:: Asian Journal of Andrology ::

Expression of germ cell nuclear factor in mouse germ cells and sperm during postnatal period

Chen Xu¹, Zong-Yao Zhou^{1, 2}, Qiang-Su Guo¹, Yi-Fei Wang¹

¹Department of Histology & Embryology, Shanghai Second Medical University, Shanghai 200025, China
²Department of Histology & Embryology, Shihezi University, Xinjiang 832002, China

Asian J Androl 2004 Sep; 6: 217-222

Keywords: germ cell nuclear factor; sperm; spatial expression; temporal expression; mouse

Abstract

Aim: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. Methods: The indirect immunofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. Results: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary spermatocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. Conclusion: GCNF may play important roles in spermatogenesis, capacitation and fertilization.

1 Introduction

The germ cell nuclear factor (GCNF) is an orphan receptor that has been implicated in spermatogenesis, neurogenesis and embryonic development. GCNF may also play a role in reproduction as in adult mouse, it is expressed predominantly in the germ cells. Northern analysis showed that two messages in testis RNA hybridized to the GCNF probe, whereas the more sensitive ribonuclease protection assay detected messenger RNA (mRNA) in the ovary [1]. In the mouse, the process of spermatogenesis spans a period of 5 weeks [2]. At the onset of puberty, the spermatogonia begin to proliferate and around postnatal day 9 - 10, they give rise to the first primary spermatocytes. During the following 12 days, these proceed through the successive stages of the first wave of meiosis [3]. In adult mouse, GCNF transcription occurs at lower levels during the meiotic prophase and increases substantially in postmeiotic germ cells, with the maximum level in stage VI-VIII round spermatids [4, 5]. Since GCNF is expressed most abundantly in testes of mouse and human [6] and the two receptor proteins reveal a sequence similarity of more than 98 % [7], it seems likely that GCNF assumes identical functions in the two mammalian species. The spatial and temporal expressions of GCNF in male mouse germ cells during postnatal development have been controversial. Besides, the expression of GCNF in mouse sperm has not yet been addressed.

The purpose of this study is to assess the spatial and temporal expression of GCNF in male mouse germ cells during postnatal development and in sperm before and after capacitation.

2 Materials and methods

2.1 Animals

BALB/c mice were purchased from the Animal Center of the Chinese Academy of Sciences. The mice were divided into nine groups according to their postnatal age (8, 10, 14 , 17, 20, 28 , 35, 70 and 420 days after birth), 4 - 6 animals in each group.

2.2 Indirect immunofluorescence staining

The testes were fixed in Bouin's solution for 10 - 18 h, embedded in paraffin, cut into 6 mm sections and mounted onto 2 % aminopropyltriethoxysilane-coated slides. After deparaffinization and rehydration, sections were washed for 10 min in PBS and incubated in 5 % BSA (bovine serum albumin) for 60 min at room temperature . The 1: 800 diluted anti-GCNF antiserum, which was raised against an Escherichia coli-expressed GCNF protein [8], was applied overnight at 4 . After three washes in PBS, the sections were incubated with FITC-conjugated goat-anti-rabbit IgG (1 : 50 diluted; Jackson ImmunoResearch Laboratories, USA) for 2 h in dark at room temperature. Finally the tissues were washed again and mounted in glycerol/PBS. As a negative control, serial sections were put through the same procedure with the preimmune serum replacing the primary antibody. Digital photographs of fluorescent sections were taken using a laser scanning confocal microscope (Carl Zeiss LSM-510, Germany) and attached software. After taking photographs, the immunofluorescence stained sections were re-stained with hematoxylin/eosin and photos taken again for cell discrimination.

