Live imaging of follicle stimulating hormone receptors in gonads and bones using near infrared II fluorophore

In vivo imaging of hormone receptors provides the opportunity to visualize target tissues under hormonal control in live animals.

Diverse peptide hormones act on their specic receptors in target cells to regulate different physiological functions. In addition to mediating hormonal actions, target cell receptors provide unique markers for imaging and identication of unique cell types in normal and malignant tissues. The follicle stimulating hormone (FSH) is a heterodimeric glycoprotein essential for gonadal development as it binds to specic receptors in granulosa cells of ovarian follicles and Sertoli cells of testicular seminiferous tubules. [1][2][3] Ovaries contain follicles as functional units. Once activated to grow, follicles develop through primordial, primary and secondary (preantral) stages until they acquire an antral cavity, followed by further growth into preovulatory follicles capable of releasing a mature egg for fertilization. 4 Imaging of ovarian follicles in patients has relied upon transvaginal ultrasound but the approach is limited in spatial resolution and only detects antral and larger follicles. 5 For infertile female patients, it is important to assess the presence of preantral follicles because recent studies have indicated the possibility to promote preantral follicle growth for infertility treatment. 6,7 In the testes, Sertoli cells are essential for spermatogenesis 8 and monitoring of FSH receptors in these cells could allow for better diagnosis of male infertility.
In addition to FSH receptors in gonads, low levels of FSH receptors were found in cultured osteoclasts and these receptors have been proposed to promote osteoporosis in postmenopausal women. 9 However, these ndings were challenged due to an inability to conrm the low expression of FSH receptors in osteoclasts 10,11 and difficulties occurred in performing in vitro FSH binding of the rigid bone structures. In Fig. 2 NIR-II imaging of ovarian follicles using follicle stimulating hormone-fluorophore CH1055 (FSH-CH) in adult female mice: time-course and specificity. (A) FSH-CH (12.5 mg) was injected into the tail vein of an adult female mouse before NIR-II imaging at different post-injection times. There was a rapid accumulation of signals in the blood vessels and kidney at 60 s. Ovarian signals begin to show up at 120 s and peak at 2-6 h after injection, showing a sustained retention for up to 24 h. Side view images are shown to focus on one ovary. (B) Quantitation of the fluorescence intensity in the ovary at different time points (n ¼ 3). (C) Imaging at high magnification at 24 h after FSH-CH injection. Ovarian NIR-II signals inside the body (in vivo) and after ovary exposure from the abdominal cavity with the uterus, vasculature and nerves connected (ex vivo) are shown. (D) Confocal image of the same ovary showing bright signals in granulosa cells inside individual follicles (arrowheads) together with background signals in the oviduct (arrow). (E) Displacement of FSH-CH binding by non-conjugated FSH in the ovary. Adult female mice were injected with FSH-CH (12.5 mg of FSH) or FSH-CH plus a 20-fold excess of unconjugated FSH (250 mg) before imaging 2 h later. Strong NIR-II signals were found in the ovary both in vivo and ex vivo whereas no signal was found in the ovary when excess FSH was injected together with FSH-CH. Light microscope pictures accompany each NIR-II image. (F) Quantitation of NIR-II signals in individual groups. Error bars indicate the standard deviation of each group. PL, photoluminescence.
view of the major health care costs incurred worldwide in both aging female and male populations, 12 it is important to further elucidate the function of FSH receptors in bones.
Recent advances in biological imaging using novel uorescent agents in the long NIR-II region have achieved a reduction of photon scattering and auto-uorescence in tissues, thus reaching deeper penetration depths in vivo. Fluorescence imaging of biological systems in the NIR-II window can probe centimeter tissue depths and achieve micron-scale spatial resolution at millimeter depths. 13,14 Using this approach, through-scalp and through-skull uorescence imaging of mouse cerebral vasculature allowed real-time assessment of blood ow anomalies in a mouse cerebral artery occlusion stroke model. 15 Here, we developed a ligand-based imaging dye by covalently linking a NIR-II uorophore to FSH for in vivo imaging of FSH receptors in live animals. In addition to detecting ovarian follicles of different sizes and testicular seminiferous tubules, we conclusively demonstrated the expression of FSH receptors in vertebrae and other bone structures.
Recombinant human FSH (35.5 kDa) was conjugated to NIR-II dye CH1055 (M.W. 0.97 kDa) based on the 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDC) method 16 (Fig. 1A). The UV-vis-NIR absorption spectrum of the FSH-CH solution in water exhibited an absorption peak at ca. 700 nm, while the uorescence emission spectrum showed a main emission peak at 1055 nm, displaying a large Stokes shi of 400 nm (Fig. 1B). FSH is responsible for follicle growth 17 and FSH receptors are expressed mainly in granulosa cells of ovarian follicles and Sertoli cells of testes tubules. 18 We obtained granulosa cells by puncturing antral follicles of immature mice pretreated with equine gonadotropin (eCG) for 2 days to stimulate follicle growth. Following the centrifugation and sonication of cell suspensions, we prepared lysates from granulosa cells containing FSH receptors. Lysates of granulosa cells were printed on plasmonic, NIR uorescenceenhancing Au slides [19][20][21] to form reverse phase microarrays before incubation with FSH-CH. As shown in Fig. 1C, granulosa cell lysates displayed bright specic NIR-II signals as compared with the negligible signals from the lysates of U87MG glioblastoma cells. Also, cultured granulosa and MDA-MB-468 (MDA) mammary cancer cells were incubated with 500 nM FSH-CH at room temperature for 4 h followed by washing out of unbound ligands. As shown in Fig. 1D, strong signals were found in granulosa cells but not in MDA cells. Furthermore, the addition of a 10-fold excess of nonconjugated FSH together with FSH-CH blocked NIR-II signals ( Fig. 1E and F), demonstrating the ability of non-conjugated FSH to compete for ligand binding.
