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De Novo Testosterone Production in Luteinizing Hormone Receptor Knockout Mice after Transplantation of Leydig Stem Cells

Scott Department of Urology (K.C.L., J.B., D.J.L.), and Department of Molecular and Cellular Biology (D.J.L.), Baylor College of Medicine, Houston, Texas 77030; and Department of Obstetrics and Gynecology (Ch.V.R., Z.L.) and Laboratory of Molecular Reproductive Biology and Medicine (Ch.V.R.), University of Louisville Health Sciences Center (Ch.V.R.), University of Louisville, Loiusville, Kentucky 40292

Address all correspondence and requests for reprints to: Dolores J. Lamb, Scott Department of Urology, Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Alkek N 730, Houston, Texas, 77030. E-mail: dlamb{at}bcm.tmc.edu.

	Abstract

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Mesenchymal stem cells or Leydig cell progenitors are rare and difficult to isolate from adult testes. The property of differential efflux of Hoechst 33342 dye by the multi-drug-like transporter enriches murine hematopoietic stem cells from bone marrow. Our work on testicular cell transplantation suggests that the "Hoechst dim" side population (SP) also contains Leydig stem cells or progenitors that proliferate and differentiate into mature functional Leydig cells. We harvested testicular cells from cryptorchid ROSA26 mice, stained them with Hoechst dye, and isolated the cell population that excludes the dye using flow cytometry. Mice with targeted deletion of the LH receptor (LHR) gene were used as the recipients of the transplanted cells. These mice are hypogonadal and infertile. Both testicular SP and non-SP cells were transplanted into the interstitium of the LHR knockout recipients’ testes. Serial serum testosterone assays revealed a significant increase in the circulating testosterone levels and restoration of spermatogenesis in the LHR-knockout recipients transplanted with the SP cells compared with that of those transplanted with non-SP. A SP cell concentration- and time-dependent increase in circulating testosterone was observed. This demonstrates the successful transplantation of functional putative Leydig stem cells into a hypogonadal recipient. The increase in testosterone concentration indicates the de novo synthesis of androgen by the transplanted SP cells. This method offers a novel technique to isolate Leydig stem cells and to study Leydig cell development.

	Introduction

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LEYDIG CELLS RESIDE in the interstitial compartment of the testis. Their primary function is androgen production essential for the development of the male phenotype and spermatogenesis. Although the postnatal development of Leydig cells is a continuous process, the distribution of the Leydig cell lineages changes as the animal reaches adulthood. The progenitors or Leydig stem cells, which are fibroblastic mesenchymal cells, predominate at birth (1). They are replaced by the immature Leydig cells during the prepubertal stage and subsequently differentiate into adult Leydig cells after puberty (2). Mitotic figures are rare in the adult Leydig cell population; hence, they are unlikely to be a significant source of new Leydig cells. However, the turnover of Leydig cells is maintained by mitosis of the persistent small population of progenitor/immature cells. This is supported by experimental work on rodents that showed spontaneous recovery of steroidogenesis after elimination of mature adult Leydig cells using ethylene dimethanesulfonate (3).

By strict definition, stem cells are capable of self-renewal, proliferation, and differentiation into functional cells. Although the immature and adult Leydig cells can be characterized by their steroidogenic markers and function, it is difficult to isolate the Leydig stem cells because of their paucity in adult testis and the lack of specific markers. The generation of LH/human chorionic gonadotropin receptor knockout (LHRKO) mouse models (4, 5) provides a functional assay to test the putative Leydig stem cells isolated from adult mice.

Our model of Leydig stem cell enrichment is based on the assumption that Leydig stem cells share similar characteristics with other types of stem cells such as hematopoietic stem cells (HSC).

In 1996, Goodell et al. (6) reported the isolation of a relatively pure population of HSC. Taking advantage of the HSC’s ability to exclude Hoechst dye via the multidrug resistance transporter protein, they enriched the adult HSC population from bone marrow cell several thousand-fold. The bone marrow cells isolated using flow cytometry analysis are designated as the side population (SP). Proteins belonging to the ATP-binding cassette (ABC) transporter superfamily (MDR-1 and Bcrp1, the murine homologue of ABCG2) are thought to be responsible for the efflux of the Hoechst dye (6, 7, 8, 9). The same purification technique has also been applied in the isolation of muscle and mammary gland stem cells (10, 11).

