The Presence of Colony-Stimulating Factor-1 and Its Receptor in Different Cells of the Testis; It Involved in the Development of Spermatogenesis In Vitro

Spermatogenesis is a complex process, in which spermatogonial cells proliferate and differentiate in the seminiferous tubules of the testis to generate sperm. This process is under the regulation of endocrine and testicular paracrine/autocrine factors. In the present study, we demonstrated that colony stimulating factor-1 (CSF-1) is produced by mouse testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells (such as CDH1-positively stained cells; a marker of spermatogonial cells). In addition, we demonstrated the presence of CSF-1 and its receptor (CSF-1R) in testicular macrophages, Leydig, Sertoli, peritubular cells and spermatogonial cells of human testis. We also show that the protein levels of CSF-1 were the highest in testis of 1-week-old mice and significantly decreased with age (2–12-week-old). However, the transcriptome levels of CSF-1 significantly increased in 2–3-week-old compared to 1-week-old, and thereafter significantly decreased with age. On the other hand, the transcriptome levels of CSF-1R was significantly higher in mouse testicular tissue of all examined ages (2–12-week-old) compared to 1-week-old. Our results demonstrate the involvement of CSF-1 in the induction the proliferation and differentiation of spermatogonial cells to meiotic and postmeiotic stages (BOULE- and ACROSIN-positive cells) under in vitro culture conditions, using methylcellulose culture system (MCS). Thus, it is possible to suggest that CSF-1 system, as a testicular paracrine/autocrine system, is involved in the development of different stages of spermatogenesis and may be used in the development of future therapeutic strategies for treatment of male infertility.

ATP activation of peritubular cells drives testicular sperm transport

Spermatogenesis, the complex process of male germ cell proliferation, differentiation, and maturation, is the basis of male fertility. In the seminiferous tubules of the testes, spermatozoa are constantly generated from spermatogonial stem cells through a stereotyped sequence of mitotic and meiotic divisions. The basic physiological principles, however, that control both maturation and luminal transport of the still immotile spermatozoa within the seminiferous tubules remain poorly, if at all, defined. Here, we show that coordinated contractions of smooth muscle-like testicular peritubular cells provide the propulsive force for luminal sperm transport toward the rete testis. Using a mouse model for in vivo imaging, we describe and quantify spontaneous tubular contractions and show a causal relationship between peritubular Ca2+ waves and peristaltic transport. Moreover, we identify P2 receptor-dependent purinergic signaling pathways as physiological triggers of tubular contractions both in vitro and in vivo. When challenged with extracellular ATP, transport of luminal content inside the seminiferous tubules displays stage-dependent directionality. We thus suggest that paracrine purinergic signaling coordinates peristaltic recurrent contractions of the mouse seminiferous tubules to propel immotile spermatozoa to the rete testis.

Proteomic Insights into Senescence of Testicular Peritubular Cells from a Nonhuman Primate Model

Age-related changes in the human testis may include morphological alterations, disturbed steroidogenesis, and impaired spermatogenesis. However, the specific impact of cell age remains poorly understood and difficult to assess. Testicular peritubular cells fulfill essential functions, including sperm transport, contributions to the spermatogonial stem cell niche, and paracrine interactions within the testis. To study their role in age-associated decline of testicular functions, we performed comprehensive proteome and secretome analyses of repeatedly passaged peritubular cells from Callithrix jacchus. This nonhuman primate model better reflects the human testicular biology than rodents and further gives access to young donors unavailable from humans. Among 5095 identified proteins, 583 were differentially abundant between samples with low and high passage numbers. The alterations indicate a reduced ability of senescent peritubular cells to contract and secrete proteins, as well as disturbances in nuclear factor (NF)-κB signaling and a reduced capacity to handle reactive oxygen species. Since this in vitro model may not exactly mirror all molecular aspects of in vivo aging, we investigated the proteomes and secretomes of testicular peritubular cells from young and old donors. Even though the age-related alterations at the protein level were less pronounced, we found evidence for impaired protein secretion, altered NF-κB signaling, and reduced contractility of these in vivo aged peritubular cells.

