Assignment 4: Summary of Oxytocin Related Published Article

Article Citation:

Petersson, Maria. 2008. Opposite effects of oxytocin on proliferation of osteosarcoma cell lines. Regulatory Peptides, 150: 50-54.

Link to Article

Summary of Introduction and Methods

Studies have shown that oxytocin plays a role in growth. Depending on the cell, oxytocin can have different effects. For instance, it has been known to:

• induce wound healing
• weight gain
• higher birth weight babies when there is a high oxytocin level in the mother’s system
• increases growth factors
• contributes to blastocyst development, and cell proliferation

However, oxytocin can also inhibit proliferation of some cells such as breast cancer and brain tumor cells.

Markers such a as Osteocalcin occurs during late osteoblast differentiation, and ALP occurs in the early stages of osteoblast differentiation can help to determine if cell differentiation phase plays a role in the response of oxytocin to the cell. This experiment explores the effects of oxytocin on cell proliferation in two human osteosarcoma cell lines, SaOS-2, TE-85, and one rat osteosarcoma cell line, UMR-106. These cell lines have different stages of differentiation.

SaOS-2 and TE-85 were maintained in McCoy’s medium, with 10% heat inactivated FBS, penicillin and streptomycin. UMR-106 cell lines were kept in alpha-MEM again with 10% heat inactivated FBS, penicillin, streptomycin, and glutamine.

They determined the effects of oxytocin on cell proliferation by:

• Measuring DNA synthesis through thymidine incorporation
• Determined the amount of reduction of uncolored tetrazolium salts into formazon derivitives (which are intensely colored) using an EZ4U kit
• Growing the cells for 48-72 hours
• The cells were cultivated and then incubated with various concentrations of oxytocin, oxytocin antagonist, or oxytocin + oxytocin antagonist for 24 or 48 hours
• Results from the EZ4U kit were analyzed as means and standard deviation. ANOVA Tests and Fishers test were used for statistical analysis with P-values 0.05 or less considered significant

Summary of Main Results

SaOS-2

Figure 1: Proliferation of SaOS-2 cells compared to a control measured using EZ4U kit and thymidine incorporation in DNA synthesis when incubated for 24 hours with oxytocin or oxytocin and an antagonist.

Increased cell proliferation significantly determined by both tests (EZ4U and Thymidine incorporation). Concentrations of 100 pmol/l increased cell proliferation by 116-122% when compared to the control. When incubated with oxytocin antagonist there was no cell proliferation. Thus, this shows that SaOS-2 cells, which have differential stages of osteoblast differentiation (ALP and osteocalcin markers) experiences proliferation when under the influence of oxytocin.

TE-85

Figure 2: Proliferation of TE-85 cells compared to a control measured using EZ4U kit and thymidine incorporation in DNA synthesis when incubated for 24 hours with oxytocin or oxytocin and an antagonist.

Proliferation decreased to 81-83% of the control when incubated with oxytocin which was determined through both tests (EZ4U and Thymidine incorporation). These effects were lost when incubated with both oxytocin and oxytocin antagonist, yet the antagonist alone had no effect on cell proliferation. The most effective concentration for decrease was 08% 7.3% of the control. This data was considered statistically significant because p<0.05.>

Because oxytocin effects on both SaOS-2 and TE-85 cell lines were diminished upon addition of the oxytocin antagonist, it can be determined that oxytocin is acting through the oxytocin receptor. As well, past studies have shown that these same results on osteosarcoma cell lines in the presence of 17B-estradiol, which is understandable as estrogens increase the:

• synthesis of oxytocin
  
• the number of oxytocin receptors
• the binding of oxytocin receptors

UMR-106

Figure 3: Proliferation of UMR-106 cells compared to a control measured using EZ4U kit and thymidine incorporation in DNA synthesis when incubated for 24 hours with oxytocin, oxytocin antagonist, or oxytocin and an antagonist.

When incubated with oxytocin cell proliferation decreased to 80-82% of the control but when incubated with the oxytocin antagonist this decrease still occurred. Alone, the oxytocin decreased cell proliferation. UMR-106 cells are similar to human TE-85 cells because they have small amounts of ALP and no osteocalcin. However, when an antagonist was added cell proliferation was inhibited. This could be a result of:

• Different origin of species
  
• The antagoginst receptor having agonistic effects to the oxytocin receptor

Considerations and Conclusions

Oxytocin may indeed play a large role in cell growth.

