Equine chorionic gonadotropin.

0163-769X/91/1201 -0027$03.00/0 Endocrine Reviews Copyright (c) 1991 by The Endocrine Society

Vol. 12, No. 1 Printed in U.S.A.

Equine Chorionic Gonadotropin BRUCE D. MURPHY AND SUSAN D. MARTINUK Reproductive Biology Research Unit, Department of Obstetrics and Gynecology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0X0

I. II. III. IV. V. VI. VII.

which attach to, invade, and phagocytose the maternal epithelium. This invasion begins on day 36 of pregnancy (Fig. 1). These patches of girdle cells form the endometrial cups, after which most of the remainder of the chorionic girdle degenerates (9). Based on its trophoblastic origin, equine chorionic gonadotropin (CG) is a more appropriate term than PMSG to describe the glycoprotein produced by the endometrial cups. A CG has been purified from at least two equids, the horse and the donkey, and can be found in hybrids of these species (10). Endometrial cups have been described in zebras (both Grevy's and Grant's), and a serum gonadotropin has been shown to be present during early gestation (11). Amino acid sequences are not homologous between species; and the placental hormone is therefore denoted herein as CG, with reference to its origin (e.g. horse CG, donkey CG, etc.) Horse CG has a number of interesting and unique characteristics. From an endocrinological view, its ability to elicit responses characteristic of both FSH and LH in species other than the horse has been known for some time (12-14). A second unusual property of horse CG is its remarkable carbohydrate content (14), which is the highest of the glycoprotein hormones, and extends its persistence in vivo. These characteristics led to its use in development of an important in vivo and in vitro model of mammalian ovarian function which results from the treatment of immature or immature hypophysectomized rats with a single low dose of horse CG to stimulate follicular development. Horse CG is of commercial value because it is readily collected and a potent agent for induction of folliculogenesis in domestic and laboratory mammals. Passeron (15) has published a fascinating account of the commercial purification and standardization of CG collected annually from as many as 12,000 mares.

Introduction Structure of equine CG Secretion of equine CG Biological activity of equine CG Persistence of equine CG in circulation Role of equine CG in equids Summary

I. Introduction

S

IXTY years ago, Cole and Hart (1) reported that serum taken from 62 mares at various stages of pregnancy and injected into immature rats and mice stimulated ovarian growth. Serum collected from mares before day 37 of pregnancy had no effect, while serum collected between days 37 and 42 induced a pronounced gonadal response. Maximal stimulation was produced by serum taken between days 43 and 80 of mare gestation, and effects could be induced by serum collected through day 131. There was little activity for the remaining 210 days of gestation. The element that stimulated the ovaries became known as PMSG. It was first assumed to be produced by the equine pituitary gland, but its localization in fetal and maternal tissues led Catchpole and Lyons (2) to conclude that it was secreted by the chorion and stored in the endometrium. It was later shown that the site of synthesis of this gonadotropin was the endometrial cups (3, 4), which are pale, circumscribed plaques of tissue that develop in the gravid horn of the mare endometrium, adjacent to the chorionic girdle of the embryo (5). The endometrial cups appear at approximately day 37 of gestation, before firm attachment of the allantochorion to the uterine epithelium (6). Clegg et al. (4) concluded that the cups were of maternal origin. In a landmark series of papers, Allen and associates (5,7,8) demonstrated that the source of the gonadotropin is the specialized trophoblast cells of the chorionic girdle,

Scope of this review

Address requests for reprints to: Dr. Bruce D. Murphy, Reproductive Biology Research Unit, Royal University Hospital, Saskatoon, Saskatchewan, Canada, S7N 0X0. * Portions of this work were funded by a Strategic Grant from the National Sciences and Engineering Research Council of Canada.

In spite of the extensive literature on equine CG, few reviews exist. There is a historical study of the discovery and partial characterization of horse CG (16), as well as 27

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MURPHY AND MARTINUK

28

Vol. 12, No. 1 Days 4 0 - 5 0

Day 37

Developing cup

Attachment and invasion of chorionic girdle cells

FIG. 1. Morphogenesis and demise of the endometrial cups in the mare. [Reproduced with permission from 0. J. Ginther: Reproductive Biology of the Mare. McNaughton and Gunn, Ann Arbor, MI, 1979 (6). Mature cup

> Days 7 0 - 8 0

1 Trophoblast (•••••I Chorionic girdle ^^™> Endometrial epithelium W$M. Endometrium Endometrial cup - — ~ Uterine milk

Sloughed cup free in uterine lumen

brief general reviews (17, 18), and a well-illustrated discussion of endometrial cup formation (6). Information on CG has been presented as part of reviews of glycoprotein hormone structure and function (14, 19, 20) and placental peptide hormones (21). Brief reviews, which include aspects of CG structure (22) and glycosylation (23), have recently appeared. The present work is an attempt to summarize recent literature on equine CG. It treats the structure, secretion, biological activity, and function in equids, as well as its biological activity and half-life in other mammals. Equids are large, expensive animals, and biological material resulting from routine slaughter is not readily available. The quantity of horse CG in circulation (24, 25) and its biological activity (26) have been shown to vary greatly between individual animals. This variability renders questionable many studies of CG in which sample numbers of animals or tissues are small. Many reports are found in conference proceedings that have not been subject to peer review, and caution should be taken in their interpretation. II. Structure of Equine CGs It is known that there is electrophoretic heterogeneity of equine gonadotropins between and within species (2729). The variation may be attributable to differences in primary structure and carbohydrate content.

Sloughed cup enclosed in allantochorionic pouch

Subunit and primary structures Gonadotropins are heterodimeric glycoprotein hormones, consisting of dissimilar a- and /3-subunits noncovalently complexed, as first reported by Papkoff and Samy (30) in studies of ovine LH. The a-subunit of glycoproteins is common to LH, FSH, and TSH in a given species and, in humans, CG shares the same asubunit with the pituitary glycoproteins (31). The j8subunits differ between hormones, and this is generally accepted to be the basis for their differential biological activity (31). Equine glycoproteins are similarly heterodimeric. Gospodarowicz (32) produced evidence for the subunit structure of horse CG. The equine a-subunit was first sequenced by Rathnam et al. (33) who reported its length to be 82 amino acids. Moore and Ward (34) developed methods for subunit separation, and Ward et al. (20) described a glycosylated horse a-subunit comprised of 96 amino acids, consistent with the known structure of asubunits of other pituitary and placental glycoproteins (31). This variation in sequence data engendered some controversy about the primary structure of equine glycoprotein a and whether it was encoded by a single gene. Isolation and cloning of the complementary DNA for the equine a-subunit resolved the controversy (35), in that it was shown that the nucleotide sequences from equine pituitary and placental libraries were identical. The dif-


February, 1991

EQUINE CHORIONIC GONADOTROPIN

PI IE PRO ASP CLY GLU PHE THR THR GLN ASP CYS PRO GLU CYS LYS ALA PRO ASP VAL GLN ASP CYS PRO GLU CYS THR PI IE PRO ASP GLY GLU PHE THR MET GLN GLY CYS PRO GLU CYS LYS LEU ARG GLU ASN LYS TYR FHE PHE LYS LEU GLY VAL PRO ILE TYR LEU GLN GLU ASN PRO PHE PHE SER GLN PRO GLY ALA PRO ILE LEU LEU LYS GLU ASN LYS TYR PHE SER LYS PRO ASP ALA PRO ILE TYR Horse a Humana Ovine a

GLN CYS LYS GLY CYS CYS PHE SER ARG ALA TYR PRO THR PRO ALA GLN CYS MET CLY CYS CYS PHE SER ARG ALA TYR PRO THR PRO LEU GLN CYS MET GLY CYS CYS PHE SER ARG ALA TYR PRO THR PRO ALA

Horse a Humana Ovine a

50 60 ARG SER ARG LYS THR MET LEU VAL PRO LYS ASN ILE THR SER GLU ARG SER LYS LYS THR MET LEU VAL GLN LYS ASN VAL THR SER GLU ARG SER LYS LYS THR MET LEU VAL PRO LYS ASN ILE THR SER GLU

Horse a I luman a Ovine a

70 SER THR CYS CYS VAL ALA LYS ALA PHE ILE ARG VAL THR VAL MET SER THR CYS CYS VAL ALA LYS SER TYR ASN ARG VAL THR VAL MET ALA THR CYS CYS VAL ALA LYS ALA PHE THR LYS ALA THR VAL MET


80 90 GLY ASN ILE LYS LEU GLU ASN HIS THR GLN CYS TYR CYS SER THR GLY GLY PHE LYS VAL GLU ASN HIS THR ALA CYS HIS CYS SER THR GLY ASN VAL ARG VAL GLU ASN HIS THR GLU CYS HIS CYS SER THR


