Deficient proliferation and apoptosis in the granulosa and theca interna cells of the bovine cystic follicle

The aim of the present study was to examine the frequencies of cell proliferation and death of granulosa and theca interna layers during development of cystic follicles in order to understand the mechanisms of cystic follicle formation. Paraffin sections of cystic follicles were immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 in order to observe proliferating and apoptotic cells, respectively. The concentrations of estradiol-17beta and progesterone in the follicular fluid of these follicles were measured by ELISA. The granulosa and theca interna layers contained both PCNA- and caspase-3-positive cells, although their numbers were limited. There was significant negative correlation between the estradiol-17beta and progesterone concentrations in the follicular fluid. Regression analysis revealed no significant correlation, except for that between the PCNA-positive cells in the theca interna and the caspase-3-positive cells in the granulosa layer. These results indicate that the granulosa and theca interna cells of the cystic follicle show weak proliferative activity and low apoptotic frequency; this implies that the cystic follicle grows slowly and then maintains a static condition without degeneration, which leads to long-term persistence of the follicle.     

Estrogen receptors alpha and beta and progesterone receptors in normal bovine ovarian follicles and cystic ovarian disease

The purpose of this study was to determine by immunohistochemistry the expression of estrogen and progesterone receptors in ovarian follicular structures from cows with cystic ovarian disease (COD) and to compare these with normal ovarian structures. Secondary, tertiary, atretic, and cystic follicles were evaluated. The follicular cysts of animals with COD presented a significantly higher expression of estrogen receptor alpha in all follicular layers than secondary, tertiary, and atretic follicles in both groups (P < .05). The intensity of estrogen receptor beta in the granulosa cell layer was stronger in tertiary than in secondary and atretic follicles in normal animals (P < .05) and in growing and cystic follicles in animals with COD (P < .05). Theca cells were scarcely stained in the 2 groups. Growing follicles and cysts from COD animals were less stained than tertiary follicles from normal animals (P < .05). Differences did not exist between the 2 groups with regard to the progesterone receptor. Ovaries of animals with COD exhibited altered estrogen receptors expression compared with that in normal animals.     

Involvement of PAPP‐A and IGFR1 in Cystic Ovarian Disease in Cattle

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra‐ovarian factors, such as members of the insulin‐like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy‐associated plasma protein A (PAPP‐A). The aim of this study was to determine IGFR1 and PAPP‐A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP‐A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP‐A present in the follicular fluid from these follicles showed no differences. Although no PAPP‐A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.     

Colocalization of DNA fragmentation and caspase-3 activation during atresia in pig antral follicles

Apoptosis is the cellular mechanism of ovarian follicular atresia. The major downstream effector of this phenomenon in many tissues is caspase-3 but little is known about its role in pig ovarian apoptosis. In the present study, we detected the localization of caspase-3 in parallel with nuclear fragmentation (TUNEL) on healthy and early atretic antral follicles. In healthy antral follicles caspase-3 and TUNEL positivity were occasionally recorded within theca layer. The incidence of DNA fragmentation, as indicated also by the biochemical detection, increased mainly in the granulosa layer of early atretic follicles. Quantitative analysis revealed, besides, that atresia was accompanied by a higher incidence of caspase-3 (57.20 +/- 20.05 versus 3.64 +/- 0.61 positive cells in atretic versus healthy follicles, respectively; P < 0.05), of TUNEL positivity (20.13 +/- 9.33 versus 0.42 +/- 0.12; P < 0.05) and simultaneous immunostaining for caspase-3 and TUNEL (15.02 +/- 6.95 versus 0.31 +/- 0.05; P < 0.05) in the granulosa layer. In detached granulosa cells isolated from the follicular fluid of early atretic follicles a further significantly increase was recorded in the percentage of TUNEL positivity and in the incidence of cells that showed colocalization of caspase-3 activity and DNA fragmentation. Granulosa cells of early atretic follicles exhibited a higher positivity for caspase-3 localized in the cytoplasm and occasionally in the nucleus area of granulosa cells. These results indicate that capsase-3 was involved and precociously activated during the process of atresia. Finally, the progressively higher incidence of TUNEL positivity and of double immunostaining in atretic cells collected within the follicular fluid seems to indicate that proteases activity leads only tardily in a detectable DNA fragmentation.     

