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Departments of Obstetrics and Gynecology, Hematology and Institute of Urology and Nephrology, University College London Medical School (M.A.H., D.J.R. R.R.C.); Weston Laboratory (H.M.) and Division of Investigative Sciences, Imperial College School of Science, Technology, and Medicine (P.M.C.), Department of Hematological Medicine (N.S.B.T.), GKT Medical School, Kings College London, London, United Kingdom WC1E 6AU; and Academic Unit of Obstetrics (R.B.), St. Mary’s Hospital, University of Manchester, Manchester, United Kingdom M13 0JH
Address all correspondence and requests for reprints to: Dr. Ratna Chatterjee, M.D., Ph.D., University College Hospital, Obstetric Hospital Reproductive Medicine Unit, London, United Kingdom WC1E 6AU. E-mail: . ratnach{at}globalnet.co.uk
Abstract
During spermatogenesis in human adults, testicular germ cells proliferate, differentiate, and die by apoptosis. However, little is known about the temporal or spatial nature of this programmed cell death. Such information may be useful for understanding prenatal developmental biology as well as spermatogenesis during adulthood, particularly in the context of germ cell disorders. We undertook this study to determine 1) whether apoptosis occurred in a cell-specific fashion in the germ cell population and the supporting somatic cells; and 2) whether apoptosis varied with gestational age. We examined human fetal testicular tissues obtained from 17 karyotypically and structurally normal fetuses of mothers who underwent spontaneous or induced abortions. Three gestational ages were defined as follows: group A, 12–13 wk gestation (n = 5); group B, 20–22 wk gestation (n = 7); and group C, 37–40 wk gestation (n = 5). Morphology in conjunction with in situ end labeling was used to identify and quantify apoptotic nuclei in fetal gonadal tissues. The results of this study suggest that gonadal apoptosis occurred in germ cells, Sertoli cells, and Leydig cells at all gestational ages. Apoptotic death was highest in the Leydig cells, followed by germ cells and Sertoli cells. There was a significant positive correlation between the apoptosis of germ cells and Sertoli cells (P < 0.01) and a negative correlation between healthy germ cells and Sertoli cells (P < 0.001). There was also a negative correlation between the intratubular cell number and the gestational age. Specifically, the proportion of Sertoli cells decreased with gestational age, although there was no significant change in the germ cell in relation to gestational age. No such relationship was found in the Leydig cell population, all of which reside outside the seminiferous tubules. These results are the first to suggest that fetal testicular apoptosis begins in the first trimester, occurs in the three major cell types, and continues throughout pregnancy. Our data also suggest that in the fetal gonad, germ and Sertoli cell proliferation and death may be controlled by a genetic program distinct from that of the Leydig cells. This information is relevant to the understanding of abnormal spermatogenesis associated with infertility and to germ cell tumors in adult life.
IT IS NOW widely accepted that apoptosis is the major mechanism of programmed cell death in a wide variety of tissues in the developing embryo (1, 2). However, the ontogeny of germ cell death in the developing human testes has not been investigated in detail. Although it is known that apoptosis is the main cause of primordial germ cell and oocyte loss in the developing mouse (3) and human fetal ovary (4), there is less information on spermatogenesis, particularly in relation to testicular developmental cell death. This may reflect the fact that the functional life span of the female gonads is defined in most species (including humans) by the size and rate of depletion of oocytes enclosed within ovarian follicles at birth, while in males spermatogenesis continues throughout adulthood. In the female, germ cells undergo atresia starting in fetal life and continuing throughout puberty and adulthood until the menopause (4). Studies using genetic-null or transgenic models have explored the functional requirement of proteins, such as the Bcl-2 family member Apaf-1, and caspases in oocyte survival and death (3, 5). These studies have demonstrated that apoptosis is of fundamental importance in gonadal development, although the potential impact of translating this information into new approaches for the clinical diagnosis and management of female infertility and the menopause is yet to be realized.
It could be argued that the idea that the distinguishing feature of male germ cells is self-renewal whereas in females it is oocyte death may be an oversimplification. Indeed, there are both animal and human data to suggest that male germ cells also undergo apoptosis during the process of biological maturation including spermatogenesis (6, 7, 8). Wang et al., (9) investigated the extent and mechanisms of male germ cell death during fetal and neonatal life by studying the testes of mice at defined fetal and postnatal ages extending from 13 d gestation (approximately equivalent to the term human infant) to 7 wk after birth. They found that spermatogenic cell apoptosis was highest at 13 d gestation and peaked again at approximately 10–13 d after birth.
