MELATONIN RECEPTORS SUBTYPES (MT1 AND MT2) IN THE UTERUS MASCULINUS OF MATURE MALE EUROPEAN BISON. BIOLOGICAL
AND SEASONAL REPRODUCTIVE ROLE

European bison (Bison bonasus, Linnaeus 1758) is an endangered species and has been placed on the Red List of Threatened Species (RL: EN A2ce, C2a) by the International Union for Conservation of Nature and Natural Resources. Although the population of European bison has grown over the last decades, it is still unknown how the fate of the Europe's largest mammal will be. Intensive activities aimed at restoring European bison population led to its increase, but more and more often, the consequences of reproduction of closely related individuals appear. The most common defects in the male reproductive system are asymmetry and hypoplasia or atrophy of the testes (1). The quality of the semen has also been reduced (2, 3). Interestingly, in the male reproductive tract of adult European bison an additional structure called a vestigial uterus (uterus masculinus) is located. The mentioned structure has been already found in some males of a few mammalian species, including hamster (3), horse and donkey (4), cat (5, 6), dog (7) as well as people (8). This phenomenon is known as the persistent Mullerian duct syndrome - PMDS (8, 9). It is still unknown what the role of uterus masculinus is, but it seems to perform secretory function or may be the target site for hormones and thereby affect the regulation of normal reproductive function.

European bison both, males and females, attain sexual maturity at the age of 3 – 4 years and are characterized by seasonality in reproduction: with the period of reproductive activity in August and September (10). One of the most important factors that determine the seasonality of animal reproduction is melatonin (N-acetyl-5-methoxytryptamine) (11, 12). This molecular indoloamine is secreted by the pinealocytes of the pineal gland, however it can also be synthesized in many tissues of the body (10). Melatonin's mechanisms of action are different, but the regulation of reproductive seasonality seems to be mainly related to melatonin MT1 and MT2 membrane receptors (13).

Therefore, the purpose of this study was to determine whether myometrial and endometrial cells of uterus masculinus could be the target site for melatonin, since the presence of MT1 and MT2 receptors in the uterus masculinus may be of cognitive significance for mechanisms regulating seasonal reproduction. Differences between MT1 and MT2 expression in the uterus masculinus between November and December will allow to observe fluid changes occurring after the period of highest reproductive activity but it may also be indicative of protective function of melatonin (14). Understanding all of the functions of testes, epididymis, accessory glands and uterus masculinus of European bison can contribute to maintaining its population. In addition, due to the clearly marked seasonality in reproductive activity of male bison, this is an excellent organism for studies related to the regulation of seasonal reproductive processes in animals.

MATERIALS AND METHODS

Animals and tissues

Biological material was obtained during selection eliminations in the seasons 2010/2011, 2011/2012 and 2012/2013 in the Bialowieza National Park (located between 23º31' and 24º21' east longitude and between 52º29' and 52º57' north latitude) mainly for several reasons, including injuries or poaching. The animals were culled according to approved guidelines for the ethical treatment of animals in accordance with Polish legal requirements. Tissue samples of uterus masculinus (Fig. 1, the place where the tissue was taken for analyses) were collected from sexually mature male European bisons and divided into 2 groups depending on the month when elimination occurred (n = 6 animals per group in November and in December). One part of collected tissues was fixed in 4% paraformaldehyde to determine the histological structure of the examined organ and the second was shocked-frozen in liquid nitrogen (–196ºC) and stored at –80ºC, until further analysis.

Fig. 1.Schematic representation of uterus masculinus anatomy in European bison with male urinary and reproductive organs; U, ureter; R, right horn of the uterus; L, left horn of the uterus; C, corpus of the uterus; B, urinary bladder; V, vesicular glands; Ur, urethra; arrow shows the area from which the analyzed tissue was obtained.

Hematoxylin and eosin staining (H&E)

Uterus masculinus tissues were fixed in 4% paraformaldehyde and embedded in Paraplast Plus (Sigma Aldrich, Saint Louis, MO, USA). Next, sections of 5 µm thick were stained with hematoxylin and eosin following standard procedures (10, 15). The microphotographs were taken using Olympus BX43 microscope equipped with soft cellSens Dimension software.

