2.7. qPCR

Total cellular RNA was isolated from mouse testes using RNAiso Plus (Takara, Shiga, Japan). cDNA was synthesized from the total RNA using ReverTra Ace (Toyobo, Tokyo, Japan) with random primers and dNTPs (Nippon gene, Tokyo, Japan) based on the protocol by the manufacturer. Real-time qPCR was performed in the Light Cycler Nano system (Roche Diagnostics, Mannheim, Germany) using SYBR green RT–PCR reagent (Toyobo) following the protocol by the manufacturer. Primers for mouse insulin-like peptide 3 (Insl3) and β-actin mRNA were designed using Primer3plus online software and prepared via custom synthesis (Sigma Genosys, Hokkaido, Japan). Primer sequences used were as follows: Insl3: 5′-CTACTGATGCTCCTGGCTCTGG-3′ (forward) and 5′-GAGATGTCTCTGCTCTAGCCAC-3′ (reverse); β-actin: 5′-CACCTTCTACAATGAGCTGCGTG-3′ (forward) and 5′-ATGGCTGGGGTGTTGAAGGTCT-3′ (reverse). The Insl3 mRNA level was normalized to that of β-actin mRNA in each sample.