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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 6 2643-2648
Copyright © 2001 by The Endocrine Society

Original Articles: Hormones and Reproductive Health

The Presence of Functional Luteinizing Hormone/Chorionic Gonadotropin Receptors in Human Sperm

A. Eblen, S. Bao1, Z. M. Lei, S. T. Nakajima and C. V. Rao

Divisions of Reproductive Endocrinology and Infertility (A.E., S.T.N.) and Basic Science Research (S.B., Z.M.L., C.V.R.), Department of Obstetrics and Gynecology, University of Louisville Health Sciences Center, Louisville, Kentucky 40292

Address correspondence and requests for reprints to: Dr. C. V. Rao, Department of Obstetrics and Gynecology, 438 MDR Building, University of Louisville, Health Sciences Center, Louisville, Kentucky 40292. E-mail: cvrao001{at}gwise.louisville.edu

Abstract

The functional receptors that bind human CG (hCG) and LH have recently been identified in a number of nongonadal human tissues. The current experiments tested the hypothesis that human ejaculated sperm may also contain them. The data revealed that they, indeed, do as determined by the presence of receptor messenger RNA and receptor protein that can bind 125I-hCG. The receptors were functional, as indicated by an increase in cyclic AMP levels and activation of sperm protein kinase A following treatment with hCG or LH. Treatment with these hormones, on the other hand, had no effect on sperm protein kinase C activity. Now that the functional LH/hCG receptors are found in human sperm, it is important to determine whether hCG treatment could improve the outcome of infertility procedures.

HUMAN CHORIONIC GONADOTROPIN (hCG) is a heterodimeric glycoprotein hormone primarily produced by human placenta (1). Its structural motif is very similar to nerve growth factor, platelet-derived growth factor-ß, and transforming growth factor-ß, which are members of the cystine-knot growth factor family (2). LH, a structural homolog of hCG, is primarily produced by anterior glands (1). Both hormones, especially hCG, are also produced in small amounts by many other tissues in the body (3, 4, 5, 6, 7, 8, 9, 10, 11). LH and hCG bind to the same receptors that are transmembrane glycoproteins, which belong to the G protein-coupled receptor family (12, 13). The members of the family have the varying lengths of extracellular domain, seven transmembrane regions, and cytoplasmic tail coupled to G-proteins. The LH/hCG receptors are not only present in gonads but also in several nongonadal tissues (14, 15, 16, 17, 18, 19, 20, 21). The nongonadal receptors were investigated by using a wide range of techniques that detect from gene transcription to signaling pathways and ultimately the biological response (14, 15, 16, 17, 18, 19, 20, 21). Nongonadal distribution of LH/hCG receptors is not species specific, as they have been found in human, monkey, rat, rabbit, mice, pig, cow, sheep, and even turkey (14, 15, 16, 17, 18, 19, 20, 21, 22). Because sperm contain steroid and peptide hormone and growth factor receptors (23, 24, 25, 26, 27, 28, 29, 30), it did not seem far-fetched to consider that human sperm may also contain LH/hCG receptors. Moreover, such a possibility was supported by a finding that epididymal rat sperm contain LH/hCG receptors (31). This study was undertaken to investigate whether donor human ejaculated sperm contain functional LH/hCG receptors.

Materials and Methods

Materials

The following were obtained: polyclonal antibody, raised against the synthetic N terminus amino acid sequence of 15–38 of the LH/hCG receptors from Dr. Patrick Roche (Mayo Clinic, Rochester, MN); a HindIII-AspHI 200-1029-bp complementary DNA (cDNA) fragment encoding human LH/hCG receptors in pBSK-SK+ plasmid from Dr. Aaron Hsueh (Stanford University School of Medicine, Palo Alto, CA); and highly purified unlabeled hCG (CR-127; 14,900 IU/mg) and human LH (AFP-0264B) from the National Hormone and Pituitary Program supported by the NIDDK, NICHHD, and USDA (Rockville, MD). The following were purchased: F12 and MEM culture medium from Sigma (St. Louis, MO); a chemiluminescence Western blot detection kit from Amersham Pharmacia Biotech (Arlington Heights, IL); [{alpha}32P] dCTP from NEN Life Science Products (Boston, MA); cAMP enzyme immunoassay kits from Cayman Chemical Co. (Ann Arbor, MI); and Taq DNA polymerase, random primer, and nonradioactive protein kinase A (PKA) and protein kinase C (PKC) activity measurement kits from Promega Corp. (Madison, WI).

