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Aberrant Expression of Cx Isoforms in the Adult Caput Epididymis exposed to Estradiol Benzoate or Flutamide at the Weaning

ABSTRACT Connexin (Cx) involves in the regulation of various physiological functions of tissue by forming a channel, a gap junction which allows direct cell-cell communication, between adjacent cells. The effect of a single subcutaneous treatment of estradiol benzoate (EB) or flutamide (Flu) at the weaning age on the expression of Cx isoforms in the adult caput epididymis was evaluated in this research. Using quantitative real-time PCR analysis, a low-dose of EB [0.015 μg/kg body weight (BW)] caused significant decreases of Cx30.3, Cx32, Cx40, Cx43, and Cx45 mRNA levels and no change of Cx26, Cx31, Cx31.1, Cx37 transcript levels. The treatment of a high-dose EB (1.5 μg/kg BW) resulted in reduced expression of Cx30.3, Cx31, Cx43, and Cx45 but increased expression of Cx37 and Cx40. Expression of all Cx isoforms examined, except Cx31, was significantly increased by the treatment of a low-dose Flu (500 μg/kg BW). However, the treatment of a high-dose Flu (5 mg/kg BW) led significant expressional suppression of Cx30.3, Cx31, Cx31.1, Cx32, Cx40, Cx43, and Cx45 but an increase of Cx37 transcript level. With the comparison of previous findings, the expression of Cx isoforms in the adult epididymis after the exposure to EB or Flu is likely differentially regulated in regional-specific and/or exposed postnatal age-specific manner.

The epididymis of the male reproductive tract is the place where sperm produced from the testis becomes mature and acquires fertilizing capacity. The epididymis has a layer of epithelial lumen surrounded by smooth layers, and the epididymal epithelium is composed of several cell types, for example principal, halo, clear, basal, and apical cells, possessing distinctive functions (Robaire & Hermo, 1988) Based on histochemical and functional aspects, the epididymis is divided into four parts, initial segment, caput epididymis, corpus epididymis, and caudal epididymis (Ro-baire & Hermo, 1988). Of these epididymal regions, the caput epididymis has some features distinct from other parts of the epididymis. These include different composition of epithelial cell types (Robaire & Hermo, 1988), thickness of the epithelium (Robaire & Hermo, 1988), and different expressional levels of certain molecules, such as androgen receptor and 5-reductase (Robaire et al., 2006). Thus, it is generally acknowledged that the caput epididymis gives an influence on sperm maturation in ways different from other epididymal segments.
In multicellular tissue, Cellular communication between adjacent cells is mostly directed by one or more types of junctional complexes, such as tight junction, adherens junction, and gap junction (Lawrence et al., 1978). However, gap junctional complex only allows the functional accordance of neighboring cells by direct exchange of small molecules, including signaling molecules, ions, and some metabolites (Valiunas et al., 2005). The gap junction is composed of six connexin (Cx) subunits, forming a channel between contiguous cells (Lawrence et al., 1978). To date, 21 Cx isoforms have been found in vertebrates, and all cells virtually possess one type of Cx isoforms at least (Goodenough & Paul, 2009). Many researches have demonstrated that certain Cx isoforms are frequently found in most of tissues and others are expressed in restricted cell types (Goodenough & Paul, 2009;Meşe et al., 2007). Other studies have also showed co-expression of more than two types of Cx isoforms and differential expression of several Cx isoforms during the development in a cell (Goodenough & Paul, 2009;Meşe et al., 2007). The existence of Cx isoforms in the male reproductive tract has been extensively examined. The expression of Cx isoforms in the epididymis has been determined by our previous researches, and the expression of a total of 9 Cx isoforms, including Cx26, Cx30.3,Cx31,Cx31.1,Cx32,Cx37,Cx40,Cx43, and Cx45, have been identified in the epididymis (Han & Lee, 2013;Lee, 2013;Seo et al., 2010). Moreover, these Cx isoforms in the epididymis have been differentially expressed according to the segment and/or age during postnatal development (Seo et al., 2010;Han & Lee, 2013;Lee, 2013). The localization of Cx43 between basal and principal cells in the rat caput epididymis has been detected by Cyr et al. (1996). However, addition information for the localization of other Cx isoforms in the epididymis is limited to date.
The expression of Cx isoforms in the epididymis is regulated by various intrinsic and/or extrinsic factors. The expression of Cx43 in the rat caudal epididymis is controlled by testis-originated factor(s) because the removal of testis results in the disappearance of immunoreactivity of Cx43 (Cyr et al., 1996). In addition, the expressional stimulation of Cx43 in human epididymis by epidermal growth factor has been reported (Dubé et al., 2012). Propylthiouracil-induced hypothyroidism at the neonatal age induces a significant decrease of Cx43 expression in the entire epididymal region, except caudal epididymis, indicating a possible role of thyroid hormone on expression of Cx43 in the epididymis (St-Pierre et al., 2003). It is well-documented that the function of epididymis is largely regulated by androgen, as well as estrogen (Robaire & Hamzeh, 2011;Schulster et al., 2016). Thus, the possibility of expressional regulation of Cx isoforms in the epididymis by androgen and/or estrogen-related compounds has been suggested. Indeed, the exposure to flutamide (Flu), an antiandrogenic substance, at the late gestational age or early postnatal age causes a decrease of Cx43 expression in the cauda epididymis (Lydka et al., 2011). In addition, our previous researches have demonstrated the effects of flutamide and estradiol benzoate (EB), an estrogenic compound, treatments at the early neonatal age on expression of Cx isoforms within different epididymal segments (Lee, 2015(Lee, , 2016a(Lee, , 2017. Moreover, the exposure of these substances at different postnatal age results in differential expression of Cx isoforms within corpus or caudal epididymal region (Lee, 2015(Lee, , 2016a(Lee, , 2016bLee & Lee, 2015). Recently, after the exposure to Flu or EB at the early neonatal age, expressional changes of Cx isoforms in the adult caput epididymis have been evaluated (Lee, 2017). But, the influence of Flu or EB treatment at different age on expression of Cx isoforms in the caput epididymis has not been studied. Thus, the aim of the present research was to evaluate the changes of expression of Cx isoforms in the adult caput epididymis treated with Flu or EB at the weaning.

