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Maternal Exposure to Octylphenol Suppresses Ovine Fetal Follicle-Stimulating Hormone Secretion, Testis Size, and Sertoli Cell Number¹

Department of Animal Husbandry and Production, Faculty of Veterinary Medicine and Conway Institute, University College Dublin (T.S., J.F.R.), Ballsbridge, Dublin 4, Ireland; and Medical Research Council, Reproductive Biology Unit (L.N.), Edinburgh, United Kingdom EH3 9EW

Address all correspondence and requests for reprints to: Dr. T. Sweeney, Department of Animal Husbandry and Production, Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland. E-mail: tsweeney{at}vetmed.ucd.ie

	Abstract

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We have tested the hypothesis that maternal exposure to octylphenol, a putative endocrine disrupting chemical, will suppress gonadotropin secretion with a concomitant decrease in testis size and Sertoli cell number during fetal life in the lamb. In Exp 1, pregnant ewes received a continuous iv infusion of diethylstilbestrol (DES; 50 µg/kg·day), octylphenol (1000 µg/kg·day), or vehicle (1:4, alcohol-saline) from days 110–115 of gestation. The fetuses were chronically catheterized in utero, and blood samples were collected every 8 h to monitor gonadotropin secretion. In Exp 2, pregnant ewes received twice weekly sc injections of DES (0.5 µg/kg·day), octylphenol (1000 µg/kg·day), or corn oil from day 70 of gestation to birth. The pituitary gland and testes were collected from the lambs at the end of the treatment period. In Exp 1, maternal exposure to octylphenol suppressed (P < 0.05) FSH concentrations without any effect (P > 0.05) on LH concentrations compared with those in control fetuses. In Exp 2, long-term maternal exposure to octylphenol or a 1000-fold lower dose of DES suppressed (P < 0.05) FSHß messenger RNA levels and the number of FSHß-immunopositive cells in the pituitary gland and reduced testis weight and the number of Sertoli cells in the testis compared with those in control lambs. We conclude that maternal exposure to octylphenol inhibits the secretion of FSH in the fetus with a concomitant decrease in testis size and Sertoli cell number at birth.

	Introduction

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CONCERNS HAVE BEEN raised about the potential adverse effects on reproductive health in humans (1, 2, 3, 4, 5) and wildlife species (6, 7) of a range of environmental chemicals that mimic the actions of estrogen. It is hypothesized that these xenoestrogens play a role in the reported decline in sperm production and the increase in testicular abnormalities that have occurred in men over the last few decades (8).

Estrogens play an important role as part of the negative feedback complex that suppresses gonadotropins in animals and man. It has been shown that a number of environmental chemicals bind to the estrogen receptor and mimic some of the endocrine effects of estrogens. The alkylphenol polyethoxylates are one group of compounds that have raised concern due to their prevalence in the environment (9, 10, 11, 12, 13, 14). They are nonionic surfactants used in the manufacture of detergents, paints, and herbicides. During sewage treatment, these compounds are broken down to short chain alkylphenol polyethoxylates, alkylphenol carboxylic acids, and alkylphenols, which bioaccumulate in the lipids of living organisms (13, 14). The estrogenic nature of octylphenol has been clearly demonstrated in cell culture (15), in a recombinant yeast screen with human estrogen receptor (16), and in animal studies (17). It competes with estradiol with equal binding affinity to and ß estrogen receptors, and it trans-activates both receptors in transient transfection assays (16). Exposure of female rats to a concentration of octylphenol similar to that used in this experiment stimulates uterine growth (17), a classical bioassay for estrogenicity.

