DHCR24-mediated sterol homeostasis during spermatogenesis is required for sperm mitochondrial sheath formation and impacts male fertility over time

Reviewer #1 (Public Review):

Summary:

The manuscript by Relovska and colleagues aims to decipher the importance of sterol homeostasis on male reproduction and, in particular, the impact of altered sterol homeostasis in sperm cells. To this end, they are generating a global line of Dhcr24 transgenic mice by mating Dhcr24fl/fl mice (overexpressing the construct in the Rosa269c gene locus) with EIla 100 CRE mice (expressing Cre recombinase in the early mouse embryo).

The data provided are robust, using a range of approaches from sperm analysis (structure, function) to lipid analysis. Results show that overexpression of DHCR24 (TG) leads to altered sterol homeostasis in spermatozoa. Sperm from TG mice have abnormal mitochondria and sperm tails. TG spermatozoa have reduced efficiency in undergoing the acrosomal reaction. Furthermore, the data suggest that TG spermatozoa have an altered metabolism with increased oxygen consumption. These data highlight that desmosterol depletion and/or altered sterol homeostasis impact sperm morphology, number, motility, and metabolism, resulting in reduced male fertility.

Strengths:

The manuscript is clear and well-written, and the results are presented in high-quality figures.

Weaknesses:

The main concern is the clear analysis of the rodent model. Indeed, the use of this particular Cre leads to whole-body overexpression, the remaining question is whether the observed effects are directly mediated by the testicular impacts of Dhcr24 overexpression. Even if it is a testicular effect, we can't conclude where it comes from. Is it at the level of spermatogenesis, as the authors speculate? Furthermore, the authors mention that normally, Dhcr24c is mainly expressed in spermatogonia, so it's not clear why they focus only on spermatozoa, which in WT males do not normally express dhcr24 according to the authors. It is worth clarifying the testicular phenotype in more detail.

The lower level of TG in aged mice could suggest an extinction of the transgene at least in sperm during aging, which could be difficult to reconcile with the observed phenotype. With this in mind, it would be interesting to define the penetrance of the phenotype during aging, to define variability between mice, and to clearly define potential correlations between sterol levels and fertility disorders, or altered sperm parameters.

Reviewer #2 (Public Review):

Summary:

Sterols, including desmosterol and cholesterol, play critical roles in male fertility including membrane rearrangements associated with sperm capacitation, steroidogenesis, and germ cell development. Relovska, Sona, et al. investigated the effects of global overexpression of classic cholesterol biosynthesis enzyme DHCR24 in a mouse model, focusing on the impacts on sperm function and male fertility. While mice were viable and did not exhibit altered plasma cholesterol levels or obesity, the authors demonstrated that concentrations of relevant sterols in sperm from transgenic mice were altered compared to WT mouse sperm, including the expected depletion of desmosterol. The transgenic males exhibited several indicators of reduced sperm function and fertility. Mitochondrial dysfunction was indicated by a noted depletion of localization in the distal middle-piece of up to approximately 20-25% of transgenic sperm flagella, and alterations in mitochondrial membrane potential and oxygen consumption rates in transgenic sperm were noted.

Strengths:

The authors demonstrate that DHCR24 overexpression was achieved and that sperm sterol levels are altered. The conclusions that global DHCR24 overexpression impacts mitochondrial localization and male fertility parameters are supported by the number of different supporting assessments utilized to reach these conclusions and this is a strength. Overall, the authors achieve their aim of demonstrating DHCR24 overexpression impacts on indicators of sperm function and fertility including reduced sperm counts and sperm motility, reduced fertility in mating trials with aged males, and reduced IVF success when sperm were capacitated in conditions of higher sperm concentrations in vitro. The authors further investigate sperm mitochondrial localization and function. While a mitochondrial sheath can form in sperm from transgenic mice, 25% of the sperm exhibit a shortened mitochondrial sheath where a distal portion of the middle piece of the sperm flagella lacks mitochondria and instead exhibits exposed outer dense fibers.

Weaknesses:

In the current study, the authors conclude that desmosterol may not act as an LXR activator in testicular cells based on assessment of relevant mRNA levels in whole testis that indicated the relevant transcripts were not altered in transgenic testes. However, caution should be taken in utilizing whole testis transcriptomics to rule out a role in specific cell populations within the testis with minor relative representation, such as macrophages or undifferentiated spermatogonia. This is an important distinction for a few reasons. The authors reveal through single-cell assessments of DHCR24 expression in WT testis that it is most highly expressed in undifferentiated spermatogonia. Further, the authors previously reported that DHCR24 over-expression in myeloid/macrophage populations did impact LXR activation impacting atherosclerosis. Taken together with emerging evidence that testis macrophages may impact spermatogonial fate decisions, the potential for DHCR24 to impact these minor testicular cell populations should not yet be ruled out. The significance of individual observations needs to be clarified through improved reporting of methodologies, specific biological and technical replicates, and statistical significance for each individual assessment. The lack of these details obfuscates the ability of the reader to interpret or replicate several reported observations which is a weakness.

(1) The fertility trials indicate a reduced number of pups/litter in aged but not younger transgenic males. However, the data for the aged males includes three data points of 0 pups, which brings to question if the data points each represent the average pups/litter for individual males or individual litters with multiple litters separately included for fertile males. Clarification could help in interpreting whether litter sizes were reduced, or if litter frequency and/or fertility of individual males was reduced. In the latter case, behavioral infertility would not be excluded from consideration.

(2) The statistical significance is not clear for altered acrosome reaction data, hyperactivated motility data, waveform analysis, mitochondrial membrane potential, and some of the sperm morphology assessments. In many assessments, the biological and technical replicates assessed need to be clarified.

(3) Methods utilized for image assessment of waveform analysis and mitochondrial membrane potential are lacking detail sufficient for replication of the assessments or for reader interpretation of how conclusions were reached.

Summary of impact:

Overall, the novel observations in this study are consistent with a role for controlled sterol concentrations being important for male fertility and indicate that this model will be useful to further investigate sterol biosynthesis contributions to testis function including steroidogenesis, spermatogenesis, and sperm function including capacitation.

Reviewer #3 (Public Review):

Summary:

The authors have created DHCR24 knockin mice and noted changes in the sperm sterol composition. Concurrently, alterations in the quantity, motility, and function of the sperm in DHCR24 knockin mice were identified.

Strengths:

The manuscript offers an intriguing perspective on how disruptions in sperm sterol composition can lead to sperm abnormalities.

Weaknesses:

From the current data, several issues remain to be clarified, including the fertility test results, which merit a more detailed presentation to ascertain whether differences stem from individual variability or overall changes. The authors suggest an increase in ROS in the sperm of DHCR24 knockin mice, leading to sperm damage, which also requires further confirmation. Moreover, the quality of some data requires verification or improvement, such as the morphological analysis of testicular sections and the OCR experiments.