CYS TYR HIS I US LYS ILE CYS TYR TYR I US LYS SER CYS TYR TYR 1US LYS SER

FlG. 2. Primary structures of the a-subunits of horse, human, and ovine glycoprotein hormones. + Indicates site for carbohydrate attachment. [Reproduced with permission from D. N. Ward and G. R. Bousfield: Glycoprotein Hormones (edited by W. W. Chin and I. Boime). Serono, Norwell, MA, 1990 (22).]

ferences in primary structure have been attributed to sequence errors in the earlier report of Rathnam et al. (33). The a-subunits of the glycoprotein hormones appear to be highly conserved (22). Equine-a displays 70-80% amino acid homology with other mammalian a-subunits (Ref. 34 and Fig. 2). There is a unique transposition, relative to a-subunits of other species, of tyrosine and histidine at amino acids 87 and 93, respectively (36), along with nonconservative substitutions at positions 33, 70, and 96, which, together, alter the hydrophobicity of the subunit (35). Horse CG/3 resembles human CG/3 because both have a glycosylated C-terminal extension (36). Determination of the amino acid structure of horse CG/3 followed that of other glycoproteins by some years, and it did not yield to enzymatic fragmentation and automated sequencing without a surprise, as horse CG and LH /3-subunits proved to be identical (37, 38). The consequent analysis (Fig. 3) demonstrated a 66% homology between the first 110 amino acids of horse CG/? and hCG/3. Homology between the primary structures of the C-terminal tails was much lower, with only three identical amino acids. Homology with LH from other species ranges from 64% relative to human LH to 77% relative to whale LH (37, 38). A recent report indicates that there is a single gene for LH/CG/? in the horse (39). Horse CG may be heterogenous with respect to its amino acid composition. It has been reported that CG secreted by horse trophoblast in vitro and CGs recovered from serum pools with variant biological activity have different N-terminal amino acids (40, 41). A similar

29

heterogeneity has been reported in other glycoproteins and may play a role in their biological activity (42). The only other equine CG about which information is available is that of the donkey, which has been isolated and purified from serum taken between 60 and 71 days of gestation. A partial cDNA for the 0-chain of donkey CG has been cloned, representing amino acids 85-146 of the molecule (43). Interestingly, the homology of the deduced amino acid sequence with horse CG for the same region is but 61%, with the greatest differences in the Cterminal extension. This disparity is presumed to be due to the presence of two nucleotides in the donkey CGj8 sequence which are not found in the horse CG/3 sequence, resulting in a reading frame shift (Ref. 43 and Fig. 3). Secondary structure The horse a-subunit has 10 half-cystine residues, the same number present in all known mammalian and nonmammalian glycoprotein hormones (22, 44). Ryan et al (45) note that the disulfide bridge assignments for asubunits of glycoproteins cannot be stipulated with certainty from the known information. However, Ward and 10 Horse LH(MCGP» SER ARG GLY PRO LEU ARG PRO LEU CYS ARG PRO ILE Donkey CGP Human CGP SER LYS GLU PRO LEU ARG PRO ARG CYS ARG PRO ILE Ovine LHP SER ARG GLY PRO LEU ARG PRO LEU CYS GLN PRO ILE 20 Horse LHp(CGp) LEU ALA ALA GLU LYS GLU ALA CYS PRO ILE CYS ILE Donkey CGP Human CGP LEU ALA VAL GLU LYS GLU GLY CYS PRO VAL CYS ILE Ovine LHp LEU ALA ALA GLU LYS GLU ALA CYS PRO VAL CYS ILE Horse LHP(CGp) THR SER ILE Donkey CGP THR THR ILE Human CGP Ovine LHP THR SER ILE

Horse Donkey CGP Human CGP Ovine LHP

CYS

ALA

GLY

TYR

CYS

PRO

40 SER

MET

VAL

ASN ALA THR ASN *ALA T11R ASN * A L A THR 30 THR PHE THR THR VAL ASN* Tl 1R PI IE THR ARC

VAI, Mhrr

CYS ALA GLY TYR CYS PRO THR MET THR ARC VAL LEU CYS ALA GLY TYR CYS PRO SER MET LYS ARG VAL LEU

50 PRO ALA ALA LEU PRO ALA ILE

60 PRO GLN PRO VAL CYS THR TYR ARG

GLN GLY VAL LEU PRO ALA LEU PRO GLN VAL VAL CYS ASN TYR ARC PRO VAL ILE LEU PRO PRO MET PRO GLN ARG VAL CYS THR TYR HIS

70 Horse LHp(CGp) GLU LEU ARG PHE ALA SER ILE ARG LEU PRO GLY CYS PRO PRO GLY Donkey CGP VAL ARG PHE GLU SER ILE ARG LEU PRO GLY CYS PRO ARG GLY Human CGp Ovine LHP LEU ARG PHE ALA SER VAL ARG LEU PRO GLY CYS PRO PRO GLY 80 Horse LHP(CGP) VAL ASP PRO MET VAL SER PHE PRO VAL Donkey CGP Human CGP VAL ASN PRO VAL VAL SER TYR ALA VAL Ovine LHP VAL ASP PRO MET VAL SER PHE PRO VAL

ALA ALA ALA ALA

LEU LEU LEU LEU

SER SKR SER SER

CYS CYS CYS CYS

HIS HIS GLN 1 US

90 CYS CYS CYS CYS

Horse LHp(CGP) Donkey CGP Human CGp Ovine LHP

GLY GLY ALA GLY

PRO PRO LEU PRO

CYS CYS CYS CYS

GLN ARG ARG ARG

ILE LEU ARG LEU

LYS LYS SER SER

THR THR THR SER

THR THR THR THR

ASP ASP ASP ASP

100 CYS CYS CYS CYS

GLY GLY GLY GLY

VAL GLY GLY PRO

PHE PRO PRO GLY

ARG ARG LYS ARG

ASP ASP ASP Tl IK

Horse LHP(CGP) Donkey CGp Human CGP Ovine LHP

GLN HIS HIS GLN

PRO PRO PRO PRO

LEU LEU LEU LEU

ALA ALA THR ALA

110 CYS CYS CYS CYS

ALA ALA ASP ASP

PRO PRO ASP HIS

GLN GLN PRO PRO

ALA THR ARG PRO

SER* SER PHE LEU

SER* SER CLN PRO

SER* SER ASP ASP

SER* CYS SER ILE

120 LYS ASP LYS ASP SER SER LEU

130 Horse LHP(CGP) PRO PRO SER GLN PRO LEU THR* SER* THR* SER* THR* PRO THR* PRO GLY Donkey CGP PRO PRO SER GLN PRO LEU THR PHE HIS ILE PRO PRO GLN LEU LEU Human CGP SER* LYS ALA PRO PRO PRO SER* LEU PRO SER PRO SER* ARG LEU PRO 140 149 Horse LHp(CGp) ALA SER ARG ARG SER SER HIS PRO LEU PRO ILE LYS THR SER Donkey CGP GLY PRO ALA ASP VAL PRO LEU ILE PRO SER GLN Human CGP GLY PRO SER* ASP THR PRO ILE LEU PRO GLN

FlG. 3. Primary structure of the /3-subunits of gonadotropic hormones. Where the structure is unknown, a dash (-) has been inserted. Horse LH/CG/J are identical (37, 38). The partial sequence of donkey CG is deduced from nucleotide sequence data (43). Ovine LH/3 from Ref. (31). * Indicates potential sites of glycosylation.


30

MURPHY AND MARTINUK

Bousfield (22) have assigned putative disulfide bonds bridging Cys 11 to Cys 36 and Cys 14 to Cys 35 in the N-terminal half of the equine a-peptide. In the C-terminal portion of the equine a-chain, the disulfide bridges have been assigned to Cys 64 with Cys 91 and Cys 87 with Cys 89. In the core of the a-subunit, Cys 32 is believed to interact with Cys 65 (22). Horse CG/3 contains 12 half-cystine residues (37), which is the usual number found in glycoprotein /?subunits (38). As with the a-subunits, the location of the half-cystine residues is highly conserved and identical between horse CG and hCG (31, 36, 37). The amino acid structure deduced from the partial cDNA of the donkey CG/3 indicates the presence of a further half-cystine in the glycosylated tail of the molecule, of unknown function and interaction (43, 46). The location of the disulfide bonds between half-cystines has been proposed by Ward and Bousfield for equine FSH/5 (22), such that there are four interactions between the N- and C-terminal regions of the molecule. No plan is available for CG/3, except that the disulfide bridges between Cys 38 and 57 and Cys 93 and 100 are believed to be present, providing the "long loop" and "determinant loop" structures which are analogous to similar structures of hCG and LH (22, 45). These regions of the 0-chain of hCG are believed to bind to the LH receptor (47). Glycosylation Horse CG is the most heavily glycosylated of the mammalian pituitary and placental glycoproteins, with approximately 45% of its mass attributable to carbohydrate moieties (27, 48-52). Both the a- and 0-subunits are glycosylated at asparagine-linked (N-glycosylation) and serine- or threonine-linked (O-linked) sites. While the points of attachment are similar to those observed in other glycoprotein hormones (53-55), the quantity and composition of the carbohydrates differ (54). The major difference can be found in the increases of Nacetyl neuraminic (sialic) acid in horse CG relative to hCG and to pituitary gonadotropins in other species (50). The earliest studies of horse CG revealed the presence of hexose sugars, hexosamine and sialic acid, differentially distributed between the a- and 0-subunits (56). Galactose, glucosamine, and sialic acid were in approximately 4-fold greater concentration on the /3-chain (27, 34). More recent studies demonstrated that the a-subunit of horse CG is about 22% carbohydrate by weight (50). It has two N-linked oligosaccharide groups at Asn 56 and Asn 82 (Refs. 20, 23, 35, 57 and Fig. 3), which together account for approximately 25% of the total carbohydrate of the heterodimer.

