Received December 24, 2001; Revised February 25, 2002; Accepted March 4, 2002. Author to whom all correspondence and reprint requests should be addressed: Dr. F. F. Casanueva, Department of Medicine, Endocrine Unit, San Fran- cisco Street s.n., PO Box 563, E-15780, Santiago de Compostela, Spain. E-mail: endocrine@usc.es Evidence of Free Leptin in Human Seminal Plasma Jesus P. Camiña, 4 Mary Lage, 4 Carmela Menendez, 4 Maria Graña, 3 Jesus García-Devesa, 1 Carlos Dieguez, 2 and Felipe F. Casanueva 4 1 Central Laboratory and Departments of 2 Physiology, 3 Gynecology, and 4 Medicine, School of Medicine and Complejo Hospitalario de Santiago, Santiago de Compostela University, Spain Endocrine, vol. 17, no. 3, 169–174, April 2002 0969–711X/02/17:169–174/$11.50 © 2002 by Humana Press Inc. All rights of any nature whatsoever reserved. 169 Leptin is an adipose tissue–secreted hormone that actively participates in the regulation of energy hom- eostasis. Besides this principal role, leptin has been implicated in a large variety of neuroendocrine, para- crine, and autocrine actions involved in the regulation of reproductive function in both experimental animals and humans. Although the participation of leptin in female reproduction is well established, any role in male reproductive function is at best tenuous. The aim of this study was to ascertain whether true leptin is present in human seminal fluid and the tissue of its production. Pooled human seminal plasma obtained from healthy donors showed by direct radioimmuno- assay (RIA) the presence of radioimmunoassayable lep- tin. Serial dilutions of unextracted semen paralleled the RIA standard curve, also devoid of interference in the assay. To prove that this activity was true leptin, seminal plasma was subjected to size-exclusion chro- matography, which showed that leptin immunore- activity eluted with the same partition coefficient as cold leptin, 125 I-leptin, and 125 I-leptin preincubated with seminal plasma. The results demonstrate that true lep- tin was present in semen in a free form, i.e., without binding proteins. The presence of leptin charge variants in seminal plasma was assessed by anion-exchange chro- matography, which showed two peaks of leptin inmuno- reactivity, while 125 I-leptin eluted as a single peak. Preincubation of 125 I-leptin with seminal fluid con- verted the single peak into a double peak, indicating that components of the seminal fluid introduce a charge variation in leptin. Leptin levels in seminal plasma of 40 healthy men were 0.95 ± 0.19 ng/mL while in 5 vas- ectomized men the levels were 0.92 ± 0.25 ng/mL, sug- gesting that testicular tissues were not the source of seminal leptin. No correlation was observed between leptin concentrations in semen and the physical char- acteristics of semen samples or physical characteris- tics of spermatozoids, such as concentration, motility, vitality, or morphology. In conclusion it was unam- biguously demonstrated that human leptin is present in seminal fluid, with at least two charge variants and no binding proteins, the most likely source being either seminal vesicles or prostate tissue. The role of seminal fluid leptin in the male reproductive function or sperm capacitation is at present unknown. Key Words: Leptin; semen; seminal plasma; body mass index. Introduction Leptin is a pleiotropic hormone produced by white adi- pose tissue, acting as the afferent loop to the hypothalamus, and contributing to energy balance by informing the brain of the amounts of adipose reserves in a given individual (1). Although it was originally thought of as the hormone regu- lating obesity, it soon became clear that leptin is actually the hormone regulating fasting, since its reduction in the situation of low energy intake puts into gear the multiple hormonal adaptations of fasting (2). In addition to its role in the regulation of energy homeostasis, leptin has been impli- cated in several neuroendocrine activities, such as regulation of growth hormone (3) and adrenal (4) and thyroid hormone secretion (5). Leptin either produces or acts in several tis- sues in a paracrine-autocrine way (6,7); however, the physi- ologic role, if any, of such actions has yet to be resolved. In addition to regulation of body composition, the physi- ologic area in which leptin has been most seriously impli- cated is reproduction. The overwhelming data on this topic can be summarized as follows: First, circulating leptin levels are higher in females than in males of any species, and this is so even at birth (8–11). Second, leptin seems to exert a per- missive role for puberty in both female experimental ani- mals (2,12) and women (13–15). Third, serum leptin levels change through pregnancy and postpartum period (16). Finally, leptin exerts regulatory actions on pituitary gona- dotropins and at the gonadal level (17–20). Interestingly, leptin also seems to be implicated in some local or paracrine actions that are still poorly understood. For example, it is present in milk crossing the neonatal intestinal barrier (21); it is present in relevant concentrations in placenta (22,23);