BIOLOGY OF REPRODUCTION 66, 877–885 (2002)
Dynamics of Testicular Germ Cell Proliferation in Normal Mice and Transgenic Mice Overexpressing Rat Androgen-Binding Protein: A Flow Cytometric Evaluation1 D.A. Jeyaraj, G. Grossman, C. Weaver, and P. Petrusz2 Department of Cell and Developmental Biology and The Laboratories for Reproductive Biology, University of North Carolina, Chapel Hill, North Carolina 27599 ABSTRACT
ABP’s established role as a binding/carrier protein for sex steroids [1], it has also been suggested that ABP may regulate fertility and spermatogenesis in a variety of experimental models [5, 6, 8–11]. The mechanism of these effects is not clear. Germ cells are capable of internalizing ABP and its homologous protein, sex hormone-binding globulin [12, 13]. Specific cell surface receptors for ABP have been described in several cell types, including germ cells [14– 16]. It has been proposed that activation of these receptors by ABP complexed with steroids may mediate the actions of ABP (and perhaps testosterone) on spermatogenesis [17, 18]. In all previous studies demonstrating ABP’s role in spermatogenesis, experimental manipulations were used that resulted in reduced concentrations of ABP, compared with physiological levels. To further analyze the possible role of ABP in spermatogenesis, transgenic mice overexpressing rat ABP (rABP) were developed [19, 20]. The testis, epididymis, and serum in transgenic (hemizygous) mice contain 20–30 times more ABP than do those of control mice [20]. Surprisingly, transgenic mice homozygous for rABP developed progressive structural and functional abnormalities in the testis, leading eventually to infertility [20–22]. Immunocytochemistry revealed intense ABP immunoreactivity in and around germ cells in all phases of development in the transgenic mice, and clusters of round spermatids showed intense immunoreactivity [20]. Selva et al. [23] reported that meiotic arrest at the level of primary spermatocytes and apoptosis of growth-arrested germ cells were the causative factors for the decline of fertility seen in the ABPtransgenic (ABP-TG) mice. Because flow cytometry allows a rapid and quantitative analysis of cell types based on their DNA content, it is particularly suitable to assess patterns of spermatogenesis. The aim of the present study was to obtain detailed quantitative information on the dynamics of germ cell proliferation in ABP-TG mice, compared with controls, from 7 to 360 days of age to asses the cellular basis of the spermatogenic impairment seen in the transgenic mice and to clarify the mechanism of action of ABP and, by implication, the action of androgens on spermatogenesis.
Transgenic mice carrying rat androgen-binding protein (ABP) genomic DNA express high amounts of testicular ABP and develop a progressive impairment of spermatogenesis. To understand the mechanism of these changes, we have studied the pattern of testicular germ cell proliferation from 7 to 360 days of age in wild-type (WT) control and transgenic homozygous (ABPTG) mice by flow cytometry after labeling DNA in isolated germ cells with propidium iodide. At all ages studied, the body weight of the ABP-TG mice was lower than that of age-matched WT controls. Significantly reduced testicular weight and total germ cell number in the ABP-TG mice were evident from Day 30 and Day 60, respectively. Flow cytometric analysis of isolated germ cells revealed that the number of germ cells undergoing proliferation (S-phase cells) was identical in WT control and ABP-TG mice up to Day 14. Subsequently, the number of germ cells in S-phase was consistently higher in ABP-TG than in WT mice. The number of primary spermatocytes was significantly increased starting from Day 60, and the numbers of round and elongated spermatids were significantly reduced in the ABP-TG animals from Day 21 and Day 60 onwards, respectively. Immunocytometry for intracellular ABP at 90 days of age revealed that the percentage of ABP-containing germ cells was greater in ABP-TG than in WT mice. The continuous presence of ABP in mouse seminiferous tubules at greater than physiological concentrations facilitates the formation of primary spermatocytes but impairs subsequent transformation to round and elongated spermatids. Based on our observations and the analysis of the available literature, the most likely mechanism for production of these effects is sustained reduction in the bioavailability of androgens.
