Evidence for the LH-releasing pathway of seminal plasma NGF in male camelids

Highlights

• NGF induces LH release in male camelids.

• NGF -induced LH secretion may be related to testosterone secretion in male llamas and alpacas.

Abstract

In the female camelid, systemic administration of NGF induces a preovulatory LH surge that results in ovulation, but the effects of seminal NGF in the male are unknown. In the present study, we tested the hypothesis that the LH-releasing pathway of NGF is present in male camelids. In Experiment 1, male llamas and alpacas were treated with NGF or GnRH (n = 2 llamas and 3 alpacas) and blood samples were collected from 1 h before to 3 h after treatment. Plasma LH concentrations increased after treatment in a surge-like fashion in both GnRH- and NGF-treated groups, but concentrations reached a maximum 2.5 times higher and remained elevated for at least 2 h longer in the NGF-treated group (treatment-by-time interaction, P = 0.01). In Experiment 2, we evaluated the LH and testosterone response to NGF vs saline treatment (n = 3 llamas and 3 alpacas). The LH response to NGF was similar to that in Experiment 1, and plasma testosterone concentrations were higher in the NGF group than in the saline group at 2, 4 and 6 h after treatment (P < 0.05). Results support the hypothesis that the LH-releasing pathway for NGF exists in male South American camelids. The LH response to NGF sustained circulating testosterone concentrations in llamas, suggesting a moderate role of NGF in testosterone secretion.

Introduction

Induced ovulators are a group of species in which ovulation is elicited by mating. Classic studies in rabbits over 100 years ago showed that ovulation was triggered by coital stimulation [1], and the putative mechanism was generalized thereafter to other induced ovulators. South American camelids are classified as induced ovulators [2], but the preovulatory LH surge and ovulation in camelids is induced by a protein present in seminal plasma [3,4], not by coital stimulation during mating [5,6]. Originally termed ovulation-inducing factor, the causative protein in seminal plasma was later identified as nerve growth factor (NGF) by peptide sequencing, protein crystallography, and in vitro cell bioassay [7]. Although seminal NGF triggers ovulation by inducing the release of LH from the anterior pituitary in vivo [8] and in vitro [9], the site of action of NGF of appears to be the hypothalamus [10].

The reproductive behavior of male camelids presents unique features. For instance, during mating the female assumes sternal recumbency and the male mounts her in half-sitting position [11]. Compared to other species, mating in camelids is a prolonged event, averaging 19 min in llamas and 17 min in alpacas, with a range of 5–40 min [12]. In a study in which ejaculation in llamas was characterized by digital palpation of urethral pulses, ejaculation occurred throughout mating, with urethral pulses occurring in clusters at a rate of approximately 1 cluster per minute [13]. The uterine lumen is the site of semen deposition during ejaculation in camelids, and the largest component of seminal plasma is NGF, representing up to 30% of total protein content [14]. The discovery that seminal plasma had a major role in triggering ovulation in camelids [3] led the discovery of its presence in the seminal plasma of several species. To date, NGF has been confirmed in the seminal plasma of bull, ram, boar, stallion, human, and rabbit [[15], [16], [17], [18], [19], [20]]. In addition, the presence of both NGF receptors have been described in sperm cells of various species [[21], [22], [23]]. Although NGF gene and protein have been found to be expressed in the prostate of male camelids [23,24], the mechanism of NGF synthesis, secretion and transport in the male reproductive tract, as well as the role of NGF on sperm function are poorly understood in camelids and other species.

Both females and males display a pulsatile pattern of LH secretion attributed to pulsatile secretion of GnRH [25,26] since immunoneutralization against GnRH blocked pulsatile secretion in ewes and rams [27,28]. However, between puberty and reproductive senescence, sexually mature females display periodic (in spontaneous ovulators) or sporadic (in induced ovulators) surges in circulating LH concentration that precede ovulation. The preovulatory LH surge results in concentrations several fold above baseline for several hours, and triggers a cascade of events that culminates in the rupture of the ovulatory follicle, release of the oocyte and subsequent formation of the corpus luteum. In contrast, males maintain a pulsatile pattern of LH secretion throughout their lifetime. While males do not have such “preovulatory surges”, pulse frequency and amplitude of plasma gonadotropins fluctuate in both the male and the female with negative feedback loops imposed by steroid hormones and inhibin [29]. Castration resulted in increased plasma LH concentration in males of a range of species, and, in seasonal species, plasma FSH and LH concentrations were greater during breeding season than non-breeding season [29]. Conversely, chronic treatment with GnRH in rams increased LH and testosterone blood concentrations, scrotal circumference, sperm production and live sperm in semen [30]. Perhaps, the LH-releasing pathway of NGF is also operant in males and plays a role regulating reproductive function.

As an initial step to evaluate the effects of NGF in male camelids, in the present study we tested the hypothesis that NGF elicits an LH secretory response in male camelids. The specific objectives were to determine the effect of treatment with NGF (of seminal plasma origin) on circulating concentrations of LH (Experiment 1) and testosterone (Experiment 2) in male llamas and alpacas.