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BOR - Papers in Press, published online ahead of print August 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020859
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BIOLOGY OF REPRODUCTION 69, 1989–1997 (2003)
DOI: 10.1095/biolreprod.103.020859
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Spermatogonial Depletion in Adult Pin1-Deficient Mice1

Fawn W. Atchison, and Anthony R. Means2

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogonia in the mouse testis arise from early postnatal gonocytes that are derived from primordial germ cells (PGCs) during embryonic development. The proliferation, self-renewal, and differentiation of spermatogonial stem cells provide the basis for the continuing integrity of spermatogenesis. We previously reported that Pin1-deficient embryos had a profoundly reduced number of PGCs and that Pin1 was critical to ensure appropriate proliferation of PGCs. The current investigation aimed to elucidate the function of Pin1 in postnatal germ cell development by analyzing spermatogenesis in adult Pin1-/- mice. Although Pin1 was ubiquitously expressed in the adult testis, we found it to be most highly expressed in spermatogonia and Sertoli cells. Correspondingly, we show here that Pin1 plays an essential role in maintaining spermatogonia in the adult testis. Germ cells in postnatal Pin1-/- testis were able to initiate and complete spermatogenesis, culminated by production of mature spermatozoa. However, there was a progressive and age-dependent degeneration of the spermatogenic cells in Pin1-/- testis that led to complete germ cell loss by 14 mo of age. This depletion of germ cells was not due to increased cell apoptosis. Rather, detailed analysis of the seminiferous tubules using a germ cell-specific marker revealed that depletion of spermatogonia was the first step in the degenerative process and led to disruption of spermatogenesis, which resulted in eventual tubule degeneration. These results reveal that the presence of Pin1 is required to regulate proliferation and/or cell fate of undifferentiated spermatogonia in the adult mouse testis.

Sertoli cells, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a highly organized cyclic process that consists of three distinct phases: mitosis, meiosis, and spermiogenesis [1, 2]. The mitotic germ cells in the adult testis are the spermatogonia, which originate from primordial germ cells (PGCs) during embryogenesis [3]. PGCs are established in the mouse embryo at 7.5 days post coitum (dpc) [4, 5]. They begin to migrate toward the embryonic gonads at 8.5 dpc and reach their destination 2 days later [6, 7]. The PGCs proliferate and expand in number between 8.5 and 13.5 dpc [8, 9]. After this time, PGCs in the male gonad enter cell cycle arrest and become gonocytes in the postnatal testis [3, 9]. Gonocytes resume proliferation around Postnatal Day 3 and give rise to spermatogonia, which enter meiosis around Day 10 and continue the first wave of spermatogenesis that results in the production of mature spermatozoa by Day 35 [10]. In the adult testis, spermatogenesis is a continual process that requires tight regulation and coordination of events among germ cells, including spermatogonia, spermatocytes, and spermatids, that is orchestrated and supported by the somatic Sertoli cells [2, 11, 12]. An integral element of this homeostatic process is the self-renewal and differentiation of the spermatogonial stem cells As (Asingle or Astem) [1, 13–16]. The As spermatogonia are considered to possess stem cell characteristics because they can divide, undergo self-renewal, differentiate to form the type A spermatogonia, and subsequently give rise to the intermediate and type B spermatogonia [1, 3, 13, 14]. The successful in vivo reconstitution of spermatogenesis after spermatogonial transplantation confirms the existence of these germline stem cells [17]. However, despite many recent advances in germline stem cell transplantation technology, the signaling events that control the proliferation, self-renewal and differentiation of spermatogonial stem cells are poorly understood [3, 16, 18]. Acquisition of this knowledge has been complicated by a lack of efficient and reliable methods of identification, isolation, enrichment, and in vitro manipulation of these critically important stem cells, unlike the methods that have contributed to recent advances in the biology of hematopoietic stem cells [18–20]. Currently, intense efforts are under way in several laboratories to develop and refine these essential techniques to advance our understanding of the mechanisms that regulate the activities of germline stem cells [21–27].

