Understanding the Spike Protein in COVID-19 Vaccine in Recombinant Vesicular Stomatitis Virus (rVSV) Using Automated Capillary Western Blots.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral agent that is responsible for the coronavirus disease-2019 (COVID-19) pandemic. One of the live virus vaccine candidates Merck and Co., Inc. was developing to help combat the pandemic was V590. V590 was a live-attenuated, replication-competent, recombinant vesicular stomatitis virus (rVSV) in which the envelope VSV glycoprotein (G protein) gene was replaced with the gene for the SARS-CoV-2 spike protein (S protein), the protein responsible for viral binding and fusion to the cell membrane. To assist with product and process development, a quantitative Simple Western (SW) assay was successfully developed and phase-appropriately qualified to quantitate the concentration of S protein expressed in V590 samples. A strong correlation was established between potency and S-protein concentration, which suggested that the S-protein SW assay could be used as a proxy for virus productivity optimization with faster data turnaround time (3 h vs 3 days). In addition, unlike potency, the SW assay was able to provide a qualitative profile assessment of the forms of S protein (S protein, S1 subunit, and S multimer) to ensure appropriate levels of S protein were maintained throughout process and product development. Finally, V590 stressed stability studies suggested that time and temperature contributed to the instability of S protein demonstrated by cleavage into its subunits, S1 and S2, and aggregation into S multimer. Both of which could potentially have a deleterious effect on the vaccine immunogenicity.

Microplastic in northern anchovies (Engraulis mordax) and common murres (Uria aalge) from the Monterey Bay, California USA - Insights into prevalence, composition, and estrogenic activity.

Microplastic (particle size <5 mm) is considered an emerging threat to the marine environment, yet data are limited for coastal ecosystems. To provide information related to microplastic in a coastal system, we used alkaline tissue digestion and Raman spectroscopy to quantify the prevalence and composition (e.g. fiber, fragment, foam, etc.) of anthropogenic microparticles in the digestive tracts of northern anchovies (Engraulis mordax, anchovy, n = 24), and common murres (Uria aalge, murre, n = 19) from the Monterey Bay, California USA. We also determined microplastic prevalence and composition in seawater (n = 12 17-h sampling periods representing ∼46,000 L sampled) from two Monterey Bay intake systems (Moss Landing, CA and Santa Cruz, CA USA). Microparticles recovered from murre digestive tracts were assessed for estrogenic activity using an in-vitro estrogen receptor activation assay. Suspected anthropogenic microparticles based on visual characteristics were recovered from all sample types with ∼2 particles per 1000 L from the seawater sampling periods, 58% prevalence in anchovies, and 100% prevalence in murres. Across samples of seawater, anchovies, and murres, the most abundant microparticle type found were fibers (78%), followed by fragments (13%), foam (6%), film (2%), and beads (1%). Raman spectroscopy identified 57% of microparticles (excluding dye-prominent and unknown) as plastic (synthetic, semi-synthetic, or blends). Almost one quarter (23%) of the murre digestive tracts contained microparticles that exhibited estrogenic activity. Our study describes the widespread occurrence, composition, and potential estrogenic activity of microplastic in the Monterey Bay and provides important information to aid in the understanding of microplastic contamination in coastal systems.

Functional profiling of Covid 19 vaccine candidate by flow virometry.

Vaccine development is a complex process, starting with selection of a promising immunogen in the discovery phase, followed by process development in the preclinical phase, and later by clinical trials in tandem with process improvements and scale up. A large suite of analytical techniques is required to gain understanding of the vaccine candidate so that a relevant immunogen is selected and subsequently manufactured consistently throughout the lifespan of the product. For viral vaccines, successful immunogen production is contingent on its maintained antigenicity and/or infectivity, as well as the ability to characterize these qualities within the context of the process, formulation, and clinical performance. In this report we show the utility of flow virometry during preclinical development of a Covid 19 vaccine candidate based on SARS-CoV-2 spike (S) protein expressed on vesicular stomatitis virus (VSV). Using a panel of monoclonal antibodies, we were able to detect the S protein on the surface of the recombinant VSV virus, monitor the expression levels, detect differences in the antigen based on S protein sequence and after virus inactivation, and monitor S protein stability. Collectively, flow virometry provided important data that helped to guide preclinical development of this vaccine candidate.

