Exposure to 4-bromodiphenyl ether during pregnancy blocks testis development in male rat fetuses.

4-Bromodiphenyl ether (BDE3) is a photodegradation product of higher polybrominated diphenyl ether flame retardants and is known as an endocrine disruptor. However, it is unclear whether and how BDE3 affects the development of fetal testes. This study aimed to investigate the effect of in utero exposure to BDE3 on fetal testicular development in rats. From gestational day (GD) 12-21, BDE3 (0, 50, 100, and 200 mg/kg) was daily gavaged to female pregnant Sprague Dawley rats. BDE3 significantly reduced serum testosterone levels of male pups starting at 50 mg/kg. BDE3 reduced fetal Leydig cell number at a dose of 200 mg/kg without affecting fetal Leydig cell cluster frequency and Sertoli cell number. In addition, BDE3 down-regulated the expression of fetal Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and their proteins at 100 and/or 200 mg/kg. RNA-seq analysis showed that genes responsive to cAMP (Ass1, Gpd1, Rpl13a) were down-regulated and hypoxia-related genes (Egln3 and P4ha1) were up-regulated at 200 mg/kg. In utero exposure to BDE3 can promote autophagy and apoptosis of fetal Leydig cells via increasing the levels of Beclin1, LC3-II, BAX, and by decreasing the levels of p62 and BCL2. In conclusion, in utero exposure to BDE3 blocks the development of fetal rat testes.

Exposure to di-n-octyl phthalate during puberty induces hypergonadotropic hypogonadism caused by Leydig cell hyperplasia but reduced steroidogenic function in male rats.

Humans are exposed to phthalates ubiquitously, which may threaten health. However, whether di-n-octyl phthalate can prevent pubertal sexual maturity is still elusive. In this study, male Sprague Dawley rats (age 35 days) were treated daily by gavage with 0, 10, 100, and 1000 mg/kg body weight of di-n-octyl phthalate from day 35 to day 49 after birth. Di-n-octyl phthalate significantly reduced serum testosterone levels at doses of 100 and 1000 mg/kg, but increased serum luteinizing hormone levels of 1000 mg/kg and decreased testosterone/luteinizing hormone ratio at ≥10 mg/kg, without affecting serum follicle-stimulating hormone levels. Di-n-octyl phthalate significantly induced Leydig cell hyperplasia (increased number of CYP11A1-positive Leydig cells) at 100 and 1000 mg/kg. Di-n-octyl phthalate down-regulates the gene expression of Cyp11a1, Hsd3b1 and Insl3 in individual Leydig cells. Di-n-octyl phthalate can also reduce the number of sperm in the epididymis. Di-n-octyl phthalate increased phosphorylated AKT1/AKT2 without affecting their total proteins, but increased the total protein and phosphorylated protein of ERK1/2 and GSK-3ß. Primary immature Leydig cells isolated from 35-day-old rats were treated with 0-50 µM di-n-octyl phthalate for 3 h. This phthalate inhibited androgen production under basal, LH-stimulated, and cAMP-stimulated conditions by 5 and 50 µM in vitro via down-regulating Cyp11a1 expression but up-regulating Srd5a1 expression in vitro. In conclusion, di-n-octyl phthalate induces hypergonadotropic hypogonadism caused by Leydig cell hyperplasia but reduced steroidogenic function and prevents sperm production.

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

Neurotrophin-3 (NT-3) acts as an important growth factor to stimulate and control tissue development. The NT-3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT-3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT-3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT-3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle-stimulating hormone levels. NT-3 increased CYP11A1-positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT-3 significantly down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT-3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT-3 dose-dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT-3 antagonist Celitinib can antagonize NT-3 action in vitro. In conclusion, the present study demonstrates that NT-3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.

Zearalenone disrupts the placental function of rats: A possible mechanism causing intrauterine growth restriction.