Other studies have illustrated that oxytocin has been shown to:

• Induce important autocrine effects in breast tumors
• Decrease a cytokine production that stimulates osteoclastogenesisIncrease prostaglandin
• E2 synthesis in osteoblast cells
• Increase plasma IGF-I levels in rats
• Acts as a mediator in IGF-I effects in the ovary
• Interact with Estradiol

How does the study relate?

Differentiation of these cells may contribute to the effect oxytocin has on cell proliferation. Through the study, it can now be theorized that oxytocin can influence proliferation of osteosarcoma cells. Thus, it may play a role in bone cancer and bone metastasis from other tissues.

Critique

Overall this paper was extremely straightforward and easy to follow. The use of real cell lines under simulated physiological conditions that represent a normal bodily state help to yield creditable results and a great protocol for future experiments involving oxytocin and the proliferation of cells. Thus, thymidine incorporation in DNA synthesis combined with an EZ4U kit can allow for a great analysis of cell growth and the influence that various factors, not only hormones, play on proliferation.

The figures were more then self explanatory, and ensured that every described result was displayed easily to the reader. The effects of oxytocin on cell proliferation of various cell line types were obvious and a quick connection relating different cell lines was possible.

The described steps required for the model were very useful at giving individuals who hope to use this protocol an expectation work and effort required to analyze how certain factors effect on cell proliferation.

Unfortunately, the paper described that the figures represented "at least three separate experiments. Similar results were obtained in all experiments." If all results were similar, then there would be no need to exclude them from the means obtained to create the figures. Thus, the study becomes questionable as readers wonder if data was selected based on the most 'positive' results, rather then a random selection of 3 groups of data from the experiment. Thus, if the researchers would include all experimental data the exact results would be clearer and more accurate. It was also briefly stated that data for the oxytocin antagonist alone for both SaOS-2 and TE-85 cell lines played no effect on cell proliferation. It would not have taken much effort to include this data in the figures, thus the authors opinion on a proliferation effect may contribute to biased data.


Possible Future Experiments

This study leads to a huge amount of possible experiments that could generate useful data.

1. If other combinations of differential cell lines are located in tissues prone to cancer, then these tissues can be used based on cell differentiation and the effects that oxytocin plays in their proliferation.

2. Can oxytocin antagonists decrease cell proliferation of cells? Could localized treatment using antagonist result be used medically as a treatment for decreasing tumor growth?

3. Further validate this experiment by including more then three samples thus proving the accuracy of the described results of oxytocin on cell proliferation in osteosarcoma differentiated cell lines.

Assignment 3: Oxytocin - Function and Pathology

Oxytocin is most known for its functions in reproductive biology. The oxytocin receptor is encoded into a single-copy gene that is expressed through uterine smooth muscle cells, and myoepithelial cells of the mammary alveoli. [1]

Oxytocin functions to contract smooth muscle cells by binding to its heptahelical G-protein receptors. When bound to the receptor, inositol triphosphate, and diacylglycerol are formed. These compounds increase intracellular calcium and activate protein kinase C which plays a role in phosphorylating membrane calcium channels which thus further increasing the levels of intracellular calcium. The increased influx of calcium results in the binding of calcium to calmodulin in muscle cells, which activates myosin and thus results in the contraction of endometrial and myoepithelial cells. [1]

Interestingly, oxytocin is said to have increased release from the neurohypophysis during sexual intercourse. This release may cause uterine contractions which is hypothesized to help the transport of sperm reach the ova. This hypothesis stems from the idea that sperm cannot reach the ova at the rate they due using simply their flagella. [2] However, this theory is speculative because a non-pregnant uterus is insensitive to oxytocin. Another hypothesis to help explain the uterine contractions is the increased amount of catecholamines during intercourse which may stimulate uterine activity. [2]

Oxytocin's role in parturition is very significant. Oxytocin concentrations heighten considerably during the final stages of labor. This increase occurs because the dilation of the cervix, and full development of the fetus can stimulate stretch receptors in the uterus to act on the hypothalamus to stimulate oxytocin release. In response the increased levels of oxytocin, the sensitivity of the myometrium of the uterus to the hormone increases. This is illustrated through the increased concentration of oxytocin receptors via estrogen increase. [2] This is important because oxytocin causes uterine contractions during labour, acting in a positive feedback loop to increasing contractions which ultimately helps to facilitate the transport of the fetus through the uterus, exiting through the vagina. [2]

