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a Institute of Reproductive Medicine of the University, D-48129 Münster, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, sperm maturation, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Germ cell transplantation has become an established experimental tool over the last few years [11–13]. Germ cell suspensions obtained after digestion of the testis of donor animals are transferred into the recipient's testicular tubules by microinjection via the rete testis [14]. Only the spermatogonia persist [15] in the host environment and are able to colonize the recipient's organ. This technique was successfully used to investigate the cause of testicular malfunction in subfertile or infertile transgenic mice [14, 16–18]. Here, we applied this technique to analyze the effects of CREM deficiency in the testis. Endpoints were both histological parameters, as used in most studies on germ cell transplantation, and genetic markers specific for the donor animals, as detected by polymerase chain reaction (PCR) assays. The status of spermatogenesis achieved after transplantation of CREM-deficient spermatogonia into a germ cell-depleted wild-type testis and of wild-type and heterozygous germ cells into deficient testes was used to determine whether the germ line or the somatic component is affected by CREM deficiency.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The animals were obtained from our institutional colony. The founders (for targeting strategy and construct details, see [4]) were raised by the research group of Dr. G. Schütz (German Cancer Research Center, Heidelberg, Germany) and housed under standard conditions. All animal experiments were in accordance with the German federal law on the Care and Use of Laboratory Animals. Experiments were performed using adult male homozygous CREM-mutant mice (ages 15–30 wk) and age-matched wild-type males as recipients and using adult CREM-deficient, wild-type, and heterozygous males of the same strain as donors. All animals were of the same genetic and immunological background (129sv:C57/Bl6) as initially described [4]. Two groups of germ cell recipients (n = 28) consisted of wild-type animals, and these mice received spermatogonia from either adult CREM-deficient donors (n = 4; 16 recipients) or adult heterozygous donors (n = 4, 12 recipients). A third group consisted of adult CREM-deficient mice (n = 16), and these mice received germ cells from adult wild-type mice (n = 4). During the experimental period, some of the recipients died and thus not all testes and epididymides were available for examination. In the group receiving cells from CREM-deficient donors, five animals died between transplantation and evaluation of the left testis and epididymis and one died between removal of the left and the right testis. Thus, 11 recipient testes and epididymides remained for examination of CREM-deficient cells after 7 wk and 10 testes and epididymides remained after 13 wk. In the group receiving cells from heterozygous donors, one animal died between transplantation and evaluation of the left testis and epididymis and one died between removal of the left and the right testis and epididymis. Thus, 11 recipient testes and epididymides were available for examination after 7 wk and 10 testes and epididymides were available after 13 wk. In the group receiving cells from wild-type donors, one animal died between transplantation and evaluation of left testis and two died between removal of the left and the right testis and epididymis. Thus, 15 recipient testes and epididymides were examined after 7 wk and 13 testes and epididymides were examined after 13 wk.
Following germ cell transfer, the host animals received three injections of a GnRH antagonist [19] (Cetrorelix, 450 µg/kg of body weight over 7 days) to improve posttransplantation recovery of the testes. As shown in previous studies using monkeys and rats, this dose leads to complete supression of testicular function [20, 21].
