Identification of candidate mitochondrial inheritance determinants using the mammalian cell-free system.

The degradation of sperm-borne mitochondria after fertilization is a conserved event. This process known as post-fertilization sperm mitophagy, ensures exclusively maternal inheritance of the mitochondria-harbored mitochondrial DNA genome. This mitochondrial degradation is in part carried out by the ubiquitin-proteasome system. In mammals, ubiquitin-binding pro-autophagic receptors such as SQSTM1 and GABARAP have also been shown to contribute to sperm mitophagy. These systems work in concert to ensure the timely degradation of the sperm-borne mitochondria after fertilization. We hypothesize that other receptors, cofactors, and substrates are involved in post-fertilization mitophagy. Mass spectrometry was used in conjunction with a porcine cell-free system to identify other autophagic cofactors involved in post-fertilization sperm mitophagy. This porcine cell-free system is able to recapitulate early fertilization proteomic interactions. Altogether, 185 proteins were identified as statistically different between control and cell-free-treated spermatozoa. Six of these proteins were further investigated, including MVP, PSMG2, PSMA3, FUNDC2, SAMM50, and BAG5. These proteins were phenotyped using porcine in vitro fertilization, cell imaging, proteomics, and the porcine cell-free system. The present data confirms the involvement of known mitophagy determinants in the regulation of mitochondrial inheritance and provides a master list of candidate mitophagy co-factors to validate in the future hypothesis-driven studies.

An Exploration of Current and Perspective Semen Analysis and Sperm Selection for Livestock Artificial Insemination.

Artificial insemination of livestock has been a staple technology for producers worldwide for over sixty years. This reproductive technology has allowed for the rapid improvement of livestock genetics, most notably in dairy cattle and pigs. This field has experienced continuous improvements over the last six decades. Though much work has been carried out to improve the efficiency of AI, there are still many areas which continue to experience improvement, including semen analysis procedures, sperm selection techniques, sperm sexing technologies, and semen storage methods. Additionally, the use of AI continues to grow in beef cattle, horses, and small ruminants as the technology continues to become more efficient and yield higher pregnancy rates. In this review, AI trends in the various livestock species as well as cutting edge improvements in the aforementioned areas will be discussed at length. Future work will continue to refine the protocols which are used for AI and continue to increase pregnancy rates within all livestock species.

Mammalian Cell-Free System Recapitulates the Early Events of Post-Fertilization Sperm Mitophagy.

Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals.

NEDD4-like ubiquitin ligase 2 protein (NEDL2) in porcine spermatozoa, oocytes, and preimplantation embryos and its role in oocyte fertilization†.

The ubiquitin-proteasome system plays diverse regulatory and homeostatic roles in mammalian reproduction. Ubiquitin ligases are the substrate-specific mediators of ubiquitin-binding to its substrate proteins. The NEDD4-like ubiquitin ligase 2 (aliases NEDL2, HECW2) is a HECT-type ubiquitin ligase that contains one N-terminal HECW ubiquitin ligase domain, one C-terminal HECT ubiquitin ligase domain, one C2 domain, and two WW protein-protein interaction modules. Beyond its predicted ubiquitin-ligase activity, its cellular functions are largely unknown. Current studies were designed to investigate the content and distribution of NEDL2 in porcine spermatozoa, oocytes, zygotes, and early preimplantation embryos, and in cumulus cells before and after in vitro maturation with oocytes, and fibroblast cells as positive control by western blot and immunocytochemistry, and to examine its roles during oocyte fertilization. Multiple isoforms of NEDL2 were identified by WB. One at approximately 52 kDa was detected only in the germinal vesicle (GV) stage and metaphase II oocytes, and in early preimplantation embryos. Other isoforms were high mass bands at 91, 136, and 155 kDa, which were only detected in somatic cells. Interestingly, ejaculated spermatozoa prominently displayed the same 52 kDa band as oocytes; they also had two minor bands of 74 and 129 kDa, which were not detected in somatic cells or oocytes. By immunofluorescence, NEDL2 showed a diffused cytoplasmic localization in all cell types and accumulated in distinct foci in the germinal vesicles (GVs) of immature oocytes, in maternal and paternal pronuclei of zygotes and nuclei of embryo blastomeres and somatic cells. In blastocysts, the labeling intensity of NEDL2 was stronger in the inner cell mass than in trophoblast, indicating higher NEDL2 content in the ICM cells than in trophectoderm. NEDL2 abundance was 10 times higher in post-maturation oocyte-surrounding cumulus cells than that of cumulus cells before in vitro maturation with hormones, indicating that NEDL2 may have a unique role in cumulus cells after ovulation. Microinjection of anti-NEDL2 antibody into oocyte before IVF did not affect the percentage of oocytes fertilized, percentage of oocytes cleaved, or blastocyst formation. However, the anti-NEDL2 antibody decreased the number of pronuclei, accelerated the formation of nuclear precursor bodies at 6 h postfertilization, inhibited sperm DNA decondensation, and resulted in more fertilized oocytes without male pronuclear formation. In summary, NEDL2 may play a key role during fertilization, especially during sperm DNA decondensation.

