Expression and Preliminary Functional Profiling of the let-7 Family during Porcine Ovary Follicle Atresia

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.     

Cholesterol side-chain cleavage cytochrome P450 and 3beta-hydroxysteroid dehydrogenase expression and the concentrations of steroid hormones in the follicular fluids of different phenotypes of healthy and atretic bovine ovarian follicles

Bovine ovarian antral follicles exhibit either one or the other of two patterns of granulosa cell death in atresia. Death can commence either from the antrum and progress toward the basal lamina (antral atresia) or the converse (basal atresia). In basal atresia, the remaining live antrally situated cells appeared to continue maturing. Beyond that, little is known about these distinct patterns of atresia. Healthy (nonatretic) follicles also exhibit either one or the other of two patterns of granulosa cell shape, follicular basal lamina ultrastructure or location of younger cells within the membrana granulosa. To examine these different phenotypes, the expression of the steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells and concentrations of steroid hormones in follicular fluid were measured in individual histologically classified bovine antral follicles. Healthy follicles first expressed SCC and 3beta-HSD in granulosa cells only when the follicles reached an approximate threshold of 10 mm in diameter. The pattern of expression in antral atretic follicles was the same as healthy follicles. Basal atretic follicles were all <5 mm. In these, the surviving antral granulosa cells expressed SCC and 3beta-HSD. In examining follicles of 3-5 mm, basal atretic follicles were found to have substantially elevated progesterone (P < 0.001) and decreased androstenedione and testosterone compared to healthy and antral atretic follicles. Estradiol was highest in the large healthy follicles, lower in the small healthy follicles, lower still in the antral atretic follicles, and lowest in the basal atretic follicles. Our findings have two major implications. First, the traditional method of identifying atretic follicles by measurement of steroid hormone concentrations may be less valid with small bovine follicles. Second, features of the two forms of follicular atresia are so different as to imply different mechanisms of initiation and regulation.     

Steroidogenesis in porcine atretic follicles: loss of aromatase activity in isolated granulosa and theca

To evaluate the mechanisms involved in the reduction of estrogen concentrations in porcine follicular fluid during atresia, nonatretic and atretic follicles ranging from 4 to 7 mm in diameter were selected. Follicular fluid estrogen concentrations were 7-16-fold less in the atretic follicles. Isolated granulosa cells from atretic follicles demonstrated a significant reduction in aromatase activity and in follicle-stimulating hormone (FSH)-induced progesterone production in vitro compared to granulosa cells from nonatretic follicles. Isolated theca from atretic follicles also demonstrated a reduction in estrogen production. However, androgen concentrations were equivalent in the follicular fluid of atretic and nonatretic follicles, and theca from atretic follicles maintained testosterone and androstenedione production in vitro. The loss of thecal aromatase activity with atresia is not secondary to a reduction in FSH responsiveness, since FSH did not increase thecal progesterone production in vitro. Cell degeneration also does not account for the reduction in thecal estrogen production, since both androgen output in vitro and follicular fluid androgen concentrations were maintained. These data thus demonstrate that a mechanism other than reduced FSH responsiveness must account for the selective loss of thecal aromatase activity in this stage of atresia.     

Mechanism of granulosa cell death during follicular atresia depends on follicular size

Changes in granulosa cell lysosomal and mitochondrial functions in relation to follicular size and to the stage of atresia were studied by fluorescent emission spectra and intensity using flow cytometry. Antral follicles were grouped by size in two groups: small, 3-6 mm and large, >6mm in diameter, and classified into three stages of atresia: non-atretic, initially atretic and advanced atretic. Differences in Rhodamine 123 (Rh123) and Acridine Orange (AO) fluorescent intensity indicated that changes in mitochondrial function are the primary mechanism of granulosa cell death in atretic follicles 3-6 mm in diameter, while its role in granulosa cell death in >6 mm atretic follicles seemed to be less important. However, modifications in lysosomal function (shown by a decrease in fluorometric intensity of AO incubated granulosa cells) were mainly associated with cell death in large atretic follicles. Our results support the hypothesis that the pathway of granulosa cell death during follicular atresia depends on the state of energy metabolism or on the production of hypoxic conditions related to follicular size. Changes in mitochondrial membrane potential and production of permeability transition pores were the main changes found in small follicles, while lysosomal function destabilization seemed to be the major cause of granulosa cell death during atresia in large follicles.     