In contrast, little is known about the ontogeny of germ cell apoptosis in the developing human fetus. This information is important, as the presence or absence of apoptosis in the male germline in fetal life may have important implications for normal as well as dysfunctional adult spermatogenesis, particularly in relation to infertility. In a recent preliminary study, Quenby et al. (10) found that in the human testis both germ cells and somatic cells undergo apoptosis in the first trimester. However, this study was largely qualitative and did not provide any quantitative comparative analysis of cell apoptosis in relation to gestational age. In the present study we have extended this work by quantifying apoptosis in the germ (GC), Sertoli (SC), and Leydig (LC) cell populations in the developing human testes in relation to gestational age.
Materials and Methods
Collection of tissues
Human fetal testicular tissues were obtained from archived materials (gonadal tissues) stored during autopsy from 17 karyotypically and structurally normal fetuses of three gestational ages: group A, spontaneous miscarriages (n = 4) or termination (n = 1), aged 12–14 wk gestation; group B, spontaneous miscarriages (n = 6) or termination (n = 1), aged 20–22 wk gestation; and group C, nonmacerated stillbirths (n = 4) or neonatal death (normal delivery, n = 1), aged 37–39 wk gestation.
The interval between fetal delivery and fixation of gonadal tissue ranged between 1 and 4 h for groups A and B and up to 48 h for group C. During the time of autopsy, parental consent was obtained for storage of archived material and for future research studies according to the hospital policy of research guidelines (Hammersmith Hospital Trust research ethics committee).
Histological preparation
All tissues were fixed in 10% buffered formaldehyde and embedded in paraffin wax. Sections were cut at 4-µm thickness, and serial sections were mounted onto silane-coated microscope slides (BDH, Dorset, UK). Hematoxylin and eosin staining was performed on one section per fetus using a standard technique for Cole’s hematoxylin, and three further serial sections of fetal gonads were used for double labeling studies for in situ end labeling (ISEL) and c-Kit (see below). These sections were lightly counterstained with methyl green.
Hematoxylin and eosin (H and E) staining
H and E staining was performed using standard techniques for Cole’s hematoxylin. Apoptotic cells were identified under a light microscope using the following morphological criteria (11): intense, uniform nuclear basophilia with either 1) chromatin condensation and nuclear shrinkage (pyknosis) or 2) fragmentation of the nucleus into several rounded and uniformly densely basophilic masses (karyorrhexis).
ISEL
ISEL was performed as described by Ansari et al. (12) with minor modifications. Briefly, three serial sections per fetus were dewaxed, incubated with 3% hydrogen peroxide for 15 min at room temperature, and then washed three times in PBS (5 min/wash). After washing, sections were digested with proteinase K 40 µg/ml (Roche Molecular Biochemicals, Lewes, UK) for 15 min at room temperature and then washed three times in PBS. After washing, sections were incubated in labeling mix (containing deoxynucleotides, biotinylated deoxy-ATP, and the Klenow fragment of DNA polymerase) for 60 min at 37 C. After further washing, biotinylated nucleotides incorporated into sections were detected using a commercial kit (Vectastain ABC, Vector Laboratories, Inc., Peterborough, UK) using nickel diaminobenzidene (Vector Laboratories, Inc.) as the substrate. Color development was observed carefully under the microscope, and reactions were quenched in PBS after 6 min.
Immunohistochemical analysis of c-Kit expression
To distinguish GC from other cells in the intratubular population, c-Kit was used as a cell surface marker. In the extratubular compartment LC are also c-Kit positive, which differentiates them from other interstitial cells. For double labeling experiments, at the end of ISEL, normal goat serum (1:20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added for 20 min at room temperature, then antibody to c-Kit (Santa Cruz Biotechnology, Inc.) was diluted 1:100 and added without washing the sections. Slides were incubated overnight at 4 C. After washing the slides in PBS, the secondary antibody (biotinylated antirabbit IgG; Santa Cruz Biotechnology, Inc.) was added for 1 h at room temperature, then the sections were washed and treated with Vectastain ABC Kit (Vector Laboratories, Inc.) for 30 min at room temperature. Finally, Vector Red (Vector Laboratories, Inc.) was added for 22 min at room temperature. Color development was observed carefully under a light microscope and was quenched in PBS. The slides were washed in distilled water for 10 min and counterstained with methyl green (Vector Laboratories, Inc.) for 3 min at room temperature, then dehydrated using Histoclear (Vector Laboratories, Inc.) and finally mounted in DPX (Merck, Lutterworth, UK).