Pin1 is a phosphorylation-directed peptidyl-prolyl isomerase that has been shown to play a role in the regulation of cell cycle progression, both in vitro and in vivo [28–34]. Many critical cell signaling phosphoproteins, including cyclinD1, c-Jun, Cdc25, p53, and ß-catenin, have been reported to be regulated by Pin1 in a variety of ways [31, 35–40]. In our previous study of Pin1-/- mice on an inbred C57BL/6J background, we found that PGCs had a prolonged cell cycle that led to decreased proliferation in the absence of Pin1 [32]. During the 5-day proliferative period, this defect resulted in a severe reduction in PGC number and therefore fewer gonocytes at birth. Pin1-/- mice had significantly reduced fertility, and Pin1-/- testes of aged animals had been reported to contain some abnormal seminiferous tubules [31, 32]. However, the nature of the spermatogenesis defect in adult Pin1-/- mice was unknown. Since many natural or targeted genetic mouse models that have a PGC phenotype, such as mutations in steel, c-Kit, gcd (germ-cell deficient)/Pog (proliferation of germ cells), and Mvh (mouse Vasa homolog), have also been shown to have spermatogenesis abnormalities [41–47], we investigated the adult Pin1-/- testis to elucidate the function of Pin1 in spermatogenesis. In this report, we present evidence that spermatogonia become progressively depleted in Pin1-/- testis, leading to complete degeneration of the spermatogenic cells by 14 mo. We also show high expression of the Pin1 protein in spermatogonia and Sertoli cells in adult mouse testis. The potential function of Pin1 in the regulation of spermatogonial proliferation and differentiation is discussed.


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

Pin1-/- mice were originally generated by Fujimori et al. [48] and obtained from Hoffman LaRoche (Nutley, NJ). The Pin1 gene deletion was transferred into a C57BL/6J background using marker-assisted speed congenic protocols by the Jackson Laboratory as previously described [32]. The age groups and the number of animals examined for histology in each age group were as follows: 1 wk (wild-type 3; Pin1-/- 4), 2–3 mo (wild-type 4; Pin1-/- 5), 7–8 mo (wild-type 4; Pin1-/- 4), and 14–15 mo (wild-type 3; Pin1-/- 4). The histology reported in Figures 1 and 2 was representative of all animals of the respective age group, although some variations in the severity of tubule degeneration existed within the animals of each age group. The micrographs in Figures 3–6 were representative of four animals of each genotype examined at 3 and 7 mo.



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  		FIG. 1. Spermatogenesis by Pin1-/- germ cells. A) Numerous spermatogonia (red stain) line the basement membrane of every testis tubule in a wild-type 1-wk-old testis. B) Some testis tubules in the 1-wk-old Pin1-/- testis are devoid of any germ cells (triangle). However, many spermatogonia (red stain) are present in some normal-appearing testis tubules (open star). C) Seminiferous tubules in different stages of spermatogenesis are present in a 3-mo-old wild-type testis. D) Some tubules in the 3-mo-old Pin1-/- testis contain multilayered seminiferous epithelia (open star) with late spermatids lining the lumen (open star). However, some degenerative tubules contain only late spermatids embedded in abnormal positions in the cytoplasm of Sertoli cells (solid star). A and B) Immunohistochemistry with antibodies to GCNA1. C and D) PAS-H histochemistry. Magnification x200



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  		FIG. 2. Progressive degeneration of the spermatogenic cells in Pin1-/- testis. A) Histology of a 14-mo wild-type testis shows tubules in different stages of normal spermatogenesis. B) Epididymis of a 14-mo-old wild-type male contains numerous sperm. C and D) A 7-mo-old Pin1-/- testis has many severely degenerative tubules interspersed with some normal-appearing tubules, whereas the epididymis contains fewer sperm. E and F) Nearly all seminiferous tubules are devoid of germ cells except for some somatic Sertoli cells in the 14-mo Pin1-/- testis. No sperm are observed in the epididymis of the 14-mo-old Pin1-/- male. PAS-H histochemistry. Magnification x200



