Effect of Antioxidants and Apoptosis Inhibitors on Cryopreservation of Murine Germ Cells Enriched for Spermatogonial Stem Cells

<div><p>Spermatogonial stem cells (SSCs) are germline stem cells that serve as the foundation of spermatogenesis to maintain fertility throughout a male’s lifetime. To treat male infertility using stem cell banking systems and transplantation, it is important to be able to preserve SSCs for long periods of time. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using antioxidants and apoptosis inhibitors in freezing medium. No differences were observed compared to controls when SSCs were cryopreserved in the presence of apoptosis inhibitors by themselves. However, mouse germ cells cryopreserved in basal medium containing the antioxidant hypotaurine (14 mM) resulted in significantly greater proliferation potential and mitochondrial activity. Furthermore, treatment groups with combinations containing 200 mM trehalose and 14 mM hypotaurine showed higher proliferation rates compared to controls. In addition, several serum free conditions were evaluated for SSC cryopreservation. Treatment media containing 10% or 20% knockout serum replacement resulted in similar cryopreservation results compared to media containing FBS. SSC transplantation was also performed to confirm the functionality of SSCs frozen in 14 mM hypotaurine. Donor SSCs formed normal spermatogenic colonies and sperm in the recipient testis. These data indicate that inclusion of 14 mM hypotaurine in cryopreservation media is an effective way to efficiently cryopreserve germ cells enriched for SSCs and that knockout serum replacement can replace FBS in germ cell cryopreservation media.</p></div

Effects of combinations of hypotaurine, Z-VAD-fmk, and trehalose on recovery rate and proliferation capacity of germ cells enriched for SSCs following cryopreservation.

<p>(A) Percentage of viable cells recovered after thawing. (B) Proliferation potential of cells recovered after thawing. Control = basal freezing medium containing 10% DMSO and 10% FBS; H = basal freezing medium with hypotaurine (14 mM); Z = basal freezing medium with Z-VAD-fmk (15 μM), T = basal freezing medium with trehalose 200 mM, H+Z = basal freezing medium with hypotaurine, and Z-VAD-fmk, H+T = basal freezing medium with hypotaurine and trehalose and H+Z+T = basal freezing medium with hypotaurine, Z-VAD-fmk and trehalose. Values are means ± SEM (n = 3 independently established cultures for each treatment). Different letters indicate significant difference (<i>P</i> < 0.05) between treatments.</p

Comparison of FBS to Sericin or KSR as cryoprotectant additives for recovery and proliferation capacity of thawed germ cells enriched for SSCs.

<p>(A) Percentage of viable cells recovered after cryopreservation in the presence of FBS or Sericin. (B) Proliferation capacity of cells after cryopreservation in the presence of FBS or Sericin. (C) Percentage of viable cells recovered after cryopreservation in the presence of FBS or KSR. (D) Proliferation capacity of cells after cryopreservation in the presence of FBS or KSR. F = basal freezing medium (containing 10% DMSO) and 10% FBS; 1S = basal freezing medium and 1% Sericin; 10SR = basal freezing medium and 10% KSR; F, H = basal freezing medium with FBS and hypotaurine (14 mM); 0.5, 1, 2S, H = basal freezing medium with 0.5, 1, 2% Sericin and Hypotaurine; 5, 10, 20SR, H = basal freezing medium with 5, 10, 20% KSR and Hypotaurine. Values are means ± SEM (n = 3 independently established cultures for each treatment). Different letters indicate significant difference (<i>P</i> < 0.05) between treatments.</p

Effect of antioxidants and apoptosis inhibitors on thawed germ cells enriched for SSCs.

<p>(A) Effect of antioxidants and apoptosis inhibitors on proliferation of germ cells enriched for SSCs after thawing. (B) Effect of antioxidants and apoptosis inhibitors on ATP production by germ cells enriched for SSCs after cryopreservation. (C-D) Characterization of germ cell colonies after cryopreservation in the presence of 14 mM hypotaurine, thawing and 7 days of culture. (C) Bright/dark-field image. (D) Immunocytochemistry for DAPI (blue), eGFP (green) and PLZF, VASA, or GFRα1 (red). Values are means ± SEM (n = 3 independently established cultures for each treatment). Different letters within each treatment group indicate significant difference (<i>P</i> < 0.05) between control and different dosages of each cryoprotectant. Scale bars: (C) = 100 μm; (D) = 75 μm;.</p

Functional SSC activity was demonstrated by counting the number of donor derived colonies after transplantation.

<p>(A) Dark-field fluorescence image of a recipient testis transplanted with germ cells cryopreserved in the presence of 14 mM hypotaurine. Colonies of donor spermatogenesis are distinct green regions of the recipient seminiferous tubules. (B, C) Dispersed seminiferous tubules from a recipient testis. Bright field (B). Dark field (C). (D) Cryosection of donor-derived germ cell colonies. Complete spermatogenesis is illustrated by the presence of sperm (white arrow) in the lumen of the seminiferous tubule. (E) The number of colonies per 10⁵ transplanted cells. Fresh: non-cryopreserved cells (294.0 ± 22.6), Control: cells cryopreserved in basal freezing media (252.0 ± 22.6), and Hypotaurine: cells cryopreserved in basal freezing media with 14 mM hypotaurine (244.0 ± 35.2). (F) The number of colonies per 10⁵ transplanted cells normalized for recovery rate and proliferation capacity. Fresh (1332.0 ± 103.0), Control (572.0 ± 51.3), and Hypotaurine (953.0 ± 137.0) (n = 2 experiments per treatment; total number of mice/testes analyzed were 10/11, 9/12, and 9/16 for fresh, control, and hypotaurine, respectively). Scale bars: (A) = 2 mm; (B, C) = 4 mm; (D) = 50 μm.</p