Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: An approach to non-surgical sterilization.

The surgical sterilization of cats and dogs has been used to prevent their unwanted breeding for decades. However, this is an expensive and invasive procedure, and often impractical in wider contexts, for example the control of feral populations. A sterilization agent that could be administered in a single injection, would not only eliminate the risks imposed by surgery but also be a much more cost-effective solution to this worldwide problem. In this study, we sought to develop a targeting peptide that would selectively bind to Leydig cells of the testes. Subsequently, after covalently attaching a cell ablation agent, Auristatin, to this peptide we aimed to apply this conjugated product (LH2Auristatin) to adult male mice in vivo, both alone and together with a previously developed Sertoli cell targeting peptide (FSH2Menadione). The application of LH2Auristatin alone resulted in an increase in sperm DNA damage, reduced mean testes weights and mean seminiferous tubule size, along with extensive germ cell apoptosis and a reduction in litter sizes. Together with FSH2Menadione there was also an increase in embryo resorptions. These promising results were observed in around a third of all treated animals. Given this variability, we discuss how these reagents might be modified in order to increase target cell ablation and improve their efficacy as sterilization agents.

Population decline: where demography, social science and biology intersect.

Since the early 1960's the world has witnessed the spectacular collapse of human fertility. As a result of this phenomenon several countries are already seeing their population numbers fall and more will follow in the coming decades. The causes of this fertility decline involve a complex interplay of socioeconomic, environmental, and biological factors that have converged to constrain fertility in posterity's wake. Since large numbers of offspring are no longer needed to compensate for high infant mortality in contemporary society, couples have opted to have small families in a quality-over-quantity investment in their progeny's future. Simultaneously, increases in female education, the enhanced participation of women in the paid workforce, and a resultant delay in childbearing has placed limits on achievable family size. Progressive urbanization, the improved availability of contraceptives and the socioeconomic pressures experienced by young adults in ageing societies, are also contributing to fertility's demise. These factors together with the individualism that pervades modern society and the increasing social acceptability of voluntary childlessness, have firmly established a low fertility ethos in most post-transition countries. Since none of these forces are about to relent, it looks as if extremely low fertility might be with us for some time to come. This may have long-term consequences. The lack of selection pressure on high fertility genotypes, the ability of ART to retain poor fertility genotypes within the population and sustained exposure to reproductive toxicants in modern industrialized environments, may all contrive to leave a permanent mark on the fecundity of our species.

The Global Decline in Human Fertility: The Post-Transition Trap Hypothesis.

Over the past half a century many countries have witnessed a rapid fall in total fertility rates, particularly in the world's most advanced economies including the industrial powerhouses of Eastern Asia and Europe. Such nations have now passed through the first and second demographic transitions and are currently exhibiting fertility rates well below the replacement threshold of 2.1, with no sign of recovery. This paper examines the factors responsible for driving these demographic transitions and considers their impact on both fertility and fecundity (our fundamental capacity to reproduce). I argue that because the first demographic transition was extremely rapid and largely driven by socioeconomic factors, it has had no lasting impact on the genetic/epigenetic underpinnings of human fecundity. However, the second demographic transition will be different. A series of conditions associated with low fertility societies, including relaxed selection pressure for high-fertility genotypes, the indiscriminate use of assisted reproductive technologies to treat human infertility, and environmental contamination with reproductive toxicants, may impact our genetic constitution in ways that compromise the future fecundity of our species. Since any fundamental change in the genetic foundations of human reproduction will be difficult to reverse, we should actively pursue methods to monitor human fecundity, as sub-replacement fertility levels become established across the globe.

Precise control of microfluidic flow conditions is critical for harnessing the in vitro transfection capability of pDNA-loaded lipid-Eudragit nanoparticles.