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  		FIG. 3. Lack of increased apoptosis in Pin1-/- testis. A) Only a few apoptotic cells (arrows) are seen in the seminiferous tubules of the 7-mo wild-type testis. B) Seminiferous tubules of the 7-mo Pin1-/- testis contain a few apoptotic cells (arrows) similar in number and position compared with wild type. C) A positive control using wild-type testis sections pretreated with DNase I to produce DNA fragmentation shows many TUNEL-positive cell clusters (arrowheads). A–C) TUNEL assay and nuclear counterstain with hematoxylin (dark brown staining shows TUNEL-positive cells). Magnification x200



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  		FIG. 6. A schematic representation of the proposed temporal progression of a normal seminiferous tubule degenerating to form an empty tubule in Pin1-/- testis. All tubules were selected from 3- and 7-mo-old Pin1-/- testes to represent the various degrees of degeneration. A) A normal-appearing tubule with spermatogonia, spermatocytes, and spermatids engaged in dynamic spermatogenesis. B) Depletion of spermatogonia results in a tubule that contains spermatocytes and spermatids but very few spermatogonia, triggering the first step in the degenerative process. C–E) The remaining spermatocytes and spermatids degenerate, resulting in a gradually thinning epithelium and, eventually, complete germ cell loss. F–G) Sertoli cells slough off in the prolonged absence of germ cells, giving rise to a virtually empty tubule in Pin1-/- testis. A and C–G) PAS-H histochemistry. B) Immunohistochemistry with antibodies to GCNA1. Original magnification x200

Histology and Immunohistochemistry

Testes and epididymides were dissected and fixed in Bouin fixative overnight at 4°C [1]. The fixed tissues were washed several times in ice-cold 70% ethanol and paraffin embedded. The tissue samples were sectioned at 7-µm intervals and stained with periodic acid-Schiff reagent and hematoxylin (PAS-H) (PolyScientific, Bay Shore, NY) for histology. Testis sections were also processed for immunohistochemistry as previously described [49]. Briefly, sections were deparaffinized and rehydrated, followed by antigen retrieval in 10 mM sodium citrate buffer. Sections were blocked in normal goat serum in PBS for 1 h at room temperature and incubated with primary antibody at 4°C overnight. After washing in PBS three times 5–10 min each, secondary antibody was applied for 1 h, followed by washing in PBS three times 5–10 min each. Staining was visualized using VectaStain and NovaRed kits according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA). Anti-germ cell nuclear antigen 1 (GCNA1) antibody 1:50 was a generous gift from G.C. Enders (University of Kansas Medical Center, Kansas City, KS) [50]. Anti-Pin1 antibody used at 1:400 dilution was generated and characterized previously in our laboratory [30].

TUNEL Assay

Apoptotic cells were detected in situ using the terminal deoxynucleotidyl transferase (TdT)-FragEL DNA fragmentation detection kit following manufacturer's instructions (Oncogene, Boston, MA). Briefly, testis sections were deparaffinized, rehydrated, and permeabilized with 20 µg/ml of proteinase K for 20 min at room temperature. For the positive control, wild-type testis sections were treated at this point in the procedure with 1 µg/µl of DNase I for 20 min at room temperature. All sections were next treated with 3% H2O2 to inactivate endogenous peroxidases and then equilibrated in 1x TdT equilibration buffer from the kit. The tissue sections were then incubated with TdT enzyme at 37°C for 90 min. The reaction was stopped with 0.5 M EDTA, and the signals detected with diaminobenzidine solution as brown precipitates. Nuclei were counterstained with hematoxylin.