Tissue preparation and isolation of total RNA from the tissue
The experimental animal at 4 months of age was euthanized by CO2 stunning, and an incision on lower abdomen was made to take out the male reproductive tract. In the cold PBS buffer, the epididymis was separated from the rest and was additionally divided into individual epididymal parts. The caput epididymis was transferred to and washed in new cold PBS buffer and was rapidly placed in liquid nitrogen. Frozen tissue was stored in -80℃ and was used for total RNA isolation.
The extraction of total RNA was began with the homogenization of tissue in the RNA extraction solution (iNtRON Biotech, Sungnam, Korea). The precipitation of total RNA was carried out with the addition of isopropanol.
After the washing in 70% DEPC-EtOH, total RNA pellet was air-dried and then resuspended in DEPC-dH2O. The amount and purity of total RNA isolated were calculated with NanoDrop Lite spectrophotomer (Thermo Scientific, Wilmington, DE), and the qualitative condition of total RNA was checked by 1.2% agarose gel electrophoresis.

Preparation of complementary DNA from total
RNA and procedure of quantitative real-time PCR A mixture of 1 μg of total RNA, oligo-dT primer, ImProm-II TM reverse transcription system (Promega, Madison, WI), and dH2O in a total volume of 20 μL was prepared to construct the first-stranded complementary DNA (cDNA).
The mixture was placed in a sequential step of 25℃ for 5 min, 42℃ for 90 min, and 70℃ for 15 min to carry out the reverse transcription reaction. The generated cDNA was utilized as a template DNA for quantitative real-time PCR analysis.
A mixture of 1 μL of cDNA, 10 pmol of each primer, 10 μL PCR master mixture (Finnzymes, Espoo, Finland), and DNase-free dH2O in a final volume of 20 μL was made for quantitative real-time PCR analysis of each Cx isoform. Table 1 shows detailed information of oligonucleotide primers and condition used for real-time PCR analysis.
The PCR was undertaken in a thermocycler (Bio-Rad Laboratories, Hercules, CA) with a series of steps as followings; a pre-denaturation step at 95℃ for 30 sec, 35 cycles of denaturation at 95℃ for 30 sec, annealing at Tm for 30 sec, and extension at 72℃ for 30 sec, and a final extension step at 72℃ for 5 min. Cyclophilin A (Ppia) was used for an internal control of quantitative real-time PCR.