It is proposed that environmental estrogens influence sperm production in the adult by disrupting the differentiation/multiplication of Sertoli cells of the fetal testis during development (8). The number of Sertoli cells determines the overall capacity to produce sperm in adult life and is fixed during the course of fetal and early neonatal development (18). Thus, disruption of Sertoli cell development could have long-term consequences for adult reproductive potential (8). Fetal endocrine status is characterized by a midgestational rise in gonadotropin secretion, which plays a crucial role in the maturation of the gonads (19). Pharmacological inhibition of the midgestational rise in gonadotropin secretion results in a 40% reduction in testis size in sheep at birth due to a reduction in the number of Sertoli cells (20). We hypothesize that environmental estrogens reduce Sertoli cell multiplication by inhibiting the synthesis and secretion of fetal pituitary gonadotropins. Two experiments were designed to test the hypothesis that maternal exposure to octylphenol would suppress fetal FSH secretion with a concomitant reduction in Sertoli cell number and testicular size.

	Materials and Methods

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Animals
Exp 1: can maternal exposure to octylphenol suppress fetal gonadotropin secretion? Ewes with a single known insemination date were used in this study (gestation is 147 days). Under general anesthesia induced with alphaxolone (0.9%) plus alphadolone (0.3%; 0.5 ml/kg, iv; Saffan, Glaxovet, Uxbridge, UK) and maintained with 2.3% halothane (May and Baker, Dagenham, UK) in oxygen, polyvinyl catheters were placed in one fetal jugular vein and carotid artery on day 106 of gestation as previously described by Brooks and White (21). After surgery, the ewe was placed in a metabolic cage and fed grass nuts, with ad libitum access to hay and water for the duration of the study. Ewes were allowed a 3-day period to recover from surgery, during which time antibiotics were administered to the fetus (10⁶ U Crystapen; Glaxovet) via the fetal jugular vein and into the amniotic fluid via an indwelling catheter and to the mother (5 ml, im; Streptopen, Glaxovet). Only plasma samples obtained from fetuses with normal arterial pH (>7.3) and paO₂ (>18 torr), as measured in a blood gas analyzer (I.L. 1306, Instrumentation Laboratories, Warrington, UK), were used in the study. Infusions of xenoestrogens were administered via a cannula inserted into the maternal jugular vein. Ewes received a continuous iv infusion of octylphenol (1000 µg/kg·day), diethylstilbestrol (50 µg/kg·day), or vehicle (20% alcohol in saline) from days 110–115 of gestation. Fetal blood samples (1 ml) were collected every 8 h from days 109–115 of gestation. All animals were then killed with sodium pentobarbitone (Euthatal, May and Baker, Dagenham, UK). Fetal pituitary glands were removed within 5–10 min of death and halved in the coronal plane. One half was frozen in OCT (Tissue-Tek, Miles Laboratories, Elkhart, IN) before storage at -70 C for subsequent in situ hybridization analysis of FSHß and LHß messenger RNA (mRNA). The other half was fixed in Bouin’s solution for 6 h before embedding in paraffin wax for immunocytochemical analysis of the specific ß-subunits of FSH and LH.

Exp 2: can maternal exposure to octylphenol suppress Sertoli cell replication during fetal life? Ewes with a single known insemination date were used in this study. Ewes were maintained indoors with ad libitum access to silage, hay, and water. Pregnant ewes were treated twice weekly with sc injections of DES (0.5 µg/kg·day), octylphenol (1000 µg/kg·day), or vehicle (corn oil) from day 70 of gestation until birth. Newborn lambs were killed as previously described, and pituitary glands were removed within 5–10 min of death. Each pituitary gland was halved in the coronal plane; one half was fixed in Bouin’s fixative for 6 h for immunohistochemical analysis, and the other half was frozen in OCT Tissue-Tek for mRNA analysis. The testes were removed, their weights were recorded, and they were fixed in glutaraldehyde. Sertoli cell number was analyzed by the dissector method, as previously described (20).

RIA
The concentrations of FSH and LH in circulation were measured in duplicate aliquots of plasma using RIAs validated for sheep plasma (22, 23). LH assay sensitivity was 0.2 ng/ml NIH oLH-S23 (NIDDK, Bethesda, MD), and the intra- and interassay coefficients of variation were 8.0% and 12.2%, respectively; the corresponding values for the FSH assay were 0.2 ng/ml NIH oFSH-RP1, 6.6%, and 11.8%.