Testis Extracts and Immunoblot

Testes were dissected from wild-type males at the indicated ages in Figure 5A and rinsed once in PBS. Whole testes were homogenized in ice-cold lysis buffer (25 mM Hepes, pH 7.4, 1 mM EDTA, 1 mM EGTA, and 0.5 mM dithiothreitol, 10% glycerol, 5 µg/ml of aprotinin, 5 µg/ml of leupeptin, 20 µg/ml of trypsin inhibitor, 100 µg/ml of Pefabloc, 25 mM NaF, and 1 mM Na Orthovanadate). The supernates were recovered after centrifugation for 20 min in a microcentrifuge at 13 000 rpm. Proteins present in tissue lysates were separated by 15% SDS-PAGE and transferred to Immobilon polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were then blocked for 1 h in 5% milk in 1x PBS/0.1% Tween-20 (PBST) and incubated in the primary antibody in 5% milk/PBST overnight at 4°C. Pin1 was detected using a rabbit polyclonal antibody (1:2000) generated previously in our laboratory [30]. Anti-actin (Sigma, St. Louis, MO) was used at 1:1000 dilution in 5% milk/PBST. The membranes were washed in PBST and incubated in secondary antibodies for 1 h at room temperature. After washing in PBST, signals were detected with enhanced chemiluminescence reagents (Amersham Pharmacia, Piscataway, NJ).



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  		FIG. 5. Pin1 protein expression in testis. A) Immunoblot of testis extracts of wild-type mice at the indicated ages. B) No Pin1 staining is detected in the Pin1-/- testis except for some nonspecific reactivity in the interstitium. C) Immunostaining of Pin1 in a wild-type testis shows ubiquitous Pin1 expression in the seminiferous tubules. However, more intense Pin1 expression is seen along the periphery of the tubule, including expression in spermatogonia (arrowheads) and Sertoli cells (arrow). D) A lower magnification image reveals stage-dependent Pin1 expression in the periphery of the seminiferous tubules. Compare the periphery of stage VII to stages XII and XI. B–D) Immunohistochemistry with antibodies to Pin1 on 3-mo testis sections. Original magnification x 100 for B and D and x400 for C.

Testis Weight and Sperm Count

Testes were dissected from wild-type and Pin1-/- males between 2 and 16 mo of age at 1–3-mo intervals, weighed, and normalized to body weight in Table 1. Epididymal sperm were counted using methods previously described by others [49]. Briefly, epididymides were dissected from wild-type and Pin1-/- males between 2 and 10 mo of age at 1–2-mo intervals and washed in PBS. The epididymides were flushed, then minced with scissors, and incubated in PBS for 10 min at room temperature. The sperm were counted using a hemacytometer.


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  		TABLE 1. Decreased sperm production in Pin1-/- males

Serum Testosterone

Serum testosterone was measured by radioimmunoassay using the kit Coat-A-Count (DPC, Los Angeles, CA). Serum was collected by centrifugation from orbital eye bleeds of live wild-type and Pin1-/- males between 2 and 10 mo of age at 1–2-month intervals. All samples were collected between 1400 and 1600 h to minimize natural daily fluctuations in serum testosterone. The radioimmunoassay was performed according to the manufacturer's instructions.

Statistics

Data were analyzed using the software program SigmaPlot (Jandel Corporation, San Rafael, CA). Statistical analysis of the wild-type and Pin1-/- groups was performed using the unpaired Student t-test. P < 0.01 was considered statistically significant. P values for each set of data are listed in Table 1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Spermatogenesis by Pin1-/- Germ Cells

