Number of germ cells and somatic cells in human fetal testes during the first weeks after sex differentiation

Eske Bendsen1,3, Anne Grete Byskov2, Steen B. Laursen1, Hans-Peter E. Larsen2, Claus Y. Andersen2 and Lars G. Westergaard1

1 Fertility Clinic, Department of Obstetrics and Gynaecology, University Hospital of Odense, Sdr. Boulevard 29, 5000 Odense C and 2 Laboratory of Reproductive Biology, Juliane Marie Center for Children, Women and Reproduction, University Hospital of Copenhagen, Rigshospitalet, Blegdamsvej 9, 2100 København Ø, Denmark 3 To whom correspondence should be addressed. e-mail: eske.bendsen{at}dadlnet.dk


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: This study presents the number of germ cells and somatic cells in human fetal testes during week 6 to week 9 post conception, i.e. the first weeks following sex differentiation of the testes. METHODS: One testis with attached mesonephros from each of 10 individual legal abortions was used. After recovery of the fetus, the testes were immediately isolated, fixed and processed for histology. The optical fractionator technique, a stereological method, was utilized to estimate the total number of germ cells in ten testes and somatic cells in six of them. RESULTS: The number of germ cells per testis increased from ~3000 in week 6 to ~30 000 in week 9. The ratio of germ cells to Sertoli cells was ~1:11 and the ratio of germ cells to somatic cells was ~1:44 throughout this period. CONCLUSIONS: For the first time, germ cell and somatic cell number have been determined during early human fetal testis development. Knowledge of the number of germ cells in this period may be very important, because several environmental pollutants are suspected to result in decreased semen quality in men born of mothers exposed to these pollutants during pregnancy.

Key words: fetal testes/first trimester/germ cells/human/in vivo


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The primordial germ cells originate in the yolk sac and migrate to the gonadal anlage early in fetal life (McKay et al., 1953Go). Sexual differentiation begins at ~6 weeks post conception (p.c.) (Witschi, 1963Go; Wartenberg, 1982Go; Lovell-Badge and Robertson, 1990Go), governed by the sex-determining region on the Y-chromosome (Lovell-Badge and Robertson, 1990Go; Koopman et al., 1990Go). At this time, the male primordial germ cells, now defined as prespermatogonia, and the Sertoli cells become enclosed in the newly formed testicular cords. Outside these cords Leydig cells differentiate (De Krester and Kerr, 1994Go). The mesonephric structures have connected to the testis in the cranial end and may influence the development and steroidogenic activity of the testis, as suggested during culture of fetal rabbit testes (Byskov and Grinsted, 1981Go; Grinsted, 1982Go).

The somatic cells of the newly differentiated testis immediately start to secrete anti-Müllerian hormone (Josso et al., 1993,Go 1998) and testosterone (Winter et al., 1977Go), securing the male differentiation of the secondary sex-organs. The prespermatogonia multiply by mitotic divisions within the cords without embarking on meiosis as observed in the fetal ovary. Also Sertoli cells and Leydig cells multiply during this period (Byskov, 1986Go).

The interaction between prespermatogonia and somatic cells in the testes is crucial for survival and proliferation of the prespermatogonia, since spermatogenic cells are unable to mature without the presence of Sertoli cells (Sharpe, 1993Go). Presumably, a certain numerical relationship between somatic cells and prespermatogonia is needed for normal testicular development. Consequently, it is of importance to have precise and unbiased estimates of the number of prespermatogonia and somatic cells in order to evaluate normal testicular development and to monitor effects of substances, which may affect or interfere with normal development (Bendsen et al., 2001Go).

The number of primordial germ cells has been evaluated in undifferentiated human gonads (Witschi, 1948Go), and the number of prespermatogonia has been estimated in human sex-differentiated testes obtained from third trimester fetuses and from boys during the first year of life (Witschi, 1948Go; Müller and Skakkebaek, 1983Go; Cortes, 1990Go). However, the number of prespermatogonia and somatic cells in sex-differentiated testes from first trimester fetuses has not yet been determined.