Lactation is stimulated by the suckling of an infant on the mother's nipple, resulting in the milk letdown, or milk ejection reflex. [1] The natural pulsatile release of oxytocin occurs after parturition when the areolae and nipples of the breast are mechanically stimulated through infanct suckling. These stimuli are conducted by afferent neural pathways to the neurohypophysis where oxytocin is synthesized and released in the paraventricular nuclei. [2] Interestingly, the neural pathways are ipsilateral, meaning they occur on the same side as the nipple being suckled. [2]

Figure 1: The milk ejection reflex stimulated by oxytocin release from the neurohypophysis.

Oxytocin plays an important role in social cognition and behaviour. Both males and females are shown to have positive social responses when administered oxytocin, even though oxytocin predominates in females during lactation and parturtion. [3] When interacting in positive social responses, and remembering positive social interactions, oxytocin levels increase. [4] Maternal behavior is often displayed and enhanced when oxytocin levels are increased. [5] While not well understood, it is obvious that an important link exists between positive social behaviors and oxytocin.

Pathology

Induced Labor:

Pregnant women whom it is deemed necessary to initiate early labor, or labor because the pregnancy has gone too long overdue, are induced. When a baby is overdue, it is still growing inside the womb. If the baby grows too large the endometrium may not be able to supply a sufficient amount of required nutrients to the baby. [5] Vaginal birth may also be difficult if the baby is very large, and a caesarean section (or removal of the fetus externally through an incision in the lower abdomen) may be necessary. Thus inducing the labor may reduce the need for a c-section. Induced labor first involves manually breaking the fluid sac, followed by the intravenous injection of oxytocin,which causes labor contractions [5] as previously described. However, there are some negative effects that oxytocin injections cause which include:

• hypertonicity
• uterine rupture
• and fetal bradycardia (or low heart rate)

The benifits of labor induction however far outweigh the risk factors. [5] Some of the complications oxytocin induced labor prevent are:

• maternal diabetes
• Rhesus isoimmunization (fetus and mother blood incompatibility)
• pre-eclampsia (Pregnancy induced hypertension because of increased serum proteins)
• premature rupture of the membranes

Thus if the pregnant individual shows signs of medical conditions that can be treated or prevented through the induction of labor, then induction should be administered. However, personal elected labor induction should be avoided as there are risk factors associated with oxytocin induced labor. [5]

NOTE: The following descriptions represent theories regarding oxytocin in relation to the pathological conditions cancer and autism. These theories are still being studied, and are described to provide a possible future outlook for oxytocin use in these conditions.

Cancer:

It was very interesting to discover that oxytocin may play a role in growth inhibition of tumors. Oxytocin receptors have recently been found in tissues other than the myo- and endometrium, and myoepithelial cells of the breast. For instance, it has been found in the vasculature, prostate, and bone tissues. [6] Oxytocin receptors have been found in breast carcinomas, and breast cancer cell lines, lung carcinomas, and a variety of other cancerous tissues. Growth inhibition is thought to occur through a non-conventional signalling pathway, cAMP-PKA, and has been shown in oxytocin treatments to neoplastic cells of the previously listed tissues. In these cases, oxytocin may actually help to stop the growth of tumors that have oxytocin receptors. [6] While the exact effects that oxytocin has on tumor growth in a variety of tissues needs to be further investigated, localized treatments of oxytocin may serve as a treatment for cell proliferation if future studies prove its effectiveness. [6]

Autism:

As previously mentioned, oxytocin plays a role in social behavior and memory. Thus it is interesting to explore the effects that oxytocin has on disorders that affect social cognition, such as autism. Autism effects speech and communication, causes impaired social functioning, and repetitive behaviors. [7]Autism patients do not have the same desire to form social bonds, which is thought to be facilitated through oxytocin, thus the connection between oxytocin and autism is obvious. Interestingly, theorists believe that oxytocin may serve as a future treatment to adult autism patients. Studies have shown that individuals with autism often fail to assign significance to speech. For example, one study tested autistic patients ability to recognize different tones such as happy, or angry in random phrases such as "The game ended at 4 o'clock." [7] Some patients were administered placebos whereas other patients were administered oxytocin. The study showed a positive result of processing and retaining social information when oxytocin was injected that was not as significant when placebos were administered. Repetitive behaviours of the individuals also were reduced upon oxytocin administration. [7]

This study suggests that oxytocin may indeed help social cognition in oxytocin individuals. While further studies are needed, the injection of oxytocin in young individuals seems promising, and may further help their cognitive development.