Quantitative analysis of apoptosis
The testicular cells (apoptotic, healthy, and total number) were counted by the naked eye by the same observer (M.A.H.). Morphology (after H and E staining) in conjunction with ISEL was used to identify and quantify apoptotic nuclei in fetal gonadal tissues. The numbers of healthy GC and SC were quantified in 3 serial sections using 15 random seminiferous tubules, whereas Leydig cells were quantified in 15 interstitial fields. In some fetuses of 12–13 wk gestation, the seminiferous tubules were not fully developed, so quantitation was limited to counting apoptosis in 15 random fields. Apoptosis was expressed as a percentage. Quantitative analysis of apoptotic testicular subtypes (GC, SC, and LC) was undertaken using this formula: X/Y x 100 = % apoptosis, where X is the number of apoptotic cells, and Y is the total cell number.
Statistical Analysis
All data were expressed as the mean ± SEM. Correlation between two variables was expressed by Spearman’s correlation coefficient, and P < 0.05 was considered statistically significant.
Results
A summary of maternal clinical data from the present study is given in Table 1
. Maternal age ranged between 19–39 yr (mean ± SEM, 31.82 ± 1.44 yr). Twelve of 17 mothers were multigravida, and 4 were primigravida. Two pregnancies were terminated, 10 miscarried spontaneously, and 5 fetuses were delivered normally, although 1 of these died soon after birth. Macroscopic appearances of the placenta were normal in all fetuses although there was evidence of congestion in 4 of them. Gonadal appearance was unremarkable in all cases, and all endocrine organs involved in reproductive development (e.g. the pituitary, thyroid, and adrenal glands) were also healthy at the histological level.
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, A and B, shows the photomicrograph of the H- and E-stained and double stained (H and E, and ISEL) fetal gonads, respectively. First the expression of c-Kit protein in the developing testes was investigated by immunohistochemistry. Only weak background staining could be seen in sections treated with preimmune serum (not shown). In contrast strong c-Kit expression could be detected on the surface of large intratubular cells with the morphological appearance of GC (Fig. 1, A and B). Similarly, c-Kit was expressed on interstitial cells (Fig. 1, A and B), previously identified as LC (13). c-Kit staining was distributed mainly on the plasma membrane, and this was completely abolished in the presence of the immunizing peptide (not shown). For quantitative analysis of defined cell populations, intratubular cells that were c-Kit positive were counted as GC, and interstitial cells (extratubular) that were c-Kit positive were counted as LC.
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). The cell population with the highest proportion of apoptosis was LC, with apoptosis ranging from 5.72–11.26% (Fig. 2A). In contrast, apoptosis of Sertoli cells did not exceed 1.58%, and germ cell apoptosis ranged from 5.33% in group B to 3.07% in group C (Fig. 2A). The number of apoptotic cells did not appear to depend on gestational age, but, rather, on cell type. Significantly, investigating the precise relationship between apoptosis in defined cell populations indicated a positive correlation between apoptosis in the GC and SC populations in every age group (P < 0.001; Fig. 2B). Correspondingly, there was a negative correlation between the number of healthy GC and SC (P < 0.001; data not shown). In contrast, there was no correlation between apoptosis in the GC and LC populations or between the SC and LC populations. When healthy cell numbers were related to fetal development, a positive correlation was observed between the proportion of healthy GC and gestational age, whereas this correlation was negative in healthy SC (P < 0.05 for each cell type; data not shown).
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), whereas the proportion of SC actually decreased (P < 0.001; Fig. 3B). Overall, with increasing gestational age, the total number of cells (germ and Sertoli) per tubule decreased (P < 0.05; Fig. 3C).
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Although it is known that apoptosis occurs in adult males during normal and abnormal spermatogenesis (7, 8), little is known about GC ontogeny in fetal life or about the developmental biology of the human testisin general. Consequently, clinical analysis of adult male fertility is largely correlative and depends on a normal range for other adult males.
A detailed understanding of the developmental biology of reproduction is dependent on a precise knowledge of the size of the founder population of male GC. The only other study of human fetal testicular apoptosis concentrated on the first trimester of pregnancy; in that study Quenby et al. (10) found that apoptosis is observed in all cellular compartments of the testis (GC, SC, and LC) at 6–8 wk of pregnancy. This coincides with the appearance of primordial GC in the yolk sac, as early as 4–6 wk postfertilization, after which they migrate to the genital ridge, where adhesion, proliferation, and survival are all important in determining final cell numbers (14, 15). As the total pool of male GC can change during gonadal development, it is equally important to know what happens to the fetal gonadal cells during the entire pregnancy. This may become particularly relevant if sperm production is halted in adult life, as in patients with infertility. Although other investigators have successfully used ISEL for identification of apoptosis in gonadal tissue in humans (6), this is the first study that has addressed the ontogeny of fetal testicular apoptosis throughout pregnancy.
