Exogenous melatonin Action on hormonal levels, implantation sites and Mel1a receptor expression and PRL-II in ovaries of pinealectomized rats induced hyperprolactinemia CURRENT STATUS: UNDER REVIEW

The study evaluated the effect of exogenous melatonin and the induction of hyperprolactinemia on estrogen, prolactin and progesterone levels, implantation sites and expression of Mel1a and PRL-II receptors in pinealectomized rats ovaries, during the third Initial pregnancy. We Used 35 rats divided into groups: I-rats Sham-pinealectomized (Sham); II – pinealectomized rats (P); III-pinealectomized rats treated with melatonin (P + Honey); IV-pinealectomized rats treated with domperidone (P + Domp); Pinealectomized V-rats treated with melatonin and domperidone (P + Honey/Domp); VI-rats treated with saline + ethanol (placebo honey) and VII-rats treated with saline solution (placebo domp). Melatonin (200μg/100g) and/or Domperidone (4mg/kg) was applied until the 7th day of gestation. The expression of the MEL1A receptor was low in the ovaries of rats P and P + Mel. The PRL-II receptor did not present differences between the groups as well as in the estrogen levels. Prolactin levels were elevated in rats treated with domperidone, while progesterone showed low levels in rats P and P + Domp. The weight and number of implantation sites were reduced in rats P and P + Mel, with no alteration in the weight of the ovaries. Thus, it is concluded that Hyperprolactinemia promotes an increase in the expression of the Mel1a receptor in the ovaries, at the beginning of pregnancy in pinealectomized rats, however without altering the expression of the PRLII receptor. In These animals, prolactin levels were kept elevated regardless of the presence or absence of melatonin, but there seems to be a synergistic effect when melatonin is associated with hyperprolactinemia and that melatonin is a preponderant factor for Maintenance of progesterone levels. Prolactin seems to an important role during the implantation process.

melatonin and is subdivided into Mel1a, Mel1b and Mel1c. The MEL1A is related to the reproductive and circadian effects of melatonin, the MEL1B is involved in the sensitivity to light by the retina and the Mel1c is found in amphibians. The Mel 2 receptor is related to N-Acetylserotonin (REPPERT, 1997;DUBOCOVICH et al., 1999). According to Pedreros et al. (2011) The treatment with melatonin in pregnant mares resulted in a normal expression of the receptor for MEL1A in the uterus and ovary and a decrease in progesterone levels.
It Is unknown the molecular events that mediate the melatonin actions in these organs, because the receptors are regulated by the Heterotrimeric G protein subunits, including serine/threonine kinase (a family of MAPK kinases -protein kinase Mitogenic Activator) involved in the transduction of signals that regulate the growth, division and differentiation of uterine and ovarian cells, and it is possible that the effects of melatonin in reproduction can be mediated by the signaling of the Cascade MAPK (protein Mitogenic activating kinase) (WOO et al., 2001). However, less than necessary studies have associated the relationship between the circulating levels of melatonin and prolactin to the development of uterine and ovarian morphophysiological alterations in non-seasonal animals (SOARES JR. et al., 2003b). Thus, the present research aimed to investigate the action of exogenous melatonin on hormonal levels, implantation sites and expression of the receptor Mel1a and PRL-II in ovaries of pinealectomized rats and induced to hyperprolactinemia, during the third Initial pregnancy.

