Xiaoheng Li

Insights into the Development of the Adult Leydig Cell Lineage from Stem Leydig Cells

Adult Leydig cells (ALCs) are the steroidogenic cells in the testes that produce testosterone. ALCs develop postnatally from a pool of stem cells, referred to as stem Leydig cells (SLCs). SLCs are spindle-shaped cells that lack steroidogenic cell markers, including luteinizing hormone (LH) receptor and 3β-hydroxysteroid dehydrogenase. The commitment of SLCs into the progenitor Leydig cells (PLCs), the first stage in the lineage, requires growth factors, including Dessert Hedgehog (DHH) and platelet-derived growth factor-AA. PLCs are still spindle-shaped, but become steroidogenic and produce mainly androsterone. The next transition in the lineage is from PLC to the immature Leydig cell (ILC). This transition requires LH, DHH, and androgen. ILCs are ovoid cells that are competent for producing a different form of androgen, androstanediol. The final stage in the developmental lineage is ALC. The transition to ALC involves the reduced expression of 5α-reductase 1, a step that is necessary to make the cells to produce testosterone as the final product. The transitions along the Leydig cell lineage are associated with the progressive down-regulation of the proliferative activity, and the up-regulation of steroidogenic capacity, with each step requiring unique regulatory signaling.

Adverse effects of di-(2-ethylhexyl) phthalate on Leydig cell regeneration in the adult rat testis.

The objective of the present study is to determine whether di-(2-ethylhexyl) phthalate (DEHP) exposure at adulthood affects regeneration of rat Leydig cells. 90-day-old Long-Evans rats received intraperitoneal injection of 75 mg/kg ethane dimethanesulfonate (EDS) to eliminate mature Leydig cells, and then were randomly divided into 3 groups, in which rats were gavaged with the corn oil (control) or 10 or 750 mg/kg DEHP daily for 35 days. Serum testosterone and luteinizing hormone levels were assessed by RIA, Leydig cell numbers and proliferation rate were evaluated, and the mRNA levels of Leydig cell specific genes were measured by qPCR. Both 10 and 750 mg/kg DEHP treatments increased Leydig cell numbers on day 14, 21 and 35 post-EDS, due to significant increase of the number of Leydig cell precursors from day 14 to 21 post-EDS. However, serum testosterone levels were halved in 10 and 750 mg/kg DEHP groups compared to control on day 35 post-EDS despite the increased Leydig cell numbers. Quantitative PCR showed that Leydig cell specific genes including Lhcgr, Cyp11a1, Hsd3b1, and Insl3 were significantly down-regulated in 750 mg/kg DEHP-treated testes on post-EDS day 21 and beyond. The present study suggests that DEHP increases Leydig cell proliferation but inhibits differentiation during the regeneration of Leydig cells.

Effects of Methoxychlor and Its Metabolite Hydroxychlor on Human Placental 3β-Hydroxysteroid Dehydrogenase 1 and Aromatase in JEG-3 Cells.

Progesterone and estradiol produced by the human placenta are critical for maintenance of pregnancy and fetal development. In the human placenta, 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) is responsible for the formation of progesterone from pregnenolone and aromatase (CYP19A1) for the production of estradiol from androgen. Insecticide methoxychlor (MXC) and its metabolite hydroxychlor (HPTE) may disrupt the activities of these 2 enzymes. In this study, we investigated the effects of MXC and HPTE on steroid production in human placental JEG-3 cells and on HSD3B1 and CYP19A1 activities. MXC and HPTE inhibited progesterone and estradiol production in JEG-3 cells. MXC and HPTE were potent HSD3B1 inhibitors with the half maximal inhibitory concentration (IC50) values of 2.339 ± 0.096 and 1.918 ± 0.078 μmol/l, respectively. MXC had no inhibition on CYP19A1 at 100 μmol/l, while HPTE was a weak inhibitor with IC50 of 97.16 ± 0.10 μmol/l. When pregnenolone was used to determine the inhibitory mode, MXC and HPTE were found to be competitive inhibitors of HSD3B1. When cofactor NAD+ was used, MXC and HPTE were the noncompetitive inhibitors of HSD3B1. When testosterone was used, HPTE was a mixed inhibitor of CYP19A1. In conclusion, MXC and HPTE are potent inhibitors of human HSD3B1, and HPTE is a weak CYP19A1 inhibitor.