Vol. 12, No. 1

The /3-subunit of horse CG is more than 50% carbohydrate and has a single N-linked, and four or more (38, 52, 55), and probably six (23, 55), O-linked carbohydrate chains. The N-linked site is at Asn 13. The O-linked units are predominately in the glycosylated tail (residues 111-149), probably at the Ser sites between 115 and 118, the Ser and Thr sites between 127 and 133, Ser residues between 137 and 141, and the Thr-Ser complex at the Cterminal pole of the molecule (Ref. 36 and Fig. 3). The major carbohydrate structures of the O-linked oligosaccharides, found only in CG/3, exist in three variants which contain galactose and sialic acid as the principal components, in three linkage patterns (23, 52). These appear to be identical to the O-linked oligosaccharides in hCG (22). N-Linked sites of horse CG are less well defined. It has been reported that the /3-subunit has JV-acetylgalactosamine, not found on the a-subunit (50). Both the aand /3-chains contain iV-acetyllactosamine polymers (58) apparently unique to horse CG (55). A series of four variants in N-linked carbohydrate chains of horse CG has been proposed (23). These are di- and triantennary oligosaccharides with a disialodiantennary chain as the major glycan (52). The sialic acid linkages present are relatively resistant to neuraminidase digestion (23, 52). There are a limited number of glycosylation sites on glycoprotein hormones. It follows that the observed difference in mol wt between horse CG and other glycoproteins is due to major structural differences between carbohydrates in different glycoproteins. The nature of these differences is, as yet, unresolved. The amount of carbohydrate present per unit of CG can vary between CG preparations. Aggarwal et al. (40) compared two pools of serum CG, one which expressed high and another, low biological activity. The former was shown to have approximately twice the total carbohydrate per unit of protein and 2-fold of each of hexosamine, hexose, and sialic acid. The quantity of sialic acid per mg of horse CG varies not only between mares but also with the stage of gestation in the same mare (59, 60). Horse LH has been shown to have similar sialic acid variation, which contributes to its microheterogeneity (61). The source of CG is a determinant of its carbohydrate content. CG derived from horse endometrial cups was first reported to have a lower carbohydrate content than that derived from serum, (12.7-30% rather than the 45% characteristic of serum CG) (27). A subsequent reevaluation, in which more purified CG from endometrial cups and serum were compared, gave the opposite result; CG from horse endometrial cups had more carbohydrate (28, 62, 63). Secreted forms are presumed to be deglycosylated before or after their appearance in serum. Horse CG secreted by equine trophoblast cells in pri-


February, 1991


mary culture has been shown to have less than half the carbohydrate content found in circulating CG (41). The significance of this finding to the in vivo glycosylation of CG is not clear. The primary "structures of horse LH and horse CG have been reported to be identical (37, 38), and the observed variation in mol wt of the two forms is due to differences in carbohydrate content. The content of hexose and hexosamine in the former is only about 65% of the latter (62, 64). The largest difference is in sialic acid content, which is twice as abundant in horse CG (62, 64). These differences in glycosylation reflect differential posttranslational processing of CG and LH. The disparity between the carbohydrate components is probably due to the site-specific occurrences of the glycotransferase enzymes, which have been shown to vary between the pituitary and placenta in other species (54). As noted below, the difference in carbohydrate content between horse LH and CG appears to influence the receptor binding activity of the two hormones. The total carbohydrate content of circulating donkey CG has been reported to be 31%, approximately twothirds of that found in horse CG, owing to lower amounts of hexosamine and sialic acid (46). The homology of donkey CG and donkey LH has not been demonstrated, but both have the same biological activity (29, 46). Donkey CG has more than twice the carbohydrate content of donkey LH, and, as with the horse, the differences are largely in sialic acid content (29, 46, 64). Folding and subunit interactions The noncovalent linkage between the subunits of horse CG, as with other glycoprotein hormones, is readily disrupted at pH of 4.0 or less (65) or by chaotropic agents such as 10 M urea (66) or 6 M guanidine HC1 (34, 65). The a- and jS-subunits of glycoproteins will reassociate to form active hormones in the pH range of 5.3-9.5 in a reaction that is time, temperature, and concentration dependent (65). Incubation of CG subunits overnight at pH 7.0 and room temperature results in their recombination (66). The intact glycoprotein hormones have not yielded to x-ray crystallographic analysis, because of the difficulties of crystallization associated with the elevated carbohydrate component (67). In spite of the recent successful crystallization of deglycosylated and desialated hCG (68, 69), the three-dimensional structure of the glycoprotein hormones is currently unknown. Nevertheless, inferences have been made from hydropathy analysis, from monoclonal antibody studies, and from observations of the activity of mutant forms of the hormones. Equine CG has received little attention; the most has been visited on hCG. Due to the similarity between hCG and horse

31

CG, it may be possible that the subunit interaction and tertiary conformation of horse CG may be inferred from the information known about hCG. The five disulfide linkages of the horse a-subunit are expected to configure the molecule into a series of five or six parallel loops or sheets as hypothesized to exist in hCG (44). Analysis of circular dichroism spectra of horse CG suggests both sheet and a-helix formation (70). The /?-unit can be expected to resemble hCG, with an overall three-dimensional structure which has some a helical components (67). From hydropathy analysis of the horse CG/3 subunit it has been concluded that there are several surface regions, including two disulfide loops, similar to those present in other glycoproteins (Refs. 37 and 38 and Fig. 5). The carbohydrates of CG are expected to have an effect on the tertiary structure of the molecule. The folding of hCG has been reported to be affected by deglycosylation, which prevents renaturation of both the a- and /3-subunits (71, 72). Deglycosylation alters the antibody binding properties of both hCG and human FSH (73). The nature of the influence of the oligosaccharide component on three-dimensional structures is not known. It is believed that a-/3 interactions of glycoproteins are not restricted to a single site (67), and the sites of attachment between the a- and /3-subunits are not known for any of the equine CGs. The hCG subunits have been reported to be connected at the /3-subunit sequence CysAla-Gly-Tyr (amino acids 34-37, Ref. 67), a highly conserved segment, which is also present in horse CG/3 (37), as well as most other known LH-like hormones, with the exception of rat LH/3 (Moyle, W.R. personal communication). A site on the a-subunit of linkage to the /?-chain may include Tyr 49, which is present in hCG (67) and horse CG (20), as well as all known glycoprotein hormone a-subunits (22). Bousfield and Ward (74) reported that the loop at 93100 of the equine LH subunit was protected from Lysdirected proteolysis when the subunits were associated. This protection was lost to the isolated /3-subunit; further, the 0-subunit with modified Lys 96 did not associate with equine a (74), suggesting a linkage site in this region of the /3-chain. The 93-100 loop of the /5-subunit appears to be a surface feature of horse CG (see Fig. 5), as with other glycoprotein hormones. In hCG, this region is believed to be involved in receptor binding (75). The supposition that it is both buried in the a-{5 subunit interaction as well as on the surface for receptor binding appears oxymoronic. Further experimentation is necessary to clarify this matter. The carbohydrate associated with Asn 52 in hCGa is necessary for the association of the a- and /3-chains, as it has been shown that site-directed mutagenesis, which deletes this amino acid, prevents the majority of subunit


32

MURPHY AND MARTINUK

interaction and impairs biological activity (76). Due to the conservation of N-linked carbohydrate sites in asubunits of gonadotropins, and the similarity of the oligosaccharide chains between horse CG and hCGa, it is reasonable to infer a similar significance of this carbohydrate in association of the a- and /?-subunits of horse CG.

III. Secretion of Equine CGs Source and pattern of secretion of CG

The first appearance of horse CG coincides with the migration of the specialized chorionic cells into the endometrium. The pattern of the presence of CG in serum is shown in Fig. 4, (24, 25) and, in general, the peak values can be observed between days 55 and 70 of gestation (24, 77, 78). The hormone levels decline gradually until approximately day 130, although CG has been found after day 200 of gestation (79). There is pronounced individual variation between mares in the 100-

IU/ML

80-

O O

604020-

20

40

60

8 0

100120140160

100-

B

IU/ML

80-

O

o

604020-

2 0

40

60

80

100120140160

DAYS OF GESTATION FIG. 4. Mean (±SE) profiles of horse CG (A) in the serum of 13 mares measured by bioassay (25) or (B) in the serum of 5 mares as determined by RIA (24). Figures redrawn.

