The methoxychlor metabolite, HPTE, inhibits rat luteal cell progesterone production.

Unlabelled: The methoxychlor metabolite, HPTE, was shown to inhibit P450-cholesterol side-chain cleavage (P450scc) activity resulting in decreased progesterone production by cultured ovarian follicular cells in previous studies. It is not known whether HPTE has any effect on progesterone formation by the corpus luteum.

Results: Exposure to 100 nM HPTE reduced progesterone production by luteal cells with progressive declines to <22% of control at 500 nM HPTE.

The methoxychlor metabolite, HPTE, directly inhibits the catalytic activity of cholesterol side-chain cleavage (P450scc) in cultured rat ovarian cells.

Exposure to the pesticide methoxychlor in rodents is linked to impaired steroid production, ovarian atrophy and reduced fertility. Following in vivo administration, it is rapidly converted by the liver to 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), the reported active metabolite. Both methoxychlor and HPTE have weak estrogenic and antiandrogenic activities, and these effects are thought to be mediated through the estrogen and androgen receptors, respectively.

Workgroup report: Implementing a national occupational reproductive research agenda--decade one and beyond.

The initial goal of occupational reproductive health research is to effectively study the many toxicants, physical agents, and biomechanical and psychosocial stressors that may constitute reproductive hazards in the workplace. Although the main objective of occupational reproductive researchers and clinicians is to prevent recognized adverse reproductive outcomes, research has expanded to include a broader spectrum of chronic health outcomes potentially affected by reproductive toxicants. To aid in achieving these goals, the National Institute for Occupational Safety and Health, along with its university, federal, industry, and labor colleagues, formed the National Occupational Research Agenda (NORA) in 1996.

In vivo exposure of young adult male rats to methoxychlor reduces serum testosterone levels and ex vivo Leydig cell testosterone formation and cholesterol side-chain cleavage activity.

Methoxychlor (MC) was developed as a replacement for the banned pesticide DDT. After in vivo administration, it is metabolized in the liver to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is proposed to be the active agent. Both MC and HPTE have been shown to exhibit weak estrogenic and antiandrogenic activities, and they are thought to exert their effects through estrogen and androgen receptors, respectively.

The reported active metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, inhibits testosterone formation by cultured Leydig cells from neonatal rats.

Methoxychlor (MC) is an insecticide that is presently used on agricultural crops, especially after the ban on the use of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) in the United States. Following administration in vivo, MC is converted to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is thought to be the active agent. However, both MC and HPTE have been reported to have weak estrogenic and antiandrogenic activities, and they are thought to exert their potential adverse (endocrine disruptive) effects through the estrogen and androgen receptors, respectively.

The effects of the reported active metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane, on testosterone formation by cultured Leydig cells from young adult rats.

Methoxychlor (MC) is an insecticide that is currently used on a variety of agricultural crops, especially following the ban of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) use in the United States. Following in vivo administration, MC is converted to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is proposed to be the active agent. Both MC and HPTE have been demonstrated to exhibit weak estrogenic and antiandrogenic activities, and they are thought to exert their effects through estrogen or androgen receptors, respectively.

An occupational reproductive research agenda for the third millennium.

There is a significant public health concern about the potential effects of occupational exposure to toxic substances on reproductive outcomes. Several toxicants with reported reproductive and developmental effects are still in regular commercial or therapeutic use and thus present potential exposure to workers. Examples of these include heavy metals, organic solvents, pesticides and herbicides, and sterilants, anesthetic gases, and anticancer drugs used in health care.

Exposure to octylphenol increases basal testosterone formation by cultured adult rat Leydig cells.

4-Tert-octylphenol (OP) is a breakdown product of 4-tert-octylphenol ethoxylate, which is a surfactant additive widely used in the manufacture of a variety of detergents and plastic products. OP has been reported to exhibit weak estrogenic activity in many assay systems. The studies described herein examined an unusual effect of OP in increasing constitutive testosterone levels of cultured Leydig cells from young adult rats.

Differential effects of octylphenol, 17beta-estradiol, endosulfan, or bisphenol A on the steroidogenic competence of cultured adult rat Leydig cells.

In the current studies, we evaluated the effects of 4-tert-octylphenol (OP), endosulfan, bisphenol A (BPA), and 17beta-estradiol on basal or hCG-stimulated testosterone formation by cultured Leydig cells from young adult male rats. Exposure of Leydig cells to increasing concentrations of OP (1 to 2000 nM), 17beta-estradiol (1 to 1000 nM), endosulfan (1 to 1000 nM) or BPA (1 to 1000 nM), alone or with 10 mIU/mL hCG for 4 or 24 h, did not lower ambient testosterone levels, although cells exposed to higher OP concentrations + hCG for 24 h often had modest declines in testosterone (10 to 20%). Of interest, exposure to the highest concentration OP (2000 nM) alone for 4 or 24 h increased testosterone levels (approximately 2-fold in 4-h exposed cells).