Assessing Marine Endocrine-Disrupting Chemicals in the Critically Endangered California Condor: Implications for Reintroduction to Coastal Environments.

Coastal reintroduction sites for California condors (Gymnogyps californianus) can lead to elevated halogenated organic compound (HOC) exposure and potential health impacts due to the consumption of scavenged marine mammals. Using nontargeted analysis based on comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/TOF-MS), we compared HOC profiles of plasma from inland and coastal scavenging California condors from the state of California (CA), and marine mammal blubber from CA and the Gulf of California off Baja California (BC), Mexico. We detected more HOCs in coastal condors (32 ± 5, mean number of HOCs ± SD, n = 7) than in inland condors (8 ± 1, n = 10) and in CA marine mammals (136 ± 87, n = 25) than in BC marine mammals (55 ± 46, n = 8). ∑DDT-related compounds, ∑PCBs, and total tris(chlorophenyl)methane (∑TCPM) were, respectively, ∼7, ∼3.5, and ∼148 times more abundant in CA than in BC marine mammals. The endocrine-disrupting potential of selected polychlorinated biphenyls (PCB) congeners, TCPM, and TCPMOH was determined by in vitro California condor estrogen receptor (ER) activation. The higher levels of HOCs in coastal condors compared to those in inland condors and lower levels of HOC contamination in Baja California marine mammals compared to those from the state of California are factors to consider in condor reintroduction efforts.

Foraging behavior, contaminant exposure risk, and the stress response in wild California condors (Gymnogyps californianus).

Wild California condors (Gymnogyps californianus) are frequently exposed to lead via lead-based ammunition ingestion, and recent studies indicate significant exposure to organochlorines (e.g. dichlorodiphenyldichloroethylene (DDE) and polychlorinated biphenyls (PCBs)) for condors feeding on beach-cast marine mammals. We investigated the influence of contaminant exposure on condor glucocorticoid response through comparisons between wild and captive populations and identification of modifiers of glucocorticoid release in wild condors. We assessed the glucocorticoid response to routine trapping and handling events through measurement of plasma corticosterone and urate glucocorticoid metabolites (GCM). Comparison of peak urate GCM levels showed wild condors exhibited higher responses to handling-associated stressors (2300 ± 1400 ng/g dry wt, average ± SD, n = 27) than captive condors (910 ± 490 ng/g dry wt., n = 6, U = 28, p = 0.003). Multiple linear regression models and an information theoretic approach (AICc) identified several extrinsic variables (e.g., time captive in flight pen before sample collection) that were negatively associated with plasma corticosterone and urate GCM levels in wild condors, which explained ~25% of glucocorticoid variation. When accounting for these extrinsic variables we found that behavioral variables associated with increased lead and organochlorine exposure risk were positively associated with GCM levels, explaining an additional 15% of glucocorticoid variation among wild condors. Days absent from management area, a variable associated with reduced survival attributed to increased lead exposure risk, had a positive influence on plasma corticosterone levels (ß = 53 ± 20 SE) and peak urate GCM levels (ß = 1090 ± 586 SE). Years observed feeding on marine mammals, a variable positively associated with DDE and PCB exposure, positively influenced peak urate GCM (ß = 1100 ± 520 SE) and the magnitude of GCM response (peak GCM - 1st urate GCM) (ß = 1050 ± 500 SE). Our findings suggest that individual propensities for contaminant-associated foraging behaviors predict higher stress-induced glucocorticoid levels in wild condors, and that accounting for variables associated with trapping and handling is essential for assessing the impact of environmental stressors such as contaminants on the condor stress response. As an abnormal glucocorticoid response to stress is associated with reduced reproduction and survival in vertebrates, this work indicates the need for further investigations into the physiological impacts of sub-lethal contaminant exposures in scavenging species worldwide.

