Treatment with cholesterol just after thawing maintains the fertility of bull sperm

Md Mazharul Islam; Takashi Umehara; Natsumi Tsujita; Masanori Koyago; Masayuki Shimada

doi:10.1093/molehr/gaad031

Treatment with cholesterol just after thawing maintains the fertility of bull sperm

Mol Hum Reprod. 2023 Aug 30;29(9):gaad031. doi: 10.1093/molehr/gaad031.

Authors

Md Mazharul Islam^{1

2}, Takashi Umehara^{1

3}, Natsumi Tsujita³, Masanori Koyago^{3

4}, Masayuki Shimada^{1

3}

Affiliations

¹ Laboratory of Reproductive Endocrinology, Department of Bioresource Science, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
² Department of Animal Breeding and Genetics, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh.
³ Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.
⁴ Livestock Improvement Association of Japan Inc., Tokyo, Japan.

Abstract

Freezing and thawing diminish sperm motility and fertility by disrupting the cholesterol balance in sperm plasma and organelle membranes. The aim of this study was to elucidate the mechanisms through which exogeneous cholesterol treatment enhances the quality of frozen-thawed bull sperm. The incorporation of cholesterol was investigated using boron-dipyrromethene (BODIPY)-cholesterol, and BODIPY signals were detected not only in the plasma membrane but also in the midpiece region immediately after thawing. The positive signal of cholesterol in the midpiece region was inhibited by a scavenger receptor class B Type I (SR-BI) inhibitor, block lipid transport 1 (BLT-1). To comprehend the role of exogenous cholesterol in the functions of the plasma membrane, propidium iodide (PI)/Annexin V and peanut agglutinin lectin (PNA) staining were performed. The results showed that treatment with exogenous cholesterol increased the number of acrosome-intact sperm and decreased the number of sperm with damage to the plasma membrane. Moreover, since BODIPY signals were also observed in the midpiece region, mitochondrial function was evaluated using a flux analyzer and a flow cytometer with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) staining, revealing an increase in the number of sperm with high-mitochondrial activity and oxygen consumption. Finally, to assess sperm fertility, computer-assisted sperm analysis (CASA) and IVF were carried out. Sperm velocities and fertilization rates in IVF were significantly enhanced by the addition of cholesterol just after thawing. Thus, the treatment with cholesterol after thawing protected the plasma membrane from the stress of thawing and maintained mitochondrial function, thereby preserving the fertilization ability of frozen-thawed bull sperm for conventional IVF and artificial insemination (AI). Therefore, the application of cholesterol just after thawing is a promising option for improving the fertility of frozen-thawed sperm.

Keywords: IVF; cholesterol; mitochondria; sperm motility; spermatozoa.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cattle
Cholesterol
Fertility
Male
Semen*
Sperm Motility*
Spermatozoa

Substances

4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
Cholesterol