1 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET and 2 Section of Child Life and Health, Department of Reproductive and Developmental Sciences, University of Edinburgh, Edinburgh EH9 1UW, UK
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Abstract |
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Key words: germ cells/GnRH antagonist/Leydig cell/Sertoli cell/spermatogonia
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Introduction |
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A major problem in addressing the issues just raised is the paucity of data on the testis itself during human childhood and a virtual absence of data on the functional status of its component cell types. This information is necessary in order to establish which cell types, if any, are active, and thus potentially at risk from therapeutic damage. Because of the obvious difficulties in obtaining such data in the human, and in particular of establishing whether any testicular cell activity can be suppressed by experimental intervention, we have turned to a non-human primate, the marmoset monkey. This primate was chosen as it exhibits the same phases of testis development as the human (Lunn et al., 1994; Sharpe et al., 2000
; McKinnell et al., 2001b
) but condensed into a period of 1518 months (Figure 1
), i.e. it descends its testes into the scrotum by birth, exhibits a neonatal/infantile period (34 months) of testicular/hormonal activity, when Sertoli cells proliferate and testosterone levels are high, followed by a period of `testis quiescence' for 78 months (`childhood'), followed by puberty. We have used various protein markers of testicular cell functional and numerical development to establish whether the period of testicular quiescence during `childhood' in the marmoset is associated with functional development and, if so, to establish whether quiescence can be induced by administration of a potent GnRH antagonist. We have also begun to evaluate the safety of the latter intervention by following treated animals through puberty and into adulthood. A further aim of these studies was to establish in the marmoset endpoints that can be applied to future human testis studies, which will determine whether, and at what age, similar functional changes occur.
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Materials and methods |
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To place findings for some testicular cell functional parameters at 35 weeks into perspective, the same endpoints were evaluated in the testes of control marmosets of various ages. The primary use of the latter animals was as controls for other studies unconnected with those presented here.
Tissue collection and processing
Animals were killed via i.p. injection of an overdose of sodium pentobarbitone (Euthatal; Rhone Merieux Ltd, Harlow, Essex, UK). Testes with epididymides attached were dissected free of connective tissue and immersion-fixed for 5.5 h in Bouin's fluid after which the testis was dissected away from the epididymis and weighed; fixed testes were then processed overnight in a Shandon processor and embedded in paraffin.
Immunohistochemical markers of testicular cell development
Several markers were used in order to gauge the status of testicular cell development. Some of these were testis cell-specific, for example sulphated glycoprotein-2 (SGP-2) and anti-müllerian hormone (AMH) for Sertoli cells and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) for Leydig cells, whereas others (e.g. androgen receptor, inhibin-) were expressed in more than one cell type. SGP-2 is one of the major secretory proteins of the Sertoli cells in the testis of the rat and human (O'Bryan et al., 1994
; McKinnell et al., 1995
) and has also been identified in the marmoset testis (O'Bryan et al., 1994
); its precise role(s) in the testis remains unclear. AMH is secreted by the fetal Sertoli cell during sexual differentiation and is responsible for inducing regression of the müllerian ducts (Josso et al., 1998
); its role in the postnatal testis is unclear, though a role in suppressing development of the adult generation of Leydig cells has been postulated (Josso et al., 1998
; Racine et al., 1998
). 3ß-HSD is one of the enzymes required for the biosynthesis of testosterone and is widely recognized as being a specific marker of Leydig cells/Leydig cell precursors (Ge et al., 1996
). Inhibin-
is one of the subunits of the dimeric protein, inhibin-B, which is secreted by Sertoli cells and possibly by Leydig cells in the testis, though various forms of the free
subunit may also be secreted (Winters and Plant, 1999
; Anderson and Sharpe, 2000
). Based on studies in the rat, androgen receptors (AR) are expressed in Sertoli cells just prior to puberty in the rat and in other somatic testicular cells, but not germ cells, at all ages (Bremner et al., 1994
). Finally, estrogen receptor-ß (ERß) was used as our studies in various species have shown that it is expressed in most cell types in the testis, including most germ cells, at all ages and its expression appears to be more or less constant in widely differing situations (McKinnell et al., 2001b
; Saunders et al., 2001
). Finally, proliferating cell nuclear antigen (PCNA), which is expressed widely during the cell cycle, and is thus a marker of `non-quiescent' cells, was used to investigate cell proliferation; PCNA has been used for similar purposes in many previous studies including in the rat (Schlatt and Weinbauer, 1994
), marmoset (Millar et al., 2000
), rhesus monkey (Schlatt and Weinbauer, 1994
) and human (Steger et al., 1998
). Specific antibodies were used to investigate the immunoexpression of these various target proteins in testicular sections from marmosets. Details of the antibodies and their use are given below.
