Differential expression and localization of four connexins in the ovary of the ayu (Plecoglossus Altivelis)

Abstract

The post-vitellogenic oocytes of teleost fish are generally unresponsive to maturation-inducing hormone (MIH) until a luteinizing hormone (LH) surge stimulates sensitivity via the acquisition of oocyte-maturational competence (OMC). Heterologous gap junctions (GJs) between granulosa cells and the oocyte have been previously implicated in the regulation of oocyte maturation in various vertebrate species. Although heterologous GJ are present in ovarian follicles of ayu (Plecoglossus altivelis), their role in maturation remains unclear. In the present study, we cloned and characterized complementary DNAs for GJ protein connexin (Cx), and examined the expression pattern of Cx messenger RNAs in the ayu ovary. Four Cx cDNAs with predicted molecular masses of 32.1 (Cx32.1), 34.9 (Cx34.9), 44.1 (Cx44.1), and 44.2 (Cx44.2) kDa, respectively, were cloned. Northern blot analysis revealed that the levels of Cx44.1 and Cx44.2 transcripts were similar during the vitellogenic and ovulatory stages. Cx32.1 transcripts were more abundant during the vitellogenic stage, but their levels declined thereafter. Cx34.9 transcript levels increased during the vitellogenic stage and remained high during the acquisition of OMC. In situ hybridization revealed that Cx44.1 and Cx44.2 signals were restricted to the oocyte, whereas the Cx32.1 and Cx34.9 signals were detected in both cellular fractions. Furthermore, a dye-transfer assay revealed the presence of functional GJs between the oocytes and follicle cells. These results suggest that Cx34.9 contributes to the formation of heterologous GJs between oocytes and granulosa cells. Moreover, GJs formed by Cx34.9 may function during the LH-dependent acquisition of OMC and the MIH-dependent resumption of meiosis in ayu.

Heterologous Gap Junctions between Granulosa Cells and Oocytes in Ayu (Plecoglossus altivelis): Formation and Role during Luteinizing Hormone-dependent Acquisition of Oocyte Maturational Competence

“…Next, we determined the site and stage of expression of each connexin messenger RNA (mRNA) in the ayu ovarian tissue using in situ and northern hybridization. Our results showed that Cx34.9 was expressed in both the follicle cells and the oocytes (Table 1), and was strongly expressed from the maturationally-incompetent stage through to mature stage (Table 1) [13]. In addition, Cx32.1 was expressed in both the follicle cells and the oocytes, and was particularly strongly expressed during the vitellogenic stage (Table 1) [13].…”

“…Our results showed that Cx34.9 was expressed in both the follicle cells and the oocytes (Table 1), and was strongly expressed from the maturationally-incompetent stage through to mature stage (Table 1) [13]. In addition, Cx32.1 was expressed in both the follicle cells and the oocytes, and was particularly strongly expressed during the vitellogenic stage (Table 1) [13]. By contrast, the expression of Cx44.1 and Cx44.2 was confirmed only in oocytes, and these connexins were present at constant levels during all stages, including after ovulation (Table 1) [13].…”

“…This method verifies the opening and closing of GJs using lucifer yellow (LY), which is a fluorescent dye capable of passing through these channels, to indicate movement of small molecules between cells [13,14]. LY that is microinjected into oocytes can diffuse into granulosa cells, thereby confirming , weak expression; ×, no expression.…”

“…In addition, Cx32.1 was expressed in both the follicle cells and the oocytes, and was particularly strongly expressed during the vitellogenic stage (Table 1) [13]. By contrast, the expression of Cx44.1 and Cx44.2 was confirmed only in oocytes, and these connexins were present at constant levels during all stages, including after ovulation (Table 1) [13]. Our findings suggested that Cx34.9-present in both follicle cells and oocytes and strongly expressed from OMC acquisition through to oocyte maturation-formed the GJs between follicle cells and oocytes that functioned in OMC acquisition.…”

“…We microinjected LY into maturationally competent follicle-enclosed oocytes, and then separated out the follicle cells and the oocytes under a binocular microscope after 2 h. Fluorescent microscopy confirmed the presence of a fluorescent signal in all of the follicle cells in this experiment. We also subjected ovarian fragments to pre-culture with the gap-junction-specific inhibitor carbenoxolone (CBX) before injecting the follicle-enclosed oocytes with LY, as described above, and separating out the follicle cells after 2 h. Fluorescent microscopy revealed that the LY diffusion was inhibited in 85.5% of the follicle cells [13]. Our findings that a low-molecular weight water-soluble substance such as LY diffused into the follicle cells from the oocytes, and that this process was significantly inhibited by CBX, confirmed the presence of functional GJs that allowed the movement of substances between granulosa cells and maturationally competent oocytes.…”