In Bactrian camels, the seminal plasmas ovulation inducing effect was reported around 30 years ago and the substance responsible was termed as ovulation-inducing factor (OIF). Studies have confirmed that in llamas and alpacas based on biological and chemical properties, OIF was identified as βNGF. OIF is a highly conserved protein and studies have revealed that presence and function of seminal OIF are conserved among species that are considered to be induced ovulators as well as those of spontaneous ovulators. Abundant amounts of OIF/NGF in seminal plasma and seminal plasma effects on ovarian function support the endocrine mode of action. The present review focuses on nature, effects and possible mechanisms of action of OIF/NGF.
Based on the type of stimulus responsible for eliciting GnRH release from the hypothalamus, mammals are classified into spontaneous or induced ovulators. In induced ovulators such as rabbit, bactrian camel, llama, alpaca, cat and ferret, copulation elicits neural signals responsible for triggering hypothalamic GnRH secretion, which leads to preovulatory discharge of LH from the pituitary (Bakker and Baum 2000). The presence of an elaborate accessory gland system in male species and induction of ovulation in females are two most intriguing mysteries in the field of biology of reproduction. Traditionally, these two processes have been thought as simply vestiges of primitive lineages (Bedford 2004; Kauffman and Rissman 2005), but current literature available about the presence of ovulation-inducing factor (OIF) in seminal plasma may provide some knowledge that links these processes together. Induction of ovulation by copulation was discovered more than 100 years ago by Walter Heape, who discovered doe rabbit permits coitus during oestrus, and withholding male at that time leads to degeneration of ova in the ovary; they are not released from the ovary (Heape 1905). In South American camels, induced ovulation phenomenon was reported in the late 1960s (England et al., 1969; San Martin et al., 1968) and about a decade later in bactrian and dromedary camels (Chen and Yuen 1979). Previous literature yielded that in >95% of females, ovulation occurs after mounting and intromission and only <14% in absence of later (Fernandez-Baca et al., 1970). Thus during copulation, physical stimulation of genitalia is primarily involved in ovulation in case of induced ovulators. Contrary to these, some previous studies in Bactrian camels revealed that some agent present in the semen was responsible for eliciting ovulation in Bactrian camels, rather than the copulation induced mechanical stimulation. The administration of Bactrian camel seminal plasma intravaginal (Chen et al., 1985; Xu et al., 1985), intramuscular or intrauterine lead to induction of ovulation in female Bactrian camels. An injection of a single intramuscular dose of seminal plasma in females of llamas and alpacas resulted ovulation in >90% females (Adams et al., 2005).
Nature of OIF
Due to LH-releasing effects (Paolicchi et al., 1999) and GnRH immuno-reactivity in seminal plasma of humans (Sokol et al., 1985) lead to the supposition that OIF may be related to the GnRH. Results documented that OIF is not a steroid, prostaglandin or GnRH rather it is a protein which is resistant to heat and enzymatic digestion with proteinase K and has a molecular mass of approximately of 30 kDa (Ratto et al., 2010). Data from X-ray diffraction revealed that OIF was similar to beta-nerve growth factor (ß-NGF) and it was revealed that biological activity of OIF was similar to NGF (Adams et al., 2016). Purified OIF from llama’s seminal plasma is a highly conserved protein ß-NGF (Ratto et al., 2012). In the seminal plasma of alpacas, ß-NGF is a protein that is expressed majorly and is involved in inducing ovulation (Kershaw-Young et al.,2012). As revealed from previous purification experiments, seminal plasma of bull is a rich source of that bovine seminal plasma NGF (Harper et al., 1982) and is likely to be produced by seminal vesicles (Hofmann and Unsicker 1982). It has also been reported in the prostate gland of bulls, rabbits and guinea pigs (Harper et al., 1979, 1982).
Effects of OIF / NGF
LH Release and Ovulation
In alpacas and llamas, injecting seminal plasma intramuscularly (equivalent to one-fourth to half of an ejaculate) resulted ovulation in 33 of 35 (94%) females as compared to 0 of 35 (0%) with saline (Ratto et al., 2005) and LH surge timing was similar to that reported after natural mating (Bravo et al., 1990). LH surge duration was prolonged after treatment with seminal plasma as compared to GnRH treatment (Adams et al., 2005). Ovulations were detected 29.3±0.7 hr after treatment with seminal plasma as revealed by ultrasonographic examination every 4 hr (Adams et al., 2005), which were similar to interval after natural mating or treatment with GnRH or LH (Ratto et al., 2006). Transcervical intrauterine deposition of seminal plasma in alpacas did not resulted in ovulation. These differences may be due to reduced absorption of OIF from the genital mucosa than intramuscular administration (Adams et al., 2005). Camelids have copulation time of 30–50 min (Bravo et al., 1990) and semen is deposited uterus, normally due to copulation, there is acute, transient inflammation of endometrium resulting from repeated abrasions by the penis (Bravo et al., 1996). In alpaca’s intrauterine treatment with endometrial curettage and twice dose of seminal plasma revealed that 41% females ovulated without curettage treatment and 67% ovulated in the curettage group (Ratto et al., 2005). Seminal plasma is more effective when given through intramuscular route as compared to intrauterine and in case of rabbits GnRH dose needed for ovulation was 10 to 20 times higher when given intravaginally than intramuscularly (Rebollar et al., 2012). It is the degree of absorption of a seminal factor from the genital mucosa into circulation (i.e. systemic dose) and not the response of tubular genitalia to physical stimulation that is responsible for ovulation (Adams et al., 2016). Seminal plasma has been used for induction of ovulation in llamas, in absence of copulation and copulation alone cannot result in ovulation without the presence of seminal plasma (Berland et al., 2016).
