OT neurons in the brain are largely aggregated in several neuroendocrine nuclei, typically the supraoptic nucleus (SON) and paraventricular nucleus (PVN). In the SON and PVN, most OT neurons send axons to the posterior pituitary and are magnocellular neuroendocrine cells. These OT neurons can release OT into the blood from OTergic terminals in the posterior pituitary and into the forebrain from OTergic axon collaterals (2). Another type of OT neurons in the CNS is parvocellular OT neurons. They are mainly present in the parvocellular division of the PVN and project to the brainstem and spinal cord but not the posterior pituitary (3). Alongside the hypothalamic OT neurons, some OT cells are also present in extrahypothalamic regions in the CNS and peripheral sites, such as amygdala, the median preoptic nucleus, uterus, placenta, amnion, corpus luteum, testis, heart and colon (4–6). Notably, OT gene expression in chorio-decidual tissues can increase three- to fourfold around the time of labor onset (7–10), supporting OT functional role in parturition. These histological features allow OT to modulate body functions at multiple levels and patterns including neuromodulation, neurosecretion, endocrine, autocrine and paracrine effects.

OT release from OT neurons is microdomain-specific (11). In response to changes in the neurochemical environment around OT neurons, changes in cytosolic Ca²⁺ level and/or firing activity occur. Increased firing rate causes OT release at the axonal terminals via excitation-secretion coupling. Increased cytosolic Ca²⁺ level triggers OT release from somata and dendrites. The somatodendritic OT release can be in synchrony with the firing activity under physiological conditions, such as suckling stimulation (12). It can be independent of the firing activity under some pathological conditions, such as maternal depression following offspring deprivation (13) and cesarean delivery (14).

The electrical activity, cytosolic Ca²⁺ level and the resultant OT secretion are under the regulation of extracellular and intracellular factors ( Figure 1 ). These factors include changes in neurochemical environment (15), tonic and clustered synaptic inputs (16, 17), intercellular junctional coupling such as connexin 36 (18), and astrocytic plasticity (19, 20). Moreover, autoregulation of OT neuronal activity is a key regulator of OT neuronal activity (21). By activation of OT receptors (OTRs), OT causes activation of a series of intracellular signaling events, such as increased cyclooxygenase-2 (21), extracellular signal-regulated protein kinases 1 and 2 (22), and protein kinase A (13). These signals can activate hyperpolarization-activated cyclic nucleotide-gated channel 3 (13, 23), which can promote OT secretion (24, 25). Under the regulation of these extracellular and intracellular factors, OT neuronal activity and OT secretion can meet the demands of body activity in response to environmental changes.

OTRs are the protein product encoded by OTR gene that is localized at 3p25-3p26.2 in the human chromosome (4). OTR belongs to the G-protein-coupled receptor superfamily and its activation is regulated by cholesterol as an allosteric modulator (26). The expression profile of OTRs is a tissue- and stage-specific, such as upregulation of the nuclear fractions in term myometrium and down-regulation in non-pregnant myometrium. The OTR gene appears to be highly methylated. Methylation around intron 1 and in intron 3 might contribute to tissue-specific suppression of the gene. OTRs are also regulated by the mechanisms of desensitization, which causes the loss of ligand-binding activity of the protein as well as suppression of OTR mRNA transcription. OTR mRNAs are present in different sizes, for instance 3.6 kb in human breast and 4.4 kb in ovary, endometrium, and myometrium. In posttranslational modifications, OTRs are further palmitoylated and glycosylated (27, 28). In addition, species differences are present and may be due to the existence of different clones of OTR genes of the myometrium and the hypothalamus at different reproductive stages (29, 30). Thus, the phenotype of OTRs from different species and different tissues of the same species could appear in different sizes.

In mammals, OTRs have been identified in a broad spectrum of tissues, including the kidney, heart, thymus, pancreas, adipocytes and other sites in addition to the CNS (4). Expressions of OTRs in the hypothalamus, uterus, and mammary glands are stimulated by estrogen (31). In females, OTRs are specifically localized in the myoepithelial cells of the mammary glands, and in the myometrium and endometrium of the uterus. Peripheral actions of OT are commonly associated with smooth muscle contraction, particularly within the female and male reproductive tracts (32).

