2 Materials and methods

2.1 Animals and RNA preparation

Male Wistar rats weighing 300-330 g were used. Castration of animals was performed via the scrotal route under ether anesthesia. The ventral prostates and the seminal vesicles were removed from at least 6 animals, and immediately used for RNA preparation in each experimental group. Tissue RNA was prepared by Trisol (GIBCO BRL). Poly(A⁺) RNA was separated by using oligotex-dT₃₀ (Takara). Procedures for animal care and their use were conducted under the Guidelines for Animal Experimentation, Faculty of Medicine, Tottori University (restricted publication) and the international laws (NIH guide for the care and use of laboratory animals, NIH publication 85-23, 1985) and policies.

2.2 cDNA cloning by reverse transcription-polymerase chain reaction (RT-PCR)

For RT-PCR cloning of rat caspase-1 cDNA, two micrograms of poly(A⁺)RNA from the ventral prostate of intact rats were reverse-transcribed in 20 L of reaction mixture containing 0.5 g of oligo-(dT)₁₅ and 200 units of Superscript (RNase H^- reverse transcriptase, GIBCO BRL). An aliquot (1 L) of the reaction mixture was added to 98 L of the PCR mixture supplemented with 50 pmol aliquots of primers (RICEF, 5GCATGGCTGACAAGATCCTGAGGG3; RICER, 5GGATTGTTCGTTTAATGTCCCGGG3) which had been synthesized according to the mice cDNA sequence^[12] to amplify a potential ORF. To amplify the 3 sequence including poly(A⁺) tail, oligo-(dT)15 instead of RICER was included in the PCR mixture. Immediately after addition of 2.5 units of Amplitaq DNA polymerase (1 L, PERKIN ELMER CETUS), the PCR reaction was started in a DNA thermal cycler (PC-700, Astek), 0.5 min at 94, 1 min at 60 and 1 min at 72, for 39 cycles. At the end of PCR, 18 L of the reaction mixture was removed and subjected to electrophoresis in a 1.8% agarose gel in TBE buffer at a constant voltage of 100 V. An amplified band of approximately 1200 bp in the gel was cut out, purified with phenol-chloroform, and then cloned into SmaI site of pUC19. E. coli JM109 was transformed with the recombinant plasmid. Sixty positive clones were sequenced by the dideoxynucleotide chain termination procedure.

2.3 Northern blot and RNA slot blot analyses

Ten g of tissue RNA from the ventral prostate and the seminal vesicles of intact or castrated animals were electrophoresed on a 1.2% denaturing agarose gel containing formaldehyde, transferred onto a nylon membrane (N⁺, Amersham), and hybridized to the ³²P-labeled caspase-1 cDNA. Fifteen g of tissue RNAs were used for RNA slot blot analysis. Conditions for hybridization, washing and exposure to X-ray films were exactly the same as described^[3].

2.4 In situ hybridization

3 Results and discussion

cDNA of rat caspase-1 from the ventral prostates was cloned and the nucleotide sequence (Figure 1) deposited in DDBJ/EMBL/GenBank with an accession number D85899. The sequence analysis of 1325 nucleotides in length predicted an open reading frame of 402 amino acids, which is highly homologous to those of mouse (91%) and human (61%). Northern blot analysis using the cDNA as probe demonstrated a transcript of 1.6 kb both in the ventral prostate and the seminal vesicles (Figure 2A). Within 48 h after castration of animals, steady state levels of the transcript increased to approximately three-fold that of the intact control (Figures 2A and 2B).

Figure 1. Nucleotide and deduced amino acid sequences of rat caspase-1.  
Figure 2.  Effect of castration on the expression of caspase-1 transcript

Finally, in situ hybridization analyses demonstrated caspase-1 transcripts in the epithelia of ventral prostate and seminal vesicles from animals castrated  for 48 h (Figure 3), where castration-induced apoptosis was observed^[1,2].

Figure 3. Expression of caspase-1 transcripts in epithelia of ventral prostate and seminal vesicle : in situ hybridization analysis. Intact or castrated (48 h) animals were used. Representative results from an anti\|sense (Left) and a sense (Right) probe. Scale bar 10 m.

In conclusion, we demonstrated for the first time the castration-induced up-regulation of caspase-1 transcripts in the epithelia of ventral prostate and seminal vesicles of rats. It becomes of particularly importance to examine the castration-induced apoptosis in male accessory sex organs by using caspase-1-deficient mice. Although caspase-1 was first isolated as a cysteine protease to cleave an inactive pro-IL-1 to generate an active IL-1^[10], it is interesting to note that the expression has been demonstrated in a variety of cell types and tissues where IL-1 is not produced^[11-13].

in the ventral prostate and the seminal vesicle: tissue RNA from at least 6 animals in each experimental group were used. (A) Northern blot (upper panel). Teng of tissue RNA was seperated on a garose gel electrophoresis. The location of 28 S and 18 S ribosomal RNA was indicated on the left of lanes as molecular size markers. The lower panel shows the electrophoretic patterns of tissue RNA. (B) RNA slot blot. Fifteen g of tissue RNA were blotted.  I: Intact animal; C24, C48, C72 and C96: animals castrated for 24, 48, 72 and 96 h, respectively.

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