spermatogenesis, testis
INTRODUCTION
Androgen-binding protein (ABP) is a secretory product of Sertoli cells, secreted into the tubular lumen and transported into the epididymis; it is present in the testis of all mammals examined [1]. Testosterone and FSH control the synthesis and secretion of ABP [2–4]. Because the hormonal requirements for ABP synthesis are very similar to those for normal spermatogenesis [5, 6], ABP is considered a biological marker for Sertoli cell function [7]. Apart from
MATERIALS AND METHODS
Chemicals
This work was supported in part by grants from The Fogarty International Center (D43 TW/HD00627 to D.A.J.) and NICHD/NIH (HD 35693 to P.P.) and through cooperative agreement U54-HD35041 as part of the Specialized Cooperative Centers Program in Reproduction Research. 2 Correspondence: Peter Petrusz, Department of Cell and Developmental Biology, University of North Carolina, CB 7090, 108 Taylor Hall, Chapel Hill, NC 27599. FAX: 919 966 1856; e-mail:
[email protected] 1
Collagenase-IV, trypsin, soybean trypsin inhibitor, pepsin, propidium iodide, BSA, ribonuclease (RNase), and deoxyribonuclease (DNase) type II were purchased from Sigma Chemical Company (St. Louis, MO). Fluorescein isothiocyanate (FITC)-conjugated affinity-purified goat anti-rabbit IgG was purchased from Jackson Immunoresearch Laboratories (West Grove, PA). The rabbit anti-ABP serum was raised in our laboratories and characterized by Feldman et al. [24].
Received: 7 June 2001. First decision: 10 July 2001. Accepted: 30 October 2001. Q 2002 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org
Animals The ABP-TG homozygous mice used in the present study were developed by Reventos et al. [19] and were propagated by crossing hemi-
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TABLE 1. Changes in body weight, testicular weight, and total number of germ cells in normal (control) and transgenic mice homozygous for ABP.* Age (days) Parameter Body weight (g) Control 5.3 ABP-TG 2.9 Testis weight (mg) Control 6.6 ABP-TG 6.0 Total no. germ cells (3106) Control 76.5 ABP-TG 62.9
7 6 0.4 6 0.7
10 4.7 6 0.5 3.7 6 0.2b
14 6.2 6 0.5 5.5 6 0.6a
21 8.2 6 1.3 6.5 6 1.1a
30
60
12.7 6 1.1 12.9 6 0.5
23.6 6 1.3 20.8 6 1.7b
6 1.7 6 1.3
13.4 6 0.9 12.2 6 1.2
17.0 6 1.5 16.8 6 1.7
33.5 6 7.6 30.8 6 7.6
97.8 6 8.5 70.0 6 11.1c
193.8 6 24.9 145.3 6 11.2c
6 22.9 6 18.2
251.0 6 29.4 236.3 6 29.4
370.6 6 63.4 389.0 6 55.5
429.8 6 48.4 414.0 6 43.9
1781.8 6 131.4 1742.1 6 168.6
3132.7 6 361.2 2493.3 6 544.9a
* Values are mean 6 SD of 5 estimations; unpaired t-test. a P , 0.05. b P , 0.01. c P , 0.001.
zygous males with homozygous females. A characteristic motor disorder of walking with high stepping and shaking of the hind limbs was consistently present in homozygous male and female mice [20]. This phenotypic feature was used in the present study to quickly identify the homozygous males. Control and ABP-TG homozygous male mice of 7, 10, 14, 21, 30, 60, 90, 120, 180, 240, 300, and 360 days of age were used in the present study. A minimum of five animals per group was analyzed. Control mice used in this experiment were from our inbred colony derived from C57BL6/6J mice (Jackson Immunoresearch). Mice were housed in an animal facility with controlled conditions and given food and water ad libitum. The Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill approved the protocols used in this study. Mice were killed with ether.
