Christopher Tubbs

Progestin, estrogen and androgen G-protein coupled receptors in fish gonads

The identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic (i.e. nonclassical) steroid actions described to date are unclear. Two novel 7-transmembrane spanning proteins, representing two distinct classes of steroid membrane receptors, membrane progestin receptor alpha (mPRalpha) and a membrane estrogen receptor (mER), GPR30, have recently been identified in several vertebrate species. Evidence that both receptors activate G-proteins and function as G-protein coupled receptors (GPCRs) is briefly reviewed. New data on progestin actions on fish gametes suggest a widespread involvement of mPRalpha in oocyte maturation and sperm hyperactivity in this vertebrate group. Information on the second messenger pathways activated upon estrogen binding to a membrane estrogen receptor in croaker gonads and preliminary evidence for the presence of a GPR30-like protein in fish gonads are discussed. Finally, initial characterization of the ligand binding, G-protein activation and molecular size of a membrane androgen receptor (mAR) in croaker ovaries suggests the presence of a third unique steroid receptor in fish gonads that also may function as a GPCR.

Binding characteristics, hormonal regulation and identity of the sperm membrane progestin receptor in Atlantic croaker

Recently a novel cDNA was discovered in spotted seatrout ovaries encoding a protein with seven transmembrane domains that has the characteristics of the membrane progestin receptor (mPR) mediating maturation-inducing steroid (MIS) induction of oocyte maturation in this species. Preliminary results suggested the MIS also activates an mPR on the spermatozoa of spotted seatrout and a closely related species, Atlantic croaker, to stimulate sperm motility. We show here that plasma membranes of croaker sperm demonstrate high affinity (Kd approximately 20 nM), limited capacity (Bmax 0.08 nM), specific and displaceable binding for progestins that is characteristic of mPRs. The MIS (17,20beta,21-trihydroxy-4-pregnen-3-one, 20beta-S) displayed the greatest binding affinity for the sperm mPR among the steroids tested. Treatment of croaker testicular tissue in vitro with gonadotropin caused a several-fold increase in sperm mPR receptor concentrations that was partially blocked in the presence of cyanoketone, which suggests this action of gonadotropin is partially mediated by stimulation of steroidogenesis. Protein bands of the predicted sizes for the mPR and its dimer (40 and 80 kDa) were detected by Western blotting of croaker sperm membranes using a specific antibody to the novel seatrout mPR (mPRalpha). Immunocytochemistry of whole croaker spermatozoa with the mPRalpha antibody showed that staining was primarily localized on the midpiece, consistent with a role of the mPRalpha in mediating MIS stimulation of sperm motility. The results suggest that the mechanism of progestin action on fish sperm involving mPRs is basically similar to that in mammals and has been evolutionarily conserved amongst vertebrates.

Functional characteristics of membrane progestin receptor alpha (mPRα) subtypes: A review with new data showing mPRα 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.

Progestin functions in vertebrate gametes mediated by membrane progestin receptors (mPRs): Identification of mPRα on human sperm and its association with sperm motility

Most of the studies on the putative membrane progestin receptor (mPR) alpha and beta subtypes that have been published in the 5 years since their discovery have supported the original hypothesis that they function as specific membrane receptors through which progestins induce rapid, nongenomic responses in target cells. Recent evidence that mPRalpha and mPRbeta have important roles in the regulation of oocyte meiotic maturation and sperm motility in both fish and mammals is reviewed. Although rapid, cell surface-initiated progestin actions on sperm to induce hyperactive motility have been demonstrated in several mammalian models, the identity of the membrane progestin receptor mediating this effect remains unclear. We demonstrate here that mPRalpha mRNA is expressed in human sperm by RT-PCR and that the mPRalpha protein is localized to the sperm membranes by Western blot analysis. Immunocytochemical staining of whole non-permeabilized human sperm confirmed the mPRalpha protein is expressed in the plasma membrane, and showed it is localized to the sperm midpiece, indicating a likely role of mPRalpha in progestin regulation of sperm motility. Moreover, the abundance of the mPRalpha protein on sperm plasma membranes from human donors that displayed low motility was significantly reduced compared to that on normal motile sperm. Finally, progestin treatment of sperm membranes caused activation of G-proteins. These results suggest that, similar to its proposed function in fishes, mPRalpha is an intermediary in progestin stimulation of sperm motility in humans by a mechanism involving G-protein activation.

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.

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.

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.

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.

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.

Gut microbiota and phytoestrogen-associated infertility in southern white rhinoceros

With recent poaching of southern white rhinoceros (Ceratotherium simum simum; SWR) 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 another rhinoceros species–the greater one-horned rhinoceros (Rhinoceros unicornis; GOHR), 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 varying 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 with respect to phytoestrogen profile, but also with 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 gut microbiota’s transformation of dietary phytoestrogens in captive SWR females.