In the present investigation we exploited a defined antigenic marker in conjunction with ISEL, morphology, and regional localization to distinguish between apoptosis in the three major gonadal cell types. The protooncogene c-Kit receptor is expressed on a variety of tissues, including GC in developing gonads. c-Kit is particularly useful as a GC marker, because this is the only intratubular cell on which it is expressed. c-Kit is thought to regulate primordial GC migration, proliferation, and apoptosis during fetal gonadal development and is also thought to be involved in spermatogonial proliferation in the adult (16). The subtle contribution of c-Kit to germ cell development is underlined by mouse mutation studies. Thus, distinct abnormalities of c-Kit gene expression (e.g. deletions, point mutations, or alternative splicing defects) can lead to different types of aberrant spermatogenesis. These include a decrease in both primordial germ cell migration and spermatogonia proliferation. In the present study we also used c-Kit for identification of LC, as these cells are extratubular and can be easily distinguished from the intratubular GC population in terms of both size and location.
The results of the present study indicate that in humans, testicular apoptosis starts as early as the first trimester of pregnancy. It occurs in all cell types and continues throughout fetal gestation. Apoptotic cell death is highest in the LC population, followed by GC and SC, and there is a negative correlation between GC and SC number and gestational age. In addition, the proportion of SC per tubule decreased with gestational age and was largely responsible for a developmentally dependent fall in the overall number of intratubular cells. As apoptosis was not age dependent, this is likely to reflect reduced rates of proliferation in one or both of the intratubular cell populations.
What controls the survival and death of germ cells remains unclear. One possibility is that survival factors (such as the c-Kit ligand) are limiting in the testes and that during GC development there is evolutionary pressure for selection of only a subpopulation of cells as in other tissues (17). Alternatively, those cells that are multiplying rapidly may be more prone to apoptosis, a safety mechanism to eliminate potentially cancerous tissue (17). In this respect, as in many other developing organs, gonadal tissues are highly proliferative before commitment to a defined cell lineage.
The gestational age-dependent decrease in total cell number in testicular cells in the current study is comparable to findings in the mouse, where SC and GC multiplication and apoptosis have been shown to be closely linked (6, 7, 18). However, the exact cause of increased apoptosis in the relatively slowly multiplying LC population remains unclear. Various intrinsic factors, including genetic modulators (6), sex steroids such as testosterone (19, 20), and pituitary gonadotropins (6, 21), may control gametogenesis in adults. FSH is the main survival factor for adult GC, and FSH and IGF-I attenuate apoptosis in cultured porcine granulosa cells (22). Stem cell factor acts as an important survival factor for GC in the adult rat testis, and the FSH prosurvival effect on GC is mediated partially through the c-Kit pathway (21). Adenophysectomy can compromise the survival of immature rat GC in testis (6, 23). Although testosterone may act as a survival factor for fetal gonads, FSH receptors are absent, at least in the first trimester (10).
The apoptosis shown in the fetal gonad in this report is physiological, as the pregnancies were uncomplicated, and gonads were derived from structurally and karyotypically normal fetuses. The termination of pregnancy was undertaken for contraceptive failures or social reasons. There was no known maternal, placental, or fetal pathology in the pregnancy failures (miscarriages or terminations).
Although the molecular and genetic mechanisms that modulate gonadal cell numbers are not yet fully understood, the implication of several pro- and antiapoptotic genes, including Apaf-1 (24), the death receptor Fas (25), and the tumor suppressor p53 (26), suggests a major role for programmed cell death in testis development. It is possible that extrinsic factors, including maternal physiology, may be equally important, and further studies are required to distinguish between the two. As apoptosis is a major mechanism for eliminating unwanted cells during normal development, the clinical challenge will be to manipulate apoptotic cell death appropriately in the pathophysiological situation without affecting normal GC development and fertility.
Acknowledgments
We are grateful to Kirsty Greenwood for excellent technical assistance with this project.
Footnotes
This work was supported in part by the Michael and Morvan Heller Charitable Trust (to R.C.) and the Garfield Weston Foundation (to H.M.).
Abbreviations: GC, Germ cells; H and E, hematoxylin and eosin; ISEL, in situ end labeling; LC, Leydig cells; SC, Sertoli cells.
Received October 10, 2000.
Accepted May 24, 2001.
References
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); second trimester, B (
); and term, C (
). The results are presented as the mean ± SEM data from five (groups A and C) or seven (group B) fetuses. B, Relationship between GC and SC apoptosis in the developing human testis. Apoptosis in the two cell populations was compared by linear regression using Spearman’s correlation coefficient. A significant correlation was found to occur between apoptosis in the two cell types (P < 0.001). The three developmental groups are indicated as follows: 
, first trimester; •, second trimester; 