Finalization of follicular atresia in sows with ... - Annals of RSCB

Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 22 – 26 Received 12 August 2016; accepted 21 December 2016.

doi: 10.ANN/RSCB-2016-0013:RSCB

Finalization of follicular atresia in sows with hyperestrogenism LIVIU BOGDAN (1), FLAVIA RUXANDA (2)*, CRISTIAN RAŢIU (3), BIANCA MATOSZ (4), VASILE RUS (2), SIDONIA BOGDAN (5), VIOREL MICLĂUŞ (2) 1 Department of Reproduction, Gynecology and Obstetrics, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania 2 Department of Histology, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania 3 Department of Dental Medicine, Faculty of Medicine and Pharmacy, University of Oradea, Romania. 4 Department of Comparative Anatomy, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania 5 Department of Surgical Propedeutics and Anesthesiology, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania *Corresponding author Flavia Ruxanda, Ph.D. Departments of Cell Biology, Histology and Embryology, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, 3-5, Calea Manǎstur Street, Cluj-Napoca 400372, Romania Phone: +40(0)264.596.384/192, Fax: +40(0)264.593.792 E-mail: [email protected]

Keywords. connective tissue, hyperestrogenism, ovary, sow. Summary in the evolving speed and the number of engaged follicles.

The aim of this study was assessing the finalization of follicular atresia process in sows presenting hyperestrogenism. Ovaries harvested from 5 sows with clinical signs of hyperestrogenism (repeated estrus, weakly expressed, infertility, vulvar edema) were processed for histological examination. Small follicles (primordial, primary) finalize the atresia process quickly and the area is invaded by stroma. In the case of medium size follicles (secondary), the oocyte and surrounding cells disappear rather quickly, but zona pellucida persists for a period of time, which in turn breaks up in smaller fragments and gradually disappears. As for the large follicles (cavitary), involution takes place in a longer period of time, the antrum is invaded by connective tissue and finally the fibrosis of the former follicle takes place. Thus, the finalization of follicular atresia depends on the size and complexity of the follicle when enetering the atresia process. Follicular atresia process takes place similarly to the physiological one in the case of hyperestrogenism. The difference consists

Introduction Atresia refers to the incapacity of a follicle to ovulate if we strictly resume to the origin of the word (in greek a=without, tresis= perforation, orifice). In a broader sphere, atresia is a degenerative process, through which over 99% of the ovarian follicles, including oocytes, are discarded from the ovaries of mammals (Manabe et al., 2004). It ensures the consumption of follicular surpus and thus the ovulation of the most healthy follicles, containing optimum quality oocytes for fertilization (Townson & Combelles, 2012). Atresia can be either physiological or pathological. Upon its morphological aspect, two types of atresia were described in bovines: antral and basal. The antral one implies the elimination of granulosa cells in the proximity of the antrum firstly and was described in all types of follicles. On the other hand, basal

The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Flavia Ruxanda, pp. 22 – 26

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Results and discussions

atresia begins with the destruction of the basal layer of the follicles and was only observed in small follicles (Irving-Rodgers et al., 2001). Removal of follicular cells is attained through apoptosis (Johnson, 2003; Matsuda-Minehata et al., 2006; Inoue et al., 2011), which ensures regression of the follicle without inflammatory response (D’Haesleer et al., 2006; Peluffo et al., 2007). Because of the fact that adult females have a limited number of follicles, and atresia unreels at a high extent, the subject is of great interest in both physiological and pathological atresia (Monniaux, 2002; Sharma, 2003; Sharma & Batra, 2008; Bhardwaj & Sharma, 2011). The morphological aspects emerged along normal (Radu et al., 2012a) and experimental (Radu et al., 2012b) atresia were studied in impuber ewes, while in sows, the stages of atresia were described (Guthrie et al., 1995; Guthrie & Garrett, 2001; Manabe et al., 1996; Pastor et al., 2001). We set out to assess the finalization of follicular atresia process in sows with hyperestrogenism.

In the case of small atretic follicles (primordial, primary), we observed oocytes and follicular cells in process of degeneration in the ovarian cortex. In small atretic follicles, there was only a smaller (Fig. 1) or larger (Fig. 2) quantity of debris, which seemed to disappear in a relatively short period of time. We did not track down any other aspects in the ovarian stroma that would suggest persistence of cellular remnants from small follicles after going through this process.

Materials and methods

Fig. 1. Primordial atretic follicle.

The biological material was represented by 5 sows, from a farm in Bihor county. The animals exhibited clinical signs of hyperestrogenism (repeated estrus, weakly expressed, infertility, vulvar edema). Ovaries were harvested and processed for histological examination. The samples were fixed in 10% buffered formalin for one week, later dehydrated in alcohol and clarified in nbutanol. The next stage was paraffin embedding and sectioning of the samples at 5 µm thickness. The slides were stained with Goldner’s trichrome method. Examination of the histological sections was performed using an Olympus BX 41 light microscope and the images were captured with an Olympus E 330 digital camera, attached to the microscope. Images were processed with the aid of Adobe Photoshop CS2 software, version 9.0.1.

Fig. 2. Primary atretic follicle.