Total RNA isolation and cDNA synthesis via reverse transcription PCR (RT-PCR)

Total RNA was isolated using column-based kit (A&A Biotechnology, Gdynia, Poland) according to manufacturer's protocol. Briefly, 20 mg of tissues were homogenized in liquid nitrogen and placed in tubes with fenozol. Next, chloroform was added and the tubes were centrifuged for 10 min at 12,000 × g. Isopropanol was added to obtained solution and loaded onto minicolumns. After centrifugation, for 1 min at 12,000 rpm, the minicolumns were washed three times with wash solution and re-centrifuged for 1 min at 12,000 × g. Dry minicolumns were placed to new tubes, and RNA eluted with 50 µl of sterile water. RNA integrity was assessed by 2% agarose gel electrophoresis, while RNA concentration was measured using Nano Drop (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Next, 1 µg of total RNA was used to prepare cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Foster City, USA) in a total volume of 20 µl according to manufacturer's instructions. Briefly, freshly prepared 10 µl of master mix (2 µl of 10×RT Buffer, 0.8 µl of 25×dNTP Mix, 2 µl of 10×RT Random Primers, 1 μl of Multiscribe Reverse Transcriptase and 3.2 µl H2O) was mixed with 1 µg RNA diluted to 10 µl with sterile water. Reverse transcription reaction was carried out in 3 steps (25ºC - 10 min, 37ºC - 120 min, 85ºC - 5 min).

MT1 and MT2 expression analysis

Primer sequences of β-actin, MT1 and MT2 were prepared according to Wang et al. (Table 1) (16). The reaction mixture for each PCR analysis contained 5 µl 2×PCR TaqNova-RED Master Mix (DNA Gdansk; Gdansk, Poland), 4 µl of primers (2 µl of each 1 µM forward and reverse primer) (Genomed; Warsaw, Poland) and 1 µl of cDNA (10 ng). Reverse transcription reaction was carried out for 35 cycles of denaturing at 95ºC for 45 s, annealing at 49ºC (MT1 and MT2) or 55ºC (β-actin) for 45 s and extension at 72ºC for 45 s, followed by final extension at 72ºC for 10 min. PCR products were electrophoretically detected on 2% agarose gel after ethidium bromide staining. The optical density was calculated using GelQuantNET software, numerically expressed as the relative density and all results were normalized to the expression of the β-actin.

Table 1. Primer sequences for genes encoding melatonin receptors (MT1 and MT2) and β-actin (ACTB) used in this study.

Protein preparation and dot blot analysis

The uterus masculinus tissues were homogenized using high-speed benchtop FastPrep 24MP homogenizer system and 2% SDS for four rounds, 20 s each. The samples were then centrifuged at 15,000 × g for 10 min, to remove cellular debris. Total protein concentration was determined using BCA assay (Thermo Scientific; Warsaw, Poland). Bovine serum albumin (BSA) was used as a calibration standard. 30 µg of proteins were used for the dot blot analysis. First proteins were loaded onto activated PVDF membranes and next dried for 30 min at RT. Membranes were blocked in 1% BSA in TBST (20 mM Tris-HCl pH 7.5, 137 mM NaCl, 0.1% Tween 20) at RT for 30 min and then incubated with antibodies (for 1 h): rabbit polyclonal anti-MT1, 1:100 (bs-0963R; Bioss; Woburn, USA), rabbit polyclonal anti-MT2, 1:200 (PA5-76652; Thermo Scientific; Warsaw, Poland) or rabbit polyclonal anti-β-actin, 1:10,000 (PA1-16889; Thermo Scientific; Warsaw, Poland) prepared in 1% BSA in TBST. Then, after 4 times washing with TBST, membranes were incubated for 30 min at RT with secondary HRP labeled anti-rabbit antibody, 1:80,000 in 1% BSA in TBST (Sigma Aldrich; Darmstadt, Germany) and washed four times with TBST again. Enhanced chemiluminescence detection kit and chemiluminescent imaging system Fusion Fx7 was used to visualize dots (BioRad; Hercules, USA). ImageJ software was used to calculate protein levels and measure the optical density of the MT1 and MT2 dots. Freeform drawing tool was used to evaluate each drop. The integrated fluorescence densities, i.e. the sum of all pixel values in the selected area, were evaluated and equivalent to the product of the area and the average gray value. The integrated fluorescence density as a relative fluorescence units is presented. All results were normalized to β-actin.

Statistical analysis

Analyzed data were presented as mean ± SD. Statistics were calculated using GraphPad Prism. In this study, we compared two independent groups with the same sample size (n = 6), therefore unpaired t-test was used. The distribution of populations was normal. Analyzed standard deviations were not equal, so Welch's correction was applied. P values < 0.05 denoted statistical significance and are displayed as *P < 0.05, **P < 0.01, ***P < 0.001.

