Yong Zhu

Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes

The structures of membrane receptors mediating rapid, nongenomic actions of steroids have not been identified. We describe the cloning of a cDNA from spotted seatrout ovaries encoding a protein that satisfies the following seven criteria for its designation as a steroid membrane receptor: plausible structure, tissue specificity, cellular distribution, steroid binding, signal transduction, hormonal regulation, and biological relevance. For plausible structure, computer modeling predicts that the protein has seven transmembrane domains, typical of G protein-coupled receptors. The mRNA (4.0 kb) is only detected in the brain and reproductive tissues on Northern blots. Antisera only detect the protein (40 kDa) in plasma membranes of reproductive tissues. The recombinant protein produced in an Escherichia coli expression system has a high affinity (Kd = 30 nM), saturable, displaceable, single binding site specific for progestins. Progestins alter signal transduction pathways, activating mitogen-activated protein kinase and inhibiting adenylyl cyclase, in a transfected mammalian cell line. Inhibition of adenylyl cyclase is pertussis toxin sensitive, suggesting the receptor may be coupled to an inhibitory G protein. Progestins and gonadotropin up-regulate both mRNA and protein levels in seatrout ovaries. Changes in receptor abundance in response to hormones and at various stages of oocyte development, its probable coupling to an inhibitory G protein and inhibition of progestin induction of oocyte maturation upon microinjection of antisense oligonucleotides are consistent with the identity of the receptor as an intermediary in oocyte maturation. These characteristics suggest the fish protein is a membrane progestin receptor mediating a “nonclassical” action of progestins to induce oocyte maturation in fish.

Identification classification and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor

Recently we discovered a previously uncharacterized gene with the characteristics of a membrane progestin receptor (mPR) in a fish model, spotted seatrout. Here, we report the identification, cloning, and characteristics of other members of this hitherto unknown family of putative mPRs from several vertebrate species, including human, mouse, pig, Xenopus, zebrafish, and Fugu, with highly conserved nucleotide and deduced amino acid sequences and similar structures to the spotted seatrout mPR. The 13 vertebrate genes identified seem to belong to an unknown gene family. Phylogenetic analysis indicates these cDNAs comprise three distinct groups (named α, β, and γ) within this gene family. Structural analyses of the translated cDNAs suggest they encode membrane proteins with seven transmembrane domains. The transcript sizes of the human α, β, and γ putative mPR mRNAs varied from 2.8 to 5.8 kb and showed distinct distributions in reproductive, neural, kidney and intestinal tissues, respectively. Recombinant human α, γ, and mouse β proteins produced in an Escherichia coli expression system demonstrated high affinity (Kd = 20–30 nM) saturable binding for progesterone. Further analysis of binding to the γ-subtype revealed binding was specific for progestins and was displaceable, with rapid rates of association and dissociation (t1/2 = 2–8 min). These results suggest this is a new family of steroid receptors unrelated to nuclear steroid receptors, but instead having characteristics of G protein-coupled receptors.

Multiple rapid progestin actions and progestin membrane receptor subtypes in fish.

Progestin hormones exert rapid, nongenomic actions on a variety of target tissues in fish. The induction of oocyte maturation and the progestin membrane receptor (mPR) that mediates this action of progestins have been well characterized in fishes. Progestins also act on Atlantic croaker spermatozoa via an mPR to rapidly increase sperm motility. Preliminary results indicate that progestins can also exert rapid actions in the preoptic anterior hypothalamus (POAH) in this species to down-regulate gonadotropin-releasing hormone (GnRH) secretion. Recently, we reported the cloning, sequencing and characterization of a novel cDNA in a closely related species, spotted seatrout, that has the characteristics of the mPR involved in the progestin induction of oocyte maturation. Three distinct mPR subtypes, named alpha, beta, and gamma, have been identified in both fishes and mammals. The tissue distribution of the mPRalpha protein in seatrout suggests the alpha-subtype mediates progestin actions on GnRH secretion, sperm motility and oocyte maturation. However, mPRbeta antisense experiments in zebrafish oocytes suggest the beta-subtype also participates in the control of oocyte maturation in zebrafish.

Progestin membrane receptors involved in the meiotic maturation of teleost oocytes: a review with some new findings.

The endocrine control of oocyte maturation in fish has proven to be a valuable model for investigating rapid, nongenomic steroid actions at the cell surface. Considerable progress has been made over the last decade in identifying and characterizing progestin membrane receptors mediating these actions in fish, in understanding the hormonal regulation and physiological roles of these receptors in oocyte maturation, in elucidating the signal transduction pathways they activate, and in determining their nature. Recent advances on these topics are briefly reviewed. New data demonstrating the involvement of pertussis toxin-sensitive inhibitory G-proteins in induction of oocyte maturation by the maturation-inducing steroid (MIS) in teleosts is also presented. In addition, the cloning strategy to isolate the MIS receptor gene from spotted seatrout ovaries and the characteristics of a novel gene and protein discovered by this approach are discussed. Current evidence suggests this G-protein-coupled receptor-like protein is the long sought after MIS receptor mediating meiotic maturation of teleost oocytes.

