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Spontaneous Germ Cell Apoptosis in Humans: Evidence for Ethnic Differences in the Susceptibility of Germ Cells to Programmed Cell Death

Division of Endocrinology, Harbor-University of California-Los Angeles Medical Center (A.P.S.H., C.W., Y.L., R.S.S.), Torrance, California 90509; the Department of Veterinary Anatomy and Public Health, Texas A&M University (L.J.), College Station, Texas 77843; and the Jiangsu Family Planning Research Institute (X.-H.W.), Nanjing, China

Address all correspondence and requests for reprints to: Ronald S. Swerdloff, M.D., Division of Endocrinology, Harbor-University of California-Los Angeles Medical Center, Box 446, 1000 West Carson Street, Torrance, California 90509. E-mail: Swerdloff{at}harbor2.humc.edu

	Abstract

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Spontaneous death of certain classes of germ cells has been shown to be a constant feature of normal spermatogenesis in a variety of mammalian species, including the human. Recent studies on various animal models have demonstrated that apoptosis is the underlying mechanism of germ cell death during normal spermatogenesis. Withdrawal of gonadotropins and/or testosterone further accelerates the germ cell apoptosis. We examined the involvement of apoptosis in the spontaneous loss of germ cells in men. Testicular samples obtained at autopsy from 5 Chinese and 9 non-Hispanic Caucasian men were analyzed. To identify individual germ cells undergoing apoptosis, we used a modified terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling technique that detects germ cell apoptosis with high sensitivity and specificity. Testicular sections from all 14 subjects exhibited spontaneous occurrence of germ cell apoptosis involving spermatogonia, spermatocytes, and spermatids (apoptotic indexes, 1.6 ± 0.4, 2.8 ± 0.6, and 5.5 ± 1.2, respectively). The incidence of spermatogonial (2.8 ± 0.8 vs. 1.0 ± 0.2) as well as spermatid (9.3 ± 2.1 vs. 3.4 ± 0.9) apoptosis was higher in Chinese than in Caucasian men. A higher incidence of spermatocyte apoptosis was also noted for Chinese (4.4 ± 1.4) compared to Caucasian (1.9 ± 0.4) men, but the difference was not statistically significant. These results suggest that germ cell death during normal spermatogenesis in men occurs via apoptosis and provide evidence for ethnic differences in the inherent susceptibility of germ cells to programmed cell death. Our data may also help to explain the greater efficacy of testosterone-induced spermatogenic suppression to azoospermia observed in Asian compared to non-Asian men.

	Introduction

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APOPTOSIS is an evolutionarily conserved, cell death process that plays a major role during normal development and homeostasis of multicellular organisms (1, 2, 3). The crucial role of this mode of cell death is increasingly being recognized in the pathogenesis of many diverse human diseases, including acquired immunodeficiency syndrome, neurodegenerative disorders, atherosclerosis, and arrhythmogenic right ventricular dysplasia (4, 5, 6).

Spermatogenesis is a complex process in which stem spermatogonia, through a series of events involving mitosis, meiosis, and cellular differentiation, become mature spermatozoa (7, 8). Of interest, germ cell death, in addition to cellular proliferation and differentiation, is conspicuous during normal spermatogenesis of various mammalian species, including men, and plays a critical role in sperm output (7, 9, 10). Extending these earlier observations, recent studies on various animal models have demonstrated that apoptosis is the underlying mechanism of germ cell death during normal spermatogenesis (11, 12, 13, 14, 15, 16). Withdrawal of gonadotropins and/or testosterone (T) further accelerates the apoptosis of germ cells (11, 12, 13, 14). These observations together with our previous findings that GnRH antagonist treatment induces azoospermia in the rat (17) suggest that apoptosis plays a major role in progressive and organized regression of spermatogenesis to azoospermia after gonadotropin deprivation.

Despite its potential for improved fertility control and clinical management of infertility in men, the mechanism of germ cell death in men is poorly understood. Recently, apoptosis was reported as a possible mechanism of spermatogonial death in prepubertal boys (18) or of 2-methoxy acetic acid-induced spermatocyte death in cultured seminiferous tubules of middle-aged (53–74 yr) human donors (19). In a preliminary study using a limited number of testicular specimens (n = 5), we reported the incidence of spontaneous apoptosis of all three classes of germ cells (spermatogonia, spermatocytes, and spermatids) in normal men (20). We also noted in that study that the rates of spermatocyte and spermatid apoptosis were significantly higher in humans than in rats. The latter finding is consistent with the observed low efficiency of spermatogenesis in humans compared to rats (21). Here we substantiate these findings using testicular samples from 14 men (5 Chinese and 9 non-Hispanic Caucasian men) and report, for the first time, the ethnic differences in the inherent susceptibility of germ cells to programmed cell death.