RESULTS

The histomorphology of uterus masculinus

The construction of the male bison's uterus is structurally similar to female and consists of three zones: i.e. myometrium, endometrium and adventitia. On the basis of microscopic observations, the myometrial layer seems to be thinnest. Myometrium was formed with myocytes in three layers: inner - oblong, middle - circular and outer - oblong. Blood vessels of the muscle type were visible in this layer. Endometrium (the mucous layer) was covered with a single-layer epithelium based on the basal membrane. The mucosal membrane formed a connective tissue with a loose collagen fibers. In the endometrium, the secretory glands with the inside large lumen were located. They were lined with one layer of cylindrical epithelium. Fibroblasts dominated the submucosal layer. Single mononuclear cells (lymphocytes, macrophages, mast cells) and blood vessels were also present. The outer layer, the adventitia, formed a connective tissue with a lightweight collagen fibers. It was covered with a single layer of epithelial cells. On the basis of microscopic observations of a minimum 6 sections of examined uterus masculinus tissues, no significant differences were found in the histological structure between November and December (Fig. 2).

Fig. 2.Hematoxylin and eosin (H&E) stained sections of uterus masculinus of European bison collected in November and December. M, myometrium; E, endometrium; Ep, epithelium; Gl, secretory glands; L, lumen; V, blood vessel. Magnification of the objective lens 10×, 20× and 40×.

Malatonin MT1 and MT2 mRNA expression in the uterus masculinus of male European bison

In the uterus masculinus of the European bison 2.782-fold higher expression of MT1 mRNA receptors was observed in November than in December and the difference was statistically significant with P < 0.01 (Fig. 3A). The comparable results were also found for MT2 receptors, where the mRNA expression was 1.695-fold increased in November than in December (P < 0.001) (Fig. 3B).

Fig. 3. Expression of MT1 and MT2 mRNA in uterus masculinus tissues collected from European bison in November and December assessed by RT-PCR method with representative images of gels. RNA was isolated from obtained tissues using column-based kit, reverse transcribed to cDNA and then RT-PCR was performed using specific primers for MT1, MT2 and ACTB. ACTB was used as a housekeeping gene. Optical density of the bands was calculated with the GelQuantNET software and results were normalized to β-actin. Values are presented as mean ± SD of n = 6. **P < 0.01, ***P < 0.001, no indication - no statistical significance.

Melatonin MT1 and MT2 protein synthesis in uterus masculinus of European bison

The measurement of MT1 protein receptors synthesis in uterus masculinus showed 3.188-fold higher level in November than in December (P < 0.001) (Fig. 4A) (molecular weight of MT1 - 38 kDa). Furthermore, the comparison of MT2 protein receptors synthesis in uterus masculinus in November and December uncovered its increased level in November where the measurements indicated a 2.382-times higher value (P < 0.001) (molecular weight of MT2 - 15 kDa) (Fig. 4B).

Fig. 4. Changes in the levels of MT1 and MT2 proteins amounts analyzed by dot blot method in uterus masculinus tissues collected in November and December. Protein lysates were obtained by tissues homogenization, concentrations verified by BCA method and equal amount of each sample was loaded onto membrane, blocked, incubated with anti-MT1 and anti-MT2 primary, anti-rabbit secondary antibodies and detected using imaging system Fusion Fx7. Optical density of drops was calculated with ImageJ. Values are presented as mean ± SD of n = 6. ***P < 0.001, no indication - no statistical significance.

DISCUSSION

The wide range of functions performed by melatonin in females allows us to assume it’s important role in the male reproductive system, also in the bison with clearly marked seasonality in reproduction. In support of such conclusions, the results of studies in females can be cited here, where melatonin upregulates LH by MT1 and MT2 in the ovary’s granulosa cells of human. Therefore, it stimulates luteinization leading to secretion of progesterone, which is crucial in maintaining the corpus luteum during pregnancy (17, 18). Melatonin was also found to affect folliculogenesis, oocyte maturation and female steroidogenesis (19, 20). MT1 expression is upregulated in ovaries and uteri of melatonin-treated rats and regulate sex steroid receptors in female reproductive tissues (21, 22). Furthermore, melatonin might have some role in fetal development (23, 24), where as a small lipophilic molecule crosses the blood barrier of the placenta, protects against free radicals, increases the amount of antioxidants, limits the level of oxytocin, as well as raises and maintains the production of progesterone (25). On the other hand, in males exposed to night pollution, a significant decrease in urinary 6-sulfatoxymelatonin concentrations has been associated with changes in daily rhythm profiles and with activation of reproductive function was observed (26).

Uterus masculinus occurs in male European bison very often and is considered as a disorder associated with development and as a result of established pathology. Formation of the uterus masculinus has been linked with insufficiency of anti-Mullerian hormone (AMH) during testis development in fetus (27). As a result of this situation, male genital organs are developed, but female genital organs are also formed (28). In male European bison, uterus masculinus is large and occurs in many individuals so it seems to perform specific functions e.g. can affect seasonality in reproduction by the light regulation. The reproductive system might be informed through environmental signals which regulate the reproductive system, providing the organisms with seasonal reproduction favorable conditions for the rearing of the offspring (29, 30). In animals with seasonal reproduction, melatonin is a key regulator of reproductive processes (25, 31). In addition, it has been shown that not only the level of melatonin, but also three genes encoding enzymes involved in pineal melatonin biosynthesis are upregulated seasonally (32).

