Hexavalent sperm-binding IgG antibody released from vaginal film for development of potent on-demand nonhormonal female contraception

Nonhormonal products for on-demand contraception are a global health technology gap; this unmet need motivated us to pursue the use of sperm-binding monoclonal antibodies to enable effective on-demand contraception. Here, using the cGMP-compliant Nicotiana-expression system, we produced an ultrapotent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody “Fab-IgG-Fab” (FIF). The Nicotiana-produced FIF had at least 10-fold greater sperm-agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulated the Nicotiana-produced FIF into a polyvinyl alcohol–based water-soluble contraceptive film and evaluated its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm were recovered from the vaginas of sheep receiving FIF Film. Our work supports the potential of multivalent contraceptive antibodies to provide safe, effective, on-demand nonhormonal contraception.

Impact of Cryopreservation on Motile Subpopulations and Tyrosine-Phosphorylated Regions of Ram Spermatozoa during Capacitating Conditions

The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm regions.

Signaling at Physical Barriers during Pollen–Pistil Interactions

In angiosperms, double fertilization requires pollen tubes to transport non-motile sperm to distant egg cells housed in a specialized female structure known as the pistil, mediating the ultimate fusion between male and female gametes. During this journey, the pollen tube encounters numerous physical barriers that must be mechanically circumvented, including the penetration of the stigmatic papillae, style, transmitting tract, and synergid cells as well as the ultimate fusion of sperm cells to the egg or central cell. Additionally, the pollen tube must maintain structural integrity in these compact environments, while responding to positional guidance cues that lead the pollen tube to its destination. Here, we discuss the nature of these physical barriers as well as efforts to genetically and cellularly identify the factors that allow pollen tubes to successfully, specifically, and quickly circumnavigate them.

Extreme climate event promotes phenological mismatch between sexes in hibernating ground squirrels

Hibernating ground squirrels rely on a short active period for breeding and mass accrual, and are thus vulnerable to extreme climate events that affect key periods in their annual cycle. Here, we document how a heatwave in March 2012 led to a phenological mismatch between sexes in Richardson’s ground squirrels (Urocitellus richardsonii). Females emerged from hibernation and commenced breeding earlier in 2012 relative to average female emergence. Although males had descended testes and pigmented scrota, it appeared that not all males were physiologically prepared to breed since 58.6% of males had non-motile sperm when breeding commenced. Body condition, relative testes size, and the relative size of accessory glands were significant predictors of sperm motility. Males with non-motile sperm had smaller accessory glands than males with motile sperm. There was no decrease in the number of juveniles that emerged in 2012 or female yearlings recruited in 2013, nor did juveniles emerge later than other years. The impact of this heatwave on male ground squirrels emphasizes the importance of assessing the consequences of climate change on the breeding success of hibernating species in both sexes, since the different sensitivity to external cues for emergence led to a mismatch in timing under this event.

PSIII-1 Influence of prenatal or postnatal exposure to gossypol from cottonseed meal on semen quality in commercial boars: a preliminary study on feral hog control

The objective was to examine the influence of prenatal (Experiment 1) or postnatal (Experiment 2) exposure to gossypol from cottonseed meal (CSM) on semen quality in commercial boars. In Experiment 1, pregnant sows (n = 5) were fed a diet containing 0% (n = 1), 0.04% (n = 2), or 0.08% (n = 2) gossypol between d 56 and 86 of gestation. Boars (n = 11) born to sows in each treatment group (0% gossypol n = 3; 0.04% gossypol n = 4; 0.08% gossypol n = 4) were fed a common diet without CSM, and semen was collected at 269±2 d of age using a live sow in estrus. In Experiment 2, boars (n = 21) were fed a diet containing 0%, 0.02%, or 0.04% gossypol between 63±1 and 105±1 d of age (Initial BW: 19.85±0.43 kg). After the treatment period, boars were fed a common diet without CSM, and semen was collected at 238±7 d of age using a breeding dummy. Sperm cell concentration, percentage of motile sperm cells, and percentage of progressively motile sperm cells were analyzed using the MIXED procedure of SAS with treatment as a fixed effect in Experiment 1 and 2 and dam as a random effect in Experiment 1. In Experiment 2, semen was not successfully collected from every boar; therefore, chi-square analysis was used to assess semen collection status between treatment groups using the FREQ procedure of SAS. In Experiment 1, there was no difference in sperm concentration (P = 0.45), percent motility (P = 0.71), or percent progressive motility (P = 0.27) between treatment groups. In Experiment 2, there was no difference in sperm concentration (P = 0.72), percent motility (P = 0.17), or percent progressive motility (P = 0.87) between treatment groups. No difference was observed in boar collection status between treatment groups (P = 0.77). In conclusion, prenatal or postnatal exposure to gossypol from CSM did not influence semen quality in commercial boars.

