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In the orchestrated environment of the testicular niche, the equilibrium between self-renewal and differentiation of spermatogonial stem cells (SSCs) is meticulously maintained, ensuring a stable stem cell reserve and robust spermatogenesis. Within this milieu, extracellular vesicles, specifically exosomes, have emerged as critical conveyors of intercellular communication. Despite their recognized significance, the implications of testicular exosomes in modulating SSC fate remain incompletely characterized. Given the fundamental support and regulatory influence of Sertoli cells (SCs) on SSCs, we were compelled to explore the role of SC-derived exosomes (SC-EXOs) in the SSC-testicular niche. Our investigation hinged on the hypothesis that SC-EXOs, secreted by SCs from the testes of 5-day-old mice-a developmental juncture marking the onset of SSC differentiation-participate in the regulation of this process. We discovered that exposure to SC-EXOs resulted in an upsurge of PLZF, MVH, and STRA8 expression in SSC cultures, concomitant with a diminution of ID4 and GFRA1 levels. Intriguingly, obstructing exosomal communication in a SC-SSC coculture system with the exosome inhibitor GW4869 attenuated SSC differentiation, suggesting that SC-EXOs may modulate this process via paracrine signaling. Further scrutiny revealed the presence of miR-493-5p within SC-EXOs, which suppresses Gdnf mRNA in SCs to indirectly restrain SSC differentiation through the modulation of GDNF expression-an indication of autocrine regulation. Collectively, our findings illuminate the complex regulatory schema by which SC-EXOs affect SSC differentiation, offering novel perspectives and laying the groundwork for future preclinical and clinical investigations., (© 2024 Wiley Periodicals LLC.)

Sertoli cells (SCs) provide an adequate environment for germ cell development. SCs possess unique features that meet germ cells' metabolic demands: they produce lactate from glucose, which is delivered as energy substrate to germ cells. SCs store fatty acids (FAs) as triacylglycerols (TAGs) in lipid droplets (LDs) and can oxidize FAs to sustain their own energetic demands. They also produce ketone bodies from FAs. It has been shown that exposure of SCs to metabolic stresses, such as glucose deprivation, triggers specific adaptive responses that sustain cell survival and preserve lactate supply to germ cells. The aim of the present study was to investigate whether there are modifications in rat SCs lipid metabolism, including LD content, FA oxidation, and ketone bodies production, as part of their adaptive response to glucose deprivation. The present study was performed in 20-day-old rat SCs cultures. We determined LD content by Oil Red O staining, FA oxidation by measuring the release of 3 H 2 O from [ 3 H] palmitate, TAGs and 3-hydroxybutyrate levels by spectrophotometric methods, and mRNA levels by RT-qPCR. Results show that the absence of glucose in SC culture medium entails: (1) a decrease in LD content and TAGs levels that is accompanied by decreased perilipin 1 mRNA levels, (2) an increase in FA oxidation that is in part mediated by AMP kinase (AMPK) activation and (3) a decrease in 3-hydroxybutyrate production. Additionally, we studied whether sestrins (SESN1, 2 and 3), proteins involved in the cellular response to stress, are regulated in glucose deprivation conditions. We show that there is an increase in SESN2 mRNA levels in deprived conditions. In conclusion, glucose deprivation affects SC lipid metabolism promoting FA mobilization from LDs to be used as energy source., (© 2023 Wiley Periodicals LLC.)

In Sertoli cells of the testis, cadherins (Cdh) are important cell-to-cell interaction proteins and contribute to the formation of the blood-testis barrier being essential for germ cells' protection. P-cadherin or Cdh3 is only expressed in Sertoli cells from embryonic to prepubertal development. Interestingly, the expression profile of Cdh3 correlates with that of activating protein-1 (AP-1) transcription factors during Sertoli cells development. To assess their potential implications in the regulation of Cdh3, different AP-1 transcription factors were overexpressed in 15P-1 Sertoli cells. We found that the overexpressions of Junb and Fosl2 activated Cdh3 promoter. ChIP-qPCR assay and luciferase reporter assay with 5' promoter deletions and site-directed mutagenesis confirmed the recruitment of Junb and Fosl2 to an AP-1 regulatory element at -47 bp in the proximal region of Cdh3 promoter in 15P-1 cells. These findings were further supported by histone modification markers and chromatin accessibility surrounding Cdh3 promoter in mouse testis. Moreover, the knockdowns of Junb and/or Fosl2 by siRNA decreased Cdh3 protein levels. Taken together, these data suggest that in 15P-1 Sertoli cells, the AP-1 family members Junb and Fosl2 are responsible for the regulation of Cdh3 expression, which requires the recruitment of both factors to the proximal region of the Cdh3 promoter., (© 2022 Wiley Periodicals LLC.)

Hybrid male sterility (HMS) is a reproductive isolation mechanism limiting the formation of fertile offspring through interspecific fertilization. Cattleyak is the interspecific hybrid presenting significant heterosis in several economic traits, but HMS restricted its wide reproduction in cettleyak breeding. In this study, we detected the specifically expressed genes of a variety of cells (undifferentiated spermatogonia, primary spermatocytes, secondary spermatocytes, haploid spermatids, sperm, Sertoli cells, Leydig cells, and macrophages) in the testis of yak and cattleyak, and found that the spermatogenesis of cattleyak might be blocked at meiosis I, and the expression of niche factors (NR5A1, GATA4, STAR, CYP11A1, CD68, TNF, and CX3CR1) in undifferentiated spermatogonia niche was abnormal. Then we isolated the undifferentiated spermatogonia and Sertoli cells from yak and cattleyak by enzyme digestion, and detected the specific genes in the two bovid testicular cells as well as the proliferation capacity of the undifferentiated spermatogonia. These results indicated that weak proliferation ability and scarce number of undifferentiated spermatogonia and abnormal gene expressions in Sertoli cells may contribute to male sterility of cattleyak., (© 2022 Wiley Periodicals LLC.)

The development of mouse spermatozoa is a continuous process from spermatogonia, spermatocytes, spermatids to mature sperm. Those developing germ cells (spermatogonia, spermatocyte, and spermatids) together with supporting sertoli cells are all enclosed inside seminiferous tubules of the testis, their identification is key to testis histology and pathology analysis. Automated segmentation of all these cells is a challenging task because of their dynamical changes in different stages. The accurate segmentation of testicular cells is critical in developing computerized spermatogenesis staging. In this paper, we present a novel segmentation model, SED-Net, which incorporates a squeeze-and-excitation (SE) module and a dense unit. The SE module optimizes and obtains features from different channels, whereas the dense unit uses fewer parameters to enhance the use of features. A human-in-the-loop strategy, named deep interactive learning, is developed to achieve better segmentation performance while reducing the workload of manual annotation and time consumption. Across a cohort of 274 seminiferous tubules from stages VI to VIII, the SED-Net achieved a pixel accuracy of 0.930, a mean pixel accuracy of 0.866, a mean intersection over union of 0.710, and a frequency weighted intersection over union of 0.878, respectively, in terms of four types of testicular cell segmentation. There is no significant difference between manual annotated tubules and segmentation results by SED-Net in cell composition analysis for tubules from stages VI to VIII. In addition, we performed cell composition analysis on 2346 segmented seminiferous tubule images from 12 segmented testicular section results. The results provided quantitation of cells of various testicular cell types across 12 stages. The rule reflects the cell variation tendency across 12 stages during development of mouse spermatozoa. The method could enable us to not only analyze cell morphology and staging during the development of mouse spermatozoa but also potentially could be applied to the study of reproductive diseases such as infertility., (© 2022 International Society for Advancement of Cytometry.)

