Functional importance of palmitoyl protein thioesterase 1 (PPT1) expression by Sertoli cells in mediating cholesterol metabolism and maintenance of sperm quality

Sertoli cells are a type of nurse cell in the seminiferous epithelium that are crucial for sustaining spermatogenesis by extending nutritional and energy support to the developing germ cells. Dysfunction of Sertoli cells could cause disordered spermatogenesis and reduced fertility in males. In this study, we focused on the expression and function of palmitoyl protein thioesterase 1 (PPT1), a lysosomal depalmitoylating enzyme, in Sertoli cells. Here, we show that PPT1 expression in Sertoli cells is responsive to cholesterol treatment and that specific knockout of Ppt1 in Sertoli cells causes male subfertility associated with poor sperm quality and a high ratio of sperm deformity. Specifically, Ppt1 deficiency leads to poor cell variably accompanied with abnormal lysosome accumulation and increased cholesterol levels in Sertoli cells. Further, Ppt1 deficiency results in poor adhesion of developing germ cells to Sertoli cells in the seminiferous epithelium, which is likely to be responsible for the reduced male fertility as a consequence of declines in sperm count and motility as well as a high incidence of sperm head deformity. In summary, PPT1 affects sperm quality and male fertility through regulating lysosomal function and cholesterol metabolism in Sertoli cells.     

Metabolic effects of L-carnitine on prepubertal rat Sertoli cells.

The role of carnitine on Sertoli cell metabolism was investigated. Carnitine effects on Sertoli cell lipid metabolism were evaluated by measuring the intracellular levels of non-esterified fatty acids (NEFA) and ketone bodies. The concentration of NEFA in Sertoli cell cultured in the presence of carnitine is significantly reduced as compared to control, while, no significant changes were observed in the concentration of ketone bodies. The functional parameters evaluated to assess the influence of carnitine on Sertoli cell carbohydrate metabolism, i.e., lactate and pyruvate production, lactate dehydrogenase activity and hexose transport, were all significantly increased following carnitine in vitro supplementation. Thus, carnitine appears to drive Sertoli cell intermediary metabolism in an intimately interrelated way, stimulating both fatty acid breakdown and glycolysis. Our results indicate that Sertoli cells are a possible target for a widespread metabolic action of carnitine and strongly support the involvement of carnitine in the regulation of Sertoli cell functions which are related with germ cell "nutrition", convincingly suggesting a direct influence of the compound at testis level.     

Follicle-stimulating hormone-induced aromatase in immature rat Sertoli cells requires an active phosphatidylinositol 3-kinase pathway and is inhibited via the mitogen-activated protein kinase signaling pathway

Postnatal development and function of testicular Sertoli cells are regulated primarily by FSH. During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development. Growth factors like epidermal growth factor (EGF) are produced in the testis and play a role in regulation of estradiol production and male fertility. Although these divergent factors modulate gonadal function, little is known about their mechanism of action in Sertoli cells. The present study investigates the intracellular events that take place down-stream of FSH and EGF receptors in Sertoli cells isolated from immature (10-d-old) rats, and examines which intracellular signals may be involved in their effects on aromatase activity and estradiol production in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were treated with FSH in combination with EGF and signaling pathway-specific inhibitors. Levels of estradiol production, aromatase mRNA (Cyp19a1), and aromatase protein (CYP19A1) were determined. Western blot analysis was performed to determine the effects of FSH and EGF on levels of activated (phosphorylated) AKT1 and p42 ERK2 and p44 ERK1, also named MAPK1 and MAPK3, respectively. The stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production were blocked by inhibition of the phosphatidylinositol 3-kinase/AKT1 signaling pathway. In contrast, inhibition of ERK signaling augmented the stimulatory effects of FSH on estradiol production, aromatase mRNA, and protein levels. Furthermore, EGF inhibited the expression of aromatase mRNA and protein in response to FSH, and these inhibitory effects of EGF were critically dependent on the activation of the ERK signaling pathway. We conclude that an active phosphatidylinositol 3-kinase /AKT signaling pathway is required for the stimulatory actions of FSH, whereas an active ERK/MAPK pathway inhibits estradiol production and aromatase expression in immature Sertoli cells.     