Interleukin-34, a Novel Paracrine/Autocrine Factor in Mouse Testis, and Its Possible Role in the Development of Spermatogonial Cells In Vitro

Spermatogenesis is the process of spermatogonial stem cell (SSC) proliferation and differentiation to generate sperm. This process is regulated by cell–cell interactions between Sertoli cells and developing SSCs by autocrine/paracrine and endocrine factors. It is also affected by cells in the interstitial compartment, such as Leydig cells and peritubular cells. Here, we demonstrate, for the first time, the presence of interleukin-34 (IL-34) in Leydig, Sertoli, and peritubular cells and in the premeiotic, meiotic, and postmeiotic cells. Its receptor, colony-stimulating factor-1 (CSF-1), has already been demonstrated in Leydig, Sertoli, premeiotic, and meiotic cells. IL-34 was detected in testicular homogenates and Sertoli cell-conditioned media, and was affected by mouse age. We showed that the addition of IL-34 in vitro to isolated cells from the seminiferous tubules of 7-day-old mice, using the methylcellulose culture system (MCS), increased the percentages and expression of the premeiotic cells (VASA), the meiotic cells (BOULE), and the meiotic/postmeiotic cells (ACROSIN) after four weeks of culture, when examined by immunofluorescence staining (IF) and qPCR analysis. It is possible to suggest that IL-34 is a novel paracrine/autocrine factor involved in the development of spermatogenesis. This factor may be used in future therapeutic strategies for the treatment of male infertility.

ATP activation of peritubular cells drives testicular sperm transport

Spermatogenesis, the complex developmental process of male germ cell proliferation, differentiation, and maturation, is the basis of male fertility and reproductive fitness. In the seminiferous tubules of the testes, spermatozoa are constantly generated from spermatogonial stem cells through a stereotyped sequence of mitotic and meiotic divisions. The basic physiological principles, however, that control both maturation and luminal transport of the still immotile spermatozoa within the seminiferous tubules remain poorly, if at all, defined. Here, we show that coordinated contractions of smooth muscle-like testicular peritubular cells provide the propulsive force for luminal sperm transport towards the rete testis and epididymis. Using a mouse model for in vivo imaging, we describe and quantify spontaneous tubular contractions and show a causal relationship between peritubular Ca2+ waves and peristaltic transport. Moreover, we identify P2 receptor-dependent purinergic signaling pathways as physiological triggers of tubular contractions both in vitro and in vivo. When challenged with extracellular ATP, transport of luminal content inside the seminiferous tubules displays stage-dependent directionality. We thus suggest that paracrine purinergic signaling coordinates peristaltic recurrent contractions of the mouse seminiferous tubules to propel immotile spermatozoa to the rete testis. Consequently, our findings could have substantial pharmaceutical implications for both infertility treatment and / or male contraception.

Palmitic Acid Targets Human Testicular Peritubular Cells and Causes a Pro-Inflammatory Response

Palmitic acid (PA) is a major fatty acid, derived from diet and endogenous production, which is being linked to inflammation. While such actions of PA at the level of the testis remain difficult to examine, we reasoned that studies in human testicular cells may be instructive. Human testicular peritubular cells (HTPCs) can be isolated from men and cultured. They have contractile properties but also produce Interleukin 6 (IL6), express the inflammasome member NLRP3, and via glia cell line derived neurotrophic factor (GDNF), they contribute to the spermatogonial stem cell niche. We found that PA at 100 µM significantly increased the levels of IL6, while NLRP3 or the related Interleukin 1 beta (IL1beta) were not affected. The contractility marker calponin (CNN1) and the growth factor GDNF were likewise not affected. ELISA studies confirmed the stimulatory PA actions on IL6. Hence, PA derived from diet and/or endogenous sources may be able to foster a pro-inflammatory milieu in the testis. A possible link of these results to diet and high fat intake and obesity is indicated by the about 12-fold elevated testicular levels of IL6 in testes of obese rhesus monkeys (n = 3), fed with a Western Style diet. They had elevated 2–5-fold increased body fat and increased circulating triglyceride levels. Further consequences of PA and obesity for testicular functions remain to be evaluated.