Zearalenone is an estrogenic mycotoxin produced by a variety of Fusarium fungi. There is evidence that exposure to zearalenone can cause intrauterine growth restriction, but little is known about the mechanism in the rat placenta caused by zearalenone. From gestational day 14-21, female Sprague Dawley rats (60 days old) were gavaged with zearalenone (0, 2.5, 5, 10, and 20 mg/kg/day body weight). Zearalenone dose-dependently reduced serum LH and FSH levels of dams at ≥ 5 mg/kg. RNA-seq and qPCR showed that zearalenone significantly down-regulated Slc38a1 expression at 2.5 mg/kg, Echs1 and Pc at 10 mg/kg, as well as Slc1a5, Cd36, Ldlr, Hadhb, and Cyp17a1 expression at a dose of 20 mg/kg, while it up-regulated the expression of Notch signal (Dvl1 and Jag 1). After zearalenone treatment, their proteins showed a similar trend. Zearalenone reduced the phosphorylation of AKT1, ERK1/2, and mTOR at 5 mg/kg or higher and 4EBP1 at 5 mg/kg. Zearalenone also increased BECLIN1, LC3B, and p62 levels and elevated BAX/BCL2 and CASP3/PROCASP3 ratios. In conclusion, zearalenone disrupts placental function such as reduction of nutrient transport and lipid metabolism possibly via AKT1/ERK1/2/mTOR-mediated autophagy and apoptosis.

Di-n-hexyl phthalate causes Leydig cell hyperplasia in rats during puberty.

Di-n-hexyl phthalate (DNHP) is commonly used as a plasticizer. However, whether DNHP influences Leydig cell development during puberty remains unexplored. In this study, DNHP (0, 10, 100, and 1000 mg/kg) was administered via gavage to 35-day-old male Sprague-Dawley rats for 21 days. Serum levels of testosterone, luteinizing hormone, follicle-stimulating hormone, Leydig cell number, the expression of Leydig and Sertoli cell genes and proteins were investigated. DNHP significantly increased serum testosterone levels at 10 mg/kg but lowered its level at 1000 mg/kg. DNHP significantly increased luteinizing hormone levels at 1000 mg/kg without affecting follicle-stimulating hormone levels. DNHP increased Leydig cell number at all doses but down-regulated the expression of Lhcgr, Hsd3b1, Hsd17b3, and Hsd11b1 in Leydig cell per se at 1000 mg/kg. DNHP elevated phosphorylation of ERK1/2 and GSK-3ß at 10 mg/kg but decreased SIRT1 and PGC-1α levels at 1000 mg/kg. In conclusion, DNHP exposure causes Leydig cell hyperplasia possibly via stimulating phosphorylation of ERK1/2 and GSK-3ß signaling pathways.

Perfluoroalkyl substances cause Leydig cell dysfunction as endocrine disruptors.

Perfluoroalkyl substances (PFASs) are a group of man-made organic substances. Some of PFASs have been classified as persistent organic pollutants and endocrine disruptors. They might interfere with the male sex endocrine system, causing the abnormal development of the male reproductive tract and failure of pubertal onset and infertility. The present review discusses the development and function of two generations of Leydig cells in rodents and the effects of PFASs on Leydig cell development after their exposure in gestational and postnatal periods. We also discuss human epidemiological data for the effects of PFASs on male sex hormone levels. The structure-activity relationship of PFASs on Leydig cell steroidogenesis and enzyme activities are also discussed.

Effects of in utero exposure to diisodecyl phthalate on fetal testicular cells in rats.

