1 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Chancellors Building, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB and 2 Section of Child Life and Health, Department of Reproductive and Developmental Sciences, University of Edinburgh, Edinburgh EH9 1LW, UK
3 To whom correspondence should be addressed. r.sharpe{at}hrsu.mrc.ac.uk
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Abstract |
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Key words: FSH/germ cells/LH/Sertoli cells/testosterone
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Introduction |
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From studies undertaken on autopsy specimens of boys who died in the first 2 years of life, it is evident that testis weight/size increases by 2-fold from birth up to 5 months of age and then remains unchanged up to age 2 years (Bidlingmaier and Hilscher, 1989
). This increase in testis size may be due in part to increase in Sertoli cell number (Cortes et al., 1987
), a change that is also indicated by the parallel rise in blood levels of inhibin-B (Andersson et al., 1998
; Anderson and Sharpe, 2000
). It is also clear that germ cell number in the human testis increases by
3-fold in the first 5 months of life (Muller and Skakkebaek, 1983
; 1984; Bidlingmaier and Hilscher, 1989
), and this observation led the researchers involved to postulate that the increase might be driven by the associated activation of gonadotrophin secretion or because of the coincident increase in intratesticular testosterone levels during the neonatal period. More recently, it has been argued that apparent deficiencies in germ cell numbers and their differentiation during the first 6 months of life in cryptorchid testes of boys are a direct result of deficiencies in intratesticular androgen production in such testes (Huff et al., 1993
; 2001; Hadziselimovic and Herzog, 2001
). Taken together, the indirect data from the various studies referred to are consistent with a role for androgens (and/or gonadotrophins) in neonatal germ cell development in the human male. However, if this is the case then the effect must be indirect as neither the Sertoli cells nor the germ cells express androgen receptors during this period in either the human male or in the non-human primate, although conversion of androgens to estrogens and their action via estrogen receptor-
is a possibility (Williams et al., 2001
; McKinnell et al., 2001
; Sharpe et al., 2003
).
As the marmoset exhibits many developmental similarities to the human in terms of testis development, including a well-defined neonatal testosterone surge coincident with Sertoli cell proliferation (Lunn et al., 1994; 1997; Sharpe et al., 2000
; McKinnell et al., 2001
), it may be a good animal model in which to clarify the issues raised above. The aim of the present study was therefore to assess whether suppression of pituitarytesticular activity for the duration of the neonatal period (
1415 weeks; Lunn et al., 1997
; McKinnell et al., 2001
) in the marmoset by treatment with a GnRH antagonist exerted significant effects on germ cell proliferation and differentiation. As marmosets show a high (
80%) dizygotic twinning rate and male co-twins are more comparable than unrelated males with regard to testicular endpoints (e.g. Kelnar et al., 2002
; Sharpe et al., 2000
; 2002), the study used a pair-wise design with one twin being treated with vehicle and the co-twin being treated with the GnRH antagonist.
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Materials and methods |
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Tissue collection and processing
Testes were dissected free of connective tissue and the epididymis and then immersion-fixed for 16 h in Sorensons fixative. Testes were then reweighed, cut into quarters and processed for embedding in epoxy resin. Epoxy resin sections 1 µm thick were stained with 1% Toluidine Blue containing 1% borax (BDH, UK) at 60°C until a suitable staining intensity was obtained as determined by microscopic examination.
Germ cell counts
Germ cells were classified as either gonocytes, pre-spermatogonia or spermatogonia according to criteria outlined by Gondos (1993) and their position within the seminiferous cord. Thus in general, cells with no observable contact with the basal lamina were classed as gonocytes, those with some point of contact as pre-spermatogonia and those with their basal aspect fully in contact with the basal lamina and underlying Sertoli cells as spermatogonia. Germ cell volume per testis was determined using the Stereologer software programme (Systems planning and Analysis Inc., USA) and utilized an Olympus BHS microscope fitted with an automatic stage (Applied Scientific Instrumentation Inc., USA).
One section from each of two blocks per animal was examined under oil immersion using an Olympus x100 SPlan Apo Objective fitted to the Olympus BH2 microscope with a 121-point eyepiece graticule. The Stereologer software was used to select random areas for cell counting, and between 20 and 143 fields were counted; the number of fields counted was determined by the software programme and was based on optimizing the accuracy and comparability of the number of cell types counted in each animal. Points falling over the nuclei of gonocytes, pre-spermatogonia and spermatogonia were scored and expressed as a percentage of the 121 points possible.
Image-Pro Plus software programme (Media Cybernetics, USA) was used to measure nuclear diameters for germ cells (100 cells per animal) and mean nuclear volumes (MNV) were then calculated using the equation (Wreford, 1995
):
Germ cell numbers were then calculated using the equation:
Tissue sections were examined and photographed using a Provis microscope (Olympus Optical, UK) fitted with a digital camera (DCS330; Eastman Kodak, USA). Captured images were transferred to a computer (G4; Apple Computer Inc., USA) and compiled using Photoshop 5.0 (Adobe Systems Inc., USA) before being printed using an Epson Stylus 870 colour printer (Seiko Epson Corp., Japan).