Vol. 12, No. 1

amounts of CG found in serum (24, 25, 80). RIAs that employ polyclonal antiserum (49, 80, 81) have resulted in minima in the range 300 ng/ml serum about day 40 of gestation. Maxima of as much as 35 tig/ml, or 2 orders of magnitude greater, have been found at the peak of secretion, day 50-70 (59, 63, 80-82). The pattern of appearance of CG in horse serum has been correlated with gross (83) and histological (6) changes in the endometrial cups (Fig. 1). Ginther (6) reported that the cups reach maximum development about day 65 of gestation. Degeneration, by cytolysis rather than phagocytosis, is initiated as early as day 60. By day 80, the cup cells have become vacoulated, and degeneration is pronounced. By day 100, some of the cups have begun to be sloughed and in some cases develop into allantochorionic pouches. By day 140, the cup cells have disappeared. The putative immunolocalization of horse CG in maternal epithelial gland cells (84) is in contrast with the prevailing view that the fetal chorionic girdle cells are the sole source of horse CG. The characteristics and cross-reactions of the antiserum used for immunolocalization in that study (84) were not reported, and there is no indication that a negative control (i.e. second antibody only) was used. However, it is fair to state that the evidence for fetal chorion cells as the sole source of horse CG is based on their ability to secrete CG in vitro, on the observation that they invade the maternal epithelium to produce the endometrial cups, the presence of CG in cup tissue, and on the information that the demise of the cups is associated with the declines in CG in circulation. None of the evidence precludes the synthesis of CG by extrachorionic tissue. Little is known about the factors that control the synthesis and secretion of the equine CG. Ultrastructural examination of the chorionic component of the endometrial cups reveals no secretory granules (84, 85) such as those found in pituitary gonadotropic cells (86). Thus, CG secretion is likely to be a constitutive function of these cells. Serum levels of CG in the horse do not undergo episodic changes (87) as do pituitary gonadotropins, further suggesting a constant mode of secretion. Injection of GnRH had no effect on CG concentrations over 4 h (87). Steroid feedback may not be important to regulation of CG secretion. In pony mares, treatment with diethylstilbesterol, a potent synthetic estrogen, or with androgens, had no apparent effect on CG concentrations in blood samples taken weekly (88). In a more recent study, no effect on circulating CG was observed in mares given frequent diethylstilbesterol injections between days 84 and 142 of gestation (89). CG secretion persists after loss or removal of the horse fetus (79, 90, 91), suggesting that the normal lifespan of the endometrial cups is not affected by fetal death or


February, 1991


removal. However, in a study where numbers were sufficient to warrant statistical analysis, it was shown that fetal loss is associated with reduced levels of CG (90), suggesting that there may be some fetal influence on the quantity of CG secreted. An alternate interpretation is that differences in blood flow to the uterus or some other pregnancy-associated factor may account for differential secretory patterns between pregnant and formerly pregnant animals. One hypothesis has been put forth to explain the longterm regulation of horse CG: that there is a maternal cell-mediated immunological reaction to paternal antigens in the fetal chorionic cells, resulting in cytolysis of the cup cells and consequent abrogation of CG synthesis and secretion (83, 92-94). This postulate was initially based on the progressive increase in leukocytic invasion of the endometrial stroma at the base of each endometrial cup (83, 95). In hybrid pregnancies, where the genetic disparity and immunological recognition are expected to be greater, the concentration of leukocytes is elevated (95). Chorionic girdle cells can persist in an apparently healthy state in vitro for periods in excess of their life span in vivo (7, 96, 97), suggesting that their demise during pregnancy can be attributed to some active impairment. Further evidence for the hypothesis can be found. It was suspected that fetal component of the endometrial cups expresses the paternal major histocompatibility complex (MHC) antigens (94). Crump et al. (98) showed that MHC antigens were produced by horse trophoblast. Antczak et al. (99) confirmed that antibodies cytotoxic to paternal lymphocytes were present in serum of foaling mares, but not geldings, stallions, or maiden mares. The first appearance of this response can be found between days 45 and 70 of gestation, corresponding with the invasion of the endometrium by the girdle cells (93, 99). Additional information can be gleaned from a study in which two pairs of chimeric horse twins of the opposite sex were mated (79). As each pair of twins is expected to be antigenically similar, the maternal immunological tolerance of the trophoblast is expected to be greater; thus, the cups were expected to persist for a longer period of time. This occurred, and CG remained detectable in circulation until 230 and 265 days of gestation in the mare of each pair, respectively. The theory of immunological regulation of CG secretion fits some of the findings. There is little doubt that a maternal immune reaction occurs at the site of the endometrial cups. However, to conclude that this is the primary mechanism that regulates CG synthesis and secretion may not be appropriate. The theory fails to explain some observations, most important, that the peripheral levels of CG increase in spite of the histological evidence for an immune reaction soon after cup

33

formation (83). The decline in peripheral CG occurs before invasion of the cups by immunological elements (95). It has been stated that the leukocyte accumulations about the cups do not represent a destructive immune response (94). Further, paternal cytotoxic responses cannot be observed in some mares (99); nonetheless it appears that CG levels decline in these animals. The serum levels of CG and the endometrial cups are not distinguishable between MHC-compatible and -incompatible pregnancies (100). In hybrid pregnancies between stallion and donkey jenny, which produce a hinny, the chorionic girdle cells in the cups persist in spite of heavy leukocyte accumulation; nevertheless, CG levels begin to decline before day 70 (95). In horse pregnancies, cytolysis of the endometrial cup cells is first seen at the lumen and in the center of the cup (6,83), points distant from the maternal lymphocytes. It is difficult to conceive that maternal cytolytic factors pass through the cup cells and exert their effects selectively upon those most removed from the site of production. Finally, proliferation of girdle cells in vitro in primary culture ceases at approximately the same time as maximal growth of the endometrial cups in vivo (96), suggesting that these cells may have a defined life span, as has been reported for mouse trophoblast cells (101). Other factors affecting the secretion of equine CG

The quantity of CG detectable in the peripheral serum varies between breeds of horses (85, 88, 102), and draft horses have lower levels of CG than do light breeds. This difference has been attributed to the dilution effect of the larger blood volume (95), although no data exist to confirm this hypothesis. Circulating concentrations of CG are considerably lower in donkeys than horses (95). In hybrids, the quantity and duration of secretion of CG are influenced by the sire (10, 95, 102), indicating that there is an effect of the paternal genome on the secretion of this hormone. In part, the variation in CG between species (donkey vs. horse) and hybrids (mule vs. hinny) can be attributed to the size of the endometrial cups. In the donkey they are small and narrow; and in mule pregnancy, they are similarly meager, and both have low CG secretion (95). In hinny pregnancies (stallion mated to donkey jenny), the endometrial cups are large and broad and CG quantities ample (95). Leukocyte invasion of the endometrial cups in mule pregnancy is believed responsible for early demise and the consequent abrogation of CG synthesis (95). The sire had demonstrable effects on CG production in intraspecific matings. A sire effect was demonstrated in a study of 227 pregnancies in 177 draft mares randomly mated to 12-different stallions (80). A highly significant


34

MURPHY AND MARTINUK

component of variability in the circulating levels of CG between days 40 and 100 of gestation was attributable to the sire (80). Sires could be divided into 2 statistically definable groups, those producing high titer CG pregnancies and those producing lower titer pregnancies. An influence of the sire on circulating concentrations of CG was confirmed in a study of 100 Standardbred mares (82). The dam profoundly influences the quantity of CG present in circulation as well. By means of mating trials carried out over four seasons, it was shown that a component of the variability in CG concentrations in serum of mares could be attributed to the mare (63). This effect of the mare was seen to override that of the stallion; i.e. the effect of sire could be reduced or eliminated by the selection of mares with history of elevated or reduced levels of CG during pregnancy. It has been conjectured that the influence of the mare on the quantity of CG produced is related to the relative convolution and consequent surface areas of the endometrium available for establishment of the endometrial cups (95). The relative importance of both the maternal expression of the gonadotropin gene and the maternal environment on the secretion of horse CG remains to be determined. Variation in the quantity of horse CG has also been attributed to the age and parity of mares, with older mares producing more (80) or less (88) CG during pregnancy. In the former experiment, the mare population had been selected for successful pregnancy, and the results may reflect this selection. The number of fetuses in the uterus has an influence on CG production, with greater amounts present in serum of mares carrying twin fetuses (103).