Diisodecyl phthalate (DIDP) is one of synthetic phthalate plasticizers. It is widely used in plastic products and is a potential endocrine disruptor. However, the effects of DIDP on fetal testicular cell development remain unclear. The objective of the present study was to determine the effects of DIDP on fetal testis development in rats after in utero exposure. Sprague Dawley dams were randomly divided into 5 groups and were daily gavaged with DIDP (0, 10, 100, 500, and 1000 mg/kg body weight) from gestational day 14-21. Serum testosterone levels, fetal Leydig cell number and distribution, testicular gene and protein expression in male pups were examined. DIDP decreased serum testosterone levels at 1000 mg/kg (1.37 ± 0.40 ng/mL, mean ± SE) when compared to the control level (3.14 ± 0.60 ng/mL). DIDP did not affect numbers of Leydig and Sertoli cells. DIDP significantly induced abnormal aggregation of fetal Leydig cells and increased the incidence of multinucleated gonocytes at 1000 mg/kg. Furthermore, DIDP down-regulated expression of Star, Cyp11a1, Hsd17b3, and Insl3 in fetal Leydig cells at 1000 mg/kg and Sox9 in Sertoli cells at 1000 mg/kg. In conclusion, the current study indicates that in utero exposure to high-dose DIDP disrupts the development of fetal testicular cells, thus affecting the male reproductive system.

Maternal exposure to zearalenone in masculinization window affects the fetal Leydig cell development in rat male fetus.

Zearalenone is a phenolic Fusarium mycotoxin, which is ubiquitous in human and animal feedstuff and often co-occurs with other mycotoxins. ZEA has been reported to disturb Leydig cell function and even cause the apoptosis to the Leydig cells. However, the effects of gestational exposure to zearalenone on fetal Leydig cells and the underlying mechanism remain unknown. Sprague Dawley dams were daily gavaged with 0, 2.5, 5, 10, and 20 mg/kg body weight ZEA from gestational day 14-21. On gestational day 21, rats were euthanized and serum testosterone levels were measured, and testes were collected for further evaluation of Leydig cell number, cell size, gene, and protein expression. Zearalenone significantly decreased anogenital distance and its index of male fetus, serum testosterone levels, Leydig cell proteins (SCARB1, STAR, CYP11A1, CYP17A1, and INSL3), and fetal Leydig cell number at 10 and/or 20 mg/kg by delaying the commitment of stem Leydig cells into the Leydig cell lineage and proliferation. Further study found that Notch signaling (RFNG, PSEN1, NOTCH1, and NOTCH3) was up-regulated by zearalenone. In conclusion, gestational exposure to high doses of zearalenone (10 and 20 mg/kg) blocks fetal Leydig cell development, thus possibly causing the anomalies of the male reproductive tract.

Acephate interferes with androgen synthesis in rat immature Leydig cells.

Acephate is an organophosphate pesticide. It is widely used. However, whether it inhibits androgen synthesis and metabolism remains unclear. In the current study, we investigated the effect of acephate on the inhibition of androgen synthetic and metabolic pathways in rat immature Leydig cells after 3-h culture. Acephate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.5 µM. It significantly inhibited luteinizing hormone and 8-Br-cAMP stimulated androgen output at 50 µM. It significantly inhibited progesterone-mediated androgen output at 50 µM. Further study demonstrated that acephate down-regulated the expression of Hsd3b1 and its protein at ≥ 0.5 µM, Lhcgr at 5 µM and Star at 50 µM. Acephate directly blocked rat testicular HSD3B1 activity at 50 µM. Acephate did not affect other androgen synthetic and metabolic enzyme activities as well as ROS production, proliferation, and apoptosis of immature Leydig cells. In conclusion, acephate targets LHCGR, STAR, and HSD3B1, thus blocking androgen synthesis in rat immature Leydig cells and HSD3B1 is being the most sensitive target of acephate.

Dimethoate blocks pubertal differentiation of Leydig cells in rats.

