A Brief Review of Luteinizing Hormone (LH, Lutropin) and

Shibayagis Rat LH ELISA KIT

Katsumi WAKABAYASHI, Ph. D.
Professor emer. Gunma University
Technical Consultant, Shibayagi Co., Ltd.
(090106)
Luteinizing hormone (LH) is also called Lutropin and Interstitial Cell Stimulating
Hormone, ICSH) from its role in males. Luteinization is limited to female mammals, however,
the name LH is used for wide range of animal species, as well as males.

What is LH?

LH is produced and stored in basophilic cells called gonadotrophs in the anterior pituitary
gland. LH is found in all vertebrates from fishes to mammals. The content of LH in rat
anterior pituitary gland is 6~7g(as NIH-LH S1)/gland in males, and 3~4 g/gland in females.
In females, the content of LH changes during the sexual cycle. Some reports indicated the
presence of LH-like substance in the testis.
LH has a heterodimer structure consisted of -subunit and -subunit with a total molecular
weight of approximately 29,000. -Subunit is a glycoprotein which is common to other
pituitary hormones follicle-stimulating hormone (FSH) and thyroid-stimulating hormone
(TSH), while -subunit is specific to LH, and is also a glycoprotein.

Amino acid sequence of rat LH

-Subunit has two oligosaccharide chains and -subunit has one chain bound to asparagine
moieties. There are many disulfide bonds within both subunits, keeping their rigid
three-dimensional structure. There precursor structures are shown below. Signal peptide
portions are shown with red characters.
-Subunit
mdcyrryaav ilvmlsmvlh ilhslpdgdl iiqgcpeckl kenkyfsklg apiyqcmgcc
fsrayptpar skktmlvpkn itseatccva ksftkatvmg narvenhtdc hcstcyyhks
Glycosylation
Glycosylation
Disulfide bond: 35-59, 38-88, 56-110, 60-112, and 87-115
-Subunit
merlqglllw lllspsvvwa srgplrplcr pvnatlaaen efcpvcitft tsicagycps
Glycosylation
mvrvlpaalp pvpqpvctyr elrfasvrlp gcppgvdpiv sfpvalscrc gpcrlsssdc
ggprtqpmtc dlphlpglll f
Disulfide bond: 29-77, 43-92, 46-130, 54-108, 58-110, and 113-120

Microheterogeneity of LH molecule

LH shows microheterogeneity deriving from oligosaccharide chains, and from the difference
of the number of sialic acids locating at the end of the chains, seven isomers or components
having different isoelectric points are present in rat pituitary gland (1,2). These components
reflect various stages of processing to the final intake of sialic acid at the end of each chain.
These components are also different in the affinity with the receptor, so that are different in
the potency to cause the androgen production in the interstitial cells (Leidig cells) in vitro.
Components with basic pIs show high potency, and those with low pIs owing to the addition

of sialic acid moieties show lower potencies with more sialic acid moieties.
The results of the observation on the pIs and relative potencies are shown below (3).
Isoelectric components
A
A
B
C
D
E
F

pI
7.9
8.4
8.8
9.1
9.3
9.6
9.8

Biological potencies*
0.133 (0.113-0.153)
0.212 (0.188-0.243)
0.403 (0.328-0.485)
0.460 (0.428-0.498)
0.613 (0.523-0.715)
0.978 (0.883-1.085)
0.868 (0.760-0.998)

*Expressed as relative potency to NIHLHS1, with 95% confidence limits

Potencies were estimated from the amounts of testosterone produced after incubation with
rat isolated Leidig cell preparation for one hour.
The components with highly basic pIs (D, E, F) occupies nearly 50% of total LH in the
female pituitary gland (about 20% in males), so the in vitro biological potency female pituitary
LH is about 4 times that of male LH. The LH in castrated male pituitary shows only 1/8 of
that of female pituitary LH. In females, because of LH surge taking place in every 4-5 days,
the pituitary gonadotrophs become almost empty, and de novo biosynthesis of LH is always
carried out. The fresh LH components have less sialic acid, and have basic pIs (3).
We also found that the addition of sialic acid moieties caused elongation of biological half
lives of the LH components. So the order of potencies obtained by in vivo androgen
productions after intravenous injection of various component were just opposite compared
with the order obtained by in vitro assay procedure (4).

