Luteinizing hormone is a gonadotropin produced by the adenohypophysis in response to GnRH stimulation, and its secretion is regulated through negative feedback by gonadal hormones (eg, testosterone or estradiol).1–3 Although LHRs are primarily found in gonadal tissues, LHR expression in reproductive extragonadal and nonreproductive tissues of swine, rabbits, rodents, cattle, humans, and dogs has been reported.2–12 Results of those studies indicate that LHRs in extragonadal tissues are functional, commonly making use of a cAMP-protein kinase A signaling pathway as well as other signaling pathways such as the protein kinase C and mitogen-activated protein kinase pathways.4 For example, in women, LHR activation results in maintenance of the uterus in a quiescent state and an increase in uterine blood flow.13 In dogs, activation of LHRs in nonreproductive tissues has been associated with an increased incidence of long-term health conditions that develop following gonadectomy (eg, urethral sphincter mechanism incompetence that leads to urinary incontinence).14–20 Luteinizing hormone–mediated reduction in urethral sphincter tone following gonadectomy may similarly contribute to an exacerbation in musculoskeletal laxity, thereby precipitating the development of hip dysplasia in dogs.21 The increased incidence of hip dysplasia in gonadectomized dogs may result from LH hypersecretion following the loss of hypothalamic-pituitary-gonadal axis negative feedback.1 Following ovariohysterectomy in dogs, urethral sphincter mechanism incompetence can be temporarily resolved after treatment with a GnRH agonist and GnRH immunization.22–24
Hip dysplasia and CCL rupture are 2 musculoskeletal conditions that have an increased incidence in gonadectomized dogs, compared with findings in sexually intact dogs.25–27 To the authors' knowledge, LHR expression in canine musculoskeletal tissues has not been evaluated. The purpose of the study reported here was to determine immunohistochemically whether LHRs are expressed in the structural support tissues of canine hip and femorotibial joints. Overall, it was hypothesized that LHR expression would be present in specimens of FHSB, RL, CCL, and FJS obtained from canine cadavers.
Materials and Methods
Sources of canine musculoskeletal tissues
Upon receiving informed organizational or owner consent, 19 canine cadavers were obtained from local animal shelters and veterinary clinics. The dogs had been euthanized by IV injection of an overdose of euthanasia solution. The sex, reproductive status, age, and breed of each dog were recorded. For shelter dogs, the age (determined on the basis of dental eruption and wear) and breed (determined on the basis of phenotypic characteristics) were approximated. Prior to tissue collection, each cadaver was examined grossly to determine its body condition score (on a scale of 1 to 9)28 and the presence or absence of hind limb muscle asymmetry. The latter assessment of biceps femoris, semitendinosus, semimembranosus, and quadriceps femoris muscles was used as an indirect measure of the presence of antemortem lameness.
Tissue specimens from the hip joint (RL and FHSB specimens) and femorotibial joint (CCL and FJS specimens) were collected from the canine cadavers. One tissue specimen was collected at each of the 4 anatomic sites in the left hind limb of each cadaver. All tissue specimens were obtained fresh and fixed in neutral-buffered 10% formalin for at least 24 hours. The FHSB specimens were decalcifieda for 7 to 14 days then replaced in formalin. Formalin-fixed canine skin tissue samples from an unrelated cadaver were collected for use as a positive experimental control during immunohistochemical analysis of musculoskeletal tissues.
Sectioning of tissue specimens
All tissue specimens from each cadaver along with a positive control sample were paraffin-embedded in the same block. To differentiate between the RL and CCL specimens, a tissue marking dye was applied to the distal end of the CCL specimen. Tissue specimens were oriented within each block to enable sectioning in the transverse plane. Sections (thickness, 6 μm) of the specimens were placed onto positively charged slides. Three sections of each tissue specimen from each cadaver were obtained; 1 section underwent immunohistochemical analysis for LHRs, 1 section was treated with negative control reagents, and 1 section was stained with H&E stain for histologic reference.