Sertoli cells are key somatic cells in the testis that form seminiferous tubules and support spermatogenesis. The isolation of pure Sertoli cells is important for their study. However, it is a difficult effort because of the close association of Sertoli cells with peritubular myoid cells surrounding seminiferous tubules. Here, we propose a novel approach to the establishment of a pure Sertoli cell culture from immature mouse testes. It is based on the staining of testicular cells for platelet-derived growth factor receptor alpha (PDGFRA), followed by fluorescence-activated cell sorting and culturing of a PDGFRA-negative cell population. Cells positive for a Sertoli cell marker WT1 accounted for more than 96% of cells in cultures from 6 to 12 days postpartum (dpp) mice. The numbers of peritubular myoid cells identified by ACTA2 staining did not exceed 4%. Cells in the cultures were also positive for Sertoli cell proteins SOX9 and DMRT1. Amh and Hsd17b3 expression decreased and Ar and Gata1 expression increased in 12 dpp cultures compared to 6 dpp cultures, which suggests that cultured Sertoli cells at least partially retained their differentiation status. This method can be employed in various applications including the analysis of differential gene expression and functional studies., (© 2022 Wiley Periodicals LLC.)

The strong hormonal dysregulation associated with obesity is responsible for the disruption of several reproductive events. Sertoli cells (SCs) function is dependent on energetic homeostasis and thus, directly associated with energy homeostasis regulating hormones. To further understand the influence of those hormones with SCs function and obesity, we hypothesize that human SCs express obesity-related genes (ORG; MC4R, GNPDA2, TMEM18, and FTO) and that they respond to energy homeostasis regulating hormones (leptin, ghrelin, and glucagon-like protein 1 [GLP-1]) stimuli. To test our hypothesis, SCs were cultured with increasing doses of leptin (0, 5, 25, or 50 ng/ml, for 24 h), ghrelin (0, 20, 100, and 500 pM, for 24 h), and GLP-1 (10, 1000, or 1 × 105 pM, for 6 h). The presence and abundance of ORG transcripts and proteins in SCs were accessed by polymerase chain reaction techniques, Western blot analysis, and immunofluorescence staining. Our results show that human SCs express MC4R, GNPDA2, TMEM18, and FTO in specific cellular locations. MC4R and FTO expression in human SCs was not responsive to the treatments. However, GNPDA2 and TMEM18 expression increased after exposure to the highest concentration of leptin and ghrelin, respectively. We highlight for the first time that human SCs express ORG and that these are responsive to energy homeostasis hormonal stimuli. GNPDA2 and TMEM18 expression respond in opposite directions according to overall energy status, mediated by energy homeostasis regulating hormones. Leptin and ghrelin control of ORG expression by human SCs can be associated with overweight-related infertility and subfertility in males., (© 2020 Wiley Periodicals LLC.)

The effect of stress on male fertility is a widespread public health issue, but less is known about the related signaling pathway. To investigate this, we established a hypercortisolism mouse model by supplementing the drinking water with corticosterone for four weeks. In the hypercortisolism mice, the serum corticosterone was much higher than in the control, and serum testosterone was significantly decreased. Moreover, corticosterone treatment induced decrease of sperm counts and increase of teratozoospermia. Increased numbers of multinucleated giant cells and apoptotic germ cells as well as downregulated meiotic markers suggested that corticosterone induced impaired spermatogenesis. Further, upregulation of macrophage-specific marker antigen F4/80 as well as inflammation-related genes suggested that corticosterone induced inflammation in the testis. Lactate content was found to be decreased in the testis and Sertoli cells after corticosterone treatment, and lactate metabolism-related genes were downregulated. In vitro phagocytosis assays showed that the phagocytic activity in corticosterone-treated Sertoli cells was downregulated and accompanied by decreased mitochondrial membrane potential, while pyruvate dehydrogenase kinase-4 inhibitor supplementation restored this process. Taken together, our results demonstrated that dysfunctional phagocytosis capacity and lactate metabolism in Sertoli cells participates in corticosterone-induced impairment of spermatogenesis., (© 2021 Wiley Periodicals LLC.)

Retinoic acid (RA), the active metabolite of vitamin A, is one of the most important factors regulating spermatogenesis. RA activates downstream pathways through its receptors (retinoic acid receptor alpha [RARA], retinoic acid receptor beta, and retinoic acid receptor gamma [RARG]) and retinoid X receptors (retinoid X receptor alpha [RXRA], retinoid X receptor beta [RXRB], and retinoid X receptor gamma [RXRG]). These receptors may serve as therapeutic targets for infertile men. However, the localization and expression of retinoid receptors in normal and infertile men were unknown. In this study, we found RARA and RARG were mostly localized in spermatocytes and round spermatids, RXRB was mainly expressed in Sertoli cells, and RXRG was expressed in most cell types in the fertile human testis. The localization of RARA, RARG, RXRB, and RXRG in men with hypospermatogenesis (HYPO) was similar to that of men with normal fertility. In addition, the messenger RNA expression levels of RARA, RARG, RXRA, RXRB, and RXRG were significantly decreased in men with Sertoli cell-only syndrome (SCOS) and maturational arrest (MA), but not in men with HYPO. These results suggest that reduced levels of RARA, RARG, RXRB, RXRA, and RXRG are more closely associated with SCOS and MA spermatogenetic failure. These results could contribute to the development of new molecular indicators of spermatogenic dysfunction and might provide novel therapeutic targets for treating male infertility., (© 2020 Wiley Periodicals LLC.)

The blood-testis barrier (BTB) separates the seminiferous epithelium into the apical and basal compartments. The BTB has to operate timely and accurately to ensure the correct migration of germ cells, meanwhile maintaining the immunological barrier. Testin was first characterized from primary Sertoli cells, it is a secretory protein and a sensitive biomarker to monitor junctions between Sertoli and germ cells. Till now, the functions of testin on BTB dynamics and the involving mechanisms are unknown. Herein, testin acts as a regulatory protein on BTB integrity. In vitro testin knockdown by RNAi caused significant damage to the Sertoli cell barrier with no apparent changes in the protein levels of several major tight junction (TJ), adhesion junction, and gap junction proteins. Also, testin RNAi caused the diffusion of two TJ structural proteins, occludin and ZO-1, diffusing away from the Sertoli cell surface into the cytoplasm. Association and colocalization between ZO-1 and occludin were decreased after testin RNAi, examined by Co-IP and coimmunofluorescent staining, respectively. Furthermore, testin RNAi induced a dramatic disruption on the arrangement of actin filament bundles and a reduced F-actin/G-actin ratio. The actin regulatory protein ARP3 appeared at the Sertoli cell interface after testin RNAi without its protein level change, whereas overexpressing testin in Sertoli cells showed no effect on TJ barrier integrity. The above findings suggest that besides as a monitor for Sertoli-germ cell junction integrity, testin is also an essential molecule to maintain Sertoli-Sertoli junctions., (© 2020 Wiley Periodicals, Inc.)

Glial cell line-derived neurotrophic factor (GDNF) and retinoic acid (RA) are two molecules crucial for the regulation of the spermatogonial compartment of the testis. During the cycle of the seminiferous epithelium, their relative concentration oscillates with lower GDNF levels in stages where RA levels are high. It has been recently shown that RA negatively regulates Gdnf expression but the mechanisms behind are so far unknown. Here, we show that RA directly downregulates Gdnf mRNA levels in primary murine Sertoli cells through binding of RARα to a novel DR5-RARE on Gdnf promoter. Pharmacological inhibition and chromatin immunoprecipitation-quantitative polymerase chain reaction analysis suggested that the underlying mechanism involved histone deacetylase activity and epigenetic repression of Gdnf promoter upon RA treatment., (© 2020 Wiley Periodicals, Inc.)