The aim of the present study was to obtain precise estimates of these numbers. We have evaluated 10 testes (from 10 fetuses) isolated from legal induced abortions performed during the last part of the first trimester of pregnancy (from and including week 6 to week 9). The number of prespermatogonia was estimated in ten testes and somatic cells in six of them by use of a stereological method, the optical fractionator technique, which is known to provide precise and unbiased estimates of cell numbers in different organs (Gundersen et al., 1988Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human fetal gonads
Human fetuses were obtained from women referred to the Department of Obstetrics and Gynaecology, University Hospital of Odense, Denmark, for legal induced abortion in the first trimester of pregnancy. The women wanted to end their pregnancy of social reasons. The women were healthy and had no history of medical treatment. In all cases the women were informed both orally and in writing about the aim and procedures of the project, before they gave their written consent to participate. The Medical Ethics Committee in the counties of Vejle and Fyn, Denmark, approved the project.

Operation technique
The operations were done according to the routine procedures in the department, with slight modifications in order to prevent damage to the fetus in utero, and carried no additional risk for the participating women (Nauert and Freeman, 1994Go). In all abortions, the fetuses were monitored with ultrasound equipment (Sonoline Prima; Siemens, Denmark). The cervical canal was dilated, and a curette (Synevac vacuum curette; Berkely Medevices, Inc., USA) was inserted. A sterile 50 ml syringe (Plastipak; Becton–Dickinson, USA) was connected to the curette and a vacuum was applied manually. After recovery, the fetus was placed in a sterile cup with culture medium (Dulbecco’s modified Eagle’s medium/Ham’s F-12; GibcoBRL Life Technologies, Denmark). All handling of the tissue was performed under sterile conditions. The fetuses were dissected under a stereomicroscope a few minutes after aspiration from the uterus. The gonads were still present and isolated in ~70% of the fetuses.

Determination of fetal age
In this study, we only used true fetal ages, i.e. the time p.c., in contrast to gestational age, i.e. the time after the first day in the last period. The fetal age was determined by measuring the length of limbs and feet (Evtouchenko et al., 1996Go).

Determination of fetal sex
The chromosomal sex was determined by PCR technique (Nakahori et al., 1991Go) and confirmed by the morphological appearance of the histological sections.

Histology
One testis–mesonephric–duct complex from each of 10 fetuses was included in the study. The decision whether to use the right testis or the left testis was done at random. However, the original position of a few testes could not be determined (the organs in the fetal abdomen had been displaced during the abortion). The remaining 10 contralateral testes were used for other research purposes. The testis–mesonephric duct complex was removed from the fetus in toto and placed in Bouin’s fixative (Bie & Berntsen a/s, Denmark) for up to 4 h, depending on the size of the complex, processed for paraffin embedding, cut into 30 µm thick serial sections (see Stereology) and stained with haematoxylin and periodic acid–Schiff reagent.

Six of the testes were cut in the longitudinal direction. Four of the testes were cut transversely to the longitudinal direction, which made it possible to determine the topographic distribution of the prespermatogonia in the testes. The topographic distribution was illustrated by the variation in the concentration of prespermatogonia between each of the serial sections, from the cranial end to the caudal end of the testes.

Identification of cell types
The cells were defined by the morphology of the nuclei (Bendsen et al., 2001Go).

Counting of cells
The number of prespermatogonia was estimated in all ten testes. The number of somatic cells was estimated in six of the testes.

Prespermatogonia
Only cells that were enclosed by the basement membrane of a testicular cord and showing prespermatogonial specific characteristics (see below) were counted as prespermatogonia. In a few places, it could not be determined whether a cell resembling a prespermatogonium was enclosed by the basement membrane of a testicular cord or not, and because Leydig cells (which are not enclosed by the cords) morphologically resemble prespermatogonia, these cells were not counted as prespermatogonia. Naturally, it is not possible to state exactly how many prespermatogonia are ‘missed’ in this way. However, in only a very few cases (<10 per testis) was there a doubt in our minds whether a particular cell was a prespermatogonium or a Leydig cell and therefore the problem is of no practical importance.

Somatic cells
The total number of somatic cells counted encompasses Sertoli cells and all cells situated outside the cords, e.g. Leydig cells, peritubular cells and mesenchymal cells. Sertoli cells were also counted separately in order to obtain precise estimates of this specific cell line and to determine the ratio between prespermatogonia and Sertoli cells.