Results
The expression of the MEL1A receptor was significantly low in the ovaries of the rats of groups P and P + Mel, when compared to the ovaries of the rats of the groups Placebo, Sham, P + Mel/Domp and P + Domp. However, it was numerically evidenced the expression in the latter Group ( Figure 1). For the PRL-II receptor, there were no significant differences in the ovaries of the rats of the studied groups, but the expression of this receptor was numerically elevated in the ovaries of the rats of the group P + Mel (Figure 2).
The Hormonal analysis revealed that there was no difference in serum estrogen levels in the rats of the experimental groups (Table I). However, prolactin levels were elevated in the rats of the P + Domp and P + Mel/Domp groups, being more pronounced in the latter, when compared with the other experimental groups (Table I). In relation to progesterone, the rats of groups P and P + Domp showed a significant decrease of this when compared with the other rats of the experimental groups (Table I). The statistical analysis related to weight and number of implantation sites had a significant reduction of these parameters in the rats of groups P and P + Mel when compared with the other groups studied. However, there were no statistical differences in the weight parameter of the ovaries (Table   II). The histological analysis of the sites of implantation of rats from the experimental groups showed that they were fully adhered to the uterine wall ( Figure 3A). However, P and P + Mel groups presented a possible delay in the development of these implantation sites ( Figure 3B). Histologically, trophoblasts were observed at different developmental stages with mitotic activity ( Figure 3C). Cytotrophoblasts with Polyploidia Were also observed. However, in the animals of the P + Domp Group, these cells were more lumping when compared with the other experimental groups ( Figure 3D).
The ovaries of all rats in the experimental groups externally presented a layer of connective tissue referring to the albugineous tunic, and the cortical and medullary layer well delimited and defined ( Figure 4A). The Group P + Mel/Domp was similar to the placebo groups with the well preserved and differentiated follicles, besides the presence of Corpus Luteum ( Figure 4B). However, the P + Domp Group presented a greater amount of corpus luteum ( Figure 4C), when compared with group P and P + Mel ( Figure 4D).