Donor Cell Isolation from the Seminiferous Epithelium
The testes of donor animals were excised and decapsulated. Testicular tissue was minced with fine scissors and transferred into culture medium (Dulbecco modified Eagle medium DMEM/F12; Gibco, Gaithersburg, MD) containing collagenase type I (Sigma, St. Louis, MO; 1 mg/ml) and DNase (Sigma; 0.5 mg/ml). Enzymatic digestion was performed at 37°C for 10 min in a shaking water bath. Interstitial cells were separated by sedimentation at unit gravity for 10 min and washed in DMEM/F12 as previously described [22]. A second digestion step was carried out in a mixture of collagenase type I (Sigma; 1 mg/ml), DNase (Sigma; 0.5 mg/ml), and hyaluronidase (Sigma, 0.5 mg/ml). The tubular fragments were aspirated several times with an automatic pipette until a single-cell suspension was obtained. The cells were rinsed twice in medium and filtered through a 50-µm nylon mesh to remove undigested remains of the tunica albuginea. While the cells were kept on ice, cell number and concentration were established microscopically in a Thoma chamber (Hecht, Sondheim, Germany). The cell suspension was centrifuged at 400 x g for 5 min. The cell pellet was then resuspended in medium/trypan blue (3 mg/ml) and adjusted to a final concentration of 1–2 x 106 cells/ml. This suspension consists of all cells present in the tubular compartment: Sertoli cells, spermatogonia, and more mature germ cell types. As demonstrated previously [15], all injected somatic and differentiating germ cells disappear from the testis, and only donor germ line stem cells, i.e., spermatogonia, remain and colonize. Therefore, we assumed that 7 and 13 wk after transfer all signals detected were derived from colonizing germ cells and did not represent cells persisting from the transplantation.
Recipient Mice
All adult recipient wild-type and CREM-deficient males were treated with busulphan (40 mg/kg of body weight, single i.p. injection) to destroy endogenous spermatogenesis [11]. Donor cells were transplanted bilaterally 6 wk after treatment. Using sterile techniques, an incision was made into the scrotum of the recipient animal and the testes were exposed. The donor cell suspension was microinjected into the rete testes under visual control. Donor cell suspension (50–80 µl) containing trypan blue was infused into each rete testis until 60–80% of the tubules visible through the surface were stained. Following transplantation, the testes were returned to the scrotum, which was sutured.
Histology
Each testis was dissected into four fragments. One fragment was frozen in liquid nitrogen and stored for optional kryosection (-80°C). Two fragments were stained with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal; Sigma). Two different protocols were used for X-Gal staining. One protocol used fixation in 4% paraformaldehyde for 1 h at 4°C, rinsing with 10 mM MgCl2 and 0.15% Triton X-100 (Sigma) in PBS (three times for 30 min each), and staining for 48 h at 37°C in staining solution of 2 mg/ml X-Gal dissolved in dimethylformamide, 5 mM MgCl2, 5 mM potassiumferrocyanide (Sigma), and 5 mM potassiumferricyanide (Sigma) in PBS. Alternatively, the fragments were stained directly for 48 h at 37°C in staining solution of 0.15% Triton X-100 and 0.1% formaldehyde in PBS. After staining, the samples were rinsed in washing buffer (10 mM MgCl2 in PBS), postfixed in 4% paraformaldehyde overnight, dehydrated, and embedded in resin (Technovit 7100; Heraeus-Kulzer, Wehrheim, Germany) or paraffin. Histological preservation of the prefixed sample was better, but the staining was stronger in the fragments directly stained even though cellular architecture was less well preserved. Positive (caput epididymis showing high endogenous lacZ activity) and negative (wild-type mouse testes) control tissues were included in each staining procedure. Some sections per sample were additionally stained with periodic acid-Schiff (PAS). At least 200 tubules of each testis from two different slides and from two different regions were analyzed histologically, and at least 120 cross sections of the epididymal tubule of each sample available were examined.
Immunohistochemistry
The fourth fragment was fixed in 4% paraformaldehyde/4% sucrose for 2 h at room temperature, rinsed in PBS, dehydrated, and embedded in paraffin for immunohistochemical analysis. Immunohistochemical staining of CREM protein was performed as described previously [6]. Sections from paraffin-embedded specimens were cut at a thickness of 5 µm. Antigen retrieval was performed by microwaving the sections in 0.05 M glycine buffer for 20 min (H2500 microwave processor; Bio-Rad Laboratories, Richmond, CA). A rabbit polyclonal antibody raised against recombinant CREM (Upstate Biotechnology, Lake Placid, NY) and recognizing all known CREM isoforms with similar affinity was used at 1:400 to 1:1200 dilutions. Five percent normal porcine serum was used to block nonspecific background staining. Biotinylated anti-rabbit IgG from swine (1:400; DAKO, Hamburg, Germany), ExtrAvidin-conjugated alkaline phosphatase (1:200; Sigma), and New-Fuchsin (DAKO) were employed for enhancement of the signal and visualization of the primary antibody. Mayer hematoxylin was used as counterstain. The sections were evaluated for CREM staining and documented as digital images using an Axioscop (Zeiss, Oberkochen, Germany) with a CCD Axiocam (Axiovision program; Zeiss).