Physiological action of LH

The receptor of LH is a transmembrane receptor of G-protein coupled type, which relates to

PKA system and penetrates the cell membrane 7 times.
In females, LH acts on maturated granulosa cells in ovarian follicle, where it causes, in
cooperation with FSH, follicular maturation, estrogen production, and ovulation, and after
ovulation, acts on corpora lutea and promotes progesterone production and secretion.
In males, LH acts on the interstitial cells (Leidig cells) in the testis, causing production and
secretion of androgens, and secondarily promotes spermatogenesis via androgen.
Deficiency of LH leads to the lower secretion of sex steroids, atrophy of interstitial cells, and
failure of ovulation and luteinization, while excessive LH causes hyperplasia of testicular
interstitial cells followed by atrophy, increased secretion of estrogen or androgen,
super-ovulation, and accelerated sexual maturation.
Human diseases showing low blood LH levels: hypo-gonadotropic eunuchoidism, primary
central amenorrhea, Sheehan syndrome, Chiari-Frommel syndrome, anorexia nervosa,
granulosa-cell tumor, adrenogenital syndrome, luteal dysfunction, craniopharyngioma, etc.
Diseases showing high blood LH levels: azospermia, Kleinfelter syndrome, Turner
syndrome, premature adolescence, premature menopause, polycistic ovary syndrome (Stein
Leventhal syndrome), ovarian growth and developmental disorder, castration, menopause,
etc.

Bioassay of LH

Biological potencies of LH preparations have been assayed originally with ovulation

induction and number of ova as markers, and later, various methods were developed as shown
below.
Ventral prostate weight method (VPW ), with the increase of ventral prostate weight in
immature male rats as marker. Greep, R.O. et al. PSEBM, 46: 644-649, 1941
Ovarian ascorbic acid depletion method (OAAD) with the decrease of ascorbic acid content
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in PMS-HCG pretreated immature rat ovary as a marker. Parlow, A. F. in Human Pituitary

Gonadotropin (Albert , A. ed.) pp.300-310, Charles C. Thomas, Springfield, 1961.
(Also described in Experimental Endocrinology A Sourcebook of Basic Techniques, by
Zarrow, M., X., Yochim, J. M. McCarthy, J. L., Academic Press, NY. 1964.
Dufau, M.L., Mendelson, C. and Catt, K.J. published an in vitro assay method ( J Clin
Endocrinol Metab, 39: 610-613) with testosterone production by isolated rat testicular
interstitial cells as a marker.
The potencies obtained for a LH preparation by different assay methods are not always
same. The reason is that LH is a mixture of molecules with different number of sialic acid
moieties, which influences the biological half life. Especially with in vivo assay, the time
period from injection of LH preparation until getting the change of the final marker relates to
the potencies obtained. Let me show an example comparing potencies of modified hCG (same
biological action to LH) obtained by OAAD and VPW.
Comparison of assay results for hCG after partial removal of sialic acid moietiesVan Hall
et al. Endocrinology 88, 456, 1971
Sialic
acid
PotencyIU/mg
removed
OAAD
VPW
RIA

0
11,290
11,740
4,430
47
1,250
220
6,020
70
86
0.7
4,100
100
56
1.2
4,830
OAAD method takes 2 hours until taking out of ovaries, while VPW needs 3 days until
prostate weight measurement because the injected hormone acts first on testicular
interstitial cells to secrete androgens which increase the weight of the ventral prostate. In the
case of RIA, the antibody recognizes only the peptide part.

