| ObjectiveThe present study was carried out to investigate effects of diethylstilbestrol on the structure of testis and spermatogenesis in pubertal rat, to detect changes of estrogen receptor expression in spermatogenic cells and analyse possible mechanism of diethylstilbestrol on impaired reproductive system in pubertal rat.Methods1. Acute toxic experimentMale SD rats, six weeks old, were housed in an animal facility. Rats were divided into four groups of six animals randomly. Group A was control group given 0 μg/kg· day diethylstilbestrol alone. Group B, C and D were administered diethylstilbestrol at the dose of 2, 10 and 50 μg/kg· day in 0.3ml corn oil on alternate days from postnatal weeks 6-10, respectively. At the end of the period, animals were killed and collected samples.2. Recovery experimentThe animals, grouping, dosage and treatment days were consistent with those of the acute toxic experiment. At the end of the acute toxic experiment, rats were bred for 60 days of recovery continually.3. Chronic toxic experimentMale SD rats, six weeks old, were housed in an animal facility. Pubertal rats were divided into four groups of six animals randomly. Group I was control groupreceived the distilled water alone. Group J, K and L were administered diethylstilbestrol at the dose of 0.02, 0.2 and 2 μg/kg·day in the distilled water for two spermatogenic cycles (96 days), respectively. At the end of the period, animals were killed and collected samples.4. Both Testes and epididymides were weighed. Daily sperm production and epididymis sperm reserves were measured. The morphological changes of testes were observed by light microscope and transmission electron microscope. The expression of estrogen receptor of spermatogenic cell nuclear was evaluated by immunohistochemical reactions.Results1. The results of body weights, testis weights, epididymal weights, daily sperm production and epididymis sperm reservesIn the acute toxic experiment, the body weight, daily sperm production and epididymis sperm reserves of group B decreased, significant differences were observed when compared to group A (P<0.01), testis weights and epididymal weights were no differences (P>0.05). Five indexes of group C and group D were all significantly decreased compared to those of group A (P<0.05).In the recovery experiment, no significant differences were observed about five indexes when group F compared to group E (P>0.05). Daily sperm production and epididymis sperm reserves of group G decreased, significant differences were observed when compared to group E (F<0.05), other three indexes were no significant differences (P>0.05). Expect the body weight, significant differences were observed about the other four indexes of group H when compared to group E(P<0.01)In the chronic toxic experiment, no significant differences were observed about five indexes when group J compared to group I (P>0.05). The body weight, daily sperm production and epididymis sperm reserves of group K decreased, significant differences were observed when compared to group I (P<0.01), othertwo indexes were no significant differences (P>0.05). The body weights, testis weights, epididymal weights, daily sperm production and epididymis sperm reserves of group L decreased dramatically compared to group I (P<0.01).2. The structure changes of rat testisIn the treated groups seminiferous tubules were atrophic, part of seminiferous tubules spermatide and sperm disappeared. With the increasing of dosage, the structure changes of rat testes became more and more severely. The increased thickness of tubular boundary was observed by transmission electron microscope, vacuolizations were common in testicular injury between spermatogonia, tubular boundary and Sertoli cell. The number of spermatogenic cells decreased.3. The expression of estrogen receptor by immunohistochemical reactionsThe percent of estrogen receptor positivestaining cell numbers in group J was 32.13% ± 3.45%, group I was 34.56% ± 1.77%, no significant differences were observed in group J and group I (P>0.05). The percent of estrogen receptor positivestaining cell numbers in group K was 28.80% ± 1.99%, lower than that of group I (P<0.01). The percent of positivestaining cell numbers in group L was 18.96% ± 1.86%, significant lower than that of group I (P<0.01).Conclusions1. Rats were exposure to DES by abdominal injecting with 2 μg/kg · day, spermatogenic epithelium were impaired and spermatogenic function were decreased. The severe impairment was detected, with the increasing of dosage. Following eight weeks recovery period, the structure of testes and spermatogenic function showed different degree recovery in 2μg/kg·day group and 10μg/kg·day group. Overall, after long-term recovery period rat fertility could be recovered, as long as the spermatogonia were not damaged.2. The oral administration of DES≥0.2 μg/kg·day of pubertal SD rats for two spermatogenic cycles could decrease rat's body weights, testis weights, epididymal weights, daily sperm production and epididymis sperm reserves. The structure oftestes were changed, including seminiferous tubule atrophy, the attenuated thickness of spermatogenic epithelium, the reductive number of spermatide and sperm.3. The oral administration of DES≥0.2 μg/kg·day of pubertal SD rats for two spermatogenic cycles could inhibit the expression of estrogen receptor, indicated that changes of estrogen receptor expression in spermatogenic cells would be the possible mechanisms of diethylstilbestrol on decreased fertility of male rat.
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