CC BY
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S.A. NOTOLLA1, S CECCONI2, G.MACHIARELLI3
MORPHOLOGICAL EFFECTS OF PESTICIDES ON THE MAMMALIAN OVARIAN FOLLICLE
Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, University "La Sapienza"1, Rome, Italy Department of Life, Health and Environmental Sciences, University of L'Aquila2,3, Italy
PhD, professor1,2,3
Annotation
In this article we have reviewed the effects of mancozeb, on the mammalian ovarian follicles. Along with the widespread use of pesticide in the world, the concerns related to their impact on human health, it is rapidly growing.
A pesticide is any substance used to kill, repel, or control certain form of plant or animal life that are considered to be pests. Pesticides include herbicides for destroying weeds and other unwanted vegetation, insecticides for controlling a wide variety of insects and fungicides, used to prevent the growth of molds and mildew (source: National Institute of Environment Health Sciences, U.S.A. )
The massive and sometimes indiscriminate use of pesticides for agricultural purposes has determined (and still determines nowadays) a definite risk for the health in humans and other animal species, although their use is now prohibited or restricted in many Countries. Pesticides may cause not only acute (from simple irritation of skin and eyes to severe neurologic disorders) but also delayed health effects in exposed subjects. Pesticides, in fact, persist in the environment, being stored in the food chain and causing an indirect and chronic exposure of the organisms. In particular, as far as the human species is concerned, it has been recently pointed out that, some environmental pollutants may have a role in determining a significant increase of chronic diseases such as different types of cancers, diabetes, neurodegenerative disorders, birth defects as well as of reproductive alterations in the exposed populations [1]. At this regard, it should be underlined that some pesticides, including organochlorine compounds, together with fungicides, industrial chemicals, phytoestrogens and pharmaceutical estrogens, are able of interfering with the endocrine and reproductive systems, mimicking or, in some cases, completely blocking the activities of endogenous estrogens. Thus, they actually function as xenoestrogens. Due to their capability of interfere with the complex homeostatic balance of the endocrine system, actually modulating its activity, xenoestrogens have been also termed «endocrine disruptors». These compounds can cause reproductive problems by affecting puberty, decreasing sperm count and quality, increasing the number of testicular germ cells and causing male breast cancer, cryptorchidism, hypospadias, miscarriages, endometriosis, irregularities of the menstrual cycle and infertility [2].
The effects of such a hormonal modulation appear particularly evident on the female reproductive system, in which the complex interactions underlying the finely tuned physiological balance seem to be particularly vulnerable to alteration by reproductive toxins [3]. Among the endocrine disruptors specifically affecting the female reproductive system, those with steroidogenic/antisteroidogenic effects have been extensively, studied and the mechanisms of toxicity clarified also at molecular level. For many others information is limited to few epidemiological data and in vivo/in vitro experiments with animal models. This is the case of dithiocarbamates, and in particular of the fungicide mancozeb, an ethylendithiocarbamate commonly used to protect fruit and vegetables, including ginseng, due to its low acute toxicity in humans. Although the mechanisms by which mancozeb may specifically act on female reproductive organs are largely unknown, data on experimental animals in vivo have highlighted that this fungicide can negatively interfere with the estrous cycle [4].It is iworthnoting that, when used in vitro at concentrations considered too low to affect human health, the fungicide impairs oocyte meiotic spindle assembly,granulosa cell morphology and embryo development in the mouse. These effects will be briefly reviewed in the next paragraphs.
Oocyte and meiotic spindle
The production of a mature oocyte competent for fertilization is the major threat of the ovary. This process occurs within the ovarian follicle, and depends on the close association between the oocyte and the surrounding somatic follicular cells [5]. In female mammals, the cortex of the ovary is housed by numerous, differently sized ovarian follicles. During follicular growth, in parallel with the increased complexity and dimensions of the follicle, the oocyte reaches its appropriate size for ovulation and ultimately undergoes a series of maturative changes that affect both nuclear and cytoplasmic domains. Nuclear changes are substantially represented by the resumption of meiosis, followed by the completion of the first meiotic division with the extrusion of the first polar body (PB1) in the perivitelline space. Afterwards, the second division immediately starts and metaphase II chromosomes arrange onto the equatorial plate of the meiotic spindle in a cortical region of the cytoplasm. Meiosis will be arrested again at this stage to be ultimately completed only at fertilization with the extrusion of the second PB (PB2). Both microtubules and microfilaments are closely associated with the reconstruction and proper positioning of chromatin during resumption of meiosis, completion of the first meiotic division and starting of the second [6]. In general, oocyte organelles -mainly mitochondria become far more abundant during final oocyte growth. Cytoplasmic ultrastructural markers of maturity in the human oocyte are: l. the occurrence of numerous and voluminous cytoplasmic aggregates,mainly formed by mitochondria and cytoplasmic membranes, and 2. the distribution of cortical granules just beneath the plasma membrane. Other organelles such as lattices, vacuoles and lipids are specie-specific cytoplasmic features in mammals other humans [7].
