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Research Article |
Department of Anatomy and Physiology,3 Swedish University of Agricultural Sciences, Centre for Reproductive Biology in Uppsala (CRU), 750 07 Uppsala, Sweden
Department of Environmental Toxicology,4 Uppsala University, Centre for Reproductive Biology in Uppsala (CRU), 752 36 Uppsala, Sweden
ABSTRACT
Environmental pollutants with estrogenic activity have a potential to disrupt estrogen-dependent developmental processes. The objective of this study was to investigate if embryonic exposure to the environmental estrogens o,p'-DDT (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane; 37, 75, 150 or 300 µg/g egg) and EE2 (17alpha-ethynyl estradiol; 60 ng/g egg) affects the reproductive system in domestic roosters. Following egg injection on Embryonic Day 4, the newly hatched chicks were sexed by cloacal inspection. A skewed phenotypic sex ratio with overrepresentation of chicks deemed as females was observed in the groups exposed to the three highest doses of o,p'-DDT but not in the EE2-exposed group. Normal sex ratios were observed in all groups at adulthood. However, a cloacal deformation seemed to remain in the adult roosters, causing an abnormal semen flow upon semen collection. Semen yield was significantly reduced in both o,p'-DDT-exposed and EE2- exposed birds, whereas semen quality was unaffected. When killed, deformations of the left testis were found in all treatment groups. Image analysis revealed a reduced seminiferous tubular area in the roosters exposed to the two highest doses of o,p'-DDT. Embryonic exposure to o,p'-DDT caused decreased comb weight and right-spur diameter, while EE2 only affected right-spur diameter. In conclusion, this study shows that embryonic exposure to estrogenic compounds can induce permanent effects in male birds. The effects of the two studied compounds were partly similar but o,p'-DDT also induced alterations not seen in the EE2-treated birds.
embryo, epididymis, estradiol, testis, toxicology
IINTRODUCTION
Avian wildlife is exposed to numerous environmental pollutants in terrestrial, limnic, and marine environments. Many pollutants have estrogenic activity and thereby the potential to disrupt estrogen-dependent developmental processes and adult physiological functions. Estrogen is directing gonadal sex differentiation in avian embryos and studies of chick embryos have shown that estrogen receptor mRNA is expressed in both male and female gonads during sex differentiation [1, 2]. In the adult male bird, the reproductive system is partly regulated by estrogen. Kwon et al. [3] found that testicular germ cells and epididymal sperm contain the enzyme aromatase, which catalyzes the conversion of androgens to estrogens. Furthermore, estrogen receptors are present in the efferent ducts and epididymis in roosters [4]. Consequently, the male reproductive system of birds is a target for estrogenic pollutants in both embryos and adults.
That exogenous hormones may influence reproductive-organ differentiation in bird embryos has been known for decades. Romanoff [5] reviews several studies on chick embryos, where egg injection of estrogen or related female hormones resulted in feminization of the left testis, inducing a so-called ovotestis. More recent studies show that injection of the synthetic estrogens diethylstibestrol (DES) and 17
-ethynyl estradiol (EE2) into the yolk of quail eggs results in a dose-dependent increase in the ovotestis frequency in male embryos and malformations of the Mullerian ducts in both sexes [6]. Likewise, in ovo exposure of chicken embryos to the 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) isomer o,p'-DDT (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane) results in ovotestis formation [7]. Similar effects are observed in quail embryos after exposure to the polycarbonate plastic monomere bisphenol A (BPA) [8] and in gull embryos exposed in ovo to DDT [9].
There are only a few studies on long-term effects of embryonic exposure to estrogenic chemicals on the reproductive system in adult male birds. Morphological malformations of the testis, such as disrupted epithelium of the seminiferous tubules, is seen in adult zebra finches exposed in ovo to estradiol benzoate [10] and in quails exposed in ovo to DES [11]. Furthermore, testis-weight asymmetry (weight of left testis/weight of right testis) is increased in adult Japanese quail following embryonic EE2 exposure [12], and o,p'-DDT exposure results in a reduced cloacal gland area [13].
The chlorinated insecticide DDT and its persistent metabolites are ubiquitously found in avian wildlife and, because of their lipophilic character, such compounds are efficiently excreted in the yolk of birds' eggs [14–17]. The aim of the present study was to investigate if embryonic exposure to the estrogenic isomer o,p'-DDT (constituting 15–20% of technical DDT) affects structure and function of the reproductive organs in roosters. For comparative purposes, the contraceptive estrogen and environmental pollutant EE2 was also examined [18]. This study, focused on the male reproductive system, is part of a larger investigation concerning effects of in ovo exposure to estrogenic substances on reproductive performance in the domestic chicken.