Regulation of endocrine systems by the microbiome: Perspectives from comparative animal models.

The microbiome regulates endocrine systems and influences many aspects of hormone signaling. Using examples from different animal taxa, we highlight the state of the science in microbiome research as it relates to endocrinology and endocrine disruption research. Using a comparative approach discussing fish, birds, and mammals, we demonstrate the bidirectional interaction between microbiota and hormone systems, presenting concepts that include (1) gastrointestinal microbiome regulation of the neuroendocrine feeding axis; (2) stress hormones and microbial communities; (3) the role of site-specific microbiota in animal reproduction; (4) microbiome effects on the neuroendocrine systems and behavior; and (5) novel mechanisms of endocrine disruption through the microbiome. This mini-review demonstrates that hormones can directly affect the richness and diversity of microbiota and conversely, microbiota can influence hormone production and mediate their functions in animals. In addition, microbiota can influence the action of a diverse range of neurotransmitters and neuropeptides in the central nervous system, which can lead to behavioral disruptions. As many animals have species-specific reproductive behaviors, it is important to understand how shifts in the microbiota relate to these complex interactions between sexes. This is especially important for captive animals on specialized diets, and there are significant implications for microbiome research in conservation and reproductive biology. For example, microbial metabolites may modify motility of gametes or modulate hormone-receptor interactions in reproductive tissues. Thus, efforts to incorporate metabolomics into the science of microbiome-endocrine relationships, both those produced by the host and those generated from microbial metabolism, are increasingly needed. These concepts have fostered an exciting emerging era in comparative endocrinology.

Identification of California condor (Gymnogyps californianus) estrogen receptor variants and their activation by xenoestrogens.

California condors released in costal sites are exposed to high levels of xenoestrogens, particularly p,p'-DDE, through scavenging of marine mammal carcasses. As a result, coastal condors carry a higher contaminant loads and experience eggshell thinning when compared to their inland counterparts. Given that condor estrogen receptors (Esrs) are activated by physiologically relevant levels of xenoestrogens, differences in vulnerability to endocrine disruption may exist depending on which Esr variant(s) an individual condor possesses. This work aims to characterize genetic polymorphisms in estrogen receptor genes (ESRs) in California condors; one identified for condor estrogen receptor 1 (ESR1) (N161S, E162D) and one in the ESR2 (T114M) gene. Each variant was confirmed in individual founder birds by direct PCR sequencing as well as in first generation offspring to understand the introduction of the alleles into the pedigree (6 birds for ESR1 and 5 birds for ESR2). Site-directed mutagenesis was performed on wild type receptors to produce each of the full-length ESR variants and activation of Esr1 and Esr2 variant and wild type receptors by xenoestrogens was compared. Maximal activation of the variant form of Esr1 was significantly higher (p < 0.05) in response to ethinyl estradiol (EE2), o,p'-DDE, p,p'-DDE, p,p'-DDT and p,p'-DDD compared to wild type Esr1. For Esr2 the wild type maximal activation was higher in response to o,p'-DDE, p,p'-DDE, o,p'-DDT, and p,p'-DDT. Although significant differences in activation of condor Esr variants by xenoestrogens occurred at high (micromolar) concentrations, they correspond to circulating concentrations previously reported in coastal birds. Release and relocation of California condors to the coast is a promising avenue for recovery, however, reproductive problems associated with xenoestrogen exposure pose a sub-lethal threat to long-term success. Based on above findings, future release decisions could be informed by ESR form(s) individual birds possess to reduce deleterious effects of xenoestrogen exposure and ultimately improve reproductive success in wild populations.