Radio-receptor assay of LH

LH can be measured also by radio-receptor assay (RRA) that located between bioassay and
immunoassay. As the source of receptor for LH, luteinized ovary obtained by treating with
PMS and hCG, or isolated testicular interstitial cells (Leidig cells) are used. Assay is carried
out based on competitive binding principle, i.e., the competition between radioiodinated LH
and non-radioactive LH in assay sample resulting in the decease of receptor-bound
radioactivity (Lee, S.E., and Ryan, R.J. Receptors for Reproductive Hormones, pp.419-430,
OMalley, B.W. and Means, A. R.(eds), Plenum Press, NY, 1973.).
Binding affinity relates, to some extent, to biological activity. So, RRA may reflect biological
potency than immunoassays. But binding to receptor does not directly relate to biological
action. For example, deglycosylated hCG binds very strongly to LH receptor, however, shows
very low activity, and plays almost as competitive inhibitor of hCG or LH (5).

Immunoassay of LH

Radioimmunoassay (RIA) kits for LH of various animal species have been provided by
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and commercial
immunoassay kits are available for human diagnostic use. In the case of NIDDK kits, a
purified preparation for radioiodine labeling (I series), antiserum (S series) and standard (RP
series) have been provided, and researchers have to radioiodinate the preparation, and set up
the RIA system by themselves.

LH standard preparations and international unit

International standard preparations of human LH
WHO and National Institute for Biological Standards and Control (NIBSC) has been
providing human LH international standard. Early preparations were not LH itself but so
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called HMG that is present in the urine of postmenopausal women derived from the pituitary
and modified by kidney. 1st IRP-HMG (HMG-24) was issued though its purity was not enough.
2nd IRP (Pergonal) was bottled with 9mg of lactose, and defined that one vial contains 40IU of
LH and 20IU of FSH because HMG was consisted of both LH and FSH. Originally it was
intended for bioassay use, but RIA kits that used this preparation as the assay standard were
commercially produced and distributed. So, in clinical diagnosis, the assay results were
expressed in terms of IU-2nd IRP. Later, NIDDK issued human LH assay kit for RIA using
LH purified from pituitary extract as the standard preparation, LER907 (48IU/mg). Both
NIDDK and commercial kits used anti-human pituitary LH. As the ratio of biological potency
to immunological potency was different between HMG and pituitary LH, the assay value of
blood LH expressed as IU with commercial kit was about 5-times higher than that obtained
with LER907. This made some trouble among researchers until commercial kits switched the
standard preparation from 2nd IRP-HMG to pituitary LH 2nd International Standard 1988
(80/552).
International standards for LH subunits have been also available.
LH- subunit: 1st International standard 1984 (78/556)
subunit: 1st International standard 1984 (78/554)
International standard preparations of animal LH
NIH-LH S1 (ovine origin) is the first biological standard preparation of animal LH, and 1
mg was defined as 1 NIH Unit. Providing succeeding lots like S-2, S-3, and so on has
continued this standard preparation. Each lot has nearly the same potency.
Standard preparations have been attached to RIA kits for various species of animals. For
rat LH, NIDDK rat LH RP-1 was provided in during early period of NIDDK RIA kit for rat.
But this standard preparation was so crude, and its potency was only 0.03 NIH Unit/mg. In
such case, the assay results should be expressed in terms of NIH Unit, however in many
reports the results were expressed as the weight of RP-1. We have to be careful in reading
such reports.
Later, for rat LH RIA, RP-2 and RP-3 were provided in place of RP-1, and those preparation
were consisted of very pure LH (same to I-series), protecting inert protein and buffer
components, and weight of the pure LH is shown on the label. We can constitute standard
solution by simply add purified water.
The potency of RP-3 is the same to I-9, and 0.9 x NIH-LH-S1 which corresponds to
888IU/mg of human 2nd IRP HMG according to the instruction paper for NIDDK rat LH RIA
kit, 19-Jan-95, by A. F. Parlow.

