Significant Benefits of Nanoparticles Containing a Necrosis Inhibitor on Mice Testicular Tissue Autografts Outcomes

Abstract

Fertility preservation for prepubertal boys relies exclusively on cryopreservation of immature testicular tissue (ITT) containing spermatogonia as the only cells with reproductive potential. Preclinical studies that used a nude mice model to evaluate the development of human transplanted ITT were characterized by important spermatogonial loss. We hypothesized that the encapsulation of testicular tissue in an alginate matrix supplemented with nanoparticles containing a necrosis inhibitor (NECINH-NPS) would improve tissue integrity and germ cells' survival in grafts. We performed orthotopic autotransplantation of 1 mm³ testicular tissue fragments recovered form mice (aged 4-5 weeks). Fragments were either non-encapsulated, encapsulated in an alginate matrix, or encapsulated in an alginate matrix containing NECINH-NPs. Grafts were recovered 5- and 21-days post-transplantation. We evaluated tissue integrity (hematoxylin-eosin staining), germ cells survival (immunohistochemistry for promyelocytic leukemia zinc-finger, VASA, and protein-boule-like), apoptosis (immunohistochemistry for active-caspase 3), and lipid peroxidation (immunohistochemistry for malondialdehyde). NECINH-NPs significantly improved testicular tissue integrity and germ cells' survival after 21 days. Oxidative stress was reduced after 5 days, regardless of nanoparticle incorporation. No effect on caspase-dependent apoptosis was observed. In conclusion, NECINH-NPs in an alginate matrix significantly improved tissue integrity and germ cells' survival in grafts with the perspective of higher reproductive outcomes.

Keywords: fertility preservation; male fertility; nanoparticles; necrosis; necrosis inhibitor; prepubertal boys; prepubertal testicular tissue; spermatogonia; tissue engineering; transplantation.

Figure 1

In vitro release of NECINH-nanoparticles (NPs) incorporated in 1% alginate hydrogel. Twenty-five microliters of NPs corresponding to 15.27 µg of NECINH were incorporated in a 50 µl alginate hydrogel and immersed in 40 mM CaCl₂ PBS (Phosphate buffer saline) for 21 days at 37 °C. The release was evaluated over time by fluorimetry (n = 4).

Figure 2

Impact of testicular fragment incorporation in NECINH nanoparticles-loaded alginate hydrogel on tissue necrosis when cultured in hypoxia. Cumulative values of fluorescence corresponding to the lactate dehydrogenase (LDH) measured in culture supernatants recovered every 48–72 h.

Figure 3

Impact of NECINH-nanoparticles-loaded hydrogel on mice testicular tissue exposed to hypoxia (1% O₂) and recovered after 5 or 21 days. Three conditions were compared for each timing (non-encapsulated, encapsulated in alginate, and encapsulated in alginate with NECINH NPs). Hematoxylin-eosin staining was used for the evaluation of STs (seminiferous tubule sections) morphology. Scale bar = 60 μm.

Figure 4

Impact of NECINH-nanoparticles-loaded hydrogel on mice testicular tissue exposed to hypoxia (1% O₂) for 21 days. Undifferentiated spermatogonial survival. Promyelocytic leukemia zinc finger (PLZF) + cells are highlighted by red arrows.

Figure 5

Impact of NECINH-nanoparticles-(NPs) loaded hydrogel on mice testicular tissue after autotransplantation for 5 and 21 days. Hematoxylin-eosin staining was used for the morphological evaluation of seminiferous tubules sections. Scale bar = 60 μm.

Figure 6

Impact of NECINH-nanoparticles-loaded hydrogel on mice testicular tissue after autotransplantation for 5 and 21 days. Tissue integrity evaluation on hematoxylin-eosin stained slides. The graph shows the percentage of intact (Score 1) STs (seminiferous tubule sections) evaluated at day 5 and day 21. * indicates p < 0.05 relative to day 21 Score 1 NECINH-NPs.

Figure 7

Impact of NECINH-nanoparticles (NPs)-loaded hydrogel on germ cells survival in mice testicular tissue after 21 days of autotransplantation. The number of positive cells/ST section for each germ cell marker (Promyelocytic leukemia zinc finger (PLZF), VASA, and protein boule-like (BOLL)) was statistically significantly higher for NECINH encapsulation compared to both alginate-only and non-encapsulation and for alginate-only encapsulation compared to non-encapsulation. Results are expressed as mean number of positive cells/seminiferous tubule section ± standard deviation. For PLZF, Non-encapsulated: 0.94 ± 0.23; Alginate: 1.80 ± 0.14; NECINH 2.37 ± 0.15. a: alginate/non-encapsulated tissue (p < 0.0001); b: NECINH NPs/non-encapsulated tissue (p < 0.0001); c: NECINH NPs/alginate (p = 0.0009). For VASA, Non-encapsulated: 2 ± 0.23; Alginate: 3.66 ± 0.43; NECINH 7.38 ± 1.56 d: alginate/non-encapsulated tissue (p = 0.0415); e: NECINH-NPs/ alginate (p = 0.0001); f: NECINH-NPs/non-encapsulated tissue (p < 0.0001). For BOLL, Non-encapsulated: 1.60 ± 0.34; Alginate: 3.16 ± 0.68; NECINH 5.60 ± 0.95. g: alginate/non-encapsulated tissue (p = 0.00111); h: NECINH-NPs/alginate (p = 0.0004); i: NECINH-NPs/non-encapsulated tissue (p < 0.0001). * statistical significant results (p < 0.05).

Figure 8

Impact of NECINH-NPs on germ cells survival in mice testicular tissue auto-transplanted for 21 days. Images show 40 X magnification of IHC for PLZF, BOLL, and VASA. Positive cells are highlighted by red arrows.

Figure 9

Impact of NECINH nanoparticles (NPs)-loaded hydrogel on lipid peroxidation in mice testicular tissue after autotransplantation for 5 and 21 days. Malondialdehyde (MDA)-positive cells were reduced on day 5 when encapsulation in alginate was performed, regardless of NPs supplementation. Positive cells are highlighted by red arrows.