MATERIALS AND METHODS
Treatment of Eggs and Birds
A total of 249 fertilized eggs from White Leghorn chickens, Gallus domesticus (strain SLU13, University breeder flock, SLU, Uppsala, Sweden), were incubated at 37.2–37.7°C and turned every third hour. On Incubation Day four, 37, 75, 150, or 300 µg/g egg (ppm) of o,p'-DDT (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane; from Dr. Ehrenstorfer GmbH, Augsburg, Germany) or 60 ng/g egg (0.060 ppm) of the positive reference substance EE2 (17-ethynyl estradiol; from Sigma Chemical Co., St. Louis, MO) was injected into the yolk via a small hole drilled at the blunt end of the egg. Both substances were dissolved in a mixture of peanut oil and lecithin, from which an emulsion in water was prepared and used as vehicle [19, 20]. Control eggs were injected with vehicle only. The injection volume was 100 µl in each egg. After injection, the shell was sealed with paraffin wax and the eggs were returned to the incubator. On Day 19 of incubation, the eggs were placed in hatching boxes at 37.5°C and 70% relative humidity. On Day 22, a total of 204 chicks were removed from the hatcher, sexed, wing banded, vaccinated against Marek disease, and reared following traditional production standards. The sex was determined by cloacal inspection by a professional chick sexer. The numbers of birds deemed as males/females at hatch and classified as males/females by the secondary sex characteristics, such as comb size, body posture, and crowing at 20 wk, are given in Table 1. The frequencies of males and females found at 20 wk were verified upon autopsy after killing the birds. The chicks were reared in three-tier cages until 16 wk of age, when they were moved to single-rooster cages. During the rearing and growing periods, the birds were fed commercial starter and grower diets. The birds were raised in 8L:16D h until 15 wk of age, after which the light period was gradually increased by 20 min/wk until 16L:8D was attained. The study was approved by the Uppsala Local Ethics Committee.
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Semen Collection and Evaluation
Between Weeks 33 and 37, cloacal inspection was performed and semen collected twice in preweighed test tubes by the abdominal-massage method [21]. All measurements on the semen samples were performed without knowledge of which treatment the roosters had been exposed to. About 30 min after collection, the test tubes were weighed (digital scale showing 0.1 mg) and then placed in a 30°C shaking water bath. For determination of the concentration of spermatozoa, 10 µl of semen was diluted 10000 times with 3% NaCl. The concentration was then measured using a hemacytometer (Counting chamber, Bürker, VWR International AB, Stockholm, Sweden), to which 20 µl of diluted semen was added. An average output of spermatozoa was estimated for each group using literature data on specific gravity of spermatozoa and seminal plasma [22] and the spermatocrit for White Leghorn [23].
For motility measurement, 10 µl of semen was diluted 1:4 with buffer (30°C) containing 150 mM NaCl and 20 mM N-tris-(hydroxymethyl)-methyl-2-aminoethanesulfonic acid (TES) and further diluted 1:1000 with buffer containing 150 mM NaCl, 60 mM TES, and 10% bovine serum albumin. The diluted semen (20 µl) was added to a motility slide [24]. Sperm motility was subjectively evaluated microscopically and rated as 0, 1, or 2 (0 = no motility, 1 = some motility, 2 = full motility). The same person made all evaluations of sperm motility.
As a further measure of semen quality, the ability of spermatozoa to bind to the inner perivitelline layer, undergo the acrosome reaction, and produce a point of hydrolysis was determined by studying the interaction of the spermatozoa with perivitelline layer from chicken eggs according to Robertson et al. [25]. This test has proven to be accurate for monitoring the likely fertilizing ability of rooster semen [26]. Semen from each rooster was diluted 1:4 in NaCl TES and stored at 40°C in a shaking water bath for 10–60 min. The diluted semen (10 µl) was added to 1 ml of Dulbecco modified Eagle medium supplemented with 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and placed in a polycarbonate vial. A piece (0.5 cm x 0.5 cm) of the inner perivitelline layer separated from the yolk by acid hydrolysis was added to the vial with semen and incubated for 5 min at 40°C. After incubation, the inner perivitelline layer was rinsed in 1% NaCl and spread onto a microscope slide. The number of holes made by the spermatozoa in the perivitelline layer was determined using dark-field optics under a 10x objective lens. Perivitelline layers from 3 eggs were used for each semen sample.