In a previous study, we showed that the newborn Pin1-/- testes contained fewer gonocytes in some tubules and no gonocytes in many tubules, as a result of the production of a reduced number of PGCs during embryogenesis [32]. To determine whether the remaining gonocytes in Pin1-/- testes were able to initiate and complete spermatogenesis, we examined postnatal testes of wild-type and Pin1-/- mice. Gonocytes resume proliferation to become spermatogonia and migrate from the center of the testis cords to the basement membrane of the testis tubules during the first few days postnatally [1, 3, 18]. In the 1-wk-old wild-type testis, numerous spermatogonia, identified by immunohistochemistry with anti-GCNA1 [50], were present along the basement membrane of every testis tubule (Fig. 1A). In contrast, the 1-wk-old Pin1-/- testis showed many Sertoli cell-only tubules (Fig. 1B, triangle), suggesting that those tubules that lacked gonocytes at birth were not repopulated by germ cells. The Sertoli cells in Pin1-/- testis had proliferated and populated normal-appearing tubules. In addition, there were some tubules in the Pin1-/- testis with many spermatogonia along the basement membrane (Fig. 1B, open star), indicating that Pin1-/- gonocytes had undergone proliferation and migration in those tubules. For the purpose of this study, we focused on these germ cell-containing regions of the testis to investigate spermatogenesis.

In the 3-mo-old wild-type testis, all of the seminiferous tubules had spermatogonia, spermatocytes, and spermatids in different stages of spermatogenesis (Fig. 1C) [1]. The Pin1-/- testis also contained tubules with multilayered seminiferous epithelia, including stage VIII tubules with mature spermatids lining the lumen (Fig. 1D, open star). This indicated that Pin1-/- germ cells were able to initiate and complete the entire spermatogenic process in a manner that produced normal sperm. Indeed, although the epididymal sperm count of Pin1-/- mice was much lower than that of wild-type mice (Table 1), the sperm were motile and functional, in accordance with our previous report that Pin1-/- males mated with wild-type females were able to sire some pups, although with greatly reduced fertility [32].

Progressive Degeneration of the Spermatogenic Cells

In addition to the normal-appearing tubules and Sertoli cell-only tubules, we also observed some tubules that contained elongated spermatids, but neither spermatocytes nor spermatogonia, in the seminiferous epithelium of the 3-mo Pin1-/- testis (Fig. 1D, solid star). In addition, the spermatids were embedded in abnormal positions within the cytoplasm of Sertoli cells, indicating that spermatogenesis had occurred in those tubules and produced spermatids, but the spermatogenic cells had subsequently degenerated (Fig. 1D, solid star). To determine the extent of this degenerative phenotype, we examined testes of older mice up to 15 mo and found that the degeneration occurred progressively in Pin1-/- testis in an age-dependent manner. Wild-type testis, even at 14 mo of age, exhibited healthy seminiferous tubules characterized by robust sperm production (Fig. 2, A and B). However, Pin1-/- testis had many more degenerative tubules and more severely affected tubules at 7 mo compared with 3 mo (Fig. 2C). Similarly, the epididymis of Pin1-/- mice contained significantly fewer sperm (Fig. 2D). By 14 mo of age, nearly all of the tubules in Pin1-/- testis were empty, and the epididymis was devoid of sperm (Fig. 2, E and F). The interstitial compartments of 3- and 7-mo Pin1-/- testes appeared normal without Leydig cell dysplasia (Figs. 1D and 2C), consistent with their normal serum testosterone level (Table 1), suggesting that the tubule degeneration was unlikely caused by Leydig cell dysfunction [2]. The apparently thicker interstitium in the 14-mo Pin1-/- testis was likely a result of secondary effects from the complete absence of spermatogenesis (Fig. 2E).

Lack of Increased Apoptosis in Pin1-/- Testis

Since many genetic mouse models with a testis degeneration phenotype have been shown to have increased germ cell apoptosis [51–55], we used TUNEL assays to detect apoptotic cells in 3- and 7-mo-old wild-type and Pin1-/- testes. Only a few apoptotic cells were seen in the wild-type testis (Fig. 3A, arrows, brown), whereas a positive control with DNase I pretreatment to produce DNA fragmentation resulted in clusters of TUNEL-positive cells (Fig. 3C, arrowheads). This result was consistent with what many others have reported using TUNEL assays in wild-type testis [55–57]. In the Pin1-/- testis, we observed only a few apoptotic cells, and the number and position of these cells were similar to those of wild type, indicating that the massive degeneration of the spermatogenic cells was not due to increased germ cell apoptosis.