Immunocytochemistry
Fixed pituitary tissue was embedded in paraffin wax, and the avidin-biotin immunoperoxidase histochemical technique (24) was used to visualize FSH- and LH-containing cells as described previously (25). Sections (5 µm) were incubated with either a rabbit antisheep FSHß (1:500; AFPC5288113) or LHß (1:500; AFP 697071P) primary antibody, both gifts from Prof. A. F. Parlow, Harbor-University of California-Los Angeles Medical Center (Torrance, CA). Point counting by direct microscopy using a 19-mm eye piece graticule on a BH-2 Olympus Corp. microscope (New Hyde Park, NY) was used to determine the percentage of immunopositive cells. Each section was examined under a light microscope with a x20 objective. The number of immunoreactive and nonimmunoreactive cells in four fields, selected relative to the posterior pituitary, were counted, thus allowing evaluation of the percentage of immunoreactive cells in the tissue.

In situ hybridization
In situ hybridization was performed on 10-µm frozen sections using a modification of a previously described method (26). Briefly, 45-mer antisense and sense oligonucleotide probes: antisense, 5'-GTGACATTCAGTGGCTACTGGGTACGTGTACAGGGAGTCTGCATG-3', complementary to bases 314–358 of the ovine FSHß gene (27); sense, 5'-CAGATGCTGGTGGTGAAAGTGATACAGACAGGGCAGGCCTCCTTC-3', complementary to bases 124–168 of the ovine LHß gene (28)] were labeled with [³⁵S]deoxyadenosine 5'-(-thio)triphosphate (1300 Ci/mmol; NEN Life Science Products-DuPont, Boston, MA) using terminal deoxynucleotidyl transferase (Roche, Indianapolis, IN). Sections were hybridized with labeled probe in hybridization buffer (40 µl; 3750 cpm/µl) overnight at 42 C. After washing, the sections were dehydrated, dipped in silver emulsion (Kodak NTB2, IBI), and stored desiccated at 4 C. They were then developed using standard procedures after 3.5 weeks.

To determine the abundance of mRNA, sections were examined using an Olympus Corp. BH-2 microscope with a x40 objective. Images were captured using a data translation quick capture card and a Macintosh 11cx computer and were quantified using the NIH Image 1.57 program. The number of silver grains in six fields, selected relative to the posterior pituitary, was evaluated. Background counts were determined by counting four random fields in the posterior pituitary, and the average of the grain counts in this region was deducted from the grain densities determined from the anterior pituitary sections. Sections from the same pituitaries were also probed with sense probes, and no differences between grain densities in the different regions of the section was observed.

Data analysis
Due to the variability in baseline LH and FSH concentrations in fetal animals, an assessment of the change in basal hormone concentrations was made by averaging the concentrations of the samples taken before the start of the infusion period. These basal values were then subtracted from the concentrations in samples taken after the start of the infusion to give the net hormone response to treatment. Data were analyzed by ANOVA followed by Duncan’s multiple range test when a significant (P < 0.05) interaction was found.

All control and experimental sections for immunocytochemistry and in situ hybridization were processed simultaneously to allow direct comparisons between experimental treatment groups. Group data for percentages of FSH- and LH-immunoreactive cells, mRNA quantification, testes size, and Sertoli cell number are presented as the mean ± SE and were statistically analyzed using ANOVA.

	Results

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Exp 1: can maternal exposure to xenoestrogens suppress fetal gonadotropin secretion?
Changes in blood plasma concentrations of fetal FSH and LH collected every 8 h for 1 day before and for 4 days during the infusion period are depicted in Fig. 1, a and b, respectively. FSH and LH concentrations remained constant (P > 0.05) in control animals over the 5-day infusion period. Maternal exposure to DES (50 µg/kg·day) suppressed (P < 0.05) both fetal FSH and LH concentrations within 24 h of the commencement of the infusion, although maternal exposure to octylphenol (1000 µg/kg·day) suppressed fetal FSH concentrations within 48 h of the commencement of the infusion (P < 0.05) without any significant effect on LH concentrations.