While the uses of oxytocin are still being discovered, this amazing hormone exhibits a promising potential to common and extreme pathologies such as cancer and autism. Because of the wide variety of social cognitive disorders, the possibilities for positive use of oxytocin may be endless. It will be exciting to see what the future holds for the use of oxytocin, and hopefully the decrease in severity of many pathological conditions!

References:

[1] Jones, Richard., and Lopez, Kristen H. 2006. Human Reproductive Biology. Elsevier Inc.: Oxford, UK.

[2] Goodman, Maurice H. 2003. Basic Medical Endocrinology. Academic Press: San Diego, California.

[3]Pfaff, Donald W. 1999. Drive: Neurobiological and Molecular Mechanisms of Sexual Motivation. MIT Press: USA.

[4]Uvnas-Moberg, Kerstin. 1998. Oxytocin may mediate the benefits of positive social interaction and emotions. Psychoneuroendocrinology: 23(8): 819–835.

[5] Dawood, M. Y. 1984. Oxytocin. Eden Press: Quebec, Canada.

[6]Kastin, Abba J. 2006. Handbook of Biologically Active Peptides. Academic Press.

[7] Hollander, E., Bartz, J., Chaplin, W., Phillips, A., Sumner, J., Soorya, L., Anagnostou, E., and Wasserman, S. 2007. Oxytocin Increases Retention of Social Cognition in Autism. Biol Psychiatry, 61: 498-503.

Assignment 2: The Structure of Oxytocin

Oxytocin is an extremely important peptide hormone that is involved in the reproduction of virtually all vertebrates. Oxytocin is a nonapeptide, meaning it has a total of nine amino acid residues which include Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly. [2] There is a disulfide bond between the cysteine residues at positions 1 and 6. [1]As a result, oxytocin is made up of a cyclic 6 amino acid part, and an alpha mediated three residue tail with a COOH terminal. [1] Oxytocin is always in association with its much larger inactive binding protein, neurophysin I when in neurosecretory granules which have an acidic pH of approximately 5.6. Thus when released into the blood which has a higher pH of approximately 7.4, the complex often dissociates. [1]

Figure 1: A three dimensional representation of oxtytocin (space filling molecule to the left of the figure) and neurophyisin (ribbon molecule to the right of the figure).

The Oxytocin receptor is a member of the rhodopsin type (class I) GPCR family. A receptor becomes active when there is an orientation change in the transmembrane domains 3 and 6 which reveals G protein binding sites for oxytocin. [1] Residues in both the transmemrane domains and extracellular domains are involved in ligand binding. The typical G protein receptor has seven trans membrane alpha helicies, three extracellular loops, and three intracellular loops. Oxytocin binds its cyclic portion of the molecule to one extracellular loop, and its 3 amino acid linear chain part to another extracellular loop. [2]

The gene encoding both oxytocin and neurophysin I is in humans is found on chromosome 20p13. The first exon of the protein contains the nine amino acid oxytocin peptide, Part of the first, second, and third exons of the gene encode neurophysin I. [1] When translated, this protien is called the pre-pro hormone and is stored in secretory granules of the paraventricual neurons of the hypothalamus. The function of neurophsyin involves the proper targeting, packaging, and storage of Oxytoxin within its nuerosecretory granules before it is released in the bloodstream. [1]

Figure 2: The coding region of oxytocin gene which codes for the nonapeptide oxytocin and its corresponding binding protein neurophysin. (Gimpl and Fahrenholz, 2001)


After searches of Oxytocin-neurophysin 1 hormone for three different species were performed, online software including BLAST (Basic Local Alignment Search Tool) was used to compare the oxytocin-neurophysin I hormone protein sequences of the three species. The results are as follows:


Click the above image to enlarge!

