Loss of protamine 1 and actin-like 7b – Investigating genes relevant for male fertility

Abstract

Sperm express a suite of genes specific to the testis. While they likely play essential roles in genes. <br /> One of the key events during spermatogenesis is the replacement of histones by protamines. This leads to hypercondensation of the chromatin, reduction of sperm nucleus size and protection of the paternal genome. In human and mice, two protamines, protamine-1 (Prm1) and protamine-2 (Prm2) are expressed in a species-specific ratio. Alterations of this ratio are associated to male sub- and infertility. Unlike PRM1, PRM2 is a precursor protein, containing a mature domain (mP2) and a cleaved domain (cP2), which is sequentially cleaved off upon DNA binding. To decipher common and unique roles of both protamines, Prm1-deficient mice were analyzed and compared to previously described Prm2-deficient mice. Whereas, loss of one allele of Prm1 (Prm1+/-) rendered male mice subfertile, Prm2+/- mice were fertile. Mice lacking Prm1 or Prm2 were infertile, showing severe DNA damage and increased histone retention. Prm1+/- mice displayed only slight DNA damage. However, they contained high levels of unprocessed PRM2 and the species-specific protamine ratio was skewed. In comparison, the protamine ratio was maintained in Prm2+/- sperm, suggesting that the ratio needs to be precisely controlled to retain full fertility. <br /> Additionally, a mouse model carrying a Prm1-Prm2 fusion gene was analyzed. Here most of the Prm1 coding sequence was fused to mP2, lacking the cP2 domain. By comparing this model to a previously described cP2-deficient mouse model we were able to narrow down the specific functions of the protamine domains and PRM1-PRM2 interaction. cP2-deficient mice were infertile, demonstrating that the cP2 domain is essential. Surprisingly, mice carrying one or two copies of the fusion allele partially retained sperm function and fertility. This suggests, that cP2 is necessary for PRM1-PRM2 interaction and that loss of cP2 might be compensated by fusing PRM1 to the mP2 domain. <br /> Moreover, Actin-like 7b (Actl7b)-deficient mice were generated and phenotypically characterized. The function of ACTL7B in spermatogenesis was unknown to date. Actl7b-/- mice were infertile, showed reduced sperm numbers and severe sperm malformations. Actl7b+/- mice appear phenotypically inconspicuous and remain fertile. First, coimmunoprecipitation and subsequent mass spectrometric analyses, imply that ACTL7B functions in the microtubule network during spermiogenesis. <br /> Together the results of this project present an important step forward in understanding the function and essentiality of a key set of sperm specific genes and their potential importance in male infertility.