Dimethoate is an organophosphate pesticide. It is widely used in agriculture. However, whether it blocks pubertal development of Leydig cells remains unknown. In the current study, we exposed male Sprague Dawley rats with 7.5 and 15 mg kg-1 dimethoate from postnatal day 35-56. We also exposed Leydig cells isolated from 35-day-old rats for 3 h. Dimethoate reduced serum testosterone levels at 7.5 and 15 mg kg-1 but increased serum luteinizing hormone and follicle stimulating hormone levels at 15 mg kg-1. Dimethoate did not influence Leydig cell number but reduced Leydig cell size and down-regulated Star, Cyp11a1, and Hsd3b1 in Leydig cells as well as their protein expression. Dimethoate inhibited basal androgen output in a dose-dependent manner with the inhibition starting at 0.05 µM. It significantly inhibited luteinizing hormone and 8Br-cAMP stimulated androgen outputs at 50 µM. It significantly inhibited 22R-hydroxycholesterol and progesterone-mediated androgen outputs at 50 µM. Further study demonstrated that dimethoate also down-regulated the expression of Star, Cyp11a1, and Hsd3b1 at 5 or 50 µM in vitro. Dimethoate did not directly inhibit rat testicular steroidogenic enzyme activities at 50 µM. In conclusion, dimethoate targets Star, Cyp11a1, and Hsd3b1 transcription, thus blocking Leydig cell differentiation during puberty.

Paraquat exposure delays late-stage Leydig cell differentiation in rats during puberty.

Paraquat is a fast and non-selective herbicide that is widely used in crop cultivation and conservation tillage systems. Animal experiments have shown that paraquat decreases sperm quality and testicular organ coefficient, but its effects on the development of Leydig cells remain unclear. The objective of the current study was to investigate the effects of paraquat exposure on the Leydig cell development in rats during puberty. Twenty-eight male 35-day-old Sprague-Dawley rats were divided into 4 groups: 0, 0.5, 2.0, and 8 mg kg-1 d-1 paraquat. Paraquat was gavaged for 10 d. Adult Leydig cells were isolated and treated with paraquat for 24 h. Paraquat in vivo significantly decreased body and testis weights at 8 mg kg-1 and lowered serum testosterone levels at 2 and 8 mg kg-1 without affecting the levels of serum luteinizing hormone and follicle-stimulating hormone. Paraquat did not alter Leydig cell number and PCNA labeling index. Real-time PCR showed that paraquat down-regulated the expression of Lhcgr, Scarb1, Cyp11a1, Cyp17a1, and Hsd17b3 genes and their proteins at 2 or 8 mg kg-1, while it up-regulated the expression of Srd5a1 at 8 mg kg-1. Paraquat increased ROS and decreased testosterone production by Leydig cells at 1 and 10 µM after in vitro 24-h exposure. Vitamin E (40 µg/ml) reversed paraquat-induced ROS and suppression of testosterone synthesis in vitro. In conclusion, paraquat directly delays Leydig cell differentiation to block testosterone synthesis via down-regulating the expression of critical testosterone synthesis-related genes and up-regulating the expression of testosterone metabolic enzyme (Srd5a1) gene and possibly via increasing ROS production.

Aconitine inhibits androgen synthesis enzymes by rat immature Leydig cells via down-regulating androgen synthetic enzyme expression in vitro.

Aconitine might have reproductive toxicity and the effects of aconitine on androgen synthesis in Leydig cells remain unclear. Here, we explore how aconitine affects androgen synthesis and metabolism in rat immature Leydig cells in vitro. Immature Leydig cells were isolated from 35-day-old male Sprague Dawley rats and cultured with 0-50 µM aconitine for 3 h in combination with LH, 8Br-cAMP, 22R-hydroxycholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively. Medium androgens were measured. The levels of Leydig cell mRNAs, Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14, were measured by qPCR. ROS and apoptosis were determined after 24-h aconitine treatment. Aconitine inhibited basal androgen production in Leydig cells at 0.05 µM and the higher concentrations. Aconitine blocked pregnenolone, progesterone, and androstenedione mediated androgen outputs without affecting 22R-hydroxycholesterol-mediated androgen production at 5 µM. Aconitine also inhibited LH and 8Br-cAMP stimulated androgen outputs at 5 µM. Further investigation showed that aconitine blocked androgen synthesis via down-regulating the expression of Scarb1, Hsd3b1, Cyp17a1, and Hsd17b3. At 50 µM, aconitine also induced ROS generation and increased apoptotic rate of Leydig cells. Aconitine lowered serum testosterone levels at 1.5 mg/kg after 7 days of oral exposure from postnatal day 35. In conclusion, aconitine inhibits androgen synthesis.