Analysis and presentation of data
The RT reactions and PCRs were independently quadru-plicated to get a mean and a standard error of an experimental group. The experimental results were reported in the relative ratios of expression between Ppia and Cx isoforms. Statistical significance of Cx expressional level among control and two experimental groups of EB or Flu treatment was determined by one-way ANOVA, followed by Duncan's test. If P < 0.05, it was considered that there was a statistical difference between two groups.
1. Changes of Cx26 and Cx30.3 expression in the caput epididymis of rat exposed to EB or Flu at the weaning As shown in Fig. 1A, there was no significant change of Cx26 transcript level with EB treatment at both low and high doses. However, a significant increase of Cx26 expression was detected with a low-dose treatment of Flu (Fig. 1A). The treatment of Flu at a high-dose didn't give a significant influence on the level of Cx26 transcript.
Expression of Cx30.3 in the adult caput epididymis was  (Fig. 1B). A significant induction of Cx30.3 expression was observed with a lowdose Flu treatment, but a high-dose Flu treatment at the weaning resulted in a decrease of Cx30.3 mRNA level in the adult caput epididymis (Fig. 1B).
2. Changes of Cx31 and Cx31.1 expression in the caput epididymis of rat exposed to EB or Flu at the weaning The level of Cx31 transcript in the adult caput epididymis was not changed by the administration of a lowdose EB at the weaning ( Fig. 2A). But, a significant reduction of Cx31 transcript level was observed with a highdose EB treatment ( Fig. 2A). Similarly, the treatment of a low-dose Flu at the weaning didn't affect the expression of Cx31 in the adult caput epididymis, while the expression of Cx31 was greatly decreased by the treatment of a highdose Flu ( Fig. 2A).
Neither a low-dose EB treatment nor a high-dose EB treatment gave any significant effect on the expressional change of Cx31.1 (Fig. 2B). The administration of a lowdose Flu at the weaning caused a clear increase of Cx31.1 transcript level in the adult caput epididymis (Fig. 2B). But, a significant decrease of Cx31.1 mRNA abundance in the adult caput epididymis was detected in the high-dose Flu treated-group (Fig. 2B).
3. Changes of Cx32 and Cx37 expression in the caput epididymis of rat exposed to EB or Flu at the weaning A significantly lower level of Cx32 in a low-dose EB treated-group at the weaning than that in the control was detected in the adult caput epididymis, even though there was no significant change of Cx32 transcript level in a high-dose EB treated-group (Fig. 3A). The level of Cx32 transcript was significantly increased by a low-dose Flu treatment, but the treatment of a high-dose Flu led to a significant decrease of Cx32 transcript level (Fig. 3A).
Expression of Cx37 in the adult caput epididymis was not changed by a low-dose EB treatment at the weaning ( Fig. 3B). However, the treatment of a high-dose EB at the weaning resulted in a significant induction of Cx37 expression (Fig. 3B). The treatment of Flu at a low-dose caused a significant increase of Cx37 transcript level (Fig. 3B).
The expressional level of Cx37 in a high-dose Flu treatedgroup was significantly higher than that in control but lower than that in a low-dose Flu treated-group (Fig. 3B).
4. Changes of Cx40, Cx43, and Cx45 expression in the caput epididymis of rat exposed to EB or Flu at the weaning The transcript level of Cx40 in the adult caput epidi-dymis was significantly decreased by a low-dose EB treatment at the weaning (Fig. 4A). However, the exposure to a high-dose EB at the weaning resulted in a significant increase of Cx40 mRNA level (Fig. 4A). An increase of Cx40 transcript level in the adult caput epididymis was detected by a low-dose Flu treatment at the weaning, while the treatment of a high-dose Flu caused a significant decrease of Cx40 mRNA amount (Fig. 4A).
The expression of Cx43 in the adult caput epididymis was significantly decreased by both low-and high-dose EB treatments at the weaning (Fig. 4B). But, the level of Cx43 transcript was significantly lower in a low-dose EB treated-group than a high-dose EB treated-group (Fig. 4B).
A great increase of Cx43 mRNA level in the adult caput epididymis was observed with a low-dose Flu treatment at the weaning (Fig. 4B). In the other hand, a high-dose Flu treatment caused a decrease of Cx43 transcript level (Fig.   4B). The treatments of both low-and high-dose EB at the weaning resulted in significant reduction of Cx45 transcript level in the adult caput epididymis (Fig. 4C). An increase of Cx45 expression in the adult caput epididymis was found in a low-dose Flu treated-group, but the treatment of a high-dose Flu at the weaning resulted in a significant decrease of Cx45 transcript level in the adult caput epididymis (Fig. 4C).    (Lee & Lee, 2015). With the same treatment, in the adult caudal epididymis, expression of Cx26 is not changed by EB treatment but significantly increased by Flu treatment at two doses (Lee, 2016b). Different expressional response of Cx43 has been observed in corpus and caudal epididymis after the same Flu or EB treatment. That is, expression of Cx43 in the adult corpus epididymis is significantly increased by a low-dose EB treatment but decreased by a high-dose EB and two-doses Flu treatments (Lee & Lee, 2015). Interestingly, expression of Cx43 in the adult caudal epididymis is significantly increased by EB or Flu treatment at the weaning age (Lee, 2016b). In the present study, the EB treatment has not affected the expre- In conclusion, the present research exhibits that expression of Cx isoforms in the adult caput epididymis is modulated by the treatment of EB or Flu at the weaning age.
In addition, the data from the current study also show that the EB or Flu treatment at different postnatal age results in differential expression of a given Cx isoform in the adult caput epididymis. However, it still remains to be answered if the abnormal expression of Cx isoforms in the epididymis by EB or Flu treatment is related with a disruption of epididymal function.