Sertoli cells (SCs) are presumed to be the center of testis differentiation because they provide both structural support and biological regulation for spermatogenesis. Previous studies suggest that SCs control germ cell (GC) count and Leydig cell (LC) development in mouse testes. However, the regulatory role of SCs on peritubular myoid (PTM) cell fate in fetal testis has not been clearly reported. Here, we employed Amh-Cre; diphtheria toxin fragment A (DTA) mouse model to selectively ablate SCs from embryonic day (E) 14.5. Results found that SC ablation in the fetal stage caused the disruption of testis cords and the massive loss of GCs. Furthermore, the number of α-smooth muscle actin-labeled PTM cells was gradually decreased from E14.5 and almost lost at E18.5 in SC ablation testis. Interestingly, some Ki67 and 3β-HSD double-positive fetal LCs could be observed in Amh-Cre; DTA testes at E16.5 and E18.5. Consistent with this phenomenon, the messenger RNA levels of Hsd3b1, Cyp11a1, Lhr, Star and the protein levels of 3β-HSD and P450Scc were significantly elevated by SC ablation. SC ablation appears to induce ectopic proliferation of fetal LCs although the total LC number appeared reduced. Together, these findings bring us a better understanding of SCs' central role in fetal testis development., (© 2020 Wiley Periodicals, Inc.)

Meiosis begins at puberty and relies on several factors, including androgens and retinoic acid in the mouse testis. CYP26B1 degrades retinoic acid in the testis during prenatal development preventing meiosis initiation. Given the concurrence of meiotic entry and completion of Sertoli cell maturation in response to androgens at puberty in the mouse, we proposed that CYP26B1 is downregulated by androgens in the Sertoli cell during this period. By immunohistochemistry, we showed that CYP26B1 declines in Sertoli cells after birth. However, luciferase reporter assays and quantitative reverse transcription-polymerase chain reaction performed in the prepubertal mouse Sertoli cell line SMAT1 revealed no changes in Cyp26b1 expression in response to androgen treatment. Furthermore, studies carried out using primary Sertoli cells of 10-day-old mice showed no changes in either Cyp26b1 or CYP26B1 expression in response to androgen treatment. In summary, the hereby reported decline in CYP26B1 expression in Sertoli cells towards pubertal onset does not appear to be caused by a direct inhibitory effect of androgens on Sertoli cells in the mouse., (© 2019 Wiley Periodicals, Inc.)

Lactate produced by glycolysis in Sertoli cells (SCs) is the main energy substrate for developing germ cells and plays a vital role in spermatogenesis. MicroRNAs (miRNAs) function as posttranscriptional regulators of gene expression in biological processes. We have previously shown that hyperthermia (43°C, 30 min) promotes lactate secretion by inhibiting phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in cultured immature boar SCs. However, it is unclear whether miRNAs are involved in AMPK-modulated glycolysis in SCs. In the present study, we identified 349 miRNAs (227 upregulated and 122 downregulated) in hyperthermia-treated boar SCs by next-generation high-throughput RNA sequencing. MiR-8-3p, which was found to be a novel upregulated miRNA in hyperthermia-treated SCs, suppressed the expression of AMPK upstream genes (protein phosphatase 2 subunit B, PPP2R5B), and further downregulated the expression of p-AMPK. The miR-8-3p mimic upregulated expression of glucose transporter 3, lactate dehydrogenase A and monocarboxylate transporter 1, and increased lactic acid dehydrogenase activity, lactate secretion, and ATP depletion in SCs; the miR-8-3p inhibitor had the opposite effects on these parameters. Our findings indicate that miR-8-3p acts as a novel regulator of AMPK-modulated lactate secretion by targeting PPP2R5B in hyperthermic boar SCs., (© 2019 Wiley-Liss, Inc., A Wiley Company.)

The term "Puberty", socially known as "Adolescence" is the transitional period from juvenile life to adulthood with functional maturation of gonads and genital organs. In this process, some remarkable developmental changes occur in morphology, physiology, and behavior leading to reproductive competence. Despite sufficient levels of gonadotropins (luteinizing hormone [LH] and follicle-stimulating hormone [FSH]), robust spermatogenesis is not initiated during infancy in primates due to the immaturity of testicular Sertoli cells. Recent studies suggest that developmental competence augmenting functional activities of receptors for androgen and FSH is acquired by Sertoli cells somewhere during the prolonged hypo-gonadotropic juvenile period. This juvenile phase is terminated with the re-awakening of hypothalamic Kisspeptin/Neurokinin B/Dynorphin neurons which induce the release of the gonadotropin-releasing hormone leading to reactivation of the hypothalamo-pituitary-testicular axis at puberty. During this period of pubertal development, FSH and LH facilitate further maturation of testicular cells (Sertoli cells and Leydig cells) triggering robust differentiation of the spermatogonial cells, ensuing the spermatogenic onset. This review aims to precisely address the evolving concepts of the pubertal regulation of hormone production with the corresponding cooperation of testicular cells for the initiation of robust spermatogenesis, which can be truly called "testicular puberty.", (© 2019 Wiley Periodicals, Inc.)

The current study was conducted to assess the relationship between testicular cells in spermatogenesis, through which the production of healthy and mature sperm is essential. However, it seems necessary to obtain more information about the three-dimensional pattern of the testis cells arrangement, which is directly related to the function of the testis after induction of diabetes. Twelve adult mice (28-30 g) were assigned into two experimental groups: (1) control and (2) diabetic (40 mg/kg STZ). The epididymal sperm collected from the tail of the epididymis and testes samples were taken for stereology, immunocytochemistry and RNA extraction. Our data showed that diabetes could notably decrease the number of testicular cells, together with a reduction of total sperm count. In addition, the results from the second-order stereology indicated the significant changes in the spatial arrangement of Sertoli cells and spermatogonial cells in the diabetic groups, in comparison with the control (P < .05). Moreover, the immunohistochemistry results showed a significant reduction in Sex-determining Region Y (SRY) box 9 gene (SOX9), vimentin, occludin, and connexin-43 positive cells in the diabetic groups compared with the control (P < .05). Furthermore, our data showed that the expression of steroidogenic acute regulatory protein steroidogenic acute regulatory protein (StAR) and peripheral benzodiazepine receptor peripheral benzodiazepine receptor (PBR) was significantly reduced in the diabetic groups, in comparison with the control (P < .05). These findings suggest that structural and functional changes of testis cells after induction of diabetes cause the alterations in the spatial arrangement of Sertoli and spermatogonial cells, ultimately influencing the normal spermatogenesis in mice., (© 2019 Wiley Periodicals, Inc.)

Heat stress is a major stressor that can lead to male reproductive dysfunction. Sertoli cells play a crucial role in spermatogenesis by providing germ cells with structural and nutritional support, and contributing to blood-testis barrier formation. Vitamin C (Vc) is an antioxidant capable of neutralizing reactive oxygen species and preventing lipid peroxidation widely used because it is inexpensive and highly accessible. In the present study, we investigated the protective effect of Vc on TM4 cells following heat stress. Pretreatment with Vc could effectively inhibit apoptosis (p < 0.01), lipid peroxidation, and lactate dehydrogenase (LDH) activity. However, a significant increase in the malondialdehyde (MDA) level and LDH activity (p < 0.01) was observed in TM4 cells without Vc-pretreatment, in conjunction with vacuole degeneration and karyopyknosis. In addition, both the messenger RNA and protein levels of CryAB, Hsp27, Hsp70, and Hsp110 substantially increased in the 3 and 12 hr recovery groups (p < 0.01). Vc also prevented microtubule aggregation following heat stress. These results suggest that pretreatment with Vc-protected TM4 cells against heat stress by reducing the level of oxidative stress and inducing heat shock protein expression., (© 2019 Wiley Periodicals, Inc.)