IV. Biological Activity of Equine CGs Necessity for intact a- and fi-subunits for biological activity

The dissociated a-subunit of horse CG has less than 4% of the activity of the intact molecule, and the /?subunit, some 6% (66). The horse LH a-subunit, which is the homolog of the horse CG a-subunit, inhibits FSHinduced events in the rat testis, suggesting that it interacts with the FSH receptors (104). Recombination of horse CGa with horse CG/3 restores some 30% of the biological activity relative to intact CG (66). Hybrid molecules formed from CG/3 (66) or equine LH/3 (105) and the a-subunits from other glycoproteins are biologically inactive. The equine a-subunit recombined with ovine LH/3 was as potent as recombined CG (66), and nearly as potent as intact equine LH. The combination of horse a with porcine LH/3 resulted in a hybrid which was the equivalent of horse LH in biological potency and nearly 50 times as potent as porcine LH in

Vol. 12, No. 1

bioassays (105). A hybrid of horse CGa with hCG/3 was more biologically active than horse CG in some bioassays (50). The potentiation of biological activity by combination of heterologous /3-chains with the horse a-subunit may be attributable to the transposition of tyrosine and histidine at positions 93 and 87 of horse a, relative to the a-subunits of other glycoprotein hormones (Fig. 2). Modification of histidine residues on horse CG dramatically reduces the biological activity of the molecule (106), lending credence to a role of histidine in the enhanced biological activity of hybrid molecules. Biological activity of equine CG in equids

The chemical similarity of horse LH and horse CG was reported more than 10 yr ago (107). As could be expected, horse CG bound to putative LH receptors in equine testis (107,108). However, this occurred at about one-tenth or less the affinity of horse LH binding (107, 109,110). The reduced receptor binding activity of horse CG compared to horse LH, found independently in three laboratories, is puzzling in light of the known amino acid homology of the two hormones (37, 38). Part of the difference may be explained by differing mass of peptide between the two hormones; i.e. 1 /xg horse CG has less protein than 1 /*g horse LH because of its greater glycosylation. As the 2 molecules are known to have different complements of carbohydrate (61, 62, 70, 111, 112), the suggestion may be made that the carbohydrate of horse CG somehow inhibits its binding to the horse LH receptor. An alternate view is that the receptor population of the horse gonad may be heterogeneous, and separate binding sites of different affinities may be capable of discriminating between horse CG and LH. In support of this view, Sairam et al. (113) have shown that the ovine testicular LH receptor has separate binding sites which can distinguish LH from hCG or horse CG. Horse CG does not bind to FSH receptors in horse follicles (108, 110) or testis (111), suggesting that CG is primarily an LH-like hormone in the horse. It is of interest that horse CG binds to donkey FSH receptors with similar affinity to its attachment to donkey LH receptors (109,110), a finding that has been interpreted to indicate that horse CG may have FSH activity in the donkey (109). The biological activity of donkey CG has been much less studied, and the homology between donkey CG and donkey LH in primary structure has not been confirmed. However, donkey LH binds to equine testis preparations with about 10% of the activity of the horse LH (29). There appears to be no information about the receptor binding or biological activity of donkey CG or LH in donkey gonadal tissues.


February, 1991


Preliminary investigations of zebra CG suggest that it resembles donkey CG in that it has primarily LH activity and little FSH activity in equine tissues (H. Papkoff, personal communication). Biological activity of equine CG in nonequid species

One of the intriguing properties of horse CG is its duality of activity in species other than the horse, recognized as early as 1940 (12). This activity can be demonstrated in the classic Steelman-Pohley bioassay for FSH and the ovarian ascorbic acid depletion assay for LH (50, 66, 111). Horse CG was first shown to bind, and to inhibit the binding of [125I]LH to ovine luteal membrane preparations by Gospodarowicz (114). Further studies demonstrated interaction with rat Sertoli cell receptors believed specific for FSH and Leydig cell receptors specific for LH (111, 115, 116), rat ovarian FSH receptors (117), pig ovarian LH receptors (107), and pig testicular FSH receptors (117). The dual activity has also been demonstrated in bioassays in vitro using various mammalian tissues and endpoints. For LH, testosterone production by murine Leydig cells has been the assay most frequently employed (14, 26, 27, 59, 104, 107, 111). FSH bioactivity has been demonstrated by measurement of cAMP production in rat seminiferous tubule homogenates (118), of estrogen production in cultures of granulosa cells from immature rat ovaries (119), and by the determination of FSH-dependent increases in plasminogen activator secretion by cultured Sertoli cells (59,117, 120) or rat granulosa cells (14, 121). Folliculogenetic potential, which is an indication of LH and FSH activity of horse CG, has been assessed by determination of the number of ovulations in immature rats (28, 60, 122). Horse LH has proven to have both FSH and LH activity in a variety of systems including RRA and in vitro bioassay (29, 60, 107, 123). FSH activity was demonstrated in vivo (124). These findings are presumed to reflect the structural homology of horse CG and horse LH (37, 38). Donkey CG has about one-third of the LH activity of horse CG as measured by RRA and in vitro bioassay, and it does not share the potent FSH activity of horse CG in nonequids (46). Donkey LH is active in nonequid systems, but donkey LH has little FSH activity (29). Zebra LH is predominantly active as LH and has even less FSH activity than does donkey LH (112). Each assay system mentioned above {in vivo or in vitro bioassay, and RRA) responds to a different characteristic of CG. In some studies, it has been shown that there is reasonable agreement in estimation of the biological activity of CG by all three methods; in others, there is no accord. In vivo bioassays measure the potency of CG relative to pituitary standards and are strongly influ-

35

enced by the circulating half-life of the hormone, which is related to its glycosylation (60). In vitro bioassays reflect the potency of the hormone over relatively short term (4-8 h; Refs. 26 and 27), or longer term (24 h; Ref. 120), and thus are not always comparable. They are also subject to variation due to the carbohydrate component of the glycoprotein being tested (125), best shown by the site-directed deletion of the a-subunit of the Asn 152linked carbohydrate in hCG which dramatically altered biological activity (126). To further complicate the matter of carbohydrate influence in vitro, it has been shown that the glycosylated tail of equine LH is not necessary for its LH activity in vitro, as it retains some 60% of its steroidogenic potency after cleavage of amino acids 121-149 (127). Neuraminidase digestion, which removes the sialic acid residues, increases the LH activity of CG in the Leydig cell assay in a manner related to the quantity of sialic acid removed (59), and LH activity increases 5-fold when sialic acid is completely removed (128). Desialation elevated FSH activity of horse CG in the rat granulosa cell bioassay (119), and increased (120) or had no effect in the Sertoli cell plasminogen activator assay (59). The horse a-subunit alone, while it has no biological activity, can suppress FSH stimulation of Sertoli cells (104). Inferences about the biological activity of CG derived from RRA assay must also be interpreted with care. Deglycosylation of LH-like hormones increases receptor binding activity but alters signal transduction and the cellular responses (129,130). Both removal of the heavily glycosylated C-terminal component of horse LH (127) or desialation of horse CG (120) increase its binding to FSH and LH receptors. Thus, variation in carbohydrate can influence receptor activity, and receptor binding changes may not be directly correlated with biological function. Further, the horse a-chain alone had approximately 10% of the binding activity of intact horse LH in immature rat granulosa cells (131), suggesting that if subunits of CG are present, they might interfere, and complicate interpretation. The exploration of the biological activity of horse CG has been further complicated by the use of a variety of standards, including ovine, bovine, human and equine gonadotropins, all with different inherent activity. In at least one case, different assays and different standards were employed in the same study (46). The concept that there is an inherent and relatively constant ratio between FSH and LH activity in horse CG has been promulgated (115, 116). In the face of the differences in assay systems and standards, the notion is not meaningful. It is clear that horse CG is heterogenous, and that its carbohydrate content can vary depending on the source of CG (cups vs. serum, 28, 41, 62, 132), preparations of CG pooled from groups of mares (18, 40),


MURPHY AND MARTINUK

36

or between mares and between stages of gestation (59). Because of this heterogeneity, horse CG can vary greatly in its biological activity, a finding first reported by Gonzalez Mencio et al. (26). Structural basis of the duality of activity The availability of and significance to human health of hCG have resulted in intensive study of its structure and receptor binding activity. This can be conceived as unfortunate, as horse CG, with its dual binding activity, seems inherently more interesting. The recent cloning of the cDNA for the rat LH (133) and FSH (134) receptors has provided deduced amino acid sequences and models for the interaction of these receptors with the gonadal plasma membrane. The putative extracellular domains of rat FSH and LH receptors, which are expected to be involved in ligand binding (135), share some 50% homology (134). Horse CG has the apparently unique capability to bind to both receptors, and the basis for this interaction is the principal enigma of the biology of horse CG. The a-subunit, the long loop, and the determinant loop have been implicated in the duality of activity.

