Sulpiride-Induced Hyperprolactinemia in Mature Female Rats: Evidence for Alterations in The Reproductive System, Pituitary and Ovarian Hormones

This experimental study was approved byUrmia University. In this research, we used 40 mature female Wistar rats, 70 days of age that weighed 160 ± 20 g. The rats were purchased from the Animal Resources Center of the Faculty of Basic Sciences , Urmia University, Urmia, Iran and were acclimatized in an environmentally controlled room (temperature: 20-23˚C and 12 hours light/12 hours dark schedule). Food and water were given ad libitum. In this study all experiments conducted on animals were in accordance with the Urmia University Guidelines of the Ethical Committee for research on laboratory animals. Following a one week acclimation period, the animals were assigned to four groups (n=10 per group): control, control-sham and two test groups. The control group rats received food and water with no treatment. The control-sham group received daily intraperitoneally (IP) injections of 0.3 ml olive oil for 28 continual days. The test subgroups received either daily low dose IP injections of SPD (20 mg/kg) or high dose IP injections of SPD (40 mg/kg), for 28 continual days.

After 28 days the animals were euthanized by a special CO2 device and the ovarian, uterine horn and mammary gland specimens were dissected out and fixed in 10% formalin fixative for histological investigations, then embedded in paraffin. Serially prepared sections (5-6 μm) were stained with hematoxylin-eosin (Merck Co, Germany). In the ovarian sections, follicles were characterized in terms of their sizes: <100, 101-200, 201-300, 301-400 and >400 μm. Follicular morphology was examined by a microscope with a ×40 objective lens (Olympus, Germany) magnification. Follicles that had a complete layer of flattened granulosa cells, oocytes with cytoplasm, and a normal nucleus were considered normal. Abnormal follicles were classified according to the presence of cytoplasmic damage, a pyknotic nucleus, and combination of damaged nucleus and cytoplasm. Follicular number was estimated by counting the number of follicles in all slides (15). We also counted the corpus luteum (CL) number for each ovary. For histomorphometric analyses, the uterine horn endometrial epithelium, endometrium, myometrium and perimetrium thicknesses were evaluated by a morphometric lens (Olympus, Germany) with a ×40 objective lens. Ultimately the glands’ distribution and numbers per one mm² were estimated.In addition, we assessed the diameters of the mammary gland lobules and histological features.

Blood samples from corresponding animals were collected directly from the heart. Serum was centrifuged at 3000 g for 5 minutes and subjected to assessments for LH, FSH, progesterone, estrogen (E2) and prolactin levels (PL).

We added a 100 μl aliquots of sera to the tubes which contained 100 μl labeled hormones with rabbit anti-sera in 0.01 M phosphate buffer (pH 7.6). Anti-rat PRL(Cisbio Bioassays, France) was diluted 1:5000, LH was diluted 1:10000 and FSH was diluted 1:2500. The mixture was incubated for 48 hours at 4˚C. Goat anti-rabbit IgG at a 1:10 dilution (200 μl) was added to the mixture. After remaining for 18 hours at 4˚C, the mixture was subsequently centrifuged at 2000×g for 30 minutes and we used a gamma counter to measure the presence of radioactivity in the pellets. The lower limit of sensitivity for the rat PRL assay was 5.2 ng/tube, for LH it was 1.3 ng/tube and for FSH it was 30 ng/tube. Intra-assay coefficient of variance for 10 times was as follows: FSH (3.56%), LH (2.64%), and PRL (5.9%). Calculated inter-assay coefficient variances for 10 times were 8.98% for FSH, 7.52% for LH and 5.9%for PRL.

Concentrations of serum estradiol were measured using CIS kits (Cisbio Bioassays, France) according to the methods given by the manufacturer. In brief, serum (300 μl) was extracted with 3 ml ethy lether. The ether layer was evaporated under (N2) gas and the extract resuspended in 300 μl of 0.04 M phosphate buffer. After the addition of 100 μl 17/3-estradiol (14000 cpm), each tube was incubated with 100 μl anti-serum raised for 18 hours at room temperature. Goat anti-rabbit r-globulin (1 ml) was subsequently added and the mixture incubated for 15 minutes at room temperature. After centrifugation, the radioactivity in the resulting pellet was counted. The sensitivity was 1.8pg/tube. In order to evaluate serum level of progesterone we mixed serum (0.1 ml), ethylether (1 ml) and propylene glycol (50 μ1). After the ether was evaporated under N2 gas, 0.5 ml phosphate buffer and 0.1 ml (20000 cpm) of iodo-progesterone were added to the tube. The mixture was incubated with 0.1 ml anti-serum raised in rabbits for 18 hours at room temperature. Then, 0.1 ml bovine serum gamma globulin and polyethylene glycol were added to the mixture followed by centrifugation for 10 minutes at 2000 g. We measured radioactivitylevels in the resulting pellet. The lower limit of sensitivity was 0.6 ng/ tube (16). The intra-assay coefficient variances were: 6.12% (for 10 times) for estradiol and 4.8% (for 10 times) for progesterone. Calculated inter-assay coefficients of variance were: estrogen (8.4% for 10 times) and progesterone (9.9% for 10 times).