Immunohistochemical analysis of LHR expression in tissue specimens
For immunohistochemical analysis, 1 section of each FHSB, RL, CCL, and FJS specimen from each cadaver was deparaffinized, rehydrated in graded ethanol baths (100%, 75%, and 50%), and subjected to heat-induced antigen retrieval with a sodium citrate antigen retrieval agent.b Endogenous peroxidase activity was inactivated with 3% hydrogen peroxide, and nonspecific binding was blocked by subsequent incubation with a serum-free protein block.c Rabbit polyclonal IgG anti-human LHR antibodyd was applied to the sections at a 1:50 dilution, followed by incubation for 30 minutes at room temperature (20°C). Negative controls for each tissue specimen from each individual cadaver were treated in a similar manner except with omission of the primary antibody. All sections were reacted with anti-rabbit IgG horseradish peroxidase polymere and then incubated with peroxidase substratef for 10 minutes. Sections were then counterstained with hematoxylin, destained in 1% lithium carbonate, dehydrated in graded ethanol baths (50%, 75%, and 100%), and cleared in xylene. Finally, cover slips were mountedg onto the slides over the sections.
Immunoexpression data analysis
For sections used in the immunohistochemical analysis, LHR immunoexpression in tissue sections was assessed in images obtained by bright-field microscopy at 400× magnification with a computerized confocal microscope.h Uniformity during image acquisition and data collection was ensured by application of the same imaging softwarei settings for intensity and exposure during all images captured. A single observer (CAK) examined all images.
Luteinizing hormone receptor immunoexpression in the FHSB and FJS sections was analyzed quantitatively by examination of 100 cells in 3 adjacent hpf (400×) for LHR-specific staining. Only bone marrow cells or synoviocytes with visible nuclei were counted. For each section, the number of stained cells was expressed as a percentage.
Luteinizing hormone receptor immunoexpression in the RL and CCL sections was analyzed in a semiquantitative manner as reported by Ciccarelli et al.29 For each RL and CCL section, 3 adjacent hpf (400×) were examined for LHR-specific staining. For all RL and CCL sections, only fibrocytes with visible nuclei were evaluated. The semiquantitative method used involved assignment of a staining score from 0 to 3 on the basis of the assessed percentage of stained cells within an hpf, as follows: 0 = no cells are stained, 1 = 1% to 10% of cells are stained, 2 = 11% to 50% of cells are stained, and 3 = > 50% of cells are stained.29 Staining intensity was also scored from 0 to 2 as follows: 0 = no staining, 1 = weak staining, and 2 = moderate to strong staining.29 The overall score for each hpf was determined from the product of the percentage staining score and staining intensity score. An overall LHR immunoexpression score was determined for each section on the basis of the mean of the products of the percentage staining score and staining intensity score of 3 adjacent hpf.29
Data handling and statistical analysis
Data normality was determined with a Shapiro-Wilk normality test prior to application of a Student t test. Applicable test assumptions were met for t test analysis for the FHSB and FJS specimen data but not for the RL and CCL specimen data. For FHSB and FJS specimens, LHR immunoexpression data are expressed as mean ± SD. A Student t test was used to compare the percentage of LHR expression in FHSB or FJS specimens between sexes and between sexually intact and gonadectomized dogs. For RL and CCL specimens, LHR immunoexpression data are expressed as median and range. A Mann-Whitney U test was used to compare the overall LHR immunoexpression scores for RL or CCL specimens between sexes and between sexually intact and gonadectomized dogs. Significance was defined as a value of P < 0.05.
Results
Dogs
Among the 19 cadavers used in the study, 11 were male dogs and 8 were female dogs. There were 3 sexually intact males, 8 gonadectomized males, 2 sexually intact females, and 6 gonadectomized females. The ages of the sexually intact males were 1 to 5 years (n = 2) and 5 to 10 years (1), and the ages of the gonadectomized males were 1 to 5 years (n = 1), 6 years (1), 5 to 10 years (4), > 10 years (1), and 13 years (1). The ages of the sexually intact females were 1 to 5 years (n = 1) and 5 to 10 years (1), and the ages of the gonadectomized females were 1 to 5 years (2), 5 to 10 years (3), and > 10 years (1). Breeds included pit bull-type (n = 7), German Shepherd Dog (2), and Labrador Retriever mix (3); several other breeds (Irish Setter, Border Collie, Akita mix, Labrador Retriever, German Shepherd Dog mix, Siberian Husky, and Golden Retriever-Poodle mix) were represented once. The dogs' body condition scores ranged from 2 to 7 (mean, 4.47; median, 4.5). Only 1 dog had evidence of hind limb muscle asymmetry (presumed to indicate antemortem lameness); the left hind limb was affected.