Kisspeptin (Kp) expression in testis has caused most of the recent research surveying its functional role in this organ. This peptide influences spermatogenesis and sperm capacitation, so it is considered as a regulator of reproduction. Kp roles exert through hypothalamic/pituitary/gonadal axis. We aimed to evaluate direct roles for Kp on proliferation and differentiation of spermatogonial cells (SCs) when the cells are cocultured with somatic cells. Somatic cells and SCs were isolated from adult azoospermic and newborn mice and then enriched using a differential attachment technique. After the evaluation of identity and colonization for SCs, the cells were cocultured with somatic cells, and three doses of Kp (10 -8 -10 -6  M) was assessed on proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c-Kit and SCP 3 , TP 1, TP 2 , and, Prm 1 markers) of the coculture system. Investigations were continued for four succeeding weeks. At the end of each level of testosterone in the culture media was also evaluated. We found positive influence from Kp on proliferative and differentiative markers in SCs cocultured with somatic cells. These effects were dose-dependent. There was no effect for Kp on testosterone level. From our findings, we simply conclude that Kp as a neuropeptide for influencing central part of reproductive axis could also positively affect peripheral processes related to spermatogenesis without having an effect on steroidogenesis., (© 2018 Wiley Periodicals, Inc.)

Adenosine monophosphate-activated protein kinase (AMPK) plays a key role in cellular energy homeostasis and cell proliferation. MicroRNAs (miRNAs) function as posttranscriptional regulators of gene expression in biological processes. It is unclear to whether miRNAs are involved in AMPK-regulated Sertoli cell (SC) proliferation. To further understand the regulation of miRNAs in the immature boar SC proliferation, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) was added to activate AMPK. By an Illumina small RNA deep sequencing, we obtained sequences and relative expression levels of 272 known mature miRNAs, among which 9 miRNAs were significantly upregulated whereas 16 miRNAs were downregulated following the AICAR treatment. The results identified 38 conserved miRNAs, with 8 significantly downregulated miRNAs whereas no upregulated miRNAs. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that miR-1285 was involved in many activities and pathways associated with cell proliferation via targeting on AMPKα2. We validated that AICAR significantly downregulated miR-1285 level in SCs. Transfection of miR-1285 mimic increased the SC viability and cell cycle progression but reduced AMPKα2 mRNA and protein levels, indicating that miR-1285 is involved in the immature boar SC proliferation via downregulating AMPKα2 expression., (© 2019 Wiley Periodicals, Inc.)

Sertoli cells provide the nutritional and metabolic support for germ cells. Wnt/β-catenin signaling is important for the development of the seminiferous epithelium during embryonic age, although after birth this pathway is downregulated. Cx43 gene codes for a protein that is critical during testicular development. The Cx43 promoter contains TCF/β-catenin binding elements (TBEs) that contribute CX43 expression in different cell types and which may also be regulating the expression of this gene in Sertoli cells. In this study, we demonstrate that 42GPA9 Sertoli cells respond to treatments that result in accumulation of β-catenin within the nucleus and in upregulation of CX43 gene transcription. β-Catenin binds to TBEs located both upstream and downstream of the transcriptional start site (TSS). Luciferase reporter experiments revealed that TBEs located upstream of the TSS are necessary for β-catenin-mediated upregulation. Our results also indicate that the Wnt/β-catenin-dependent upregulation of the Cx43 gene in Sertoli cells is accompanied by changes in epigenetic parameters that may be directly contributing to generating a chromatin environment that facilitates the establishment of the transcriptional machinery at this promoter., (© 2018 Wiley Periodicals, Inc.)

Within the seminiferous tubules of the testis, Gja1-encoded connexin43 plays a critical role in intercellular communication between Sertoli cells. These cells nurture, protect and stimulate the developing germ cells and spermatids. SOX transcription factors are known to play an important role in male fertility and sex determination; however, their physiological function and the identity of their target genes in postnatal Sertoli cells remain to be defined. Members of the activating protein-1 (AP-1) family have been shown to regulate Gja1 expression in myometrial and testicular cells and to physically interact with SOX members, suggesting that these transcription factors may regulate its expression within the testis. Hence, we performed co-transfections of expression plasmids encoding SOX4, SOX8, SOX9 and cJUN with different mouse Gja1 promoter/luciferase reporter constructs within TM4 and 15P-1 Sertoli cells. We showed that a functional cooperation between cJUN and SOX8 or SOX9 regulates Gja1 expression and may involve DNA regulatory elements located between -132 and -26 bp. Such synergy relies on the recruitment of cJUN to the -47 base pair (bp) AP-1 DNA regulatory element of the mouse Gja1 promoter. Hence, SOX and AP-1 members cooperate to regulate Gja1 within testicular Sertoli cells., (© 2018 Wiley Periodicals, Inc.)

MicroRNAs (miRNAs) have been shown to play a key role in spermatogenesis. However, whether the miRNAs influence androgen/androgen receptor (AR) signaling during spermatogenesis remains unclear. Using a bioinformatic approach, a potential miRNA, miR-130a, which could bind to Ar-3'untranslated region directly was identified. The expression pattern of miR-130a was further characterized by quantitative real-time polymerase chain reaction. It was found that miR-130a was abundant in testis and its expression level was negatively correlated with age. Overexpression of miR-130a could inhibit AR expression both in vitro and in vivo. Moreover, the mice with an intratesticular injection of miR-130a showed defects in spermatogenesis and increased germ cell apoptosis. Taken together, these results suggest that miR-130a could negatively regulate AR expression in mouse Sertoli cell, which further cause defects in spermatogenesis., (© 2018 Wiley Periodicals, Inc.)

The somatic component of seminiferous epithelium, the Sertoli cells (Sc) respond to Follicle stimulating hormone (FSH), and Testosterone (T) to produce factors which are necessary for germ cell (Gc) survival and differentiation. Infant Sc do not support spermatogenesis in spite of sufficient hormonal milieu, a situation similar to that found in male idiopathic infertility. Sc maturation during pubertal period involves expression of some genes which may be important for initiation of spermatogenesis. Analysis of differentially expressed genes, one by one, in infant and pubertal Sc might provide useful information about the regulation of spermatogenesis. DNA microarray based analysis of mRNA from 5-days (infant) and 12-days (pubertal) old rat Sc revealed increased expression of Nor-1 by pubertal Sc. NOR-1 is an orphan nuclear receptor involved in maintaining cellular homeostasis and disease. We generated transgenic mice using shRNA cloned under Pem (Rhox5) promoter which is activated at puberty in Sc. Such transgenic mice had reduced Nor-1 expression and increased Tgfβ1, Tgfβ3, and Smad3 expression. Moreover, an increase in β-catenin expression was observed in NOR-1 knockdown testes. High β-catenin in such transgenic mice was found to be associated with disruption of Sc maturation characterized by elevated expression of Anti Mullerian hormone, Cytokeratin 18, and Sox9. This disruption of Sc maturation resulted in Gc apoptosis. Such NOR-1 knockdown mice showed reduced sperm count and litter size. We report for the first time that NOR-1 plays a crucial role in regulating sperm count and male fertility., (© 2018 Wiley Periodicals, Inc.)

Maternal nutrition during critical gestation periods impacts on offspring in later life; effects of high-starch maternal diet on testicular development in lambs were addressed. Dairy ewes were fed diets providing either 27% (Starch, S) or 11% (Fiber, F) of starch from mating to lambing (∼147 days; S147, F147) or for the last 75 days of gestation (S75, F75). Testes of single male lambs were measured and then sampled for histological and gene expression analyses at selected ages. Testicular dimensions and weight were similar among groups, but the total area of seminiferous tubules increased with age and tended to be higher (p = 0.057) in lambs from starch- than fiber-fed ewes. Sertoli and germ cells number increased with age, but was not influenced by maternal diet. Transcript abundances of androgen receptor (AR), insulin-like growth factor 1 (IGF1), and hydroxysteroid (17-beta) dehydrogenase 3 (HSD17B3) was similar between S147 and F147 lambs (p > 0.1). Abundance of luteinizing hormone/choriogonadotropin receptor (LHCGR) and steroidogenic acute regulatory protein (STAR) was higher in young vs older lambs, whereas insulin-like growth factor 2 (IGF2) levels increased with age. The expression of vascular endothelial growth factor A (VEGFA), Anti-Müllerian hormone (AMH), IGF1, follicle stimulating hormone receptor (FSHR), and insulin-like growth factor 2 receptor (IGF2R) was not influenced by maternal diet or lamb age (p > 0.1). In conclusion, a high-starch maternal diet did not influence gene expression, but may have affected testicular structure in infant offspring, as seen by an increase in the total area of seminiferous tubules., (© 2018 Wiley Periodicals, Inc.)