As shown in Fig. 2A and Video S1, † the injection of FSH-CH into adult female mice led to an initial accumulation of signals (at 60 seconds) in the kidney, the site of FSH metabolism. 22 At 120 seconds aer injection, signals appeared in the ovary, reaching their highest levels at 6 h and lasting for 24 h (Fig. 2B). Under high magnication in vivo, strong signals could be detected in ovarian follicles (Fig. 2C, in vivo) as conrmed by removing the ovary outside the body (Fig. 2C, ex vivo). Under confocal uorescence NIR-II imaging using a home-built instrument, strong signals were found in granulosa cells of antral and smaller follicles (Fig. 2D, arrowheads). Furthermore, injection of FSH-CH together with a 20-fold excess of nonconjugated FSH led to lower signals in the ovary either in vivo or aer its removal from the body (ex vivo) (Fig. 2E). Quantitation of the NIR-II uorescence intensity indicated a major suppression of signals aer blocking with excess nonconjugated FSH (Fig. 2F), demonstrating the hormonal speci-city of FSH-CH binding.
To further demonstrate the ability of FSH-CH to image preantral follicles, we injected FSH-CH into mice at 17 days of age, the ovaries of which contained only preantral or smaller follicles. As shown in Fig. 3A and B, strong signals were found in the ovary at both 2 h and 24 h aer FSH-CH injection. Under confocal uorescence imaging at high magnication, strong NIR-II signals were evident in secondary follicles ( Fig. 3C and D,  arrows).
To investigate FSH-CH binding to testes, adult male mice were injected with FSH-CH, followed by live imaging (Fig. 4A). Quantitation of NIR-II signals demonstrated accumulation of FSH-CH in both testes in a time-dependent manner with bright signals detected from 120 s onward, which peaked at 0.5 h and lasted for at least 2 h (Fig. 4B, dashed circles). Strong signals were conrmed aer removing the testes from the body (Fig. 4C; in vitro). As shown in Fig. 4D, confocal uorescence NIR-II imaging allowed ex vivo detection of seminiferous tubules. Furthermore, an injection of a 20-fold excess of FSH together with FSH-CH led to negligible signals in the testes (Fig. 4E), demonstrating the hormonal specicity of FSH-CH binding.
Aer injecting FSH-CH in both adult female and male mice, we found strong NIR-II signals in vertebrae and other bones. As shown in Fig. 5A, NIR-II signals were found in the spine, thighbones, tibia and ovaries in the female animals. In addition, nonspecic signals were found in the kidney and liver, the organs of FSH-CH metabolism. In the male animals, NIR-II signals were also found in the spine and thighbones (Fig. 5B). Under high magnication, NIR-II signals were detected in the vertebrae and thighbone of a female, together with the radius, ulna, and hand bones as well as the tibia and foot bones in a male (Fig. 5C). Furthermore, an injection of FSH-CH together with a 20-fold excess of FSH decreased the signals in the bones in both sexes (Fig. S2 †), demonstrating the hormonal specicity of FSH-CH binding. Pharmacokinetics of bone (Fig. S3A †), based on NIR-II imaging using FSH-CH, has been shown. In addition, quantitation of the uorescence intensity in the bone at each time point in both sexes is presented in Fig. S3D. † The maximum uorescence signal strength of a structure imaged using a ligand-conjugated uorophore is dictated by parameters including the ligand-receptor binding kinetics, the permeation of the imaging agent into a given tissue, imaging depth, and background autouorescence of the imaging agent's optical properties. In addition to the use of a NIR-II uorophore that confers improvements in the spatial resolution due to the reduction in scattering and better penetration of signals at longer wavelengths, the present use of a FSH-conjugated NIR-II uorophore allows high affinity and specic targeting of the uorophore to unique cell types in gonads and bones expressing FSH receptors. The small size of the uorophore does not interfere with the high affinity binding of FSH to its cognate receptors nor impedes FSH permeation to target cells. FSH receptors are expressed mainly in ovarian granulosa cells in females and Sertoli cells in testicular seminiferous tubules in males. In addition to providing the rst in vivo imaging of FSH receptors in gonads, we further demonstrated FSH binding signals in vertebrae and other bones, consistent with earlier ndings in isolated osteoclasts. 9 In addition, NIR-II signals for FSH-CH were concentrated in the kidney and liver, the sites of FSH and CH1055 metabolism, respectively. 22 Although antral preovulatory follicles could be stimulated by gonadotropins to release mature oocytes capable of undergoing fertilization for pregnancy, recent studies indicate that many infertile patients still possess preantral follicles in their ovaries which could respond to an In Vitro Activation (IVA) therapy to develop into larger follicles and to generate mature eggs for pregnancy. 6,7 Ultrasound and MRI are commonly used in clinics to diagnose follicle growth and ovarian tumors. 