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Endocrinology Vol. 140, No. 4 1875-1883
Copyright © 1999 by The Endocrine Society

ARTICLES

Cytoplasmic Liberation of Protein Gene Product 9.5 during the Seasonal Regulation of Spermatogenesis in the Monkey (Macaca fuscata)1

Yoshimitsu Tokunaga, Shinji Imai, Ryuzo Torii and Toshihiro Maeda

Department of Anatomy (Y.T., S.I., T.M.) and Institute of Experimental Animals (R.T.), Shiga University of Medical Science, Otsu 520-2192, Japan

Address all correspondence and requests for reprints to: Yoshimitsu Tokunaga, Department of Anatomy, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520-2192, Shiga, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Primate spermatogenesis is distinguished by yet unidentified mechanisms to regulate its spermatogenetic activity. In contrast to the well documented hormonal regulators, the cellular events responsible for the regulation of the spermatogenesis has not been addressed. By using PGP 9.5-immunohistochemistry, our previous study demonstrated that the monkey spermatogonia are divided into two distinct sub-populations, i.e. cytoplasmic PGP 9.5-positive and cytoplasmic PGP 9.5-negative spermatogonia. By comparing the cytoplasmic expression of PGP 9.5 between the breeding and nonbreeding seasons of the Japanese monkey (Macaca fuscata) in association with PCNA labeling, the present study demonstrates that the cytoplasmic PGP 9.5-positive Ap spermatogonia significantly increases when the spermatogenetic activity declines in the nonbreeding season. An ultrastructural subcellular localization of PGP 9.5 suggests that the increase of the cytoplasmic PGP 9.5 expression is due to a liberation of PGP 9.5 molecule from the nucleus into the cytoplasm. The results provide a theoretical basis by which PGP 9.5 serves as a novel marker for spermatogonial subtypes, which will have further implications for future studies on spermatogenesis. The analysis using this novel marker suggests that the Ap spermatogonia is a key stage to regulate the amount of the sperm produced in response to the hormonal regulators, and the cytoplasmic liberation of PGP 9.5 may serve as a pivotal phenomenon that enables the fully restorable, transient suppression of spermatogenesis in primate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PITUITARY GONADOTROPIC hormones, LH FSH, are the prime regulators of primate spermatogenesis (1, 2, 3). LH affects indirectly the seminiferous tubules via inducing testosterone production by the Leydig cells (2), and testosterone sustains continuous production of spermatozoa (2). Role of FSH, in turn, has been studied in adult monkeys. Immunization against FSH markedly reduces germ cell population (4) and even causes infertility (5). In the GnRH antagonist-treated monkey, supplement of FSH maintains qualitatively normal spermatogenesis (6). Both LH and FSH are, thus, required to attain normal spermatogenesis. However, cellular mechanism responsible for the accurate regulation of primate spermatogenesis has not been determined.

The primate spermatogonia, including those of human, have been generally divided into three subpopulations, which are termed as type Ad, type Ap, and type B spermatogonia (7, 8). Type Ad spermatogonia have been considered to be quiescent reservoir cells, which give rise to type Ap. Type Ap spermatogonia proliferate and provide type B. Type B spermatogonia are considered to proliferate continuously and differentiate further into the primary spermatocytes (7, 9). Following the production of the primary spermatocytes, only a 4-fold increase in germ cell population is possible through two meiotic divisions (9). As the candidate mechanism to regulate spermatogenesis, 1) ratio of type Ap cells derived from type Ad cells; 2) proliferation rate of the type Ap cells, and/or 3) ratio of type B cells derived from type Ap cells can be proposed (9). It has been suggested by the studies using hypophysectomized or gonadotropin-suppressed monkeys that FSH specifically stimulates the proliferation of type Ap spermatogonia (6, 10). With the aid of testosterone, FSH can also increase the number of type B spermatogonia (11). Taken together, the ratios of both type Ap and type B spermatogonia can be regulated collaboratively by FSH, LH and testosterone.

It has been suggested that the primates produce relatively small amount of sperm as compared to the rodents (3, 12). Moreover, the regulation of spermatogenesis among the primates can be characterized by an yet unidentified cellular mechanism to suppress spermatogenesis when reproduction activity is low (7, 9, 13). Japanese monkey (Macaca fuscata) has been reported to be the seasonal breeder (14, 15, 16); their testicular function, e.g. serum testosterone level, testicular size, and sperm count, is annually suppressed in the nonbreeding season, but most characteristically it returns to the fully functional level in the next breeding season (15, 16, 17). We thus used this animal to study the cellular mechanism to regulate spermatogenesis in primate.

Protein gene product (PGP) 9.5 has been reported to be a sensitive pan-neuronal marker, the physiologic function of which remains to be determined (18). In our previous study, we have demonstrated that PGP 9.5 is indeed expressed in the spermatogonia of Japanese monkey (19). In the present study, we attempt to show that PGP 9.5 is present in the cytoplasm of the primate spermatogonia when their proliferation activity is suppressed. In turn, PGP 9.5 is absent from the cytoplasm when the proliferation activity is at the normal level. Using this novel marker of the primate spermatogenesis, we attempt to characterize the cell kinetics during the seasonal suppression of the primate spermatogenesis. The elucidated data provide an important insight for a better understanding of the regulatory mechanism in the primate spermatogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sixteen male Japanese monkeys (Macaca fuscata) were investigated from September 1994 to August 1997. The animals were estimated to be over 10 yr old with body weight ranging from 11.6–13.5 kg, and all had reached sexual maturity. Eight of the monkeys were studied during the breeding season (September to February), whereas the remaining eight during the nonbreeding season (March to August). All animals were maintained in accordance with the guidance of the Japanese Association for Laboratory Animal Science, the Primate Society of Japan, and the provisions laid out in the Guide for Animal Experimentation at Shiga University of Medical Science and Guidelines for the Husbandry and Management of Laboratory Animals. All monkeys were housed individually, and maintained under a controlled photoperiod (lights on 0800–2000) and at an ambient temperature of 23 ± 1°C. Each monkey was fed 20 g/kg·day of commercial pellet monkey chow (CMK-1, Japan CLEA, Tokyo, Japan) supplemented with sweet potatoes and bananas everyday. Water was provided ad libitum.

