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International Journal of Reproductive BioMedicine
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences of Yazd
ISSN: 1680-6433 EISSN: 2008-2177
Vol. 7, Num. 2, 2009, pp. 91-95

Iranian Journal of Reproductive Medicine Vol. 7, No. 2, Spring, 2009, pp. 91-95

Short communication 

Effect of oxytocin injection on folliculogenesis, ovulation and endometrial growth in mice

Leila Roshangar1 Ph.D., Jafar Soleimani Rad1Ph.D., Parisa Nikpoo2 M.Sc. student, Manijeh Sayyah Melli3 M.D.

1 Department of Embryology and Histology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.
2Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
3Gynecologist, Tabriz University of Medical Sciences, Alzahra Hospital, Tabriz, Iran. 
Corresponding Author: Jafar Soleimani Rad, Department of Embryology and Histology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. E-mail:Soleimanirj@yahoo.com

Received: 3 September 2008; accepted: 27 May 2009

Code Number: rm09016

Abstract

Background: Induction of ovulation in ART is necessary for superovulation and the side effects of superovulatory drugs are debated. Oxytocin as a natural hormone, have receptors and is synthesized by several reproductive organs. Preovulatory presence of oxytocin receptor mRNAs in granulosa cells indicating a role for oxytocin in follicular development.
Objective: The aim of the present study was to investigate the effect of exogenous oxytocin injection on folliculogenesis, ovulation and endometrial growth in mice. 
Materials and Methods: Forty adult female mice were divided into two groups as control and experimental. The mice at their sterous cycle received 1 IU/gr oxytocin, in experimental, and the same volume of solvent in control groups. Half of the mice in each group are sacrificed at 24 hours post injection and the other half, 48 hours after the injection. Ovarian samples fixed in 10% formalin, embedded in paraffin and sections were stained with H and E and studied using stereological techniques. The data were analyzed with Man – Whitney test.
Results: Microscopic examination revealed that the number and morphological features of follicles at different stages were similar at 24 and 48 hours post injection in both groups. The volumes of the ovaries were similar in both groups at 24 hour. However, at 48 hour, the volume of ovaries, corpora lutea and endometrial thickness, in experimental group, were significantly higher than those in control group (p< 0.05).
Conclusion: According to the increased volume of corpus luteum in the experimental group, it is concluded that oxytocin injection has a stimulatory effect on induction of ovulation.                                                                                                                                                                          

Key words: Oxytocin, Folliculogenesis, Ovulation, Endometrium, Mouse. 

Introduction 

Ovulation induction and acceleration of endometrial maturation are important factors in assisted   reproductive technologies  (ART).  Using synthetic drugs for superovulation could result in hyperstimulation syndrome, production of atretic follicles and impairment of endometrial maturation which would result in imbalanced synchronization between endometrial maturation and blastocyst development and consequently implantation failure (1- 3). Therefore, a substance lacking such side effects would highly be preferred. It appears that oxytocin, a peptide hormone, mainly produced by neurohypophysis is involved in reproduction biology (4-7).

Recent studies suggest that oxytocin is also synthesized in corpus luteum and endometrial epithelial cells. Oxytocin mRNA increases more than 150 times during gestation and at term (8). It is shown that this hormone has receptors on granulosa cells, cumulus cells, endometrial and luteal cells (8-11). Presence of oxytocin receptor mRNA in granulosa cells, before ovulation, indicate that oxytocin may involved in follicular development (12).

There are also evidences that oxytocin has a role on regulation of GnRH secretion, synthesis and release of LH (8, 13-16) and progesterone secretion (15,17). On the other hand, administration of oxytocin antagonist resulted in decreasing of LH concentration (17, 18). In spite of the above evidences that oxytocin is involved in reproduction, the role of this hormone on endometrial growth and maturation is not exactly known. Furthermore, its effect on follicular development and ovulation is controversial (19, 20). That is, continuous injection of oxytocin to heifers, for 12 days (1.9 mg/d), did not change the number of growing follicles and had no effect on the size of the ovulating follicles (19). On the other hand, slow-releasing injection of 10 IU oxytocin to Marino ewes showed that after 10 days of oxytocin treatment, the ovulation rate was higher in oxytocin –treated group (20). It is obvious that finding of a role for oxytocin in these basic steps of pregnancy would provide a chance for its application in ART.  The aim of the present study was to investigate the effect of exogenous oxytocin injection on folliculogenesis, ovulation and endometrial growth. 

Materials and methods

In this study, 40 female balb/c mice, 2.5-3 months old with 25-30 gr weight were used. The mice were obtained from Tabriz University of Medical Sciences’ animal-house and kept in an air conditioned room with a 12h light/12h dark cycle. All animals’ procedures complied with an approved Tabriz University of Medical Sciences’ animal care and use committee.

