RARs only heterodimerize with RXRs. These homodimers or heterodimers can bind to appropriate RA response elements or retinoid X response elements to regulate gene expressions [22] and consequently regulate divergent cellular processes [23].

Genetic studies have shown that RAR{alpha} and RXRß are critical for normal spermatogenesis. Both RAR{alpha} [24] and RXRß [25] knockout male mice are sterile. Spermatogenesis in vitamin A-deficient (VAD) mice and rats is blocked such that most of the germ cells in later stages of differentiation are lost from the seminiferous tubules [18, 19]. The only germ cells remaining in the VAD seminiferous tubules are the undifferentiated A spermatogonia and a few preleptotene spermatocytes. Spermatogenesis can be restored in VAD rats by feeding retinol or repeatedly injecting tRA [26, 27].

In contrast with the positive effects of RA on testis function in adults, it was shown previously that tRA inhibited testicular cord formation in the XY gonads from E13 embryos [28]. These seemingly opposite, paradoxical actions of RA on fetal testis versus adult testis have been noted in a recent review [29]. At the molecular level, tRA diminished the deposition of laminin and fibronectin in the basement membrane of E14 testis in organ culture [30]. However, the specific cellular effect of tRA on the fetal Sertoli cell differentiation, gonocyte survival, and mesonephric cell migration, which are required for the testicular cord formation, is not known. In the current study, we investigated the effect of tRA on these cellular events involved in the XY gonad development and the involvement of RAR{alpha} in these tRA effects using an organ culture method [28].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Timed pregnant female Sprague-Dawley rats were obtained from Charles River Laboratories (Hollister, CA). Plug date was considered Embryonic Day 0 (E0). Animal experimentation was approved by the Institutional Animal Care and Use Committee and conducted in accordance with the highest standards of humane animal care as outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Organ Cultures

E13 gonads were dissected out with the mesonephroi and cultured on Millicell CM filters (Millipore, Bedford, MA) floating on the surface of CMRL 1066 medium (Life Technologies, Rockville, MD), supplemented with penicillin-streptomycin, insulin (10 µg/ml), and transferrin (10 µg/ml) at 37°C with 5% CO2:95% air. To determine the sex of E13 embryos, polymerase chain reaction analysis for Sry gene was conducted on the tail genomic DNA isolated from E13 embryos [28]. One gonad was cultured in medium containing tRA (Sigma, St. Louis, MO) or RAR{alpha}-selective agonist Am580 (Biomol Research Lab, Plymouth Meeting, PA). The selectivity of Am580 for RARs has been established by using COS-7 cells transfected with expression vectors for each of the RARs [31]. Am580 has a higher binding affinity for RAR{alpha} (8 nM) than RARß (131 nM) or RAR{gamma} (450 nM). The dissociation constant of tRA for RAR{alpha} is 19.5 nM and that of Am580 is 8 nM [32]. The specificity of the biological effect of Am580 has also been studied in both cultured cells and organ cultures of embryonic testis [32]. In addition, a RAR{alpha}-selective antagonist Ro 41-5253 [33] was added 30 min before the treatment with tRA. Transactivation experiments have demonstrated that a 2- to 10-fold excess of Ro 41-5253 was necessary to suppress the activation of RAR{alpha} [33]. The other gonad from the same fetus was cultured in medium containing an equivalent volume of dimethyl sulfoxide (solvent for tRA) as a control. The organs were maintained for 3 days in culture, at which time testicular cords were well developed in the control gonads. The culture medium was changed every day. At the end of culture, the tissues were fixed for 1 h at room temperature in Bouin solution, embedded in paraffin, and sectioned. At least three independent organ culture experiments were conducted, with each experiment consisting of 4–6 pairs of XY gonads.