OT expression during pregnancy and parturition in females has some unique features unseen in males. Changes in brain OT binding sites during pregnancy may influence the sudden onset of maternal behavior in female rats at parturition. Transcriptional regulation of OTR gene expression mediates changes in receptor density in the brain in a region-specific manner during pregnancy, such as the uterus (33), the PVN, SON, the bed nucleus of stria terminalis (BNST) and the medial preoptic area (mPOA) (34). Moreover, peak OTR mRNA expression was observed at parturition in the SON, brainstem regions, mPOA, BNST, and olfactory bulbs. Postpartum OTR expression in all brain regions returned to levels observed in virgin rats (35). These features of OTR expression are in agreement with the demands of establishing maternal behaviors and parturition.

Correspondingly, the uterus transitions from a quiescent non-contractile state to an active contractile state at the end of pregnancy. This is in association with increased OT/OTR signals (10, 36). Uterine quiescence requires prevention of excessive Ca²⁺ influx through voltage-dependent Ca²⁺channels at the human myometrial smooth muscle cells. Through most time of pregnancy, the K⁺ leak current is dominant and maintains the cell at a sufficiently negative membrane potential to prevent premature uterine contraction. However, increased myometrial OT/OTR expression near the term (10, 36) significantly increases OTR-associated Gαq-protein activation of protein kinase C (33), which then inhibits the activity of sodium-activated, high-conductance, K⁺ leak channel. This results in depolarization of the uterine smooth muscle cells and calcium entry that causes uterine contraction (37). This effect is associated with the induction of cyclooxygenase 2, production of prostaglandin F2α and connexin 43 of the uterus (38, 39).

The functions of OT depend on the distribution of OTRs and relative change in OT levels. In the brain and spinal cord, activation of OTRs is associated with a variety mental activities, such as social memory, pair-bonding, maternal behavior, and aggression and instinctive behaviors such as sexual activity, anxiety, feeding and pain perception (40–42). In the circulation, OT can facilitate parturition and the milk-ejection reflex and regulate activities of other organ systems (43, 44). Locally-produced OT can promote the differentiation of thymic cells, inhibit inflammation (45), protect the heart from ischemic injury (46), and suppress metastasis of colorectal cancer (5) among many other functions (1, 4).

In the CNS sites, OT can regulate social activity, instinctive behaviors, and visceral functions. For example, by activation of inhibitory neurotransmission in the medial frontal cortex, the amygdala and hippocampus, OT can promote social recognition and pro-social behaviors while reducing stress and fear (47–49). Released from parvocellular OT neurons in the PVN, OT can act on the ventral tegmental area (VTA) to activate rewarding process. OT can suppress nociception ad pain by acting on the periaqueductal gray and spinal cord. By acting on the dorsal vagal complex, OT can regulate visceral activities via vagus. By innervating median eminence and median preoptic area, OT can increase gonadotropin-releasing hormone (GnRH) release and the activity of HPG axis (1, 50). In addition, intrahypothalamic OT can inhibit corticotrophin-releasing hormone neurons and social stress via acting on the parvocellular division of the PVN (51).

By contrast, circulating and locally-produced OT can influence body functions at cellular, tissue, organ and system levels. For instance, OT can promote insulin secretion (52) and immunological homeostasis (53), protects cardiovascular system (46), suppresses colorectal cancer migration (5), and potentially antagonizes COVID-19 pathogenesis (54). In addition, OT is a natriuretic hormone that acts directly on the kidneys (55). On the other hand, its synthesis and secretion are regulated by changes in plasma osmolality and blood volume (56, 57). This process proceeds in an estrogen-dependent manner in females (58, 59). Thus, OT extensively modulates body functions.

In studies on OT functions, significant difference between males and females emerges, which is particularly significant in psychological and reproductive functions.