Stereology
The number of cells was estimated using the optical fractionator technique (Gundersen et al., 1988Go). This technique provides precise and unbiased estimates of the total number of cells in an organ, by counting cells in only a fraction of that organ (West and Gundersen, 1990Go; West et al., 1991Go, 1996; Feinstein et al., 1996Go). The cells are counted in so-called ‘optical dissectors’, based on parallel thin optical sections inside a thick section (25–50 µm) of the testis (see below). Even though the concentration of cells in testicular tissue is relatively large, it is possible to ‘look through’ these thick sections. The precision of this technique is affected by variation in the thickness of the sections. Our experience is that a section thickness of 30 µm is optimal for human fetal testicular tissue, i.e. a stable thickness from section to section is obtained. The Computer-Assisted Stereological Toolbox (CAST)–grid system (Olympus, A/S, Denmark) was used for all counting.

Sections
Cells were estimated from 10 to 15 sections that were selected at equally spaced intervals along the entire extent of the testis. The first section was randomly selected between the first two to six sections, depending of the size of the testis, and thereafter every second to sixth section was sampled.

Sectional area
By positioning an unbiased counting frame of known area, 134–668 µm2, at the co-ordinates of a rectangular lattice superimposed on the section, a systematic random sample of the area of each of the sections was achieved.

Section thickness
The thickness of each of the sections, used in the analysis, was measured at every fifth point selected from the co-ordinates used to position the dissector samples, where cells were counted in the counting frame. A known fraction of the thickness of the sections was sampled with optical dissectors at each position of the lattice. The counting frame was moved a known distance (h), 10 µm, through the thickness of the section.

Counting
Optical dissector counting rules (Gundersen et al., 1988Go; West and Gundersen, 1990Go), based on the original physical dissector counting rules (Sterio, 1984Go; Gundersen, 1986Go), were used to count the number of cells in the optical dissectors. The counting unit was the nucleus of the cell.

Estimates
Estimates of the total number of cells in the gonad were calculated as the product of the number of cells counted with the optical dissectors and the reciprocals of the fraction of sections sampled, the fraction of the sectional area sampled, and the fraction of the section thickness sampled.

The efficiency of the fractionator is expressed by a ‘coefficient of variation’ (CV) of the individual number estimates (N). CV depends on (i) homogeneity of the cell density in the testis, (ii) variation in the section thickness, and (iii) the number of sections sampled. On the basis of similar analysis performed on other structures (Gundersen and Jensen, 1987Go; West 1993Go, 1996), we decided that a CV (N) of 0.1 would be adequate.

Counting of prespermatogonia
The actual number of prespermatogonia counted per testis was 108–386 dispersed in 10–15 sections. CV (N) was 0.06–0.10%.

Counting of Sertoli cells and other somatic cells
The actual number of Sertoli cells counted per testis was 110–272 dispersed in 10–15 sections. CV (N) was 0.06–0.10%. The actual number of somatic cells (including Sertoli cells) counted per testis was 135–312 dispersed in 10–15 sections. CV (N) was 0.07–0.10%.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Morphology of the testes
The length of the testes was ~2 mm in week 6 (Figure 1) and increasing to >3 mm in week 9. The diameter of the testes increased from ~0.3 mm in week 6 (Figure 1) to ~1 mm in week 9. Testicular cords were present in all testes, although better defined after week 6. Two zones, a peripheral and a central one, were present (Figure 1). The centre was occupied with a network of closely packed, coiled cell-cords with a diameter of ~20–45 µm without prespermatogonia, the rete testis cords (Figures 1 and 1). The cords consisted of irregular cells with elongated nuclei 3–7 µm in diameter, and were confined by a basement membrane. In the periphery, solid testicular cords, consisting of Sertoli cells and prespermatogonia and surrounded by a basement membrane, were present (Figures 1, 1 and 1). The outside of the testicular cords was lined with spindle-shaped cells exhibiting dense nuclei. The diameter of the testicular cords was in the range of 40–130 µm. At some places, in particular in testes older than 6 weeks, the Sertoli cells were elongated and formed a columnar epithelium. At other places the Sertoli cells were more irregular and appeared as a multi-layered, irregular epithelium. The nuclei measured up to 15 µm in length and 3–5 µm in cross-section. The basement membrane lining the rete testis cords continued in the basement membrane lining the testicular cords. Prespermatogonia showed spherical nuclei with a diameter up to 9 µm. The nuclei had one large irregular nucleolus and smaller nucleoli. Leydig-like cells were seen between testicular cords (Figure 1). Based on morphological characteristics, only a very few prespermatogonia were pyknotic (<20 per testis).



