Evaluating The Effect of Melatonin on HAS2 , and PGR Expression,
as Well as Cumulus Expansion, and Fertility Potential in Mice
Infertility is a worldwide health problem which affects approximately 15% of sexually active couples. One of
the factors influencing the fertility is melatonin. Also, protection of oocytes and embryos from oxidative stress inducing
chemicals in the culture medium is important. The aim of the present study was to investigate if melatonin could
regulate hyaluronan synthase-2 (
Materials and Methods
In this experimental study, for this purpose, 30 adult female mice and 15 adult male mice
were used. The female mice were superovulated using 10 U of pregnant mare serum gonadotropin (PMSG) and 24
hours later, 10 U of human chorionic gonadotropin (hCG) were injected. Next, cumulus oocyte complexes (COCs) were
collected from the oviducts of the female mice by using a matrix-flushing method. The cumulus cells were cultured with
melatonin 10 µM for 6 hours and for real-time reverse transcription-polymerase chain reaction (RT-PCR) was used
for evaluation of
The results of this study showed that
To improve the oocyte quality and provide new approaches for infertility treatment, administration of
melatonin as an antioxidant, showed promising results. Thus, it is concluded that fertility outcomes can be improved by
melatonin it enhances
As defined by world health organization (WHO) infertility is as the inability of a couple of child-bearing age to conceive over 12 months of regular unprotected sexual intercourse and is considered as a public health problem (1). About 10-15% of young couples have been reported to suffer from this conditions. Of these, 40-55, 20-30, and 15-17% are due to female factors, male factors and unexplained conditions, respectively (2, 3).
For 10% of couples trying to conceive underlying causeof the infertility are not easily identifiable, though all theirrelevant tests are normal. In this case,
Oxidative stress can be reduced and culture conditions can
be promoted an antioxidant or a radical scavenger is added to
Several studies have demonstrated that melatonin is a
powerful direct free-radical scavenger. In contrast to a
majority of other known radical scavengers, melatonin is a
universal multifunctional antioxidant. Melatonin has been
reported to act as a hydrophilic and hydrophobic antioxidant
since it is both lipid and water soluble (9). Also, the presence
of melatonin receptors in the ovary at multiple sites, indicates
that melatonin may influence the reproductive system (10).
Melatonin concentrations in ovarian follicles increase with
follicular growth (11). Furthermore, melatonin treatmentenhances the hCG-stimulated progesterone secretion
(12). Earlier studies indicated that progesterone, a steroidhormone is a key player in ovulation. The biological effectsof progesterone are mediated via
It is generally accepted that
Materials and Methods
This experimental study was approved by the Research Committee of Tabriz Medical University according to the rules of the Ethics Committee. Forty five adult mice (15 male and 30 females, 30-35 g and 6-8 weeks old) were obtained from the animal house of Tabriz University of Medical Sciences (TUMS). Animals were kept in a 12-hour light/12hour dark cycle with an unrestricted access to food and water at room temperature for 2 weeks.
Preparation of melatonin stock solution was done using an ethanol/TCM199 system. For this purpose 23.23 mg of melatonin (Sigma, United states) was dissolved in 1 ml of 0.1% absolute ethanol and diluted with TCM199 and serial dilutions were prepared. Using this method, a stock solution of melatonin 10 µM was prepared (22) and stored in a refrigerator at 4°C at most for 2 weeks. In our experiment, ethanol amount was 0.1% in the maturation medium.
All the animals were treated in accordance with the guidelines of University Ethics Committee for care and use of laboratory animals. The female mice were superovulated using an intraperitoneal injection of 10 IU human menopausal gonadotropin (HMG) (NV Organon, Oss, The Netherlands) and after 24 hours, another intraperitoneal injection of 10 IU of human chorionic gonadotropin (hCG) (NV Organon, Oss, Holland) was done.
The female mice were sacrificed within 48 hours of hCG injection, and the ovaries and oviducts were removed after. Next, the mice ovaries (n=60) and oviducts were placed in sterile phosphate-buffered saline (PBS, Sigma, USA). Then, samples were transported to the Tissue Engineering Laboratory. Upon removing the stromal tissues surrounding the oviducts, the oocytes were collected from the uterine tube using the flushing method (universal medium of Azar Panam). By using a head sampler, the oocytes were drawn and poured into a dish. Finally liquid oil was added to prevent the culture medium evaporation.