Dose Related Effects of OIF/NGF on Ovulatory Responses
Studies have revealed that OIF has a dose-related effect on the ovulatory mechanism and this effect is pronounced at various physiologically relevant doses, which may be as little as 1/100th that present in an ejaculate. It has also been observed that dose of OIF required to elicit pituitary and ovarian responses was higher when administered by intrauterine infusion than by intramuscular or intravenous routes (Silva et al., 2015). Surge in plasma LH levels and rates of ovulation were similar in female llamas that were injected with 2 mg OIF intramuscularly or intravenously, however, those treated by intrauterine deposition did not resulted in LH response and ovulations. But when intrauterine dose was increased to the total amount present in an average llama ejaculate (i.e. 5 ml seminal plasma or 20 mg of OIF), resulted a surge in LH concentration as compared to that yielded with lower intramuscular or intravenous dose (Adams et al., 2016).
Luteotrophic Effect of OIF/NGF
A functional corpus luteum (CL) is indispensible for maintenance of pregnancy) throughout gestation in camelids (Al-Eknah et al., 2001). In llamas, maintenance of functional CL between 8 and 10 days after ovulation has been implicated in maternal recognition of pregnancy (Adams et al., 1991), and early luteogenic processes may be associated with the ability of CL to respond early pregnancy signals. It has been seen that CL was larger in female llamas that were treated intramuscularly with a conservative dose of homologous seminal plasma; moreover CL regressed later and produced more than two times progesterone than CL resulting from GnRH-induced ovulation luteotrophic effect may be attributed to elicitation of LH secretion pattern by seminal plasma. Seminal plasma induced LH surge was sustained beyond the 8-hr sampling period in animals treated with OIF and after GnRH treatment; concentrations of LH were basal by 6 hr. The surge in plasma LH concentration triggered by seminal plasma was sustained beyond the 8-hr sampling period in OIF-treated animals, whereas LH concentrations were basal by 6 hr after GnRH treatment (Adams et al., 2016). Angiogenesis plays a pivotal role during the formation of CL, due to the fact that CL receives highest blood supply per unit of tissue than any other organ of the body (Wiltbank et al., 1988).
OIF/NGF treated llamas had greater blood flow to the pre-ovulatory follicle 4 hr post treatment as compared to GnRH treated ones. In addition to this OIF/NGF treated llamas had also higher vascular flow to the CL and greater concentrations of plasma progesterone 6 days after treatment (Adams et al., 2016). Giving two doses of OIF/ NGF, given just before and at the time of ovulation resulted in development of larger CL with greater vascularization and also produced higher progesterone concentrations as compared to single pre-ovulatory dose induced CL (Fernandez et al., 2014). In cattle (a spontaneous ovulator), both camelid and bovine seminal plasma were found to be luteotrophic, although, no measurable increase in LH concentrations in plasma were detected (Tribulo et al., 2015). Recent findings in cattle revealed that OIF/NGF induced luteotrophic effect is mediated by directly at the level of the ovary via interaction with trkA receptors of theca and granulosa cells of developing CL and dominant follicle (Carrasco et al., 2016). NGF may be an important mediator of ovulation due to seminal plasma in llamas due to the fact that ovulation does not take place if blood βNGF levels do not increase. This increase in blood βNGF levels occurs after copulation with an intact male or intrauterine infusion of seminal plasma (Berland et al., 2016).