Effects of L-carnitine and its derivatives on postischemic cardiac function,ventricular fibrillation and necrotic and apoptotic cardiomyocyte death in isolated rat hearts

The study aimed to examine whether L-carnitine and its derivatives, acetyl-L-carnitine and propionyl-L-carnitine, were equally effective and able to improve postischemic cardiac function, reduce the incidence of reperfusion-induced ventricular fibrillation, infarct size, and apoptotic cell death in ischemic/reperfused isolated rat hearts. There are several studies indicating that L-carnitine, a naturally occurring amino acid and an essential cofactor, can improve mechanical function and substrate metabolism not only in hypertrophied or failing myocardium but also in ischemic/reperfused hearts. The effects of L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine, on the recovery of heart function, incidence of reperfusion-induced ventricular fibrillation (VF), infarct size, and apoptotic cell death after 30 min ischemia followed by 120 min reperfusion were studied in isolated working rat hearts. Hearts were perfused with various concentrations of L-carnitine (0.5 and 5 mM), acetyl-L-carnitine (0.5 and 5 mM), and propionyl-L-carnitine (0.05, 0.5, and 5 mM), respectively, for 10 min before the induction of ischemia. Postischemic recovery of CF, AF, and LVDP was significantly improved in all groups perfused with 5 mM of L-carnitine, acetyl-L-carnitine, and propionyl-L-carnitine. Significant postischemic ventricular recovery was noticed in the hearts perfused with 0.5 mM of propionyl-L-carnitine, but not with the same concentration of L-carnitine or L-acetyl carnitine. The incidence of reperfusion VF was reduced from its control value of 90 to 10% (p < 0.05) in hearts perfused with 5 mM of propionyl-L-carnitine only. Other doses of various carnitines failed to reduce the incidence of VF. The protection in CF, AF, LVDP, and VF reflected in a reduction in infarct size and apoptotic cell death in hearts treated with various concentrations of carnitine derivatives. The difference between effectiveness of various carnitines on the recovery of postischemic myocardium may be explained by different membrane permeability properties of carnitine and its derivatives.     

The ATP-binding cassette transporter 1 mediates lipid efflux from Sertoli cells and influences male fertility

The liver X receptor/retinoid X receptor (LXR/RXR)-regulated gene ABCA1 effluxes cellular cholesterol and phospholipid to apolipoprotein A1 (apoA1), which is the rate-limiting step in high-density lipoprotein synthesis. The RXR pathway plays a critical role in testicular lipid trafficking, and RXRbeta-deficient male mice are sterile and accumulate lipids in Sertoli cells. Here, we demonstrate that ABCA1 mRNA and protein are abundant in Sertoli cells, whereas germ cells express little ABCA1. LXR/RXR agonists stimulate ABCA1 expression in cultured Sertoli MSC1 and Leydig TM3 cell lines. However, Sertoli TM4 cells lack ABCA1, and TM4 cells or primary Sertoli cells cultured from ABCA1(-/-) mice both fail to efflux cholesterol to apoA1. Expression of exogenous ABCA1 restores apoA1-dependent cholesterol efflux in Sertoli TM4 cells. In vivo, ABCA1-deficient mice exhibit lipid accumulation in Sertoli cells and depletion of normal lipid droplets from Leydig cells by 2 months of age. By 6 months of age, intratesticular testosterone levels and sperm counts are significantly reduced in ABCA1(-/-) mice compared with wild-type (WT) controls. Finally, a 21% decrease (P = 0.01) in fertility was observed between ABCA1(-/-) males compared with WT controls across their reproductive lifespans. These results show that ABCA1 plays an important role in lipid transport in Sertoli cells and influences male fertility.     