In order to evaluate the effect of SPD on natural mating and fertilizing ability in different groups, we randomly chose 5 rats from each group. The chosen rats were adjoined with 1 mature normal male rat for 7 days. The vaginal plugs (white coagulum) were checked every day for 7 days and the vaginal smear process was conducted on cases with vaginal plugs in order to clarify the presence or absence of sperm. The day of sperm detection in smears was considered as day 0 of pregnancy and after 21-23 days (pregnancy period in rats) the numbers of rats that were born were counted.

All results were expressed as mean ± SD. Experimental data were analyzed using analysis of variance and Duncan’s multiple range test (SPSS version 16.00, Chicago, IL, USA). Correlation between the serum levels of PRL with the SPD-administered dosewas analyzed on an Indigo-2 O2 Work Station (Silicon Graphics, Mountain View, CA, USA) using Matlab (MathWorks Inc., Natick, MA, USA). P<0.05 was considered to be statistically significant.

We observed no changes in total weight gain of control, control-sham and low dose SPD group sat the end of the treatment period. Animals in high dose SPD group had significantly less weight gain (Fig 1A, B).

Histological analyses showed that in SPD groups the total number of follicles significantly (p<0.05) decreased in comparison to control and control-sham animals. Both left and right ovaries from the control and control-sham groups contained follicles in various stages of development that included primary, secondary and tertiary follicles of different sizes (<100 ìm to >400 ìm), whereas there was no large antral follicle (>400 ìm) in the high dose SPD group (Fig 2A-C). In SPD-administered animals the cortex of the ovaries (left and right) were covered with small antral atretic follicles (<100-200 ìm). Atresia was mostly present in follicles <100 ìm and additionally observed in 201-300 ìm follicles in high dose SPD rats (Table 1).

Comparing the rate of normal follicles between the control, control-sham and test groups revealed a significant (p<0.05) decrease in the SPD groups. This reduction was dose-dependent; animals that received high dose SPD had the lowest number of normal follicles. The highest rate of normal follicles between the test groups was observed in the low dose SPD group. The data for normal follicles are depicted in table 2. We observed that animals who received SPD exhibited significantly higher numbers of CL compared to the control and control-sham groups (Fig 3).

Comparing the uterine horn endometrial epithelium height (left and right) between all groups showed that in SPD-administered groups the epithelial height significantly (p<0.05) increased compared to control and control-sham animals. This impairment was SPD dose dependent as the group that received a high dose of SPD showed the highest endometrial epithelium. Simultaneously the endometrial, myometrium and perimetrium thicknesses in both side uterine hornswere remarkably (p<0.05) increased in both the low and high SPD-administered groups (Fig 4A-D). In SPD groups, the numbers of endometrial glands per mm² of the endometrium were considerably (p<0.05) increased. Animals in control and control-sham groups had remarkably lower numbers of endometrial glands per mm² of endometrium (Fig 5). The data for histomorphometric analyses are presented in table 3.

Light microscopic analyses showed that the lactating alveolus diameter in glands and the distribution of lactiferous ducts remarkably (p<0.05) increased in SPD-administered groups. The alveolar epithelium height significantly increased in animals treated with SPD, which was dose dependently increased in SPD-treated animals. Histological analyses showed that simultaneous with remarkable secretory alveoli and ductal development,there were fatty globules observed in the secretory and intra-lobular ducts of the SPD-administered rats’ mammary glands (Fig 6A-D). The glands of the control and control-sham animals had inactive lobules and secretory ducts. The data for histomorphometric analyses are shown in table 4.

Biochemical analyses showed that the serum levels of PRL significantly (p<0.05) increased in animals that received low and high doses of SPD. In contrast, control and control-sham animals showed constant PRL levels. A comparison of LH and FSH serum levels between SPD and control groups showed that serum LH and FSH levels remarkably (p<0.05) decreased in animals that received either low or high doses of SPD. Although reduction in LH and FSH levels progresse daccording to SPD administration doses, we observed no statistically significant (p>0.05) difference between low and high dose SPD-administered animals. The serum levels of estradiol significantly decreased, while the progesterone level remarkably increased in animals treated with SPD. The high dose SPD-administered group showed the lowest serum level of estradiol and the highest level of progesterone compared to the low dose SPD, control and control-sham groups. The data for hormonal analyses are presented in Figures 7A-C. The data for correlation between PRL, LH and FSH are presented in Figures 8-A and 8-B.

Analysis of the fertilizing index in the control, control-sham and SPD test groups showed that the SPD-administered groups had a negative fertilizing index with no neonates. In contrast the control and control-sham animals showed positive fertilizing indexes with 31 and 19 neonates, respectively (Table 5).