Subjects and housing
Sexually experienced Wistar male rats were used (250–300 g/bw). Males were living in a room with an inverted light-dark cycle (12–12 hr, lights off at 0800 hrs). Every rat was manipulated daily, hence they were habituated to experimenter contact and to the environment in order to avoid the stress-induced PRL release. Ovariectomized females were used, and their sexual receptivity was induced with steroids, dissolved in sesame oil. Accordingly, subcutaneous injections of estradiol benzoate (10 μg) and then progesterone (2 mg) were administered 48 and 4 hrs before tests, respectively. Rats were housed in plastic cages (50 × 30 × 20 cm) containing wood chip bedding and rooms were under a controlled temperature (22 ± 2°C), with food (Harlan Mexico rodent chow) and water available ad libitum. Every surgical intervention and manipulation of rats was guided by the Society for Neuroscience Policy on the Use of Animals in Neuroscience Research.
This experiment was designed to obtain the baseline levels of serum PRL in the dark phase. Four groups were formed: G8 (n = 10), G12 (n = 12), G16 (n = 12), and G20 (n = 12). The number indicates the hour of the day on which blood was collected, such that the term 'G12' would indicate the sample was taken at 12:00 hrs. Males were cannulated for blood collection (see below) two days before the beginning of the experiment. Blood samples (0.2 ml) were collected once per animal on the hour indicated by the group, but samples at 08:00 hrs were taken about 5 minutes after the designated hour, and samples at 20:00 hrs were taken about 5 minutes before the designated hour.
Males were cannulated for blood collection. They were anesthetized with sodium pentobarbital (Smith Kline, Mexico; 30 mg/kg bw, i.p.), and the right jugular vein was exposed. The vein was half-cut and 2.5 cm of a cannula (Dow Corning, USA, 0.30 mm ID and 0.64 mm OD) introduced to leave the tip in the right atrium. The cannula and vein were fixed and a small loop of the cannula was positioned in the ventral neck area to avoid displacement during head movements. The other side of the cannula was guided subcutaneously to the back and exposed through a hole made in the skin. The skin in the neck was sutured, covering the cannula and loop. Therefore, only the end of the cannula in the back was exposed. It was filled with saline solution with a Hamilton syringe, the tip was closed with a small metal plug, and it was covered with a belt (1.5 cm wide) that surrounded the thoracic area of the body. The belt was lab-made for this experiment and was designed for two purposes: to protect the cannula from male scratching movements, and to allow the male to copulate without any interference. Cannulated males were placed alone in a cage. To collect blood, a small drop of a heparin solution was placed in a Hamilton syringe. The syringe was connected to the tip of the cannula and blood extracted and mixed inside the syringe with the heparin solution. After blood collection, a second Hamilton syringe was connected to the cannula to inject 0.2 ml of saline solution to wash most of the blood left inside the cannula and to avoid coagulated plugs. Blood samples were centrifuged, and the plasma obtained was stored at -20°C until assayed.
PRL levels in plasma samples were measured using the ELISA procedure. A 96 well plate was coated with PRL (NIDDK-rPRL-I-6) at a 10 ng/50 μl concentration in carbonate buffer (sodium carbonate 0.1 M, and sodium bicarbonate 0.035 M), and incubated at 4°C for 24 hr. On the other hand, using eppendorf tubes, PRL standard curves were done in triplicate at several concentrations in PBS 0.1 M and Tween 20 0.05% (concentrations: 120, 60, 30, 25 and 7.5 ng/ml). Also, both diluted plasma samples (1:6) and standard curves were incubated in parallel with the first antibody (1/100, NIDDK-anti-rPRL-RP-3) overnight at 4°C. Then, the plate was washed (3 times, 5 min each) with PBS-Tween 20 solution, followed by the adding of bovine serum albumin 1% for an hour, and washed once for 3 min with the PBS-Tween 20 solution. 50 μl of standard curve and plasma sample were took, added to the wells, and incubated at 4°C for 24 hr. Again, the plate was washed (3 times, 3 min each) with PBS-Tween 20 solution, incubated with the second peroxidase-conjugated antibody (1/800) at room temperature for 2 hr, and washed (3 times, 5 min each) with PBS-Tween 20 solution. The plate was developed with orthophenildiamine (10 mg) and hydrogen peroxide (3%) in citrate buffer (citric acid 0.05 M, and dibasic sodium phosphate 0.1 M) for 10 min, and stopped with cold sulphuric acid (4 N). The plate was read at 490 nm. The sensitivity was about 3 ng/ml, and intra and interassay coefficient variations were 9.2 and 15.5% respectively.
The experimental paradigm of this study was used to determine the effect of consecutive ejaculations (up to 4) on the level of serum PRL and on the histology of the ventral and dorsal prostate. Males were prepared for blood collection two days before the beginning of tests, and females were treated appropriately to induce a state of sexual receptivity. Experiments were carried out under red light between 12:00 and 16:00 hrs, the time considered ideal for the proper execution of sexual behaviour in rats . A male rat was taken from its cage and a blood sample obtained (0.2 ml). It was then placed in a Plexiglas arena (60 cm diameter × 60 cm high), and a receptive female was introduced five minutes later. Parameters detailing the sexual behaviour of the male were recorded immediately , but only the Hit Rate parameter is showed in results for the last experiment; it is a proportion obtained after the computation of the number of intromissions divided by the sum of the number of intromissions and the number of mounts . This is a good indicator of the appropriate execution of male's sexual behaviour.