Samples

This study was approved by our University Human Studies Committee. Semen samples were obtained from healthy 25- to 32-yr-old graduate, medical, and dental student donors by masturbation. Some of the donors were single and, thus, have not yet fathered children. Informed consent was obtained. The samples were centrifuged for 10 min at 500 x g and washed two times with F12 culture medium. More than 25 individual donor semen samples with normal sperm count, motility, and morphology were used in these studies.

Granulosa cells, which were used as receptor-positive control cells, were collected during oocyte retrieval for in vitro fertilization and embryo transfer in our fertility center. After they were separated from red blood cells by centrifugation for 10 min at 220 x g in 50% Percoll gradient, they were plated in MEM medium containing 10% FCS and incubated overnight in CO2 incubator at 37 C.

RT-PCR

Total RNA was isolated using single-step acid-guanidinium-thiocyanate-chloroform-extraction method (32). Five-microgram RNA aliquots were annealed for 15 min at 65 C with 2.5 µg random primer and reverse transcribed into cDNA for 60 min at 42 C with 15-mM units of avian myeloblastosis virus reverse transcriptase in 10 mM Tris-HCI (pH 9.0) containing 0.1% Triton X-100, 50 mM KCl, 5 mM MgCI2, and 1 mM each dNTP. PCR was performed with 10 µL reverse transcribed cDNA, 2.5 U Taq DNA polymerase, 50 pmol primer pair (forward, 5'-GCAGAAGATGCACAATGGAG-3'; reverse, 5'-CTCTCAGCAAGCATGGAAGA-3') in 10 mM Tris-HCL (pH 9.0) containing 0.1% Triton X-100, 50 mM KCl, 1.5 mM MgCl2, and 0.25 mM each dNTP. The reactions were carried out for 35 cycles, with each consisting of denaturation for 0.5 min at 94 C, annealing for 2 min at 55 C, and extension for 1 min at 72 C. After electrophoresis in 2.5% agarose gels, PCR products were denatured, renatured, and then blotted onto nylon membranes. The 123-bp DNA ladder was used for determining the molecular size of amplified products. The omission of template RNA served as a negative procedural control, and human granulosa cells served as a positive control.

Southern blotting

The PCR-amplified products were transferred to nitrocellulose membranes, which were prehybridized for 4 h at 42 C in 6x SSC, 10x Denhardt’s solution, 0.5% SDS, 50% formamide, and 100 µg/mL sheared salmon sperm DNA. Then, the hybridization was performed overnight at 42 C in the same buffer with LH/hCG receptor cDNA labeled with [32P] dCTP by the random priming method using the Prime-a-Gene Labeling System kit (Promega Corp.). The membranes were then washed first for 45 min at 50 C with 2x SSC containing 0.1% SDS, then with 1x SSC, and finally with 0.1x SSC for 30 min at the same temperature. The washed blots were exposed for 3 h to Kodak XAR-2 film (Eastman Kodak Co., Rochester, NY).