Isolation of Germ Cells Testicular germ cells from control and ABP-TG mice were isolated following the methods described by O’Brien [25]. The tunica albuginea was removed, and the decapsulated tubules were incubated in PBS containing 0.5 mg/ml collagenase for 15 min at 328C in a shaking water bath. The tubules were washed twice with PBS containing 1.0 mg/ml DNase. The washed tubules were incubated with 1.0 mg/ml trypsin in PBS for 15 min at 328C. An equal amount of soybean trypsin inhibitor was added, and the suspension was mixed by gentle pipetting with a plastic transfer pipette for 3–5 min and was filtered through an 80-mm nylon mesh (50mm mesh if prepubertal). The resulting monocellular filtrate was washed twice with PBS, and cells were counted in a hemocytometer, fixed in 70% ice-cold ethanol, and stored at 48C until flow cytometry analysis.
Propidium Iodide Staining and Flow Cytometry The method described by Krishnamurthy et al. [26] was followed to stain the DNA in isolated germ cells. Ethanol-fixed germ cells (1–2 3 106) were washed twice in PBS and incubated in 500 ml of 0.2% pepsin (dissolved in 0.9% saline, pH 2.0) for 10 min at 378C. After centrifugation, the cells were stained with staining solution containing 25 mg/ml propidium iodide, 40 mg/ml RNase, and 0.3% Tween-20 in PBS at room temperature for 20 min. The propidium iodide-stained cells were analyzed in a FACScan flow cytometer (Becton-Dickinson Immunocytometry, San Jose, CA). The fluorescent signals of propidium iodide-stained cells were recorded, and a cytogram of DNA area vs. cell count was used to select cell populations on the basis of their DNA content. A total of 10 000 events were recorded for each histogram. The relative numbers of testicular cell types, i.e., elongated spermatids (H 5 hypostainability of elongated spermatids due to condensation of nuclear DNA), round spermatids (1C 5 haploid), secondary spermatocytes and spermatogonia (2C 5 diploid), primary spermatocytes (4C 5 tetraploid), and G-2 spermatogonia (S-phase 5 spermatogonia synthesizing DNA) were calculated using the software Summit (Cytomation, Fort Collins, CO). The total number of each germ cell type was calculated as follows: number of particular cell type detected in sample 3 total germ cell count. total number of cells in sample
ABP Immunocytometry of Germ Cells Freshly isolated germ cells (106 cells) from 90-day-old control and ABP-TG mice were fixed in 1.0% buffered paraformaldehyde for 30 min
at 48C. The cells were washed twice in 3.0 ml of staining buffer (1.0% BSA in PBS) and resuspended in 0.3% Triton X-100 and kept for 5 min at room temperature. The cells were then washed twice with 3.0 ml of staining buffer and resuspended in 100 ml of staining buffer containing rabbit anti-rat ABP serum (1:1000) and incubated at room temperature for 1 h. After incubation, the cells were washed twice in staining buffer and incubated in the dark for 30 min at room temperature with 100 ml of staining buffer containing a 1:100 dilution of fluorosceinated goat antirabbit IgG. After two washes in staining buffer, the cells were counterstained with propidium iodide: 25 mg/ml in 1.0 ml of staining buffer containing 40 mg/ml RNase. The intensities of the green fluorescence for FITC and the red fluorescence for propidium iodide were measured in the FACScan flow cytometer. A total of 10 000 cells were analyzed for each sample. Control cells were processed in the same manner but without the primary antibody. A marker line was drawn in the flow cytometry dot plot (FITC on y-axis and DNA [propidium iodide] on x-axis) separating background signals from positive staining, as determined from the control samples. Cells were considered positive for the presence of ABP if their fluorescence intensity exceeded that of the marker. The percentage of positive cells was quantified using the Summit software.
Immunohistochemistry Testes removed from control and ABP-TG mice were immersed and fixed overnight in Bouin fixative, embedded in paraffin, and sectioned at 8 mm. The sections were deparaffinized and rehydrated. A polyclonal antirat ABP antibody raised in rabbit was used as primary antibody. The double peroxidase-antiperoxidase method described by Ordronneau et al. [27] was employed to demonstrate immunoreactive ABP using diaminobenzidine as chromogen, resulting in a dark brown reaction product. The sections were counterstained with toluidine blue, dehydrated in graded concentrations of ethanol and xylene, and viewed with a Nikon Eclipse 600 microscope equipped with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI).
