Zong-Yao Zhou^{1, 3}, Chen Xu¹, Qiang-Su Guo¹, Yuan-Xin Hu², Yong-Lian Zhang², Yi-Fei Wang¹

The germ cell nuclear factor (GCNF), also known as retinoid acid receptor-related testis-associated receptor (RTR) or NR6A1, is an orphan receptor of the nuclear receptor superfamily. Originally isolated from mouse cDNA libraries, homologs of GCNF have been identified in humans, Xenopus laevis and zebrafish [1-4]. During embryonic development, GCNF gene is expressed in early embryonic stem cells, trophoblasts and neuronal precursor cells, suggesting that the receptor may participate in the regulation of neurogenesis and be of importance to the embryonic development [5-7]. In the adult, GCNF expression is abundant in the testis and ovary [8-11]. In the testis, GCNF is most abundant in round spermatids. Therefore, GCNF may participate in the regulation of gene transcription during a specific stage of spermato-genesis. Two transcripts of approximately 7.4 Kb and 2.3 Kb are present in the testis, but only the larger one is found in somatic cells [10]. Recently, we found that GCNF is also expressed in the epithelia of mouse epididymis. Apart from the 7.4 Kb transcript, another 3.1 Kb transcript is also present in the mouse epididymis. The expression levels of both GCNF transcripts in mouse epididymis are down-regulated by androgen treatment and the two mRNAs (7.4 & 3.1 Kb) encode for the same protein. In situ hybridization and indirect immuno-fluorescence results show that GCNF is abundant in the proximal corpus of adult mouse epididymis [12].

The epididymis is a multifunctional male accessory organ. It provides an adequate environment for the final maturation of sperm. Gross morphology shows that the highly convoluted single tubule of the adult epididymis is divided into four major segments: initial segment, caput, corpus and cauda. Each region has distinct functions and expresses specific proteins. The proper development and functional maintenance of the epididymis are essential for its normal function. The epididymis develops from the Wolffian duct during embryogenesis. During postnatal development, epididymis epithelial cells undergo rapid proliferation and expansion [13]. Beginning with an undifferentiated epithelium, the epididymis undergoes a series of changes resulting in the establishment of a fully differentiated epithelium, which differs in morphology, gene expression and function throughout its whole length [14]. The spatial and temporal patterns of gene expression are critical to the development and maintenance of a fully functional epididymis and a normal intact epididymis is essential for the functional maturation of the sperm [14, 15].

The purpose of this study is to investigate the spatial and temporal pattern of GCNF expression in murine epididymis during the postnatal period, in order to highlight the possible relationship between GCNF expression and epididymal development and function.

BALB/c mice and SD rats were purchased from the Animal Center of the Chinese Academy of Sciences and divided into nine groups of 4-6 animals each according to their postnatal age, i.e. at day 8, 10, 12, 14, 28, 35, 49 and 70 and month14 after birth.

Rabbit polyclonal anti-GCNF antiserum was prepared as described previously [12, 16].

In the mouse, there were no GCNF positive cells in the epididymis on day 8-10 (data not shown). On day 12, the onset of GCNF expression was detected in only a few positive nuclei in the low columnar undifferentiated cells in the caput, corpus and cauda (Figure 1b, c and d). It is not a non-specific staining as the negative control stained with preimmune serum did not show any staining (Figure 1a). By day 28 and 35, the number of GCNF positive cells increased progressively and was distributed evenly throughout the whole epididymis (Figure 1e, f, g, and h). On day 35, most cells in the corpus epididymis had GCNF positive staining (Figure 1g), while the number of GCNF positive cells started to decline in the distal cauda (Figure 1h). On day 49, GCNF expression in the initial segment, caput and proximal corpus of the epididymis reached the highest levels and the number of GCNF positive cells declined dramatically in the distal corpus and cauda (Figure 1 i, j, k and l). From day 70 to month 14, the GCNF expression pattern in the epididymis was the same as that of the adult (56 days) as we formerly reported [12]. It was mainly expressed in proximal corpus epididymis (Figure 1m, n, o and p), i.e., most cells in the proximal corpus showed strong positive staining (Figure 1o).