Our experiment has been completely unique due to the lack of any information in this topic as well as the unavailability of biological material. In this study, for the first time, we provide data about the differential level of MT1 and MT2 receptors expression in European bison uterus masculinus. Our results have demonstrated that the expression of mRNA and protein synthesis for MT1 and MT2 receptors in adult male uterus masculinus has been upregulated during the period following the highest reproductive activity. That may indicate that melatonin is one of the most important regulators in the seasonal reproduction of male European bison. Membrane receptors for melatonin MT1 and MT2 have been also found in secretory glandular cells and myometrium cells of uterus in female, where they are responsible for potentiation of contractility (33) and inhibition of cAMP signaling (34). Also, it has been shown that polymorphism of MT1A gene may also have an impact on seasonal reproductive activity (35). Because they play such an important role in female uterus, we suggest that their presence is associated with a significant role in the uterus masculinus of male European bison, too. It has been known, that melatonin interacts with the reproductive system through melatonin receptors and the supreme regulatory function in this area is the hypothalamic-pituitary-gonadal axis (36, 37). So it seems that melatonin can be involved in the local reproductive processes of the European bison reproductive system by acting with the MT1 and MT2 receptors of the uterus masculinus. We have observed increased expression of MT1 and MT2 mRNA as well as protein synthesis in the uterus masculinus in November when compared to December. Thus, in November, melatonin may be more active in local processes, play protective action against oxidative damage and support the reproductive system of the adult male European bison. Melatonin as a potent antioxidant (sweat free radicals) can protect against cell death and oxidative stress (38, 39). It has been also noted that in uterus exposed to continuous light, the apoptosis may decrease (38). In addition, in vitro and in vivo studies have showed a protective function of melatonin against the toxic ionizing radiation-mediated effects (19). Based on this data, we consider that melatonin probably plays protective function in uterus masculinus of the European bison and thus indirectly affects their whole reproductive tract. November in European bisons is a period associated with more intense processes but is intended to completely suppress reproductive functions in males during following months. On the other hand, reduced expression of MT1 and MT2 in December may be due to the reduction of energy expenditure in the reproductive system. June is the period when the highest reproductive activity of European bison starts, and our results are supported by the earlier presented paper by Levine et al. (40). Moreover, the quality of bison semen during the period of the highest reproductive activity is correlated with the function of melatonin. The concentration, count and motility of semen in the summer were lower than in the winter, when melatonin level was low. Thus, the low level of melatonin associated with long exposure to light during the summer, negatively affects the quality of semen (41). Since, it has been proven earlier, melatonin is derived not only from the pineal gland but may be locally synthesized in the gonads (42), so we supposed that the presence of MT1 and MT2 receptors in the uterus masculinus and other reproductive organs of European bisons (4) is associated with the paracrine effect of melatonin. Previously, it has been repeatedly demonstrated, that melatonin can indirectly affect many different systems and regulate mechanisms of their action (43). MT1 and MT2 are involved in circadian rhythm and correct reproductive functions regulation (44, 45). Our previous research has shown that in the testes of mature male bisons in December, e.g. after the period of the most intense reproductive activity, the level of mRNA expression of MT1 and MT2 receptors and protein synthesis is higher when compared to June, when the period of preparation for reproductive season begins in bisons. Melatonin affects the release of gonadotropins that determine the regulation of gonadal function through the hypothalamic-pituitary axis. If there is a lot of light and the night is short, the low secretion of melatonin affects the release of GnRH (gonadotropin-releasing hormone) and then gonadotrophins (41). Besides we suppose, that melatonin may also play an important immunoprotective function, which would secure male bison germ cells during intense reproductive activity (10, 46).

In conclusion, both melatonin receptors subtypes are of crucial importance in the uterus masculinus of male European bison. Our results open up new, interesting perspectives for further studies on the molecular mechanism of melatonin and the biological significance of its receptors. Because the existence of European bison is threatened by many different factors, such as parasites (47) or infectious diseases (48), further detailed research is desirable to preserve this unique treat.

Authors contribution: A. Tabecka-Lonczynska contributed materials, performed the experiments, carried out data interpretation, wrote the paper, conceived and designed the experiments; J. Mytych performed the experiments, carried out data interpretation; P. Solek performed the experiments and analyzed the data; M. Kulpa-Greszta performed the experiments; M. Koziorowski carried out data interpretation. All authors read and approved the final version of this manuscript.

J. Mytych and P. Solek contributed equally to this work.

Acknowledgements: The authors are grateful to the Management and Personnel of the Bialowieza National Park for their assistance in collecting of the materials.

Conflict of interests: None declared.

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