In mouse testes, Sertoli cells support the continuous process of spermatogenesis, which is dependent on seminiferous epithelial cycles along the longitudinal axis of the seminiferous tubule. Sertoli cell function is modulated partly by local cytokines and/or growth factors derived from adjacent tissues such as blood vessels, macrophages, rete testis, etc. However, the spatial activation patterns by local signals in vivo remain unclear. In this study, we focused on Signal Transducers and Activators of Transcription (STAT) signaling in Sertoli cells, because STAT is a major crucial cytokine transducer for somatic cyst cell regulation in Drosophila testis niches. In mouse testes, STAT3 was ubiquitously expressed in Sertoli cells throughout the seminiferous tubules. Phosphorylated STAT3 (p-STAT3) was predominantly observed in the Sertoli cells within the valve-like structure adjacent to the rete testis (i.e., the Sertoli valve [SV]) in the terminal segment of the proximal seminiferous tubules. In the distal seminiferous tubules with active spermatogenesis, most Sertoli cells were negative for anti-p-STAT3 staining. Albeit rarely, a small patch of several p-STAT3-positive Sertoli cells was detected frequently in seminiferous epithelial cycle stages I-VI. Such p-STAT3-positive ratios in the convoluted seminiferous epithelia were significantly increased in germ cell-less testes than in the wild-type testes, but with considerably lower ratios than in the SV region. These findings imply that regionally distinct patterns of STAT3 phosphorylation in the Sertoli cells depend on either location or spermatogenic activity in normal healthy testes in vivo, highlighting a novel entry point to understanding STAT signaling in mammalian spermatogenesis., (© 2018 Wiley Periodicals, Inc.)

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, little is known about its role in sperm flagella formation and male fertility. IFT140 is a component of IFT-A complex. In mouse, it is highly expressed in the testis. Ift140 gene was inactivated specifically in mouse spermatocytes/spermatids. The mutant mice did not show any gross abnormalities, but all were infertile and associated with significantly reduced sperm number and motility. Multiple sperm morphological abnormalities were discovered, including amorphous heads, short/bent flagella and swollen tail tips, as well as vesicles along the flagella due to spermiogenesis defects. The epididymides contained round bodies of cytoplasm derived from the sloughing of the cytoplasmic lobes and residual bodies. Knockout of Ift140 did not significantly affect testicular expression levels of selective IFT components but localization of IFT27 and IFT88, two components of IFT-B complex, was changed. Our findings demonstrate that IFT140 is a key regulator for male fertility and normal spermiogenesis in mice. It not only plays a role in sperm flagella assembling, but is also involved in critical assembly of proteins that interface between the germ cell plasma and the Sertoli cell., (© 2017 Wiley Periodicals, Inc.)

Macrophage migration inhibitory factor (MIF) is a multifunctional molecule. MIF was originally identified as a T-cell-derived factor responsible for the inhibition of macrophage migration. In testicular tissue of adult rats, MIF is constitutively expressed by Leydig cells under physiological conditions. The aim of this study was to examine MIF levels in testicular homogenates from different aged mice, and the capacity of Sertoli cells to produce it. We also examined MIF involvement in spermatogonial cell migration. Similar levels of MIF protein were detected in testicular homogenates of mice of different ages (1-8-week-old). However, the RNA expression levels of MIF were high in 1-week-old mice and significantly decreased with age compared to 1-week-old mice. MIF was stained in Sertoli, Leydig cells, and developed germ cells in the seminiferous tubules. Isolated Sertoli cells from 1-week-old mice stained to MIF. Cultures of Sertoli cells from 1-week-old mice produced and expressed high levels of MIF which significantly decreased with age. MIF was localized in the cytoplasm and nucleus of Sertoli cell cultures isolated from 1-week-old mice; however, it was localized only in the cytoplasm and branches of cultures isolated from 8-week-old mice. MIFR was detected in GFRα1 and Sertoli cells. MIF could induce migration of spermatogonial cells, and this effect was synergistic with glial cell-line neurotrophic factor. Our results show, for the first time, the capacity of Sertoli cells to produce MIF under normal conditions and that MIFR expressed in GFRα1 and Sertoli cells. We also showed that MIF induced spermatogonial cell migration. J. Cell. Physiol. 232: 2869-2877, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The sperm cell has a unique, polarized, and segregated surface that is modified extensively by the changing environments in both the male and the female reproductive tracts. The sperm cannot refresh its surface, as protein translation and membrane recycling by intracellular vesicular transport have ceased upon its maturation. So, how is the sperm surface modified in the reproductive tracts and how do these processes affect fertilization? This review traces these modifications as boar sperm travels from their liberation from the Sertoli cell into the lumen of seminiferous tubules of the testis to the site of fertilization in the ampulla of the oviduct in the sow, via an artificial insemination route. The effect of sperm dilution for artificial insemination, as well as more extensive sperm processing for in vitro fertilization, cryopreservation, or sex sorting, are also discussed with respect to how these procedures affect sperm surface organization and fertilization capacity., (© 2017 Wiley Periodicals, Inc.)

The transcription factor FOXJ3 (Forkhead box J3) is highly expressed in spermatogonia and meiotic spermatocytes within mouse testes. Here, we addressed how FOXJ3 might participate in spermatogenesis using two conditional knockout mouse models in which Foxj3 was deleted from either spermatogonia or meiotic spermatocytes. Both models exhibited complete male sterility, but distinct etiologies: Deleting FOXJ3 from spermatogonia using Foxj3 flox/flox , Mvh-Cre mice caused Sertoli-cell-only syndrome in males. Foxj3-deficient spermatogonia were lost as early as postnatal Day 4, partially due to the accumulation of DNA double-stranded breaks. In contrast, loss of FOXJ3 in spermatocytes using Foxj3 flox/flox , Stra8-Cre mice led to meiotic arrest. Indeed, the mRNA abundance of meiotic arrest-related proteins (Rad51, Dmc1, Brca1, Brca2, Brit1, Eif4g3, Hop2, Hormad1, and Rnf212) was significantly reduced in Foxj3 flox/flox , Stra8-Cre spermatocytes. Thus, we conclude that FOXJ3 is required for the survival of spermatogonia and participates in spermatocyte meiosis. Mol. Reprod. Dev. 83: 1060-1069, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Formation and maintenance of testis cords during embryogenesis are essential for establishing testicular structure and function in adults. At least five genes (Wt1, Dhh, Sox8/Sox9, and Dax1) appear to be required for the maintenance of testis cord integrity in mice. Here, we report that RFX1 is specifically expressed in fetal Sertoli cells. Mouse embryos conditionally deficient in Rfx1 (Rfx1(flox/flox) , Amh-Cre) possessed disrupted testis cords, as the basal lamina lining was fragmented or completely absent in some areas of the testes. Spermatogenesis was blocked, leading to complete infertility. Expression of integrin alpha-6 was significantly decreased in Rfx1-deficient testes compared to control testes; indeed, luciferase and chromatin immunoprecipitation assays indicated that RFX1 directly activates transcription of Itga6 (the gene coding for integrin alpha-6). Taken together, RFX1 transcriptionally targets Itga6 in Sertoli cells, thereby, helping maintain the integrity of the basal lamina during testis cord development. Mol. Reprod. Dev. 83: 606-614, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Besides its capacity to inhibit the 1,4,5-trisphosphate (IP3) receptor, the regulatory protein IRBIT (IP3 receptor binding protein released with IP3) is also able to control the activity of numerous ion channels and electrolyte transporters and thereby creates an optimal electrolyte composition of various biological fluids. Since a reliable execution of spermatogenesis and sperm maturation critically depends on the establishment of an adequate microenvironment, the expression of IRBIT in male reproductive tissue was examined using immunohistochemical approaches combined with biochemical fractionation methods. The present study documents that IRBIT is expressed in Leydig and Sertoli cells. In addition, pronounced IRBIT expression was detected in sperm precursors during early stages of spermatogenesis as well as in spermatozoa. Analyzing tissue sections of rodent epididymides, IRBIT was found to co-localize with the proton pumping V-ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of narrow and clear cells. A similar co-localization of IRBIT with CFTR was also observed for Sertoli cells and developing germ cells. Remarkably, assaying caudal sperm in immunogold electron microscopy, IRBIT was found to localize to the acrosomal cap and the flagellum as well as to the sperm nucleus; moreover, a prominent oligomerization was observed for spermatozoa. The pronounced occurrence of IRBIT in the male reproductive system and mature spermatozoa indicates a potential role for IRBIT in establishing the essential luminal environment for a faithful execution of spermatogenesis and epididymal sperm maturation, and suggest a participation of IRBIT during maturation steps after ejaculation and/or the final fertilization process., (© 2015 Wiley Periodicals, Inc.)