The modified method for sperm capacitation was employed as described by Cross et al [9]. Briefly, mouse spermatozoa were removed from the caput and cauda epididymis and incubated in Tyrode's solution for 15 min at 37 . The supernatant was centrifuged at 1 000 g for 5 min and the pellet was resuspended in 1 mL Tyrodes solution containing BSA (16 mg/mL). Spermatozoa smears were prepared (spermatozoa before capacitation). The resuspended spermatozoa were incubated for 2 h at 37, 5 % CO₂ and were then smeared onto other slides (spermatozoa following capacitation). Smeared spermatozoa were fixed in acetone for 20 min at 4 , stained for the presence of GCNF as described above and those positive for the immunostain were counted. The smears were washed three times in PBS, then FITC-conjugated goat-anti-rabbit IgG (1 : 50 diluted) and Rhodamine-labeled pisum sativum agglutinin (Rh-PSA, 1 : 200 diluted, Vector) were incubated with the smears for 2 h at room temperature. Digital photographs of fluorescent smears were taken using a Carl Zeiss photograph. The images were edited with Adobe Photoshop 6.0 for presentation purposes only. The degree of staining was not altered using this software.

3 Results

3.1 GCNF expression in postnatal testes

At day 8, there were mainly Sertoli cells and spermatogonia in seminiferous tubules and only a few early primary spermatocytes could be identified as shown in Figure 1A and 1B. GCNF was first detected in the nuclei of spermatogonia and a few early primary spermatocytes. From day 10 to day 14, the number of GCNF-positive spermatogonia was increased and most of the early primary spermatocytes were GCNF-positive at day 14 (Figure 1C, 1D). At day 17, round spermatids could be first identified in a few seminiferous tubules. These round spermatids were all strong GCNF-positive. Besides, some nuclei of late pachytene spermatocytes were GCNF-positive. At this stage, spermatogonia and early primary spermatocytes were all GCNF-negative (Figure 1E, 1F). At day 20, GCNF-positive cells were increased due to proliferation of the round spermatids. Most of the round spermatids were GCNF-positive (Figure 2A, 2B). At day 28, GCNF expression was mainly in round spermatids and a part of the late pachytene spermatocytes (Figure 2C, 2D). At day 35 (Figure 2E, 2F), GCNF expression was increased dramatically and the stable expression level continued to the old mice (420-day-old). From day 35 to day 420, GCNF was indicated in the nuclei of late pachytene spermatocytes and round spermatids. However , Sertoli cells, spermatogonia and elongating spermatids were all GCNF-negative in adult mice (Table 1).

Figure 1. GCNF expression in mouse testis during postnatal period. Mouse testis sections stained by indirect immunofluorescence using anti-GCNF polyclonal antibody. A: Mouse testis at postnatal day 8 showing GCNF-positive staining in nuclei of spermatogonia (g) and early stage spermatocytes (e). C: Mouse testis at postnatal day 14, showing GCNF-positive staining in nuclei of spermatogonia (g) and early stage spermatocytes (e). E: Mouse testis at postnatal day 17 show GCNF-positive staining in nuclei of round spermatids (r). B, D, F: The same sections to A, C, E, restaining by HE after immunofluorescence staining. Scale bar = 20 m.

Figure 2. GCNF expression in mouse testis during postnatal period. Mouse testis sections stained by indirect immunofluorescence using anti-GCNF polyclonal antibody. A: Mouse testis at postnatal day 20 showing GCNF-positive staining in nuclei of round spermatids (r). C: Mouse testis at postnatal day 28 showing GCNF-positive staining in nuclei of pachytene spermatocytes (p) and round spermatids (r). E: Mouse testis at postnatal day 35 showing GCNF-positive staining in nuclei of pachytene spermatocytes (p) and round spermatids (r). B, D, F: the same sections as A, C, E, restaining by HE after immunofluorescence staining. G: The negative control corresponding serial section to E, staining with preimmune serum. Scale bar=20 mm.

Table 1. The number and intensity (within parentheses) of GCNF-positive cell in mouse testis. Annotation. <-> : negative; <+>: a few positive cells or low intensity; <++>: moderate number of positive cells or moderate intensity; <+++>: most cells positive or strong intensity; </>: germ cells not found.

