Taylor Malachowski

The foundation of mammalian spermatogenesis is provided by undifferentiated spermatogonia, which comprise of spermatogonial stem cells (SSCs) and transit-amplifying progenitors that differentiate in response to retinoic acid (RA) and are committed to enter meiosis. Our laboratory recently reported that the foundational populations of SSCs, undifferentiated progenitors, and differentiating spermatogonia are formed in the neonatal testis in part based on their differential responsiveness to RA. Here, we expand on those findings to define the extent to which RA responsiveness during steady-state spermatogenesis in the adult testis regulates the spermatogonial fate. Our results reveal that both progenitor and differentiating spermatogonia throughout the testis are capable of responding to exogenous RA, but their resulting fates were quite distinct-undifferentiated progenitors precociously differentiated and proceeded into meiosis on a normal timeline, while differentiating spermatogonia were unable to hasten their entry into meiosis. This reveals that the spermatogonia responding to RA must still complete the 8.6 day differentiation program prior to their entry into meiosis. Addition of exogenous RA enriched testes with preleptotene and pachytene spermatocytes one and two seminiferous cycles later, respectively, supporting recent clinical studies reporting increased sperm production and enhanced fertility in subfertile men on long-term RA analog treatment. Collectively, our results reveal that a well-buffered system exists within mammalian testes to regulate spermatogonial RA exposure, that exposed undifferentiated progenitors can precociously differentiate, but must complete a normal-length differentiation program prior to entering meiosis, and that daily RA treatments increased the numbers of advanced germ cells by directing undifferentiated progenitors to continuously differentiate.

ABSTRACT In mammalian testes, premeiotic spermatogonia respond to retinoic acid by completing an essential lengthy differentiation program before initiating meiosis. The molecular and cellular changes directing these developmental processes remain largely undefined. This wide gap in knowledge is due to two unresolved technical challenges: (1) lack of robust and reliable in vitro models to study differentiation and meiotic initiation; and (2) lack of methods to isolate large and pure populations of male germ cells at each stage of differentiation and at meiotic initiation. Here, we report a facile in vitro differentiation and meiotic initiation system that can be readily manipulated, including the use of chemical agents that cannot be safely administered to live animals. In addition, we present a transgenic mouse model enabling fluorescence-activated cell sorting-based isolation of millions of spermatogonia at specific developmental stages as well as meiotic spermatocytes.

Abstract Despite breakthroughs in cancer treatment, chemotherapy-induced osteo-toxicity is a major problem that compromises the quality of life and overall survival of cancer patients regardless of cancer type. Recently we demonstrated that chemotherapy-induced senescence drives bone loss by both limiting mineralization of new bone and increasing bone resorption. However, the underlying mechanism of action remains elusive. Therefore, it is critical that we understand the mechanisms that drive these toxicities and develop approaches to mitigate their severity. To establish whether senescent bone resident cells or systemic responses to chemotherapy drove therapy-induced bone loss, we used a vertebral body transplant (vossicle) model. Using this approach, we found that the specific elimination of senescent cells in L4 and L5 donor vossicles (INK-ATTAC) implanted into wildtype mice, protects from chemotherapy-induced bone loss within the vossicles but not the femur of the recipient mice. This demonstrated that chemotherapy-induced senescence in resident bone cells is responsible for bone loss. To determine which bone resident cell(s) underwent senescence in response to chemotherapy and how their gene expression was impacted, we used the p16-CreERT2-tdTomato mouse model and found that chemotherapy triggers senescence in bone marrow adipocytes. Subsequently, we observed that postnatal fat ablation in adipoqCre-inducible DTR transgenic mice (iDTRADQ) prevented chemotherapy-induced bone loss, indicating senescent adipocytes trigger bone loss. Furthermore, we observed chemotherapy-induced bone loss is attributed to RANKL-mediated high osteoclasts activity. Collectively, our data demonstrate that chemotherapy causes senescence in marrow adipocytes which in turn triggers bone loss via RANKL-mediated osteoclasts activation and bone loss can be protected by eliminating senescent cells. Citation Format: Ganesh Kumar Raut, Taylor Malachowski, Taylor Holt, Renata Oliveira, Xianmin Luo, Douglas Faget, Qihao Ren, Sheila Stewart. Chemotherapy-induced adipocyte senescence triggers bone loss through osteoclast activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2956.

Chemotherapy-induced bone loss is a debilitating and common side effect of cancer treatment, though its underlying mechanisms remain poorly understood. Here, we show that, despite the systemic administration of chemotherapy, cellular senescence is restricted to bone marrow adipo-lineage cells specifically Cxcl12-abundant reticular (CAR) cells and bone marrow adipocytes (BMAds). Induction of senescence within these populations promotes RANK ligand (RANKL)-mediated osteoclastogenesis, leading to significant bone loss. Notably, we find that inhibition of the p38MAPK-MK2 pathway suppresses the senescence-associated secretory phenotype (SASP), including RANKL production abrogating bone loss. Furthermore, treatment with the senolytic combination dasatinib and quercetin (D + Q) selectively eliminates senescent CAR cells and BMAds, effectively preventing chemotherapy-induced bone loss. Given that nearly all chemotherapy treated patients experience bone loss and associated fracture risk, our findings offer a promising therapeutic avenue to preserve bone integrity and improve quality of life for cancer patients.