4-Bromodiphenyl Ether Causes Adrenal Gland Dysfunction in Rats during Puberty.

Polybrominated diphenyl ethers (PBDEs) are a group of flame retardants with two or more bromines attached. They are endocrine disruptors. PBDEs photodegrade into 4-bromodiphenyl ether (BDE3). Whether BDE3 impairs adrenal cortical cell function during postnatal development still remains unknown. The aim of the current study was to investigate the influence of BDE3 on adrenal cortical cell function. Sprague-Dawley rats (35 days of age, male) were orally administered with BDE3 (0, 50, 100, and 200 mg/kg/day body weight) for 21 days. BDE3 significantly increased serum aldosterone and corticosterone levels at 200 mg/kg without affecting adrenocorticotropic hormone level. Further study showed that BDE3 up-regulated Cyp11b1 at 100 and 200 mg/kg and Scarb1, Star, Cyp11b2, Cyp21, and Nr5a1 mRNA levels in the 200 mg/kg group. BDE3 also decreased the phosphorylation of AMP-activated protein kinase (AMPK) at 200 mg/kg and increased PGC-1α and phosphorylated cyclic AMP-responsive element-binding protein (CREB)/CREB at 200 mg/kg. Taken together, these findings demonstrate that BDE3 stimulates adrenal cell function likely through decreasing phosphorylation of AMPK and increasing phosphorylation of CREB.

Flurbiprofen Inhibits Androgen Productions in Rat Immature Leydig Cells.

Flurbiprofen is one of the nonsteroidal anti-inflammatory drugs. Whether flurbiprofen affects androgen biosynthesis in Leydig cells is still unknown. Immature Leydig cells (ILCs) isolated from 35-day-old male Sprague-Dawley rats were cultured with 0-100 µM flurbiprofen for 24 h and medium androgen levels and Leydig cell mRNA levels were measured. Immature Leydig cells were also incubated with 100 µM flurbiprofen for 3 h in combination with luteinizing hormone (LH), 8bromo-cAMP, 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively, and medium androgen levels were measured. The ROS generation and apoptosis rate were also investigated. The direct effects of flurbiprofen on androgen biosynthetic and metabolizing enzyme activities were measured. Flurbiprofen significantly inhibited basal, LH, and 8bromo-cAMP stimulated androgen production at 10 and 100 µM. Further study demonstrated that flurbiprofen competitively inhibited rat and human testis 3ß-hydroxysteroid dehydrogenase (HSD3B) activity with the half maximal inhibitory concentration (IC50) values of 0.95 µM for rat enzyme and 6.31 µM for human enzyme. In addition, flurbiprofen down-regulated the expression of Srd5a1 and Akr1c14 at 1, 10, and 100 µM. Flurbiprofen also down-regulated Lhcgr expression at 100 µM. Flurbiprofen at 10 and 100 µM increased ROS production and apoptosis rate of rat Leydig cells. In conclusion, flurbiprofen directly inhibits HSD3B activity and the expression levels of Srd5a1 and Akr1c14 in rat Leydig cells, thus leading to the reduction of androgen secretion.

Pubertal exposure to tebuconazole increases testosterone production via inhibiting testicular aromatase activity in rats.