Antibodies
Immunolocalization of AR utilized a rabbit polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) raised against an epitope at the N-terminus of human AR, and was used at a dilution of 1:2000. ERß was immunolocalized using an affinity-purified, polyclonal anti-peptide IgG raised in sheep against a specific peptide in the hinge (D) domain of human ERß, as previously described in detail (Saunders et al., 2000); it was used at a dilution of 1:1000. Rigorous evaluation of the specificity of the antibodies used, based on Western analyses and pre-absorption, has been detailed in our previous studies (Saunders et al., 2000
; Williams et al., 2000
; McKinnell et al., 2001a). SGP-2 was immunolocalized using a polyclonal antibody raised in sheep against human SGP-2 (gift from Dr J.McRae), used at a dilution of 1:3000. Immunolocalization of 3ß-HSD and AMH utilized rabbit polyclonal antibodies against the human proteins (gifts from Professors I.Mason and N.Josso respectively); they were used at a dilution of 1:2000 and 1:1000 respectively. Proliferating cell nuclear antigen (PCNA) was immunolocalized utilizing a monoclonal antibody (PC10; Dako) used at a dilution of 1:100. Immunolocalization of inhibin-
utilized a monoclonal antibody (173/9K) described previously (Groome et al., 1990
; Majdic et al., 1996
), and used at a dilution of 1:1000.
Immunohistochemistry
Unless otherwise stated, all incubations were performed at room temperature. Sections were deparaffinized in Histoclear (National Diagnostics, Hull, UK), rehydrated in graded ethanols and washed in water. For AR, ERß, AMH and inhibin-, a temperature-induced antigen retrieval step (Norton et al., 1994
) was used prior to immunohistochemistry. The method was optimized for each antibody and therefore varied slightly according to the antibody used. Thus, sections were subjected to antigen retrieval in either 0.01 mol/l citrate buffer, pH 6.0 (for AR, AMH and inhibin-
), or 0.05 mol/l glycine, pH 3.5, and 0.01% (w/v) EDTA (for ERß). After pressure cooking for 5 min at full pressure, sections were left to stand, undisturbed, for 20 min, then cooled under running tapwater before being washed twice (5 min each) in Tris-buffered saline (TBS; 0.05 mol/l TrisHCl, pH 7.4, 0.85% w/v NaCl). Endogenous peroxidase activity was blocked by immersing all sections in 3% (v/v) H2O2 in methanol (both from BDH Laboratory Supplies, Poole, Dorset, UK) for 30 min, which was followed by two 5 min washes in TBS. To block non-specific binding sites, sections were incubated for 30 min with the appropriate normal serum diluted 1:5 in TBS containing 5% (w/v) bovine serum albumin (BSA; Sigma, Poole, Dorset, UK). For AR, 3ß-HSD and AMH, normal swine serum (NSW) was used and for ERß, SGP-2, PCNA and inhibin-
, normal rabbit serum (NRS) was used (both from Diagnostics Scotland, Carluke, UK). Primary antibodies were added to the sections at the appropriate dilution in either NSW/TBS/BSA (for AR, 3ß-HSD and AMH) or NRS/TBS/BSA (for ERß, SGP-2, PCNA and inhibin-
) and incubated overnight at 4°C in a humidified chamber, followed by two 5 min washes in TBS. For AR, 3ß-HSD, AMH and inhibin-
, sections were then incubated for 30 min with horseradish peroxidase (HRP)-labelled polymer (EnVision; Dako). For other antibodies, sections were incubated for 30 min with either biotinylated rabbit anti-sheep IgG (Vector Laboratories, Peterborough, UK) in the case of ERß and SGP-2, or biotinylated rabbit anti-mouse IgG (Dako) in the case of PCNA, then washed twice (5 min each) in TBS, and incubated for a further 30 min with avidinbiotin conjugated to horseradish peroxidase (Dako) diluted in 0.05 mol/l TrisHCl, pH 7.4, according to the manufacturer's instructions. All sections were then washed twice (5 min each) in TBS, and immunostaining was developed using Liquid DAB-plus (Dako) until staining was optimal, when the reaction was stopped by immersing sections in distilled water. All sections were then lightly counterstained with haematoxylin, dehydrated in graded ethanols, cleared in xylene and coverslipped using Pertex mounting medium (CellPath plc, Hemel Hempstead, UK). As negative controls, slides were processed as above except that the appropriate normal serum was substituted for the primary antibody. To ensure the reproducibility of findings, tissue sections from four or five animals at each age or treatment group were evaluated, and this was performed on at least two separate occasions.