With respect to the testis, three recent reports present conflicting results regarding the presence of spermatogonial stem cells (SSC) in the testicular SP. Brinster’s group (12) concludes that testicular SP cells do not share the same characteristics as their putative SSC, whereas Fouchet and Vicini’s (13, 14) laboratories report successful colonization of the recipient seminiferous tubules after transplantation of the SP cells. One of the potential explanations for this discrepancy is that the SP cell population contains different types of stem cells (SSC and Leydig stem cells). The sorting mechanisms and donor types (whole vs. cryptorchid testes) can also influence the heterogeneity of the resulting cell population.

Our model was designed to test whether Leydig stem cells are present in the testicular SP by transplanting the flow cytometry-sorted testicular SP cells into the interstitium of recipient mice testes. We examined the ability of testicular SP to colonize the recipient’s testis. In addition, we hypothesized that the engrafted cells can restore testosterone production and spermatogenesis in the LHRKO mice.

	Materials and Methods

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The animal protocol was reviewed and approved by the institutional animal review board at Baylor College of Medicine. Care was taken to ensure the proper maintenance and treatment of the experimental animals.

Donor testicular cell isolation
Cells for transplantation were isolated from the testes of the transgenic mouse line ROSA26 (originally from The Jackson Laboratory, Bar Harbor, ME), which are maintained on a C57BL/6 x 129/Sv genetic background and express the Escherichia coli LacZ gene. Many cell types, including all stages of germ cell differentiation, stain positively for ß-galactosidase (ß-gal), a characteristic that allows accurate tracking of the transplanted cells. The male pups were surgically made cryptorchid at age 6–8 wk to minimize the number of haploid germ cells in the donor cell population. On average, six to eight donor mice were killed and their testes were harvested 6–8 wk postoperatively.

A single cell suspension was prepared using a two-step enzymatic digestion procedure as described previously (15). The cells were resuspended in medium containing DMEM, 5% HEPES, and 10% fetal bovine serum.

Hoechst 33342 dye staining and flow cytometry analysis and sorting
The procedure employed for flow cytometry cell sorting of SP cells is described elsewhere (6). Briefly, the cells were stained with Hoechst vital dye (bis-benzimide H 33342; Sigma-Aldrich, Inc., St. Louis, MO) at a concentration of 5 µg/ml for 90 min at 37 C. To demonstrate that sorted SP cells are capable of Hoechst dye exclusion, we added verapamil (75 µg/ml; Sigma-Aldrich, Inc.), which inhibits Hoechst efflux by blocking the ABC transporter, to an aliquot of the cell solution just before the Hoechst staining as a negative control (Fig. 1). After staining, cells were resuspended in media at 4 C. Propidium iodide (2 µg/ml; Sigma-Aldrich, Inc.) was added to the final suspension before flow cytometry sorting to exclude dead cells from the flow cytometry profile.

View larger version (43K): [in this window] [in a new window]   FIG. 1. Flow cytometry sorting of "Hoechst dim" SP. A, SP and Non-SP cells were isolated as shown in the gated blocks. B, Verapamil (75 µg/ml or 150 µM) was added to the testicular cell solution to block the ABC transporters before Hoechst staining. Note the SP cells have decreased due to their inability to extrude the Hoechst dye.

Recipients and SP cell transplantation
Two recipient models were used in this study. The first model, WBB6F1/J-Kit^w/Kit^w-v mice (designated as W/Wv), was purchased from The Jackson Laboratory. They are sterile with a Sertoli-cell-only testicular phenotype. The second type of recipient was the LHRKO transgenic mouse line that results from the targeted deletion of 4-kb pairs of the LHR gene that contain the promoter region and most of the sequence of exon 1 (4). Three wild-type littermates and nine LHRKO mice were transferred from the University of Louisville to Baylor College of Medicine for this study. Phenotypically, the null mice were severely undervirilized. The serum testosterone concentration was low (<20 ng/dl), and the males were infertile.