Identification of Motile Sperm Domain Containing 1 (MOSPD1) As a Novel Target of the Wnt/β-Catenin Signaling Pathway in Colorectal Cancer

Aberrant activation of the Wnt/β-catenin signaling pathway plays a crucial role in the development and progression of colorectal cancer. Previously, we identified a set of candidate genes that were regulated by this signaling pathway, and we focused on MOSPD1, motile sperm domain containing 1, in this study. Immunohistochemical staining revealed that the expression of MOSPD1 was elevated in tumorous cells of colorectal cancer tissues compared with non-tumorous cells. Using ChIP-seq data and JASPAR database, we searched for the regulatory region(s) in the MOSPD1 gene as a target of the Wnt/β-catenin signaling, and identified a region containing three putative TCF-binding motifs in the 3’-flanking region. Additional analyses using reporter assay and ChIP-qPCR suggested that this region harbors an enhancer activity through an interaction with TCF7L2 and β-catenin. These data have clarified that MOSPD1 is a novel direct target of the Wnt/β-catenin signaling. In addition, the identification of its enhancer region may be helpful for the future studies of precise regulatory mechanisms of MOSPD1.

Application of high hydrostatic pressure (HHP) to improve cryopreservation of young bull semen

The objective of the study was to determine the effect of high hydrostatic pressure (HHP) on quality of cryopreserved semen of young bulls. Semen for this study was collected from 8 bulls aged between 13 and 18 months at monthly intervals, from June to September. After collection, semen was diluted in a commercial Bioxcell® extender (one part at 1:1 and a second part to give a sperm concentration of 20 million/0.2 mL), filled into straws and treated with HHP at 30 MPa for 90 min. After HHP treatment, pre-diluted semen (1:1) was diluted to a sperm concentration 20 million/0.2 mL and filled into straws. In addition, part of the semen diluted to a concentration of 20 million/0.2 mL was not treated with HHP (control). All of it was held at +4°C and frozen in a freezer after 2.5-h equilibration. Semen was thawed in a water bath at 38°C and subjected to estimation of the percentage of motile sperm both subjectively and using a computer-assisted semen analyzer and cytometric assessment of sperm cell membrane integrity. Subjective motility and fast progressive motility were significantly higher with pre-diluted (1:1) and HHP treated semen compared to control (P<0.05). No significant differences were observed in percentage of membrane-intact spermatozoa between control and experimental groups. Additionally, the influence of HHP on the sperm of individual bulls was assessed. In bull number 2, the HHP treatment after semen pre-dilution significantly improved progressive motility from 54.1 to 63.4 percent (P <0.05). In bull number 4, the HHP treatment after semen pre-dilution significantly improved subjective motility, rapid motility and progressive motility by 12.5, 16.8 and 16.3 percent, respectively (P<0.05). No effect was seen for 6 bulls. It is concluded that for some bulls, the application of HHP before semen freezing may improve the cryopreservation outcome. However, this requires further research in this area, also to determine the fertilizing capacity of bull semen exposed to high hydrostatic pressure.

Rheotaxis-based sperm separation using a biomimicry microfluidic device

Sperm selection is crucial to assisted reproduction, influencing the success rate of the treatment cycle and offspring health. However, in the current clinical sperm selection practices, bypassing almost all the natural selection barriers is a major concern. Here, we present a biomimicry microfluidic method, inspired by the anatomy of the female reproductive tract, that separates motile sperm based on their rheotaxis behavior to swim against the flow into low shear rate regions. The device includes micropocket geometries that recall the oval-shaped microstructures of the female fallopian tube to create shear protected zones for sperm separation. Clinical tests with human samples indicate that the device is capable of isolating viable and highly motile sperm based on their rheotaxis responses, resulting in a separation efficiency of 100%. The device presents an automated alternative for the current sperm selection practices in assisted reproduction.

Using Gum Arabic in Place of Egg Yolk as Cryoprotectant for Cryopreservation of the Buck Semen

Background: Egg yolk (EY) is well known to be toxic for buck spermatozoa, which creates restrictions of its use in cryopreservation. Therefore, this study is to compare the effect of different levels of gum Arabic (GA) in an extender on quality and fertility of cryopreserved buck sperm. Methods: Each ejaculate of six bucks was frozen in Tris with one of concentrations of GA which contained 3, 6, 9 and 12 gm/ 100 ml in place of the EY. Control was Tris extender containing 2.5% of EY. Result: A percentages of total motile sperm (54.92%; P less than 0.05) and progressively motile sperm (26.22%; P less than 0.05) of semen was frozen in Tris containing 9% of GA. Similar to control group, the pregnancy rate of does inseminated with extender containing 9% (50.0%) were significantly higher than those of does inseminated with extender containing 6% (8.33%), 3% (0.0%) and 12% (0.0%). Semen evaluation and fertility rate were similar when replacing the EY with GA in the Tris cryodiluent, after cryopreservation of buck semen. The present study shows that high motility rate of frozen semen and acceptable pregnancy rate can be obtained when using GA in place of EY for cryopreserving the buck sperm.