Dissection and Tissue Preparation
A total of 89 roosters were killed at 48 wk of age by an intravenous injection of pentobarbital sodium (60 mg/ml, Apoteket AB, Umeå, Sweden). Weight and shape of testis/epididymis, body and comb weights, as well as length and diameter of spurs were recorded. The testis/epididymis and ductus deferens were also examined for gross abnormalities. Gonado-somatic index (100 x testis weight/body weight) and testis weight asymmetry (weight of left testis/weight of right testis) were calculated.
Two transverse tissue slices (about 2 mm thick) were cut from each testis/epididymis. One slice was fixed in 5% paraformaldehyde in 67 mM phosphate buffer (pH 7.2) and the other in 2.5% glutaraldehyde in 67 mM phosphate buffer (pH 7.2). Fixation time was 24 h at 4°C for both fixatives. After dehydration in increasing concentrations of ethanol, the samples were embedded in paraffin wax for future immunohistochemical evaluation and in water-soluble resin (Historesin, Leica Microsystems Nussloch GmbH, Heidelberg, Germany) for histology. Resin sections (2 µm) from testis/epididymis were cut with a microtome (Leica RM 2165, Leica Instruments GmbH, Germany) using glass knives. The sections were stained with hematoxylin-eosin.
Image Analysis
Digital images of sections from the left testis of 11 birds in each treatment were taken with a Nikon Microphot-FXA imaging system (4x objective lens; Bergström Instrument AB, Stockholm, Sweden). Five images randomly spaced beneath the testicular curvature from different areas in one section/bird were used. Each image area was 1.20 mm2. The number of seminiferous tubules and their total area were measured using an image analysis software (Easy Image Measurements 2000, Bergström Instrument AB, Stockholm, Sweden). The average area of seminiferous tubules and proportion of seminiferous tubules were calculated for each bird. Mean values were then calculated for each treatment. All slides were coded and the analysis was performed by one person.
Statistics
Data were subjected to analysis of variance using the General Linear Model procedure of statistics, Kernel Release 6.0 (StatSoft, Inc., 2004, STATISTICA, version 7). The differences in body weight, comb weight, semen weight, testis weight asymmetry, gonado-somatic index, spur length and diameter, the ability of spermatozoa to make holes in the inner perivitelline layer, as well as the number, proportion, and area of the seminiferous tubules between the treatment groups and the control were tested using the Duncan test. Statistical significance was defined as P < 0.05, and values are presented as means ± SEM.
The sex ratios at hatch and at 20 wk, the frequencies of defects in cloacas, and the frequencies of deformed testes in the treated groups were compared with the control values using the Fisher exact test (two-tailed).
RESULTS
Cloacal Inspection and Semen Evaluation
At hatch, sex determination by cloacal inspection showed a significantly skewed sex ratio, with overrepresentation of chicks deemed as females in the groups treated with 75, 150, or 300 µg o,p'-DDT/g egg, but not in the group treated with 60 ng/g egg of the positive reference substance EE2 (Table 1). When the sex was determined based on secondary sex characteristics of the birds at puberty (20 wk), the sex ratio was normal in all groups. However, cloacal inspection of the adult birds showed a significant increase in the frequency of cloacal defects among the roosters treated with 75, 150, or 300 µg o,p'-DDT/g egg (Table 1). Defects such as outgrowths, deformations, or a small phallic groove resulted in an abnormal semen flow upon collection. Semen flow was classified as abnormal when semen was flowing on either side of the phallic groove instead of through the groove. The alteration in semen flow did not, however, render collection of semen more difficult.
Semen weight was significantly lower in the groups treated with 75, 150, or 300 µg o,p'-DDT/g egg and in the group treated with 60 ng EE2/g egg compared with the weight in the control group (Fig. 1a). An estimated average output of spermatozoa from two samplings was 3.0 x 109 (control), 2.7 x 109 (37 µg o,p'-DDT), 1.2 x 109 (75 µg o,p'-DDT), 0.8 x 109 (150 µg o,p'-DDT), 0.4 x 109 (300 µg o,p'-DDT), and 1.2 x 109 (60 ng EE2). No significant difference in concentration of spermatozoa between the groups of treated birds and the controls could be detected (Fig. 1b). No significant differences in sperm motility could be detected and, in general, the motility was high (= 2 on the scale used). None of the treatment groups differed from the control group regarding the ability of spermatozoa to make holes in the inner perivitelline layer (Fig. 2).