Octylphenol inhibits testosterone biosynthesis by cultured precursor and immature Leydig cells from rat testes.

4-tert-octyphenol (OP) is a surfactant additive widely used in the manufacture of a variety of detergents and plastic products. OP has been reported to mimic the actions of estrogen in many cellular systems. The present studies evaluated the direct effects of OP on human chorionic gonadotropin (hCG)-stimulated testosterone biosynthesis by cultured precursor and immature Leydig cells from 23-day old (prepubertal) rats.

Biphasic effects of octylphenol on testosterone biosynthesis by cultured Leydig cells from neonatal rats.

The present studies evaluated the suitability of using cultured dispersed testicular cells from neonatal rats as a source for fetal Leydig cells and the use of these cells to examine direct toxic effects of environmental/occupational chemicals on androgen biosynthesis. For the current studies, the direct actions of octylphenol (OP), a surfactant additive widely used in the manufacture of various detergents, on testosterone biosynthesis by cultured rat neonatal Leydig cells were examined. Octylphenol is considered a xenoestrogen and has been reported to mimic the actions of estrogen in many cellular systems.

Temperature and germ cell regulation of Leydig cell proliferation stimulated by Sertoli cell-secreted mitogenic factor: a possible role in cryptorchidism.

Local control of Leydig cell morphology and function by seminiferous tubules was suggested in previous in vivo studies, especially those that used experimental cryptorchid rat testis as a model. These studies reported changes in morphology, increases in cell number and mitotic index and decreases in testosterone formation and luteinizing hormone/human chorionic gonadotropin receptor levels of Leydig cells. However, little is known about how these changes are mediated.

Evidence that α5β1 integrins mediate Leydig cell binding to fibronectin and enhance Leydig cell proliferation stimulated by a Sertoli cell-secreted mitogenic factor in vitro.

We reported previously that coculture of immature rat Sertoli cells with Leydig cells or the addition of a concentrate from Sertoli cell-conditioned medium (SCCM) stimulated Leydig cell [(3)H]-thymidine incorporation, increased cell number, and altered Leydig cell morphology (Wu and Murono, 1994). In the present studies, the effect of various extracellular matrix proteins on immature Leydig cell binding, proliferation and response to SCCM concentrate was investigated. Pretreatment of culture wells with 50 μg/mL collagen I or 10 μg/mL laminin inhibited Leydig cell binding to culture wells about 95 and 89%, respectively; however, 5 μg/mL fibronectin did not change the level of attachment.

A Sertoli cell-secreted paracrine factor(s) stimulates proliferation and inhibits steroidogenesis of rat Leydig cells.

Previous studies have shown that disruption or damage to the seminiferous tubules by radiation, antiandrogen, vitamin A deficiency or experimental cryptorchidism causes Leydig cell hypertrophy and hyperplasia, suggesting that Sertoli cells secrete a mitogenic factor(s) that stimulates Leydig cell proliferation. To study the possible paracrine regulation of Leydig cell proliferation by Sertoli cells, highly purified Leydig cells and Sertoli cells were co-cultured in a two-chambered co-culture system. Our results revealed that co-culture of immature rat Sertoli cells with Leydig cells stimulated Leydig cell DNA synthesis by 19-fold, increased cell number by about 3.

Enhanced stimulation of 5 alpha-reductase activity in cultured Leydig cell precursors by human chorionic gonadotropin.

Previous studies have demonstrated that the increase in number of Leydig cells during prepubertal maturation results, in part, from the differentiation of mesenchymal precursors between the second and fourth week of postnatal life. After conversion to immature Leydig cells, they actively synthesize testosterone, but this androgen does not accumulate because high 5 alpha-reductase activity rapidly converts testosterone to 5 alpha-reduced metabolites. The present studies examined whether the conversion of precursor cells to immature Leydig cells in vitro by human chorionic gonadotropin (hCG), as characterized by progressive increases in testosterone formation and 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) activity, is associated similarly with an enhanced stimulation of 5 alpha-reductase activity.

Evidence that both receptor- and heparan sulfate proteoglycan-bound basic fibroblast growth factor are internalized by cultured immature Leydig cells.

The present studies examined how 125I-labeled basic fibroblast growth factor (bFGF) bound to high affinity receptors and with lower affinity to heparan sulfate proteoglycans (HSPG) of cultured immature rat Leydig cells was processed. Following incubation for 2 h at 4 degrees C with 125I-bFGF, cells were washed to remove unbound radioactivity. Fresh medium was added, and cells were incubated at 4 degrees and/or 37 degrees C.

