Role of Nitric Oxide on the Generation of Atretic Follicles in the Rat Ovaries
The aim of this study was to investigate the role
of Nitric Oxide (NO) in the atresia of ovarian follicles in an animal
model. Twenty adult, female rats (90 days old with body weights of 210 ± 10
g in the beginning of the experiments) were divided into 4 groups of 5
each. They were treated twice daily from the subcutaneous route for 21
successive days with either of the following chemicals: nitroglycerine,
L-arginine, L-NAME, or saline. On day 22, all animals were sacrificed.
Ovaries were dissected out free of connected tissue and were fixed in
formaline 10%. Later, paraffine blocks were prepared and serial sections
were made by means of H and E routine staining method. Intact and atretic
follicles were counted separately. In addition, damages were analyzed
qualitatively from the points of view of appearance and morphologic changes.
In the evaluation of ovarian follicular structures, different types of
healthy as well as atretic follicles were observed. In most of atretic
follicles, the oocytes were abnormally elongated and increnation of their
outlines were obvious. There were numerous macrophages around and inside
of the atretic follicles. Our investigation regarding the distribution
of atretic follicles in the ovaries of test groups revealed that atretic
follicles in the L-NAME treated group were increased in comparison to
the control group. Conversely, however, in the arginine-treated group,
the atretic follicles were reduced compared to the control animals. Treatment
with nitroglycerine of the rats decreased the number of atretic follicles
significantly (p<0.05) in comparison to the control group. In conclusion,
enhanced NO, either from endogenous or exogenous origins, prevents atresia
phenomenon, while inhibition of NO exerts an opposite effect.
Nitric Oxide (NO) is a chemical mediator present
in different tissues as in human reproductive system (Calka, 2006). It
is involved in the regulation of many functions in the reproductive system,
including parturition, pregnancy, implantation, ovular activities, steroidogenesis,
folliculogenesis and the regulation of LHRH (Jain et al., 1993).
NO was reported to increase the levels of LH significantly with no effect
on the production of prolactin (Russell et al., 2005). In addition,
decreased levels of NO enhanced the contraction of smooth muscle in the
fallopian tubes, hence, increasing egg transport in the ovum (Ekerhord
et al., 1999). NO increases endometrial secretions during the oogenesis
phase in female animals (Morlin and Hammarstrom, 2005), increases blood
circulation and plays a role in follicle development, oocyte maturation
and steroidogenesis (Philip et al., 2001).
In this study, we investigated the role of this putative
mediator on follicular atresia in the rat. For this purpose, animals were
treated with either of the following substances: saline (control), nitroglycerine
(NTG, an NO donor), L-arginine (LARG, precursor of NO), or N[g]-nitro-L-arginine
methyl ester (L-NAME, an inhibitor of NO synthase [NOS]).
MATERIALS AND METHODS
This study was performed in the year 2006 in Urmia
University Faculty of Sciences, Urmia, Iran.
Animals: Twenty adult, female rats were used in this study.
They were 90 days old, with body weights of 210±10 g in the beginning
of the experiments. They were fed with commercial chow and tap water ad
lib and kept at room temperature with 12 h artificial light in 24
Experimental protocol: Animals were divided into 4 groups of 5 each. They
were treated twice daily from the subcutaneous route for 21 successive
days with either of the following chemicals: Group A with nitroglycerine
(an NO donor), group B with L-arginine (the precursor of NO), group C
with L-NAME (an inhibitor of NO synthase) and group D with saline solution
(control). On day 22, all animals were sacrificed. Ovaries were dissected
out free of connected tissue and were fixed in formaline 10%. Later, paraffine
blocks were prepared and serial sections were made by means of H and E
routine staining method. Intact and atretic follicles were counted separately.
In addition, damages were analyzed qualitatively from the points of view
of appearance and morphologic changes.
Chemicals: L-NAME, LARG and NTG were products of Sigma (St.
Luis, USA), Merck (Darmstadt, Germany) and Daroupakhsh Pharmaceutical
Company (Tehran, Iran), respectively.
Statistics: Differences in the number of atretic follicles were
first analyzed by one-way ANOVA and thereafter by Bonferroni`s t-test.
A p-value smaller than 0.05 was considered as the level of significance.
