Proteome profile of the cerebellum from α7 nicotinic acetylcholine receptor deficient mice.

The alpha7 nicotinic acetylcholine receptor (α7 nAChR; CHRNA7) is expressed in the nervous system and in non-neuronal tissues. Within the central nervous system, it is involved in various cognitive and sensory processes such as learning, attention, and memory. It is also expressed in the cerebellum, where its roles are; however, not as well understood as in the other brain regions. To investigate the consequences of absence of CHRNA7 on the cerebellum proteome, we performed a quantitative nano-LC-MS/MS analysis of samples from CHRNA7 knockout (KO) mice and corresponding wild type (WT) controls. Liver, an organ which does not express this receptor, was analyzed, in comparison. While the liver proteome remained relatively unaltered (three proteins more abundant in KOs), 90 more and 20 less abundant proteins were detected in the cerebellum proteome of the KO mice. The gene ontology analysis of the differentially abundant proteins indicates that the absence of CHRNA7 leads to alterations in the glutamatergic system and myelin sheath in the cerebellum. In conclusion, our dataset provides new insights in the role of CHRNA7 in the cerebellum, which may serve as a basis for future in depth-investigations.

An ovarian phenotype of alpha 7 nicotinic receptor knockout mice.

In brief: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is expressed by various murine ovarian cells. Morphological and molecular investigations, including a proteomic study of adult Chrna7 knockout (KO) mouse ovaries, reveal the roles of these receptors in the local regulation of the ovary. Abstract: Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is involved in cellular functions ranging from synaptic transmission in neurons to regulation of inflammation, cell growth and metabolism to cell death in other cells. Our qPCR results and other studies indicated that nAChRa7 is expressed in the adult mouse ovary, while in situ hybridization and single-cell sequencing data suggested this expression may be shared by several ovarian cells, including fibroblast-like and steroidogenic stroma cells, macrophages and oocytes of small follicles. To explore a possible involvement of nAChRa7 in ovarian functions, we evaluated ovarian morphology of Chrna7-null mutant adult mice (KO) and wildtype mice (WT; 3 months, metestrus) by performing immunohistochemistry, qPCR studies, measurements of serum progesterone and proteomic analyses. The evaluation of serial sections indicated fewer primordial follicles but similar numbers of primary, secondary and tertiary follicles, as well as corpora lutea in KO and WT mice. Atresia was unchanged. Serum progesterone and mRNA levels of proliferation and most apoptosis markers were not changed, yet two typical macrophage markers were elevated. Furthermore, the proteomes of KO ovaries were significantly altered with 96 proteins increased and 32 decreased in abundance in KOs compared to WTs. Among the elevated proteins were markers for stroma cells. Hence, the lack of nAChRa7 causes changes in small follicle counts and alterations of the ovarian stroma cells. The ovarian phenotype of Chrna7 mutant mice links this channel protein to the local regulation of ovarian cells, including stroma cells.

Muscarinic receptors in the rat ovary are involved in follicular development but not in steroid secretion.

Acetylcholine (ACh) may be involved in the regulation of ovarian functions. A previous systemic study in rats showed that a 4-week, intrabursal local delivery of the ACh-esterase blocker Huperzine-A increased intraovarian ACh levels and changed ovarian follicular development, as evidenced by increased healthy antral follicle numbers and corpora lutea, as well as enhanced fertility. To further characterize the ovarian cholinergic system in the rat, we studied whether innervation may contribute to intraovarian ACh. We explored the cellular distribution of three muscarinic receptors (MRs; M1, M3, and M5), analyzed the involvement of MRs in ovarian steroidogenesis, and examined their roles in ovarian follicular development in normal conditions and in animals exposed to stressful conditions by employing the muscarinic antagonist, atropine. Denervation studies decreased ovarian norepinephrine, but ovarian ACh was not affected, evidencing a local, nonneuronal source of ACh. M1 was located on granulosa cells (GCs), especially in large antral follicles. M5 was associated with the ovarian vascular system and only traces of M3 were found. Ex vivo ovary organo-typic incubations showed that the MR agonist Carbachol did not modify steroid production or expression of steroid biosynthetic enzymes. Intrabursal, in vivo application of atropine (an MR antagonist) for 4 weeks, however, increased atresia of the secondary follicles. The results support the existence of an intraovarian cholinergic system in the rat ovary, located mainly in follicular GCs, which is not involved in steroid production but rather via MRs exerts trophic functions and regulates follicular atresia.

Intratesticular xenografting of Klinefelter pre-pubertal testis tissue as potential model to study testicular fibrosis.

RESEARCH QUESTION: Is intratesticular xenotransplantation a potential ex-vivo model for studying testicular fibrosis related to Klinefelter syndrome? STUDY DESIGN: First, a feasibility study of an ex-vivo model to study testicular fibrosis in patients with Klinefelter syndrome was performed. Testis tissue from boys with pre-pubertal Klinefelter syndrome (n = 3) and controls (n = 2) (<18 years) was grafted to the mouse testis (n = 12) and recovered after 2, 4, 6 and 8 weeks. Part two of this study consisted of a validation of this model, evaluating the effects of the mast cell blocker ketotifen on the histology of the grafts of Klinefelter syndrome (n = 5) and controls (n = 3), transplanted to mice (n = 10), after 4 weeks of ketotifen or saline treatment. Immunohistochemistry determined the histology of the grafts and the presence of mast cells and spermatogonia. RESULTS: The feasibility study showed that 4 weeks after transplantation, all Klinefelter syndrome grafts could be recovered. Later, degeneration was observed. Most recovered grafts showed an intact histology, with 67 ± 12% intact tubules for the Klinefelter syndrome grafts and 65 ± 15% of intact tubules for the control grafts. In the few remaining Klinefelter syndrome grafts, treatment with ketotifen improved testicular histology compared with non-treated grafts. Graft survival was patient dependent. No germ cell loss was observed after transplantation. CONCLUSION: Xenografting could become a model for the longitudinal study of the fibrotic process related to Klinefelter syndrome; however, the current model has a limited survival period and patient-specific differences in histology.