Immunostained sections were examined and photographed using a Provis microscope (Olympus Optical, London, UK) fitted with a digital camera (DCS330; Eastman Kodak, Rochester, NY, USA). Captured images were transferred to a computer (G4; Apple Computer Inc., Cupertino, CA, USA) and compiled using Photoshop 5.0 (Adobe Systems Inc., Mountain View, CA, USA) before being printed using an Epson Stylus 870 colour printer (Seiko Epson Corp., Nagano, Japan).
Determination of PCNA-labelled germ cell volume per testis and the spermatogonial labelling index
The volume per testis of PCNA-labelled cell nuclei was determined by standard point-counting. Cross-sections of testes from all marmosets in each treatment group were examined under oil-immersion using a Leitz x63 plan apo objective fitted to a Leitz laborlux microscope and a 121-point eyepiece graticule. Using a systematic clock-face sampling pattern from a random starting point, 16 fields were counted. Points falling over the nuclei of PCNA-positive cells were scored and expressed as a percentage of the total points counted. For each animal, the values for percentage nuclear volume were converted to absolute nuclear volumes per testis by reference to testis volume (= weight) as shrinkage was minimal, i.e. testis weights before and after fixation were comparable in each treatment group. All PCNA-positive cells detected within the seminiferous tubules in this study were identified as being germ cells; no labelled Sertoli cells were observed.
Point-counting enabled comparative determination of the relative proportions of non-quiescent cells in the testes of control and GnRH antagonist-treated marmosets, but did not discriminate between germ cell types (some spermatogonia and some spermatocytes were PCNA-labelled). As it is the proliferating spermatogonia that are thought to be most at risk from toxic effects of cancer therapy, the PCNA-labelling index of spermatogonia was determined separately. The method used the Area Fraction Probe in the Stereologer software programme (Systems Planning and Analysis Inc., Alexandria, VA, USA) and utilized an Olympus BHS microscope fitted with an automatic stage (Applied Scientific Instrumentation Inc, Eugene, OR, USA). The area fraction probe places a grid in the frame and the Object Area fraction is determined by clicking each `x' that touches the object of interest (in this case, the cytoplasm or nucleus of a spermatogonium). Each spermatogonium was classed as PCNA-positive or -negative. Spermatogonia that were in contact with the basement membrane of the seminiferous tubule were classified as germ cells. Spermatogonial nuclei were easily distinguished from Sertoli cell nuclei but it was decided that no attempt would be made to exclude preleptotene spermatocytes (which may still be in contact with the basement membrane) from this analysis as these cells (especially unlabelled) could not be reliably identified under the present circumstances. The PCNA-labelling index of spermatogonia was calculated as a percentage of the total of labelled + unlabelled cells.
Determination of Leydig cell volume per testis
Leydig cell volume per testis was determined by point counting on sections of Bouin-fixed testes which had been immunostained for 3ß-HSD using modifications of methods described previously (Sharpe et al., 1998, 2000
). The method used the Area Fraction Probe in the Stereologer software programme as described above. Once completed, Stereologer automatically displays the results including area fraction and coefficient of error. The values for area fraction were then converted to absolute volumes per testis by reference to testis volume (= weight).
Plasma levels of testosterone
Levels of testosterone in plasma were measured using an enzyme-linked immunosorbent assay adapted from an earlier radioimmunoassay method (Corker and Davidson, 1981) as detailed elsewhere (Atanassova et al., 1999
). The limit of detection was ~12 pg/ml.
Statistical analysis
Comparison of data from control and GnRH antagonist-treated marmosets used either analysis of variance or Student's t-test. Where comparison of endpoints was made for co-twin marmosets (n = 4) at week 35, the paired t-test was used. In all of the latter comparisons, as well as for plasma testosterone levels during progression through puberty, data were log-transformed prior to analysis as there were unequal variances in control and GnRH antagonist-treated groups.
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Results |
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Germ cell development
Control twins had more numerous and more advanced germ cell types (some early spermatocytes were apparent) in their seminiferous tubules than did their GnRH antagonist-treated co-twins, though there was considerable variability between individual control males (Figure 4). To highlight this difference and to provide an indication of cell proliferation, immunostaining with PCNA was applied (Figure 4
). Virtually all cells within the seminiferous tubules that labelled with PCNA were germ cells and these were evidently more numerous in control twins than in their GnRH antagonist-treated co-twin brothers. Most germ cells, as well as Sertoli cells, immunoexpressed ERß in their nuclei and the intensity of this expression did not change detectably in GnRH antagonist-treated compared with control marmosets (Figure 4
).