Spermatogonial Depletion in Adult Pin1-/- Testis

Besides germ cell apoptosis, other abnormal events during spermatogenesis could also cause degeneration of the spermatogenic cells. These events include arrest in meiosis, a block in the progression of spermiogenesis, or abnormal spermatogonial cell fate decisions [2]. To evaluate how spermatogenesis was disrupted in Pin1-/- testis, we used the GCNA1 antibody to distinguish different types of germ cells in situ. Spermatogonia have been shown to react very strongly with the GCNA1 antibody, whereas pachytene spermatocytes and round spermatids stain weakly with this reagent [50]. We performed immunohistochemistry with anti-GCNA1 on 3- and 7-mo wild-type and Pin1-/- testis sections and analyzed in detail those tubules that still contained germ cells and had not undergone extensive degeneration. For these analyses, tubules were staged according to the criteria of Russell et al. [1] but were within one stage error because acrosomes were not stained. In a wild-type stage VII tubule (Fig. 4A, open star), spermatogonia stained intensely for GCNA1 (Fig. 4A, open arrow), whereas pachytene spermatocytes stained only weakly (Fig. 4A, solid arrow). In contrast, we discovered many tubules in Pin1-/- testes that lacked both spermatocytes and spermatogonia but contained round and elongated spermatids, suggesting that meiosis had been completed, but the spermatogonia and spermatocytes had been lost from the tubules (see a comparable "stage VII" tubule in Fig. 4B, open star; quote marks used to indicate not a true stage VII because of the abnormalities that exist). In addition, we also found tubules that contained spermatocytes and spermatids but few or no spermatogonia in Pin1-/- testes. For example, a wild-type stage X tubule contained spermatogonia, spermatocytes, and elongating spermatids (Fig. 4C, solid star). However, in the Pin1-/- testis, whereas a comparable "stage X" tubule (Fig. 4D, solid star) had pachytene spermatocytes and elongating spermatids, very few zygotene spermatocytes and spermatogonia (arrow) were present (Fig. 4D, solid star). Moreover, in the wild-type stage X tubule, numerous oval-shaped spermatogonia, characteristic of undifferentiated type A spermatogonia [1, 58], were present along the basement membrane (Fig. 4C, arrowheads). In contrast, an apparently normal stage X tubule in the Pin1-/- testis lacked these morphologically distinct undifferentiated type A spermatogonia, even though it contained differentiated spermatogonia and spermatocytes (Fig. 4D, triangle). These findings strongly suggest that the principle cause of spermatogenesis disruption in adult Pin1-/- testis was spermatogonial depletion.



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  		FIG. 4. Spermatogonial depletion in Pin1-/- testis. A–D) Immunohistochemistry using antibodies to GCNA1 (red staining) on 7-mo wild-type and Pin1-/- testis sections. Stages are plus or minus one stage according to criteria established in Russell et al., see text. A) A stage VII wild-type seminiferous tubule (open star) contains intensely stained spermatogonia (open arrow), weakly stained pachytene spermatocytes (solid arrow), and lightly stained spermatids. B) A comparable "stage VII" tubule (open star) in Pin1-/- testis contains only lightly stained spermatids but neither spermatogonia nor spermatocytes. C) Darkly stained spermatogonia and zygotene spermatocytes, lightly stained pachytene spermatocytes, and elongating spermatids are present in a stage X tubule in the wild-type testis (solid star). In addition, several oval GCNA1-positive cells (arrowheads) characteristic of undifferentiated type A spermatogonia are present along the basement membrane of the seminiferous tubule. D) A comparable "stage X" tubule in the Pin1-/- testis (solid star) contains pachytene spermatocytes and elongated spermatids but very few zygotene spermatocytes and spermatogonia (open arrow). Moreover, a relatively normal-appearing stage X tubule (triangle) contains spermatogonia along with spermatocytes and spermatids but lacks the oval type A spermatogonia like those seen in C. Magnification x200