Vol. 12, No. 1

Hydrophilicity 5.0 -

0

-t4

-5.0 -

Surface Probability 1.0 -

05

0

-

Flexibility 1.25 -

1.0 —

A

r\

A

A

0.75

Antigenic Index 1.0 -

The a-subunit Biological activity of gonadotropins requires the intact hormone, including the a- and /?-subunits and the carbohydrate component. Studies with synthetic peptides suggest that binding of hCG to receptors is an interaction of at least two regions on each of the subunits (44). The increased activity of LH /?-subunits when combined with horse a-chains (105), and the capability of horse a alone to antagonize FSH activity in the rat seminiferous tubule (104) suggest that the duality of biological activity may be related in part to the equine a-subunit. However, the a-subunit alone does not convey the FSH activity to the molecule, as equine a alone or hybrids of equine a and ovine LH /3-subunits from other species express little FSH activity in vitro (131). The long loop The duality of biological activity of horse CG has often been attributed to the 0-subunit of the molecule. At least two regions of glycoprotein jS-chains have been implicated in biological function, the so-called long loop, formed by the disulfide bond between amino acids 38 and 57 of hCG and horse CG (67) and the so-called determinant loop, formed by the disulfide interaction between amino acids 93 and 100 of glycoproteins (14). Structure-function studies and hydropathy analysis suggest that portions of the long loop of hCG (67), FSH (136, 137), and horse CG (Fig. 5) are on the surface of

-1.0 -

50

100

FIG. 5. Surface and antigenic components of horse CG/3 as derived by hydropathy analysis using the program Mac Vector. The upper panel represents the proposed disulfide interactions. Regions with positive values (>0) in hydrophilicity and antigenicity and >0.5 in surface probability are presumed to be on the surface of the intact hormone. The long loop (amino acids 38-57) and the determinant loop (amino acids 93-100) both have surface components by this analysis.

these molecules. Antibodies against the long loop of hCG bind to the intact molecule, further indicating its surface location (138). The importance of the long loop to biological activity can be inferred from the capability of the peptide sequence alone to induce androgen synthesis in Leydig cells (139). Further, the corresponding long loop of FSH (Cys 32 to Cys 51) appears to be a surface structure (137), binds to the FSH receptor (140), and can induce estradiol synthesis by Sertoli cells (141). If the long loop is significant to the duality of activity, some structural similarity to both the long loops of LH and FSH should be present. Comparison of long loop sequences between hCG, horse LH/CG, and ovine LH suggest reasonable homology; 12 of 20 amino acids are the same (Figs. 3 and 6). There is little similarity between horse LH/CG ancTovine FSH (Fig. 6), as only the two Cys and one each or the highly conserved Val and Gly



February, 1991 ALA

ALA

\ PRO MET VAL ARG

GLY

LEU \ PRO \ ALA \ ILE

I

I

PRO

VAL

I

1

GLN PRO

MET \ SER \ PRO

/ VAL

VAL

/ GLN

VAL / PRO

LEU PRO

LEU

ALA

/ ARG

\ LEU

I

PRO

LYS

/ GLN

\ MET

I MET

VAL

THR

I

\ PRO

VAL

/

\

CYS 1

CYS

HORSE CG (38-57)

ILE

\

LEU PRO

\

/ VAL | \RG •

PRO

\ MET 1

1

1 PRO

1

GLN

\ SER

ARG

/ VAL

\

CYS 1

CYS

hCG

M-57)

/ LYS

\

\ PRO

/

CYS

1

PRO

ASP

/ LEU

/ VAL

THR

37

CYS

OVINE LH (38-57)

\ ALA

/ TYR / VAL / LEU

\ ARG

\

PRO

\ ASN I

1

1

ASP

1ILE

1

GLN

ARG

\ THR

LYS / THR

\

TYR \

/

CYS

1

CYS

OVINE FSH ( 3 2 - 5 1 )

FlG. 6. Comparison of the long loop from the /3-subunits of horse CG (37, 38), hCG (31), and ovine FSH (Ref. 22 and D. N. Ward, personal communication). The structures are based on the assumption of interaction of the half-cystine residues at amino acids 38 and 57 of horse CG, hCG, and ovine LH and at 32 and 51 of ovine FSH.

residues are the same. The amino acids believed important for LH binding, such as Arg 43 and Pro 50 (75), are disparate between horse CG and FSH. Chimeric molecules, in which portions of the hCG j8subunit have been altered by site-directed mutagenesis, have been used to study biological activity. When the long loop of hCG/3 was replaced by the corresponding long loop of human FSH, the consequent molecule bound only to LH receptors (142). The sum of the information suggests that the long loop of the /3-chain is not the source of the dual LH/FSH activity of horse CG. The determinant loop The determination loop hypothesis, first espoused by Ward and Moore (14), states that the 93-100 disulfide loop of the /3-subunit of glycoproteins is substituted between hormones to allow for net charge differences for this region, which are then reflected in the biological activity of the molecule. For example, LH-active molecules have a net charge for the determinant loop region of 0 or +1, while FSH-active molecules have a net charge of - 3 . The peptide sequence for FSH or substitution of amino acids to result in a negative charge of the 93-100 loop abrogates binding to the LH receptor. However, replacement of the LH sequence of hCG with the FSH sequence in chimeric proteins does not alter the binding of the chimeric hormone to the LH receptor (142). Sitedirected mutagenesis in which Arg 94 in hCG was replaced by Asp resulted in 50% reduction in LH receptor binding and in vitro biological activity, indicating the importance of the region to LH activity (143).

Horse CG has a net charge on the determinant loop of 0 (37). The determinant loop of the deduced amino acid sequence of donkey CG, which appears to express little FSH activity in nonequids (46), has a net positive charge (43). According to the determinant loop hypothesis, both horse CG and donkey CG should express LH activity, as they clearly do. However, the FSH activity of horse CG remains unexplained by this theory. Leigh and Stewart (43) sought differences between horse CG, donkey CG, and FSH 0-subunits as potential explanations for the FSH activity of horse CG. An obvious difference, which may be of significance, is the presence of Val 102 in FSH sequences and horse CG and its absence in LH-active hormones, including donkey CG. It may be that the region from 100-111 is significant to the dual action of horse CG. The sum of the available information suggests that horse CG binding to FSH and LH receptors is too complicated to be explained by either the long loop or by charge differences in the determinant loop. As both subunits, and at least four different amino acid sequences, appear involved in the binding of glycoprotein hormones to their receptors, the explanation for dual biological activity most likely can be found in the threedimensional conformation of the horse CG molecule and the LH and FSH receptors.

V. Persistence of Equine CGs in Circulation The glycosylation of horse CG results in its persistence in the blood of the horse and other species for longer periods than other glycoprotein hormones. Kamerling


38

MURPHY AND MARTINUK

et al. (23) have attributed the long half-life to the form of sialic acid linkages present in CG, which are relatively resistant to neuraminidases. The first reports of the clearance of horse CG indicated that it had a half-life of 6 days in gelding and 24-26 h in rabbit circulation (144). Using relatively insensitive bioassays, Cole et al. (145) determined the half-life to be of the same order for horse CG in mares. Cole et al. (145) reported that approximately 1% of the horse CG can be found in the urine and 0.2% in the milk. This led them to conclude that the fate of the majority of CG (85+%) was metabolism and degradation rather than renal filtration. By RIA it was confirmed that urine from mares at 90 days of gestation contained little CG, relative to serum (49). However, this fraction of CG was detectable by RIA and ELISA using a high affinity monoclonal antibody against bovine LH, and the pattern of occurrence reflects its appearance in serum (146,147). Levels in urine (nanograms per mg creatinine) were approximately 1% of those observed in circulation (nanograms per ml), consistent with the view that filtration is not the principal means of loss of CG from circulation. It should be noted that the monoclonal antibody employed by Roser and Lofstedt (146, 147) is believed to react with a single epitope of the CG molecule and may recognize only a fragment or a metabolite of CG. Other studies of the persistence of horse CG in circulation of nonequid mammals have been carried out, some to determine the role of sialic acid in the clearance of this molecule. In an early study in which a physiologically heterogenous group of ewes was infused with one of two doses of horse CG, the disappearance of the hormone was assigned to a two-compartment model, with a clearance half-life of 21.2 ± 1.1 h. (148). Subsequent studies further suggest the presence of a two-compartment model in sheep (28, 60), rats (149), and cattle (150). The first component of the disappearance curve represents the distribution phase and is characterized by the rapid removal of the hormone from circulation (28, 60, 151). The elimination component of the disappearance curve represents the return of the hormone from the extravascular compartments and its subsequent metabolism and excretion. The half-life for the distribution phase is from 0.3 h in rats (149, 151) to 3-4 h in the ewe (28, 60), and 45.6 h in the cow (150). The half-life for the elimination phase ranges from 6 h in rats (151) to 61 h in the ewe (28, 60) and 121 h in the cow (150). Removal of some 99% of the sialic acid component of horse CG by neuraminidase treatment increases both compartments of its clearance rate in the rat (151). The extent of removal of sialic acid from CG is a function of the time for which the hormone is incubated with neuraminidase; thus decremental detachment is possible (59). Administration to ewes of horse CG from which none,