When mouse oocytes are matured in vitro with or without increasing concentrations of the fungicide mancozeb (from 0.001 to 1 |jg/ml), the percentage of oocyte extruding PB1 at the end of the maturation period is similar in all the conditions tested (about 95%). However, when oocytes are analyzed by immunofluorescence in order to determine whether the fungicide could
Background
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ЭКСПЕРИМЕНТАЛЬНЫЕ И КЛИНИЧЕСКИЕ ИССЛЕДОВАНИЯ ^
affect spindle microtubule integrity or chromosome alignment on metaphase plate, untreated (control) oocytes mostly (98%) show normally assembled spindles, located at the periphery of the cell, with well-aligned chromosomes on methaphaseplate. Conversely, mancozeb induces a dose-dependent increase in spindle morphology alterations, mainly represented by elongation of microtubules [8]. Thus, in vitro maturation of mouse oocyte and spindle analysis can be used as a sensitive test for assessing cellinjury due to pesticides, even when used at low concentration.
Oocyte competence to fertilization
At fertilization, after the extrusion of PB2, the chromosomes retained by the egg become surrounded by an envelope deriving from the coalescence of cytoplasmic membranes, forming the female pronucleus. At the same time as the formation of the female pronucleus, the sperm nucleus incorporated in the ooplasmexpands and decondences to form the male pronucleus. Once the female and male pronuclei are formed, they migrate to the center of the egg, where they become closely associated each other. After pronuclear association the nuclear envelope breaks down and the andmaternal and paternal chromosomes finally come together, thus establishing the genetic pattern of the new individual. This event, called syngamy, is considered to be the expression of the end of the fertilization process: Now, the first embryo cleavage occurs.
Oocyte competence to fertilization is affected by exposure to mancozeb, both in vivo and in vitro. In particular, when female mice are treated with 50 and 500 mg/Kg of mancozeb from gestational day 2 to postnatal day 20, it has been demonstrated that, only at the highest dose, mancozeb induces a significant decrease of ovulated eggs and a decrease of fertilizability related to a reduction of the formation of male and female pronuclei [9]. Mouse oocytes matured in vitro in presence of 0.1 and 1 |jg/ ml mancozeb show a significant reduction in the formation of male and female pronuclei even in culture conditions. All these abnormal features can be prognostic of subsequent embryo developmental failure. Thus, when used in vitro at concentrations considered too low to cause human health injuries, the fungicide mancozeb impairs in the mouse not only meiotic spindle assembly, likely perturbing microtubular organization, but also early embryo development, ultimately interfering with one or more steps related with normal cell cycle progression and successful reproduction.
Granulosa cells
During follicular growth, the follicular cells encircling and interacting with the oocyte undergo a series of modifications in size, number and shape. In detail, at the primordial- to primary- follicle transition, follicular cells transform from a monolayer of flattened cells into cuboid-low columnar cells. At this time, deposition of the zonapellucida (ZP) at the oocyte interface, as well as a concentric arrangement of stromal cells to form the thecal layers joust outside the basement membrane that outlines the follicular layer are other crucial events that characterize ovarian follicles when exit the resting phase to start growth. Further follicular growth is caused by both the simultaneous enlargement of the oocyte and stratification of the follicular cells (secondary follicle stage). During the subsequent antral phase, whereas the oocyte has reached its appropriate size for ovulation, the follicular cells continue to proliferate and differentiate, becoming granulosa cells. The formation of the antral cavity segregates two different subpopulations in the granulosa compartment: the cumulus oophorus, whose inner layer is formed by alongated cells called corona radiata- closely arranged around the oocyte and its covering ZP-, and the polyhedral parietal granulosa cells, joined together by a thick stalk. Mature preovulatory follicles have reached terminal stages of growth and maturation and appear as large vesicles bulging from the ovarian surface. Hormone secretion (estrogens and progesterone) is another major function of the ovary during the menstrual cycle. Estrogen production is primarily due to the close interplay between granulosa and inner thecal cells before ovulation, whereas progesterone is secreted by the corpus luteum, which derives from the post-ovulatory remodeling of the follicle wall.
Granulosa cells, when cultured in vitro, show a polygonal shape and a flattened cytoplasm with a central prominent nucleus. Cell surface shows microvilli, rare blebs and thin or large, flat prolongations, often contacting neighbouring cells. Scanning electron microscopic observations on cultured mouse granulosa cells treated with 0.01 jg/ml mancozeb for 24 hours do not reveal significant surface changes as those typically observed in apoptotic cells, such as cell shrinkage and extensive surface blebbing. However, a reduction of cell medium diameters of granulosa cells and of intercellular attachments in exposed respect to control cells indicated that these cells may be reactive to a low dose of mancozeb. This sensitivity is confirmed by immunofluorescence studies, which reveal that cultured granulosa cells exposed to mancozeb undergo time- and dose-dependent modifications of their morphology. In fact when mouse granulosa cells are cultured for 1, 24 and 36 hours in the presence of increasing concentrations of the fungicide (0.001 - 1 jg/ml), no changes in cortical actin distribution are observed after 1 hour treatment, at any concentration. By contrast, a 24-hour treatment with 0.1 and 1 jg/ml induces cell reshaping (transition from an epithelisl-like to a spindle-like shape) and changes in the actin cortical cytoskeleton. These changes become more evident after 36 hours, even when the pesticide is administered at low concentrations (0.01 jg/ml). Despite these findings, this series of experiments confirms that no apoptotic changes are induced by mancozeb in cultured mouse granulosa cells, even if exposed to the highest concentrations of the pesticide (1 jg/ml) for 36 hours [10].
