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A sperm-specific phospholipase (PL) C, termed PLCζ, is proposed to be the soluble sperm factor that induces Ca2+ oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCζ mRNA have reduced amounts of PLCζ protein. Sperm derived from these transgenic mice trigger patterns of Ca2+ oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca2+ oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCζ is the physiological trigger of Ca2+ oscillations required for activation of development.

In all species studied, fertilization induces intracellular Ca21 ([Ca21]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca21]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca21]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca21]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca21]i oscillations to injection of stallion sperm, and [Ca21]i monitoring was performed. Such pairs consistently displayed [Ca21]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species. assisted reproductive technology, calcium, fertilization, gamete biology

We have previously demonstrated that initiation of intracellular calcium ([Ca 21 ]i) oscillations in mouse eggs signals activation or apoptotic death depending on the age of the eggs in which the oscillations are induced. To extend these studies, mouse eggs were aged in vitro to 24, 32, and 40 h post-hCG and injected with sperm cytosolic factor (SF), adenophostin A, or sperm (intracytoplasmic sperm injection), and the times at which signs of apoptosis first appeared were examined. These treatments, which induced [Ca 21 ]i oscillations, caused fragmentation and other signs of programmed cell death in eggs as early as 32 h post-hCG. The susceptibility of aged eggs to apoptosis appeared to be due to cytoplasmic deficiencies, because fusion of recently ovulated eggs with aged, SF-injected eggs prevented fragmentation. Evaluation of mRNA and protein levels of the apoptotic regulatory proteins Bcl-2 and Bax showed a prominent decrease in the amounts of Bcl-2 mRNA and protein in aged eggs, whereas Bax mRNA levels did not appear to be changed. Lastly, the Ca 21 responses induced by the aforementioned Ca 21 agonists ceased in advance in aged eggs. Together, these results suggest that one or several critical cytosolic molecules involved in the regulation of Ca 21 homeostasis, and in maintaining the equilibrium between anti- and proapoptotic proteins, is either lost or inactivated during postovulatory egg aging, rendering the fertilizing Ca 21 signal into an apoptosis-inducing signal.

A sperm-specific phospholipase (PL) C, termed PLCzeta, is proposed to be the soluble sperm factor that induces Ca(2+) oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCzeta mRNA have reduced amounts of PLCzeta protein. Sperm derived from these transgenic mice trigger patterns of Ca(2+) oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca(2+) oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCzeta is the physiological trigger of Ca(2+) oscillations required for activation of development.

In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.

We have previously demonstrated that initiation of intracellular calcium ([Ca2+]i) oscillations in mouse eggs signals activation or apoptotic death depending on the age of the eggs in which the oscillations are induced. To extend these studies, mouse eggs were aged in vitro to 24, 32, and 40 h post-hCG and injected with sperm cytosolic factor (SF), adenophostin A, or sperm (intracytoplasmic sperm injection), and the times at which signs of apoptosis first appeared were examined. These treatments, which induced [Ca2+]i oscillations, caused fragmentation and other signs of programmed cell death in eggs as early as 32 h post-hCG. The susceptibility of aged eggs to apoptosis appeared to be due to cytoplasmic deficiencies, because fusion of recently ovulated eggs with aged, SF-injected eggs prevented fragmentation. Evaluation of mRNA and protein levels of the apoptotic regulatory proteins Bcl-2 and Bax showed a prominent decrease in the amounts of Bcl-2 mRNA and protein in aged eggs, whereas Bax mRNA levels did not appear to be changed. Lastly, the Ca2+ responses induced by the aforementioned Ca2+ agonists ceased in advance in aged eggs. Together, these results suggest that one or several critical cytosolic molecules involved in the regulation of Ca2+ homeostasis, and in maintaining the equilibrium between anti- and proapoptotic proteins, is either lost or inactivated during postovulatory egg aging, rendering the fertilizing Ca2+ signal into an apoptosis-inducing signal.