2019 Luteinizing hormone receptor is immunoexpressed within the canine thyroid Luteinizing hormone receptor is immunoexpressed within the canine thyroid Khawla H. Zwida, Michelle A. Kutzler Department of Animal and Rangeland Sciences, Oregon State University, Corvallis, OR Abstract Gonadectomy has been implicated as a risk factor for development of canine hypothyroidism, due to loss of negative feedback to the anterior pituitary, resulting in persistently elevated luteinizing hormone concentrations. Receptors for luteinizing hormone have been identified in various canine gonadal and extragonadal tissues, including bladder and skin. The aim was to investigate if luteinizing hormone receptors were also expressed in the canine thyroid. Formalin-fixed, paraffin-embedded thyroid tissues from eight dogs of various sexes and breeds were subjected to routine immunohistochemical methods. Expression of luteinizing hormone receptors was evident in thyrocytes from all thyroid tissue sections examined; the percentage of positive luteinizing hormone receptors cellular expression was 46.5 ± 23.8% (mean ± standard deviation). Immunoexpression of luteinizing hormone receptors was localized to thyroid follicular epithelial cells, with no immunoreactivity in other thyroid constitutive structures or in any negative control tissue sections. This is apparently the first report to demonstrate luteinizing hormone receptors expression in the canine thyroid gland. Functional studies are needed to determine whether unregulated hypersecretion of luteinizing hormone after gonadectomy affects thyroid function. Keywords: Dog, hypothyroidism, immunohistochemistry, luteinizing hormone receptor Introduction Hypothyroidism is the most common endocrine disorder of dogs, affecting 0.2 - 0.8% of dogs.1-3 The most common presenting dermatological and metabolic symptoms are alopecia, dry/poor quality coat, seborrhea, lethargy, exercise intolerance, weight gain, skin hyperpigmentation, pyoderma and cold intolerance. These symptoms occur in combination more than 60% of the time.1,3,4 Hypothyroidism is more common with advancing age;5 the mean age at diagnosis is 7.2 years (range, 0.5 - 15 years).3 The risk of developing hypothyroidism is higher in certain breeds, including Doberman Pinschers, Miniature Schnauzers, Golden Retrievers, Cocker Spaniels, Shetland Sheepdogs, Irish Setters and Dachshunds.3,4,6,7 In these high-risk breeds, young dogs may also develop hypothyroidism.7 One gene has been implicated in hypothyroidism and lymphocytic thyroiditis in several dog breeds, including the Doberman Pinscher, Rhodesian Ridgeback, English Setter and Giant Schnauzer. This gene is the major histocompatibility complex dog leukocyte antigen haplotype or allele associations.8,9 This is in contrast to humans, in which there are several genetic risk factors for development of autoimmune thyroid disease,1 including a polygenic predisposition or autoimmune hypothyroidism resulting from one of seven genes (AIRE, FOXP3, IL2RA, ITCH, LRBA, STAT1, and STAT3).10,11 Gonadectomized dogs have a higher risk for developing hypothyroidism, with an overall incidence of 30%, significantly higher than the general dog population.7 It is noteworthy that hypothyroidism also affects 10 - 15% of postmenopausal women,12 an endocrine state with many hormonal similarities to gonadectomized dogs. Following gonadectomy or menopause, circulating luteinizing hormone (LH) concentrations increase significantly and remain persistently elevated, due to a lack of negative hormonal feedback. Previous research has demonstrated the presence of LH receptors (LHR) within normal and adenomatous human thyroid glands.13 However, effects of LHR activation in the human thyroid has not been well described, but may involve intracellular signaling similar to thyroid stimulating hormone (TSH) receptor activation (e.g. adenylate cyclase). To our knowledge, the presence of LHR in the canine thyroid gland has not been reported. We hypothesized that LH receptors are present in the canine thyroid and that LHR localizes to cells with TSH receptors. Material and methods Formalin-fixed, paraffin-embedded archived canine thyroid tissues from 8 dogs were provided by the Oregon State University Veterinary Diagnostic Laboratory and used for this investigation (Table). In Clinical Theriogenology • Volume 11 Number 1 • March 201923 addition, formalin-fixed paraffin-embedded archived skin tissue from another dog was used as a positive control, as canine skin expresses abundant LHR.14 Serial 6-μm sections were cut from paraffin blocks to determine LHR and TSH receptor expression on adjacent tissue sections. Sections were mounted on poly- l-lysine-coated slides, deparaffinized in xylene, rehydrated in a graded ethanol series (100, 75, and 50%, respectively) and subjected to heat-induced epitope retrieval (#S1700, Dako, Carpinteria, CA). For this, slides were placed in boiling sodium citrate in a Nordicware® tender cooker and boiling continued in a microwave for a total of 10 minutes. Thereafter, slides were left in solution and allowed to cool to room temperature for ~ 20 minutes. Tissue-specific endogenous peroxidase activity was inhibited by incubating slides in 3% hydrogen peroxide and nonspecific binding blocked with 1% horse serum. Goat polyclonal anti-human LH receptor (SC-26341, Santa Cruz Biotechnology, Dallas, TX) or mouse monoclonal IgG1 TSH receptor (SC-53542, Santa Cruz Biotechnology) were applied at dilutions of 1:50 and 1:100, respectively. Primary antibodies were incubated for 1 hour at room temperature. Negative controls from each tissue were similarly treated, but without primary antibody. Slides were then reacted for 30 minutes with biotinylated horse anti-goat IgG (SK-5300 Vector Laboratories, Burlingame, CA) and incubated for 30 minutes with avidin-biotin-peroxidase complex (PK6105, ABC kit, Vector Laboratories) followed by a brief incubation with Nova Red Peroxidase substrate (SK4800, Vector Laboratories,). Slides were counter-stained with hematoxylin, dehydrated and mounted. Cells positively expressing LH and TSH receptors were those with red staining in the cytoplasm. Cells were counted from five randomly selected fields per antibody per dog with a Leica DM4000B microscope using bright field microscopy at 400X magnification. The number of cells evaluated per dog in these fields was 281.3 ± 142.3 (mean ± SD). Representative images from each dog were digitally captured using a QImaging camera (QICAM 12-bit, #QIC-F-M-12-C, QImaging, Surrey, BC) and QCapturePro image capturing software (QImaging). Data were reported as mean ± SD. Results Immunostaining for LHR was strong in epidermis and hair bulbs (Figure 1). However, there was no TSH receptor expressed in skin (data not shown). Expression of LHR was evident in all thyroid tissue sections, with a mean ± SD percentage of positive cellular expression of 46.5 ± 23.8%. The pattern of LH receptor immunoexpression was localized to thyroid follicular epithelial cells, with no signs of immunoreactivity in other thyroid constitutive structures (Figure 2). Some cells had cap-like immunostaining. It was noteworthy that LHR immunostaining in the canine thyroid could not be differentiated from that of TSH receptor (Figure 3), whose immunoexpression has been previously reported in cows and rats.15 Thyroid stimulating hormone receptor expression was 56.0 ± 13.3% (mean ± SD). There was no positive staining evident in any negative control tissue section (Figures 1-3). Discussion Immunoexpression of LHR has been described in many canine tissues including the skin,14,16 lower urinary tract (bladder and urethra),17,18 and adrenal cortex,19 lymph node,20 and musculoskeletal tissues (ligaments, synovia, subchondral bone).21 However, this was apparently the first report that the canine thyroid gland expressed LHR. Using a cut-off of > 25% of cells staining positive for LHR, 16.7% of normal human thyroids were positive for LHR.13 If a cut-off of > 25% of cells staining positive were applied for dogs, 6 out of 8 (75%) thyroids examined in the current study would be positive for LHR. Molecular mechanisms underlying regulation of LHR expression in gonadal and non-gonadal tissues have been investigated. In chickens, LH increases ovarian LHR gene expression, whereas activin A prevents LH-induced increases in LHR gene expression.22 Expression of LHR is inhibited post- transcriptionally in rat luteal cells by mevalonate kinase.23 Estrogen reduced ovine LHR gene expression in several extra-gonadal tissues, including medulla oblongata, hypothalamus, ruminant stomach (except reticulum), intestinal tissues (except colon), pancreas, liver, kidney, and uterus. In contrast, progesterone increased