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Discussion |
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There are various data which together suggest that the prepubertal human testis may be `quietly active' as opposed to truly quiescent (Wu et al., 1990, 1991
, 1996
; Clark et al., 1997
; Chemes, 2001
). For example, germ cell number per testis increases ~3-fold during childhood along with a significant increase in testis volume/weight (Muller and Skakkebaek, 1983
; Paniagua and Nistal, 1984
), and increases in Leydig cell development (Nistal et al., 1986
) and in the intratesticular (Chemes, 1996
) and spermatic vein levels of testosterone (Santoro et al., 1981
) have been reported between 2 and 4 and between 6 and 8 years of age. The latter changes may coincide with night-time activation of pulsatile LH secretion (Wu et al., 1990
, 1991
, 1996
; Hayes and Crowley, 1998
). There is also evidence that formation of spermatocytes, and even occasional spermatids, is a normal feature of the childhood testis (Muller and Skakkebaek, 1983
; Paniagua and Nistal, 1984
; Rey et al., 1993
; Chemes, 2001
), but all of these cells degenerate because of the lack of appropriate maturation or functional support from the Sertoli and Leydig cells (Chemes, 2001
). The latter observations imply that germ cell proliferation and differentiation are more widespread than is revealed simply by counting the numerical increase in germ cells. All of these described changes occur at least 45 years prior to the onset of puberty (Chemes, 2001
), and although they are of small magnitude when compared with the change in the same parameters that occur during puberty, it may be these changes that are responsible for the susceptibility of the testis of boys to damage by some cancer therapies.
The present studies in the marmoset reinforce the impression gained from the limited human studies by demonstrating that significant testicular cell activity is detectable at an age (35 weeks) when the testis is considered to be `quiescent' and which is considerably in advance of the first signs of normal puberty in our colony of marmosets (5060 weeks), based on the blood levels of testosterone (Lunn et al., 1994, 1997
; McKinnell et al., 2001b
; this study). This activity extended to Sertoli, Leydig and germ cells based on various functional markers of their maturational development and/or proliferation. Moreover, our studies show that this cellular activation is largely, if not completely, dependent on gonadotrophin stimulation as the functional cell changes were almost completely inhibited by treatment for 10 weeks with a GnRH antagonist. With the exception of expression of low levels of 3ß-HSD in Leydig/precursor cells, the marmoset testis at age 2025 weeks shows none of the cellular activity found at 35 weeks (this study plus our unpublished data). This suggests that development of the testis towards its adult status and function is initiated much earlier than `at puberty'. Moreover, the early changes occurring during the `quiescent' phase are without any observable manifestation outside of the testis itself, as has been suggested (Chemes, 2001
). Indeed, even within the testis, the clear impression morphologically was that Sertoli cells were still immature, yet evaluation of their functional status via SGP-2 indicated that their functional differentiation was already under way. If such findings in the marmoset are predictive of the human, then they imply that functional development of testicular cells is likely to be initiated at some point during childhood. It is not possible to predict accurately in what age range such changes might occur because of the considerably greater length of human childhood compared with the equivalent period in the marmoset. However, other data (Wu et al., 1990
, 1991
, 1996
) suggest that this could be by mid-childhood. As the present studies have identified a number of protein markers of testicular cell functional development, application of these to human testicular autopsy specimens should enable us to establish if, and when, comparable changes in expression of these markers occurs during human testis development. Such studies are in progress.
The potential importance and usefulness of the present findings depends on the extent to which the marmoset provides an appropriate model for development of the human testis during childhood. As this period has been poorly studied thus far, it is difficult to draw definitive conclusions. In most other respects the marmoset does appear to be a reasonable model for the human. Thus the marmoset descends its testes into the scrotum by birth, exhibits neonatal proliferation of Sertoli cells and a testosterone surge followed by infantile `quiescence', a pattern of events comparable with those in the human male (Lunn et al., 1994, 1997
; Sharpe et al., 2000
; McKinnell et al., 2001b
). Finally, in adulthood the organization of spermatogenesis in the marmoset shows many of the features typical of the human (Millar et al., 2000
; Sharpe et al., 2000
). Other non-human primates, for example the Cebus monkey, show comparable changes with the marmoset and human (Rey et al., 1993
), though the more commonly used Rhesus monkey shows some notable differences in the timing of testicular descent and neonatal Sertoli cell proliferation (Sharpe et al., 2000
), but does show a neonatal testosterone surge (Mann et al., 1997
; Weinbauer and Nieschlag, 1999
) as in the human (Andersson et al., 1998
) and marmoset (Lunn et al., 1994
; Weinbauer and Nieschlag, 1999
; Sharpe et al., 2000
; McKinnell et al., 2001b
).
