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
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Abstract |
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Key words: fetal testes/first trimester/germ cells/human/in vivo
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Introduction |
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The somatic cells of the newly differentiated testis immediately start to secrete anti-Müllerian hormone (Josso et al., 1993, 1998) and testosterone (Winter et al., 1977
), 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, 1986
).
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, 1993). 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., 2001
).
The number of primordial germ cells has been evaluated in undifferentiated human gonads (Witschi, 1948), 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, 1948
; Müller and Skakkebaek, 1983
; Cortes, 1990
). 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., 1988).
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Materials and methods |
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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, 1994). 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; BectonDickinson, 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 (Dulbeccos modified Eagles medium/Hams 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., 1996).
Determination of fetal sex
The chromosomal sex was determined by PCR technique (Nakahori et al., 1991) and confirmed by the morphological appearance of the histological sections.
Histology
One testismesonephricduct 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 testismesonephric duct complex was removed from the fetus in toto and placed in Bouins 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 acidSchiff 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., 2001).
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., 1988). 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, 1990
; West et al., 1991
, 1996; Feinstein et al., 1996
). The cells are counted in so-called optical dissectors, based on parallel thin optical sections inside a thick section (2550 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, 134668 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., 1988; West and Gundersen, 1990
), based on the original physical dissector counting rules (Sterio, 1984
; Gundersen, 1986
), 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, 1987; West 1993
, 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 108386 dispersed in 1015 sections. CV (N) was 0.060.10%.
Counting of Sertoli cells and other somatic cells
The actual number of Sertoli cells counted per testis was 110272 dispersed in 1015 sections. CV (N) was 0.060.10%. The actual number of somatic cells (including Sertoli cells) counted per testis was 135312 dispersed in 1015 sections. CV (N) was 0.070.10%.
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Results |
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Discussion |
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The number of primordial germ cells has been evaluated in two human embryos <6 weeks of age and estimated to be 4501400 per embryo (Witschi, 1948
). 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 00011 300 000) (Cortes, 1990
). In boys <1 year old, the number has been estimated as 6 500 000 per testis (range: 2 200 00018 000 000) (Müller and Skakkebaek, 1983
).
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., 2001).
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Acknowledgements |
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References |
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Submitted on October 2001; resubmitted on August 8, 2002. accepted on October 15, 2002