Blood levels of LH
Factors promoting LH secretion
LHRH
Physiologically, lowered sex steroids blood levels, sexual cycle especially positive feedback
of estrogen at preovulatory period causing LH surge via LHRH and leading to ovulation,
postmenopausal period, aging in men.
Factors suppressing LH secretion
Increased sex steroids blood levels, opioid peptides especially endorphin, childhood,
pregnancy, childbed.
Human
Adult males: ~3mIU/ml( WHO 1st IRP-LH)
Females:
Ovulatory period ~14mIU/ml, Follicular period ~4mIU/ml, Luteal period ~2.5mIU/ml
Menopausal ~18mIU/ml

From here on, the author will show his observation made in his laboratory (Hormone Assay
Center, Institute of Endocrinology, Gunma University)
Serum LH levels in healthy human subjects in relation to age (6)
Age
05
6 10
11 15
16 20
21 30
31 40
41 50
51 60
61 70
71
16 40
51
Aged women
51 75

Number
18
20
14
14
26
23
17
17
24
17
63
58
30

Serum LH ngxLER907/ml
Mean
SE
6.3
1.2
9.0
1.4
12.9
1.8
16.0
1.8
23.9
1.8
21.4
2.3
32.5
4.7
57.4
5.5
69.87
11.1
60.9
6.0
22.3
1.4
63.4
6.2
163.0

11.5

Serum FSH ngx(LER907)/ml

Mean
SE
14.6
4.0
28.9
4.4
84.5
7.9
71.2
7.3
80.6
5.4
102.2
7.2
156.3
19.8
242.3
33.7
368.5
42.0
297.9
22.3
89.5
4.2
336.1
33.4
1,138.5

75.1

LER907: LH preparation extracted and purified from human pituitaries by Dr. L. E.

Reichert. It was provided by NIDDK as the standard preparation for human LH RIA kit.
The biological potency is 48IU/mg.
Rat
Males: ~0.5ng/ml
Females: nearly the same to males except in the LH surge on proestrous day.
Recommended dilution for supernatant of pituitary homogenate (if the range of the assay
system is 0.25~32ng/ml)
Mature male: Homogenize one pituitary with 1ml buffer. Freeze and thaw once, then
centrifuge. The supernatant should diluted to 1,000~2,000x for assay.
Mature female: 400~500x
Serum LH and testosterone levels of male rats during sexual maturation (meanSE) (7)
Age
Number Serum T
Serum LH
Seminal vesicles
Ventral prostate
(weeks)
(ng/ml)
(ng/ml)
(mg)
(mg)
0.610.02
6.540.37
12.641.43
3
5 0.050.01
0.320.05
23.101.87
32.102.98
5
5 0.190.03
0.480.07
153.8022.10
81.026.60
7
5 1.340.21
10
5 1.280.20
0.420.12
626.0622.65
182.3012.34
32
5 1.540.27
0.380.09
1427.5040.96
242.7822.47
At 3 weeks of age, blood testosterone (T) levels are very low, and this makes LH
comparatively high LH levels. Maybe at this age the responsiveness of Leidig cells to LH is
low, and do not produce enough T to suppress LH secretion.
We examined threshold of LH suppressing effect of T by hypodermic implantation of
various amounts of T sealed in silicone tubing to castrated rats of various ages immediately
after the operation, and checked LH and T levels one week later, and prepared a graph
showing the relationship between logarithmic concentration of serum T and LH decreasing
percentage. The X-intercept shows the threshold while the slope indicates responsiveness.
Threshold and responsiveness were calculated for each age group, and results are shown in
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the figures below. The threshold was very low at 3 weeks of age, and then gradually going up,
reaching maximum at 7 weeks of age, and then decrease. Responsiveness, on the other hand,
was rather high at 3 weeks of age and gradually lowered until 7 weeks of age then increase.
It is suggested that while sexual maturation proceed, low responsiveness and high
threshold allow the high blood level of T under loose negative feedback action. After the
sexual maturation, negative feedback system works effectively under low negative feedback
threshold and high responsiveness.
Responsiveness of LH decreasing effect caused
by testosterone in castrated rats
120
LH-decreasing ratio/Logserum T)

Serum testosterone, ng/ml

Threshold of LH decreasing effect caused by

testosterone in castrated rats in relation to age
0.4

0.3

0.2

0.1

0
3

7
Age in weeks

10

100
80
60
40
20
0

32

7
10
Age in weeks

32

How rapid does pituitary-testicular system work?