	Spermatogonia	Early spermatocytes	Pachytene spermatocytes	Round spermatids	Elongating spermatids	Sertoli cells
8 day	+ (+)	+ (+)	/	/	/	- (-)
14 day	++ (+)	++ (++)	/	/	/	- (-)
17 day	- (-)	- (-)	+ (+)	+ (++)	/	- (-)
20 day	- (-)	- (-)	+ (+)	++ (++)	/	- (-)
28 day	- (-)	- (-)	++ (++)	++ (+++)	/	- (-)
35 day	- (-)	- (-)	++ (+++)	+++ (+++)	- (-)	- (-)
70 day	- (-)	- (-)	++ (+++)	+++ (+++)	- (-)	- (-)
420 day	- (-)	- (-)	++ (+++)	+++ (+++)	- (-)	- (-)

3.2 GCNF expression in sperm before and after capacitation

Before capacitation, 98 % spermatozoa were stained by both anti-GCNF indirect immunofluorescence (in acrosomal cap region) and Rh-PSA (in whole acrosome) (Figure 3A, 3B). When the antiserum was replaced by preimmune serum, the immunoreactivity of the spermatozoa was negative. Following capacitation, however, only about 20 % spermatozoa were GCNF-positive in the acrosomal cap region, whereas the intensity of Rh-PSA staining was stronger than other sperm (Figure 3C, 3D).

Figure 3. Localization of GCNF in mouse sperm. A: Sperm from epididymis stained by indirect immunofluorescence using anti-GCNF polyclonal antibody. Scale bar = 20 mm. B: Sperm from epididymis (before capacitation) stained by anti-GCNF indirect immunofluorescence(green) and Rh-PSA (red) (2.5 mg/mL). Scale bar = 20 mm. C: Sperm after capacitation stained by GCNF indirect immunofluorescence, showing GCNF-negative in most sperm, while a few sperm (about 20 %) still GCNF-positive(f). Scale bar=10 mm. D: Sperm (after capacitation) stained by GCNF indirect immunofluorescence and Rh-PSA, showing GCNF-positive in acrosomal cap region (sperm non- capacitation) was the sperm with strong staining of Rh-PSA (red) (h), while GCNF-negative sperm with less intensity of Rh-PSA at acrosomal cap region (p) may undergo capacitation. Scale bar = 10 mm. E: Negative control sperm stained with preimmune serum. Scale bar=10 m.

The localization of GCNF immunostaining in caput and cauda spermatozoa was similar.

4 Discussion

The GCNF gene was reported to be predominantly expressed as two transcripts (7.4 and 2.3 Kb) in germ cells of adult mice [1, 6] and the same protein is translated from both transcripts [10]. When germ cells at defined stages of spermatogenesis were used for Northern analysis, GCNF mRNA was abundant in round spermatids but was not detected in spermatogonia or spermatocytes isolated from juvenile mice [11]. Katz et al reported that GCNF expression first occurs in the testis of 20-day-old mice, when round spermatids first emerge [5]. Another investigation showed that GCNF mRNA was detected in the testes from mice at 8 days of age, when spermatid do not exist [4]. An immunofluorescence study showed that GCNF protein was present only in primary spermatocytes, but not in round spermatids [12]. However , Lan et al reported recently that using anti-GCNF polyclonal antibodies, the highest level of GCNF expression was observed in elongating spermatids and no staining was observed in spermatogonia or pachytene spermatocytes in the seminiferous tubules of adult mouse [13]. Therefore, expression of GCNF protein in the mouse testis remains to be clarified.

It was shown in this study that GCNF was first detected in the nuclei of spermatogonia and early primary spermatocytes in 8-day-old mice. Since the 7.4-Kb GCNF transcript is detected earlier during prepubertal development in the mouse [4], our results suggested that this larger transcript begins to express in 8-day-old mouse. On the other hand, the 2.3-Kb GCNF transcript is expressed predominantly in haploid round spermatids [7]. According to our results, the smaller transcript expression may be increased in 17-day-old mouse. Considering the 2.3-Kb GCNF message appears specific to the testis and the 7.4-Kb GCNF message, although predominantly expressed in the testis, is also detected in certain other organs such as brain, liver, lung, kidney [1, 6] and epididymis [14], the 7.4-Kb GCNF protein may be mainly involved in spermatogenesis, neurogenesis and differen-tiation. However, the 2.3-Kb GCNF protein may be involved in spermiogenesis, especially in the transition proteins [15].