The mice were cocycled and used at proestrus phase, which was determined based on cytological morphology and proportion in vaginal smear (19).   Then, they were divided into two groups of control and experimental. The mice in experimental group were received 10 mlU/gr oxytocin as IP injection according to Robinson and Evans (13). Oxytocin was purchased from Menoro Pharmaceutical Co. (Tehran, Iran) as 1ml vial and diluted in 1.5 ml distilled water. The mice, in control group, received the same volume of solvent. Ten out of 20 mice, in experimental group, were sacrificed 24 hours post-injection and the rest at 48 hours after the injection. According to estrous cycle in mice, the 24 and 48 hours was regarded as equivalent to follicular and luteal phases respectively.

A similar number of control mice were sacrificed accordingly. The mice were anesthetized with chloroform, and 1-1.5 ml blood was obtained from the heart for biochemical analysis. Ovaries and uteri were removed and fixed in 10% formalin, and embedded in paraffin. Ovarian specimens were sectioned serially with 50µm intervals and 5µm thick sections of ovary and uterine were stained with H and E and studied with light microscope. The 50µm interval was chosen based on the size of corpora lutea and graffian follicles. The follicular and luteal phases were reconfirmed according to uterine endometrial morphology.                                               

Stereological studies                                                                                          

Ovarian volume from control and experimental groups were calculated as mm3 using point grid counting in serial sections according to v=T×a⁄f ×∑p. Where, v=volume, T=distance between sections, a/f =area/field and ∑p=the number of points hitting the image. In the sections, from follicular phase, the number of ovarian follicles was counted in 10 sections from each specimen and the total number of follicles in an ovary was calculated according to the following equation:

               ∑Q  

N (v) =  ───     

              a/f×h

Where, ∑Q = number of follicles, h=distance between selected sections, and a/f=area/field. For counting of follicular number, in Motic software, the photographs of sections were transferred to monitor and counting was carried out on the monitor.

In the sections, from luteal phase, the volume of ovaries and volume of corpora lutea were calculated in a similar manner.

For determination of endometrial thickness the least and maximum thickness of endometrium was measured in each specimen and the mean values was calculated for each group.                                                                   

In the collected blood, the sera were separated by centrifugation and concentration of LH and estradiol was determined by radioimmunoassay technique. The collected data were compared between control and experimental groups.

Statistical analysis

The data were analyzed using Mann Whitney test and p<0.05 level was considered significant.

Results

Histological study of ovarian sections from both control and experimental groups revealed that the quality of follicles, including; oocytes, granulosa cells, and thecal layers were similar. Based on morphometric studies, the number of follicles at different stages of development is summarized in table I. As it is shown in the table, there are no differences between control and experimental groups regarding the number of primordial, primary, growing, secondary, and antral ( including graffian follicles ) and the volume of ovary on follicular phase. However, on luteal phase, as indicated in table II, the volume of corpora lutea and ovaries and endometrial thickness were increased significantly (p<0.05) in experimental group in comparison to control group. Histological studies are shown in figures 1 and 2. As it is shown in the figures, the ovary in experimental group (Figure 1) is larger than the ovary in control group (Figure 2). Furthermore, the corpora lutea is also very extensive in experimental group compared with the control group. Morphometric determination of endometrial thickness revealed that endometrium, in experimental group was significantly thicker than the endometrium in the control group (p<0.03). Concentrations of estradiol and LH are shown in tables III at 24 and 48 hours after oxytocin injection. As it is indicated in table III, the concentration of estradiol, in control group, from 24 hour to 48 hour decreased (p<0.04). On the contrary ,       in        experimental       group ,      its concentration at 48 hour in relevance to 24 hour increased significantly (p<0.04). However, the changes of LH from 24 hour to 48 hours were similar in both groups and did not differ significantly.

Table I. Number of different follicles and volume of ovary (mm3) in control and experimental groups at follicular phase.

Follicular phase

Experimental

Control

p-value

Primordial follicle

65.31 ± 5.3

78.12± 4.68

0.66

Primary follicle

166.66 ± 20.83

144.53±30.07

0.41

Growing primary follicle

1453.57 ± 206.78

1890.62±281.06

0.47

Secondary follicle

366.007±57.74

365.62±51.05

0.96

Antral follicle

98.75±22.46

112.81±34.81

0.23

Ovarian volume

83.55±1161(mm3)

91.04±101.95(mm3)

0.81

p< 0.05 is significant

The aim of the present study was to investigate the effect of oxytocin on folliculogenesis, ovulation and endometrial thickness. Our results showed that at follicular phase, the number of follicles at different stages of development did not differ between control and experimental groups. This finding indicates that oxytocin injection has not a significant effect on the number of follicules. In agreement with our findings, it is shown that injection of oxytocin in rat (22) and calf (19) have no effect on folliculogenesis or size of the follicules.