Gut Microbiota and Phytoestrogen-Associated Infertility in Southern White Rhinoceros.

With recent poaching of southern white rhinoceros (SWR [Ceratotherium simum simum]) reaching record levels, the need for a robust assurance population is urgent. However, the global captive SWR population is not currently self-sustaining due to the reproductive failure of captive-born females. Dietary phytoestrogens have been proposed to play a role in this phenomenon, and recent work has demonstrated a negative relationship between diet estrogenicity and fertility of captive-born female SWR. To further examine this relationship, we compared gut microbial communities, fecal phytoestrogens, and fertility of SWR to those of another rhinoceros species-the greater one-horned rhinoceros (GOHR [Rhinoceros unicornis]), which consumes a similar diet but exhibits high levels of fertility in captivity. Using 16S rRNA amplicon sequencing and mass spectrometry, we identified a species-specific fecal microbiota and three dominant fecal phytoestrogen profiles. These profiles exhibited various levels of estrogenicity when tested in an in vitro estrogen receptor activation assay for both rhinoceros species, with profiles dominated by the microbial metabolite equol stimulating the highest levels of receptor activation. Finally, we found that SWR fertility varies significantly not only with respect to phytoestrogen profile, but also with respect to the abundance of several bacterial taxa and microbially derived phytoestrogen metabolites. Taken together, these data suggest that in addition to species differences in estrogen receptor sensitivity to phytoestrogens, reproductive outcomes may be driven by the gut microbiota's transformation of dietary phytoestrogens in captive SWR females.IMPORTANCE Southern white rhinoceros (SWR) poaching has reached record levels, and captive infertility has rendered SWR assurance populations no longer self-sustaining. Previous work has identified dietary phytoestrogens as a likely cause of this problem. Here, we investigate the role of gut microbiota in this phenomenon by comparing two rhinoceros species to provide the first characterizations of gut microbiomes for any rhinoceros species. To our knowledge, our approach, combining parallel sequencing, mass spectrometry, and estrogen receptor activation assays, provides insight into the relationship between microbially mediated phytoestrogen metabolism and fertility that is novel for any vertebrate species. With this information, we plan to direct future work aimed at developing strategies to improve captive reproduction in the hope of alleviating their threat of extinction.

Induction of sperm hypermotility through membrane progestin receptor alpha (mPRα): A teleost model of rapid, multifaceted, nongenomic progestin signaling.

Rapid progestin effects on sperm physiology have been described in a variety of vertebrate species. Here, we briefly review the signaling pathways mediating rapid progestin induction of sperm hypermotility and increased fertility in two teleost species, Atlantic croaker and southern flounder. Acute in vitro treatment of teleost sperm with the progestin hormone, 20ß-S, causes activation of progestin membrane receptor alpha (mPRα, or Paqr7) coupled to a stimulatory olfactory G protein (Golf), resulting in increased cAMP and calcium concentrations and hypermotility upon activation in a hyperosmotic medium. Pharmacological tools were used to investigate the involvement of mPRα and several intracellular signaling pathways in the hypermotility response. Evidence was obtained using the specific mPRα agonist, Org OD 02-0, that this progestin action is mediated through mPRα and not through the nuclear PR. The results indicate that progestins induce hypermotility through activation of a membrane adenylyl cyclase (Acy)/cAMP pathway, an epidermal growth factor receptor (Egfr)/Mapkinase pathway, and a Pi3kinase/Akt/phosphodiesterase (Pde) pathway which result in increased sperm calcium concentrations within 10 s. The finding that inhibition of any one of these pathways is sufficient to prevent hypermotility along with the calcium increase suggests that activation of all of them and the associated calcium increase are required for the progestin hypermotility response. On the basis of these findings a model of progestin induction of sperm hypermotility in teleosts is proposed. As teleosts lack CatSper, the model described here is a non-CatSper mediated one and may therefore be applicable to a wide variety of nonmammalian vertebrates.