LHR gene expression in various tissues, including the hypophysis, olfactory bulb, rumen, small intestine, kidney, oviduct, and uterus.24 Regulation of LHR expression in the thyroid gland needs additional investigation. In prepubertal Clinical Theriogenology • Volume 11 Number 1 • March 2019 24 rats with PTU (6-N-propyl-2-thiouracil) induced hypothyroidism, thyroid LH receptor gene expression was not significantly different from controls.25 In humans, there was increased thyroid LHR expression in women with high serum LH concentrations.13 However, thyroid glands from these patients were adenomatous and it is not known if the women were hypothyroid at sample collection. In the current study, serum tetraiodothyronine (T4) and LH concentrations in these dogs were unknown, due to use of archived tissues. In women who have undergone gonadectomy, LH concentrations are three times higher after ovary removal, whereas both triiodothyronine and T4 concentrations are significantly reduced after ovary removal.26 This observation provides evidence that a causal relationship could exist in dogs as well. That gonadectomized dogs have significantly elevated circulating LH concentrations and are at greater risk for developing hypothyroidism7 is an impetus to determine effect of reducing LH concentrations on thyroid hormone secretion. The current study provided evidence that LH receptors may have a role in thyroid hormone secretion. However, more research is needed to determine whether LHR expression is increased in gonadectomized dogs. Acknowledgement The authors thank Kay Fischer and the Oregon State University Histology Laboratory for technical assistance. Authors’ contributions Zwida contributed to the design of the study, performed all of the immunohistochemistry experiments, and image acquisitions. Kutzler conceived and contributed to the design of the study and oversaw immunohistochemistry and image acquisition. Both authors drafted the manuscript, critically revised it, gave final approval and agreed to be accountable for all aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Conflict of interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The authors thank the Ministry of Scientific Research and Higher Education in Libya for graduate school financial assistance and Dr. Howard Meyer of Chippewa Kennels for funding research supplies. References 1. Mooney CT: Canine hypothyroidism: a review of aetiology and diagnosis. N Z Vet J 2011;59:105-114. 2. Johnson MB, Hattersley AT, Flanagan SE: Monogenic autoimmune diseases of the endocrine system. Lancet Diabetes Endocrinol 2016;4:862-872. 3. Panciera DL: Hypothyroidism in dogs: 66 cases (1987-1992). J Am Vet Med Assoc1994:204:761-767. 4. Dixon RM, Mooney CT: Canine serum thyroglobulin autoantibodies in health, hypothyroidism and nonthyroidal illness. Res Vet Sci 1999;66:243-246. 5. Bagchi N, Brown TR, Parish RF: Thyroid dysfunction in adults over age 55 years. A study in an urban US community. JAMA Intern Med 1990;150:785-787. 6. Nesbitt GH, Izzo J, Peterson L, et al: Canine hypothyroidism: a retrospective study of 108 cases. J Am Vet Med Assoc 1980;177:1117-1122. 7. Milne KL, Hayes HM Jr: Epidemiologic features of canine hypothyroidism. Cornell vet 1981;71:3-14. 8. Kennedy LJ, Huson HJ, Leonard J, et al: Association of hypothyroid disease in Doberman Pinscher dogs with a rare major histocompatibility complex DLA class II haplotype. Tissue Antigens 2006;67:53-56. 9. Wilbe M, Sundberg K, Hansen IR, et al: Increased genetic risk or protection for canine autoimmune lymphocytic thyroiditis in Giant Schnauzers depends on DLA class II genotype. Tissue Antigens 2010;75:712-719. 10. Johnson MB, Hattersley AT, Flanagan SE: Monogenic autoimmune diseases of the endocrine system. Lancet Diabetes Endocrinol 2016;4:862-872. Clinical Theriogenology • Volume 11 Number 1 • March 201925 11. Sanyal D, Raychaudhuri M: Hypothyroidism and obesity: An intriguing link. Indian J Endocrinol Metab 2016;20:554- 557. 12. Giri A, Edwards TL, LeGrys VA, et al: Subclinical hypothyroidism and risk for incident ischemic stroke among postmenopausal women. Thyroid 2014;24:1210-1217. 13. Liu J, Chen G, et al: Serum levels of sex hormones and expression of their receptors in thyroid tissue in female patients with various types of thyroid neoplasms. Pathol Res Pract 2014;210:830-835. 