Comparison of glucose-6-phosphate dehydrogenase activity in healthy and atretic follicles in rat ovary

Changes in the glucose-6-phosphate dehydrogenase activity have been determined in relation to atresia of Graafian follicles in the rat ovary. Induction of atresia in follicles either due to absence of hCG in the hormonally stimulated immature ovaries or by repeated injections of pentobarbitone sodium to proestrous rats caused significant rise in the enzyme activity. Measurement of enzyme activity in isolated follicular compartments of healthy and atretic follicles revealed that it is significantly higher in the thecal tissue than the granulosa. Increase in enzyme activity in the atretic follicles than the healthy ones occurs due to its rise both in theca and granulosa cells. The significance of these changes in the enzyme activity in healthy and atretic follicles are discussed in relation to the precocious luteinization of cells in the follicular envelope with the onset of atresia.     

Chemotactic detection of incipient ovarian follicular atresia.

Chemotactic activity was measured in the follicular fluid collected from normal and atretic Graafian follicles isolated from the rat ovaries. The atresia of Graafian follicles was induced by pentobarbitone injections for 3 days beginning the day of proestrous. The chemotactic activity, as measured by direct morphological evaluation of cellular locomotion of individual cells and Boyden leading front assay, was significantly higher in follicular fluid from atretic follicles and it showed a progressive increase from day 1 to day 3 of blockade of ovulation. In vitro exposure of blocked follicles to PMSG and hCG on day 1, 2 and 3 failed to alter the chemotactic response of leukocytes towards follicular fluid of atretic follicles. Increased chemotaxis in the follicular fluid after 24 hr of blockade of ovulation appears to form an important criterion to identify atretic follicles well in advance, before the morphological symptoms of degeneration become apparent and the incipient change once induced in follicles is not reversed by gonadotropins.     

Follicular atresia and LH concentrations during the follicular phase of the estrous cycle in the goat (Capra hircus)

The objective of the study was to identify the effects of LH on the final follicle maturation process as well as the incidence of atresia during the follicular phase of the goat's estrous cycle. In Experiment 1, concentrations of the LH were measured during the follicular phase of a synchronized cycle in 8 Canary goats. In Experiment 2, the same animals were synchronized again. On each day of a 4-day experimental period (day 0=day of sponges withdrawal), 2 of the goats were bilaterally ovariectomized. Follicles with a diameter >1 mm were dissected out to obtain qualitative histological data in normal, early atretic I, early atretic II, advanced atretic I and advanced atretic II follicles. The total interval from sponge withdrawal to LH peak was 77.5±9.8 h. LH peak concentration averaged 44±5.3 ng/ml and the mean length of the preovulatory surge (amounts over 10 ng/ml) was 8.9±0.9 h. During the total follicular phase, there were more atretic follicles than normal follicles (58 vs. 30, P<0.05). The number of early and advanced atretic follicles was similar. There were more early atresia I than early atresia II follicles (23 vs. 6, P<0.05). On day 2, the number of advanced atretic follicles was greater than early atretic follicles (10 vs. 4, P<0.05). There was an increase in the number of early atretic follicles from day 2 to day 4 (4 vs. 9, P<0.05), which was consistent with the effects of the preovulatory LH surge.     