Porcine model for the study of sperm capacitation, fertilization and male fertility.

Mammalian fertilization remains a poorly understood event with the vast majority of studies done in the mouse model. The purpose of this review is to revise the current knowledge about semen deposition, sperm transport, sperm capacitation, gamete interactions and early embryonic development with a focus on the porcine model as a relevant, alternative model organism to humans. The review provides a thorough overview of post-ejaculation events inside the sow's reproductive tract including comparisons with humans and implications for human fertilization and assisted reproductive therapy (ART). Porcine methodology for sperm handling, preservation, in vitro capacitation, oocyte in vitro maturation, in vitro fertilization and intra-cytoplasmic sperm injection that are routinely used in pig research laboratories can be successfully translated into ART to treat human infertility. Last, but not least, new knowledge about mitochondrial inheritance in the pig can provide an insight into human mitochondrial diseases and new knowledge on polyspermy defense mechanisms could contribute to the development of new male contraceptives.

The domestic pig as a model for the study of mitochondrial inheritance.

Maternal mitochondrial inheritance is a fundamental paradigm within reproductive biology, yet the molecular mechanisms which underlie this process remain poorly understood. The ubiquitin proteasome system (UPS) and branches of the autophagic pathway have been implicated in taking part in the active degradation of sperm mitochondria post-fertilization. Despite this knowledge, there remains much unknown about this process, including the cofactors and substrates involved, as well as the implications of what occurs when these systems of degradation fail. Mitochondrial inheritance research has utilized a variety of animal models. However, one model that is of particular importance, especially when attempting to link mitochondrial inheritance research to humans, is the domestic pig. Pigs offer relatively easy collection of gametes which are similar to those of humans. Furthermore, pigs are physiologically and anatomically more similar to humans than the majority of other model systems available. Porcine in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and novel cell-free systems are research tools which can be exploited to provide greater insight into the processes behind sperm mitochondrial degradation. In the future studies of mitochondrial inheritance, pigs will likely play a crucial role as an animal model system.

Boar semen improvement through sperm capacitation management, with emphasis on zinc ion homeostasis.

Critical to fertilization success, sperm capacitation within the female oviductal sperm reservoir endows mammalian spermatozoa with hyperactivated motility and capacity to fertilize. An elaborate cascade of signaling events during capacitation guides the redistribution of sperm plasma membrane seminolipid and cholesterol, Ca-influx and increases tyrosine phosphorylation to promote hyperactivated motility. Such events result in the remodeling of the sperm acrosome, increased fluidity and fusability of the plasma membrane, shedding of surface-adsorbed seminal plasma proteins that glue sperm heads to the oviductal epithelium and ultimately the release of hyperactivated spermatozoa from the oviductal sperm reservoir. Discovered recently, the capacitation-induced sperm zinc ion efflux and resultant zinc signatures are reflective of sperm capacitation status and fertilizing ability, inspiring the retrospection of zinc ion functions in the physiology and fertility of boar sperm and that of other species. This review also highlights the merit of the domestic boar as a biomedical model for spermatology and fertilization research. Relevant to the quest for better fertility management in the livestock industries, the benefits of zinc ion supplementation through nutrition and direct addition to extended semen are discussed in the context of artificial insemination (AI). Ideas are shared on future technologies for zinc management in AI doses and research on the sperm zinc-interacting proteome.