View larger version (18K): [in this window] [in a new window]   Figure 1. Exp 1: plasma concentrations of FSH (a) and LH (b) in blood samples collected from chronically catheterized fetal sheep every 8 h for 1 day before and 4 days after maternal iv infusion of octylphenol (1000 µg/kg·day; ), DES (50 µg/kg·day; •), or vehicle (1:4, ethanol-saline; ). FSH and LH concentrations in the three samples taken before the start of the infusion were averaged and subtracted from all samples to give the change in gonadotropin concentration. As the gonadotropin response to xenoestrogens was similar between males and female fetuses, data from both sexes have been combined. All values are the mean ± SEM of eight animals per group.

View larger version (100K): [in this window] [in a new window]   Figure 2. Exp 2: radioactive in situ hybridization analysis of FSHß subunit mRNA in 10-µm frozen coronal pituitary sections collected from fetuses after maternal exposure to vehicle control (A), octylphenol (1000 µg/kg·day, B), or DES (0.5 µg/kg·day; C). Quantitative analysis of grain density (D) revealed a decrease (means with different superscripts are significantly different, P < 0.05) in FSHß mRNA after maternal exposure to octylphenol and DES.

View larger version (25K): [in this window] [in a new window]   Figure 4. Exp 2: analysis of LHß subunit mRNA and the percentage of immunoreactive LHß cells revealed no effect (P > 0.05) of maternal exposure to octylphenol (1000 µg/kg·day) or DES (0.5 µg/kg·day) on LHß mRNA or the percentage of immunopositive cells.

View larger version (139K): [in this window] [in a new window]   Figure 3. Exp 2: localization of immunoreactive FSHß in 5-µm coronal pituitary sections collected from fetuses after maternal exposure to vehicle (A), octylphenol (1000 µg/kg·day; B), or DES (C). Quantitative analysis of the percentage of immunopositive cells (D) revealed a significant (means with different superscripts are significantly different, P < 0.05) decrease in FSHß-immunopositive cells after maternal exposure to octylphenol and DES.

View larger version (23K): [in this window] [in a new window]   Figure 5. Exp 2: testicular weight and the number of Sertoli cells were reduced (means with different superscripts are significantly different, P < 0.05) after maternal exposure to octylphenol (1000 µg/kg·day) and DES (0.5 µg/kg·day).

	Discussion

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These findings provide the first demonstration that maternal exposure to the xenoestrogen, octylphenol, inhibits fetal FSH secretion. This ability to selectively inhibit FSH provides good evidence that this xenoestrogen, at this dose, is not having a general toxic effect in the pituitary gland, but, rather, is acting as a discrete endocrine disrupter. Fetal endocrine status in many species, including humans and sheep, is characterized by a midgestational rise in gonadotropin secretion that plays a crucial role in maturation of the fetal testis (19). We have previously shown that pharmacological inhibition of fetal gonadotropin secretion in sheep causes a 40% reduction in the size of the testis at birth with an associated reduction in Sertoli cell number (20). Similarly, in rats, suppression of FSH secretion during the critical period of Sertoli cell multiplication in early neonatal life reduces Sertoli cell number, testicular weight, and sperm output in the adult rat (29, 30). As we have found that short-term exposure to octylphenol is able to inhibit fetal FSH secretion, we reasoned that prolonged exposure throughout the latter half of pregnancy would decrease the rate of Sertoli cell replication with a consequent reduction in testis size at birth.

The second experiment extended the initial observation that maternal exposure to octylphenol selectively suppresses FSH synthesis and secretion, and showed that these changes were associated with disrupted testicular development. Maternal exposure of ewes to DES over the same period of gestation resulted in significant reductions in testicular weight and Sertoli cell number at birth; the magnitudes of these reductions were the same as those seen after octylphenol treatment (albeit at a 2000-fold lower dose than octylphenol).