DEHP Impairs Zebrafish Reproduction by Affecting Critical Factors in Oogenesis

Public concerns on phthalates distributions in the environment have been increasing since they can cause liver cancer, structural abnormalities and reduce sperm counts in male reproductive system. However, few data are actually available on the effects of Di-(2-ethylhexyl)-phthalate (DEHP) in female reproductive system. The aim of this study was to assess the impacts of DEHP on zebrafish oogenesis and embryo production. Female Danio rerio were exposed to environmentally relevant doses of DEHP and a significant decrease in ovulation and embryo production was observed. The effects of DEHP on several key regulators of oocyte maturation and ovulation including bone morphogenetic protein-15 (BMP15), luteinizing hormone receptor (LHR), membrane progesterone receptors (mPRs) and cyclooxygenase (COX)-2 (ptgs2) were determined by real time PCR. The expressions of BMP15 and mPR proteins were further determined by Western analyses to strengthen molecular findings. Moreover, plasma vitellogenin (vtg) titers were assayed by an ELISA procedure to determine the estrogenic effects of DEHP and its effects on oocyte growth. A significant reduction of fecundity in fish exposed to DEHP was observed. The reduced reproductive capacity was associated with an increase in ovarian BMP15 levels. This rise, in turn, was concomitant with a significant reduction in LHR and mPRβ levels. Finally, ptgs2 expression, the final trigger of ovulation, was also decreased by DEHP. By an in vitro maturation assay, the inhibitory effect of DEHP on germinal vesicle breakdown was further confirmed. In conclusion, DEHP affecting signals involved in oocyte growth (vtg), maturation (BMP15, LHR, mPRs,) and ovulation (ptgs2), deeply impairs ovarian functions with serious consequences on embryo production. Since there is a significant genetic similarity between D.rerio and humans, the harmful effects observed at oocyte level may be relevant for further molecular studies on humans.

Characterization and Expression of the Nuclear Progestin Receptor in Zebrafish Gonads and Brain

Abstract The zebrafish nuclear progestin receptor (nPR; official symbol PGR) was identified and characterized to better understand its role in regulating reproduction in this well-established teleost model. A full-length cDNA was identified that encoded a 617-amino acid residue protein with high homology to PGRs in other vertebrates, and contained five domains characteristic of nuclear steroid receptors. In contrast to the multiplicity of steroid receptors often found in euteleosts and attributed to probable genome duplication, only a single locus encoding the full-length zebrafish pgr was identified. Cytosolic proteins from pgr-transfected cells showed a high affinity (Kd = 2 nM), saturable, single-binding site specific for a native progestin in euteleosts, 4-pregnen-17,20beta-diol-3-one (17,20beta-DHP). Both 17,20beta-DHP and progesterone were potent inducers of transcriptional activity in cells transiently transfected with pgr in a dual luciferase reporter assay, whereas androgens and estrogens had little potency. The pgr transcript and protein were abundant in the ovaries, testis, and brain and were scarce or undetectable in the intestine, muscle, and gills. Further analyses indicate that Pgr was expressed robustly in the preoptic region of the hypothalamus in the brain; proliferating spermatogonia and early spermatocytes in the testis; and in follicular cells and early-stage oocytes (stages I and II), with very low levels within maturationally competent late-stage oocytes (IV) in the ovary. The localization of Pgr suggests that it mediates progestin regulation of reproductive signaling in the brain, early germ cell proliferation in testis, and ovarian follicular functions, but not final oocyte or sperm maturation.

Candidates for membrane progestin receptors--past approaches and future challenges.

Progestins have a broad range of functions in reproductive biology. Many rapid nongenomic actions of progestins have been identified, including induction of oocyte maturation, modulation of reproductive signaling in the brain, rapid activation of breast cancer cell signaling, induction of the acrosomal reaction and hypermotility in mammalian sperm. Currently, there are three receptor candidates for mediating rapid progestin actions: (1) membrane progestin receptors (mPRs); (2) progestin receptor membrane components (PGRMCs); and (3) nuclear progestin receptors (nPRs). The recently-described mPR family of proteins has seven integral transmembrane domains and mediates signaling via G-protein coupled pathways. The PGRMCs have a single transmembrane with putative Src homology domains for potential activation of second messengers. The classical nPRs, in addition to having well defined transcriptional activity, can also mediate rapid activation of intracellular signaling pathways. However, details of the mechanisms by which these three classes of progestin receptors mediate rapid intracellular signaling and their subcellular localization remain unclear. In addition, mPRs, nPRs and PGRMCs exhibit overlapping expression and functions in multiple tissues, implying potential interactions during oocyte maturation, parturition, and breast cancer signaling in individual cells. However, the overwhelming majority of studies to date have focused on the functions of one of these groups of receptors in isolation. This review will summarize recent findings on the three major progestin receptor candidates, emphasizing the different approaches used, some experimental pitfalls, and current controversies. We will also review evidence for the involvement of mPRs and nPRs in one of the most well-characterized nongenomic steroid actions in basal vertebrates, oocyte maturation, and conclude by suggesting some future areas of research. Clarification of the controversies surrounding the identities and localization of membrane progestin receptors may help direct future research that could advance our understanding of rapid actions of steroids.