Figure 1. GCNF expression in mouse epididymis during postnatal period.
Epididymis sections stained by indirect immunofluorescence using anti-GCNF polyclonal antibodies. b-d: caput (b), corpus (c) and cauda (d) on day 12 mice. e-h: initial segment (e), caput (f), proximal corpus (g) and proximal cauda (h) of mouse epididymis on day 35. i-l: initial segment (i), caput (j), corpus (k) and cauda (l) of on day 49 mice. m-p: initial segment(m), caput (n), proximal corpus (o) and proximal cauda (p) on day 70 mice. Arrowhead indicates clear cells in the cauda with GCNF positive staining. q: initial segment of on day 49 mice, showing narrow cell (N) with strong GCNF positive staining in nucleus and cytoplasm. r: the same section as q, but re-stained with HE after GCNF immunofluorescence reaction. a, s: negative control are corresponding serial section to c and q, staining with preimmune serum. Scale bar =50 m.

The GCNF expression pattern in the rat epididymis was a little different from that of the mouse. First, GCNF expression started on day 14 (Figure 2a, b, c and d) instead of on day 12 in the mouse. Second, on day 35, almost all the cells in the rat epididymis had strong GCNF positive staining (Figure 2e, f, g and h). Third, from day 49 to month 14, most cells in the initial segment of caput and proximal corpus showed strong positive staining and there were almost no GCNF positive cells in the distal corpus and cauda (Figure 2i, j, k, l, m, n, o and p). The number of GCNF expressing cells in the mouse and rat epididymal segments during postnatal period is shown in Table 1.

Figure 2. GCNF expression in rat epididymis during postnatal period.
Epididymis sections stained by indirect immunofluorescence using anti-GCNF polyclonal antibodies. b-d: caput (b), corpus (c) and cauda (d) on day 14 rats. e-h: initial segment (e), caput (f), proximal corpus (g) and proximal cauda (h) on day 35 rats. i-l: initial segment (i), caput (j), corpus (k) and cauda (l) on day 49 rats. m-p: initial segment (m), caput (n), proximal corpus (o) and proximal cauda (p) at month 14 rats. Arrowhead indicates narrow cell (N) with strong GCNF positive staining in nucleus and cytoplasm (m). q: corpus at month 14 rats, showing clear cell (C) nucleus with positive staining. r: the same section as q, re-stained with HE after GCNF immunofluorescence reaction. a, s: negative controls are corresponding serial section to c and q, staining with preimmune serum. Scale bar = 50 m.

The cell types of GCNF expression could be easily identified on the same section used for immunofluorescence and then stained with hematoxylin and eosin. From the postnatal day 35 to month 14, positive GCNF staining could be detected in the nuclei of all cell types (principal, basal, apical, narrow, clear and halo cells) in the epididymis of mice and rats (Figure 1q, r and s and Figure 2q, r and s). The number of the GCNF positive narrow cells and clear cells did not obviously change during the postnatal period (data not shown). In adults (70 days) and old (14 months) rats, most clear cells in the distal corpus and cauda still showed strong positive staining for GCNF (Figure 2q, r and s), while principal cells in this region were almost negative (data not shown). Interestingly, GCNF positive staining could be found in both the nuclei and the cytoplasm of the narrow cells (Figure 1q, r and s and Figure 2m).

A unique spatial and temporal pattern of GCNF expression in murine epididymis was described in this study. GCNF begins to be expressed in the mouse epididymis on day 12 or 14 in rats. At this time the epididymal epithelium is essentially undifferentiated. When the epididymal epithelium begins to differentiate (day 15-28), GCNF expression increases gradually. The highest expression of GCNF was observed on day 35 in both mice and rats and at that time dramatic morphological changes occurred in the epididymal epithelium [1, 17, 18]. From day 49 until adult, epididymal epithelia were fully differentiated and GCNF exhibited a region-specific expression, i.e., it was expressed predominantly in the initial segmen-t, the caput and proximal corpus of the epididymis. Our results indicate that GCNF expression during the postnatal period is closely associated with the differentiation of the epididymal epithelium.

Although the precise nature of the contribution by the epididymal epithelium to maturation of spermatozoa is not completely understood, the gene products synthesized in the epithelium are thought to play region-specific roles in creating the microenvironment conducive to acquisition of fertilizing capability. It is generally accepted that the initial segment, the caput and proximal corpus are crucial for spermatozoa to acquire their motility and fertilizing capability [2, 19]. So the regional expression pattern of GCNF in mouse and rat epididymides suggests that it might be involved in the regulation of target gene expression, which might be important for the maturation of spermatozoa.