RIA of hormones
To evaluate the breeding activity by hormonal assay, blood samples (1 ml) were collected from the cephalic vein. Immediately after collection, the samples were centrifuged, and the separated sera were stored at -30 C until assay. Serum concentration of testosterone and LH was measured using RIAs as previously described (20).

Tissue preparation
The monkeys were deeply anesthetized with an im injection of ketamine hydrochloride (25 mg/kg) and an intravenous injection of sodium pentobarbital (10 mg/kg). The respiration was maintained by intratracheal tubing with manual ventilation. Through median sternotomy, the ascending aorta was cannulated. After a flush with 0.01 M PBS (pH 7.4), the animals were perfused with 4% paraformaldehyde, 0.5% glutaraldehyde, and 0.2% picric acid in 0.1 M phosphate buffer (PB; pH 7.4). The perfusion pressure was adjusted to the expected normal mean arterial blood pressure as previously published (19, 21). After perfusion, the testes were removed. The central nervous, digestive, urinary, and skeletal systems were also used for other studies. The mean length of the major axis was measured for both testes and represented testicular size. The testes were cut into 5-mm blocks, and immersed for 48 h at 4 C in the same fixative minus glutaraldehyde. After fixation, the blocks were rinsed and preserved in 0.1 M PB containing 15% sucrose. Some of the sample blocks were re-fixed in Bouin’s solution overnight at room temperature (RT), and prepared for paraffin sections (5-µm thick). Other blocks were cut using a vibratome (60-µm thick) for electron microscopic observation.

PGP 9.5 immunohistochemistry
Paired 5-µm paraffin sections were prepared. One section was immunostained for PGP 9.5, and the other was stained with periodic acid-Schiff (PAS) with hematoxylin to determine the seminiferous epithelial stage. The seminiferous epithelium was staged according to Nagato (22). Endogenous peroxidase activity was blocked by immersing in 0.1% hydrogen peroxide and in 0.1% phenylhydrazine (15 min, RT). The sections were incubated with rabbit polyclonal antibody against human PGP 9.5 (diluted to 1:1000; Ultraclone, Cambridge, UK; 3 h at RT), biotinylated goat antirabbit IgG (1:1000; Vector Laboratories, Inc. Burlingame, CA; 1 h at RT), and avidine biotin-peroxidase (ABC; 1:1000; Vector; 1 hr at RT). The peroxidase activity was developed in a solution containing 0.01% 3,3'-diaminobenzidine tetrahydrochloride (DAB), 1% nickel ammonium sulfate (NAS), and 0.0003% hydrogen peroxide in a 50 mM Tris-HCl buffer, pH 7.6. The antibodies and the ABC were diluted with 0.1 M PBS, pH 7.4 containing 0.3% Triton X-100. The primary antibody solution contained 10% BSA.

Intensification of the chromogen development by NAS facilitates identification of PGP 9.5-immunoreactivity by producing dark violet color. In order to characterize the spermatogonia that were once stained for PGP 9.5, the intense chromogen color was reduced by periodic acid. An incubation in 0.5% periodic acid aqueous solution (for 5 min) deconjugates only NAS, and produces faint brown color for the PGP 9.5-positive cells. Nuclear staining with hematoxylin was then added to enable typing of the cells that were once identified as either PGP 9.5-positive or negative.

Vibratome sections were immunostained for PGP 9.5 in the same manner as paraffin sections above and prepared for EM. The immunostained sections were postfixed in 1% osmium tetroxide (1 h, 4 C), and dehydrated and flat-embedded in an epoxy resin. One-micrometer semithin sections were stained with methylene blue and studied using a Nomarsky’s interference microscopy. When antiserum to PGP 9.5 was omitted, or absorbed with human PGP 9.5 obtained from the antibody manufacturer, immunostaining was abolished.

Preembedding electron microscopic immunocytochemistry
Vibratome sections were incubated in anti-PGP 9.5 antibody (1:5000; 3 days, 4 C). Then, the sections were incubated in ultra-small gold conjugated goat antirabbit IgG (1:100; Aurion, Wageningen, The Netherlands) with 5% normal goat serum overnight at 4 C. After fixing with 2% glutaraldehyde (10 min, RT), silver intensification was performed using InteSE M (Amersham International, Buckinghamshire, UK). The immunostained sections were postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide for 90 min at 4 C (23, 24, 25). After flat-embedding in epoxy resin, ultrathin sections were cut, stained with uranyl acetate and lead citrate, and studied using an electron microscope (H-7100TE, Hitachi, Tokyo, Japan). When the primary antibody was preabsorbed, immunogold-silver staining was completely abolished.

PCNA immunohistochemistry
For assessment of cell proliferation, proliferating cell nuclear antigen (PCNA) immunohistochemistry was performed. Five-micrometer paraffin sections were incubated with mouse monoclonal antibody against recombinant PCNA (Clone PC 10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:100) with 10% BSA, followed by the incubation with horse antimouse IgG (1:1000) and ABC. The chromogen was developed in the same solution as described above. PCNA labeling cells in a unit area (1 mm2) were counted and compared between the breeding and non-breeding seasons.

Double immunohistochemistry
In order to evaluate the density of PGP 9.5-expressing cells/Sertoli cells, double immunohistochemistry for PGP 9.5 and vimentin was performed. The Sertoli cells have been shown to be selectively labeled with vimentin (26), which is concentrated around the nucleus of mature Sertoli cells (45). Paraffin sections were incubated with primary antibody solution containing antibody against PGP 9.5 (1:1000) and mouse antibody against porcine vimentin (1:100; DAKO, Glostrup, Denmark; 3 h, RT). After immunostaining for PGP 9.5, the sections were thoroughly washed and incubated with horse antimouse IgG (1:1000) followed by incubation in ABC. The second peroxidase activity was developed in solution containing 0.1% DAB and 0.001% hydrogen peroxide. Because the Sertoli cells are likely to alter the size of their cell soma during the testicular regression, we counted only the vimentin-positive Sertoli cells that contain the immunoreactivity-void nucelei. The present method of enumeration hereby attempted to exclude the possible bias due to the altered size of cell soma, although it does not exclude another theoretically possible bias due to an altered size of the nucleus of the Sertoli cell.

To investigate the relation between the expression of PGP 9.5 and cell proliferation, double immunostaining of PGP 9.5 and PCNA was performed. Sections were incubated in primary antibodies against PGP 9.5 and PCNA. After detecting PGP 9.5 by DAB and NAS (dark violet), the sections were thoroughly washed and incubated with a horse antimouse IgG 1:1000 followed by incubation in ABC. The second peroxidase activity was developed in solution containing 0.1% DAB and 0.001% hydrogen peroxide (brown). Thus, immunoreactivity for PGP 9.5 displays dark violet, whereas PCNA displays brown.

Calculation of PGP 9.5-expressing spermatogonia
Density of PGP 9.5-expressing cells were calculated using the double immunostained sections, and number of PGP 9.5-expressing cells/100 Sertoli cells represented the density. Density of PGP 9.5-expressing cell and PGP 9.5-positive cell ratios of three types of spermatogonia (i.e. Ad, Ap, B) at each seminiferous epithelial stage were calculated.

Statistics
The Mann-Whitney U test for unpaired observations was used and P values <0.01 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of PGP 9.5 in the primate spermatogonia
The previous light-microscopic characterization has shown that PGP 9.5 is expressed in the cytoplasm of 1) all of the type Ad spermatogonia; 2) a group of the type Ap spermatogonia; and 3) none of the type B spermatogonia (19). In order to further identify the cell types of the PGP 9.5-expressing spermatogonia, the expression of PGP 9.5 was investigated using both semithin and ultrathin sections. Ultrastructural classification of the monkey spermatogonia followed the previous report by Cavicchia and Dym (27). Briefly, the type Ad spermatogonia was characterized by 1) intensely stained nucleoplasma, 2) a narrow clear zone lining the inner aspect of the nuclear envelope, and 3) nucleolus attached to the nuclear envelope and surrounded by a narrow clear zone; the resting type Ap spermatogonia was characterized by 1) lightly stained nucleoplasma, 2) nucleoli removed from the nuclear periphery, and 3) loose and abundant nucleolonema; the proliferating type Ad spermatogonia was characterized by 1) increased size of nucleus and nucleolus, and 2) chromatin flakes along the nuclear envelope; and the type B spermatogonia was characterized by 1) discrete clumps of chromatin, and 2) limited contact with basal lamina.

The semithin sections revealed the intracellular localization of PGP 9.5. An intense PGP 9.5 immunoreactivity was localized in the cytoplasm of the type Ad spermatogonia, whereas a weak immunoreactivity was localized in the nucleus of the type B spermatogonia (Fig. 1AGo). These localizations of PGP 9.5 in type Ad and B cells were not altered for the breeding and nonbreeding seasons. As regards the type Ap spermatogonia, two patterns of the localization were noted for the breeding season; i.e. pattern 1 (cytoplasmic PGP 9.5-positive): the cytoplasm was selectively stained like that of the type Ad (arrowhead, Fig. 1AGo), and pattern 2 (cytoplasmic PGP 9.5-negative): both the cytoplasm and nucleus were weakly stained (arrow, Fig. 1AGo). In the nonbreeding season, almost all of the type Ap spermatogonia were cytoplasmic PGP 9.5-positive, and the cytoplasmic PGP 9.5-negative Ap were seldom noted (Fig. 1BGo). Identification of the cell type by the nuclear staining was carried out using the same sections after decoloration.



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  		Figure 1. Semithin sections of monkey seminiferous epithelium immunostained with PGP 9.5. In the breeding season (A), two distinct patterns are noted in the Ap spermatogonia; pattern 1: PGP 9.5 is strongly stained in the cytoplasm (arrowhead), or pattern 2: PGP 9.5 is faintly stained in both the cytoplasm and nucleus (arrow). In the non-breeding season (B), almost Ap cells are strongly stained by PGP 9.5 in the cytoplasm (arrowheads). Ad and B indicate type Ad spermatogonia and type B spermatogonia, respectively. Magnification, x550.

 
The ultrathin sections using immunogold silver staining further characterized the intracellular distribution of PGP 9.5. The PGP-immunoreactive (ir) silver grains were present in the cytoplasm of the type Ad spermatogonia (Fig. 2AGo). Type B spermatogonia localized the silver grains in the nucleus but barely in the cytoplasm (Fig. 2DGo). Regarding the type Ap spermatogonia, the resting form of type Ap cells (27) had the silver grains in the cytoplasm and the nucleus (Fig. 2BGo). The proliferating form of type Ap cells (27), in contrast, localized the silver grains in the nucleus (Fig. 2CGo). The cytoplasmic PGP 9.5-negative Ap spermatogonia on the light-microscopic level probably correspond to the proliferating Ap spermatogonia, whereas the cytoplasmic PGP 9.5-positive Ap cells correspond to the resting Ap cells (Fig. 2Go, B and C).



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  		Figure 2. Electron micrographs of monkey spermatogonia immunostained with PGP 9.5. A, In type Ad spermatogonia, PGP 9.5-ir silver grains are localized in the cytoplasm. B, The PGP 9.5 are localized preferentially in the cytoplasm of the resting form of type Ap, whereas PGP 9.5 are localized mainly in the nucleus of the proliferating form of Ap (C). D, In type B spermatogonia, PGP 9.5-ir silver grains are localized in the nucleus but barely in the cytoplasm. Magnification, x4,600.

 
General changes of the spermatogenesis in the nonbreeding season
The testicular size was decreased in the nonbreeding season as compared with that of the breeding season (mean ± SEM: 31.6 ± 2.1 mm vs. 47.2 ± 1.2 mm, P < 0.01). The seminiferous epithelium was composed of only 2–4 spermatogenic cell layers in the nonbreeding season, whereas 4–6 layers in the breeding season (Fig. 3Go, A and B).



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  		Figure 3. Seasonal changes of the seminiferous epithelium and proliferating germ cells. The epithelium of the nonbreeding season (A) is thinner than that of the breeding season (B). PCNA-labeling germ cells are decreased in the non-breeding season (D) as compared with those in the breeding season (C). Magnification, x100.

 
We used eight animals for breeding season, and eight animals for nonbreeding season. We examined approximately 1000 spermatogonia out of 50 tubules per animal of the breeding season (total 8321 spermatogonia of 426 tubules). For nonbreeding season, 350 spermatogonia out of 30 tubules were examined per animal (total 3454 spermatogonia out of 237 tubules).

Seasonal changes in serum levels of testosterone and LH
All of the measurement was performed at the same time. For LH measurements the intra- and inter-assay coefficients of variation were 3.8% and 2.9%, respectively, and lower limit of the sensitivity was 1 ng (5 ng/ml). For testosterone measurements the intraassay and interassay coefficients of variation were 4.8% and 7.3%, respectively, and lower limit of sensitivity was 5 pg (250–1000 pg/ml). In the nonbreeding season, serum level of testosterone significantly decreased to 0.7 ± 0.1 ng/ml from 8.3 ± 0.8 ng/ml in the breeding season (mean ± SEM, P < 0.01). Serum LH level also decreased in the nonbreeding season to 29.6 ± 1.0 ng/ml from 38.5 ± 1.3 ng/ml in the breeding season (mean ± SEM, P < 0.01).

Seasonal change in proliferation activity studied by PCNA expression
When studied by the PCNA immunohistochemistry and morphometry, cell proliferation activity was generally decreased in the nonbreeding season. An intense PCNA immunoreactivity was seen in the spermatogonia and the primary spermatocytes of the breeding season (Fig. 3CGo). The density of the PCNA-labeling germ cells was decreased to 461.6 ± 27.3/mm2 in the nonbreeding season as compared with 1487.0 ± 88.0/mm2 in the breeding season (mean ± SEM, P < 0.01, also compare Fig. 3Go, C and D).

Seasonal changes in the densities of PGP 9.5-ir spermatogonia
PGP 9.5-immunoreactivity was noted in the spermatogonia, and other constitutional cells in the seminiferous epithelium and the interstitial cells were not stained by PGP 9.5-immunohistochemistry. The Sertoli cells are specifically identified by vimentin-staining (26), and the double staining of vimentin and PGP 9.5 clearly indicated that the Sertoli cells were not stained with PGP 9.5 (Fig. 4CGo and 5DGo).



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  		Figure 4. Seasonal changes of PGP 9.5 immunohistochemistry. (A and B) PGP 9.5 single staining. PGP 9.5-positive spermatogonia are more abundant in the non-breeding season than those in the breeding season. Magnification, x130. C and D, Double immunostaining with PGP 9.5 and vimentin. PGP 9.5-positive spermatogonia (dark violet) are situated on the basement membrane of the seminiferous tubules. The Sertoli cells are clearly identified by vimentin immunostaining (brown). Magnification, x200.

 


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  		Figure 5. Double immunostaining with PCNA and PGP 9.5. Immunoreactivity for PGP 9.5 displays dark violet, whereas PCNA displays brown. Breeding (A) and nonbreeding seasons (B). The cytoplasmic PGP 9.5-expressing cells (arrowheads) do not overlap with the nuclear PCNA-expressing cells (arrows) for both the seasons. Magnification, x660.

 
The density of the cytoplasmic PGP 9.5-positive spermatogonia was represented as a density against the Sertoli cells, i.e. the number of cytoplasmic PGP 9.5-positive cells/100 Sertoli cells. The density of the cytoplasmic PGP 9.5-positive spermatogonia increased to 58.1 ± 0.8 in the nonbreeding season from 33.7 ± 0.8 in the breeding season (mean ± SEM, P < 0.01, also see Fig. 4Go).

PGP 9.5 and spermatogonial proliferation
Expression of PGP 9.5 was studied in association with the proliferation activity of the spermatogonia by the double immunostaining with PGP 9.5 and PCNA (Fig. 5Go). We evaluated 2620 spermatogonia in the breeding season and 1380 in the nonbreeding season and confirmed that the expression of cytoplasmic PGP 9.5 and nuclear PCNA seldom overlapped throughout the year; only 0.5% of the spermatogonia expressed the both antigens in the nonbreeding season and 0.6% in the breeding season. The proliferating cells decreased in the non-breeding season as shown by the PCNA labeling (Fig. 3Go), and the cytoplasmic PGP 9.5-positive cells increased.

We calculated percentages of the three spermatogonia, i.e. type Ad, type Ap, and type B spermatogonia for both breeding and nonbreeding seasons. In the nonbreeding season, there was an increase in the type Ad cells and a decrease in the type B cells. We then calculated the percentages of cytoplasmic PGP 9.5-positive Ap spermatogonia for both seasons (Fig. 6Go). Although no difference was noted in the percentage of the total Ap cells, the percentage of the cytoplasmic PGP 9.5-positive cells clearly increased in the non-breeding season (Fig. 6Go, asterisk).



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  		Figure 6. Proportional change of the cytoplasmic PGP 9.5-positive spermatogonia between the breeding and nonbreeding seasons. Despite no change in the percentages of total number of Ap spermatogonia, the percentage of cytoplasmic PGP 9.5-positive Ap prominently increases in the nonbreeding season (asterisk).

 
Epithelial stage-dependent expression of PGP 9.5 by the type Ap spermatogonia
The staging of the seminiferous epithelium was performed according to Nagato’s description (22). In the breeding season, the percentages of the cytoplasmic PGP 9.5-positive Ap spermatogonia were considerably low in the late epithelial stages (stages VI–VIII, Fig. 7AGo). The Ap spermatogonia of the stages VI to VIII are known to proliferate for self-renewal (8, 22). Because PGP 9.5 is hardly expressed in the cytoplasm by proliferating cells (i.e. PCNA-labeled cells), the low percentages of the cytoplasmic PGP 9.5 expression in the stages VI–VIII are probably due to an active self-renewal of the type Ap spermatogonia. There was no Ap cells in the stage IX and X of the breeding season (Fig. 7AGo). The Ap spermatogonia of the stage IX and X are known to differentiation into the type B spermatogonia (8, 22). Thus, the disappearance of the Ap cells is probably due to an active differentiation into the type B cells.



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  		Figure 7. Cytoplasmic PGP 9.5-positive rate in the type Ap spermatogonia during the epithelial cycle. In the breeding season (A) a stage-dependent change is noted, whereas over 88% of Ap spermatogonia are positive throughout the epithelial cycle in the nonbreeding season (B).

 
The most of type Ap spermatogonia were cytoplasmic PGP 9.5-positive in the all epithelial stages of the nonbreeding season, and numerous cytoplasmic PGP 9.5-positive Ap cells were noted even in the stages IX and X (Fig. 7BGo). These cytoplasmic PGP 9.5-positive Ap spermatogonia are not likely to proliferate or differentiate but remain to be the resting Ap cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using PGP 9.5 immunolabeling and morphometry, the present study demonstrates a drastic change of the cell kinetics during the seasonal suppression of the primate spermatogenesis. The seasonality in spermatogenetic activity has been shown to result from the hormonal changes of the hypothalamus-pituitary-gonadal system (13, 15, 28). The present study also confirms that serum levels of testosterone and LH significantly decrease in the nonbreeding season (i.e. 0.7 ± 0.1 ng/ml from 8.3 ± 0.8 ng/ml for testosterone, and 29.6 ± 1.0 ng/ml from 38.5 ± 1.3 ng/ml for LH). Although the decrease of the LH level in the nonbreeding season is rather small as compared with that of testosterone, there would perhaps be unidentified mechanisms to manifest enhanced responses in the biological activities of the hormones. For instance, the pituitary responsiveness to GnRH has been shown to be down-regulated in the nonbreeding season, and the LH level is correspondingly lowered (28). As a result of the complexed hormonal regulatory mechanisms, though not fully elucidated, there would finally be the drastic change of the spermatogenetic kinetics during the seasonal suppression of primate spermatogenesis (Fig. 8Go). The manifested changes of the spermatogenetic kinetics is likely to result from a change of Ap spermatogenesis (29, 30).



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  		Figure 8. A model of action of PGP 9.5 in regulation of spermatogonial proliferation. Ub, Ubiquitin; CDK, cyclin-kinase.

 
The most noteworthy aspect concerning the physiologic suppression of the spermatogenetic activity is its reversibility. GnRH antagonist-induced suppression of spermatogenesis has also been shown to be reversible, i.e. the spermatogenetic activity returns to the fully functional level after withdrawal of the GnRH antagonist (31). Taken together, the regulation of spermatogenesis at the level of type Ap may be a pivotal phenomenon that enables the complete reversibility of the spermatogenesis in primate. However, the cellular mechanism leading to the reversible suppression of type Ap proliferation has not been elucidated.

PGP 9.5 has been considered as a sensitive pan-neuronal marker of an unknown physiologic role (18, 32, 33). Our previous study demonstrated the presence of PGP 9.5 in the spermatogonia of Japanese monkey (19). In the present study, we demonstrate that the intracellular localization of PGP 9.5 in the spermatogonia is closely associated with proliferation activity. PCNA is a widely used marker for cell proliferation (34, 35, 36), and the expression of PCNA is drastically decreased in the nonbreeding season. Interestingly, the cytoplasmic localization of PGP 9.5 and nuclear expression of PCNA seldom overlapped, and the cytoplasmic localization of PGP 9.5 is predominantly seen in the type Ap cells of the nonbreeding season. The rat spermatogonia, which have been reported to proliferate unceasingly, do not possess PGP 9.5 (32, 37). These observations lead us to a hypothesis that the cytoplasmic localization of PGP 9.5 may have an important role in suppression of proliferation and may serve as a marker for the non-proliferating spermatogonia of primates.

PGP 9.5 has recently been identified as ubiquitin carboxyl-terminal hydrolase (38), the known function of which is to release ubiquitins from their ligand proteins (38, 39). The present study shows that PGP 9.5 is present in the nuclei of both the resting and proliferating cells (see Fig. 2Go). Inside the nucleus, the release of ubiquitin from the chromatin fibers has been shown to result in the chromatin condensation in vitro (40, 41), suggesting its role in the initial process of cell division (39, 40; also see the proliferating cells in Fig. 8Go). In the nucleus of the resting cell, however, it has not been elucidated whether PGP 9.5 still acts to promote chromatin condensation (i.e. for cell division), or whether an yet unknown mechanism exists to block the PGP 9.5’s action of releasing ubiquitin (also see the resting cells in Fig. 8Go).

The substrates for PGP 9.5 are also present in the cytoplasm. The PGP 9.5 molecule liberated into the cytoplasm has been recently shown to deconjugate ubiquitins from the cell cycle motor complex consisting of cyclin and cyclin-dependent kinase (CDK) (38, 42, 43; also see Fig. 8Go). The release of ubiquitin from the CDK inhibitor blocks the action of the cell cycle motor complex, thereby the cell cycle arrests (46; also see the resting cells in Fig. 8Go). The type Ad spermatogonia are considered to be quiescent reservoir cells, and it is also noteworthy that the Ad cells have PGP 9.5 richly in the cytoplasm. In the absence of PGP 9.5, in contrast, the cell cycle motor is released from the CDK inhibitor via a process called ubiquitin-dependent proteolysis (47). Thus, the cell proliferation is likely to be promoted when PGP 9.5 is not liberated into the cytoplasm (the proliferating cells in Fig. 8Go). The type B spermatogonia continuously proliferate and differentiate into spermatocytes, and it should be noted that the B cells do not have PGP 9.5 in the cytoplasm.

Type Ap spermatogonia, in turn, are divided into the cytoplasmic PGP 9.5-positive and negative cells (19). This observation may imply that type Ap spermatogonia consist of two distinct groups. One is resting, and the other is proliferating. The resting Ap spermatogonia may have liberated PGP 9.5 into the cytoplasm, thereby their cell cycle is to be suspended. This hypothesis is coherent with the observation that the cytoplasmic PGP 9.5-positive Ap (resting) spermatogonia predominate in the early seminiferous stages of the breeding season and in all stages of the nonbreeding season. The cytoplasmic PGP 9.5-negative Ap (proliferating) have not liberated PGP 9.5 into the cytoplasm, thus their DNA replication is to be activated. This speculation is also supported by the fact that the cytoplasmic PGP 9.5-negative Ap cells predominate in the late seminiferous stages of the breeding season. Ultrastructural assessment of the proliferative features of the type Ap spermatogonia also supports this hypothesis. In conclusion, the type Ap spermatogonia appear to liberate the PGP 9.5 according to the seasonal changes of the hypothalamus-pituitary-gonadal axis and perhaps of some other regulators, and may alter their proliferation activity.

Accumulating research data suggest that type Ap spermatogonia can give rise to type Ad spermatogonia when spermatogenesis is suppressed (44). The percentage of cytoplasmic PGP 9.5-positive Ap (resting) spermatogonia increases in the nonbreeding season, and the cell group that liberates the PGP 9.5 into the cytoplasm may become the source of future Ad spermatogonia in the nonbreeding season.

It is very tempting to speculate that the reversible quiescence of type Ap spermatogonia is mediated by PGP 9.5, which regulates cell cycle activities via deconjugating ubiquitins from the ligand proteins. Together with the fact that the unceasingly proliferating rat spermatogonia do not possess PGP 9.5 (32, 37), the present observation suggests that the regulation of Ap spermatogenesis by utilizing PGP 9.5 molecule is an evolved mechanism to protect the genetic integrity of the stem cells in primate.


   Acknowledgments
 
The authors thankfully acknowledge an excellent technical support provided by Noriko Kirihata and the staff of the Central Research Laboratory, Shiga University of Medical Science.


   Footnotes
 
1 This work was supported by a Grant-in-Aid for Encouragement of Young Scientists from the Minister of Education, Science, Sports and Culture of Japan (09770008). Back

Received May 15, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Weinbauer GF, Nieschlag E 1997 Endocrine control of germ cell proliferation in the primate testis. Adv Exp Med Biol 424:51–58[Medline]
  2. Zirkin BR 1993 Regulation of spermatogenesis in the adult mammal: gonadotropins and androgens. In: Desjardins C, Ewing LL (eds) Cell and Molecular Biology of the Testis, Oxford University Press, Oxford, pp 166–188
  3. Sharpe RM 1994 Regulation of spermatogenesis. In: Knobil E, Neill JD (eds) The Physiology of Reproduction, Raven Press, New York, vol. 1, pp 1363–1434
  4. Aravindan GR, Gopalakrishnan K, Ravindranath N, Moudgal NR 1993 Effect of altering endogenous gonadotrophin concentrations on the kinetics of testicular germ cell turnover in the bonnet monkey (Macaca radiata). J Endocrinol 137:485–495[Abstract/Free Full Text]
  5. Moudgal NR, Ravindranath N, Murthy GS, Dighe RR, Aravindan GR, Martin F 1992 Long-term contraceptive efficacy of vaccine of ovine follicle-stimulating hormone in male bonnet monkeys (Macaca radiata). J Reprod Fertil 96:91–102[Abstract/Free Full Text]
  6. Weinbauer GF, Behre HM, Fingscheidt U, Nieschlag E 1991 Human follicle-stimulating hormone exerts a stimulatory effect on spermatogenesis, testicular size, and serum inhibin levels in the gonadotropin-releasing hormone antagonist-treated nonhuman primate (Macaca fascicularis). Endocrinology 129:1831–1839[Abstract/Free Full Text]
  7. de Rooij DG, van Dissel-Emiliani FMF, van Pelt AMM 1989 Regulation of spermatogonial proliferation. Ann NY Acad Sci 564:140–153[Medline]
  8. Clermont Y, Antar M 1973 Duration of the cycle of the seminiferous epithelium and the spermatogonial renewal in the monkey Macaca arctoides. Am J Anat 136:153–166[CrossRef][Medline]
  9. Meistrich ML, van Beek MEAB 1993 Spermatogonial stem cells. In: Desjardins C, Ewing LL (eds) Cell and Molecular Biology of the Testis, Oxford University Press, Oxford, pp 266–295
  10. van Alphen MMA, van de Kant HJG, de Rooij DG 1988 Follicle-stimulating hormone stimulates spermatogenesis in the adult monkey. Endocrinology 123:1449–1455[Abstract/Free Full Text]
  11. Marshall GR, Zorub DS, Plant TM 1995 Follicle-stimulating hormone amplifies the population of differentiated spermatogonia in the hypophysectomized testosterone-replaced adult rhesus monkey (Macaca mulatta). Endocrinology 136:3504–3511[Abstract]
  12. Russell LD, Peterson RN 1984 Determination of the elongate spermatid-Sertoli cell ratio in various mammals. J Reprod Fertil 70:635–641[Abstract/Free Full Text]
  13. Wickings EJ, Nieschlag E 1980 Seasonality in endocrine and exocrine testicular function of the adult rhesus monkey (Macaca mulatta) maintained in a controlled laboratory environment. Int J Androl 3:87–104[Medline]
  14. Aso T, Tominaga T, Oshima K, Matsubayashi K 1977 Seasonal changes of plasma estradiol and progesterone in the Japanese monkey (Macaca fuscata fuscata). Endocrinology 100:745–750[Abstract/Free Full Text]
  15. Matsubayashi K, Watanabe G, Taya K, Katakai Y, Sasamoto S, Suzuki J, Nozaki M 1991 Seasonal changes in plasma concentrations of immunoreactive inhibin and testicular activity in male Japanese monkeys. Biol Reprod 44:822–826[Abstract]
  16. Enomoto T, Matsubayashi K, Nagato Y, Nakano M 1994 Seasonal changes in the spermatogenic epithelium of adult Japanese macaques (Macaca fuscata fuscata). Primates 35:465–472[CrossRef]
  17. Matsubayashi K, Enomoto T 1983 Longitudinal studies on annual changes in plasma testosterone, body weight and spermatogenesis in adult Japanese monkeys (Macaca fuscata fuscata) under laboratory conditions. Primates 24:521–529 (Abstract 284)[CrossRef]
  18. Thompson RJ, Doran JF, Jackson P, Dhillon AP, Rode J 1983 PGP 9.5—a new marker for vertebrate neurons and neuroendocrine cells. Brain Res 278:224–228[CrossRef][Medline]
  19. Tokunaga Y, Fujimiya M, Maeda T 1997 The presence of PGP 9.5 in the spermatogonia is associated with the spermatogenesis of the Japanese macaque (Macaca fuscata). In: Motta PM (ed) Recent Advances in Microscopy of Cells, Tissues and Organs. Kluwer Academic Publishers, Boston, pp 585–590
  20. Torii R, Kitagawa N, Nigi H, Ohsawa N 1993 Effect of repeated restraint stress at 30-minute intervals during 24-hour on serum testosterone, LH and glucocorticoids levels in male Japanese monkeys. Exp Anim 42:67–73
  21. Yamashita T, Maeda T, Tokunaga Y, Mano T 1997 Fine structure of crest synapses in the locus coeruleus of the Japanese macaque (Macaca fuscata), with special reference of noradrenergic neurons. Acta Anat Nippon 72:199–208
  22. Nagato Y, Enomoto T, Matsubayashi K 1994 Observation of the cycle of the seminiferous epithelium in the Japanese macaque (Macaca fuscata) using semithin sections. Primates 35:455–464[CrossRef]
  23. Karnovsky MJ Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. Proceeding of the 11th Meeting of American Society of Cell Biology, New Orleans, 1971, p 146
  24. White DL, Mazurkiewicz JE, Barrnett RJ 1979 A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures. J Histochem Cytochem 27:1084–1091[Abstract]
  25. Tokunaga Y, Maeda T 1996 An improved method for pre-embedding immunoelectron microscopy using Triton X-100 combined with reduced osmium and platinous diaminobenzidine. Acta Histochem Cytochem [Suppl] 29:579–580
  26. Aumuler G, Steinbruck M, Krause W, Wagner HJ 1988 Distribution of vimentin-type intermediate filaments in Sertoli cells of human testis, normal and pathologic. Anat Embryol 178:129–136[CrossRef][Medline]
  27. Cavicchia JC, Dym M 1978 Ultrastructural characteristics of monkey spermatogonia and preleptotene spermatocytes. Biol Reprod 18:219–228[Abstract]
  28. Torii R, Nigi H 1994 Hypothalamo-pituitary-testicular function in male Japanese monkeys (Macaca fuscata) in non-mating season. Exp Anim 43:381–387
  29. Weinbauer GF, Schubert J, Yeung CH, Rosiepen G, Nieschlag E 1998 Gonadotrophin-releasing hormone antagonist arrests premeiotic germ cell proliferation but dose not inhibit meiosis in the male monkey: a quantitative analysis using 5-bromodeoxyuridine and dual parameter flow cytometry. J Endocrinol 156:23–34[Abstract]
  30. Zhengwei Y, Wreford NG, Schlatt S, Weinbauer GF, Nieschlag E, Mc Lachlan RI 1998 Acute and specific impairment of spermatogonial development by GnRH antagonist-induced gonadotrophin withdrawal in the adult macaque (Macaca fascicularis). J Reprod Fertil 112:139–147[Abstract/Free Full Text]
  31. Weinbauer GF, Limberger A, Behre HM, Nieschlag E 1994 Can testosterone alone maintain the gonadotrophin-releasing hormone antagonist-induced suppression of spermatogenesis in the non-human primate? J Endocrinol 142:485–495[Abstract/Free Full Text]
  32. Wilson POG, Barber PC, Hamid QA, Power BF, Dhillon AP, Rode J, Day INM, Thompson RJ, Polak JM 1988 The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies. Br J Exp Path 69:91–104[Medline]
  33. Bradbury JM, Thompson RJ 1985 Immunoassay of the neuronal and neuroendocrine marker PGP 9.5 in human tissue. J Neurochem 44:651–653[CrossRef][Medline]
  34. Schlatt S, Weinbauer GF 1994 Immunohistochemical localization of proliferating cell nuclear antigen as a tool to study cell proliferation in rodent and primate testes. Int J Androl 17:214–222[Medline]
  35. Connolly KM, Bogdanffy MS 1993 Evaluation of proliferating cell nuclear antigen (PCNA) as an endogenous marker of cell proliferation in rat liver: a dual-stain comparison with 5-bromo-2'-deoxyuridine. J Histochem Cytochem 41:1–6[Abstract]
  36. Wrobel KH, Bickel D, Kujat R 1996 Immunohistochemical study of seminiferous epithelium in adult bovine testis using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen (PCNA). Cell Tissue Res 283:191–201[CrossRef][Medline]
  37. Properzi G, Cordeschi G, Francavilla S 1992 Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat. Histochemistry 97:61–68[CrossRef][Medline]
  38. Wilkinson KD, Lee K, Deshpande S, Duerksen-Hughes P, Boss JM, Pohl J 1989 The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science 246:670–673[Abstract/Free Full Text]
  39. Wilkinson KD 1997 Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J 11:1245–1256[Abstract]
  40. Matsui S, Seon BK, Sandberg AA 1979 Disappearance of a structural chromatin protein A24 in mitosis: implications for molecular basis of chromatin condensation. Proc Natl Acad Sci USA 76:6386–6390[Abstract/Free Full Text]
  41. Bradbury EM 1992 Reversible histone modifications and the chromosome cell cycle. Bioessays 14:9–16[CrossRef][Medline]
  42. Lam YA, Xu W, DeMartino GN, Cohen RE 1997 Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature 385:737–740[CrossRef][Medline]
  43. Zhu Y, Carroll M, Papa FR, Hochstrasser M, D’Andrea AD 1996 DUB-1, a deubiquitinating enzyme with growth-suppressing activity. Proc Natl Acad Sci USA 93:3275–3279[Abstract/Free Full Text]
  44. Fouquet JP, Dadoune JP 1986 Renewal of spermatogonia in the monkey (Macaca fascicularis). Biol Reprod 35:199–207[Abstract]
  45. Vogl AW, Pfeiffer DC, Redenbach DM, Grove BD 1993 Sertoli cell cytoskeleton. In: Russel LD, Griswold MD (eds) The Sertoli cell. Cache River Clearwater, pp 39–86
  46. Sherr CJ, Roberts J 1995 Inhibitors of mammalian G1 cyclin-dependent kinases. Gene Dev 9:1149–1163[Free Full Text]
  47. Pagana M, Tam SM, Theodoras AM, Beer-Romero P, Del Sal G, Chau V, Yew PR, Draetta GF, Rolf M 1995 Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science 269:682–685[Abstract/Free Full Text]



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