A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin.

Sherman, Gary; Wolfe, Michael W.; Farmerie, Todd A.; Clay, Colin M.; Threadgill, Deborah S.; Sharp, Daru; Nilson, John H.

doi:10.1210/mend.6.6.1379674

Cited by 55 publications

(11 citation statements)

References 18 publications

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“…1). In contrast to humans, no gene duplication occurred in the horse; the LH␤ gene lacking the CTP does not exist in equids, and the developmental pathway of the eLH/CG␤ CTP-bearing gene was proposed, based on sequence alignment with the LH␤ DNA of humans and cattle (7,10). The analyses showed that in humans and horses, frameshift deletion in the region of codons 112-115 of the ancestral LH␤ gene (deletion of one nucleotide in codon 114 and 10 bp subsequent to codon 111 in primates and equids, respectively) accounted for extension of the reading frame (4, 7).…”

Section: Resultsmentioning

confidence: 99%

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The LHβ Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla

Nakav

Jablonka‐Shariff

Kaner

et al. 2005

Journal of Biological Chemistry

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The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) ␤ to the ␤-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LH␤ gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LH␤ gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LH␤ gene (boCTP) would be sufficient to generate the LH␤ species of a ruminant with properties typical to the CG␤ subunit. The mutated bovine LH␤-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CG␤ CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CG␤ subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCG␤-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCG␤ subunit, a sorting function attributed to the Oglycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LH␤ to CG␤ evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LH␤ gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.The family of glycoprotein hormones includes lutropin (LH), 1 follitropin, and thyrotropin, as expressed by the pituitary, and chorionic gonadotropin (CG), which is produced by the placenta of primates and equids (1). Each hormone is a noncovalent heterodimer composed of a common ␣-subunit and a unique ␤-subunit that confers receptor specificity. The ␤-subunits have substantial sequence similarities, including conserved location of cysteine residues that suggests an origin from a common ancestral gene (2). The human (h) CG␤ genes have evolved from an ancestral LH␤ gene by frameshift mutations that resulted in a readthrough into a previously untranslated region and the extension of the reading frame (3, 4) (Fig. 1A). As a result, a short carboxyl-terminal sequence of hLH␤ is replaced by a longer carboxyl-terminal peptide (CTP) in hCG␤ (3-6). In the equine (e), both the placental CG␤ and pituitary LH␤ subunits are products of the same gene (eLH/CG␤) and contain a CTP that is presumed to have evolved by a distinct mechanism (7) (for further explanations see "Results"). Except for primates and equids, no other identified animal species b...

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Section: Resultsmentioning

confidence: 99%

“…3). Because the 3Ј region of the equine LH/CG␤ and bovine LH␤ genes was similar, we chose a strategy that mimicked the proposed developmental paradigm of the LH/ CG␤ gene subunit of the horse and donkey (7,10). The bovine LH␤ cDNA was modified by eliminating 10 bp in the 112-115 codons to shift the reading frame.…”

Section: Resultsmentioning

confidence: 99%

The LHβ Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla

Nakav

Jablonka‐Shariff

Kaner

et al. 2005

Journal of Biological Chemistry

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“…EMSA was performed using in vitro translated SF-1 and the 3ЈGSE oligonucleotide probe with an increasing molar excess of unlabeled 3ЈGSE or 5ЈGSE oligonucleotides (38), and human (39) are aligned with the core SF-1 binding site (GSE sequence) as defined in the human glycoprotein ␣-subunit and with the cognate Egr-1 site (1, 31). Nucleotide numbers are relative to the transcriptional start site of the rat sequence as defined by Jameson et.…”

Section: Sf-1 Binds Specifically To the Putative 3јgsementioning

confidence: 99%

Steroidogenic Factor-1 and Early Growth Response Protein 1 Act through Two Composite DNA Binding Sites to Regulate Luteinizing Hormone β-Subunit Gene Expression

Halvorson¹,

Ito²,

Jameson³

et al. 1998

Journal of Biological Chemistry

122

111

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Recent in vivo and in vitro studies have implicated the orphan nuclear receptor, steroidogenic factor-1 (SF-1), and the early growth response protein 1 (Egr-1) in the transcriptional regulation of the luteinizing hormone ␤-subunit (LH␤) gene. We have previously demonstrated the ability of SF-1 to bind to and transactivate the rat LH␤ gene promoter acting at a consensus gonadotropespecific element (GSE) located at position ؊127. We have now identified a second functional GSE site at position ؊59. In addition, based on electrophoretic mobility shift assay, in vitro translated Egr-1 is shown to bind to two putative Egr-1 binding sites (positions ؊112 and ؊50), which appear to be paired with the identified GSE sites. By transient transfection assay in pituitary-derived GH 3 cells, it was seen that Egr-1 increases promoter activity of region ؊207/؉5 of the rat LH␤ gene promoter through action at both Egr-1 sites. Furthermore, LH␤ gene promoter activity is markedly augmented in the presence of both factors together relative to activity in the presence of SF-1 or Egr-1 alone (150-fold versus 14-fold and 12-fold, respectively). These data define two composite SF-1-Egr-1 response-elements in the proximal LH␤ gene promoter and suggest that SF-1 and Egr-1 act synergistically to increase expression of the LH␤ gene in the gonadotrope.Precise regulation of gonadotropin gene expression is required for normal reproductive function. The pituitary gonadotropins, luteinizing hormone and follicle-stimulating hormone, are composed of a common ␣-subunit linked to one of two unique ␤-subunits, LH␤ 1 or FSH␤. The common ␣-subunit can also associate with thyroid-stimulating hormone ␤-subunit in pituitary thyrotropes or, in humans, with placentally derived chorionic gonadotropin ␤-subunit.Studies of the ␣-subunit gene promoter have identified a number of transcription factors and cognate cis-acting DNA elements that provide basal, tissue-specific, and hormonally mediated regulation of gene expression. In particular, a gonadotrope-specific element (GSE) is believed to be important for conferring gonadotrope-specific expression of the ␣-subunit gene (1, 2).The GSE, or Ad4 response element, regulates expression of a number of genes with a role in steroidogenesis, sexual differentiation, and adult reproductive function (3). The GSE/Ad4 site has been shown to interact with the transcription factor, steroidogenic factor-1 (SF-1), resulting in transcriptional activation of a variety of genes, including the steroidogenic P450, the Mullerian inhibiting substance, and the aromatase genes, among others (4 -6).SF-1 is a member of the nuclear hormone receptor superfamily. Although it was previously considered to be an orphan member of this family, it has recently been reported that SF-1-dependent transcriptional activity is increased in the presence of cholesterol metabolites, particularly 25-OH-cholesterol (7). It is currently unknown whether this putative ligand is important for SF-1 function in nonsteroidogenic tissues, such as the pituitary gland...