Effects of in Utero Exposure to Dicyclohexyl Phthalate on Rat Fetal Leydig Cells

Dicyclohexyl phthalate (DCHP) is one of the phthalate plasticizers. The objective of the present study was to investigate the effects of DCHP on fetal Leydig cell distribution and function as well as testis development. Female pregnant Sprague Dawley dams orally received vehicle (corn oil, control) or DCHP (10, 100, and 500 mg/kg/day) from gestational day (GD) 12 to GD 21. At GD 21.5, testicular testosterone production, fetal Leydig cell number and distribution, testicular gene and protein expression levels were examined. DCHP administration produced a dose-dependent increase of the incidence of multinucleated gonocytes at ≥100 mg/kg. DCHP dose-dependently increased abnormal fetal Leydig cell aggregation and decreased fetal Leydig cell size, cytoplasmic size, and nuclear size at ≥10 mg/kg. DCHP reduced the expression levels of steroidogenesis-related genes (including Star, Hsd3b1, and Hsd17b3) and testis-descent related gene Insl3 as well as protein levels of 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and insulin-like 3 (INSL3) at ≥10 mg/kg. DCHP significantly inhibited testicular testosterone levels at ≥100 mg/kg. The results indicate that in utero exposure to DCHP affects the expression levels of fetal Leydig cell steroidogenic genes and results in the occurrence of multinucleated gonocytes and Leydig cell aggregation.

Effects of methoxychlor and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane on human and rat 17α-hydroxylase/17,20-lyase activity.

Exposure to methoxychlor, an agricultural pesticide, has been associated with reduced testicular androgen secretion. However, methoxychlor is converted to 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) in the liver, which then acts as its biologically active metabolite. Both methoxychlor and HPTE have been credited with estrogenic properties and have a weak anti-androgenic activity. However, the exact mechanisms of steroidogenic enzyme inhibition remain to be clarified. In the present study, human and rat testis microsomes were employed to investigate the inhibitory activities of methoxychlor and HPTE on 17α-hydroxylase/17,20-lyase (CYP17A1). The CYP17A1 enzyme is critical for androgen biosynthesis and catalyzes conversion of progesterone into androstenedione. The results demonstrated that HPTE directly inhibited human and rat CYP17A1 activities, while methoxychlor had no effects on this enzyme activity even at a concentration of 100 μM. The IC50 values of HPTE were 1.13±0.10 (human) and 6.87±0.13 μM (rat), respectively. When HPTE was incubated with rat immature Leydig cells, it also inhibited CYP17A1 activity with an IC50 value of 6.29±0.1 μM. Results of enzyme inhibition were supported by the observation that HPTE inhibited luteinizing hormone-stimulated 5α-androstane-3α,17β-diol and testosterone secretion by immature Leydig cells with IC50 values of 6.61±0.03 and 3.78±0.003 μM, respectively. The mode of action of HPTE on CYP17A1 activity was determined to be uncompetitive with the substrate progesterone. In conclusion, HPTE, the metabolite of MXC, directly inhibited human and rat testis CYP17A1 activities.

Effects of Ziram on Rat and Human 11β-Hydroxysteroid Dehydrogenase Isoforms.

Ziram is a widely used fungicide for crops. Its endocrine disrupting action is largely unknown. 11β-Hydroxysteroid dehydrogenases, isoforms 1 (HSD11B1) and 2 (HSD11B2), have been demonstrated to be the regulators of the local levels of active glucocorticoids, which have broad physiological actions. In the present study, the potency of ziram was tested for its inhibition of rat and human HSD11B1 and HSD11B2. Ziram showed the inhibition of rat HSD11B1 reductase with IC50 of 87.07 μM but no inhibition of human enzyme at 100 μM. Ziram showed the inhibition of both rat and human HSD11B2 with IC50 of 90.26 and 34.93 μM, respectively. Ziram exerted competitive inhibition of rat HSD11B1 when 11-dehydrocorticosterone was used and mixed inhibition when NADPH was supplied. Ziram exerted a noncompetitive inhibition of both rat and human HSD11B2 when steroid substrates were used and an uncompetitive inhibition when NAD(+) was supplied. Increased DTT concentrations antagonized rat and human HSD11B2 activities, suggesting that the cysteine residues are associated with the inhibition of ziram. In conclusion, for humans, ziram is a selective inhibitor of HSD11B2, implying that this agent may cause excessive glucocorticoid action in local tissues such as the kidney, brain, and placenta.