The cumulus cells surrounding the oocytes, (i.e., cumulus oocyte complexes (COCs) were transferred to another dish containing the medium and then PBS was added for washing. In the experimental groups, the cumulus cells were cultured in the medium supplement with 10 µM melatonin for 6 hours. For evaluation of cumulus expansion, COCs were morphologically classified at recovery as having a compact or expanded investment. COCs were cultured in 200 mµ of a universal IVF medium and then incubated with sterile mineral oil at 37°C with 5% CO2 for 6 hours. After this incubation period, the medium was centrifuged twice at 3000 rpm for 5 minutes. Afterwards, the pellet of the cumulus cells was transferred to -20°C for 1 hour and then stored at -80°C until RNA isolation.
In vitro fertilization
The animals selected for IVF were divided into control and experimental groups. After superovulation, the femalemice were killed by cervical dislocation, their oocytes were collected by uterine tube flushing. Next, sperms were collectedfrom the caudal epididymis of male mice by incubating the pieces of epididymis with Ham’s F-10 medium cultureusing a CO2 incubator at 37°C for 20 minutes. The spermsamples were added to the collected oocytes of the controland experimental groups. The rate of fertility success was evaluated based on embryo formation associated with more cleavages and morula.
Real time reverse transcription-polymerase chain reaction
|Gene||Sequence (5ˊ-3ˊ)||Start||Stop||Product length|
Using SPSS software, version 22 and the sample t-test, all the statistical analyses were performed. P<0.05 were considered statistically significant.
Effect of melatonin on
HAS2 and PGR expression in
Melatonin effect on fertilization rate and cumulus expansion
IVF was carried out in both control and experimental groups and 100 oocytes were evaluated from each group. By counting the number of embryos, it was revealed that more oocytes were developed into the embryos in the experimental group receiving melatonin for 6 hours as compared to the control group which did not receive melatonin (P<0.05). The embryos developed in the experimental and control groups are shown in Figures 3 and 4, respectively. Moreover, compact cumulus was seen to be tightly attached to the cells surrounding the smooth-surfaced oocytes over the cumulus hillock as shown by evaluating of the uniformity of ooplasm. On the other hand, expanded cumulus cells were detached from the oocytes with a matrix visible between the cumulus cells. The numbers of the expanded and compact COCs are demonstrated in Table 2. The statistical analysis showed that embryo formation in the experimental group was significantly increased (P<0.05, Table 2,).
|- PGR gene expression in isolated cumulus cell in control groups (C) and in groups received melatonin (D).|
|- HAS2 gene expression in isolated cumulus cell in control groups (A) and in groups received melatonin (B).|
|Oocyte||Experimental Group (%)||Control Group (%)||P value|
|Oocyte expansion rate||64||36||<0.05|
It is generally accepted that melatonin and the presence of its receptors in granulosa cells can potentially exert beneficial effects on the ovarian function (22). As melatonin is a powerful free-radicals scavenger which is even more potent than vitamin E (23), it might be able to protect granulosa cells from the cytotoxicity of free- radicals that could be produced following long-term in vitro culture (24, 25).
The results of the present research are in agreement with previous histological and immunohistochemical studies, which showed that free-radicals scavenger could promote the qualities of the cumulus-oocyte complexes through inducing a uniform distribution of follicle cells covering the oocytes in the ovaries (26). These findings are in line with those reported the study of Bahadori et al. (9), describing the detrimental effect of oxidative stress on oocyte microenvironment and subsequently on implantation, follicular development, ovulation, oocyte quality, and early embryonic development. Also, the results of the study conducted by Ishizuka et al. (27) is consistent with those of the current research indicating that melatonin supports both mice fertilization and early development of embryonic tissue in the culture medium.
However, no relationships between oxidative stress
markers and pregnancy rate were reported by Jozwick et
It is concluded that melatonin can improve IVF
outcome by increasing cumulus cell expansion and
The authors of this article are grateful to the Tissue Engineering Center of Tabriz University of Medical Sciences for its financial support. This paper was a research proposal approved by the Research Deputy of Tabriz University of Medical Sciences. The authors declare that they have no conflicts of interest.
M.E.; Participated in executing the plot. L.R.; Contributed to the design and executive process. M.E., L.R.; Involved in writing and editing the manuscript and preparation of figures. J.S.R.; Participated in the design and conception. N.K.; Conducted in real time RTPCR technique. All authors read and approved the final manuscript.