Key:

* Identical Residues
: Conserved Substitutions
. Semi-Conserved Substitutions


The above image investigates the similiarity of oxytocin and neurophysin between each of three species, Homo sapiens (human), Rattus norvegicus (rat), and Bos taurus(pig). The score in the clustalw score table represents the percent identity between oxytocin in each species. Thus, as shown from the image, evolution of oxytocin in humans differs equally when compared to the rat and pig. The high score of 88 shows that humans are closely related to both pig and rat, which is not surprising considering they are both mammals. However, their oxytocin-neurophysin proteins do differ in structure. Rat and pig are more closely related to each other then they are to humans which can be illustrated through their score of 90, which means a 90% similarity. This can also be shown by the below diagram which was obtained from BLAST, and is a phylogram illustration the phylogentic relationship between the three species, showing that rat and pig are more closely related than humans. (Click on the image to enlarge)

To conclude, BLAST is a great tool to use when comparing protein and nucleotide sequences between different species. It helps to compare evolutionary history, and how protiens differ between species.

References:

[1] Gimpl, Gerald., and Fahrenholz, Falk. 2001. The Oxytocin Receptor System: Structure, Function, and Regulation. Physiological Reviews, 82(2): 629-683.

[2] Liu, D., Seuthe, A. B., Ehrler, O. T., Zhang, X., Wyttenbach, T., Hsu, J. F., and Bowers, M. T. 2005. Oxytocin-Receptor Binding: Why Divalent Metals Are Essential. J. Am. Chem. Soc., 127: 2024-2025.

[3] Portland State University. Department of Chemistry. Retreived October 20, 2008 from http://chem.pdx.edu/research/‏.

Assignment 1: My Favorite Hormone

Oxytocin, a mammalian hormone, is extremely interesting and has a variety of functions. Oxytocin was one of the first completely chemically characterized peptide hormones [2]. It is believed that oxytocin evolved from an ancient precursor hormone approximately 600 million years ago, around the time of vertebrate and invertebrate divergance. [1]

Oxytocin is a nonapeptide hormone, meaning it has an amino acid chain length of 9 residues. [2] As seen in the above image (Figure 1), a disulfide bond exists between the peptide's cysteine molecules at positions 1 and 6. This disulfide bond gives oxytocin a circular shape with a tri-peptide tail. [2]

The gene responsible for oxtocin's amino acid sequence is much larger then the actual hormone. It contains:

• an amino terminal signal peptide


• the oxytocin amino acid sequence


• neurophysin I amino acid sequence
  
• co-peptin - a carboxyl terminal peptide

The final products of this large gene after synthesis and cleavage include only oxytocin and neurophysin I.

Synthesis:

Oxytocin is a neurohypophyseal hormone meaning stored in the pars nervosa of the neurohypophysis. We most often refer to this tissue site as the posterior pituitary. [3]

It was originally believed that oxytocin was synthesized in the neurohypophysis, however, in the 1930's it was revealed that they are actually synthesized in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus, with a higher concentration of ocytocin synthesizing cells in the PVN. [2] These regions are clusters of cell bodies. [3]

Figure 2: Anatomy of the pituitary gland, the PVN and SON can be seen extending into the hypothalamus.

Active oxytocin is accompanied by an inactive binding protein called neurophysin I, or oxyphysin.

Synthesis occurs in the following steps:

• Translation into a into a large precursor protein, called pre-pro-oxytocin-neurophysin I (prepro-OT-NPI)


• Prepro-OT-NPI is stored in granules, called Herring bodies, and are carried along by axonal transport to the neurohypophysis (NOTE: the granules contain not only Prepro-OT-NPI, but also the enzymes that cleave the peptide chain to create the active hormone)


• Transport to the nuerohypophysis takes two hours and during this time the enzymes cleave the precursor to yield oxytocin and neurophysin which are then stored in the axonal endings of the pars nervosa


• Afferent impulses travel along the spinal cord to the brain stem, where they reach the PVN and SON and cause a depolarization whereby the action potential propogates along the hypothalamico-neurohypophyseal tract to the nerve terminals in the posterior pituitary


• A Ca2+ influx into the nerve terminal causes the exocytosis of the secretory granules, releasing oxytocin into the blood stream

Oxytocin release is regulated through independent nervous control. Nipple suckling, the activation of uterine stretch receptors, and even certain emotinal states have an effect on the secretion of oxytocin. These nervous responses will be discussed in the following section.

Function:

Oxytocin has many diverse functions which include:

• Stimulation of the uterus to begin contraction during childbirth


• The post pregnancy secretion of breast milk


• Affiliation with love: contributing to maternal behavior and positive social responses

Parturition

Oxytocin is known to play a very important role during pregnancy, particularly during birth. During pregnancy ooestrogens cause the secretion of oxytocin to be steadily increased, and also increase the concentration of oxytocin receptors in myometrium smooth muscle cells. [2] However, the actions of oxytocin are not in full effect until the end of the pregnancy because throughout pregnancy there is a high concentration of progesterone which overrides oxytocin receptor synthesis until its concentration decreases at the end of the pregnancy [3].