It is logically presumed that the ability of GnRH antagonist administration to suppress testicular cell activation in the marmoset during `childhood' in the present studies results from suppression of gonadotrophin secretion. However, this could not be shown directly because of the continued unavailability of assays for the detection of gonadotrophins in the marmoset. However, this observation demonstrates the feasibility of being able to suppress such testicular cell activity. This is widely considered to be a prerequisite for any method of protecting spermatogenic potential in situ in boys with cancer who are at risk of future infertility because of their cancer therapy (Howell and Shalet, 1999; Grundy et al., 2001
). As the present findings demonstrate functional activation of Sertoli and Leydig cells, as well as of germ cells, in the `childhood' marmoset testis, they do not provide an unequivocal answer as to which cell type(s) in the testes of children might confer susceptibility to damage by cancer therapies, though it is presumed that loss of stem spermatogonia is ultimately responsible for the loss of sperm production in adulthood in such children (Howell and Shalet, 1999
; Grundy et al., 2001
). In this regard, the present data demonstrate that spermatogonial proliferation, based on PCNA-labelling, was apparently not suppressed in 35 week marmosets by GnRH antagonist administration, though the absolute volume per testis of spermatogonia was reduced. These findings suggest that the survival of spermatogonia may be partially gonadotrophin-dependent whereas the proliferation of surviving spermatogonia is largely or completely gonadotrophin-independent. If the latter involves spermatogonial stem cells, then GnRH antagonist therapy is likely to be ineffectual in providing protection against cancer therapies that will affect these cells. Nevertheless, current interest in testicular germ cell transplantation means that understanding of the factors regulating spermatogonial stem cell proliferation and spermatogonial differentiation are moving ahead fast, and this new understanding is likely to open new interventionist possibilities.
For the reasons voiced above, it is considered essential that several key questions are answered sequentially before GnRH agonist/antagonist or other intervention therapies in childhood could be considered clinically as a means of protecting the testis from cytotoxic therapy. First, it has to be demonstrated that such a therapeutic intervention is safe and does not itself induce unacceptable long-lasting problems. We are undertaking such studies in the marmoset by tracking control and GnRH antagonist-treated animals through puberty and into adulthood. Data for the pubertal rise in testosterone levels, and its delay in GnRH antagonist-treated marmosets, are presented in the current study. Fertility of these animals is being assessed and eventually the animals will be subjected to a full autopsy. If these findings do not indicate any major long-term effects of the GnRH antagonist treatment, the logical next question to ask is whether this treatment really does confer testicular protection against potentially damaging cancer therapy. For this purpose, we would test whether GnRH antagonist treatment, as undertaken in the present studies, is able to protect against local testicular irradiation. However, the present findings indicating continued (and presumably gonadotrophin-independent) proliferation of spermatogonia, based on PCNA-labelling, cast doubt on whether such a strategy would be successful. It therefore appears that improved understanding of the factors regulating spermatogonial proliferation will be necessary before protection strategies for the human testis in childhood can be considered seriously.
Though our findings raise questions about the likelihood of success of GnRH antagonist intervention therapy as a protection for the testis in boys with cancer, they provide important new information that strongly supports the concept that the testis in childhood is quietly active (Chemes, 2001). The present studies have examined this activity in the `childhood' marmoset testis using various protein markers, and we have studies in progress to evaluate immunoexpression of the same markers in the human testis throughout childhood. Combined with more detailed studies of spermatogonial development, these studies should provide much-needed information on the status of cell activity in the human testis in childhood and thus allow determination of the age range when intervention `testis-quietening' therapy is potentially viable. At the very least, such studies should improve our understanding of cell development and cellcell interactions in the childhood testis and help to elucidate why the testis at this time is susceptible to toxic damage by some cancer therapies.
In summary, the present findings add to the growing impression that the `childhood' testis is not quiescent and that activation of testicular cell function occurs well before the onset of puberty (gonadarche). These changes are largely gonadotrophin-dependent as they can be mostly prevented by GnRH antagonist administration, though an important exception appears to be spermatogonial proliferation. These findings identify that the regulation of GnRH (gonadotrophin)-independent spermatogonial proliferation requires elucidation, but provide new insight into the unexplained susceptibility of the `quiescent' childhood testis to damage by certain cancer therapies and open the possibility of designing interventionist protection strategies.