Migration Assay

After the dissection of XY gonad plus mesonephros from E13 embryos, the gonad was separated from the mesonephros using a fine needle. The mesonephroi were incubated in culture medium containing 10 µM 5(6)-carboxyfluorescein diacetate, succinimidyl ester (Molecular Probes, Eugene, OR), which has been used for cell migration assay in the organ culture system of Embryonic Day 13 testis [34, 35], for 20 min at 37°C. Then mesonephroi were washed in fresh culture medium for 30 min at 37°C, assembled with its respective gonad, and cultured for 3 days in the absence or presence of tRA, Am580, or Ro 41-5253.

Immunohistochemistry

Immunohistochemistry was performed as described previously [36]. The tissue sections were deparaffinized, rehydrated, microwaved in 10 mM sodium citrate, treated with 0.3% H2O2 to quench endogenous peroxidases, and incubated in 10% rabbit or goat normal serum for 10 min at room temperature to block nonspecific protein binding. For detection of MIS and RARs, the sections were incubated with goat or rabbit polyclonal antibodies (MIS, 1:300; RAR{alpha}, RARß, or RAR{gamma}, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA) in a humidified chamber overnight at 4°C. For detection of mouse vasa homologue (MVH), a rabbit polyclonal antibody against MVH (1:1000) was used [37]. Sections were washed with PBS and treated with biotinylated rabbit anti-goat secondary antibody (Zymed Laboratories, South San Francisco, CA) for MIS or biotinylated goat anti-rabbit (Vector Laboratories, Burlingame, CA) for MVH and RARs, followed by incubation with peroxidase-conjugated streptavidin substrate-chromagen mixture containing aminoethyl carbazole from Zymed Laboratories. As negative controls, serial sections were incubated with control immunoglobulin G from the same species or primary antibodies preadsorbed with 50-fold excess of synthetic immunizing peptide (Santa Cruz Biotechnology).

Assay for Apoptosis

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay was performed as described by the manufacturer of the Apoptosis Detection System (Promega Biotech Corporation, Madison, WI). This assay detects fragmented DNA in apoptotic cells by catalytically incorporating fluorescein-12-dUTP at the 3'-OH DNA ends using the TdT enzyme. The fluorescein-12-dUTP-labeled DNA can then be visualized directly under a fluorescence microscope. The TUNEL-positive cells in the entire cross section were counted. At least nine cross sections were analyzed.

Digital Images

All digital images were obtained using Leitz DMRB with epifluorescence and a Magnafire digital camera (Optronics, Goleta, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
All-Trans-Retinoic Acid Inhibits Testicular Cord Formation and Cell Migration from the Adjacent Mesonephros into XY Gonad

The effects of tRA on testicular cord formation and mesonephric cell migration were assessed using organ cultures of XY gonads from E13 embryos. Testicular cords formed normally in the control XY gonads from E13 embryos cultured for 3 days (Fig. 1A). In contrast, treatment with 1 µM tRA completely inhibited the testicular cord formation (Fig. 1D), which is consistent with the previous data [28]. Although the treatment with lower concentrations of tRA could not completely inhibit the formation of testicular cords, nonetheless, organs treated with either 0.01 µM or 0.1 µM tRA formed testicular cords with an abnormally enlarged diameter (Fig. 1, B and C). The tRA increased the mean diameter of the testicular cords from 39.4 ± 1.1 µm in the control to 49.1 ± 1.2 µm at 0.01 µM (n = 3; P < 0.05) and 59.0 ± 2.0 µm at 0.1 µM (n = 3; p <0.05).



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  		FIG. 1. Dose effect of all-trans-retinoic acid (tRA) on testicular cord formation in organ cultures of Embryonic Day 13 (E13) XY gonads. E13 gonads plus mesonephroi were cultured for 3 days in the absence (A) or presence of 0.01 µM (B), 0.1 µM (C), and 1 µM (D) tRA. t, Testis; m, mesonephros. Bar = 500 µm for A–D. Data are representative of three independent experiments

To determine the potential cellular mechanism for tRA-induced inhibition of testicular cord formation, the effect of tRA on cell migration from the adjacent mesonephros into the XY gonad, which is required for normal testicular cord formation [3, 4, 38], was assessed. Extensive cell migration was detected in the control gonads (Fig. 2A). On the contrary, virtually no mesonephric cell migration was observed in the gonads treated with 1 µM tRA (Fig. 2B).