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Testis and Secondary Sex Characteristics
Roosters with deformation of one or both testes were found in all treatment groups. The frequency of deformed left testis was significantly higher in all treatment groups compared with the control and the frequency of deformed right testis was significantly increased in the groups treated with 150 or 300 µg o,p'-DDT/g egg but not in the EE2-treated group (Table 1).
The deformed testes were irregularly shaped, with an uneven surface and a stunted epididymis (Fig. 3). Testes from more than one third of the exposed birds displayed blisters filled with transparent fluid. In the control group, the testes were bean-shaped, with a smooth surface and a clearly visible epididymis (Fig. 3), and no deformations could be detected.
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Roosters treated with 75, 150, or 300 µg o,p'-DDT/g egg as well as those treated with 60 ng EE2/g egg had significantly smaller right-spur diameter compared with the controls. The average right-spur diameter (± SEM) was 8.8 ± 0.2, 8.7 ± 0.3, 8.9 ± 0.2, and 8.7 ± 0.3 mm, respectively, compared with 9.7 ± 0.2 mm in the control group. Left-spur diameter and spur length were unaffected. The average comb weight was significantly lower in roosters treated with 150 or 300 µg o,p'-DDT/g egg (67.6 ± 5.1 g and 64.9 ± 4.3 g) compared with the comb weight of the control roosters (81.8 ± 4.3 g). EE2 had no significant effect on the comb weight. No significant differences in body weight, testis weight asymmetry, or gonado-somatic index were found between any of the different treatment groups and the control group.
Image Analysis
There were no significant differences in the number of seminiferous tubules per mm2 between any of the treatments. The seminiferous tubules constituted a significantly lower proportion of the testis area in roosters treated with 300 µg o,p'-DDT/g egg compared with the controls (Table 2). The average area of the seminiferous tubules was also significantly smaller in roosters treated with 150 or 300 µg o,p'-DDT/g egg. The roosters exposed to EE2 were unaffected regarding both proportion and average area of the seminiferous tubules (Table 2).
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DISCUSSION
Roosters exposed to the three highest doses of o,p'-DDT or to EE2 during their embryonic development had a significantly reduced semen production. The mean weight of the semen samples in the group treated with the highest dose of o,p'-DDT was only 12% of the weight of the control samples. There were no significant differences in concentration of spermatozoa between the groups of treated birds and the control birds, and consequently the output of spermatozoa was decreased. This finding shows that embryonic exposure to o,p'-DDT or EE2 induced a persistent effect on testicular function. Even though semen quality was not significantly affected by the treatments, fertility would probably be impaired because of the reduced output of spermatozoa.
Our measurements showed a significant reduction in the average area of the seminiferous tubules of the left testis in roosters exposed to the two highest doses of o,p'-DDT, but not to EE2. The proportion of seminiferous tubules also decreased after treatment with 300 µg o,p'-DDT/g egg, indicating that an increased amount of interstitial tissue in the testis accompanies the decrease in tubular area. No similar effect on the seminiferous tubules was present in quail exposed to 150 µg o,p'-DDT/g egg [13]. A positive correlation between the diameter of the seminiferous tubules and Sertoli cell size has been observed in Syrian hamster [27], and experiments in rats have shown that Sertoli cell development is modulated by estrogen [28]. Thus, the decreased seminiferous tubular diameter observed in the present study could be a consequence of a primary effect on the Sertoli cells. This presumption is supported by the finding that rats neonatally exposed to estrogen had fewer Sertoli cells and a decreased diameter of the seminiferous tubules [29, 30]. A positive correlation between Sertoli cell number and testicular size has also been observed in birds, as reviewed by Etches [31]. The testis weight of the treated roosters in this study was, however, unaffected. An extended, and more detailed, microscopic evaluation of testis and epididymis is currently being undertaken in our laboratory and preliminary results (not shown) indicate an effect of embryonic exposure to both o,p'-DDT and EE2 on testicular spermiogenesis and epididymal histology.
Testicular deformations such as abnormal shape and blisters and a stunted epididymis were evident in all treatment groups. These effects were mainly seen in the left testis, whereas the right testis was affected only after exposure to the two highest doses of o,p'-DDT. The left testis has an ambisexual potential and is more sensitive to estrogen than the right testis. It is well known that exposure to o,p'-DDT causes feminization of the left testis in bird embryos, as shown in gulls, quail, and chickens [7, 9]. A stunted epididymis has also been reported in quail embryonically exposed to DES [11].