	Materials and Methods

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Testicular specimens

Normal testicular samples from five Chinese men between 18–43 yr of age and nine non-Hispanic Caucasian men between 25–40 yr of age were examined. Tissues were obtained at autopsy within 15 h after sudden traumatic death, fixed with 5% glutaraldehyde in 0.05 mol/L sodium cacodylate buffer, cut into small pieces (2 x 4 x 4 mm), and processed for routine paraffin embedding. Specimens came from China (Jiangsu Family Planning Research Institute, Nanjing, China) and the United States (Texas A&M University, College Station, TX). These subjects had no prior history of medical illness or chronic drug or alcohol abuse. The collection of testes at autopsy was approved by the respective institutional ethical review committees. The choice of fixative was based on the results of our animal studies which showed that glutaraldehyde fixation significantly improved both the specificity and the sensitivity of the in situ 3'-end labeling of apoptotic DNA fragmentation while maintaining excellent morphological preservation (22). The adjacent testicular slice from each subject was further diced into small pieces, postfixed in 1% osmium tetroxide, and processed for routine epon embedding.

In situ detection of apoptotic germ cells

In situ detection of cells with DNA strand breaks was performed in glutaraldehyde-fixed, paraffin-embedded testicular sections (6 µm) using an ApopTag-peroxidase kit (Oncor, Gaithersburg, MD). The technique uses terminal deoxynucleotidyl transferase (TdT) to catalyze template-independent addition of digoxigenin-deoxy (d)-UTP and -dATP to 3'-hydroxy ends of fragmented DNA generated by internucleosomal cleavage. The incorporated nucleotides form a random heteropolymer of digoxigenin-dUTP and -dATP. An antidigoxigenin antibody conjugated to peroxidase is added, which generates an intense signal for chromogenic substrates. The ApopTag kit has been validated for in situ detection of germ cell apoptosis in a variety of mammalian species, including the human by us (11, 14, 15, 16, 20, 22). In brief, after deparaffinization and rehydration, tissue sections were incubated with proteinase K (20 µg/mL) for 20 min at 37 C, washed in distilled water, and then treated with 2% hydrogen peroxide in phosphate-buffered saline for 5 min at room temperature to quench endogenous peroxidase activity. Sections were incubated with a mixture containing digoxigenin-conjugated nucleotides and TdT in a humidified chamber at 37 C for 1 h and subsequently treated with antidigoxigenin-peroxidase for 30 min at room temperature. Immunoreactive cells were detected by incubating the sections with a mixture of 0.05% diaminobenzidine and 0.01% H₂O₂ for 6 min. Sections were stained with or without 0.5% methyl green in 0.1 mol/L sodium acetate buffer (pH 4.0) as a counterstain, dehydrated in 100% butanol, cleared in xylene, and mounted with Permount (Fischer Scientific Corp., Fairlawn, NJ).

Negative and positive controls were carried out in every assay. As negative controls, tissue sections were processed in an identical manner, except the TdT enzyme was substituted by the same volume of distilled water. Testicular tissues from an adult rat 5 or 7 days after GnRH antagonist treatment, in which apoptosis is well characterized (11, 14, 22), were used as a positive control.

In addition to the in situ end-labeling technique, glutaraldehyde-fixed epoxy-embedded testicular specimens were examined for morphological signs of germ cell death (7, 11).