Size-frequency analysis of atresia in cycling rats

The purpose of this study was to delineate when, during follicular growth, the alternative developmental pathways leading to ovulation or atresia diverge. By using computerized image analysis techniques, random samples of healthy and atretic follicles in ovaries of cycling rats were subjected to size-frequency analysis. The vast preponderance of atretic follicles were of the early antral size class (approximately 300-350 micron diameter, 800-1000 granulosa cells in the largest cross-section); atretic small follicles (less than 250 granulosa cells in the largest cross-section) were rare. Follicles in early stages of atresia were uncommon in ovaries of animals killed at estrus, but were found with great frequency in ovaries of animals killed the following day (metestrus). These results suggest that, under normal cyclic conditions, there may be only one major branching point during follicular development when growing follicles become susceptible to atresia. The alternative developmental pathways leading to ovulation and atresia may not diverge until the penultimate stage of growth, immediately preceding the final transformation into a preovulatory follicle.     

Multiparametric study of atresia in ewe antral follicles: histology, flow cytometry, internucleosomal DNA fragmentation, and lysosomal enzyme activities in granulosa cells and follicular fluid

The differential quantitative participation of apoptosis and necrosis in ewe antral follicles of two different sizes, separated in four stages of atresia using macroscopic, histologic, and esteroid quantification methods was assessed. Annexin V binding and propidium iodide (PI) uptake was used to detect healthy live cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V+/PI-), and necrotic or late apoptotic cells (PI+). Additionally we used internucleosomal DNA fragmentation as a quantitative estimate of apoptosis. Presence and distribution of lysosomal enzymes in follicular fluid and granulosa cells was used as a measure of necrotic cell death. DNA flow cytometry and gel electrophoresis were positively correlated with the progression of atresia, small atretic follicles tend to have higher percentages of internucleosomal cleaved DNA than follicles >6 mm. Annexin/PI binding also indicates that apoptosis and necrosis increase with atresia progression, generally apoptosis outweighs necrosis in small follicles. Acid phosphatase and glucosaminidase in follicular fluid of 3-6 mm follicles showed no significant modifications between healthy and initially atretic follicles, and only a small, but significant increase in activity in advancedly atretic follicles. On the contrary, lysosomal enzyme activity in follicles >6 mm showed positive correlation between atresia stages and the activities of acid phosphatase and glucosaminidase in follicular fluid. A similar size-differential behavior was found in free or membrane-bound lysosomal enzyme activity of granulosa cells. Necrosis, but principally apoptosis, were present during all stages of follicular maturation indicating that growth and maturation of ovarian follicles involves a continuous renewal of granulosa cells, regulated by apoptosis. Mechanisms regulating this equilibrium may participate in the final destiny, whether ovulation or atresia of ovarian follicles.     

Differential steroid and gonadotropin response by individual tertiary porcine follicles in vitro. Possible physiologic role of atretic follicles

Since atretic follicles contain significant amounts of androgen and/or progesterone in their follicular fluid, we examined whether they also contribute to ovarian steroid secretion. Steroid secretion by atretic porcine follicles and their responsiveness to FSH was assessed by a perifusion system that allows for separate dynamic incubation of whole follicles in vitro. Identically treated nonatretic follicles of comparable size served as a reference group. The extent of granulosal pyknosis, on which the staging of atresia was based, was inversely related to follicular estradiol (E2) secretion and its responsiveness to FSH. Both basal and FSH-stimulated secretion of testosterone (T), androstenedione (A), and progesterone (P) were maintained by follicles in all stages of atresia. Secretion of A by late atretic follicles was greater than that in earlier stages or by nonatretic follicles. Atretic follicles may therefore release comparable or larger amounts of androgen and P into their intraovarian environment than do nonatretic follicles. We examined whether steroids secreted by atretic follicles in vitro could be utilized by nonatretic follicles. A static incubation system was used that allows for simultaneous incubation of a number of individual follicles. When nonatretic follicles were exposed to A, T, or P in physiologic concentrations (10(-7)-10(-5) M), their secretion of E2 increased 2-8-fold. Doses of FSH or LH that stimulated follicular steroid in vitro had no additional stimulatory effect when combined with A or P treatment, respectively. In conclusion, atretic follicles may contribute significantly to intraovarian levels of androgen and P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of gelatinase on follicular atresia in the bovine ovary