Pin1 Expression in the Testis

We previously reported that newborn gonocytes had high expression of the Pin1 protein [32]. To explore whether postnatal testes continue to express Pin1, we performed immunoblot analysis of testis extracts prepared at several different ages from Postnatal Day 8 to 6 mo (Fig. 5A). We found that Pin1 expression in the testis was constant from early postnatal period to adulthood (Fig. 5A). To investigate the sites of Pin1 expression in the adult testis, we performed immunohistochemistry with anti-Pin1 antibodies on 3- and 7-mo-old testis sections. As expected, no Pin1 staining was detected in the Pin1-/- testis except for some nonspecific reactivity in the interstitium (Fig. 5B). Staining in the wild-type testis revealed that Pin1 was ubiquitously expressed in the testis but had the highest expression in the region near the basal compartment of the tubules (Fig. 5C), with intense expression in spermatogonia (arrowheads), spermatocytes, and Sertoli cells (arrow). In addition, Pin1 expression appeared to be higher in earlier stage tubules (I–VIII) and less in later stages such as XII (Fig. 5D, compare the tubule periphery in VII to XII and XI). Since type Aaligned (Aal) spermatogonia become A1 spermatogonia at stages VII/VIII, the number of Aal is lower in the later stages from IX to XII [3, 15]. On the other hand, As and Apaired (Apr) spermatogonia are thought to remain constant throughout the different stages [3, 15]. Thus, the higher Pin1 expression in the periphery of earlier stage tubules suggests that the As, Apr, and Aal subpopulation of spermatogonia express Pin1 more avidly than do the differentiated A1-B types of spermatogonia.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Based on our results, we propose that depletion of spermatogonia is the first step in the degenerative process in Pin1-/- testis. A schematic diagram assembled from seminiferous tubules in 3- and 7-mo Pin1-/- testis is represented in Figure 6 to illustrate the proposed temporal changes that occur during transition from a healthy tubule to an empty tubule in the absence of Pin1. Germ cells in Pin1-/- testis initially accomplish the mitosis, meiosis, and spermiogenesis phases of spermatogenesis (Fig. 6A). However, spermatogonia become depleted in those tubules at some point during adult life (Fig. 6B). The remaining spermatocytes, round and elongated spermatids, then degenerate and presumably are phagocytized by Sertoli cells [1] (Fig. 6, C–E). Sertoli cells apparently eventually slough off, resulting in virtually empty tubules in the Pin1-/- testis (Fig. 6, F and G). The defect in spermatogenesis and tubule degeneration in Pin1-/- testis was nonuniform but progressive in the context of the whole organ. The segmental nature of tubule degeneration is not without precedent, as others have reported spermatogenesis defects in genetic mouse models with heterogeneity of defective tubules dispersed among normal tubules [59–62]. Unfortunately, such a heterogeneous alteration in spermatogenesis adds considerable complexity to any attempts designed at elucidating the underlying mechanism responsible for spermatogonial depletion in the absence of Pin1.