Statistical Analysis Changes in different parameters were compared using unpaired t-tests and the software GraphPad Instat (San Diego, CA). P values of #0.05 were considered significant. Quantitative results are stated as the mean 6 SD.
RESULTS
Body Weights
The body weights of the ABP-TG mice were lower than those of age-matched controls throughout this study (Fig. 1A and Table 1). The differences were significant from the 10th day onwards. Testicular Weights
Testicular weights for the ABP-TG mice were significantly lower than those for age-matched controls (Fig. 1B and Table 1). The reduction in the testicular weight was
879
GERM CELL PROLIFERATION IN NORMAL AND ABP TRANSGENIC MICE TABLE 1. Extended. Age (days) Parameter
90
Body weight (g) Control 26.3 6 ABP-TG 22.7 6 Testis weight (mg) Control 220.1 6 ABP-TG 163.9 6 Total no. germ cells (3106) Control 3631.7 6 ABP-TG 2513.6 6
120
180
240
300
360
2.1 3.0a
28.1 6 1.1 25.2 6 1.9b
31.8 6 1.8 28.3 6 0.6b
34.8 6 1.7 27.5 6 2.8c
32.1 6 1.4 27.4 6 0.8c
33.1 6 1.4 27.8 6 2.4c
13.7 24.6c
225.5 6 25.7 165.0 6 18.0b
234.5 6 23.7 156.2 6 20.8c
228.4 6 40.3 175.1 6 21.6b
265.2 6 29.6 198.7 6 14.2c
247.2 6 17.0 149.5 6 14.8c
279.5 241.8c
3732.4 6 342.4 2693.4 6 224.3c
3187.8 6 84.8 1819.6 6 373.4c
3274.6 6 283.7 2164.6 6 244.9c
3464.4 6 252.3 2356.4 6 369.8c
3841.3 6 104.9 1999.2 6 392.8c
first seen at Day 30 and persisted throughout the study. The difference in testicular weight between control and ABPTG mice ranged from 28% on Day 30 to 40% on Day 360.
S-phase was consistently higher in ABP-TG than in control mice (Fig. 3B and Table 2). The difference was significant at Days 21, 60, 90, and 240 (Table 2).
Germ Cell Number
Primary Spermatocytes (4C)
Even though the reduction in the testicular weight was seen from Day 30 onwards, the total germ cell number started declining in the ABP-TG mice from Day 60 (2493.3 3 106 6 544.9 3 106 in ABP-TG vs. 3132.7 3 106 6 361.2 3 106 in WT). The extent of the reduction in the total germ cell number (the percentage difference between control and ABP-TG mice) increased gradually with age and reached its maximum (48%) on Day 360 (Fig. 1C and Table 1).
The number of primary spermatocytes (4C) in control and ABP-TG mice followed a similar pattern of increase until Day 30. However, starting from Day 60 (but with the
Flow Cytometric Analysis
Figure 2 shows the distribution histograms of propidium iodide-stained testicular germ cells based on DNA flow cytometry for mice of selected ages (7, 21, 60, and 360 days) as examples. Depending on the DNA content, five populations of cells were distinguished and quantified: 1) primary spermatocytes (4C), 2) spermatogonia synthesizing DNA (S-phase), 3) spermatogonia, secondary spermatocytes, and testicular somatic cells (2C), 4) round spermatids (1C), and 5) elongating and elongated spermatids (H). Somatic cells (Sertoli cells, peritubular myoid cells, Leydig cells, and others) represent only about 3% of the total testicular cells [28], and they are included in the 2C population. The different cell populations are discussed in the sequence of their development in the seminiferous tubules rather that in the order they appear in the flow cytometric profile. Spermatogonia and Secondary Spermatocytes (2C)
Because secondary spermatocytes are very short lived [29], we assume that the majority of the 2C cells detected represent nondividing spermatogonia. In control mice, the total 2C cell population was 64.3 3 106 6 21.8 3 106/ testis pair on Day 7 and increased to 208.9 3 106 6 31.3 3 106 on Day 10 (Fig. 3A and Table 2). Although the pattern of changes in ABP-TG mice was the same, there was a slight but significant decrease in the total 2C cell population on Days 180, 300, and 360 in the ABP-TG mice. Spermatogonia Synthesizing DNA (S-phase)
The number of spermatogonia synthesizing DNA was practically identical in control and ABP-TG mice up to Day 14. Starting from Day 21, however, the number of cells in
FIG. 1. Changes in body weight (A), testicular weight (B), and total germ cell number (C) in control and ABP-TG mice. A decrease was noticed in the body weight and testicular weights of ABP-TG mice from Days 10 and 30 onwards, respectively. The difference in the total number of germ cells between control and ABP-TG mice was evident from Day 60 and increased gradually. Values are the mean 6 SD of five estimations.
