How one gene shakes up our understanding of male infertility

In a recent article published in the journal Developmentresearchers are investigating the role of the ACTL7B gene in sperm formation Actl7b-deficient mice.

Study: Actl7b deficiency leads to mislocalization of LC8-type dynein light chains and disruption of mouse spermatogenesis. Image credits: Komsan Loonprom / Shutterstock.com

Background

ACTL7B, a testis-specific actin-related protein (Arp), shares up to 60% amino acid identity with conventional actins and is highly conserved among rodents and primates. In mice and humans ACTL7B is expressed exclusively in the testis, suggesting a role in spermatogenesis.

Several animal studies have been linked ACTL7B with fertility, while studies in human subjects reveal the presence of single nucleotide polymorphisms (SNPs) in the coding sequence of ACTL7B in cohorts of infertile men.

However, these studies do not directly address the ACTL7B gene with infertility. Furthermore, there remains a lack of studies elucidating its molecular function ACTL7B.

About the study

In the current study, researchers apply clustered regularly spaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing in zygotes of C57Bl/6J, a common inbred strain of laboratory mice, to Actlt7b-deficient mice to analyze the role of ACTL7B in spermatogenesis.

Two kinds Actlt7bKnockout (KO) mice were generated, including heterozygous (Actl7b^+/-) and homozygous (Actl7b^−/−) mice. A genotyping polymerase chain reaction (PCR) assay was used to make the distinction Actl7bΔ allele from wild-type mice (Actl7b^+/+) with alleles of Actlt7b– Or mice.

Immunohistochemical staining against ACTL7B was performed on tissue sections obtained from the testis, caput, and cauda epididymis of heterozygous (Actl7b^+/-), homozygous (Actl7b^−/−), and wild type (Actl7b^+/+) mice.

The presence of any structural defects including acrosome biogenesis, DNA condensation, cuffing, and sperm tail formation in the sperm cells of Actl7b-deficient male mice, was also determined using transmission electron micrography (TEM).

ACTL7B protein interaction changes in the testicular proteome of Actl7b-deficient mice were also assessed. To this end, the researchers coupled anti-ACTL7B antibody to Dynabeads and used uncoupled beads as a control. Following this co-immunoprecipitation experiment, mass spectrometry (MS) was used to identify eluted proteins from whole testes of five mice from homozygous, heterozygous and wild-type groups.

Principal component analysis (PCA) showed differential clustering of all three samples, while differential abundance analysis (DA) revealed protein abundance Actl7b^+/- in comparison with Actl7b^+/+ samples.

An evolutionary analysis of ACTL7A And ACTL7B genes were also performed, as these genes show sequence similarity and are testis specific. This analysis allowed the researchers to compare the conservation of these genes and predict their essence.

Findings of the study

Spermatids from Actl7b-deficient mice were arrested during development, which subsequently led to the development of several abnormalities after step nine of spermatogenesis, including malformed flagella. Consequently, most spermatids were aborted.

Some of these degrading spermatids accumulated in the lumen of the seminiferous tubules along with other immature spermatids that were eliminated by Sertoli cells. This was confirmed by increasing levels of autophagy marker proteins in Actl7b^−/− testes.

MS analyzes revealed that ACTL7B specifically interacted with the dynein light chains LC8-Type 1 and Type 2 (DYNLL1 and DYNLL2), which appear in step nine of spermatogenesis. Furthermore, ACTL7B appears to exert its effects through interactions with the microtubule network or the dynein 1 motor complex, rather than with the actin cytoskeleton.

Other way around, Actl7b+/− male mice showed reduced ACTL7B levels and remained fertile similarly to wild-type mice.

Earlier, Actlt7b-KO mice were reported to be infertile due to severe oligoteratozoospermia, as well as malformed sperm tails and heads, and a tenfold reduction in sperm count compared to wild-type mice. However, in the current study, the sperm count in KO mice was approximately 32,000, which was a thousand-fold reduction compared to the approximately 32,000,000 sperm counts observed in wild-type mice. This is likely due to phenotypic differences in the parent mouse strains used.

Both models showed reduced germ cell loss, lower epididymal sperm count, and reduced release of germ cells into and out of the testis, along with several structural sperm abnormalities.

Conclusions

As human and mouse ACTL7B genes are very similar, the study results suggest that ACTL7B variants may lead to failed spermatogenesis and male infertility in humans. Moreover, since ACTL7B could help distinguish between obstructive and non-obstructive azoospermia with high accuracy at translational and transcriptional levels, the presence of this gene could potentially be used as a biomarker for male infertility in the future.