Genetic Association in the Maintenance of the Mitochondrial Microenvironment and Sperm Capacity.

Sperm motility is one of the major determinants of male fertility. Since sperm need a great deal of energy to support their fast movement by active metabolism, they are thus extremely vulnerable to oxidative damage by the reactive oxygen species (ROS) and other free radicals generated as byproducts in the electron transport chain. The present study is aimed at understanding the impact of a mitochondrial oxidizing/reducing microenvironment in the etiopathology of male infertility. We detected the mitochondrial DNA (mtDNA) 4,977 bp deletion in human sperm. We examined the gene mutation of ATP synthase 6 (ATPase6 m.T8993G) in ATP generation, the gene polymorphisms of uncoupling protein 2 (UCP2, G-866A) in the uncoupling of oxidative phosphorylation, the role of genes such as manganese superoxide dismutase (MnSOD, C47T) and catalase (CAT, C-262T) in the scavenging system in neutralizing reactive oxygen species, and the role of human 8-oxoguanine DNA glycosylase (hOGG1, C1245G) in 8-hydroxy-2'-deoxyguanosine (8-OHdG) repair. We found that the sperm with higher motility were found to have a higher mitochondrial membrane potential and mitochondrial bioenergetics. The genotype frequencies of UCP2 G-866A, MnSOD C47T, and CAT C-262T were found to be significantly different among the fertile subjects, the infertile subjects with more than 50% motility, and the infertile subjects with less than 50% motility. A higher prevalence of the mtDNA 4,977 bp deletion was found in the subjects with impaired sperm motility and fertility. Furthermore, we found that there were significant differences between the occurrences of the mtDNA 4,977 bp deletion and MnSOD (C47T) and hOGG1 (C1245G). In conclusion, the maintenance of the mitochondrial redox microenvironment and genome integrity is an important issue in sperm motility and fertility.

Mitochondrial Function in Modulating Human Granulosa Cell Steroidogenesis and Female Fertility.

Ovarian follicle steroidogenesis associated with embryo quality results in a successful pregnancy. Each follicle consists of an oocyte surrounded by granulosa cells, which secrete several steroid and peptide hormones. Follicles harvested from women who conceived after assisted reproductive therapy (ART) had significantly higher estradiol levels in follicular fluids than the follicles from women who failed to conceive after ART. The higher follicular estradiol levels correlate well with successful fertilization following ART. Mitochondria are the central sites for steroid hormone biosynthesis. The first and rate-limiting step in the biosynthesis of steroid hormones occurs in the mitochondria of granulosa cells. In the present study, we hypothesized that the mitochondria in granulosa cells are critical for maintaining oocyte quality and fertility capacity. This study aims to clarify the relationship between mitochondrial function and granulosa cell steroidogenesis, and the relationship between hormone levels and fertility capacity. Sera, follicular fluids and granulosa cells were obtained from individuals undergoing IVF-ET treatment. The oocyte numbers, oocyte quality, fertilization rate, and pregnancy rate were also recorded. The patients who provided the granulosa cells were further classified into four groups: endometriosis, ovarian endometrioma, endometriosis without ovarian endometrioma, and polycystic ovary syndrome (PCOS); patients with other female factor infertility and male factor infertility were used as controls. We measured the levels of estradiol (E2) by radioimmunoassay. Concurrently, we analyzed the mitochondrial mass and membrane potential, and apoptosis by flow cytometry using nonyl acridine orange, TMRE, Annexin V-FITC and PI. Mitochondrial morphology was visualized by transfection with pLV-mitoDsRed. In addition, we assessed the protein levels of steroidogenic enzymes, steroidogenic acute regulatory protein (StAR) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) by Western blot. The results showed significantly decreased serum E2 and follicular E2 levels, and decreased IVF outcomes, in the patients with endometriosis. Reduced mitochondrial mass and decreased mitochondrial membrane potential were correlated with lower E2. Furthermore, a significant decrease in StAR and 3ß-HSD was found in patients with ovarian endometrioma. The enzyme levels of StAR and 3ß-HSD were highly correlated with E2 levels. Finally, elevated cumulus cell apoptosis was found in the patient group with ovarian endometrioma and PCOS. In conclusion, mitochondrial dysfunction of human granulosa cells may contribute to the decline of steroidogenesis, decreased fertilization rate, oocyte maturation rate, and oocyte quality, and it can ultimately jeopardize fertility.