Glucocorticoid measurement in plasma, urates, and feathers from California condors (Gymnogyps californianus) in response to a human-induced stressor.

Vertebrates respond to stressful stimuli with the secretion of glucocorticoid (GC) hormones, such as corticosterone (CORT), and measurements of these hormones in wild species can provide insight into physiological responses to environmental and human-induced stressors. California condors (Gymnogyps californianus) are a critically endangered and intensively managed avian species for which information on GC response to stress is lacking. Here we evaluated a commercially available I125 double antibody radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA) kit for measurement of CORT and GC metabolites (GCM) in California condor plasma, urate, and feather samples. The precision and accuracy of the RIA assay outperformed the ELISA for CORT and GCM measurements, and CORT and GCM values were not comparable between the two assays for any sample type. RIA measurements of total CORT in condor plasma collected from 41 condors within 15 minutes of a handling stressor were highly variable (median = 70 ng/mL, range = 1-189 ng/mL) and significantly different between wild and captive condors (p = 0.02, two-tailed t-test, n = 10 wild and 11 captive). Urate GCM levels (median = 620 ng/g dry wt., range = 0.74-7200 ng/g dry wt., n = 216) significantly increased within 2 hr of the acute handling stressor (p = 0.032, n = 11 condors, one-tailed paired t-test), while feather section CORT concentrations (median = 18 pg/mm, range = 6.3-68 ng/g, n = 37) also varied widely within and between feathers. Comparison of multiple regression linear models shows condor age as a significant predictors of plasma CORT levels, while age, sex, and plasma CORT levels predicted GCM levels in urates collected within 30 min of the start of handling. Our findings highlight the need for validation when selecting an immunoassay for use with a new species, and suggest that non-invasively collected urates and feathers hold promise for assessing condor responses to acute or chronic environmental and human-induced stressors.

Reproductive Impacts of Endocrine-Disrupting Chemicals on Wildlife Species: Implications for Conservation of Endangered Species.

Wildlife have proven valuable to our understanding of the potential effects of endocrine-disrupting chemicals (EDCs) on human health by contributing considerably to our understanding of the mechanisms and consequences of EDC exposure. But the threats EDCs present to populations of wildlife species themselves are significant, particularly for endangered species whose existence is vulnerable to any reproductive perturbation. However, few studies address the threats EDCs pose to endangered species owing to challenges associated with their study. Here, we highlight those barriers and review the available literature concerning EDC effects on endangered species. Drawing from other investigations into nonthreatened wildlife species, we highlight opportunities for new approaches to advance our understanding and potentially mitigate the effects of EDCs on endangered species to enhance their fertility.

Estrogenicity of captive southern white rhinoceros diets and their association with fertility.

The captive southern white rhinoceros (SWR) population is not currently self-sustaining, primarily due to poor or absent reproduction of captive-born (F1+) females. In this study, we investigate the role of dietary phytoestrogens in this reproductive phenomenon by characterizing activation of SWR estrogen receptors (ESRs) 1 and 2 by diet items from nine North American institutions and comparing female SWR fertility to total diet estrogenicity. Of the diet items tested, alfalfa hay and soy and alfalfa-based commercial pellets were found to be the most potent activators of SWR ESRs. In contrast, most grass hays tested were not estrogenic. The estrogenicity of total diets varied across the institutions surveyed and the degree of diet estrogenicity was positively associated with the percentage of the total diet comprised by pellets. Comparisons of fertility records of the institutions surveyed showed no significant relationship between diet estrogenicity and fertility for female SWR conceived or born in the wild (F0). However, for F1+ females, there was a significant negative relationship between institutional diet estrogenicity and fertility. Taken together, these data suggest that developmental exposure to phytoestrogens may be the cause of poor fertility in captive-born female SWR. Whether the low fertility of the current population of captive-born female SWR is permanent or can be reversed by removing phytoestrogens from the diet remains unclear. However, our findings suggest that in order for the SWR population to become self-sustaining, the development and feeding of low phytoestrogen diets should be strongly considered.

