In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone loss1. Although oestrogen normally counteracts excessive bone resorption by promoting bone formation, this sex steroid drops precipitously during this postpartum period. Here we report that brain-derived cellular communication network factor 3 (CCN3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to build bone in lactating females. We began by showing that our previously reported female-specific, dense bone phenotype2 originates from a humoral factor that promotes bone mass and acts on skeletal stem cells to increase their frequency and osteochondrogenic potential. This circulatory factor was then identified as CCN3, a brain-derived hormone from ARCKISS1 neurons that is able to stimulate mouse and human skeletal stem cell activity, increase bone remodelling and accelerate fracture repair in young and old mice of both sexes. The role of CCN3 in normal female physiology was revealed after detecting a burst of CCN3 expression in ARCKISS1 neurons coincident with lactation. After reducing CCN3 in ARCKISS1 neurons, lactating mothers lost bone and failed to sustain their progeny when challenged with a low-calcium diet. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone for both sexes and define a new maternal brain hormone for ensuring species survival in mammals. A brain-derived hormone, CCN3, is newly identified to have a role as an osteoanabolic factor to build bone in lactating females and in the viability of offspring.

In lactating mothers, the high calcium (Ca2+) demand for milk production triggers significant bone resorption. While estrogen would normally counteract excessive bone loss and maintain sufficient bone formation during this postpartum period, this sex steroid drops precipitously after giving birth. Here, we report that brain-derived CCN3 (Cellular Communication Network factor 3) secreted from KISS1 neurons of the arcuate nucleus (ARCKISS1) fills this void and functions as a potent osteoanabolic factor to promote bone mass in lactating females. Using parabiosis and bone transplant methods, we first established that a humoral factor accounts for the female-specific, high bone mass previously observed by our group after deleting estrogen receptor alpha (ERα) from ARCKISS1 neurons1. This exceptional bone phenotype in mutant females can be traced back to skeletal stem cells (SSCs), as reflected by their increased frequency and osteochondrogenic potential. Based on multiple assays, CCN3 emerged as the most promising secreted pro-osteogenic factor from ARCKISS1 neurons, acting on mouse and human SSCs at low subnanomolar concentrations independent of age or sex. That brain-derived CCN3 promotes bone formation was further confirmed by in vivo gain- and loss-of-function studies. Notably, a transient rise in CCN3 appears in ARCKISS1 neurons in estrogen-depleted lactating females coincident with increased bone remodeling and high calcium demand. Our findings establish CCN3 as a potentially new therapeutic osteoanabolic hormone that defines a novel female-specific brain-bone axis for ensuring mammalian species survival.

Abstract Disclosure: T.Y. Hu: None. M.E. Babey: None. I. Niu: None. S.M. Nanavati: None. E.J. Murphy: None. Background: Serum estradiol levels have been studied in pre-menopausal and post-menopausal women. There is limited literature on ovarian vein estradiol concentrations. Most data are from sampling done in the setting of hyperandrogenism and primarily in post-menopausal women. This case illustrates hormone concentrations in a pre-menopausal woman who underwent ovarian vein sampling in the workup of hyperandrogenism. Clinical Case: A 33-year G1P1 woman with hypertension and prediabetes presented with three years of menstrual irregularity and infertility after IUD removal. She reported rapid weight gain and hirsutism over one year. Exam was consistent with hyperandrogenism with virilization (modified Ferriman-Gallwey score of 30). Labs showed elevated total testosterone ranging from 144-208 ng/dL (ref 2-45 ng/dL), free testosterone 32.7-35 pg/mL (ref 1.3-9.2 pg/mL), and androstenedione 2.053 ng/ml (ref 0.26-2.14 ng/ml). Cortisol post 1 mg dexamethasone suppression test, 24-hour urine free cortisol, and DHEA-S were normal. Morning unstimulated 17-hydroxyprogesterone was 80.41 ng/dL (ref follicular phase 15-70 ng/dL, luteal phase 35-290 ng/dL). Given normal DHEA-S, an adrenal etiology such as adrenocortical carcinoma was less likely. CT showed no adrenal or ovarian masses. Transvaginal ultrasound revealed numerous bilateral peripheral follicles without adnexal masses or evidence of hyperthecosis and appearance suggestive of PCOS. While the testosterone elevation could be consistent with PCOS, the clinical picture was more concerning for an androgen-secreting tumor. Given the concern for ovarian etiology and patient’s desire for fertility, ovarian vein sampling was performed by a radiologist experienced with adrenal vein sampling to localize a suspected tumor. The right ovarian vein is generally harder to sample than the left. Results were consistent with androgen hypersecretion on the right (right: total testosterone 5,817 ng/dL, androstenedione >40 ng/mL, DHEA-S 20.084 ng/mL; left: total testosterone 189 ng/dL, androstenedione 1.798 ng/mL, DHEA-S 1.893 ng/mL). Estradiol levels were notably elevated on the right at 1945 pg/mL compared to 70 pg/mL on the left. The patient underwent right oophorectomy with pathology showing nodular stromal hyperthecosis and a large corpus luteum cyst. Hyperandrogenism resolved post-operatively with total testosterone 14 ng/dL. Conclusion: We report hormonal profiles from ovarian vein sampling in a pre-menopausal woman with unilateral stromal hyperthecosis. There is limited literature on ovarian vein in this entity and no prior data on estradiol levels. We hypothesize estradiol levels were elevated due to aromatase activity in the right ovary. Testing from the presumably unaffected left ovarian vein helps to characterize ovarian vein estradiol levels in a pre-menopausal woman with suspected hyperandrogenic anovulation. Presentation Date: Saturday, June 17, 2023