Altered differentiation and clustering of Sertoli cells in transgenic mice showing a Sertoli cell specific knockout of the connexin 43 gene

Histological analysis revealed that Sertoli cell specific knockout of the predominant testicular gap junction protein connexin 43 results in a spermatogenic arrest at the level of spermatogonia or Sertoli cell-only syndrome, intratubular cell clusters and still proliferating adult Sertoli cells, implying an important role for connexin 43 in the Sertoli and germ cell development. This study aimed to determine the (1) Sertoli cell maturation state, (2) time of occurrence and (3) composition, differentiation and fate of clustered cells in knockout mice. Using immunohistochemistry connexin 43 deficient Sertoli cells showed an accurate start of the mature markers androgen receptor and GATA-1 during puberty and a vimentin expression from neonatal to adult. Expression of anti-Muellerian hormone, as a marker of Sertoli cell immaturity, was finally down-regulated during puberty, but its disappearance was delayed. This observed extended anti-Müllerian hormone synthesis during puberty was confirmed by western blot and Real-Time PCR and suggests a partial alteration in the Sertoli cell differentiation program. Additionally, Sertoli cells of adult knockouts showed a permanent and uniform expression of GATA-1 at protein and mRNA level, maybe caused by the lack of maturing germ cells and missing negative feedback signals. At ultrastructural level, basally located adult Sertoli cells obtained their mature appearance, demonstrated by the tripartite nucleolus as a typical feature of differentiated Sertoli cells. Intratubular clustered cells were mainly formed by abnormal Sertoli cells and single attached apoptotic germ cells, verified by immunohistochemistry, TUNEL staining and transmission electron microscopy. Clusters first appeared during puberty and became more numerous in adulthood with increasing cell numbers per cluster suggesting an age-related process. In conclusion, adult connexin 43 deficient Sertoli cells seem to proliferate while maintaining expression of mature markers and their adult morphology, indicating a unique and abnormal intermediate phenotype with characteristics common to both undifferentiated and differentiated Sertoli cells.     

Epididymal protease inhibitor (EPPIN) is differentially expressed in the male rat reproductive tract and immunolocalized in maturing spermatozoa

EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine‐rich protein containing both Kunitz and whey acidic protein (WAP)‐type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down‐regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen‐dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility. Mol. Reprod. Dev. 79: 832–842, 2012. © 2012 Wiley Periodicals, Inc.     

Calcium/phospholipid-dependent protein kinases in rat Sertoli cells: regulation of androgen receptor messenger ribonucleic acid

The possibility that Sertoli cell responses to testosterone are modulated by the calcium/phospholipid-dependent protein kinase (protein kinase C; PKC) was examined in rat Sertoli cells in culture. Both soluble and particulate cell fractions showed low constitutive phosphotransferase activity. Incubation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) was associated with a transient induction in both cell fractions of calcium/phosphatidylserine-dependent PKC activity, which was elevated from 15 min to 1 h. Consistent with this, mRNAs for the calcium/phospholipid-dependent isomeric forms of PKC (alpha, beta, and gamma) were detected. The expression levels of mRNAs for PKCalpha and PKCbeta were also up-regulated (2.5- to 3-fold) by TPA (10(-7) M), but these effects were much slower (peaking after 12 h) than those on phosphotransferase activity. In the presence of TPA (10(-7) M), expression of androgen receptor (AR) mRNA showed a transient time-dependent down-regulation ( approximately 70%), in which the nadir was reached after 6 h and baseline expression was again obtained after 12 h. The regulatory effect of PKC activation on AR mRNA was confirmed by the absence of response to a biologically inactive phorbol ester. A concentration-dependent decrease (half-maximal effect at approximately 10(-8) M TPA) of AR mRNA was also observed. These data suggest that Sertoli cell responses to testosterone may be inhibited by a transiently active PKC with a wide intracellular distribution.     

Fas ligand expression in TM4 Sertoli cells is enhanced by estradiol "in situ" production

The testis is an immunologically privileged site of the body where Sertoli cells work on to favor local immune tolerance by testicular autoantigens segregation and immunosuppressive factors secretion. Fas/Fas Ligand (FasL) system, expressed prevalently in Sertoli cells, has been considered to be one of the central mechanisms in testis immunological homeostasis. In different cell lines it has been reported that the proapoptotic protein FasL is regulated by 17-beta estradiol (E2). Thus, using as experimental model mouse Sertoli cells TM4, which conserve a large spectrum of functional features present in native Sertoli cells, like aromatase activity, we investigated if estradiol "in situ" production may influence FasL expression. Our results demonstrate that an aromatizable androgen like androst-4-ene-3,17-dione (Delta4) enhanced FasL mRNA, protein content and promoter activity in TM4 cells. The treatment with N(6),2'-O-dibutyryladenosine-3'-5'-cyclic monophosphate [(Bu)(2)cAMP] (simulating FSH action), that is well known to stimulate aromatase activity in Sertoli cells, amplified Delta4 induced FasL expression. Functional studies of mutagenesis, electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays revealed that the Sp-1 motif on FasL promoter was required for E2 enhanced FasL expression in TM4 cells. These data let us to recruit FasL among those genes whose expression is up-regulated by E2 through a direct interaction of ERalpha with Sp-1 protein. Finally, evidence that an aromatizable androgen is able to increase FasL expression suggests that E2 production by aromatase activity may contribute to maintain the immunoprivilege status of Sertoli cells.     