Previous experiments in the rat have provided inconclusive evidence that in utero or lactational exposure of rats to xenobiotic estrogens such as octylphenol or butylbenzyl phthalate suppresses testicular weight and sperm counts (31, 32, 33). Sharpe and colleagues (33) discuss the possibility that other factors, such as water quality, may change over time, resulting in differences in the experimental outcome of these publications. Our results support the initial findings of Sharpe and colleagues (31) that in utero exposure to octylphenol suppresses testicular development and further demonstrate that fetal FSH secretion is disrupted, which highlights one mechanism through which exposure to endocrine-disrupting chemicals could disrupt testicular development. It is, of course, possible that these compounds could be having a direct effect on testicular cells. Such direct inhibitory effects of steroids on Leydig cells has previously been demonstrated (34).

In this experiment DES was used as a positive control treatment. DES is a synthetic nonsteroidal estrogen that was administered to pregnant women to prevent preterm labor between 1948 and 1971. Human males prenatally exposed to DES suffered decreased fertility, abnormalities in quality and quantity of sperm, and an increased incidence of epididymal cysts compared with unexposed adult males (35, 36). Although DES is known to be an estrogenic compound, the mechanism by which it affects a wide range of cell types is unknown, and it probably does so through a variety of different routes. Our data suggest that maternal exposure to DES can suppress fetal FSH and LH synthesis and secretion in utero. The doses used in this experiment are well within the range of those to which humans were exposed. It is essential to identify which of the above abnormalities diagnosed in humans can be caused by prenatal suppression of FSH and/or LH.

One of the interesting outcomes of these experiments was the differential effect of octylphenol on FSH and LH secretion during fetal/early postnatal life. A number of factors, in particular the inhibins, activin, and follistatins, differentially influence FSH and LH secretion (37, 38). It remains to be determined whether the synthesis of these factors have been influenced by in utero exposure to octylphenol.

A key question in relation to the broader significance of these results is how do the levels of octylphenol in the environment relate to the concentration of octylphenol administered to the animals in this experiment. The answer to this question is confounded by two major problems. Firstly, few data exist on the levels of octylphenol in the environment. However, the intermediate and final breakdown products of the alkylphenol polyethoxylates have been identified in waterways throughout Europe and the U.S. (11, 12). The reported concentrations are extremely variable and range from nanograms to milligrams per liter. Thus, heavily polluted water systems would provide concentrations similar to those used in this experiment. Secondly, alkylphenols bioaccumulate in body fat (13, 14). Thus, although concentrations of alkylphenols in the environment may be lower than that used in this experiment, significant concentrations may be stored in the body and mobilized during fasting or energetically expensive physiological conditions, such as pregnancy. In fact, the release of pesticide residues from fat stores during fasting has been shown to have estrogenic effects in mice (39).

In conclusion, we report that maternal exposure of pregnant ewes to octylphenol inhibits the normal FSH profile of the fetus in utero, with a resultant decrease in Sertoli cell number and testis size at birth. These data add to a growing body of evidence supporting the concept that exposure to endocrine-disrupting chemicals during fetal development may lead to adverse effects on reproductive health. The challenge now is to determine the biological relevance of these observations to man by first determining the level of exposure of humans to this and related compounds, and second establishing whether these levels are sufficient to cause long-term harmful effects on testicular function in the adult.

	Acknowledgments

 
We are extremely grateful to Dr. D. Howe, N. Anderson and J. Docherty for expert technical assistance. Antibodies for RIA and immunohistochemistry were kindly donated by the National Hormone and Pituitary Program (University of Maryland, School of Medicine, NIDDK).

	Footnotes

 
¹ This work was supported by a European Science Foundation Postdoctoral Research Grant and an Irish Health Board Postdoctoral Research Grant (to T.S.). Exp 1 was funded by the United Kingdom Medical Research Council. Exp 2 was funded by the Irish Health Research Board.

² Present address: AstraZeneca Central Toxicology Laboratory, Molecular Endocrinology Group, Alderley Park, Macclesfield, Cheshire, United Kingdom SK10 4TJ.

Received November 18, 1999.

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