Conclusions
In conclusion, all these morphological data indicate that, besides the different mechanism of action, ethylendithiocarbamates can exert important toxic effects on mammalian oocytes, and their role as inducers of infertility should not be underestimated. In addition, these findings demonstrate that mancozeb can negatively affect mammalian granulosa cells even at very low concentrations.
Further, the possibility that the oocyte and its somatic microenvironment could be specific targets of mancozeb suggests a role for thos largely used fungicide as candidate inductor of infertility also in exposed human females. The idea that mancozeb may affect human reproduction is also supported by the detection of birth defects in three infants exposed to mancozeb during pregnancy. Thus, the accurate characterization of the human reproductive hazard potential of mancozeb may represent a toxicological priority, and strategies aimed to preventing the indiscriminate use of this fungicide should be taken into careful consideration.
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ТУЙ1Н
С.А. НОТОЛЛА1, С. СЕККОНИ2, Г. МАККИАРЕЛЛИ3
СУТКОРЕКТ1ЛЕРД1И ФОЛЛИКУЛАЛАРЫНА ПЕСТИЦИДТЕР ЭСЕРШЩ МОРФОЛОГИЯЛЫК ТИ1МД1Л1Г1
«LaSapienza» Университет^, Рим, Италия «L'Acuila» университет^3, Италия PhD, профессор1,2,3
Элемде пестицидтердi колданумен 6ipre олардыч адам денсаулыгына байланысты, сондай-ак репродуктивтi денсаулык сурактары бойынша мэселелер тез есуде. Бул макалада авторлар манкозеблч тиiмдiлiгiн карастырган, кеп жагдайда фунгицид сYткоректiлердiч жумыртка фолликулаларында, эаресе гранулезаныч жумырткаклеткалары мен клеткаларында осы заттыч эсерiнен болатын морфологиялык езгерютерге катысты жемiстер мен кекенiстердi коргау Yшiн колданылады. Егер эйелдiч репродуктивт агзасына менкозебпен накты эсер етуге болатын механизмдер белгiлi бiр сатыда аныкталмаса, жануарларга табиги жагдайда жасалган сараптамалар фунгицидтiч циклде бiршама езгерютерге алып келетiнiн керсеттi. Сонымен катар, пробиркще концентрат колдану барысында оныч адам денсаулыгына зияны аз екендИне кез жеттi, бул фунгицид тышкандар жумырткаклеткаларын урыктандырады жэне морфодинамикасыныч гранулез клеткаларын меотикалык жиынтыктыч жумырткаклеткаларын эларетедк Барлык мэлiметтер фунгицидтiч адам популяциясын элсiрететiн бедеулiк индукторы болуы мYмкiн екендiгiн дэлелдейдi.
Нег'зг'! свздер: жумырткаклеткалар, урыктандыру, гранулеза клеткалары, манкоцеб, жука курылым, CYmwpeKmmep.
РЕЗЮМЕ
С.А.НОТОЛЛА1, С.СЕККОНИ2, Г.МАККИАРЕЛЛИ3
МОРФОЛОГИЧЕСКИЕ ЭФФЕКТЫ ВОЗДЕЙСТВИЯ ПЕСТИЦИДОВ НА ФОЛЛИКУЛЫ МЛЕКОПИТАЮЩИХ
Университет «LaSapienza» 1, Рим, Италия Университет «L'Acuila»23, Италия PhD, профессор1,2,3
Наряду с широким использованием пестицидов в мире, проблемы, связанные с их влиянием на здоровье человека, а также по вопросам репродуктивного здоровья увеличиваются быстро. В этой статье мы рассмотрели эффекты манкоцеба. Обычно фунгицид используется для защиты фолликулов яичников млекопитающих от фруктов и овощей, особенно в отношении морфологических изменений, вызванных этим веществом на яйцеклетки и клетки гранулезы. Если механизмы, посредством которых манкоцеб может конкретно действовать на женские репродуктивные органы, в значительной степени неизвестны, то эксперименты на животных в естественных условиях показывают, что фунгицид может вызвать некоторые изменения в цикле. Кроме того, при использовании в пробирке концентрации мы убедились в том, что фунгицид вреден для здоровья человека, ухудшает оплодотворение яйцеклеток и клеток гранулезы морфодинамики у мышей. Все эти данные свидетельствуют о роли фунгицида как вероятного индуктора бесплодия, который подвергает всю человеческую популяцию.
Ключевые слова: яйцеклетки, оплодотворение, клетки гранулезы, манкоцеб, тонкая структура, млекопитающие.
Батыс Цазацстан медицина журналы №1-2 (38) 2013 ж.