However, unlike superovulatory drugs, that could affect oocyte maturation and consequently pregnancy rate (23, 24), oxytocin has no adverse effect on oocyte maturation (25). The fact that, at follicular phase, ovarian volume in experimental group in comparison to control group, did not change, is another confirmation for the above mentioned results (Table I). On the other hand, in luteal phase, after oxytocin injection, the volume of ovaries and corpora lutea increased significantly (p<0.05).

It means that oxytocin did stimulate ovulation and resulted in increasing of corpora lutea and ovarian volume. In support of our findings, there are several reports suggesting that oxytocin may have a role on ovulation (26-29).

Our findings in luteal phase, together with the results from follicular phase, means that stimulatory effect of oxytocin on ovulation is a gradual effect rather than a sudden effect. In agreement with our hypothesis, Tallam and Walton (2000) have shown that continuous infusion of oxytocin, in heifers, delays luteal regression without inhibiting follicular development (19). Increasing of endometrial thickness in oxytocin injected animals was another finding of the present study.

This finding, well correlates with stimulatory effect of oxytocin on ovulation, i.e. oxytocin injection increased ovulation and consequently the formation of corpora lutea is increased and resulted in an elevation of progesterone secretion. The luteinizing effect of progesterone on endometrium is well known (11). The above mentioned morphological changes well correlated with hormonal changes in our experiment. Hormonal assay in the present study showed that in control group, estradiol concentration decreases from 24 hour to 48 hour post injection, which is an indication of stop of folliculogenesis. While, in experimental group the concentration of estradiol increased at the same time interval, meaning that follicular growth continued or the period of folliculogenesis is prolonged.

The effect of oxytocin on steroidogenesis has already been shown. Progesterone secretion is stimulated by oxytocin in cultured granulosa cells isolated from preovulatory follicles (30). Furthermore, it is shown that oxytocin regulates progesterone release in human granulosa-lutein cells in culture medium (31). However, the changes of LH concentration, in control and experimental groups, were similar.                                                                                        Since the commonly used superovulatory drugs cause a sudden increase in folliculogenesis and formation of numerous graffian follicles, it increases the chance for hyperstimulation syndrome (1). The main point of our finding is that, oxytocin, by acting in a gradual manner would not result in hyperstimulation syndrome.

Conclusion

Taking together, the stimulatory effects of oxytocin on esteroidogenesis, ovulation and endometrial luteinization, it is concluded that oxytocin promotes several different mechanisms on reproductive system which go together and keep pace. In this sense, it is not similar to superovulatory drugs that act isolately on a single event.

