Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ⁰ cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ⁰ mouse melanoma cells into syngeneic C57BL/6N^su9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ⁰ cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer.

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 3β-hydroxysteroid dehydrogenase (3β-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 3β-HSD was found in patients with ovarian endometrioma. The enzyme levels of StAR and 3β-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.

In vitro spermatogenesis (IVS) has already been successfully achieved in rodents by organotypic and soft matrix culture systems. However, the former does not allow single cell input, and the latter presents as a simple thick layer in which all cells are embedded. We explored a new culture system using a mouse model by employing an alginate-based hydrogel and 3D bioprinting, to control scaffold design and cell deposition. We produced testicular constructs consisting of printed cell-free scaffolds (CFS) with prepubertal testicular cells (TC) in their easy-to-access macropores. Here, the pores represented the only cell compartment (TC/CFS). Double-cell compartment testicular constructs were achieved by culturing magnetic-activated cell sorting-enriched epithelial cells in the pores of interstitial cell-laden scaffolds (CD49f ⁺ /CLS). Cell spheres formed in the pores in the weeks following cell seeding on both CFS and CLS. Although restoration of the tubular architecture was not observed, patches of post-meiotic cells including elongated spermatids were found in 66% of TC/CFS. Differentiation up to the level of round spermatids and elongated spermatids was observed in all and 33% of CD49f ⁺ /CLS constructs, respectively. Organ culture served as the reference method for IVS, with complete spermatogenesis identified in 80% of cultivated prepubertal tissue fragments. So far, this is the first report applying a 3D bioprinting approach for IVS. Further optimization of the scaffold design and seeding parameters might be permissive for tubular architecture recreation and thereby increase the efficiency of IVS in printed testicular constructs. While it remains to be tested whether the gametes generated on the alginate-based scaffolds can support embryogenesis following IVF, this IVS approach might be useful for (patho)physiological studies and drug-screening applications.

The crucial role that oestrogens play in male reproduction has been generally accepted; however, the exact mechanism of their action is not entirely clear and there is still much more to be clarified. The oestrogen response is mediated through oestrogen receptors, as well as classical oestrogen receptors’ variants, and their specific co-expression plays a critical role. The importance of oestrogen signalling in male fertility is indicated by the adverse effects of selected oestrogen-like compounds, and their interaction with oestrogen receptors was proven to cause pathologies. The aims of this review are to summarise the current knowledge on oestrogen signalling during spermatogenesis and sperm maturation and discuss the available information on oestrogen receptors and their splice variants. An overview is given of species-specific differences including in humans, along with a detailed summary of the methodology outcome, including all the genetically manipulated models available to date. This review provides coherent information on the recently discovered mechanisms of oestrogens’ and oestrogen receptors’ effects and action in both testicular somatic and germ cells, as well as in mature sperm, available for mammals, including humans.