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  		FIG. 2. Effect of tRA on cell migration from the mesonephros into the XY gonad. A pair of E13 gonads plus mesonephroi from the same fetus was cultured for 3 days in the absence (A) or presence of 1 µM tRA (B). t, Testis; m, mesonephros. Arrows indicate mesonephric cell migration. Bar = 500 µm for A and B. Data are representative of three independent experiments

All-Trans-Retinoic Acid Reduces the Number of Gonocytes by Increasing Apoptosis

Hematoxylin-eosin-stained sections displayed cellular organization in the E13 XY gonads treated with various concentrations of tRA (Fig. 3, A–D). Sertoli cells, which are identifiable by elongated, irregular shapes, were observed in the periphery of testicular cords, and gonocytes, which are characterized by their round shape, were primarily observed in the center of testicular cords in the control gonads (Fig. 3A). In contrast, this organization of cells deteriorated with increasing concentrations (0.01–1 µM) of tRA (Fig. 3, B, C, and D). By 0.1 µM tRA, there was a visible sign of cell degeneration within testicular cords, as shown by clear areas and fewer gonocytes observed in the treated gonads compared with the number seen in the control gonad (Fig. 3, compare C with A). By 1 µM tRA, different types of cells are difficult to discern due to the disorganization of testicular cords (Fig. 3D).



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  		FIG. 3. Dose effect of tRA on gonocyte survival. E13 gonads plus mesonephroi were cultured for 3 days in the absence (A and E) or presence of 0.01 µM tRA (B and F), 0.1 µM tRA (C and G), and 1 µM tRA (D and H). Sections were stained with hematoxylin and eosin (A–D) or immunostained with an antibody against MVH (E–H). Arrows indicate gonocytes. Bar = 25 µm for A–D and bar = 25 µm for E–H. Data are representative of three independent experiments

MVH protein has been shown to be expressed exclusively in the cytoplasm of gonocytes soon after their colonization of embryonic gonad [37], and thus, it can be used to identify gonocytes. In the control gonads, MVH-positive gonocytes were found abundantly within the testicular cords (Fig. 3E). On the other hand, tRA decreased the number of MVH-positive gonocytes in a dose-dependent manner (Fig. 3, F–H).

To determine whether tRA-induced loss of gonocytes represented activation of apoptosis, TUNEL assay was conducted on the XY gonad sections. tRA at 0.1 µM (Fig. 4B) and 1 µM (Fig. 4C) significantly increased the number of apoptotic cells in the E13 gonads (Fig. 4D) compared with few apoptotic cells in untreated E13 gonads (Fig. 4A).



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  		FIG. 4. Dose effect of tRA on apoptosis in E13 XY gonads. E13 gonads plus mesonephroi were cultured for 3 days in the absence (A) or presence of 0.1 µM (B), and 1 µM tRA (C), followed by TUNEL assay. Bar = 25 µm for A–C. TUNEL-positive cells were counted and shown in D. Values are mean ± SEM. *, Significant at P < 0.05; **, significant at P < 0.01 compared with the controls. Data are representative of three independent experiments

All-Trans-Retinoic Acid Decreases MIS Expression in Sertoli Cells

To determine whether tRA affected Sertoli cell differentiation in the organ cultures of E13 gonads, MIS immunohistochemistry was conducted. MIS is expressed exclusively in Sertoli cells in the mouse beginning between E11.5 and E12.5 until shortly after birth [39], and thus, MIS has been used as a marker of fetal Sertoli cell differentiation [40]. The control gonads showed robust MIS immunostaining, especially in the highly organized Sertoli cells lining the periphery of the testicular cords (Fig. 5, A and C). Greatly different from this pattern of MIS expression in the controls, the amount of MIS was severely reduced in Sertoli cells, which were not organized but scattered throughout the gonads treated with tRA (Fig. 5, B and D).



