The development of new biomarkers of spermatozoa quality in cattle

There is a current need for new biomarkers of spermatozoa quality, that consistently and correctly identify spermatozoa that will successfully contribute to subsequent embryo development. This could improve the standardization of semen analysis, decrease early embryo mortality, and use these biomarkers as a selection tool before servicing females. This study utilized imaging techniques to identify potential biomarkers of sperm quality, using sires previously classified as high (n = 4) or low (n = 4) performing at producing blastocysts in vitro. Spermatozoa were assessed before and following a gradient purification protocol, to understand how populations of cells are impacted by such protocols and may differ between in vivo and in vitro use. Pre-gradient samples from low-performing sires had an increased incidence of DNA damage, although post-gradient samples from high-performing sires were found to have an increased incidence of DNA damage. When evaluating morphology via fluorescent microscopy, the most prevalent defects in pre-gradient samples from high-performing sires were tail defects, which are successfully removed during purification processing. The most prevalent defects in pre-gradient samples from low-performing sires were aggresome defects located in the head, which would be brought into an oocyte upon fertilization and may impair embryo development. Image-based flow cytometry (IBFC) was employed to quantify defect prevalence to evaluate a greater sample size decreasing the variability that exists in manual assessments. Using IBFC, aggresome defects were again identified in the heads of spermatozoa from low-performing sires. Post-gradient samples from low-performing sires had a significantly greater (p < 0.05) incidence of aggresome defects than post-gradient samples from high-performing sires. Additionally, IBFC was used to evaluate spermatozoa viability following gradient purification. Distinct populations of sperm cells were identified. High-performing sires had more spermatozoa in the population deemed most viable than low-performing sires. This study demonstrated that spermatozoa defects vary in populations before and following gradient purification, indicating that it may be beneficial to separately evaluate semen for in vivo and in vitro use. Furthermore, a prevalent defect in low-performing sires that could explain a discrepancy between successful fertilization and embryo development was identified. Therefore, elucidating a malfunction regulated by sire, that could potentially affect early embryo development

A Non-synonymous point mutation in a WD-40 domain repeat of EML5 leads to decreased bovine sperm quality and fertility.

This study is part of a concerted effort to identify and phenotype rare, deleterious mutations that adversely affect sperm quality, or convey high developmental and fertility potential to embryos and ensuing progeny. A rare, homozygous mutation in EML5 (EML5R1654W), which encodes a microtubule-associated protein with high expression in testis and brain was identified in an Angus bull used extensively in artificial insemination (AI) for its outstanding progeny production traits. The bull?s fertility was low in cross-breeding timed AI (TAI) (Pregnancy/TAI = 25.2%; n = 222) and, in general, AI breeding to Nellore cows (41%; n = 822). A search of the 1,000 Bull Genomes Run9 database revealed an additional 74 heterozygous animals and 8 homozygous animals harboring this exact mutation across several different breeds (0.7% frequency within the 6,191 sequenced animals). Phenotypically, spermatozoa from the homozygous Angus bull displayed prominent piriform and tapered heads, and outwardly protruding knobbed acrosomes. Additionally, an increased retention of EML5 was also observed in the sperm head of both homozygous and heterozygous Angus bulls compared to wild-type animals. This non-synonymous point mutation is located within a WD40 signaling domain repeat of EML5 and is predicted to be detrimental to overall protein function by genomic single nucleotide polymorphism (SNP) analysis and protein modeling. Future work will examine how this rare mutation affects field AI fertility and will characterize the role of EML5 in spermatogenesis

Pharmacologic treatment with CPI-613 and PS48 decreases mitochondrial membrane potential and increases quantity of autolysosomes in porcine fibroblasts

A metabolic phenomenon known as the Warburg effect has been characterized in certain cancerous cells, embryonic stem cells, and other rapidly proliferative cell types. Previously, our attempts to induce a Warburg-like state pharmaceutically via CPI-613 and PS48 treatment did augment metabolite production and gene expression; however, this treatment demonstrated a Reverse Warburg effect phenotype observed in cancer-associated stroma. In the current study, we inquired whether the mitochondria were affected by the aforementioned pharmaceutical treatment as observed in cancerous stromal fibroblasts. While the pharmaceutical agents decreased mitochondrial membrane potential in porcine fetal fibroblasts, the number and size of mitochondria were similar, as was the overall cell size. Moreover, the fibroblasts that were treated with CPI-613 and PS48 for a week had increased numbers of large autolysosome vesicles. This coincided with increased intensity of LysoTracker staining in treated cells as observed by flow cytometry. Treated fibroblasts thus may utilize changes in metabolism and autophagy to mitigate the damage of treatment with pharmaceutical agents. These findings shed light on how these pharmaceutical agents interact and how treated cells augment metabolism to sustain viability. c2019, The Author(s).Includes bibliographical references

The Testicular and Epididymal Expression Profile of PLCζ in Mouse and Human Does Not Support Its Role as a Sperm-Borne Oocyte Activating Factor

Phospholipase C zeta (PLCζ) is a candidate sperm-borne oocyte activating factor (SOAF) which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT) and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF) revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides

Identification of the Inorganic Pyrophosphate Metabolizing, ATP Substituting Pathway in Mammalian Spermatozoa

Inorganic pyrophosphate (PPi) is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1) in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF) rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS) and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies

Evidence for Paternal Leakage in Hybrid Periodical Cicadas (Hemiptera: Magicicada spp.)

