The role of tyrosine hydroxylase within dapoxetine-assisted therapy against premature ejaculation

There are several investigations that have revealed that cerebral dopamine (DA) plays a pivotal role in the occurrence of premature ejaculation (PE). Although tyrosine hydroxylase (TH) is an essential enzyme for the synthesis of DA, only few investigations have described the role of TH in regulation mechanisms for ejaculation till now. To investigate whether there is a correlation between TH expression level in the brain and different ejaculation behavior in rats. Then explore whether the TH expression in the brain will change after acute dapoxetine treatment in rats with Rapid ejaculation. Rats (male, S-D rats, 6–8 weeks) were separated into three groups based on their ejaculation frequency: Rapid, Normal, and Sluggish. Expression level of DA in the brain was determined by enzyme-linked immune sorbent assay (ELISA) kit, TH expression level in the brain was determined by immunohistochemistry and Western Blot (WB) techniques. Among the three groups, DA and TH expression level were the highest in the Rapid ejaculation group, while the lowest was the Sluggish ejaculation group. The results also showed that TH level was positively associated with ejaculation frequency (r = 0.8038, P < 0.001) and negatively associated with ejaculation latency (r=-0.6199, P = 0.018). Furthermore, acute dapoxetine therapy in rats with Rapid ejaculation downregulated TH level in the brain. Changes in ejaculation behavior were significantly linked with TH level. Upregulated TH in selected brain regions related with ejaculation could cause rapid ejaculation. The effect of dapoxetine in prolonging ejaculation could be related to TH downregulation within the brain.

dopaminergic drugs facilitate ejaculation [13], while thioridazine [14] and chlorprothixene [15] delay ejaculation by blocking central DA receptors. Previous studies have found that dysregulations within central DA levels are an important factor affecting ejaculation [16]. Tyrosine hydroxylase (TH) is present in all dopaminergic cells, which is a highly specific and stable protein with a reported half-life of 17-34 h in various catecholamine-producing cells [17,18].TH is a crucial enzyme in the brain which can catalyze DA synthesis, thereby affecting the regulation of ejaculation [19].
Selective serotonin reuptake inhibitors (SSRIs) can reduce the reuptake of 5-HT through presynaptic nerve terminals by blocking the 5-HT transporter (5-HTT) selectively [20]. Dapoxetine, a kind of short-acting SSRIs, is the sole officially licensed oral medication for the treatment of PE in several countries [21,22]. Previous studies have confirmed [23] that dapoxetine can improve quality of sexual activity in PE patients. Moreover, several studies suggest [24][25][26] that SSRIs can also inhibit dopaminergic neurotransmission by affecting dopamine secretion / recapture or through dopaminergic receptors. Since TH plays an important role in the synthesis of DA, we speculated that the regulation of TH level by SSRIs in the brain might be a way to affect the synthesis of DA.
PE, as a type of sexual dysfunction condition, has become a prominent study topic in recent years, although there are few overall studies and lacking in research based on large-scale, natural screening of animal models. Ejaculation research can only be conducted on animals due to ethical reason. The genomics and biological behavioral features of rats are highly comparable to human, while rats are also relatively inexpensive and easy to procure. On considering the above factors, there are clear benefits to replace and validate PE using rat models. To further study the pathophysiology of PE, male rat models were applied to simulate human rapid ejaculation / PE in this study.
Presently, there is no literature suggesting the role of TH within the regulation of ejaculation. This study probed whether rats with different ejaculation behaviors were correlated with TH expression in the brain, and evaluated TH expression in different brain areas. Meanwhile, this study also probed whether dapoxetine could affect ejaculation behavior by downregulating TH level within the brain. Therefore, this research could open up a novel possibility for exploring the pathogenesis of PE.