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  		FIG. 5. Effect of tRA on MIS expression in Sertoli cell. A pair of E13 gonads plus mesonephroi from the same fetus were cultured for 3 days in the absence (A and C) or presence of 1 µM tRA (B and D). C and D are higher magnifications of A and B, respectively. Arrows indicate Sertoli cells. Bar = 50 µm for A and B and bar = 25 µm for C and D. Data are representative of three independent experiments

The RAR{alpha} Agonist Mimics the Inhibitory Effects of tRA, While the RAR{alpha} Antagonist Suppresses the Inhibitory Ability of tRA

To determine whether tRA exerted its action, at least partially, through RAR{alpha}, RAR{alpha}-selective agonist (Am580) and antagonist (Ro 41-5253) were used. Treatment with Am580 at 1 nM completely inhibited the testicular cord formation (Fig. 6A) and impaired the mesonephric cell migration (Fig. 6B) in the cultured E13 XY gonads. In addition, Am580 dramatically decreased the number of germ cells, as demonstrated by immunohistochemistry of MVH (Fig. 6C). Previously, this agonist was shown to be at least 1000-fold more potent than tRA on both the cultured cells and the organ cultures of embryonic testis [32]. Inversely, Ro 41-5253, a RAR{alpha}-selective antagonist, at 5 µM, counteracted the inhibitory effect of tRA on testicular cord formation (Fig. 6D), cell migration from the mesonephros (Fig. 6E), and germ cell survival (Fig. 6F). Ro 41-5253 alone did not disturb these cellular processes (data not shown).



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  		FIG. 6. Effect of RAR{alpha}-selective agonist (Am580) and antagonist (Ro 41-5253) on the testicular cord formation (A and D), mesonephric cell migration (B and E), and gonocyte survival (C and F) in the E13 gonad organ cultures. The E13 gonads were treated with Am580 at 1 nM (A–C) or Ro 41-5253 at 5 µM (D–F) in the presence of 1 µM tRA. Arrows indicate gonocytes. Bar = 500 µm for A, B, D, and E and bar = 25 µm for C and F

Immunolocalization of RARs in E13 Gonads in Organ Culture

The cellular localization of RAR proteins was examined using immunohistochemistry on sections from the E13 XY gonads in organ cultures. RAR{alpha} immunoreactivity was detected exclusively in some of the peritubular cells and interstitial cells (Fig. 7, A and C). RARß immunoreactivity, on the other hand, was primarily present within the testicular cords, in the cytoplasm of gonocytes in the E13 gonads (Fig. 7, B and D). No RAR{gamma} immunoreactivity was detected in the E13 gonads (data not shown).



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  		FIG. 7. Immunohistochemistry for RAR{alpha} and RARß in E13 XY gonads. RAR{alpha} (A and C) and RARß (B and D) immunoreactivities were detected in the E13 XY gonads organ cultures. C and D are higher magnifications of A and B, respectively. Arrows in C indicate interstitial cells; arrows in D indicate gonocyte. Bar = 25 µm for (A and B) and bar = 25 µm for (C and D). Data are representative of three independent experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Testicular cord formation is the earliest clearly defined morphological change in testis development. This process involves a complex series of cellular events [41]. One critical event is the migration of cells from the adjacent mesonephros into the XY gonad that is induced by the differentiating gonadal cells [3, 4]. Another event established to be important is the differentiation of fetal Sertoli cells initiated by Sry [1, 42, 43]. In this study, we demonstrated that tRA blocked the mesonephric cell migration into the XY gonad. This expands the earlier finding that treatment of the E13 gonads with tRA inhibited testicular cord formation [28]. Because it has been recognized that testicular cord formation does not occur if mesonephric cell migration is blocked [3], the tRA-induced disruption of testicular cord formation is likely due to the failure of mesonephric cell migration into the XY gonad.