Tebuconazole is a triazole compound used agriculturally to treat plant pathogenic fungi. However, whether pubertal exposure to tebuconazole affects Leydig cell development remains unknown. Here, we exposed male Sprague-Dawley rats at 35 days of age to 0, 25, 50, or 100 mg kg-1 day-1 tebuconazole for 21 days. Tebuconazole exposure increased serum testosterone level but lowered estradiol level at a dose of 100 mg kg-1, without affecting serum luteinizing hormone and follicle-stimulating hormone concentrations. Tebuconazole up-regulated the expression of testicular Cyp11a1, Hsd11b1, and Fshr genes as well as their proteins at a dose of 100 mg kg-1. However, tebuconazole did not stimulate the proliferation of Leydig cells. Tebuconazole in vitro inhibits aromatase activity in primary rat Leydig cells with IC50 value of 40 µmol/L. In conclusion, tebuconazole exposure stimulates pubertal Leydig cell differentiation via inhibiting aromatase activity.

Paraquat exposure delays stem/progenitor Leydig cell regeneration in the adult rat testis.

Paraquat, a widely used nonselective herbicide, is a serious hazard to human health. However, the effects of paraquat on the male reproductive system remain unclear. In this study, adult male Sprague Dawley rats were intraperitoneally injected ethane dimethane sulfonate (EDS, 75 mg/kg) to initiate a regeneration of Leydig cells. EDS-treated rats were orally exposed to paraquat (0.5, 2, 8 mg/kg/day) from post-EDS day 17 to day 28 and effects of paraquat on Leydig and Sertoli cell functions on post-EDS day 35 and day 56 were investigated. Paraquat significantly decreased serum testosterone levels at 2 and 8 mg/kg. Paraquat lowered Leydig cell Hsd17b3, Srd5a1, and Hsd11b1 mRNA levels but increased Hsd3b1 on post-EDS day 35. Paraquat lowered Cyp11a1, Cyp17a1, and Hsd11b1 but increased Srd5a1 on post-EDS day 56. However, paraquat did not alter Leydig cell number and PCNA labeling index. Epididymal staining showed that few sperms were observed in paraquat-treated rats. Primary culture of adult Leydig cells showed that paraquat diminished testosterone output and induced reactive oxygen species generation at 1 and 10 µM and apoptosis rate at 10 µM. In conclusion, a short-term exposure to paraquat delays Leydig cell regeneration from stem/progenitor Leydig cells, causing low production of testosterone and an arrest of spermatogenesis.

A nanocomposite consisting of gold nanobipyramids and multiwalled carbon nanotubes for amperometric nonenzymatic sensing of glucose and hydrogen peroxide.

Gold nanobipyramids were synthesized by a seed-mediated growth method and then supported by multi-walled carbon nanotubes (denoted as AuNBP/MWCNTs). The electrocatalytic activity of the AuNBP/MWCNTs on a glassy carbon electrode (GCE) towards direct glucose oxidation and hydrogen peroxide reduction was superior to that of AuNBPs and MWCNTs. The modified GCE, operated at a typical working voltage of +0.15 V (vs. SCE) and in 0.1 M NaOH solution, exhibits a linear response in the 10 µM to 36.7 mM glucose concentration range with a 3.0 µM detection limit (at S/N = 3) and a sensitivity of 101.2 µA mM-1 cm-2. It also demonstrates good sensitivity towards hydrogen peroxide in at pH 7 solution at a working potential of -0.50 V (vs. SCE), with a linear response range from 5.0 µM to 47.3 mM, a sensitivity of 170.6 µA mM-1 cm-2 and a detection limit of 1.5 µM. Graphical abstract A electrochemical sensing platform based on the use of gold nanobipyramids and multi-walled carbon nanotubes nanocomposites (AuNBP/MWCNTs) is described for the determination of glucose and hydrogen peroxide.

Food components and environmental chemicals of inhibiting human placental aromatase.