Discussion
The Literature reports that the pinealectomy in sheep (seasonal animal) promotes a decrease in the expression of the Mel1a receptor in the reproductive organs, especially in the ovaries (PANDI-PERUMAL et al., 2008). Although, Okatani et al. (2001), studying non-seasonal pinealectomized animals and treated with melatonin at a dose of 0, 4mg/kg reported a small expression in the Mel1a receptor in the ovary, but when the same author used exogenous melatonin at the dose of 4mg/kg There was an increase in the expression of this receptor. Thus, the lower expression of this receptor in the ovaries of rats in group P + Mel similar to group P verified in this study may be related to the dose of melatonin (200μg/100g), suggesting a dose-dependent effect of this hormone in the expression of this Receiver.
In addition, Lee et al. (2003) reported that the presence of endogenous prolactin in sheep is capable of stimulating the expression of the Mel1a melatonin receptor, and that pinealectomized ewes submitted to Hyperprolactinemia, presented regulation of the Gonoidal functions, as well as increased expression of this receptor in the ovaries (NISWENDER et al., 2000;PERKS et al., 2003), The expression of the Mel1a receptor due to hyperprolactinemia is due to a possible stimulus in the production of P450SCC protein, which activates the dissociation of G proteins in α and βγ dimers.
Interacting with several effector molecules involved in the transmission of Cell signaling, stimulating the expression of the Mel1a receptor (MASANA et al., 2001;PANDI-PERUMAL et al., 2008;TAMURA et al., 2008). Explaining the numerical increase of the Mel1a receptor expression in the ovaries of rats in the P + Domp group.
The PRL-II receptor did not present significant differences in the ovaries of the rats between the experimental groups, but the expression of this receptor was numerically elevated in the ovaries of the rats of the group P + Mel. According to Lee et al. (2003) Pinealectomized Rats submitted to hyperprolactinemia present numerically low expression of the receptor PRL-II during the beginning of gestation, reaching a greater expression when the placenta is formed. Reese et al. (2000) Studying mice (females) submitted to hyperprolactinemia report that the receptor PRL-II is expressed in the uterus. In addition, Jabbour; Critchley (2001) studying pinealectomized and melatonin-treated rats demonstrated that there was a greater expression of the PRL II receptor in the uterus of these animals, and that when they were still submitted to hyperprolactinemia the expression of the PRL II receptor It decreased, which may explain the values observed for the experimental group P + Domp and P + Mel + Domp.
The Hormonal analysis of serum estrogen levels in the rats of the experimental groups confirmed the results already cited in the literature, because according to Torres-Farfan et al. (2003) pinealectomized rats treated with melatonin and/or subjected to Hyperprolactinemia does not present alterations in the production of estrogen in the early stages of gestation.
It is established that oral administration of melatonin at a dose of 4 mg/kg in pinealectomized rats causes an abrupt increase in the concentration of prolactin after 30 min of application (DIAZ et al., 1999). Studies conducted with pregnant ewes, treated daily with melatonin at a dose of 2mg/kg showed that this hormone was able to elevate the plasma concentration of PRL (DICKS, 2000). In addition, studies with goats reported that the treatment with exogenous melatonin is able to delay the fall time of the PRL levels, keeping them elevated (SANTIAGO-MORENO et al., 2004). However, the elevated levels of prolactin observed in the present study were only seen in the rats of the P + Domp and P + Mel/Domp groups, being more pronounced in the latter. As there was no increase in the PRL levels in the rats of the P + Mel groups, we can suggest that the amount of melatonin administered in the present study at the beginning of pregnancy was not sufficient to maintain elevated PRL levels and that there seems to be a synergistic effect of Melatonin and Domperidone.
The levels of progesterone in rats of groups P and P + Domp showed a significant decrease of this when compared with the other rats of the experimental groups. According to Grasselli et al. (2008) Ewes treated daily with 2, 5mg/Kg of melatonin promoted the resumption of ovarian activity, stimulated by the increase of progesterone. In Addition, Coelho et al. (2006) reported that pinealectomized lambs and treated with melatonin at a dose of 0, 8mg/kg showed an increase in the plasma concentration of progesterone. Bonnefond et al. (2006) also reported that the increase in progesterone may be influenced by the presence of melatonin. And McConneli and Hinds (1995) reported that high levels of prolactin during pregnancy may cause a negative feedback on the increase in progesterone. This suggests a regulating effect of melatonin on the production of progesterone during pregnancy, with or without prolactin.
It is Known that the reduction of circulating melatonin caused by pinealectomy induces an increase in oxidative stress, but does not inhibit the implantation of the blastocyst in the endometrial epithelium.
Pinealectomized rats present a reduction in the number of implanted sites. Whereas, pinealectomized rats subjected to doses of 10mg/kg of exogenous melatonin for 3 weeks present an increase in the rate of blastocyst implantation, by stimulating the production of gonadotrophic hormones, with consequent stimulus of the production of Progesterone and greater development of Corpus lutetes (KOCH et al., 2010). Sandyk et al. (2000) also reported that when non-pinealectomized pregnant rats are treated with melatonin there is a stimulus in the production of progesterone, which prevents the immunological rejection of trophoblasts, thus facilitating the implantation, and stimulating the greater Corpus luteum Development. This fact was evidenced in the results presented in the present study, because the rats of the experimental groups P and P + Mel presented the lowest means of implanted sites and less development of them, while the rats of the experimental groups P + Domp and P + Mel/Domp exhibited a greater number of implantation sites and a greater amount of corpus lutetes.
Thus, it seems that although the literature reports that melatonin administration increases the implantation rate, the dosage of 200μg/100g of melatonin was not sufficient to stimulate such increase.
In relation to cytotrophoblasts cells It was evidenced that these in the P + Domp group were more Mice submitted to hyperprolactinemia present proliferation of trophoblastic cells, with an increase in the mitotic index as well as its volume. Rossi et al. (2002) showed that mice submitted to hyperprolactinemia had more proliferated trophoblastic cells and with a higher volume, being morphologically more developed, in the pregnancy phase.
Thus, it is concluded that Hyperprolactinemia promotes an increase in the expression of the Mel1a receptor in the ovaries, at the beginning of pregnancy in pinealectomized rats, however without altering the expression of the PRLII receptor. In These animals, prolactin levels were kept elevated regardless of the presence or absence of melatonin, but there seems to be a synergistic effect when melatonin is associated with hyperprolactinemia and that melatonin is a preponderant factor for Maintenance of progesterone levels. Prolactin seems to play an important role during the implantation process. Furthermore, treatment with melatonin seems to retard the implantation process in the uterine wall as well as provoke a lower development of corpus luteum in the ovary, having treatment with domperidone contrarian effects in non-seasonal animals.

Conclusions
In conclusion, the prolactin levels were kept elevated regardless of the presence or absence of melatonin, but there seems to be a synergistic effect when melatonin is associated with hyperprolactinemia and that melatonin is a preponderant factor for Maintenance of progesterone levels. Prolactin seems to play an important role during the implantation process.
After the formation of the respective groups, the rats were treated for 7 days. The Experimental Protocol was approved by the institutional Ethics of Committee to Universidade Federal Rural de Pernambuco n º. 23082.009629/2010.