References           

  1. Opsahl MS, Robins ED, O’Connor DM, Scott RT, Fritz MA. Characteristics of gonadotropin response, follicular development and endometrial growth and maturation across consecutive cycles of clomiphene citrate treatment. Fertil Steril 1996; 66:533.
  2. Tarlatzis BC, Kolibianakis EM. GnRH agonists vs antagonists. Best Pract Res Clin Obstet Gynaecol 2007; 21: 57-65.
  3. Csemiczky G, Wramsly H, Johannisson E. Importance Of endometrial guality in women with tubal infertility during a natural menstrual cycle for the outcome of IVF treatment. J Assist Reprod Genet 1998; 15:55-61.
  4. Burns PD, Mendesjr OB, Yemm RS, Clay CM, Nelson SE, Hayes SH. Cellular mechanisms by which oxytocin mediates ovine endometrial prostaglandin F2α synthesis: Role of Gi proteins and nitrogen-activated protein kinases. Biology of reproduction 2001; 65:1150-1155.
  5. Khan-Dawood FS. Immunocytochemical localization of oxytocin and neurophysin in human corpora lutea. Am J Anat 1987; 179:18-24.
  6. Liedman R, Hansson SR, Howe D, Igidbasian S, Mcleod A, Russell RJ, et al. Reproduction hormones in plasma over the menstrual cycle in primary dysmenorrheal compared with healthy subjects. Gynecol Endocrinol 2008; 24: 508-513.
  7. Rodgers RJ, O'Shwa JD, Findlay JK, Flint APE, Sheldrick EL. Large luteal cells: the sourco of luteal oxytocin in the sheep. Endocrinology 1983; 113: 2302-2304.
  8. Gerald G, Falk F. The Oxytocin Receptor System: Structure, Function, and Regulation. Physiol Rev 2001; 81: 629-683.
  9. Okuda K, Uenoyama Y, Fujita Y, Iga K, Sakamoto K, Kimura T. Functional oxytocin receptors in bovine granulosa cells. Biol Reprod 1997; 56: 625-631.
  10. Furuya K, Mizumoto Y, Makimura N, Mitsui C, Murakami M, Tokuka S, et al. A novel biological aspect of ovarian oxytocin: gene expression of oxytocin and oxytocin receptor in cumulus/Iuteal cells and the effect of oxytocin on embryogenesis in fertilized occytes. Adv Exp Med Biol 1995; 395: 523-528.
  11. Skarzynski DJ, Ferreira-Dias G, Okuda K. Regulation of  luteal function and corpus luteum regression in cow: hormonal control, immune mechanisms and intercellular communication. Reprod Domest Anim 2008; 43: suppl: 57-65.
  12. Einspanier A, Ivell R, Hodges JK. Oxytocin: a follicular Iuteinisation factor in the marmoset monkey. Adv Exp Med Biol 1995; 395: 517-522.
  13. Robinson G, Evans JJ, Catt KJ. Oxytocin stimulates    LH production by the anterior pituitary gland of the rat. Endocrinology 1992; 132: 277-283.
  14. Evans J.J. Peptides Interact in Gonadotrophin Regulation. Arch Physiol Biochem 2002; 110:154 - 116.
  15. Mass S, Jarry H, Teichmann A, Rath W, KuhnW, Wuttke W. Paracrine actions of oxytocin, prostaglandin Fand estradiol within the human corpus luteum. J Clin Endocrinol Metab 1992; 74: 306-312.
  16. Evans JJ, Reid RA, Wakeman SA, Croft LB, Benny PS. Evidence that oxytocin is a physiological component of LH regulation in non-pregnant women. Human Reproduction 2003; 18: 1428 -1431.
  17. Hull ML, Reid RA, Evans JJ, Benny PS, Aickin DR. Pre-ovulatory oxytocin administration promotes the onset of the luteinizing hormone surge in human females. Hum Reprod 1995; 10: 2266-2269.
  18. Einspanier A, Jarry H, Pitzel L, Holtz W, Wuttke W. Determination of secretion of estradiol, progesterone, oxytocin, and angiotensin II from tertiary follicles and freshly formed corpora lutea in freely moving sows. Endocrinology 1991; 129:3403-3409.
  19. Tallam SK, Walton JS. Effects of oxytocin on follicular development and duration of the estrous cycle in heifers. Therigenology 2000; 53: 951-962.
  20. King PR, Coetzer WA. The effect of treatment with a slow-releasing oxytocin preparation at the onset of oestrus on the ovulation rate of Merino ewes. J S Afr Vet Assoc 1997, 68: 16-27.
  21. Drickamer LC. Dtermination of oestrous condition in female mice is dependent upon time of day. J Reprod Fertil 1987; 79: 659-662.
  22. Robinson G, Evans JJ. Oxytocin influences preovulatory follicular development and advances ovulation in rats. Acta Endocrinol (copenh) jun 1991; 125 :109 -112.
  23. Wramsby H, Fredga K, Liedholm P. Chromosome analysis of human oocytes from preovulatory follicles in stimulated cycles. New Engl J Med 1987; 316: 121-124.
  24. Homburg R, Armar NA, Eshel A, Adam J, Jacobs HS. Influence of serum luteinising hormone concentrations on ovulation, conception, and early pregnancy loss in polycystic ovary syndrome. Br Med J 1988; 297: 1024-1026.
  25. Okuda K, Uenoyama Y, Fujita Y, Iga K, Sakamoto K, Kimura T. Functional oxytocin receptors in bovine granulosa cells. Biol Reprod 1997; 56: 625-631.
  26. Gimpl G, Fahrenholz F. The oxytocin receptor system: structure, function, and regulation. Physiol Rev 2001; 81: 629-683.
  27. Copland JA, Zlatnik MG, Ives KL, Soloff MS. Oxytocin receptor regulation and action in a human granulosa-lutein cell line. Biol Reprod 2002; 66: 1230-1236.
  28. Amico JA, Seif SM, Robinson AG. Elevation of oxytocin and oxytocin-associated neurophysin in the plasma of normal woman during midcycle. J Clin Endocrinol Metab 1981; 53: 1229-1232.
  29. Sayyah Melli M, Tagavi S, Alizadeh M, Ghojazadeh M, Kazemi Sheshvan M. Comparison the effect of oxytocin and human chorionic gonadotropin on ovulation. J Med Sci 2007; 7: 1126- 1134.
  30. Aladin-Chandrasekher Y, Fortune JE. Effects of oxytocin on steroidogenesis by bovine theca and granulosa cells in vitro. Endocrinology 1990; 127: 926-933.
  31. Mass S, Jarry H, Teichmann A, Rath W, Kuhn W, Wuttke W. Paracrine actions of oxytocin, prostaglandin F2 alpha, and estradiol within the human corpus luteum.  Jornal of clinical Endocrinol Metaboli 1992; 74, 306-312.

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