Statistical analysis
As predominantly co-twins were used for this study, paired t-test comparison of testis weight and germ cell numbers in vehicle-treated (controls) and GnRH antagonist-treated co-twins was utilized. Comparison of testis weight and germ cell numbers in control animals at birth and at 1824 weeks of age used Students t-test. For all comparisons, data for germ cell numbers was log-transformed prior to analysis because of the considerable variation between animals and because of heterogeneity of variance.
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Results |
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Discussion |
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Based on the present findings in the marmoset, we consider that the postulated link between deficiencies in testosterone levels in cryptorchid testes in human neonates and subnormal germ cell numbers and/or differentiation (Hadziselimovic and Herzog, 2001; Huff et al., 2001
) is unlikely to reflect a causal relationship. However, this conclusion must remain tentative as there is a potentially important difference in the timing of early germ cell differentiation in the human and marmoset. In the testes of newborn boys, many of the germ cells present have already differentiated into spermatogonia or pre-spermatogonia even though gonocytes are still present (Fukuda et al., 1975
; Paniagua and Nistal, 1984
; Gondos, 1993
), and a similar picture is evident in the cynomolgus monkey (Fouquet, 1982
; Kluin et al., 1983
). This is clearly not the case in the marmoset in which spermatogonia are not evident in fetal life and gonocytes still remain the predominant germ cell type, even at the end of the neonatal period. It cannot be excluded that this difference could be important in the context of the present study, though a more rational interpretation is that marmosets exhibit slightly delayed timing of germ cell proliferation and migration/differentiation in perinatal life in comparison with the human. It is the completion of gonocyte transformation into spermatogonia that has been suggested as being promoted by intratesticular androgens in the human in the neonatal period (Hadziselimovic and Herzog, 2001
; Huff et al., 2001
), and this process was only attenuated in the present studies in marmosets in which the neonatal testosterone surge was suppressed. The fact that the relative proportions of the different germ cell types remained unchanged from controls in the GnRH antagonist-treated marmosets, leads us to conclude that the loss of hormones neonatally has an overall rather than a spermatogonia-specific effect, and this would be more consistent with an effect of gonadotrophin deprivation on the Sertoli cells. Furthermore, it is already established that suppression of the neonatal testosterone surge in the marmoset has no effect on fertility in adulthood (Lunn et al., 1997
) and only modest effects on spermatogenesis and germ cell volume per testis (Sharpe et al., 2000
).
Reliable assays for LH and FSH (and inhibin-B) are not available for the marmoset and this places obvious limitations on confirming beyond doubt the effectiveness of the GnRH antagonist treatment in suppressing gonadotrophin secretion. However, there are several pieces of indirect evidence that point to the effectiveness of the GnRH antagonist treatment regimen in the neonatal marmoset. Thus, the neonatal testosterone rise is completely suppressed (Lunn et al., 1994; 1997; McKinnell et al., 2001
) coincident with atrophy of the Leydig cells (Prince et al., 1998
), and Sertoli cell proliferation (which is primarily FSH-driven in the neonatal period) is suppressed (Sharpe et al., 2000
). Moreover, similar treatment of other neonatal primates or rodents with the same or similar long-acting GnRH antagonists is well-documented to cause major or complete suppression of gonadotrophin secretion (e.g. Mann et al., 1989
; 1999; Sharpe et al., 2000
). It is therefore reasonable to conclude that similar effective suppression was achieved in the neonatal marmoset. Consequently, it can be concluded with a degree of certainty that germ cell proliferation and differentiation in the neonatal period in the marmoset is largely gonadotrophin- and testosterone (and estrogen)-independent. Indeed, we would argue that this independence might be absolute, for although neonatal GnRH antagonist treatment undoubtedly reduced total germ cell numbers by the end of the neonatal period, it is likely that this resulted from altered germ cell survival (due to suppressed Sertoli cell function) rather than from altered proliferation or differentiation. We reached a similar conclusion regarding germ cell proliferation in the infantile marmoset testis, just prior to the onset of puberty, as this also appears to be gonado trophin- and testosterone-independent (Kelnar et al., 2002
; M.F.H. Brougham, unpublished data). The important question as to what factors are responsible for germ cell proliferation and differentiation in the neonatal and infantile periods remains unanswered.
In conclusion, the present findings in the marmoset suggest that the neonatal period of pituitarytesticular activity in primates plays only a minor, if any, direct role in regulating the germ cell proliferation and differentiation that occurs neonatally. Assuming that these findings are directly relevant to the human, it seems unlikely that the postulated causal relationship between reduced intratesticular testosterone and abnormal germ cell development in the neonatal cryptorchid testis in boys is tenable. Instead, the latter association may reflect an inherent defect in Sertoli and Leydig cell development in fetal life that results both in impaired germ cell development and in reduced intratesticular testosterone levels (Lee et al., 2001; Skakkebaek et al., 2001
; Sharpe et al., 2003
).
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Acknowledgements |
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References |
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Submitted on April 7, 2003; accepted on June 26, 2003.