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Luteinizing hormone regulates the phosphorylation and localization of the mitochondrial effector dynamin-related protein-1 (DRP1) and steroidogenesis in the bovine corpus luteum.

ABSTRACT: The corpus luteum is an endocrine gland that synthesizes and secretes progesterone. Luteinizing hormone (LH) activates protein kinase A (PKA) signaling in luteal cells, increasing delivery of substrate to mitochondria for progesterone production. Mitochondria maintain a highly regulated equilibrium between fusion and fission in order to sustain biological function. Dynamin-related protein 1 (DRP1), is a key mediator of mitochondrial fission. The mechanism by which DRP1 is regulated in the ovary is largely unknown. We hypothesize that LH via PKA differentially regulates the phosphorylation of DRP1 on Ser616 and Ser637 in bovine luteal cells. In primary cultures of steroidogenic small luteal cells (SLCs), LH, and forskolin stimulated phosphorylation of DRP1 (Ser 637), and inhibited phosphorylation of DRP1 (Ser 616). Overexpression of a PKA inhibitor blocked the effects of LH and forskolin on DRP1 phosphorylation. In addition, LH decreased the association of DRP1 with the mitochondria. Genetic knockdown of the DRP1 mitochondria receptor, and a small molecule inhibitor of DRP1 increased basal and LH-induced progesterone production. Studies with a general Dynamin inhibitor and siRNA knockdown of DRP1 showed that DRP1 is required for optimal LH-induced progesterone biosynthesis. Taken together, the findings place DRP1 as an important target downstream of PKA in steroidogenic luteal cells.

SUBMITTER: Plewes MR

PROVIDER: S-EPMC7136153 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundBovine luteal parenchymal cells express class II major histocompatibility complex (MHC) molecules and stimulate class II MHC-dependent activation of T cells in vitro. The ability of a class II MHC-expressing cell type to elicit a response from T cells in vivo is also dependent on expression of costimulatory molecules by the antigen presenting cell and delivery of a costimulatory signal to the T cell. Whether bovine luteal parenchymal cells express costimulatory molecules and can deliver the costimulatory signal is currently unknown.MethodsBovine luteal tissue was collected during the early (day 5; day of estrus = day 0), mid (day 11-12), or late (day 18) luteal phase of the estrous cycle, and at 0, 0.5, 1, 4, 12 or 24 hours following administration of PGF2alpha to cows on day 10 of the estrous cycle. Northern analysis was used to measure CD80 or CD86 mRNA concentrations in luteal tissue samples. Mixed luteal parenchymal cell cultures and purified luteal endothelial cell cultures were prepared, and real-time RT-PCR was used to examine the presence of CD80 and CD86 mRNA in each culture type. Monoclonal antibodies to CD80 and CD86 were added to a mixed luteal parenchymal cell-T cell co-culture in vitro T cell proliferation assay to assess the functional significance of costimulatory molecules on activation of T lymphocytes by luteal parenchymal cells.ResultsNorthern analysis revealed CD80 and CD86 mRNAs in luteal tissue, with greatest steady-state concentrations at midcycle. CD80 and CD86 mRNAs were detected in mixed luteal parenchymal cell cultures, but only slight amounts of CD80 (and not CD86) mRNA were detected in cultures of luteal endothelial cells. Luteinizing hormone, PGF2alpha and TNF-alpha were without effect on concentrations of CD80 or CD86 mRNA in mixed luteal parenchymal cells cultures. Anti-CD80 or anti-CD86 monoclonal antibodies inhibited T cell proliferation in the in vitro T cell proliferation assay.ConclusionIt can be concluded from this study that parenchymal cells within the bovine CL express functional costimulatory molecules that facilitate interactions between with T cells, and these components of the antigen presentation pathway are expressed maximally in the midcycle CL.

Project description:BackgroundIn higher primates, during non-pregnant cycles, it is indisputable that circulating LH is essential for maintenance of corpus luteum (CL) function. On the other hand, during pregnancy, CL function gets rescued by the LH analogue, chorionic gonadotropin (CG). The molecular mechanisms involved in the control of luteal function during spontaneous luteolysis and rescue processes are not completely understood. Emerging evidence suggests that LH/CGR activation triggers proliferation and transformation of target cells by various signaling molecules as evident from studies demonstrating participation of Src family of tyrosine kinases (SFKs) and MAP kinases in hCG-mediated actions in Leydig cells. Since circulating LH concentration does not vary during luteal regression, it was hypothesized that decreased responsiveness of luteal cells to LH might occur due to changes in LH/CGR expression dynamics, modulation of SFKs or interference with steroid biosynthesis.MethodsSince, maintenance of structure and function of CL is dependent on the presence of functional LH/CGR its expression dynamics as well as mRNA and protein expressions of SFKs were determined throughout the luteal phase. Employing well characterized luteolysis and CL rescue animal models, activities of SFKs, cAMP phosphodiesterase (cAMP-PDE) and expression of SR-B1 (a membrane receptor associated with trafficking of cholesterol ester) were examined. Also, studies were carried out to investigate the mechanisms responsible for decline in progesterone biosynthesis in CL during the latter part of the non-pregnant cycle.Results and discussionThe decreased responsiveness of CL to LH during late luteal phase could not be accounted for by changes in LH/CGR mRNA levels, its transcript variants or protein. Results obtained employing model systems depicting different functional states of CL revealed increased activity of SFKs [pSrc (Y-416)] and PDE as well as decreased expression of SR-B1 correlating with initiation of spontaneous luteolysis. However, CG, by virtue of its heroic efforts, perhaps by inhibition of SFKs and PDE activation, prevents CL from undergoing regression during pregnancy.ConclusionsThe results indicated participation of activated Src and increased activity of cAMP-PDE in the control of luteal function in vivo. That the exogenous hCG treatment caused decreased activation of Src and cAMP-PDE activity with increased circulating progesterone might explain the transient CL rescue that occurs during early pregnancy.

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Luteinizing hormone regulates the phosphorylation and localization of the mitochondrial effector dynamin-related protein-1 (DRP1) and steroidogenesis in the bovine corpus luteum.

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