LHR immunoexpression in specimens of structural support tissues of canine hip and femorotibial joints
Luteinizing hormone receptor immunoexpression data were obtained for specimens of FHSB, RL, CCL, and FJS from the left hind limb of all 19 cadavers. All FHSB samples examined expressed LHRs (Figure 1). There was no significant difference in percentage of bone marrow cells of FHSB specimens with LHR expression between female and male dogs (32.1 ± 34.1% vs 24.7 ± 22.0%, respectively; P = 0.32) or between sexually intact and gonadectomized dogs (27.0 ± 29.1% vs 27.0 ± 25.4%, respectively; P = 0.50). With respect to fibrocytes in RL samples, female dogs had greater (P = 0.03) overall LHR expression scores (median, 6 [range, 1 to 6]), compared with findings for male dogs (all specimens, 0; Figure 2). However, there was no significant (P > 0.99) difference in overall LHR expression score for RL fibrocytes between sexually intact (median, 3 [range, 0 to 6]) and gonadectomized (median, 0.5 [range, 0 to 6]) dogs. The LHR expression scores for fibrocytes in CCL specimens did not differ significantly between female (median, 5 [range, 4 to 6]) and male (median, 3 [range, 0 to 6]) dogs (P > 0.99) or between sexually intact (all specimens, 6) and gonadectomized (median, 4 [range, 0 to 6]) dogs (P > 0.99; Figure 3). The percentage of synoviocytes of the FJS specimens with LHR expression did not differ significantly between sexes (31.3 ± 34.0% for female dogs vs 27.7 ± 29.6% for male dogs; P = 0.44) or reproductive statuses (27.7 ± 29.6% for sexually intact dogs vs 32.7 ± 37.0% for gonadectomized dogs; P = 0.43; Figure 4). There was no nonspecific staining in the negative control tissue sections. In addition, the adnexal units of canine epithelial hair follicles in all canine skin control sections were positive for LHR, as has been previously reported.30
Representative photomicrograph of a transverse section of an FHSB specimen from a gonadectomized male dog following immunohistochemical staining for LHRs. On the basis of LHR-specific staining, LHRs (arrows) were present and were localized to the subchondral trabecular bone. Luteinizing hormone receptor–specific immunostain with hematoxylin counterstain; bar = 10 μm.
Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.565
Representative photomicrograph of a transverse section of an RL specimen from a gonadectomized female dog following immunohistochemical staining for LHRs. Luteinizing hormone receptors were present (arrows) and were localized to fibrocytes and collagen fibers within the dense connective tissue of the RL. Luteinizing hormone receptor–specific immunostain with hematoxylin counterstain; bar = 10 μm.
Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.565
Representative photomicrograph of a transverse section of a CCL specimen from a gonadectomized male dog following immunohistochemical staining for LHRs. Luteinizing hormone receptors were present (arrows) and were localized to fibrocytes and collagen fibers within the dense connective tissue of the CCL. Luteinizing hormone receptor–specific immunostain with hematoxylin counterstain; bar = 10 μm.
Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.565
Representative photomicrograph of a transverse section of an FJS specimen from a gonadectomized female dog following immunohistochemical staining for LHRs. Luteinizing hormone receptors were present (arrows) and were localized to the synoviocytes in the joint capsule. Luteinizing hormone receptor–specific immunostain with hematoxylin counterstain; bar = 10 μm.
Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.565
Discussion
The objective of the present study was to determine whether LHRs were present in the structural support tissues of canine hip and femorotibial joints. Specimens of the FHSB, RL, CCL, and FJS obtained from the left hind limbs of 19 cadavers were evaluated immunohistochemically for LHRs. As hypothesized, variable expression of LHRs was identified in the fibrocytes of the RL specimens and bone marrow cells of the FHSB specimens as well as in the fibrocytes of the CCL specimens and synoviocytes of the FJS specimens. With the exception of greater expression of LHRs in the RL specimens from female dogs, compared with findings for male dogs, there were no significant differences in receptor abundance in the examined tissues between sexes or between sexually intact and gonadectomized dogs. To the authors' knowledge, this is the first study of LHR expression in canine musculoskeletal tissues. There is a need for additional investigation into the sex-associated difference for LHR expression in canine RL fibrocytes. Further studies are also warranted to investigate LHR expression in diseased tissues (eg, in specimens collected from dogs with hip dysplasia or CCL injury).
