1 The University Laboratory of Seminology and Immunology of Reproduction, Dept of Medical Pathophysiology, University of Rome `La Sapienza', Policlinico `Umberto I', 00161, Rome and 2 Dept of Clinical Biochemistry, Istituto Superiore di Sanità, Rome, Italy
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Abstract |
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Key words: human semen/immature germ cells/Percoll gradient
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Introduction |
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Immature germ cells are always present in human semen in variable numbers not necessarily related to spermatozoa concentration, although some authors have reported an inverse correlation (Sperling and Kaden, 1971). Recently, various attempts have been made in order to identify specific sub-populations of immature germ cells for clinical purposes. In particular, spermatids from human semen in patients with non-obstructive azooospermia have been collected and used in intracytoplasmic sperm injection (ICSI) programmes, resulting in normal births (Tesarik et al., 1995
). For the same clinical purpose, a simple Percoll gradient has allowed other authors to collect spermatids identified both by staining and by evaluating haploidy using fluorescence in-situ hybridization (Angelopoulos et al., 1997
). More recently, a purified population of immature germ cells was obtained from the testicular tissue of azoospermic men employing a fluorescent activated cell sorter (Aslam et al., 1998
).
The aim of this paper is to report on the use of a discontinuous Percoll gradient method, modified to enable the best separation possible of immature germ cells from the other cells found in the ejaculate, in order to obtain a cellular suspension free of spermatozoa for diagnostic and experimental purposes.
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Materials and methods |
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The ejaculates were divided into two aliquots to evaluate intra-assay variation by duplicates, except cases 6 and 7, owing to the low volume of the semen.
Discontinuous Percoll gradient
Isotonic 100% Percoll (Sigma Chemical Co, St Louis, MO, USA) was obtained by adding nine parts of Percoll to one part of Earle's salt solution 10X (Imperial, UK). The Percoll 100% was diluted with Earle's salt solution to obtain the following dilutions: 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 100%. The gradient column was prepared in a 15-ml Falcon tube by gently layering 1 ml of each of the above-mentioned solutions, starting from the 100% fraction at the bottom (0.5 ml for each dilution was used when the semen volume available was 0.5ml). One millilitre of the semen (or the whole semen in cases with less than 1 ml of ejaculate) was diluted with Earle's solution (1:2) and centrifuged at 400 g for 15 min at 18°C. The semen cell pellet was resuspended in 0.5 ml of Earle's solution. The semen cell suspension was gently stratified on top of the discontinuous Percoll gradient and centrifuged for 25 min at 800 g at 18°C. The single Percoll fractions were separated and each was put into a single test tube. The single fractions were analysed in order to select the ones with the greatest concentration of immature germ cells. The fractions which contained the majority of the immature germ cells (30%, 35%, 40% and 45%) were mixed with Earle's solution (1:2) and centrifuged at 150 g for 10 min at 18°C. The pellet was resuspended in 1 ml of Earle's solution and the cell concentration was evaluated.
Cell identification
Immature germ cells were counted using a Thoma counting chamber, evaluating at least 100 cells. The May-Grünwald-Giemsa staining technique was used to identify various kind of germ cells (spermatogonia, spermatocytes I and II, spermatids) and leukocytes (Schenck and Schill, 1988; Foresta et al., 1992
). To further define the leukocyte contamination, the pool of the cell fractions containing the major quantity of germ cells (from 30% to 45%) was tested with an immunofluorescence microscopic technique using anti-CD45 fluorescein isothiocyanate (FITC) monoclonal antibodies (mAb) (El-Demiry et al., 1986
). A sample of 100 µl of the cell suspension obtained by pool of the fractions from 30% to 45% was mixed with 10 µl of mAb (CD45 anti-Hle-1; Becton Dickinson, Mountain View, CA, USA) conjugated to FITC. After a 30-min incubation at room temperature in the dark, the cells were washed twice with PBS, resuspended in 100 µl of PBS and analysed (at least 100 germ cells) using fluorescence microscopy (Leica Dialux 22, 50x and 100x) with the following filter combination: 490 nm excitation and 530 nm barrier filters.