Maternal behavior begins before breastfeeding near the term and is clearly associated with the actions of OT. For example, in OT knockout mice, maternal behavior is disrupted (83) and maternal behavior is reduced significantly when OTR signaling is blocked (84). By contrast, IAO rapidly increases maternal care in mice (85). It is believed that OT neurons in the PVN are sensitive for the smell of offspring in lactating and multiple-parturient rats (86). OT neurons in the PVN and SON project to the hippocampus, amygdala, ventral striatum, hypothalamus, nucleus accumbens and brainstem nuclei; they can extensively modulate maternal behaviors (2, 3). By modulation of the maternal behaviors, OT convincingly promotes human development as recently reviewed (87, 88).

Notably, women with peripartum exposure to synthetic OT have a higher relative risk of depressive or anxiety disorder diagnosis or antidepressant/anxiolytic prescription within the first year postpartum than women without synthetic OT exposure (89). This is likely due to the suppression of excessive OT on OT neuronal activity via a mechanism of post-excitation inhibition of OT neuronal activity (14, 23, 90).

Under pathological conditions, reduced OT release or disorders in OTR signaling can cause many diseases, such as social stress (51), schizophrenia and depression (91), obesity (92), lactation failure, postpartum depression (13, 13) and even mammary tumor (93). For instance, schizophrenia is a form of mental disorders and involves dysregulation of the OT system. From animal models to human studies, observations have revealed that OT can improve the psychopathology of patients with schizophrenia by regulating social cognition and behavior (94). Postpartum depression is associated with disorders on maternal OT system. As reported in rats, lack of suckling stimulation and bolus OT release during lactation can decrease maternal blood OT levels and cause milk insufficiency and maternal depression in lactating mothers deprived of offspring (13, 95) or following cesarean delivery (14). However, disrupted maternal behavior and lactation performance can be largely improved by IAO during offspring deprivation (13) or following cesarean delivery (14). Obviously, disorder in OT system activity contributes to disorders in mental activity while IAO has the potential to correct abnormal social behaviors such as those in schizophrenia and postpartum depression.

Notably, in mediating OT actions, the efficiency of OTRs depends on their gene polymorphisms, expression levels and posttranslational modification (96–98). For example, the GG homozygotes of OTR rs2254298 are associated with childhood adversity (99); DNA methylation in the 1st intron of the OTR gene causes common learning and behavioral impairments (100). Thus, OT-associated diseases are not only associated with disorders in OT secretion but also with abnormality of OT/OTR signaling.

It is well established that a menstrual cycle is regulated by HPG axis. However, studies also highlight a pivotal role of OT in menstrual cycle. For instance, increased brain OT level can increase GnRH secretion, specifically at pre-ovulation stage (101, 102). Consistently, plasma OT is significantly higher during ovulatory phases than the luteal phase in ovulating women (103). In rats, c-Fos expression in the SON is significantly higher and OT neurons-associated astrocytic processes retract significantly during the proestrus (104, 105). These findings support functional activation of OT neurons and increased OT release before ovulation. Thus, the stimulatory effect of OT on GnRH secretion can promote luteinizing hormone (LH) secretion, facilitate ovulation and prepare uterus environment for pregnancy.

By contrast, exogenous OT can shorten postpartum estrous interval but triggering a delay in ovulation while a higher dose of OT could stimulate the growth of small, medium, and total follicles in postpartum buffaloes (106). Estradiol level increase is correlated with OT release from the pituitary and causes more luteal OT secretion (107). Thus, OT secretion and estrogen release before luteal phase can form a positive feedback loop and they together facilitate ovulation ( Figure 2A ).

During pregnancy, persistent release of uterine progesterone and estrogen interrupts the periodical activity of the HPG axis. Moreover, increased progesterone also inhibits OT release (108). They together cause the cessation of menstrual cycles. Following parturition, breastfeeding delays the resumption of normal ovarian cycles by disrupting the pattern of pulsatile release of GnRH from the hypothalamus. Intermittent bolus release of OT during suckling, its disruption of normal interaction of OT with the HPG axis and inhibition of energy intake also play a key role in lactation-associated amenorrhea (92). When suckling stimulus declines near weanling, preovulatory LH surge restores and ovulation takes place with the formation of a corpus luteum of variable normality.

