Factors From Damaged Sperm Affect Its DNA Integrity and Its Ability to Promote Embryo Implantation in Mice

Abstract

Endogenous nucleases in mouse sperm can be activated by freeze-thawing the spermatozoa in media without cryoprotection and cleaving spermatozoa DNA. The role of sperm chromatin integrity during intracytoplasmic sperm injection (ICSI) is of critical importance. We analyzed in the B6D2 mouse the proportion of DNA-fragmented spermatozoa (DFS) produced by incubation in conditioned medium (CM) generated by freeze-thawing sperm in the absence of cryoprotection. We then examined the subsequent development, implantation, and offspring obtained after ICSI with incubated spermatozoa. When fresh sperm cells were incubated for 90 minutes in this CM, a significant increase in the amount of DFS was detected by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay (27% vs 4.5% in fresh sperm). After ICSI of fresh and incubated spermatozoa, embryos were cultured in vitro to either the 2-cell or blastocyst stage before they were transferred into pseudopregnant CD1 females. On day 14, recipients were sacrificed, and implantation rates, estimated as the number of live fetuses plus resorptions, were determined. When ICSI was performed with sperm incubated in CM, no effects on fertilization, embryo cleavage, blastocyst rate, or blastocyst morphology were detected; however, the quality of the embryos was affected because the total implantation rate decreased significantly (P , .05) when 2-cell embryos or blastocysts were transferred. Independently of sperm pretreatment, in vitro cultures significantly affected the percentage of live fetuses present on day 14 of pregnancy. These results demonstrated that there are factors released from fragmented spermatozoa capable of inducing DNA fragmentation in intact sperm that may compromise, to some extent, birth rates after ICSI.

Fertilizability, developmental competence, and chromosomal integrity of oocytes microinjected with pre-treated spermatozoa in mice

“…Therefore, detailed observation of embryos derived from pre-treated spermatozoa should be carried out during pre-and post-implantation. The present results revealed that embryos derived from damaged (pre-treated) spermatozoa frequently developed up to the blastocyst stage with active mitosis (Table 2), in agreement with another study (Perez-Crespo et al 2008). However, DNA-damaged spermatozoa reduced blastocyst formation depending on the disintegration level (Ahmadi & Ng 1999a, 1999b.…”

“…Chromosomal damage of pre-treated spermatozoa a number of membrane-damaged spermatozoa, since it is reported that the factor released from the spermatozoa induced DNA nicks (Perez-Crespo et al 2008). Nevertheless, ICSI oocytes with incubated or pre-treated spermatozoa have much higher rates of chromosome damage than that of the control group.…”

Factors Affecting Fertilization and Embryonic Development During Intracytoplasmic Sperm Injection in Pigs

“…The endogenous sperm nucleases are released from plasma membrane-damaged spermatozoa [44] and activated with divalent cations such as Mg 2+ and Ca 2+ [45]. Furthermore, it has been reported that incubation of spermatozoa with endogenous nuclease released from sperm leads to an increase in the proportion of DNA fragmented spermatozoa [39]. In our study, supplementation with EGTA, a chelating agent for the cations, in the buffer can inhibit the sperm DNA fragmentation and improved embryonic development after ICSI [38].…”

“…It has been reported that DNA-fragmented spermatozoa had adverse affect on in vitro embryonic development [38], where we used the DNA-fragmented sperm caused significantly after freeze-drying. There also reported the impaired embryo implantation and fetus development after ICSI in mice [39,40]. DNA fragmentation in human spermatozoa is one of the causes of failure of embryonic development and pregnancy [41].…”

Heterologous murine and bovine IVF using bottlenose dolphin (Tursiops truncatus) spermatozoa

“…Frozen 235 dolphin spermatozoa were thawed the day of the experiment whereas murine M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 9 spermatozoa were obtained from fresh semen. Murine semen was collected from a total of five 9-month old B6D2F1 mice [33]. Mice were fed ad libitum with a standard diet (Harland Ibérica, Barcelona, Spain) and maintained in a temperature and lightcontrolled room (23 °C; 14 hours light: 10 hours dark).…”