A novel progestin receptor (mPR) with seven-transmembrane domains was recently discovered in spotted seatrout and homologous genes were identified in other vertebrates. We show that cDNAs for the mPR ␣ subtypes from spotted seatrout A LTHOUGH THE IMPORTANCE of rapid (i.e. nonclassical) steroid actions initiated at the cell surface through binding to steroid membrane receptors has become more widely accepted within the past few years, details of the initial steroid-mediated events, including the identities of the steroid membrane receptors and their mechanisms of action, remain unclear and are surrounded by controversy (1-3). There is clear evidence that a variety of receptor proteins are involved in initiating these nonclassical steroid actions in different cell models, including nuclear steroid receptors or nuclear steroid receptor-like forms (1, 2, 4), receptors for other ligands that also bind steroids (2, 5), and unidentified receptors with different characteristics from those of any known receptors (2, 6). Recently, a novel cDNA was discovered in spotted seatrout ovaries that has several characteristics of the progestin membrane receptor (mPR) mediating progestin induction of oocyte maturation in this species by a nongenomic mechanism (7). The seatrout cDNA (st-mPR␣) encodes a 40 kDa protein, which has seven transmembrane domains, and receptor activation alters pertussis toxin-sensitive adenylyl cyclase activity, both of which suggest stmPR␣ is a G protein-coupled receptor (GPCR) or GPCR-like protein (7). More than 20 closely related genes have been cloned from other vertebrate species, including three mPR subtypes in humans, named ␣, ␤, and ␥, which show high levels of expression in human reproductive, brain, and kidney tissues, respectively (8). The identification of a new class of putative steroid receptors, unrelated to nuclear steroid receptors, but instead related to GPCRs, provides a plausible explanation of how steroids can initiate rapid hormonal responses in target cells by activating receptors on the cell surface. There has been broad recognition of the potential significance of these findings (1, 9, 10) and also an extensive research effort to determine the distribution, hormonal regulation, and biological roles of the mPRs in various vertebrate models (11-16). However, critical information is still lacking on several key features of mPRs essential for clearly establishing this proposed alternative model of steroid action and for understanding its likely evolutionary origins.The st-mPR␣ protein has been localized to the plasma membrane of seatrout oocytes (7), but progestin binding and activation of signal transduction pathways in the plasma membranes of cells transfected with the st-mPR␣ and human mPRs remain to be demonstrated. To date, progestin binding has only First Published Online November 9, 2006Abbreviations: GPCR, G protein-coupled receptor; HLY3, hemolysin 3; hu-mPR␣, human membrane progestin receptor ␣; MMD, monocyte to macrophage differentiation protein; mPR, membrane progestin rece...

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.

No abstract

No abstract

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.