Molecular and Cellular Endocrinology 252 (2006) 136–141
The steroidogenic effect of single-chain bovine LH analogs
in cultured bovine follicular cells
Ruth Braw-Tal
a
, Svetlana Pen
a
, Moran Grinberg
b
, Sigal Nakav
b
, David Ben-Menahem
b,∗
a
Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Beit Dagan 50250, Israel
b
Department of Clinical Pharmacology, Faculty of Health Sciences, Ben–Gurion University of the Negev, Beer-Sheva 84105, Israel
Abstract
Single-chain gonadotropin analogs had been constructed for the purpose of structure–function studies and analog design. Incorporation of a
spacer derived from the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) subunit between the tethered subunit domains of the
human gonadotropins is beneficial for the secretion of the single-chain variants without compromising biocactivity. Although the CG subunit
containing the CTP domain is expressed only in primates and equids, a CTP-like sequence exists in the untranslated region of the LH gene of
several mammalian species, including the bovine species. The CTP encrypted in the bovine LH DNA (designated as ‘boCTP’) and the CTP derived
from the human CG subunit (denoted as ‘huCTP’) served as a linker sequence in the design of bovine single-chain luteinizing hormone (LH)
analogs. The purpose of the present study was to evaluate steroidogenesis in cultured bovine theca cells following stimulation with these single-
chain analogs. The concentration of the LHboCTP and LHhuCTP analogs in the conditioned media of the expressing CHO cells was three-
to six-fold higher than that of the “linkerless” LH and LH111 variants. The four analogs induced androstenedione and progesterone secretion
from the primary theca cells in a dose-dependent manner, but differences in the steroidogenic response were observed. The LHboCTP analog
(10 ng/ml) effectively induced androstenedione and progesterone secretion over unstimulated levels (4.0- and 4.4-fold increase for androstenedione
and progesterone, respectively). The response to the pituitary bovine LH standard (10 ng/ml) was less pronounced for both steroids (two- to
three-fold increase over basal levels). The activities of LHhuCTP, LH and LH111 were comparable and sightly reduced relative to the
LHboCTP activity. The data suggested that LHboCTP was ranked as the most potent and this was even more prominent when analogs
were used at a lower dose (1ng/ml). These data suggest that the design, including the huCTP or boCTP linker, is favorable for the production
of single-chain bovine LH analogs. Furthermore, spacing of the tethered subunit domains with the cryptic boCTP sequence that originated from
the bovine LH gene appears advantageous for the purpose of stimulating steroid production in the species-specific bioassay. Thus, an effective
strategy to produce bioactive single-chain LH analogs in non-primate, non-equid species would be the mutatation of the LH genes with the aim
of expressing the cryptic CTP sequence as a spacer derived from the DNA of the same organism.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Bovine LH; Single chain analogs; CTP; Cryptic CTP; Theca cells; Species-specific bioassay
1. Introduction
The pituitary gonadotropins, luteinizing hormone (LH), fol-
licle stimulating hormones (FSH) and the placental chorionic
gonadotropin (CG) that is produced in primates and equids,
are regulators of steroid production in the ovary and testis.
Each of these glycoproteins is a non-covalent heterodimer, com-
posed of a common subunit and a unique subunit that
confers the receptor-specificity. The heterodimer, but not the
monomeric subunits, efficiently binds and activates the cognate
receptor (Pierce and Parsons, 1981). In order to bypass the rate-
∗
Corresponding author. Tel.: +972 8 6477485; fax: +972 8 6477629.
E-mail address: dbm@bgumail.bgu.ac.il (D. Ben-Menahem).
limiting assembly step, to expand the arsenal of muteins for
structure–function studies and analog design, and to combine
various activities in a single polypeptide single chain, analogs of
the human glycoprotein hormones were genetically engineered
by in tandem linking of the genes encoding the monomeric
subunits (Narayan et al., 1995; Sugahara et al., 1995, 1996;
Grossman et al., 1997; Kanda et al., 1999; Garcia-Campayo
et al., 2005). Recently, the tethering approach was exploited
to gonadotropins of additional species including equine, ovine,
bovine and tilapia (Galet et al., 2001; Dirnberger et al., 2001;
Fidler et al., 2003; Min et al., 2003; Kasuto and Levavi-Sivan,
2005; Nakav et al., in press).
We previously engineered genetically the single-chain bovine
LH variants, in which the and subunit domains were linked
directly or via the heterologous huCTP (derived from the human
0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2006.03.011