Possible Mechanisms of Action
Although it is clear that seminal OIF/NGF responsible for ovulation mediates the effect on through surge release of LH into circulation, however it is not clear whether site of action of OIF is primarily at the site of hypothalamus or also involves the pituitary gland. Study revealed that GnRH antagonist (cetrorelix) pretreatment of llamas resulted in blockade of LH release and ovulation, which suggest a direct or indirect effect of OIF on GnRH releasing neurons in hypothalamus (Silva et al., 2011). In ovariectomized llamas, response of LH to OIF/NGF treatment was muted and after pre-treatment with oestradiol resulted in partial restoration of LH response, in agreement with the hypothesis that hypothalamus is involved in the pathway of OIF/NGF (Adams et al., 2016). Numerous in vitro studies documented that effect of OIF/NGF on pituitary gonadotrophs is direct. An induced release of LH secretion was reported following treatment of primary cultures of llama and bovine anterior pituitary cells and the magnitude of LH release was proportional to treatment dose (Bogle et al., 2012). Purified OIF or seminal plasma from Bactrian camels or alpacas addition to primary culture of rat pituitary cells caused the secretion of LH (Zhao et al., 2001). Binding with specific (trkA) and non-specific (p75) receptors is involved in effect of OIF/NGF on the hypothalamo-pituitary-ovarian axis. In induced ovulators, neural pathways involved in the activation of GnRH neurons are not well understood. Initial studies of the pattern of distribution revealed that around 60% of GnRH neurons were located in the anterior and medio-basal hypothalamus and were scattered widely rather than in focal accumulations or nuclei (Carrasco 2016). OIF/NGF molecule with over 100 amino acids and molecular mass of 26 kDa, is so big that it cannot diffuse through the blood–brain barrier (Banks 2009) and hence needs an active transport mechanism. In rats, NGF receptors have been found within the hypothalamus (Gibbs and Plaff 1994). It has been observed that elicitation of the LH surge using exogenous OIF/NGF begins and peaks 1-2 hr after that elicitation by exogenous GnRH (Adams et al., 2016). The delayed response with OIF/NGF may be due to an intermediate step in the pathway needed for release of GnRH/LH. The LH surge magnitude was dose-dependent following treatment with the two peptides (Silva et al., 2012; Tanco et al., 2011). Some other possible pathways reveal direct action on terminals of GnRH neurons or a direct action on pituitary gonadotrophs. In 75% of LH-containing gonadotroph cells and 44% of those cells with high-affinity NGF receptor trkA in anterior pituitary cells of rat (Patterson and Childs 1994). Some school of thought suggests β1 tanycytes are involved in pulsatile release of GnRH into the portal blood (Rodriguez et al., 2005). In the lateral regions of the median eminence, GnRH nerve fibres and their endings are concentrated and separate from the perivascular space by unique cells called tanycytes, which line the floor of third ventricle (Rodriguez et al., 1979). Within the median eminence, OIF/NGF stimulates the release of molecules that regulate the transient and cyclic release at the GnRH terminals (Prevot, 2002). Another hypothesis is that organum vasculosum of the lamina terminalis (OVLT) outside of the blood-brain barrier may be involved in sensing the travelling molecules in the systemic blood (Herde et al., 2011; Rodriguez et al.,2010).
OIF/ NGF in the Male
NGF was initially reported from prostate gland of guinea pig (Harper et al., 1979); later on research was carried out to explore the source and abundance of NGF in the male accessory glands of other species. Among the prostate glands of the guinea pig, the rabbit and bull, the highest concentrations of NGF were found to be contained in prostates of guinea pig. Recent researches have revealed that at least one male accessory gland in all species contains OIF/NGF (Bogle 2016). Prostate gland appears to be main source of OIF/NGF in llamas, rabbits, guinea pigs and white-tailed deer and ampullae and seminal vesicles in cattle and bison. On the basis of response to in vivo bioassay (ovulation), in vitro bioassay (PC12 cells) and immunoassay (ELISA, RIA, immuno-histochemistry; Bogle 2016; Tribulo et al., 2015; Maranesi et al., 2015; Casares-Crespo et al., 2016), highest levels of OIF/NGF appears to be in camelids and rabbits. In llamas, 90-100% of ovulation rate was reported on treatment with seminal plasma of camelid or rabbit (Adams et al., 2005; Silva et al., 2011) and ovulation rates were 26% with bull semen (Ratto et al., 2006), and 18%, 29% using stallion and boar semen, respectively (Bogle et al., 2011). When compared to llama seminal plasma, concentration of OIF/NGF in bovine seminal plasma was only 10 to 20% and female llamas treated with bovine seminal plasma, with a dose of OIF/NGF equivalent to that found in llama seminal plasma yielded ovulation rates similar to that an equal dose of OIF/NGF induced by llama seminal plasma (Tribulo et al., 2015).
The ovulation inducing effect of seminal plasma in camels is reported to be due to presence of protein known as OIF, was identified as βNGF. In reproductive tract, the presence and function of an OIF/NGF system has been reported in many species of animals including various spontaneous and induced ovulators. Seminal plasma effects on ovarian function and presence of abundant OIF/NGF in it, supports endocrine mode of action of OIF. Ovulation is brought about by LH surge in the llamas and alpacas, and it is the function of the degree of absorption of a seminal factor from the genital mucosa into circulation. The magnitude of LH releases (central mechanism) and changes in the expression of specific receptors in ovarian follicles (local mechanism) may be responsible for luteotrophic effect of OIF/NGF. Seminal plasma of camels and rabbits has highest levels of OIF/NGF but has been identified in at least one of the male accessory glands in all species. Further studies are needed to determine the prevalence and function of OIF among various species and to test some unexplained causes of infertility which may be based on alterations in levels of OIF.