Germ cell differentiation in the marmoset (Callithrix jacchus) during fetal and neonatal life closely parallels that in the human

Abstract

Background: Testicular germ cell tumours (TGCT) are thought to originate from fetal germ cells that fail to differentiate normally, but no animal model for these events has been described. We evaluated the marmoset (Callithrix jacchus) as a model by comparing perinatal germ cell differentiation with that in humans.

Methods: Immunohistochemical profiling was used to investigate germ cell differentiation (OCT4, NANOG, AP-2gamma, MAGE-A4, VASA, NANOS-1) and proliferation (Ki67) in fetal and neonatal marmoset testes in comparison with the human and, to a lesser extent, the rat.

Results: In marmosets and humans, differentiation of gonocytes into spermatogonia is associated with the gradual loss of pluripotency markers such as OCT4 and NANOG, and the expression of germ cell-specific proteins such as VASA. This differentiation occurs asynchronously within individual cords during fetal and early postnatal life. This contrasts with rapid and synchronous germ cell differentiation within and between cords in the rat. Similarly, germ cell proliferation in the marmoset and human occurs throughout perinatal life, in contrast to rats in which proliferation ceases during this period.

Conclusions: The marmoset provides a good model for normal human germ cell differentiation and proliferation. The perinatal marmoset may be a useful model in which to establish factors that lead to failure of normal germ cell differentiation and the origins of TGCT.

Figure 1:

General structure and organization of the fetal and early postnatal marmoset (Callithrix jacchus) testis as illustrated by immunoexpression of anti-Müllerian hormone (AMH) in Sertoli cells within seminiferous cords (upper panels) and 3β-hydroxysteroid dehydrogenase (3β-HSD) in Leydig cells in the interstitium (lower panels). In the top panels, the clear, unstained cytoplasm outlines the nuclei of germ cells within seminiferous cords (inset). Scale bar 50 µm. wks, weeks; d, days.

Figure 2:

Immunoexpression of pluripotency factors (AP-2γ, NANOG, OCT4) that are markers of undifferentiated germ cells (gonocytes) in the marmoset testis and comparison with the human. Note that the proportions of germ cells expressing these markers decreases with increasing age. Note also the similar pattern of OCT4 expression in the marmoset compared to the human. Scale bar 50 µm.

Figure 3:

Immunoexpression of protein markers (MAGE-A4, VASA) of differentiated germ cells (pre-spermatogonia/spermatogonia) in the marmoset testis and comparison with the human. Note the increasing proportion of germ cells expressing these markers in the marmoset with increasing age and the similarity with VASA expression in the human. Scale bar 50 µm.

Figure 4:

Distinction of undifferentiated germ cells, immunoexpressing AP-2γ (red, nuclear localization), from differentiated germ cells, immunoexpressing VASA (green, cytoplasmic localization), in the marmoset and human perinatal testis using confocal microscopy. Note that all germ cells are visualized with this combination of markers. A progressive increase with age in the proportion of germ cells expressing VASA is seen as the proportion of AP-2γ-positive germ cells decreases. A small proportion of germ cells co-expressed both markers (arrows) and are presumed to reflect a transition state between undifferentiated and differentiated. Sections conterstained with TO-PRO-3 (blue). m, months.

Figure 5:

Comparison of perinatal germ cell differentiation in the marmoset, human and rat using protein markers of undifferentiated (OCT4: red, nuclear localization) and differentiated (VASA: green, cytoplasmic localization) germ cells and confocal microscopy. Note that in seminiferous cords in both the marmoset and human at each age studied there is a mixed population of germ cells based on the expression of these two markers, including occasional cells in which the markers were co-localized (arrows). This is in contrast to the rat, in which OCT4 is co-expressed with VASA in all germ cells at embryonic day 15.5 (e15.5) but was no longer detected within any of the germ cells at e19.5 (and was largely absent at e17.5, not shown). wks, weeks.

Figure 6:

(A) Comparison of the immunoexpression of NANOS-1 (red, nuclear and cytoplasmic localization) and VASA (green, cytoplasmic localization) in germ cells in the testis during fetal and postnatal life in the marmoset (top panel) and human (lower panel) using confocal microscopy. Note that neither protein is expressed in early gestation in either species but begin to be co-expressed (yellow) in the final third of gestation in the marmoset and in the second trimester in the human testis. In postnatal life, these markers continue to be co-expressed and co-localization in the peri-nuclear region is seen in most germ cells. (B) Immunoexpression of NANOS-1 (green, nuclear and cytoplasmic localization) and AP-2γ (red, nuclear localization) in the marmoset and human testis for comparison with VASA/AP-2γ expression (Fig. 4). NANOS-1 is expressed in most of the germ cells in the 1-day-old marmoset and 31-week-gestation human testis, with the remainder expressing AP-2γ. However, in contrast to VASA/AP-2γ (Fig. 4), no germ cells showed co-localization of NANOS-1 and AP-2γ. All sections conterstained with TO-PRO-3 (blue).

Figure 7:

(A) Germ cell proliferation in the marmoset testis in fetal and early postnatal life as visualized by immunoexpression of Ki67 (brown) and (B) as quantified to generate a proliferation index. Germ cell proliferation (arrows) occurs in a proportion of germ cells throughout fetal (F) and postnatal life in the marmoset. At all ages a variable proportion of germ cells are not proliferating (arrowheads). Note that some Sertoli cells are also proliferating in the sections in A. In (B), bars represent mean ± SEM (n = 3 for each age). Scale bar 50 µm.

Figure 8:

Germ cell proliferation in the fetal rat testis as visualized by immunoexpression of the proliferation marker, Ki67 (brown). Germ cell proliferation (arrows) occurs in the majority of germ cells at e15.5 but has ceased completely by e19.5 when all germ cells are negative for Ki67 (arrowhead). Note that, at both ages, some Sertoli cells (peripherally located) are immunopositive for Ki67. Scale bar 50 µm.

Figure 9:

Comparison of germ cell protein expression in the marmoset and human from fetal (F) into postnatal life. The dimensions of the grey bars illustrate the relative proportion of germ cells expressing an individual marker at a given age.