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Acknowledgements |
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Notes |
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References |
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Andersson, A-M., Toppari, J., Haavisto, A-M., Petersen, J.H., Simell, T. and Skakkebaek, N.E. (1998) Longitudinal reproductive hormones in infants: peak of inhibin B levels in boys exceeds levels in adult men. J. Clin. Endocrinol. Metab. , 83, 675681.
Atanassova, N.N., McKinnell, C., Walker, M., Turner, K.J., Fisher, J.S., Morley, M., Millar, M.R., Groome, N.P. and Sharpe, R.M. (1999) Permanent effects of neonatal estrogen exposure in rats on reproductive hormone levels, Sertoli cell number and the efficiency of spermatogenesis in adulthood. Endocrinology, 140, 53645373.
Bath, L.E., Wallace, W.H.B. and Kelnar, C.J.H. (1998) Disorders of growth and development in the child treated for cancer. In Kelnar C.J.H., Savage, M.O., Stirling, H.F. and Saenger, P. (eds), Growth DisordersPathophysiology and Treatment. Chapman & Hall, London, pp. 641660.
Bremner, W.J., Millar, M.R., Sharpe, R.M. and Saunders, P.T.K. (1994) Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens. Endocrinology, 135, 12271234.[Abstract]
Chemes, H.E. (1996) Leydig cell development in humans. In Payne, A.H., Hardy, M.P. and Russell, L.D. (eds), The Leydig Cell. Cache River Press, Vienna, IL, USA, pp. 175202.
Chemes, H.E. (2001) Infancy is not a quiescent period of testicular development. Int. J. Androl. , 24, 27.[ISI][Medline]
Clark, P.A., Iranmanesh, A., Veldhuis, J.D. and Rogol, A.D. (1997) Comparison of pulsatile luteinizing hormone secretion between prepubertal children and young adults: evidence for a mass/amplitude-dependent difference without gender or day/night contrasts. J. Clin. Endocrinol. Metab. , 82, 29502955.
Corker, C.S. and Davidson, D.W. (1981) Radioimmunoassay of testosterone in various biological fluids without chromatography. J. Steroid Biochem. , 9, 319323.
Ge, R-S., Shan, L-X. and Hardy, M.P. (1996) Pubertal development of Leydig cells. In Payne, A.H., Hardy, M.P. and Russell, L.D. (eds), The Leydig Cell. Cache River Press, Vienna, IL, USA, pp. 159172.
Groome, N.P., Hancock, J., Betteridge, A. and Waks, M. (1990) Monoclonal and polyclonal antibodies reactive with the 132 amino terminal sequence of the alpha subunit of human 32K inhibin. Hybridoma, 9, 3135.[ISI][Medline]
Grundy, R., Gosden, R.G., Hewit, M., Larcher, V., Leiper, A, Spoudeas, H.A., Walker, D. and Wallace, W.H. (2001) Fertility preservation for children treated for cancer (1): scientific advances and research dilemmas. Arch. Dis. Child. , 84, 355359.
Hayes, F.J. and Crowley, W.F. Jr (1998) Gonadotrophin pulsations across development. Horm. Res. , 49, 163168.[ISI][Medline]
Howell, S.J. and, Shalet, S.M. (1999) Pharmacological protection of the gonads. Med. Pediatr. Oncol. , 33, 4145.[ISI][Medline]
Josso, N., Racine, C., di Clemente, N., Rey, R. and Xavier, F. (1998) The role of anti-mullerian hormone in gonadal development. Mol. Cell. Endocrinol. , 145, 37.[ISI][Medline]
Lunn, S.F., Recio, R., Morris, K.D. and Fraser, H.M. (1994) Blockade of the neonatal rise in testosterone by a gonadotrophin-releasing hormone antagonist: effects on timing of puberty and sexual behaviour in the male marmoset monkey. J. Endocrinol. , 141, 439447.[Abstract]
Lunn, S.F., Cowen, G.M. and Fraser, H.M. (1997) Blockade of the neonatal increase in testosterone by a GnRH antagonist: the free androgen index, reproductive capacity and postmortem findings in the male marmoset monkey. J. Endocrinol. , 154, 125131.[Abstract]
Majdic, G., Sharpe, R.M., O'Shaughnessy, P.J. and Saunders, P.T.K. (1996) Expression of cytochrome P450 17-hydroxylase/C17-20 lyase (P450c17) in the fetal rat testis is reduced by maternal exposure to exogenous estrogens. Endocrinology, 137, 10631070.[Abstract]
Mann, D.R., Akinbami, M.A., Wallen, K., Gould, K.G., Groome, N.P., Swanston, I.A., McNeilly, A.S. and Fraser, H.M. (1997) Inhibin-B in the male Rhesus monkey: impact of neonatal gonadotropin-releasing hormone antagonist treatment and sexual development. J. Clin. Endocrinol. Metab. , 82, 19281933.