Genotyping
Genotyping of mice was performed by PCR on animals 6–10 wk of age. Genomic DNA was isolated from the tail using proteinase K (0.5 mg/ml; Merck, Darmstadt, Germany) and subsequent ethanol precipitation, and the precipitate was resuspended in 150 µl of Tris-EDTA buffer (pH 7.5). The following primers were used: CREM 5', 5'-TGGATTGTGCTGGGAGGTTGTTC-3'; CREM 3', 5'-TCTTTGAGGGCCTTGAGTTCCTC-3';lacZ, 5'-CGCCATTCGCCATTCAGGCTGC-3'. Primer CREM 5' binds to a sequence present in the wild-type and mutated allele. Primer lacZ binds to a sequence present only in the targeting construct. Primer CREM 3' binds to a sequence only present in the wild-type allele. Primer combination CREM 5'- CREM 3' generates a 173-base pair (bp) amplicon indicating the wild-type allele, and primer pair CREM 5'-lacZ yields 332 bp indicating the mutated allele. PCR conditions (25 µl total volume) were 1 µl genomic DNA, 2.5 µl 10x PCR buffer without MgCl2, 2 mM MgCl2, 0.5 mM primer CREM 5', 0.25 mM primers lacZ and CREM 3' or CREM 5', respectively, 1 µM dNTPs, 0.5 µl Taq polymerase (5 U/µl; Promega, Madison, WI), and sterile water. Thirty-two cycles were run at 94°C for 30 sec, 62°C for 40 sec, and at 72°C for 60 sec.
For genotyping of testicular tissue, genomic DNA was isolated from fragments of testis tissue frozen in liquid nitrogen and stored at -80°C. A small amount of DNA (5 µl) was used for amplification to detect very low amounts of DNA in the sample. For genotyping of epidydimal eluates, the epididymides were dissected into proximal and distal parts and rinsed with PBS buffer through an incision between the corpus and the cauda, and the sample was retrieved from one incision site in the most distal part of the tubule in the cauda epididymis. The content of the cauda epididymis was flushed out using a micropipette for injection of PBS and collection of fluid. Genotyping of the flushout samples was performed by PCR as described above with the exception of increasing amounts of DNA up to 5 µl.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Histological analysis revealed that 6% and 8% of the transplanted testes available for examination were colonized by wild-type spermatogonia after 7 wk and 13 wk, respectively (Table 1). Elongated spermatids were never observed in untreated CREM-deficient mice but were present in the transplanted animals at both time points analyzed. In the remaining majority of the testes (96% and 92%), the RSMA phenotype was exclusively found (Fig. 1, A and B, and Table 1). In those testes colonized sucessfully with donor cells, restoration up to the level of qualitatively complete spermatogenesis was observed focally (Fig. 1, C and D). Most of the epididymides showed the CREM-deficient phenotype and contained only round cells (Table 1). Histological analysis of the epididymides supports the data obtained from the testes (Table 1). In 6% (7 wk) and 15% (13 wk) of the organs, some cross sections of the epididymal tubule contained morphologically normal sperm after transplantation (Fig. 1E). The immunohistochemical detection of the wild-type CREM protein proved that the round spermatids in areas of complete spermatogenesis are of wild-type origin (Fig. 1F). The PCR screening of testis tissue and epididymal eluates allowed both alleles (wild type and mutated) of the CREM gene to be detected and confirmed the histological observations (Figs. 2 and 3 and Tables 2 and 3). About one-third of the eluates contained genomic DNA from the wild-type CREM locus (Table 2).