With the same aim, leukocyte identification by dual-colour immunophenotyping was performed using the following Becton Dickinson matched murine monoclonal antibody reagents directly conjugated to phycoerythrin (PE) or fluorescein isothiocyanate (FITC): anti-Leu M3 PE (CD14)/anti-Hle-1 FITC (CD45) (monocytes and leukocytes); anti-Leu 15/CR3 PE (CD11b) (granulocytes); and anti-Leu 12 FITC (CD19)/anti-Leu-4 PE (CD3) (B and T lymphocytes). A sample of 100 µl of the cell suspension obtained by pooling of the fractions from 30% to 45% was stained using 5 µl of monoclonal antibody reagents. This suspension was incubated for 20 min, at room temperature, in the dark. After incubation the cells were washed three times with PBS and analysed (evaluating 25 000 cells) using a Ortho Cytoron Absolute 4 flow cytometer (Ortho Instruments).
Data obtained from side scatter against forward scatter allowed us to study cellular population. Debris noise was eliminated with the discriminator. The data were reported as the percentage positivity of the total population.
Statistical analysis
Mean values of duplicates of the immature germ cell concentrations were used for each sample. Descriptive analysis of the immature germ cell concentrations was performed in the four groups of ejaculates using range, mean and SD.
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Results |
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The immature germ cell types most frequently found were primary spermatocytes and spermatids. In the ejaculates of patients suffering from spermatogenesis arrest (cases 9 and 10), we found spermatocytes, but an absence of spermatids in the one case of primary spermatocyte arrest. However, in the case of spermatid arrest, we found all the germ cells, including spermatids. This agrees perfectly with the results of testicular cytology and histology.
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Discussion |
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Primary spermatocytes are more numerous than secondary spermatocytes, perhaps owing to the shorter life of the latter. Spermatids are the most frequent cells seen in the ejaculate both in fertile and infertile subjects, obviously excluding cases of spermatogenic arrest (Fedder et al., 1993). Spermatogonia have a scanty cytoplasm with a round nucleus 67 µm width and 1 or 2 nucleoli. Primary spermatocytes are broad cells with a large central nucleus 89 µm in diameter in which it is possible to recognize the chromosomal spindle and a large central nucleolus 12 µm in diameter. They are very fragile cells, easily damaged by preparation techniques. Secondary spermatocytes are small round cells, with a central condensed nucleus 67 µm in diameter (sometimes two nuclei). Initial spermatids are small round cells with an condensed nucleus (sometimes two nuclei). Elongated spermatids have characteristics that, depending on their maturity, can make them similar to spermatozoa.
As mentioned in the Introduction, to date very little attention has been given to the evaluation of immature germ cells in the semen and yet the separation of these cells could be extremely useful for research and diagnostic purposes. Two simple examples can be given to support this. The study of immature germ cells in the ejaculate would help to increase our knowledge of the normal and pathological chromosomal arrangements of the spermatogenic line. In fact, it is now possible to employ molecular probes to identify specific DNA fragments of interphasic cells (interphasic cytogenetics), using in-situ hybridization. This technique allows us to study single chromosomes and their numeric (aneuploidia) and structural alterations (deletions, translocations). Fertile and infertile subjects with various andrological pathologies (cryptorchidism, testicular cancer, etc.) can be evaluated. For example, data regarding the hyperploidia of chromosome 1 in seminoma and in carcinoma in situ of the testis could permit early diagnosis by studying the semen instead of requiring testicular biopsy (Giwercman et al., 1987; Skakkebæk et al., 1987
; Meng et al., 1996
; Salanova et al., 1996
). These complex cytogenetic techniques must employ cellular samples, as much as possible free not only of debris and leukocytes but also of spermatozoa, in order to avoid cellular overlapping that can make microscopic evaluation difficult.