Testicular SP and non-SP cells were transplanted into the interstitium of W/Wv mice testes at age 2 months and into LHRKO mice testes at age 4–5 months. The donor cell solution was loaded into a micropipette. The testicular capsule was punctured with a 27-gauge needle, and the solution was injected into the interstitium via the opening. The rate of injection was regulated by the Transjector 5246 (Eppendorf, Hamburg, Germany). Five percent trypan blue dye was added to the cell solution to assist in visualizing the flow between the seminiferous tubules. Three W/Wv mice were injected with the SP cells (5–15 µl per testis) with a cell concentration of 10⁵ cells/ml. Due to the small number of testicular SP cells present for the LHRKO transplantation, all sorted SP cells were injected into the testes of one null recipient during each experiment to maximize the efficiency of engraftment. An equal number of non-SP cells were transplanted into another null mouse. Overall, five LHRKO were injected with SP cells, whereas the other four null mice received the non-SP cells.

Analysis of recipient testes after transplantation
W/Wv recipients.
The W/Wv mice were killed 3 months after transplantation. The testes were immediately fixed in CHO’s fixative (3% paraformaldehyde, 0.2% glutaraldehyde, and 2% sucrose in PBS, pH 7.5) at 4 C for 2 h. Whole-mount colorimetric ß-gal staining was performed using the modified protocols from Specialty Media (Phillipsburg, NJ). Because it is difficult to stain the tissue evenly with this method, we bivalved the testes for optimal staining. After rinsing with PBS and washing with buffer solutions, the testes were incubated in COMPLETE ß-Gal Tissue Stain Solution (Specialty Media) for 3 h at 37 C. The testes were then refixed with CHO’s fixative for 12 h (or overnight) before being embedded in paraffin. Sections of the paraffinized blocks were made at 5 µm thickness and counterstained with nuclear fast red solution for analysis. Positive colonization with donor cells appears blue with this assay.

To demonstrate the steroidogenic ability of the colonized donor cells, we costained the W/Wv recipient testis slides with a polyclonal rabbit antimouse cytochrome P450 side chain cleavage (P450scc) antibody (Chemicon, Temecula, CA) using standard immunohistochemical techniques. The steroidogenic cells appear brown with this staining.

LHRKO recipients.
The transplanted LHRKO mice were housed in individual cages. Serum testosterone concentrations from the wild-type and transplanted LHRKO mice were examined at 6, 12, 16, 20 wk after transplantation. Trained veterinary technicians performed the retroorbital bleeding after administration of anesthesia. Serum testosterone was assayed using a modified testosterone RIA system (DSL-4100; Diagnostic Systems Laboratories, Inc., Webster, TX) in triplicate.

The transplanted LHRKO mice were killed at 20 wk after transplantation. The harvested testes were fixed immediately with CHO’s fixative overnight at 4 C. The tissues were then dehydrated, embedded in paraffin, and sectioned at 5 µm thickness. Hematoxylin and eosin staining was performed on every fifth testicular section to examine the architecture of the seminiferous tubules and interstitial cells. To identify the colonization of transplanted donor cell, immunohistochemistry (IHC) using antirabbit primary antibody against ß-gal (1:2000; Biodesign, Saco, ME) was performed on the adjacent slides that were stained with hematoxylin and eosin. The rationale for examining these sections with IHC is to preserve the testicular histology and to be able to stain evenly throughout the section to detect the presence of donor cells. A modified IHC technique was used. Briefly, after deparaffinization, the sections were rehydrated and the surface antigen was retrieved in citrate buffer at pH 6.0 for 2 min. Endogenous enzyme activity was blocked with 3% hydrogen peroxide. Dako Envision+ system (Dako Cytomation) was used, followed by staining with 3–3'-diaminobenzidine tetrahydrochloride and counterstaining with hematoxylin. Cells that express ß-gal stain brown.