Pinealectomy
The Pinealectomy was performed in animals previously anesthetized with pentobarbital 40mg/kg intraperitoneal route LACERDA et al., 2007). Afterwards, the trichotomy and asepsis of the dorsal area of the head were performed. An incision was made on the dorsal midline of the head with a low-speed micromotor and a dentist drill No. 05, a circular fragment of the cap was removed. This fragment was placed in saline solution at 0.9%. After removal of the bone fragment, venous sinus ligation was performed for the removal of the pineal (KUSZAK; RODIN, 1977). Next, the bone fragment was replaced and the skin was sutured. For Post-surgical pain prevention, buprenorphine (Tengesic®) was administered at a dose of 0.05 mg/kg, by subcutaneous route every 12 hours and 30mg of Ampicillin intramuscularly (MI), both for a period of five days, such as pain prevention and encephalopathy Resulting from surgical procedures (i.e., pinealectomy and Shampinealectomy) (GUVENAL et al., 2001;KREIMER et al., 2005;YAMATOGI et al., 2005).

Colpocytological Examination
For

Weight of the Ovaries and Count and Weight of the Implantation Sites
The ovaries and uterus were weighed on a precision analytical scale. Subsequently, the implantation sites were counted.

RNA Extraction
The Samples of the ovaries, duly identified, were packaged in a freezer-80 º C for posterior RNA extraction. At the time of extraction, the sample was transferred to a N2-cooled mortar to undergo a maceration process. After This step, the material was transferred to an Eppendorf tube, receiving as a complement of Trizol 1000uL. RNA was extracted under the conditions recommended by the manufacturer.
The extracted RNA was quantified in Spectrophotometer (Bionate3 of Thermoscientific) and analyzed in agarose gel at 1.5%, (Pronadisa) stained with Syber Green II (RNA-LCG Biotecnolia), analyzed in ultraviolet light and photographed (Olympus -Digital Chamber -C-7070 Wide) to check its quality.

Reverse Transcription (RT-PCR)
The first cDNA tape synthesized by reverse transcriptase was binded to the RT-PCR system using an adapator 3 ´-Oligo (dT) 18 (Quiagen), 1 Μ g total RNA and 1U reverse transcriptase (  1 -The ASSIGNOR (S) declares that the work referred to above is original and of its exclusive authorship; that it is not being submitted to any means of communication for the purpose of publication and that all authorizations have been obtained for citing sources, being responsible to the ASSIGNEE for any violations or offenses against the rights of third parties, possibly resulting from the content of the work or characterized by it, it is copyright or any other rights, exempting the ASSIGNEE from any responsibility or participation in these acts and their consequences and effects. Thus, as the holder (s) and holder (s) of all the copyright in the work, the ASSIGNOR (S) hereby assigns and transfers to the ASSIGNEE, on a definitive basis, exclusive and free of charge, the totality of the author's patrimonial rights over it, including the images of its property contained in the work.
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Ass. ASSIGNOR: -Availability of supporting data The researchers present in this current work guarantee, attest and confirm all the necessary data and material availability that the journal Biology Direct needs. Expression of the Mel1a receptor in the ovaries of the rats of the different experimental groups presenting mean ± standard deviation (P = 0,0028) (Sham-pinealectomized (Sham); pinealectomized (P); pinealectomized and treated with melatonin (P + Honey); Pinealectomized and induced to Hyperprolactinemia by Domperidone (P + Domp); pinealectomized, treated with melatonin and induced to hyperprolactinemia by Domperidone (P + Mel + Domp); treated with saline + ethanol (placebo honey); Treated with saline solution (placebo domp). . * Averages followed by the same letter do not differ significantly from each other by the Kruskal-Wallis test with post-hoc Dunn (p < 0.05).

Figure 2
Expression of the receptor PRL-II in the ovaries of the rats of the different experimental groups presenting mean ± standard deviation (P = 0,7601) (Sham-pinealectomized (Sham); pinealectomized (P); pinealectomized and treated with melatonin (P + Honey); Pinealectomized and induced to Hyperprolactinemia by Domperidone (P + Domp);p inealectomized, treated with melatonin and induced to hyperprolactinemia by Domperidone (P + Mel + Domp); treated with saline + ethanol (placebo honey); Treated with saline solution (placebo domp)). * Averages followed by the same letter do not differ significantly from each other by the Kruskal-Wallis test with post-hoc Dunn (p < 0.05).