Identification of California Condor Estrogen Receptors 1 and 2 and Their Activation by Endocrine Disrupting Chemicals.

Recently, California condors (Gymnogyps californianus) have been reintroduced to coastal regions of California where they feed on marine mammal carcasses. There is evidence that coastal-dwelling condors experience reproductive issues, such as eggshell thinning, likely resulting from exposure to endocrine-disrupting chemicals (EDCs). To address this problem, we have identified and cloned condor estrogen receptors (ESRs) 1 and 2 and characterized their activation by EDCs present in the coastal habitats where condors reside. Dichlorodiphenyltrichloroethane (DDT) and its metabolites all activated ESR1 and ESR2, although their relative potency differed between the receptors. Bisphenol A, dieldrin, trans-nonachlor, and polychlorinated biphenyl 52 (PCB52) moderately activated both ESRs, whereas PCB138 and PCB153 stimulated little to no activation. Overall, EDC activation of condor ESR2, which is the first ESR2 cloned from a raptor species, was greater than that of ESR1. Significant activation of both condor ESRs by EDCs occurred at high concentrations (≥1µM), which are within the range of plasma levels of certain EDCs (eg, dichlorodiphenyldichloroethylene [p'p-DDE]) in coastal-dwelling condors. Finally, phylogenetic analyses of ESRs of 41 avian species identified a single amino acid position in ESR2 under positive selection. Mutation of this amino acid affected receptor activation by EDCs, suggesting the identity of this amino acid may influence EDC sensitivity of avian species. Together, these findings broaden our understanding of EDC interactions with ESRs in avian species. For condors specifically, these data could be used to evaluate EDC exposure risk at future release sites to identify those least likely to compromise the continued recovery of this species.

Advances in conservation endocrinology: the application of molecular approaches to the conservation of endangered species.

Among the numerous societal benefits of comparative endocrinology is the application of our collective knowledge of hormone signaling towards the conservation of threatened and endangered species - conservation endocrinology. For several decades endocrinologists have used longitudinal hormone profiles to monitor reproductive status in a multitude of species. Knowledge of reproductive status among individuals has been used to assist in the management of captive and free-ranging populations. More recently, researchers have begun utilizing molecular and cell-based techniques to gain a more complete understanding of hormone signaling in wildlife species, and to identify potential causes of disrupted hormone signaling. In this review we examine various in vitro approaches we have used to compare estrogen receptor binding and activation by endogenous hormones and phytoestrogens in two species of rhinoceros; southern white and greater one-horned. We have found many of these techniques valuable and practical in species where access to research subjects and/or tissues is limited due to their conservation status. From cell-free, competitive binding assays to full-length receptor activation assays; each technique has strengths and weaknesses related to cost, sensitivity, complexity of the protocols, and relevance to in vivo signaling. We then present a novel approach, in which receptor activation assays are performed in primary cell lines derived from the species of interest, to minimize the artifacts of traditional heterologous expression systems. Finally, we speculate on the promise of next generation sequencing and transcriptome profiling as tools for characterizing hormone signaling in threatened and endangered species.

Activation of southern white rhinoceros (Ceratotherium simum simum) estrogen receptors by phytoestrogens: potential role in the reproductive failure of captive-born females?