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Figure 1. Testis–mesonephric duct complex from a fetus (6 weeks) fixed immediately after the abortion. The gonad (g) is seen at the top. Rete testis (rt), mesonephros (M), Wolffian duct (Wo) and Müllerian duct (Mü) are seen at the bottom. Figures 1–5. Counting was performed on 30 µm thick sections. However, when photographing these thick sections many of the cells would have looked blurred, and therefore the sections illustrated here are only 5 µm thick.

Figure 2. Higher magnification of Figure 1 showing several testicular cords confined by basement membranes (arrows). The cords consist of prespermatogonia (p) and Sertoli cells.

Figure 3. Testis from a fetus (8 weeks) fixed immediately after the abortion. The testicular cord has a prespermatogonium in mitosis (m).

Figure 4. Same testis as illustrated in Figure 3. A rete testis cord is confined by a basement membrane (arrows) without prespermatogonia.

Figure 5. Same testis as illustrated in Figure 3. A testicular cord is confined by a basement membrane (arrow). The morphology of prespermatogonia (p) and Leydig cells (L) is similar and the cells cannot be separated from each other on morphological characteristics. However, prespermatogonia are situated inside the testicular cords and Leydig cells outside. Notice the morphological similarity between the two cells.

 
Counting of prespermatogonia
In week 6 the number of prespermatogonia per testis was ~3000 and increased to ~30 000 in week 9 (Table I). The topographic distribution of prespermatogonia was determined and showed that prespermatogonia were evenly distributed throughout the long axis of the testis (Table II).


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Table I. Number of prespermatogonia and somatic cells in human fetal testes
 

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Table II. Mean concentration (mean number of cells per counting frame) of prespermatogonia in the cranial end and in the caudal end in human fetal testes
 
Counting of Sertoli cells and other somatic cells
In week 7 the total number of somatic cells was ~600 000 and increased to ~1 750 000 in week 9 (Table I). In week 7 the number of Sertoli cells was ~150 000 and increased to ~450 000 in week 9 (Table I). The ratio between the number of prespermatogonia and Sertoli cells, and the ratio between prespermatogonia and the total number of somatic cells, remained constant during the first trimester of pregnancy (Table III).


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Table III. Ratio between prespermatogonia (P) and Sertoli (S) cells (Ratio 1), and ratio between prespermatogonia and total number of somatic cells (Ratio 2)
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study is the first to describe the number of prespermatogonia and somatic cells in sex-differentiated first trimester fetuses from weeks 6 to 9 inclusive. We have shown that ~3000 prespermatogonia are present at week 6 and that the number increases to ~30 000 at week 9 (p.c.).

The number of primordial germ cells has been evaluated in two human embryos <6 weeks of age and estimated to be ~450–1400 per embryo (Witschi, 1948Go). The number of prespermatogonia in human testes from second trimester fetuses is presently unknown. The number of prespermatogonia in third trimester fetuses has been estimated as 3 800 000 per testis (range: 1 500 000–11 300 000) (Cortes, 1990Go). In boys <1 year old, the number has been estimated as 6 500 000 per testis (range: 2 200 000–18 000 000) (Müller and Skakkebaek, 1983Go).

In the present study, the ratio between prespermatogonia and Sertoli cells was ~1:11 and the ratio between prespermatogonia and the total number of somatic cells was ~1:44. These ratios were constant throughout the period studied, indicating that the rate of proliferation of prespermatogonia and somatic cells are of the same magnitude.

Knowledge of the numbers of germ cells as well as somatic cells described in this study are important, since these cells represent the basis for further development and function of the testis. The number of germ cells in the developing testis may be negatively affected if exposed to environmental pollutants with a suspected negative impact on sperm counts later in life. In fact, recently we have shown that octylphenol, a chemical present in the environment and capable of mimicking certain effects of estrogen, can harm human prespermatogonia in vitro (Bendsen et al., 2001Go).


    Acknowledgements
 
The excellent technical assistance of Inga Husum, Laboratory of Reproductive Biology, University Hospital of Copenhagen, is greatly acknowledged. Christian Olesen and Nanna D.Rendorff, Department of Medical Genetics, Institute of Medical Biochemistry and Genetics, University of Copenhagen, are acknowledged for their help with sex determination of the testes by PCR technique. The study was sponsored by the Faculty of Health Sciences, University of Southern Denmark, by the Danish Medical Research Council, nos. 9700832 and 9502022, and by the Danish Environmental Research program (Center for Environmental Estrogen Research).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Submitted on October 2001; resubmitted on August 8, 2002. accepted on October 15, 2002