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FIG. 2. Representative DNA flow cytogram of propidium iodide-stained testicular germ cell distribution pattern of 7-, 21-, 60-, and 360-day-old control and ABP-TG mice. H, Elongated spermatids; 1C, round spermatids; 2C, secondary spermatocytes and Somatic cells; S-Phase, spermatogonial cells synthesizing DNA; 4C, primary spermatocytes. Means are presented in Table 2.
exceptions of Days 120 and 360), the testes of the ABPTG mice consistently contained significantly higher numbers of primary spermatocytes than did testes from control mice (Fig. 3C and Table 2). Round Spermatids (1C)
Round spermatids in control mice were first detected on Day 21 (37.4 3 106 6 3.8 3 106), and their numbers peaked on Day 120 (1845.4 3 106 6 165.6 3 106). This peak was followed by a slight decline and approximately steady levels thereafter. Although the pattern in ABP-TG mice was similar, the total number of round spermatids in ABP-TG mice was consistently and significantly lower in all age groups 21 days or older (Fig. 3D and Table 2).
Elongating and Elongated Spermatids (H) In control mice, the elongated spermatid cell population started developing between Day 30 and Day 60, reached its peak on Day 90 (1109.8 3 106 6 96.2 3 106), and remained below but close to this level until Day 360 (Fig. 3E and Table 2). In ABP-TG mice, the elongated spermatid population appeared at the same time, but in contrast to the controls, the total number remained significantly lower from 60 days of age onward. In addition, there was a steep decline between Days 300 and 360. The differences in the elongated spermatid populations between control and ABPTG mice were 40.5% on Day 60 and 90% on Day 360. ABP Immunocytometry In control mice, only 3.15% 6 0.90% of total germ cells were positive for ABP. Most of these positive cells be-
GERM CELL PROLIFERATION IN NORMAL AND ABP TRANSGENIC MICE
881
longed to the 2C (spermatogonia and secondary spermatocytes) population. In ABP-TG mice, the percentage of total germ cells positive for ABP was increased to 22.00% 6 4.19% and included all types of germ cells. (Fig. 4). Immunohistochemistry
No immunoreactive ABP was found in the testis of 21day-old WT control mice (Fig. 5A). In contrast, the testes of ABP-TG mice contained detectable levels of ABP already at Day 7 (data not shown); at 21 days of age, immunoreactive ABP was detected in Sertoli cells and occasionally in germ cells (Fig. 5B). In 180-day-old control mice, immunoreactive ABP was found only in the lumina of seminiferous tubules and attached to sperm tails (Fig. 5C). In 180-day-old ABP-TG mice, Sertoli cell stained heavily for ABP, especially the processes surrounding spermatids in the adluminal compartment. A few germ cells also contained immunoreactive ABP (Fig. 5D). In 360-dayold ABP-TG mice, extensive ABP immunoreactivity was found in Sertoli cell cytoplasm and in or around many germ cells (Fig. 5F). DISCUSSION
The results of this study confirm those of many earlier reports suggesting that both reduced [5, 6, 8–11] and increased [18, 20–23] amounts of testicular ABP have deleterious effects on spermatogenesis. One of the important outcomes of our study is the precise quantitative description of the dynamics of spermatogenesis in normal mice from 7 through 360 days of age. Flow cytometric analysis of testicular germ cells revealed that on Days 7–10 about 85% of the germ cells in normal mice (64.3 3 106 6 21.8 3 106 cells of 76.5 3 106 6 22.9 3 106 cells) were in diploid (2C) state along with a small population of tetraploid (4C) primary spermatocytes (4.14 3 106 6 1.53 3 106 cells, 5.5%) and cells in