In the case of medium-size atretic follicles, with a somewhat more complex structure (secondary), we observed that after the disappearance of the oocyte, zona pellucida persists a while longer and appears collapsed because of the degeneration of oocyte (Fig. 3). In a more advanced stage, zona pellucida fragments and the resulted debris is phagocytized by macrophages (Fig. 4).


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Fig. 3. Secondary atretic follicle – fragmentation of zona pellucida.

Fig. 5. Atretic antral follicle – Glassy membrane.

Fig. 6. Aretic antral follicle with fibrosis.

Fig. 4. Secondary atretic follicle – macrophage phagocytizing zona pellucida.

Small sized follicles disappear from the ovarian cortex without leaving any trace. Apparently, after degeneration of follicular cells and oocyte, the area is invaded by surrounding connective tissue (stroma). No matter whether atresia begins with oocyte degeneration, follicular cells or both at the same time, the ending will be the same – total disappearance of the former follicle. Similar results with the ones noticed by us were reportend by other authors in physiological atresia. They affirm that in the case of small follicles, the components degenerate and disappear from the ovary, and the area where the former follicle was present, is no longer visible in the ovarian stroma (Adlersberg et al., 1955). Same aspects were signaled by Ross & Wojciech (2006), who mention that the space occupied by the follicle is invaded by stromal cells. In the case of medium size follicles, in pathological atresia, the oocyte and surrounding cells disappear rather quickly.

In the case of large atretic follicles (cavitary), we observed young connective tissue, delimited by the glassy membrane (Fig. 5). In late atresia, the connective tissue in antrum proliferates and consolidates further, so that the area of the former antrum contains fibrous tissue (Fig. 6). Some follicles can present a small cavity even in an advanced stage of fibrosis.


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We can still observe oocyte remnants in process of degeneration for a relatively short period of time. Zona pellucida is the most persistent component, which collapses because of the disappearance of the oocyte, it thickens and then gradually breaks up. The fragments resulted from zona pellucida’s degradation are phagocytized by macrophages. Ross & Wojciech (2006) observed similar aspects in physiological atresia, mentioning that zona pellucida collapses and thickens after oocyte disappearance. Other authors affirm that there is only a granular material left from the former oocyte, which is aggregated towards the central area and zona pellucida outlines the structure. As atresia advances, zona pellucida appears collapsed, even curved (Radu et al., 2012a). In some species, zona pellucida can persist for long periods of time incorporated in stroma (Peters & McNatty, 1980), aspect present in sows taken into study. Other authors signal the rapid involution of small cavitary follicles with oocyte shrinkage, surrounded by a peripheric sudanophilic zone, which sometimes encloses the central mass (Sharma et al., 1992). In the case of large follicles (cavitary), involution takes place in longer periods of time, depending on how developed they were when entering atresia. The process lasts longer because of the structural complexity of the follicle: oocyte, zona pellucida, corona radiata, cumulus oophorus, membrana granulosa, theca interna and externa etc. After oocyte degeneration and phagocytosis of the resulted debris by macrophages, the antrum is invaded by young connective tissue. The glossy membrane forms in the area occupied by the follicular basement membrane (Ross & Wojciech, 2006), aspect which we also observed in the case of pathological atresia of antral follicles. The authors assert that it is actually the basement membrane between the granular cells and theca interna, which thickens, forming a wavy hyaline layer and is a characteristic of large cavitary follicles (Ross & Wojciech, 2006; Vlcjova et al., 2012). In sheep, in physiological atresia, antrum of the atretic cavitary follicles collapses, forming folds; theca interna suffers a total hyalinization process, while theca

externa dedifferentiates and membrana granulosa undergoes a fibrosis process (Vlcjova et al., 2012). Other authors signal the cicatrization of the whole follicle in the final stage of atresia in cavitary follicles (Diculescu et al., 1971). Formation of glassy membrane and the final fibrosis of the whole follicle took place in pathological atresia in sows, in the present study. Some researchers tracked down debris from collapsed follicles in mouse ovaries as stroma and interstitial tissue, five cycles after the follicle became atretic. As the animal ages, the follicular debris accumulates in the ovary because its ability to remove the degenerated follicles decreases (Peters & McNatty, 1980). We can state that finalization of follicular atresia depends on the size and complexity of the follicle when entering atresia. Hyperestrogenism did not determine interference of new mechanisms that would provoke cell or ovarian tissue destruction along with those of follicular atresia, normally unreeled in ovary. The basic mechanisms through which the follicular reserve was reduced were the same as in physiological atresia. Nevertheless, the difference comprised a larger number of atretic follicles and higher intensity and extent of the events. The results show that substances possessing an estrogenic action, unbalance the process of follicular consumption through atresia (an irreversible process) with consequences on the follicular pool, thus being able to compromise the female reproductively.

Conclusions In sows with hyperestrogenism, the process of follicular atresia unfolds similarly to the physiological one, with approximately same stages, except that it unreels at a higher intensity and the number of follicles undergoing this process is clearly larger. Finalization of atresia is comparable in both pathological and physiological atresia, being in direct relation with the structural complexity of each follicle upon entering this process.


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References

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