McKinnell, C., Brackenbury, E.T., Qureshi,S.J., Hargreave, T.B. and Sharpe, R.M. (1995) Comparative analysis of proteins secreted in vitro by isolated seminiferous tubules from man and the rat. Int. J. Androl. , 18, 103111.[ISI][Medline]
McKinnell, C., Atanassova, N., Williams, K., Fisher, J.S., Walker, M., Turner, K.J., Saunders, P.T.K. and Sharpe, R.M. (2001a) Suppression of androgen action and the induction of gross abnormalities of the reproductive tract in male rats treated neonatally with diethylstilbestrol. J. Androl. , 22, 323338.
McKinnell, C., Saunders, P.T.K., Fraser, H.M., Lunn, S.F., Kelnar, C.J.H., Civlin, C., Morris, K.D. and Sharpe, R.M. (2001b) Comparison of androgen receptor (AR) and oestrogen receptor-ß immunoexpression in the testes of marmosets from birth to adulthood: low AR immunoexpression in Sertoli cells during the neonatal testosterone rise. Reproduction, 122, 419429.
Meistrich, M.L., Wilson, G., Zhang, Y., Kurdoglu, B. and Terry, N.H. (1997) Protection from procarbazine-induced testicular damage by hormonal pretreatment does not involve arrest of spermatogonial proliferation. Cancer Res., 15, 10911097.
Meistrich, M.L., Wilson, G., Kangasniemi, M. and Huhtaniemi, I.P. (2000) Mechanism of protection of rat spermatogenesis by hormonal pretreatment: stimulation of spermatogonial differentiation after irradiation. J. Androl. , 21, 464469.
Millar, M.R., Sharpe, R.M., Weinbauer, G.F., Fraser, H.M. and Saunders, P.T.K. (2000) Marmoset spermatogenesis: organisational similarities to the human. Int. J. Androl. , 23, 266277.[ISI][Medline]
Muller, J. and Skakkebaek, N.E. (1983) Quantification of germ cells and seminiferous tubules by stereological examination of the testicles from 50 boys who suffered from sudden death. Int. J. Androl. , 6, 143156.[ISI][Medline]
Nistal, M., Paniagua, R., Regadera, J., Santamaria, L. and Amat, P. (1986) A quantitative morphological study of human Leydig cells from birth to adulthood. Cell Tissue Res., 246, 229236.[ISI][Medline]
Norton, A.J., Jordan, S. and Yeomans, P. (1994) Brief, high-temperature heat denaturation (pressure cooking): a simple and effective method of antigen retrieval for routinely processed tissues. J. Path. , 173, 371379.[ISI][Medline]
O'Bryan, M.K., Mallidis, C., Murphy, B.F. and Baker, H.W.G. (1994) Immunohistological localisation of clusterin in the male genital tract in humans and marmosets. Biol. Reprod. , 50, 502509.[Abstract]
Paniagua, R. and Nistal, M. (1984) Morphological and histometric study of human spermatogonia from birth to the onset of puberty. J. Anat. , 139, 535552.[ISI][Medline]
Racine, C., Rey, R., Forest, M.G., Louis, F., Ferre, A., Huhtaniemi, I.P., Josso, N. and di Clemente, N. (1998) Receptors for anti-mullerian hormone on Leydig cells are responsible for its effects on steroidogenesis and cell differentiation. Proc. Natl. Acad. Sci. USA , 95, 594599.
Rey, R.A., Campo, S.M., Bedecarras, P., Nagle, C.A. and Chemes, H.E. (1993) Is infancy a quiescent period of testicular development? Histological, morphometric and functional study of the seminiferous tubules of the Cebus monkey from birth to puberty. J. Clin. Endocrinol. Metab. , 76, 13251331.[Abstract]
Santoro, S., Boninsegni, R., Bassi, F., Pampaloni, A., Grisola, G.A., Forti, G. and Serio, M. (1981) Testosterone concentrations in spermatic venous blood plasma of prepubertal boys. Int. J. Androl. , 4, 8285.[ISI][Medline]
Saunders, P.T.K., Millar, M.R., Williams, K., Macpherson, S., Harkiss, D., Anderson, R.A., Orr, B., Groome, N.P., Scobie, G. and Fraser, H.M. (2000) Differential expression of estrogen receptor-alpha and -beta and androgen receptor in the ovaries of marmoset and human. Biol. Reprod. , 63, 10981105.