Previously, we showed that Pin1 regulates the proliferation of mammalian PGCs [32]. In the current study, an additional role for Pin1 in germ cell biology was identified. Thus, whereas postnatal gonocytes and spermatogonia in prepubertal Pin1-/- testis are able to proliferate and differentiate to form mature spermatozoa, the maintenance of spermatogonia in adult testes requires Pin1. This apparent variance in the requirement for Pin1 may be explained by recent discoveries that some differences exist in niche and properties of postnatal gonocytes/spermatogonia and adult spermatogonia [16, 17, 21, 23]. For instance, spermatogonia from prepubertal testis have been shown to differ from adult spermatogonia in the proportion of their Kit-negative (repopulating) populations and cell cycle characteristics [21]. In fact, even among gonocytes of the same stage in the rat, reconstitution assays identified two distinct populations of spermatogonia based on differences in morphology and function [63]. Therefore, the regulatory signals that control the initiation of spermatogenesis and the maintenance of spermatogenesis may differ, and our results indicate that Pin1 participates in the latter signaling pathways. Thus, we speculate that Pin1 can regulate spermatogonial proliferation and/or self-renewal and differentiation in the adult testis.

Determining precisely how Pin1 regulates the maintenance of spermatogonia is not a simple matter. However, we can propose several possibilities. First, given the role of Pin1 in regulating the timing of PGC proliferation [32], it is conceivable that Pin1 can also regulate the cell cycle timing of spermatogonia in general and undifferentiated spermatogonia in particular. Type As, Apr, and Aal spermatogonia are thought to divide in a cyclic manner, with periods of mitotic activity interspersed with periods of cell cycle quiescence throughout the cycle of the seminiferous epithelium [3, 15]. We speculate that misregulation in the timing of proliferation of these spermatogonial populations may result in missed "windows of opportunity" in cell division during any given cycle of spermatogenesis. The consequence of such an event would be the presence of fewer spermatogonia in each ensuing cycle, which would in turn lead to gradual depletion of these cells. The observation that fewer GCNA1-positive spermatogonia and fewer morphologically distinct type A spermatogonia are found in some Pin1-/- tubules is consistent with this hypothesis. Second, it may be possible that As spermatogonia exhaust their stem cell potential sooner in Pin1-/- testis, perhaps as a result of accelerated proliferative cycles in response to a higher demand for spermatogenesis due to the initial reduced allocation of germ cells among the tubules. In this regard, a similar type of local feedback regulatory mechanism has been reported to exist in which the proliferation of undifferentiated spermatogonia is prolonged when inadequate numbers of A1 spermatogonia are produced [3, 64]. Accelerated exhaustion of stem cell potential would contribute to defective germline stem cell self-renewal and eventual spermatogonial depletion. Finally, the differentiation pathway of spermatogonia may be improperly favored in the absence of Pin1, giving rise to depletion of undifferentiated spermatogonia, a phenotype not readily distinguishable from those predicted by the other hypotheses based solely on morphological analysis. Indeed, this mechanism would most likely be secondary to changes in gene expression [11]. Although few genes have been demonstrated to regulate cell fate decision of undifferentiated spermatogonia, Sertoli cell-derived factors have been shown to be important [11]. The dosage of a Sertoli cell-secreted factor, glial cell line-derived neurotrophic factor, has been shown to control spermatogonial cell fate based on analysis of mutant and transgenic mice [65]. Since Pin1 is expressed in Sertoli cells and spermatogonia, the possibility exists that Pin1 might act through Sertoli cell or in concert with its action in spermatogonia to regulate maintenance of spermatogonia. Future research efforts, capitalizing on advances in germline stem cell characterization, isolation, and in vitro manipulation, will help to illuminate the mechanisms underlying spermatogonial depletion in adult Pin1-/- testis.


   ACKNOWLEDGMENTS
 
We thank G.C. Enders (University of Kansas Medical Center, Kansas City, KS) for providing the GCNA1 antibody. We are grateful to Takafumi Uchida for the initial generation of Pin1 Null Mice [48].


   FOOTNOTES
 
1 This work was supported by National Institutes of Health grant CA-82845 (to A.R.M.) and by the Medical Scientist Training Program from the National Institutes of Health (to F.W.A.). Back

2 Correspondence: Anthony R. Means, Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC 27710. FAX: 919 681 7767; means001{at}mc.duke.edu Back

Received: 30 June 2003.

First decision: 15 July 2003.

Accepted: 6 August 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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