The mesenchymal cells, which migrate into the XY gonad from the adjacent mesonephros, are believed to contribute to the peritubular myoid cells and vascular endothelial cells in the XY gonads. A single layer of flattened peritubular myoid cells envelopes Sertoli cells and gonocytes to form testicular cords. These peritubular myoid cells, together with Sertoli cells, are responsible for the production of extracellular matrix components that are required for organizing the basal lamina [44, 45], postulated to be important in supporting the structural integrity of testicular cords. Therefore, the inhibition of the mesonephric cell migration by tRA could potentially reduce the peritubular myoid cell population in the gonad and, as a result, produce insufficient amounts of extracellular matrix components, leading to disruption of the structural integrity of testicular cords. This is consistent with the previous finding that tRA decreased the deposition of laminin and fibronectin in the basement membrane of E14 gonad in organ culture [30].

Moreover, available evidence strongly indicates that the expression of Sry in the Sertoli cell precursors [43] is pivotal for early testis development [4, 46]. Sertoli cells that express Sry differentiate and act as an organization center for testis formation by directing other cell types into their respective lineages [47]. Interestingly, treatment with tRA decreased the production of MIS, a well-established functional marker of Sertoli cell differentiation in the embryonic testis [12], suggestive of an impaired differentiated function for fetal Sertoli cells.

In addition, Sertoli cells were found to be scattered in the gonad treated with 1 µM tRA, with no evidence of any cell aggregation. These results suggest that the fetal Sertoli cells in the presence of tRA are unable to aggregate and organize PGCs, presumably because the differentiated functions of Sertoli cells are impaired. These results reiterate the importance of the normal, differentiated fetal Sertoli cells in the XY gonadal development [47].

Furthermore, it is known that PGCs that are aggregated by Sertoli cells are able to develop into gonocytes within the testicular cords [48], and Sertoli cells are believed to be essential for this process. Thus, when the differentiated functions of fetal Sertoli cells are impaired in the presence of tRA, PGCs may die because they could not develop further into gonocytes. Similar arrest in gonocyte development that leads to apoptosis occurs at the neonatal stage of testis development. For instance, gonocytes die if they remain in the center of the seminiferous tubule and fail to proceed to mitosis and migrate to the basal area of the seminiferous tubule to become stem cell spermatogonia or differentiated spermatogonia at the neonatal stage [49, 50].

In this light, it was not surprising that tRA markedly reduced the number of gonocytes in the fetal gonads and increased the number of cells undergoing apoptosis, suggesting that tRA could regulate survival of gonocytes in the embryos. However, more intriguing are the results that tRA at 0.1 µM can reduce the number of gonocytes by increasing apoptosis but cannot completely inhibit the formation of testicular cords. These results suggest that the loss of gonocytes is unlikely to be a secondary effect of the inhibition of mesonephric cell migration. Instead, it may be a distinctively separate effect from the tRA-induced inhibitory effect on cell migration. This is in agreement with the previous report, which showed that the testicular cord formed normally in the absence of germ cells [51]. Regardless, to our knowledge, our studies are the first to show that tRA can inhibit both the gonocyte survival and the mesonephric cell migration, two important cellular events that are definitely required for the development of functional XY gonads. Previously, Livera et al. [52] had shown that tRA greatly decreased the number of gonocytes in the E14 testis, in which the testicular cords have already formed.