The serine/arginine-rich splicing actor 4 (SRSF4) is essential for pre-mRNA splicing and can influence alternative-splice-site choice. Little is known about the specific function of this gene in the reproductive system, although a recent study identified a SRSF4 polymorphism significantly associated with a decreased risk of non-obstructive azoospermia in Chinese men. We previously found that the expression of Srsf4 was up-regulated in the testes of Sertoli-cell-selective androgen receptor knockout (S-Ar(-/y)) mice compared to wild-type mice using digital gene expression analysis. In this study, we confirmed and extended the selective gene expression data: SRSF4 was mainly located in the nucleus of Sertoli cells, and Srsf4 expression in the Sertoli-cell-derived cell line TM4 is down-regulation by testosterone. Moreover, androgen receptor directly binds the androgen-responsive element of the Srsf4 promoter. Taken together, these results demonstrate that Srsf4 is a direct downstream target of the androgen receptor in mouse Sertoli cells., (© 2015 Wiley Periodicals, Inc.)

Lipopolysaccharide (LPS) induces germ cell apoptosis, but its mechanism of action is not clear. One possibility is that LPS regulates the expression of FAS ligand (FASLG) in Sertoli cells, which will then influence germ cell apoptosis. In this study, LPS reduced the viability of cultured, immature boar Sertoli cells in a time- and dose-dependent manner; enhanced the production of pro-inflammatory cytokines including tumor necrosis factor α (TNFA), interleukin-1β (IL1B), nitric oxide (NO), and transforming growth factor-β (TGFB); and increased the expression of FASLG in a dose-dependent manner. While 10 μg/ml LPS enhanced the expression of FASLG, reduced cell cycle progression, and impaired the ultrastructure of Sertoli cells, this dose did not induce apoptosis. LPS also had no effect on the activity or expression of matrix metalloproteinases 2 or 9 (MMP2 or MMP9). In contrast, the expression of ssc-miR-187 increased following LPS challenge, and inhibition of ssc-miR-187 blocked LPS-induced expression of FASLG. Our results therefore suggest that LPS reduces the viability of and enhances FASLG expression in cultured, immature boar Sertoli cells through elevated secretion of TNFA, IL1B, NO, and TGFB as well as through the regulation of ssc-miR-187 potency., (© 2015 Wiley Periodicals, Inc.)

A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general-purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock-in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP-mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1-Cre-transgenic (tg) embryos almost equally as efficiently as from Nr5a1-hCD271-tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh-Cre-tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types., (© 2015 Wiley Periodicals, Inc.)

Mammalian target of rapamycin (mTOR) is known to be involved in mammalian cell proliferation, while S-phase kinase-associated protein 2 (SKP2) plays a vital role in the cell cycle. Within the testis, estrogen also plays an important role in Sertoli cell proliferation, although it is not clear how. The present study asked if mTOR is involved in 17β-estradiol-dependent Sertoli cell proliferation. We specifically assessed if extracellular signal-regulated kinase 1/2 (ERK1/2) and/or phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) exert convergent effects toward the activation of mTOR signaling, and if this signaling regulates the expression of SKP2 through retinoblastoma (RB) and early mitotic inhibitor 1 (EMI1) protein and on CCNE1 and CCND1 mRNA levels. Treatment with 17β-estradiol for 15-90 min activated mTOR, with mTOR phosphorylation peaking after 30 min. U0126 (5 μM), a specific inhibitor of (MEK1/2), and 10-DEBC (2 μM), a selective inhibitor of AKT, both significantly reduced 17β-estradiol-induced phosphorylation of mTOR. Rapamycin suppressed 17β-estradiol-induced Sertoli cell proliferation, appearing to act by reducing the abundance of SKP2, CCND1, and CCNE1 mRNA as well as RB and EMI1 protein. These data indicated that 17β-estradiol enhances Sertoli cell proliferation via mTOR activation, which involves both ERK1/2 and PI3K/AKT signaling. Activated mTOR subsequently increases SKP2 mRNA and protein expression by enhancing the expression of CCND1 and CCNE1, and inhibits SKP2 protein degradation by increasing EMI1 abundance., (© 2015 Wiley Periodicals, Inc.)

Thyroid hormone is required for the timely transition of Sertoli cells from proliferative to differentiating and maturing. This transition takes place during a critical developmental period in mammals, which in mice is the first post-natal week. In order to identify the underlying molecular mechanisms of this differentiation process, we used Cre/loxP technology to selectively block the function of the thyroid hormone receptor TRα1 in Sertoli cells. We then used RNA-seq to analyze the changes in gene expression induced in the post-natal testis. This differential analysis provides genetic clues to the initial testicular defects resulting from disrupted thyroid hormone signaling, and suggests that Sertoli cells influence germ cells soon after their birth., (© 2014 Wiley Periodicals, Inc.)

Androgen signaling plays a crucial role in spermatogenesis, yet few downstream targets for this signaling pathway have been identified. In the current study, we found that the expression of heat-shock transcription factor 1 (Hsf1) was increased in the testes of Sertoli cell-selective androgen receptor knockout (S-AR(-/y) ) mice compared with wild-type mice by quantitative real-time PCR, and the expression of HSF1 in the S-AR(-/y) Sertoli cells was significantly increased, based on immunofluorescence analysis. In vitro cell-culture studies showed that testosterone repressed the expression of Hsf1 in TM4 cells, a mouse Sertoli cell line. Moreover, a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay showed that testosterone repressed Hsf1 expression by facilitating the binding of androgen receptor to the Hsf1 promoter. Our experiments also demonstrated that testosterone-mediated inhibition of Hsf1 transcription down-regulated the expression of heat-shock proteins HSP105 and HSP60. Taken together, these results reveal that Hsf1 is a novel target of androgen receptor in mouse Sertoli cells, and testosterone and its receptor regulate the process of spermatogenesis partially by inhibiting Hsf1 expression., (© 2014 Wiley Periodicals, Inc.)

