1. Male mice lacking either the Ppp3cc or Ppp3r2 genes, which encode parts of the sperm-specific calcineurin protein, produced sperm with rigid midpieces and were infertile.
2. Wild-type mice treated with cyclosporine A (CsA) or FK506, nonspecific calcineurin inhibitors, produced similarly defective sperm and displayed infertility which was reversible within a week of stopping drug treatment.
Evidence Rating Level: 2 (Good)
Study Rundown: Calcineurin is a phosphatase enzyme which activates T cells in the immune response. As a result, calcineurin inhibitors such as CsA and FK506 are used as immunosuppressants during certain medical procedures. Here, researchers characterized the effects of CsA and FK506 on male fertility in mice, with the eventual goal of developing of a male contraceptive based on calcineurin inhibition.
The calcineurin protein is made of two parts, or subunits. PPP3CC and PPP3R2, which are particular forms of the catalytic and regulatory calcineurin subunits, respectively, were found specifically in differentiating germ cells in male mice. Male mice lacking either one of the corresponding genes were infertile. Because their sperm displayed inflexible midpieces that could not curve during sperm hyperactivation, the sperm did not penetrate the zona pellucida (ZP) that surrounds eggs. Indeed, in vitro fertilization (IVF) with sperm from animals lacking PPP3CC successfully produced embryos only when performed in media containing glutathione, which destabilizes the ZP. Treating wild-type male mice daily with CsA or FK506 led to infertility within two weeks, and their sperm were visually similar to those of the genetic knockouts within 4-6 days. Fertility of these mice was recovered within a week after stopping drug treatment. Final experiments demonstrated that human sperm also specifically contained PPP3CC and PPP3R2 as opposed to other forms of the calcineurin subunits.
Translating these results to clinical development of a reversible male contraceptive depends on the identification of a drug which can specifically inhibit PPP3CC or PPP3R2 without affecting the systemic immune response. If successful, this work may lay the foundation for the long-awaited male contraceptive.
Relevant Reading: Effect of Prograf (FK506) on spermatogenesis in rats
In-Depth [animal study]: Western blotting showed that PPP3CC and PPP3R2 were localized in the testes, epididymides and sperm of wild-type mice. C-Kitw/wv mice, which lack differentiating germ cells, did not have these proteins in their epididymides and testes, indicating that PPP3CC and PPP3R2 are specific to spermatogenic cells.
Male knockout mice lacking the gene for PPP3CC were infertile compared to control mice (n=3-6 males and 19-49 vaginal plugs indicating copulation, per group). IVF using their sperm was unsuccessful compared to normal sperm (p<0.001, n=5 males and 303-396 eggs per group), but succeeded when performed in medium containing glutathione. Sperm from knockout mice displayed reduced hyperactivity (p<0.01-0.05 at 10 and 120 minutes following sperm suspension in media, n=9 males per group), and had rigid midpieces which could not functionally bend during hyperactivity to penetrate the ZP (p<0.001 at 10 and 120 minutes, n=305-367 sperm from 3 males per group). Male knockout mice lacking the gene for PPP3R2 showed similar infertility phenotypes.
Treating wild-type male mice with 80 mg/kg CsA or 8 mg/kg FK506 per day for 2 weeks resulted in infertility measured both in vivo (p<0.001 for each drug vs. control mice, n=3-4 males and 108-156 eggs per group) and using IVF (p<0.001 for each, n=4-5 males and 189-240 eggs per group). Nearly 100% of sperm from these mice showed midpiece rigidity (p<0.001 vs. sperm from control mice, n=312-493 sperm from 3-5 males per group), an effect which began to appear within 4-6 days of treatment. Fertility recovered by 1 week after stopping treatment.
Finally, western blotting showed that only the PPP3CC and PPP3R2 forms of the calcineurin subunits were present in human sperm. This result suggests that selectively targeting PPP3CC or PPP3R2 may effectively compromise male fertility in the clinical setting.
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