Intravenous testosterone injection to castrated male rats decreased the blood LH level to
50% within 5 minutes.
Intravenous injection of LH doubled the blood level of testosterone about 10minutes later.

Proestrous LH surge and ovulation

In female rats, a large amount of LH is released on the afternoon of proestrus, so-called LH
surge. The blood levels show the peak around 15:00 ~18:00. Around 13:00 of proestrous day is
called critical period, and if rats are anesthetized by barbiturates for 1-2 hours, the LH surge
and ovulation is cancelled. The surge will be postponed until the next day. Not only LH surge
but also prolactin surge is delayed. (Unpublished data)

Changes in serum LH levels and pituitary LH contents after orchidectomy

Male rats of Wistar strain were orchidectomized at 8 weeks of age, and serum LH and
pituitary LH during 6-24 hours (6 rats) and 1-42 days (5 rats) after the operation. LH was
measured using NIDDK rat LH RIA kit. Results are expressed as NIH-LH-S1 equivalent.
Mean and SE are shown. (Unpublished data)
S erum L H levels

8
7
6
5
4
3
2
1
0

LH x S1, ng/ml

LH x S1, ng/ml

Serum LH levels
4
3
2
1
0
0

14

21

28

35

42

Days after castration

12

24

Hours after castration

Pituitary LH content

Pituitary LH content
20

60

LH x S1, mg/pit

LH x S1, g/pit

50
40
30
20
10
0

15
10
5
0

14

21

28

35

42

Days after castration

12

Hours after castration

24

Blood LH levels showed only a slight increase after 6 hours, however, significantly
increased after 12 hours, and showed further increase after 24 hours. Increased LH levels
went lower after 3 days and lowest levels were seen after 7 days, then the levels increased and
stayed nearly the same levels to those of after 24 hours.
The pituitary LH contents showed the minimum levels after 3 days, and then continued to
increase until 35 days when the levels reach a plateau.

References

1) Wakabayashi, K. (1977)
Heterogeneity of rat luteinizing hormone revealed by radioimmunoassy and electrofocusing
studies.
Endocrinol. Japon. 24: 473-485

2)Hattori, M-A., Ozawa, K., and Wakabayashi, K. (1985)

Sialic acid moiety is responsible for the charge heterogeneity and the biological potency of
rat lutropin.
Biochem. Biophys. Res. Commun. 127:501-508
3) Hattori, M-A.., Sakamoto, K., and Wakabayashi, K. (1983)
The presence of LH components having different ratios of bioactivity to immunoreactivity
In the rat pituitary glands.
Endocrinol. Japon. 30: 289-296.
4) Shioya, N. and Wakabayashi, K. (1998)
In vivo bioactivities and kinetic parameters of rat luteinizing hormone components:
Discrepancy between in vitro and in vivo assays.
Endocrine J 45: 307-314
5) Hattori, M., Hachisu, T., Shimohigashi, Y., and Wakabayashi, K. (1988)
Conformation of the subunit of deglycosylated human chorionic gonadotropin in the interaction
at receptor sites.
Mol. Cell. Endocrinol., 57, 17-23

6) Isurugi, K., Fukutani, K., Takayasu, H., Wakabayashi, K. and Tamaoki, B. (1974)
Age-related changes in serum luteinizing hormone (LH) and follicle-stimulating hormone
(FSH) levels in normal men.
J. Clin. Endocrinol. Metab. 39: 955-957
7) Takikawa, M. and Wakabayashi, K. (1994)
Quantitative analysis of hypothalamic-hypophyseal-testicular system: Why testosterone
can act under negative feedback control.
Endocrine J. 41: 257-265
8) Chiba, K., Kobayashi, H., and Wakabayashi, K. (1997)
Isolation and partial characterization of LH, FSH and TSH from canine pituitary gland.
Endocrine Journal, 44:205-218