No signs of an ovotestis were seen in the adult roosters following embryonic exposure to o,p'-DDT or EE2. This observation is in agreement with several earlier studies on quail and rooster embryonically exposed to DES, in which the ovotestis did not persist until adulthood [12, 32, 33]. Likewise, quail exposed embryonically to 150 µg o,p'-DDT/g egg did not show ovotestis as adults [13]. Thus, ovotestis formation appears to be a transient effect of embryonic exposure to estrogenic substances.
Embryonic exposure to o,p'-DDT, but not EE2, resulted in cloacal defects affecting sex determination of the young chicks and semen flow as they reached sexual maturity. Using cloacal inspection, it was impossible to distinguish newly hatched male chicks from the females in the groups treated with 75, 150, or 300 µg o,p'-DDT/g egg. The location of the malformation in the cloacal region is not known but may involve the genital tubercle, which is an anatomical structure that can be used to distinguish the sexes at an early age [5] and is considered to be the forerunner to the phallus of adult roosters [34]. A malformation of the cloaca was observed in the adult o,p'-DDT-treated roosters, resulting in an abnormal semen flow upon collection, with semen flowing on either side of the groove. In adult roosters, the groove is formed by lymphatic fluid from the vascular body [35]. Interestingly, estrogen receptors are present in both the vascular body and the genital tubercle of early chick embryos [1, 36, 37], suggesting that the developmentally induced cloacal malformation was a consequence of interaction by o,p'-DDT with estrogen receptors in the cloacal region.
Effects on secondary sex characteristics like comb and spurs were observed in this experiment. The average comb weight was significantly lower in the roosters treated with 150 or 300 µg o,p'-DDT/g egg compared with the comb weight of the control roosters, whereas birds exposed to EE2 had normal comb weight. A decreased comb weight has also been observed in male chicks treated with DDT 2–3 mo from Day 8 after hatching [38] or with BPA from 2 wk after hatching to Week 16 [39]. Roosters treated with the three highest doses of o,p'-DDT, as well as with EE2, had significantly smaller right-spur diameter compared with control roosters. It is suggested that female birds choose a mate based on secondary sex characteristics because these are indicators of fitness [40]. For example, in pheasants, male fitness is correlated with spur length and females prefer to mate with long-spurred males [41]. Thus, embryonic exposure to estrogenic pollutants may decrease reproductive success not only by disrupting sex organ function but also by affecting secondary sex characteristics.
In conclusion, embryonic exposure of domestic rooster to either the synthetic estrogen EE2 or the environmental estrogenic pollutant o,p'-DDT resulted in persistent effects on epididymal-testicular structure and function. Consequently, embryonic xeno-estrogen exposure can result in impaired reproductive performance of the adult male bird. Most effects by the two studied compounds were similar, but o,p'-DDT also caused effects not seen after treatment with EE2, such as anomalies of the cloaca and reduced comb size. Whether these differences in effect by the two compounds were related to differences in dose, kinetics, or mode of action remains to be clarified.
ACKNOWLEDGMENTS
The authors are grateful to Mrs. Marianne Ekwall and Mrs. Margareta Mattson for excellent technical assistance.
FOOTNOTES
2 Correspondence: A. Blomqvist, Department of Anatomy and Physiology, Swedish University of Agricultural Sciences, Box 7011, S-750 07 Uppsala, Sweden. FAX: 46 0 18 672111; alexandra.blomqvist{at}afys.slu.se 
1 Supported by the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning.
Received: 30 June 2005.
First decision: 28 August 2005.
Accepted: 8 November 2005.
REFERENCES
This article has been cited by other articles:
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  A. Mattsson, J. A Olsson, and B. Brunstrom Selective estrogen receptor {alpha} activation disrupts sex organ differentiation and induces expression of vitellogenin II and very low-density apolipoprotein II in Japanese quail embryos Reproduction, August 1, 2008; 136(2): 175 - 186. [Abstract] [Full Text] [PDF]
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 C. Bredhult, L. Sahlin, and M. Olovsson Gene expression analysis of human endometrial endothelial cells exposed to op'-DDT Mol. Hum. Reprod., February 1, 2008; 14(2): 97 - 106. [Abstract] [Full Text] [PDF]
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K. H Brown, I. R Schultz, and J. J Nagler Reduced embryonic survival in rainbow trout resulting from paternal exposure to the environmental estrogen 17{alpha}-ethynylestradiol during late sexual maturation Reproduction, November 1, 2007; 134(5): 659 - 666. [Abstract] [Full Text] [PDF]
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