When the fetus is fully developed it causes the uterus to stretch, which signal uterine stretch recptors to act on the hypothalamus ordering it to release oxytocin [3]. Oxytocin then travels to to the uterine muscles where it binds to receptors and increases the intensity of uterine contractions. The result is a positive feedback loop, whereby increased contractions stimulate the uterine stretch receptors even more, resulting in further oxytocin secretion. [5] Oxytocin also causes the myometrial smooth muscle cells to secrete prostoglandins, which also funtion to aid the myometrium in contraction [3].

Once contractions cause the fetus to reach the cervix, the pressure stimulates mechanoreceptors which also have nervous connections to the hypothalamus, again increasing the release of oxytocin. This positive feedback causes the cervix to dialate, allowing the fetus to move through the cervic and vagina, thus resulting in birth. [3]

Lactation

Lactation occurs after parturition, and is the process of secreting milk for young through the breast. This is a very important trait to possess because milk is extremely important to the nutrition of mammalian newborns [3]. Oxytocin plays an extremely important role in lactation. When a newborn suckles for milk, the richly innervated nipple undergoes mechanical stimulation and sends the stimuli to the central nervous system which stimulates the release of oxytocin from the neurohypophysis. Oxytocin then travels to the myoepithelial cells surrounding the breast alveoli (where milk is stored) causing them to contract. [4] This contraction squeezes the alveolus forcing milk into milk ducks and finally exiting through the nipple. [3]

While continuous suckling creates a positive feedback on oxytocin release, the body can also develop a type of condition reflex that initiates oxytocin release. So when a mother hears a baby's cry, the nervous system will immediatly stimulate the release of oxytocin. Another interesing fact is that stiumlation of a males nipples also heighten plasma oxytocin levels, however the reasons behind this action are unknown. [3]

Social Functions

Oxytocin has actually been described as the neurohormone of affiliation resulting in maternal behavior and positive social responses. [6] Various studies have shown that when injected with oxytocin, many animals behave more friendly, and have reduced agression. Oxytocin expression is predominant in females as its primary functions are to allow for sucessful female reproduction, yet experiments show that males too have positive social responses when administered oxytocin. [6]

Oxytocin has also been linked to memory associated with positive social responses. For instance, when a positive social memory is created, and stimuli cause an individual to recall that memory, there is an accompanied increase in plasma oxytocin levels. Oxytocin levels also increase during positive social interactions such as touching. [8]

Maternal behavior has been shown to increase upon the injection of oxytocin. For instance, rats injected with oxytocin would group foster pups together in a corner, lick them, build nests, and retrieve pups if they were removed from the group. These behaviors were shown to be absent in rats who were not induced with oxytocin. [7]

This concludes my discussion of the brilliant hormone oxytocin. While many questions still remain regarding the social effects of oxytocin, it is clear that its high importance to mammalian physiology is definite. I hope this dicussion helped you to gain a great understanding and appreciation for this amazing hormone!

References

[1]Dirks, R W., Van Kesteren, R E., Smit, A B., With, N D., Geraerts, W P., and Joosse, J. 1992. Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis. Endocrinology Proc. Natl. Acad. Sci. USA: (89) 4593-4597.

[2] Hadley, Mac E. 1998. Prentice Hall Inc.: New Jersey, USA.

[3] Hill, Richard W., Wyse, Gordon A., and Anderson, Margaret. 2004. Animal Physiology. Sinauer Associates: Sunderland, MA, USA.

[4] Gard, Paul. 1998. Human Endocrinology. Taylor and Francis: London, UK.

[5] Jones, Richard., and Lopez, Kristen H. 2006. Human Reproductive Biology. Elsevier Inc.: Oxford, UK.

[6] Pfaff, Donald W. 1999. Drive: Neurobiological and Molecular Mechanisms of Sexual Motivation. MIT Press:USA.

[7] Dawood, M. Y. 1984. Oxytocin. Eden Press: Quebec, Canada.

[8]Uvnas-Moberg, Kerstin. 1998. Oxytocin may mediate the benefits of positive social interaction and emotions. Psychoneuroendocrinology: 23(8): 819–835.