Saunders, P.T.K., Williams, K., Macpherson, S., Urquhart, H., Irvine, D.S., Sharpe, R.M., Millar, M.R. (2001) Differential expression of oestrogen receptor alpha and beta proteins in the testes and male reproductive tract of human and non-human primates. Mol. Hum. Reprod. , 7, 227236.
Schlatt, S. and Weinbauer, G.F. (1994) Immunohistochemical localization of proliferating cell nuclear antigen as a tool to study cell proliferation in rodent and primate testes. Int. J. Androl. , 17, 214222.[ISI][Medline]
Sharpe, R.M., Atanassova, N.N., McKinnell, C., Parte, P., Turner, K.J., Fisher, J.S., Kerr, J.B., Groome, N.B., Mcpherson, S., Millar, M.R. et al. (1998) Abnormalities in functional development of the Sertoli cells in rats treated neonatally with diethylstilbestrol: a possible role for estrogens in Sertoli cell development. Biol. Reprod. , 59, 10841094.
Sharpe, R.M., Turner, K.J., McKinnell, C., Groome, N.P., Atanassova, N.N., Millar, M.R., Buchanan, D.L. and Cooke, P.S. (1999) Inhibin B levels in plasma of the male rat from birth to adulthood: effect of experimental manipulation of Sertoli cell number. J. Androl. , 20, 94101.
Sharpe, R.M., Walker, M., Millar, M.R., Morris, K., McKinnell, C., Saunders, P.T.K. and Fraser, H.M. (2000) Effect of neonatal GnRH antagonist administration on Sertoli cell number and testicular development in the marmoset: comparison with the rat. Biol. Reprod. , 62, 16851693.
Shetty, G., Wilson, G., Huhtaniemei, I. P., Shuttlesworth, G.A., Reissmann, T. and Meistrich, M.L. (2000) Gonadotropin-releasing hormone analogs stimulate and testosterone inhibits the recovery of spermatogenesis in irradiated rats. Endocrinology, 141, 17351745.
Steger, K., Aleithe, I., Behre, H. and Bergmann, M. (1998) The proliferation of spermatogonia in normal and pathological human seminiferous epithelium: an immunohistochemical study using monoclonal antibodies against Ki-67 and proliferating cell nuclear antigen. Mol. Hum. Reprod. , 4, 227233.[Abstract]
Weinbauer, G.F. and Nieschlag, E. (1999) Testicular physiology of primates. In Weinbauer, G.F. and Korte, R. (eds), Reproduction in Non-human Primates: A Model System for Human Reproductive Physiology and Toxicology. Waxmann Verlag GmbH, New York, pp. 1326.
Williams, K., Saunders, P.T.K., Atanassova, N., Fisher, J.S., Turner, K.J., Millar, M.R., McKinnell, C. and Sharpe, R.M. (2000) Induction of progesterone receptor immunoexpression in stromal tissue throughout the male reproductive tract after neonatal estrogen treatment of rats. Mol. Cell. Endocrinol. , 164, 117131.[ISI][Medline]
Winters, S.J. and Plant, T.M. (1999) Partial characterization of circulating inhibin-B and pro-alphaC during development in the male rhesus monkey. Endocrinology, 140, 54975504.
Wu, F.C.W., Butler, G.E., Kelnar, C.J.H. and Sellar, R.E. (1990) Patterns of pulsatile LH secretion before and during the onset of puberty in boys|a study using an immunoradiometric assay. J. Clin. Endocrinol. Metab. , 70, 629637.[Abstract]
Wu, F.C.W., Butler, G.E., Kelnar, C.J.H., Stirling, H.F. and Huhtaniemi, I.P. (1991) Patterns of pulsatile luteinizing hormone and follicle-stimulating hormone secretion in prepubertal (midchildhood) boys and girls and patients with idiopathic hypogonadotropic hypogonadism (Kallmann's syndrome): a study using an ultra-sensitive time-resolved immunofluorometric assay. J. Clin. Endocrinol. Metab. , 72, 12291237.[Abstract]
Wu, F.C.W., Butler, G.E., Kelnar, C.J.H., Huhtaniemi, I.P. and Veldhuis, J.D. (1996) Ontogeny of pulsatile gonadotropin releasing hormone (GnRH) secretion from midchildhood through puberty to adulthood in the human male: a study using deconvolution analyses and an ultrasensitive immunofluorometric assay. J. Clin. Endocrinol. Metab. , 81, 17981805.[Abstract]
Submitted on September 17, 2001; accepted on December 7, 2001.