Role of the neonatal period of pituitary–testicular activity in germ cell proliferation and differentiation in the primate testis

R.M. Sharpe1,3, H.M. Fraser1, M.F.H. Brougham2, C. McKinnell1, K.D. Morris1, C.J.H. Kelnar2, W.H.B. Wallace2 and M. Walker1

1 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Chancellor’s 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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The neonatal period of pituitary–testicular activity (NPTA) in human males has been hypothesized to play a role in germ cell proliferation and differentiation and to be defective in cryptorchid testes. The present study was carried out to establish in the marmoset if suppression of the NPTA, by treatment with a GnRH antagonist, results in impaired germ cell proliferation and/or differentiation. METHODS: Comparison of germ cell (GC) numbers and differentiation from gonocytes to pre-spermatogonia and spermatogonia, at birth (in controls) and at the end of the NPTA in marmoset co-twin males treated from birth to age 14 weeks with vehicle or GnRH antagonist. RESULTS: From birth to age 18–24 weeks, testis weight increased ~5-fold and GC number ~10-fold, including increased numbers of gonocytes and pre-spermatogonia and the first appearance of spermatogonia. Treatment with GnRH antagonist attenuated the increase in testis weight and GC numbers, but the effect was only partial (24–30% reduction), and the relative proportions of gonocytes, pre-spermatogonia and spermatogonia in the GnRH antagonist-treated group were unchanged from control values. CONCLUSIONS: The NPTA plays only a minor, if any, role in GC proliferation and differentiation in the marmoset. The changes in GnRH antagonist-treated co-twins may reflect impaired GC survival due to withdrawal of gonadotrophin support for Sertoli cells. These findings do not support a pivotal role for the NPTA in neonatal GC development in primates.

Key words: FSH/germ cells/LH/Sertoli cells/testosterone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In primates, including the human, males exhibit a neonatal period of ‘hypothalamic–pituitary–testicular activity’ which is characterized by secretion of FSH and LH and elevation of blood levels of inhibin-B and testosterone towards or into the adult range (Winters et al., 1975Go; Forest, 1990Go; Mann and Fraser, 1996Go; Andersson et al., 1998Go). In neonatal human females, activation of gonadotrophin secretion also occurs but this is not accompanied by elevation of sex steroid secretion, as in boys in whom testosterone levels are elevated for a period of ~5–6 months (Winters et al., 1975Go; Forest, 1990Go). However, the physiological role of this so-called ‘neonatal testosterone surge’ is unclear (Mann and Fraser, 1996Go).

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, 1989Go). This increase in testis size may be due in part to increase in Sertoli cell number (Cortes et al., 1987Go), a change that is also indicated by the parallel rise in blood levels of inhibin-B (Andersson et al., 1998Go; Anderson and Sharpe, 2000Go). 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, 1983Go; 1984; Bidlingmaier and Hilscher, 1989Go), 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., 1993Go; 2001; Hadziselimovic and Herzog, 2001Go). 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-{beta} is a possibility (Williams et al., 2001Go; McKinnell et al., 2001Go; Sharpe et al., 2003Go).

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., 1994Go; 1997; Sharpe et al., 2000Go; McKinnell et al., 2001Go), 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 pituitary–testicular activity for the duration of the neonatal period (~14–15 weeks; Lunn et al., 1997Go; McKinnell et al., 2001Go) 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., 2002Go; Sharpe et al., 2000Go; 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and their treatment
Animals were captive-bred common marmoset monkeys (Callithrix jacchus), maintained in a colony that has been self-sustaining since 1973. For the present studies, a total of 13 newborn male marmosets were used, of which three were studied at age 1–2 days to provide baseline measurements of the testis prior to the onset of the period of neonatal pituitary–testicular activity; these animals resulted from triplet births and were killed as part of animal husbandry. The remaining animals consisted of five pairs of male co-twins which were killed at 18–24 weeks (i.e. 4–10 weeks after the end of the period of neonatal testicular activity, when the testis is quiescent). Marmosets show considerable between-animal variability that would normally necessitate the use in experiments of larger numbers of animals, based on power calculations. However, as co-twins tend to be highly comparable, this enabled pair-wise design of the present study and thus minimized the number of animals used. Starting on the day of birth, co-twins were injected s.c. with either 10 mg/kg GnRH antagonist (Antide: Contraceptive development branch, Center for Population Research, NICHD) in propylene glycol:water (1:1, v:v) or with the vehicle alone (control). This treatment was then repeated on days 3 and 7 and then at weekly intervals until week 14 when the final injection was given. The primary use of all of the animals used in the present studies was for other investigations (Lunn et al., 1997Go; Mann et al., 1999Go) unconnected with those presented here. An earlier study of the present animals (Mann et al., 1999Go), and related studies (Lunn et al., 1997Go; McKinnell et al., 2001Go), have shown that the described GnRH antagonist treatment regimen abolishes the normal neonatal rise in testosterone levels, presumably due to suppression of LH secretion. These studies were approved by the local ethics committee for studies in primates and were performed according to the Animal Scientific Procedures (UK) Act (1986) under Project Licence approval by the UK Home Office.