Autoregulation of transformer-2 alternative splicing is necessary for normal male fertility in Drosophila.

In the male germline of Drosophila the transformer-2 protein is required for differential splicing of pre-mRNAs from the exuperantia and att genes and autoregulates alternative splicing of its own pre-mRNA. Autoregulation of TRA-2 splicing results in production of two mRNAs that differ by the splicing/retention of the M1 intron and encode functionally distinct protein isoforms. Splicing of the intron produces an mRNA encoding TRA-2(226), which is necessary and sufficient for both male fertility and regulation of downstream target RNAs. When the intron is retained, an mRNA is produced encoding TRA-2(179), a protein with no known function. We have previously shown that repression of M1 splicing is dependent on TRA-2(226), suggesting that this protein quantitatively limits its own expression through a negative feedback mechanism at the level of splicing. Here we examine this idea, by testing the effect that variations in the level of tra-2 expression have on the splicing of M1 and on male fertility. Consistent with our hypothesis, we observe that as tra-2 gene dosage is increased, smaller proportions of TRA-2(226) mRNA are produced, limiting expression of this isoform. Feedback regulation is critical for male fertility, since it is significantly decreased by a transgene in which repression of M1 splicing cannot occur and TRA-2(226) mRNA is constitutively produced. The effect of this transgene becomes more severe as its dosage is increased, indicating that fertility is sensitive to an excess of TRA-2(226). Our results suggest that autoregulation of TRA-2(226) expression in male germ cells is necessary for normal spermatogenesis.     

Expression and function of class B scavenger receptor type I on both apical and basolateral sides of the plasma membrane of polarized testicular Sertoli cells of the rat

Class B scavenger receptor type I (SR-BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR-BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR-BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle-stimulating hormone (FSH). When the expression of SR-BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first-round spermatogenesis and continued to be detectable thereafter. The level of SR-BI mRNA expression in Sertoli cells was lower at spermatogenic stages I-VI than at other stages. SR-BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR-BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR-BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR-BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.     

Androgen binding protein is tissue-specifically expressed and biologically active in transgenic mice

In view of the inconclusive data concerning the role of androgen-binding protein (ABP) in male reproductive physiology, we thought it would be pertinent to make several transgenic mouse lines overexpressing the rat ABP gene to unravel its role in Sertoli cell and epididymal homeostasis. Heterozygote transgenic mouse lines carrying the 5.5 kb ABP rat genomic DNA were produced by pronuclear microinjection. Northern blot analysis showed overexpression of rat ABP (rABP) mRNA in the testis of transgenic mice compared to rat testis control. rABP was appropriately expressed in Sertoli cells as demonstrated by in situ hybridization analysis. Sertoli cell number is increased in the seminiferous tubules of mice overexpressing rABP compared to non-transgenic littermates and scattered Sertoli cells present vacuolated-like cytoplasms, PAS and osmium negative. Compared to the wild type, the transgenic mice exhibited reduced fertility and focal damage in seminiferous epithelium characterized by morphological features compatible with programmed cell death.     