Western blotting

This procedure was performed by homogenizing sperm in 25 mM HEPES buffer, (pH 7.4) containing 10 µg/mL leupeptin and aprotinin and 50 µg/mL 4-(2aminoethyl)-benzenesulfonyl-fluoride. Thirty-microgram protein aliquots were dissolved in loading buffer [125 mM Tris-HCL (pH 6.8) containing 4% SDS and 20% glycerol] and separated by 8% discontinuous SDS-PAGE under reducing conditions. The separated proteins were electroblotted onto Immobilon P membranes (33). After blocking the nonspecific binding sites with 5% nonfat dry milk in 5 mM Tris-HCI (pH 7.4) containing 136 mM NaCI and 0.1% Tween 20, the blots were incubated overnight at 4 C with 1:1000 dilution of the LH/hCG receptor antibody and washed three times 10 min each time with 5 mM Tris-HCI (pH 7.4) containing 136 mM NaCI and 0.1% Tween 20 buffer. The washed blots were incubated again for 1 h at 22 C with a 1:1000 dilution of horseradish peroxidase-labeled second antibody, and then the binding of LH/hCG receptor was detected by an enhanced chemiluminescence detection system. The molecular size of LH/hCG receptor protein was determined by running molecular size marker proteins in an adjacent lane. The primary antibody was preabsorbed with excess receptor peptide in the procedural controls.

Immunocytochemistry

Sperm samples were dried on microscope slides and fixed in Bouin’s solution. The slides were then treated with 0.3% hydrogen peroxide in methanol to block endogenous peroxidase activity. After three washes with phosphate-buffered saline, nonspecific binding sites were blocked with BSA. The samples were incubated overnight at 4 C with a 1:300 dilution of the LH/hCG receptor antibody (34). Then, the sections were washed and incubated with a biotinylated secondary antibody, an avidin-biotin-immunoperoxidase complex, and exposed to diaminobenzidine and hydrogen peroxide. The receptor antibody was preabsorbed with excess receptor peptide for the procedural control.

Covalent receptor cross-linking

Fifty-microgram protein aliquots of sperm homogenates were incubated for 30 min at 37 C with 2 x 106 cpm/mL of 125I-hCG in the presence and absence of 30 µg/mL unlabeled hCG. The hCG was radioiodinated by a lactoperoxidase technique to a specific activity of 80 µCi/µg (35). The samples were then centrifuged at 27,000 x g for 30 min at 4 C, and pellets containing bound 125I-hCG were solubilized in 1% Triton X-100 and separated on 8% SDS-PAGE under nonreducing conditions. The gels were fixed, dried, and exposed for 3 days to Kodak XAP-5 film (Eastman Kodak Co.) with intensifying screens at -70 C.

Measurement of cAMP

Sperm samples (2 x 106 cells/well) were incubated in the presence or absence of hCG for increasing lengths of time, and then 500 µL media were removed for cAMP measurements using a commercial kit. The kit instructions were followed.

Measurement of PKA and PKC activities

Sperm samples (2 x 106 cells/well) were incubated for 2 h in the presence or absence of 100 ng/mL hCG or LH, and then lysed by sonication. The samples were then frozen and thawed in 200 µL of 25 mM Tris-HC1 (pH 7.5) containing 1 mM EDTA, 1 mM dithiothreitol, 20 mM NaC1, 0.5 mM phenylmethanesulfonyl fluoride, 1 µM aprotinin, and 50 µM leupeptin. The PK activities were determined by incubating for 30 min at 30 C with 5 µg lysate protein with fluorescent-labeled A1 peptide for the PKA assay and fluorescent-labeled C1 peptide for the PKC assay. The nonphosphorylated and phosphorylated fluorescent peptides were separated on 0.8% agarose gels. The phosphorylated fluorescent peptide bands were excised and eluted, and the optical density at 570 nm was measured using a 96-well plate reader. PK activities were calculated from the densitometric values, using the instructions provided by the kit manufacturer. The positive and negative controls supplied in the kit were assayed at the same time as sperm samples.

Replication of experiments and statistical analysis

Each experiment was repeated on three to four different sperm samples. Each sample was assayed in triplicate for cAMP and PK measurements. The data presented were the means and their SE. ANOVA with Duncan’s multiple range test was used in data analysis (36).