Amino acid sequence of LH in various animals

LH subunit
Rat (P11962)
mdcyrryaav ilvmlsmvlh ilhslpdgdl iiqgcpeckl kenkyfsklg apiyqcmgcc
fsrayptpar skktmlvpkn itseatccva ksftkatvmg narvenhtdc hcstcyyhks
Mouse (P01216)
mdyyrkyaav ilvmlsmflh ilhslpdgdf iiqgcpeckl kenkyfsklg apiyqcmgcc
fsrayptpar skktmlvpkn itseatccva kaftkatvmg narvenhtec hcstcyyhks
Human (P01215)
mdyyrkyaai flvtlsvflh vlhsapdvqd cpectlqenp ffsqpgapil qcmgccfsra
yptplrskkt mlvqknvtse stccvaksyn rvtvmggfkv enhtachcst cyyhks
Sheep (P01218)
mdyyrkyaaa ilailslflq ilhsfpdgef tmqgcpeckl kenkyfskpd apiyqcmgcc
fsrayptpar skktmlvpkn itseatccva kaftkatvmg nvrvenhtec hcstcyyhks
Dog (Q9XSW8)
mdcyrkyaav ilaalsvflh ilhsfpdgef tmqgcpeckl kenkyfsklg apiyqcmgcc
fsrayptpar skktmlvpkn itseatccva kaftkatvmg nakvenhtec hcstcyyhks
Cat (Q52R91)
mdyyrkyaav ilailsvflh ilhsfpdgef tmqgcpeckl kenkyfsklg apiyqcmgcc
fsrayptpar skktmlvpkn itseatccva kaftkatvmg nakvenhtec hcstcyhhki

LH subunit
Rat
Isomer 1 (NP036990)
merlqglllw lllspsvvwa srgplrplcr pvnatlaaen efcpvcitft tsicagycps
mvrvlpaalp pvpqpvctyr elrfasvrlp gcppgvdpiv sfpvalscrc gpcrlsssdc
ggprtqpmtc dlphlpglll f
Isomer 2 (NP001029147)
msqglllwll lspsvvwasr gplrplcrpv natlaaenef cpvcitftts icagycpsmv
rvlpaalppv pqpvctyrel rfasvrlpgc ppgvdpivsf pvalscrcgp crlsssdcgg
prtqpmtcdl phlpglllf
Isomer 2 has essentially same amino acid sequence except that 2 amino acids in signal
peptide region are lacking. So, the mature peptide of LH of isomer 2 has same amino acid
sequence to isomer 1.
Chin,W.W., Godine,J.E., Klein,D.R., Chang,A.S., Tan,L.K. and Habener,J.F.
Nucleotide sequence of the cDNA encoding the precursor of the beta subunit of rat lutropin

Proc. Natl. Acad. Sci. U.S.A. 80 (15), 4649-4653 (1983)

PUBMED

6192440

Mouse (O09108)
merlqglllw lllspsvvwa srgplrplcr pvnatlaaen efcpvcitft tsicagycps
mvrvlpaalp pvpqpvctyr elafasvrlp gcppgvdpiv sfpvalscrc gpcrlsssdc
ggprtqpmac dlphlpglll l
Human (P01229)
memlqgllll lllsmggawa sreplrpwch pinailavek egcpvcitvn tticagycpt
mmrvlqavlp plpqvvctyr dvrfesirlp gcprgvdpvv sfpvalscrc gpcrrstsdc

ggpkdhpltc dhpqlsgllf l
Sheep (P01231)
memlqglllw lllgvagvwa srgplrplcq pinatlaaek eacpvcitft tsicagycls
mkrvlpvilp pmpqrvctyh elrfasvrlp gcppgvdpmv sfpvalschc gpcrlsstdc
ggprtqplac dhpplpdilf l
Dog (P18842)
lqglllwlll svggvwasrg plrplcrpin atlaaeneac pvcitfttti cagycpsmvr
vlpaalppvp qpvctyhelh fasirlpgcp pgvdpmvsfp valscrcgpc rlsnsdcggp
raqslacdrp llpgllfl
Cat (O77805)
memlqgllll wllllnvggv wtsreplrpl crpinatlaa eneacpvcvt fttticagyc
psmmrvlpaa lppvpqpvct yrelrfasvr lpgcppgvdp vvsfpvalsc rcgpcrlsss
dcggpraqpl acdrpplpgl lfl
End

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