Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP‐dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross‐activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP‐dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI‐deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.

Mast-cell products can stimulate fibroblast proliferation, implying that these cells are key players in fibrosis. One mast-cell product, the serine protease tryptase, is known to activate protease-activated receptor 2 (PAR2) and cause proliferation of fibroblasts. We found that recombinant tryptase, human mast-cell (HMC-1) supernatant, which contains tryptase, and the PAR2-activating peptide SLIGKV exert fibroproliferative actions in human fibroblasts. Here we report insights into this action, which after activation of PAR2 leads to increased expression of cyclooxygenase 2 (COX2), a key enzyme in the biosynthesis of prostaglandins, and consequently to enhanced prostaglandin synthesis. Subsequent cell proliferation is mediated by the prostaglandin 15-deoxy-Δ^12,14-prostaglandin J₂, which acts via the nuclear peroxisome proliferator-activated receptor γ (PPARγ). Fibroblast proliferation induced by tryptase and PAR2 agonist peptide can be blocked by antagonists of COX2 and PPARγ, implying that the proliferative effect of tryptase is PAR2-initiated but depends on COX2, 15-deoxy-Δ^12,14-prostaglandin J₂, and PPARγ. This previously uncharacterized pathway could be of relevance for human fibrotic diseases. For instance, increased numbers of activated mast cells are correlated with fibrosis in testes of infertile men. In these cases all components of the signaling pathway of tryptase were detected as well as expression of COX2. Therefore, our study describes as-yet-unknown interactions between mast cells and fibroblasts, which could be relevant for human fibrotic diseases.

Aging men display reduced reproductive health, however testis aging is poorly understood at the molecular and genomic level. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from both young and older men, and examined age-related changes in germline development and in the testicular somatic cells. Age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and somatic cells ranged from moderate to severe. Altered pathways included signaling and inflammation in multiple cell types, metabolic signaling in Sertoli cells, hedgehog signaling and testosterone production in Leydig cells, cell death and growth in testicular peritubular cells, and possible developmental regression in both Leydig and peritubular cells. Remarkably, the extent of dysregulation correlated with body mass index in older, but not younger men. Collectively, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging, and their possible exacerbation by concurrent chronic conditions such as obesity.

In healthy men, several layers of inconspicuously flat cells and extracellular matrix (ECM) proteins build the wall of the seminiferous tubules. The cells of this wall, peritubular cells, are not well characterized. They are smooth-muscle-like and contractile and transport immotile sperm, a function important for male fertility. However, their full functional importance, especially their potential contribution to the paracrine regulation of the male gonad, is unknown. In men with impaired spermatogenesis, the architecture of the tubular wall is frequently altered. Deposits of ECM and morphological changes of peritubular cells imply that functions of peritubular cells may be fundamentally altered. To be able to study human peritubular cells and their functions, a culture method was established. It is based on small biopsies of patients with obstructive azoospermia but normal spermatogenesis (human testicular peritubular cells, HTPCs) and non-obstructive azoospermia, impaired spermatogenesis, and testicular fibrosis (HTPCFs). Results obtained from cellular studies and parallel examinations of biopsies provide insights into the repertoire of the secretion products, contractile properties, and plasticity of human peritubular cells. They produce ECM components, including the proteoglycan decorin, which may influence paracrine signaling between testicular cells. They may contribute to the spermatogonial stem cell niche via secreted factors. They are regulated by mast cell and macrophage products, and in response produce factors that can fuel inflammatory changes. They possess a high degree of plasticity, which results in hypertrophy and loss of contractile abilities. The data collectively indicate important roles of inconspicuous testicular peritubular cells in human male fertility and infertility.

Besides the hypothalamus and pituitary, melatonin action at the testicular level has been recently suggested. Therefore, we investigated in the Syrian hamster, a well-characterized seasonal breeder, melatonin action on Leydig cells, testicular expression of melatonergic receptors, and possible interactions between melatonin receptors and the previously identified testicular serotoninergic and CRH systems. In isolated Leydig cells from active testes of adult hamsters kept in a long-day (14 h light, 10 h dark) photoperiod and from regressed testes of adult animals exposed to a short-day photoperiod during 16 wk (6 h light, 18 h dark), melatonin significantly reduced human chorionic gonadotropin-stimulated production of cAMP and the main androgens: testosterone and androstane-3alpha,17beta-diol, respectively, and decreased the expression of steroidogenic acute regulatory protein, P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase. In Leydig cells exposed to a short-day photoperiod during 16 wk, melatonin stimulated the conversion of testosterone into 5alpha-reduced androgens by inducing 5alpha-reductase isoform 1, and controlled androstane-3alpha,17beta-diol production by inhibiting 3alpha-hydroxysteroid dehydrogenase expression. Melatonin subtype (mel1a) receptors were detected in Leydig cells. Although the local serotonin system did not mediate melatonin action on androgen production, melatonergic effect on steroidogenesis involved the interaction between mel1a receptors and the inhibitory CRH system. Moreover, melatonin significantly increased CRH mRNA levels and production in hamster Leydig cells expressing CRH subtype 1 receptors. Our studies indicate that melatonin may act as a local inhibitor of human chorionic gonadotropin-stimulated cAMP and androgen production through mel1a receptors, down-regulation of steroidogenic acute regulatory protein, and key steroidogenic enzymes expression and its interaction with the local CRH system.