Table 1.
Description of donors.
Table 2.
Histology of the biopsies obtained from donors.
Fig 1.
Comparison of the transcriptomes of testes with complete spermatogenesis with the transcriptomes of testes with SCO syndrome.
(A) K means cluster analysis comparing the transcriptomes of testes with complete spermatogenesis with testes with SCO syndrome. Genes were clustered under 5 Gene Ontology terms; one or two representative terms per cluster are listed here. (B) The abundance in of a few well-characterized transcripts considered markers of three testicular somatic cells: Sertoli cells (VIM), peritubular myoid cells (ACTA2), and Leydig cells (INHA). Also shown are transcripts encoding receptors for three hormones required for normal testis function: FSHR, AR, and LHCGR. (C) Abundance of transcripts expressed by human SSCs (GFRA1, RET, ZBTB16, UCHL1, ENO2), by spermatocytes (SYCP2, SYCP3) and by round spermatids (PRM1, PRM2). Data in panels B and C are presented as CPM values in the total testis transcriptome divided by the CPM value for beta actin in the same sample. Asterisks over a pair of bars identify significant differences between testes with complete spermatogenesis and SCO testes (p<0.005).
Fig 2.
The expression of Sertoli cell signature transcripts in testes with complete spermatogenesis and in testes with SCO syndrome.
(A) PCA analysis of the expression of Sertoli cell signature transcripts in individual testes. Data were obtained from 4 testes with complete spermatogenesis (C1-4, green symbols) and seven SCO testes (SCO5, SCO7-12, red symbols) (B). A comparison of the mean level of expression of each Sertoli cell signature transcript in testes with complete spermatogenesis and in testes with SCO syndrome. Transcripts that were expressed in SCO testes at significantly higher levels (FDR<0.05), the same level or significantly lower levels are identified by blue, white and black dots, respectively.
Fig 3.
Expression of Sertoli cell signature transcripts encoding proteins that form adhesive or gap junctions or that organize and polarize the Sertoli cell plasma membrane.
(A) CLDN11 and JAM3 are tight junction adhesive proteins. Nectin2 and CDH2 are adhesive proteins of ectoplasmic specializations. GJB2 encodes the gap junction protein, Connexin 26. (B) Transcripts that encode proteins that organize or polarize the plasma membrane. CRB2 is a cell polarity component protein that defines the site of separation of the apical and basal-lateral plasma membrane domains. TJP3 is an adaptor protein that links the adhesive proteins of a tight junction to the actin cytoskeleton. SPTB, SPTPN4 and SPTPN5 are scaffolding proteins that concentrate beneath the plasma membrane, seed formation of membrane microdomains, and link those domains to the actin cytoskeleton. All data in this figure are expressed as mean + SEM of CPMs. The presence of an asterisk over a pair of bars indicates a significant difference between testes with complete spermatogenesis and testes with SCO syndrome (FDR≤0.05).
Fig 4.
Expression of Sertoli cell signature transcripts encoding regulators of vesicular trafficking.
(A) Expression of RRAS2 and HRAS and the catalytic subunit of phosphoinositide 3-kinase (PI3K), PIK3CA. Binding of RRAS2 and HRAS to PI3KCA stimulates its catalytic activity. (B) Expression of regulatory subunits of PI3K. (C) Small GTPase family members that regulate vesicular trafficking. RAB20 regulates endocytic trafficking; RAB40B and RAB3D regulate exocytic trafficking. Activities of these small GTPases are stimulated by PI3K. Data are expressed as mean + SEM of CPMs for each transcript. The presence of an asterisk over a pair of bars indicates a significant difference between testes with complete spermatogenesis and testes with SCO syndrome (FDR<0.05).
Fig 5.
Expression of Sertoli cell signature transcripts that encode growth factors that are known regulators of SSCs and/or progenitor spermatogonia.
Data are expressed as mean + SEM of CPMs. An asterisk over a pair of bars indicates a significant difference (FDR≤0.05).
Fig 6.
Confirmation that FGF8 is a product of human Sertoli cells and verification that expressions of both FGF8 protein and mRNA are reduced in SCO testes.
Testes with complete spermatogenesis (A) and with SCO syndrome (B) were dispersed into single cells and then incubated with antibodies for the Sertoli cell marker SOX9 and for FGF8. Negative control cells (C) were not incubated with antibodies. Cells were then analyzed FACs. These data are representative of three independent analyses. (D) Quantification by real-time PCR of levels of FGF8 mRNA in biopsies of human testes with complete spermatogenesis and in testes with SCO syndrome. Data (mean + SEM; n = 3) were normalized for expression of beta actin in the same sample (p<0.001).
Fig 7.
Summary: Deficits in Sertoli cell gene expression that are associated with specific aspects of human SCO syndrome.
(A) Human testis with complete spermatogenesis. Polarity complex proteins (red) and tight junctions (gray) polarize the plasma membrane thereby generating apical and basal-lateral domains (identified by up and down arrows). Additional tight junctions, ectoplasmic specialization (yellow) and gap junctions (blue and green) are concentrated at sites of cell-cell contact. Vesicular trafficking (VT; tan circles) of adhesive and gap junction proteins facilitates the assembly and disassembly of junctions, controlled by a pathway that includes Ras GTPases, PI3K and RAB small GTPases (purple shading). Sertoli cells express normal amounts of GDNF, FGF8 and BMP4, growth factors that regulate replication and differentiation of SSCs. (B) The infertile SCO testis. Inadequate expression of polarity and adaptor proteins renders Sertoli cells incapable of properly polarizing their plasma membranes. There is reduced, inefficient vesicular trafficking of proteins that form tight junctions, ectoplasmic specializations and gap junctions, stranding these proteins in vesicles within the cytoplasm. Finally, there is reduced expression of GDNF, FGF8 and BMP4. (C) The effects of GDNF, FGF8 and BMP4 on SSCs and progenitor spermatogonia. GDNF and FGF8 promote self-renewing replication of SSCs, while BMP4 stimulates differentiation of SSCs into progenitor spermatogonia and progenitors into fully differentiated B spermatogonia.