Functional assessment of centrosomes of spermatozoa and spermatids microinjected into rabbit oocytes

Abstract

Although intracytoplasmic sperm injection (ICSI) is a widely used assisted reproductive technique, the fertilization rates and pregnancy rates of immature spermatids especially in round spermatid injection (ROSI) remain very low. During mammalian fertilization, the sperm typically introduces its own centrosome which then acts as a microtubule organizing center (MTOC) and is essential for the male and female genome union. In order to evaluate the function of immature germ cell centrosomes, we used the rabbit gamete model because rabbit fertilization follows paternal pattern of centrosome inheritance. First, rabbit spermatids and spermatozoa were injected into oocytes using a piezo-micromanipulator. Next, the centrosomal function to form a sperm aster was determined. Furthermore, two functional centrosome proteins (gamma-tubulin and centrin) of the rabbit spermatogenic cells were examined. Our results show that the oocyte activation rates by spermatozoa, elongated spermatids, and round spermatids were 86% (30/35), 30% (11/36), and 5% (1/22), respectively. Sperm aster formation rates after spermatozoa, elongated spermatids, and round spermatids injections were 47% (14/30), 27% (3/11), and 0% (0/1), respectively. The aster formation rate of the injected elongating/elongated spermatids was significantly lower than that of the mature spermatozoa (P = 0.0242). Moreover, sperm asters were not observed in round spermatid injection even after artificial activation. These data suggest that poor centrosomal function, as measured by diminished aster formation rates, is related to the poor fertilization rates when immature spermatogenic cells are injected.

The Effect on Intracytoplasmic Sperm Injection Outcome of Genotype, Male Germ Cell Stage and Freeze-Thawing in Mice

“…For example, in rabbits no or very few pups were born following ROSI because of a high incidence of aneuploid embryos [13], [14]. This might be caused by the inability of rabbit round spermatids to form a microtubule-organizing center (MTOC) in the ooplasm [15]. In mice, by contrast, the MTOC is formed by the ooplasmic asters so ROSI does not always cause aneuploidy [16], [17], although some aberrant DNA methylation and histone acetylation of the paternal genome has been reported [18], [19].…”

Development of feline embryos produced by Piezo-actuated intracytoplasmic sperm injection of elongated spermatids

“…In rabbits, over 75% of oocytes injected with elongated spermatids were activated and Ca 2+ oscillations were induced [34]. For this reason, artificial activation has not been carried out by microinjection using elongated spermatids in rabbits [21, 35]. Although 89% of oocytes injected with elongated spermatids were activated in mice, Ca 2+ oscillations were not observed and only a few Ca 2+ rises observed [34].…”

Assisted Reproductive Technologies (ART) With Baboons Generate Live Offspring: A Nonhuman Primate Model for ART and Reproductive Sciences

“…This is important for several reasons, including the closer genetic proximity to humans of baboons compared to macaques like the well studied rhesus monkey. Furthermore, discoveries using discarded specimens from infertile patient-couples are leading to novel approaches for diagnosing idiopathic forms of human infertility (Tachibana et al, 2009b), and primate confirmations are important. Finally, the availability of the baboon as a research resource complements the more limited macaque.…”