Cell Biology

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

Sona Relovska
Huafeng Wang
Xinbo Zhang
Pablo Fernández-Tussy
Kyung Jo Jeong
Jungmin Choi
Yajaira Suárez
Jeffrey G. McDonald
Carlos Fernández-Hernando author has email address
Jean-Ju Chung author has email address

Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA
Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06510, USA
Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, CT 06510, USA
Department of Biomedical Sciences, Korea University College of Medicine, Seoul, South Korea
Department of Genetics, Yale School of Medicine, Yale University, New Haven, Connecticut, USA
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA
Center for Human Nutrition, University of Texas Southwestern Medical Center, Dallas, TX 75390
Department of Gynecology and Obstetrics, Yale School of Medicine, New Haven, CT 06510, USA

https://doi.org/10.7554/eLife.95627.1

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Figures and data

DHCR24 overexpression leads to age-dependent decline in male fertility.
(A) Diagram showing the involvement of DHCR24 in two parallel pathways to produce cholesterol: the Bloch (left) and Kandutsch-Russel (right) pathways. (B) Litter sizes resulting from wild-type (WT) and Dhcr24-TG (TG) crosses of young adult (YA) and mature adult (MA) mice. Error bars SD, statistical analysis one-way ANOVA. (C-D) Dhcr24 mRNA expression in human (C) and mouse (D) testicular cells using human (GSE109037) and mouse (GSE109033) testis single-cell RNA (scRNA)-seq datasets. UMAP plots represent 7 and 11 clusters of testicular cells in human and mouse (left) and nebula plots of Dhcr24 mRNA expression density in these clusters (right). EC, endothelial cell; PTM, peritubular myoid cell. (E) Western blot analysis of DHCR24 protein levels in epididymal spermatozoa from caput (Ca), corpus (Co), or cauda (Cd) of WT and Dhcr24-TG young and mature adults. YA, young adult (2-4 months old); MA, mature adult (5-7 months old). Illustration created with BioRender.com.
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Dysregulated sterol biosynthesis affects sperm plasma membrane of Dhcr24-TG males.
(A and B) Lipidomics analysis of WT and Dhcr24-TG (TG) sperm from mature adult mice for lipid species in Bloch (A) and Kandutsch-Russel (B) pathways. Statistical analysis student’s t-test. (C) Cholesterol levels from the testis of WT or Dhcr24-TG young adult and mature adult animals. (D) Acrosome reaction progression by IZUMO staining. Spermatozoa from young adult WT and Dhcr24-TG (TG) animals were incubated under non-capacitating (uncap) or capacitating (cap) conditions in the absence (-) or presence (+) of methyl-β-cyclodextrin (MBCD). (E) In vitro fertilization using spermatozoa of WT and Dhcr24-TG (TG) young adult animals capacitated at high (∼20×10⁶ sperm/mL) or standard (2×10⁶ sperm/mL) cell concentration. All error bars SD.

Dhcr24-TG males have decreased sperm count and motility.
(A) Sperm counts from young and mature adult WT and Dhcr24-TG (TG) male mice. Statistical analysis one way ANOVA. (B) Hematoxylin and eosin staining of testis and epididymis from mature adult WT and Dhcr24-TG mice. (C) Total and hyperactivated motility from spermatozoa of young and mature adult WT and Dhcr24-TG males before and after capacitation (n=4). Statistical analysis two-way ANOVA. (D) Flagellar waveform analysis of WT and Dhcr24-TG spermatozoa before and after capacitation (young adult). (E) Brightfield images of spermatozoa from young adult WT and Dhcr24-TG animals. White arrow points to thinner area in the flagellum in Dhcr24-TG sperm. YA, young adult (2 – 4 months old); MA, mature adult (5 – 7 months old). All error bars SD.

Sperm from Dhcr24-TG mice show abnormal head bending and incomplete mitochondrial packing.
(A) Immunofluorescence images of WT and Dhcr24-TG spermatozoa from young adult animals stained with antibodies against Tom20 (red), ODF (green) and counterstained with Hoechst detecting nucleus (blue). (B) Scanning electron microscopy (SEM) images of WT and Dhcr24-TG spermatozoa from young (WT – bottom, Dhcr24-TG – top right, bottom two) and mature adult (WT – top, Dhcr24-TG – top left) animals. The bending of the head towards the midpiece can be observed in Dhcr24-TG spermatozoa. White arrows indicate the exposed outer dense fiber (ODF) in the midpiece of Dhcr24-TG spermatozoa. (C) Quantification of sperm defects in SEM images of sperm from young and mature adult WT and Dhcr24-TG animals. Left, percentage of sperm with shortened mitochondrial sheath; right, percentage of sperm with tail bending at the connecting piece (CP), midpiece (MP), both connecting piece and midpiece (CP+MP), or at the junction of midpiece-principal piece (MP-PP). (D) Transmission Electron Microscopy (TEM) images of WT and Dhcr24-TG spermatozoa from young (WT – top) and mature adult (WT – bottom, Dhcr24-TG – all) animals. White arrows indicate the exposed outer dense fiber (ODF) in the midpiece of Dhcr24-TG spermatozoa. The erratic mitochondrial packing along the sperm tail results in ODF exposure in the distal region of Dhcr24-TG spermatozoa. YA, young adult (2 – 4 months old); MA, mature adult (5 – 7 months old).

DHCR24 overexpression affects spermatozoan metabolism.
(A) Dynamic representative images of mitochondrial membrane potential indicated by MitoTracker™ Red CMXRos in uncapacitated WT and Dhcr24-TG sperm upon antimycin treatment. (B) Transition of the mitochondrial membrane potential. The changes of fluorescence intensity were calculated as ΔF/F₀ (F₀, the mean fluorescence intensity of the sperm midpiece before adding antimycin A (t = 10 s); F, the fluorescence of the midpiece after adding antimycin A; ΔF=F− F₀). Error bars SD, n=4. (C) Fold change in basal respiratory oxygen consumption rate from Seahorse analysis, measurements normalized to WT uncapacitated sperm. Spermatozoa from middle aged adult. Error bars SD.

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