	Results

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Colonization of the donor testicular SP cells
Immunostaining of the transplanted W/Wv mice testes showed clusters of donor cells in the interstitial and peritubular space (Fig. 2A). This demonstrates successful colonization with expansion of the transplanted testicular SP cells. When costained with P450scc antibody, most of the donor cells also stained positive and confirmed their steroidogenic ability (Fig. 2B). This suggests that the SP cells have differentiated into mature functional Leydig cells. The nonsteroidogenic donor cells (small arrow) may represent quiescent Leydig stem cells or other types of stem cells if the testicular SP is not a homogenous cell population.

View larger version (78K): [in this window] [in a new window]   FIG. 2. Successful interstitial colonization of transplanted SP cells and their steroidogenic potential. A, Colorimetric staining of the W/Wv recipient testis with X-gal shows the abundance of donor cells (blue) in the interstitial space. The black arrow indicates the Sertoli-cell-only phenotype of the recipient’s seminiferous tubules. B, Costaining of the sections with cytochrome P450scc, a steroidogenic marker (stains brown for positive cells) demonstrates that the donor cells (**, stains both blue and brown) are capable of producing androgen. This indicates that the SP cells contain Leydig stem cells or progenitors. The exclusively blue stain in some cells (small arrow) reflects the nonhomogenous nature of the isolated SP cells.

View larger version (127K): [in this window] [in a new window]   FIG. 3. Successful transplantation of testicular SP cells in the LHRKO mouse testis and the restoration of spermatogenesis. Brown staining of the interstitial cells (brown arrows, positive for anti-ß-gal) indicates the donor (ROSA26) origin of the cells. Adjacent tubules show layers of germ cells including elongated spermatids.

View larger version (25K): [in this window] [in a new window]   FIG. 4. De novo testosterone production in the testicular SP transplanted LHRKO mice. A, Mean circulating testosterone levels of the mice. Wt, Wild type (n = 3); –/– SP, LHRKO mice transplanted with the testicular SP cells (n = 3); –/– Non-SP, LHRKO mice transplanted with non-SP cells (n = 4). Significant difference is apparent between the serum testosterone levels of SP-transplanted and non-SP-transplanted recipients. (*, P < 0.01). B, A cell concentration- and time-dependent increase in serum testosterone in the testicular SP-transplanted mice.

Spontaneous fertility has not been observed in the recipients to date despite this partial restoration of spermatogenesis, a result likely due to the absence of masculinization of the brain during development and undervirilization of the genital tract secondary to the lack of androgen during development. Indeed, the reproductive organs of the recipients were small compared with those of the wild-type littermates.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We used the technique of differential Hoechst dye efflux between stem cells and mature cells to isolate potential progenitors or Leydig stem cells. In addition, we successfully engrafted these cells into the interstitial space of testes of W/Wv and LHRKO mice, and showed functional activity—androgen production—in the LHRKO recipients. This suggests that adult Leydig cell progenitors are present in the interstitial space of the testis and exhibit the stem cell characteristics of self-renewal, repopulation, and differentiation into a functional Leydig cell. The flow cytometry technique, based upon exclusion of the Hoechst dye, successfully enriched these testicular cells.

Circulating testosterone concentrations did not rise immediately, but followed a transplanted cell concentration- and time-dependent pattern. This indicates that the donor cells need to multiply and differentiate into androgen-producing cells. Because mature Leydig cells do not divide and multiply, this observation also suggests that the donor cells are more likely to be Leydig stem cells or progenitors.

This technique offers a novel way to isolate Leydig stem cells and further characterizes testicular stem cells. Brinster’s group (12) reports the isolation of testis SP cells using a Hoechst dye efflux method similar to the one described here. Their transplantation assay demonstrates that the testis SP and SSC are distinct populations. Highly enriched SSC activity was found in the major histocompatability class (MHC)-I^–Thy-1⁺c-kit^– cell fraction of the mouse cryptorchid testis, and the surface phenotype of the testis SP was found to be MHC-I⁺Thy-1^–Sca-1⁺. This result supports our hypothesis that testis SP cells are likely of mesenchymal origin, and our data suggest that these cells may be predominantly Leydig stem cells rather than SSC.