Experimental animals
The experimental animals were 7-8week-old S-D rats (110 males, 250-350 g and 110 females, 200-300 g), obtained from the Animal Management Center of Anhui Medical University, and housed under routine conditions (light cycle from 7:00 am to 7:00 pm, 20 ± 2℃, relative humidity of 40 ± 5%). To reduce error, all rats were reared for at least two weeks to acclimatize prior to commence experiments. Purified water and standard laboratory feeds were readily available. The bilateral ovaries of female rats were removed post-anesthesia with pentobarbital sodium (30 mg/ Kg), and allowed to recover for at least for one week prior to behavioral test. Estradiol benzoate (10 µg, dissolved in 0.1mL sesame oil, incubated at 60 °C for one hour) was injected subcutaneously to female rats, 48 h prior to mating experiment and progesterone (50 µg) was administered (as described above) 4 h prior to the mating experiment, to receive the artificial estrus. Each female rat was induced once-weekly, which would be replaced if the estrus did not succeed.

Behavioral tests
Prior to commence mating experiment, male rats were transferred to acrylic boxes (50 × 40 × 30 cm) alone, to adapt at dim lighting for 15 min. Consequently, the male and female rats were allowed to mate freely within the cage for 60 min. Mating experiments and sexual behavior indicators were monitored by motion camera (EZVIZ S2®). During the sixweek (T1-6) mating experiment, each male rat mated with a fixed female rat only once-weekly. In order to obtain stable sexual behavior indicators, the first three weeks (T1-3) were deemed as the adaptation period. Upon completion of the six-week observation period, the following sexual behavior indicators were recorded: mount latency (ML) was the time from when the rats were transferred into one cage to the first mount; intromission latency (IL) was the time from when the rats were transferred into one cage to the first intromission; ejaculation latency (EL) recorded the time from first intromission to ejaculation; mount frequency (MF) were the times of mount prior to each ejaculation; intromission frequency (IF) were the times of intromission prior to ejaculation; ejaculation frequency (EF) were the times of ejaculation within one hour; postejaculatory interval (PEI) recorded the time from ejaculation to the next intromission; intromission ratio (IR) was calculated by IF/(IF + MF). This study divided the male rats into three groups using mean EF value of the final three mating experiments (T4-6). Male rats with EF value within lowest 10% were classified as the Sluggish ejaculation group, the intermediate 45-55% was the Normal ejaculation group, while the highest 10% for EF value were classified as Rapid ejaculation group.

Treatment
According to the above behavioral tests, this study selected Rapid ejaculation rats (n = 6) from a novel group of rats (n = 60), labelled the dapoxetine and 0.9% saline water into serial number as per randomization list, then the above selected rats were consequently divided into two groups using the random number table: treatment group (n = 3, dapoxetine, 60 mg/Kg) and control group (n = 3, 0.9% saline water), besides, the research staff in this study were not involved in the conduct of this trial. The two groups underwent the final mating test again within an identical environment. The treatment group rats were administered dapoxetine by gavage (60 mg/Kg, dissolved in 0.9% saline water) 3 h prior to the mating experiment. Meanwhile, the control group rats were administered 0.9% saline water in the same way. Finally, these rats were exposed to trans-cardiac perfusion with 200 mL 0.9% saline water post-mating experiment, consequently having the brain removed and stored at -80 °C.

DA detection by ELISA
Brain tissue samples were fragmented by ultrasound, and consequently centrifuged to separate supernatant (2000 g/ min, 10 min, 4 °C). ELISA kit was used for the detection of DA level in brain tissue supernatant, as per kit protocol.

TH protein analysis: western blotting
Brain tissue was removed post-euthanasia and consequently stored at -80 °C. Proteomic level was determined by BCA protein determination kit®(P0010S). The protocol was as follows: Protein was separated by 10% SDS polyacrylamide gel electrophoresis, consequently transferred to nitrocellulose (NC) membrane. Non-specific binding was blocked by incubating with 5% milk in TBS-T for 1 h at 25 °C. The membranes were subsequently incubated with antibodies against TH (1:200) and GAPDH (1:2000), and consequently detected using peroxidase-conjugated rabbit anti-goat IgG (1:5000) and goat anti-rabbit IgG (1: 5000), enhanced using chemiluminescence detection reagents (Beyo™ ECL Moon®). The specific TH proteomic signal was detected by the chemiluminescence imaging system ChemiScope 5600®(Clinx Science Instruments™, China™and analyzed by Image Pro Plus 6.0® (National Institute of Health, USA).

