https://www.clevelandclinic.org/ReproductiveResearchCenter/docs/agradoc114.pdf

Role of reactive oxygen species in the pathophysiology of human reproduction

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2002.tb02599.x

Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques.

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca2+ oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca2+ release. The ‘sperm factor’ hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca2+ transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLCζ, that triggers Ca2+ oscillations in mouse eggs indistinguishable from those at fertilisation. PLCζ removal from sperm extracts abolishes Ca2+ release in eggs. Moreover, the PLCζ content of a single sperm was sufficient to produce Ca2+ oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLCζ as the molecular trigger for development of a fertilised egg into an embryo.

PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development.

Spermatogenesis is a productive and highly organized process that generates virtually unlimited numbers of sperm during adulthood. Continuous proliferation and differentiation of germ cells occur in a delicate balance with other testicular compartments, especially the supporting Sertoli cells1. Many complex aspects of testis function in humans and large animals have remained elusive because of a lack of suitable in vitro or in vivo models. Germ cell transplantation has produced complete donor-derived spermatogenesis in rodents2,3,4,5,6 but not in other mammalian species7,8,9. Production of sperm in grafted tissue from immature mammalian testes and across species has not yet been accomplished. Here we report the establishment of complete spermatogenesis by grafting testis tissue from newborn mice, pigs or goats into mouse hosts. This approach maintains structural integrity and provides the accessibility that is essential for studying and manipulating the function of testes and for preserving the male germ line. Our results indicate that this approach is applicable to diverse mammalian species.

Sperm from neonatal mammalian testes grafted in mice

At fertilization in mammals, the sperm induces a characteristic series of Ca2+ oscillations in the egg which serve as the essential trigger for egg activation and early development of the embryo. It is not known how the sperm initiates this fundamental process, however, nor has any pathway linking sperm-egg membrane-receptor binding with intracellular Ca2+ release been demonstrated. Microinjection of sperm extracts into mammalian eggs elicits Ca2+ oscillations identical to those occurring at fertilization, which suggests that sperm may introduce a Ca2+ oscillation-inducing factor into the egg on gamete membrane fusion. Here we identify a soluble sperm protein that exhibits Ca2+ oscillation-inducing ('oscillogen') activity in eggs. Sperm oscillogen exists as an oligomer with a subunit of M(r) 33K and a specific intracellular localization at the equatorial segment of the sperm head. Cloning of the 33K oscillogen complementary DNA indicates similarity with a hexose phosphate isomerase found in prokaryotes. This sperm-derived oscillogen, termed oscillin, may represent the physiological trigger for development in mammals.

Calcium oscillations in mammalian eggs triggered by a soluble sperm protein

The aim of this study was to investigate whether the human spermatozoon participates in the activation of human oocytes following intracytoplasmic sperm injection (ICSI) and if so, by what mechanism. In the first series of experiments, we randomized human oocytes which had remained unfertilized after in-vitro fertilization (IVF) or ICSI, for intracytoplasmic injection with live spermatozoa, spermatozoa presumed to be dead and no spermatozoa. Secondly, unfertilized human oocytes and freshly ovulated mouse oocytes were randomized for intracytoplasmic and sub-zonal injection with human sperm cytosolic fraction (CF) before and after heat treatment. We found that oocyte injection with initially motile spermatozoa induces human oocyte activation at a significantly higher rate than injection with dead spermatozoa (61 versus 0%; P < 0.001) or injection without a spermatozoon (61 versus 14%; P < 0.001). Intracytoplasmic injection of CF activated both human and mouse oocytes at the same rate as sperm injection of human oocytes (activation rates of 70 and 65% respectively). This effect was greatly reduced by heat treatment of the CF. From these experiments we conclude firstly that the human spermatozoon injected intracytoplasmically contributes to human oocyte activation and secondly that the spermatozoon releases into the oocyte a heat-sensitive, intracellularly active factor, which is not species-specific.

Human oocyte activation following intracytoplasmic injection: the role of the sperm cell.

Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity

In the present study we investigated the relevance of sperm immobilization prior to intracytoplasmic sperm injection (ICSI) in the fertilization process. Using supravital staining of the spermatozoa with eosin and studying sperm decondensation with 2 mM dithiothreitol (DTT) in conditions imitating sperm handling during ICSI, we demonstrated that immobilization of the spermatozoon by squeezing its tail between the glass pipette and the bottom of the dish damages the sperm plasma membrane. Polyvinylpyrrolidone (PVP), which is usually present in the drop with the spermatozoon to facilitate its handling, was found to impede the access of both eosin and DTT to the sperm nucleus. We conclude that (i) sperm immobilization prior to ICSI damages the sperm plasma membrane, that (ii) this damage is sufficient for thiol-reducing agents to gain access to the sperm nucleus, and finally that (iii) PVP possibly interferes with sperm nucleus decondensation.

Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation

When intracytoplasmic sperm injection (ICSI) is performed, it is important to know the capacity of sperm cells to activate the oocytes, although knowledge of their ability to fuse with the oocytes is not vital. Hamster oocytes are not suitable for this purpose because they are easily activated by the injection procedure Itself. We therefore investigated whether mouse oocytes could be used to assess the activation properties of human spermatozoa. Mouse oocytes were randomized for injection with initially motile spermatozoa, medium, heat-treated or salt-extracted spermatozoa, and the survival and activation rates were examined. About half of the mouse oocytes survived the intracytoplasmic injection of a human sperm cell. Unlike hamster oocytes, the rate of activation provoked by the injection procedure itself was acceptably low (20%), resembling in this respect the behaviour of human oocytes. Following the injection of initially motile human spermatozoa, all mouse oocytes were activated. The injection of heat-treated or salt-extracted human spermatozoa resulted in activation rates of 14 and 15% respectively, comparable with the results following sham ICSI. These data support the hypothesis of a sperm-associated oocyte activation factor. In most activated oocytes, the human sperm nucleus decondensed to form a male pronucleus. Cytogenetic analysis at the first metaphase revealed that human sperm chromosomes were able to undergo replication in a heterologous environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracytoplasmic injection of human spermatozoa into mouse oocytes : a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.

/pdf/sperm-associated-oocyte-activating-factor-is-released-from-7mfdl9oh0h.pdf

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.

Andrei Rybouchkin

Papers

Human oocyte activation following intracytoplasmic injection: the role of the sperm cell.

Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity

Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation

Intracytoplasmic injection of human spermatozoa into mouse oocytes : a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.