In the evaluation of ovarian follicular structures, different types
of healthy as well as atretic follicles were observed. The number of atretic
follicles were enhanced slightly after L-NAME treatment while L-arginine
and nitroglycerine had an opposite effect; the effect of the latter reached
the level of significance (p<0.05, Fig. 1). In most of atretic follicles,
the oocytes were abnormally elongated and increnation of their outlines
were obvious. Follicles with 2-3 granulosa cells were exhibiting dispersed
antral spaces between them (precautions antral formation) as a sign of
follicular atresia. In some primary and secondary follicles, signs of
luteinization were seen in granulosa cells, which is also a potential
sign of follicular atresia. In atretic follicles pyknotic index in granulose
cells were high, further revealing follicular atresia. There were numerous
macrophages around and inside of the atretic follicles; by the progression
of follicular atresia, their population increased accordingly. The structure
of Zona Pleucida (ZP) was affected in atretic follicles and signs of breakdown
perforation and fragmentation were seen in ZP structure of atretic follicles.
Present investigation regarding the distribution of atretic follicles
in the ovaries of test groups revealed that atretic follicles in the L-NAME
treated group were increased in comparison to the control group. Conversely,
however, in the arginine-treated group, the atretic follicles were reduced
compared to the control animals. Treatment with nitroglycerine of the
rats decreased the number of atretic follicles significantly (p<0.05)
in comparison to the control group (Fig. 1). In the atretic
follicles, the following changes were observed: cleavage-like divisions
in oocyte nucleus (Fig. 2) and malformed oocyte with
shrinkage and luteinized granulosa follicle (Fig. 3).
Comparison of the
avenge numbers of atretic follicles between control and treated
experimental groups. As seen in the figure, increased levels of
NO by L-Arg and TNG (p<0.05) decreased the number of atretic
follicles, while inhibition by L-NAME of NO production had the opposite
Oocyte nucleus shows
cleavage-like divisions (1) and a macrophage is seen inside the
Arrow number 1 shows
a malformed oocyte with shrinkage and number 2 demonstrates granulosa
follicle that has been luteinized
Although research activities have long been done
over follicular atresia, the underlying mechanisms are not fully understood
yet. Whilst some believe that early signs are observed in the oocyte,
others claim that it is initiated from granulosa cells and then spread
to other parts (Asami, 1920; Sturgis, 1949; Ingram, 1962; Pincus and Enzman,
1975; Daud et al., 1988). Follicular atresia is a process which
may occur during any stage of the follicular growth (Byskov and Rasmussen,
1974). Differential diagnosis between normal and atretic follicles is
a difficult task as all follicles do not follow a similar process during
the phenomenon (Byskov, 1979; Grimes et al., 1987).
Of numerous number of the follicles in an ovary, only
a limited number succeed in growth, maturity and, finally, ovulation.
The rest become atretic. Atresia may occur in different forms, including
picnosis of granulosa cells (Hirshfield, 1989), segmentation of basal
layer (Bagavandoss et al., 1983), floating the granulosa cells
in the antral fluid (Hay et al., 1976), shrinkage, segmentation
and disappearance of the nucleus and nucleolus (Ojeda et al.,
1992; Hirshfield and Midyley, 1978; Peters and Ball, 1987).
Various forms of atresia were observed in the present
study. The interesting finding, however, was that many atretic follicles
possessed a few characteristics of atresia so that, especially in larger
follicles, more than 6 important signs of atresia phenomenon were observed.
In cytologic criteria, picnosis and chromatinization
of granulosa cells, disrupture between them and existence of floating
granulosa cells in the antrum with picnotic nuclei are evident signs of
follicular atresia (Byskov and Rasmussen, 1974).
During recent years, studies have shown that macrophages
have a great impact on the regulation of the ovarian functions (Wu et
al., 2004). They reported that macrophages and their secretory products
can lead to disturbances in the ovaries, including polycystic ovary syndrome,
endometriosis and early failure of ovaries. In addition, the role of these
cells in the destruction of cellular remnants in various tissues of the
body has already been shown. Therefore, it can be concluded that in the
ovarian tissue, whenever atresia or cell death occurs, macrophages are
In this study, too, the presence of macrophages was shown
in different areas of the ovarian tissue. No macrophages were seen around
healthy follicles but they had an active presence around atretic ones.
It was possible to locate many macrophages in the antrum of antral follicles
and many of them were seen in the connective tissue surrounding non-healthy
(atretic and cystic) follicles. Following ovulation, the cellular complex
of the follicles undergoes alterations that finally lead to the formation
of the corpus luteum (Byskov, 1979). The granulosa and internal tech participate
in the formation of corpus luteum. The granulosa cells are larger and
paler while the thecal cells are smaller and denser than the others (Dellman
and Eurell, 1998). Based on what was said, it can be concluded that any
luteinization of the follicular wall that occurs before ovulation is a
non-physiologic process and can be considered as atretic processes.
Some other important signs of atresia may be manifested
in the oocyte of which the mitotic division and cleavage-like changes
and segmentation of oocyte are common (Talukar et al., 1991). Similar
changes were observed in this study as well. Mitotic and cleavage-like
divisions were demonstrated in the follicles larger than 200 μm.