A translational cellular model for the study of peritubular cells of the testis

Testicular peritubular cells (TPCs) are smooth muscle-like cells, which form a compartment surrounding the seminiferous tubules. Previous studies employing isolated human testicular peritubular cells (HTPCs) indicated that their roles in the testis go beyond sperm transport and include paracrine and immunological contributions. Peritubular cells from a non-human primate (MKTPCs), the common marmoset monkey, Callithrix jacchus, share a high degree of homology with HTPCs. However, like their human counterparts these cells age in vitro and replicative senescence limits in-depth functional or mechanistic studies. Therefore, a stable cellular model was established. MKTPCs of a young adult animal were immortalized by piggyBac transposition of human telomerase (hTERT), that is, without the expression of viral oncogenes. Immortalized MKTPCs (iMKTPCs) grew without discernable changes for more than 50 passages. An initial characterization revealed typical genes expressed by peritubular cells (androgen receptor (AR), smooth-muscle actin (ACTA2), calponin (CNN1)). A proteome analysis of the primary MKTPCs and the derived immortalized cell line confirmed that the cells almost completely retained their phenotype. To test whether they respond in a similar way as HTPCs, iMKTPCs were challenged with forskolin (FSK) and ATP. As HTPCs, they showed increased expression level of the StAR protein (StAR) after FSK stimulation, indicating steroidogenic capacity. ATP increased the expression of pro-inflammatory factors (e.g. IL1B; CCL7), as it is the case in HTPCs. Finally, we confirmed that iMKTPCs can efficiently be transfected. Therefore, they represent a highly relevant translational model, which allows mechanistic studies for further exploration of the roles of testicular peritubular cells.

The Glucocorticoid Receptor NR3C1 in Testicular Peritubular Cells is Developmentally Regulated and Linked to the Smooth Muscle-Like Cellular Phenotype

Whether glucocorticoids (GC) can directly affect human testicular functions is not well understood. A predominant site of GC receptor (GR; NR3C1) expression in the adult testis are peritubular smooth muscle-like cells, which express smooth muscle actin (ACTA2), contract and thereby are involved in sperm transport. In contrast to the adult, neither GR nor ACTA2, or elastin (ELN) were detected in the peritubular compartment before puberty in non-human primate testes. In isolated human testicular peritubular cells (HTPCs), activation of GR by dexamethasone (Dex) caused the translocation of GR to the nucleus and stimulated expression of ACTA2 and ELN, without affecting the expression of collagens. Cytoskeletal ACTA2-rearrangements were observed and were associated with an increased ability to contract. Our results indicate post-pubertal testicular roles of GC in the maintenance of the contractile, smooth muscle-like phenotype of peritubular cells.

Insights into replicative senescence of human testicular peritubular cells

There is evidence for an age-related decline in male reproductive functions, yet how the human testis may age is not understood. Human testicular peritubular cells (HTPCs) transport sperm, contribute to the spermatogonial stem cell (SSC) niche and immune surveillance, and can be isolated and studied in vitro. Consequences of replicative senescence of HTPCs were evaluated to gain partial insights into human testicular aging. To this end, early and advanced HTPC passages, in which replicative senescence was indicated by increased cell size, altered nuclear morphology, enhanced β-galactosidase activity, telomere attrition and reduced mitochondrial DNA (mtDNA), were compared. These alterations are typical for senescent cells, in general. To examine HTPC-specific changes, focused ion beam scanning electron microscopy (FIB/SEM) tomography was employed, which revealed a reduced mitochondrial network and an increased lysosome population. The results coincide with the data of a parallel proteomic analysis and indicate deranged proteostasis. The mRNA levels of typical contractility markers and growth factors, important for the SSC niche, were not significantly altered. A secretome analysis identified, however, elevated levels of macrophage migration inhibitory factor (MIF) and dipeptidyl peptidase 4 (DPP4), which may play a role in spermatogenesis. Testicular DPP4 may further represent a possible drug target.