By contrast, women with a history of primary or secondary dysmenorrhea have lower blood OT concentrations during menses, which is associated with worse menstrual pain and pain-related behavior (61). In women with early life sexual abuse, OT can positively modulate menstruation-related mood disorders (109). In addition, OT can enhance conditioned safety memory (73), elicit maternal behavior towards alien pups in virgin females (110), alleviate chronic pelvic pain (111) and rehabilitate anorexia nervosa (112). Thus, OT extensively modulates mental and physical activities and menstruation-associated diseases.

In all mammals, OT is a hormone necessary for mothers to nurse their offspring through the milk-ejection reflex. Clearly, in OT-knockout mice, the pups cannot obtain milk from the mother because the dams fail to eject milk for the offspring to obtain through suckling the nipples (119). Consistently, conditioned OTR knockout dams experience high rate of pup mortality (120).

Successful breastfeeding depends on coordinated activities of numerous humoral factors. For instance, the increased hypothalamic OT release during suckling promotes prolactin secretion from the anterior pituitary. They together make milk production and ejection available for the baby during breastfeeding (121). This effect is different from the increased prolactin during pregnancy that acts to maintain the corpus luteum of the ovary and helps to sustain pregnancy but has no direct association with OT release (122). Under physiological condition and normal nutrition, breastfeeding in normally developed women relies on the milk-ejection reflex. In this reflex, suckling stimulation at the nipples activates OT neurons, causes OT release in a bolus into the blood, and results in the ejection of milk from the teat ( Figure 2C ). The activation of OT neurons appears as intermittently recurrent and simultaneous increase in the firing rate in a large pool of OT neurons over several seconds, which causes the bolus release of OT at OTergic neural terminals in the posterior pituitary (44). Without this synchronized burst firing of OT neurons, OT release from one or a few of OT neurons or only one side of the hypothalamus cannot trigger full milk ejections (123, 124). In the synchronized activation of OT neurons, simultaneous retraction of astrocytic processes from OT neurons, shared synapses, increased gap junctional coupling and cellular apposition are logically important contributors (44). However, for coordinated inter-nuclear burst synchrony among OT neurons, a synchronized signal from the mammillary body complex in the ventroposterior hypothalamus is the most important event. This is because disruption of it but not other brain regions disrupts inter-nuclear burst synchrony (125, 126).

Under the drive of exogenous factors including synaptic input, astrocytic plasticity and interneuronal interactions ( Figure 1 ), a series of OTR-associated signaling events are activated during suckling. These events include mobilization of βɣ subunits (16), induction of cyclooxygenase 2 and prostaglandin production (21), phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (22), activation of protein kinase A (13, 127), and filamentous actin reorganization (21, 22) among many others (44). These factors express in a different spatiotemporal order to modulate ion channel activity on the membrane (128), such as hyperpolarization-activated cyclic nucleotide-gated channel 3 (13, 23), and thus trigger burst firing in OT neurons and OT release. Nevertheless, detailed neural circuit between OT neurons and their regulatory network and the cellular mechanisms underlying burst firing remain to be explored.

About lactation-associated health issue, it has been extensively accepted that normal breastfeeding can reduce the incidence of postpartum depression, maternal obesity, diabetes, and even breast cancer. OT is necessary for these benefits of breastfeeding. As revealed in animal study, maternal behavior in OT knockout mice is incomplete (83). Lack of suckling stimulation and bolus OT release during lactation can decrease maternal blood OT levels and cause hypogalactia and maternal depression (13, 95). By contrast, IAO can largely restore maternal behavior and lactation performance as stated above. Importantly, OT can also suppress breast cancer. This is because OT can reduce the oxidative stress of the mammary glands and the occurrence of pre-cancer lesions in the mammary glands (129). Moreover, OT can down-regulate the NF-κB and up-regulation of miR-195. These molecules can promote cell apoptosis, inhibit cell proliferation and consequently, decrease the mammary tumor volume and weight (130). Thus, OT is not only essential for normal breastfeeding but also for the long-term health benefits of women.

Menopause is the end of women’s menstrual cycles following decrease of reproductive hormones. From the middle age, blood OT levels decrease gradually (131). Decreased levels of OT can largely account for diminished sexual ability and vagal activity and reduced estrogen levels ( Figure 2D ).