Moreover, recent progress in the field of assisted reproduction has substantially modified the management of azoospermic patients as well as the concept of azoospermia itself. In fact, it is now possible to use spermatozoa obtained from the epididymis and from the testis (Craft et al., 1993; Silber, 1994
). In addition, immature haploid germ cells (spermatids) can now be taken from the testis and from semen for microinjection techniques (Edwards et al., 1994
; Silber and Lenahan, 1995
; Tesarik et al., 1995
, 1996
, Tesarik and Mendoza, 1996
). Also with this technique, it is important to have as pure a sample as possible, in order to identify easily the spermatids necessary for the microinjection procedure.
Various methods have been proposed in order to select different cellular populations; discontinous gradient separation is one of the most frequently used techniques. This method employs the centrifugation of a gradient column constituted by a viscous liquid whose density increases gradually from top to bottom of the test tube. Such procedures are based on the differences of cell dimensions and on the speed of centrifugation. In fact, a sample, stratified on the gradient column and centrifuged for a sufficient time, passes through the gradients forming zones of sedimentation at the gradient interfaces, each one containing cells characterized by a specific velocity of sedimentation. This velocity depends on the density and dimension of the cell, on the density and viscosity of the medium and how much the cell differs from the spheric form. For these reasons we used a gradient comprising numerous fractions of Percoll with minimum differences of density. This allowed us to separate not only immature germ cells from spermatozoa, which are very different in form and density, but also to separate immature germ cells from leukocytes which are sometimes very similar.
In seminology, the Percoll gradient is the most widely used separation technique. In order to obtain the best population of spermatozoa, Berger et al. (1985) proposed the use of 40, 55, 70, 80, 90 and 100% gradients. However, round cells, leukocytes and immature germ cells were packed together in the upper fractions. Recently, a Percoll gradient with only three fractions (5070100%) was proposed by Angelopoulos et al. (1997). In this paper, a good separation of round spermatids was obtained in fraction 70%. Difficulties were seen when the spermatid concentration was low. Germ cells were then obtained by pooling the 70% fraction and the interface of fraction 50%. Moreover, this was possible only if the leukocyte concentration was low; in all the other cases, leukocyte contamination made the cell identification difficult.
In this paper we show that, in the patients analysed, the number of immature germ cells isolated from the samples varied considerably both in fertile and infertile subjects and between the various groups of andrological pathologies. In particular, the highest concentrations were found in subjects with genital tract infection. In this case, we postulate that the inflammation induced a greater exfoliation of immature germ cells from seminiferous tubules. However, also in these samples, our method achieved a good separation of immature germ cells with scanty leukocyte contamination.
Our study demonstrates that the fractions from 30 to 45% showed the highest concentration of immature germ cells and the lowest leukocyte contamination. These data are supported by the use of permanent staining techniques that give a morphological definition even if they require a high level of experience from the analyst. In fact, spermatids and spermatocytes can seem, in some cases, very similar to leukocytes and, due to the presence of more than one nucleus, they can seem like polymorphonuclear neutrophils with a multilobulated nucleus. To overcome these possible mistakes, we also used immunofluorescence microscopy and flow cytometry analysis: rapid and simple methods that were particularly useful to discriminate the various subpopulations of leukocytes in the Percoll fractions.
These data indicate that our method can separate most of the immature germ cells, not only spermatids, from the other cells with a low level of leukocyte contamination (6%). The method also allows the successive identification of the different spermatogenic cellular types and, consequently, the confirmation of cytologic- and histologic-diagnosed maturative arrest (cases 6 and 7).
In conclusion, many situations require the separation of immature germ cells from spermatozoa, leukocytes and debris in order to enable the best unstained identification of immature germ cells. This separation could also serve to improve our understanding of spermatogenetic disorders. The modified Percoll gradient set up and described above can provide the purified immature germ cell suspensions necessary for certain diagnostic and research purposes.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 26, 1998; accepted on December 7, 1998.