Immunohistochemistry
This experiment was conducted to detect the distribution of TH in various brain regions. Firstly, the paraffin embedded tissue sections (4 μm) were deparaffinized by xylene, and consequently rehydrated through an ethanol gradient (100%, 95%, 85% and 75%, 1 × 5 min each). Sections were exposed to acetone for 10 min at -20 °C, and consequently retrieved antigens using citric acid in a microwave oven at full-intensity for 3 min until boiling point was reached, and subsequently at 20% intensity for 10 min. Following such steps, all sections were blocked by 10% normal goat serum (90 min), then washed with deionized water. Consequently, the sections were incubated with antibodies as follow: TH(1:200, AF6113, Affinity®)overnight for 4 °C, thrice-washed in PBS, incubated with biotinylated goat anti-rabbit antibody for 90 min in a humidity chamber, then washed. Avidin-biotin peroxidase was used to detect the antibody-antigen system. Stained sections were photographed through a microscope (DM1000®, Leica™), and the relevant parts of samples were collected by the Leica Application Suite® imaging system, and finally quantified through Image Pro Plus 6.0®.

Statistical analysis
All data were analyzed by SPSS 25.0® software. The Shapiro-Wilk test was used to evaluate whether the data conformed to normal distribution. T test was used for comparison between two groups, One-way ANOVA followed by Bonferroni post hoc test was used for multiple comparison. The data reflected mean ± standard deviation (SD). P < 0.05 was considered to be statistically significant.
group (0.9% saline water). The sexual parameters of the treatment group altered significantly compared to the control group, which were showed in Table 2. The treatment group showed less EF (P = 0.003) and longer EL (P = 0.032) compared to the control group. ML and IL were considerably prolonged post-dapoxetine treatment (P < 0.0001), although there was no statistically significant difference in MF (P = 0.451) and IF (P = 0.642) between these two groups.

Expression of DA and TH in rats with differing ejaculation behaviors
Nine rats (3 cases of Rapid, Normal, Sluggish, separately) were used for assessing DA expression based on ELISA technique, illustrated in Fig. 1. The results indicated that there were statistical variations within DA expression level among the three groups, the concentration of Rapid, Normal and Sluggish groups decreased accordingly.
Consequently, Immunohistochemistry was performed on 9 rats (3 cases of Rapid, Normal, Sluggish, separately) to detect the expression of TH protein. Western blotting (WB) was performed on 9 rats (3 cases of Rapid, Normal, Sluggish, separately) to detect the expression of TH protein.
From the results that TH expression showed statistically difference among the three groups. Rats with Rapid ejaculation had a higher level of TH protein than Normal rats and rats with Sluggish ejaculation (Fig. 2). The results (Fig. 3) for correlation analyses showed that TH expression was positively linked with EF (r = 0.8038 P < 0.001) and negatively correlated with EL (r=-0.6199 P = 0.018). The expression level of TH within hippocampus and raphe nucleus among the three groups was shown in Fig. 4. In addition, the TH expression level of Rapid, Normal and Sluggish groups decreased with statistical significance.

Effect of dapoxetine on DA and TH levels in the brain of rapid ejaculation rats
Post-treating with dapoxetine, DA and TH expression in Rapid ejaculation rats was considerably lower than the control group (Figs. 5 and 6).

Behavior characteristics in male rats
Eighteen rats were removed from the 6-week mating experiment since they did not demonstrate any insertion behavior (16.36%,18/110). Post-obtaining the mean EF from the final three weeks (T4-6), rats were divided into three groups: Rapid ejaculation group(n = 12), Normal ejaculation group(n = 10™and Sluggish ejaculation group (n = 11). The sexual behavior parameters of different groups were showed in Table 1. Compared to Normal ejaculation group, Rapid ejaculation group had a shorter EL and more EF, whereas Sluggish ejaculation group had opposing features. Rapid ejaculation group showed the shortest ML, IL and EL among the three groups, with statistically significant variations. Likewise, the three groups had significant differences in MF and IF, although no significant changes in IR.

Effect of dapoxetine on sexual parameters of rapid ejaculation rats
To study the effect of dapoxetine on the regulation of ejaculation, the Rapid ejaculation rats were divided into two groups: treatment group (dapoxetine, 30 mg/rat) and control systems, the cause of premature ejaculation (PE) has not clear yet. More and more attention has been paid to the role of DA in the pathogenesis of ejaculation disorder [27,28].

