The functional role of the copper transporter 1 in spermatogenesis and in cisplatin-induced testicular injury

Cisplatin (cis-diamminedichloroplatinum(II) (cDDP)) is a highly effective chemotherapeutic drug used for the treatment of various cancers including ovarian, bladder, cervical, head and neck, small-cell and non-small-cell lung cancer. Unfortunately, cDDP is encumbered by its dose-limiting toxicity including prolonged azoospermia and, in some cases, permanent male infertility. Since patients that undergo cDDP based chemotherapy for these types of cancers are mostly young men in their prime reproductive age (ages 15-44), one of the major concerns after their recovery is infertility which would affect their quality of life. Therefore, it important to understand the mechanism of this reproductive toxicity in order to develop strategies to alleviate cDDP induced testicular injury that results after chemotherapy exposure. It has been well established that cDDP induces apoptosis in tumor and normal cell types, however, mechanisms leading to its long-lasting reproductive toxicity is less understood. Whether cDDP-induced impairment of spermatogenesis is a result of direct drug exposure to the germ cells (GCs) or results from a secondary effect to the somatic Sertoli cells (SCs) function remains unclear. An increasing body of evidence has implicated the major role of the high-affinity copper (Cu) transporter 1 (SLC31A1, CTR1), which functions in the uptake of Cu into mammalian cells, in cDDP sensitivity. The level of Ctr1 expression is recognized to be highly associated with cDDP accumulation and sensitivity in eukaryotic cells. The testis is one of the organs that express high Ctr1 mRNA and protein levels, which suggests that CTR1 protein may play a significant role in cDDP induced toxicity in testis. Moreover, given the essential role of CTR1 in Cu homeostasis, very little is known on the Cu transport mechanism and the physiological role of CTR1 protein in testis. Thus, this dissertation is focused on deciphering the role of CTR1 in testicular Cu homeostasis and its functional role in cDDP-induced testicular injury. In this thesis, two independent mouse models were generated with the conditional knockout of Ctr1 in either GCs (GC [superscript ΔCtr1]) or SCs (SC [superscript ΔCtr1]) using the Cre-Lox system. Loss of Ctr1 in GCs revealed the essential role of its expression in GCs to maintain functional spermatogenesis. GC [superscript ΔCtr1] mice exhibited a significant reduction of testicular mass by a severe progressive loss of GCs starting at postnatal day (PND) 28 leading to testis hypoplasia by adulthood. No spermatogenic recovery was observed in GC [superscript ΔCtr1] testis beyond PND 41, despite the presence of undifferentiated spermatogonial cells. On the other hand, SC [superscript ΔCtr1] mice were indistinguishable from their WT littermates with respect to fertility, spermatogenesis, growth, and development. Upon examination of testicular Cu level, SC [superscript ΔCtr1] mice exhibited significant reduced Cu and Cu-dependent cellular activities. The results from the two independent mouse models reveal, for the first time, the differential cell-specific role of CTR1 expression by testicular cell types (GCs and SCs) in maintaining functional spermatogenesis and Cu homeostasis. Finally, the role of CTR1 expression by SCs in cDDP-induced testicular injury was examined. Due to the testicular hypoplasia of the adult GC [superscript ΔCtr1] mouse testis, cDDP treatment of GC [superscript ΔCtr1] mice was not performed. Acute treatment of cDDP (5mg/kg) on SC [superscript ΔCtr1] mice was done and analyzed. Interestingly, SC [superscript ΔCtr1] mice testis exhibited higher resistance to cDDP-induced GC apoptosis than WT mice. Moreover, cDDP treated SC [superscript ΔCtr1] mice testis displayed significantly less platinum levels than their treated WT littermates. The results in this study provide the first evidence that loss of CTR1 expression by SCs enhances testis resistance to cDDP toxicity by reducing the accumulation of cDDP in testis. Taken together, the observations described within this thesis demonstrate the physiological role of CTR1 for functional spermatogenesis and Cu homeostasis in testis. Furthermore, loss of CTR1 expression in SCs revealed a newly identified pathway for cDDP uptake and toxicity. The mechanism(s) underlying the Cu transport system in testis and its effect on fertility will provide insights into the possible development of targeted therapies to improve fertility in individuals who are affected from diseases connected to an imbalance of Cu metabolism. Furthermore, the involvement of CTR1 in cDDP injury to the SCs sets the foundation to further understand and design strategies to protect or alleviate long-lasting reproductive injury following cDDP therapy