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Transplantation of Germ Cells from Heterozygous Donors into Wild-Type Recipients
Spontaneous spermatogenic recovery up to the level of complete spermatogenesis, but no colonization of transplanted cells, was histologically observed in these wild-type recipient testes (Table 1). However, donor-derived cells were localized in the testicular tissue at both time points by PCR analysis (64%, 7 wk; 20%, 13 wk; Table 3 and Fig. 3). After 13 wk, 20% of the epididymides investigated contained X-Gal-positive cells (Table 1). A similar rate of successful transplantation was detected by PCR of epididymal eluates (Table 2 and Fig. 2).
Transplantation of CREM-Deficient Germ Cells into Wild-Type Recipients
Spontaneous recovery up to the level of qualitatively complete spermatogenesis was exclusively detected by histology in the testes and mature sperm were seen in these wild-type recipient epididymides (Table 1). No X-Gal staining was present. PCR analysis of frozen testicular tissue revealed that the mutated CREM locus was marginally detectable only in one testis 13 wk after transfer (Fig. 3 and Table 3). No mutant alleles were found in epididymal eluates (Table 2 and Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Some CREM-dependent genes expressed in round spermatids have been described [3, 10]. There is also strong evidence for the importance of CREM during the development and maturation of the male germ cells in primates [3, 6]. The transcription factor CREM is expressed in germ cells and Sertoli cells [3, 6–9]. The CREM gene consists of several exons encoding different functional domains of the CREM protein [10, 24–26]. CREM exists in various isoforms [25]. CREM-deficient mice are deficient for all CREM proteins containing the DNA-binding domain [4, 5]. By using gene targeting, the exons encoding for the CREM DNA binding domain were selectively eliminated and replaced with a construct encoding for the lacZ gene.
Our results indicate that the germ cell but not the somatic testicular environment is responsible for spermatogenic arrest under the CREM-deficient condition. The transplanted wild-type spermatogonia colonize the testes of the CREM-deficient recipients successfully and produce mature sperm. We postulate that CREM deficiency does not disturb Sertoli cell function because support of wild-type germ cells during maturation is qualitatively maintained in the absence of CREM [27]. However, we cannot exclude an effect of CREM on the kinetics of germ cell development or the efficiency of germ cell production.
Complete spermatogenesis in CREM-deficient recipients was observed as early as 7 wk after germ cell transfer. Similar to the first wave of spermatogenesis during puberty in mice [28], the initiation of spermatogenesis after transplantation appears to be as fast as the steady state development of germ cells during adulthood [15, 23, 29, 30]. The course of germ cell development after germ cell transplantation indicates that colonization by spermatogonial stem cells starts immediately after their infusion into the testicular microenvironment and in parallel with the spontaneous recovery of the endogeneous germ cells.
The histological appearance of elongated spermatids is a valid argument for the capability of the CREM-deficient testis to support complete spermatogenesis. The differentiation of germ cells occurred often in a pattern similar to that described previously following germ cell transplantation in rats [31]. The authors of the rat study concluded that the spermatogenic cycle of the donor-derived germ cells is regulated by the intraluminal microenvironment. Our observation of qualitatively normal spermatogenesis in the tubules of CREM-deficient testes indicates that the tubular microenvironment of the testis is intact and can support complete maturation of germ cells.
The colonization efficiency of CREM-deficient cells was extremely low in comparison to heterozygous or wild-type donor cells. Some mechanisms may exist that eliminate or hinder the CREM-deficient germ cells from colonization and differentiation in the wild-type host.
Our study shows that the RSMA seen in the CREM-deficient testes is caused by a germ cell-intrinsic problem and not by the somatic testicular environment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: E. Nieschlag, Institute of Reproductive Medicine of the University, Domagkstr. 11, D-48129 Münster, Germany. FAX: 492518356093; nieschl{at}uni-muenster.de
Received: 18 December 2001.
First decision: 3 January 2002.
Accepted: 22 April 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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