We concluded that these abnormalities are seen solely in tertiary and
graffian follicles, whereas small follicles at early stages of growth
were free of these disturbances.
Shrinkage and malformation of the oocyte are other prominent
signs of atresia (Hirshfield, 1989). In normal follicles, oocytes are
spherical with a round shape seen in their cross sections. The nucleus
is located in the centre and is seen clearly together with the nucleolus
and the oocyte has a completely smooth wall. However, we observed a number
of oocytes with serrated and rough surroundings. Oocyte was crumpled and
shrunken form. Surroundings of the oocyte had a shape of serrated, folded
and dented appearance. In most cases, this cell was detached from the
granulosa cells and from its cumulous mass in the antral follicles and
was floating in the follicular fluid. This abnormality was observed in
all stages of follicular growth, including antral and pre-antral stages.
It should be stated that any change in the structure
of ZP can affect the health of oocyte, but it should be also mentioned
that degenerative changes in the ZP take place following degenerative
changes in the oocytes and granulosa cells (Hasanzadeh and Sadrkhanloo,
2002). The present research work demonstrated several cases of atresia,
related to ZP. This abnormality was seen in the follicles of more progressive
stage. From the disturbances seen in the ZP, the followings can be numbered:
deletion of some parts of the membrane, perforation, laminating, hyalinization,
breakage, incarnation and folding in ZP.
Studies performed on the role of NO in the rabbit shows
that this substance has effects on ovulation, oocyte maturation and production
of sex steroids. By relaxing the vascular smooth muscle, NO increases
blood flow in the ovary, easing the rupture of the follicle and ovulation.
In addition, endogenous NO and NO-related substances inhibit steroid production
by the luteal and granulosa cells (Yamauchi et al., 1997). In
vitro studies suggest a clear role for NO in the persistence of the
ovum as well as on ovulation. The effects of NOS inhibitors in the body,
however, are not identical with those seen under in vitro conditions;
it is possible that these effects in the body are mediated by extra-follicular
factors as well (Mitchel et al., 2004). After ovulation, homogenous
endothelial NOS can be seen in corpus luteum. During investigations performed
on mouse ovary, it was observed that eNOS-derived NO is the mediator of
oocyte miotic maturation. Besides, NO is involved in the PSRAFT (regression)
of the luteal function via inhibition of steroidogenesis. NO may induce
rupture of follicles in the rabbit during the ovulation process and this
is probably accomplished via prostaglandin production (Tsafriri et
Based on what seen in the present findings, L-NAME and
L-arginine, respectively, have dramatically increased and decreased the
number the atretic follicles. Other findings have revealed that NO is
significantly implicated in apoptosis. NO may decrease apoptosis by nitrosylating
cysteine and hence, inhibition of caspase-3 (Lothar and Cherer, 1999).
Studies have demonstrated that endogenous NO, produced by granulosa cells
of immature follicles, may decline apoptosis and, therefore, prevent atresia
development in follicles. Indeed, elevated levels of NO have been shown
to decrease apoptosis in the granulose cells in pigs and calves (Tamanini
et al., 2003). This could be one of the underlying mechanisms to
decline atresia of the follicles in the animal model used in this study.
1: Asami, G., 1920. Observations on the follicular atresia in the rabbit ovary. Anat. Rec., 18: 323-343.
2: Bagavandoss, P., A.R.J. Midgley and M. Wicha, 1983. Developmental changes in the ovarian follicular basal lamina detected by immunofluorescence and electron microscopy. J. Histochem. Cytochem., 31: 633-640.
Direct Link |
3: Byskov, A.G. and G. Rasmussen, 1974. Ultrastructural Studies of the Developing Follicle in the Development and Maturation of the Ovary and its Function. Eport Medical Amsterdam, UK., pp: 55-62.
4: Byskov, A.G., 1979. Atresia, Ovarian Follicular Development and Function. Raven Press, New York, pp: 111-118.
5: Calka, J., 2006. The role of nitric oxide in the hypothalamic control of LHRH and oxytocine release, sexual behavior and aging of the LHRH and oxytocine neurons. Folia Histochem. Cytobiol., 44: 3-12.
Direct Link |
6: Daud, A.L., F.M. Bumpus and A. Husain, 1988. Evidence for selective expression of angiotensin 2 receptors on atretic follicle in the rat ovary and auto radiographic study. Endocrinology, 122: 2727-2734.
7: Dellman, H.D. and J.A. Eurell, 1998. Text Book of Veterinary Histology. 5th Edn., Williams and Wilkins, USA., pp: 247-270.