Comparison of the Effects of Dibutyl and Monobutyl Phthalates on the Steroidogenesis of Rat Immature Leydig Cells

Dibutyl phthalate (DBP) is a widely used synthetic phthalic diester and monobutyl phthalate (MBP) is its main metabolite. DBP can be released into the environment and potentially disrupting mammalian male reproductive endocrine system. However, the potencies of DBP and MBP to inhibit Leydig cell steroidogenesis and their possible mechanisms are not clear. Immature Leydig cells isolated from rats were cultured with 0.05–50 μM DBP or MBP for 3 h in combination with testosterone synthesis regulator or intermediate. The concentrations of 5α-androstanediol and testosterone in the media were measured, and the mRNA levels of the androgen biosynthetic genes were detected by qPCR. The direct actions of DBP or MBP on CYP11A1, CYP17A1, SRD5A1, and AKR1C14 activities were measured. MBP inhibited androgen production by the immature Leydig cell at as low as 50 nM, while 50 μM was required for DBP to suppress its androgen production. MBP mainly downregulated Cyp11a1 and Hsd3b1 expression levels at 50 nM. However, 50 μM DBP downregulated Star, Hsd3b1, and Hsd17b3 expression levels and directly inhibited CYP11A1 and CYP17A1 activities. In conclusion, DBP is metabolized to more potent inhibitor MBP that downregulated the expression levels of some androgen biosynthetic enzymes.

Effects of butylated hydroxyanisole on the steroidogenesis of rat immature Leydig cells.

Abstract Butylated hydroxyanisole (BHA) is a synthetic antioxidant used for food preservation. Whether BHA affects testosterone biosynthesis is still unclear. The effects of BHA on the steroidogenesis in rat immature Leydig cells were investigated. Rat immature Leydig cells were isolated from 35-old-day rats and cultured with BHA (50 μM) for 3 h in combination with 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone or dihydrotestosterone, and the concentrations of 5α-androstanediol and testosterone in the media were measured. Leydig cells were cultured with BHA (0.05–50 μM) for 3 h. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1 and Akr1c14. The testis microsomes were prepared to detect the direct action of BHA on 3β-hydroxysteroid dehydrogenase 1 (HSD3B1), 17α-hydroxylase (CYP17A1) and 17β-hydroxysteroid dehydrogenase 3 activities. In Leydig cells, BHA (50 μM) significantly inhibited LH- and 8Br-cAMP-mediated androgen production. BHA directly inhibited rat testis CYP17A1 and HSD3B1 activities. At 50 μM, it also reduced the expression levels of Hsd17b3 and Srd5a1 and their protein levels. In conclusion, BHA directly inhibits the activities of CYP17A1 and HSD3B1, and the expression levels of Hsd17b3 and Srd5a1, leading to the lower production of androgen in Leydig cells.

Determination of acacetin in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study.

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of acacetin in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 2.0 min and the elution of acacetin was at 0.83 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization (ESI) by multiple reaction monitoring (MRM) of the transitions at m/z 285.3→242.2 for acacetin and m/z 237.2→194.3 for carbamazepine (internal standard). The calibration curve was linear over the range of 1-1600 ng/mL with a lower limit of quantitation (LLOQ) of 1.0 ng/mL. Mean recovery of acacetin in plasma was in the range of 78.4-85.2%. Intra-day and inter-day precision were both <10.5%. This method was successfully applied in pharmacokinetic study after intravenous administration of 5.0mg/kg acacetin in rats.

A Short-Term Exposure to Tributyltin Blocks Leydig Cell Regeneration in the Adult Rat Testis

Background: Tributyltin (TBT) is widely used as an antifouling agent that may cause reproductive toxicity. The mechanism of TBT on Leydig cell development is still unknown. The objective of the present study was to investigate whether a brief exposure to low doses of TBT permanently affects Leydig cell development and to clarify the underlying mechanism. Methods: Adult male Sprague Dawley rats were randomly assigned into four groups and gavaged normal saline (control), 0.1, 1.0, or 10.0 mg/kg/day TBT for a consecutive 10 days, respectively. At the end of TBT treatment, all rats received a single intraperitoneal injection of 75 mg/kg ethane dimethane sulfonate (EDS) to eliminate all of adult Leydig cells. Leydig cells began a developmental regeneration process on post-EDS day 35. The Leydig cell regeneration was evaluated by measuring serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels on post-EDS day 7, 35, and 56, the expression levels of Leydig cell genes, Leydig cell morphology and number and proliferation on post-EDS day 56. Results: TBT significantly reduced serum testosterone levels on post-EDS day 35 and 56 and increased serum luteinizing hormone and follicle-stimulating hormone levels on post-EDS day 56 at ≥1 mg/kg/day. Immunohistochemical staining showed that there were fewer regenerated Leydig cells in the TBT-treated testis on post-EDS day 56. Further study demonstrated that the mRNA or protein levels of Leydig (Lhcgr, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and Sertoli cells (Fshr, Dhh, and Sox9) were significantly down-regulated in the TBT-treated testes when compared to the control. Immunofluorescent staining showed that TBT inhibited Leydig cell proliferation as judged by the reduced number of proliferating cyclin nuclear antigen-positive Leydig cells on post-EDS day 35. Conclusion: The present study demonstrated that a short-term TBT exposure blocked Leydig cell developmental regeneration process via down-regulating steroidogenesis-related proteins and inhibiting the proliferation of Leydig cells.