Vol. 12, No. 1

20, 53, or 80% of the sialic acid had been removed revealed that neither the distribution nor the elimination phase half-lives were significantly increased until 80% of the sialic acid was not present (60). However, both the distribution of CG to the tissues and the clearance rates was progressively increased with the proportion of sialic acid removed (60). This kinetic pattern is different from that seen with hCG, where the distribution to the tissues remains constant after deglycosylation or desialation, but rate constants for the two compartments are markedly increased by carbohydrate removal (152). The altered sialic acid is believed to render CG vulnerable to hepatic metabolism via the asialoglycoprotein system (153). The standard pharmacokinetic analysis does not take into account the targeting of CG metabolism by the liver, and conclusions about tissue distribution should be interpreted with caution. IV. Role of Equine CG in Equids Despite all that is known about the structure and biological activity of equine CG, it is not currently possible to make an unequivocal statement about its function, or, for that matter, its role in pregnancy in the horse. The critical experiment with which to establish the significance of horse CG to pregnancy would be the removal (surgically or by immunoneutralization) of all of the cup tissue, followed by observation of the endocrine and other effects during gestation. For a variety of reasons, these experiments are impractical at this time. It has been conjectured that horse CG, because of its high sialic acid content, may serve to isolate the trophoblast from recognition by the maternal immune system (83). Subsequent studies demonstrated that the maternal immune system responds to paternal antigens between days 45 and 70 after ovulation (99), suggesting that masking of fetal antigens by CG is ineffective. Horse CG function has also been thought to cause the formation and/or maintenance of luteal structures, which appear at approximately day 40 of gestation (12, 154). These are known as the secondary corpora lutea and are derived from ovulations or from luteinization of ovarian follicles that develop between days 17 and 50 of gestation (6, 155, 156, 157). Both the primary and secondary corpora lutea persist to days 160-180 of gestation (6,156), and both continue to secrete progesterone during this period (157). The follicles which are the precursors of the secondary corpora lutea develop well in advance of the invasion of the endometrium by the chorionic girdle cells, indicating that this folliculogenesis is independent of CG (158, 159). However, the secondary corpora lutea form and levels of progesterone increase soon after the first appearance



February, 1991

of CG in serum, findings which have resulted in the suggestion that the placental gonadotropin is responsible for ovulation or luteinization of follicles (160, 161). Further, secondary corpora lutea were not found in bred mares that underwent hysterectomy, which led Squires et al. (156) to conclude that CG contributes to ovulation and the maintenance of these corpora lutea. Horse CG can induce ovulation in nonequid species (162). Levels of CG in the mare at day 39 are in the range of 1-5 Mg/ml serum (63) and levels of LH associated with the preovulatory surge range from 5-130 ng/ml (6). CG levels in the horse are therefore likely to be sufficient to induce ovulation in the horse in spite of the fact that CG has lower receptor binding activity to horse gonads relative to horse LH (70, 107-109). The elevations in progesterone concentrations in circulation of the mare, which have been associated with the presence of CG (109,160), suggest a luteotrophic role for CG in maintenance of the primary and secondary corpora lutea. In support of this hypothesis, CG will stimulate progesterone production in vitro in slices of primary corpora lutea and secondary corpora lutea derived from both ovulated and unovulated follicles (160). Further, dose-dependent enhancement of progesterone occurs in equine granulosa cells incubated with horse CG (P. F. Flood and K. J. Betteridge, unpublished data and Fig. 7), in spite of the assertion that CG may not be luteotrophic in the horse corpus luteum (95). The role and necessity of CG in equid pregnancy have been inferred from interspecies pregnancies and reciprocal transfer of donkey and horse embryos (92, 95). 1000

100 CD

2 to O)

o

10

m

0

0.128

0.32

0.8

2.0

5.0

eCG concentration (i.u./m|) FlG. 7. Progesterone accumulation by collagenase-dissociated horse luteal cells (1.0 x 106 per well) in the presence of horse CG. Cells were incubated for 4 h under an atmosphere of 95% CO2-5% CO2 and progesterone measured by RIA. Note logarithmic y axis. (Flood, P. F., and K. G. Betteridge, unpublished observation).

39

Because of the small sample sizes and numerous treatments and conditions, the information, for the most part, must be regarded as anecdotal. Hinny pregnancies (resulting from stallion bred to donkey) and pregnancies resulting from transfer of horse embryos to the donkey uterus are characterized by ample endometrial cup tissue and CG production far in excess of that present in donkey pregnancy (92, 95,161). Progesterone concentrations are also elevated in hinnies (92, 95, 161), as is the total amount of luteal tissue (109), suggestive of an effect of CG on the formation and maintenance of secondary corpora lutea. In a study with reasonable sample size (n = 6-30), mule pregnancies (donkey jack bred to mare) were characterized by low levels of CG, while progestagen levels did not differ from intraspecific pregnancies in the mare or donkey (109). In that study, progestagen elevations at the time of inception of CG production suggested that CG is involved in the establishment and/or maintenance of the secondary luteal tissue (109). In support of the view that CG may not be necessary to equine pregnancy, donkey embryos transferred to uteri of four horses did not produce detectable CG, and all but one aborted (95). Administration of varying doses of horse CG to a few mares at varying times after transfer of a donkey embryo did not result in increases in circulating progesterone, or obviate abortion (92, 95). This result is difficult to interpret because some animals were also treated with synthetic progestins, with no apparent effect. Allen and co-workers (92, 95,163) concluded that CG plays a minor or no role as a luteotropin in mare gestation, and based on parturitions of horse embryos transplanted to the donkey uterus, that CG may not be necessary. However, since abortions of donkey embryos from the horse uterus were due, at least in part, to a failure of placental formation (92, 163), the validity of the model is questionable. It has not been determined whether the secondary corpora lutea are necessary for the support of gestation in the horse. Holtan et al. (164) reported that pregnancy continued in some mares ovariectomized as early as day 50 of gestation. The equine uterus and placenta contribute progesterone to the circulation beginning by at least day 80 of gestation (164). From the aggregate of available information, it is concluded that the role of CG in equids is to induce ovulation or luteinization of the secondary corpora lutea and to serve as a luteotropin to the primary and secondary corpora lutea. CG and the consequent ovarian structures may serve as a redundant system to maintain pregnancy until placental support is sufficient to do so and may be essential to pregnancy in a proportion of horses.


MURPHY AND MARTINUK

40

VII. Summary Cells from the chorionic girdle of the equine trophoblast invade the maternal endometrium at day 36 of gestation and become established as secretory elements known as the endometrial cups. These structures, which persist for 40-60 days, produce a gonadotropin which can be found in circulation until about day 130 of gestation. This glycoprotein has been identified in the horse and the donkey, with the former having received much better characterization. It consists of 2 noncovalently linked peptide chains; an a-subunit of 96 amino acids, which is common to that found in other horse glycoprotein hormones. The /3-subunit of 149 amino acids is identical to horse LH/3. Horse CG is the most heavily glycosylated of the known pituitary and placental glycoprotein hormones. The a-subunit has two and the /3-subunit one N-linked glycosylation site, and the 0-chain has in excess of four O-linked glycosylation sites. The N-linked glycans have some oligosaccharides that are not found on other glycoprotein hormones. The sialic component of glycosylation confers an exceptionally long half-life on CG compared to other glycoprotein hormones. Horse CG has LH-like activity in horse receptor and in vitro bioassays. In spite of the amino acid homology, it has lower LH activity than does horse LH. Its most intriguing, and as yet unexplained, characteristic is its pronounced FSH and LH activity in species other than the horse. Horse CG binds to FSH receptors of virtually all mammalian species, other than the horse, in which it has been tested and will produce biological effects peculiar to FSH. It has similar and potent interaction with LH receptors. The structural basis of this duality is not known but may be related to the region 90-110 of the j8chain. Horse CG is believed to be constitutively expressed by the trophoblastic cells until the endometrial cups degenerate. The role of CG in equine gestation is not completely understood. It is believed to act as an LH-like hormone to induce supplementary ovulation and/or luteinization of follicles in the mare. It has not been established whether CG or the accessory corpora lutea are necessary for successful horse pregnancy. They may serve as a redundant system to assure that there is sufficient secretion of the primary corpus luteum to maintain pregnancy until the placenta assumes its role as the principal steroidogenic organ of gestation. Acknowledgments We are grateful to the following for their critical review of and helpful comments on the manuscript: Drs. J. U. Baenziger, P. F. Flood, W. R. Moyle, and M. Redmond. We thank Drs. P. F. Flood and K. J. Betteridge for access to their unpublished results, Dr. X-M. Li for aid with the figures, Dr. M. Redmond for computer analysis of CG struc-

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ture, and L. Godart for her extensive work in preparation of the manuscript.