It has previously been shown that testicular cord formation was blocked in the E13 gonad cultured with a RAR pan agonist [28], suggesting that the action of tRA on the testicular cord formation may be mediated through any of the three RARs. In this study, tRA inhibitory effects on XY gonad development were reproduced by Am580, a RAR{alpha}-selective agonist, and abrogated by a RAR{alpha}-selective antagonist. Indeed, both RAR{alpha} and RARß proteins were observed in the organ cultures of E13 testes. RAR{alpha} is expressed in the peritubular cells and in the cells of interstitium, whereas RARß is primarily found in the cytoplasm of cells within the testicular cords. Thus, these observations suggest that the RAR{alpha} signaling pathway in the interstitial cells may have a critical role in the XY gonad development. However, it should be pointed out that our results certainly do not mean that RARß found in the seminiferous cord is unimportant. Altogether, these results indicate that the effect of tRA on the XY gonad development involves complex intertwined actions and responses among interstitial cells, Sertoli cells, and germ cells, as well as involves different retinoid receptors in these cells.

In conclusion, we have identified early cellular events that are inhibited by tRA during the XY gonad development, including mesonephric cell migration, fetal Sertoli cell differentiation, and survival of gonocytes. These inhibitory effects could sufficiently account for tRA-induced disruption of testicular cord formation observed previously. However, it is yet to be determined what molecular mechanisms may underlie the tRA-mediated disruption of these cellular processes in the formation of testicular cords. Our results suggest that RAR{alpha} may be involved in mediating the molecular effects of tRA in the XY gonad development.


   ACKNOWLEDGMENTS
 
We thank Dr. Toshiaki Noce (Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan) for generously providing an antibody against MVH.


   FOOTNOTES
 
1 Correspondence: FAX: 509 335 1907; khkim{at}wsu.edu Back

Received: 11 September 2003.

First decision: 6 October 2003.

Accepted: 10 November 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R. Male development of chromosomally female mice transgenic for Sry. Nature 1991 351:117-121[CrossRef][Medline]
  2. Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990 346:240-244[CrossRef][Medline]
  3. Buehr M, Gu S, McLaren A. Mesonephric contribution to testis differentiation in the fetal mouse. Development 1993 117:273-281[Abstract]
  4. Martineau J, Nordqvist K, Tilmann C, Lovell-Badge R, Capel B. Male-specific cell migration into the developing gonad. Curr Biol 1997 7:958-968[CrossRef][Medline]
  5. Magre S, Jost A. The initial phases of testicular organogenesis in the rat. An electron microscope study. Arch Anat Microsc Morphol Exp 1980 69:297-318[Medline]
  6. Jost A, Magre S, Agelopoulou R. Early stages of testicular differentiation in the rat. Hum Genet 1981 58:59-63[Medline]
  7. Magre S, Jost A. Sertoli cells and testicular differentiation in the rat fetus. J Electron Microsc Technol 1991 19:172-188[CrossRef][Medline]
  8. Josso N, Picard JY. Anti-Mullerian hormone. Physiol Rev 1986 66:1038-1090[Free Full Text]
  9. Josso N, Cate RL, Picard JY, Vigier B, di Clemente N, Wilson C, Imbeaud S, Pepinsky RB, Guerrier D, Boussin L, Legeai L, Carre-Eusebe D. Anti-Mullerian hormone: the Jost factor. Recent Prog Horm Res 1993 48:1-59[Medline]
  10. Behringer RR. The Mullerian inhibitor and mammalian sexual development. Philos Trans R Soc London B, Biol Sci 1995 350:285-288[Medline]
  11. Roberts LM, Hirokawa Y, Nachtigal MW, Ingraham HA. Paracrine-mediated apoptosis in reproductive tract development. Dev Biol 1999 208:110-122[CrossRef][Medline]
  12. Behringer RR, Finegold MJ, Cate RL. Mullerian-inhibiting substance function during mammalian sexual development. Cell 1994 79:415-425[CrossRef][Medline]
  13. Mishina Y, Rey R, Finegold MJ, Matzuk MM, Josso N, Cate RL, Behringer RR. Genetic analysis of the Mullerian-inhibiting substance signal transduction pathway in mammalian sexual differentiation. Genes Dev 1996 10:2577-2587[Abstract/Free Full Text]
  14. Chytil F. Retinoids in lung development. FASEB J 1996 10:986-992[Abstract]
  15. Kim KH, Wang Z. Action of vitamin A on the testis: role of the Sertoli cell. In: Russell LD, Griswold MD (eds.), The Sertoli Cell. Clearwater, FL: Cache River Press; 1993:517–536
  16. Maden M. Vitamin A in embryonic development. Nutr Rev 1994 52:S3-12[Medline]
  17. Sulik KK, Cook CS, Webster WS. Teratogens and craniofacial malformations: relationship to cell death. Development 1988 103:suppl213-231[Medline]
  18. Mason KE. Differences in testes injury and repair after vitamin deficiency, vitamin E deficiency and inanition. Am J Anat 1933 52:153-239[CrossRef]
  19. Mitranond V, Sobhon P, Tosukhowong P, Chindaduangrat W. Cytological changes in the testes of vitamin A-deficient rats. I. Quantitation of germinal cells in the seminiferous tubules. Acta Anat (Basel) 1979 103:159-168[Medline]
  20. Chambon P. A decade of molecular biology of retinoic acid receptors. FASEB J 1996 10:940-954[Abstract]
  21. Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell 1995 83:841-850[CrossRef][Medline]
  22. Pfahl M, Chytil F. Regulation of metabolism by retinoic acid and its nuclear receptors. Annu Rev Nutr 1996 16:257-283[CrossRef][Medline]
  23. Giguere V. Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling. Endocr Rev 1994 15:61-79[CrossRef][Medline]
  24. Lufkin T, Lohnes D, Mark M, Dierich A, Gorry P, Gaub MP, LeMeur M, Chambon P. High postnatal lethality and testis degeneration in retinoic acid receptor {alpha} mutant mice. Proc Natl Acad Sci U S A 1993 90:7225-7229[Abstract/Free Full Text]
  25. Kastner P, Mark M, Leid M, Gansmuller A, Chin W, Grondona JM, Decimo D, Krezel W, Dierich A, Chambon P. Abnormal spermatogenesis in RXR-ß mutant mice. Genes Dev 1996 10:80-92[Abstract/Free Full Text]
  26. Huang HF, Dyrenfurth I, Hembree WC. Endocrine changes associated with germ cell loss during vitamin A-induced recovery of spermatogenesis. Endocrinology 1983 112:1163-1171[Medline]
  27. Van Pelt AM, de Rooij DG. Retinoic acid is able to reinitiate spermatogenesis in vitamin A deficient rats and high replicate doses support the full development of spermatogenic cell. Endocrinology 1991 128:697-704[Abstract]
  28. Cupp AS, Dufour JM, Kim G, Skinner MK, Kim KH. Action of retinoids on embryonic and early postnatal testis development. Endocrinology 1999 140:2343-2352[Abstract/Free Full Text]