However, two recent publications (13, 14) show that the testicular SP does contain an enriched population of SSC as demonstrated by the colonization of donor cells and spermatogenesis in the recipient seminiferous tubules. There are several potential explanations for this discrepancy. First, the donor selection (cryptorchid vs. normal) results in "different" populations as Lassalle et al. (14) suggest. Second, the toxicity of the Hoechst dye may have affected the viability of the sorted SP cells, hence the lack of colonization and spermatogenesis in the study by Kubota et al. (12). Finally, the SP population may, in our opinion, contain different types of stem cells—SCC, Leydig, and perhaps myoid stem cells. This is suggested by the results from Vicini’s group (13) that different regions of the testicular SP may contain different immunophenotypes (see Fig. 1 from Ref.13). In fact, our sorted "SP" cells seem to fall within the R2 region of the testicular SP of Falciatori et al. Furthermore, our results in the W/Wv transplantation show that not all the engrafted cells have steroidogenic ability. This further supports the heterogeneity of the testicular SP.

Our results reveal partial restoration of spermatogenesis in both SP- and non-SP-transplanted mice. This highlights the importance of local testicular testosterone levels, which are vital in maintaining spermatogenesis. A recent report by Zhang et al. (16) shows qualitative full spermatogenesis in 58% of the seminiferous tubules of their LHR-knockout model at 12 months of age. They suggest that, contrary to current dogma, spermatogenesis is possible without a LH-stimulated high level of intratesticular testosterone. Persistent FSH is sufficient to stimulate spermatogenesis up to the postmeiotic round spermatid stage in their LHR-knockout mice, which is similar to the gonadotropin-deficient rodent models treated with FSH. In our model, the 1-yr-old null mice testes have 1–15% of tubules with elongated spermatids and no mature sperm, whereas the transplanted LHRKO mice have elongated spermatids in between 20 and 80% of the seminiferous tubules. This discrepancy can simply reflect the different targeted deletion strategies of the LHR gene in the generation of the two different knockout models. Nevertheless, low gonadotropin-independent constitutive production of testicular testosterone may indeed be sufficient to maintain spermatogenesis, as suggested. Due to the limited number of LHRKO mice available, we have not yet assayed the intratesticular testosterone levels in the SP-engrafted mice. It will be interesting to correlate the testicular testosterone levels in the transplanted testis with the level of spermatogenesis.

In an experiment in which exogenous testosterone was administered to neonatal and adult LHRKO mice, spermatogenesis was observed in both groups (17). However, only the mice that received testosterone treatment at the neonatal period gained the capacity for successful in vivo fertilization. The adult LHRKO mice that received testosterone replacement could only conceive using in vitro methods. Likewise, no spontaneous fertility has been observed in our engrafted animals to date. This may result from behavioral deficits, as well as the undervirilization that leads to the reproductive defects in the knockout mice. Future transplantation studies using neonatal recipients will clarify this point.

Conclusion
To our knowledge, this is the first report of Leydig stem cell transplantation and successful restoration of serum testosterone in the LHRKO mice. This technique will enable Leydig stem cell enrichment and will provide an ideal model to study the regulation of Leydig cell differentiation and development.

	Acknowledgments

 
We thank Dr. Margaret Goodell from the Baylor College of Medicine stem cell core laboratory for her technical advice regarding the testis SP isolation protocol, Mr. Mike Cubbage and Christopher Threeton for the flow cytometry application, Mr. Wilson Chuang for careful reading of the manuscript, and Ms. Deanna Killen for the IHC staining.

	Footnotes

 
This work was supported in part by grants from the American Foundation for Urologic Disease (to K.C.L.), National Institutes of Health Grants P01 HD 36289 (to D.J.L.) and R03 HD 40223 (to Z.L. and Ch.V.R.).

Abbreviations: ABC, ATP-binding cassette; ß-gal, ß-galactosidase; HSC, hematopoietic stem cells; IHC, immunohistochemistry; LHRKO, LH/human chorionic gonadotropin receptor knockout; MHC, major histocompatability class; P450scc, cytochrome P450 side chain cleavage; SP, side population; SSC, spermatogonial stem cells.

Received December 22, 2003.

Accepted for publication April 22, 2004.

	References

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