How a single gene is shaking 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 using Rule 7b-missing rats.

Lesson: Actl7b deficiency leads to impaired activation of LC8-type light chains and impaired murine spermatogenesis. Photo Credit: Komsan Loonprom / Shutterstock.com

It’s the background

ACTL7B, an actin-specific actin-related protein (Arp), shares up to 60% amino acid identity with normal actins and is highly conserved in mice and monkeys. In mice and humans, ACTL7B it is specifically expressed in the testes, thus suggesting a role in spermatogenesis.

Several animal studies have been corroborated ACTL7B reproductively, while studies involving human subjects have reported the presence of single nucleotide polymorphisms (SNPs) in the coding sequence ACTL7B in groups of sterile men.

However, these studies did not directly affect ACTL7B the sterile gene. In addition, there remains a lack of studies that clarify the molecular function ACTL7B.

About research

In the present study, the researchers used short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene in zygotes of C57Bl/6J, a standard breed of laboratory mice, to produce. Rule 7b-null mice to analyze the role of ACTL7B in spermatogenesis.

Two types of Rule 7b-knockout (KO) mice were generated, consisting of heterozygous (Rule 7b^+/−and homozygous (Rule 7b^−/−) rats. A genotyping polymerase chain reaction (PCR) test was used for discrimination Actl7bΔ allele from wild-type mice (Rule 7b^+/+) and alleles appear Rule 7b-KO mice.

Immunohistochemical staining against ACTL7B was performed on tissue sections obtained from testis, caput, and cauda epididymis from heterozygous (Rule 7b^+/−homozygous (Rule 7b^−/−), and the wild type (Rule 7b^+/+) rats.

The presence of any structural defects, including acrosome biogenesis, DNA condensation, manchette formation, and sperm tail formation in sperm. Rule 7b-deficient male mice, were also determined using transmission electron micrography (TEM).

ACTL7B-modifying protein interactions in the testicular proteome Rule 7b-deficient mice were also tested. To date, researchers have conjugated an anti-ACTL7B antibody to Dynabeads and used unconjugated beads as a control. Following this co-immunoprecipitation experiment, mass spectrometry (MS) was used to identify proteins extracted from all testes of five mice from the 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 Rule 7b^+/− as compared Rule 7b^+/+ samples.

Evolutionary analysis of ACTL7A again ACTL7B genes are also made, as these genes show sequence similarity and are specific to the testis. This analysis allowed the researchers to compare the conservation of these genes and predict their importance.

Research findings

The spermatids Rule 7b-deficient mice were arrested during development, which led to the formation of several abnormalities after the ninth step of spermatogenesis, including defective flagella. Therefore, the number of spermatids decreases.

Some of these degrading spermatids are collected in the lumen of the seminiferous tubules and other immature spermatids are released by the Sertoli cells. This was confirmed by increased levels of autophagy marker proteins in Rule 7b^−/− testicles.

MS analysis revealed that ACTL7B interacts specifically with dynein light chains LC8-Type 1 and Type 2 (DYNLL1 and DYNLL2), which appear in the ninth step of spermatogenesis. In addition, ACTL7B appears to exert its effects by interacting with the microtubule network or the dynein 1 motor complex, rather than the actin cytoskeleton.

In contrast, Actl7b+/− male mice showed reduced levels of ACTL7B and remained fertile similarly to wild-type mice.

Previously, Rule 7b-KO mice are reported to be infertile due to severe oligoteratozoospermia, as well as abnormal sperm tails and heads, and a 10-fold reduction in sperm count compared to wild-type mice. However, in the current study, sperm counts in KO mice were approximately 32,000, which was a 1,000-fold decrease compared to the approximately 32,000,000 sperm cells observed in wild-type mice. This may be due to phenotypical differences in the parental mouse strain used.

Both models showed decreased germ cell loss, epididymal sperm count, and immature germ cell release in and from the testis, as well as multiple sperm structural abnormalities.

Conclusions

Like a man and a mouse ACTL7B genes are very similar, research results suggest that ACTL7B variants can lead to sperm failure and male infertility. Moreover, since ACTL7B can help distinguish between obstructive and non-obstructive azoospermia with high accuracy at the translation and transcription levels, the presence of this gene may be used as a biomarker of male infertility in the future.