The influence of gonadal hormones on the mechanical properties of musculoskeletal tissues in various experimental models, including those involving dogs, has been well defined.31–46 Estrogen, relaxin, progesterone, and androgen receptors have also been detected in CCL specimens obtained from dogs, rodents, rabbits, and humans.47–49 In an investigation of the multifactorial etiopathogenesis of CCL injury, Slauterbeck et al50 determined that both gonadal hormones and tissue remodeling are important factors affecting applied mechanical load and magnitude of load on ligament failure pathways. The established association between gonadal hormones and musculoskeletal pathological changes suggests that the role of LHR activation in structural support tissues of canine hip and femorotibial joints, particularly in gonadectomized individuals, should be further investigated. Although the expression of LHR in these musculoskeletal tissues was not significantly different between sexually intact and gonadectomized dogs in the present study, sustained elevated plasma LH concentrations in gonadectomized dogs would likely result in increased LHR activation.1 Increased LHR activation may be a contributory factor in the reportedly increased incidence of hip dysplasia and CCL injury in gonadectomized dogs, compared with findings for sexually intact dogs.51,52
Hip dysplasia and CCL injury are the 2 most common musculoskeletal diseases in dogs.51 Canine hip dysplasia is a multifactorial disease involving a combination of genetic and environmental risk factors. Canine CCL injury is an acquired disease involving acute or chronic biomechanical stress.52 It is important to note that dogs affected by hip dysplasia are born with anatomically normal hip joint conformation.53 Joint laxity predisposes to the development of hip dysplasia and secondary degenerative joint disease. Similarly, joint laxity predisposes to CCL injury. Osteoarthritis secondary to hip dysplasia and CCL injury is the primary factor contributing to chronic lameness in dogs.54 The importance of medical management for the prevention and treatment of these orthopedic diseases requires further study to determine the efficacy of LH-reducing treatments, such as administration of GnRH agonists or vaccines, in dogs.
Acknowledgments
Supported in part by Dr. Howard Meyer and the Agricultural Research Foundation, as well as the Oregon State University Honors College. Funding sources did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.
The authors declare that there were no conflicts of interest.
Preliminary immunohistochemical analysis results presented in abstract format at the International Symposium on Canine and Feline Reproduction in Paris, France, June 2016, and the Northwest Reproductive Sciences Symposium in Philomath, Ore, June 2016. Final immunohistochemical analysis results presented in abstract format at the Alliance for Contraception in Cats and Dogs Symposium in Boston, Mass, July 2018, and as an honors undergraduate thesis in Corvallis, Ore, May 2018.
The authors thank Dr. Christiane Loehr for assistance with histological analysis. The authors also thank Khawla Zwida and Dr. Valerio Moccia for assistance with developing the immunohistochemical protocols.
ABBREVIATIONS
| CCL | Cranial cruciate ligament |
| FHSB | Femoral head subchondral bone |
| FJS | Femorotibial joint synovium |
| GnRH | Gonadotropin-releasing hormone |
| LH | Luteinizing hormone |
| LHR | Luteinizing hormone receptor |
| RL | Round ligament of the hip joint |
Footnotes
Cal-Ex decalcifier, Fisher Chemical, Waltham, Mass.
Ready-To-Use S1700, Dako, Carpinteria, Calif.
Ready-to-Use 0909, Dako, Carpinteria, Calif.
NLS1436, Novus Biologicals, Littleton, Colo.
One-Step HRP polymer anti-rabbit IgG (H+L), ImmunoBioscience Corp, Mukilteo, Wash.
Vector NovaRED peroxidase (HRP) substrate kit SK-4800, Vector Laboratories Inc, Burlingame, Calif.
Richard-Allan Scientific Cytoseal XYL 8312-4, Thermo Fisher Scientific, Waltham, Mass.
Leica CTR 6500 Imaging Software, Leica Microsystems, Buffalo Grove, Ill.
Leica Application Suite X, Leica Microsystems, Buffalo Grove, Ill.
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