Distribution of Cytochrome P450-side Chain Cleavage in the Theca Interna Layers of Bovine Small Antral and Cystic Follicles

Cystic follicle is anovulatory follicular structure that is caused by an endocrine imbalance. The activity of cytochrome P450‐side chain cleavage (P450scc) is essential for the initiation of steroidogenesis in the follicle. The present study was designed to compare the frequency of cells containing P450scc between healthy and atretic small antral follicles, and among several types (I, II and III, classified based on the presence of granulosa layer) of cystic follicles. Paraffin sections of healthy (2–5 mm in diameter), atretic (2–5 mm) and cystic follicles (>25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine P450scc. The P450scc‐positive cells were counted in four different regions of the follicles from the apical to the basal side. In small antral follicles and cystic follicles, P450scc‐positive cells were localized in the theca interna layers but not granulosa layers. The P450scc‐positive cell populations decreased in the late atretic follicles compared with the early and advanced atretic follicles at all the regions of follicle. Type III cystic follicles showed significantly lower frequencies of P450scc‐positive cells than those in the types I and II cystic follicles. These results suggest that in both small and cystic follicles in cows, total loss of granulosa cells may be associated with the reduction of frequency of P450scc‐positive cells in theca interna layer.     

Intraovarian localization of growth factors in induced cystic ovaries in rats

We hypothesized that the special hormonal environment present in animals with cystic ovarian disease (COD) interferes with cellular production of growth factors (GFs). The objective of the present study was to characterize the expression of insulin-like growth factor (IGF)-I, fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) in induced COD using immunohistochemistry. We used an experimental model based on the exposure to constant light of adult rats during 15 weeks. We quantified the expression of GFs in cystic and normal ovaries by the Immunohistochemical Stained Area (IHCSA). In animals with COD, a significant reduction in the IHCSA of IGF-I in the follicular fluid, theca and granulosa layers of cysts occurred; and an increase in the interstitial tissue with regard to the control group. We found moderate immunoreactivity of FGF-2 in granulosa and theca layers of secondary and tertiary follicles and lower expression in the granulosa and theca interna layers of cystic follicles. Immunoexpression of VEGF was found in granulosa and theca cells of secondary and tertiary follicles. This study shows changes in the ovarian expression of IGF-I, FGF-2 and VEGF in induced COD. We can propose that an alteration in the control of the follicular dynamic, through the GFs, added to other features, could be involved in the ovarian cyst pathogenesis.     

Altered Expression of Transforming Growth Factor‐Beta Isoforms in Bovine Cystic Ovarian Disease

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra‐ovarian factors may contribute to follicular persistence. Transforming growth factor‐beta (TGFB) isoforms are important paracrine and autocrine signalling molecules that regulate ovarian follicle growth and physiology. Considering the importance of these factors in the ovarian physiology, in this study, we examined the expression of TGFB isoforms (TGFB1, TGFB2 and TGFB3) in the ovary of healthy cows and animals with spontaneous and adrenocorticotrophic hormone (ACTH)‐induced COD. In the oestrous‐synchronized control group, the expression of TGFB1 in granulosa and theca cells was higher in spontaneous cysts than in atretic or tertiary follicles. When we compared TGFB2 expression in granulosa cells from atretic or tertiary follicles from the oestrous‐synchronized control group with that in ACTH‐induced or spontaneous follicular cysts, we found a higher expression in the latter. The expression of the TGFB isoforms studied was also altered during folliculogenesis in both the spontaneous and ACTH‐induced COD groups. As it has been previously shown that TGFB influences steroidogenesis, ovarian follicular proliferation and apoptosis, an alteration in its expression may contribute to the pathogenesis of this disease.     