Microfluidics is widely regarded as a leading technology for industrial-scale manufacture of multicomponent, gene-based nanomedicines in a reproducible manner. Yet, very few investigations detail the impact of flow conditions on the biological performance of the product, particularly biocompatibility and therapeutic efficiency. Herein, this study investigated the engineering of a novel lipid-Eudragit hybrid nanoparticle in a bifurcating microfluidics micromixer for plasmid DNA (pDNA) delivery. Nanoparticles of ~150 nm in size, with uniform polydispersity index (PDI = 0.2) and ξ-potential of 5-11 mV were formed across flow rate ratios (FRR, aqueous to organic phase) of 3:1 and 5:1, respectively. The hybrid nanoparticles maintained colloidal stability and structural integrity of loaded pDNA following recovery by ultracentrifugation. Importantly, in vitro testing in human embryonic kidney cell line (HEK293T) revealed significant differences in biocompatibility and transfection efficiency (TE). Lipid-Eudragit nanoparticles produced at FRR 3:1 displayed high cellular toxicity (0-30% viability), compared with nanoparticles prepared at FRR 5:1 (50-100% viability). Red fluorescent protein (RFP) expression was sustained for 24-72 h following exposure of cells to nanoparticles, indicating controlled release of pDNA and trafficking to the nucleus. Nanoparticles produced at FRR 5:1 resulted in markedly higher TE (12%) compared with those prepared at FRR 3:1 (2%). Notably, nanoparticles produced using the bench-scale nanoprecipitation method resulted in lower biocompatibility (30-90%) but higher RFP expression (25-38%). These findings emphasize the need for in-depth analysis of the effect of formulation and flow conditions on the physicochemical and biological performance of gene nanomedicines when transitioning from bench to clinic.

Development of a model for studying the developmental consequences of oxidative sperm DNA damage by targeting redox-cycling naphthoquinones to the Sertoli cell population.

Oxidative stress can be induced in the testes by a wide range of factors, including scrotal hyperthermia, varicocele, environmental toxicants, obesity and infection. The clinical consequences of such stress include the induction of genetic damage in the male germ line which may, in turn, have serious implications for the health and wellbeing of the progeny. In order to confirm the transgenerational impact of oxidative stress in the testes, we sought to develop an animal model in which this process could be analysed. Our primary approach to this problem was to induce Sertoli cells (robust, terminally differentiated, tissue-specific testicular cells whose radioresistance indicates significant resistance to oxidative stress) to generate high levels of reactive oxygen species (ROS) within the testes. To achieve this aim, six follicle-stimulating hormone (FSH) peptides were developed and compared for selective targeting to Sertoli cells both in vitro and in vivo. Menadione, a redox-cycling agent, was then conjugated to the most promising FSH candidate using a linker that had been optimised to enable maximum production of ROS in the targeted cells. A TM4 Sertoli cell line co-incubated with the FSH-menadione conjugate in vitro exhibited significantly higher levels of mitochondrial ROS generation (10-fold), lipid peroxidation (2-fold) and oxidative DNA damage (2-fold) than the vehicle control. Additionally, in a proof-of-concept study, ten weeks after a single injection of the FSH-menadione conjugate in vivo, injected male mice were found to exhibit a 1.6 fold increase in DNA double strand breaks and 13-fold increase in oxidative DNA damage to their spermatozoa while still retaining their ability to initiate a pregnancy. We suggest this model could now be used to study the influence of chronic oxidative stress on testicular function with emphasis on the impact of DNA damage in the male germ line on the mutational profile and health of future generations.

Predicting the Outcome of Equine Artificial Inseminations Using Chilled Semen.

This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm® Equine portable device. After the initial evaluation, a subpopulation of cells was subjected to a migration assay through a 5 µm polycarbonate filter within a Samson™ isolation chamber over a 15 min period. The cells were assessed for their concentration, motility parameters, and ability to reduce the membrane impermeant tetrazolium salt WST-1. The data, combined with the stallion and mare's ages, were used to predict the likelihood of pregnancy, as confirmed by rectal ultrasound sonography performed 14 days post ovulation. The criteria used to predict pregnancy were optimized for each individual stallion, resulting in an overall accuracy of 87.9% if analyzed pre-chilling and 95% if analyzed post-chilling. This study suggests that an analysis of stallion ejaculate can be used to predict the likelihood of pregnancy resulting from artificial insemination in mares with a high level of accuracy.

Establishment and characterization of oviductal organoids from farm and companion animals†.