8: Ekerhord, E., M. Brannstrom, B. Weijdegard and A. Norstrom, 1999. Localization of nitric oxide synthase and effects of nitric oxide donors on the human fallopian tube. Mol. Hum. Reprod., 5: 1040-1041.
Direct Link |
9: Grimes, R.W., P. Matton and J.J. Irland, 1987. A comparison of histological and non-histological indices of atresia and follicular function. Biol. Reprod., 37: 82-88.
10: Hasanzadeh, S. and R. Sadrkhanloo, 2002. Study of ovarian follicular atresia in makoyee ews in different stages of estrous cycle and different seasons of a year. J. Fac. Vet. Med. Univ. Tehran, Iran, 55: 81-86.
11: Hay, M.F., P.G. Cran and R.M. Moor, 1976. Structural changes occurring during atresia in sheep ovarian follicles. Cell Tissue Res., 169: 515-529.
12: Hirshfield, A.N. and A.R. Midyley, 1978. Morphologic analysis of follicular development in the rat. Biol. Reprod., 190: 597-605.
13: Hirshfield, A.N., 1989. Rescue of atresic follicles in vitro and in vivo. Biol. Reprod., 40: 181-190.
14: Ingram, D.L., 1962. Atresia in the Ovary. Vol. 7, Academic Press, New York, pp: 247-275.
15: Jain, B., I. Rubinstein, R.A. Robbins, K.L. Leise and J.H. Sission, 1993. Modulation of airway epithelial cell ciliary beat frequency by nitric oxide. Biochem. Biophys. Res. Commun., 191: 83-88.
16: Lothar, R. and B.F. Cherer, 1999. Nitric oxide inhibits caspase-3 by s-nitrosation in vivo. J. Biol. Chem., 274: 6823-6826.
17: Mitchel, L.M., C.R. Kennedy and G.M. Hartshorne, 2004. Pharmacological manipulation of nitric oxide levels in mouse follicle cultures demonstrates key role of extra follicular control of ovulation. Hum. Reprod., 19: 1705-1712.
Direct Link |
18: Morlin, B. and M. Hammarstrom, 2005. Nitric oxide increases endocervical secretion at the ovulatory phase in the female. Acta obstetricia Gynecol. Scandinavica, 84: 883-886.
Direct Link |
19: Ojeda, S.R., G.A. Dissen and M.P. Junnier, 1992. Neurotrophic Factors and Female Sexual Development. In: Frontiers in Neuroendocrinology, Ganong, W.F. and L. Martin (Eds.). Raven Press, New York, pp: 120-162.
20: Peters, A.R. and P.H.J. Ball, 1987. Reproduction in Cattle. Butterworth, London, pp: 141-142.
21: Philip, P.M.W., R. Pathansali, R. Iddenden and F. Bath, 2001. The effect of transdermal glyceryl trinitrate, a nitric oxide denor, on blood pressure and platelet function in acute stroke. Cerebrovasc Dis., 11: 265-272.
Direct Link |
22: Pincus, G. and E.V. Enzman, 1975. The growth, maturation and atresia of ovarian eggs in the rabbit. Morphology, 61: 351-383.
23: Russell, J.M., E. Murphree, J. Janik and P. Callahan, 2005. Effect of steroids and nitric oxide on pituitary hormone release in ovariectomized, peripubertal rats. Reproduction, 129: 497-504.
Direct Link |
24: Sturgis, H.S., 1949. Rate and Significance of atresia in the ovarian follicle of the rhesus monkey. Embryol. Corneg. Inst., 33: 67-70.
25: Talukar, S.R., C.C. Bordoli and S. Ahmed, 1991. Histological and histochemical studies of atretic follicles in ovaries of Assam local coat (Capra hicus) in early post natal life. Indian Vet. J., 68: 245-248.
26: Tamanini, C., G. Basini, F. Rasselli and M. Tirelli, 2003. Nitric oxide and the ovary. J. Anim. Sci., 81: E1-E7.
27: Tsafriri, A., S.Y. Chun, A.J.W. Hsueh and M. Conti, 1996. Oocyte maturation involves compartmentalization and opposing changes of cAmp levels in follicular somatic and germ cells: Studies using selective phosphodiesterase inhibitors. Dev. Biol., 178: 393-402.
28: Wu, R., K.H. Vander Hock, N.K. Rayan, R.J. Norman and R.L. Robker, 2004. Macrophage contribution to ovarian function. Hum. Reprod. Update, 10: 113-119.
Direct Link |
29: Yamauchi, J., T. Miyuzaki, S.H. Iwasaki, H. Kishi, M. Kuroshima, C.H. Tei and Y. Yoshimura, 1997. Effect of nitric oxide on ovulation and ovarian steroidogenesis and prostaglandin production in the Rabbit. Endocrinology, 138: 3630-3637.