The number of consecutive ejaculations marked the assigned group of the animal. Males from group E1 (n = 17) were allowed to ejaculate once, and then a post-ejaculatory blood sample was obtained. Group E2 (n = 13) males were allowed to ejaculate twice, group E3 (n = 12) males were allowed three ejaculations, and group E4 (n = 12) males were allowed four ejaculations, with post-ejaculatory blood samples being obtained after the final ejaculatory event of each male. Every animal was thus sampled twice per test: once before and once after its ejaculatory series, and samples analyzed with the ELISA procedure. On the other hand, about 50% of males in each group were killed after its respective ejaculation, the prostate obtained, weighed and prepared for histology with the hematoxilin-eosin stain.
The stain with hematoxilin-eosin started by immersing the prostate for 24 h in Helly fixative (Mercuric Chloride 5%, Potassium Dichromate 2.5%, Sodium Sulfate 1%, and Formalin 37%); then the fixative was washed with tap water for 30 min. Dehydration was carried out by immersing the prostate in ethanol 70% (1 hr), 80% (1 hr), 96% (3 times, 2 hrs each), absolute ethanol (overnight), and absolute ethanol (2 times, 1 hr each). Clearing was done by immersing the prostate in xylene (3 times, 1 hr each). Both dehydration and clearing were carried out under continuous shaking. Then, the prostate was embedded in melted paraffin (2 times, 2 hr each at 57°C) and a block was done by using a microtome cassette. The paraffin block was sectioned on a Leica microtome (5 μm). Sections were placed on a mounting bath with gelatinized water at 52°C and tissues mounted on slides that were placed for 1 hr in an oven (~58°C). For staining, tissues were immersed in xylene (3 times, 5 min each), absolute ethanol/xylene 1:1 (5 min), ethanol 96% (3 min), iodine alcohol (5 min), sodium thiosulfate (10 min), tap water (2 min), Mayer's Hematoxilin (10 min), tap water (30 sec), acid ethanol (fast bath), tap water (10 sec), lithium carbonate (30 sec), tap water (10 sec), Eosin Y (4 fast baths), ethanol 96% (3 min), absolute ethanol (2 min), absolute ethanol/xylene 1:1 (2 min), and xylene (5 min). Finally, sections were coverslipped with undiluted Permount. Each slide had sections from just one region of the prostate, i.e., we obtained ventral slides and dorsolateral slides. The slides were observed in an Olympus Provis AX-70 microscope, and images obtained and analyzed with the Image-Pro Plus software. Measures obtained were the height of epithelial cells and the area of the alveoli.
This experiment was done to determine the effect on the prostate of treatments that are known to increase PRL serum level and decrease sexual behaviour. Three groups of males were used (n = 6 each): control males (without treatment); males treated with haloperidol; and males with a pituitary graft under the kidney capsule. After 15 days of treatment, males were killed, the prostate obtained, weighed and prepared for histology with the hematoxilin-eosin stain. Blood samples were also obtained from these males to get the profile of PRL in serum. Haloperidol was administered with implants that consisted of silastic capsules (10 mm long, 1.58 mm i.d., 3.06 mm o.d.) filled with the crystalline drug (~9 mg). The capsule was placed subcutaneously in the nape of the neck under ether anaesthesia. Pituitary grafts were obtained from a donor male that was killed immediately after pituitary removal.
In this experiment, a controlled administration of exogenous PRL was used to determine the effect on the prostate of sexual behaving males. Three groups of males were used (n = 30 each): Intacts with no treatment, Vehicle injected and PRL injected. Treated males were injected subcutaneously with two daily doses (10:00 and 18:00 hrs) of vehicle or ovine PRL. On day 3 after the beginning of treatments, every male was tested for sexual behaviour. Then, 6 males of each group were killed, the prostate obtained, weighed and prepared for histology with the hematoxilin-eosin stain. Blood samples were also obtained from these males to get the profile of PRL in serum. Remaining males were returned to the animal room and the same procedure repeated on days 6, 9, 12 and 15.
Ovine PRL was obtained in bottles of 33.3 μg (Sigma Chemical, Mexico) that were diluted in injectable grade water (14.5 ml) to obtain a concentration of 100 μM. 100 μl of this stock solution was diluted (1:4) in the injectable water. 100 μl of this final solution were in each dose injected to experimental males (~50 μg of PRL per dose).
Nonparametric statistics were used in Experiments 1 and 2. Between-group comparisons of data from Experiment 1 were made with Kruskal-Wallis (KW) analysis of variance, and paired contrasts were obtained with Mann-Whitney (MW) tests. PRL data from Experiment 2, comparing pre-and post-ejaculatory results of correlated groups, were analyzed with the Wilcoxon Signed-Rank test. Experiment 3 and 4 were analyzed with a One Way ANOVA followed by the post hoc Dunnet test when the F value showed significant differences at p < 0.05. In every statistics, significant differences were inferred when p < 0.05.