Expression of membrane progestin receptors in zebrafish (Danio rerio) oocytes, testis and pituitary

A distinct family of membrane progestin receptors (mPRalpha, mPRbeta, and mPRgamma) that mediate rapid, nongenomic actions of progestins has been identified and characterized in several fish species as well as in frogs, rats, pigs, and humans. However, few studies to date have thoroughly examined tissue specific expression of mPR protein and transcripts in any model species. In the present study, the expression of both mPRalpha and mPRbeta in zebrafish was examined by RT-PCR, Western blotting and immunohistochemistry using mPR specific primers and antibodies. The proteins and mRNAs of mPRalpha and mPRbeta were co-localized in the major reproductive organs, including the ovary, testis, and pituitary. Both mPRalpha and mPRbeta were found in scattered cells in the pituitary for the first time. In the testis, immunostaining of mPRalpha was restricted to the sperm, while mPRbeta was found in spermatocytes and spermatogonia. In the ovary, both mPRalpha and mPRbeta were detected in denude oocytes and follicular layer cells. Furthermore, mPRalpha and mPRbeta proteins were localized at or near the oocyte membrane of maturationally competent stage IV oocytes in a probable location for mediating progestin-induced nongenomic signaling in oocytes.

Elevations of Somatolactin in Plasma and Pituitaries and Increased α-MSH Cell Activity in Red Drum Exposed to Black Background and Decreased Illumination

The effects of different backgrounds and illumination levels on somatolactin (SL) concentrations in juvenile red drum (Sciaenops ocellatus) plasma and pituitaries were investigated using a homologous radioimmunoassay. Both plasma and pituitary SL concentrations were significantly higher in red drum exposed for 1 week to black backgrounds than in fish exposed to light backgrounds. Plasma SL levels decreased from 5.39 +/- 1.18 ng/ml in red drum adapted to a dark-blue background to 0.73 +/- 0.05 ng/ml within 1 hr of transfer to light-background tanks. Plasma SL concentrations were significantly elevated in fish 3 hr after transfer from light-background tanks (0.41-0.99 ng/ml) to black-background tanks (5.38 +/- 1.60 ng/ml) or black-background tanks without illumination (3.54 +/- 1.34 ng/ml). Maximum circulating SL levels (9.47 +/- 0.78 ng/ml) were observed 1 day after transfer of red drum to a black-background tank without illumination. Fish exposed to black backgrounds had the darkest body coloration, the greatest dispersion of melanin in their melanophore cells, and the most numerous and active alpha-melanophore-stimulating hormone (alpha-MSH) cells. These results provide evidence that SL may be involved in adaptation of red drum to different backgrounds and illuminations. However, the relative importance of SL and alpha-MSH in the regulation of this process remains to be elucidated.

Controls of meiotic signaling by membrane or nuclear progestin receptor in zebrafish follicle-enclosed oocytes.

Both membrane progestin receptors (mPRs) and the nuclear progestin receptor (nPR or Pgr) decode the non-genomic progestin signaling (NGPS) in vertebrates. However, the receptor for deciphering extracellular NGPS and initiating meiosis resumption in vertebrate oocytes is still contested hotly. We studied the roles of nPR and mPRs by determining their localization, changes of expression, and activation of NGPS during final oocyte maturation (FOM) in zebrafish. The nPR transcript and protein were expressed abundantly in follicular cells that were surrounding stage IV oocytes, but nPR transcript appeared absent within stage IV oocytes. The most significant daily changes of nPR transcript were observed in stage IV follicular cells, with the highest level observed just prior to ovulation. In contrast, the expressions of mPRα and mPRβ transcripts and proteins were abundant and increased significantly in late stage denuded oocytes prior to oocyte maturation, consistent with the purported role of mPRs in interpreting NGPS. Moreover, over-expression of mPRα in follicle-enclosed oocytes significantly increased the activity of MAPK, the production of cyclin B protein, and the number of oocytes that underwent FOM without exogenous progestin, while over-expression of mPRβ or nPR alone had no such effect. Intriguingly, significant acceleration of FOM was observed when follicle-enclosed oocytes were incubated with the maturation inducing steroid, 4-pregnen-17, 20β-diol-3-one (DHP) following over-expression of nPR or mPRα. Interestingly, this acceleration in oocyte maturation was observed approximately 1h later in oocytes over-expressing nPR compared to those over-expressing mPRα. Importantly, the acceleration of maturation in the nPR injected group was blocked by treatment with the transcription inhibitor actinomycin D, implying a requirement of the genomic signaling pathway, while the same treatment did not affect the accelerated rate of maturation in mPRα injected oocytes. Taken together, these results imply that nPR and mPRβ are unlikely receptors for inducing FOM, while mPRα is the long-sought-after nongenomic progestin receptor that deciphers extracellular NGPS to initiate meiosis resumption in follicle-enclosed zebrafish oocytes.