Immunohistochemical Expression of Growth Factors in the Follicular Wall of Normal and Cystic Ovaries of Sows

The expression of growth factors was evaluated immunohistochemically in normal and cystic ovaries of sows. The immunohistochemically stained area (IHCSA) was quantified by image analysis to analyse the expression of these proteins in the follicular wall of secondary, tertiary and cystic follicles. IGF‐I immunoreactivity was strong in the granulosa cell layer (GC), moderate in the theca interna (TI) and mild in the theca externa (TE) of the normal follicles. There was severe reduction of the labelling to IGF‐I in the GC of the follicular and luteinized cysts. In the normal follicles, the reactivity for IGF‐II was very similar to pattern noted in IGF‐I. There was reduction of the IHCSAs in the GC of the follicular and luteinized cysts, but the decrease was not significant. The staining of the IGF‐II in the TI and TE of the cysts was increased, in comparison with normal follicles. The IHCSAs for VEGF were higher in the GC and TE of the normal follicles in contrast to TI, but this difference was noted only in the tertiary follicle. The VEGF reactivity increased in the GC of the cysts, in relation to normal follicles. The results of the current study show that the formation of ovarian cysts in sows is associated with alterations in the immunohistochemical expression of some growth factors.     

Apoptosis in the Antral Follicles of Swamp Buffalo and Cattle Ovary: TUNEL and Caspase-3 Histochemistry

The present study was carried out to investigate the pattern of apoptosis in the healthy antral and atretic follicles of Philippine swamp buffaloes (BU) in comparison with Holstein-Friesian (HF) cows. Paraffin sections of healthy follicles and various stages of atretic follicles were stained using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labelling (TUNEL) method to detect DNA fragmentation and cleaved caspase-3 antibody to detect cells committed to undergo apoptosis. Five equidistant areas of a follicle were counted for the presence of TUNEL- and caspase-3-positive cells. Healthy follicles of BU and HF contained no TUNEL-positive cells in the granulosa and theca layer but showed some caspase-3 positivity. The granulosa layer of advanced atretic follicles showed a significantly higher frequency of caspase-3 positivity than the healthy and early atretic follicles in both breeds. The frequency of caspase-3-positive cells of BU was significantly higher than HF in the granulosa layer of healthy, early atretic and advanced atretic follicles. In the theca interna layer, BU and HF showed a significantly lower and higher frequency of TUNEL-positive cells in the late atretic follicles compared with advanced atretic follicle, respectively. However, the frequency of caspase-3-positive cells of both BU and HF in the late atretic follicles was significantly higher than the advanced atretic follicles in the theca interna layer. These results indicate that caspase-3 and DNA fragmentation is involved in the buffalo ovarian apoptotic process.     

The Influence of Ovarian Stromal/Theca Cells During In Vitro Culture on Steroidogenesis,Proliferation and Apoptosis of Granulosa Cells Derived from the Goat Ovary

Early follicular development is closely related to oocyte‐granulosa cells‐ovarian stromal cells/theca cells. The aim of the present study was to investigate the effects of ovarian cortical, medullary stromal and theca cells on oestradiol and progesterone biosynthesis, proliferation and apoptosis of goat ovary granulosa cells in vitro. Using Transwell coculture system, we evaluated steroidogenesis, cell proliferation and apoptosis, and some molecular expressions regarding steroidogenic enzyme, luteinizing hormone receptor and apoptosis‐related genes in granulosa cells. The results indicated that ovarian stromal/theca cells were able to stimulate oestradiol and progesterone production, promote cell proliferation and inhibit apoptosis of granulosa cells. Among all the three kinds of cells, theca cells affected strongly on granulosa cell function, and ovarian medullary stromal cells had the weakest effect on granulosa cells. These findings would provide an important knowledge of cell interaction among follicular cells during follicular development.     

Regulation mechanism of selective atresia in porcine follicles: regulation of granulosa cell apoptosis during atresia

