A new study published in Development recently examined the role of the ACTL7B gene in sperm production. To do this, the researchers used mice that were lacking the Actl7b gene.
Historical Context
A protein called ACTL7B, belonging to the actin-related family, has up to 60% amino acid similarity to typical actins and is very similar between mice, humans and other primates. This protein is expressed only in the testes, indicating that it has a purpose in spermatogenesis.
Results from experiments conducted on animals suggest that ACTL7B relates to fertility, while research done with humans revealed single nucleotide polymorphisms (SNPs) in the coding sequence of ACTL7B in groups of men who are not able to have children.
Despite the fact that no research has been able to specifically link ACTL7B to infertility, there is still a need for investigations into what its molecular function is.
Investigating the topic
This study is an exploration of the subject.
For this research, scientists utilized CRISPR/Cas9-mediated gene editing to develop Actlt7b-lacking mice of the C57Bl/6J strain, a typical inbred type of lab mice, to investigate the part of ACTL7B in spermatogenesis.
A genotyping polymerase chain reaction (PCR) was performed to differentiate between wild-type ( Actl7b +/+) and Actlt7b -knockout (KO) mice, which were represented by heterozygous ( Actl7b +/−) and homozygous ( Actl7b −/−) mice.
Immunohistochemical tests were done on tissue sections taken from the testis, caput, and cauda epididymis of heterozygous ( Actl7b +/−), homozygous ( Actl7b −/−), and wild-type ( Actl7b +/+) mice. These tests were done using ACTL7B as the antigen.
Using transmission electron micrography (TEM), the structural integrity of sperms from Actl7b -deficient male mice was investigated. This included analyzing for any abnormalities in acrosome biogenesis, DNA condensation, manchette formation, and sperm tail formation.
To investigate the effects of Actl7b deficiency on protein interactions, the researchers employed an anti-ACTL7B antibody coupled to Dynabeads. As a reference, uncoupled beads were used. Subsequently, mass spectrometry (MS) was employed to detect proteins that were eluted from the whole testes of five mice from homozygous, heterozygous, and wild-type groups.
Analysis through PCA demonstrated that all three samples had different clustering patterns, and DA analysis exposed a variation in the amount of protein in Actl7b +/− as opposed to Actl7b +/+ samples.
The researchers conducted an evolutionary study of ACTL7A and ACTL7B, as they show sequence resemblance and are expressed only in the testis. This analysis enabled them to examine the conservation of the genes and guess their criticality.
Research results
The results of the study have been presented.
In Actl7b-deficient mice, spermatids encountered an impasse in their development, resulting in a number of malformations in the flagella after the ninth step of spermatogenesis. This ultimately led to the destruction of the majority of the spermatids.
The lumen of the seminiferous tubules contained diminishing spermatids in combination with immature spermatids that were eradicated by Sertoli cells. This was supported by the higher levels of autophagy marker proteins in Actl7b −/− testes.
An MS study showed that ACTL7B forms a specific association with the dynein light chains, LC8-Type 1 and Type 2 (DYNLL1 and DYNLL2), which are found in the ninth phase of spermatogenesis. Moreover, it appears that the effects of ACTL7B are exerted via its interaction with the microtubule network or the dynein 1 motor complex, instead of the actin cytoskeleton.
In contrast, male Actl7b+/ − mice had less ACTL7B and maintained the same level of fertility as wild-type mice.
In previous studies, Actlt7b -KO mice were said to suffer from infertility, caused by a severe oligoteratozoospermia, as well as malformed sperm heads and tails, and a 10-fold decrease in sperm counts when compared to wild-type mice. Nevertheless, in this study, sperm numbers in KO mice were around 32,000, representing a 1,000-fold reduction when compared to the approximately 32,000,000 sperm cells seen in wild-type mice. This is likely attributed to distinctions in the parental mouse strains utilized.
The two models both showed a decrease in germ cell loss, the amount of sperm in the epididymis, and immature germ cells being released from the testis, in addition to some structural sperm irregularities.
Overall,
In conclusion, it can be seen that…
The research demonstrates that alterations in ACTL7B gene may cause spermatogenesis impairment and male infertility in humans, since it is so similar to the mouse gene. Moreover, since ACTL7B could be used with high precision to separate obstructive from non-obstructive azoospermia at the translational and transcriptional levels, there is potential for this gene to be used as a biomarker for male infertility in the future.
- Merges, G. E., Arevalo, L., Kovacevic, A., et al. (2023). Investigation of the consequences of Actl7b deficiency reveals misplacement of LC8 type dynein light chains and disruption of mouse spermatogenesis. Development 150 (21). doi:10.1242/dev.201593