Organoid technology has provided a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of disease. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. This is particularly true for the field of reproduction and the application of assisted reproductive technologies (ART). Here, we have developed a platform to grow oviductal organoids from five domestic species-bovine, porcine, equine, feline, and canine. The organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected infundibular/fimbrial samples. The addition of WNT, TGFß, BMP, ROCK, and Notch signaling pathway activators or inhibitors to the organoid culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were subsequently observed and described. After 21 days, well-defined and synchronized motile ciliated cells were observed in organoids. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have carried out a detailed cross-species comparison of oviductal organoids, which would be valuable in advancing our knowledge of oviduct physiology and, potentially, help in increasing the success of ART.

DNA damage in testicular germ cells and spermatozoa. When and how is it induced? How should we measure it? What does it mean?

This review surveys the causes and consequences of DNA damage in the male germ line from spermatogonial stem cells to fully differentiated spermatozoa. Within the stem cell population, DNA integrity is well maintained as a result of excellent DNA surveillance and repair; however, a progressive increase in background mutation rates does occur with paternal age possibly as a result of aberrant DNA repair as well as replication error. Once a germ cell has committed to spermatogenesis, it responds to genetic damage via a range of DNA repair pathways or, if this process fails, by the induction of apoptosis. When fully-differentiated spermatozoa are stressed, they also activate a truncated intrinsic apoptotic pathway which results in the activation of nucleases in the mitochondria and cytoplasm; however, the physical architecture of these cells prevents these enzymes from translocating to the nucleus to induce DNA fragmentation. Conversely, hydrogen peroxide released from the sperm midpiece during apoptosis is able to penetrate the nucleus and induce DNA damage. The base excision repair pathway responds to such damage by cleaving oxidized bases from the DNA, leaving abasic sites that are alkali-labile and readily detected with the comet assay. As levels of oxidative stress increase and these cells enter the perimortem, topoisomerase integrated into the sperm chromatin becomes activated by SUMOylation. Such activation may initially facilitate DNA repair by reannealing double strand breaks but ultimately prepares the DNA for destruction by nucleases released from the male reproductive tract. The abasic sites and oxidized base lesions found in live spermatozoa are mutagenic and may increase the mutational load carried by the offspring, particularly in the context of assisted conception. A variety of strategies are described for managing patients expressing high levels of DNA damage in their spermatozoa, to reduce the risks such lesions might pose to offspring health.

Predicting the outcome of Thoroughbred stallion matings on the basis of dismount semen sample analyses.

In brief: A capacity to predict the likelihood of pregnancy following natural matings would be of considerable benefit to the Thoroughbred horse breeding industry. In this article, we describe a strategy for achieving this outcome through the analysis of dismount samples, that achieved an overall accuracy of 94.6%. Abstract: The purpose of this study was to determine whether the analysis of dismount semen samples from Thoroughbred stallions could be used to predict whether a given mating would result in a pregnancy. The analysis was based on 143 matings of 141 mares by a cohort of 7 Thoroughbred stallions over a 4-week period at an Australian Stud. The criteria of semen quality utilized in this analysis involved a preliminary assessment of the raw dismount sample in terms of semen volume, sperm number, and sperm movement characteristics using an iSperm® Equine portable device. Following this initial assessment, a subpopulation of progressively motile spermatozoa was isolated by virtue of the cells ability to migrate across a 5 µm polycarbonate filter in a Samson™ isolation chamber over a 15-minute period. These isolated cells were again evaluated for their number and quality of movement using the iSperm® system and, in addition, assessed for their ability to reduce WST-1, a membrane impermeant tetrazolium salt. These data were then combined with additional information describing the ages of the animals used in this study, their historical breeding records, and mating frequency during the breeding season. The total data set was then used to predict the occurrence of pregnancy, as confirmed by ultrasound at ~14 days post mating. The criteria used to predict fertility in the ensuing multivariate discriminant analysis were optimized for each individual stallion. Using this strategy, we were able to successfully predict the outcome of a cover with an overall accuracy of 94.6%.

Sperm cryopreservation: current status and future developments.