In the testis, F-actin structures are involved in spermatid nuclear remodeling and cytoplasm reduction, maintenance of the blood-testis barrier, support of the spermatogonial stem cell niche, and release of spermatids into the tubular lumen. To gain a better understanding of actin regulation in Sertoli-germ cell interactions, we investigated the expression of the Palladin (Palld) gene, which encodes a widely expressed phosphoprotein that localizes to actin-rich cytoplasmic structures, including focal adhesions, cell-cell junctions, podosomes, and stress fibers, and serves as a molecular scaffold to bundle actin fibers. In germ cells, PALLD was concentrated along the tubulin- and F-actin-containing cytoplasmic manchette that forms adjacent to the elongating spermatid nucleus during spermiogenesis. To our surprise, PALLD relocated from the cytoplasm to the nucleus of Sertoli cells in the juvenile testis, coincident with the onset of puberty, and this localization was maintained in the adult. We provide evidence that the 140 kDa isoform of PALLD predominates in Sertoli cells, and that it is apparently cleaved, with the C-terminus localizing to the nucleus while the N-terminus remains cytoplasmic. We investigated the nuclear localization of the C-terminus of PALLD and found that it is regulated by a putative nuclear export signal. These results provide the foundation for future work employing Sertoli cell- and spermatid-specific Palld-knockout mice to study diverse roles of PALLD as both a nuclear-actin regulatory protein and as a potential regulator of manchette formation during spermatogenesis., (Copyright © 2013 Wiley Periodicals, Inc.)

Carcinoembryonic antigen (CEA) family, a subgroup of the immunoglobulin (Ig) superfamily, is divided into two sub-families: the CEA-related cell adhesion molecules (CEACAM) and the pregnancy-specific glycoproteins. The isoform CEACAM2 is expressed in mouse testis; in this study, we identified a novel isoform of Ceacam2, Ceacam2-Long (Ceacam2-L). CEACAM2-L is different from CEACAM2 in that it has much longer cytoplasmic tail region. Ceacam2-L starts to appear faintly in mouse testis after 3 weeks of postnatal development, and its expression level increased after 5 weeks. Immunoblot analysis confirmed the expression of CEACAM2-L in the seminiferous epithelium of mouse testis. Immunohistochemical data showed that CEACAM2-L was not observed on spermatogonia, spermatocytes, round spermatids, or Sertoli cells, but was seen at the plasma membrane of elongating spermatids in contact with extended cytoplasmic processes of Sertoli cells. CEACAM2-L was not detected at the head region of elongating spermatids, where the apical ectoplasmic specialization is constructed. These data suggest that CEACAM2-L might be a novel adhesion molecule contributing to cell-to-cell adhesion between elongating spermatids and Sertoli cells within the seminiferous epithelium., (Copyright © 2012 Wiley Periodicals, Inc.)

In eukaryotes, mRNA is actively exported to the cytoplasm by a family of nuclear RNA export factors (NXF). Four Nxf genes have been identified in the mouse: Nxf1, Nxf2, Nxf3, and Nxf7. Inactivation of Nxf2, a germ cell-specific gene, causes defects in spermatogenesis. Here we report that Nxf3 is expressed exclusively in Sertoli cells of the postnatal testis, in a developmentally regulated manner. Expression of Nxf3 coincides with the cessation of Sertoli cell proliferation and the beginning of their differentiation. Continued expression of Nxf3 in mature Sertoli cells of the adult is spermatogenesis stage-independent. Nxf3 is not essential for spermatogenesis, however, suggesting functional redundancy among Nxf family members. With its unique expression pattern in the testis, the promoter of Nxf3 can be used to drive postnatal Sertoli cell-specific expression of other proteins such as Cre recombinase., (Copyright © 2011 Wiley-Liss, Inc.)

Dufd1 (DUF729 domain containing 1) is related to Mtfr1 (mitochondrial fission regulator 1), a gene involved in the regulation of antioxidant activity in the mouse testis. The present study was undertaken to better understand their role in regulating mitochondrial architecture and function in the mouse. We show that Dufd1 is expressed as a 2 kb mRNA and has a more specific tissue pattern compared to Mtfr1, with highest level of expression in testes, lower level in spleen, and negligible levels in other organs and/or tissues. In the male gonad, Dufd1 mRNA expression increases during postnatal development, similarly to Mtfr1. In situ hybridization and real-time PCR analyses show that Dufd1 is expressed in the seminiferous tubules by middle-late pachytene spermatocytes and spermatids. In transfected cells, the Dufd1-tagged protein is located in mitochondria, associated with the tips of mitochondrial tubules and to tubules constrictions, and induces mitochondrial fission although with a lesser efficiency than Mtfr1. We also found that both endogenous Dufd1 and Mtfr1 proteins are associated with membrane-enriched subcellular fractions, including mitochondria. Inhibition of Mtfr1 and/or Dufd1 expression, in a testicular germ cells line, severely impairs O(2) consumption and indicates that both genes are required for mitochondrial respiration. Accordingly, analysis of testes mitochondria from Mtfr1-deficient mice reveals severely reduced O(2) consumption and ATP synthesis compared to wt animals. These data show that, in murine testis, Dufd1 and Mtfr1 have redundant functions related to mitochondrial physiology and represent genes with a potential role in testicular function., (© 2010 Wiley-Liss, Inc.)

We report that full-length and truncated transcripts of Fyn tyrosine protein kinase are expressed during testicular development. Truncated Fyn (tr-Fyn) transcripts encode a 24 kDa protein with a N-terminal (NT) domain, a complete Src homology (SH) 3 domain and an incomplete SH2 domain. The kinase domain is missing in tr-Fyn. In contrast, full-length Fyn transcripts encode a 59-55 kDa protein. Fractionated spermatids by centrifugal elutriation express tr-Fyn transcripts and protein, but not full-length Fyn transcripts and protein. Neither full-length Fyn nor tr-Fyn transcripts and encoded proteins are detected in elutriated pachytene spermatocytes. Sertoli cells express full-length and truncated Fyn throughout testicular development. In contrast, sperm contain full-length Fyn transcripts and protein but not the truncated form. tr-Fyn protein is visualized at the cytosolic side of Golgi membranes, derived proacrosomal vesicles, along the outer acrosome membrane and the inner acrosome membrane-acroplaxome complex anchoring the acrosome to the spermatid nuclear envelope. Fyn and phosphotyrosine immunoreactivity coexist in the tail of capacitated sperm. During fertilization, the Fyn-containing acroplaxome seen in the egg-bound and egg-fused sperm is no longer detected upon decondensation of the sperm nucleus. tr-Fyn expands the catalog of truncated tyrosine protein kinases expressed during spermiogenesis. We suggest that the NT and SH3 domains of tr-Fyn may recruit adaptor and effector proteins, in particular GTPase activating proteins, required for acrosome-acroplaxome biogenesis, acroplaxome F-actin dynamics and Sertoli cell function. During fertilization, full-length Fyn in the acroplaxome may contribute to a transient local signaling burst during the early events of sperm-egg interaction.

Ets-related molecule (Erm) is a member of the Ets transcription factor family. Erm is known to be an important factor for the self-renewal of Spermatogonial stem cells (SSCs) and the maintenance of spermatogenesis. We investigated the molecular mechanism of Erm regulation on SDF-1 in TM4 Sertoli cells. Erm and Sdf-1 levels were up-regulated after FGF2 treatment in TM4 cells, whereas these levels were significantly decreased by FGF2 in ST2 bone marrow stromal cells. Knockdown of Erm by siRNA in the presence of FGF2 decreased the Sdf-1 levels in TM4 cells. The expression levels of Erm were similar and Erm overexpression increased the Sdf-1 in both TM4 and ST2 cells. FGFR subtype analysis revealed that FGFR4 was expressed in TM4 cells but not in ST2 cells. A blocking experiment also confirmed that FGFR4 is partly responsible for the up-regulation of Erm and SDF-1 induced by FGF2 stimulation in TM4 cells. FGF2 and ERM increased the activity of Sdf-1 gene promoter region in a dose-dependent manner. EMSA revealed that ERM strongly binds to the -846 to -851 nucleotide region of the potential Ets binding site (EBS) in the Sdf-1 promoter. In addition, CXCR4, the SDF-1 receptor, was expressed in spermatogonia and Sertoli cells in the seminiferous tubules of the mouse testis. Our results indicate that ERM directly regulates Sdf-1 gene expression by interacting with its cis-acting element in response to FGF2 stimulation in TM4 cells.