synthetic phase (1.36 3 106 6 0.42 3 106 cells, 1.8%). By Day 14, there was a large reduction in the relative abundance of the 2C population (from 85% to 37%) accompanied by an increase in the number (and proportion) of spermatogonia in S-phase and of 4C primary spermatocytes. Round spermatids (1C) first appeared on Day 21, and their number increased dramatically between Day 21 and 30 (from 37.4 3 106 6 3.8 3 106 cells to 1131.5 3 106 6 116.0 3 106 cells). These changes reflect the initial phases of the first wave of spermatogenesis. The elongated spermatid (H) population in control mice appeared between Days 30 and 60, reflecting the transformation of round spermatids to mature spermatozoa during this period. The total number of elongated spermatids reached its maximum on Day 90 (1109.8 3 106 6 95.2 3 106 cells). Beyond 60–90 days of age, the total numbers of 2C, S-phase, 4C, 1C, and H cells in control mice did not show any substantial changes. Our data also reveal that the testis develops and spermatogenesis begins normally in the presence of excess transgenic ABP. Although some testicular abnormalities were evident as early as Day 21, the severity of the changes increased with age. This progressive testicular impairment (reduction in testicular weight and total germ cell number) is consistent with earlier reports that transgenic mice overproducing rABP had progressively reduced fertility [20– 23]. Whereas in a previous study [20] there was marked reduction (10–25%) in the seminiferous tubular diameter, Esteban et al. [22] reported no difference in tubular diameter between control and ABP-TG mice. However, these
FIG. 3. Total numbers of spermatogonia and secondary spermatocytes (A), spermatogonia synthesizing DNA (S-Phase cells) (B), primary spermatocytes (A), round spermatids (D), and elongating and elongated spermatids (E) in control and ABP-TG mice at various ages. Values are the mean 6 SD of five estimations.
authors [22] reported an increase in the number of Sertoli cells in the ABP-TG mice over that seen in controls. Sertoli cell numbers were not assessed in our study; however, the significantly reduced germ cell numbers in ABP-TG mice after 60 days of age would make the relative number of Sertoli cells appear higher. To interpret correctly the changes in spermatogenesis observed in the transgenic mice, it is important to determine whether these changes are indeed caused by overproduction of ABP or by some other mechanism unrelated to the functions of ABP, e.g., insertional mutation (see also Selva et al. [23]). Arguments in favor of the first explanation are the
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TABLE 2. Changes in testicular germ cell proliferation in normal (control) and transgenic mice homozygous for ABP.* Age (days) Parameter
7
Spermatogonia (2C) Control 64.3 6 21.8 ABP-TG 50.2 6 16.1 S-Phase cells Control 1.4 6 0.4 ABP-TG 1.3 6 0.5 Primary spermatocytes (4C) Control 4.1 6 1.5 ABP-TG 3.1 6 1.6 Round spermatids (IC) Control — ABP-TG — Elongated spermatids (H) Control — ABP-TG —
10
14
21
30
60
208.9 6 31.3 198.8 6 32.5
138.8 6 34.93 141.7 6 19.8
129.93 6 14.0 143.3 6 24.8
240.4 6 41.2 293.0 6 9.9a
225.0 6 32.2 200.4 6 34.7
4.9 6 0.9 4.0 6 1.4
19.0 6 6.6 20.2 6 3.7
14.9 6 0.8 23.6 6 3.2c
29.6 6 4.5 39.7 6 13.3
28.1 6 4.3 36.77 6 4.14c
14.0 6 2.2 11.2 6 2.2
158.4 6 30.7 197.4 6 40.3
198.6 6 19.7 202.2 6 52.9
310.6 6 67.5 325.4 6 40.9
403.3 6 40.3 624.7 6 86.6c
— —
— —
37.4 6 3.8 10.1 6 3.9c
1131.5 6 116.0 938.1 6 123.4a
1505.6 6 256.6 876.9 6 61.5c
— —
— —
— —
— —
668.4 6 96.2 404.5 6 85.4c
* Values are mean 6 SD of 5 estimations; unpaired t-test. a P , 0.05. b P , 0.01. c P , 0.001.