Association study and expression analysis of porcine ESR1 as a candidate gene for boar fertility and sperm quality

Male fertility is impaired through the lack of ESR1 (Estrogen Receptor 1) but little is known about the ESR1 roles in boar spermatogenesis and fertility. Therefore, this research was aimed at investigating the association with sperm quality and boar fertility traits in a total of 300 boars both from purebred Pietrain and Pietrain × Hampshire crosses. A SNP in coding region of ESR1g.672C>T in exon 1 was associated with sperm motility (P<0.05) and plasma droplet rate (P<0.01) while the polymorphism in non-coding region of ESR1g.35756T>C in inton 1 was associated with non-return rate (P<0.05). Furthermore, to analyse the mRNA and protein expression of ESR1 in boar reproductive tissues, a total of six boars were divided into two groups [Group I (G-I) and Group II (G-II)], where G-I had relatively better sperm quality. ESR1 expression was higher in tissues collected from G-I boars than those of collected from G-II boars, and the difference in mRNA expression was significant (P<0.01) in head of epididymis. The ESR1 protein expression results from western blot coincided with the results of qRT-PCR. The ESR1 protein localization observed a strong staining in the cytoplasm of Sertoli cell in the testis, in the epithelial cells in head and tail of epididymis, in smooth muscle in tail of epididymis, and in the post acrosomal region and tail of the spermatozoa. These results will improve the understanding of the functions of the ESR1 in spermatogenesis within the reproductive tract and will shed light on ESR1 as a candidate in the selection of boar with good sperm quality and fertility.     

Actions of extracellular matrix on Sertoli cell morphology and function

Sertoli cells were isolated and cultured in the absence or presence of extracellular matrix (ECM) to determine whether ECM may influence Sertoli cell function on a molecular level. As previously described, a morphological analysis of the cells indicated that ECM allows the expression of a columnar histotype and the formation of junctional complexes. The combined actions of ECM and hormones were found to have a profound effect in promoting the expression of a polarized Sertoli cell morphology. In our investigation of the effects of ECM on Sertoli cells, we used transferrin and androgen-binding protein (ABP) production as biochemical markers of Sertoli cell function. The presence of ECM was found to cause a 25% increase in the basal level of transferrin production; however, ECM had no effect on the basal level of ABP production by Sertoli cells. Regulatory agents such as follicle-stimulating hormone (FSH) and a combination of FSH, insulin, retinol, and testosterone stimulated the production of both transferrin and ABP. The ability of hormones to stimulate these Sertoli cell functions was not influenced by the presence of ECM. Similar results were obtained with 2-microns- or 50-microns-thick ECM and with a seminiferous tubule biomatrix preparation. ECM was found to increase the maintenance of long-term Sertoli cell cultures; however, the decline in Sertoli cell functional integrity, which occurs during cell culture, was not affected by the presence of ECM. An additional functional parameter examined was the radiolabeled proteins secreted by Sertoli cells. ECM did not promote the production or affect the electrophoretic profile of Sertoli cell-secreted proteins under basal or hormonally stimulated conditions. Combined results indicated that although ECM allowed the expression of a normal Sertoli cell histotype, ECM had no major effects on the Sertoli cell functions analyzed nor on the hormonal regulation of these functions. The inability of ECM to affect Sertoli cell function on a molecular level is discussed with regard to environmental as opposed to regulatory cellular interactions. Our observations imply that dramatic effects of ECM on cell morphology do not necessarily correlate to subsequent effects on cellular function.     

MicroRNA-143 (miR-143) regulates cancer glycolysis via targeting hexokinase 2 gene

High glycolysis, well known as "Warburg effect," is frequently observed in a variety of cancers. Whether the deregulation of miRNAs contributes to the Warburg effect remains largely unknown. Because miRNA regulates gene expression at both mRNA and protein levels, we constructed a gene functional association network, which allows us to detect the gene activity instead of gene expression, to integratively analyze the microarray data for gene expression and miRNA expression profiling and identify glycolysis-related gene-miRNA pairs deregulated in cancer. Hexokinase 2 (HK2), coding for the first rate-limiting enzyme of glycolysis, is among the top list of genes predicted and potentially regulated by multiple miRNAs including miR-143. Interestingly, miR-143 expression was inversely associated with HK2 protein level but not mRNA level in human lung cancer samples. miR-143, down-regulated by mammalian target of rapamycin activation, reduces glucose metabolism and inhibits cancer cell proliferation and tumor formation through targeting HK2. Collectively, we have not only established a novel methodology for gene-miRNA pair prediction but also identified miR-143 as an essential regulator of cancer glycolysis via targeting HK2.