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136. Santa Coloma TA, Reichert Jr LE 1990 Identification of a folliclestimulating hormone receptor-binding region in hFSH-/3-(8l-95) using synthetic peptides. J Biol Chem 265:5037 137. Vakharia DD, Dias JA, Thakur AN, Anderson TT, O'Shea A 1990 Mapping of an assembled epitope of human follicle-stimulating hormone-/? using monoclonal antibodies, synthetic peptides and hormone-receptor inhibition. Endocrinology 127:658 138. Moyle WR, Matzuk MM, Campbell RK, Cogliani E, Dean-Emig DM, Krichevsky A, Barnett RW, Boime I 1990 Localization of residues that confer antibody binding specificity using human chorionic gonadotropin/luteinizing hormone /? subunit chimeras and mutants. J Biol Chem 265:8511 139. Keutmann HT, Hua QX, Weiss MA. Spectroscopy of a receptorbinding sequence in hLH/hCG beta subunit. Proceedings of the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990 (Abstract) 140. Schneyer AL, Sluss PM, Huston JS, Ridge RJ, Reichert Jr LE 1988 Identification of a receptor binding region on the /3 subunit of human follicle-stimulating hormone. Biochemistry 27:666 141. Santa Coloma TA, Dattatreyamurty B, Reichert Jr LE 1990 A synthetic peptide corresponding to human FSH /3-subunit 33-53 binds to FSH receptor, stimulates basal estradiol biosynthesis, and is a partial antagonist of FSH. Biochemistry 29:1194 142. Campbell RK, Moyle WR, Use of synthetic genes to characterize glycoprotein hormone structure and function. Proceedings of the 71st Annual Meeting of the Endocrine Society, Seattle, WA, 1989, (Abstract) 143. Chen F, El-Deiry S, Puett D Site-directed mutagenesis, expression, and characterization of human chorionic gonadotropin-beta. Proceedings of the 72nd Annual Meeting of the Endocrine Society, Atlanta, GA, 1990 (Abstract) 144. Catchpole HR, Cole HH, Pearson PG 1935 Studies of the rate of disappearance and fate of mare gonadotropic hormone following intravenous injection. Am J Physiol 112:21 145. Cole-HH, Bigelow M, Finkel J, Rupp GR 1967 Biological half-life of endogenous PMS following hysterectomy and studies on losses in urine and milk. Endocrinology 81:927 146. Roser JF, Le T, Lofstedt RL 1988 The presence of an eCG-like molecule in the urine of pregnant mares. Biol Reprod [Suppl 1] 38:139 147. Roser JF, Lofstedt RM 1989 Urinary eCG patterns in the mare during pregnancy. Theriogenology 32:607 148. Mclntosh JEA, Moor RM, Allen WR 1975 Pregnant mare serum gonadotrophin: rate of clearance from the circulation of sheep. J Reprod Fertil 44:95 149. Aggarwal BB, Papkoff H 1981 Studies on the disappearance of equine chorionic gonadotropin from the circulation in the rat: tissue uptake and degradation. Endocrinology 109:1242 150. Schams D, Menzer C, Schallenberger E, Hoffmann B, Hahn J, Hahn R 1978 Some studies on pregnant mare serum gonadotropin and on endocrine responses after application for superovulation in cattle. In: Sreenan JM (ed) Control of Reproduction in the Cow. Martinus Nijhoff, The Hague, p 122 151. Aggarwal BB, Papkoff H 1985 Plasma clearance and tissue uptake of native and desialylated equine gonadotropins. Domest Anim Endocrinol 2:173 152. Liu L, Southers JL, Cassels Jr JW, Banks SM, Wehmann RE, Blithe DL, Chen H-C, Nisula BC 1989 Structure-kinetic relationships of choriogonadotropin and related molecules. Am J Physiol 256:E721 153. Weiss P, Ashwell G 1989 The asialoglycoprotein receptor: properties and modulation by ligand. Prog Clin Biol Res 300:169 154. Amoroso EC, Hancock JL, Rowlands IW 1948 Ovarian activity in the pregnant mare. Nature 161:159 155. Van Rensburg SJ, van Neikerk CH 1968 Ovarian function, follicular estradiol 17/? and luteal progesterone and 20a-hydroxy-pregn4-3-one in cycling and pregnant equines. Onderstepoort J Vet Res 35:301 156. Squires EL, Garcia MC, Ginther OJ 1974 Effects of pregnancy and hysterectomy on the ovaries of pony mares. J Anim Sci 38:823


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157. Squires EL, Ginther OJ 1975 Collection technique and progesterone concentration of ovarian and uterine venous blood in mares. J Anim Sci 40:275 158. Allen WE 1975 Ovarian changes during early pregnancy in pony mares in relation to PMSG production. J Reprod Fertil [Suppl] 23:425 159. Evans MJ, Irvine CHG 1975 Serum concentrations of FSH, LH and progesterone during the oestrous cycle and early pregnancy in the mare. J Reprod Fertil [Suppl] 23:193 160. Squires EL, Stevens WB, Pickett BW, Nett TM 1979 Role of pregnant mare serum gonadotropin in luteal function of pregnant mares. Am J Vet Res 40:889

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161. Urwin VE, Allen WR 1982 Pituitary and chorionic gonadotrophic control of ovarian function during early pregnancy in equids. J Reprod Fertil [Suppl] 32:371 162. Murphy BD, Hunter DB, Onderka DK, Hazlewood J 1987 Use of equine chorionic gonadotrophin in female mink. Theriogenology 28:667 163. Allen WR, Kydd JH, Antczak DF 1990 Xenogenic donkey-inhorse pregnancy created by embryo transfer: immunological aspects of a model of early abortion. In: Choauat G (ed) Immunology of the Fetus. CRC Press, Boca Raton, p 267 164. Holtan DW, Squires EL, Lupin DR, Ginther OJ 1979 Effect of ovariectomy on pregnancy in mares. J Reprod Fertil [Suppl] 27:457

2nd International Conference on New Actions of Parathyroid Hormone Pisa, Italy, May 22-25, 1991 Scientific Committee: M. L. Brandi (Italy), T. Fujita (Japan), C. Gennari (Italy), R. D. Hesch (Germany), S. G. Massry (USA), D. A. McCarron (USA), H. Mori (Japan), A. Pinchera (Italy), M. Rosenblatt (USA). The program will focus on molecular and biological aspects of the effects of PTH on: aging, cardiovascular system, endocrine system, hematopoietic system, immune system, kidney, nervous system, neurotransmitters, and pain. It will also feature presentations on the mechanisms and processing of synthesis, secretion, and metabolism of PTH and PTH-related protein. The final program will include lectures by invited speakers, selected oral communications and poster presentations. The deadline for submission of abstracts is March 1st, 1991. For further information please contact: Dr. Claudio Marcocci, Istituto di Endocrinologia, Universita di Pisa Viale del Tirreno 64, 56018 Tirrenia-Pisa, Italy Tel: 50-33274. Fax: 50-33433


Ovarian stimulation with human chorionic gonadotropin and equine chorionic gonadotropin affects prostacyclin and its receptor expression in the porcine oviduct.

Physical-chemical and biological characterization of different preparations of equine chorionic gonadotropin.

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

Induction of follicular luteinization by equine chorionic gonadotropin in cyclic guinea pigs.

PMSG): antigenic determinants on alpha- and beta-subunits.

Effects of equine chorionic gonadotropin on reproductive performance in anestrous mink.

Structure-function relationships during preovulatory development of porcine follicles following equine chorionic gonadotropin stimulation.

Luteoprotective role of equine chorionic gonadotropin (eCG) during pregnancy in the mare.

Determination of human chorionic gonadotropin.

Free β-human chorionic gonadotropin, total human chorionic gonadotropin and maternal risk of breast cancer.

Feedback mechanism of human chorionic gonadotropin secretion.

Carboxypeptidase digestion of human chorionic gonadotropin.

Serum-chorionic-gonadotropin assay and ectopic pregnancy.

Ectopic human chorionic gonadotropin in breast carcinoma.

Beta-human Chorionic Gonadotropin-secreting Lung Adenocarcinoma.

Zebra chorionic gonadotropin: partial purification and characterization.

A chorionic gonadotropin-secreting human pituitary cell.

Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high-gradient magnetic separation.

Equine Chorionic Gonadotropin Modulates the Expression of Genes Related to the Structure and Function of the Bovine Corpus Luteum.

Beta-human Chorionic Gonadotropin-producing Renal Cell Carcinoma.

Human chorionic gonadotropin and ovarian and placental steroidogenesis.

[Ectopic production of chorionic gonadotropin in apudomas (author's transl)].

Tumor necrosis factor-alpha inhibits human chorionic gonadotropin secretion.

Ovarian clear-cell carcinoma producing estradiol and human chorionic gonadotropin.