Male and female BALB/c mice (25 g, 8-10 weeks old) were obtained from the Choongang Animal Institute (Korea), and bred in our animal facility. Testes (n = 3-5/group) at 1, 2, 4, and 8 weeks after birth were used for Western blot analysis and histology (n = 3-5 samples). All of the experiments were carried out in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals (USA).

Testes from mouse were isolated, dissected, and homogenized in lysis buffer for protein analysis. The opposite testis was fixed in 4% paraformaldehyde in phosphate buffer and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin, and used for immunohistochemistry.

The anti-PLD antibody was generated against the C-terminal 12 amino acid residues 1063-1074 (TKEAIVP MEVWT) of the rat PLD1. The antiserum recognizes both PLD1 and PLD2 both PLD2 and PLD1 because the seven C-terminal amino acids of PLD2 and PLD1 are identical [16]. For affinity purification of the antibodies, the peptide was coupled to Affi-Gel 15 (Bio-Rad, USA) according to the manufacturer's instructions. The anti-PLD antibody used in this study has been shown to label reactive astrocytes and macrophages in rats with EAE [1], ischemic brain injury [14], and clip compression injury [9], as well as ganglion cells and glial cells, including Muller cells, in the developing and adult retina of the rat [12,13].

Tissue samples were homogenized in immunoprecipitation assay buffer [20 mM HEPES (pH 7.2), 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 150 mM NaCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride]. After incubation for 30 min in an ice-bath, the homogenates were centrifuged, and the lysate supernatant was precleared by incubation with preimmune IgG and protein A-Sepharose for 30 min. Precleared cell lysates were incubated with the anti-PLD antibody and 30 µl of the 50% slurry of protein A-Sepharose for 4 h. The immune complex was collected by centrifugation and washed five times with ice-cold buffer that contained 20 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 2 mM Na₃VO₄, 10% glycerol, and 1% Nonidet P-40. SDS sample buffer was added and the mixture was boiled. The recovered proteins were separated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore, USA), blocked in 5% nonfat dried milk, and probed with the anti-PLD antibody. Immunoreactive bands were visualized by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies and the ECL reagents (Amersham Biosciences, USA).

Sections of paraffin-embedded tissues (5 µm thick) were deparaffinized and allowed to react with the affinity-purified anti-PLD antibody. Immunoreactivity was visualized with the avidin-biotin peroxidase reaction (Vector Elite; Vector, USA). Peroxidase was developed with diaminobenzidine (Vector, USA). The sections were counterstained with hematoxylin before being mounted. As a control, the primary antibody was omitted for a few test sections, and no specific labelling was found in these sections (Fig. 2C).

To investigate the developmental expression patterns of PLD isozymes in the mouse testis at postnatal ages of 1, 2, 4, and 8 weeks, lysates from mouse testis were immunoprecipitated and analyzed by Western blotting using antibody to PLD (Fig. 1). Both PLD1 and PLD2 were detected in the testis at postnatal week 1, and the expression of two PLD isozymes increased gradually until postnatal week 4 and was enhanced further at postnatal week 8. Considering the initiation of germ cell development around postnatal week 4, PLD expression appears to be closely associated with spermatogenesis.

At 1 (Fig. 2A) and 2 (Fig. 2B) weeks after birth, the seminiferous tubules consist of single or multiple layers of undifferentiated cells. Immunohistochemistry showed weak PLD immunostaining in some undifferentiated cells in the seminiferous tubules. Intense PLD immunostaining was seen in interstitial Leydig cells in the testis of both 1- and 2-week-old mice (Fig. 2A & B).

In the mouse testis at 4 weeks after birth, primary spermatocytes are visualized in the seminiferous tubules. PLD immunoreactivity was visualized in some small primary spermatocytes along the basement membrane (Fig. 2D), where PLD was also localized in the nucleus of primary spermatocytes (Fig. 2D, arrowheads). This nuclear staining of PLD was slightly different from that of 1- or 2-week-old mice. PLD was slightly expressed in Sertoli cells, while PLD was intensely localized in Leydig cells (Fig. 2D, asterisk).

In the mature testis at 8 weeks after birth, intense PLD immunostaining was seen in the primary spermatocytes, with characteristic nuclear localization (Fig. 2E, arrowheads) and in the residual bodies (Fig. 2E, arrows) in the spermatids of the seminiferous tubules. PLD was variably immunostained in spermatogonia in this study. PLD intensely immunostained the Leydig cells in the interstitial spaces (Fig. 2E, asterisk).