Mitochondrial inheritance is generally assumed to be maternal. However, there is increasing evidence of exceptions to this rule, especially in hybrid crosses. In these cases, mitochondria are also inherited paternally, so “paternal leakage” of mitochondria occurs. It is important to understand these exceptions better, since they potentially complicate or invalidate studies that make use of mitochondrial markers. We surveyed F1 offspring of experimental hybrid crosses of the 17-year periodical cicadas Magicicada septendecim, M. septendecula, and M. cassini for the presence of paternal mitochondrial markers at various times during development (1-day eggs; 3-, 6-, 9-week eggs; 16-month old 1st and 2nd instar nymphs). We found evidence of paternal leakage in both reciprocal hybrid crosses in all of these samples. The relative difficulty of detecting paternal mtDNA in the youngest eggs and ease of detecting leakage in older eggs and in nymphs suggests that paternal mitochondria proliferate as the eggs develop. Our data support recent theoretical predictions that paternal leakage may be more common than previously estimated

Doubly Uniparental Inheritance of Mitochondria As a Model System for Studying Germ Line Formation

BACKGROUND: Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos. METHODOLOGY/PRINCIPAL FINDINGS: We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow. CONCLUSIONS/SIGNIFICANCE: In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans

Paternal mtDNA and Maleness Are Co-Inherited but Not Causally Linked in Mytilid Mussels

BACKGROUND: In marine mussels of the genus Mytilus there are two mitochondrial genomes. One is transmitted through the female parent, which is the normal transmission route in animals, and the other is transmitted through the male parent which is an unusual phenomenon. In males the germ cell line is dominated by the paternal mitochondrial genome and the somatic cell line by the maternal. Research to date has not allowed a clear answer to the question of whether inheritance of the paternal genome is causally related to maleness. METHODOLOGY/PRINCIPAL FINDINGS: Here we present results from hybrid crosses, from triploid mussels and from observations of sperm mitochondria in fertilized eggs which clearly show that maleness and presence of the paternal mitochondrial genome can be decoupled. These same results show that the female mussel has exclusive control of whether her progeny will inherit the mitochondrial genome of the male parent. CONCLUSIONS/SIGNIFICANCE: These findings are important in our efforts to understand the mechanistic basis of this unusual mode of mitochondrial DNA inheritance that is common among bivalves

Biparental inheritance of plastidial and mitochondrial DNA and hybrid variegation in Pelargonium

Plastidial (pt) and mitochondrial (mt) genes usually show maternal inheritance. Non-Mendelian, biparental inheritance of plastids was first described by Baur (Z Indukt Abstamm Vererbungslehre 1:330–351, 1909) for crosses between Pelargonium cultivars. We have analyzed the inheritance of pt and mtDNA by examining the progeny from reciprocal crosses of Pelargoniumzonale and P. inquinans using nucleotide sequence polymorphisms of selected pt and mt genes. Sequence analysis of the progeny revealed biparental inheritance of both pt and mtDNA. Hybrid plants exhibited variegation: our data demonstrate that the inquinans chloroplasts, but not the zonale chloroplasts bleach out, presumably due to incompatibility of the former with the hybrid nuclear genome. Different distribution of maternal and paternal sequences could be observed in different sectors of the same leaf, in different leaves of the same plant, and in different plants indicating random segregation and sorting-out of maternal and paternal plastids and mitochondria in the hybrids. The substantial transmission of both maternal and paternal mitochondria to the progeny turns Pelargonium into a particular interesting subject for studies on the inheritance, segregation and recombination of mt genes

Inheritance of mitochondrial DNA in the rotifer Brachionus plicatilis

By crossing Brachionus plicatilis s.s. NH1L strain and German strain, we obtained two types of hybrids, NH1L female × German male designated as NXG and German female × NH1L male designated as GXN. To confirm the crossing of the two hybrid strains at the genetic level, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis using 10 kinds of primers (10 and 12 mers) was carried out. Some amplified DNA fragments from RAPD of the hybrid strain showed mixed patterns of both parental strains, thus confirming that both hybrids were crossbreeds of the NH1L and German strains. Using these hybrids, we investigated the mode of mitochondrial inheritance in B. plicatilis. Full length mtDNA of the four strains was amplified by PCR, and digested with restriction enzymes to obtain restriction fragment length polymorphism (RFLP) patterns. Both hybrid strains had the same RFLP patterns as their female parents. This result shows that mitochondrial inheritance in rotifers is maternal