38,39 For follicles smaller than the secondary follicle (diameter < 100 mm), which are the majority of follicles, both ultrasound and MRI are ineffective. Because the prevailing transvaginal ultrasound approach 5 does not allow imaging of preantral follicles, attempts have been made to use serum Anti-Mullerian Hormone (AMH) levels to predict the presence of preantral follicles. 23 But in clinics, some patients with undetectable AMH levels could still respond to the IVA treatment due to the existence of residual preantral follicles. 24 We used ultrasound and magnetic resonance imaging (MRI) approaches (ESI Fig. S4A and B †) to perform in vivo imaging of a mouse ovary but only obtained a low quality outline of large preovulatory follicles. We also used non-targeting single-walled carbon nanotubes (SWNCTs) 15 to achieve live NIR-II imaging of the overall vascularity in vivo (Fig. S4C †). Although blood vessels surrounding ovarian follicles were detected, the signals were not specic and were found throughout all vasculature. The present use of specic FSH-CH allows for noninvasive imaging of FSH receptors in both antral and preantral follicles in vivo, thus providing the most sensitive and specic approach to detect follicles. Further improvement of the present FSH-CH approach and design of a portable transvaginal NIR-II probe could allow live imaging of preantral follicles in infertile female patients to be used as a diagnostic tool. For male infertile patients, NIR-II imaging of testes seminiferous tubules could also allow for better diagnosis of Sertoli cell functions in patients with oligospermia due to Sertoli cell defects. 25 An earlier study demonstrated that osteoclasts and their precursors possess Gi2a-coupled FSH receptors that activate MEK/Erk, NF-kB, and Akt to enhance osteoclast formation and promote bone loss. 9 Due to the difficulties involved in studying osteoclasts embedded in the rigid bone structure and a lack of in vivo tools to perform live imaging of bones expressing FSH receptors, earlier studies relied upon the use of isolated osteoclasts and their precursors expressing extremely low levels of FSH receptors. In addition, these ndings have been challenged by another group showing no FSH receptor expression in mouse cultured osteoclasts or bones. 10,11 Consistent with the expression of functional FSH receptors in osteoclasts, 9 our study using live animals provides direct evidence for the binding of the FSH-CH ligand to FSH receptors in vertebrae and other bones in both sexes. To our knowledge, this is the rst in vivo molecular imaging of the FSH receptor in bones.
Menopause-associated diminishment of bone density and bone fracture is a major health issue in modern society. 26 The present CH1055 uorophore has a low cell toxicity and short half-life in vivo. 16 Aer ruling out potential side effects of the present uorophore, the present NIR-II approach could provide valuable tools for in vivo evaluation of osteoclast functions in bones under different clinical conditions. Although estrogen replacement is routinely used to minimize fracture risks, side effects of estrogen include coronary heart disease, stroke, thromboembolic events, and breast cancer. 27 Because FSH blocking antibodies prevent bone loss by inhibiting bone resorption and stimulating bone synthesis, 28 our demonstration of bone FSH receptors in vivo provides the basis for designing FSH receptor antagonists to prevent bone loss in postmenopausal women contraindicated for the estrogen replacement therapy. The FSH receptor has also been shown to affect stemness and proliferation in mesenchymal stem cells (MSC) associated with osteoclast prevalence in bone marrow. 29 FSH receptors are also expressed by endothelial cells in the blood vessels of a wide range of tumors in patients. 30,31 The present approach could provide new tools for monitoring tumor progression in vivo. 32 There are only a few studies dealing with molecular imaging reagents with sensitivity to the FSH receptor, 33 the others focus on FSH receptor expressing cancer cells/tumors and vasculature associated with tumor development and progression.
The FSH receptor belongs to a subgroup of G-proteincoupled, seven-transmembrane receptors, 34 consisting of receptors for the luteinizing hormone (LH) 35 and thyroid stimulating hormone (TSH). 36 Based on the present approach, the conjugation of a NIR-II uorophore to the paralogous LH or TSH in the future could allow imaging of specic target cells expressing LH receptors (theca, luteal, and mature granulosa cells in the ovary and Leydig cells in the testes) and TSH receptors (thyroid nodules). Evaluation of LH conjugated NIR-II uorophores could allow better monitoring of Leydig cell tumors whereas TSH conjugated uorophores allow the evaluation of malignant and benign neoplasms of the thyroid in patients. 37 Because the present uorophore CH1055 has minimal cell toxicity and is rapidly excreted from the body, 16 further renement of the present NIR-II uorophore conjugation approach could open ways to perform in vivo live imaging of receptors for diverse peptide and protein hormones in their target tissues.