14. Welle MM, Reichler IM, Barth A, et al: Immunohistochemical localization and quantitative assessment of GnRH-, FSH-, and LH-receptor mRNA Expression in canine skin: a powerful tool to study the pathogenesis of side effects after spaying. Histochem Cell Biol 2006;126:527-535. 15. Schmid KW, Jasani B, Morgan JM, et al: Light microscopic immunocytochemical demonstration of thyroid-stimulating hormone (TSH) receptors on normal rat thyroid cells. J Histochem Cytochem 1988;36:977-982. 16. Venencie PY, Méduri G, Pissard S, et al: Luteinizing hormone/human chorionic gonadotrophin receptors in various epidermal structures. Br J Dermatol 1999;141:438-446. 17. Ponglowhapan S, Church DB, Scaramuzzi RJ, et al: Luteinizing hormone and follicle-stimulating hormone receptors and their transcribed genes (mRNA) are present in the lower urinary tract of intact male and female dogs. Theriogenology 2007;67:353-366. 18. Ponglowhapan S, Church DB, Khalid M: Differences in the expression of luteinizing hormone and follicle-stimulating hormone receptors in the lower urinary tract between intact and gonadectomised male and female dogs. Domest Anim Endocrinol 2008;34:339-351. 19. Galac S, Kars VJ, Klarenbeek S, et al: Expression of receptors for luteinizing hormone, gastric-inhibitory polypeptide, and vasopressin in normal adrenal glands and cortisol-secreting adrenocortical tumors in dogs. Domest Anim Endocrinol 2010;39:63-75. 20. Ettinger A, Zwida K, Kutzler M: Normal and neoplastic canine lymphocytes express luteinizing hormone receptors. Clin Theriogenology 2017;428. 21. Kiefel C, Kutzler M: Luteinizing hormone receptor expression in canine anterior cruciate and femoral head ligaments. ISCFR VIII 2016; p.151. 22. Davis AJ, Brooks CF, Johnson PA: Activin A and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNA in avian granulosa cells. Biol Reprod 2001;65:1352-1358. 23. Nair AK, Menon KM: Regulation of luteinizing hormone receptor expression: evidence of translational suppression in vitro by a hormonally regulated mRNA-binding protein and its endogenous association with luteinizing hormone receptor mRNA in the ovary. J Biol Chem 2005;280:42809-42816. 24. Wang LH, Zhang W, Gao QX, Wang F: Expression of the luteinizing hormone receptor (LHR) gene in ovine non- gonadal tissues during estrous cycle. Genet Mol Res 2012;11:3766-3780. 25. Rao JN, Liang JY, Chakraborti P, et al: Effect of thyroid hormone on the development and gene expression of hormone receptors in rat testes in vivo. J Endocrinol Invest 2003;26:435-443. 26. De Leo V, D'Antona D, Lanzetta D: Thyrotropin secretion before and after ovariectomy in premenopausal women. Gynecol Endocrinol 1993;7:279-283. Clinical Theriogenology • Volume 11 Number 1 • March 2019 26 Table. Signalment of the subjects used to determine LH receptor immunoexpression in canine thyroid tissue. The expression of thyrocytes varied considerably among individuals. Breed Reproductive Status Age Thyroid cells expressing LH receptors Labrador Retriever Spayed Female 2 months 5% American Pit Bull Terrier Intact Male 5 months 51% Labrador Retriever Spayed Female 6 months 19% Maltese Spayed Female 2 years 62% Rottweiler Intact Male 6 years 66% Irish Setter Castrated Male 10 years 38% Cairn Terrier Spayed Female 12 years 60% Clinical Theriogenology • Volume 11 Number 1 • March 201927 Figure 1. Representative image of immunohistochemical localization; arrows indicate immunoexpression for LH receptor in the canine epidermis (scale bar = 10 μm). Inset image is negative control for LH receptor immunostaining. Figure 2. Representative image of immunohistochemical localization; arrows indicate a low level of immunoexpression for LH receptor in the canine thyroid (scale bar = 10 μm). Inset image is negative control for LH receptor immunostaining. Clinical Theriogenology • Volume 11 Number 1 • March 2019 28 Figure 3. Representative image of immunohistochemical localization; arrows indicate a high level of immunoexpression for LH receptor in the canine thyroid (scale bar = 10 μm). Inset image is negative control for LH receptor immunostaining. Figure 4. Representative image of immunohistochemical localization; arrows indicate a low level of immunoexpression for TSH receptor in the canine thyroid (scale bar = 10 μm). Inset image is negative control for TSH receptor immunostaining. 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