Egg Development After In Vitro Insemination in Japanese Quail (Coturnix japonica)

Abstract

In vitro fertilization has been widely used to produce offspring in several mammalian species. We previously successfully produced Japanese quail chicks using intracytoplasmic sperm injection (ICSI), whereas in vitro insemination was not successful. This may be due to the difficulties associated with mimicking the sperm-egg fusion process and subsequent events in physiological polyspermic fertilization in vitro. In the present study, we observed egg development after in vitro insemination and investigated the inactivation of metaphase-promoting factor (MPF) and cytostatic factor (CSF), which are downstream of the Ca²⁺ signaling pathway in the egg, due to fertilizing sperm. We found a sperm number-dependent increase in hole formation caused by sperm penetration of the perivitelline membrane, the extracellular coat surrounding the egg. Egg development was observed following in vitro insemination; however, the developmental rate and stages after 24-h culture were inferior to those of ICSI eggs, even when insemination was performed with a high number of sperm (2 × 10⁴). We also noted the downregulation of inositol 1,4,5-trisphosphate receptor-1, ryanodine receptor-3, cyclin B1, and c-MOS, which are important regulatory components of MPF and CSF in the egg, which was dependent on the number of sperm used for insemination. However, the decreases observed in these components did not reach the levels observed in the ICSI eggs. Collectively, the present results suggest that a sperm number higher than 2 × 10⁴ is required for the progression of the Ca²⁺ signaling pathway, which initiates subsequent egg development in Japanese quail.

Keywords: Blastoderm; Cytostatic factor; In vitro insemination; Japanese quail; Metaphase-promoting factor.

Fig. 1.

Hole formation on the pvm located on the germinal disc following in vitro insemination with different sperm numbers. (A–C) The pvm was isolated from an egg incubated with 2 × 10² (A), 2 × 10³ (B), or 2 × 10⁴ (C) sperm, subjected to Schiff’s staining, and observed under a light microscope. (D) The number of holes per 3 mm² area was calculated and expressed as mean ± SD. Values were obtained from three samples. Scale bar = 200 μm.

Fig. 2.

Presence of CCNB1, c-MOS, ITPR1, and RYR3 proteins in quail eggs and their down-regulated expression after ICSI. (A) Detection of CCNB1, c-MOS, ITPR1, and RYR3 proteins in ovulated quail eggs. (B) Western blot analysis 3 h after ICSI with injection of PLCZ1, CS, and ACO2 cRNA or the solvent alone. (C) Western blot analysis 3 h after ICSI with PLCZ1 cRNA alone, CS and ACO2 cRNAs, or the solvent alone. (D) Quantification of immunoreactivity after ICSI. Band intensities were quantified and expressed as the means ± standard deviations of three samples. Values with different letters are significantly different (P < 0.01).

Fig. 3.

Changes in ITPR1, RYR3, CCNB1, and c-MOS protein expression levels in quail eggs following in vitro insemination with different sperm numbers. (A–C) Western blot analysis 3 h after in vitro insemination with 2 × 10² (A), 2 × 10³ (B), or 2 × 10⁴ (C) sperm, respectively. Numbers 1–6 represent each of the six eggs treated with sperm. Egg extracts after ICSI or PBS treatment were used as the control. (D) Quantification of immunoreactivity after in vitro insemination. Band intensities were quantified and are expressed as means ± standard deviations. The sample number of PBS- and ICSI-treated eggs is three each. Band intensities in these three samples were measured in each membrane.