following. Overproduction of ABP is one of the fundamental phenotypic characteristics of the ABP-TG mice, and another transgenic line also demonstrated both increased production of ABP and impaired spermatogenesis [19]. Furthermore, the progressive nature of the abnormalities parallels the progressively increasing amounts of ABP FIG. 4. Immunocytometry of testicular germ cells. Bivariate analysis of ABP (FITC on y-axis) and DNA (PI on x-axis) represented as dot plots with arrows indicating germ cells positive for the presence of ABP. 1C, Spermatids; 2C, secondary spermatocytes; S-Phase, spermatogonia synthesizing DNA; 4C, primary spermatocytes.
detectable in the testes of the transgenic mice (e.g., see Fig. 5). In the transgenic mice, rABP mRNA is expressed specifically in the testis but not in other tissues or organs, the correct transcription start site is utilized, and expression within the testis is restricted to the correct cell type, i.e., the Sertoli cells [19–21]. These arguments strongly indicate
883
GERM CELL PROLIFERATION IN NORMAL AND ABP TRANSGENIC MICE TABLE 2. Extended. Age (days) Parameter
90
Spermatogonia (2C) Control 227.2 6 ABP-TG 208.2 6 S-Phase cells Control 33.2 6 ABP-TG 48.68 6 Primary spermatocytes (4C) Control 377.5 6 ABP-TG 662.0 6 Round spermatids (1C) Control 1629.9 6 ABP-TG 826.1 6 Elongated spermatids (H) Control 1109.0 6 ABP-TG 415.2 6
120
180
240
300
360
28.9 54.8
224.6 6 15.1 241.2 6 38.9
253.0 6 13.7 169.4 6 42.6a
284.9 6 35.9 227.5 6 42.7
275.8 6 30.3 200.9 6 32.1a
277.2 6 5.3 195.5 6 72.3a
5.1 8.20b
40.5 6 5.3 42.40 6 6.56
39.5 6 2.0 41.65 6 7.00
34.1 6 4.9 51.89 6 4.00c
34.2 6 7.8 47.61 6 10.12
39.1 6 4.1 42.69 6 17.57
18.5 102.5c
500.7 6 125.9 594.7 6 66.9
379.9 6 20.2 523.2 6 124.3a
372.3 6 38.8 523.5 6 107.0a
408.8 6 53.2 583.8 6 109.7a
404.1 6 30.3 453.9 6 149.8
57.3 227.7c
1845.4 6 165.5 1170.0 6 190.0c
1491.2 6 54.5 724.1 6 110.3c
1391.1 6 72.5 844.8 6 147.1c
1438.7 6 145.1 1039.1 6 202.6a
1609.8 6 69.8 1047.8 6 124.5b
96.2 69.5c
859.6 6 183.3 346.7 6 59.9c
826.9 6 73.3 341.4 6 63.0c
939.0 6 149.0 271.7 6 49.1c
823.9 6 111.1 297.4 6 137.4c
983.7 6 55.4 94.2 6 33.8c
that the only genetic abnormality in the ABP-TG mice is overproduction of ABP, and the other abnormalities seen are the results of this overproduction, i.e., the presence and actions of high amounts of ABP. Starting on Day 21 and continuing throughout the study, the number of proliferating spermatogonia (S-phase) was higher in the ABP-TG mice than that in age-matched controls. Starting from Day 60, the number of primary spermatocytes was also consistently higher in the ABP-TG mice than in controls. However, the numbers of round spermatids and elongated spermatids, starting from Days 30 and 60, respectively, were significantly lower in ABP-TG mice than in controls. This quantitative pattern is consistent with a reduced transformation of spermatocytes to spermatids in the ABP-TG mice (meiotic arrest), as suggested by Selva et al. [23]. However, the increased rate of spermatogonial divisions observed in the present study may also explain, at least in part, the ‘‘accumulation’’ of primary spermatocytes. The reduction in testicular weight, total germ cell number, and the absolute numbers of round and elongated spermatids began in the ABP-TG mice around the same time, between 30 and 60 days of age. The apparent accumulation of primary spermatocytes also started during this period and persisted throughout the study. This time period in male rodents represents the onset of puberty, the time when testicular functions are activated by gonadotropins and testosterone [30, 31]. The striking coincidence of these hormonal changes with the first appearance of the spermatogenetic impairment in the ABP-TG mice strongly suggests that excess ABP causes these impairments by sequestration of