  29. Livera G, Rouiller-Fabre V, Pairault C, Levacher C, Habert R. Regulation and perturbation of testicular functions by vitamin A. Reproduction 2002 124:173-180[Abstract]
  30. Marinos E, Kulukussa M, Zotos A, Kittas C. Retinoic acid affects basement membrane formation of the seminiferous cords in 14-day male rat gonads in vitro. Differentiation 1995 59:87-94[CrossRef][Medline]
  31. Szondy Z, Reichert U, Bernardon JM, Michel S, Toth R, Karaszi E, Fesus L. Inhibition of activation-induced apoptosis of thymocytes by all-trans- and 9-cis-retinoic acid is mediated via retinoic acid receptor alpha. Biochem J 1998 331:767-774[Medline]
  32. Livera G, Rouiller-Fabre V, Habert R. Retinoid receptors involved in the effects of retinoic acid on rat testis development. Biol Reprod 2001 64:1307-1314[Abstract/Free Full Text]
  33. Apfel C, Bauer F, Crettaz M, Forni L, Kamber M, Kaufmann F, LeMotte P, Pirson W, Klaus M. A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects. Proc Natl Acad Sci U S A 1992 89:7129-7133[Abstract/Free Full Text]
  34. Cupp AS, Uzumcu M, Skinner MK. Chemotactic role of neurotropin 3 in the embryonic testis that facilitates male sex determination. Biol Reprod 2003 68:2033-2037[Abstract/Free Full Text]
  35. Uzumcu M, Westfall SD, Dirks KA, Skinner MK. Embryonic testis cord formation and mesonephric cell migration requires the phosphatidylinositol 3-kinase signaling pathway. Biol Reprod 2002 67:1927-1935[Abstract/Free Full Text]
  36. Dufour JM, Kim KH. Cellular and subcellular localization of six retinoid receptors in rat testis during postnatal development: identification of potential heterodimeric receptors. Biol Reprod 1999 61:1300-1308[Abstract/Free Full Text]
  37. Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M, Noce T. Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mech Dev 2000 93:139-149[CrossRef][Medline]
  38. Tilmann C, Capel B. Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad. Development 1999 126:2883-2890[Abstract]
  39. Munsterberg A, Lovell-Badge R. Expression of the mouse anti-Mullerian hormone gene suggests a role in both male and female sexual differentiation. Development 1991 113:613-624[Abstract]
  40. Colvin JS, Green RP, Schmahl J, Capel B, Ornitz DM. Male-to-female sex reversal in mice lacking fibroblast growth factor 9. Cell 2001 104:875-889[CrossRef][Medline]
  41. Capel B. The battle of the sexes. Mech Dev 2000 92:89-103[CrossRef][Medline]
  42. Burgoyne PS, Buehr M, Koopman P, Rossant J, McLaren A. Cell-autonomous action of the testis-determining gene: Sertoli cells are exclusively XY in XX {longleftrightarrow} XY chimeric mouse testes. Development 1988 102:443-450[Abstract]
  43. Albrecht KH, Eicher EM. Evidence that Sry is expressed in preSertoli cells and Sertoli and granulosa cells have a common precursor. Dev Biol 2001 240:92-107[CrossRef][Medline]
  44. Skinner MK, Tung PS, Fritz IB. Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components. J Cell Biol 1985 100:1941-1947[Abstract/Free Full Text]
  45. Tung PS, Fritz IB. Morphogenetic restructuring and formation of basement membranes by Sertoli cells and testis peritubular cells in co-culture: inhibition of the morphogenetic cascade by cyclic AMP derivatives and by blocking direct cell contact. Dev Biol 1987 120:139-153[CrossRef][Medline]
  46. Capel B, Albrecht KH, Washburn LL, Eicher EM. Migration of mesonephric cells into the mammalian gonad depends on Sry. Mech Dev 1999 84:127-131[CrossRef][Medline]
  47. McLaren A. Germ and somatic cell lineages in the developing gonad. Mol Cell Endocrinol 2000 163:3-9[CrossRef][Medline]
  48. de Rooij DG, Grootegoed JA. Spermatogonial stem cells. Curr Opin Cell Biol 1998 10:694-701[CrossRef][Medline]
  49. Orth JM, Jester WF, Li LH, Laslett AL. Gonocyte-Sertoli cell interactions during development of the neonatal rodent testis. Curr Top Dev Biol 2000 50:103-124[CrossRef][Medline]
  50. Nagano R, Tabata S, Nakanishi Y, Ohsako S, Kurohmaru M, Hayashi Y. Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis. Anat Rec 2000 258:210-220[Medline]
  51. Merchant H. Rat gonadal and ovarian organogenesis with and without germ cells. An ultrastructural study. Dev Biol 1975 44:1-21[CrossRef][Medline]
  52. Livera G, Rouiller-Fabre V, Durand P, Habert R. Multiple effects of retinoids on the development of Sertoli, germ, and Leydig cells of fetal and neonatal rat testis in culture. Biol Reprod 2000 62:1303-1314[Abstract/Free Full Text]



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