The cryopreservation of spermatozoa is an important reproductive technology for the preservation of fertility in man and animals. Since the serendipitous discovery of glycerol as an effective cryoprotectant in 1947, sperm cryopreservation has undergone many changes in terms of the freezing methods employed, the rates at which samples are frozen and thawed, and the media used to preserve sperm functionality and DNA integrity. An extensive literature survey has been conducted addressing the cryoprotectants employed for both animal and human semen and the freezing protocols utilised. The results indicate that glycerol remains the dominant cryoprotective agent, usually incorporated into a balanced salt solution containing energy substrates, buffers, osmolytes and protein in the form of human serum albumin (human) or skimmed milk (animal). Realisation that some of the damage observed in cryostored cells involves the generation of reactive oxygen species during the thawing process, has prompted many studies to assess the relative merits of incorporating antioxidants into the cryopreservation media. However, in the absence of systematic comparisons, there is currently no consensus as to which antioxidant combination might be the most effective. Utilising our fundamental understanding of cryodamage to optimise cryopreservation protocols for each species will be important in the future.

Role of sperm DNA damage in creating de-novo mutations in human offspring: the 'post-meiotic oocyte collusion' hypothesis.

Spermatogonial stem cells exhibit a low level of spontaneous mutation that is heavily impacted by paternal age via mechanisms that appear to involve the aberrant repair of DNA damage. This background de-novo mutation frequency can be increased 1000-fold by mutations affecting a key signal transduction pathway that confers upon its descendants a selective advantage, leading to clonal expansion and nests of mutant germ cells in the testes of ageing males. This 'selfish selection' model effectively explains the origin of several dominant developmental disorders, such as achondroplasia and Apert syndrome, but cannot be generalized to account for the majority of de-novo mutations where no selective advantage is apparent. In this article, an additional germline mutation pathway is proposed that recognizes the unique susceptibility of spermatozoa to DNA damage and the importance of the oocyte in repairing these lesions prior to the S phase of the first mitotic division. Any deficiency or inaccuracy on the part of the oocyte in effecting this repair process has the potential to fix paternal DNA damage as a de-novo mutation in the embryo. Such a mechanism supports emerging data indicating that assisted conception procedures may enhance the mutational load carried by ART offspring.

Causative mechanisms and functional correlates of MTT reduction in stallion spermatozoa.

MTT is a commonly used cell vitality probe, due to its ability to form insoluble formazan deposits at cellular locations of intense oxidoreductase activity. Although this response is considered a reflection of mitochondrial redox activity, extra-mitochondrial sites of MTT reduction have been recognized within the spermatozoa of several mammalian species. Therefore, the aim of this study was to determine the major sites and causative mechanisms of MTT reduction in stallion spermatozoa. Our results show that stallion spermatozoa displayed substantial mitochondrial formazan deposition, as well as a single extra-mitochondrial formazan deposit in various locations on the sperm head in approximately 20% of cells. The quality and capacitation status of stallion spermatozoa were positively correlated with the presence of an extra-mitochondrial formazan granule. Additionally, extra-mitochondrial formazan deposition was suppressed by the presence of an NADPH oxidase (NOX) inhibitor (VAS2870; active against NOX2, NOX4 and NOX5), MnTMPyP (SOD mimetic) and zinc (NOX5 inhibitor) suggesting that extra-mitochondrial MTT reduction may be facilitated by NOX-mediated ROS generating activity, conceivably NOX5 or NOX2. When comparing MTT to resazurin, another well-known probe used to detect metabolically active cells, MTT reduction had a higher correlation with sperm concentration and motility parameters (R2= 0.91), than resazurin reduction (R2 = 0.76). We conclude that MTT reduction in stallion spermatozoa follows a species-specific pattern due to a high dependence on oxidative phosphorylation and a degree of NOX activity. As such, MTT reduction is a useful diagnostic tool to assess extra-mitochondrial redox activity, and therefore, the functional qualities of stallion spermatozoa.

Rapid impact of COVID-19 infection on semen quality: a case report.

A unique opportunity to conduct a longitudinal analysis of semen quality in a male subject immediately before, during and after COVID-19 infection, has revealed new insights into the impact of this virus on male reproductive function. A moderate COVID infection that did not require hospitalization resulted in a state of azoospermia that persisted for 4 weeks. Given that the duration of spermatogenesis and epididymal sperm maturation in the human is 78 days, we calculate that a viral attack on the germ line was initiated at or before the patient was symptomatic and may have been signalled by a sudden reduction in sperm count and motility, several weeks earlier. Before the virus had been fully cleared, reinitiation of spermatogenesis occurred as evidenced by spermatozoa reappearing in the ejaculate exhibiting high levels of motility but significant levels of oxidative DNA damage as measured by a modified 8-OHdG assay protocol. These unique data indicate that even a moderate COVID-19 infection is capable of rapidly inducing a state of azoospermia that rapidly reverses as the infection wanes.