Evidence that biphasic effects of basic fibroblast growth factor on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity in cultured immature Leydig cells are mediated by binding to heparan sulfate proteoglycans.

The present studies examined the effects of heparin, heparinase, insulin or insulin-like growth factor-I on basic fibroblast growth factor actions on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity of cultured immature rat Leydig cells. Treatment with basic fibroblast growth factor alone (0.025-20 ng/ml) for 2 days had a biphasic effect on enzyme activity, with lower concentrations (0.

Basic fibroblast growth factor-induced increase in 125I-human chorionic gonadotropin binding to luteinizing hormone receptors in cultured immature Leydig cells is mediated by binding to heparan sulfate proteoglycans.

Previous studies have shown that basic fibroblast growth (bFGF) has a biphasic effect on 125I-hCG binding to LH receptors in cultured Leydig cells from immature rats. Low concentrations of bFGF (0.1-1.

Effects of acidic fibroblast growth factor on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase and 5 alpha-reductase activities and [125I]human chorionic gonadotrophin binding in cultured immature Leydig cells.

The present studies examined the effects of acidic fibroblast growth factor (aFGF) on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) and 5 alpha-reductase activities and [125I]human chorionic gonadotrophin ([125I]hCG) binding in cultured immature rat Leydig cells. Increasing concentrations of aFGF (0.1-20 ng/ml) progressively decreased basal 3 beta-HSD activity from 0.

Biphasic effect of basic fibroblast growth factor on 125I-human chorionic gonadotropin binding to cultured immature Leydig cells.

The present studies examined the effects of basic fibroblast growth factor (bFGF or FGF-2) on 125I-human chorionic gonadotropin (hCG) binding to cultured immature rat Leydig cells. We found that low concentrations of bFGF (0.1-1.

Evidence for basic fibroblast growth factor receptors in cultured immature Leydig cells.

Previous studies have shown that basic fibroblast growth factor (bFGF) can modulate basal and luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated Leydig cell functions. It has not been ascertained whether these actions are due to direct or indirect effects on Leydig cells. To resolve this question, a multi-step procedure was used to isolate highly-purified Leydig cells from immature rats.

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in two distinct density Leydig cells from immature rats. Differences in responsiveness to human chorionic gonadotropin or 8-bromoadenosine 3',5'-monophosphate.

The present studies examined the responsiveness to human chorionic gonadotropin (hCG) or 8-bromoadenosine 3',5'-monophosphate (8-Br-cAMP) of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity of cultured immature Band 2 (low density) or Band 3 (high density) Leydig cells isolated on Percoll gradients. Enzyme activity increased in relation to the dose of hCG or 8-Br-cAMP in both bands; however, activity in Band 2 cells increased about 200% above control, while activity in Band 3 cells increased only about 30-60% above control following 6 days of treatment. Maximal responses were observed 4-6 days following exposure to hCG or 8-Br-cAMP in both bands.

Evidence that Leydig precursors localize in immature band two cells isolated on Percoll gradients.

The present studies examined responses to hCG and/or insulin of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase activity (3 beta-HSD) in cultured Band 2 and Band 3 cells from 25- to 40-day-old rats isolated on Percoll gradients. In Band 2 cells, from 25-day-old rats enzyme activity increased about 3- and 2.5-fold, after 6 days of exposure to hCG or insulin, respectively.

Platelet derived growth factor inhibits 5 alpha-reductase and delta 5-3 beta-hydroxysteroid dehydrogenase activities in cultured immature Leydig cells.

Platelet derived growth factor inhibited both hCG- and 8-Br-cAMP-stimulated 5 alpha-reductase activity in cultured immature Leydig cells in a dose-dependent manner, while not significantly inhibiting basal enzyme activity. Platelet derived growth factor also inhibited basal delta 5-3 beta-hydroxysteroid dehydrogenase activity and hCG-stimulated testosterone formation. Maximal inhibitions were achieved with 10 ng/ml of platelet derived growth factor.

Regulation of 5 alpha-reductase activity in cultured immature leydig cells by human chorionic gonadotropin.

The present studies examined the hormonal regulation of 5 alpha-reductase activity in cultured immature rat Leydig cells. Within the testis 5 alpha-reductase was concentrated in the interstitial cell compartment, and among interstitial cells, the enzyme was localized primarily in Band 3 of Percoll density gradients, which contains the majority of Leydig cells. Among various factors reported previously to stimulate testicular 5 alpha-reductase activity when administered in vivo to immature rats (LH/hCG, FSH, luteinizing hormone releasing hormone or prolactin), only LH/hCG directly stimulated 5 alpha-reductase activity of cultured immature Band 3 cells.