Enumeration of apoptotic germ cells

Testicular sections were examined with an American Optical microscope (Buffalo, NY) with x40 objective and a x10 eyepiece. A square grid fitted within one eyepiece provided a reference area of 62,500 µm². The method used for apoptotic germ cell count was essentially similar to those originally proposed for germ cell quantitation in humans (23, 24). In brief, apoptotic spermatogonia (sum of A long, dark-type A, pale-type A, and B spermatogonia), spermatocytes (sum of preleptolene, leptotene, zygotene, pachytene, diplotene, and dividing spermatocytes), spermatids (sum of Sa, Sb₁, Sb₂, Sc, Sd₁, and Sd₂), and viable Sertoli cell nuclei with distinct nucleoli within the frame of the grid were counted. The identification of various apoptotic germ cells was based on their morphological characteristics, including cell and nuclear sizes, chromatin pattern and distribution, and staining affinity (23, 24, 25, 26). For each testis, at least 100 grid fields were examined. The rate of germ cell apoptosis (apoptotic index) was expressed as the number of apoptotic germ cells per 100 Sertoli cells.

Statistical analysis

Results were expressed as the mean ± SEM. Statistical analysis was performed using the StatView (4.01) software package (Abacus Concepts, Berkeley, CA; 1992) on a Macintosh II Si computer (Apple Computer Inc., Cupertino, CA). Differences between means were evaluated using the unpaired t test. P < 0.05 was considered statistically significant.

	Results

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Testicular sections from all 14 subjects (5 Chinese and 9 Caucasian men) exhibited spontaneous occurrence of germ cell apoptosis as revealed by the in situ 3'-end DNA-labeling technique (Fig. 1, A–C, and Table 1). Unlike in rat (11, 14) and hamster (16) testes, apoptotic cells involving all three classes of germ cells (spermatogonia, spermatocytes, and spermatids) were readily observed in human testes. Apoptosis usually occurred in isolated or small groups of germ cells. Occasional multinucleated giant cells involving a few apoptotic round spermatids were found in a testicular section from 1 of the 5 Chinese men (Fig. 1D). Sertoli cell apoptosis was never observed in these subjects.

View larger version (125K): [in this window] [in a new window]   Figure 1. In situ 3'-end labeling of DNA strand breaks in apoptotic germ cells in glutaraldehyde-fixed paraffin-embedded testicular sections of normal men. Spontaneous apoptosis of spermatogonia (SPG), spermatocytes (SPC), and spermatids (STD) are readily observed in normal human testes (A–D). Methyl green was used as a counterstain, except in D. Magnification, x1200. Scale bar = 10 µm.

View larger version (31K): [in this window] [in a new window]   Figure 2. A comparison of mean apoptotic index (AI) of germ cells between Chinese and Caucasian men. Values are the mean ± SEM; the number in the parentheses represents the number of the subjects. *, Significantly different.

	Discussion

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Using this technique, which detects apoptosis with high sensitivity and specificity, we now provide evidence for spontaneous apoptosis of spermatogonia, spermatocytes, and spermatids in normal adult men. The incidence of apoptosis varies among the subjects as well as between the ethnic backgrounds. The higher incidence of germ cell apoptosis in the Chinese than in the Caucasian men is intriguing. The triggering factors for spontaneous germ cell apoptosis during normal spermatogenesis are not known. It is also uncertain why the Chinese exhibited higher incidence of germ cell apoptosis than Caucasians. Available evidence from animal studies suggest that germ cell apoptosis is a hormonally regulated process (11, 14, 32), but can be modulated by many extrinsic and intrinsic factors, including growth factors, cytokines, and death inducer and repressor genes (33, 34, 35, 36). We anticipate that future studies directed toward the identification of these modulatory factors in the testes of Chinese and Caucasian men will explain the observed ethnic differences in the inherent susceptibility of germ cells to programmed cell death.

Experimental hormonal male contraceptive approaches have used various agents to suppress gonadotropin levels and induce azoospermia and severe oligospermia. Based on earlier information about the suppression of spermatogenesis in the rat through accelerated apoptosis in response to a lack of hormonal stimulation (11, 12, 13, 14), it is our belief that germ cell apoptosis is an important determinant of hormonal male contraceptive-induced spermatogenic suppression in men. A multicenter studies demonstrated a greater efficacy of T-induced spermatogenic suppression to azoospermia in Asian (predominantly Chinese) men than that in Caucasians (37). These differences in suppression rates could not be explained by differences in physical characteristics, routine clinical chemistry screening, or hormonal and semen profiles (38). The observed incidence of accelerated apoptosis could be an important determinant of these differences.

	Footnotes

 
¹ Supported by a fellowship (MFF-95–05) from the Contraceptive Research and Development Program.

Received July 30, 1997.

Revised September 16, 1997.

Accepted September 25, 1997.

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