More than 99% of follicles undergo a degenerative process known as "atresia", in mammalian ovaries, and only a few follicles ovulate during ovarian follicular development. We have investigated the molecular mechanism of selective follicular atresia in mammalian ovaries, and have reported that follicular selection dominantly depends on granulosa cell apoptosis. However, we have little knowledge of the molecular mechanisms that control apoptotic cell death in granulosa cells during follicle selection. To date, at least five cell death ligand-receptor systems [tumor necrosis factor (TNF)alpha and receptors, Fas (also called APO-1/CD95) ligand and receptors, TNF-related apoptosis-inducing ligand (TRAIL; also called APO-2) and receptors, APO-3 ligand and receptors, and PFG-5 ligand and receptors] have been reported in granulosa cells of porcine ovaries. Some cell death ligand-receptor systems have "decoy" receptors, which act as inhibitors of cell death ligand-induced apoptosis in granulosa cells. Moreover, we showed that the porcine granulosa cell is a type II apoptotic cell, which has the mitochondrion-dependent apoptosis-signaling pathway. Briefly, the cell death receptor-mediated apoptosis signaling pathway in granulosa cells has been suggested to be as follows. (1) A cell death ligand binds to the extracellular domain of a cell death receptor, which contains an intracellular death domain (DD). (2) The intracellular DD of the cell death receptor interacts with the DD of the adaptor protein (Fas-associated death domain: FADD) through a homophilic DD interaction. (3) FADD activates an initiator caspase (procaspase-8; also called FLICE), which is a bipartite molecule, containing an N-terminal death effector domain (DED) and a C-terminal DD. (4) Procaspase-8 begins auto-proteolytic cleavage and activation. (5) The auto-activated caspase-8 cleaves Bid protein. (6) The truncated Bid releases cytochrome c from mitochondrion. (7) Cytochrome c and ATP-dependent oligimerization of apoptotic protease-activating factor-1 (Apaf-1) allows recruitment of procaspase-9 into the apoptosome complex. Activation of procaspase-9 is mediated by means of a conformational change. (8) The activated caspase-9 cleaves downstream effector caspases (caspase-3). (9) Finally, apoptosis is induced. Recently, we found two intracellular inhibitor proteins [cellular FLICE-like inhibitory protein short form (cFLIPS) and long form (cFLIPL)], which were strongly expressed in granulosa cells, and they may act as anti-apoptotic/survival factors. Further in vivo and in vitro studies will elucidate the largely unknown molecular mechanisms, e. g. which cell death ligand-receptor system is the dominant factor controlling the granulosa cell apoptosis of selective follicular atresia in mammalian ovaries. If we could elucidate the molecular mechanism of granulosa cell apoptosis (follicular selection), we could accurately diagnose the healthy ovulating follicles and precisely evaluate the oocyte quality. We hope that the mechanism will be clarified and lead to an integrated understanding of the regulation mechanism.     

Lectin histochemical pattern on the normal and cystic ovaries of sows

The lectin histochemical pattern (LHP) was characterized and compared in normal and cystic ovaries of sows. Six biotinylated lectins (PNA, SBA, WGA, RCA‐1, DBA and UEA‐1) were used on tissue sections. In the normal ovaries, the reaction to UEA‐1 and SBA was mild to moderate in mesothelial and endothelial cells. RCA‐1 staining was mild to moderate in theca interna of growing follicles, corpora luteum and mesothelium. In addition, this lectin presented strong reaction in endothelial cells, granulosa cells of atretic follicles, zona pellucida of growing follicles and plasma. DBA showed strong intensity in mesothelial and endothelial cells. There was mild to moderate reactivity to WGA in granulosa cells, corpus luteum and theca interna of follicles in development, and moderate in zona pellucida, in granulosa cells of atretic follicles and mesothelium. PNA staining was mild to moderate in oocytes and in the adventitia and media of medullary arteries. Changes in the LHP of the cystic ovaries were noted; however, there were no differences in these findings between the follicular and luteinized cysts. UEA‐1 reactivity in the cystic ovaries was moderately reduced in the mesothelial and endothelial cells, whereas there was mild reduction in the DBA staining in the granulosa cells. Reaction to RCA‐1 and WGA in the cysts also was decreased in theca interna, zona pellucida and granulosa cells of atretic follicles. Furthermore, endothelium and theca interna in the cystic ovaries presented mild reduction of marcation to SBA, whereas there was decreased reactivity to PNA in the oocytes and adventitia and media layers of the medullary arteries. The results of the current study show that cysts modify the LHP in swine ovaries. These changes of glycoconjugates in many ovarian structures could modify diverse process and may be one of the reasons for decreased fertility in sows.