Fibroblast growth factor receptor 3 is highly expressed in rarely dividing human type A spermatogonia

Abstract

Human spermatogonia (Spg) and their fetal precursors express fibroblast growth factor receptor 3 (FGFR3). To further elucidate the role of FGFR3 in the control of Spg self-renewal, proliferation, and/or differentiation, and to narrow down the FGFR3-positive cell type(s) in the normal adult human testis, tissue sections and whole mount preparations of seminiferous tubules were analyzed combining immunofluorescence and confocal fluorescence microscopy. FGFR3 protein was chiefly observed in cellular membranes and cytoplasmic vesicles of a subpopulation of type A Spg, which comprised the chromatin rarefaction zone-containing type A(dark). Cytoplasmic expression of FGFR3 and nuclear expression of proliferation-associated antigen KI-67 were mutually exclusive. Similarly, FGFR3-positive Spg were negative for Doublesex and Mab-3 related transcription factor 1 (DMRT1). By contrast, undifferentiated embryonic cell transcription factor 1 (UTF1) and survival time-associated PHD finger in ovarian cancer 1 protein (SPOC1) were co-expressed in the nuclei of FGFR3-positive Spg. Whole mounted seminiferous tubules illustrated the clonogenic arrangement of the FGFR3/UTF1 double-positive Spg, which mainly occurred as pairs or quadruplets and, different from the KIT-positive Spg, showed no overlap with KI-67 labeled clusters. Taken together, in the adult human testis, FGFR3 expression is a feature of small clones of rarely dividing type A Spg which resemble "undifferentiated" Spg, including the spermatogonial stem cells.

Biglycan is a novel binding partner of fibroblast growth factor receptor 3c (FGFR3c) in the human testis

“…Previously, we characterized the FGFR3 protein in human spermatogonia (Juul et al, 2007) and found that only the FGFR3c isoform was expressed in these cells throughout development starting from the second trimester of gestation (Ewen et al, 2013). In the adult human testis, FGFR3 localizes to the A-type spermatogonia, and is preferentially expressed in non-proliferating cells (Ewen et al, 2013;von Kopylow et al, 2012). Importantly, FGFR3-activating mutations have been identified in spermatocytic seminoma (Goriely et al, 2009), which is a rare germ cell tumor thought to originate from clonal expansion of spermatogonia (Rajpert-De Meyts et al, 2003).…”

Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes

“…Although there were variable ischemic changes in some of the testes, most were histologically normal (SI Appendix, Table S1). The spermatogonial origin of these mutations is indicated by the strong immunoreactivity of cells within putative clones to MAGEA4, FGFR3, and pAKT, antigens characteristic of premeiotic germ cells (30)(31)(32)(33). Microdissection of putative clones, with the aim of maximizing the relative mutant DNA content of the sample (up to ±50%), enabled the simultaneous screening of ∼300,000 nucleotides in over 100 candidate genes, in comparison with only 19 nucleotides (across 6 genes) that were investigated in all previous sperm (16,(26)(27)(28)(29) and testis piece (11)(12)(13)(14)(15) studies combined.…”

“…We focused this work on the testes of older men, reasoning that PAE mutations would be more readily detected in this age group. In an earlier immunohistochemical survey, we reported that a small fraction of seminiferous tubules (which we termed "immunopositive tubules") exhibit increased numbers of spermatogonia displaying strong immunoreactivity to antibodies against MAGEA4 (melanoma antigen A4, a spermatogonial marker of unknown function) (30), FGFR3 (a known PAE protein expressed in spermatogonia) (26,31), and pAKT (phospho-v-akt Significance A major goal in genetics is to understand the processes that shape the frequency of new mutations, particularly those causing human disease. Here, we focus on specific mutations in the male germline that, although initially rare, confer a growth or survival advantage to the stem cell, leading to clonal expansion over time: a process similar to early tumor growth and currently described only in humans.…”

Comparison of enzymatic digestion and mechanical dissociation of human testicular tissues

“…The FGFR3 is a marker for undifferentiated spermatogonia, and colocalization studies have revealed that it is only expressed by a subpopulation of UTF1-positive spermatogonia. Furthermore, SALL4 has been shown to be expressed by a population of undifferentiated spermatogonia (2,3), whereas MAGE A4 is expressed in spermatogonia and spermatocytes (4,5). In contrast, vimentin (VIM) and smooth muscle actin (SMA) facilitate detection of somatic Sertoli, peritubular, and mesenchymal cells; and peritubular and endothelial cells, respectively (6,7).…”

“…As only small testicular biopsies containing a limited number of SSCs can be obtained from prepubertal patients, the following processes need to be optimized to obtain the highest possible number of viable SSCs from each biopsy: [1] Cryopreservation protocols, [2] the dissociation procedure of testicular tissues, [3] the enrichment of spermatogonia, and [4] the propagation of SSCs. With regard to process 1, comparative results were obtained comparing cell viability after enzymatic digestion of testicular tissues cryopreserved in four commonly used cryoprotective agents (11).…”