The protamines are encoded by a family of highly conserved genes, Prm 1 and Prm 2, which are important in spermiogenesis. These genes encode small, highly basic proteins that replace the histones in spermatid chromatin structure. The replacement of histones by protamines is thought to be important for the compaction and condensation of spermatid chromatin. The protamines are expressed only in the postmeiotic, haploid spermatids [15]. In humans and mice, there are two protamine genes, P1 and P2. An improper P1 / P2 ratio in humans, caused by a reduction in P2 expression, is thought to be one of the causes of some idiopathic infertility syndromes [16]. In mice, premature translation of P1 leads to an arrest in spermiogenesis and infertility [17]. Furthermore, a sequence to which GCNF can bind, which consist of a direct repeat of the core halfsite AGGTCA with zero base pairs spacing the repeat (DR0), has been identified. Several genes that contain DR0 sequences in their 5?promoter regions have been identified, including protamines. The mouse protamine 1 and 2 genes, therefore, are potential target genes for GCNF regulation [15].

So far, GCNF expression in spermatozoa and the relation of GCNF to sperm capacitation and sperm acrosome reaction remain unknown. According to our indirect immunofluorescence staining, anti-GCNF antiserum reacted with the acrosomal cap region, which we identified by the shape of the stained area. It was difficult to conclude whether the area observed was the surface or the interior of the acrosomal cap. It has been found that 98 % sperm were immuno-positive for GCNF in the acrosomal cap region before capacitation, but the immunoreactivity was not found in the acrosomes of elongating spermatids. It has been suggested that GCNF might be localized on sperm membranes (corresponding to acrosomal cap region) rather than in the acrosomal contents, since about 80 % of capacitated spermatozoa, but not yet acrosome reacted, were immuno-negative for GCNF. The disappearance of GCNF expression in the spermatozoa after capacitation may be due to the rearrangement of the sperm membrane proteins during capacitation. It was well known that during capacitation, some proteins on sperm membrane will be masked and some will be unmasked. The protein hidden and exposure is at least in part significant for sperm-ovum recognition and fertilization. The spermatozoa remaining GCNF-positive may be the spermatozoa non-capacitated. It was reported that a sperm protein, P34H located at the acrosomal cap region, is involved in sperm-zona pellucida interaction [18]. The hamster P26h and the human P34H are both located at the same region of sperm, and antibodies recognizing them interfered with sperm-zona pellucida interaction in vitro [19]. Therefore, GCNF may be involved in the regulation of male fertility by regulating capacitation and fertilization. Sperm acrosomes can bind many antibodies nonspecifically, so the immunofluorescence staining results do not exclude the possibility of nonspecific staining. We are preparing to do a Western blot to verify the immunofluorescence staining results. The molecular mechanism of GCNF functioning in sperm capacitation and sperm-ovum fertilization needs further research.

Acknowledgements

The work was supported by the National Natural Sciences Foundation of China (No. 30070391), the ?73?Basic Research Funding Scheme of China (No. G 199905501), the Science and Technology Development Foundation of Shanghai Population and Family Planning Commission (No. 03JG 05009).

We thank Dr. Yong-Lian Zhang and Dr. Yuan-Xin Hu for helpful discussion and providing anti-GCNF antiserum; JM Wang and SX Qin for their excellent technical assistance; Dr. Barry T. Hinton for reviewing and linguistic revision of the manuscript.

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Correspondence to: Dr. Yi-Fei Wang, Department of Histology & Embryology, Shanghai Second Medical University, 280 South Chongqing Road, Shanghai 200025, China.
Tel: +86-21-6445 3260, Fax: +86-21-6466 3160
Email: wangyf@shsmu.edu.cn
Received 2003-12-19 Accepted 2004-06-03

Keywords: germ cell nuclear factor; sperm; spatial expression; temporal expression; mouse

Abstract

Spermatogonia

Pachytene spermatocytes