The captive southern white rhinoceros (SWR; Ceratotherium simum simum) population serves as an important genetic reservoir critical to the conservation of this vulnerable species. Unfortunately, captive populations are declining due to the poor reproductive success of captive-born females. Captive female SWR exhibit reproductive problems suggested to result from continual ovarian follicular activity and prolonged exposure to endogenous estrogen. However, we investigated the potential role of exogenous dietary phytoestrogens in the reproductive failure of SWR by cloning and characterizing in vitro phytoestrogen binding and activation of recombinant SWR estrogen receptors (ESR). We compared those characteristics with recombinant greater one-horned rhinoceros (GOHR; Rhinoceros unicornis) ESR, a species that receives similar captive diets yet reproduces relatively well. Our results indicate that phytoestrogens bind rhino ESR in a manner similar to other vertebrate species, but there are no differences found in phytoestrogen binding affinity of SWR ESR compared with GOHR ESR. However, species-specific differences in ESR activation by phytoestrogens were detected. The phytoestrogen coumestrol stimulated greater maximal activation of SWR ESR1 than GOHR ESR1. SWR ESR2 were also more sensitive to phytoestrogens and were activated to a greater extent by both coumestrol and daidzein. The concentrations in which significant differences in ESR activation occurred (10(-7) to 10(-5) m) are consistent with circulating concentrations measured in other vertebrate species. Taken together, these findings suggest that phytoestrogens potentially pose a risk to the reproductive health of captive SWR. However, additional studies are needed to further clarify the physiological role of dietary phytoestrogens in the reduced fertility of this species.

Identification of 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S) receptor binding and membrane progestin receptor alpha on southern flounder sperm (Paralichthys lethostigma) and their likely role in 20ß-S stimulation of sperm hypermotility.

The existence of direct progestin actions on teleost sperm to stimulate hypermotility is not widely acknowledged because it has only been demonstrated in members of the family Sciaenidae. In the present study, progestin stimulation of sperm hypermotility was investigated in a non-sciaenid, southern flounder, and the potential role of membrane progestin receptor alpha (mPRα or Paqr7b) in mediating this action was examined. The major progestin produced in vitro by flounder testicular fragments co-migrated with 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S) during thin-layer chromatography. Treatment of flounder sperm with 5 nM-100 nM 20ß-S significantly increased sperm velocity in vitro, whereas 17,20ß-dihydroxy-4-pregnen-3-one and other steroids were ineffective. A single class of high affinity (K(d) 22.95 nM), saturable, limited-capacity binding sites (B(max) 0.013 nM) specific for 20ß-S was identified on sperm membranes. Treatment of sperm membranes with guanosine 5'-(3-O-thio)triphosphate reduced [(3)H]-20ß-S binding, suggesting the 20ß-S receptor couples to a G protein. The membrane adenylyl cyclase inhibitor 2',5'-dideoxyadenosine blocked 20ß-S-induced sperm hypermotility, indicating 20ß-S activates stimulatory G proteins. Finally, flounder paqr7b was cloned and characterized from testicular tissues. The Paqr7b protein is expressed on the midpiece of flounder sperm and is more abundant in individuals with high sperm motility than low motility donors. These findings suggest that 20ß-S stimulates sperm hypermotility in flounder through activation of stimulatory G proteins, likely through Paqr7b. The finding that progestins directly stimulate sperm hypermotility in a flatfish, a highly derived species not belonging to the teleost family Sciaenidae, suggests this phenomenon is widespread among advanced fishes.

Expression and gonadotropin regulation of membrane progestin receptor alpha in Atlantic croaker (Micropogonias undulatus) gonads: role in gamete maturation.