Results

We first began looking for LH/hCG receptor messenger RNA in human sperm by RT-PCR. Ethidium bromide-stained agarose gels did not show any bands, and the amplification cycle number could not be increased due to the appearance of nonspecific products. The lack of detection in agarose cells could be due to low levels, and, if so, Southern blotting would be able to detect it. Indeed, as shown in Fig. 1Go, Southern blotting detected an expected 342-bp fragment in all four sperm samples, just as in receptor-positive human granulosa cells. Template omission did not show the fragment. This finding, coupled with amplification of the expected size fragment not only from receptor-rich human granulosa cells but also from human sperm, suggests that the procedure was specific.



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  		Figure 1. Southern blot analysis of RT-PCR products of LH/hCG receptor amplification from human sperm samples. Four different sperm samples were analyzed along with negative procedural control and receptor positive human granulosa cells.

 
Western blot analysis detected an 80-kDa protein in all three sperm samples just as in human granulosa cells (Fig. 2Go). This protein was not detected when receptor antibody was preabsorbed with excess receptor peptide.



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  		Figure 2. Western blot analysis for LH/hCG receptors in human sperm samples. Three different sperm samples along with human granulosa cells were analyzed with native receptor antibody and preabsorbed receptor antibody.

 
Immunocytochemistry demonstrated that LH/hCG receptor immunostaining was mostly confined to the sperm head, to some extent to the midpiece, and very little to the tail region (Fig. 3AGo). Receptor immunostaining was variable among sperm. Receptor immunostaining was not detected when the receptor antibody was preabsorbed with excess receptor peptide (Fig. 3BGo).



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  		Figure 3. Immunocytochemistry for LH/hCG receptors in human sperm samples. B, A control in which receptor antibody preabsorbed with excess receptor peptide was used. Magnification, x1500.

 
Covalent receptor cross-linking studies demonstrated the formation of a 125-kDa complex in all three sperm samples just as in human granulosa cells (Fig. 4Go). This formation was inhibited when excess unlabeled hCG was present in the incubation medium. This 125-kDa protein is a receptor-125I-hCG complex, and the 45 kDa size difference between the free receptor and the complex represent the size of unbound 125I-hCG.



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  		Figure 4. Covalent receptor cross-linking in human sperm samples. Three different sperm samples along with human granulosa cells were analyzed. All lanes contain 125I-hCG, and lanes 1, 3, 5, and 7 also contain excess unlabeled hCG. The 125 kDa represents the receptor-125I-hCG complex, and 45 kDa represents free 125I-hCG.

 
Treatment of sperm samples with highly purified hCG resulted in a significant increase in cAMP levels at 100 ng/mL compared with the control (Fig. 5AGo). Treatment with 100 ng/mL hCG resulted in a significant increase in cAMP levels at 120 min, followed by a nonsignificant decline at 240 min of incubation (Fig. 5BGo).



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  		Figure 5. Dose (A) and time (B) dependency of hCG effect on cAMP production by sperm. A, Sperm samples were incubated for 2 h in the presence or absence of increasing concentrations of hCG. B, Sperm samples were incubated for increasing lengths of time in the presence or absence of 100 ng/mL hCG. In both cases, media cAMP levels were measured. The levels in control samples in B did not significantly vary during incubation; therefore, the levels across all time points were averaged and considered as 100%. *, P < 0.05 compared with control.

 
The cAMP response suggests that hCG or LH treatment would activate sperm PKA. Consistent with this expectation, treatment with 100 ng/mL hCG or LH resulted in a significant increase in PKA activity compared with control sperm (Fig. 6AGo). This activation is specific, as hCG or LH treatment had no effect on sperm PKC activity (Fig. 6BGo).



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  		Figure 6. Activation of sperm PKA (A), but not PKC (B), by hCG and LH. Sperm samples were incubated for 2 h in the presence or absence of 100 ng/mL hCG or LH. *, P < 0.05 compared with control.