testosterone, i.e., reducing testosterone’s availability for interaction with its molecular targets, primarily the androgen receptor. This interpretation of our results is also consistent with conclusions from many earlier studies that addressed the effects of testosterone (or its withdrawal) on spermatogenesis. Increased spermatogonial proliferation (parallel with reduced germ cell apoptosis) in response to reduced testosterone levels was described by Shuttlesworth et al. [32]. The completion of meiosis and the formation of round spermatids are androgen-dependent processes in the genetically gonadotropin-deficient (hpg) mouse [33]. Perhaps the strongest evidence in favor of reduced availability of androgens in the ABP-TG mice is the strong reduction in the transformation from round to elongated spermatids. This trans-
formation is the main androgen-dependent step in spermatogenesis [34–36]. Still another change indicating testosterone deficiency in the ABP-TG mice is the large-scale detachment of clusters of round spermatids observed in older animals [20], another known consequence of testosterone withdrawal [37]. In an in vitro study, Roberts and Zirkin [38] found that ABP was capable of inhibiting the action of testosterone, presumably by preventing its access to the androgen receptor. Excess ABP within the seminiferous tubules may also act by reducing the availability of testosterone as a substrate for the enzymes 5a-reductase and aromatase, thus leading to reduced formation of dihydrotestosterone and estradiol. Levallet et al. [39] and Saunders et al. [40] demonstrated the expression of p450 aromatase and that of estrogen receptor b, respectively, in germ cells of rat testis, suggesting a possible role for estrogens in spermatogenesis [41]. Analysis of testicular steroid, aromatase, and 5a-reductase levels in transgenic mice might help to clarify these potentially complex mechanisms. As an alternative explanation, the possible direct effect of ABP on testicular germ cells cannot be ruled out. In the present study, the percentage of ABP-containing germ cells was much greater in ABP-TG than in WT control mice at 90 days of age (even though the total germ cell number was significantly reduced). This finding is consistent with earlier immunocytochemical observations describing intense ABP immunoreactivity in and around all types of germ cells in ABP-TG mice [20]. In an in vitro study using isolated germ cells, ABP was bound and internalized by such cells and modified protein synthesis in a manner dependent on the presence or absence of sex steroids [17]. ABP (or ABP complexed with steroids) may thus function as an intercellular messenger mediating communication among Sertoli cells (source of ABP), Leydig cells (source of testosterone), and germ cells. Because germ cells are not thought to possess androgen receptor [42, 43], ABP’s role may be the activation of a putative receptor on the membrane of the germ cell, initiating a signal transduction cascade leading to a cellular response. Much work will be needed before we can understand how this mode of action of ABP, if operative, could bring about the changes in spermatogenesis seen in the transgenic mice. ACKNOWLEDGMENTS The authors thank Dr. Larry W. Arnold for the use of the UNC flow cytometry facility and the anonymous reviewers of this paper for their valuable comments.
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FIG. 5. Immunohistochemical localization of ABP in the testes of 21-day-old (A and B), 180-day-old (C and D), and 360-day-old (E and F) WT control and ABP-TG mice. More ABP immunoreactivity was present in Sertoli cells and in or around germ cells in all phases of their development in ABP-TG mice than in WT controls.
GERM CELL PROLIFERATION IN NORMAL AND ABP TRANSGENIC MICE
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