Discussion
Ejaculation is a complex process involving numerous neural The results showed that the expression trend of TH in the hippocampus was similar to that in raphe nucleus within same group, and there were significant difference in TH expression among the three groups. Thereinto, the expression of TH was the highest in the Rapid ejaculation rats and lowest in Sluggish ejaculation rats. Based on these results, we hypothesized that the occurrence of PE could be related to the high expression of TH in corresponding brain regions. SSRIs are first-line drugs licensed for the treatment of PE, which can delay ejaculation by inhibiting the synthesis of DA [26], we speculated that TH could play an important role. However, the association between SSRIs and TH in the treatment of PE has not been studied. Dapoxetine, as a type of short-acting SSRIs, was administered to rats with Rapid ejaculation to observe the changes of TH expression in this study, so as to explore the association between SSRIs and TH. Compared with the control group (0.9% Normal saline), the expression of TH in the brain was significantly reduced post-treating with dapoxetine. Therefore, we hypothesized that dapoxetine reduced the synthesis of DA by downregulating TH, thus delayed ejaculation.
Nevertheless, there were still a few shortcomings in this study. First, we administered the dapoxetine to rats by gavage, although normal saline was administered in the same way to the control group, its effect on mating was unknown. Furthermore, at present, sexual behavior testing was only carried out on natural screening rats, which can be verified by gene knockout rats in the future.

Conclusion
The expression of TH in the brain is one of the key factors in regulating ejaculation. The TH expression in rats with Rapid ejaculation group was significantly increased compared with the Normal ejaculation group. Additionally, dapoxetine could delay ejaculation by downregulating TH expression in the brain. This research consequently suggests a novel concept and direction for the treatment of PE.
In this study, the TH expression level in brain of rats with different ejaculation behavior did exhibit variations, comparable with DA expression level within the brain. These results are in agreement with the previously reported data, that rats with rapid ejaculation showed an increased TH level in the dopaminergic pathway [29]. Therefore, we speculated that changes of ejaculation behavior could be related to TH level in the brain.
In our study, male rats were divided into three groups according to mean EF values (T4-6): Rapid, Normal, and Sluggish. There were statistical differences among the three groups in mean values of ML, IL, EL. As a sexual-drive index, the ML in Rapid ejaculation group was shorter than Normal group, while the ML was the longest in Sluggish ejaculation group among these three groups. Putnam SK et al. [30] showed that DA related to the initiation of sexual behavior, therefore, we speculated that the reason for the shortening of ML in Rapid ejaculation rats could be caused by the upregulation of DA in the brain. Furthermore, as one of the indicators to assess erectile function [31], IR showed no statistical difference among three groups, which could eliminate the effects caused by erectile dysfunction (ED).
Several studies have found that DA level was closely linked to PE [8,32]. The mesolimbic dopaminergic pathway plays a facilitative role in male sexual behavior [33]. Within this study, Rapid ejaculation rats showed the highest DA expression, while the Sluggish ejaculation rats showed the lowest DA expression level. Furthermore, Tyrosine hydroxylase (TH), as a key enzyme in DA synthesis, promotes the synthesis of DA. According to another study [34], inhibiting the phosphorylation of TH at Ser40 could decrease the DA expression. It is reasonable to conclude that DA expression level is affected by TH expression in the brain. This hypothesis is also supported by this study. The trend of TH expression was similar to that of DA among three groups, TH expression was higher in Rapid ejaculation group and lower in Sluggish ejaculation group compared with Normal ejaculation group. In addition, TH level was positively correlated with EF and negatively correlated with ML in sexual behavior index of rats. Based on the results, we speculated that delay of ejaculation might be related to the low TH expression in the brain.
It is well known that ejaculation is a centrally integrated and highly coordinated process that involves many areas of the brain. The amplitude of low-frequency fluctuation (ALFF) of patients with PE was reduced significantly in bilateral hippocampus through the assessment based on imaging technology [35]. Meanwhile, compared with the healthy control group, the volume of gray matter in striatum was significantly reduced in LPE patients [36]. In this study, the expression of TH protein in the brain regions associated with ejaculation was detected by immunohistochemistry. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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