Tissue collection and processing
Testes were dissected free of connective tissue and the epididymis and then immersion-fixed for ~16 h in Sorenson’s 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 (1993Go) 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, 1995Go):


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 18–24 weeks of age used Student’s 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Testis growth and germ cell development during the neonatal period
Between birth and 18–24 weeks of age there was a ~5-fold increase in testis weight and nearly a 10-fold increase in germ cell number per testis (Figure 1). At birth, 69% of germ cells were classified as gonocytes and 31% as pre-spermatogonia, whereas no spermatogonia were evident (Figure 1 and Figure 2). By 18–24 weeks, the absolute numbers of gonocytes and pre-spermatogonia per testis had increased by an average of 7.2- and 11.6-fold respectively and small numbers of spermatogonia were now evident, though they accounted for only 3.7% of all germ cells (Figure 1 and Figure 2). At 18–24 weeks of age though the majority (55.8%) of germ cells within the cords were still classed as being gonocytes, it was evident that movement of these cells to the basal lamina and their differentiation into spermatogonia was occurring (Figure 1 and Figure 2).



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Figure 1. Changes in testis weight and germ cell numbers between birth and early infancy (18–24 weeks of age) in the marmoset. Bars represent means ± SD for n = 3 (birth) or n = 5 (18–24 weeks). Note that the animals providing data at 18–24 weeks are the vehicle-treated co-twins of GnRH antagonist-treated males shown in Figures 2–4. ***P < 0.001, in comparison with respective value at birth.

 


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Figure 2. Representative morphology of the testicular cords at birth in controls and at age 22 weeks in control and GnRH antagonist-treated co-twins to illustrate the classification of germ cells based on several factors, including their position in the cords. Gonocytes (G) are mainly centrally located and have no contact with the basal lamina. Pre-spermatogonia (PSg) have made contact with the basal lamina. Spermatogonia (Sg) have all of their basal surface in contact with the basal lamina and lie beneath Sertoli cells. Note the generally similar appearance and distribution of germ cells in the cords of the control and GnRH antagonist-treated animals. SC = Sertoli cell nuclei. Scale bar = 100 µm.

 
Effect of GnRH antagonist-mediated suppression of the neonatal period of pituitary–testicular activity
At 18–24 weeks of age, testis weight and germ cell number per testis varied considerably between different control twins, though the two parameters appeared closely related (Figure 3). Co-twins treated with GnRH antagonist showed a variable but significant reduction in testis weight and, though germ cell number was also reduced by a similar proportion, an actual decrease was only observed in four of the five twins, so this difference did not achieve statistical significance (Figure 3). In all control and GnRH antagonist-treated animals, gonocytes, pre-spermatogonia and spermatogonia were evident, though the proportions varied considerably between animals (Figure 4). This variation was most pronounced for spermatogonial numbers in GnRH antagonist-treated co-twins (Figure 4). Pair-wise comparison of germ cell types revealed mean reductions of 11, 35 and 45% for the numbers per testis of gonocytes, pre-spermatogonia and spermatogonia respectively in GnRH antagonist-treated co-twins, though only the difference for pre-spermatogonia achieved statistical significance (Figure 4). Comparison of the proportion of germ cells that were in each category revealed no major difference in mean values between control and GnRH antagonist- treated groups, though there was considerably greater variability in GnRH antagonist-treated than in control co-twins (Table I).



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Figure 3. Total germ cell number per testis and testis weight in individual vehicle- and GnRH antagonist-treated co-twins at age 18–24 weeks. Bars to the extreme right show mean values and P-values derived from the paired t-test.

 


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Figure 4. Numbers of gonocytes, pre-spermatogonia and spermatogonia per testis in individual vehicle- and GnRH antagonist-treated co-twins at age 18–24 weeks. Bars to the extreme right show mean values and P-values derived from the paired t-test. Note the wide variation between individual animals/co-twins, especially for spermatogonia.

 

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Table I. Proportionate distribution of germ cell types at the end of the neonatal period in control and GnRH antagonist-treated co-twin marmosets
 
Does neonatal GnRH antagonist treatment inhibit germ cell development in the neonatal period?
The results shown in Figure 4 are suggestive of a partial inhibitory effect of GnRH antagonist treatment on germ cell numbers at weeks 18–24, though there was considerable variation between co-twins. To put this in perspective, the magnitude of increase in numbers of each germ cell type since birth was calculated for each twin, using the mean data for germ cell numbers at birth as the reference starting point. This analysis (Table II) revealed that, though the relative increase in GnRH antagonist-treated co-twins was lower than the control co-twin in most (but not all) instances for each germ cell type, in every GnRH antagonist-treated animal there was a real increase in all germ cell types relative to birth. This analysis also highlighted that GnRH antagonist-treated co-twin 2 was the only GnRH antagonist-treated animal not to show a major inhibition of increase in spermatogonial numbers (Table II). However, in all other respects, data for this co-twin was comparable with others in this treatment group and it was confirmed that testosterone levels in this animal were suppressed to baseline in the neonatal period, including up to the time of death (data not shown). Exclusion of data for co-twins 2 did not materially alter mean values except for lowering the relative increase in spermatogonial numbers since birth in the GnRH antagonist group (Table II). There was no consistent relationship between the age when killed (18–24 weeks) and the absolute numbers of (Figure 4), or relative increase since birth (Table II) in, germ cells/cell types in either control or GnRH antagonist-treated animals.


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Table II. Magnitude of increase in germ cell numbers from birth to weeks 18–24 in individual marmosets, enabling comparison of the variability of the effect of GnRH antagonist treatment in co-twins
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The purpose of the present studies was to establish, in the marmoset, if the hormonal changes that occur during the neonatal period of pituitary–testicular activity were linked with proliferation and differentiation of germ cells in the testis. The study has shown that, as occurs in the human (Muller and Skakkebaek, 1983Go; 1984; Bidlingmaier and Hilscher, 1989Go) and in other non-human primates (Mann et al., 1989Go; Rey et al., 1993Go), there is a significant increase in both testis weight and germ cell number during the neonatal period in the marmoset, changes that can be partly prevented by GnRH antagonist-mediated suppression of the pituitary–testicular axis for the duration of the neonatal period. However, it is emphasized that the latter treatment had only a relatively minor effect as overall germ cell number still increased by 6.9-fold in animals in which the neonatal hormonal changes were suppressed, compared with an 8.9-fold increase in controls. A similar outcome was evident when germ cell movement/differentiation was considered. Thus, although GnRH antagonist treatment reduced the absolute numbers of pre-spermatogonia and spermatogonia, this was only a partial effect and, overall, the relative proportions of these germ cell types remained unchanged from control values at the end of the neonatal period in the GnRH antagonist-treated group of males. However, because of the small numbers of animals available for study and the considerable variation in germ cell numbers between animals, our findings do not completely rule out a role for neonatal hormones in promoting differentiation into spermatogonia. Nevertheless, based on the data as a whole, we conclude that the neonatal hormonal changes in male primates play only a minor, if any, role in germ cell proliferation and differentiation during this period of life. The relatively small changes that we have observed in GnRH antagonist-treated males are perhaps more likely to reflect altered germ cell survival, as the result of withdrawal of gonadotrophin support for the Sertoli cells (see below).

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, 2001Go; Huff et al., 2001Go) 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., 1975Go; Paniagua and Nistal, 1984Go; Gondos, 1993Go), and a similar picture is evident in the cynomolgus monkey (Fouquet, 1982Go; Kluin et al., 1983Go). 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, 2001Go; Huff et al., 2001Go), 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., 1997Go) and only modest effects on spermatogenesis and germ cell volume per testis (Sharpe et al., 2000Go).

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., 1994Go; 1997; McKinnell et al., 2001Go) coincident with atrophy of the Leydig cells (Prince et al., 1998Go), and Sertoli cell proliferation (which is primarily FSH-driven in the neonatal period) is suppressed (Sharpe et al., 2000Go). 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., 1989Go; 1999; Sharpe et al., 2000Go). 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., 2002Go; 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 pituitary–testicular 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., 2001Go; Skakkebaek et al., 2001Go; Sharpe et al., 2003Go).


    Acknowledgements
 
Antide was synthesised at the Salk Institute (under contract N01-HD-0-2906 with NIH) and made available by the Contraceptive Development Branch, Center for Population Research, NICHD. We are also grateful to Dr Steve Lunn for assistance.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 7, 2003; accepted on June 26, 2003.