Human placental CYP19A1 catalyzes the estrogen synthesis from androgens. The enzyme is encoded by CYP19A1 gene located in chromosome 15q21. This enzyme is a monooxygenase in the smooth endoplasmic reticulum. The various promoters of the CYP19A1 gene determine its expression in different tissues and the distal promoter I.1 controls its expression in the placenta and retinoids can regulate the expression. Many food components and environmental chemicals inhibit CYP19A1 activity via different modes of action. These chemicals include gossypol, flavones, flavanones, chalconoids, resveratrol, and tobacco alkaloids derived from foods as well as phthalates, insecticides, fungicides, and biocides in the contaminated foods. The inhibition of placental CYP19A1 could impair pregnancy.

Dehydroepiandrosterone and Its CYP7B1 Metabolite 7α-Hydroxydehydroepiandrosterone Regulates 11ß-Hydroxysteroid Dehydrogenase 1 Directions in Rat Leydig Cells.

Background: The purpose of this study was to investigate cytochrome P450-7B1 (CYP7B1) in the human and rat testes to regulate 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) activity. We hypothesized that dehydroepiandrosterone (DHEA) and its product 7α-hydroxydehydroepiandrosterone (7αOHD) after catalysis of CYP7B1 played a critical role in driving the direction of 11ß-HSD1, because 7αOHD is an alternative substrate for 11ß-HSD1. Methods: We examined the influence of DHEA and 7αOHD on 11ß-HSD1 activities in both intact Leydig cells and microsomes using radioactive substrates and identified the location of CYP7B1 in Leydig cells using immunohistochemical staining, Western blot, and qPCR. Results: We found that DHEA stimulated 11ß-HSD1 oxidase activity in intact cells (EC50 = 0.97 ± 0.11 µM) and inhibited its reductase activity (IC50 = 1.04 ± 0.06 µM). In microsomes, DHEA was a competitive inhibitor of the reductase activity. The 11ß-HSD1 oxidase activity in intact cells was inhibited by 7αOHD (IC50 = 1.18 ± 0.12 µM), and the reductase activity was enhanced (EC50 = 0.7 ± 0.04 µM). 7αOHD was a competitive inhibitor of 11ß-HSD1 oxidase. CYP7B1 was present in rat Leydig cells, as shown by immunohistochemistry, Western blotting, and qPCR analysis. Conclusion: Our results are consistent with a conclusion that DHEA in the circulation driving 11ß-HSD1 toward an oxidase in Leydig cells mainly through inhibiting the reductase of the enzyme, while 7αOHD (CYP7B1 catalytic product of DHEA) drives the enzyme toward the opposite direction.

Dicyclohexyl phthalate blocks Leydig cell regeneration in adult rat testis.

Dicyclohexyl phthalate (DCHP) is a phthalate plasticizer with a ring structure in the alcohol moiety. The objective to the current study was to determine the effects of DCHP on Leydig cell regeneration in the adult rat-testis. Adult male Sprague Dawley rats received intraperitoneally an injection of ethane dimethane sulfone (EDS) to eliminate all Leydig cells in the testis and then were divided into 4 groups of 0 (control), 10, 100, and 1000 mg/kg/day DCHP. Rats were gavaged either vehicle (corn oil, control) or DCHP from post-EDS day 7 to day 21 and 28. On post-EDS day 21 and day 28, rats were euthanized and serum testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH) levels were measured, and Leydig cell number, cell size, gene, and protein expression were evaluated. During the course of exposure, DCHP did not cause the general toxicity to rats. On post-EDS day 21, DCHP significantly increased serum testosterone level at 10 and 100 mg/kg and increased Leydig cell number at 10 mg/kg via stimulating their mitosis. On post-EDS day 28, DCHP lowered serum testosterone levels and Leydig cell number at 1000 mg/kg. DCHP dose-dependently down-regulated the expression of many Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, and Insl3) and their proteins, especially at 1000 mg/kg. DCHP also lowered the pAKT1/AKT1 and pERK1/2/ERK1/2 ratios. In conclusion, DCHP at low doses (10 and 100 mg/kg) increased Leydig cell number during the initial regeneration and inhibited Leydig cell regeneration during the course of its exposure.