Follicular atresia, like follicular growth and ovulation, is characterized by excessive tissue remodeling. It is hypothesized that probably one of the tissue-remodeling enzymes, such as the gelatinases, could be playing an important role in this process. The present study was undertaken to determine the role of gelatinase on follicular atresia in the cow. Follicles of 2-6 mm in diameter were dissected from ovaries, and follicular fluid was categorized according to the morphological appearance of the cumulus-oocyte complexes. Gelatinase activity within the follicular fluid was analyzed by gelatin zymography, and film in situ zymography was employed in order to localize gelatinase. TUNEL was performed on cryosectioned ovaries to understand follicular health. The concentrations of steroids in follicular fluid were also measured by solid phase fluoroimmunoassay. ProMMP-2 was detected in all normal and atretic categories of follicular fluid. The active form of MMP-2 and an additional band of proMMP-9 were detected only in atretic follicular fluid. Gelatinase activity was recorded in both granulosa cells (GCs) and theca cells (TCs) but were found in comparatively higher numbers in those follicles that exhibited a thinned and partially detached granulosa layer. TUNEL confirmed that apoptosis had commenced in the GCs of follicles of the latter category. The estradiol-17beta (E(2)):progesterone (P(4)) ratio was found to be significantly lower in atretic follicles than in normal follicles. These results suggest a plausible role for gelatinase in follicular health, especially the active form of MMP-2 and proMMP-9, and that bovine follicular fluid may be a key indicator of atresia.     

Morphological and biochemical correlates of equine ovarian follicles as a function of their state of viability or atresia.

The histological features and hormonal content of follicular fluid of antral follicles during oestrus were correlated. As a result it was possible to characterize several categories of viable and atretic follicles. A seemingly important stage in maturation appeared to be at 3 cm in diameter since follicular oestrogens and androgens underwent a 3-fold increase in concentration at that size. Evidence was obtained to suggest that oestrogens are anti-atretogenic. However, a drop in oestrogens was not the cause of atresia since degeneration commenced when levels were high. Contrary to the concept that androgens are atretogenic in some species, it was also evident that elevated androgens did not precipitate spontaneous atresia. Theca epithelioid cells not only underwent histological luteinization in viable follicles as they matured toward ovulation but occasionally in atretic follicles as well. Elevated prostaglandin F levels were associated with follicles in the transitory states of either luteinization or atresia. Granulosa cells of viable follicles only were capable of specifically binding hCG. It was not determined whether loss of binding capacity or atresia occurred first. Follicular atresia in the mare appears to be a gradual process of which the initiating cause remains unknown.     

Oxygen consumption by rat oocytes and cumulus cells during induced atresia

Oxygen consumption was measured in denuded oocytes and oocyte-cumulus complexes isolated from atretic rat follicles. Adult cyclic rats or immature PMSG-treated rats were used, and follicular atresia was induced by hypophysectomy on the morning of pro-oestrus or by repeated pentobarbitone injections beginning on the day of pro-oestrus. The later stages of atresia were accompanied by meiosis-like changes in the oocytes. Oxygen consumption by oocytes that had resumed meiosis (germinal vesicle breakdown, GVB) was higher than in oocytes with an intact germinal vesicle, a change similar to that observed in oocytes maturing in healthy follicles. This may indicate that the meiotic process in the atretic follicles is similar to that in normal ones. Oxygen consumption by the cumulus cells was not altered during pentobarbitone-induced atresia. Hypophysectomy led to a rapid and marked increase in cumulus oxygen consumption in cyclic rats but there was no change in PMSG-treated young animals. Since both pentobarbitone-treatment and hypophysectomy result in follicular atresia, but changes in cumulus respiration occurred only in hypophysectomized adult rats, it is concluded that an increase in cumulus respiration is not inherent to the atretic process.     

Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

Development and physiology of follicular atresia during ovarian growth in the house fly, Musca domestica

Atretic follicles regularly occur in the ovary of the house fly, Musca domestica. The frequency of ovarian follicular atresia and the proportion of atretic follicles per ovary are related to the stage of oögenesis and to the age of the females. Only vitellogenic follicles may become atretic. The atresia may occur at any stage of vitellogenesis, though most follicles become atretic in mid-vitellogenesis. Atretic follicles are completely resorbed within 24–36 hr. The follicle cells may play a synthesizing role during growth and disintegrating one during follicle resorption. The induction of glycogen synthesis by the cessation of RNA and protein synthesis and by vitellogenesis in normal follicles is discussed. The same processes occur prematurely in the atretic follicle which can be thus distinguished by a high content of glycogen.     

A comparison of histological and non-histological indices of atresia and follicular function

Histological indices of atresia for bovine follicles greater than or equal to 5 mm in diameter were compared with potential non-histological indices of atresia such as opaqueness of the exposed surface of non-excised follicles, concentrations of steroids in follicular fluid (FF) and specific binding of gonadotropins by granulosal cells. Each non-excised follicle was classified as clear (n=86), intermediate (n=79), or opaque (n=115), on the basis of the appearance of its exposed surface. A section of tissue from each follicle was evaluated histologically for atresia and assigned to one of the following categories: non-atretic, intermediately atretic, strongly atretic, or luteinized-atretic. Concentrations of estradiol (E), progesterone (P), and testosterone (T) and capacity of granulosal cells to bind radioactive ovine follicle-stimulating hormone (oFSH) and human chorionic gonadotropin (hCG) were determined for each follicle. Overall incidence of atresia was similar for clear (n=66%), intermediate (60%), and opaque (72%) follicles. Opaque follicles, however, were more likely to be strongly atretic (42%) than were clear (21%) or intermediate (23%) follicles. Non-atretic and intermediately atretic follicles had similar concentrations of E, P, and T and similar capacities to bind gonadotropins. Strongly atretic and luteinized-atretic follicles contained a higher concentration of P, lower E, and a reduced capacity of granulosal cells to bind oFSH than non-atretic and intermediately atretic follicles. A ratio of P:E in FF greater than or equal to 10 usually (greater than 90%) indicated that a follicle was atretic. However, lesser ratios of P:E did not accurately indicate whether follicles were atretic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis and ovarian function: novel perspectives from the teleosts

Apoptosis is a fundamental mechanism in follicular atresia and postovulatory regression in mammals, but its role in teleost ovarian function is currently unknown. This study tested the hypotheses that apoptosis mediates follicular atresia in teleosts and is inducible in vitro by incubation in serum-free conditions. Vitellogenic follicles from rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) were incubated overnight in serum-free medium and examined for apoptosis by 3'-end-labeling and/or TUNEL analysis. Primary, postovulatory, and oocytectomized vitellogenic trout follicles and atretic goldfish follicles were evaluated in similar fashion. Overall, goldfish follicles had lower levels of DNA fragmentation than trout follicles. The DNA fragmentation in atretic goldfish follicles was similar to that measured in healthy vitellogenic and prematurational follicles; DNA fragmentation did not change after incubation. In the trout, postovulatory and oocytectomized vitellogenic follicles showed significantly greater in vitro susceptibility to apoptosis than intact vitellogenic follicles, whereas primary follicles were least susceptible. The TUNEL analyses revealed that in trout vitellogenic follicles, more thecal/epithelial cells than granulosa cells showed fragmented DNA in vivo, but incubation (24 h) did not result in increased apoptosis in cells of either type. These results indicate that apoptosis is involved in normal ovarian growth and postovulatory regression in teleosts, but that it does not appear to be an early event in teleost follicular atresia.     

Involvement of cell proliferation in the process of follicular atresia in the guinea pig

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs.