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Original Article |
Research Laboratory, Garrahan Pediatric Hospital, Buenos Aires 1245, Argentina
Address all correspondence and requests for reprints to: Alicia Belgorosky, Hospital de Pediatria Garrahan, C. de los Pozos 1881, Buens Aires 1245, Argentina. E-mail: abelgo{at}elsitio.net.
Abstract
Programmed cell death and proliferation are evolutionary conserved processes that play a major role during normal development and homeostasis. In the testis, during the fetal and newborn periods, they might determine final adult size and fertility potential. In the present study, we have measured the relative number of testicular cells in apoptosis and in active proliferation in the seminiferous cords and in the interstitium, at different age periods of prepubertal testicular development in humans. Testes from 44 prepubertal subjects without endocrine and metabolic abnormalities were collected at necropsy. They were divided in three age groups (Gr): Gr 1, newborn (1- to 21-d-old neonates), n = 18, mean (±SD) age 0.3 ± 0.23 months; Gr 2, post natal activation (1- to 6-month-old infants), n = 13, mean age 3.93 ± 1.90 months; and Gr 3, early childhood period (1- to <6-yr-old boys), n = 13, mean age 31.5 ± 18.9 months. Apoptosis was detected in 5-µm tissue sections using a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and cell proliferation was assessed by Ki-67 immunohistochemistry. Evaluation of apoptosis was confirmed by estimation of active caspase-3. Mean (±SD) testicular weight was 0.38 ± 0.20, 0.54 ± 0.35, and 0.51 ± 0.11 g in Gr 1, Gr 2, and Gr 3, respectively. In Gr 1, there was a significant positive correlation between age and testis weight (P = 0.02).
Mean (±SD) germ cell apoptotic index, AI, (% of apoptotic cells out of total cell number) was 15.0 ± 6.60, 27.0 ± 8.80 and 33.4 ± 11.4 in Gr 1, Gr 2, and Gr 3, respectively. In Sertoli cells, it was 6.60 ± 4.07, 22.0 ± 14.0 and 27.5 ± 19.8, respectively. In interstitial cells, it was 10.2 ± 6.38, 18.0 ± 6.70 and 25.7 ± 15.5, respectively. In the three types of cells, AI in Gr 1 was significantly lower than in Gr 2 or Gr 3 (P < 0.05). Mean (±SD) germ cell proliferation index, PI, was 18.6 ± 13.0, 10.0 ± 6.50 and 10.9 ± 6.24% in Gr 1, Gr 2, and Gr 3, respectively. In Sertoli cells and in interstitial cells PI was similar in the three age groups. The PI/AI ratio was used to compare relative differences among age groups. The PI/AI ratio of germ cells, Sertoli cells and interstitial cells in Gr 1 was significantly higher than in Gr 2 or Gr 3 (P < 0.05). It is concluded that, in normal subjects, there is a vigorous growth of the testis during the newborn period with subsequent stabilization during the first years of prepuberty. This cell growth seems to be mainly mediated by decreased apoptosis. The factors that modulate apoptosis of testicular cells are not known, but it is remarkable that this change takes place before the testosterone peak of the post natal gonadal activation of the first trimester of life. These changes taking place during the newborn period might be important to define testicular function in adults.
THE DEVELOPMENT OF the human testis goes through periods of profound morphological (1) and functional changes (2, 3) from birth to maturity. To study the prepubertal testicular period of development, in previous reports (4, 5) we have divided our study subjects in age groups of different functional activity. This is in line with several reports indicating that childhood, and particularly infancy, are not quiescent periods of testicular development, both in the interstitial (6) and in the seminiferous cord compartments (7, 8). The cellular and functional activity of the prepubertal testis, however, differs markedly from that of the sexually mature testis in the two compartments, particularly in the seminiferous epithelium, which does not show development of spermatogenesis before adolescence (1).
The seminiferous epithelium of the newborn human testis is composed of solid cords about 50 µm in diameter where two main types of cells are differentiable. The most numerous are the precursors of Sertoli cells that form a multilayer epithelium. The second type of cells belong to the germinal line: gonocytes in newborns and primitive A spermatogonia after the second month of life (1). In the interstitium, fetal type Leydig cells are seen in the newborn testis, and an increase in the number of Leydig cells, postnatal Leydig cell population, have been described during the postnatal serum testosterone peak (9). Leydig cells regress in early infancy (10). Juvenile fibroblasts are then the predominant type of cell in the intertubular spaces (1).
Death along with growth and differentiation is a critical part of the life cycle of the cell. Homeostatic control of cell number is the result of the dynamic balance between cell proliferation and cell death. Cells in apoptosis present a characteristic internucleosomal cleavage, and this DNA fragmentation is an important part of the programmed cell death mechanism. In situ end-labeling of human testicular tissue has been used in several studies to estimate apoptosis (11).
Erkkila et al. (12) demonstrate that apoptosis is a normal, hormonally controlled phenomenon in the adult human testis. In seminiferous tubules, it can be induced in vitro, under serum-free conditions, but it is suppressed by testosterone, suggesting that local testosterone is a critical germ cell survival factor. Studies in patients with cryptorchidism, also suggest that apoptosis is a hormonally controlled, normal phenomenon of the human prepubertal testis, both in the seminiferous cords and interstitial areas (13).
Both gonadotropins and androgens are important for optimal cell growth and differentiation during development. Tapanainen et al. (14) demonstrate that hypophysectomy-induced seminiferous tubules and interstitial cells degeneration is mediated by apoptosis and that pituitary gonadotropins and androgens play an essential role in preventing apoptotic cell death in the immature rat testis. In nonhuman primates, proliferation of Sertoli cells takes place during the neonatal-juvenile transition (15, 16). However, the mechanism by which the testicular cell mass is established soon after birth has not been addressed in humans.
In the present study, we measured the relative number of testicular cells in apoptosis and in active proliferation in the seminiferous cords and in the interstitium, at different age periods of prepubertal testicular development. We compared these findings with changes in testicular weight. We found that the first month of life (newborn period), i.e. before the period of post natal Leydig cell activation, is characterized by a higher rate of testis growth than later in prepuberty. This growth was mainly secondary to decreased apoptosis in several types of testicular cells.
Materials and Methods
Study subjects
Similar to that reported previously (5), testes were divided into age groups on the basis of testicular histology: Group (Gr) 1, newborn period (1- to 21-d-old neonates), n = 18 mean (±SD) age 0.3 ± 0.23 months; Gr 2, postnatal activation period (1- to 6-month-old infants), n = 13 mean age 3.93 ± 1.90 months; and Gr 3, early childhood period (1- to <6-yr-old children), n = 13 mean age 31.5 ± 18.9 months. A total of 44 testes from prepubertal patients without endocrine or metabolic diseases were studied. Necropsy was carried out after written consent from the closest relatives had been obtained. The study was approved by the Institutional Review Board of the Garrahan Pediatric Hospital. Death was secondary to multiple diseases in every group of subjects, but congenital cardiac malformation was responsible for death in approximately 60% of cases in Gr 1 and 30% of cases in Gr 2 and Gr 3. Cells collected from most of these testes had been used for cell culture studies (4, 5). They were able to secrete testosterone and inhibin B during culture and to respond to gonadotropin stimulation.
All patients had died in our hospital. Cadavers were placed in a refrigerator within 1 h after death. Testes collected at necropsy were fixed in 4% formalin in PBS, and embedded in paraffin. Five-micrometer tissue sections, placed on silanized microscope slides, were used for detection of apoptotic and of proliferating cells. In each group of subjects, Sertoli, germ, and interstitial cell apoptosis and proliferation was studied.
Detection of apoptosis
Apoptosis was detected using the DeadEnd Colorimetric Apoptosis Detection System (Promega Corp., Madison, WI). This system end-labels the fragmented DNA of apoptotic cells using a modified terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. It consists in the incorporation of biotinylated nucleotides at the 3'-OH DNA ends, using the enzyme terminal deoxynucleotidyl transferase (TdT). Horseradish-peroxidase-labeled streptavidin is then bound to these biotinylated nucleotides. Peroxidase is detected using the peroxidase substrate, hydrogen peroxide and the stable chromogen diaminobenzidine (DAB). With this procedure, apoptotic nuclei are stained dark brown. Briefly, the tissue sections were deparaffinized, rehydrated, and fixed in 4% paraformaldehyde solution in PBS. Proteinase K (20 µg/ml) treatment for 20 min was followed by a second fixation in 4% paraformaldehyde solution in PBS. Optimal time and concentration of proteinase K was studied, and defined experimentally. One hundred microliters of TdT reaction mixture, consisting in equilibration buffer, biotinylated nucleotide mix and TdT enzyme, were used to cover each tissue section. The reaction was left for 1 h, at 37 C, in a humidified chamber. Then, the slides were immersed in 0.3% hydrogen peroxide to block endogenous peroxidases, and incubated 30 min in the streptavidin-HRP solution. Finally, 100 µl DAB were added to each slide and incubated for 10 min to develop the color reaction. The slides were counterstained with hematoxylin, dehydrated, and mounted. Positive nuclei stain brown, whereas nonapoptotic nuclei stain blue.
A positive control for detection of DNA fragmentation was included in each experiment, by adding deoxyribonuclease I solution (1 µg/ml) and incubating for 15 min at 37 C, previously to the TdT reaction. A negative control was also performed in which no TdT enzyme was present in the reaction mixture. Results were evaluated microscopically. At least 100 nuclei of interstitial, Sertoli and germ cells (total 300 nuclei) were counted for each subject. The apoptotic index (AI) was defined as the percentage of positive brown nuclei out of total cell number, for each cell type.
Postmortem time was defined as the time between death and removal of testes. It ranged from 6–50 h, but in 40 of 47 cases it was 24 h or less. Mean ± SD postmortem time was 20 ± 11.4 h. The possibility of an artificial elevation of AI as a result of DNA degradation during this period was evaluated. First, no correlation was found between postmortem time and AI in Sertoli, germ, or interstitial cells (r = 0.04, 0.11, and 0.11, respectively, n = 44, p = NS) implicating that no increment in AI took place during postmortem time. This lack of significant correlation was also observed when the same analysis was carried out for the three age groups separately. Furthermore, it was found that mean (±SD) postmortem time was similar in the three age groups studied: Gr 1, 21.1 ± 9.63, Gr 2, 20.1 ± 12.6 and Gr 3, 18.1 ± 12.9 h. Second, no focal areas of necrosis, characterized by sections of intra cord or intracellular cell death were observed using hematoxylin and eosin cell staining. Third, testicular cell apoptosis was studied after orchidectomy in three patients not included in the study (patient age was within that of Gr 1 in one and of Gr 3 in two other patients). In these cases, fixation was carried out immediately after removal of testes. AI was determined in the 3 cell types of the 3 testes. In 8 of the 9 comparisons, AI was within the 95% confidence interval of AI for the corresponding cell type and age group. It was concluded that no degradation of DNA took place during postmortem time.
Immunohistochemical analysis of expression of caspase-3. In most samples of Gr 1 (n = 10), Gr 2 (n = 9), and Gr 3 (n = 13), active caspase-3 was analyzed to verify the TUNEL method with another method for the estimation of apoptosis. An affinity-purified goat polyclonal antibody (caspase-3 p20) raised against a peptide mapping at the amino terminus of caspase-3 subunit (SC-1226, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used. Formalin-fixed, paraffin-embedded testicular sections were deparaffinized and rehydrated. Tissue sections were then immersed into a target retrieval solution (DAKO Corp., Carpinteria, CA; code no. S1700) and heated at 95–99 C for 20 min. Endogenous biotin activity was blocked with Biotin Blocking System (DAKO Corp. X0590). The staining protocol (DAKO Corp., Catalyzed Signal Amplification System K 1500) was preceded by the following steps: treatment with hydrogen peroxide, incubation with a blocking protein and incubation with the primary antibody (20 ng/ml). A rabbit antigoat antibody was added, followed by the streptavidin-biotin complex, the amplification reagent and the streptavidin-peroxidase conjugate. After addition of DAB, specimens were dehydrated and mounted. Nuclei were counterstained with hematoxylin. A normal goat serum (DAKO Corp. code no. 0510) was used as a negative control. Positive immunostaining was estimated in the cytoplasm. Results were evaluated by counting the number of positive cells out of 100 cells, for each cell type.
Detection of proliferation
To detect proliferating cells, a monoclonal mouse antibody anti-Ki-67 (DAKO Corp., Clone Ki-S5, code no. M 7187) was used. The Ki-67 antigen is expressed during G1, S, G2, and M phase of the cell cycle, while resting, noncycling cells (G0 phase), lack the Ki-67 antigen.
Formalin-fixed, paraffin-embedded testicular sections were used. Tissue slides were deparaffinized and rehydrated. Formaldehyde is known to induce conformational changes in the antigen molecules by forming intermolecular cross-linkages. Excessive formalin fixation can mask antigenic sites and diminish specific staining. Then target retrieval procedure is necessary. For this, tissue sections were immersed into a Target Retrieval Solution (DAKO Corp. code no. S1700) and heated to 95–99 C for 20 min. To permeabilize tissues, the same procedure with Proteinase K, as described above, was applied. The staining protocol consisted in a first step in hydrogen peroxide to block endogenous peroxidases, incubation with the primary antibody, followed by the biotinylated linked antibody (containing antimouse immunoglobulins), addition of streptavidin conjugated with horseradish peroxidase, and addition of DAB. After hematoxylin counterstaining, the specimens were mounted. Positive nuclei from cycling cells were stained brown, whereas nuclei form noncycling cells were stained blue (hematoxylin staining). Tissue from a lymphatic ganglion was used as a positive control. As negative control, tissues fixed, processed and embedded in an identical manner to study samples, but with no primary antibody were used.
Proliferation index (PI) was calculated as the percentage of positive cells out of total cell number, for each cell type (interstitial, Sertoli, or germ cells).
PI/AI ratio in the three age groups
The rate of proliferation tends to increase cell number, whereas the rate of programmed cell death tends to decrease it, the two modulating the cell number pool. In the different age groups, we have calculated the PI/AI ratio as a relative parameter of the effect of these opposite processes on testicular growth, assuming no significant age changes in the time course of proliferation or apoptosis.
Statistical analyses
All means and SD were calculated from individual values in each group. ANOVA, followed by Student-Newmann-Keuls test, and linear regression analysis was used for data analysis.
Results
Testicular weight in the three age groups
Mean (±SD) testis weight in Gr 1, 2, and 3 was 0.38 (±0.20), 0.54 (±0.35) and 0.51 (±0.11) g, respectively. Weight was significantly lower in Gr 1 (P < 0.05) than in Gr 2 or 3. Means in Gr 2 and Gr 3 were equal. The oldest subject in Gr 1 was 0.7-month-old. Figure 1
shows that there was a significant positive correlation between age and testis weight in Gr 1 (weight in g = 0.24 + 0.45 x age in months, r = 0.54, P = 0.02). No correlation was found in Gr 2 or Gr 3 (inset).
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Figure 2 shows microphotographs of representative testis sections of Gr 1 and Gr 3 after in situ labeling to detect apoptotic cells. A lower incidence of apoptosis in Sertoli, germ and interstitial cells was apparent, at visual examination, in Gr 1. To corroborate this impression, apoptotic cell counting was carried out.
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). Similarly, mean germ cell AI in Gr 1 (15.0 ± 6.60) was significantly lower than in Gr 2 (27.0 ± 8.80) and in Gr 3 (33.4 ± 11.4), P < 0.05. In interstitial cells, mean AI index was also significantly lower in Gr 1 (10.2 ± 6.38) than in Gr 2 (18.0 ± 6.70), P < 0.05, and than in Gr 3 (25.7 ± 15.5), P < 0.05. Theses results show that apoptosis of the three cell types increases after the newborn period.
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shows microphotographs of representative testis sections of Gr 1 and Gr 2 after in situ labeling to detect proliferating cells. Positive brown staining was observed in the three cell types of the two age groups.
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).
PI/AI ratio of Sertoli cells in Gr 1 (2.24 ± 1.80) was significantly higher than in Gr 2 (0.78 ± 0.45), P < 0.05, and significantly higher than in Gr 3 (0.61 ± 0.57), P < 0.05. Similarly, PI/AI ratio of germ cells in Gr 1 (1.42 ± 1.33) was significantly higher than in Gr 2 (0.40 ± 0.26), P < 0.05, and than in Gr 3 (0.45 ± 0.45), P < 0.05. Finally, PI/AI ratio of interstitial cells in Gr 1 (2.10 ± 2.02) was also significantly higher than in Gr 2 (0.65 ± 0.33), and significantly higher than in Gr 3 (0.59 ± 0.41), P < 0.05 (Fig. 4).
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Immunohistochemical analysis of active caspase-3 in Sertoli, germ, and interstitial cells of Gr 1, Gr 2, and Gr 3
Table 2 shows percentage positive staining for active caspase-3 in the three cell types of testes of Gr 1, Gr 2, and Gr 3. Caspase-3 positive staining in Gr 1 was significantly lower than in Gr 2 or Gr 3 in the three cell types (P < 0.05), indicating that differences in the rate of apoptosis among groups was confirmed by differences in active caspase-3.
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Müller and Skakkebaek (17) studied human testis growth during the first 10 yr of life. They found that median weight of testes, in 53 boys who suffered from sudden death, increased from 0.57 during the first year of life to 1.5 g during the 5- to 9.9-yr period. In the present study, our subject material allowed for an analysis during the first year of life separating newborns (Gr 1) from the rest of infancy (Gr 2). We found a vigorous testicular growth during the first 3 wk of life that waned during the rest the first year. In fact, we could not detect further growth during early childhood (Gr 3). Therefore, we conclude that testis size doubles during the first month of life and then remains practicably stable for at least 5 yr.
It has been previously reported that apoptosis occurs normally in the cryptorchid human testis prepubertally, both in germ cells and somatic cells (13). In that study, apoptotic cells in the seminiferous cords were identified as being mostly spermatogonia, even though Sertoli cells were also detected. We are not aware of any study of cell proliferation in the human testis. However, we believed that the combined detection of apoptosis and cell proliferation improves the quality of information to estimate increasing or decreasing cell populations in tissues. The extent of testicular cell proliferation during fetal and neonatal development determines the final adult testis size and potential for sperm output in the rat (18), and possibly in the human.
Our results showed that the newborn period is characterized by a lower rate of apoptosis in germ cells, Sertoli cells, and interstitial cells compared with that of the rest of early prepuberty. On the other hand, the rate of proliferation of germ cells was higher in newborns than in later periods. We have used the ratio PI/AI to estimate the combined effect of proliferation and apoptosis in a particular age period. In newborns, this ratio was significantly higher than in the rest of prepuberty, indicating that this is a period of central importance to increase cell mass in the two compartments of the testis. The higher ratio found in neonates is in line with changes in weight that we detected in our testis material during these periods. Our studies also suggest that the main mechanism for modulation of cell number in the prepubertal testis is the regulation of apoptotic cell death. It has been published that apoptosis has a crucial role in human adult germ cell development (19) and in human fetal testicular cell ontogeny (20).
Even though gonadotropins start to increase during the first month of life, it is remarkable that the peak of the activation of the gonadotropin testicular axis that takes place during the second and third month of life (21) was not associated with a lower rate of apoptosis, or with increases in testis weight. What are the factors that control prepubertal testicular changes in apoptosis and cell proliferation is not clear at this point. One possibility is that hormonal or growth factors present in the feto-placental unit might influence testicular cell growth for a few weeks after birth. Most studies on the hormonal control of apoptosis in adult rat (14) and adult human testes (12) have been carried out in maturing germ cells (such as spermatocytes and spermatids) that are not present during prepuberty. In the study of Heiskanen et al. (13) in the prepubertal cryptorchid testis, apoptosis was actually increased by human chorionic gonadotropin treatment during the first month but returned to the initial level later on. These authors put forward the controversial interpretation that it was human chorionic gonadotropin and/or testosterone withdrawal after treatment that increased apoptosis. Our data suggest that gonadotropins might not be the only modulators of cell growth during the first months of life. A role for activin A, follistatin, and FSH in Sertoli cell differentiation has been also proposed in the rat (22). Plant and Marshall (23) proposed two temporal patterns of Sertoli cell proliferation before puberty in higher primates: an insidious proliferation throughout the entire prepubertal period or one that is restricted to infancy. Our data are in favor of the second hypothesis, but cell proliferation would take place in the newborn period.
Other candidate for modulating testicular growth during the newborn period is GH or prolactin, acting through the GH receptor or through its own receptor. Indeed, a surge in GH and prolactin secretions has been reported to occur soon after birth (24, 25, 26). This hypersomatotropism, however, is associated with low serum IGF-I levels. Patient with isolated GH deficiency or with GH insensitivity have small testis and delayed puberty (27, 28), although they might be fertile. On the other hand, we have reported that recombinant human GH stimulates testosterone secretion in cultures of cells isolated from testes of infants and children, stressing the capacity of testicular cells to respond to GH (4).
In conclusion, we have found that during the newborn period there is a vigorous increment in testicular cell populations. This increase is secondary to decreased apoptosis relative to cell proliferation. Factors that might modulate this growth are not known, but this phenomenon is previous to the post natal peak in the secretion of gonadotropins and testosterone.
Acknowledgments
Footnotes
This work was supported by The National Council of Scientific and Technical Investigation (CONICET), Secretary of Science and Technology (FONCYT), and Ministry of Health (Beca Carrillo-Oñativia) of Argentina.
Abbreviations: AI, Apoptotic index; DAB, diaminobenzidine; Gr, group; PI, proliferation index; TdT, terminal deoxynucleotidyl transferase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.
Received January 14, 2002.
Accepted August 7, 2002.
References
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