The future of assessing bull fertility: Can the 'omics fields identify usable biomarkers?†.

Breeding soundness examinations for bulls rely heavily on the subjective, visual assessment of sperm motility and morphology. Although these criteria have the potential to identify infertile males, they cannot be used to guarantee fertility or provide information about varying degrees of bull fertility. Male factor fertility is complex, and the success of the male gamete is not necessarily realized until well after the spermatozoon enters the oocyte. This paper reviews our existing knowledge of the bull's contribution from a standpoint of the sperm's cargo and the impact that this can have on fertilization and the development of the embryo. There has been a plethora of recent research characterizing the many molecular attributes that can affect the functional competence of a spermatozoon. A better understanding of the molecular factors influencing fertilization and embryo development in cattle will lead to the identification of biomarkers for the selection of bulls of superior fertility, which will have major implications for livestock production. To see this improvement in reproductive performance, we believe incorporation of modern technology into breeding soundness examinations will be necessary-although many of the discussed technologies are not ready for large-scale field application. Each of the 'omics fields discussed in this review have shown promise for the identification of biomarkers of fertility, with certain families of biomarkers appearing to be better suited to different evaluations throughout a bull's lifetime. Further research is needed for the proposed biomarkers to be of diagnostic or predictive value.

Male Infertility and Oxidative Stress: A Focus on the Underlying Mechanisms.

Reactive oxygen species (ROS) play a critical role in defining the functional competence of human spermatozoa. When generated in moderate amounts, ROS promote sperm capacitation by facilitating cholesterol efflux from the plasma membrane, enhancing cAMP generation, inducing cytoplasmic alkalinization, increasing intracellular calcium levels, and stimulating the protein phosphorylation events that drive the attainment of a capacitated state. However, when ROS generation is excessive and/or the antioxidant defences of the reproductive system are compromised, a state of oxidative stress may be induced that disrupts the fertilizing capacity of the spermatozoa and the structural integrity of their DNA. This article focusses on the sources of ROS within this system and examines the circumstances under which the adequacy of antioxidant protection might become a limiting factor. Seminal leukocyte contamination can contribute to oxidative stress in the ejaculate while, in the germ line, the dysregulation of electron transport in the sperm mitochondria, elevated NADPH oxidase activity, or the excessive stimulation of amino acid oxidase action are all potential contributors to oxidative stress. A knowledge of the mechanisms responsible for creating such stress within the human ejaculate is essential in order to develop better antioxidant strategies that avoid the unintentional creation of its reductive counterpart.

Proteomic analysis of spermatozoa reveals caseins play a pivotal role in preventing short-term periods of subfertility in stallions†.

Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days postbreeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations and to determine the biological mechanisms governing such differences. Using liquid chromatography-mass spectrometry (LC-MS/MS), we compared the proteomic profile of semen samples collected from commercially "fertile" stallions, during high- and low-fertility periods. A total of 1702 proteins were identified, of which, 38 showed a significant change in abundance (P ≤ 0.05). Assessment of intra- and interstallion variability revealed that caseins (namely κ-, α-S1-, and α-S2-casein) were significantly more abundant during "high-fertility" periods, while several epididymal, and seminal plasma proteins (chiefly, epididymal sperm binding protein 1 [ELSPbP1], horse seminal plasma protein 1 [HSP-1], and clusterin), were significantly more abundant during "low-fertility" periods. We hypothesized that an increased abundance of caseins offers greater protection from potentially harmful seminal plasma proteins, thereby preserving cell functionality and fertility. In vitro exposure of spermatozoa to casein resulted in decreased levels of lipid scrambling (Merocyanine 540), higher abundance of sperm-bound caseins (α-S1-, α-S2-, and κ-casein), and lower abundance of sperm-bound HSP-1 (P ≤ 0.05). This study demonstrates key pathways governing short-term fertility fluctuations in the stallion, thereby providing a platform to develop robust, fertility assessment strategies into the future.