 
Discussion

Sperm are the latest addition to the list of nongonadal tissues and cells that contain functional LH/hCG receptors. Although much remains to be learned on the importance of nongonadal LH/hCG signaling, recent inactivation of LH receptors by gene targeting in embryonic stem cells will help further advance this research area (37). In fact, it is already beginning to do so. For example, spermatogenic cells in null animals are receptor negative compared with wild-type and heterozygous littermates supporting the concept that sperm contain functional LH receptors (37). In addition, null animals have nongonadal phenotype. Gonadal steroid hormone replacement therapy to determine whether this phenotype was due to a decreased gonadal steroid hormone levels is beginning to indicate that LH signaling is important (37).

The present study demonstrated by using a combination of techniques that human ejaculated sperm contain LH/hCG receptors. The sperm preparations had no detectable contaminating reproductive tract epithelial or white blood cells. Thus, the receptor messenger RNA, receptor protein, and 125I-hCG binding, detected by RT-PCR, Western blotting, and covalent receptor cross-linking, respectively, must be due to sperm themselves. However, the potential minor contribution by contaminating cells cannot completely be ruled out.

The receptors were mostly in the sperm head, which is the region that undergoes morphological and biochemical changes during capacitation. This pattern is similar to epidermal growth factor receptors, which are also localized to the head (30) and quite different from steroid hormone receptors, which are mostly located in sperm midpiece (24).

The receptor expression not only varied between semen samples but also between sperm in the same sample. These variations should hardly be surprising considering the wide variability among various parameters of sperm function. We have not investigated whether sperm LH/hCG receptor expression correlate with sperm motility or morphology.

Using a wide variety of approaches, a number of previous studies have demonstrated that the cAMP/PKA signaling pathway promotes sperm capacitation and motility (38, 39, 40, 41, 42). Although we do not know whether hCG or LH could mimic cAMP/PKA, logically, they would be expected considering that the sperm hCG/LH receptors are functionally coupled to increasing cAMP production and activating PKA. Nevertheless, direct hCG testing is required, which we plan to do in the near future.

There are two potential sources of hCG and LH that act on sperm. One is seminal plasma, and the other is the female reproductive tract. Seminal plasma contains hCG and LH, sometimes in association with sperm (43, 44, 45, 46, 47, 48, 49). This association could be reflecting hCG binding to sperm receptors. Prostates, seminal vesicles, and even testes are the potential sources of seminal plasma hCG and LH (3, 7, 9, 11, 49). Concerning the reproductive tract, both oviduct and uterus can synthesize hCG with tubal mucosal cells and endometrial glands being the primary source (6, 10).

It is possible that seminal plasma hCG and LH subserve different functions than the female reproductive tract’s hCG. For example, other substances present in seminal plasma may prevent premature sperm activation by hCG and LH. The seminal plasma hCG and LH, however, may not totally be without a function because they could be involved in regulating testosterone synthesis by sperm (50). The finding that seminal plasma hCG was positively correlated with seminal plasma testosterone levels supports such a possibility (49). Once sperm is deposited in the female reproductive tract, local hCG may begin acting to stimulate sperm motility and capacitation, both of which are obviously required for successful fertilization.

hCG/LH receptors and hCG are also present in human oocytes (51). Thus, the presence of these molecules in reproductive tract (6, 10) and female (51) and male gametes may allow bidirectional communication between gametes and gametes-female reproductive tract.

Additional studies to investigate the importance of LH/hCG receptors in sperm functions are required. If hCG/LH promote sperm motility, morphology, capacitation, and fertilization, then it may be possible to treat poor semen samples with hCG to ultimately increase sperm competence to fertilize oocyte. Perhaps much also can be learned from determining sperm characteristics in men with activating and inactivating LH receptor mutations and also sperm from LH receptor knockout mice. The findings may suggest new areas of clinical significance on sperm LH receptors.

In summary, this is the first demonstration of human sperm containing functional hCG/LH receptors. Additional studies to test whether hCG can be used to improve the quality of poor semen samples and the outcome of infertility procedures are required.

Footnotes

1 Present address: Department of Physiology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, Georgia 30310. Back

Received August 16, 2000.

Revised October 16, 2000.

Accepted December 10, 2000.

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