Recent results suggest that membrane progestin receptor alpha (mPRalpha) mediates nongenomic actions of progestin hormones to induce oocyte maturation and sperm hypermotility in several teleost species. The role of mPRalpha in gamete and gonadal physiology was further evaluated in the present study by examining gonadal expression of mPRalpha during gamete maturation in Atlantic croaker (Micropogonias undulatus), a well-characterized teleost model of oocyte maturation and sperm motility. Sequencing of the croaker mPRalpha gene isolated from croaker ovaries showed it is 98% homologous at the nucleotide level to spotted seatrout mPRalpha. The mPRalpha mRNA and protein were detected in both somatic and gonadal tissues. In croaker ovaries, the mPRalpha protein was present throughout the gonadal cycle and was upregulated by gonadotropin in vitro, coincident with the acquisition of oocyte maturational competence (i.e., ability to respond to progestin hormones and complete oocyte maturation). Both mPRalpha mRNA and protein were also expressed in croaker testes throughout the gonadal cycle. Expression of mPRalpha protein was weakly upregulated in testes after 18 h of in vitro gonadotropin treatment. Immunocytochemical staining showed mPRalpha was localized to both germ and interstitial cells. Finally, elevated levels of mPRalpha protein in croaker sperm were associated with high sperm motility. Taken together, these data strongly support the hypothesis that mPRalpha mediates progestin induction of oocyte maturation and upregulation of sperm motility in teleosts.

Progestin signaling through an olfactory G protein and membrane progestin receptor-alpha in Atlantic croaker sperm: potential role in induction of sperm hypermotility.

Progestin stimulation of sperm hypermotility remains poorly understood despite having been described in numerous vertebrate species. We show here that progestin stimulation of sperm hypermotility in a teleost, the Atlantic croaker (Micropogonias undulatus) is associated with activation of an olfactory G protein (Golf). Furthermore, we provide evidence that this progestin action is mediated by membrane progestin receptor-alpha (mPRalpha). Golf was identified in croaker sperm membranes and was specifically activated after treatment with the progestin 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S). Treatment of sperm membranes with 20beta-S caused an increase in cAMP production, which was blocked by pretreatment with cholera toxin and two membrane adenylyl cyclase inhibitors: 2',5'-dideoxyadenosine and SQ22536. Moreover, preincubation of croaker sperm with 2',5'-dideoxyadenosine and SQ22536 resulted in a significant inhibition of 20beta-S-stimulated hypermotility. Binding of [3H]20beta-S to sperm membranes was decreased after pretreatment with GTPgammaS but not pertussis toxin, suggesting the receptor is coupled to a pertussis toxin-insensitive G protein. Golf and mPRalpha were coexpressed on the sperm midpiece and flagella and were coimmunoprecipitated from sperm membranes. Finally, expression of mPRalpha protein on sperm increased after in vivo treatment with LHRH and was associated with increased induction of sperm motility by 20beta-S. These results suggest that 20beta-S activates mPRalpha in croaker sperm, which in turn activates Golf and membrane adenylyl cyclase to stimulate sperm hypermotility. Taken together these findings provide a plausible mechanism by which progestins stimulate sperm hypermotility in croaker and provide the first evidence of hormonal activation of Golf in any species.

Functional characteristics of membrane progestin receptor alpha (mPRalpha) subtypes: a review with new data showing mPRalpha expression in seatrout sperm and its association with sperm motility.

A novel cDNA with characteristics of the membrane progestin receptor regulating induction of oocyte maturation by a non-genomic mechanism, named st-mPRalpha, was recently discovered in seatrout. Subsequently, both recombinant and native mPRalphas have been localized to the plasma membrane in several vertebrate models where they have been shown to bind progestins specifically, resulting in activation of G proteins. Non-genomic actions of progestins to stimulate hypermotility and the acrosome reaction in sperm have been identified, but the receptors which mediate these processes are unknown. Here, we demonstrate progestin stimulation of sperm motility in seatrout and expression of st-mPRalpha mRNA and protein in sperm with the receptor localized on the plasma membrane. Immunocytochemical staining of non-permeabilized sperm shows st-mPRalpha is localized to the midpiece with an extracellular N-terminal region, indicating its likely role in progestin regulation of sperm motility. Moreover, the abundance of the st-mPRalpha protein on sperm membranes from seatrout donors with low motility was significantly reduced compared to that of normal motile sperm. Finally, progestin treatment of sperm membranes caused activation of G proteins. These results suggest that st-mPRalpha is an intermediary in progestin stimulation of sperm motility in seatrout by an unknown mechanism involving G protein activation.