Effects of oncotherapy on testicular stem cells and niche

Editor's note

The authors of the MHR paper “Irradiation affects germ and somatic cells in prepubertal monkey testis xenografts” were given the opportunity to comment on this Letter to the Editor, but declined to make a formal response.

Sir,

We read with interest the article recently published in MHR (Tröndle et al., 2017). In addition to well reported germ cells loss in response to irradiation of macaque testicular tissue biopsies, these authors show that testicular somatic cells, in particular peritubular myoid cells, are affected. Testicular fragments from two pre-pubertal macaques were irradiated and later transplanted on to the back of nude mice. The fragments were studied after 6.5 months and they found that CXCL11 expressed by smooth muscle cells of blood vessels and seminiferous tubules was down-regulated at both transcript and protein level. No effect was observed in CXCL12 which is a Sertoli cell marker.

We were disappointed that our study reporting the effect of chemotherapy on testicular somatic cells (Anand et al., 2016) was not cited in their article. Both studies show an effect of radiotherapy and chemotherapy on the somatic compartment in the testis. These findings have significant clinical relevance in the field of oncofertility for cancer survivors. We isolated Sertoli cells from normal and chemoablated mouse testis (busulphan 25 mg/kg) and subjected to microarray analysis. Our results showed an altered expression of Gdnf, Scf, Fgf, Bmp4, androgen-binding protein, components of blood-testis-barrier and also stem cells related signaling pathways, including Wnt. Thus in addition to germ cells loss, even the Sertoli cells function is compromised after chemotherapy. Analysis of the whole transcriptome of a pure population of Sertoli cells gives a bigger and better picture of the effects of oncotherapy on the somatic cells that constitute a niche for testicular stem cells compared to studying few cells specific markers.

In addition, we have reported that a novel population of pluripotent, very small embryonic-like stem cells (VSELs) survive chemotherapy in the testis due to their relatively quiescent nature (Anand et al., 2014, 2016; Patel and Bhartiya, 2016), their numbers increase initially, we hypothesize as an attempt to restore spermatogenesis but since the niche (source of growth factors and cytokines for normal stem cell function) is compromised, the surviving VSELs are unable to differentiate further to generate sperm. VSELs in adult gonads were recently reviewed (Bhartiya et al., 2016). Similarly, Kurkure et al. (2015) have reported the presence of VSELs along with Sertoli cells in azoospermic testicular biopsies collected from seven men survivors of childhood cancer. The results, suggesting a compromised niche in response to irradiation and chemotherapy, also explain much of the published data describing attempts at transplanting germ cells in chemoablated testis that result only in colonization and proliferation of transplanted viable germ cells but fail to differentiate further. This situation can easily be compared to a ‘seed and soil’ concept wherein one may keep transplanting seeds but they will fail to germinate since the soil is compromised—something reproductive biologists have been attempting to do over last two decades since the landmark paper by Brinster's group (Brinster and Zimmermann, 1994).

By transplanting niche cells (Sertoli or bone marrow mesenchymal cells) via the inter-tubular route into a chemoablated testis, spermatogenesis was completely restored from the endogenous VSELs that survive chemotherapy and sperm isolated from the cauda fertilized eggs in vitro (Anand et al., 2014, 2016). A similar beneficial effect of transplanting mesenchymal cells in chemoablated testis resulting in improved testicular function and fertile offspring has been reported by several other groups as well although these groups do not acknowledge the presence of testicular VSELs (Supplementary Table 1 in Anand et al., 2016; reviewed in Kadam et al., 2017). It is noteworthy to point out here that even chemoablated ovaries become functional and live births are reported on transplanting mesenchymal cells (Bhartiya, 2017; reviewed in Kadam et al., 2017).

In contrast to our findings (Anand et al., 2016) and those reported by Tröndle et al. (2017); Kanatsu-Shinohara et al. (2016) reported that there is no effect of chemotherapy on the testicular somatic cells as judged by immuno-expression of (WT-1, CLDN11 and SOX-9) and Leydig (PDGFRA) cells specific markers. They report down-regulation of SOX-9 only; however, a careful look at the representative images in their manuscript suggests altered CLDN11 and WT-1 expression in busulphan treated testis.

Kadam et al. (2017) suggest that transplanting mesenchymal stem cells (MSCs) could improve spermatogonial stem cells (SSCs) transplantation efficiency by their paracrine action. In contrast, we have shown that there is no need to transplant SSCs at all as VSELs survive in chemoablated testis and will differentiate into SSCs. This group describes MSCs as LIN-CD45-SCA-1+ cells and HSCs as LIN-CD45+SCA-1+. However, this leads to confusion since VSELs also have similar surface phenotype of LIN-CD45-SCA-1+ (Anand et al., 2016; Shaikh et al., 2017). In fact, MSCs are not to be considered stem cells as discussed recently by Caplan (2017).

It is crucial that we arrive at a consensus of the presence of VSELs in adult mammalian testis (protocols to study them are provided as a supplement in Bhartiya et al., 2016), their survival after oncotherapy and how these endogenous VSELs can be manipulated to restore spermatogenesis by transplanting healthy niche cells (mesenchymal cells) via the inter-tubular route, rather than the technically challenging intra-tubular transplantation of germ cells. Results reported by Anand et al. (2016) suggest that there may be no need to cryopreserve testicular tissue prior to oncotherapy and will provide a paradigm shift in the field of oncofertility in future. Based on the promising results of several animal studies reporting the birth of fertile pups (Supplementary Table 1 in Anand et al., 2016; reviewed in Kadam et al., 2017), the strategy to restore spermatogenesis by transplanting mesenchymal cells (provide paracrine support to the surviving, endogenous VSELs to undergo spermatogenesis) in azoospermic testis is ripe for translation. Testicular function could be recovered in pediatric cancer survivors early on in life to support proper growth and later as a source of sperm to procreate.

Conflict of interest

None declared.

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