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“…As a heterodimer, it is composed of two subunits, an alpha (CGα), and a beta (CGβ), both are encoded by a single copy gene in both primates and equids (Pierce & Parsons, 1981). However, contrary to primates and hCG, the eCGβ and equine luteinizing hormone (eLH) are encoded by a single gene and also share a common proximal promoter region (Sherman et al, 1992). Both eCGα and eCGβ contain DNA regulatory elements that allow pituitary and placenta-specific expression (Talmadge et al, 1984a).…”

Section: Ecgmentioning

confidence: 99%

“…and LHβ polypeptides are almost identical and encoded by the same gene, indicating the coordinated regulation between the pituitary and placenta in horses (Talmadge & Fiddes, 1984b) (Bousfield et al, 1987). Also, it has been established that a single gene CGβ is evolved independently in primates and equids, thus using different mechanisms in their patterns of placenta-specific expression (Sherman et al, 1992).…”

Section: Ecg Productionmentioning

confidence: 99%

90 Establishment and characterization of Day 30 equine chorionic girdle and allantochorion cell lines

Salman

Asghar

Magee

et al. 2020

Reprod. Fertil. Dev.

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Establishing cell lines is a good model for experimental applications to study molecular mechanisms and cell-specific gene expression. Equids have a diffuse epitheliochorial placenta, where the invasive trophoblast is represented by the chorionic girdle (CG) and the noninvasive trophoblast by the allantochorion (AC). Embryonic CG cells are unique to horses and have a crucial role in equine chorionic gonadotropin (eCG) production and maintenance of pregnancy during the first trimester. This study had three objectives: (1) establishing a stable cell line from Day 30 CG cells and AC using lentivirus encoding hTERT; (2) characterisation of Day 30 CG cells and AC cell morphology and expression of eCG α (eCGA) and β (eCGB) subunits, major histocompatibility complex class II (MHCII), and Kisspeptin receptor (KISS1R) in CG and AC cells; (3) investigating eCG protein production invitro from Day 30 CG and AC cells. Three mares (n=3) were used to collect Day 30 conceptuses by non-surgical uterine lavage on Day 30 of pregnancy. All 3 conceptuses were dissected for CG and AC cells then cultured invitro to confluency in cell culture plates. Second-generation lentiviral particles were generated using a three-vector system including transfer vector pLV-hTERT-IRES-hygro, and human telomerase reverse transcriptase (hTERT) lentivirus was utilised to establish stable hygromycin-resistant equine embryonic cell lines. Reverse-transcription PCR (RT-PCR) was used to study gene expression in cells and radioimmunoassay was used to investigate protein presence in the media. We established a hygromycin-resistant Day 30 CG and AC cell lines that express eCGA, eCGB, and hTERT and confirmed using RT-PCR yielding the predicted bands. The cell lines were maintained for 16 passages (7±2 days/passage), 10 of which were cultured after the lentiviral infection steps. Also, we characterised CG cells as fast-growing, large, binucleated, and epithelioid, and AC cells as rapid-growing showing smaller, squamous, mononucleate, epithelioid, and elongated fibroblastic cells. The RT-PCR results showed eCGA and eCGB subunits are expressed by both Day 30 CG and AC cells, but MHCII and KISS1R genes were not expressed in either of cells. Moreover, radioimmunoassay results showed that Day 30 CG cells did produce eCG protein (35.42ngmL−1) invitro earlier than what previous literature has shown. However, Day 30 AC cells did not produce eCG protein (0.042ngmL−1) invitro, and both CG and AC cell lines stopped secreting eCG in the media after the lentiviral infection. To conclude, establishing stable and hygromycin-resistant cell lines from Day 30 equine CG and AC cells using lentivirus encoding pLV-hTERT-IRES-hygro is attainable. Also, equine chorionic gonadotropin eCG protein is produced invitro as early as Day 30 from CG cells.

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A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin.

Cited by 55 publications

References 18 publications

The LHβ Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla

The LHβ Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla

Steroidogenic Factor-1 and Early Growth Response Protein 1 Act through Two Composite DNA Binding Sites to Regulate Luteinizing Hormone β-Subunit Gene Expression

90 Establishment and characterization of Day 30 equine chorionic girdle and allantochorion cell lines

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