Vitamin C is an essential micronutrient for the development of male germ cells. In the gonad, the germ cells are isolated from the systemic circulation by the blood-testis barrier, which consists of a basal layer of Sertoli cells that communicate through an extensive array of tight junction complexes. To study the behavior of Sertoli cells as a first approach to the molecular and functional characterization of the vitamin C transporters in this barrier, we used the 42GPA9 cell line immortalized from mouse Sertoli cells. To date, there is no available information on the mechanism of vitamin C transport across the blood-testis barrier. This work describe the molecular identity of the transporters involved in vitamin C transport in these cells, which we hope will improve our understanding of how germ cells obtain vitamin C, transported from the plasma into the adluminal compartment of the seminiferous tubules. RT-PCR analyses revealed that 42GPA9 cells express both vitamin C transport systems, a finding that was confirmed by immunocytochemical and immunoblotting analysis. The kinetic assays using radioactive vitamin C revealed that both ascorbic acid (AA) transporters, SVCT1 and SVCT2, are functionally active. Moreover, the kinetic characteristics of dehydroascorbic acid (DHA) and 3-methylglucose (OMG) transport by 42GPA9 Sertoli cells correspond to facilitative hexose transporters GLUT1, GLUT2 and GLUT3 expressed in these cells. This data is consistent with the concept that Sertoli cells have the ability to take up vitamin C. It is an important finding and contributes to our knowledge of the physiology of male germ cells.

The cAMP response element binding protein/activating transcription factor (CREB/ATF) family of transcription factors is hormone responsive and critical for nearly all mammalian cell types. The basic helix-loop-helix (bHLH) family of transcription factors is important during the development and differentiation of a wide variety of cell types. Independent studies of the role of the bHLH protein scleraxis in testicular Sertoli cells and paraxis in muscle development using yeast-2-hybrid screens provided the novel observation that bHLH proteins can directly interact with ATF/CREB family members. Analysis of the interactions demonstrated the helix-loop-helix domain of bHLH proteins directly interacts with the leucine zipper (ZIP) region of CREB2/ATF4 to form heterodimers. The direct bHLH-CREB2 binding interactions were supported using co-immunoprecipitation of recombinant proteins. Structural analysis of bHLH and ATF4 heterodimer using previous crystal structures demonstrated the heterodimer likely involves the HLH and Zip domains and has the potential capacity to bind DNA. Transfection assays demonstrated CREB2/ATF4 over-expression blocked stimulatory actions of scleraxis or paraxis. CREB1 inhibited MyoD induced myogenic conversion of C3H10T1/2 cells. CREB2/ATF4 and scleraxis are expressed throughout embryonic and postnatal testis development, with scleraxis specifically expressed in Sertoli cells. ATF4 and scleraxis null mutant mice both had similar adult testis phenotypes of reduced spermatogenic capacity. In summary, bHLH and CREB family members were found to directly heterodimerize and inhibit the actions of bHLH dimers on Sertoli cells and myogenic precursor cells. The observations suggest a mechanism for direct cross-talk between cAMP induced and bHLH controlled cellular differentiation.

Outer dense fibers are a major constituent of the sperm tail and outer dense fiber 2 (ODF2) protein is one of their major components. ODF2 shares partial homology with cenexin 1 and cenexin 2, regarded as centriolar proteins. We show that ODF2 and cenexin 2 transcripts are the product of differential splicing of a single gene, designated Cenexin/ODF2 and that cenexin 1 is an incomplete clone of ODF2. ODF2 terminates in exon 20b whereas in cenexin 2 this exon is spliced out and translation terminates in exon 24. We demonstrate a transcriptional switch during rat testicular development, from somatic-type to testis-type ODF2 and cenexin transcripts during the onset of meiosis. The switch is completed when spermiogenesis is established. ODF2 immunoreactive sites were visualized in the acroplaxome, along the sperm tail and the centrosome-derived sperm head-to-tail coupling apparatus. An unexpected finding was the presence of ODF2 antigenic sites, but not cenexin antigenic sites, in the dense fibrillar component of the nucleolus of Sertoli cells, spermatogonia and primary spermatocytes. The characterization of the genomic origin, processing and developmental expression of ODF2 transcript isoforms and their protein products can help reconcile differences in the literature on the role of ODF2 and cenexin in the centrosome. Furthermore, the finding of ODF2 in the dense fibrillar component of the nucleolus suggests that this protein, in addition to its presence in sperm outer dense fibers and centrosome, highlights and adds to the nucleolar function during spermatogenesis and early embryogenesis.

In mammalian testes Sertoli cells form tight junctions whose function is fundamental for the maintenance of a normal spermatogenesis. Hepatocyte growth factor (HGF) is a cytokine influencing the cellular tight junctions either in normal or in tumor cells. We have previously demonstrated that HGF is expressed in the rat testis and influences many functional activities of somatic and germ cells. We now report that HGF decreases the levels of testicular occludin and influences the position of the molecule in the tight junctions as demonstrated by confocal microscopy analysis. In fact in the presence of the factor occludin was mainly localized in the suprabasal region of the tubules whereas in its absence occludin was prevalently localized in the basal region. Occludin production is known to be regulated by different cytokines including TGFbeta. We have investigated the role of HGF in the regulation of the levels of TGFbeta and we report that HGF significantly increases the amount of the active fraction of the factor without affecting the amount of the total TGFbeta. Urokinase type plasminogen activator (uPA) is closely related with the tight junctions and is one of the molecules able to activate the inactive TGF-beta. We found that HGF significantly increases the amount of uPA present in the testis suggesting that HGF regulates the amount of active TGFbeta via uPA levels. In conclusion we report that in the testis HGF regulates Sertoli-Sertoli tight junctions inducing a reduction and redistribution of occludin possibly modulating the levels of uPA and active TGFbeta., ((c) 2008 Wiley-Liss, Inc.)

In mammals, Sry, Sox9, and M33 act as regulators at a chromatin level and promote the maturation of embryonic gonads into testes. Recently, it was shown that transcriptional regulation by DNA methylation plays crucial roles in gene expression during the differentiation and development of various cell types. To determine the involvement of DNA methylation in sex determination of the gonad, we developed and performed organ culture of gonad with the DNA methyltransferase inhibitor 5-azacytidine to induce global DNA methylation status changes. In vitro treatment with 5-azacytidine specifically inhibited testicular cord formation in a dose-dependent manner; however, no appreciable defect was observed in ovarian explants. Inhibition of testicular cord was observed only in gonads from 11.5 days post-coitus embryos. These effects were not observed in 5-azacytidine-treated gonads from 12.0 days post-coitus embryos. To determine the effect of 5-azacytidine on Sertoli and Leydig cell differentiation in the testis, we performed whole mount in situ hybridization analysis. The Leydig and stromal cell marker genes Lhx9, Mfge8, and 3beta-Hsd were normally induced in 5-azaytidine-treated testicular explants. Sertoli cell marker genes, Sox9 and MIS were normally induced, but Col9a3, encoding an extracellular matrix component, was inhibited in 5-azacytidine-treated testicular explants. Thus, our data show that DNA methylation is involved in testicular cord formation and Sertoli cell differentiation, acting directly on the gonad at 11.5 days post-coitus.